David Miklos, M.D., Ph.D.

All Publications

• Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-ß production. Blood Du, J., Paz, K., Flynn, R., Vulic, A., Robinson, T. M., Lineburg, K. E., Alexander, K. A., Meng, J., Roy, S., Panoskaltsis-Mortari, A., Loschi, M., Hill, G. R., Serody, J. S., Maillard, I., Miklos, D., Koreth, J., Cutler, C. S., Antin, J. H., Ritz, J., MacDonald, K. P., Schacker, T. W., Luznik, L., Blazar, B. R. 2017

  Abstract

  Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.

  View details for DOI 10.1182/blood-2017-01-758854

  View details for PubMedID 28254742

  View details for PubMedCentralID PMC5418639

• Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT. Blood Ryan, C. E., Sahaf, B., Logan, A. C., O'Brien, S., Byrd, J. C., Hillmen, P., Brown, J. R., Dyer, M. J., Mato, A. R., Keating, M. J., Jaglowski, S., Clow, F., Rezvani, A. R., Styles, L., Coutre, S. E., Miklos, D. B. 2016

  Abstract

  Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism-associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell-mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre-germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.

  View details for PubMedID 27802969

  View details for PubMedCentralID PMC5179333

• The Biology of Chronic Graft-Versus-Host Disease: a Task Force Report From the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-Versus-Host Disease. Biology of blood and marrow transplantation Cooke, K. R., Luznik, L., Sarantopoulos, S., Hakim, F. T., Jagasia, M., Fowler, D. H., van den Brink, M. R., Hansen, J. A., Parkman, R., Miklos, D. B., Martin, P. J., paczesny, s., Vogelsang, G., Pavletic, S., Ritz, J., Schultz, K. R., Blazar, B. R. 2016

  Abstract

  Chronic graft-versus-host disease (GVHD) is the leading cause of late, nonrelapse mortality and disability in allogeneic hematopoietic cell transplantation recipients and a major obstacle to improving outcomes. The biology of chronic GVHD remains enigmatic, but understanding the underpinnings of the immunologic mechanisms responsible for the initiation and progression of disease is fundamental to developing effective prevention and treatment strategies. The goals of this task force review are as follows: This document is intended as a review of our understanding of chronic GVHD biology and therapies resulting from preclinical studies, and as a platform for developing innovative clinical strategies to prevent and treat chronic GVHD.

  View details for DOI 10.1016/j.bbmt.2016.09.023

  View details for PubMedID 27713092

• High-throughput allogeneic antibody detection using protein microarrays JOURNAL OF IMMUNOLOGICAL METHODS Paul, J., Sahaf, B., Perloff, S., Schoenrock, K., Wu, F., Nakasone, H., Coller, J., Miklos, D. 2016; 432: 57-64

  Abstract

  Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform.

  View details for DOI 10.1016/j.jim.2016.02.009

  View details for Web of Science ID 000375886900008

  View details for PubMedID 26902899

• A Randomized Phase II Crossover Study of Imatinib or Rituximab for Cutaneous Sclerosis after Hematopoietic Cell Transplantation. Clinical cancer research Arai, S., Pidala, J., Pusic, I., Chai, X., Jaglowski, S., Khera, N., Palmer, J., Chen, G. L., Jagasia, M. H., Mayer, S. A., Wood, W. A., Green, M., Hyun, T. S., Inamoto, Y., Storer, B. E., Miklos, D. B., Shulman, H. M., Martin, P. J., Sarantopoulos, S., Lee, S. J., Flowers, M. E. 2016; 22 (2): 319-327

  Abstract

  Cutaneous sclerosis occurs in 20% of patients with chronic graft-versus-host disease (GVHD) and can compromise mobility and quality of life.We conducted a prospective, multicenter, randomized, two-arm phase II crossover trial of imatinib (200 mg daily) or rituximab (375 mg/m(2) i.v. weekly × 4 doses, repeatable after 3 months) for treatment of cutaneous sclerosis diagnosed within 18 months (NCT01309997). The primary endpoint was significant clinical response (SCR) at 6 months, defined as quantitative improvement in skin sclerosis or joint range of motion. Treatment success was defined as SCR at 6 months without crossover, recurrent malignancy or death. Secondary endpoints included changes of B-cell profiles in blood (BAFF levels and cellular subsets), patient-reported outcomes, and histopathology between responders and nonresponders with each therapy.SCR was observed in 9 of 35 [26%; 95% confidence interval (CI); 13%-43%] participants randomized to imatinib and 10 of 37 (27%; 95% CI, 14%-44%) randomized to rituximab. Six (17%; 95% CI, 7%-34%) patients in the imatinib arm and 5 (14%; 95% CI, 5%-29%) in the rituximab arm had treatment success. Higher percentages of activated B cells (CD27(+)) were seen at enrollment in rituximab-treated patients who had treatment success (P = 0.01), but not in imatinib-treated patients.These results support the need for more effective therapies for cutaneous sclerosis and suggest that activated B cells define a subgroup of patients with cutaneous sclerosis who are more likely to respond to rituximab.

  View details for DOI 10.1158/1078-0432.CCR-15-1443

  View details for PubMedID 26378033

• Risks and benefits of sex-mismatched hematopoietic cell transplantation differ according to conditioning strategy. Haematologica Nakasone, H., Remberger, M., Tian, L., Brodin, P., Sahaf, B., Wu, F., Mattsson, J., Lowsky, R., Negrin, R., Miklos, D. B., Meyer, E. 2015; 100 (11): 1477-1485

  Abstract

  Sex-mismatched hematopoietic cell transplantation is linked to increased graft-versus-host disease and mortality in myeloablative conditioning. Here we evaluated outcomes of 1,041 adult transplant recipients at two centers between 2006 and 2013 and investigated how the effect of sex-mismatching differed in myeloablative, reduced-intensity, and non-myeloablative total lymphoid irradiation with anti-thymocyte globulin conditioning. Among patients who underwent myeloablative conditioning, male recipients with female donors had increased chronic graft-versus-host disease (hazard ratio 1.83, P<0.01), increased non-relapse mortality (hazard ratio 1.84, P=0.022) and inferior overall survival (hazard ratio 1.59, P=0.018). In contrast, among patients who received reduced-intensity conditioning, male recipients with female donors had increased acute graft-versus-host disease (hazard ratio 1.96, P<0.01) but no difference in non-relapse mortality or overall survival. Among the patients who underwent total lymphoid irradiation with anti-thymocyte globulin, male recipients with female donors showed no increase in graft-versus-host disease or non-relapse mortality. Notably, only in the cohort receiving total lymphoid irradiation with anti-thymocyte globulin were male recipients with female donors significantly associated with reduced relapse (hazard ratio 0.64, P<0.01), and allo-antibody responses against H-Y antigens were predictive of reduced relapse. In the cohort given total lymphoid irradiation with anti-thymocyte globulin, the graft-versus-leukemia effect resulted in superior overall survival in recipients of sex-mismatched grafts (HR 0.69, P=0.037). In addition, only in the cohort treated with total lymphoid irradiation with anti-thymocyte globulin were female recipients with male donors associated with reduced relapse (hazard ratio 0.59, P<0.01) and superior survival (hazard ratio 0.61, P=0.014) compared with sex-matched pairs. We conclude that the risks and benefits of sex-mismatched transplants appear to differ according to conditioning strategy and this could affect donor selection.

  View details for DOI 10.3324/haematol.2015.125294

  View details for PubMedID 26250581

• Allogeneic hematopoietic cell transplantation after failed autologous transplant for lymphoma using TLI and anti-thymocyte globulin conditioning BONE MARROW TRANSPLANTATION Rezvani, A. R., Kanate, A. S., Efron, B., Chhabra, S., Kohrt, H. E., Shizuru, J. A., Laport, G. G., Miklos, D. B., Benjamin, J. E., JOHNSTON, L. J., Arai, S., Weng, W., Negrin, R. S., Strober, S., Lowsky, R. 2015; 50 (10): 1286-1292

  Abstract

  We describe 47 patients with lymphoma and failed prior autologous hematopoietic cell transplantation (HCT) who received TLI-ATG (anti-thymocyte globulin) conditioning followed by allogeneic HCT. Thirty-two patients had non-Hodgkin lymphoma (NHL; diffuse large B-cell lymphoma (n=19), T-cell NHL (n=6), mantle cell lymphoma (n=4) or other B-cell subtypes (n=3)), and 15 had Hodgkin lymphoma. The median follow-up was 4.9 (range, 2.1-11.9) years. The cumulative incidence of grade II-IV acute GvHD at day +100 was 12%, and the cumulative incidence of extensive chronic GvHD at 1 year was 36%. The 3-year cumulative incidences of overall survival (OS), PFS and non-relapse mortality (NRM) were 81%, 44% and 7%, respectively. Fifteen patients died (relapse, n=10; NRM, n=5). Among the 25 patients with relapse after allogeneic HCT, 11 (44%) achieved durable (>1 year) CRs following donor lymphocyte infusion or chemoradiotherapy. The majority of surviving patients (75%; n=24) were able to discontinue all immunosuppression. For patients with relapsed lymphoma after autologous HCT, allogeneic HCT using TLI-ATG conditioning is a well-tolerated, predominantly outpatient therapy with low NRM (7% at 3 years), a low incidence of GvHD, durable disease control and excellent OS (81% at 3 years).

  View details for DOI 10.1038/bmt.2015.149

  View details for Web of Science ID 000362497400004

  View details for PubMedID 26146806

  View details for PubMedCentralID PMC4699844

• Red blood cell transfusions are associated with HLA class I but not H-Y alloantibodies in children with sickle cell disease. British journal of haematology Nickel, R. S., Hendrickson, J. E., Yee, M. M., Bray, R. A., Gebel, H. M., Kean, L. S., Miklos, D. B., Horan, J. T. 2015; 170 (2): 247-256

  Abstract

  Blood transfusions can induce alloantibodies to antigens on red blood cells (RBCs), white blood cells and platelets, with these alloantibodies affecting transfusion and transplantation. While transfusion-related alloimmunization against RBC antigens and human leucocyte antigens (HLA) have been studied, transfusion-related alloimmunization to minor histocompatibility antigens (mHA), such as H-Y antigens, has not been clinically characterized. We conducted a cross-sectional study of 114 children with sickle cell disease (SCD) and tested for antibodies to 5 H-Y antigens and to HLA class I and class II. Few patients had H-Y antibodies, with no significant differences in the prevalence of any H-Y antibody observed among transfused females (7%), transfused males (6%) and never transfused females (4%). In contrast, HLA class I, but not HLA class II, antibodies were more prevalent among transfused than never transfused patients (class I: 33% vs. 13%, P = 0·046; class II: 7% vs. 8%, P = 0·67). Among transfused patients, RBC alloantibody history but not amount of transfusion exposure was associated with a high (>25%) HLA class I panel reactive antibody (Odds ratio 6·8, 95% confidence interval 2·1-22·3). These results are consistent with immunological responder and non-responder phenotypes, wherein a subset of patients with SCD may be at higher risk for transfusion-related alloimmunization.

  View details for DOI 10.1111/bjh.13424

  View details for PubMedID 25891976

• Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing. Blood Kurtz, D. M., Green, M. R., Bratman, S. V., Scherer, F., Liu, C. L., Kunder, C. A., Takahashi, K., Glover, C., Keane, C., Kihira, S., Visser, B., Callahan, J., Kong, K. A., Faham, M., Corbelli, K. S., Miklos, D., Advani, R. H., Levy, R., Hicks, R. J., Hertzberg, M., Ohgami, R. S., Gandhi, M. K., Diehn, M., Alizadeh, A. A. 2015; 125 (24): 3679-3687

  Abstract

  Recent studies have shown limited utility of routine surveillance imaging for diffuse large B-cell lymphoma (DLBCL) patients achieving remission. Detection of molecular disease by immunoglobulin high-throughput sequencing (Ig-HTS) from peripheral blood provides an alternate strategy for surveillance. We prospectively evaluated the utility of Ig-HTS within 311 blood and 105 tumor samples from 75 patients with DLBCL, comparing Ig-HTS from the cellular (circulating leukocytes) and acellular (plasma cell-free DNA) compartments of peripheral blood to clinical outcomes and (18)fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET/CT; n = 173). Clonotypic immunoglobulin rearrangements were detected in 83% of patients with adequate tumor samples to enable subsequent monitoring in peripheral blood. Molecular disease measured from plasma, compared with circulating leukocytes, was more abundant and better correlated with radiographic disease burden. Before treatment, molecular disease was detected in the plasma of 82% of patients compared with 71% in circulating cells (P = .68). However, molecular disease was detected significantly more frequently in the plasma at time of relapse (100% vs 30%; P = .001). Detection of molecular disease in the plasma often preceded PET/CT detection of relapse in patients initially achieving remission. During surveillance time points before relapse, plasma Ig-HTS demonstrated improved specificity (100% vs 56%, P < .0001) and similar sensitivity (31% vs 55%, P = .4) compared with PET/CT. Given its high specificity, Ig-HTS from plasma has potential clinical utility for surveillance after complete remission.

  View details for DOI 10.1182/blood-2015-03-635169

  View details for PubMedID 25887775

  View details for PubMedCentralID PMC4463733

• Allogeneic HY antibodies detected 3 months after female-to-male HCT predict chronic GVHD and nonrelapse mortality in humans BLOOD Nakasone, H., Tian, L., Sahaf, B., Kawase, T., Schoenrock, K., Perloff, S., Ryan, C. E., Paul, J., Popli, R., Wu, F., Otani, J. M., Coller, J., Warren, E. H., Miklos, D. B. 2015; 125 (20): 3193-3201

  Abstract

  Allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome (HY-Abs) develop after hematopoietic cell transplant (HCT) of male recipients with female donors (F→M). However, the temporal association between HY-Ab development and chronic graft-versus-host disease (cGVHD) has yet to be elucidated. We studied 136 adult F→M HCT patients, with plasma prospectively collected through 3 years posttransplant, and measured immunoglobulin G against 6 H-Y antigens. Multiple HY-Abs were frequently detected beginning at 3 months posttransplant: 78 (57%) of F→M patients were seropositive for at least 1 of the 6 HY-Abs, and 3-month seropositivity for each HY-Ab was associated with a persistent seropositive response throughout the posttransplant follow-up period (P < .001 in each). There were no associations between pretransplant features and 3-month overall HY-Ab development. Detection of multiple HY-Abs at 3 months (represented by HY score) was significantly associated with an increased risk of cGVHD (P < .0001) and nonrelapse mortality (P < .01). Compared to clinical factors alone, the addition of HY score to clinical factors improved the predictive potential of cGVHD (P < .01). Monitoring HY-Ab development thus stratifies cGVHD risk in F→M HCT patients and may support preemptive prophylaxis therapy for cGVHD beginning at 3 months posttransplant.

  View details for DOI 10.1182/blood-2014-11-613323

  View details for Web of Science ID 000355689000024

  View details for PubMedID 25766725

  View details for PubMedCentralID PMC4432013

• Therapeutic benefits targeting B-cells in chronic graft-versus-host disease INTERNATIONAL JOURNAL OF HEMATOLOGY Nakasone, H., Sahaf, B., Miklos, D. B. 2015; 101 (5): 438-451

  Abstract

  Allogeneic hematopoietic cell transplantation (allo-HCT) can be a curative strategy for hematological diseases, and the indications for allo-HCT have broadened widely due to recent progress in supportive strategies. However, patients must overcome various complications and chronic graft-versus-host disease (cGVHD) remains the most common allo-HCT cause of long-term morbidity and mortality. cGVHD is difficult to biologically assess due to the heterogeneity of cGVHD symptoms, and the pathogenesis of cGVHD has yet to be established. Recent experimental model progress has suggested that B-cells play a critical role in cGVHD development. Consistent with these experimental results, some clinical studies investigating B-cell depletion and modulation of B-cell signaling pathways have decreased cGVHD incidence and provided some therapeutic benefit. However, randomized control studies are necessary to confirm the efficacy of B-cell targeting drugs for cGVHD. Here, we review the pathophysiology of cGVHD, especially focusing on the role of B-cell immunity, and discuss the efficacy of both B-cell depletion and modulation of B-cell signaling pathways in human cGVHD prevention, initial treatment, and salvage treatment.

  View details for DOI 10.1007/s12185-015-1782-4

  View details for Web of Science ID 000354213800004

  View details for PubMedID 25812839

• National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: III. The 2014 Biomarker Working Group Report BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Paczesny, S., Hakim, F. T., Pidala, J., Cooke, K. R., Lathrop, J., Griffith, L. M., Hansen, J., Jagasia, M., Miklos, D., Pavletic, S., Parkman, R., Russek-Cohen, E., Flowers, M. E., Lee, S., Martin, P., Vogelsang, G., Walton, M., Schultz, K. R. 2015; 21 (5): 780-792

  Abstract

  Biology-based markers to confirm or aid in the diagnosis or prognosis of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation or monitor its progression are critically needed to facilitate evaluation of new therapies. Biomarkers have been defined as any characteristic that is objectively measured and evaluated as an indicator of a normal biological or pathogenic process, or of a pharmacologic response to a therapeutic intervention. Applications of biomarkers in chronic GVHD clinical trials or patient management include the following: (1) diagnosis and assessment of chronic GVHD disease activity, including distinguishing irreversible damage from continued disease activity; (2) prognostic risk to develop chronic GVHD; and (3) prediction of response to therapy. Sample collection for chronic GVHD biomarkers studies should be well documented following established quality control guidelines for sample acquisition, processing, preservation, and testing, at intervals that are both calendar and event driven. The consistent therapeutic treatment of subjects and standardized documentation needed to support biomarker studies are most likely to be provided in prospective clinical trials. To date, no chronic GVHD biomarkers have been qualified for use in clinical applications. Since our previous chronic GVHD Biomarkers Working Group report in 2005, an increasing number of chronic GVHD candidate biomarkers are available for further investigation. This paper provides a 4-part framework for biomarker investigations: identification, verification, qualification, and application with terminology based on Food and Drug Administration and European Medicines Agency guidelines.

  View details for DOI 10.1016/j.bbna.2015.01.003

  View details for Web of Science ID 000353856700002

  View details for PubMedID 25644957

• ABO Mismatch Is Associated with Increased Nonrelapse Mortality after Allogeneic Hematopoietic Cell Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Logan, A. C., Wang, Z., Alimoghaddam, K., Wong, R. M., Lai, T., Negrin, R. S., Grumet, C., Logan, B. R., Zhang, M., Spellman, S. R., Lee, S. J., Miklos, D. B. 2015; 21 (4): 746-754

  Abstract

  We evaluated ABO associated outcomes in 1737 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT) at Stanford University between January 1986 and July 2011. Grafts were 61% ABO matched, 18% major mismatched (MM), 17% minor MM, and 4% bidirectional MM. Median follow-up was 6 years. In multivariate analysis, overall survival (OS) was inferior in minor MM hematopoietic cell transplantations (median 2.1 versus 6.3 years; hazard ratio [HR], 1.56; 95% confidence interval [CI], 1.19 to 2.05; P = .001) in comparison with ABO-matched grafts. ABO minor MM was associated with an increase in early nonrelapse mortality (NRM) (18% versus 13%; HR, 1.48; 95% CI, 1.06 to 2.06; P = .02). In an independent Center for International Blood and Marrow Transplant Research (CIBMTR) analysis of 435 lymphoma patients receiving mobilized peripheral blood grafts, impairment of OS (HR, 1.55; 95% CI, 1.07 to 2.25; P = .021) and increased NRM (HR, 1.72; 95% CI, 1.11 to 2.68; P = .03) were observed in recipients of ABO minor-MM grafts. A second independent analysis of a CIBMTR data set including 5179 patients with acute myeloid leukemia and myelodysplastic syndrome identified a nonsignificant trend toward decreased OS in recipients of ABO minor-MM grafts and also found ABO major MM to be significantly associated with decreased OS (HR, 1.19; 95% CI, 1.08 to 1.31; P < .001) and increased NRM (HR, 1.23; 95% CI, 1.08 to 1.4; P = .002). ABO minor and major MM are risk factors for worse transplantation outcomes, although the associated hazards may not be uniform across different transplantation populations. Further study is warranted to determine which patient populations are at greatest risk, and whether this risk can be modified by anti-B cell therapy or other peri-transplantation treatments.

  View details for DOI 10.1016/j.bbmt.2014.12.036

  View details for Web of Science ID 000351790300025

  View details for PubMedID 25572032

• A Reduced-Toxicity Regimen Is Associated with Durable Engraftment and Clinical Cure of Nonmalignant Genetic Diseases among Children Undergoing Blood and Marrow Transplantation with an HLA-Matched Related Donor. Biology of blood and marrow transplantation Mahadeo, K. M., Weinberg, K. I., Abdel-Azim, H., Miklos, D. B., Killen, R., Kohn, D., Crooks, G. M., Shah, A. J., Kharbanda, S., Agarwal, R., Kapoor, N. 2015; 21 (3): 440-444

  Abstract

  Blood and marrow transplantation (BMT) is a standard curative therapy for patients with nonmalignant genetic diseases. Myeloablative conditioning has been associated with significant regimen-related toxicity (RRT), whereas reduced-intensity conditioning regimens have been associated with graft failure. In this prospective pilot trial conducted at 2 centers between 2006 and 2013, we report the outcome of 22 patients with nonmalignant genetic diseases who were conditioned with a novel reduced-toxicity regimen: i.v. busulfan (16 mg/kg), alemtuzumab (52 mg/m(2)), fludarabine (140 mg/m(2)), and cyclophosphamide (105 mg/kg). The median age of the study population was 3.5 years (range, 5 months to 26 years). No cases of sinusoidal obstruction syndrome, severe or chronic graft-versus-host disease (GVHD), or primary graft failure were reported. Median time to neutrophil engraftment (>500 cells/μL) and platelet engraftment (>20K cells/μL) were 19 (range, 12 to 50) and 23.5 (range, 14 to 134) days, respectively. The median length of follow-up was 3 years (range, .2 to 6.3). The overall survival rates were 95% at 100 days (95% confidence interval, .72 to .99) and 90% at 6 years (95% confidence interval, .68 to .98). RRT and chronic GVHD are significant barriers to BMT for patients with nonmalignant genetic diseases. This alemtuzumab-based reduced-toxicity regimen appears to be promising with durable engraftment, effective cure of clinical disease, low rates of RRT, and no observed chronic GVHD.

  View details for DOI 10.1016/j.bbmt.2014.11.005

  View details for PubMedID 25459642

• Immunoglobulin and T cell receptor gene high-throughput sequencing quantifies minimal residual disease in acute lymphoblastic leukemia and predicts post-transplantation relapse and survival. Biology of blood and marrow transplantation Logan, A. C., Vashi, N., Faham, M., Carlton, V., Kong, K., Buño, I., Zheng, J., Moorhead, M., Klinger, M., Zhang, B., Waqar, A., Zehnder, J. L., Miklos, D. B. 2014; 20 (9): 1307-1313

  Abstract

  Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10(-4) after induction predicts subsequent relapse. Likewise, MRD ≥ 10(-4) in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10(-6) or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10(-4) before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10(-6) had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.

  View details for DOI 10.1016/j.bbmt.2014.04.018

  View details for PubMedID 24769317

• Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1. Bone marrow transplantation Fleischhauer, K., Fernandez-Viña, M. A., Wang, T., Haagenson, M., Battiwalla, M., Baxter-Lowe, L. A., Ciceri, F., Dehn, J., Gajewski, J., Hale, G. A., Heemskerk, M. B., Marino, S. R., McCarthy, P. L., Miklos, D., Oudshoorn, M., Pollack, M. S., Reddy, V., Senitzer, D., Shaw, B. E., Waller, E. K., Lee, S. J., Spellman, S. R. 2014; 49 (9): 1176-1183

  Abstract

  HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.

  View details for DOI 10.1038/bmt.2014.122

  View details for PubMedID 24955785

• Total lymphoid irradiation-antithymocyte globulin conditioning and allogeneic transplantation for patients with myelodysplastic syndromes and myeloproliferative neoplasms. Biology of blood and marrow transplantation Benjamin, J., Chhabra, S., Kohrt, H. E., Lavori, P., Laport, G. G., Arai, S., Johnston, L., Miklos, D. B., Shizuru, J. A., Weng, W., Negrin, R. S., Lowsky, R. 2014; 20 (6): 837-843

  Abstract

  Allogeneic hematopoietic cell transplantation (allo HCT) is the only curative therapy for the myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), but treatment toxicity has been a barrier to its more widespread use. The nonmyeloablative regimen of total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) permits the establishment of donor hematopoiesis necessary for the graft-versus-malignancy effect and is protective against acute graft-versus-host disease (aGVHD), but it has minimal direct cytotoxicity against myeloid diseases. We explored the use of TLI-ATG conditioning to treat 61 patients with allo HCT for MDS (n = 32), therapy-related myeloid neoplasms (n = 15), MPN (n = 9), and chronic myelomonocytic leukemia (n = 5). The median age of all patients was 63 years (range, 50 to 73). The cumulative incidence of aGVHD grades II to IV was 14% (95% confidence interval [CI], 4% to 23%) and for grades III to IV, 4% (95% CI, 0 to 9%), and it did not differ between patients who received allografts from related or unrelated donors. The cumulative incidence of nonrelapse mortality (NRM) at 100 days, 12 months, and 36 months was 0%, 7%, and 11%. Overall survival and progression-free survival were 41% (95% CI, 29% to 53%) and 35% (95% CI, 23% to 48%), respectively. The safety and tolerability of TLI-ATG, as exemplified by its low NRM, provides a foundation for further risk-adapted or prophylactic interventions to prevent disease progression.

  View details for DOI 10.1016/j.bbmt.2014.02.023

  View details for PubMedID 24607552

• Clinical impact of H-Y alloimmunity. Immunologic research Popli, R., Sahaf, B., Nakasone, H., Lee, J. Y., Miklos, D. B. 2014; 58 (2-3): 249-258

  Abstract

  H-Y antigens are a group of minor histocompatibility antigens encoded on the Y-chromosome with homologous H-X antigens on the X-chromosome. The disparate regions of the H-Y antigens are highly immunogenic and play an important role in understanding human alloimmunity. In this review, we investigate the history of H-Y antigen discovery along with their critical contributions in transplantation and pregnancy. In hematopoietic cell transplantation, male recipients with female donors who become seropositive for B-cell responses as H-Y antibodies following transplantation have increased rates of chronic graft-versus-host disease and decreased rates of relapse. Conversely, female patients who receive male kidney allografts are more likely than other gender combinations to develop H-Y antibodies and reject their allografts. Finally, in the setting of pregnancy, mothers who initially gave birth to boys are more likely to have subsequent pregnancy complications, including miscarriages, in association with H-Y antibody development. H-Y antigens continue to serve as a model for alloimmunity in new clinical scenarios. Our development of more sensitive antibody detection and next-generation DNA sequencing promises to further advance our understanding and better predict the clinical consequences of alloimmunity.

  View details for DOI 10.1007/s12026-014-8514-3

  View details for PubMedID 24781195

• Minimal residual disease quantification using consensus primers and high- throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia LEUKEMIA Logan, A. C., Zhang, B., Narasimhan, B., Carlton, V., Zheng, J., Moorhead, M., Krampf, M. R., Jones, C. D., Waqar, A. N., Faham, M., Zehnder, J. L., Miklos, D. B. 2013; 27 (8): 1659-1665

  Abstract

  Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH-HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10(-4) and 20% in those with MRD 10(-4) (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5-32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden 10(-5) was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH-HTS method may facilitate routine MRD quantification in clinical trials.Leukemia advance online publication, 12 March 2013; doi:10.1038/leu.2013.52.

  View details for DOI 10.1038/leu.2013.52

  View details for Web of Science ID 000322823200006

  View details for PubMedID 23419792

• 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., Löhr, 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 PubMedID 23652802

• 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

• Birth Order and Transplantation Outcome in HLA-Identical Sibling Stem Cell Transplantation: An Analysis on Behalf of the Center for International Blood and Marrow Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Dobbelstein, C., Ahn, K. W., Haagenson, M., Hale, G. A., Van Rood, J. J., Miklos, D., Waller, E. K., Spellman, S. R., Fernandez-Vina, M., Ganser, A., Aljurf, M., Bornhaeuser, M., Gupta, V., Marino, S. R., Pollack, M. S., Reddy, V., Eder, M., Lee, S. J. 2013; 19 (5): 741-745

  Abstract

  Allogeneic stem cell transplantation (SCT) is the most effective treatment option for many hematologic malignancies, but graft-versus-host disease (GVHD) remains a major cause of treatment failure. Along with well-established risk factors for transplantation outcomes, recent single-center studies have identified a birth order effect in HLA-identical sibling SCT, with lower rates of acute and chronic GVHD and improved overall survival when the donor is younger than the recipient. One hypothesized mechanism for this effect is microchimerism due to fetomaternal and transmaternal sibling cell trafficking during pregnancy as the donor is exposed to recipient antigens in utero. The aim of the present study was to validate previously reported single-center data in a large, multicenter cohort provided by the Center for International Blood and Marrow Transplantation. All adult and pediatric patients (n = 11,365) with a hematologic malignancy who underwent allogeneic SCT with a graft from an HLA-identical sibling donor between 1990 and 2007 were included. When donors were younger than recipients, there was a significantly lower rate of acute GVHD grade II-IV and chronic GVHD in children, as well as a lower rate of chronic GVHD in adolescents. However, the hypothesized overall positive effect of lower relapse and better survival when donors are younger than recipients was not observed. Our data suggest that if otherwise equally matched, a graft from a younger sibling may be superior to a graft from an older sibling for children and adolescents undergoing SCT.

  View details for DOI 10.1016/j.bbmt.2013.01.020

  View details for Web of Science ID 000318132500010

  View details for PubMedID 23380341

• H-Y antigen-binding B cells develop in male recipients of female hematopoietic cells and associate with chronic graft vs. host disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sahaf, B., Yang, Y., Arai, S., Herzenberg, L. A., Herzenberg, L. A., Miklos, D. B. 2013; 110 (8): 3005-3010

  Abstract

  B cells are known to play an important role in pathogenesis of human chronic graft vs. host disease (cGVHD). Our group has previously shown that IgG allo-antibodies recognize Y chromosome-encoded proteins (H-Y) and a dominant H-Y epitope, DEAD box protein (DBY-2) detectable 6-12 mo after transplant in male patients who receive grafts from female donors (F→M) hematopoietic cell transplantation (HCT). Here we present FACS studies of peripheral blood mononuclear cells collected 6 mo after transplant showing that 16 of 28 (57%) F→M HCT patients have circulating donor B cells that express B-cell receptor (mainly IgM and Igλ) specific for DBY-2. The detection of these DBY-2 B cells 6 mo after HCT are associated with cGVHD development (P = 0.004). Specifically, 15 of 16 F→M with DBY-2 B cells developed cGVHD. In contrast, cGVHD developed in only 5 of the 12 who did not have DBY-2 B cells detected. This demonstrates circulating human B cells binding an alloantigen (DBY-2) and that these DBY-2-specific B cells appear before development of cGVHD in roughly half of the F→M patients. Our study suggests that detection of anti-DBY-2 B cells may predict cGVHD and that this prediction may have clinical utility. Validation of this hypothesis will require larger prospective studies.

  View details for DOI 10.1073/pnas.1222900110

  View details for Web of Science ID 000315954400081

  View details for PubMedID 23382226

• Humoral Immunity to Cytomegalovirus and Chronic Graft-Versus-Host Disease VIRAL IMMUNOLOGY Namboodiri, A. M., Nietert, P. J., Wadia, P. P., Miklos, D. B., Pandey, J. P. 2012; 25 (4): 338-340

  Abstract

  Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in patients with chronic graft-versus-host disease (cGVHD), but the underlying mechanisms are not understood. The aim of this investigation was to determine whether humoral immune responses to the HCMV antigens were quantitatively different in hematopoietic cell transplant (HCT) recipients who developed cGVHD from those who did not. Antibodies to HCMV and its proteins UL94 and UL70 were quantitated in 79 cGVHD and 30 non-cGVHD patients by enzyme-linked immunosorbent assays (ELISAs). Mean levels of antibodies to the whole HCMV and to its protein UL94 were not significantly different between the cGVHD and the non-cGVHD subjects. However, the levels of antibodies to HCMV UL70 were significantly higher in non-cGVHD subjects than in those with cGVHD (20.91±15.63 versus 15.00±10.35 ng/mL; p=0.03). This suggests that anti-UL70 antibodies might play a protective role in the development of cGVHD.

  View details for DOI 10.1089/vim.2012.0013

  View details for Web of Science ID 000307729400014

  View details for PubMedID 22803743

• 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

• 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

• High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Logan, A. C., Gao, H., Wang, C., Sahaf, B., Jones, C. D., Marshall, E. L., Buno, I., Armstrong, R., Fire, A. Z., Weinberg, K. I., Mindrinos, M., Zehnder, J. L., Boyd, S. D., Xiao, W., Davis, R. W., Miklos, D. B. 2011; 108 (52): 21194-21199

  Abstract

  The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

  View details for DOI 10.1073/pnas.1118357109

  View details for Web of Science ID 000298479900065

  View details for PubMedID 22160699

• Clonally identical Hodgkin's disease develops after allogeneic hematopoietic cell transplant for CLL BONE MARROW TRANSPLANTATION Tseng, D., Jones, C. D., Anderson, M., Warnke, R., Zehnder, J. L., Miklos, D. B. 2011; 46 (12): 1576-1578

  View details for DOI 10.1038/bmt.2010.340

  View details for Web of Science ID 000298326500015

  View details for PubMedID 21258419

• 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

• A phase 1 study of imatinib for corticosteroid-dependent/refractory chronic graft-versus-host disease: response does not correlate with anti-PDGFRA antibodies BLOOD Chen, G. L., Arai, S., Flowers, M. E., Otani, J. M., Qiu, J., Cheng, E. C., McMillan, A., Johnston, L. J., Shizuru, J. A., Miklos, D. B. 2011; 118 (15): 4070-4078

  Abstract

  Stimulatory antiplatelet derived growth factor receptor α (PDGFRA) antibodies have been associated with extensive chronic graft-versus-host disease (cGVHD). We performed a phase 1 dose escalation trial of imatinib in corticosteroid-dependent/refractory cGVHD to assess the safety of imatinib and test the hypothesis that abrogation of PDGFRA signaling can ameliorate the manifestations of cGVHD. Fifteen patients were enrolled. Mean follow-up time was 56.6 weeks (range, 18-82.4 weeks). Imatinib 400 mg daily was associated with more frequent moderate to life-threatening adverse events than 200 mg daily. The main adverse events were nausea, edema, confusion, diarrhea, liver function test elevation, fatigue, and myalgia. The overall response rate was 40% (6 of 15). The treatment failure rate was 40% (6 of 15). Twenty percent (3 of 15) of subjects had stable disease. Of 4 subjects with phospho-PDGFRA and phospho-PDGFRB immunohistochemistry studies before and after treatment, inhibition of phosphorylation was observed in 3 but correlated with response in one. Anti-PDGFRA antibodies were observed in 7 of 11 evaluable subjects but correlated with clinical activity in 4. We conclude that cGVHD responds to imatinib through multiple pathways that may include PDGFRA signal transduction. This study is registered at www.clinicaltrials.gov as #NCT00760981.

  View details for DOI 10.1182/blood-2011-03-341693

  View details for Web of Science ID 000296282200013

  View details for PubMedID 21828142

• Combined CD4 T-Cell and Antibody Response to Human Minor Histocompatibility Antigen DBY After Allogeneic Stem-Cell Transplantation TRANSPLANTATION Porcheray, F., Miklos, D. B., Floyd, B. H., Sarantopoulos, S., Bellucci, R., Soiffer, R. J., Antin, J. H., Alyea, E. P., Ritz, J., Zorn, E. 2011; 92 (3): 359-365

  Abstract

  Antibody responses to HY antigens in male recipients are frequent after transplantation of stem cells from female donors (Miklos et al., Blood 2005; 105: 2973; Miklos et al., Blood 2004; 103: 353). However, evidence that this B-cell immunity is accompanied by T-cell responses to the cognate antigens is scarce. Here, we examined T- and B-cell responses to DBY antigen in a male patient who received hematopoietic stem cells from a human leukocyte antigen-identical female sibling.We used 93 overlapping peptides representing the entire DBY protein to detect and characterize T-cell and antibody responses to DBY by enzyme-linked immunosorbent spot (ELISPOT) and enzyme-linked immunosorbent assay.High frequency CD4+ T cells specific for a unique DBY peptide were detected in the patient blood. We isolated the corresponding T-cell clone and characterized the recognized epitope as an 18-mer peptide restricted by human leukocyte antigen-DRB1*0101. Upon stimulation, this clone produced cytokines with no evidence of Th1 or Th2 polarization. Remarkably, this clone also recognized the DBX homologue peptide and responded to female donor dendritic cells stimulated with poly I/C or lipopolysaccharide, indicating that the peptide was endogenously processed in these cells. High titer DBY-specific antibodies were also found in the patient serum which, in contrast to the T-cell response, did not cross-react with DBX.We show here the development of a coordinated B and T-cell response to DBY in a recipient of sex mismatched allogeneic hematopoietic stem-cell transplantation. Our findings support a role for CD4+ T cells in the development of humoral immunity to minor histocompatibility antigens.

  View details for DOI 10.1097/TP.0b013e3182244cc3

  View details for Web of Science ID 000293176300021

  View details for PubMedID 21709606

• The Outcomes of Family Haploidentical Hematopoietic Stem Cell Transplantation in Hematologic Malignancies Are Not Associated with Patient Age BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Dong, L., Wu, T., Gao, Z., Zhang, M., Kan, F., Spellman, S. R., Tan, X., Zhao, Y., Wang, J., Lu, D., Miklos, D., Petersdorf, E., Fernandez-Vina, M., Lee, S. J. 2011; 17 (8): 1205-1213

  Abstract

  Haploidentical hematopoietic cell transplantation (HCT) has been used to treat hematologic malignancies, but it is unknown whether the procedure is more effective in adults or children. To address this question, we analyzed patients aged 1 to 65 years old receiving myeloablative conditioning regimens followed by family 2 to 3 antigen HLA-mismatched HCT and reported to the Center for International Blood and Marrow Transplant Research (CIBMTR; n = 137) or performed in Dao-Pei Hospital in China, China (n = 181). The Dao-Pei cohort had more acute and chronic graft-versus-host disease (GVHD), less relapse, lower transplant-related mortality (TRM), and better leukemia-free survival (LFS) than the CIBMTR cohort. Overall survival (OS) and outcomes were similar between adults and children. In the CIBMTR cohort receiving ex vivo T cell depletion (TCD), adults had higher TRM (relative risk [RR] 2.71, 95% confidence interval [CI] 1.29-5.69, P = .008) and lower OS (RR 1.75, 95% CI 1.08-2.84, P = .023) than children. In the CIBMTR subset that did not receive ex vivo TCD, relapse was lower in adults compared to children (RR 0.24, 95% CI 0.07-0.80, P = .020), but TRM, LFS, and OS were similar. We conclude that outcomes in adults and children are similar overall, although children have better survival than adults if ex vivo TCD is used.

  View details for DOI 10.1016/j.bbmt.2010.12.703

  View details for Web of Science ID 000293429600013

  View details for PubMedID 21193055

• B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies NATURE MEDICINE Winer, D. A., Winer, S., Shen, L., Wadia, P. P., Yantha, J., Paltser, G., Tsui, H., Wu, P., Davidson, M. G., Alonso, M. N., Leong, H. X., Glassford, A., Caimol, M., Kenkel, J. A., Tedder, T. F., McLaughlin, T., Miklos, D. B., Dosch, H., Engleman, E. G. 2011; 17 (5): 610-U134

  Abstract

  Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B cell-depleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease.

  View details for DOI 10.1038/nm.2353

  View details for Web of Science ID 000290250400038

  View details for PubMedID 21499269

  View details for PubMedCentralID PMC3270885

• Protein Microarrays Discover Angiotensinogen and PRKRIP1 as Novel Targets for Autoantibodies in Chronic Renal Disease MOLECULAR & CELLULAR PROTEOMICS Butte, A. J., Sigdel, T. K., Wadia, P. P., Miklos, D. B., Sarwal, M. M. 2011; 10 (3)

  Abstract

  Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may also hold utility in the discovery of auto-antibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a noninvasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, because of the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, four were validated in 71 individuals, with (n=50) and without (n=21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against angiotensinogen and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate of progression of chronic kidney disease.

  View details for DOI 10.1074/mcp.M110.000497

  View details for Web of Science ID 000287847200001

  View details for PubMedID 21183621

• 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

• Recombinant antigen microarrays for serum/plasma antibody detection. Methods in molecular biology (Clifton, N.J.) Wadia, P. P., Sahaf, B., Miklos, D. B. 2011; 723: 81-104

  Abstract

  Recombinant antigen arrays represent a new frontier in parallel analysis of multiple immune response profiles requiring only minute blood samples. In this article, we review the benefits and pitfalls of recombinant antigen microarrays developed for multiplexed antibody quantification. In particular, we describe the development of antigen arrays presenting a set of Y chromosome-encoded antigens, called H-Y antigens. These H-Y antigens are immunologically recognized as minor histocompatibility antigens (mHA) following allogeneic blood and organ transplantation. Clinically relevant B-cell responses against H-Y antigens have been demonstrated in male patients receiving female hematopoietic stem cell grafts and are associated with chronic graft versus host development. This chapter discusses our recombinant antigen microarray methods to measure these clinically relevant allo-antibodies.

  View details for DOI 10.1007/978-1-61779-043-0_7

  View details for PubMedID 21370061

• Outcomes of pediatric bone marrow transplantation for leukemia and myelodysplasia using matched sibling, mismatched related, or matched unrelated donors BLOOD Shaw, P. J., Kan, F., Ahn, K. W., Spellman, S. R., Aljurf, M., Ayas, M., Burke, M., Cairo, M. S., Chen, A. R., Davies, S. M., Frangoul, H., Gajewski, J., Gale, R. P., Godder, K., Hale, G. A., Heemskerk, M. B., Horan, J., Kamani, N., Kasow, K. A., Chan, K. W., Lee, S. J., Leung, W. H., Lewis, V. A., Miklos, D., Oudshoorn, M., Petersdorf, E. W., Ringden, O., Sanders, J., Schultz, K. R., Seber, A., Setterholm, M., Wall, D. A., Yu, L., Pulsipher, M. A. 2010; 116 (19): 4007-4015

  Abstract

  Although some trials have allowed matched or single human leukocyte antigen (HLA)-mismatched related donors (mmRDs) along with HLA-matched sibling donors (MSDs) for pediatric bone marrow transplantation in early-stage hematologic malignancies, whether mmRD grafts lead to similar outcomes is not known. We compared patients < 18 years old reported to the Center for International Blood and Marrow Transplant Research with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome undergoing allogeneic T-replete, myeloablative bone marrow transplantation between 1993 and 2006. In total, patients receiving bone marrow from 1208 MSDs, 266 8/8 allelic-matched unrelated donors (URDs), and 151 0-1 HLA-antigen mmRDs were studied. Multivariate analysis showed that recipients of MSD transplants had less transplantation-related mortality, acute graft-versus-host disease (GVHD), and chronic GVHD, along with better disease-free and overall survival than the URD and mmRD groups. No differences were observed in transplant-related mortality, acute and chronic GVHD, relapse, disease-free survival, or overall survival between the mmRD and URD groups. These data show that mmRD and 8/8 URD outcomes are similar, whereas MSD outcomes are superior to the other 2 sources. Whether allele level typing could identify mmRD recipients with better outcomes will not be known unless centers alter practice and type mmRD at the allele level.

  View details for DOI 10.1182/blood-2010-01-261958

  View details for Web of Science ID 000284110400040

  View details for PubMedID 20671124

• H-Y antibody titers are increased in unexplained secondary recurrent miscarriage patients and associated with low male : female ratio in subsequent live births HUMAN REPRODUCTION Nielsen, H. S., Wu, F., Aghai, Z., Steffensen, R., van Halteren, A. G., Spierings, E., Christiansen, O. B., Miklos, D., Goulmy, E. 2010; 25 (11): 2745-2752

  Abstract

  The birth of a boy is significantly more common than a girl prior to secondary recurrent miscarriage (SRM) and is associated with a poorer chance of a subsequent live birth. Children born after SRM are more likely to be girls. High-titer antisera specific for male antigens (H-Y) have been shown to arrest development of male bovine embryos efficiently. We consequently questioned the role of H-Y antibodies in women with SRM.Serum samples from patients with unexplained SRM (n = 84), unexplained primary recurrent miscarriage (PRM) (n = 12) and healthy women (n = 37) were obtained. The samples were taken during pregnancy (gestational weeks 4-5) for 77 (80%) of the patients. Enzyme-linked immunosorbent assay was used to detect immunoglobulin G antibodies that specifically recognized any of the five recombinant H-Y proteins (EIF1AY, RPS4Y1, ZFY, DDX3Y and UTY) and their H-X homologs.H-Y-specific antibodies were more frequent in SRM patients (46%) compared with female controls (19%, P = 0.004) and PRM patients (8%, P = 0.01). The presence of H-Y antibodies in early pregnancy was associated with a low male: female birth ratio among the subsequent live births, as only 12% of children born to H-Y antibody-positive patients were boys compared with 44% boys born to H-Y antibody negative patients (P = 0.03).The high frequency of H-Y antibody-positive SRM patients and the association between the presence of these antibodies in early pregnancy and the low number of male offspring, suggest that maternal immune responses against H-Y antigens can cause pregnancy losses. Further exploring these mechanisms may increase our understanding of unexplained SRM.

  View details for DOI 10.1093/humrep/deq242

  View details for Web of Science ID 000283124000007

  View details for PubMedID 20823116

• Complete donor T-cell engraftment 30 days after allogeneic transplantation predicts molecular remission in high-risk chronic lymphocytic leukaemia BRITISH JOURNAL OF HAEMATOLOGY Jones, C. D., Arai, S., Lowsky, R., Tyan, D. B., Zehnder, J. L., Miklos, D. B. 2010; 150 (5): 637-639

  View details for DOI 10.1111/j.1365-2141.2010.08252.x

  View details for Web of Science ID 000281060700020

  View details for PubMedID 20528878

• Allogeneic T cells impair engraftment and hematopoiesis after stem cell transplantation. Proceedings of the National Academy of Sciences of the United States of America Müller, A. M., Linderman, J. A., Florek, M., Miklos, D., Shizuru, J. A. 2010; 107 (33): 14721-14726

  Abstract

  Because of the perception that depleting hematopoietic grafts of T cells will result in poorer immune recovery and in increased risk of graft rejection, pure hematopoietic stem cells (HSC), which avoid the potentially lethal complication of graft-versus-host disease (GVHD), have not been used for allogeneic hematopoietic cell transplantation (HCT) in humans. Ideal grafts should contain HSC plus mature cells that confer only the benefits of protection from pathogens and suppression of malignancies. This goal requires better understanding of the effects of each blood cell type and its interactions during engraftment and immune regeneration. Here, we studied hematopoietic reconstitution post-HCT, comparing grafts of purified HSC with grafts supplemented with T cells in a minor histocompatibility antigen (mHA)-mismatched mouse model. Cell counts, composition, and chimerism of blood and lymphoid organs were evaluated and followed intensively through the first month, and then subsequently for up to 1 yr. Throughout this period, recipients of pure HSC demonstrated superior total cell recovery and lymphoid reconstitution compared with recipients of T cell-containing grafts. In the latter, rapid expansion of T cells occurred, and suppression of hematopoiesis derived from donor HSC was observed. Our findings demonstrate that even early post-HCT, T cells retard donor HSC engraftment and immune recovery. These observations contradict the postulation that mature donor T cells provide important transient immunity and facilitate HSC engraftment.

  View details for DOI 10.1073/pnas.1009220107

  View details for PubMedID 20679222

• Allogeneic T cells impair engraftment and hematopoiesis after stem cell transplantation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mueller, A. M., Linderman, J. A., Florek, M., Miklos, D., Shizuru, J. A. 2010; 107 (33): 14721-14726

  View details for DOI 10.1073/pnas.1009220107

  View details for Web of Science ID 000281287600038

• 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

• Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements JOURNAL OF IMMUNOLOGY Boyd, S. D., Gaeta, B. A., Jackson, K. J., Fire, A. Z., Marshall, E. L., Merker, J. D., Maniar, J. M., Zhang, L. N., Sahaf, B., Jones, C. D., Simen, B. B., Hanczaruk, B., Nguyen, K. D., Nadeau, K. C., Egholm, M., Miklos, D. B., Zehnder, J. L., Collins, A. M. 2010; 184 (12): 6986-6992

  Abstract

  Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

  View details for DOI 10.4049/jimmunol.1000445

  View details for Web of Science ID 000278516700047

  View details for PubMedID 20495067

• Rituximab in hematopoietic cell transplantation EXPERT OPINION ON BIOLOGICAL THERAPY Arai, S., Miklos, D. B. 2010; 10 (6): 971-982

  Abstract

  The success of rituximab therapy in managing B cell malignancies supports its widespread application in both autologous and allogeneic hematopoietic cell transplantation.We searched the PubMed database using the terms rituximab, stem cell transplant, autologous, or allogeneic and limited the search to clinical trials in English. In total, 92 trials were identified and 16 were reviewed in detail for significance of rituximab intervention. In this review, we will examine rituximab's emerging roles in: i) in vivo graft purging; ii) maintenance following autologous transplantation; iii) allogeneic transplant conditioning; and iv) the rationale for its use in the treatment/prevention of chronic graft-versus-host disease and post-transplant lymphoproliferative disorder.The reader will gain an understanding of the use of rituximab not only in transplants for B cell malignancies, but also its extension to other diseases where we are learning that B cells are involved in the pathogenesis.With rituximab firmly established in the non-transplant therapy of B cell malignancies, the new challenge in transplantation is how to incorporate the drug for optimum efficacy in those patients coming to transplant with relapse after rituximab-containing therapy.

  View details for DOI 10.1517/14712598.2010.485982

  View details for Web of Science ID 000277392300011

  View details for PubMedID 20420511

• Antibodies specifically target AML antigen NuSAP1 after allogeneic bone marrow transplantation BLOOD Wadia, P. P., Coram, M., Armstrong, R. J., Mindrinos, M., Butte, A. J., Miklos, D. B. 2010; 115 (10): 2077-2087

  Abstract

  Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify nucleolar and spindle-associated protein 1 (NuSAP1) and chromatin assembly factor 1, subunit B (p60; CHAF1b) as targets of new antibody responses that developed after allogeneic HCT. Western blots and enzyme-linked immunosorbent assays (ELISA) validated their post-HCT recognition and enabled ELISA testing of 120 other patients with various malignancies who underwent allo-HCT. CHAF1b-specific antibodies were predominantly detected in patients with acute myeloid leukemia (AML), whereas NuSAP1-specific antibodies were exclusively detected in patients with AML 1 year after transplantation (P < .001). Complete genomic exon sequencing failed to identify a nonsynonymous single nucleotide polymorphism (SNP) for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and reverse transcriptase-polymerase chain reaction (RT-PCR) showed NuSAP1 was predominately expressed in the bone marrow CD34(+)CD90(+) hematopoietic stem cells, leukemic cell lines, and B lymphoblasts compared with other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% of patients with AML following allogeneic HCT and suggests a tumor antigen role.

  View details for DOI 10.1182/blood-2009-03-211375

  View details for Web of Science ID 000275751300033

  View details for PubMedID 20053754

• 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

  View details for PubMedCentralID PMC2721787

• Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and "antibodyome'' measures PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, L., Wadia, P., Chen, R., Kambham, N., Naesens, M., Sigdel, T. K., Miklos, D. B., Sarwal, M. M., Butte, A. J. 2009; 106 (11): 4148-4153

  Abstract

  We have conducted an integrative genomics analysis of serological responses to non-HLA targets after renal transplantation, with the aim of identifying the tissue specificity and types of immunogenic non-HLA antigenic targets after transplantation. Posttransplant antibody responses were measured by paired comparative analysis of pretransplant and posttransplant serum samples from 18 pediatric renal transplant recipients, measured against 5,056 unique protein targets on the ProtoArray platform. The specificity of antibody responses were measured against gene expression levels specific to the kidney, and 2 other randomly selected organs (heart and pancreas), by integrated genomics, employing the mapping of transcription and ProtoArray platform measures, using AILUN. The likelihood of posttransplant non-HLA targets being recognized preferentially in any of 7 microdissected kidney compartments was also examined. In addition to HLA targets, non-HLA immune responses, including anti-MICA antibodies, were detected against kidney compartment-specific antigens, with highest posttransplant recognition for renal pelvis and cortex specific antigens. The compartment specificity of selected antibodies was confirmed by IHC. In conclusion, this study provides an immunogenic and anatomic roadmap of the most likely non-HLA antigens that can generate serological responses after renal transplantation. Correlation of the most significant non-HLA antibody responses with transplant health and dysfunction are currently underway.

  View details for DOI 10.1073/pnas.0900563106

  View details for Web of Science ID 000264278800020

  View details for PubMedID 19251643

  View details for PubMedCentralID PMC2657434

• B Cells and Transplantation: An Educational Resource Tandem Meeting on Biology of Blood and Marrow Transplantation Small, T. N., Robinson, W. H., Miklos, D. B. ELSEVIER SCIENCE INC. 2009: 104–13

  View details for DOI 10.1016/j.bbmt.2008.10.016

  View details for Web of Science ID 000263034300022

  View details for PubMedID 19147088

• H-Y antibody development associates with acute rejection in female patients with male kidney transplants TRANSPLANTATION Tan, J. C., Wadia, P. P., Coram, M., Grumet, F. C., Kambham, N., Miller, K., Pereira, S., Vayntrub, T., Miklos, D. B. 2008; 86 (1): 75-81

  Abstract

  Human minor histocompatibility antigens (mHA) and clinically relevant immune responses to them have not been well defined in organ transplantation. We hypothesized that women with male kidney transplants would develop antibodies against H-Y, the mHA encoded on the Y-chromosome, in association with graft rejection.We tested sera from 118 consecutive transplant recipients with kidney biopsies. Antibodies that specifically recognized the recombinant H-Y antigens RPS4Y1 or DDX3Y were detected by IgG enzyme-linked immunosorbent assay and western blotting. Immunogenic epitopes were further identified using overlapping H-Y antigen peptides for both the H-Y proteins.In the 26 female recipients of male kidneys, H-Y antibody development posttransplant (1) was more frequent (46%) than in other gender combinations (P<0.001), (2) showed strong correlation with acute rejection (P=0.00048), (3) correlated with plasma cell infiltrates in biopsied kidneys (P=0.04), and (4) did not correlate with C4d deposition or donor-specific anti-human leukocyte antigen (HLA) antibodies. Of the two H-Y antigens, RPS4Y1 was more frequently recognized (P=0.005).This first demonstration of a strong association between H-Y antibody development and acute rejection in kidney transplant recipients shows that in solid organ allografts, humoral immune responses against well defined mHA have clear clinical correlates, can be easily monitored, and warrant study for possible effects on long-term graft function.

  View details for DOI 10.1097/TP.0b013e31817352b9

  View details for Web of Science ID 000257790400014

  View details for PubMedID 18622281

  View details for PubMedCentralID PMC2943873

• Renal pathology in hematopoietic cell transplantation recipients 96th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Troxell, M. L., Pilapil, M., Miklos, D. B., Higgins, J. P., Kambham, N. NATURE PUBLISHING GROUP. 2008: 396–406

  Abstract

  Hematopoietic cell transplantation-associated renal injury may be related to a combination of factors including chemotherapy, radiation, infection, immunosuppressive agents, ischemia, and graft-versus-host disease. Renal biopsy specimens from hematopoietic cell transplant recipients at two institutions (Stanford University Medical Center and Oregon Health & Science University) were reviewed in correlation with clinical data. Fifteen cases were identified (post hematopoietic cell transplant time 0.7-14.5 years), including six with autologous hematopoietic cell transplant. Indications for renal biopsy included proteinuria (n=13; nephrotic range in 8), increased serum creatinine (n=10), or both (n=6). Many patients had multiple pathologic findings on renal biopsy. Membranous glomerulonephritis was the most common diagnosis (n=7), including two patients with autologous hematopoietic cell transplant and five with evidence of chronic graft-versus-host disease elsewhere. Four membranous glomerulonephritis patients achieved sustained remission with rituximab therapy. Other glomerular pathology included focal segmental glomerulosclerosis (n=1) and minimal change disease (n=1). Evidence of thrombotic microangiopathy was common (in isolation or combined with other pathology), as was acute tubular necrosis and tubulointerstitial nephritis. Of 14 patients with follow-up (2-64 months, mean 19 months), 6 had chronic renal insufficiency (serum creatinine >1.5 mg/dl), 2 had end stage renal disease, and 6 had essentially normal renal function. Our retrospective study shows that renal dysfunction in hematopoietic cell transplant recipients is often multifactorial, and biopsy may reveal treatable causes. Membranous glomerulonephritis is seen in autologous and allogeneic hematopoietic cell transplant recipients, and may respond to anti-B-cell therapy, which has implications regarding pathogenesis and relationship to graft-versus-host disease.

  View details for DOI 10.1038/modpathol.3801011

  View details for Web of Science ID 000254288500005

  View details for PubMedID 18223556

• Rituximab for steroid-refractory chronic graft-versus-host disease Joint Meeting of the American-Society-for-Blood-and-Marrow-Transplantation/Center-for-International-Blood-and-Marrow-Transplant-Research Cutler, C., Miklos, D., Kim, H. T., Treister, N., Woo, S., Bienfang, D., Klickstein, L. B., Levin, J., Miller, K., Reynolds, C., Macdonell, R., Pasek, M., Lee, S. J., Ho, V., Soiffer, R., Antin, J. H., Ritz, J., Alyea, E. AMER SOC HEMATOLOGY. 2006: 756–62

  Abstract

  B cells may be implicated in the pathophysiology of chronic graft-versus-host disease (GVHD), as evidenced by antibody production against sex-mismatched, Y chromosome-encoded minor HLA antigens in association with chronic GVHD. We therefore designed a phase 1/2 study of anti-B-cell therapy with rituximab in steroid-refractory chronic GVHD. Twenty-one patients were treated with 38 cycles of rituximab. Rituximab was tolerated well, and toxicity was limited to infectious events. The clinical response rate was 70%, including 2 patients with complete responses. Responses were limited to patients with cutaneous and musculoskeletal manifestations of chronic GVHD and were durable through 1 year after therapy. The median dose of prednisone among treated subjects fell from 40 mg/day to 10 mg/day, 1 year after rituximab therapy (P < .001). A chronic GVHD symptom score improved in the majority of treated patients. Antibody titers against Y chromosome-encoded minor HLA antigens fell and remained low, whereas titers against infectious antigens (EBV, tetanus) remained stable or rose during the treatment period. We conclude that specific anti-B-cell therapy with rituximab may be beneficial for patients with steroidrefractory chronic GVHD. This trial was registered at www.clinicaltrials.gov as #NCT00136396.

  View details for DOI 10.1182/blood-2006-01-0233

  View details for Web of Science ID 000239129500057

  View details for PubMedID 16551963

• Toward biomarkers for chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on criteria for clinical trials in chronic graft-versus-host disease: III. Biomarker working group report BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Schultz, K. R., Miklos, D. B., Fowler, D., Cooke, K., Shizuru, J., Zorn, E., Holler, E., Ferrara, Y., Shulman, H., Lee, S. Y., Martin, P., Filipovich, A. H., Flowers, M. E., Weisdorf, D., Couriel, D., Lachenbruch, P. A., Mittleman, B., Vogelsang, G. B., Pavletic, S. Z. 2006; 12 (2): 126-137

  Abstract

  Biology-based markers that can be used to confirm the diagnosis of chronic graft-versus-host disease (GVHD) or monitor progression of the disease could help in the evaluation of new therapies. Biomarkers have been defined as any characteristic that is objectively measured and evaluated as an indicator of a normal biologic or pathogenic process, a pharmacologic response to a therapeutic intervention, or a surrogate end point intended to substitute for a clinical end point. The following applications of biomarkers could be useful in chronic GVHD clinical trials or management: (1) predicting response to therapy; (2) measuring disease activity and distinguishing irreversible damage from continued disease activity; (3) predicting the risk of developing chronic GVHD; (4) diagnosing chronic GVHD: (5) predicting the prognosis of chronic GVHD; (6) evaluating the balance between GVHD and graft-versus-leukemia effects (graft-versus-leukemia or GVT); and (7) serving as a surrogate end point for therapeutic response. Such biomarkers can be identified by either hypothesis-driven testing or by high-throughput discovery-based methods. To date, no validated biomarkers have been established for chronic GVHD, although several candidate biomarkers have been identified from limited hypothesis-driven studies. Both approaches have merit and should be pursued. The consistent treatment and standardized documentation needed to support biomarker studies are most likely to be satisfied in prospective clinical trials.

  View details for DOI 10.1016/j.bbmt.2005.11.010

  View details for Web of Science ID 000235284900002

  View details for PubMedID 16443511

• Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Miklos, D. B., Kim, H. T., Miller, K. H., Guo, L. X., Zorn, E., Lee, S. J., Hochberg, E. P., Wu, C. J., Alyea, E. P., Cutler, C., Ho, V., Soiffer, R. J., Antin, J. H., Ritz, J. AMER SOC HEMATOLOGY. 2005: 2973–78

  Abstract

  Minor histocompatibility antigens (mHAs) are known targets of donor T cells after allogeneic hematopoietic stem cell transplantation (HSCT). In contrast, B-cell responses to mHAs have not been extensively characterized and the clinical significance of antibodies to mHAs is unknown. We tested 121 patients who underwent HSCT and 134 healthy donors for immunoglobulin G (IgG) antibodies against 5 mHAs encoded by genes on the Y chromosome (DBY, UTY, ZFY, RPS4Y, and EIF1AY). Antibodies to at least one H-Y protein developed in 52% of male patients with female donors compared with 8.7% of male patients with male donors (P < .0001), and in 41.4% of healthy females compared with 7.8% of healthy males (P < .0001). H-Y antibodies develop 4 to 12 months after transplantation and persist for long periods. The clinical significance of H-Y antibodies was characterized in 75 male patients with hematologic malignancies who received stem cells from female donors (F --> M HSCT). The presence of H-Y antibodies correlated with chronic graft-versus-host disease (GVHD) by univariate (odds ratio [OR] = 15.5; P < .0001) and multivariable logistic regression analysis (OR = 56.5; P < .0001). Antibody response to Y-chromosome encoded histocompatibility antigens (H-Y antigens) was also associated with maintenance of disease remission (P < .0001). B cells may provide a new target for immune intervention in chronic GVHD.

  View details for Web of Science ID 000228042900059

  View details for PubMedID 15613541

• Antibody response to DBY minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors 44th Annual Meeting of the American-Society-of-Hematology Miklos, D. B., Kim, H. T., Zorn, E., Hochberg, E. P., Guo, L. X., Mattes-Ritz, A., Viatte, S., Soiffer, R. J., Antin, J. H., Ritz, J. AMER SOC HEMATOLOGY. 2004: 353–59

  Abstract

  Minor histocompatibility antigens (mHAs) recognized by donor T cells play a central role as immunologic targets of graft-versus-host disease (GVHD) and graft versus leukemia after allogeneic hematopoietic stem cell transplantation (HSCT). Men who have undergone sex-mismatched allogeneic HSCT are at high risk for GVHD because of immune responses directed against mHAs encoded by genes on the Y chromosome (termed H-Y antigens). We hypothesized that the immunogenicity of mHAs results in a coordinated response involving B cells as well as T cells. To test this, we measured antibody responses to a well-characterized H-Y antigen, dead box RNA helicase Y (DBY), and its homolog, DBX, in 150 HSCT patients. Using Western blot and enzyme-linked immunosorbent assay (ELISA), we found that 50% of male patients who received stem cell grafts from female donors developed antibody responses to recombinant DBY protein. Antibodies to DBY were also detected in 17% of healthy women, but not in healthy men. Antibody responses were directed primarily against areas of amino acid disparity between DBY and DBX. These studies demonstrate that the immune response to mHA includes the generation of specific antibodies and suggests that the serologic response to these antigens may also be useful in the identification of new mHAs.

  View details for DOI 10.1182/blood-2003-03-0984

  View details for Web of Science ID 000187573000060

  View details for PubMedID 14512314