Bio

Clinical Focus


  • Blood and Marrow Transplantation
  • Hematology
  • Cancer > Hematology
  • Cancer > Blood and Marrow Transplant

Academic Appointments


Administrative Appointments


  • Medical Director, Clinical Bone Marrow Transplantation Laboratory (1990 - Present)
  • Division Chief, Blood and Marrow Transplant Program, Stanford University (2000 - Present)

Honors & Awards


  • Fellowship, Damon Runyon-Walter Winchell Cancer Fund (1988-1991)
  • Fellow, Jose Carreras International Leukemia Foundation (1993-1996)
  • President, International Society of Cellular Therapy (2000-2002)
  • Distinguished Clinical Scientist Award, Doris Duke Foundation (2004-2009)
  • President, American Society of Blood and Marrow Transplantation (2006-2007)
  • Member, Association of American Physicians (AAP) (2008)
  • Associate Editor, Blood (2010-present)

Professional Education


  • Residency:Stanford University School of Medicine (1987) CA
  • Internship:Stanford University School of Medicine (1985) CA
  • Fellowship:Stanford University School of Medicine (1987) CA
  • Board Certification: Hematology, American Board of Internal Medicine (1992)
  • Board Certification: Internal Medicine, American Board of Internal Medicine (1987)
  • Medical Education:Harvard Medical School (1984) MA
  • BA, Univ. California, Berkeley, Biochemistry (1977)
  • MD, Harvard Medical School, Medicine (1984)

Community and International Work


  • Volunteer

    Topic

    Fund raising for research and patient support

    Partnering Organization(s)

    Leukemia and Lymphoma Society

    Location

    US

    Ongoing Project

    Yes

    Opportunities for Student Involvement

    No

Research & Scholarship

Current Research and Scholarly Interests


Research projects in our laboratory are aimed at studying the biology of cell populations capable of inducing or suppressing graft vs host disease as well as cells capable of promoting a graft vs tumor effect. In particular we are studying:

1) The clinical utility of expanded cytotoxic cells for immunotherapy. We have developed animal models utilizing mice with severe combined immunodeficiency (SCID) which will accept human tumor cells or murine model systems of syngeneic and allogeneic transplantation. In some instances the tumor cells are transfected with the bioluminescent marker luciferase so that the tumor cell growth can be quantitatively tracked in living animals. Using these model system we are studying the clinical efficacy of the expanded cytotoxic cells.

2) The interaction between the cytotoxic effector cells and a variety of tumor cell targets is under study. The role of granzyme/perforin and fas mediated pathways as well as the cell surface molecule NKG2D in cytotoxicity is under study.

3) We are exploring the biological impact of phenotypically defined populations of regulatory T cells on graft vs host disease and graft vs tumor reactions.

4) We are utilizing bioluminescent techniques to study these complex biological processes by either labelling the tumor or effector cell populations with the light emitting luciferase gene such that small numbers of cells can be tracked non-invasively, sensively and quantitatively.

Clinical Trials


  • Protocol For A Research Database For Hematopoietic Stem Cell Transplantation, Other Cellular Therapies and Marrow Toxic Injuries Recruiting

    The primary purpose of the Research Database is to have a comprehensive source of observational data that can be used to study HSC transplantation. A secondary purpose of the Research Database is to have a comprehensive source of data to study marrow toxic injuries. Objectives: To learn more about what makes stem cell transplants work well, such as determining the following: - how well recipients recover from their transplant - how recovery after a transplant can be improved - how access to transplant for different groups of patients can be improved - how well donors recover from the collection procedures

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  • Bone Marrow Grafting for Leukemia and Lymphoma Recruiting

    Bone Marrow Grafting for Leukemia and Lymphoma

    View full details

  • High Dose Sequential Therapy and Autologous Stem Cell Rescue for Multiple Myeloma Not Recruiting

    To assess the role of autologous hematopoietic cell rescue in the treatment of multiple myeloma.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

    View full details

  • Transplantation for Patients With Chronic Lymphocytic Leukemia Not Recruiting

    To evaluate the role of high dose therapy and autologous or allogeneic hematopoietic cell transplantation for the treatment of chronic lymphocytic leukemia.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Fluticasone Propionate, Azithromycin, and Montelukast Sodium in Treating Patients With Bronchiolitis Obliterans Who Previously Underwent Stem Cell Transplant Not Recruiting

    This phase II trial studies how well giving fluticasone propionate, azithromycin, and montelukast sodium (FAM) together works in treating patients with bronchiolitis obliterans who previously underwent stem cell transplant. FAM may be an effective treatment for bronchiolitis obliterans

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, 650-723-0822.

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  • Prophylactic Use of Maribavir for the Prevention of Cytomegalovirus (CMV) Disease in Stem Cell Transplant Recipients Not Recruiting

    The purpose of this research study is to investigate whether or not maribavir is safe and effective for preventing CMV disease when taken by mouth for up to 12 weeks in patients who have had a stem cell transplant.

    Stanford is currently not accepting patients for this trial. For more information, please contact Janice Brown, (650) 723 - 0822.

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  • Post T-plant Infusion of Allogeneic Cytokine Induced Killer Cells as Consolidative Therapy in Myelodysplastic Syndromes/Myeloproliferative Disorders Recruiting

    This study examines CIK (Cytokine Induced Killer Cells) as Consolidative Therapy after Non-Myeloablative Allogeneic Transplantation.

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  • Acute Graft-versus-Host Disease Treatment (BMT CTN 0802) Not Recruiting

    The study is a Phase III, randomized double blind, placebo controlled, and trial evaluating the addition of MMF vs. placebo to systemic corticosteroids as initial therapy for acute GVHD. The primary endpoint will be GVHD free survival at Day 56 post randomization.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Nilotinib and Imatinib Mesylate After Donor Stem Cell Transplant in Treating Patients With Acute Lymphoblastic Leukemia or Chronic Myelogenous Leukemia Not Recruiting

    This phase I/II trial is studying the side effects and best way to give nilotinib when given together with imatinib mesylate after donor stem cell transplant in treating patients with acute lymphoblastic leukemia or chronic myelogenous leukemia. Nilotinib and imatinib mesylate may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth.

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, (650) 723 - 0822.

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  • Autologous Bone Marrow Transplantation in Acute Non-Lymphoblastic Leukemia During First or Subsequent Remission Not Recruiting

    Evaluate the role of high dose chemotherapy with autologous hematopoietic cell transplantation for AML.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Haploid Allogeneic Transplant Using the CliniMACS System Not Recruiting

    To assess the proportion of patients with donor neutrophil engraftment on or before day 30 post transplant. To assess the incidence of acute GvHD during the first 100 days after transplantation.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • TLI & ATG for Non-Myeloablative Allogeneic Transplantation for MDS and MPD Not Recruiting

    To evaluate the feasibility and safety of TLI/ATG conditioning for allogeneic HCT for elderly patients with advanced stage MDS and MPD.

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, 650-723-0822.

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  • Phase II Poor Risk Diffuse Large B-cell Lymphoma (DLBCL) of Total Lymphoid Irradiation (TLI) and Antithymocyte Globulin (ATG) Followed by Matched Allogeneic Hematopoietic Transplantation as Consolidation to Autologous Hematopoietic Cell Transplantation (AHCT) Not Recruiting

    The purpose of this study is to develop an alternative treatment for patients with relapsed diffuse large B cell lymphoma who are not likely to be cured by the conventional transplantation regimen.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Allo BMT in Advanced Leukemia or High Grade Lymphoma Not Recruiting

    To evaluate the role of ablative allogeneic hematopoietic cell transplantation in the treatment of advanced leukemia or lymphoma.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Cyclosporine Eye Drops in Preventing Graft-Versus-Host Disease of the Eye in Patients Who Have Undergone Donor Stem Cell Transplant for Hematologic Cancer or Bone Marrow Failure Disorder Not Recruiting

    RATIONALE: Cyclosporine eye drops may prevent graft-versus-host disease of the eye in patients who have undergone donor stem cell transplant for hematologic cancer or bone marrow failure disorder. PURPOSE: This randomized phase I trial is studying how well cyclosporine eye drops work in preventing graft-versus-host disease of the eye in patients who have undergone donor stem cell transplant for hematologic cancer or bone marrow failure disorder.

    Stanford is currently not accepting patients for this trial. For more information, please contact Joanne Otani, (650) 721 - 2372.

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  • A Phase 3 Study of Brentuximab Vedotin (SGN-35) in Patients at High Risk of Residual Hodgkin Lymphoma Following Stem Cell Transplant (The AETHERA Trial) Not Recruiting

    This is a randomized, double-blind, placebo-controlled, multicenter phase 3 trial to evaluate the efficacy and safety of brentuximab vedotin (SGN-35) and best supportive care (BSC) compared to placebo and BSC in treatment of residual Hodgkin lymphoma (HL) following autologous stem cell transplant (ASCT).

    Stanford is currently not accepting patients for this trial. For more information, please contact Sarah Robeson, (650) 725 - 1647.

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  • Non-Myeloablative Allogeneic Transplant for Myelodysplastic Syndromes and Myeloproliferative Disorders Not Recruiting

    To improve survival outcomes for patients with MDS and MPD with a nonmyeloablative allogeneic hematopoietic cell transplant.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Post-transplant Autologous Cytokine-induced Killer (CIK) Cells for Treatment of High Risk Hematologic Malignancies Not Recruiting

    The purpose of the study is to conduct a phase I study of adoptive immunotherapy with autologous, ex-vivo expanded cytokine-induced killer (CIK) cells to reduce the relapse rate in autologous stem cell transplant patients with high-risk hematologic malignancies.

    Stanford is currently not accepting patients for this trial. For more information, please contact Sherry Moore, (650) 725 - 7951.

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  • Allogeneic HCT Using Nonmyeloablative Host Conditioning With TLI & ATG vs SOC in AML Not Recruiting

    Acute myeloid leukemia (AML) is a cancer of the bone marrow that mostly affects older adults. Even with the best chemotherapy, two-year disease-free survival is achieved in a minority of patients. Bone marrow transplantation from a sibling donor may improve cure rates; however, patients over 50 years of age have a high risk of complications and therefore generally are excluded from this treatment option. Recently our group developed a transplantation strategy for older cancer patients that protects against transplant-associated complications, yet does not interfere with the ability of the transplanted donor cells to destroy cancer cells. With this new method, we can now safely evaluate transplantation as a curative therapy for AML patients over the age of 50. We have assembled clinical and scientific researchers throughout the state of California to study and compare bone marrow transplantation using our new approach with the best standard of care chemotherapy in AML patients over the age of 50. The results of this study have the potential to establish a new treatment standard that will improve survival of older AML patients.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, 650-725-1647.

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  • Allogeneic Transplantation From Related Haploidentical Donors in Older Patients With Indolent Hematologic Malignancies Not Recruiting

    The purpose of the study is to evaluate the feasibility and safety of transplanting CD34+ selected hematopoietic cells from a haploidentical related donor following a nonmyeloablative regimen of total lymphoid irradiation (TLI) and antithymocyte globulin (ATG).

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • 90Y-IBRITUMOMAB Tiuxetan and AHCI With HD Chemotherapy and Autologous Transplantation for Relapsed or Resistant NHL Not Recruiting

    To test a new way to approach hematopoietic stem cell transplantation for Relapsed or Resistant Non-Hodgkin's Lymphoma.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Intravenous Administration of RGI-2001 in Patient Undergoing Allogenic Hematopoietic Stem Cell Transplantation (AHSCT) Recruiting

    The clinical trial is a Phase 1/2a, open-label, multi-center, dose-escalation study to evaluate the safety, tolerability and pharmacokinetic profile of RGI-2001 in patients undergoing AHSCT, with radiation or non-radiation myeloablative preparative treatment. The study will be separated into two parts; a dose escalation phase to assess safety, followed by a large expansion phase to further evaluate the pharmacologic effects of either a Maximum Tolerated Dose, Maximum Feasible Dose or optimal pharmacologically active dose of RGI-2001. The initial dose escalation safety portion of the study (Part 1) will include higher risk patients and limit the unrelated donor transplants. After safety is established in part 1 of the study, the second portion of the study will expand the enrollment criteria and allow transplantation by either related or unrelated donors. This study will endeavor to identify the dose range at which RGI-2001 has an acceptable safety profile, at which biologic activity is observed, and to guide possible dose levels to utilize in later phase studies based on biological activity.

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  • Phase I/II MAHCT w/ TCell Depleted Graft w/ Simultaneous Infusion Conventional and Regulatory T Cell Recruiting

    For patients with hematologic malignancies undergoing allogeneic myeloablative (MA) HCT with a T cell depleted graft, the infusion of naturally occurring regulatory T cells with conventional T cells (T cell addback) in pre-defined doses and ratios will reduce the incidence of acute graft vs host disease while augmenting the graft vs leukemia effect and improving immune reconstitution.

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  • Phase I/II of a CpG-Activated Whole Cell Vaccine Followed by Autologous Immunotransplant for MCL Recruiting

    While autologous hematopoietic stem cell transplant (AHCT) has been shown to cure some subtypes of non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL) still has a prognosis marked by relapse. This study will evaluate the safety and efficacy of treating newly diagnosed MCL patients with an autologous, tumor-derived vaccine followed by re-infusion of vaccine-primed T cells combined with standard autologous hematopoietic stem cell transplant (AHCT). CpG-MCL vaccine is derived from each patient's own tumor, treated in vitro with the immunostimulatory CpG-enriched oligodeoxynucleotide - PF-3512676 - and irradiated. The overall treatment schema is that patients receive: induction chemotherapy, priming vaccinations, leukapheresis to harvest vaccine-primed T cells, preparative myeloablative chemotherapy followed by AHCT, re-infusion of vaccine-primed T-cells ('immunotransplant') and repeat vaccinations zero and three months post-AHCT.

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  • Cytokine Induced Killer Cells as Post-Transplant Immunotherapy Following Allogeneic Hematopoietic Cell Transplantation Not Recruiting

    The purpose of the study is to determine if the use of activated T cells can effectively treat relapsed disease following allogeneic hematopoietic cell transplantation without causing GVHD.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Mixed Chimera Allogeneic Transplantation From Matched Unrelated Donors For The Treatment Of Multiple Myeloma Not Recruiting

    The purpose of the study is to determine the toxicity and feasibility of non-myeloablative allogeneic hematopoietic cell transplants for multiple myeloma from unrelated donors.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Sirolimus and Mycophenolate Mofetil as Graft Versus Host Disease Prophylaxis in Myeloablative Matched Related Donor Hematopoietic Cell Transplant Not Recruiting

    To evaluate the incidence of grade II-IV acute GVHD with sirolimus and mycophenolate mofetil GVHD prophylaxis.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • A Phase II Trial of Rituximab and Corticosteroid Therapy for Newly Diagnosed Chronic Graft Versus Host Disease Not Recruiting

    We hypothesize the addition of rituximab to prednisone for the initial treatment of chronic GVHD will increase the overall response rate, and enable a more rapid and effective steroid taper.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Double Cord Versus Haploidentical (Blood and Marrow Transplant Clinical Trials Network #1101) Recruiting

    Hematopoietic cell transplants (HCT)are one treatment option for people with leukemia or lymphoma. Family members,unrelated donors or banked umbilical cordblood units with similar tissue type can be used for HCT. This study will compare the effectiveness of two new types of bone marrow transplants in people with leukemia or lymphoma: one that uses bone marrow donated from family members with only partially matched bone marrow; and, one that uses two partially matched cord blood units.

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  • Defibrotide for Patients With Hepatic Veno-occlusive Disease: A Treatment IND Study Recruiting

    Single arm, open-label study to provide Defibrotide to patients diagnosed with VOD. Defibrotide is no longer available though the Emergency Use IND mechanism (also known as compassionate use, or single patient named use). This protocol is the only mechanism by which Defibrotide can be made available to patients in the U.S.

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  • Defibrotide for the Treatment of Severe Hepatic Veno-Occlusive Disease in Hematopoetic Stem Cell Transplant Patients Not Recruiting

    The purpose of this study is to (1) demonstrate the efficacy and safety (toxicity) of 25 mg/kg/day of Defibrotide in patients with severe veno-occlusive disease (sVOD) and (2) evaluate serum and endothelial markers of VOD through the analysis of blood samples.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Enrichment & Purging of Stem Cells in Non-Hodgkin's Lymphoma Not Recruiting

    To evaluate the role of purging the hematopoietic cell graft on outcomes for non-Hodgkin's Lymphoma.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Refferals, (650) 723 - 0822.

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  • Stem Cell Transplant With Lenalidomide Maintenance in Patients With Multiple Myeloma (BMT CTN 0702) Not Recruiting

    The study is designed as a Phase III, multicenter trial of tandem autologous transplants plus maintenance therapy versus the strategy of single autologous transplant plus consolidation therapy with lenalidomide, bortezomib and dexamethasone (RVD) followed by maintenance therapy or single autologous transplant plus maintenance therapy as part of upfront treatment of multiple myeloma (MM). Lenalidomide will be used as maintenance therapy for three years in all arms.

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, (650) 723 - 0822.

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  • Phase II Trial of Prophylactic Rituximab Therapy for Prevention of CGVHD Not Recruiting

    To determine if Rituximab administered after allogeneic transplantation decreases the incidence of chronic GvHD

    Stanford is currently not accepting patients for this trial. For more information, please contact Kate Tierney, (650) 725 - 7063.

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  • Autologous Followed by Non-myeloablative Allogeneic Transplantation for Non-Hodgkin's Lymphoma Recruiting

    The purpose of this trial is to develop an alternative treatment for patients with poor risk non-Hodgkin's lymphoma. This trial uses a combination of high dose chemotherapy with stem cell transplant using the patient's own cells. This is followed with non-myeloablative transplant using stem cells from a related or unrelated donor to try and generate an anti-lymphoma response from the new immune system.

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  • Stem Cell Transplant From Matched Unrelated or Partially Matched Related Donors Not Recruiting

    To evaluate the use of unrelated donors for hematopoietic cell transplantation in the treatment of hematologic and lymphoid malignancies.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • A Pilot Study of Imatinib Mesylate in Steroid Refractory Chronic Graft Versus Host Disease Not Recruiting

    To determine if subjects with steroid refractory cGVHD can tolerate imatinib mesylate and whether their cGVHD responds to imatinib mesylate.

    Stanford is currently not accepting patients for this trial. For more information, please contact Joanne Otani, (650) 721 - 2372.

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  • Chronic Graft-versus-Host Disease Treatment (BMT CTN 0801) Not Recruiting

    This study is designed as a combined Phase II/III, randomized, open label, multicenter, prospective comparative study of sirolimus plus prednisone versus sirolimus/calcineurin-inhibitor plus prednisone for the treatment of chronic GVHD. Patients will be stratified by transplant center and will be randomized to an experimental arm of one of the two pre-specified experimental arms (sirolimus + prednisone or the comparator arm of sirolimus + calcineurin inhibitor + prednisone) in a 1:1 ratio.

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, 650-723-0822.

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  • Allogeneic Transplantation for Patients With Acute Leukemia or Chronic Myelogenous Leukemia (CML) Not Recruiting

    The purpose of the study is to evaluate the overall and disease free survival of recipients who have received G-CSF mobilized stem cells from HLA matched sibling donors.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • A Randomized, Double-Blind, Placebo-Controlled Trial to Evaluate Palifermin (rHuKGF) in the Reduction of Acute Graft Versus Host Disease in Subjects With Hematologic Malignancies Undergoing Allogeneic Marrow/PBPC Transplantation Not Recruiting

    The purpose of this study is to reduce the incidence of grade 2-4 GVHD and WHO grades 3-4 Oral Mucositis.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Sirolimus & Mycophenolate Mofetil as GVHD Prophylaxis in Myeloablative, Matched Related Donor HCT Not Recruiting

    GVHD prophylaxis of sirolimus and mycophenolate mofetil for patients undergoing matched related allogeneic transplant for acute and chronic leukemia, MDS, high risk NHL and HL

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

    View full details

  • Ph II of Autologous Followed by Nonmyeloablative Allogeneic Transplantation Using TLI & ATG Not Recruiting

    To evaluate the toxicity and tolerability of this tandem autologous/allogeneic transplant approach for patients with advanced stage multiple myeloma.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

    View full details

  • Sibling and Unrelated Donor Hematopoietic Cell Transplant in Hematologic Malignancies Not Recruiting

    The purpose of this study is to determine the tolerability and efficacy in treating patients aged 51-60 with acute leukemia and in treating myelodysplastic syndromes (MDS) or myeloproliferative disorders (MPD).

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Autologous Stem Cell Rescue for Primary Amyloidosis Not Recruiting

    To evaluate the role of high dose therapy and autologous hematopoietic cell transplant for amyloidosis.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • High -Dose Sequential Therapy and Single Autologous Transplantation for Multiple Myeloma Not Recruiting

    This study uses a sequence of high-dose chemotherapy drugs and a stem cell transplant to treat multiple myeloma. The study is being performed to evaluate the efficacy and side effects of treatment. Specifically, the study is designed to reduce the risk of interstitial pneumonitis.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Comparing Peripheral Blood Stem Cell Transplantation Versus Bone Marrow Transplantation in Individuals With Hematologic Cancers Not Recruiting

    The study is designed as a Phase III, randomized, open label, multicenter, prospective, comparative trial of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) versus marrow from unrelated donors for transplantation in patients with hematologic malignancies. Recipients will be stratified by transplant center and disease risk and will be randomized to either the PBSC or marrow arm in a 1:1 ratio.

    Stanford is currently not accepting patients for this trial. For more information, please contact Kate Tierney, (650) 725 - 7063.

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  • Sirolimus as Treatment of Steroid-Refractory or Steroid-Dependent Chronic Graft-Versus-Host Disease Not Recruiting

    To study the effectiveness of an immunosuppressive drug, sirolimus in the treatment of chronic graft versus host disease in combination with prednisone.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

    View full details

  • Allogeneic Transplantation Using TL1 & ATG for Older Patients With Hematologic Malignancies Not Recruiting

    To measure how frequently and to what degree a complication of transplant cell acute graft versus host disease (GV/HD) occurs.

    Stanford is currently not accepting patients for this trial. For more information, please contact Physician Referrals, 650-723-0822.

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  • Ph II of Non-myeloablative Allogeneic Transplantation Using TLI & ATG In Patients w/ Cutaneous T Cell Lymphoma Recruiting

    Non-myeloablative approach for allogeneic transplant is a reasonable option, especially given that the median age at diagnosis is 55-60 years and frequently present compromised skin in these patients, which increases the risk of infection. Therefore, we propose a clinical study with allogeneic HSCT using a unique non-myeloablative preparative regimen, TLI/ATG, to treat advanced MF/SS.

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  • Sirolimus/Tacrolimus Versus Tacrolimus/Methotrexate for Preventing Graft-Versus-Host Disease (GVHD) Not Recruiting

    The study is designed as a phase III, randomized, open label, multicenter, prospective, comparative trial of sirolimus and tacrolimus versus tacrolimus and methotrexate as graft-versus-host disease (GVHD) prophylaxis after human leukocyte antigen (HLA)-matched, related, peripheral blood stem cell transplantation in individuals with hematologic cancer. Participants will be stratified by transplant center and will be randomly assigned to the sirolimus/tacrolimus or tacrolimus/methotrexate arms at a 1:1 ratio.

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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  • Gemcitabine and Hodgkin's Disease Chemotherapy Followed by Peripheral Blood Stem Cell Rescue for Hodgkin's Disease Not Recruiting

    Phase II Gemcitabine + HD Chemotherapy Followed by PBSC Rescue for HD

    Stanford is currently not accepting patients for this trial. For more information, please contact BMT Referrals, (650) 723 - 0822.

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Teaching

2013-14 Courses


Publications

Journal Articles


  • Role of lymphocyte activation gene-3 (lag-3) in conventional and regulatory T cell function in allogeneic transplantation. PloS one Sega, E. I., Leveson-Gower, D. B., Florek, M., Schneidawind, D., Luong, R. H., Negrin, R. S. 2014; 9 (1)

    Abstract

    Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.

    View details for DOI 10.1371/journal.pone.0086551

    View details for PubMedID 24475140

  • Mast cells suppress murine GVHD in a mechanism independent of CD4+CD25+ regulatory T cells. Blood Leveson-Gower, D. B., Sega, E. I., Kalesnikoff, J., Florek, M., Pan, Y., Pierini, A., Galli, S. J., Negrin, R. S. 2013; 122 (22): 3659-3665

    Abstract

    To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001). The increased mortality risk in C57BL/6-Kit(W-sh/W-sh) hosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P < .001). We did not detect any deficit in the number or function of CD4(+)CD25(+) regulatory T cells (Tregs) in C57BL/6-Kit(W-sh/W-sh) mice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-Kit(W-sh/W-sh) recipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-Kit(W-sh/W-sh) mice during GVHD was significantly improved if the mice were engrafted with bone marrow-derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10-deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.

    View details for DOI 10.1182/blood-2013-08-519157

    View details for PubMedID 24030387

  • IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease after Bone Marrow Transplantation. Biology of blood and marrow transplantation Colonna, L., Florek, M., Leveson-Gower, D. B., Sega, E. I., Baker, J., Smith, A. T., Negrin, R. S. 2013; 19 (11): 1557-1565

    Abstract

    Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4(+)CD25(+)Foxp3(+) natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17-producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17-deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation.

    View details for DOI 10.1016/j.bbmt.2013.07.024

    View details for PubMedID 23921175

  • Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation. Blood Schneidawind, D., Pierini, A., Negrin, R. S. 2013; 122 (18): 3116-3121

    Abstract

    Alloreactivity of donor lymphocytes leads to graft-versus-host disease (GVHD) contributing to significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Within the past decade, significant progress has been made in elucidating the mechanisms underlying the immunologic dysregulation characteristic of GVHD. The recent discoveries of different cell subpopulations with immune regulatory function has led to a number of studies aimed at understanding their role in allogeneic HCT and possible application for the prevention and treatment of GVHD and a host of other immune-mediated diseases. Preclinical animal modeling has helped define the potential roles of distinct populations of regulatory cells that have progressed to clinical translation with promising early results.

    View details for DOI 10.1182/blood-2013-08-453126

    View details for PubMedID 24068494

  • A distinct evolution of the T-cell repertoire categorizes treatment refractory gastrointestinal acute graft-versus-host disease BLOOD Meyer, E. H., Hsu, A. R., Liliental, J., Loehr, A., Florek, M., Zehnder, J. L., Strober, S., Lavori, P., Miklos, D. B., Johnson, D. S., Negrin, R. S. 2013; 121 (24): 4955-4962

    Abstract

    Steroid refractory gastrointestinal (GI) acute graft versus host disease (aGVHD) is a major cause of mortality in hematopoietic stem cell transplantation (HCT) without immune markers to establish a diagnosis or guide therapy. We found that T cell receptor β (TCRβ) CDR3 repertoire sequencing reveals patterns that could eventually serve as a disease biomarker of T cell alloreactivity in aGVHD. We identified T cell clones in GI biopsies in a heterogeneous group of 15 allogeneic HCT patients with GI aGVHD symptoms. Seven steroid-refractory aGVHD patients showed a more conserved TCRβ clonal structure between different biopsy sites in the GI tract than eight primary-therapy responsive patients. Tracking GI clones identified at endoscopy longitudinally in the blood also revealed an increased clonal expansion in patients with steroid-refractory disease. Immune repertoire sequencing-based methods could enable a novel personalized way to guide diagnosis and therapy in diseases where T cell activity is a major determinant.

    View details for DOI 10.1182/blood-2013-03-489757

    View details for Web of Science ID 000321896300024

  • A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model BMC MEDICINE Baeuerlein, C. A., Riedel, S. S., Baker, J., Brede, C., Garrote, A. J., Chopra, M., Ritz, M., Beilhack, G. F., Schulz, S., Zeiser, R., Schlegel, P. G., Einsele, H., Negrin, R. S., Beilhack, A. 2013; 11
  • A new approach for eradication of residual lymphoma cells by host nonreactive anti-third-party central memory CD8 T cells BLOOD Lask, A., Ophir, E., Or-Geva, N., Cohen-Fredarow, A., Afik, R., Eidelstein, Y., Reich-Zeliger, S., Nathansohn, B., Edinger, M., Negrin, R. S., Hagin, D., Reisner, Y. 2013; 121 (15): 3033-3040

    Abstract

    Generation of T cells endowed with graft-versus-leukemia (GVL) and depleted of graft-versus-host (GVH) activity represents a highly desirable goal in bone marrow transplantation (BMT). Here, we demonstrate that donor anti-third-party CD8 T cells with central memory phenotype (Tcm) exhibit marked GVL reactivity through a unique T-cell receptor-independent mechanism. Thus, in a residual disease mouse model, Tcm therapy following autologous BMT led to significant survival prolongation, with 30% to 40% of the treated mice displaying long-term tumor-free survival. A more impressive finding was that infusion of donor Tcm in an allogeneic model rapidly eliminated residual lymphoma cells and led to long-term survival of 100% in the absence of GVH disease. Collectively, the strong GVL reactivity of anti-third-party Tcm, coupled with their demonstrated enhancement of bone marrow allografting, suggests that the use of Tcm therapy in conjunction with allogeneic T-cell-depleted BMT could be of particular benefit in patients with B-cell malignancies who cannot tolerate intensive myeloablative conditioning.

    View details for DOI 10.1182/blood-2012-06-432443

    View details for Web of Science ID 000321825700028

    View details for PubMedID 23446736

  • Murine anti-third-party central-memory CD8(+) T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells BLOOD Ophir, E., Or-Geva, N., Gurevich, I., Tal, O., Eidelstein, Y., Shezen, E., Margalit, R., Lask, A., Shakhar, G., Hagin, D., Bachar-Lustig, E., Reich-Zeliger, S., Beilhack, A., Negrin, R., Reisner, Y. 2013; 121 (7): 1220-1228

    Abstract

    Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

    View details for DOI 10.1182/blood-2012-07-441493

    View details for Web of Science ID 000314870700026

    View details for PubMedID 23223359

  • A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model. BMC medicine Bäuerlein, C. A., Riedel, S. S., Baker, J., Brede, C., Garrote, A. J., Chopra, M., Ritz, M., Beilhack, G. F., Schulz, S., Zeiser, R., Schlegel, P. G., Einsele, H., Negrin, R. S., Beilhack, A. 2013; 11: 134-?

    Abstract

    Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention.Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles.We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD.Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.

    View details for DOI 10.1186/1741-7015-11-134

    View details for PubMedID 23692886

  • Cancer Vaccines and T Cell Therapy BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Rezvani, K., Brody, J. D., Kohrt, H. E., Logan, A. C., Advani, R., Czerwinski, D. K., Weng, W., Negrin, R. S., Carlton, V., Faham, M., Levy, R., Barrett, J. 2013; 19 (1): S97-S101

    View details for DOI 10.1016/j.bbmt.2012.09.020

    View details for Web of Science ID 000313998100024

    View details for PubMedID 23041602

  • Mapping immune processes in intact tissues at cellular resolution. journal of clinical investigation Brede, C., Friedrich, M., Jordán-Garrote, A., Riedel, S. S., Bäuerlein, C. A., Heinze, K. G., Bopp, T., Schulz, S., Mottok, A., Kiesel, C., Mattenheimer, K., Ritz, M., von Krosigk, V., Rosenwald, A., Einsele, H., Negrin, R. S., Harms, G. S., Beilhack, A. 2012; 122 (12): 4439-4446

    Abstract

    Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

    View details for DOI 10.1172/JCI65100

    View details for PubMedID 23143304

  • Mapping immune processes in intact tissues at cellular resolution JOURNAL OF CLINICAL INVESTIGATION Brede, C., Friedrich, M., Jordan-Garrote, A., Riedel, S. S., Baeuerlein, C. A., Heinze, K. G., Bopp, T., Schulz, S., Mottok, A., Kiesel, C., Mattenheimer, K., Ritz, M., von Krosigk, V., Rosenwald, A., Einsele, H., Negrin, R. S., Harms, G. S., Beilhack, A. 2012; 122 (12): 4439-4446

    View details for DOI 10.1172/JCI65100

    View details for Web of Science ID 000311926200020

  • Prophylactic rituximab after allogeneic transplantation decreases B-cell alloimmunity with low chronic GVHD incidence BLOOD Arai, S., Sahaf, B., Narasimhan, B., Chen, G. L., Jones, C. D., Lowsky, R., Shizuru, J. A., Johnston, L. J., Laport, G. G., Weng, W., Benjamin, J. E., Schaenman, J., Brown, J., Ramirez, J., Zehnder, J. L., Negrin, R. S., Miklos, D. B. 2012; 119 (25): 6145-6154

    Abstract

    B cells are involved in the pathogenesis of chronic GVHD (cGVHD). We hypothesized that prophylactic anti-B-cell therapy delivered 2 months after transplantation would decrease allogeneic donor B-cell immunity and possibly the incidence of cGVHD. Therefore, in the present study, patients with high-risk chronic lymphocytic leukemia (n = 22) and mantle-cell lymphoma (n = 13) received a total lymphoid irradiation of 80 cGy for 10 days and antithymocyte globulin 1.5 mg/kg/d for 5 days. Rituximab (375 mg/m(2)) was infused weekly on days 56, 63, 70, and 77 after transplantation. The incidence of acute GVHD was 6%. The cumulative incidence of cGVHD was 20%. Nonrelapse mortality was 3%. Rituximab treatment after allogeneic transplantation significantly reduced B-cell allogeneic immunity, with complete prevention of alloreactive H-Y Ab development in male patients with female donors (P = .01). Overall survival and freedom from progression at 4 years for chronic lymphocytic leukemia patients were 73% and 47%, respectively; for mantle-cell lymphoma patients, they were 69% and 53%, respectively.

    View details for DOI 10.1182/blood-2011-12-395970

    View details for Web of Science ID 000307398700030

    View details for PubMedID 22563089

  • Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells BLOOD Gill, S., Vasey, A. E., De Souza, A., Baker, J., Smith, A. T., Kohrt, H. E., Florek, M., Gibbs, K. D., Tate, K., Ritchie, D. S., Negrin, R. S. 2012; 119 (24): 5758-5768

    Abstract

    Natural killer (NK) cells are potent anti-viral and antitumor "first responders" endowed with natural cytotoxicity and cytokine production capabilities. To date, attempts to translate these promising biologic functions through the adoptive transfer of NK cells for the treatment of cancer have been of limited benefit. Here we trace the fate of adoptively transferred murine NK cells and make the surprising observation that NK cells traffic to tumor sites yet fail to control tumor growth or improve survival. This dysfunction is related to a rapid down-regulation of activating receptor expression and loss of important effector functions. Loss of interferon (IFN)? production occurs early after transfer, whereas loss of cytotoxicity progresses with homeostatic proliferation and tumor exposure. The dysfunctional phenotype is accompanied by down-regulation of the transcription factors Eomesodermin and T-bet, and can be partially reversed by the forced overexpression of Eomesodermin. These results provide the first demonstration of NK-cell exhaustion and suggest that the NK-cell first-response capability is intrinsically limited. Further, novel approaches may be required to circumvent the described dysfunctional phenotype.

    View details for DOI 10.1182/blood-2012-03-415364

    View details for Web of Science ID 000307396500028

    View details for PubMedID 22544698

  • Co-transplantation of pure blood stem cells with antigen-specific but not bulk T cells augments functional immunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mueller, A. M., Shashidhar, S., Kuepper, N. J., Kohrt, H. E., Florek, M., Negrin, R. S., Brown, J. M., Shizuru, J. A. 2012; 109 (15): 5820-5825

    Abstract

    Impaired immunity is a fundamental obstacle to successful allogeneic hematopoietic cell transplantation. Mature graft T cells are thought to provide protection from infections early after transplantation, but can cause life-threatening graft-vs.-host disease. Human CMV is a major pathogen after transplantation. We studied reactivity against the mouse homologue, murine CMV (MCMV), in lethally irradiated mice given allogeneic purified hematopoietic stem cells (HSCs) or HSCs supplemented with T cells or T-cell subsets. Unexpectedly, recipients of purified HSCs mounted superior antiviral responses compared with recipients of HSC plus unselected bulk T cells. Furthermore, supplementation of purified HSC grafts with CD8(+) memory or MCMV-specific T cells resulted in enhanced antiviral reactivity. Posttransplantation lymphopenia promoted massive expansion of MCMV-specific T cells when no competing donor T cells were present. In recipients of pure HSCs, naive and memory T cells and innate lymphoid cell populations developed. In contrast, the lymphoid pool in recipients of bulk T cells was dominated by effector memory cells. These studies show that pure HSC transplantations allow superior protective immunity against a viral pathogen compared with unselected mature T cells. This reductionist transplant model reveals the impact of graft composition on regeneration of host, newly generated, and mature transferred T cells, and underscores the deleterious effects of bulk donor T cells. Our findings lead us to conclude that grafts composed of purified HSCs provide an optimal platform for in vivo expansion of selected antigen-specific cells while allowing the reconstitution of a naive T-cell pool.

    View details for DOI 10.1073/pnas.1120237109

    View details for Web of Science ID 000302533500055

    View details for PubMedID 22440752

  • Sirolimus and mycophenolate mofetil as GVHD prophylaxis in myeloablative, matched-related donor hematopoietic cell transplantation BONE MARROW TRANSPLANTATION Johnston, L., Florek, M., Armstrong, R., McCune, J. S., Arai, S., Brown, J., Laport, G., Lowsky, R., Miklos, D., Shizuru, J., Sheehan, K., Lavori, P., Negrin, R. 2012; 47 (4): 581-588

    Abstract

    We investigated sirolimus and mycophenolate mofetil (MMF) as GVHD prophylaxis in patients with advanced hematological malignancies receiving myeloablative hematopoietic cell transplantation (HCT) from HLA-identical sibling donors. On the basis of pre-study stopping rules, the trial was closed to accrual after enrollment of 11 adult patients. In all, 7 of the 11 patients received BU-containing preparative regimens. Sirolimus was discontinued in three patients because of the toxicity-related events of severe sinusoidal obstructive syndrome, portal vein thrombosis, altered mental status and in one patient because of the risk of poor wound healing. In all, 6 of the 11 patients developed grade II-IV acute GVHD (AGVHD) a median of 15.5 days post HCT. Two of three patients with grade IV AGVHD had sirolimus discontinued by 9 days post HCT. All patients responded to AGVHD therapy without GVHD-related deaths. There were two non-relapse- and two relapse-related deaths. At a median follow-up of 38 months (2-47 months), 7 of 11 patients were alive without disease. MMF and sirolimus GVHD prophylaxis did not reduce the risk of AGVHD, however, there were no GVHD-related deaths. The severe toxicities in the patients receiving the BU-containing preparative regimens limited the continued use of sirolimus and MMF for the prevention of AGVHD.

    View details for DOI 10.1038/bmt.2011.104

    View details for Web of Science ID 000302576700018

    View details for PubMedID 21552302

  • Tandem chemo-mobilization followed by high-dose melphalan and carmustine with single autologous hematopoietic cell transplantation for multiple myeloma BONE MARROW TRANSPLANTATION Chen, A. I., Negrin, R. S., McMillan, A., Shizuru, J. A., JOHNSTON, L. J., Lowsky, R., Miklos, D. B., Arai, S., Weng, W., Laport, G. G., Stockerl-Goldstein, K. 2012; 47 (4): 516-521

    Abstract

    Single autologous hematopoietic cell transplant (AHCT) with high-dose melphalan prolongs survival in patients with multiple myeloma but is not curative. We conducted a study of intensive single AHCT using tandem chemo-mobilization with CY and etoposide followed by high-dose conditioning with melphalan 200 mg/m(2) plus carmustine 15 mg/kg. One hundred and eighteen patients in first consolidation (CON1) and 58 patients in relapse (REL) were transplanted using this intensified approach. Disease response improved from 32% very good PR (VGPR)+CR pre-mobilization to 76% VGPR+CR post transplant in CON1. With a median follow-up of 4.7 years, the median EFS was 2.8 years, and the median OS was 5.1 years in CON1. OS from time of transplant was significantly shorter for REL (3.4 years) compared with CON1 (5.1 years; P=0.02). However, OS from time of diagnosis was similar in REL (6.1 years) and CON1 (6.0 years; P=0.80). The 100-day non-relapse mortality in the CON1 and REL groups was 0% and 7%, respectively. In summary, intensified single AHCT with tandem chemo-mobilization and augmented high-dose therapy is feasible in multiple myeloma and leads to high-quality response rates.

    View details for DOI 10.1038/bmt.2011.106

    View details for Web of Science ID 000302576700008

    View details for PubMedID 21602899

  • Long-term outcomes after transplantation of HLA-identical related G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow BLOOD Mielcarek, M., Storer, B., Martin, P. J., Forman, S. J., Negrin, R. S., Flowers, M. E., Inamoto, Y., Chauncey, T. R., Storb, R., Appelbaum, F. R., Bensinger, W. I. 2012; 119 (11): 2675-2678

    Abstract

    Between 1996 and 1999, 172 patients (median age, 42 years) with hematologic malignancies were randomly assigned to receive either HLA-identical related bone marrow or G-CSF-mobilized peripheral blood mononuclear cells (G-PBMCs) after myeloablative conditioning. Early results showed that transplantation of G-PBMCs, compared with marrow, was associated with significantly superior 2-year disease-free survival (DFS) and overall survival. Ten-year follow-up showed a sustained DFS benefit associated with G-PBMCs (mortality or relapse hazard ratio, 0.64; 95% confidence interval, 0.4-1.0; P = .03), although the likelihood of overall survival was not significantly different between the 2 groups (mortality hazard ratio, 0.75; 95% confidence interval, 0.5-1.2; P = .20). The 10-year cumulative incidence of chronic GVHD and the duration of systemic immunosuppression were similar in the 2 groups. In summary, transplantation of HLA-identical related G-PBMCs, compared with marrow, was associated with superior short-term and long-term DFS, and there was no evidence that this benefit was outweighed by GVHD-related late mortality.

    View details for DOI 10.1182/blood-2011-12-396275

    View details for Web of Science ID 000301941700037

    View details for PubMedID 22308289

  • Cytomegalovirus Viral Load and Virus-Specific Immune Reconstitution after Peripheral Blood Stem Cell versus Bone Marrow Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Guerrero, A., Riddell, S. R., Storek, J., Stevens-Ayers, T., Storer, B., Zaia, J. A., Forman, S., Negrin, R. S., Chauncey, T., Bensinger, W., Boeckh, M. 2012; 18 (1): 66-75

    Abstract

    Peripheral blood stem cell (PBSC) products contain more T cells and monocytes when compared with bone marrow (BM), leading to fewer bacterial and fungal infections. Cytomegelovirus (CMV) viral load and disease as well as CMV-specific immune reconstitution were compared in patients enrolled in a randomized trial comparing PSBC and BM transplantation. There was a higher rate of CMV infection and disease during the first 100 days after transplantation among PBSC recipients (any antigenemia/DNAemia: PBSC, 63% vs BM, 42%, P = .04; CMV disease: PBSC, 17% vs BM, 4%, P = .03). By 2 years, CMV disease rates were similar. The early increase in CMV events correlated temporarily with lower CMV-specific CD4(+) T helper and CD8(+) cytotoxic T lymphocyte function at 30 days after transplantation in PBSC recipients. By 3 months after transplantation and thereafter, CMV-specific immune responses were similar between BM and PBSC recipients. In conclusion, higher CMV infection and disease rates occurred in PBSC transplant recipients early after transplantation. These differences may be because of a transient delay in CMV-specific immune reconstitution following PBSC transplantation.

    View details for DOI 10.1016/j.bbmt.2011.05.010

    View details for Web of Science ID 000303140200008

    View details for PubMedID 21664286

  • Long-Term Outcome of Patients with Metastatic Breast Cancer Treated with High-Dose Chemotherapy and Transplantation of Purified Autologous Hematopoietic Stem Cells BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Mueller, A. M., Kohrt, H. E., Cha, S., Laport, G., Klein, J., Guardino, A. E., Johnston, L. J., Stockerl-Goldstein, K. E., Hanania, E., Juttner, C., Blume, K. G., Negrin, R. S., Weissman, I. L., Shizuru, J. A. 2012; 18 (1): 125-133

    Abstract

    Metastatic breast cancer remains a major treatment challenge. The use of high-dose chemotherapy (HDCT) with rescue by autologous mobilized peripheral blood (MPB) is controversial, in part because of contamination of MPB by circulating tumor cells. CD34(+)Thy-1(+) selected hematopoietic stem cells (HSC) represent a graft source with a greater than 250,000-fold reduction in cancer cells. Here, we present the long-term outcome of a pilot study to determine feasibility and engraftment using HDCT and purified HSC in patients with metastatic breast cancer. Twenty-two patients who had been treated with standard chemotherapy were enrolled into a phase I/II trial between December 1996 and February 1998, and underwent HDCT followed by rescue with CD34(+)Thy-1(+) HSC isolated from autologous MPB. More than 12 years after the end of the study, 23% (5 of 22) of HSC recipients are alive, and 18% (4 of 22) are free of recurrence with normal hematopoietic function. Median progression-free survival (PFS) was 16 months, and median overall survival (OS) was 60 months. Retrospective comparison with 74 patients transplanted between February 1995 and June 1999 with the identical HDCT regimen but rescue with unmanipulated MPB indicated that 9% of patients are alive, and 7% are without disease. Median PFS was 10 months, and median OS was 28 months. In conclusion, cancer-depleted HSC following HDCT resulted in better than expected 12- to 14-year PFS and OS in a cohort of metastatic breast cancer patients. These data prompt us to look once again at purified HSC transplantation in a protocol powered to test for efficacy in advanced-stage breast cancer patients.

    View details for DOI 10.1016/j.bbmt.2011.07.009

    View details for Web of Science ID 000303140200015

    View details for PubMedID 21767515

  • Adoptive Immunotherapy with Cytokine-Induced Killer Cells for Patients with Relapsed Hematologic Malignancies after Allogeneic Hematopoietic Cell Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Laport, G. G., Sheehan, K., Baker, J., Armstrong, R., Wong, R. M., Lowsky, R., Johnston, L. J., Shizuru, J. A., Miklos, D., Arai, S., Benjamin, J. E., Weng, W., Negrin, R. S. 2011; 17 (11): 1679-1687

    Abstract

    Donor leukocyte infusions induce remissions in some patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (HCT); however, graft-versus-host disease (GVHD) remains the major complication of this strategy. Cytokine-induced killer (CIK) cells are a unique population of cytotoxic T lymphocytes that express the CD3(+)CD56(+) phenotype and show marked up-regulation of the natural killer cell receptor NKG2D (CD314). CIK cells are non-major histocompatibility complex-restricted and NKG2D-dependent in target recognition and cytotoxicity. We explored the feasibility of ex vivo expansion of allogeneic CIK cells in patients with relapsed hematologic malignancies after allogeneic HCT. Eighteen patients (median age, 53 years; range, 20-69 years) received CIK cell infusions at escalating doses of 1 × 10(7) CD3(+) cells/kg (n = 4), 5 × 10(7) CD3(+) cells/kg (n = 6), and 1 × 10(8) CD3(+) cells/kg (n = 8). The median expansion of CD3(+) cells was 12-fold (range, 4- to 91-fold). CD3(+)CD56(+) cells represented a median of 11% (range, 4%-44%) of the harvested cells, with a median 31-fold (range, 7- to 515-fold) expansion. Median CD3(+)CD314(+) cell expression was 53% (range, 32%-78%) of harvested cells. Significant cytotoxicity was demonstrated in vitro against a panel of human tumor cell lines. Acute GVHD grade I-II was seen in 2 patients, and 1 patient had limited chronic GVHD. After a median follow-up of 20 months (range, 1-69 months) from CIK infusion, the median overall survival was 28 months, and the median event-free survival was 4 months. All deaths were due to relapsed disease; however, 5 patients had longer remissions after infusion of CIK cells than from allogeneic HCT to relapse. Our findings indicate that this form of adoptive immunotherapy is well tolerated and induces a low incidence of GVHD, supporting further investigation as an upfront modality to enhance graft-versus-tumor responses in high-risk patient populations.

    View details for DOI 10.1016/j.bbmt.2011.05.012

    View details for Web of Science ID 000296829000016

    View details for PubMedID 21664472

  • Role of regulatory T cell populations in controlling graft vs host disease BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY Negrin, R. S. 2011; 24 (3): 453-457

    Abstract

    Immune function is critical in health and disease. The control and regulation of immune reactions is an area of intense investigation that has important implications for allogeneic hematopoietic cell transplantation. Immune reactions are regulated in a number of important ways. Compartmentalization of immune responses and the production of both pro-inflammatory and anti-inflammatory cytokines play a major role. More recently several populations of T cells that regulate immune responses termed regulatory T cells have been identified. This manuscript will focus on CD4+CD25+FoxP3+ natural regulatory T cells (T(reg)) and ??TCR+CD4+NK1.1+ natural killer T (NK-T) cells which both suppress graft vs host disease but appear to function by distinct mechanisms.

    View details for DOI 10.1016/j.beha.2011.05.006

    View details for Web of Science ID 000295655400015

    View details for PubMedID 21925098

  • Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4(+)CD25(+)Foxp3(+) regulatory T cells BLOOD Shin, H., Baker, J., Leveson-Gower, D. B., Smith, A. T., Sega, E. I., Negrin, R. S. 2011; 118 (8): 2342-2350

    Abstract

    Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4?CD25?Foxp3? regulatory T-cell (Treg) proliferation and function in vitro. We investigated whether the combination of RAPA plus IL-2 could impact acute GVHD induction after bone marrow transplantation (BMT). RAPA plus IL-2 resulted in improved survival and a reduction in acute GVHD lethality associated with an increased expansion of donor type CD4?Foxp3? Tregs and reduced CD4?CD25? conventional T cells (Tcons). RAPA plus IL-2, but not either drug alone, increased both expansion of donor natural Tregs and conversion of induced Tregs from donor CD25? Tcons while IL-2 alone increased conversion of Tregs from CD25? Tcon. RAPA plus IL-2 treatment resulted in less production of IFN-? and TNF, cytokines known to be important in the initiation of acute GVHD. These studies indicate that the pharmacologic stimulation of T cells with IL-2 and the suppression of Tcon proliferation with RAPA result in a selective expansion of functional Tregs and suppression of acute GVHD.

    View details for DOI 10.1182/blood-2010-10-313684

    View details for Web of Science ID 000294258000037

    View details for PubMedID 21734238

  • Autologous versus Reduced-Intensity Allogeneic Hematopoietic Cell Transplantation for Patients with Chemosensitive Follicular Non-Hodgkin Lymphoma beyond First Complete Response or First Partial Response BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Tomblyn, M. R., Ewell, M., Bredeson, C., Kahl, B. S., Goodman, S. A., Horowitz, M. M., Vose, J. M., Negrin, R. S., Laport, G. G. 2011; 17 (7): 1051-1057

    Abstract

    Patients with follicular lymphoma (FL) typically experience an indolent course; however, the disease is rarely curable with conventional chemotherapy. Autologous hematopoietic cell transplantation (HCT) can extend progression-free survival (PFS) and overall survival (OS), but relapse is the primary cause of failure. Allogeneic HCT confers lower relapse rates due to a graft-versus-lymphoma effect. Reduced-intensity conditioning (RIC) allows the performance of allogeneic HCT with lower toxicity. The Blood and Marrow Transplant Clinical Trials Network conducted a prospective multicenter trial comparing these two strategies in patients with relapsed, chemotherapy-sensitive FL. Patients were assigned to a treatment arm based on the availability of an HLA-matched sibling donor (MSD). Those with an MSD underwent allogeneic HCT (n = 8) with the FCR preparative regimen (fludarabine, cyclophosphamide [Cy], rituximab [RTX]) and received tacrolimus and methotrexate for graft-versus-host disease (GVHD) prophylaxis. Those without an MSD (n = 22) underwent mobilization with Cy, RTX, and filgrastim and received a conditioning regimen of either CBV (Cy, carmustine, Etoposide [VP16]) or total body irradiation with Cy and VP16. Patients undergoing autologous HCT received 4 doses of weekly maintenance RTX (375 mg/m²) starting on day +42 post-HCT. Sixteen patients were in complete remission, 10 patients were in partial remission, and 1 patient had stable disease after salvage therapy and before HCT. Median follow-up was 36 months (range, 1-51 months). OS was 73% in autologous HCT versus 100% in allogeneic HCT, and PFS was 63% in autologous HCT versus 86% in allogeneic HCT. No patient had grade II-IV acute GVHD; two patients developed extensive chronic GVHD. Three autologous recipients died from nonrelapse causes. This trial closed early because of slow accrual. We show that the FCR regimen is well tolerated, and that both allogeneic and autologous HCT result in promising 3-year OS and PFS in patients with relapsed FL.

    View details for DOI 10.1016/j.bbmt.2010.11.004

    View details for Web of Science ID 000292059300014

    View details for PubMedID 21073974

  • Low doses of natural killer T cells provide protection from acute graft-versus-host disease via an IL-4-dependent mechanism BLOOD Leveson-Gower, D. B., Olson, J. A., Sega, E. I., Luong, R. H., Baker, J., Zeiser, R., Negrin, R. S. 2011; 117 (11): 3220-3229

    Abstract

    CD4(+) natural killer T (NKT) cells, along with CD4(+)CD25(+) regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4(+)NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 10(4) cells suppress GVHD caused by 5.0 × 10(5) Tcons in an interleukin-4 (IL-4)-dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-? (IFN-?) and tumor necrosis factor-? (TNF-?) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4(+)NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.

    View details for DOI 10.1182/blood-2010-08-303008

    View details for Web of Science ID 000288496300034

    View details for PubMedID 21258007

  • CD8(+)CD44(hi) but not CD4(+)CD44(hi) memory T cells mediate potent graft antilymphoma activity without GVHD BLOOD Dutt, S., Baker, J., Kohrt, H. E., Kambham, N., Sanyal, M., Negrin, R. S., Strober, S. 2011; 117 (11): 3230-3239

    Abstract

    Allogeneic hematopoietic cell transplantation can be curative in patients with leukemia and lymphoma. However, progressive growth of malignant cells, relapse after transplantation, and graft-versus-host disease (GVHD) remain important problems. The goal of the current murine study was to select a freshly isolated donor T-cell subset for infusion that separates antilymphoma activity from GVHD, and to determine whether the selected subset could effectively prevent or treat progressive growth of a naturally occurring B-cell lymphoma (BCL(1)) without GVHD after recipients were given T cell-depleted bone marrow transplantations from major histocompatibility complex-mismatched donors. Lethal GVHD was observed when total T cells, naive CD4(+) T cells, or naive CD8(+) T cells were used. Memory CD4(+)CD44(hi) and CD8(+)CD44(hi) T cells containing both central and effector memory cells did not induce lethal GVHD, but only memory CD8(+) T cells had potent antilymphoma activity and promoted complete chimerism. Infusion of CD8(+) memory T cells after transplantation was able to eradicate the BCL(1) lymphoma even after progressive growth without inducing severe GVHD. In conclusion, the memory CD8(+) T-cell subset separated graft antilymphoma activity from GVHD more effectively than naive T cells, memory CD4(+) T cells, or memory total T cells.

    View details for DOI 10.1182/blood-2010-10-312751

    View details for Web of Science ID 000288496300035

    View details for PubMedID 21239702

  • Short-Term Immunosuppression Promotes Engraftment of Embryonic and Induced Pluripotent Stem Cells CELL STEM CELL Pearl, J. I., Lee, A. S., Leveson-Gower, D. B., Sun, N., Ghosh, Z., Lan, F., Ransohoff, J., Negrin, R. S., Davis, M. M., Wu, J. C. 2011; 8 (3): 309-317

    Abstract

    Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation.

    View details for DOI 10.1016/j.stem.2011.01.012

    View details for Web of Science ID 000288404400012

    View details for PubMedID 21362570

  • Long-term outcomes in patients with high-risk myeloid malignancies following matched related donor hematopoietic cell transplantation with myeloablative conditioning of BU, etoposide and CY BONE MARROW TRANSPLANTATION Naik, S., Wong, R., Arai, S., Brown, J., Laport, G., Lowsky, R., Miklos, D., Shizuru, J., Blume, K., Negrin, R., Johnston, L. 2011; 46 (2): 192-199

    Abstract

    Patients with high-risk or advanced myeloid malignancies have limited effective treatment options. These include high-dose therapy followed by allogeneic hematopoietic cell transplantation (HCT). We report a single-institution, long-term follow-up of 96 patients, median age 50 (range, 20-60) years, who received HLA-matched related HCT between 1992 and 2007. All patients were treated with a uniform preparatory regimen intended to enhance the widely used regimen of BU and CY that included: BU 16.0?mg/kg (days -8 to -5), etoposide 60?mg/kg (day -4), CY 60?mg/kg (day -2) with GVHD prophylaxis of CsA or FK506 and prednisone. Disease status at transplantation was high-risk AML (n=41), CML in second chronic phase or blast crisis (n=8), myelofibrosis and myeloproliferative disorders (n=8), and myelodysplasia (n=39). Thirty-six percent (n=35) of patients received BM whereas 64% (n=61) received G-CSF-mobilized PBPC. With a median follow-up of 5.6 years (range, 1.6-14.6 years) actuarial 5-year OS was 32% (95% CI 22-42) and 5-year EFS was 31% (95% CI 21-41). Relapse rate was 24% (95% CI 15-33) at 2 and 5 years. Nonrelapse mortality was 29% (95% CI 20-38) at day 100 and 38% (95% CI 29-47) at 1 year. Cumulative incidence of acute (grade II-IV) and extensive chronic GVHD was 27% (95% CI 18-36) and 29% (95% CI 18-40), respectively. There was no statistically significant difference in OS (31 vs 32%, P=0.89) or relapse rates (17 vs 28%, P=0.22) for recipients of BM vs PBPC, respectively. These results confirm that patients with high-risk or advanced myeloid malignancies can achieve long-term survival following myeloablative allogeneic HCT with aggressive conditioning.

    View details for DOI 10.1038/bmt.2010.114

    View details for Web of Science ID 000287190700004

    View details for PubMedID 20498648

  • Natural and Expanded CD4(+) CD25(+) Regulatory T Cells in Bone Marrow Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Colonna, L., Sega, E. I., Negrin, R. S. 2011; 17 (1): S58-S62

    View details for DOI 10.1016/j.bbmt.2010.10.020

    View details for Web of Science ID 000292877000013

    View details for PubMedID 21195311

  • CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment after Allogeneic Transplantation of Hematopoietic Cells CANCER RESEARCH Duerr, C., Pfeifer, D., Claus, R., Schmitt-Graeff, A., Gerlach, U. V., Graeser, R., Krueger, S., Gerbitz, A., Negrin, R. S., Finke, J., Zeiser, R. 2010; 70 (24): 10170-10181

    Abstract

    Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT.

    View details for DOI 10.1158/0008-5472.CAN-10-1943

    View details for Web of Science ID 000285334200019

    View details for PubMedID 21159639

  • Definition of an Enhanced Immune Cell Therapy in Mice That Can Target Stem-Like Lymphoma Cells CANCER RESEARCH Contag, C. H., Sikorski, R., Negrin, R. S., Schmidt, T., Fan, A. C., Bachireddy, P., Felsher, D. W., Thorne, S. H. 2010; 70 (23): 9837-9845

    Abstract

    Current treatments of high-grade lymphoma often have curative potential, but unfortunately many patients relapse and develop therapeutic resistance. Thus, there remains a need for novel therapeutics that can target the residual cancer cells whose phenotypes are distinct from the bulk tumor and that are capable of reforming tumors from very few cells. Oncolytic viruses offer an approach to destroy tumors by multiple mechanisms, but they cannot effectively reach residual disease or micrometastases, especially within the lymphatic system. To address these limitations, we have generated immune cells infected with oncolytic viruses as a therapeutic strategy that can combine effective cellular delivery with synergistic tumor killing. In this study, we tested this approach against minimal disease states of lymphomas characterized by the persistence of cancer cells that display stem cell-like properties and resistance to conventional therapies. We found that the immune cells were capable of trafficking to and targeting residual cancer cells. The combination biotherapy used prevented relapse by creating a long-term, disease-free state, with acquired immunity to the tumor functioning as an essential mediator of this effect. Immune components necessary for this acquired immunity were identified. We further demonstrated that the dual biotherapy could be applied before or after conventional therapy. Our approach offers a potentially powerful new way to clear residual cancer cells, showing how restoring immune surveillance is critical for maintenance of a disease-free state.

    View details for DOI 10.1158/0008-5472.CAN-10-2650

    View details for Web of Science ID 000285045900033

    View details for PubMedID 20935221

  • Phase I/II Trial of GN-BVC, a Gemcitabine and Vinorelbine-Containing Conditioning Regimen for Autologous Hematopoietic Cell Transplantation in Recurrent and Refractory Hodgkin Lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Arai, S., Letsinger, R., Wong, R. M., Johnston, L. J., Laport, G. G., Lowsky, R., Miklos, D. B., Shizuru, J. A., Weng, W., Lavori, P. W., Blume, K. G., Negrin, R. S., Horning, S. J. 2010; 16 (8): 1145-1154

    Abstract

    Autologous hematopoietic cell transplantation with augmented BCNU regimens is effective treatment for recurrent or refractory Hodgkin lymphoma (HL); however, BCNU-related toxicity and disease recurrence remain challenges. We designed a conditioning regimen with gemcitabine in combination with vinorelbine in an effort to reduce the BCNU dose and toxicity without compromising efficacy. In this phase I/II dose escalation study, the gemcitabine maximum tolerated dose (MTD) was determined at 1250 mg/m(2), and a total of 92 patients were treated at this dose to establish safety and efficacy. The primary endpoint was the incidence of BCNU-related toxicity. Secondary endpoints included 2-year freedom from progression (FFP), event-free survival (EFS), and overall survival (OS). Sixty-eight patients (74%) had 1 or more previously defined adverse risk factors for transplant (stage IV at relapse, B symptoms at relapse, greater than minimal disease pretransplant). The incidence of BCNU-related toxicity was 15% (95% confidence interval, 9%-24%). Only 2% of patients had a documented reduction in diffusing capacity of 20% or greater. With a median follow-up of 29 months, the FFP at 2 years was 71% and the OS at 2 years was 83%. Two-year FFP was 96%, 72%, 67%, and 14% for patients with 0 (n = 24), 1 (n = 37), 2 (n = 23), or 3 (n = 8) risk factors, respectively. Regression analysis identified PET status pretransplant and B symptoms at relapse as significant prognostic factors for FFP. This new transplant regimen for HL resulted in decreased BCNU toxicity with encouraging FFP and OS. A prospective, risk-modeled comparison of this new combination with other conditioning regimens is warranted.

    View details for DOI 10.1016/j.bbmt.2010.02.022

    View details for Web of Science ID 000280137800013

    View details for PubMedID 20197102

  • NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects BLOOD Olson, J. A., Leveson-Gower, D. B., Gill, S., Baker, J., Beilhack, A., Negrin, R. S. 2010; 115 (21): 4293-4301

    Abstract

    Natural killer (NK) cells suppress graft-versus-host disease (GVHD) without causing GVHD themselves. Our previous studies demonstrated that allogeneic T cells and NK cells traffic similarly after allogeneic bone marrow transplantation (BMT). We therefore investigated the impact of donor NK cells on donor alloreactive T cells in GVHD induction. Animals receiving donor NK and T cells showed improved survival and decreased GVHD score compared with controls receiving donor T cells alone. Donor T cells exhibited less proliferation, lower CD25 expression, and decreased interferon-gamma (IFN-gamma) production in the presence of NK cells. In vivo, we observed perforin- and Fas ligand (FasL)-mediated reduction of donor T cell proliferation and increased T cell apoptosis in the presence of NK cells. Further, activated NK cells mediated direct lysis of reisolated GVHD-inducing T cells in vitro. The graft-versus-tumor (GVT) effect was retained in the presence of donor NK cells. We demonstrate a novel mechanism of NK cell-mediated GVHD reduction whereby donor NK cells inhibit and lyse autologous donor T cells activated during the initiation of GVHD.

    View details for DOI 10.1182/blood-2009-05-222190

    View details for Web of Science ID 000278117900021

    View details for PubMedID 20233969

  • Biology and clinical effects of natural killer cells in allogeneic transplantation CURRENT OPINION IN ONCOLOGY Benjamin, J. E., Gill, S., Negrin, R. S. 2010; 22 (2): 130-137

    Abstract

    Following allogeneic hematopoietic cell transplantation, donor-derived natural killer (NK) cells target recipient hematopoietic cells, resulting in an antileukemia effect and a lower incidence of graft rejection. NK cells do not mediate and may diminish graft versus host disease. Here we review the determinants of NK cell alloreactivity and their implications for adoptive NK cell therapy.NK cell alloreactivity has been defined by the absence of recipient MHC class I ligands for donor inhibitory killer immunoglobulin-like receptor (KIR) receptors, as predicted by a number of algorithms. Recently, the role of activating NK receptors and their cognate ligands has received more attention. The beneficial clinical effect of NK-cell alloreactivity has not been uniformly demonstrated, likely reflecting differences in conditioning regimens, graft components and posttransplant immune suppression. Investigations of NK cell phenotype and function after transplantation have helped demonstrate which NK cell subsets mediate the graft versus leukemia effect. These advances have proceeded in parallel with increasing facility in GMP-grade bulk purification and administration of NK cell preparations.NK cells are a heterogeneous population of lymphocytes with diverse patterns of target-cell recognition and effector function. Further clinical and functional correlations will help maximize their potential for clinical benefit.

    View details for DOI 10.1097/CCO.0b013e328335a559

    View details for Web of Science ID 000275198100009

    View details for PubMedID 20010294

  • Long-term follow-up of patients with diffuse large B-cell non-Hodgkin's lymphoma receiving purged autografts after induction failure BONE MARROW TRANSPLANTATION Benjamin, J. E., Chen, G. L., Cao, T. M., Cao, P. D., Wong, R. M., Sheehan, K., Shizuru, J. A., JOHNSTON, L. J., Negrin, R. S., Lowsky, R., Laport, G. G. 2010; 45 (2): 303-309

    Abstract

    Patients with diffuse large B-cell lymphoma (DLBCL) who do not achieve a complete response to front-line combination chemotherapy are often offered high-dose therapy and autologous hematopoietic cell transplantation (AHCT). However, the efficacy of this therapy in this patient population has been addressed in only a few published reports. We retrospectively analyzed the outcomes of patients with a diagnosis of de novo DLBCL who underwent AHCT at our center between 1988 and 2002, and identified 43 consecutive patients who had not achieved a CR before AHCT, although most showed at least a partial response (PR) to either induction or subsequent salvage chemotherapy. A total of 15 patients received a conditioning regimen that included high-dose chemotherapy with fractionated TBI (FTBI), whereas 28 patients received high-dose chemotherapy only. All autografts were treated ex vivo with MoAbs and complement in an effort to remove any residual malignant B cells. A total of 33 (77%) patients achieved a CR after AHCT. With a median follow-up of 7.3 years, the 5-year OS was 69% and EFS was 59%. Four patients died from non-relapse mortality. By univariate analyses, the following characteristics did not significantly impact OS: disease stage at diagnosis, age-adjusted IPI (International Prognostic Index) score, age > or =40 years, earlier radiotherapy and the use of FTBI in the conditioning regimen. These results confirm the long-term efficacy of AHCT for patients with DLBCL after induction failure.

    View details for DOI 10.1038/bmt.2009.152

    View details for Web of Science ID 000274397400013

    View details for PubMedID 19597427

  • Immunoglobulin G Fc Receptor Fc gamma RIIIa 158 V/F Polymorphism Correlates With Rituximab-Induced Neutropenia After Autologous Transplantation in Patients With Non-Hodgkin's Lymphoma JOURNAL OF CLINICAL ONCOLOGY Weng, W., Negrin, R. S., Lavori, P., Horning, S. J. 2010; 28 (2): 279-284

    Abstract

    Rituximab has been given after autologous hematopoietic cell transplantation for recurrent or refractory B-cell lymphoma with the goal of eradicating minimal residual disease. Our previous report showed that administration of two courses of rituximab after transplantation is feasible, with encouraging clinical outcomes after a short follow-up. However, neutropenia after the first or second post-transplantation rituximab treatment occurred in 52% of patients. We previously reported that polymorphisms of two immunoglobulin G Fc receptors predict rituximab response, presumably because of their role in antibody-dependent cellular cytotoxicity. In the current report, we determine whether FcgammaR polymorphisms are correlated with clinical outcomes in 33 patients with B-cell non-Hodgkin's lymphoma who received post-transplantation rituximab.Genomic DNA was used for FcgammaRIIIa V/F or the FcgammaRIIa H/R genotyping. The FcgammaR polymorphisms were then correlated with the incidence of rituximab-induced neutropenia, event-free survival (EFS), and overall survival (OS).The FcgammaRIIIa 158 V allele dose was correlated with a higher incidence of rituximab-induced neutropenia. The odds of neutropenia after the first or second post-transplantation rituximab increased three-fold with each V allele (robust z = 2.08, P = .038). The FcgammaRIIa polymorphism had no impact on rituximab-induced neutropenia. We did not observe a correlation of either FcgammaRIIIa or FcgammaRIIa polymorphism with EFS or OS.The high affinity FcgammaRIIIa 158 V allele is associated with rituximab-induced neutropenia after autologous transplantation. This is a potential tool to identify a high-risk population for developing neutropenia after antibody therapy.

    View details for DOI 10.1200/JCO.2009.25.0274

    View details for Web of Science ID 000273418000017

    View details for PubMedID 19933905

  • Direct Imaging of Immune Rejection and Memory Induction by Allogeneic Mesenchymal Stromal Cells STEM CELLS Zangi, L., Margalit, R., Reich-Zeliger, S., Bachar-Lustig, E., Beilhack, A., Negrin, R., Reisner, Y. 2009; 27 (11): 2865-2874

    Abstract

    Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase-labeled MSCs (Luc(+) MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune-deficient Balb-Nude or non-obese diabetic severe combined immunodeficiency (NOD-SCID) recipients. Graft rejection in re-challenge experiments infusing Luc(+) Fib into mice, which had previously rejected Luc(+) MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T-cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re-challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8(+) or TEA CD4(+) T cells exhibited a memory phenotype (CD122(+), CD44(+), and CD62L(low)). Collectively, these results demonstrate that MSCs are not intrinsically immune-privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party ("off the shelf") MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long-term survival of allogeneic MSCs likely represents a major challenge.

    View details for DOI 10.1002/stem.217

    View details for Web of Science ID 000271830700024

    View details for PubMedID 19750539

  • Tissue-Specific Homing and Expansion of Donor NK Cells in Allogeneic Bone Marrow Transplantation JOURNAL OF IMMUNOLOGY Olson, J. A., Zeiser, R., Beilhack, A., Goldman, J. J., Negrin, R. S. 2009; 183 (5): 3219-3228

    Abstract

    NK cells have potential therapeutic impact in suppressing graft-versus-host disease (GVHD) and enhancing antitumor effects as a cellular therapy for hematologic malignancies. However, few studies have addressed the trafficking and in vivo behavior of NK cells in murine models of bone marrow transplantation (BMT). We investigated NK cell trafficking and survival following allogeneic and syngeneic BMT using a novel bioluminescence-based imaging strategy. Transplantation of luciferase-expressing NK cells revealed CD62L-mediated trafficking to lymphoid organs and trafficking to GVHD target tissues, as evidenced by in vivo and ex vivo bioluminescence imaging. The NK cells persisted for approximately 4 wk after transplantation in allogeneic recipients, but were not detectable in syngeneic recipients. CFSE-labeling studies showed extensive NK cell proliferation in vivo. Transplanted NK cells up-regulated molecules necessary for homing to the lymph nodes, gastrointestinal tract, and skin, yet did not cause clinical GVHD. This expansion and tissue-specific homing was not solely due to the conditioning regimen, as NK cells proliferated and reached lymphoid and GVHD target tissue in unconditioned allogeneic RAG2(-/-) gamma-chain(-/-) recipients. IL-2 enhanced expansion and antitumor activity of NK cells. These results provide significant insight into the behavior and potential therapeutic impact of NK cells in BMT.

    View details for DOI 10.4049/jimmunol.0804268

    View details for Web of Science ID 000269391400040

    View details for PubMedID 19657090

  • TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors BLOOD Kohrt, H. E., Turnbull, B. B., Heydari, K., Shizuru, J. A., Laport, G. G., Miklos, D. B., Johnston, L. J., Arai, S., Weng, W., Hoppe, R. T., Lavori, P. W., Blume, K. G., Negrin, R. S., Strober, S., Lowsky, R. 2009; 114 (5): 1099-1109

    Abstract

    A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versus-host disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)-matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and event-free survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graft-versus-tumor reactions. Active trial registration currently at clinicaltrials.gov under IDs of NCT00185640 and NCT00186615.

    View details for DOI 10.1182/blood-2009-03-211441

    View details for Web of Science ID 000268491100025

    View details for PubMedID 19423725

  • Natural Killer Cells in Allogeneic Transplantation: Effect on Engraftment, Graft- versus-Tumor, and Graft-versus-Host Responses BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Gill, S., Olson, J. A., Negrin, R. S. 2009; 15 (7): 765-776

    Abstract

    Natural killer (NK) cells are effectors of the innate immune system and recognize cells transformed by viruses or neoplasia. Their response to "missing self" signals was described 3 decades ago, but the recent discovery of a panoply of activating receptors has made it clear that NK cell reactivity arises from a combination of inhibitory and activating signals. Successful clinical exploitation of NK cell reactivity was demonstrated in allogeneic transplantation for acute myelogenous leukemia from HLA-haploidentical donors when matched donors were not available. Multiple clinical studies have since attempted to use NK reactivity in the setting of both HLA-matched and -mismatched transplantation, with varying results. This review summarizes the heterogeneous clinical results and explains them based on a succinct description of NK cell biology.

    View details for DOI 10.1016/j.bbmt.2009.01.019

    View details for Web of Science ID 000267588300001

    View details for PubMedID 19539207

  • Regulation of different inflammatory diseases by impacting the mevalonate pathway IMMUNOLOGY Zeiser, R., Maas, K., Youssef, S., Duerr, C., Steinman, L., Negrin, R. S. 2009; 127 (1): 18-25

    Abstract

    The 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) interfere with the mevalonate pathway. While initially developed for their lipid-lowering properties, statins have been extensively investigated with respect to their impact on autoantigen and alloantigen driven immune responses. Mechanistically it was shown that statins modify immune responses on several levels, including effects on dendritic cells, endothelial cells, macrophages, B cells and T cells. Several lines of evidence suggest that statins act in a disease-specific manner and are not effective in each immune disorder. This review discusses possible modes of action of statins in modulating immunity towards autoantigens and alloantigens.

    View details for DOI 10.1111/j.1365-2567.2008.03011.x

    View details for Web of Science ID 000264784600003

    View details for PubMedID 19191903

  • Glycogen synthase kinase 3 beta missplicing contributes to leukemia stem cell generation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Abrahamsson, A. E., Geron, I., Gotlib, J., Dao, K. T., Barroga, C. F., Newton, I. G., Giles, F. J., Durocher, J., Creusot, R. S., Karimi, M., Jones, C., Zehnder, J. L., Keating, A., Negrin, R. S., Weissman, I. L., Jamieson, C. H. 2009; 106 (10): 3925-3929

    Abstract

    Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

    View details for DOI 10.1073/pnas.0900189106

    View details for Web of Science ID 000264036900051

    View details for PubMedID 19237556

  • Follicular lymphoma B cells induce the conversion of conventional CD4(+) T cells to T-regulatory cells INTERNATIONAL JOURNAL OF CANCER Ai, W. Z., Hou, L., Zeiser, R., Czerwinski, D., Negrin, R. S., Levy, R. 2009; 124 (1): 239-244

    Abstract

    There has been accumulating evidence that CD4(+)CD25(+) FoxP3 expressing regulatory T cells (Treg) are highly concentrated in tumors, thereby fostering an immune-privileged microenvironment. Some studies have shown that T-cell receptor (TCR) stimulation can convert conventional T cells into Treg. Follicular lymphoma (FL) B cells can enhance this Treg conversion. We investigated whether FL tumor B cells, as opposed to normal B cells, are unique in their ability to convert effector T cells into Treg. We found that tumor B cells alone, without artificial TCR stimulation, could induce conventional T cells to express FoxP3 and to acquire regulatory function. In contrast to their malignant counterpart, normal B cells did not induce Treg conversion. Treg conversion was independent of the T cell background, as T cells isolated from FL or normal peripheral blood were equally susceptible to being converted by tumor B cells. Our study provides evidence for a tumor-specific mechanism by which FL tumor cells promote immune escape through the induction of Treg.

    View details for DOI 10.1002/ijc.23881

    View details for Web of Science ID 000261664600031

    View details for PubMedID 18814264

  • Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation JOURNAL OF IMMUNOLOGY Reichardt, W., Duerr, C., von Elverfeldt, D., Juettner, E., Gerlach, U. V., Yamada, M., Smith, B., Negrin, R. S., Zeiser, R. 2008; 181 (7): 4770-4779

    Abstract

    Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

    View details for Web of Science ID 000259755700040

    View details for PubMedID 18802080

  • In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity BLOOD Nishimura, R., Baker, J., Beilhack, A., Zeiser, R., Olson, J. A., Sega, E. I., Karimi, M., Negrin, R. S. 2008; 112 (6): 2563-2574

    Abstract

    Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

    View details for DOI 10.1182/blood-2007-06-092817

    View details for Web of Science ID 000259088000053

    View details for PubMedID 18565854

  • Plasmacytoid dendritic cells take up opsonized antigen leading to CD4(+) and CD8(+) T cell activation in vivo JOURNAL OF IMMUNOLOGY Bjorck, P., Beilhack, A., Herman, E. I., Negrin, R. S., Engleman, E. G. 2008; 181 (6): 3811-3817

    Abstract

    Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.

    View details for Web of Science ID 000259250400014

    View details for PubMedID 18768834

  • Long-term remission of Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic hematopoietic cell transplantation from matched sibling donors: a 20-year experience with the fractionated total body irradiation-etoposide regimen BLOOD Laport, G. G., Alvarnas, J. C., Palmer, J. M., Snyder, D. S., Slovak, M. L., Cherry, A. M., Wong, R. M., Negrin, R. S., Blume, K. G., Forman, S. J. 2008; 112 (3): 903-909

    Abstract

    Allogeneic hematopoietic cell transplantation (HCT) is the only known curative modality for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL). Sixty-seven patients with HLA-matched sibling donors received fractionated total body irradiation (FTBI) and high-dose VP16, whereas 11 patients received FTBI/VP16/cyclophosphamide, and 1 patient received FTBI/VP16/busulfan. The median age was 36 years. At the time of HCT, 49 patients (62%) were in first complete remission (CR1) and 30 patients (38%) were beyond CR1 (> CR1). The median follow-up was 75 months (range, 14-245 months). The 10-year overall survival for the CR1 and beyond CR1 patients was 54% and 29% (P = .01), respectively, and event-free survival was 48% and 26% (P = .02), respectively. There was no significant difference in relapse incidence (28% vs 41%, P = .28), but nonrelapse mortality was significantly higher in the beyond CR1 patients, (31% vs 54%, P = .03, respectively). By univariate analysis, factors affecting event-free and overall survival were white blood cell count at diagnosis (< 30 x 10(9)/L vs > 30 x 10(9)/L) and disease status (CR1 vs > CR1). The median time to relapse for CR1 and for beyond CR1 patients was 12 months and 9 months, respectively. Our results indicate that FTBI/VP16 with or without cyclophosphamide confers long-term survival in Ph(+) ALL patients and that disease status at the time of HCT is an important predictor of outcome.

    View details for DOI 10.1182/blood-2008-03-143115

    View details for Web of Science ID 000258257900062

    View details for PubMedID 18519812

  • Long-term results of autologous hematopoietic cell transplantation for peripheral T cell lymphoma: The Stanford experience BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Chen, A. I., McMillan, A., Neprin, R. S., Horning, S. J., Laport, G. G. 2008; 14 (7): 741-747

    Abstract

    The peripheral T cell lymphomas (PTCL) carry a worse prognosis compared to B cell non-Hodgkin lymphoma. There is no uniform standard therapy for PTCL, and autologous hematopoietic cell transplant (AHCT) is often offered as consolidation in first remission or at relapse because of the poor outcomes with conventional therapy. We conducted a retrospective review of patients who underwent AHCT for PTCL from 1989 to 2006. Fifty-three cases were identified consisting of systemic anaplastic large cell (n = 18), PTCL unspecified (n = 17), angioimmunoblastic (n = 9), nasal type extranodal NK/T (n = 7), hepatosplenic (n = 2), and adult T cell leukemia/lymphoma (n = 1). Fifteen patients were transplanted in first complete or partial response (CR1/PR1), 32 in second or beyond CR or PR (CR2/PR2+), and 11 with primary refractory disease (REF). With a median follow-up was 5 years (range: 1.0-11.5), the 5-year progression-free survival (PFS) and overall survival (OS) were 25% and 48%, respectively. Disease status at AHCT had a significant impact on PFS and OS. The 5-year PFS for patients in CR1/PR1, CR2/PR2+, and REF was 51%, 12%, and 0%, respectively, and the corresponding figures for OS were 76%, 40%, and 30%, respectively. The pretransplant factors that impacted survival were disease status and the number of prior regimens. Histology, age, sex, stage, B symptoms, bone marrow involvement, and duration of first response did not significantly affect PFS or OS. Based on these results, AHCT as consolidation therapy in first complete or partial response may offer a durable survival benefit. However, AHCT with conventional salvage chemotherapy has minimal durable benefit in patients with relapsed or refractory PTCL, and thus novel strategies and/or allogeneic HCT should be more aggressively explored in lieu of AHCT for relapsed/ refractory PTCL.

    View details for DOI 10.1016/j.bbmt.2008.04.004

    View details for Web of Science ID 000256971000002

    View details for PubMedID 18541192

  • Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs BLOOD Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Baker, E. M., Landan, G., Herman, E. I., Butcher, E. C., Contag, C. H., Negrin, R. S. 2008; 111 (5): 2919-2928

    Abstract

    In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

    View details for DOI 10.1182/blood-2007-09-112789

    View details for Web of Science ID 000253671600062

    View details for PubMedID 17989315

  • Interleukin-2 receptor downstream events in regulatory T cells - Implications for the choice of immunosuppressive drug therapy CELL CYCLE Zeiser, R., Negrin, R. S. 2008; 7 (4): 458-462

    Abstract

    Naturally occurring CD4+CD25(high)FOXP3+ regulatory T cells (Tregs) constitute a powerful mechanism of immune regulation and therefore, have important therapeutic potential for disorders such as autoimmune diseases, allograft rejection and graft-versus-host disease. Disruption of the IL-2R signalling pathway by genetic defects of the interleukin (IL)-2 gene or components of the IL-2 receptor (R) complex results in severe T cell-mediated autoimmunity rather than immunodeficiency, indicating a crucial role for IL-2R signalling for Treg development and function. Signalling downstream of the IL-2R can act through the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway, the Janus kinase (JAK)/Signal transducers and Activators of Transcription (STAT) pathway and the mitogen-activated protein kinase (MAPK) pathway. In this report we focus on the relevance of these pathways as well as the impact of immunosuppressive drugs that may affect or enhance Treg function by targeting IL-2R signalling.

    View details for Web of Science ID 000254364800008

    View details for PubMedID 18235249

  • The impact of regulatory T cells on T-cell immunity following hematopoietic cell transplantation BLOOD Nguyen, V. H., Shashidhar, S., Chang, D. S., Ho, L., Kambham, N., Bachmann, M., Brown, J. M., Negrin, R. S. 2008; 111 (2): 945-953

    Abstract

    Regulatory T cells (Tregs) prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tcons). However, the impact of Tregs on T-cell development and immunity following hematopoietic cell transplantation (HCT) is unknown. Using a murine GvHD model induced by Tcons, we demonstrate that adoptive transfer of Tregs leads to (1) abrogration of GvHD, (2) preservation of thymic and peripheral lymph node architecture, and (3) an accelerated donor lymphoid reconstitution of a diverse TCR-Vbeta repertoire. The resultant enhanced lymphoid reconstitution in Treg recipients protects them from lethal cytomegalovirus (MCMV) infection. By contrast, mice that receive Tcons alone have disrupted lymphoid organs from GvHD and remain lymphopenic with a restricted TCR-Vbeta repertoire and rapid death on MCMV challenge. Lymphocytes from previously infected Treg recipients generate secondary response specific to MCMV, indicating long-term protective immunity with transferred Tregs. Thymectomy significantly reduces survival after MCMV challenge in Treg recipients compared with euthymic controls. Our results indicate that Tregs enhance immune reconstitution by preventing GvHD-induced damage of the thymic and secondary lymphoid microenvironment. These findings provide new insights into the role of Tregs in affording protection to lymphoid stromal elements important for T-cell immunity.

    View details for DOI 10.1182/blood-2007-07-103895

    View details for Web of Science ID 000252458700071

    View details for PubMedID 17916743

  • Differential impact of mammalian target of rapamycin inhibition on CD4(+)CD25(+)Foxp3(+) regulatory T cells compared with conventional CD4(+) T cells BLOOD Zeiser, R., Leveson-Gower, D. B., Zambricki, E. A., Kambham, N., Beilhack, A., Loh, J., Hou, J., Negrin, R. S. 2008; 111 (1): 453-462

    Abstract

    Based on their ability to control T-cell homeostasis, Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) are being considered for treatment of autoimmune disorders and acute graft-versus-host disease (aGVHD). When combining Tregs with the immunosuppressant rapamycin (RAPA), we observed reduced alloreactive conventional T-cell (Tconv) expansion and aGVHD lethality compared with each treatment alone. This synergistic in vivo protection was paralleled by intact expansion of polyclonal Tregs with conserved high FoxP3 expression. In contrast to Tconv, activation of Tregs with alloantigen and interleukin-2 preferentially led to signal transducer and activator of transcription 5 (STAT5) phosphorylation and not phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activity. Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR pathway, remained high in Tregs but not Tconv during stimulation. Conversely, targeted deletion of PTEN increased susceptibility of Tregs to mTOR inhibition by RAPA. Differential impact of RAPA as a result of reduced usage of the mTOR pathway in Tregs compared with conventional T cells explains the synergistic effect of RAPA and Tregs in aGVHD protection, which has important implications for clinical trials using Tregs.

    View details for DOI 10.1182/blood-2007-06-094482

    View details for Web of Science ID 000252002000063

    View details for PubMedID 17967941

  • Preemptive HMG-CoA reductase inhibition provides graft-versus-host disease protection by Th-2 polarization while sparing graft-versus-leukemia activity BLOOD Zeiser, R., Youssef, S., Baker, J., Kambham, N., Steinman, L., Negrin, R. S. 2007; 110 (13): 4588-4598

    Abstract

    We investigated whether atorvastatin (AT) was capable of protecting animals from acute graft-versus-host disease (aGVHD) across major histocompatibility complex (MHC) mismatch barriers. AT treatment of the donor induced a Th-2 cytokine profile in the adoptively transferred T cells and reduced their in vivo expansion, which translated into significantly reduced aGVHD lethality. Host treatment down-regulated costimulatory molecules and MHC class II expression on recipient antigen-presenting cells (APCs) and enhanced the protective statin effect, without impacting graft-versus-leukemia (GVL) activity. The AT effect was partially reversed in STAT6(-/-) donors and abrogated by L-mevalonate, indicating the relevance of STAT6 signaling and the L-mevalonate pathway for AT-mediated aGVHD protection. AT reduced prenylation levels of GTPases, abolished T-bet expression, and increased c-MAF and GATA-3 protein in vivo. Thus, AT has significant protective impact on aGVHD lethality by Th-2 polarization and inhibition of an uncontrolled Th-1 response while maintaining GVL activity, which is of great clinical relevance given the modest toxicity profile of AT.

    View details for DOI 10.1182/blood-2007-08-106005

    View details for Web of Science ID 000252001200068

    View details for PubMedID 17827390

  • Host-derived interleukin-18 differentially impacts regulatory and conventional T cell expansion during acute graft-versus-host disease BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Zeiser, R., Zambricki, E. A., Leveson-Gower, D., Kambham, N., Beilhack, A., Negrin, R. S. 2007; 13 (12): 1427-1438

    Abstract

    Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses, and has been shown to modulate acute graft-versus-host disease (aGVHD). It is still unknown whether increased IL-18 levels during aGVHD are of host or donor origin, and how the absence of IL-18 has an impact on migration and expansion of conventional CD4(+)CD25(-)(Tconv) and CD4(+)CD25(+) regulatory (Treg) T cells in vivo. By utilizing IL-18 gene-deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, whereas donor hematopoietic cells contributed significantly less. By generating IL-18(-/-) luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. Although migration to secondary lymphoid organs did not have a significantly impact from the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-gamma levels in the early phase after transplantation. We conclude that host-derived IL-18 is a major factor for IFN-gamma production that may have a protective effect on CD4(+)-mediated aGVHD, but is nonessential for Treg expansion in an allogeneic environment.

    View details for DOI 10.1016/j.bbmt.2007.08.041

    View details for Web of Science ID 000251495900004

    View details for PubMedID 18022572

  • A novel technique for the enrichment of primary ovarian cancer cells AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY Chan, J. K., Hamilton, C. A., Anderson, E. M., Cheung, M. K., Baker, J., Husain, A., Teng, N. N., Kong, C. S., Negrin, R. S. 2007; 197 (5)

    Abstract

    Primary cancer cells that are extracted from ovarian tumors can serve as an optimal substrate to study the biologic characteristics of ovarian cancer. We describe an efficient and effective method of enriching ovarian tumor cells from ascitic fluid using an immunomagnetic-based method.Mononuclear cells were isolated from ascites specimens by Ficoll gradient separation. Epithelial ovarian cancer cells were labeled magnetically with monoclonal human epithelial antigen-125 that is conjugated to microbeads. After immunomagnetic separation, the purity of tumor cells before and after purification was quantified by cytologic analysis and confirmed by fluorescence-activated cell sorter analysis.Peritoneal ascites specimens were obtained from 6 patients with ovarian cancer. The median age of our patients was 61.5 years (range, 46-79 years). Three patients had papillary serous carcinoma; 2 patients had clear cell carcinoma, and 1 patient had an undifferentiated adenocarcinoma. The mean tumor purity was only 22.8% +/- 10% (range, 1%-60%) before separation. After enrichment, the purity improved to 82.3% +/- 4.0% (range, 70%-90%). Our enrichment technique increased the tumor purity by 59.5% +/- 8.4%. The mean percent yield after positive enrichment was 30.1% +/- 14.5%.The immunomagnetic cell separation technique is an efficient and effective method for isolating and purifying ovarian tumor cells from ascites. Results from experiments with fresh tumor cells rather than cancer cell lines may be more relevant for clinical application.

    View details for DOI 10.1016/j.ajog.2007.05.006

    View details for Web of Science ID 000250915500021

    View details for PubMedID 17980191

  • CD101 surface expression discriminates potency among murine FoxP3(+) regulatory T cells JOURNAL OF IMMUNOLOGY Fernandez, I., Zeiser, R., Karsunky, H., Kambham, N., Beilhack, A., Soderstrom, K., Negrin, R. S., Engleman, E. 2007; 179 (5): 2808-2814

    Abstract

    CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.

    View details for Web of Science ID 000248991800020

    View details for PubMedID 17709494

  • In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation BLOOD Nguyen, V. H., Zeiser, R., daSilva, D. L., Chang, D. S., Beilhack, A., Contag, C. H., Negrin, R. S. 2007; 109 (6): 2649-2656

    Abstract

    CD4(+)CD25(+) regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

    View details for DOI 10.1182/blood-2006-08-044529

    View details for Web of Science ID 000245004700060

    View details for PubMedID 17095616

  • Early CD30 signaling is critical for adoptively transferred CD4(+)CD25(+) regulatory T cells in prevention of acute graft-versus-host disease BLOOD Zeiser, R., Nguyen, V. H., Hou, J., Beilhack, A., Zambricki, E., Buess, M., Contag, C. H., Negrin, R. S. 2007; 109 (5): 2225-2233

    Abstract

    Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30-/-) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30-/- animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.

    View details for DOI 10.1182/blood-2006-07-038455

    View details for Web of Science ID 000244641100067

    View details for PubMedID 17068147

  • The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia BLOOD Oehler, V. G., Gooley, T., Snyder, D. S., Johnston, L., Lin, A., Cummings, C. C., Chu, S., Bhatia, R., Forman, S. J., Negrin, R. S., Appelbaum, F. R., Radich, J. P. 2007; 109 (4): 1782-1789

    Abstract

    The impact of imatinib mesylate (IM) treatment for chronic myeloid leukemia (CML) on subsequent allogeneic transplantation is uncertain. To better understand this relationship, we retrospectively compared 145 patients with CML receiving IM for a minimum of 3 months before allogeneic hematopoietic cell transplantation (HCT) to 231 patients with CML who did not. IM treatment was associated with no increase in early hepatotoxicity or engraftment delay after HCT compared with the historical cohort. In addition, there was no statistically significant difference in the IM-treated cohort compared with the historical cohort with regard to overall survival, disease-free survival, relapse, and nonrelapse mortality. For chronic-phase (CP) patients, IM response prior to HCT was associated with post-HCT outcome. Patients who underwent transplantation in CP with a suboptimal response or a loss of response on IM had a statistically significant higher hazard of mortality when compared with CP patients who achieved a complete cytogenetic response (CCR) or major cytogenetic response (MCR) on IM (HR=5.31, 95% confidence interval [CI] 1.13-25.05, P=.03). These data indicate that pre-HCT IM is not associated with increased transplant-related morbidity (TRM) or poorer outcomes. However, patients with a suboptimal or loss of IM response before HCT do worse, suggesting a more aggressive disease course for these patients.

    View details for Web of Science ID 000244219400061

    View details for PubMedID 17062727

  • Promise and challenges of human regulatory T cells in the clinic BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Negrin, R. S., Hou, J. 2007; 13 (1): 12-16
  • Role of naturally arising regulatory T cells in hematopoietic cell transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Nguyen, V. H., Zeiser, R., Negrin, R. S. 2006; 12 (10): 995-1009

    Abstract

    Naturally arising CD4(+)CD25(+) regulatory T cells (Tregs) have the potential to suppress aberrant immune responses and to regulate peripheral T-cell homeostasis. In murine models of bone marrow transplantation, Tregs promote donor bone marrow engraftment and decrease the incidence and severity of graft-versus-host-disease without abrogating the beneficial graft-versus-tumor immunologic effect. These findings, in concert with observations that Tregs in mice and humans share phenotypic and functional characteristics, have led to active investigations into the use of these cells to decrease complications associated with human hematopoietic cell transplantation. Early human studies suggest that an imbalance of Tregs and effector T cells may contribute to the development of graft-versus-host-disease. However, the mechanisms of immunoregulation, in particular the allorecognition properties of Tregs, their effects on and interaction with other immune cells, and their sites of suppressive activity, are not well understood. In this review, we discuss the current knowledge of Treg biology and the potential therapeutic strategies and barriers of Treg immunotherapy in human hematopoietic cell transplantation.

    View details for DOI 10.1016/j.bbmt.2006.04.009

    View details for Web of Science ID 000241305900001

    View details for PubMedID 17084366

  • High-dose carmustine, etoposide, and cyclophosphamide followed by allogeneic hematopoietic cell transplantation for non-Hodgkin lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Law, L. Y., Horning, S. J., Wong, R. M., Johnston, L. J., Laport, G. G., Lowsky, R., Shizuru, J. A., Blume, K. G., Negrin, R. S., Stockerl-Goldstein, K. E. 2006; 12 (7): 703-711

    Abstract

    Allogeneic hematopoietic cell transplantation (HCT) has been shown to be curative in a group of patients with aggressive non-Hodgkin lymphoma (NHL). A previous study has demonstrated equivalent outcomes with a conditioning regimen based on total body irradiation and another not based on total body irradiation with preparative therapy using cyclophosphamide, carmustine, and etoposide (CBV) in autologous HCT. We investigated the safety and efficacy of using CBV in an allogeneic setting. Patients were required to have relapsed or be at high risk for subsequent relapse of NHL. All patients had a fully HLA-matched sibling donor. Patients received carmustine (15 mg/kg), etoposide (60 mg/kg), and cyclophosphamide (100 mg/kg) on days -6, -4, and -2, respectively, followed by allogeneic HCT. All patients were treated with cyclosporine and methylprednisolone as prophylaxis for graft-versus-host disease (GVHD). Thirty-one patients (median age, 46 years) who were felt to be inappropriate candidates for autologous transplantation were enrolled. Each subject had a median of 3 previous chemotherapy regimens. All patients engrafted. Fifteen of 31 patients are alive. Median follow-up time was 11.5 months (range, .4-126). There were 8 deaths due to relapse. Nonrelapse mortality (n = 8) included infection (n = 3), GVHD (n = 2), diffuse alveolar hemorrhage (n = 1), veno-occlusive disease in the setting of concurrent acute GVHD of the liver (n = 1), and leukoencephalopathy (n = 1). Probabilities of event-free survival and overall survival were, respectively, 44% (95% confidence interval, 26%-62%) and 51% (33%-69%) at 1 year and 44% (26%-62%) and 47% (29%-65%) at 5 years. Probability of relapse was 33% (15%-51%) at 1 year and 5 years. Probability of nonrelapse mortality was 31% (13%-49%) at 1 year and 5 years. Incidences were 29% for acute GVHD and 39% for chronic GVHD. None of the 12 patients who developed chronic GVHD has disease recurrence. Patients who had required >3 previous chemotherapy regimens before HCT had an increased probability of relapse. CBV is an effective preparative regimen for patients with aggressive NHL who undergo allogeneic HCT.

    View details for DOI 10.1016/j.bbmt.2006.02.009

    View details for Web of Science ID 000238774800002

    View details for PubMedID 16785059

  • Inhibition of CD4(+)CD25(+) regulatory T-cell function by calcineurin-dependent interleukin-2 production BLOOD Zeiser, R., Nguyen, V. H., Beilhack, A., Buess, M., Schulz, S., Baker, J., Contag, C. H., Negrin, R. S. 2006; 108 (1): 390-399

    Abstract

    CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.

    View details for DOI 10.1182/blood-2006-01-0329

    View details for Web of Science ID 000238596900060

    View details for PubMedID 16522809

  • In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease. Nature reviews. Immunology Negrin, R. S., Contag, C. H. 2006; 6 (6): 484-490

    Abstract

    Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions.

    View details for PubMedID 16724101

  • Innovation - In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease NATURE REVIEWS IMMUNOLOGY Negrin, R. S., Contag, C. H. 2006; 6 (6): 484-U2

    View details for DOI 10.1038/nri1879

    View details for Web of Science ID 000238529700017

  • Acute graft-versus-host disease-challenge for a broader application of allogeneic hematopoietic cell transplantation. Current stem cell research & therapy Zeiser, R., Beilhack, A., Negrin, R. S. 2006; 1 (2): 203-212

    Abstract

    Allogeneic hematopoietic cell transplanation (aHCT) has been a successful treatment option for malignant disease based on the graft-versus-tumor effect. However, the overall clinical success of aHCT is impaired by the high morbidity and mortality caused by acute graft-versus-host disease (aGVHD). aGVHD can also be seen as the major limitation of aHCT for a broader clinical applicability of this treatment, particularly for non-malignant disease conditions. Recent murine studies have shed more light on the kinetics of aGVHD development by tracking donor T cells in vivo. These data define two functionally distinct stages in aGVHD pathogenesis taking place in different anatomical compartments. The aGVHD initiation phase is confined to T cell areas in secondary lymphoid organs in contrast to the later aGVHD effector phase at target sites. This temporal pattern may explain the clinical observation that when acute aGVHD is clinically overt, treatment with intensified immunosuppression often remains ineffective. This review will focus on the immune-pathogenesis of aGVHD, conventional and novel treatment strategies including the removal of naïve T cells, tolerance induction by mesenchymal stem cells, regulatory T cells, genetic manipulation of donor cells and the potential of memory T cells for improving immune reconstitution without aGVHD. A better understanding of the mechanisms involved in aGVHD pathogenesis might allow for a broader application of novel stem cell therapies.

    View details for PubMedID 18220867

  • Synergistic antitumor effects of immune cell-viral biotherapy SCIENCE Thorne, S. H., Negrin, R. S., Contag, C. H. 2006; 311 (5768): 1780-1784

    Abstract

    Targeted biological therapies hold tremendous potential for treatment of cancer, yet their use has been limited by constraints on delivery and effective tumor targeting. We combined an immune effector cell population [cytokine-induced killer (CIK) cells] with an oncolytic viral therapy to achieve directed delivery to, and regression of, tumors in both immunodeficient and immunocompetent mouse models. Preinfection of CIK cells with modified vaccinia virus resulted in a prolonged eclipse phase with the virus remaining hidden until interaction with the tumor. Whole-body imaging revealed that the cells retained their ability to traffic to and to infiltrate the tumor effectively before releasing the virus. These results illustrate the potential of combining biotherapeutics for synergistic effects that more effectively treat cancer.

    View details for DOI 10.1126/science.1121411

    View details for Web of Science ID 000236204300048

    View details for PubMedID 16556847

  • Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: A preclinical study CLINICAL CANCER RESEARCH Chan, J. K., Hamilton, C. A., Cheung, M. K., Karimi, M., Baker, J., Gall, J. M., Schulz, S., Thorne, S. H., Teng, N. N., Contag, C. H., Lum, L. G., Negrin, R. S. 2006; 12 (6): 1859-1867

    Abstract

    Cytokine-induced killer (CIK) cells are ex vivo activated and expanded CD8+ natural killer T cells that have been shown to have antitumor activity. This is the first study exploring cell killing of primary ovarian carcinoma cells with and without bispecific antibodies. Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. Bispecific antibodies against cancer antigen-125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On fluorescence-activated cell sorting analysis, the expansion of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by bispecific antibodies, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 +/- 0.3% to 89.4 +/- 2.1% at an E:T ratio of 100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001 and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.

    View details for DOI 10.1158/1078-0432.CCR-05-2019

    View details for Web of Science ID 000236458800028

    View details for PubMedID 16551871

  • The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab CYTOTHERAPY Kornacker, M., Verneris, M. R., Kornacker, B., Ganten, T. M., Scheffold, C., Negrin, R. S. 2006; 8 (1): 13-23

    Abstract

    Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb.Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells.When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity.bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.

    View details for DOI 10.1080/14653240500518264

    View details for Web of Science ID 000235427100003

    View details for PubMedID 16627341

  • Total lymphoid irradiation and transplantation tolerance CURRENT OPINION IN ORGAN TRANSPLANTATION Lowsky, R., Negrin, R. S. 2006; 11 (1): 54-61
  • Protective conditioning for acute graft-versus-host disease - Reply NEW ENGLAND JOURNAL OF MEDICINE Lowsky, R., Negrin, R. S., Strober, S. 2005; 353 (25): 2718-2718
  • Silencing human NKG2D, DAP10, and DAP12 reduces cytotoxicity of activated CD8(+) T cells and NK cells JOURNAL OF IMMUNOLOGY Karimi, M., Cao, T. M., Baker, J. A., Verneris, M. R., Soares, L., Negrin, R. S. 2005; 175 (12): 7819-7828

    Abstract

    Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.

    View details for Web of Science ID 000234030400008

    View details for PubMedID 16339517

  • Ex vivo expanded dendritic cells home to T-cell zones of lymphoid organs and survive in vivo after allogeneic bone marrow transplantation AMERICAN JOURNAL OF PATHOLOGY Schimmelpfennig, C. H., Schulz, S., Arber, C., Baker, J., Tarner, I., McBride, J., Contag, C. H., Negrin, R. S. 2005; 167 (5): 1321-1331

    Abstract

    Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

    View details for Web of Science ID 000232970200014

    View details for PubMedID 16251416

  • Protective conditioning for acute graft-versus-host disease NEW ENGLAND JOURNAL OF MEDICINE Lowsky, R., Takahashi, T., Liu, Y. P., Dejbakhsh-Jones, S., GRUMET, F. C., Shizuru, J. A., Laport, G. G., Stockerl-Goldstein, K. E., JOHNSTON, L. J., Hoppe, R. T., Bloch, D. A., Blume, K. G., Negrin, R. S., Strober, S. 2005; 353 (13): 1321-1331

    Abstract

    Conditioning with total lymphoid irradiation plus antithymocyte serum protects mice against acute graft-versus-host disease (GVHD) after hematopoietic-cell transplantation. We tested this strategy in humans.Thirty-seven patients with lymphoid malignant diseases or acute leukemia underwent an experimental conditioning regimen with 10 doses of total lymphoid irradiation (80 cGy each) plus antithymocyte globulin, followed by an infusion of HLA-matched peripheral-blood mononuclear cells from related or unrelated donors who received granulocyte colony-stimulating factor.Of the 37 transplant recipients, only 2 had acute GVHD after hematopoietic-cell transplantation. Potent antitumor effects in patients with lymphoid malignant diseases were shown by the change from partial to complete remission. In the transplant recipients who underwent conditioning with total lymphoid irradiation and antithymocyte globulin, the fraction of donor CD4+ T cells that produced interleukin-4 after in vitro stimulation increased by a factor of five, and the proliferative response to alloantigens in vitro was reduced, as compared with normal control subjects and control subjects who underwent conditioning with a single dose of total-body irradiation (200 cGy).A regimen of total lymphoid irradiation plus antithymocyte globulin decreases the incidence of acute GVHD and allows graft antitumor activity in patients with lymphoid malignant diseases or acute leukemia treated with hematopoietic-cell transplantation.

    View details for Web of Science ID 000232146200004

    View details for PubMedID 16192477

  • In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets BLOOD Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Wieland, C. B., Herman, E. I., Baker, E. M., Cao, Y. A., Contag, C. H., Negrin, R. S. 2005; 106 (3): 1113-1122

    Abstract

    Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (T(EM)) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

    View details for DOI 10.1182/blood-2005-02-0509

    View details for Web of Science ID 000230949100061

    View details for PubMedID 15855275

  • Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation TRANSPLANTATION Cao, Y. A., Bachmann, M. H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R. J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T. C., Fathman, C. G., Robbins, R. C., Herzenberg, L. A., Negrin, R. S., Contag, C. H. 2005; 80 (1): 134-139

    Abstract

    Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.

    View details for DOI 10.1097/01.TP.0000164347.50559.A3

    View details for Web of Science ID 000230473800023

    View details for PubMedID 16003245

  • Low levels of Her2/neu expressed by Ewing's family tumor cell lines can redirect cytokine-induced killer cells CLINICAL CANCER RESEARCH Verneris, M. R., Arshi, A., Edinger, M., Kornacker, M., Natkunam, Y., Karami, M., Cao, Y. A., Marina, N., Contag, C. H., Negrin, R. S. 2005; 11 (12): 4561-4570

    Abstract

    To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression.Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging.EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas).CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

    View details for Web of Science ID 000229725900037

    View details for PubMedID 15958642

  • A phase I trial of autologous cytokine-induced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Leemhuis, T., Wells, S., Scheffold, C., Edinger, M., Negrin, R. S. 2005; 11 (3): 181-187

    Abstract

    We have previously reported on the ex vivo generation of cytotoxic effector cells, termed cytokine-induced killer (CIK) cells, that have both in vitro and in vivo antitumor activity in murine models. We now report on our efforts for the large-scale expansion of CIK cells and also present preliminary results from a phase I clinical trial. Nine patients with advanced Hodgkin disease (n = 7) and non-Hodgkin lymphoma (n = 2), all of whom had relapsed after an autologous transplantation, were treated with escalating doses of CIK cells (3 patients at each dose level of 1 x 10(9) , 5 x 10(9) , or 1 x 10(10) cells). The CIK cells were produced by culturing unselected cells from steady-state apheresis products with interferon gamma, OKT3, and interleukin 2. After 21 days in culture, with the addition of fresh media and interleukin 2 every 3 to 4 days, the median culture was 97% viable (range, 61%-100%), 98% CD3 + (range, 66%-99%), 76% CD8 + (range, 27%-96%), 23% CD4 + (range, 6%-78%), 20% CD3 + CD56 + (range, 8%-58%), and <1% CD16 + 56 + (range, 0.2%-7.7%). The CD3 + CD56 + cells have previously been shown to exhibit the most cytotoxic activity. The absolute number of CD3 + CD56 + cells typically expanded 290-fold (range, 3- to 4000-fold) under these culture conditions. In vitro cytotoxic activity was measured against a human B-cell tumor line (OCI-Ly8). At a 40:1 effector-target cell ratio, CIK cells killed 32% (range, 2%-69%) of the target cells. A total of 21 infusions were administered to 9 patients. The number of CIK cells infused ranged from 1.0 x 10(9) to 1.0 x 10(10) per treatment. Toxicity was minimal, and there were no immediate adverse reactions to the infusions. Two patients had partial responses, and 2 patients had stabilization of disease: 1 for more than 18 months. Considering that these were heavily pretreated patients with advanced hematologic malignancies, we believe that CIK cells expanded in this fashion may have utility for the treatment of high-risk patients with evidence of minimal residual disease after autologous transplantation.

    View details for DOI 10.1016/j.bbmt.2004.11.019

    View details for Web of Science ID 000227958300003

    View details for PubMedID 15744236

  • Randomized trial of allogeneic related bone marrow transplantation versus peripheral blood stem cell transplantation for chronic myeloid leukemia BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Oehler, V. G., Radich, J. P., Storer, I., Blume, K. G., Chauncey, T., Clift, R., Snyder, D. S., Forman, S. J., Flowers, M. E., Martin, P., Guthrie, K. A., Negrin, R. S., Appelbaum, F. R., Bensinger, W. 2005; 11 (2): 85-92

    Abstract

    Seventy-two chronic myeloid leukemia patients were enrolled as part of a larger randomized trial at 3 centers between March 1996 and July 2001 to undergo either HLA-matched related allogeneic bone marrow (BM) or filgrastim (granulocyte colony-stimulating factor)-mobilized peripheral blood stem cell (PBSC) transplantation. Forty patients received BM, and 32 patients received PBSCs. There was no statistically significant difference in the incidence of acute or chronic graft-versus-host disease (GVHD), overall survival, disease-free survival, or non-relapse-related mortality between patients receiving BM or PBSC transplants. The cumulative incidence of grade II to IV acute GVHD was 49% in BM and 55% in PBSC recipients ( P = .48). The cumulative incidence of clinical extensive chronic GVHD was 50% in BM and 59% in PBSC recipients ( P = .46). Among 62 chronic phase chronic myeloid leukemia patients, there was no significant difference in overall survival (87% versus 81%; P = .59), disease-free survival (80% versus 81%; P = .61), or non-relapse-related mortality (13% versus 19%; P = .60) by cell source (BM versus PBSCs). Among chronic phase patients, however, there was a trend toward a higher cumulative incidence of relapse at 3 years in BM recipients (7% versus 0%; P = .10) and a higher cumulative incidence of chronic GVHD in PBSC recipients (59% versus 40%; P = .11). The trend toward a higher relapse incidence in BM recipients persisted with a longer follow-up.

    View details for DOI 10.1016/j.bbmt.2004.09.010

    View details for Web of Science ID 000227233100002

    View details for PubMedID 15682068

  • Hematopoietic stem and progenitor cells: Clinical and preclinical regeneration of the hematolymphoid system ANNUAL REVIEW OF MEDICINE Shizuru, J. A., Negrin, R. S., Weissman, I. L. 2005; 56: 509-538

    Abstract

    A vast literature exists on the biology of blood formation and regeneration under experimental and clinical conditions. The field of hematopoiesis was recently advanced by the capacity to purify to homogeneity primitive hematopoietic stem and progenitor cells. Isolation of cells at defined maturational stages has enhanced the understanding of the fundamental nature of stem cells, including how cell fate decisions are made, and this understanding is relevant to the development of other normal as well as malignant tissues. This review updates the basic biology of hematopoietic stem cells (HSC) and progenitors, the evolving use of purified HSC as grafts for clinical hematopoietic cell transplantation (HCT) including immune tolerance induction, and the application of HSC biology to other stem cell fields.

    View details for DOI 10.1146/annurev.med.54.101601.152334

    View details for Web of Science ID 000227504100028

    View details for PubMedID 15660525

  • Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8(+) T cells BLOOD Verneris, M. R., Karami, M., Baker, J., Jayaswal, A., Negrin, R. S. 2004; 103 (8): 3065-3072

    Abstract

    Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)-unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling.

    View details for Web of Science ID 000222163500040

    View details for PubMedID 15070686

  • Evaluation of effector cell fate and function by in vivo bioluminescence imaging METHODS Edinger, M., Hoffmann, P., Contag, C. H., Negrin, R. S. 2003; 31 (2): 172-179

    Abstract

    The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.

    View details for DOI 10.1016/S1046-2023(03)00127-0

    View details for Web of Science ID 000185379400009

    View details for PubMedID 12957575

  • CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation NATURE MEDICINE Edinger, M., Hoffmann, P., Ermann, J., Drago, K., Fathman, C. G., Strober, S., Negrin, R. S. 2003; 9 (9): 1144-1150

    Abstract

    Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.

    View details for DOI 10.1038/nm915

    View details for Web of Science ID 000185061600026

    View details for PubMedID 12925844

  • Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging BLOOD Edinger, M., Cao, Y. A., Verneris, M. R., Bachmann, M. H., Contag, C. H., Negrin, R. S. 2003; 101 (2): 640-648

    Abstract

    Cancer therapeutics have achieved success in the treatment of a variety of malignancies, however, relapse of disease from small numbers of persistent tumor cells remains a major obstacle. Advancement of treatment regimens that effectively control minimal residual disease and prevent relapse would be greatly accelerated if sensitive and noninvasive assays were used to quantitatively assess tumor burden in animal models of minimal residual disease that are predictive of the human response. In vivo bioluminescence imaging (BLI) is an assay for the detection of small numbers of cells noninvasively and enables the quantification of tumor growth within internal organs. Fusion genes that encode bioluminescent and fluorescent reporter proteins effectively couple the powerful in vivo capabilities of BLI with the subset-discriminating capabilities of fluorescence-activated cell sorting. We labeled 2 murine lymphoma cell lines with dual function reporter genes and monitored radiation and chemotherapy as well as immune-based strategies that employ the tumorcidal activity of ex vivo-expanded CD8(+) natural killer (NK)-T cells. Using BLI we were able to visualize the entire course of malignant disease including engraftment, expansion, metastasis, response to therapy, and unique patterns of relapse. We also labeled the effector NK-T cells and monitored their homing to the sites of tumor growth followed by tumor eradication. These studies reveal the efficacy of immune cell therapies and the tempo of NK-T cell trafficking in vivo. The complex cellular processes in bone marrow transplantation and antitumor immunotherapy, previously inaccessible to investigation, can now be revealed in real time in living animals.

    View details for DOI 10.1182/blood-2002-06-1751

    View details for Web of Science ID 000180384800039

    View details for PubMedID 12393519

  • Advancing animal models of neoplasia through in vivo bioluminescence imaging EUROPEAN JOURNAL OF CANCER Edinger, M., Cao, Y. A., Hornig, Y. S., Jenkins, D. E., Verneris, M. R., Bachmann, M. H., Negrin, R. S., Contag, C. H. 2002; 38 (16): 2128-2136

    Abstract

    Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.

    View details for Web of Science ID 000179242500008

    View details for PubMedID 12387838

  • Visualization of effective tumor targeting by CD8+natural killer T cells redirected with bispecific antibody F(ab ')(2)HER2xCD3 CANCER RESEARCH Scheffold, C., Kornacker, M., Scheffold, Y. C., Contag, C. H., Negrin, R. S. 2002; 62 (20): 5785-5791

    Abstract

    HER2 is an attractive immunotherapeutic target for neoplastic disease because this cell surface molecule is overexpressed on a large fraction of malignant tumor cells. To directly assess therapeutic responses to targeted therapy by noninvasive in vivo imaging in small animals, human HER2-expressing ovarian carcinoma cells were genetically modified with a firefly luciferase gene, and light emission was used for visualization of tumor growth and response to therapy. This imaging approach was able to demonstrate in real-time tumor regression in a HER2 xenograft mouse model by adoptive transfer of in vitro induced and expanded cytotoxic CD8+ natural killer T (NKT) cells retargeted with a humanized bispecific antibody F(ab')(2)HER2xCD3. Immunotherapy with effector cells alone or a humanized monoclonal antibody anti-p185(HER2) (4D5-8) resulted in significant but slower reduction in tumor burden. Long-term survival of tumor xenografts correlated inversely with visible residual tumor burden. In vitro, F(ab')(2)HER2xCD3 substantially augmented cytotoxic activity of CD8+ NKT cells. By flow-sorting, CD8+ NKT cells coexpressing CD56 were found to have the highest redirected killing ability. Treatment with concanamycin A or EGTA abrogated CD8+ NKT cytotoxicity indicating that perforin is a major pathway of tumor cell lysis. In contrast, when CD8+ NKT cell were cross-linked with F(ab')(2)HER2xCD3 neither the immunosuppressants cyclosporine A and FK506, nor the increase of intracellular cyclic AMP by dibutyryl cyclic AMP were able to inhibit cytotoxicity demonstrating that signaling via the CD3 antigen changes the biological activity of non-MHC-restricted effector cells. These studies have demonstrated that CD8+ NKT cells can be successfully redirected to tumor cells using bispecific antibodies and offer a promising strategy for adoptive immunotherapy of neoplastic diseases.

    View details for Web of Science ID 000178693400027

    View details for PubMedID 12384539

  • Studies of ex vivo activated and expanded CD8(+) NK-T cells in humans and mice JOURNAL OF CLINICAL IMMUNOLOGY Verneris, M. R., Baker, J., Edinger, M., Negrin, R. S. 2002; 22 (3): 131-136

    Abstract

    Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. Here we describe studies of both human and murine expanded CIK cells or CD8+ NK-T cells. These related populations of cells are ex vivo activated and expanded T cells that express both T and NK markers. They can be generated from patients with malignancies and mediate cytotoxicity against autologous hematopoietic malignancies. Recent work in murine models show that these cells mediate cytotoxicity by using a perforin-granzyme and not through Fas ligand. In allogeneic stem cell transplantation experiments, large numbers of expanded CD8+ NK-T cells could be transplanted across major histocompatibility barriers without causing severe GVHD and GVT effects were retained. We conclude that expanded CD8+ NK-T cells are a promising form of cellular therapy in the allogeneic setting.

    View details for Web of Science ID 000175503300003

    View details for PubMedID 12078854

  • Understanding immune cell trafficking patterns via in vivo bioluminescence imaging JOURNAL OF CELLULAR BIOCHEMISTRY Mandl, S., Schimmelpfennig, C., Edinger, M., Negrin, R. S., Contag, C. H. 2002: 239-248

    Abstract

    Cell migration is a key aspect of the development of the immune system and mediating an immune response. There is extensive and continual redistribution of cells to different anatomic sites throughout the body. These trafficking patterns control immune function, tissue regeneration, and host responses to insult. The ability to monitor the fate and function of cells, therefore, is imperative to both understanding the role of specific cells in disease processes and to devising rational therapeutic strategies. Determining the fate of immune cells and understanding the functional changes associated with migration and proliferation require effective means of obtaining in vivo measurements in the context of intact organ systems. A variety of imaging methods are available to provide structural information, such as X-ray CT and MRI, but only recently new tools have been developed that reveal cellular and molecular changes as they occur within living animals. We have pioneered one of these techniques that is based on the observations that light passes through mammalian tissues, and that luciferases can serve as internal biological sources of light in the living body. This method, called in vivo bioluminescence imaging, is a rapid and noninvasive functional imaging method that employs light-emitting reporters and external photon detection to follow biological processes in living animals in real time. This imaging strategy enables the studies of trafficking patterns for a variety of cell types in live animal models of human biology and disease. Using this approach we have elucidated the spatiotemporal trafficking patterns of lymphocytes within the body. In models of autoimmune disease we have used the migration of "pathogenic" immune cells to diseased tissues as a means to locally deliver and express therapeutic proteins. Similarly, we have determined the tempo of NK-T cell migration to neoplastic lesions and measured their life span in vivo. Using bioluminescence imaging individual groups of animals can be followed over time significantly reducing the number of animals per experiment, and improving the statistical significance of a study since changes in a given population can be studied over time. Such rapid assays that reveal cell fates in vivo will increase our basic understanding of the molecular signals that control these migratory pathways and will substantially speed up the development and evaluation of therapies.

    View details for DOI 10.1002/jcb.10454

    View details for Web of Science ID 000180777600028

    View details for PubMedID 12552623

  • Visualization of tumor growth and response to NK-T cell based immunotherapy using bioluminescence ANNALS OF HEMATOLOGY Negrin, R. S., Edinger, M., Verneris, M., Cao, Y. A., Bachmann, M., Contag, C. H. 2002; 81: S44-S45

    View details for Web of Science ID 000178591900015

    View details for PubMedID 12611073

  • Bioluminescence imaging of lymphocyte trafficking in vivo EXPERIMENTAL HEMATOLOGY Hardy, J., Edinger, M., Bachmann, M. H., Negrin, R. S., Fathman, C. G., Contag, C. H. 2001; 29 (12): 1353-1360

    Abstract

    Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

    View details for Web of Science ID 000172949100001

    View details for PubMedID 11750093

  • Hodgkin disease-derived cell lines expressing ubiquitous mitochondrial creatine kinase show growth inhibition by cyclocreatine treatment independent of apoptosis INTERNATIONAL JOURNAL OF CANCER Kornacker, M., Schlattner, U., Wallimann, T., Verneris, M. R., Negrin, R. S., Kornacker, B., Staratschek-Jox, A., Diehl, V., Wolf, J. 2001; 94 (4): 513-519

    Abstract

    Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. Although these results support a role of CK activity in the inhibitory action of cCr, it remains open whether the cCr effect is due to its inhibition of CK-linked energy metabolism or if alternative mechanisms have to be considered. Because the anti-proliferative effect of cCr was not due to induction of apoptosis, in contrast to most other anticancer agents, treatment with the creatine analogue cCr may represent an advantageous therapeutic approach for cells resistant to programmed cell death.

    View details for Web of Science ID 000171542700008

    View details for PubMedID 11745437

  • Rapid engraftment after allogeneic transplantation of density-enriched peripheral blood CD34+cells in patients with advanced hematologic malignancies CANCER Cao, T. M., Kusnierz-Glaz, C., Valone, F., Stockerl-Goldstein, K. E., Hu, W. W., Johnston, L., Blume, K. G., Strober, S., Negrin, R. S. 2001; 91 (12): 2205-2213

    Abstract

    Acute graft versus host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Preclinical studies have suggested that a T-cell subset with a CD4-/CD8- double-negative (DN) T-cell phenotype is capable of suppressing GVHD. Double-negative T cells can be mobilized into the peripheral blood with granulocyte colony-stimulating factor (G-CSF) and enriched by density centrifugation. The current study was performed to study the feasibility and safety of applying a density gradient separation technique for enrichment of CD34+ and DN T cells, while depleting CD4+ and CD8+ single-positive (SP) T cells from peripheral blood progenitor cells (PBPCs) for the purpose of allogeneic transplantation.Twenty-five patients with advanced hematologic malignancies were treated with a myeloablative preparative regimen consisting of fractionated total body irradiation, etoposide, and cyclophosphamide. Human leukocyte antigen identical donors were mobilized with G-CSF PBPC collected by apheresis. The apheresis product was applied to a single-step density gradient, and the low-density cell population was collected. The low-density cell population was infused as the sole source of allogeneic cells after myeloablative therapy. Graft versus host disease prophylaxis consisted of cyclosporine with or without prednisone.CD34 cell recovery was efficient with a median 72% yield, providing for a median CD34+ cell dose of 6.5 x 10(6)/kg (range,1.0- 13.9 x 10(6)/kg). CD3+CD4+ or CD3+CD8+ SP T cells were depleted by a median of 94.4% (range, 58.8- 99.2%), and the ratio of CD34+:SP T cells increased 10-fold. Double-negative T cells were depleted by 92% (range, 18.8- 99.4%), thus the ratio of DN:SP T cells increased less than 2-fold in 71% of apheresis samples tested. Hematopoietic engraftment was rapid, and there was no occurrence of graft failure in examinable patients. Median time to absolute neutrophil count greater than 0.5 x 10(9)/L and platelet count greater than 20 x 10(9)/L was 10.5 and 12 days, respectively. The incidence of Grade 2-4 acute GVHD was 26% (95% confidence interval [CI], 6-45%), although not all patients were examinable due to an unexpectedly high nonrecurrence mortality that at Day 180 was 62% (95% CI, 40-83%).These data suggest that T-cell subset manipulation via density gradient separation is a safe procedure and allowed rapid hematopoietic recovery. Selective enrichment of a donor DN T-cell subset was observed in only a few and was not associated with a reduced incidence of GVHD. However, the low-density selected cells still resulted in GVHD, and there was a high treatment-related mortality.

    View details for Web of Science ID 000169348200001

    View details for PubMedID 11413507

  • Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production BLOOD Baker, J., Verneris, M. R., Ito, M., Shizuru, J. A., Negrin, R. S. 2001; 97 (10): 2923-2931

    Abstract

    T cells with natural killer cell phenotype and function (NKT cells) have been described in both human and murine tissues. In this study, culture conditions were developed that resulted in the expansion of CD8(+) NKT cells from bone marrow, thymus, and spleen by the timed addition of interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and anti-CD3 monoclonal antibody. After 14 to 21 days in culture, dramatic expansion of CD3(+), CD8(+), alphabetaT-cell receptor(+) T cells resulted with approximately 20% to 50% of the cells also expressing the NK markers NK1.1 and DX5. The CD8(+) NKT cells demonstrated lytic activity against several tumor target cells with more than 90% lysis by day 14 to day 21 of culture. Cytotoxicity was observed against both syngeneic and allogeneic tumor cell targets with the greatest lytic activity by the cells expressing either NK1.1 or DX5. The expanded CD8(+) NKT cells produce T(H)1-type cytokines with high levels of IFN-gamma and tumor necrosis factor alpha. Expansion of the CD8(+) NKT cells was independent of CD1d. Ly49 molecules were expressed on only a minority of cells. A single injection of expanded CD8(+) NKT cells was capable of protecting syngeneic animals from an otherwise lethal dose of Bcl1 leukemia cells. Expanded CD8(+) NKT cells produced far less graft-versus-host disease (GVHD) than splenocytes across major histocompatibility barriers, even when 10 times the number of CD8(+) NKT cells as compared to splenocytes were injected. This reduction in GVHD was related to IFN-gamma production since cells expanded from IFN-gamma knock-out animals caused acute lethal GVHD, whereas cells expanded from animals defective in fas ligand, fas, IL-2, and perforin did not. These data indicate that CD8(+) NKT cells expanded in this fashion could be useful for preserving graft-versus-leukemia activity without causing GVHD.

    View details for Web of Science ID 000170301300001

    View details for PubMedID 11342413

  • Survivin expression correlates with apoptosis resistance after lymphocyte activation and is found preferentially in memory T cells IMMUNOLOGY LETTERS Kornacker, M., Verneris, M. R., Kornacker, B., Scheffold, C., Negrin, R. S. 2001; 76 (3): 169-173

    Abstract

    The prevention of apoptosis may be critical for immunological function. Survivin is a recently cloned member of the inhibitor of apoptosis protein family. We analyzed survivin expression before and after lymphocyte activation in isolated cell populations. Prior to activation, survivin was undetectable. After activation with IL-2 and OKT-3, CD3(+) cells expressed survivin. Next, we correlated survivin expression with Fas, FasL and the amount of apoptosis over time in culture. After activation, survivin was readily detected by day 2 and decreased thereafter. Prior to activation (day 0), Fas was present on 60% of the cells and on 100% by days 2-6. Peak FasL mRNA expression was at day 2. During peak survivin expression (days 2-4) the apoptotic fraction was low, but when survivin expression decreased the apoptotic fraction increased rapidly. Finally, we found that CD45RO(+) memory T cells showed a higher expression of survivin than did CD45RA(+) naive T cells after activation. These results suggest that survivin may contribute to T-cell survival early in T-cell responses as well as in memory immune responses.

    View details for Web of Science ID 000168371500004

    View details for PubMedID 11306144

  • CD58/LFA-3 and IL-12 provided by activated monocytes are critical in the in vitro expansion of CD56(+) T cells CANCER IMMUNOLOGY IMMUNOTHERAPY Lopez, R. D., Waller, E. K., Lu, P. H., Negrin, R. S. 2001; 49 (12): 629-640

    Abstract

    A small proportion of human CD3+ T lymphocytes are known to co-express CD56, an antigen usually restricted in its expression to natural killer (NK) cells. Whereas the in vivo function of CD3+ CD56+ T cells remains unknown, we and others have previously shown that both in vitro and in vivo, these cells can mediate a significantly greater degree of MHC-unrestricted cytotoxicitv against a variety of human tumor cells when compared to either CD3+ CD56- T cells or lymphokine activated killer (LAK) cells. While the mechanismns regulating the in vivo expansion of CD56+ T cells are not known, here we demonstrate the importance of CD2-mediated IL-12-dependent signals in the in vitro expansion of CD56+ T cells. Specifically, we show that activated monocytes provide a contact dependent factor (CD58/LFA-3) and a soluble factor (IL-12), both critical for the in vitro expansion of CD56+ T cells. The biological and therapeutic implications of these findings are discussed.

    View details for Web of Science ID 000167181700001

    View details for PubMedID 11258789

  • Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. NEW ENGLAND JOURNAL OF MEDICINE Bensinger, W. I., Martin, P. J., Storer, B., Clift, R., Forman, S. J., Negrin, R., Kashyap, A., Flowers, M. E., Lilleby, K., Chauncey, T. R., STORB, R., Appelbaum, F. R., Rowley, S., Heimfeld, S., Blume, K. 2001; 344 (3): 175-181

    Abstract

    In recipients of allogeneic hematopoietic-cell transplants, peripheral-blood cells mobilized with the use of filgrastim (recombinant granulocyte colony-stimulating factor) engraft more rapidly than bone marrow. However, the relative effects of these techniques on the rates of acute and chronic graft-versus-host disease, overall survival, and disease-free survival have not been determined in randomized studies.Between March 1996 and July 1999, 172 patients (12 to 55 years of age) with hematologic cancer were randomly assigned to receive either bone marrow or filgrastim-mobilized peripheral-blood cells from HLA-identical relatives for hematopoietic rescue after the treatment of hematologic cancer with high doses of chemotherapy, with or without radiation.The recovery of both neutrophils and platelets was faster with peripheral-blood cells than with marrow (P<0.001 for both comparisons). The cumulative incidence of grade II, III, or IV acute graft-versus-host disease at 100 days was 64 percent with peripheral-blood cells and 57 percent with marrow (hazard ratio, 1.21; 95 percent confidence interval, 0.81 to 1.81; P=0.35). The cumulative incidence of chronic graft-versus-host disease was 46 percent with peripheral-blood cells and 35 percent with marrow (hazard ratio, 1.16; 95 percent confidence interval, 0.71 to 1.90; P=0.54). The estimated overall probability of survival at two years was 66 percent with peripheral-blood cells and 54 percent with marrow (hazard ratio for death, 0.62; 95 percent confidence interval, 0.38 to 1.02; P=0.06). The rate of disease-free survival at two years was 65 percent with peripheral-blood cells and 45 percent with marrow (hazard ratio for relapse or death, 0.60; 95 percent confidence interval, 0.38 to 0.95; P=0.03).In patients given high-dose chemotherapy, with or without radiation, for the treatment of hematologic cancer, allogeneic peripheral-blood cells used for hematopoietic rescue restore blood counts faster than allogeneic bone marrow, without increasing the risk of graft-versus-host disease.

    View details for Web of Science ID 000166402800003

    View details for PubMedID 11172139

  • High-dose therapy and autologous bone marrow transplantation for follicular lymphoma in first complete or partial remission: results of a phase II clinical trial BLOOD Horning, S. J., Negrin, R. S., Hoppe, R. T., Rosenberg, S. A., Chao, N. J., Long, G. D., Brown, B. W., Blume, K. G. 2001; 97 (2): 404-409

    Abstract

    Advanced stage follicular small cleaved and mixed cell lymphoma is characterized by relapse from remission and survival ranging from 6 to 12 years. Because young patients have the greatest compromise in longevity, the efficacy and toxicity of high-dose radiochemotherapy and bone marrow transplantation after conventional chemotherapy was evaluated in a prospective phase II clinical trial. Thirty-seven patients in a minimal disease state after conventional chemotherapy received fractionated total body irradiation and high-dose etoposide and cyclophosphamide, followed by purged autologous bone marrow. A reference sample of 188 patients of similar age, stage, and histology managed at this institution before 1988 was identified for comparison of patient characteristics and outcomes. Compared with reference patients, transplant recipients had a higher tumor burden at diagnosis. With a median follow-up of 6.5 years, the estimated 10-year survival after transplantation was 86%. There was a single lymphoma death yielding a 10-year disease-specific survival of 97%. There were 2 early transplant-related deaths and 2 late acute leukemia deaths. Ten patients relapsed, one with microscopic disease only. High tumor burden at diagnosis and incomplete response to chemotherapy adversely influenced survival in the reference but not in the transplanted patients. The estimated risk of death of 14% and relapse of 30% at 10 years in our transplanted follicular lymphoma patients, the majority of whom had high tumor burdens, compares favorably with our observations in appropriately matched reference patients.

    View details for Web of Science ID 000166388000011

    View details for PubMedID 11154216

  • High-dose therapy and autologous hematopoietic-cell transplantation for follicular lymphoma beyond first remission: The Stanford University experience BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Cao, T. M., Horning, S. F., Negrin, R. S., Hu, W. W., Johnston, L. F., Taylor, T. L., Shizuru, J. A., Hoppe, R. T., Brown, B. W., Blume, K. G., Stockerl-Goldstein, K. 2001; 7 (5): 294-301

    Abstract

    A retrospective analysis was performed to investigate the outcome of high-dose therapy (HDT) and autologous hematopoietic cell transplantation in patients with follicular lymphomas beyond first remission. Ninety-two patients with primary induction failure or relapsed follicular low-grade lymphoma (FLGL), follicular large cell lymphoma (FLCL), and transformed follicular lymphoma (TFL) were treated with myeloablative therapy consisting of etoposide (60 mg/kg), cyclophosphamide (100 mg/kg), and either carmustine (BCNU;15 mg/kg) or fractionated total body irradiation (FTBI; 1200 cGy) followed by transplantation of purged autologous bone marrow or peripheral blood hematopoietic cells. For the 49 patients with relapsed FLGL, the median age was 49 years and the median interval from diagnosis to HDT was 30 months. The 4-year estimate of overall survival (OS) was 60% (95% confidence interval [CI], 45%-75%) and of disease-free survival (DFS) was 44% (95% CI, 29%-59%). Treatment with the FTBI-containing HDT regimen was associated with significantly longer DFS (P = .04) and OS (P = .04) in our multivariate analysis. OS was also significantly longer among those treated with 3 or fewer chemotherapy regimens. For the 26 FLCL patients, the median age was 51 years and in 31% the indication for HDT was primary induction failure. For FLCL patients, the 4-year estimate of OS was 58% (95% CI, 37%-79%) and of DFS was 51% (95% CI, 30%-72%). Among the 17 patients with TFL, 13 (76%) transformed at first relapse, and only 6 patients (35%) achieved complete remission with salvage therapy prior to HDT. For TFL patients, the 4-year estimate of OS was 50% (95% CI, 24%-76%) and of DFS 49% (95% CI, 20%-78%). There were 3 occurrences of myelodysplasia (1 after treatment with TBI, 2 after BCNU treatment), yielding an estimated incidence of 7% (95% CI, 0%-16%) at 56 months. This analysis shows that relapsed FLGL patients treated with 3 or fewer different chemotherapy regimens show inferior survival. The HDT regimen containing FTBI appears to be superior to the BCNU-based regimen for relapsed FLGL, although longer follow-up is needed to evaluate late effects. Lastly, patients with TFL or induction failure and relapsed FLCL can achieve survival outcome comparable to those observed with the indolent follicular lymphomas.

    View details for Web of Science ID 000169118600007

    View details for PubMedID 11400952

  • Expansion of cytotoxic CD3(+)CD56(+) cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Alvarnas, J. C., Linn, Y. C., Hope, E. G., Negrin, R. S. 2001; 7 (4): 216-222

    Abstract

    Immunotherapy may potentially improve the outcome of autologous hematopoietic cell transplantation (HCT). Poor effector cell proliferation and marginal antitumor activity limit attempts to use immunotherapy. We have characterized the ex vivo expansion, up to 1000-fold, of CD3+ CD56+ lymphocytes from the peripheral blood lymphocytes (PBL) of healthy donors. Expanded cells termed cytokine-induced killer (CIK) cells induce non-major histocompatibility complex-restricted lysis of tumor cells and demonstrate cytolytic activity superior to lymphokine-activated killer cells without the requirement of interleukin (IL)-2 treatment in vivo. To determine whether cytolytic cells could be expanded from patient material, we evaluated samples of peripheral blood progenitor cells (PBPCs) from 25 patients undergoing autologous HCT. The PBPCs were expanded by priming with interferon-gamma followed by anti-CD3 monoclonal antibody and IL-2 the next day. Fluorescence-activated cell sorting analysis was performed on days 0, 15, 21, and 28 of cell culture. The median T-cell content rose from 15.3% (range, 1.1% to 89.7%) on day 0 to 97.2% (range, 83.6% to 99.5%) by day 15. By day 21, T cells expanded 21.8-fold (range, 1.7- to 420.0-fold) and CD3+ CD56+ cells expanded 44.8-fold (range, 5.1- to 747.0-fold). CIK cells were used as effector cells against B-cell lymphoma targets (OCI-Ly8) with a median of 24% (range, 3% to 67%) and 42% (range, 6% to 96%) specific lysis of target cells on days 21 and 28, respectively. CIK cells derived from PBL of 2 additional patients with acute myelogenous leukemia demonstrated 39% and 78% specific lysis of OCI-Ly8 and 26% and 58% specific lysis of autologous leukemic blasts at an effector:target ratio of 40:1. CIK cells may be expanded from granulocyte colony-stimulating factor-mobilized PBPCs of patients undergoing autologous HCT. CIK cells may provide a potent tool for use in posttransplantation adoptive immunotherapy.

    View details for Web of Science ID 000168435400004

    View details for PubMedID 11349808

  • CD31 mismatching affects marrow transplantation outcome BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION GRUMET, F. C., Hiraki, D. D., Brown, B. W., Zehnder, J. L., Zacks, E. S., Draksharapu, A., Parnes, J., Negrin, R. S. 2001; 7 (9): 503-512

    Abstract

    Graft-versus-host disease (GVHD) complicating allogeneic bone marrow transplantation (BMT) is often attributed to mismatched minor histocompatibility antigens (mHags), which are poorly defined in humans. CD31 is a candidate human mHag relevant to acute GVHD, but reports disagree about its level of significance, the role of HLA restriction, and the relative importance of different polymorphic codons within the molecule. We therefore examined in greater detail the impact of CD31-matching on BMT outcome in a prospective study from a single institution. Samples of recipient and donor DNA were collected pretransplantation for all patients receiving unmanipulated bone marrow from an HLA-identical sibling over a 45-month period at our institution. CD31 DNA typing of alleles at the 3 polymorphic codons 125 (L or V), 563 (N or S), and 670 (R or G) was performed for 118 patient-donor pairs plus 2 additional pairs who had codon 125 typing only. Donor-recipient CD31 nonidentity was tested for correlation with BMT clinical outcome measures of severe acute GVHD, chronic GVHD, relapse, and survival. Gene frequencies of approximately 0.5 for each allele at all 3 codons were comparable to previous reports. Because complete association was seen for 563N with 670G and for 563S with 670R, nonidentity for those codons was analyzed as a single genetic marker designated codon 563/670. Donor-recipient CD31 nonidentity was a significant risk factor for overall survival, both at codon 563/670 (hazard ratio [hr] = 2.58, P = .005) and at codon 125 (hr = 1.07, P = .036). Similar results held for disease-free survival. Nonidentity at codon 563/670 was also a significant risk factor (odds ratio [OR] = 11.15, P = .011) for severe (grades III, IV) versus no (grade 0) acute GVHD. Nonidentity at codon 125 posed less but still significant risk (OR = 9.30, P = .030). When the comparison group without severe acute GVHD was expanded to include grade I as well as grade 0 patients, the risk from CD31 nonidentity increased for both codon 563/670 (OR = 12.31, P = .010) and codon 125 (OR = 11.24, P = .011). CD31 nonidentity remained a significant independent risk factor for survival and for severe acute GVHD when tested in multivariate analysis with the covariates of adulthood, recipient-donor sex difference, ethnic group, disease, pretransplantation risk category, HLA-A2 type, B44-like types, and GVHD prophylactic regimen. CD31 nonidentity showed a trend but failed to achieve statistical significance as a risk factor for relapse and for chronic GVHD. In conclusion, donor-recipient CD31 nonidentity is a significant risk factor for survival and for severe acute GVHD in HLA-identical sibling BMT. The stronger associations with codon 563/670 suggest that polymorphism may be more important than the linked polymorphism at codon 125.

    View details for Web of Science ID 000171449200004

    View details for PubMedID 11669217

  • Efficacy and toxicity of a CCNU-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory Hodgkin's disease BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Stuart, M. J., Chao, N. S., Horning, S. J., Wong, R. M., Negrin, R. S., JOHNSTON, L. J., Shizuru, J. A., Long, G. D., Blume, K. G., Stockerl-Goldstein, K. E., Goldstein, S. 2001; 7 (10): 552-560

    Abstract

    High-dose CBV (cyclophosphamide, carmustine, and etoposide) in combination with autologous HCT achieves survival rates of approximately 50% at 5 years in recurrent or refractory Hodgkin's disease (HD). However, carmustine (BCNU) dose-dependent pulmonary toxicity occurs in 20% to 30% of patients. A decreased incidence of interstitial pneumonitis as well as a possible benefit in efficacy has been reported with lomustine (CCNU) compared to BCNU in the standard dose setting. In a dose-escalation study, we substituted CCNU for BCNU in the CBV regimen for 16 patients with HD (n = 12) or non-Hodgkin's lymphoma (n = 4). Based on the promising results, an additional 47 consecutive patients with HD were treated with the following regimen: CCNU (15 mg/kg) orally on day -6, etoposide (60 mg/kg) intravenously on day -4, and cyclophosphamide (100 mg/kg) intravenously on day -2. Peripheral blood progenitor cells and/or bone marrow were infused on day 0. With a median follow-up for the surviving patients of 3.2 years (range, 0.8-9.9 years), the 3-year overall survival rate was 57% (CI, +/-15%), event-free survival was 52% (CI, +/-14%), and freedom from progression was 68% (CI, +/-14%). There were 21 deaths, 10 due to HD. Six patients died due to respiratory failure. Interstitial pneumonitis occurred in 63% of patients and could not be correlated with prior chest radiotherapy. This regimen demonstrated survival rates similar to those of historical studies that used the CBV regimen. However, the incidence of interstitial pneumonitis was in excess of expected.

    View details for Web of Science ID 000172275500004

    View details for PubMedID 11760087

  • Engineering hematopoietic grafts: Purified allogeneic hematopoietic stem cells plus expanded CD8(+) NK-T cells in the treatment of lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Verneris, M. R., Ito, M., Baker, E., Arshi, A., Negrin, R. S., Shizuru, J. A. 2001; 7 (10): 532-542

    Abstract

    A major benefit of allogeneic hematopoietic cell transplantation (HCT) in the treatment of malignancies is the graft-versus-tumor (GVT) effect conferred by lymphocytes contained within the graft. However, lymphocytes can also induce the potentially lethal complication of graft-versus-host disease (GVHD). We have previously reported a method of generating large numbers of ex vivo activated and expanded T cells with antitumor activity after culture with interferon-y, cross-linking antibodies to CD3, and interleukin-2. Murine splenocytes expanded under these conditions are a heterogeneous population of which approximately 20% to 60% of cells express natural killer (NK)-cell markers (NK1.1 and DX5) and display major histocompatibility complex (MHC)-unrestricted antitumor activity. Here we demonstrate the in vivo antitumor activity of this population of expanded CD8+ NK-T cells when transplanted across MHC barriers into tumor-bearing hosts. In cotransfer studies with purified allogeneic hematopoietic stem cells, expanded CD8+ NK-T cells confer GVT activity with minimal to no GVHD. In vitro studies show that, although expanded NK-T cells lyse normal allogeneic bone marrow cells, they preferentially mediate cytolysis against tumor targets. These cells persist in the peripheral circulation of host animals for at least 3 weeks posttransfer. GVT activity is dependent on perforin, but not on Fas-ligand. We conclude that expanded CD8+ NK-T cells may serve as a valuable adjuvant population for allogeneic HCT because they mediate GVT effects with minimal GVHD.

    View details for Web of Science ID 000172275500002

    View details for PubMedID 11760085

  • CD2-mediated IL-12-dependent signals render human gamma delta-T cells resistant to mitogen-induced apoptosis, permitting the large-scale ex vivo expansion of functionally distinct lymphocytes: implications for the development of adoptive immunotherapy strategies BLOOD Lopez, R. D., Xu, S., Guo, B., Negrin, R. S., Waller, E. K. 2000; 96 (12): 3827-3837

    Abstract

    The ability of human gamma delta-T cells to mediate a number of in vitro functions, including innate antitumor and antiviral activity, suggests these cells can be exploited in selected examples of adoptive immunotherapy. To date, however, studies to examine such issues on a clinical scale have not been possible, owing in large measure to the difficulty of obtaining sufficient numbers of viable human gamma delta-T cells given their relative infrequency in readily available tissues. Standard methods used to expand human T cells often use a combination of mitogens, such as anti-T-cell receptor antibody OKT3 and interleukin (IL)-2. These stimuli, though promoting the expansion of alpha beta-T cells, usually do not promote the efficient expansion of gamma delta-T cells. CD2-mediated, IL-12-dependent signals that result in the selective expansion of human gamma delta-T cells from cultures of mitogen-stimulated human peripheral blood mononuclear cells are identified. It is first established that human gamma delta-T cells are exquisitely sensitive to apoptosis induced by T-cell mitogens OKT3 and IL-2. Next it is shown that the CD2-mediated IL-12-dependent signals, which lead to the expansion of gamma delta-T cells, do so by selectively protecting subsets of human gamma delta-T cells from mitogen-induced apoptosis. Finally, it is demonstrated that apoptosis-resistant gamma delta-T cells are capable of mediating significant antitumor cytotoxicity against a panel of human-derived tumor cell lines in vitro. Both the biologic and the practical implications of induced resistance to apoptosis in gamma delta-T cells are considered and discussed because these findings may play a role in the development of new forms of adoptive cellular immunotherapy. (Blood. 2000;96:3827-3837)

    View details for Web of Science ID 000165514000023

    View details for PubMedID 11090067

  • Resistance of ex vivo expanded CD3(+)CD56(+) T cells to Fas-mediated apoptosis CANCER IMMUNOLOGY IMMUNOTHERAPY Verneris, M. R., Kornacker, M., Mailander, V., Negrin, R. S. 2000; 49 (6): 335-345

    Abstract

    A variety of malignancies express Fas ligand (FasL), which can induce apoptosis in effector lymphocytes and may limit the success of cellular immunotherapy. Our laboratory has been investigating a population of ex vivo activated T cells, termed cytokine-induced killer (CIK) cells. These cells share functional and phenotypic properties with natural killer cells and a subset of cytolytic cells have the phenotype CD3+CD56+. CIK cells expand in culture, have significant antitumor activity and are presently being tested in phase I/II clinical trials. In this study, we investigated the sensitivity of CIK cells to Fas-mediated apoptosis. Fas engagement leads to apoptosis in small numbers of CIK cells and does not significantly influence antitumor cytotoxicity. CIK cells will undergo apoptosis following Fas engagement when protein synthesis is inhibited, suggesting the expression of antiapoptotic genes. Evaluation of antiapoptotic gene transcripts shows an upregulation in the expression of cFLIP, Bcl-2, Bcl-xL, DAD1 and survivin. Resistance to Fas-mediated apoptosis may come about through an in vitro selection for Fas resistance, since CIK cells synthesize FasL and supernatant from CIK cultures contains biologically active soluble FasL, which can be inhibited with Fas:Fc. These results indicate that CIK cells are a suitable form of immunotherapy against FasL-positive tumors.

    View details for Web of Science ID 000088614100007

    View details for PubMedID 10946816

  • Effect of oral glutamine supplementation during bone marrow transplantation JOURNAL OF PARENTERAL AND ENTERAL NUTRITION Dickson, T. M., Wong, R. M., Negrin, R. S., Shizuru, J. A., JOHNSTON, L. J., Hu, W. W., Blume, K. G., Stockerl-Goldstein, K. E. 2000; 24 (2): 61-66

    Abstract

    Because all patients receiving bone marrow transplant (BMT) and peripheral blood progenitor cell transplant (PBPCT) experience gastrointestinal (GI) toxicity from the preparative regimen of chemotherapy, with or without radiation, oral glutamine was administered during the preparatory regimen and after transplant to maintain GI structure and function.To evaluate effects of oral glutamine on nutritional status and overall outcome, a prospective, randomized, double-blinded study was performed on 58 autologous and allogeneic BMT patients. Patients received 30 g of oral glutamine or placebo daily.The trends of decreased median length of stay and the median number of days of total parenteral nutrition (TPN) were seen in the group supplemented with the >0.285-g/kg (the recommended dosage) dose of glutamine; however, there was no statistically significant difference in the nutritional status and overall patient outcome as assessed by days receiving TPN, number of days required until oral intake resumed, length of hospitalization, number of days and highest grade of mucositis, and quantity and number of days of diarrhea.This study does not support the hypothesis that oral glutamine may offer benefit. Further investigation is required regarding clinical tools for determining effectiveness, administration for tolerance and compliance, dosage, and potential of oral glutamine usage.

    View details for Web of Science ID 000089389000004

    View details for PubMedID 10772184

  • Favorable treatment outcome in non-Hodgkin's lymphoma patients with "poor" mobilization of peripheral blood progenitor cells BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Stockerl-Goldstein, K. E., Reddy, S. A., Horning, S. J., Blume, K. G., Chao, N. J., Hu, W. W., JOHNSTON, L. J., Long, G. D., Strober, S., Wong, R. M., Feiner, R. H., Kohler, S., Negrin, R. S. 2000; 6 (5): 506-512

    Abstract

    Our purpose was to evaluate the outcome and costs of high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation in patients with the inability to mobilize sufficient numbers of PBPCs to allow rapid engraftment after PBPC transplantation. We treated 172 consecutive non-Hodgkin's lymphoma (NHL) patients with cyclophosphamide and granulocyte colony-stimulating factor followed by apheresis to collect PBPCs. The cells were separated on a Percoll gradient and purged with monoclonal antibodies and complement. The patients were categorized as "good" mobilizers if a collection of > or =2 x 10(6) CD34+ cells/kg was obtained (n = 138, 80%) or "poor" mobilizers if <2 x 10(6) CD34+ cells/kg were obtained (n = 34, 20%). With a median follow-up of 3.5 years, there is no statistically significant difference in actuarial event-free survival, overall survival, or relapse for good mobilizers compared with poor mobilizers. However, there was a trend toward increasing nonrelapse, transplantation-related mortality of 11.8% for poor mobilizers versus 3.6% for good mobilizers (P = .08) and early death from all causes including relapse within 120 days (poor 20.6% versus good 8.7%, P = .06). The total cost for bone marrow transplantation-related care was significantly higher, at $140,264 for poor mobilizers versus $80,833 for good mobilizers (P = .0001). The population of patients with NHL who mobilize PBPCs poorly into the circulation have a higher cost for posttransplant support. However, there is no significant difference in relapse, event-free survival, or overall survival for such patients compared with those who mobilize PBPCs easily.

    View details for Web of Science ID 000090049700004

    View details for PubMedID 11063379

  • A phase I trial of recombinant human thrombopoietin in patients with delayed platelet recovery after hematopoietic stem cell transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Nash, R. A., Kurzrock, R., DiPersio, J., Vose, J., Linker, C., Maharaj, D., Nademanee, A. P., Negrin, R., Nimer, S., Shulman, H., Ashby, M., Jones, D., Appelbaum, F. R., Champlin, R. 2000; 6 (1): 25-34

    Abstract

    Delayed platelet recovery is a significant complication after both autologous and allogeneic hematopoietic stem cell transplantation (HSCT). A multicenter, phase I dose-escalation study of recombinant human thrombopoietin (rhTPO) was conducted to assess its safety and to obtain preliminary data on its efficacy in patients with persistent severe thrombocytopenia (<20,000/microL) >35 days after HSCT. Thirty-eight patients, 37 of whom were evaluable, were enrolled in the study from April 1996 through January 1997. rhTPO was administered at doses of 0.6, 1.2, and 2.4 microg/kg as a single dose (group A) or in multiple doses every 3 days for a total of 5 doses (group B). No significant adverse effects were observed. Ten patients had recovery of platelet counts during the 28-day study period; 3 of these 10 had an increase in marrow megakaryocyte content 7 days after completing treatment with rhTPO. When all baseline marrows were compared with samples after rhTPO treatment, there was no difference in marrow megakaryocyte content (P = 0.49). This study design could not answer the question of whether the recoveries of platelet counts observed in some patients were spontaneous or influenced by rhTPO treatment; nonetheless, the authors found no correlation between the dose of rhTPO and the recovery of platelet counts. Increases in serum TPO levels were dose-dependent and remained significantly elevated for up to 72 hours after treatment. To evaluate response, further studies of treatment strategies with rhTPO in patients with delayed platelet recovery are required.

    View details for Web of Science ID 000090022000003

    View details for PubMedID 10707996

  • Transplantation of highly purified CD34(+)Thy-I+ hematopoietic stem cells in patients with metastatic breast cancer BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Negrin, R. S., Atkinson, K., Leemhuis, T., Hanania, E., Juttner, C., Tierney, K., Hu, W. W., JOHNSTON, L. J., Shizuru, J. A., Stockerl-Goldstein, K. E., Blume, K. G., Weissman, I. L., Bower, S., Baynes, R., Dansey, R., Karanes, C., Peters, W., Klein, J. 2000; 6 (3): 262-271

    Abstract

    We report here the transplantation of extensively purified "mobilized" peripheral blood CD34Thy-1 hematopoietic stem cells from 22 patients with recurrent or metastatic breast cancer. Patients were mobilized with either high-dose granulocyte colony-stimulating factor (G-CSF) alone or cyclophosphamide plus G-CSE Median purity of the stem cell product at cryopreservation was 95.3% (range, 91.1%-98.3%), and viability was 98.6% (range, 96.5%-100%). After high-dose chemotherapy with carmustine, cisplatin, and cyclophosphamide, CD34+Thy-1 cells at a median dose of 11.3 x 10(5) per kilogram (range, 4.7-163 x 10(5) per kilogram) were infused. No infusion-related toxicity was observed. Neutrophil recovery was prompt, with median absolute neutrophil count >500/microL by day 10 (range, 8-15 days) and >1000/microL by day 11 (range, 8-17 days). Median platelet recovery (>20,000/microL) was observed by day 14 (range, 9-42 days) and >50,000/microL by day 17 (range, 11-49 days). Tumor cell depletion below the limits of detection of a sensitive immunofluorescence-based assay was accomplished in all patients who had detectable tumor cells in apheresis products before processing. Although CD4+ T-cell reconstitution was slow, no unusual infections were observed. Neither early nor late graft failure was observed, and no patient required infusion of unmanipulated backup cells. At a median follow-up of approximately 1.4 years and a maximum follow-up of 2.5 years, 16 of the 22 patients remain alive, with 9 free of disease progression, and have stable blood counts. In summary, highly purified CD34+Thy-1+ cells used as the sole source of the hematopoietic graft result in rapid and sustained hematopoietic engraftment.

    View details for Web of Science ID 000090022300006

    View details for PubMedID 10871151

  • Use of reporter genes for optical measurements of neoplastic disease in vivo NEOPLASIA Contag, C. H., Jenkins, D., Contag, F. R., Negrin, R. S. 2000; 2 (1-2): 41-52

    Abstract

    Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies.

    View details for Web of Science ID 000086519700004

    View details for PubMedID 10933067

  • Four-cycle high-dose therapy with hematopoietic support for metastatic breast cancer: No improvement in outcomes compared with single-course high-dose therapy BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Hu, W. W., Negrin, R. S., Stockerl-Goldstein, K., JOHNSTON, L. J., Shizuru, J. A., Wong, R. M., Chao, N. J., Long, G. D., Feiner, R. H., Blume, K. G. 2000; 6 (1): 58-69

    Abstract

    Multiple-cycle high-dose therapy with autologous hematopoietic progenitor cell (AHPC) support has been used to deliver dose-intensive therapy. We have used this approach as well as single-cycle high-dose therapy in treating patients with metastatic breast cancer. We present the outcomes of multiple-cycle high-dose therapies and compare them with those resulting from single-course high-dose therapies performed at a single institution. Fifty-five patients received 4 cycles of intensive chemotherapy with AHPC support. Three multicycle regimens were sequentially applied. Twenty patients were enrolled to receive 4 cycles of high-dose mitoxantrone, thiotepa, and cyclophosphamide. Nineteen subsequent patients received this regimen modified by the incorporation of paclitaxel. Sixteen patients received 2 cycles of high-dose melphalan, thiotepa, and paclitaxel and 2 cycles of mitoxantrone, thiotepa, and paclitaxel. The results of all 3 multiple-cycle therapies are compared with those of 55 contemporaneous patients with metastatic breast cancer who received a single course of high-dose cyclophosphamide and thiotepa or cyclophosphamide, cisplatin, and BCNU (carmustine) with hematopoietic cell rescue. Multiple-cycle therapy was associated with more infectious complications, increased transfusion requirements, and increased hospital admissions. However, there were no significant differences in outcomes between the groups. For 55 patients who received multiple-cycle therapy, the actuarial 3-year overall survival rate was 36% (95% confidence interval [CI] 23%-49%); freedom from progression and event-free survival were both 15% (CI 5%-25%). The median time to disease progression and median survival were 1.0 and 1.6 years, respectively. For the 55 patients who underwent a single course of high-dose therapy, the 3-year overall survival was also 36% (CI 18%-54%), whereas freedom from progression and event-free survival were both 19% (CI 7%-31%). The median time to progression and median survival were 0.8 and 2.2 years, respectively. Within the constraints of this patient population, the outcomes of 4 cycles of high-dose therapy with AHPC support were not superior to those resulting from single courses of high-dose therapy in the treatment of patients with metastatic breast cancer.

    View details for Web of Science ID 000090022000007

    View details for PubMedID 10708000

  • High-dose therapy with hematopoietic cell transplantation for patients with central nervous system involvement by non-Hodgkin's lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Alvarnas, J. C., Negrin, R. S., Horning, S. J., Hu, W. W., Long, G. D., Schriber, J. R., Stockerl-Goldstein, K., Tierney, K., Wong, R., Blume, K. G., Chao, N. J. 2000; 6 (3A): 352-358

    Abstract

    Central nervous system (CNS) involvement by non-Hodgkin's lymphoma (NHL) carries a poor patient prognosis whether it occurs as a primary site of disease or secondarily in patients with systemic disease. In a group of 481 patients undergoing high-dose therapy with hematopoietic cell transplantation (HCT) for NHL, 15 patients (3.1%) were identified with CNS involvement. Two patients had primary CNS lymphoma, and 13 had secondary disease. All patients received intrathecal chemotherapy, and 13 received CNS radiotherapy before transplantation. Fourteen patients received systemic chemotherapy. At the time of transplantation, both patients with primary CNS lymphoma and 8 patients with secondary disease had achieved a complete response, 3 patients had achieved a partial response, 1 had failed induction therapy, and 1 had progression of CNS disease before high-dose therapy. Fourteen patients received carmustine, etoposide, and cyclophosphamide as the preparative regimen, and 1 patient received fractionated total body irradiation instead of carmustine. The 2 patients with primary CNS lymphoma were alive and free of disease, 1 at 1,085 days after HCT and 1 at 3,704 days after HCT. The actuarial 5-year event-free survival (EFS) was 46% +/- 26%, and overall survival (OS) was 41% +/- 28%. The median EFS and OS were 2.2 and 1.5 years, respectively. Three patients experienced symptomatic memory loss or intellectual decline after therapy, 1 patient developed paraplegia, and 1 patient had a thrombotic stroke 20 months after HCT. Despite treatment-related toxicities, 7 patients responding to quality-of-life questions at approximately 1 year after HCT gave their overall quality of life a median rating of 9 out of a possible 10 (range, 6-10). High-dose therapy with autologous HCT can produce extended EFS in patients with secondary CNS lymphoma and possibly in those with primary CNS NHL.

    View details for Web of Science ID 000090022500008

    View details for PubMedID 10905773

  • Toxicity of high-dose sequential chemotherapy and purged autologous hematopoietic tell transplantation precludes its use in refractory/recurrent non-Hodgkin's lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Johnston, L. J., Stockerl-Goldstein, K. E., Hu, W. W., Negrin, R. S., Hoppe, R. T., Blume, K. G., Horning, S. J. 2000; 6 (5A): 555-562

    Abstract

    We conducted a pilot study in 20 patients with high-risk or recurrent/refractory non-Hodgkin's lymphoma (NHL) using high-dose sequential chemotherapy (HDSC) and autologous hematopoietic cell transplantation (AHCT). After cytoreduction with standard salvage therapy, HDSC/AHCT was administered in 4 phases at 2- to 4-week intervals. Phase 1 consisted of cyclophosphamide 7 g/m2 followed by granulocyte colony-stimulating factor (G-CSF) at 10 microg/kg per day and leukapheresis upon recovery from white blood cell nadir. The hematopoietic cell product was enriched by Percoll gradient separation and purged with a B-cell or T-cell monoclonal antibody panel and complement. Phase 2 consisted of methotrexate 8 g/m2 with leucovorin rescue and vincristine 1.4 mg/m2. Phase 3 was etoposide 2 g/m2 with G-CSF 5 microg/kg per day. In phase 4, the preparative regimen of mitoxantrone 60 mg/m2 and melphalan 180 mg/m2 was administered followed by AHCT. The NHL histologies were diffuse large cell, follicular/diffuse mixed, small noncleaved cell, T-cell-rich B-cell, lymphoblastic, and peripheral T cell. The remission status was first partial remission (PR1; n = 1) or beyond first complete remission (post-CR1; n = 19). Of the 20 patients enrolled, 11 proceeded through all 4 phases. Nine were removed from the study after the first or second phase because of progressive disease (n = 5), poor hematopoietic cell mobilization (n = 1), excessive toxicity (n = 2), and chronic active hepatitis C (n = 1). Treatment-related toxicities in the remaining 11 transplant recipients were cardiomyopathy, hemorrhagic cystitis, persistent cytopenias, acute renal failure, abnormal liver function test results, and infectious complications. There were no treatment-related deaths. Eight of the 11 transplant recipients were alive, 6 without disease, at a median follow-up of 2.7 years. The estimated median 2-year event-free survival was 55%, and overall survival was 70%. We conclude that HDSC/AHCT in refractory/recurrent NHL is associated with considerable acute and chronic toxicities. Given the toxicity profile, efficacy data were not sufficiently promising to warrant further study.

    View details for Web of Science ID 000090107100004

    View details for PubMedID 11071261

  • Pulmonary toxicity syndrome in breast cancer patients undergoing BCNU-containing high-dose chemotherapy and autologous hematopoietic cell transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Cao, T. M., Negrin, R. S., Stockerl-Goldstein, K. E., JOHNSTON, L. J., Shizuru, J. A., Taylor, T. L., Rizk, N. W., Wong, R. M., Blume, K. G., Hu, W. W. 2000; 6 (4): 387-394

    Abstract

    We performed a retrospective review to investigate pulmonary toxicity syndrome (PTS) in a cohort of breast cancer patients undergoing BCNU-containing high-dose chemotherapy (HDC). Our aim was to characterize presentation, identify risk factors, determine outcome following therapy, and find any association with differences in survival. We reviewed the data of 152 patients with stage II or III or metastatic breast cancer treated with cyclophosphamide 5625 mg/m2, cisplatin 165 mg/m2, and BCNU 600 mg/m2 followed by autologous peripheral blood hematopoietic cell transplantation. During follow-up, PTS was diagnosed when the following criteria were met: (1) presentation with typical clinical symptoms of PTS, (2) an absolute carbon monoxide diffusion capacity (DLCO) decline of 10% compared with pre-HDC DLCO, and (3) no clinical evidence of active pulmonary infection. Patients were then treated with a course of corticosteroid therapy. The incidence of PTS for all 152 patients was 59%, with a median onset at 45 days (range, 21-149 days) post-HDC. The median absolute DLCO decrement was 26% (range, 10%-73%) at diagnosis of PTS. There was no significant correlation between patient age, stage of breast cancer, pre-HDC chemotherapy regimen, pre-HDC chest wall radiotherapy, tobacco use, prior lung disease, or baseline pulmonary function test results and the development of PTS. We did observe an interesting association between PTS and the development of a noncholestatic elevation of transaminases. Of PTS patients treated with prednisone therapy for a median of 105.5 days (range, 44-300 days), 91% achieved resolution of their PTS without pulmonary sequelae. At 3 years, the overall survival (OS) of stage II or III patients who developed PTS was 84% (95% confidence interval [CI], 73%-95%); of metastatic breast cancer patients with PTS, the OS was 58% (95% CI, 38%-78%). These values were not significantly different from those of patients who did not develop PTS (91% [95% confidence interval [CI], 81%-100%] and 53% [95% CI, 32%-74%], respectively). No significant differences in disease-free or event-free survival were observed between patients with and without PTS. The incidence of PTS in breast cancer patients treated with a BCNU-containing HDC regimen can be remarkably high. Treatment with a course of corticosteroid therapy is successful in the vast majority.

    View details for Web of Science ID 000090022700005

    View details for PubMedID 10917574

  • Equivalence of 2 effective graft-versus-host disease prophylaxis regimens: Results of a prospective double-blind randomized trial BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Chao, N. J., Snyder, D. S., Jain, M., Wong, R. M., Niland, J. C., Negrin, R. S., Long, G. D., Hu, W. W., Stockerl-Goldstein, K. E., JOHNSTON, L. J., Amylon, M. D., Tierney, D. K., O'Donnell, M. R., Nademanee, A. P., Parker, P., Stein, A., Molina, A., Fung, H., Kashyap, A., Kohler, S., Spielberger, R., Krishnan, A., Rodriguez, R., Forman, S. J., Blume, K. G. 2000; 6 (3): 254-261

    Abstract

    We have previously demonstrated a decrease in the incidence of acute graft-versus-host disease (GVHD) with the addition of methotrexate (MTX) to cyclosporine (CSP) and prednisone (PSE) chemotherapy in patients with leukemia. We have now completed a prospective randomized trial comparing the 3-drug regimen (CSP/MTX/PSE, including 3 doses of MTX) to the standard 2-drug regimen (CSP/MTX, including 4 doses of MTX) to investigate the benefit of PSE used up front for the prevention of acute and chronic GVHD. In the trial, 193 patients were randomized and 186 were included in the final analysis. All patients received a bone marrow graft from a fully histocompatible sibling donor. The preparatory regimen consisted of fractionated total-body irradiation (fTBI) and etoposide in all but 13 patients, who received fTBI and cyclophosphamide. The patients were randomized to receive either CSP/MTX/PSE or CSP/MTX. The 2 groups were well balanced with respect to diagnosis, disease stage, age, donor-recipient sex, and parity. In an intent-to-treat analysis, the incidence of acute GVHD was 18% (95% confidence interval [CI] 12-28) for the CSP/MTX/PSE group compared with 20% (CI 10-26) for the CSP/,MTX group (P = .60), with a median follow up of 2.2 years. Overall survival was 65% for those receiving CSP/MTX/PSE and 72% for those receiving CSP/MTX (P = .10); the relapse rate was 15% for the CSP/MTX/PSE group and 12% for the CSP/MTX group (P = .83). The incidence of chronic GVHD was similar (46% versus 52%; P = .38), with a follow-up of 0.7 to 6.0 years. Of interest, 21 patients went off study due to GVHD (5 in the CSP/MTX/PSE group and 16 in the CSP/MITX group [P = .02]), and 11 patients went off study because of alveolar hemorrhage (3 in the CSP/MTX/PSE group and 8 in the CSP/MTX group [P = .22]). The addition of PSE did not result in a higher incidence of infectious complications, bacterial (66% versus 58%), viral (77% versus 66%), or fungal (20% versus 20%), in those receiving CSP/MTX/PSE versus CSP/MTX, respectively. These data suggest that the addition of PSE was associated with a somewhat lower incidence of early posttransplantation complications but did not have a positive impact on the incidence of acute or chronic GVHD or event-free or overall survival.

    View details for Web of Science ID 000090022300005

    View details for PubMedID 10871150

  • Long-term follow-up of 23 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with allogeneic bone marrow transplant in first complete remission LEUKEMIA Snyder, D. S., Nademanee, A. P., O'Donnell, M. R., Parker, P. M., Stein, A. S., Margolin, K., Somlo, G., Molina, A., Spielberger, R., Kashyap, A., Fung, H., Slovak, M. L., Dagis, A., Negrin, R. S., Amylon, M. D., Blume, K. G., Forman, S. J. 1999; 13 (12): 2053-2058

    Abstract

    Between 1984 and 1997, 23 consecutive patients with Philadelphia chromosome-positive acute lymphoblastic leukemia in first complete remission were treated with allogeneic bone marrow transplants from HLA-matched siblings. All patients but one were conditioned with fractionated total body irradiation (1320 cGy) and high-dose etoposide (60 mg/kg). One patient received high-dose cyclophosphamide instead of etoposide, and another patient received both drugs. Nine patients died following BMT, two from relapsed leukemia, and seven from transplant-related causes. The 3-year probabilities of disease-free survival and relapse are 65% and 12%, respectively. For patients transplanted after 1992, these probabilities are 81% (48-95%, 95% confidence interval) and 11% (2-50%), respectively. The relatively low relapse rate in this group of patients compared to published reports may reflect the enhanced anti-leukemic activity of etoposide in combination with FTBI compared to other conditioning regimens. The enhancement in overall survival for patients transplanted after 1992 may reflect improvements in supportive care, in particular, the prophylaxis of serious fungal and viral infections.

    View details for Web of Science ID 000084116700018

    View details for PubMedID 10602428

  • A feasibility study of multiple cycle therapy with melphalan, thiotepa, and paclitaxel followed by mitoxantrone, thiotepa, and paclitaxel with autologous hematopoietic cell support for metastatic breast cancer CLINICAL CANCER RESEARCH Hu, W. W., Long, G. D., Stockerl-Goldstein, K. E., JOHNSTON, L. J., Chao, N. J., Negrin, R. S., Blume, K. G. 1999; 5 (11): 3411-3418

    Abstract

    Dose-intensive chemotherapy appears to be important in the treatment of patients with recurrent solid tumors. Expanding upon our prior experience, we report the results of our most recent approach to administering dose-intensive therapy using four cycles of moderately high-dose chemotherapy with hematopoietic cell support for patients with metastatic breast cancer. This outpatient therapy includes high-dose melphalan, thiotepa, and paclitaxel for two cycles followed by mitoxantrone, thiotepa, and paclitaxel for two cycles, with each cycle supported with autologous peripheral blood progenitor cells (PBPCs). Between December 1994 and June 1996, 16 patients with recurrent or refractory breast cancer were enrolled in this prospective study. They had received a median of two previous chemotherapy regimens, with a median of nine prior cycles of chemotherapy. For mobilization of autologous PBPCs, patients received cyclophosphamide, 4 g/m2, followed by granulocyte colony-stimulating factor (G-CSF). PBPCs were collected by apheresis. Each day's collection was divided into four equal fractions, and each fraction was infused after each cycle of combination therapy. Cycles 1 and 2 consisted of melphalan, 80 mg/m2, thiotepa, 300 mg/m2, and paclitaxel, 200 mg/m2. Cycles 3 and 4 were comprised of mitoxantrone, 30 mg/m2, and thiotepa and paclitaxel at the same doses as in the first two cycles. The cyclophosphamide infusion was administered in the hospital, whereas all subsequent infusions of chemotherapy and PBPCs were performed on an outpatient basis. The first seven patients were randomized to receive alternate cycle G-CSF or placebo on day +1 of each cycle. Including the initial pulse of cyclophosphamide, 67 (84%) of a planned 80 total courses of chemotherapy were delivered. Of the planned 64 cycles of high-dose combination chemotherapy, 52 cycles (81%) were delivered. Treatment was discontinued for progressive disease (one patient) or morbidity (five patients). Twelve of 16 patients completed at least three cycles of therapy. Nine patients completed all four cycles. One death resulted from fungal sepsis. In 20 cycles delivered to the first seven patients, day +1 G-CSF versus placebo was administered, with a median WBC recovery of 10 versus 13 days, respectively (P = 0.048 in cycle 1). The median duration of response was almost 9 months, and the median survival was 18 months after therapy. With a median follow-up of 1.5 years and longest follow-up of 4.2 years, two patients continue to be without evidence of disease. The 3-year event-free survival, freedom from progression, and overall survival are 19%, 20%, and 31%, respectively. This four-cycle regimen of high-dose combination therapy supported with hematopoietic progenitor cells is feasible, but it is associated with a range of posttransplant complications. The efficacy of such a treatment would have to be substantially superior to that of other currently available therapies, including single autologous transplant procedures, to justify the prolonged period of treatment, multiple episodes of pancytopenia, and associated toxicities, including infectious risks. G-CSF administration after each PBPC infusion appears to accelerate time to neutrophil recovery but does not affect red cell or platelet engraftment.

    View details for Web of Science ID 000083853200012

    View details for PubMedID 10589752

  • Visualizing the kinetics of tumor-cell clearance in living animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W. S., Cao, Y. A., Negrin, R. S., Contag, C. H. 1999; 96 (21): 12044-12049

    Abstract

    Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.

    View details for Web of Science ID 000083166800066

    View details for PubMedID 10518573

  • Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia Edinger, M., Sweeney, T. J., Tucker, A. A., Olomu, A. B., Negrin, R. S., Contag, C. H. 1999; 1 (4): 303-310

    Abstract

    Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

    View details for PubMedID 10935484

  • ISHAGE Scientific Committee Report - Assays to determine hematopoietic stem cell content in blood or marrow grafts CYTOTHERAPY Verfaillie, C. M., Ploemacher, R., di Persio, J., Sutherland, R., Serke, S., Johnsen, H., Noga, S., Negrin, R. 1999; 1 (1): 41-49
  • Cyclosporine, methotrexate, and prednisone compared with cyclosporine and prednisone for prevention of acute graft-vs.-host disease: effect on chronic graft-vs.-host disease and long-term survival. Biology of blood and marrow transplantation Ross, M., Schmidt, G. M., Niland, J. C., Amylon, M. D., Dagis, A. C., Long, G. D., Nademanee, A. P., Negrin, R. S., O'Donnell, M. R., Parker, P. M., Smith, E. P., Snyder, D. S., Stein, A. S., Wong, R. M., Forman, S. J., Blume, K. G., Chao, N. J. 1999; 5 (5): 285-291

    Abstract

    Graft-vs.-host disease (GVHD) is a major predictor of outcome following allogeneic bone marrow transplantation (BMT). For patients alive at day 100 after BMT, the presence or absence of chronic GVHD is one of the most important determinants of survival and quality of life. We wished to determine the effects on chronic GVHD of two regimens used for the prophylaxis of acute GVHD: cyclosporine, methotrexate, and prednisone (CSA/MTX/PSE) and cyclosporine and prednisone (CSA/PSE). One hundred forty-nine evaluable patients were entered into the acute GVHD study. As of 31 March 1997, 63 months after the last patient underwent BMT, the median survival time was 4.5 years (range 0.09-9.9). The incidence of chronic GVHD was independent of the prophylactic regimen (55 vs. 54%), and extensive chronic GVHD occurred in 25 and 24% of patients receiving CSA/MTX/PSE and CSA/PSE, respectively. Of note, the median Karnofsky performance status of both groups was 100% (range 70-100%), reflecting the low incidence of extensive chronic GVHD. Survival rates free of chronic GVHD were 52 vs. 42% (p = 0.29) for patients receiving CSA/MTX/PSE vs. CSA/PSE. The incidence of relapse was also similar in both groups of patients. These data suggest that the combinations of CSA/MTX/PSE and CSA/PSE result in comparable chronic GVHD-free survival without an increase in leukemic relapse.

    View details for PubMedID 10534058

  • Impact of admission body weight and chemotherapy dose adjustment on the outcome of autologous bone marrow transplantation. Biology of blood and marrow transplantation Dickson, T. M., Kusnierz-Glaz, C. R., Blume, K. G., Negrin, R. S., Hu, W. W., Shizuru, J. A., Johnston, L. L., Wong, R. M., Stockerl-Goldstein, K. E. 1999; 5 (5): 299-305

    Abstract

    We performed a retrospective analysis of 473 consecutive adult patients undergoing autologous bone marrow transplantation for hematologic malignancies between 1988 and 1995. The analysis examined whether significant deviation from ideal body mass index is associated with a decrease in event-free survival (EFS), an increase in nonrelapse mortality (NRM) including late toxicities and second malignancies, or relapse. Chemotherapy dosing in underweight and overweight patients is administered based on the relationship of admission body weight (ABW) to ideal body weight (IBW). Doses were adjusted for obesity; however, the adjustment did not obviate increased risk for NRM. Patients were categorized into five groups according to the relationship of ABW to age-adjusted body mass index (aBMI) as a percent of actual BMI, as follows: group I, 70-79%; group II, 80-99%; group III, 100-119%; group IV, 120-139%; and group V, 140-199% aBMI. When body weight was expressed as percent BMI adjusted for age, there was a significantly increased risk for NRM in groups I and IV (p = 0.03 and 0.02, respectively). A trend toward greater NRM in group V (p = 0.10) was also noted. Multivariate analysis confirmed that the risk of NRM for extremely underweight and overweight patients is almost three times that of patients close to ideal body weight. Age-adjusted BMI was an independent predictive factor for NRM but not associated with increased relapse. We determined that dose adjustment could be safely used without significant increase of relapse. In patients with significant deviation of BMI from aBMI, dose adjustment and possible weight normalization should be considered.

    View details for PubMedID 10534060

  • Assays to determine hematopoietic stem cell content in blood or marrow grafts. Cytotherapy Verfaillie, C. M., Ploemacher, R., di Persio, J., Sutherland, R., Serke, S., Johnsen, H., Noga, S., Negrin, R. 1999; 1 (1): 41-49

    View details for PubMedID 19746648

  • Expansion of Philadelphia chromosome-negative CD3(+)CD56(+) cytotoxic cells from chronic myeloid leukemia patients: In vitro and in vivo efficacy in severe combined immunodeficiency disease mice BLOOD Hoyle, C., Bangs, C. D., Chang, P., Kamel, O., Mehta, B., Negrin, R. S. 1998; 92 (9): 3318-3327

    Abstract

    We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.

    View details for Web of Science ID 000076692200037

    View details for PubMedID 9787169

  • Minimal residual disease. Current opinion in hematology Negrin, R. S. 1998; 5 (6): 488-493

    Abstract

    A major limitation in the treatment of malignant disorders is the accurate and sensitive detection of minimal residual disease. It is clear that routine radiographic and pathologic studies are extremely important, however, lacking in sensitivity. Clearly, the goal of minimal residual disease detection is to make individual treatment decisions such that those patients who require more aggressive approaches are treated promptly and to avoid toxic and expensive treatments for those patients who do not require them. To reach this goal two milestones are required. The first is to develop and standardize sensitive and specific tools to detect minimal residual disease. The second is to determine whether these tools in fact predict clinical outcome. This review is divided into two parts. In the first section, recent advances in the detection of minimal residual disease will be discussed. This will be followed by studies reported in the past year that have attempted to determine whether those assays are reliable and predict clinically relevant endpoints.

    View details for PubMedID 9814661

  • Engraftment of chronic myeloid leukemia in SCID mice HEMATOLOGICAL ONCOLOGY Hoyle, C. F., Negrin, R. S. 1998; 16 (3): 87-100

    Abstract

    Chronic myeloid leukemia (CML) is a clonal disorder of primitive hematopoietic stem cells characterized by a reciprocal translocation between chromosomes 9 and 22. Animal models of CML would be useful to study the biology and potential therapies in this disease. Mice with severe combined immunodeficiency (SCID) which will accept human xenografts have been useful in the study of a variety of human malignancies. CML has been difficult to establish in SCID mice possibly due to the lack of a functioning human stroma and relevant cytokines. To facilitate engraftment we injected cells in matrigel which is a soluble extract of basement membranes; is liquid below 22 degrees C and gels at 37 degrees C. CD34+ myeloid blast crisis cells (2 x 10(6)) were mixed in matrigel and injected subcutaneously into 10 SCID mice. All mice developed large tumours which spread to the mouse BM and spleen. However the percentage of human cells in the mouse BM and spleen was variable and ranged from 1 to 50 per cent. In contrast chronic phase (CP) CML cells mixed in matrigel did not form subcutaneous tumours and spread to the BM and spleen was detectable by PCR and not macroscopically. Groups of mice were injected with matrigel containing 1-20 x 10(7) MNC (2-20 x 10(5) CD34+ cells) from five patients with CP CMP. Bcr-abl sequences were detected by RT-PCR in the peripheral blood (PB) of 38/84 (45 per cent) mice at 3-10 weeks following injection of the CML cells but rarely at later time points. In addition, 33/75 (44 per cent) of mice sacrificed between 7 and 35 weeks following injection of CML cells were bcr/abl positive in the bone marrow and 17/70 (24 per cent) were positive in the spleen. Bcr-abl positive human CFU-GM colonies were also cultured from the murine bone marrow of several mice indicating that hematopoietic progenitor cells were able to migrate from the matrigel and engraft in murine hematopoietic organs. Engraftment of CP-CML was more successful in mice given higher numbers of CD34+ cells. Histological examination revealed that myeloid cells grow locally in the matrigel for several weeks, during which time the matrigel is infiltrated by blood vessels which may allow for the migration of CML progenitors to the murine bone marrow. This model system may be useful for studying the role of immunotherapy after allogeneic and autologous bone marrow transplantation.

    View details for Web of Science ID 000080050000001

    View details for PubMedID 10235067

  • A multicenter study of platelet recovery and utilization in patients after myeloablative therapy and hematopoietic stem cell transplantation BLOOD Bernstein, S. H., Nademanee, A. P., Vose, J. M., Tricot, G., Fay, J. W., Negrin, R. S., DiPersio, J., Rondon, G., Champlin, R., Barnett, M. J., Cornetta, K., Herzig, G. P., Vaughan, W., Geils, G., Keating, J., Messner, H., Wolff, S. N., Miller, K. B., Linker, C., Cairo, M., Hellmann, S., Ashby, M., Stryker, S., Nash, R. A. 1998; 91 (9): 3509-3517

    Abstract

    An observational study was conducted at 18 transplant centers in the United States and Canada to characterize the platelet recovery of patients receiving myeloablative therapy and stem cell transplantation and to determine the clinical variables influencing recovery, determine platelet utilization and cost, and incidence of hemorrhagic events. The study included 789 evaluable patients transplanted in 1995. Clinical, laboratory, and outcome data were obtained from the medical records. Variables associated with accelerated recovery in multivariate models included (1) higher CD34 count; (2) higher platelet count at the start of myeloablative therapy; (3) graft from an HLA-identical sibling donor; and (4) prior stem cell transplant. Variables associated with delayed recovery were (1) prior radiation therapy; (2) posttransplant fever; (3) hepatic veno-occlusive disease; and (4) use of posttransplant growth factors. Disease type also influenced recovery. Recipients of peripheral blood stem cells (PBSC) had faster recovery and fewer platelet transfusion days than recipients of bone marrow (BM). The estimated average 60-day platelet transfusion cost per patient was $4,000 for autologous PBSC and $11,000 for allogeneic BM transplants. It was found that 11% of all patients had a significant hemorrhagic event during the first 60 days posttransplant, contributing to death in 2% of patients. In conclusion, clinical variables influencing platelet recovery should be considered in the design and interpretation of clinical strategies to accelerate recovery. Enhancing platelet recovery is not likely to have a significant impact on 60-day mortality but could significantly decrease health care costs and potentially improve patient quality of life.

    View details for Web of Science ID 000073274500051

    View details for PubMedID 9558412

  • Fractionated total-body irradiation, etoposide, and cyclophosphamide followed by allogeneic bone marrow transplantation for patients with high-risk or advanced-stage hematological malignancies. Biology of blood and marrow transplantation Long, G. D., Amylon, M. D., Stockerl-Goldstein, K. E., Negrin, R. S., Chao, N. J., Hu, W. W., Nademanee, A. P., Snyder, D. S., Hoppe, R. T., Vora, N., Wong, R., Niland, J., Reichardt, V. L., Forman, S. J., Blume, K. G. 1997; 3 (6): 324-330

    Abstract

    Myeloablative therapy followed by allogeneic bone marrow transplantation (BMT) has proven to be curative therapy in patients with hematologic malignancies. Relapse, however, remains a major cause of treatment failure for patients with advanced disease. During the past 15 years, we have gained considerable experience with the combination of fractionated total-body irradiation (FTBI) and etoposide followed by allogeneic BMT for hematologic malignancies. In an attempt to decrease post-transplant relapse rates, 67 patients under the age of 50 years with high-risk or advanced-stage hematological malignancies received an intensified regimen of FTBI and etoposide plus cyclophosphamide followed by BMT from a genotypically-matched related donor. The regimen consisted of 1320 cGy of FTBI in 11 fractions, 60 mg/kg of etoposide (VP-16), and 60 mg/kg of cyclophosphamide (CY). Fifty-three patients received cyclosporine and prednisone for graft-vs.-host disease (GVHD) prophylaxis and 14 patients received cyclosporine, methotrexate, and prednisone. Diagnosis at BMT included 45 patients with acute leukemia, 7 patients with chronic leukemia, and 15 patients with high-grade non-Hodgkin's lymphoma (NHL). Actuarial disease-free survival (DFS) at 3 years was 42% +/- 12% for the entire group with a median follow-up of 50 months (range 20-74) for 28 patients who remain alive in continued complete remission (CR). Actuarial 3-year-DFS was 38% +/- 14% in 52 patients with acute or chronic leukemia and 60% +/- 25% in 15 patients with NHL with relapse rates of 45% +/- 16% and 21% +/- 11%, respectively. DFS at 3 years was 40% +/- 18% in 32 patients with acute leukemia in 1st relapse or 2nd CR or chronic myelogenous leukemia in accelerated phase, and was 32% +/- 22% in 20 patients with more advanced disease. Regimen related mortality occurred in 9 patients (4, veno-occlusive disease of the liver; 2, multi-organ failure; 1, diffuse alveolar hemorrhage; 1, central nervous system (CNS) hemorrhage; 1, adult respiratory distress syndrome (ARDS). The combination of FTBI, etoposide, and cyclophosphamide followed by allogeneic BMT is an effective and relatively well-tolerated regimen for patients with advanced hematologic malignancies. The role for this regimen should be further defined by prospective clinical trials.

    View details for PubMedID 9502300

  • Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model BRITISH JOURNAL OF HAEMATOLOGY HELLSTROMLINDBERG, E., Negrin, R., Stein, R., Krantz, S., Lindberg, G., Vardiman, J., Ost, A., Greenberg, P. 1997; 99 (2): 344-351

    Abstract

    Previous studies have shown that approximately 40% of patients with myelodysplastic syndrome (MDS) and anaemia respond to treatment with human recombinant granulocyte-CSF (G-CSF) plus erythropoietin (epo). The present study was designed to investigate pre-treatment variables for their ability to predict erythroid responses to this treatment. 98 patients with MDS (30 RA, 31 RARS, 32 RAEB, five RAEB-t) were treated with a combination of G-CSF (0.3-3.0 microg/kg/d, s.c.) and epo (60-300 U/kg/d, s.c.) for at least 10 weeks. Minimum criteria for erythroid response was a 100% reduction of red blood cell (RBC) transfusion need or an increase in haemoglobin level of > or = 1.5 g/dl. 35 patients (36%) showed responses to treatment. Medium duration of response was 11-24 months. In multivariate analysis, serum erythropoietin levels and initial RBC-transfusion need retained high statistical significance (P < 0.01). Using pre-treatment serum epo levels as a ternary variable (< 100, 100-500 or > 500 U/l) and RBC transfusion need as a binary variable (< 2 or > or = 2 units per month), the analysis provided a predictive score for erythroid response. This score divided patients into three groups: one group with a high probability of erythroid responses (74%), one intermediate group (23%) and one group with poor responses to treatment (7%). This predictive scoring system could be used in decisions regarding use of these cytokines for treating the anaemia of MDS, both for defining patients who should not be given the treatment and for selecting patients for inclusion in prospective trials.

    View details for Web of Science ID A1997YG53500019

    View details for PubMedID 9375752

  • Cellular immunotherapy following autologous hematopoietic progenitor cell transplantation. Biology of blood and marrow transplantation Lu, P. H., Negrin, R. S. 1997; 3 (3): 113-121

    View details for PubMedID 9310188

  • Cellular immunotherapy and autologous transplantation for hematologic malignancy IMMUNOLOGICAL REVIEWS Margolin, K. A., Negrin, R. S., Wong, K. K., Chatterjee, S., Wright, C., Forman, S. J. 1997; 157: 231-240

    Abstract

    The success of allogeneic transplantation is in part due to the immunotherapeutic effect mediated by the graft. Autologous transplantation is hampered by the absence of this effect, leading to a higher relapse rate. We have conducted a series of studies designed to augment the immunologic activity of the graft after autologous transplant with a view towards introducing an autologous graft-versus-tumor effect that could decrease the rate of relapse after autologous transplant. These studies have included IL-2 activation of marrow followed by post-transplant infusional IL-2, the development of a novel protocol for the generation of highly efficient cytotoxic effector cells, termed cytokine-induced killer (CIK) cells, with broad and potent antitumor activity. In order to determine the potential for generating peptide-specific cytolytic T cells, studies have been conducted upon transducing antigen-presenting cells (APC) with AAV vector-mediated gene transfer, a vector capable of transducing non-proliferating target cells. Transduction of human monocytes and macrophages resulted in high expression of the transduced gene. This latter study forms the basis for determining whether genetic modification of APC can potentiate specific immune responses to tumor-specific gene products. Taken together, these strategies will hopefully increase the therapeutic efficacy of autologous transplantation.

    View details for Web of Science ID A1997XL05000017

    View details for PubMedID 9255634

  • Granulocyte colony-stimulating factor-induced comobilization of CD4(-)CD8(-) T cells and hematopoietic progenitor cells (CD34(+)) in the blood of normal donors BLOOD KUSNIERZGLAZ, C. R., Still, B. J., Amano, M., Zukor, J. D., Negrin, R. S., Blume, K. G., Strober, S. 1997; 89 (7): 2586-2595

    Abstract

    The feasibility of transplantation of HLA-matched hematopoietic progenitor cells from the blood of normal donors given granulocyte colony-stimulating factor (G-CSF) has been reported recently. In the current study, the changes in T-cell subsets as well as CD34+ cells were determined in one blood volume leukapheresis products of six normal individuals given G-CSF. Examination of the T-cell subsets in the leukapheresis products showed three different patterns: one in which a discrete population of CD4- CD8- alphabeta T cells was found in addition to the typical CD4+ and CD8+ T cells in the unfractionated as well as in high- and low-density cells; a second in which the discrete population of CD4- CD8- alphabeta T cells was predominant only in the low-density fractions; and a third in which a discrete population of CD4- CD8- T cells was not observed. The median yield of CD4- CD8- T cells was about fourfold to fivefold higher than the calculated number present in one blood volume (5L) from normal individuals. The ratios of CD34+ cells to CD4+ and CD8+ T cells, and of CD4- CD8- T cells to CD4+ and CD8+ T cells, were highest in the low-density fractions. These fractions suppressed the mixed leukocyte, and may ameliorate graft-versus-host disease as compared with unfractionated cells.

    View details for Web of Science ID A1997WQ35900041

    View details for PubMedID 9116306

  • High-dose therapy and autologous hematopoietic progenitor cell transplantation for recurrent or refractory Hodgkin's disease: Analysis of the Stanford University results and prognostic indices BLOOD Horning, S. J., Chao, N. J., Negrin, R. S., Hoppe, R. T., Long, G. D., Hu, W. W., Wong, R. M., Brown, B. W., Blume, K. G. 1997; 89 (3): 801-813

    Abstract

    One hundred nineteen patients with relapsed or refractory Hodgkin's disease (HD) received high-dose therapy followed by autologous hematopoietic progenitor cell transplantation. Three preparatory regimens, selected on the basis of prior therapy and pulmonary status, were employed. Twenty-six patients without a history of prior chest or pelvic irradiation were treated with fractionated total body irradiation, etoposide (VP) 60 mg/kg and cyclophosphamide (Cy) 100 mg/kg. Seventy-four patients received BCNU 15 mg/kg with identical doses of VP and Cy. A group of 19 patients with a limited diffusing capacity or history of pneumonitis received a novel high-dose regimen consisting of CCNU 15 mg/kg, VP 60 mg/kg and Cy 100 mg/kg. Twenty-nine patients (24%) had failed induction therapy and 35 (29%) had progressive HD within 1 year of initial chemotherapy. At 4 years actuarial survival was 52%, event-free survival was 48% and freedom from progression (FFP) was 62%. No significant differences were seen in survival data with the three preparatory regimens. Six patients died within 100 days of transplantation and 5 died at a later date of transplant-related complications. Secondary malignancies have developed in 6 patients, including myelodysplasia/leukemia in four patients and solid tumors in two patients. Regression analysis identified systemic symptoms at relapse, disseminated pulmonary or bone marrow disease at relapse and more than minimal disease at the time of transplantation as significant prognostic factors for overall and event-free survival and FFP. Patients with none of these factors enjoyed an 85% FFP at 4 years compared with 41% for patients with one or more unfavorable prognostic factors (P = .0001). Our results confirm the efficacy of high-dose therapy and autografting in recurrent or refractory HD. Although longer follow-up is necessary to address ultimate cure rates and toxicity, our data indicate that a desire to reduce late effects should drive future research efforts in favorable patients whereas new initiatives are needed for those with less favorable prognoses.

    View details for Web of Science ID A1997WG07300008

    View details for PubMedID 9028311

  • Activated T cells and cytokine-induced CD3(+)CD56(+) killer cells ANNALS OF HEMATOLOGY SCHMIDTWOLF, G. D., Negrin, R. S., SCHMIDTWOLF, I. G. 1997; 74 (2): 51-56

    Abstract

    Over the past two decades, attempts have been made to develop immunotherapy for patients with cancer. A significant obstacle to the development of successful adoptive immunotherapy has been the availability of appropriate cytotoxic cells. Immunologic effector cells such as lymphokine-activated killer (LAK) cells, activated T cells such as tumor-infiltrating lymphocytes (TILs), and cytokine-induced killer (CIK) cells may be suitable to remove residual tumor cells.

    View details for Web of Science ID A1997WL90200001

    View details for PubMedID 9063373

  • High dose etoposide-based myeloablative therapy followed by autologous blood progenitor cell rescue in the treatment of multiple myeloma CANCER Long, G. D., Chao, N. J., Hu, W. W., Negrin, R. S., Wong, R. M., Blume, K. G. 1996; 78 (12): 2502-2509

    Abstract

    A number of studies have demonstrated that high dose chemotherapy, with or without radiotherapy, with autologous marrow and/or peripheral blood progenitor cell support can result in improved overall and complete response rates in patients with multiple myeloma, and a minority of patients become long term survivors. Based on their favorable experience with high dose etoposide-based regimens in patients with Hodgkin's disease and non-Hodgkin's lymphoma, the authors explored the use of these regimens prior to autologous progenitor cell rescue in patients with multiple myeloma.Thirty-four patients (median age, 49 years; range, 38-65) with multiple myeloma who were responsive to standard chemotherapy were enrolled in this study. Blood progenitor cells were collected after treatment with cyclophosphamide at a dose of 4 g/m2 followed by granulocyte-colony stimulating factor (G-CSF) at a dose of approximately 10 micrograms/kg/day subcutaneously, and the collection continued daily until the target number of mononuclear cells had been obtained. The preparative regimen consisted of fractionated total body irradiation (FTBI) of 1200 centigray in 10 fractions on Day -8 to Day -5, etoposide 60 mg/kg intravenously (i.v.) on Day -4, and cyclophosphamide 100 mg/kg i.v. on Day -2. Day 0 was the day of progenitor cell infusion. Patients who were older than 50 years or had received prior radiation therapy that precluded FTBI received carmustine 15 mg/kg i.v. or lomustine 15 mg/kg orally on Day -6 rather than FTBI. G-CSF (5 micrograms/kg/day) was begun the day after progenitor cell infusion and continued until engraftment.Recovery of granulocytes to 500/microL occurred at a median of 9 days (range, 7-13), and platelet recovery to 20,000/microL occurred without transfusion at 9 days (range, 6-88) after progenitor cell infusion. Thirty-two patients were evaluable for response. Eleven patients (34%) achieved a complete remission, 17 (53%) achieved a partial remission, and 4 (13%) had stable disease following high dose therapy and progenitor cell rescue. The actuarial event free survival at 4 years for the entire group was 26%, and overall survival was 36%. The median time to progression of disease was 13 months (range, 2-42). Two patients died of regimen-related toxicity, one of venoocclusive disease of the liver and the other of multiorgan failure. In a multivariate analysis, only the extent of prior therapy was a significant prognostic factor for event free survival, and no significant factors were identified for overall survival.High dose etoposide-based myeloablative regimens followed by autologous blood progenitor cell rescue are relatively well tolerated and effective for the treatment of multiple myeloma, and a minority of patients become long term disease free survivors.

    View details for Web of Science ID A1996VW71400009

    View details for PubMedID 8952558

  • Maintenance treatment of the anemia of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor and erythropoietin: Evidence for in vivo synergy BLOOD Negrin, R. S., Stein, R., Doherty, K., Cornwell, J., Vardiman, J., Krantz, S., Greenberg, P. L. 1996; 87 (10): 4076-4081

    Abstract

    Patients with myelodysplastic syndromes (MDS) have refractory cytopenias leading to transfusion requirements and infectious complications. In vitro marrow culture data have indicated that granulocyte colony stimulating factor (G-CSF) synergizes with erythropoietin (EPO) for the production of erythroid precursors. In an effort to treat the anemia and neutropenia in this disorder, MDS patients were treated with a combination of recombinant human EPO and recombinant human G-CSF. Fifty-five patients were enrolled in the study of which 53 (96%) had a neutrophil response. Forty-four patients were evaluable for an erythroid response of which 21 (48%) responded. An erythroid response was significantly more likely in those patients with relatively low serum EPO levels, higher absolute basal reticulocyte counts and normal cytogenetics at study entry. Seventeen (81%) of the patients who responded to combined G-CSF plus EPO therapy continued to respond during an 8-week maintenance phase. G-CSF was then discontinued and all patients' neutrophil responses were diminished, whereas 8 continued to have an erythroid response to EPO alone. In 7 of the remaining 9 patients, resumption of G-CSF was required for recurrent erythroid responses. The median duration of erythroid responses to these cytokines was 11 months, with 6 patients having relatively prolonged and durable responses for 15 to 36 months. Our results also indicate that approximately one half of responding patients require both G-CSF and EPO to maintain an effective erythroid response, suggesting that synergy between G-CSF and EPO exists in vivo for the production of red blood cells in MDS.

    View details for Web of Science ID A1996UK87900007

    View details for PubMedID 8639764

  • Paradoxical effect of thalidomide prophylaxis on chronic graft-vs.-host disease. Biology of blood and marrow transplantation Chao, N. J., Parker, P. M., Niland, J. C., Wong, R. M., Dagis, A., Long, G. D., Nademanee, A. P., Negrin, R. S., Snyder, D. S., Hu, W. W., Gould, K. A., Tierney, D. K., Zwingenberger, K., Forman, S. J., Blume, K. G. 1996; 2 (2): 86-92

    Abstract

    Thalidomide has been reported to be an effective agent for the treatment of chronic graft-vs.-host disease (GVHD). To determine its efficacy as a prophylactic agent for the prevention of chronic GVHD, a prospective randomized double-blind study was performed. A total of 59 patients were randomized to receive either placebo or thalidomide (200 mg orally twice a day) beginning 80 days after allogeneic bone marrow transplantation (BMT). Fifty-four evaluable patients were analyzed, 26 received placebo, and 28 received thalidomide. The characteristics of patients were well-balanced between the two groups. Following the first interim analysis conducted by the Data Safety Monitoring Board using an intent-to-treat approach, a statistically significant difference in the incidence of chronic GVHD was found. Patients receiving thalidomide developed chronic GVHD more often than patients receiving placebo (p = 0.06). Moreover, an apparent overall survival advantage was noted for patients receiving placebo compared to those receiving thalidomide (p = 0.006). Adjustment for possible confounding factors did not eliminate these negative effects of thalidomide. These results demonstrate that while thalidomide is an effective agent for the therapy of chronic GVHD, its use at the doses administered for the prophylaxis of chronic GVHD resulted in a paradoxical outcome with a higher incidence of chronic GVHD and a lower overall survival. We conclude that the early use of thalidomide results in a shift in the balance between GVHD and induction of tolerance. These data demonstrate again the importance of phase III double-blind controlled randomized studies.

    View details for PubMedID 9118303

  • Influence of preparatory regimen and source of hematopoietic cells on outcome of autotransplantation for non-Hodgkin's lymphoma. Biology of blood and marrow transplantation Stockerl-Goldstein, K. E., Horning, S. J., Negrin, R. S., Chao, N. J., Hu, W. W., Long, G. D., Hoppe, R. T., Amylon, M. D., BROWN, B. W., Wong, R. M., Blume, K. G. 1996; 2 (2): 76-85

    Abstract

    The use of high-dose chemotherapy with or without total-body irradiation (TBI) followed by autologous hematopoietic cell transplantation is associated with improved survival for relapsed or refractory non-Hodgkin's lymphoma (NHL). Previous reports comparing preparatory regimens with or without TBI followed by autologous bone marrow transplantation (ABMT) or peripheral blood progenitor cell transplantation (PBPCT) for these patients did not demonstrate any survival difference between the different modalities. No randomized studies comparing survival for patients with NHL transplanted with radiochemotherapy vs. chemotherapy alone have been reported. We treated 221 patients with high-risk, relapsed or refractory NHL with either chemotherapy alone or radiochemotherapy followed by ABMT or PBPCT. The patients were assigned preparatory regimens in a non-randomized manner and this analysis was performed to evaluate differences in outcome with the two preparatory regimens. Actuarial five-year event-free survival (EFS) was similar in patients receiving fractionated total-body irradiation (FTBI) plus etoposide (VP-16) and cyclophosphamide (Cy) compared with chemotherapy alone consisting of carmustine (BCNU) plus identical doses of VP-16 and Cy (52% vs. 46%, p = 0.08). Overall survival (OS) favored radiochemotherapy (61%) compared with chemotherapy alone (53%, p = 0.02). The relapse rate was the same in both groups (41%), whereas the transplantation-related mortality (TRM) was similar in patients receiving chemotherapy alone and those receiving radiochemotherapy (13% vs. 7% respectively, p = 0.30). Proportional hazards analysis of significant variables including preparatory regimen found only the number of prior relapses to be predictive of EFS. Fewer number of prior relapses, radiochemotherapy and PBPCT were significant predictors of favorable OS. In additional analyses, the improved OS of the radiochemotherapy regimen was confirmed only for patients receiving ABMT but was not a significant predictor of outcome in patients transplanted with PBPCT. From these retrospective data we conclude: 1) PBPCT resulted in survival superior to that of ABMT; 2) the risk of relapse is similar with either preparatory regimen; 3) patients with fewer prior relapses enjoyed superior overall and event-free survival as well as fewer relapses; and 4) there were no significant differences in the two preparatory regimens when combined with PBPCT in relapsed or refractory NHL.

    View details for PubMedID 9118302

  • Sensitivity of multidrug-resistant tumor cell lines to immunologic effector cells CELLULAR IMMUNOLOGY SCHMIDTWOLF, I. G., Lefterova, P., Johnston, V., Scheffold, C., Csipai, M., Mehta, B. A., Tsuruo, T., Huhn, D., Negrin, R. S. 1996; 169 (1): 85-90

    Abstract

    The ability of malignant cells to survive exposure to cytotoxic agents is a major obstacle to cure in patients with cancer. Multidrug resistance and the expression of P-glycoprotein are emerging as a cause of chemotherapy failure. Immunologic effector cells such as lymphokine-activated killer (LAK) cells or cytokine-induced killer (CIK) cells are capable of killing a broad range of tumor cell lines or freshly isolated tumor cells. As demonstrated here, LAK, and CIK cells possess a high level of cytotoxic activity against tumor cell lines both resistant and sensitive to chemotherapeutic agents such as doxorubicin or vinblastine. CIK cells possessed a higher level of cytotoxic activity than LAK cells as determined by 51Cr release and a tumor colony assay. Monoclonal antibodies against P-glycoprotein did not block the lysis of tumor cells resistant to chemotherapy by CIK cells. In contrast, antibodies to LFA-1 and ICAM-1 blocked CIK cell-mediated tumor cell lysis. These data demonstrate that immunological approaches to cancer therapy may be useful in overcoming disease caused by drug resistance.

    View details for Web of Science ID A1996UF06500012

    View details for PubMedID 8612299

  • Yttrium-90-labeled anti-CD20 monoclonal antibody therapy of recurrent B-cell lymphoma CLINICAL CANCER RESEARCH Knox, S. J., Goris, M. L., Trisler, K., Negrin, R., Davis, T., Liles, T. M., GRILLOLOPEZ, A., Chinn, P., Varns, C., Ning, S. C., Fowler, S., Deb, N., Becker, M., Marquez, C., Levy, R. 1996; 2 (3): 457-470

    Abstract

    A Phase I/II dose escalation study of 90Y-murine anti-CD20 monoclonal antibody (mAb) in patients with recurrent B-cell lymphoma was performed. The primary objectives of the study were: (a) to determine the effect of the preinfusion of unlabeled anti-CD20 mAb on the biodistribution of 111In-anti-CD20 mAb; (b) to determine the maximal tolerated dose of 90Y-anti-CD20 mAb that does not require bone marrow transplantation; and (c) to evaluate the safety and antitumor effect of 90Y-anti-CD20 mAb in patients with recurrent B-cell lymphoma. Eighteen patients with relapsed low- or intermediate-grade non-Hodgkin's lymphoma were treated. Biodistribution studies with 111In-anti-CD20 mAb were performed prior to therapy. Groups of three or four patients were treated at dose levels of approximately 13.5, 20, 30, 40, and 50 mCi 90Y-anti-CD20 mAb. Three patients were retreated at the 40-mCi dose level. The use of unlabeled antibody affected the biodistribution favorably. Nonhematological toxicity was minimal. The only significant toxicity was myelosuppression. The overall response rate following a single dose of 90Y-anti-CD20 mAb therapy was 72%, with six complete responses and seven partial responses and freedom from progression of 3-29+ months following treatment. Radioimmunotherapy with

    View details for Web of Science ID A1996TY62000004

    View details for PubMedID 9816191

  • BUSULFAN/CYCLOPHOSPHAMIDE AS CONDITIONING REGIMEN FOR ALLOGENEIC BONE-MARROW TRANSPLANTATION FOR MYELODYSPLASIA JOURNAL OF CLINICAL ONCOLOGY ODONNELL, M. R., Long, G. D., Parker, P. M., Niland, J., Nademanee, A., Amylon, M., Chao, N., Negrin, R. S., Schmidt, G. M., Slovak, M. L., Smith, E. P., Snyder, D. S., Stein, A. S., Traweek, T., Blume, K. G., Forman, S. J. 1995; 13 (12): 2973-2979

    Abstract

    A non-radiation-containing regimen of busulfan and cyclophosphamide (BU/CY) was evaluated for toxicity, relapse, and long-term survival in patients who received allogeneic bone marrow transplantation (BMT) for myelodysplasia (MDS).Thirty-eight patients with MDS, including eight with therapy-related MDS, were prepared for BMT using BU/CY.Fourteen patients remain in first remission 18 to 60 months posttransplant. Five patients relapsed after BMT, and four of these patients died. Eight additional patients died of acute or chronic graft-versus-host disease (GVHD), and 11 died of regimen-related toxicity, primarily systemic mycoses. Overall survival rate at 2 years was 45% (95% confidence interval [CI], 0.30 to 0.61), with a 24% probability of relapse (95% CI, 0.10 to 0.49). Regimen-related toxicity was manifested primarily as hepatic dysfunction in 72% of patients, with 16% developing overt venoocclusive disease (VOD).Non-radiation-containing preparative regimens offer long-term survival in allogeneic BMT for MDS that is comparable to that of radiation-containing regimens, and are useful in patients with therapy-related MDS. Monitoring BU levels may reduce regimen-related mortality and improve survival.

    View details for Web of Science ID A1995TK25600017

    View details for PubMedID 8523063

  • THALIDOMIDE AS SALVAGE THERAPY FOR CHRONIC GRAFT-VERSUS-HOST DISEASE BLOOD Parker, P. M., Chao, N., Nademanee, A., ODONNELL, M. R., Schmidt, G. M., Snyder, D. S., Stein, A. S., Smith, E. P., Molina, A., Stepan, D. E., Kashyap, A., Planas, I., Spielberger, R., Somlo, G., Margolin, K., Zwingenberger, K., WILSMAN, K., Negrin, R. S., Long, G. D., Niland, J. C., Blume, K. G., Forman, S. J. 1995; 86 (9): 3604-3609

    Abstract

    Thalidomide has been reported to be an effective agent for treatment of chronic graft-versus-host disease (CGVHD). To determine the efficacy of this agent in patients with refractory CGVHD a total of 80 patients who failed to respond to prednisone (PSE) or PSE and cyclosporine (CSA) were treated with thalidomide. Sixteen patients (20%) had a sustained response, 9 with a complete remission and 7 with a partial response. Twenty-nine patients (36%) had thalidomide discontinued because of side effects, which included sedation, constipation, neuritis, skin rash, and neutropenia. Side effects were reversible with drug discontinuation except for mild residual neuritis in one case. Rashes and neutropenia have not previously been reported as thalidomide side effects when used for CGVHD treatment. We conclude thalidomide is immunosuppressive and active in the treatment of CGVHD. A high incidence of reversible side effects limited dose intensity and reduced the number of patients who could benefit from treatment.

    View details for Web of Science ID A1995TB16300043

    View details for PubMedID 7579470

  • 2 PATHWAYS OF EXOCYTOSIS OF CYTOPLASMIC GRANULE CONTENTS AND TARGET-CELL KILLING BY CYTOKINE-INDUCED CD3(+)CD56(+) KILLER-CELLS BLOOD Mehta, B. A., SCHMIDTWOLF, G. H., Weissman, I. L., Negrin, R. S. 1995; 86 (9): 3493-3499

    Abstract

    Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell-induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3-mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP-sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be-identified target cell surface molecules.

    View details for Web of Science ID A1995TB16300028

    View details for PubMedID 7579455

  • MULTIPLE CYCLES OF HIGH-DOSE CHEMOTHERAPY SUPPORTED BY HEMATOPOIETIC PROGENITOR CELLS AS TREATMENT FOR PATIENTS WITH ADVANCED MALIGNANCIES CANCER Long, G. D., Negrin, R. S., Hoyle, C. F., KUSNIERZGLAZ, C. R., Schriber, J. R., Blume, K. G., Chao, N. J. 1995; 76 (5): 860-868

    Abstract

    Retrospective studies suggest that dose intensity is an important determinant of outcome in the treatment of patients with a variety of malignant diseases such as breast cancer, ovarian cancer, and lymphoma. Unfortunately, these results have not been clearly substantiated in prospective randomized trials. One problem with these studies may be that the degree of dose escalation is not sufficient to result in an improved outcome because the chemotherapy doses are limited by hematopoietic toxicity. In an attempt to deliver more dose-intensive therapy, the feasibility of the administration of multiple cycles of high dose chemotherapy with hematopoietic progenitor cell and growth factor support was investigated in patients with advanced malignancies.Nineteen patients with metastatic breast cancer and six patients with refractory non-Hodgkin's lymphoma were initially treated with etoposide (VP-16) (2 gm/m2) and granulocyte-colony stimulating factor (G-CSF). Peripheral blood hematopoietic progenitor cells were collected by leukapheresis and cryopreserved as the patients' leukocyte counts recovered from the nadir induced by VP-16. Patients were then treated with four cycles of mitoxantrone (18 mg/m2), thiotepa (150-200 mg/m2) and cyclophosphamide (4500-5000 mg/m2) as a 48-72 hour continuous infusion followed by infusion of one-quarter of their progenitor cells 48 hours later. All patients also received G-CSF (5 micrograms/kg/day) until engraftment.A total of 88 of a planned 100 cycles of therapy were administered to these 25 patients. The median time to recovery of an absolute neutrophil count of 500/microliters or greater was 13-14 days (range, 7-18 days) and time to recovery of a platelet count of 20,000/microliters or greater was 13-14 days (range, 7-16 days) after the initiation of each cycle of chemotherapy. The median number of platelet transfusions required after each cycle was 2-3 (range, 0-18 transfusions) and the number of erythrocyte transfusions was 4 (range, 0-10). The most common toxicity was diarrhea. Prophylactic intravenous antibiotics were administered to avoid fever with neutropenia. Two patients developed interstitial pneumonitis and one patient died. One heavily pretreated patient failed to engraft after the first cycle. Reversible veno-occlusive disease of the liver developed in one patient after the fourth cycle of therapy. Four patients progressed while on therapy. Eight patients were disease free and 13 patients had a partial response or had a positive bone scan as the only evidence of disease at the completion of therapy. Seven patients, two with lymphoma and five with breast cancer (28%), remain progression free with a median follow-up of 24.7 months (range, 17-28 months).Support with hematopoietic progenitor cells and growth factors allows the timely administration of repetitive cycles of high dose chemotherapy, resulting in a significant increase in dose intensity with acceptable toxicity.

    View details for Web of Science ID A1995RP85100020

    View details for PubMedID 8625190

  • ENRICHMENT OF BONE-MARROW AND BLOOD PROGENITOR (CD34(+)) CELLS BY DENSITY GRADIENTS WITH SUFFICIENT YIELDS FOR TRANSPLANTATION EXPERIMENTAL HEMATOLOGY Schriber, J. R., DEJBAKHSHJONES, S., KUSNIERZGLAZ, C. R., Ginzton, N., Still, B., Negrin, R. S., Greenberg, P., Strober, S. 1995; 23 (9): 1024-1029

    Abstract

    We have evaluated the use of iso-osmolar Percoll density gradients to enrich CD34+ hematopoietic progenitor cells and to reduce T cells for purposes of bone marrow or mobilized peripheral blood cell transplantation (BMT or PBCT). Samples from 12 normal BM donors and 11 patients undergoing mobilization of PB cells using chemotherapy and G-CSF were placed over a five-step density gradient from 40 to 50% Percoll. In BM, low-density fractions 1 to 3 (40 to 45% Percoll) accounted for 3% of starting nucleated cells with a 10- to 20-fold enrichment of hematopoietic progenitors (CD34+ cells) and a 20-fold reduction of CD4+ and CD8+ T cells. In PB, fractions 1 to 3 accounted for 20 to 30% of the starting nucleated cells with a five-fold enrichment of hematopoietic progenitors. Based on these values, such populations have been used for clinical transplantation using a single apheresis. The reduced cell numbers in the low-density fractions can facilitate tumor purging, and the reduced T cell numbers present in the marrow may ameliorate graft-vs.-host disease.

    View details for Web of Science ID A1995RP11800011

    View details for PubMedID 7543414

  • TRANSPLANTATION OF ENRICHED AND PURGED PERIPHERAL-BLOOD PROGENITOR CELLS FROM A SINGLE APHERESIS PRODUCT IN PATIENTS WITH NON-HODGKINS-LYMPHOMA BLOOD Negrin, R. S., KUSNIERZGLAZ, C. R., Still, B. J., Schriber, J. R., Chao, N. J., Long, G. D., Hoyle, C., Hu, W. W., Horning, S. J., Brown, B. W., Blume, K. G., Strober, S. 1995; 85 (11): 3334-3341

    Abstract

    High-dose chemotherapy with or without radiotherapy followed by autologous transplantation of hematopoietic progenitor cells is an effective treatment for patients with high-risk or relapsed non-Hodgkin's lymphoma. Chemotherapy and/or hematopoietic growth factors have been used to mobilize progenitor cells in the peripheral blood for transplantation. However, the mobilized blood cell products have been found to be frequently contaminated with tumor cells, and techniques have not been developed to purge tumor cells from these products. In addition, the minimum number of hematopoietic progenitor cells required for engraftment has not yet been fully elucidated. We treated 21 patients with a single infusion of cyclophosphamide (4 g/m2) followed by daily administration of granulocyte colony-stimulating factor (G-CSF). After recovery of the white blood cell count, a single 3-hour apheresis collection was performed. The apheresis product was then applied to a discontinuous Percoll gradient. The low-density fractions resulting from this separation procedure were enriched for CD34+ progenitor cells (total cell yield, 19.5%; CD34+ cell recovery, 81.2%). These enriched cellular products were treated with a panel of anti-B cell or anti-T cell monoclonal antibodies and complement in an effort to remove residual tumor cells. After treatment of the patient with myeloablative therapies, the enriched and purged cells were reinfused. Hematologic recovery was rapid, with median neutrophil engraftment in 10 days [absolute neutrophil count (ANC), greater than 0.5 x 10(9)/L] and 11 days (ANC, greater than 1.0 x 10(9)/L). Median platelet transfusion independence required 13 days. The rapidity of multilineage engraftment correlated with the number of CD34+ cells per kilogram that were infused. Patients who received more than 2 x 10(6) CD34+ cells per kilogram had rapid hematologic engraftment, whereas those patients transplanted with less than 2 x 10(6) CD34+ cells per kilogram had slower platelet recovery. Modeling studies using a lymphoma cell line with a t(14; 18) chromosomal translocation demonstrated the successful removal of tumor cells assayed using the polymerase chain reaction (PCR) after the processing and purging. Four of the 21 patients had PCR-detectable lymphoma cells in the bone marrow and peripheral blood; however, the enriched and purged blood products reinfused in all four did not contain detectable tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1995RA13600043

    View details for PubMedID 7538824

  • IS NITROUS-OXIDE SAFE FOR BONE-MARROW HARVEST ANESTHESIA AND ANALGESIA Lederhaas, G., BROCKUTNE, J. G., Negrin, R. S., Riley, E., Brodsky, J. B. 1995; 80 (4): 770-772

    Abstract

    Patients with non-Hodgkins lymphoma undergoing autologous bone marrow harvest were studied in a prospective, randomized fashion. All patients received a general anesthetic consisting of intravenous thiopental, fentanyl, and vecuronium and were ventilated with oxygen and isoflurane. Group I (19) patients also were ventilated with nitrous oxide (70%) whereas patients in Group II (19) did not receive nitrous oxide. Bone marrow samples were obtained at the beginning and end of the harvest. Viability of bone marrow mononuclear cells was assessed with a colony-forming unit-granulocyte macrophage (CFU-GM) assay, CFU-GM growth is a marker for myeloid progenitor cells and is dependent on intact deoxyribonucleic acid synthesis. Rate of neutrophil engraftment after autologous bone marrow transplantation was also studied. Both groups of patients were statistically similar in age, weight, anesthetic duration, CFU-GM counts at both sample draws, and the time for successful engraftment. There appears to be no difference in bone marrow viability as assayed by both CFU-GM colony growth and engraftment in human bone marrow exposed to a general anesthetic with nitrous oxide.

    View details for Web of Science ID A1995QP46200021

    View details for PubMedID 7893033

  • A NEW PREPARATORY REGIMEN FOR AUTOLOGOUS BONE-MARROW TRANSPLANTATION FOR PATIENTS WITH LYMPHOMA CANCER Chao, N. J., Kastrissios, H., Long, G. D., Negrin, R. S., Horning, S. J., Wong, R. M., Blaschke, T. F., Blume, K. G. 1995; 75 (6): 1354-1359

    Abstract

    This trial studied the feasibility and efficacy of a new preparatory regimen for autologous bone marrow transplantation for patients with advanced lymphoid malignancies.Twenty-one patients with Hodgkin's disease (n = 12) and non-Hodgkin's lymphoma (n = 9) were treated in this study. Lomustine was substituted for carmustine) in a dose-escalation study with an initial dose of 6 mg/kg and increasing by 3 mg/kg in groups of four patients. The preparatory regimen consisted of lomustine (6-15 mg/kg) orally on Day -6, etoposide (60 mg/kg) intravenously (i.v.) on Day -4, and cyclophosphamide (100 mg/kg) i.v. on Day -2. Peripheral blood progenitor cells and/or bone marrow were infused on Day 0.Lomustine was well tolerated in all patients with no significant toxicity specific to this drug. Engraftment was prompt: the time to achieving greater than or equal to 500 granulocytes/microliters was 12 days (range, 9-16 days) and the time to achieving greater than or equal to 25,000 platelets/microliters without transfusion support was 16 days (range, 9-22 days). Five patients experienced interstitial pneumonitis, three of whom had active or recent interstitial pneumonitis before bone marrow transplantation, and one who just completed mantle irradiation. Three patients died from this preparatory regimen, one of progressive interstitial pneumonitis, one of Legionella pneumonia, and one of multiorgan failure. Three patients with non-Hodgkin's lymphoma relapsed. Fourteen patients are currently alive and disease free to date. The actuarial are currently alive and disease free to date. The actuarial disease free survival was 57%, with a median follow-up of 23 months (range, 1-48 months).The preparatory regimen consisting of lomustine/etoposide/cyclophosphamide is active in treating patients with lymphomas. Further trials with high doses of lomustine are warranted.

    View details for Web of Science ID A1995QL72800017

    View details for PubMedID 7882286

  • ENRICHMENT OF HUMAN HEMATOPOIETIC STEM-CELL ACTIVITY IN THE CD34(+)THY-1(+)LIN(-) SUBPOPULATION FROM MOBILIZED PERIPHERAL-BLOOD BLOOD Murray, L., Chen, B., Galy, A., Chen, S., Tushinski, R., Uchida, N., Negrin, R., Tricot, G., Jagannath, S., Vesole, D., Barlogie, B., Hoffman, R., Tsukamoto, A. 1995; 85 (2): 368-378

    Abstract

    The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy-1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell-containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1995QB54600009

    View details for PubMedID 7529060

  • EFFECTS OF GRANULOCYTE-COLONY-STIMULATING FACTOR THERAPY ON IN-VITRO HEMATOPOIESIS IN MYELODYSPLASTIC SYNDROMES LEUKEMIA Nagler, A., MacKichan, M. L., Negrin, R. S., Donlon, T., Greenberg, P. L. 1995; 9 (1): 30-39

    Abstract

    We evaluated the effects of 2 months of G-CSF treatment on in vitro hematopoiesis in 17 patients with myelodysplastic syndromes (MDS). Although in vitro marrow myeloid progenitor cell (CFU-GM) growth stimulated by G-CSF generally remained subnormal, in the majority of neutrophil responders significantly augmented incremental change (termed AIC) of CFU-GM numbers occurred after treatment, as did neutrophilic differentiation. The neutrophil non-responders had less prominent in vitro myeloid responses and lower basal neutrophil levels (p < 0.05). Following G-CSF treatment, the initially subnormal erythroid burst-forming unit (BFU-E) values underwent AIC in five of 11 patients along with increased reticulocyte responses in vivo, whereas four of the five patients who lacked AIC of BFU-E did not. Three patients with persisting cytogenetic abnormalities and increased neutrophilic differentiation in vitro also responded in vivo, suggesting that G-CSF induced in vivo cellular differentiation from the abnormal clone. Two of the three patients who developed blastic responses in vivo had increased CFU-GM growth pre- and post-therapy. These results indicate in vivo-in vitro correlations for myeloid and erythroid responses of MDS marrow cells which related to treatment with G-CSF.

    View details for Web of Science ID A1995QH98700007

    View details for PubMedID 7531261

  • FRACTIONATED TOTAL-BODY IRRADIATION, ETOPOSIDE, AND CYCLOPHOSPHAMIDE PLUS AUTOGRAFTING IN HODGKINS-DISEASE AND NON-HODGKINS-LYMPHOMA JOURNAL OF CLINICAL ONCOLOGY Horning, S. J., Negrin, R. S., Chao, N. J., Long, G. D., Hoppe, R. T., Blume, K. G. 1994; 12 (12): 2552-2558

    Abstract

    High-dose etoposide was incorporated into a regimen of fractionated total-body irradiation (FTBI) and high-dose cyclophosphamide before autologous transplant with the goal to enhance the antitumor effect of the myeloablative regimen in poor-risk lymphoid malignancies.Ninety-six patients, 24 with recurrent or refractory Hodgkin's disease and 72 with poor-risk non-Hodgkin's lymphoma (NHL), were treated on this study. Cytoreduction with conventional therapy was attempted before administration of the preparatory regimen. The preparatory regimen consisted of 12 Gy total-body irradiation administered in 10 1.2-Gy fractions on day -8 through day -5, etoposide 60 mg/kg on day -4, and cyclophosphamide 100 mg/kg on day -2. Patients with NHL received bone marrow purged with a panel of monoclonal antibodies and complement on day 0, while patients with Hodgkin's disease received peripheral-blood stem cells alone or with unmanipulated bone marrow.The major morbidities of transplant were mucositis and skin toxicity. Eight patients (8.6%) died of regimen-related toxicities within 100 days of transplant. Engraftment was related to the rescue product; the median time to a neutrophil count more than 500/microL was 10 days for patients with Hodgkin's disease and 16 days for NHL patients. With a maximum follow-up duration of longer than 5 years, the 3-year actuarial survival rate is 57%. At 3 years, the actuarial freedom from progression (FFP) rate is 55% and the event-free survival rate is 47% for patients with Hodgkin's disease, while the respective figures for NHL patients are 60% and 53%. Among 32 patients with intermediate- and high-grade lymphoma transplanted subsequent to first relapse, 70% are free of lymphoma and 60% are event-free at > or = 3 years.The preparatory regimen consisting of FTBI, etoposide, and cyclophosphamide demonstrates relative efficacy in patients with Hodgkin's disease and NHL selected for high-dose therapy. Longer follow-up duration is needed to determine the rate of cure and to assess late complications. Major remaining challenges for high-dose therapy are a more inclusive strategy for all poor-risk patients and the need to reduce posttransplant relapses.

    View details for Web of Science ID A1994PV81100006

    View details for PubMedID 7989928

  • FRACTIONATED TOTAL-BODY IRRADIATION AND HIGH-DOSE ETOPOSIDE AS A PREPARATORY REGIMEN FOR BONE-MARROW TRANSPLANTATION FOR 94 PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA IN CHRONIC PHASE BLOOD Snyder, D. S., Negrin, R. S., ODONNELL, M. R., Chao, N. J., Amylon, M. D., Long, G. D., Nademanee, A. P., Stein, A. S., Parker, P. M., Smith, E. P., Somlo, G., Margolin, K., Molina, A., Stepan, D. E., LIPSETT, J. A., Hoppe, R. T., Slovak, M. L., Niland, J. C., Dagis, A. C., Wong, R. M., Forman, S. J., Blume, K. G. 1994; 84 (5): 1672-1679

    Abstract

    Ninety-four consecutive patients with chronic myelogenous leukemia in first clinical chronic phase, median age of 34.0 years (range, 6.8 to 52.4 years), with a histocompatible sibling donor, were treated with fractionated total body irradiation (1,320 cGy) and high-dose etoposide (60 mg/kg) followed by allogeneic bone marrow transplantation (BMT). The median time from diagnosis to BMT was 7.0 months (range, 2.3 to 72.0 months). Sixty patients were treated before BMT with hydroxyurea alone, four patients with busulfan alone, one patient with interferon alone, and the other 29 patients were treated with various combinations of these drugs. Cumulative probabilities of overall survival, event-free survival, and relapse at 5 years were 73%, 64%, and 14%, respectively. The median follow-up time for surviving patients was 38 months, ranging from 12 to 88 months. By stepwise Cox regression analysis, significant prognostic variables were age at transplant, acute graft-versus-host disease > or = grade II, cytomegalovirus-associated interstitial pneumonitis, and years from diagnosis to BMT.

    View details for Web of Science ID A1994PE38700040

    View details for PubMedID 8068956

  • GRANULOCYTE-COLONY-STIMULATING FACTOR AFTER ALLOGENEIC BONE-MARROW TRANSPLANTATION BLOOD Schriber, J. R., Chao, N. J., Long, G. D., Negrin, R. S., Tierney, D. K., KUSNIERZGLAZ, C., Lucas, K. S., Blume, K. G. 1994; 84 (5): 1680-1684

    Abstract

    Hematopoietic growth factors have been shown to be effective in reducing the period of neutropenia after autologous bone marrow transplantation (BMT). Initial concerns over potential aggravation of graft-versus-host disease (GVHD) and increase in the incidence of relapse in patients with myeloid leukemias influenced the number of studies using hematopoietic growth factors after allogeneic BMT. We report the experience with 50 patients treated at a single institution using granulocyte colony-stimulating factor (G-CSF) after allogeneic sibling (n = 30) and matched unrelated (n = 20) BMT. The time to an absolute neutrophil count > or = 500/microL was significantly faster in patients who received G-CSF and cyclosporine and prednisone for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis (10 v 13 days, P < .01). A similar accelerated myeloid engraftment was observed for those patients who received the addition of methotrexate for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis regimen (16 v 19 days, P < .05). The median time to engraftment for patients receiving a matched unrelated BMT and G-CSF was 17 days (range 13 to 26). We did not observe any increase in GVHD or early mortality in the matched related sibling BMT. The incidence of acute GVHD in the matched unrelated BMT recipients was also low at 21%; however, 9 patients (45%) died within 100 days of the date of BMT, similar to the experience reported with granulocyte-macrophage CSF. This study confirms the efficacy of G-CSF in accelerating myeloid engraftment after allogeneic matched sibling BMT. The higher early mortality associated with patients receiving matched unrelated BMT suggests that randomized controlled trials using G-CSF after allogeneic BMT should be performed.

    View details for Web of Science ID A1994PE38700041

    View details for PubMedID 7520782

  • A NOVEL POPULATION OF EXPANDED HUMAN CD3+CD56+ CELLS DERIVED FROM T-CELLS WITH POTENT IN-VIVO ANTITUMOR-ACTIVITY IN MICE WITH SEVERE COMBINED IMMUNODEFICIENCY JOURNAL OF IMMUNOLOGY Lu, P. H., Negrin, R. S. 1994; 153 (4): 1687-1696

    Abstract

    Recently, we have reported a novel protocol for the generation of highly efficient cytotoxic effector cells by culturing PBLs in the presence of IFN-gamma, IL-2, mAb against CD3, and IL-1 alpha. We have termed these cultures cytokine-induced killer (CIK) cells because the phenotype of the cells with the greatest cytotoxicity expresses both the T cell marker CD3 and the NK cell marker CD56. Cells with this phenotype are rare (approximately 1 to approximately 5%) in uncultured PBLs. CD3+CD56+ cells expand nearly 1000-fold under these culture conditions. The majority of the CD3+CD56+ cytotoxic cells in CIK cultures were derived from CD3+CD56- T cells, and not CD3-CD56+ NK cells. Expression of CD56, but not CD8, on CD3+ cells correlated with the greatest cytotoxicity against various cellular targets. We have used mice with severe combined immunodeficiency (SCID) injected with human lymphoma cells to evaluate the in vivo antitumor effects of CIK vs lymphokine-activated killer (LAK) cells. Groups of animals inoculated with 1 x 10(6) SU-DHL4 cells (a human B lymphoma cell line with a t(14;18) chromosomal translocation), injected 1 day later with CIK cells either i.v. or i.p., had significantly prolonged survival compared with control animals injected with tumor cells alone (median survival 90 days vs 58 days, p < 0.001) or animals treated with LAK cells (median survival 90 days vs 68 days, p < 0.002). Approximately 30% of the SCID mice challenged with SU-DHL4 cells and treated with CIK cells became long-term survivors compared with none of the animals treated with LAK cells. No molecular evidence of occult lymphoma was found in the CIK cell-treated long-term survivors when their bone marrow, spleen, liver, and lung were analyzed by t(14;18) PCR at the end of 6 mo. By using these culture conditions, a novel population of cytotoxic cells can be generated readily from T cells that have superior in vivo antitumor activity in SCID mice, as compared with LAK cells.

    View details for Web of Science ID A1994PB40600028

    View details for PubMedID 7519209

  • GENERATION OF T-CELLS FROM CYTOKINE-MOBILIZED PERIPHERAL-BLOOD AND ADULT BONE-MARROW CD34+ CELLS BLOOD Galy, A. H., Webb, S., CEN, D. H., Murray, L. J., CONDINO, J., Negrin, R. S., Chen, B. P. 1994; 84 (1): 104-110

    Abstract

    The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

    View details for Web of Science ID A1994NV95900014

    View details for PubMedID 7517204

  • PROPAGATION OF LARGE NUMBERS OF T-CELLS WITH NATURAL-KILLER-CELL MARKERS BRITISH JOURNAL OF HAEMATOLOGY SCHMIDTWOLF, I. G., Lefterova, P., Johnston, V., Huhn, D., Blume, K. G., Negrin, R. S. 1994; 87 (3): 453-458

    Abstract

    Previously, a subset of T cells co-expressing the NK cell antigen CD56 has been described. These CD3+CD56+ cells are rare in peripheral blood collections and have been poorly characterized. We have developed culture conditions which allow for the rapid expansion of CD3+CD56+ cells. The protocol for cellular expansion includes the addition of interferon-gamma on day 0, interleukin-1, interleukin-2 and a monoclonal antibody against CD3 on day 1 to peripheral blood lymphocytes. Cells of the CD3+CD56+ phenotype increased up to 6000-fold using this protocol after 16 d in culture. These cells have been characterized by flow cytometry and have been found to express the alpha, beta T cell receptor, co-express the CD5 and CD8 antigens and do not express the CD16 antigen. Morphologically, these cells cannot be distinguished from NK cells. CD3+CD56+ killer cells lyse a variety of tumour cells with intermediate activity between CD3-CD56+ NK cells and CD3+CD56- T cells.

    View details for Web of Science ID A1994NW74800002

    View details for PubMedID 7527643

  • A RANDOMIZED STUDY OF ERYTHROPOIETIN AND GRANULOCYTE-COLONY-STIMULATING FACTOR (G-CSF) VERSUS PLACEBO AND G-CSF FOR PATIENTS WITH HODGKINS AND NON-HODGKINS-LYMPHOMA UNDERGOING AUTOLOGOUS BONE-MARROW TRANSPLANTATION BLOOD Chao, N. J., Schriber, J. R., Long, G. D., Negrin, R. S., CATOLICO, M., Brown, B. W., MILLER, L. L., Blume, K. G. 1994; 83 (10): 2823-2828

    Abstract

    Anemia is a universal finding in patients undergoing autologous bone marrow transplantation (BMT). Effective therapies to increase the number of autologous red blood cells could result in a lower morbidity and mortality associated with red blood cell transfusions. We examined whether the addition of erythropoietin (Epo) to intensive therapy supported by progenitor cell transplantation and granulocyte colony-stimulating factor (G-CSF) would result in a lower requirement for red blood cell transfusions. Thirty-five patients with lymphoma were randomized to receive Epo versus placebo. Epo (600 U/kg three times per week) or placebo was begun 3 weeks before administration of high-dose therapy. Epo was held during the week of the preparatory regimen, and restarted on the day after BMT. All patients also received G-CSF following BMT. No significant differences were noted between the two groups in terms of patient characteristics at pretreatment or post-BMT evaluation. There were no differences in the total number of red blood cell units transfused (median Epo: 8 v placebo: 6, P = .22) nor the number of platelet transfusions given (median Epo: 12 v placebo 5, P = .14). Engraftment of granulocytes (absolute neutrophil count > or = 500/microL) occurred in a median of 12 days (range, 9 to 33) for the patients receiving Epo and G-CSF, compared with a median of 10 days (range, 8 to 22) for those receiving placebo and G-CSF (P = .70). Likewise, there were no differences in the time to platelet count > or = 20,000/microL without further transfusions with a median of 22 days (range, 15 to 150+) for those receiving Epo and G-CSF compared with a median of 20 days (range, 11 to 54) for those patients receiving placebo and G-CSF (P = .28). The combination of G-CSF and Epo as administered in this study appears to be safe but does not result in an improvement in the total number of red blood cell transfusions or total number of single donor platelet units transfused.

    View details for Web of Science ID A1994NL60700008

    View details for PubMedID 7514046

  • DETECTION OF TUMOR-CELLS IN PURGED BONE-MARROW AND PERIPHERAL-BLOOD MONONUCLEAR-CELLS BY POLYMERASE CHAIN-REACTION AMPLIFICATION OF BCL-2 TRANSLOCATIONS JOURNAL OF CLINICAL ONCOLOGY Negrin, R. S., PESANDO, J. 1994; 12 (5): 1021-1027

    Abstract

    To compare bone marrow (BM) before and after purging with monoclonal antibodies (MAbs) and complement with peripheral-blood mononuclear cells (PBMNCs) for tumor-cell contamination by amplification of t(14;18) sequences using the polymerase chain reaction (PCR).Sixty patients with non-Hodgkin's lymphoma (NHL) undergoing autologous BM transplantation were evaluated. Six BM biopsies were performed at the time of harvesting and evaluated morphologically for tumor involvement. The harvested BM was treated with a panel of anti-B-cell MAbs directed against CD9, CD10, CD19, and CD20, followed by rabbit complement. Clonogenic assays were performed before and after purging. DNA was extracted and t(14;18) sequences amplified by PCR. PBMNCs collected by apheresis for back-up purposes were similarly evaluated.Fifteen patients (25%) were PCR-positive before BM purging. Following MAb- and complement-mediated purging, there was a reduction in the PCR-amplified signal in 10 patients (67%). There was no reduction in colony-forming unit granulocyte-macrophage (CFU-GM) colony growth following purging. Eight of these 15 patients (53%) had morphologic evidence of BM involvement at the time of harvesting. In these eight patients, only three had a reduction in the PCR-amplified products, as compared with all seven who were morphologically negative at the time of BM harvesting (P = .026). Fourteen of these 15 patients had PBMNCs collected near the time of BM harvesting and 12 (86%) were PCR-positive.BM purging with MAbs and complement results in reduction in the number of t(14;18)-positive tumor cells, especially in those patients who have no morphologic evidence of BM disease at the time of harvesting. Purged BM was less contaminated with t(14;18)-positive cells than unpurged PBMNCs, which were frequently contaminated with tumor cells.

    View details for Web of Science ID A1994NL03800022

    View details for PubMedID 8164025

  • THE ROLE OF N-LINKED CARBOHYDRATE RESIDUES IN LYMPHOKINE-ACTIVATED KILLER CELL-MEDIATED CYTOLYSIS CELLULAR IMMUNOLOGY Mehta, B. A., Collard, H. R., Negrin, R. S. 1994; 155 (1): 95-110

    Abstract

    We evaluated LAK cell cytotoxicity toward a sensitive B cell lymphoma and several resistant EBV-transformed cell lines in order to explore the mechanism by which some cells are preferentially recognized as targets. Cytolysis of the sensitive cells was inhibited by monoclonal antibodies against the surface proteins LFA-1 and ICAM-1; however, surface expression of ICAM-1 was similar on the resistant and sensitive cell lines. Prevention of post-translational addition of N-linked oligosaccharides by treatment of the resistant cells with tunicamycin resulted in a dramatic enhancement in LAK cell cytotoxicity which was partially inhibited by antibodies against LFA-1 and ICAM-1. Treatment of the resistant cells with the endoglycosidase N-glycanase also increased LAK cell sensitivity. Tunicamycin treatment caused a decrease in the molecular weight of ICAM-1 from approximately 95,000 to 50,000 Da. Conjugate formation between the LAK cells and the sensitive and resistant target cells was similar before and after deglycosylation. We conclude that carbohydrate modification of ICAM-1 or an alternative glycoprotein confers resistance to LAK cell cytotoxicity in some cell lines.

    View details for Web of Science ID A1994NH46100008

    View details for PubMedID 7909499

  • MUTATIONS IN THE RAS PROTOONCOGENES IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES LEUKEMIA Neubauer, A., Greenberg, P., Negrin, R., Ginzton, N., Liu, E. 1994; 8 (4): 638-641

    Abstract

    Activation of the N- and K-ras proto-oncogenes is the most common molecular abnormality in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In retrospective studies, approximately 3-36% of MDS patients were reported to harbor a mutated ras proto-oncogene, with some series suggesting the presence of ras-mutations are associated with progressive disease and a poor prognosis. Since hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) are currently used for therapy in MDS but may stimulate the proliferation of leukemic cells, we assessed the frequency and significance of ras mutations in 27 MDS patients, 15 of whom underwent G-CSF therapy. Patients were analyzed for the presence of mutations in codons 12, 13, and 61 of the N- and K-ras proto-oncogenes. Only three patients (11%, two refractory anemia with excess of blasts (RAEB), one RAEB in transformation (RAEB-T)) harbored activated ras oncogenes with the mutations localized in N-ras codons 12 and 61. Patients were followed for periods of up to 4 years or until death supervened. Patients exhibiting ras mutations were no more likely to develop AML compared to ras-negative patients (1/3 vs. 10/24) or to have decreased survival (p = 0.64). These data indicate that, in this group of MDS patients, ras mutations do not appear to correlate with a poor prognosis, and do not adversely interact with exogenously administered G-CSF.

    View details for Web of Science ID A1994NJ51700016

    View details for PubMedID 7512175

  • ANALYSIS OF HUMAN HEMATOPOIETIC STEM-CELL POPULATIONS BLOOD CELLS Murray, L., DiGiusto, D., Chen, B., Chen, S., Combs, J., Conti, A., Galy, A., Negrin, R., Tricot, G., Tsukamoto, A. 1994; 20 (2-3): 364-370

    Abstract

    Purification of human hematopoietic stem cells (HSC) may be useful clinically for preparation of tumor-free grafts to be used for autologous transplantation and as targets for gene therapy. To analyze the phenotype of the human HSC, assays were used that measure the unique properties of stem cells, i.e., their long-term repopulating ability and their multilineage potential. These assays include: (1) an in vitro long-term hematopoietic culture system, using the murine bone marrow stromal cell line SyS1, which supports both B lymphopoiesis and myelopoiesis; (2) fetal human bone grafts implanted in SCID-hu mice, in which maintenance of CD34+ cells and B and myeloid differentiative capacity of candidate stem cell populations may be measured; (3) fetal human thymus grafts in SCID-hu mice, which allow the analysis of in vivo T-cell potential of a candidate stem cell population. Stem cells in adult bone marrow (ABM) or cytokine-mobilized peripheral blood (MPB) are thought to express CD34 but lack expression of markers indicating lineage commitment. This CD34+ Lineage- (Lin-) subpopulation has been isolated by fluorescence-activated cell sorting and tested for activity in the assays described here. CD34+ Lin- cells from both ABM and MPB demonstrated long-term engraftment in the SCID-hu bone model. This CD34+ Lin- population can be subfractionated further using an antibody to Thy-1. The Thy-1+ subset of CD34+Lin- cells is enriched for both long-term culture-initiating cells (LTC-1C) and has the ability to engraft in vivo.

    View details for Web of Science ID A1994PZ37500023

    View details for PubMedID 7538340

  • PHENOTYPIC CHARACTERIZATION AND IDENTIFICATION OF EFFECTOR-CELLS INVOLVED IN TUMOR-CELL RECOGNITION OF CYTOKINE-INDUCED KILLER-CELLS EXPERIMENTAL HEMATOLOGY SCHMIDTWOLF, I. G., Lefterova, P., Mehta, B. A., Fernandez, L. P., HUHN, L. D., Blume, K. G., Weissman, I. L., Negrin, R. S. 1993; 21 (13): 1673-1679

    Abstract

    Cytokine-induced killer (CIK) cells are highly efficient cytotoxic effector cells capable of lysing tumor cell targets. Cultures of human CIK cells have been shown to have enhanced cytotoxicity and to proliferate more rapidly than lymphokine activated killer (LAK) cells by both in vitro and in vivo studies. In this report, we have further characterized the phenotype of CIK cells and explored the molecular structures involved in CIK-mediated cell lysis of tumor target cells. The dominant cell phenotype in CIK cell cultures expresses the alpha, beta T cell receptor (TCR-alpha/beta). In addition, CD56 is expressed on the main effector cell on a per-cell basis. Interestingly, the total number of CD56+ cells increases more than 1000-fold during the generation of CIK cells, mainly due to expansion of CD56+ cells coexpressing CD3. The higher lytic activity of CIK cells as compared to LAK cells is mainly due to the higher proliferation of CD3+CD56+ cells and to the cytotoxic activity of TCR-alpha/beta+ cells in CIK cell cultures. CIK-mediated cellular lysis is non-major histocompatibility antigen (MHC) restricted. The cytotoxic effect of CIK cells against tumor targets is blocked by antibodies directed against lymphocyte function-associated antigen (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1).

    View details for Web of Science ID A1993MW61900009

    View details for PubMedID 7694868

  • FRACTIONATED TOTAL-BODY IRRADIATION AND HIGH-DOSE ETOPOSIDE AS A PREPARATORY REGIMEN FOR BONE-MARROW TRANSPLANTATION FOR 99 PATIENTS WITH ACUTE-LEUKEMIA IN 1ST COMPLETE REMISSION BLOOD Snyder, D. S., Chao, N. J., Amylon, M. D., Taguchi, J., Long, G. D., Negrin, R. S., Nademanee, A. P., ODONNELL, M. R., Schmidt, G. M., Stein, A. S., Parker, P. M., Smith, E. P., Stepan, D. E., Molina, A., LIPSETT, J. A., Hoppe, R. T., Niland, J. C., Dagis, A. C., Wong, R. M., Forman, S. J., Blume, K. G. 1993; 82 (9): 2920-2928

    Abstract

    Ninety-nine consecutive patients with acute leukemia in first complete remission under age 50 (median age 27 years; age range 1 to 47 years) with a histocompatible sibling donor were treated with fractionated total body irradiation (1,320 cGy) and high-dose etoposide (60 mg/kg) followed by allogeneic bone marrow transplantation. Sixty-one patients were diagnosed with acute myelogenous leukemia (AML), 34 patients with acute lymphoblastic leukemia (ALL), 3 patients with biphenotypic acute leukemia, and 1 patient with acute undifferentiated leukemia. Thirty of the 34 patients with ALL had at least one of the following high-risk factors: age greater than 30, white blood cell count at presentation > 25,000/microL, extramedullary disease, certain chromosomal translocations, or the need for greater than 4 weeks of induction chemotherapy to achieve first complete remission. Cumulative probabilities of disease-free survival and relapse at 3 years were 61% and 12%, respectively, for the 61 patients with AML and 64% and 12%, respectively, for the 34 patients with ALL. By stepwise Cox regression analysis, significant prognostic variables for patients with acute myelogenous leukemia were the presence of acute graft-versus-host disease and increasing age, whereas for patients with acute lymphoblastic leukemia, significant variables were age and the development of cytomegalovirus-associated interstitial pneumonia. Complications related to graft-versus-host disease and relapse of leukemia were the major causes of death.

    View details for Web of Science ID A1993ME65700039

  • EFFICIENT GENERATION AND CHARACTERIZATION OF TUMOR-CELL SUBCLONES WITH DIFFERENT ADHESION PATHWAYS INVOLVED IN CELL-LYSIS HAEMATOLOGICA Lefterova, P., Negrin, R., Neubauer, A., Huhn, D., Blume, K., SCHMIDTWOLF, I. 1993; 78 (6): 353-358

    Abstract

    Specific tumor cell recognition is required for optimal tumor directed therapy. Lymphokine activated killer (LAK) cells are able to recognize tumor targets specifically because LAK cells can distinguish between normal and tumor cells. This study was aimed at analyzing receptor molecules on tumor cells and their counter-receptor molecules on LAK cells. Cell lines which differ in the pathway by which LAK cell lysis is mediated are important for an analysis of receptor molecules.We adapted a novel method for efficient production of K562 clones in order to analyze the mechanisms by which target and effector receptor molecules mediate different LAK cell interactions. K562 cells were exposed to ethyl methanesulfonate (EMS) for mutagenization and addition of two rounds of irradiated LAK cells. Prior data demonstrated that irradiation does not effect cytolysis. Surviving cells were plated in methylcellulose and single colonies were obtained after ten days. Cells were washed, resuspended in medium, expanded and tested as targets in a 51Cr-release assay.With this procedure a variety of clones could be generated easily and time-savingly. All twelve clones expressed the bcr/abl transcript, as determined by PCR, and were sensitive to LAK cell lysis. However, cell blockage studies revealed that K562 clones were generated with LAK cell recognition differing from parental K562. Antibody blockage showed that lysis of clone 5 (LEF 5) is partially mediated via the LFA-1/ICAM-1 pathway. ICAM-1 expression of this clone was similar to expression on K562, as determined by flow cytometry.These clones are of great value for studying the receptor molecules involved in LAK--tumor cell interactions.

    View details for Web of Science ID A1993MR46800002

    View details for PubMedID 8175030

  • CYCLOSPORINE, METHOTREXATE, AND PREDNISONE COMPARED WITH CYCLOSPORINE AND PREDNISONE FOR PROPHYLAXIS OF ACUTE GRAFT-VERSUS-HOST DISEASE NEW ENGLAND JOURNAL OF MEDICINE Chao, N. J., Schmidt, G. M., Niland, J. C., Amylon, M. D., Dagis, A. C., Long, G. D., Nademanee, A. P., Negrin, R. S., ODONNELL, M. R., Parker, P. M., Smith, E. P., Snyder, D. S., Stein, A. S., Wong, R. M., Blume, K. G., Forman, S. J. 1993; 329 (17): 1225-1230

    Abstract

    Acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation remains a serious problem. In a clinical trial, we tested the combination of cyclosporine and prednisone with and without methotrexate for the prevention of GVHD.One hundred fifty patients with either acute leukemia in first complete remission, chronic myelogenous leukemia in first chronic phase, or lymphoblastic lymphoma in first complete remission were enrolled in the study. All the patients were given fractionated total-body irradiation (1320 cGy) and etoposide (60 mg per kilogram of body weight) in preparation for transplantation, and received bone marrow from genotypically histocompatible donors. To prevent GVHD, they were randomly assigned to prophylactic treatment with either cyclosporine, methotrexate, and prednisone or cyclosporine and prednisone without methotrexate. All the patients received standardized supportive care after transplantation, including intravenous gamma globulin.Patients receiving cyclosporine, methotrexate, and prednisone had a significantly lower incidence of acute GVHD of grades II to IV (9 percent) than those receiving cyclosporine and prednisone (23 percent, P = 0.02). Multivariate regression analysis demonstrated that an increased risk of acute GVHD was associated with an elevated serum creatinine concentration (P = 0.006) and treatment with cyclosporine and prednisone alone (P = 0.02). The lower incidence of acute GVHD was not associated with a higher rate of relapse of leukemia or lymphoma. There was no significant difference in disease-free survival at three years between the two treatment groups (64 percent with the three-drug regimen vs. 59 percent with the two-drug regimen, P = 0.57).The combination of cyclosporine, methotrexate, and prednisone was more effective in preventing acute GVHD of grades II to IV than was the combination of cyclosporine and prednisone without methotrexate.

    View details for Web of Science ID A1993MB98800003

    View details for PubMedID 8413388

  • THE EFFICACY OF GRANULOCYTE-COLONY-STIMULATING FACTOR FOLLOWING AUTOLOGOUS BONE-MARROW TRANSPLANTATION FOR NON-HODGKINS-LYMPHOMA WITH MONOCLONAL-ANTIBODY PURGED BONE-MARROW LEUKEMIA Schriber, J. R., Negrin, R. S., Chao, N. J., Long, G. D., Horning, S. J., Blume, K. G. 1993; 7 (10): 1491-1495

    Abstract

    Cloned colony-stimulating factors have been shown to accelerate myeloid recovery following autologous bone marrow transplantation. Studies with granulocyte-macrophage colony-stimulating factor (GM-CSF) have demonstrated efficacy in accelerating neutrophil recovery in patients rescued from myeloablative therapy. In our previous study, however, the subset of patients who received monoclonal antibody and complement purged bone marrow grafts followed by GM-CSF recovered neutrophil counts at the same rate as placebo-treated patients. We have now performed a phase II trial to assess whether granulocyte colony-stimulating factor (G-CSF) results in accelerated engraftment in this group of patients. Twenty-three consecutive patients with recurrent non-Hodgkin's lymphoma received G-CSF (10.5 +/- 1.2 micrograms/kg per day) following myeloablative therapy consisting of etoposide (60 mg/kg), cyclophosphamide (100 mg/kg), and either carmustine (15 mg/kg) or fractionated total body irradiation (1200 cGy). All patients received bone marrow grafts which had been purged with a panel of monoclonal antibodies directed against either B or T cell determinants plus complement. Peripheral blood mononuclear cells (PBMC) were not administered to any of the patients in this study. Twenty-one patients engrafted at a median absolute neutrophil count (ANC) greater than 500/microliters at day 12 and ANC greater than 1000/microliters at day 14. The time to myeloid engraftment was significantly shortened compared to our previous experience with either GM-CSF or placebo following identical preparatory regimens (p < 0.01). G-CSF is capable of accelerating myeloid engraftment in patients receiving monoclonal antibody purged bone marrow grafts following myeloablative therapy when compared to historical control groups treated with placebo or GM-CSF.

    View details for Web of Science ID A1993MD20600001

    View details for PubMedID 7692189

  • TREATMENT OF THE ANEMIA OF MYELODYSPLASTIC SYNDROMES USING RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR IN COMBINATION WITH ERYTHROPOIETIN BLOOD Negrin, R. S., Stein, R., Vardiman, J., Doherty, K., Cornwell, J., Krantz, S., Greenberg, P. L. 1993; 82 (3): 737-743

    Abstract

    We treated myelodysplastic syndrome patients (MDS) with both recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human erythropoietin (EPO) to determine whether such combination therapy resulted in improvement of their anemias. Twenty-four of 28 patients begun on study completed the protocol and were evaluable for erythroid responses. Therapy was initiated with G-CSF at 1 micrograms/kg administered by daily subcutaneous injection and adjusted to either normalize or double the neutrophil count. EPO was then administered by daily subcutaneous injection at a dose of 100 U/kg and dose-escalated to 150 and 300 U/kg every 4 weeks while continuing the G-CSF. Changes in absolute reticulocyte count, hematocrit level, and need for RBC transfusions were compared with pretreatment values as well as other blood cell counts. Ten of 24 patients (42%) had erythroid responses, whereas all patients had neutrophil responses. Six previously transfused patients no longer required RBC transfusions during the treatment period. Erythroid responses were found to be independent of patient age, French-American-British subtype, duration of disease, prior RBC transfusion requirements, or cytogenetic abnormalities at presentation. Pretreatment serum EPO levels were lower in erythroid-responding as compared with nonresponding patients (median 157 v 600 U/L; P = .05). The combined treatment modality was generally well tolerated. We conclude that a substantial percentage of MDS patients had both erythroid and myeloid responses when treated with the combination of G-CSF and EPO.

    View details for Web of Science ID A1993LQ74100008

    View details for PubMedID 7687889

  • USE AND TOXICITY OF THE COLONY-STIMULATING FACTORS DRUG SAFETY Schriber, J. R., Negrin, R. S. 1993; 8 (6): 457-468

    Abstract

    The colony-stimulating factors (CSFs) have emerged as effective drugs in a variety of clinical situations. These drugs stimulate the production and activity of haematopoietic cells in vitro and in vivo. Two members of this group, granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF), have been approved in the US and Europe for use following cytotoxic chemotherapy and autologous bone marrow transplantation. Other uses of the CSFs include myelodysplastic syndromes, aplastic anaemia, the acquired immunodeficiency syndrome (AIDS) and cyclic and congenital neutropenias. Although CSFs have generally been well tolerated in clinical use there are a number of theoretical concerns, including disease acceleration, biased stem cell commitment and bone marrow exhaustion. New CSFs are currently under development. Combinations of growth factors in the future may maximise effectiveness while minimising toxicity.

    View details for Web of Science ID A1993LF04000006

    View details for PubMedID 7687134

  • GRANULOCYTE COLONY-STIMULATING FACTOR MOBILIZED PERIPHERAL-BLOOD PROGENITOR CELLS ACCELERATE GRANULOCYTE AND PLATELET RECOVERY AFTER HIGH-DOSE CHEMOTHERAPY BLOOD Chao, N. J., Schriber, J. R., Grimes, K., Long, G. D., Negrin, R. S., RAIMONDI, C. M., Horning, S. J., Brown, S. L., Miller, L., Blume, K. G. 1993; 81 (8): 2031-2035

    Abstract

    Hematopoietic growth factors have been used to accelerate engraftment after bone marrow transplantation and to "mobilize" peripheral blood progenitor cells (PBPC). We report on the data in 85 consecutive patients with Hodgkin's disease who were treated in a single institution using different methods to obtain PB progenitor cells. Use of granulocyte colony-stimulating factor for mobilization resulted in a significantly accelerated time to recovery of granulocytes (10 days v 12 days, P < .01) when compared with "nonmobilized" PBPC recipients. Similarly, use of mobilized PBPC resulted in a significantly accelerated time to platelet engraftment (13 days v 30 days, P < .001) when compared with "nonmobilized" recipients. Moreover, there was a statistically significant difference in total costs in favor of the group receiving "mobilized" PBPC.

    View details for Web of Science ID A1993KY00500008

    View details for PubMedID 7682454

  • BUSULFAN ETOPOSIDE - INITIAL EXPERIENCE WITH A NEW PREPARATORY REGIMEN FOR AUTOLOGOUS BONE-MARROW TRANSPLANTATION IN PATIENTS WITH ACUTE NONLYMPHOBLASTIC LEUKEMIA BLOOD Chao, N. J., Stein, A. S., Long, G. D., Negrin, R. S., Amylon, M. D., Wong, R. M., Forman, S. J., Blume, K. G. 1993; 81 (2): 319-323

    Abstract

    Current intensive chemotherapy for acute nonlymphoblastic leukemia (ANLL) results in a complete remission in the majority of patients. Unfortunately, the duration of remission is short and most of the patients will experience a relapse of their underlying disease. Autologous bone marrow (BM) transplantation is being explored as a treatment modality designed to improve relapse-free survival. We have conducted a phase II trial exploring the combination of busulfan (16 mg/kg) and etoposide (60 mg/kg) in an attempt to improve antitumor efficacy using this novel preparative regimen. To date, 50 patients (48 with ANLL and 2 patients with biphenotypic acute leukemia) have been treated. The first 20 patients received unmanipulated BM; 28 patients subsequently received 4-hydroperoxycyclophosphamide (4-HC) (60 micrograms/mL)-purged bone marrow, and 2 patients with biphenotypic acute leukemia received both 4-HC (60 micrograms/mL) and etoposide (5 micrograms/mL)-purged BM. Thirty-four patients were in first complete remission (CR1), 12 patients in second complete remission (CR2), and 4 patients in relapse. The median time from first complete remission to BM harvest was 3 months (range, 0.8 to 4) compared with median time of 2 months (range, 1.5 to 5.0) for patients in second complete remission. The median time from harvest to transplant was 1 month for both groups (range, 0.4 to 36). A median of 0.7 x 10(8) (range, 0.2 to 1.4) mononuclear cells were infused. Patients achieved an absolute neutrophil count of > or = 500/microL at a median of 26 days (range, 13 to 96), an untransfused platelet count > or = 20,000/microL at a median of 56 days (range, 15 to 278) and a sustained hematocrit > or = 30% at a median of 50 days (range, 19 to 116). Twenty-six patients are alive and in continued CR. Follow-up of the surviving patients ranged from 6 months to 66 months with a median follow-up of 31 months. Patients receiving purged BM have an actuarial disease-free survival of 57% with a relapse rate of 28% compared with patients receiving unpurged BM whose actuarial disease-free survival is 32% with a relapse rate of 62% (P = .06 for relapse rate). The most significant extramedullary toxicities for this regimen are hepatic and cutaneous (including mucositis). The BU/VP-16 regimen is associated with a significant proportion of patients surviving disease free, especially in the group receiving purged BM. Whether this regimen offers a substantial improvement in disease-free survival over currently used regimens will require a prospective randomized study.

    View details for Web of Science ID A1993KH51200006

    View details for PubMedID 8422458

  • THE USE OF COLONY STIMULATING FACTORS IN CLINICAL BONE-MARROW TRANSPLANTATION CURRENT OPINION IN IMMUNOLOGY Negrin, R. S., Greenberg, P. L. 1992; 4 (5): 567-570

    Abstract

    The use of colony stimulating factors in clinical bone marrow transplantation is rapidly evolving. Both granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor have shown efficacy in bone marrow transplant patients in accelerating the rate of myeloid recovery. The use of colony stimulating factor primed peripheral blood progenitor cells appears to accelerate platelet recovery as well as myeloid recovery.

    View details for Web of Science ID A1992KN23800005

    View details for PubMedID 1418720

  • DYNAMIC ASSESSMENT OF QUALITY-OF-LIFE AFTER AUTOLOGOUS BONE-MARROW TRANSPLANTATION BLOOD Chao, N. J., Tierney, D. K., Bloom, J. R., Long, G. D., Barr, T. A., STALLBAUM, B. A., Wong, R. M., Negrin, R. S., Horning, S. J., Blume, K. G. 1992; 80 (3): 825-830

    Abstract

    To determine the quality of life in adult patients after autologous bone marrow transplantation (BMT), we administered a questionnaire to a cohort of patients seen at a single referral-based center. The sample included adults 18 years and older during the 1 year following an autologous BMT. Both disease-free patients and those who relapsed with 1-year of follow-up data available were included. Of 59 eligible patients, 58 (98%) responded to the questionnaire. Patients completed a telephone questionnaire administered by a nurse specialist in the field of BMT approximately every 90 days. At the time of initial contact on day +90, the mean quality of life was 7.8 (range, 1 to 10) on a scale of 1 to 10, with 10 being the best. By the end of the first year of follow-up, the mean quality of life was 8.9 (range, 3 to 10). Seventy-eight percent of the patients were employed. Twenty-one percent lost weight during the first year, with the majority reporting voluntary weight loss. Fourteen percent reported difficulties with sexual activity. Only 5% reported difficulty with sleeping or with frequent colds. One patient felt that her appearance was worse, and none of the patients reported a poor appetite. Eighty-eight percent of surviving adult patients reported an above-average to excellent quality of life 1 year following autologous BMT. This outcome is encouraging and suggests that this procedure is not associated with long-term morbidity in the surviving adult patient.

    View details for Web of Science ID A1992JF85500033

    View details for PubMedID 1638031

  • G-CSF AND PERIPHERAL-BLOOD PROGENITOR CELLS LANCET Chao, N. J., Long, G. D., Negrin, R. S., Schriber, J. R., RAIMONDI, C. M., Brown, S. L., Blume, K. G. 1992; 339 (8806): 1410-1410

    View details for Web of Science ID A1992HX63600025

    View details for PubMedID 1375969

  • Use of the polymerase chain reaction for the detection of tumor cell involvement of bone marrow and peripheral blood: implications for purging. Journal of hematotherapy Negrin, R. S. 1992; 1 (4): 361-368

    Abstract

    Bone marrow purging is being performed increasingly in an effort to deplete residual tumor cells from the graft prior to reinfusion. Several studies have suggested that the removal of tumor cells is an important clinical goal. In this review the utility of the polymerase chain reaction (PCR) for the detection of small numbers of tumor cells in bone marrow and peripheral blood is discussed. Using sensitive assays such as PCR, it is expected that the efficacy of bone marrow purging strategies will be improved and this will hopefully result in decreased relapse rates following autologous bone marrow transplantation.

    View details for PubMedID 1345678

  • INVITRO AND INVIVO ACTIVITY OF MURINE LYMPHOKINE-ACTIVATED KILLER-CELLS AFTER CRYOPRESERVATION TRANSFUSION SCHMIDTWOLF, I. G., Aihara, M., Negrin, R. S., Blume, K. G., Chao, N. J. 1992; 32 (1): 42-45

    Abstract

    The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B-cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.

    View details for Web of Science ID A1992HA66000010

    View details for PubMedID 1731434

  • Therapy of hematopoietic disorders with recombinant colony-stimulating factors. Advances in pharmacology (San Diego, Calif.) Negrin, R. S., Greenberg, P. L. 1992; 23: 263-296

    View details for PubMedID 1540537

  • USE OF A SCID MOUSE HUMAN LYMPHOMA MODEL TO EVALUATE CYTOKINE-INDUCED KILLER-CELLS WITH POTENT ANTITUMOR CELL-ACTIVITY JOURNAL OF EXPERIMENTAL MEDICINE SCHMIDTWOLF, I. G., Negrin, R. S., Kiem, H. P., Blume, K. G., Weissman, I. L. 1991; 174 (1): 139-149

    Abstract

    C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.

    View details for Web of Science ID A1991FU89700018

    View details for PubMedID 1711560

  • USE OF THE POLYMERASE CHAIN-REACTION TO MONITOR THE EFFECTIVENESS OF EX VIVO TUMOR-CELL PURGING BLOOD Negrin, R. S., Kiem, H. P., SCHMIDTWOLF, I. G., Blume, K. G., Cleary, M. L. 1991; 77 (3): 654-660

    Abstract

    The polymerase chain reaction (PCR) was used to detect residual malignant disease before and after ex vivo purging with monoclonal antibodies and complement or immunomagnetic treatment of BM samples contaminated with known numbers of t(14;18)-carrying tumor cells. Sensitivity of the PCR was demonstrated by detecting a specific t(14;18) amplification product in DNA extracted from a preparation consisting of one tumor cell among 10(5) normal cells. When BM contaminated with 1% to 5% t(14;18)-carrying cells from the B-cell lymphoma line SU-DHL-4 was subjected to two rounds of anti-B-cell pool of antibodies and complement (Ab-C) treatment a 3- to 4-log reduction of the pretreatment PCR signal was observed. A similar log-cell kill was detected using an independent clonogenic assay confirming the utility of the PCR approach. BM contaminated with a second B-cell lymphoma cell line, OCI-Ly8, was more resistant because a third cycle of Ab-C treatment was required to obtain a similar reduction in the PCR signal. A similar 4 logs of tumor cell removal was obtained using anti-B-cell antibodies conjugated to magnetic beads. These studies demonstrate that the t(14;18) PCR can be used to detect levels of tumor cells as low as 0.001%. This approach can be used to determine the effectiveness of BM purging in patients undergoing autologous BM transplantation as well as to assess the biologic role of minimal marrow disease.

    View details for Web of Science ID A1991EV33200031

    View details for PubMedID 1991175

  • THE STANFORD EXPERIENCE WITH HIGH-DOSE ETOPOSIDE CYTOREDUCTIVE REGIMENS AND AUTOLOGOUS BONE-MARROW TRANSPLANTATION IN HODGKINS-DISEASE AND NON-HODGKINS-LYMPHOMA - PRELIMINARY DATA ANNALS OF ONCOLOGY Horning, S. J., Chao, N. J., Negrin, R. S., Hoppe, R. T., Kwak, L. W., Long, G. D., STALLBAUM, B., OCONNOR, P., Blume, K. G. 1991; 2: 47-50

    Abstract

    Seventy-seven Hodgkin's disease and non-Hodgkin's lymphoma (NHL) patients received high-dose etoposide in combination with cyclophosphamide and either fractionated total body irradiation (TBI) (n = 28) or carmustine (n = 49) prior to autologous bone marrow transplantation. Marrow from NHL patients was purged in vitro with a panel of monoclonal B- and T-cell antibodies and complement. Six toxic deaths (8%) occurred, all in patients who received carmustine. With a median follow-up of 1 year, 57 patients are alive and free from progressive disease. The 1-year actuarial survival and freedom from progression are 85 and 73% in fractionated TBI/etoposide/cyclophosphamide-treated patients and 79 and 72% in carmustine/etoposide/cyclophosphamide-treated patients. Forty-five of these patients participated in prospective trials for which eligibility criteria were (1) less than 25% curability with conventional therapy; (2) achievement of minimal disease state with conventional therapy; and (3) transplantation early in the course of disease. One-year actuarial survival for 18 patients with relapsed Hodgkin's disease is 80% and for 21 relapsed intermediate and high-grade NHL patients, 70%. One NHL Burkitt's patient was transplanted on a protocol for high-risk intermediate and high-grade NHL in first remission. Five patients with follicular mixed or small cleaved NHL were also transplanted in first remission.

    View details for Web of Science ID A1991EV30000010

    View details for PubMedID 2043498

  • IMPACT OF MARROW CYTOGENETICS AND MORPHOLOGY ON INVITRO HEMATOPOIESIS IN THE MYELODYSPLASTIC SYNDROMES - COMPARISON BETWEEN RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR (CSF) AND GRANULOCYTE-MONOCYTE CSF BLOOD Nagler, A., Binet, C., MacKichan, M. L., Negrin, R., Bangs, C., Donlon, T., Greenberg, P. 1990; 76 (7): 1299-1307

    Abstract

    Marrow cells from 36 patients with myelodysplastic syndromes (MDS) (13 refractory anemia [RA], 14 refractory anemia with excess of blasts [RAEB], 9 RAEB in transformation [RAEB-T]) were evaluated for their in vitro proliferative and differentiative responsiveness to recombinant human granulocyte colony-stimulating factor (G-CSF) or granulocyte-monocyte CSF (GM-CSF). GM-CSF exerted a stronger proliferative stimulus than G-CSF for marrow myeloid clonal growth (CFU-GM) in these patients (44 v 12 colonies per 10(5) nonadherent buoyant bone marrow cells [NAB], respectively, P less than .025). GM-CSF stimulated increased CFU-GM growth in the 16 patients with abnormal marrow cytogenetics in comparison with the 20 patients who had normal cytogenetics (52 and 30 colonies per 10(5) NAB, respectively, P less than .05), whereas no such difference could be demonstrated with G-CSF (11 and 16 colonies per 10(5) NAB, respectively). In contrast, granulocytic differentiation of marrow cells was induced in liquid culture by G-CSF in 15 of 32 (47% patients), while GM-CSF did so in only 4 of 18 (22%) patients (P less than .025) including, for RAEB/RAEB-T patients: 9 of 18 versus 0 of 9, respectively (P less than .025). For MDS patients with normal cytogenetics, G-CSF- and GM-CSF-induced marrow cell granulocytic differentiation in 12 of 18 (67%) versus 3 of 11 (27%), respectively (P less than .025), contrasted with granulocytic induction in only 3 of 14 (21%) and 1 of 7 (14%) patients with abnormal cytogenetics, respectively. We conclude that G-CSF has greater granulocytic differentiative and less proliferative activity for MDS marrow cells than GM-CSF in vitro, particularly for RAEB/RAEB-T patients and those with normal cytogenetics.

    View details for Web of Science ID A1990EB07800006

    View details for PubMedID 1698477

  • MAINTENANCE TREATMENT OF PATIENTS WITH MYELODYSPLASTIC SYNDROMES USING RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR BLOOD Negrin, R. S., HAEUBER, D. H., Nagler, A., Kobayashi, Y., Sklar, J., Donlon, T., Vincent, M., Greenberg, P. L. 1990; 76 (1): 36-43

    Abstract

    Myelodysplastic syndromes (MDS) are characterized by chronic refractory cytopenias resulting in increased risk of infection, bleeding, and conversion to acute leukemia. In an effort to improve these cytopenias we have treated 18 patients over a 6- to 8-week period with increasing daily subcutaneous doses of recombinant human granulocyte colony-stimulating factor (G-CSF). Sixteen patients responded with improvement in neutrophil counts. On cessation of treatment these counts returned to baseline values over a 2- to 4-week period. To maintain these improved blood counts 11 patients were treated with G-CSF for more prolonged periods. Ten patients again responded with an increase in total leukocyte counts (1.6- to 6.4-fold) and absolute neutrophil counts (ANC) (3.6- to 16.3-fold), with responses persisting for 3 to 16 months. A significantly decreased risk of developing bacterial infections was noted during periods with ANC greater than 1,500/mm3 as compared with periods of time with ANC less than 1,500/mm3. Two anemic patients had a greater than 20% rise in hematocrit over the study period, and 2 additional patients had a decrease in red blood cell transfusion requirements during G-CSF treatment. Bone marrow myeloid maturation improved in 7 of 9 maintenance phase patients. Three patients progressed to acute myeloid leukemia during treatment. The drug was generally well-tolerated and no severe toxicities were noted. These data demonstrated that G-CSF administered to MDS patients by daily subcutaneous administration was well-tolerated and effective in causing persistent improvement of the neutrophil levels and marrow myeloid maturation. These effects were associated with a decreased risk of infection and, in some patients, with decreased red blood cell transfusion requirements.

    View details for Web of Science ID A1990DM15500005

    View details for PubMedID 1694702

  • EFFECTS OF RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR AND GRANULOCYTE-MONOCYTE COLONY STIMULATING FACTOR ON INVITRO HEMATOPOIESIS IN THE MYELODYSPLASTIC SYNDROMES LEUKEMIA Nagler, A., Ginzton, N., Negrin, R., Bang, D., Donlon, T., Greenberg, P. 1990; 4 (3): 193-202

    Abstract

    We evaluated the effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) and granulocyte-monocyte colony stimulating factor (rhGM-CSF) on the in vitro proliferative, differentiative, and regenerative responsiveness of marrow cells from myelodysplastic syndrome patients (MDS) in comparison to those from normal individuals. Our studies showed decreased primary clonogenicity of myeloid (CFU-GM) and erythroid (BFU-E) hemopoietic progenitor cells from the MDS patients. rhGM-CSF had more potent stimulatory effects than rhG-CSF for MDS marrow CFU-GM growth; no enhanced cellular proliferation in the MDS patients was observed in liquid culture with either rhGM-CSF or rhG-CSF. Decreased myeloid clonal cell self-generation and/or recruitment occurred in the MDS patients upon exposure to either rhG-CSF or rhGM-CSF. rhG-CSF demonstrated more potent granulocytic differentiation effects than rhGM-CSF both for normals and MDS patients using marrow enriched for immature myeloid cells with lesser differentiation noted for MDS. Cytogenetic abnormalities, present with or without additional normal karyotypes in native marrow of four MDS patients, persisted after culture with rhG-CSF, indicating induced differentiation of both normal and abnormal clones. Although proliferative and differentiative effects were seen with both factors these data show MDS marrow cells in vitro to have predominantly differentiative responsiveness to rhG-CSF and proliferative responsiveness to rhGM-CSF.

    View details for Web of Science ID A1990DB45100007

    View details for PubMedID 1690318

  • Bone marrow transplantation for hematologic malignancies: the Stanford experience. Clinical transplants Chao, N. J., Amylon, M. D., Long, G. D., Negrin, R. S., Hoppe, R. T., Horning, S. J., Blume, K. G. 1990: 157-163

    Abstract

    Allogeneic and autologous BMTs are highly effective and successful treatment modalities for selected patients. Use of BMT earlier in the course of disease yields better results when compared to patients with more advanced disease. Recent advances such as use of cloned growth factors, cytokines, etc..., will continue to contribute to lessen morbidity and mortality. Finally, as investigators understand, prevent, and treat expected side effects from BMTs, the patients' burden in terms of physical, psychological, and financial costs should lessen substantially.

    View details for PubMedID 2103141

  • THE USE OF HEMATOPOIETIC GROWTH-FACTORS IN THE TREATMENT OF MYELODYSPLASTIC SYNDROMES CANCER SURVEYS Greenberg, P. L., Negrin, R., Nagler, A. 1990; 9 (1): 199-212

    Abstract

    Based on the results of preclinical and in vitro studies demonstrating enhanced granulocytic proliferation and differentiation induced by granulocyte-monocyte and granulocyte-colony stimulating factors (GM-CSF and G-CSF), these recombinant human haemopoietic growth factors have been used to treat cytopenic patients with myelodysplastic syndromes (MDS). Laboratory investigations have shown responsiveness of enriched haemopoietic precursors in vitro to the proliferative and granulocytic differentiative stimuli of G-CSF, generally without increased clonal regeneration. To date, five short-term phase I/II clinical trials using GM-CSF have demonstrated that 38 of 45 treated patients had improvements in neutrophil counts, 14 had increased reticulocyte counts, with three of these patients having decreased red blood cell transfusion requirements, and eight had a transient increase in platelets. In 12 patients an increase in marrow and/or peripheral blood blasts was noted. Seven patients progressed to acute myeloid leukaemia (AML), particularly patients with greater than 15% marrow blasts. In a longer term study, five patients received GM-CSF for two to nine weeks, although only one maintained increased neutrophil counts, one developed antibodies to GM-CSF and one's condition evolved into AML. Eighteen patients have been treated for two months in phase I/II clinical trials with G-CSF, 16 of whom had normalization of neutrophil counts with improved marrow maturation, five had increased reticulocyte counts with three having decreased transfusion requirements, four had transient increases in blasts and no substantial changes in platelet counts were noted. Eleven patients have received maintenance therapy with G-CSF for 6-16 months and 10 had persistent increases in neutrophil counts with enhanced marrow myeloid maturation. Decreased infectious episodes were noted in these patients at times at neutrophil improvements. Four of the 18 patients have subsequently developed AML after 6-16 months. Both CSFs were well tolerated, although the incidence of fever, myalgias and bone pain was more prominent in patients receiving GM-CSF at higher doses. In vitro correlates with these in vivo results were demonstrated as laboratory studies showed that G-CSF had greater myeloid differentiative and less proliferative effects for MDS marrow than did GM-CSF. Marrow cytogenetic studies after treatment generally indicated persistence of the initial normal and/or abnormal clones. These studies have demonstrated that both G-CSF and GM-CSF improve neutrophil counts in a high proportion of patients with MDS and that chronic administration of G-CSF elicits persistent neutrophil responses and may decrease infections. Phase III controlled trials are required to determine whether the natural history of this disorder will be altered by use of colony stimulating factors.

    View details for Web of Science ID A1990EK32300008

    View details for PubMedID 2276114

  • LYMPHOKINE-ACTIVATED KILLER CELL-ACTIVITY AFTER CRYOPRESERVATION JOURNAL OF IMMUNOLOGICAL METHODS SCHMIDTWOLF, I. G., Aihara, M., Negrin, R. S., Blume, K. G., Chao, N. J. 1989; 125 (1-2): 185-189

    Abstract

    The effect of cryopreservation on the cytotoxic activity of lymphokine-activated killer (LAK) cells was studied. LAK cells were generated by incubating peripheral blood lymphocytes for 3-5 days with recombinant interleukin-2 (rIL-2) and then cryopreserved using a programmed freezer. Cytotoxicity was determined in a 51Cr release assay. After thawing, the LAK cells had reduced cytotoxicity (25.5-39.1% as compared to the original lytic units). Cytotoxic activity could be restored to pre-cryopreserved levels by reincubation with rIL-2 for 2 days after thawing. Thus, maximal cytotoxicity of cryopreserved LAK cells could be achieved by incubation with rIL-2 before and after the freezing process. The level of cytotoxicity was comparable to that of LAK cells from fresh peripheral blood lymphocytes. Cryopreserved LAK cells may have potential in adoptive immunotherapy.

    View details for Web of Science ID A1989CF02900023

    View details for PubMedID 2607152

  • TREATMENT OF MYELODYSPLASTIC SYNDROMES WITH RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR - A PHASE I-II TRIAL ANNALS OF INTERNAL MEDICINE Negrin, R. S., HAEUBER, D. H., Nagler, A., Olds, L. C., Donlon, T., SOUZA, L. M., Greenberg, P. L. 1989; 110 (12): 976-984

    Abstract

    To determine the hematopoietic effects and toxicity of recombinant human granulocyte colony-stimulating factor (G-CSF) in patients with myelodysplastic syndromes.The G-CSF was administered by daily subcutaneous injection to outpatients in a phase I-II trial. Dose was escalated every 2 weeks between 0.1 to 3.0 micrograms/kg body weight.d over an 8-week treatment period.Outpatient clinical research center at a university hospital.Twelve consecutive patients with myelodysplastic syndromes: two refractory anemia, seven refractory anemia with excess of blasts, three refractory anemia with excess of blasts in transformation.In 10 of 12 patients, elevations in blood leukocyte counts (2- to 10-fold) and absolute neutrophil counts (5- to 40-fold) were seen over the 8-week treatment period. Five of seven severely neutropenic patients (absolute neutrophil count, less than 0.5 x 10(9)/L) had a rise in count to 1.2 to 16.3 x 10(9)/L. Increased reticulocyte counts occurred in 5 patients, and were associated with decreased transfusion requirements in 2 of 9 erythrocyte transfusion-dependent patients. Treatment with G-CSF enhanced marrow myeloid cell maturation in 9 of 11 evaluable patients. Neutrophil chemotaxis and phagocytosis in vitro were improved or unchanged after treatment in 6 of 8 patients tested. In 11 of 12 patients, there were no substantial changes in platelet, lymphocyte, eosinophil, or monocyte counts. Three responding patients initially had abnormal cytogenetics that persisted after G-CSF therapy, suggesting induced differentiation of the abnormal clone. The therapy was associated with minimal toxicity. None of the patients' conditions converted to acute leukemia during treatment or in short-term follow-up.Treatment with G-CSF administered by subcutaneous injection is well tolerated and effective for improving the neutropenia, and less commonly the transfusion-dependent anemia, over 6 to 8 weeks in patients with myelodysplastic syndromes.

    View details for Web of Science ID A1989AB29600006

    View details for PubMedID 2471429

  • NASAL CONTINUOUS POSITIVE AIRWAY PRESSURE IN ATELECTASIS CHEST Duncan, S. R., Negrin, R. S., Mihm, F. G., Guilleminault, C., Raffin, T. A. 1987; 92 (4): 621-624

    Abstract

    Nasal continuous positive airway pressure (CPAP) has been widely and safely used in the treatment of sleep disorders but has not been previously utilized for therapy of pulmonary atelectasis in adults. We observed three patients with significant atelectasis which was refractory to conventional chest physiotherapy. Bronchoscopy was not a viable therapeutic option in any patient. Therapy with continuous nasal CPAP was initiated at 10 to 15 cm H2O. The patients tolerated the therapy well and had prompt resolution of atelectasis. Nasal CPAP may be an effective modality for therapy of pulmonary atelectasis in spontaneously breathing patients, particularly when conventional therapies are not tolerated or are ineffectual.

    View details for Web of Science ID A1987K327500012

    View details for PubMedID 3308344

Conference Proceedings


  • CD11B+CD11C+DENDRITIC CELLS INTERACT WITH ALLOREACTIVE T CELLS IN THE INTESTINAL MUCOSA IN ACUTE GRAFT-VERSUS-HOST DISEASE Garrote, A. L., Brede, C., Baeuerlein, C., Riedel, S., Kiesel, C., Grether, M., Mattenheimer, K., Ritz, M., Baker, J., Negrin, R., Einsele, H., Beilhack, A., Schulz, S. FERRATA STORTI FOUNDATION. 2010: 480-480
  • DIFFERENCES IN ALLOREACTIVE T CELL MIGRATION IN MHC MATCHED VERSUS MHC MISMATCHED HCT ARE CAUSED BY WAVES OF T CELL EXPANSION Baeuerlein, C., Riedel, S., Brede, C., Garrote, A. L., Kiesel, C., Ritz, M., Schulz, S., Grether, M., Beilhack, G., Negrin, R., Einsele, H., Beilhack, A. FERRATA STORTI FOUNDATION. 2010: 479-479
  • Simultaneous blockade of several endothelial ligands is required to prevent alloreactive T-cell trafficking to target tissues Beilhack, A., Baker, J., Riedel, S., Brede, C., Baeuerlein, C. A., Jordan-Garrote, A. L., Kiesel, C., Monteforte, R., Ritz, M., Mattenheimer, K., Grether, M., Beilhack, G. F., Einsele, H., Negrin, R. S., Schulz, S. NATURE PUBLISHING GROUP. 2010: S15-S15
  • Influence of age on the outcome of 500 autologous bone marrow transplant procedures for hematologic malignancies KUSNIERZGLAZ, C. R., Schlegel, P. G., Wong, R. M., Schriber, J. R., Chao, N. J., Amylon, M. D., Hu, W. W., Negrin, R. S., Lee, Y. S., Blume, K. G., Long, G. D. AMER SOC CLINICAL ONCOLOGY. 1997: 18-25

    Abstract

    To determine the effect of age on the outcome of autologous bone marrow transplantation (ABMT) and/or peripheral-blood progenitor-cell (PBPC) transplantation.A retrospective analysis was performed on 500 consecutive patients who ranged in age from 1 to 65 years (median, 40) with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), multiple myeloma (MM), or acute nonlymphoblastic leukemia (AML) who underwent autologous hematopoietic-cell transplant procedures at Stanford University Medical Center.The actuarial 5-year event-free survival (EFS) rate was 44%, the relapse rate 47%, and the regimen-related mortality (RRM) rate 8.6%. Disease status at time of transplantation, categorized as either minimal or advanced disease, was the strongest predictive factor for EFS (relative risk (RR) for advanced-disease group, 1.8; P < .0003) and relapse rate (RR for advanced-disease group, 1.9; P < .0004). Patients with minimal or advanced disease had an EFS rate of 48% and 30% and relapse rates of 43% and 72%, respectively. The EFS rate of patients less than 50 years verus > or = 50 years of age was 46% versus 34% (P = .03). Cox regression analysis showed that age was predictive for EFS (RR for patients 50 to 65 years, 1.4; P = .03). The actuarial RRM rate for these age groups was 7.4% versus 12.7% (P = .07), respectively. Multivariate analysis demonstrated that age (odds ratio [OR] for patients 50 to 65 years, 1.9; P < .05) and period of transplantation (OR for most recent years [1991 to 1995], 0.6; P = .06) were the most predictive factors for RRM.Although age greater than 50 years is associated with an inferior outcome following autologous hematopoietic-cell transplantation, it does not appear to be warranted to limit this potentially curative procedure based solely on age. The upper age limit of high-dose therapy with autologous progenitor-cell and/ or bone marrow support remains to be defined.

    View details for Web of Science ID A1997WB90700005

    View details for PubMedID 8996120

  • Enrichment of allogeneic CD34+ cells and T cell depletion by percoll density gradient centrifugation Negrin, R. S., KUSNIERZGLAZ, C., Blume, K. G., Strober, S. STOCKTON PRESS. 1996: S31-S33

    View details for Web of Science ID A1996UG02500008

    View details for PubMedID 8722331

  • ROLE OF ETOPOSIDE (VP-16) IN PREPARATORY REGIMENS FOR PATIENTS WITH LEUKEMIA OR LYMPHOMA UNDERGOING ALLOGENEIC BONE-MARROW TRANSPLANTATION Blume, K. G., Long, G. D., Negrin, R. S., Chao, N. J., KUSNIERZGLAZ, C., Amylon, M. D. STOCKTON PRESS. 1994: S9-S10

    Abstract

    In 1983, we began a series of clinical trials with the goal of reducing the relapse rate following allogeneic BMT for hematologic malignancies. Because of its anti-leukemic activity, the drug VP-16 was chosen and combined with total body irradiation (TBI). The first series (trial I) consisted of patients who had advanced leukemia. This trial showed a relapse rate of 32% and a disease-free survival rate of 43%. Thereafter, this regimen was tested in a randomized trial (trial II) under the auspices of the Southwest Oncology Group (SWOG study 8612). The FTBI/VP-16 regimen was compared with the combination of busulfan and cyclophosphamide (BU/CY). A recent analysis indicates a disease-free advantage for patients prepared with FTBI/VP-16; however this difference is not statistically significant. In another trial (trial III), patients in their first remission of leukemia were prepared with the FTBI/VP-16 regimen and long-term disease-free survival was found to be 60-70% with a relapse rate of approximately 10%. These results compare favorably with data obtained with alternative preparatory regimens. The FTBI/VP-16 regimen is currently being compared to the 'standard' regimen, FTBI/CY, in a prospective trial (trial IV). Since the regimen-related toxicity has been relatively low, we have added one dose of CY 60 mg/kg to the FTBI/VP-16 combination. This regimen (trial V) is currently being tested in patients with advanced leukemia. The preliminary results of this ongoing trial indicate further improvement in disease-free survival through a reduction of the post-transplant relapse rate.

    View details for Web of Science ID A1994QE92500004

    View details for PubMedID 7728133

  • EFFECTS OF CSFS IN PRELEUKEMIA Greenberg, P. L., Negrin, R., Nagler, A. STOCKTON PRESS. 1990: 121-126

    Abstract

    Based on pre-clinical and in vitro studies demonstrating enhanced granulocytic proliferation and differentiation induced by granulocyte-monocyte and granulocyte-colony stimulating factors (GM-CSF and G-CSF), these recombinant human hormones have been used to treat cytopenic patients with preleukemia [i.e., myelodysplastic syndromes (MDS)]. To date, five studies have been reported using GM-CSF short-term (generally 7-14 days, x 1-5 courses). Thirty-eight of 45 treated patients had improvements in neutrophil counts, 14 had increased reticulocyte counts with three of these individuals having decreased RBC transfusion requirements, and eight had transient increases in platelets. In 12 patients an increase in marrow and/or peripheral blood blasts was noted. Seven patients progressed to acute myeloid leukemia (AML), particularly patients with greater than 15% marrow blasts. In a longer term study, five patients received GM-CSF for 2 to 9 weeks, although only one individual maintained increased neutrophil counts, one developed antibodies to GM-CSF and one evolved into AML. Eighteen patients have been treated for 2 months with G-CSF, 16 of whom had normalization of neutrophil counts with improved marrow maturation, five had increased reticulocyte counts with three having decreased transfusion requirements, no substantial changes in platelet counts were noted. Eleven patients have received maintenance therapy with G-CSF for 6-16 months, ten had persistent increases in neutrophil counts with enhanced marrow myeloid maturation and five had increased reticulocytes. Decreased infectious episodes were notedat times of neutrophil improvements. Four of the 18 individuals have subsequently developed AML after 6-16 months.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1990DU04800030

    View details for PubMedID 1697191

  • EFFECTS OF PROLONGED TREATMENT OF MYELODYSPLASTIC SYNDROMES WITH RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR Greenberg, P., Negrin, R., Nagler, A., Vincent, M., Donlon, T. ALPHAMED PRESS. 1990: 293-302

    Abstract

    In vitro marrow hemopoietic cultures were utilized to determine the possible efficacy of recombinant human granulocyte colony-stimulating factor (G-CSF) for treating the refractory cytopenias present in the myelodysplastic syndromes (MDS). Our studies showed responsiveness of enriched hemopoietic precursors in vitro to the proliferative and granulocytic differentiative stimuli of G-CSF, generally without increased clonal self-generation. These in vitro parameters correlated with in vivo hematologic responses in our Phase I and II clinical trials. In this study 18 patients were treated for two months with s.c. administration (0.1-3 micrograms/kg/day) of G-CSF, escalating doses every two weeks. This study indicated normalization of neutrophil courses in 16 patients and reticulocyte responses with decreased red blood cell (RBC) transfusion requirements in three of 12 transfusion-dependent patients. Marrow myeloid maturation improved in the responding patients. Extended treatment for additional six- to 16-month periods has indicated persisting neutrophil responses. The relative risk of developing bacterial infections was significantly decreased in patients whose neutrophil level normalized (absolute neutrophil count greater than 1,500/mm3) during G-CSF therapy, compared to such episodes in their pretreatment neutropenic period. This therapy was well-tolerated, without serious toxicity being noted. In vitro neutrophil function (chemotaxis and phagocytosis) remained normal or improved in six of the eight tested patients. Transformation to acute myelogenous leukemia occurred in two patients with refractory anemia with excess blasts in transformation (RAEB-T) during or within a month of the treatment period. Marrow cytogenetic studies indicate persistence of the initial normal and/or abnormal clones.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1990CT55300028

    View details for PubMedID 1691248

  • EFFECTS OF TREATMENT OF MYELODYSPLASTIC SYNDROMES WITH RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR Greenberg, P., Negrin, R., Nagler, A., Souza, L., Donlon, T. WILEY-LISS, INC. 1990: 151-161

    View details for Web of Science ID A1990BR58Q00012

    View details for PubMedID 1693211

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