Dr. Natkunam is an expert in the diagnosis of hematopoietic tumors including lymphoma and leukemia, and has over 15-years of experience. As the Director of Hematopathology, she oversees all hematopathology diagnostic services for Stanford Health and Stanford Children’s Health. Her research focuses on refining criteria for the diagnosis of hematopoietic tumors through discovery and application of biomarkers, an approach that has furnished novel reagents and guidelines for clinical practice. She currently serves on the editorial boards of the American Journal of Surgical Pathology and Human Pathology, and on the Eastern Cooperative Oncology Group and the Lunenburg Lymphoma Biomarker Consortium.

Clinical Focus

  • Hematopathology
  • Anatomic Pathology
  • Pathology and Laboratory Medicine

Academic Appointments

Administrative Appointments

  • Co-Director, Immunodiagnosis Laboratory, Stanford (2005 - Present)
  • Director of Faculty Development and Diversity, Stanford (2009 - Present)
  • Director of Hematopathology, Stanford (2009 - Present)

Professional Education

  • Residency:Stanford University School of Medicine Registrar (2000) CA
  • Medical Education:Mt Sinai School of Medicine (1995) NY
  • Internship:Stanford University School of Medicine (1996) CA
  • Board Certification: Anatomic Pathology, American Board of Pathology (1998)
  • PhD, Mt.Sinai School of Medicine, Molecular Biology (1993)
  • AB, Bryn Mawr College, Biology (1987)

Research & Scholarship

Current Research and Scholarly Interests

My laboratory studies the tissue expression pattern of proteins that are of potential interest for the diagnosis, prognostic stratification and therapy of patients with lymphoma and leukemia. We are currently investigating prognostic markers in diffuse large B-cell lymphoma such that risk groups can be better defined. We are also interested in the immunodiagnostic and molecular characterization of rare and aggressive hematopoietic tumors such as Natural Killer/T-cell lymphomas, histiocytic malignancies and childhood lymphomas in addition to immunodeficiency-associated lymphoproliferative disorders.

Clinical Trials

  • Genome, Proteome and Tissue Microarray in Childhood Acute Leukemia Not Recruiting

    We will study gene and protein expression in leukemia cells of children diagnosed with acute leukemia. We hope to identify genes or proteins which can help us grade leukemia at diagnosis in order to: (a) develop better means of diagnosis and (b) more accurately choose the best therapy for each patient.

    Stanford is currently not accepting patients for this trial. For more information, please contact Norman J Lacayo, 650-723-5535.

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  • Phase 2 Fludarabine, Cytoxan and FCCAM <Alemtuzumab> in Untreated B-Cell Chronic Lymphocytic Leukemia Not Recruiting

    The primary objective of this study was to evaluate the safety and efficacy of the combination of fludarabine and cyclophosphamide in previously untreated CLL patients. Participants will receive fludarabine and cyclophosphamide on days 1, 2, and 3 of six 28-day cycles.

    Stanford is currently not accepting patients for this trial. For more information, please contact Nini Estevez, (650) 725 - 4041.

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  • Phase 2 Study of Atorvastatin Safety and Antitumor Effects in Non-Hodgkin's Lymphoma Not Recruiting

    This is an approach which can inflict significant toxicity. An alternative is to block expression of oncogenes which are over-expressed only in cancer cells, a therapeutic approach which could reduce toxicity to the host while maximizing destruction of the oncogene-dependent malignant cells.

    Stanford is currently not accepting patients for this trial. For more information, please contact Alice Fan, 650-736-1285.

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  • Phase I Intratumoral Dendritic Cell Immunotherapy in Thermally Ablated Liver Metastases Not Recruiting

    Up to twenty-two patients will be enrolled in this study to receive autologous dendritic cells (DCs) administered intratumorally into liver metastases following radiofrequency thermal ablation of those lesions. Patients will receive two vaccinations of DCs at monthly intervals. A dose escalation study of DCs will be included in this study in an attempt to define the maximum tolerated dose of administered DCs.

    Stanford is currently not accepting patients for this trial. For more information, please contact Jenna Rogers, (650) 723 - 4467.

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  • Phase I/II Intratumoral DC Immunotherapy With Gemcitabine & XRT in Unresectable Pancreatic Cancer Not Recruiting

    To determine the safety, feasibility and appropriate dendritic cell dose to vaccinate patients with pancreas cancer

    Stanford is currently not accepting patients for this trial. For more information, please contact Jenna Rogers, (650) 723 - 4467.

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2017-18 Courses


All Publications

  • Immunohistochemical Profile of MYC Protein in Pediatric Small Round Blue Cell Tumors. Pediatric and developmental pathology Chisholm, K. M., Krishnan, C., Heerema-McKenney, A., Natkunam, Y. 2017; 20 (3): 213-223


    Deregulation of MYC oncoprotein in cancers can result from multiple oncogenic mechanisms. Although MYC translocations define Burkitt lymphoma and MYC protein expression is a poor prognostic factor in undifferentiated neuroblastomas, the distribution of MYC protein (c-MYC) across other pediatric small round blue cell tumors (SRBCT) has not been well characterized. We undertook this study to assess MYC protein expression in a large cohort of pediatric lymphomas, sarcomas, and other SRBCT. Tissue microarrays containing 302 SRBCT were successfully evaluated by immunohistochemistry using anti-MYC clone Y69, with nuclear positivity scored as 0%, 1%-25%, 26%-50%, 51%-75%, or 76%-100%. MYC protein staining of >50% of lesional cells was identified in 60% of Burkitt lymphomas, 50% of B lymphoblastic lymphomas, 33% of T lymphoblastic lymphomas, 31% of rhabdomyosarcomas, 33% of Ewing sarcomas, and 25% of soft tissue sarcomas, not otherwise specified. Only 14% of neuroblastomas showed >50% staining, and of these, if known, MYCN was not amplified. No cases of Wilms tumor, synovial sarcoma, or desmoplastic small round cell tumor had >50% staining. Recurrences and metastases often had the same percentage of MYC staining (15/30). In conclusion, MYC protein exhibited variable expression across and within pediatric SRBCT subtypes. Overall, these findings provide a baseline for MYC expression in pediatric SRBCT and suggest that there may be multiple mechanisms of MYC upregulation in these different neoplasms.

    View details for DOI 10.1177/1093526616689642

    View details for PubMedID 28521631

  • Unifying mechanism for different fibrotic diseases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Chen, S., Cui, L., Van Neste, C., Tsai, J. M., Kambham, N., Vogel, H., Natkunam, Y., Gilliland, D. G., Nolan, G., Weissman, I. L. 2017; 114 (18): 4757-4762


    Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

    View details for DOI 10.1073/pnas.1621375114

    View details for Web of Science ID 000400358000057

    View details for PubMedID 28424250

    View details for PubMedCentralID PMC5422830

  • Molecular impact of selective NFKB1 and NFKB2 signaling on DLBCL phenotype. Oncogene Guo, X., KOFF, J. L., MOFFITT, A. B., Cinar, M., Ramachandiran, S., Chen, Z., Switchenko, J. M., Mosunjac, M., Neill, S. G., Mann, K. P., Bagirov, M., Du, Y., Natkunam, Y., Khoury, H. J., Rossi, M. R., Harris, W., Flowers, C. R., Lossos, I. S., Boise, L. H., Dave, S. S., Kowalski, J., Bernal-Mizrachi, L. 2017


    Diffuse large B-cell lymphoma (DLBCL) has been categorized into two molecular subtypes that have prognostic significance, namely germinal center B-cell like (GCB) and activated B-cell like (ABC). Although ABC-DLBCL has been associated with NF-κB activation, the relationships between activation of specific NF-κB signals and DLBCL phenotype remain unclear. Application of novel gene expression classifiers identified two new DLBCL categories characterized by selective p100 (NF-κB2) and p105 (NF-κB1) signaling. Interestingly, our molecular studies showed that p105 signaling is predominantly associated with GCB subtype and histone mutations. Conversely, most tumors with p100 signaling displayed ABC phenotype and harbored ABC-associated mutations in genes such as MYD88 and PIM1. In vitro, MYD88 L265P mutation promoted p100 signaling through TAK1/IKKα and GSK3/Fbxw7a pathways, suggesting a novel role for this protein as an upstream regulator of p100. p100 signaling was engaged during activation of normal B cells, suggesting p100's role in ABC phenotype development. Additionally, silencing p100 in ABC-DLBCL cells resulted in a GCB-like phenotype, with suppression of Blimp, IRF4 and XBP1 and upregulation of BCL6, whereas introduction of p52 or p100 into GC cells resulted in differentiation toward an ABC-like phenotype. Together, these findings identify specific roles for p100 and p105 signaling in defining DLBCL molecular subtypes and posit MYD88/p100 signaling as a regulator for B-cell activation.

    View details for DOI 10.1038/onc.2017.90

    View details for PubMedID 28368397

  • Epstein-Barr virus-positive follicular lymphoma. Modern pathology Mackrides, N., Campuzano-Zuluaga, G., Maque-Acosta, Y., Moul, A., Hijazi, N., Ikpatt, F. O., Levy, R., Verdun, R. E., Kunkalla, K., Natkunam, Y., Lossos, I. S., Vega, F., Chapman, J. 2017; 30 (4): 519-529


    Epstein-Barr virus (EBV) -associated follicular lymphoma is only rarely reported. Herein, we report the largest series analyzing prevalence and clinicopathologic characteristics of EBV-associated follicular lymphoma occurring in unselected cases. Out of 382 analyzed cases, 10 EBV-positive follicular lymphomas were identified (prevalence=2.6%, 95% confidence interval 1.3-4.0%). All EBV-positive follicular lymphomas showed EBV-encoded small RNA-positive lymphoma cells present in a follicular distribution. Of these, eight also had tissue available for testing of expression of latent membrane protein 1 (LMP1), out of which six (75%) were positive. There was a significant association with grades 3A-3B follicular lymphoma (P<0.0001) and CD30 expression (P=0.0002). EBV-positive follicular lymphomas were otherwise morphologically and immunophenotypically indistinguishable from EBV-negative cases of similar grade. Nine of the EBV-positive follicular lymphomas occurred in patients with no known history of immunosuppression, while one patient had a history of hydroxychloroquine administration for Sjögren's syndrome. The mean age in the EBV-positive and -negative follicular lymphomas was 56 (range 31-83 years) and 49 years (range 25-92 years), respectively, with no statistically significant difference. Seven of the patients with EBV-positive follicular lymphoma had additional biopsies from different time points available for review, all of which showed progression of disease in the form of progression of tumor grade. Five of these progressed to diffuse large B-cell lymphoma, one of which had tissue available for testing and was EBV-positive. Our findings suggest that EBV infection may have a role in lymphomagenesis and/or disease progression in a subset of follicular lymphomas, thereby expanding the spectrum of recognized EBV-associated B-cell lymphomas.

    View details for DOI 10.1038/modpathol.2016.214

    View details for PubMedID 27982024

  • Epstein-Barr virus-positive follicular lymphoma MODERN PATHOLOGY Mackrides, N., Campuzano-Zuluaga, G., Maque-Acosta, Y., Moul, A., Hijazi, N., Ikpatt, F. O., Levy, R., Verdun, R. E., Kunkalla, K., Natkunam, Y., Lossos, I. S., Vega, F., Chapman, J. 2017; 30 (4): 519-529
  • B-Cell and Classical Hodgkin Lymphomas Associated With Immunodeficiency 2015 SH/EAHP Workshop Report-Part 2 AMERICAN JOURNAL OF CLINICAL PATHOLOGY De Jong, D., Roemer, M. G., Chan, J. K., Goodlad, J., Gratzinger, D., Chadburn, A., Jaffe, E. S., Said, J., Natkunam, Y. 2017; 147 (2): 153-170


    The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology submitted small and large B-cell lymphomas (BCLs), including classical Hodgkin lymphoma (CHL), in the context of immunodeficiency.Clinicopathologic and molecular features were studied to explore unifying concepts in malignant B-cell proliferations across immunodeficiency settings.Cases submitted to the workshop spanned small BCLs presenting as nodal or extranodal marginal zone lymphoma and lymphoplasmacytic lymphoma, Epstein-Barr virus (EBV) positive in 75% of cases. Submitted large BCLs formed a spectrum from diffuse large B-cell lymphoma (DLBCL) to CHL across immunodeficiency settings. Additional studies demonstrated overexpression of PD-L1 and molecular 9p24 alterations in the large BCL spectrum and across different immunodeficiency settings.Small BCLs occur in all immunodeficiency settings, and EBV positivity is essential for their recognition as immunodeficiency related. Large BCLs include a spectrum from DLBCL to CHL across all immunodeficiency settings; immunohistochemical and molecular features are suggestive of shared pathogenetic mechanisms involving PD-L1 immune checkpoints.

    View details for DOI 10.1093/ajcp/aqw216

    View details for Web of Science ID 000397108600003

    View details for PubMedID 28395108

  • Primary/Congenital Immunodeficiency 2015 SH/EAHP Workshop Report-Part 5 AMERICAN JOURNAL OF CLINICAL PATHOLOGY Gratzinger, D., Jaffe, E. S., Chadburn, A., Chan, J. K., De Jong, D., Goodlad, J. R., Said, J., Natkunam, Y. 2017; 147 (2): 204-216


    The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review primary immunodeficiency and related lymphoproliferations.Primary immunodeficiencies were divided into immune dysregulation, DNA repair defects, low immunoglobulins, and combined immunodeficiencies.Autoimmune lymphoproliferative syndrome (ALPS) is a prototypical immune dysregulation-type immunodeficiency, with defects in T-cell signaling or apoptosis, expansion of T-cell subsets, and predisposition to hemophagocytic lymphohistiocytosis. DNA repair defects directly predispose to malignancy. Low immunoglobulin immunodeficiencies such as common variable immunodeficiency (CVID) have underlying T-cell repertoire abnormalities predisposing to autoimmunity and B-cell lymphoproliferations. The full spectrum of B-cell lymphoproliferative disorders occurs in primary immunodeficiency.Lymphoproliferations in primary immunodeficiency mirror those in other immunodeficiency settings, with monomorphic B- and sometimes T lymphoproliferative disorders enriched in DNA repair defects. Distinctive T-cell subset expansions in ALPS, CVID, and related entities can mimic lymphoma, and recognition of double-negative T-cell or cytotoxic T-cell expansions is key to avoid overdiagnosis.

    View details for DOI 10.1093/ajcp/aqw215

    View details for Web of Science ID 000397108600006

    View details for PubMedID 28395106

  • EBV-Positive B-Cell Proliferations of Varied Malignant Potential 2015 SH/EAHP Workshop Report-Part 1 AMERICAN JOURNAL OF CLINICAL PATHOLOGY Natkunam, Y., Goodlad, J. R., Chadburn, A., De Jong, D., Gratzinger, D., Chan, J. K., Said, J., Jaffe, E. S. 2017; 147 (2): 129-152


    The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review B-cell proliferations of varied malignant potential associated with immunodeficiency.The Workshop Panel reviewed all cases of B-cell hyperplasias, polymorphic B-lymphoproliferative disorders, Epstein-Barr virus (EBV)-positive mucocutaneous ulcer, and large B-cell proliferations associated with chronic inflammation and rendered consensus diagnoses. Disease definitions, boundaries with more aggressive B-cell proliferations, and association with EBV were explored.B-cell proliferations of varied malignant potential occurred in all immunodeficiency backgrounds. Presentation early in the course of immunodeficiency and in younger age groups and regression with reduction of immunosuppression were characteristic features. EBV positivity was essential for diagnosis in some hyperplasias where other specific defining features were absent.This spectrum of B-cell proliferations show similarities across immunodeficiency backgrounds. Localized forms of immunodeficiency disorders arise in immunocompetent patients most likely due to chronic immune stimulation and, despite aggressive histologic features, often show indolent clinical behavior.

    View details for DOI 10.1093/ajcp/aqw214

    View details for Web of Science ID 000397108600002

    View details for PubMedID 28395107

  • T- and NK-Cell Lymphomas and Systemic Lymphoproliferative Disorders and the Immunodeficiency Setting 2015 SH/EAHP Workshop Report-Part 4 AMERICAN JOURNAL OF CLINICAL PATHOLOGY Gratzinger, D., De Jong, D., Jaffe, E. S., Chadburn, A., Chan, J. K., Goodlad, J. R., Said, J., Natkunam, Y. 2017; 147 (2): 188-203


    The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review immunodeficiency-related T- and natural killer (NK)-cell lymphoproliferations.The Workshop Panel reviewed 88 T- or NK-cell lymphoproliferations and rendered consensus diagnoses.Hyperplasias of T-cell subsets may be clonal; retained architecture and the clinical setting support a benign diagnosis. Specific associations include hepatosplenic T-cell lymphoma with iatrogenic immunosuppression and breast implants with an indolent variant of anaplastic large cell lymphoma. Epstein-Barr virus (EBV)-positive T-cell lymphomas rarely occur in the acquired immunodeficiency setting. Systemic T- and NK-cell lymphoma of childhood overlaps with chronic active EBV and reversible hemophagocytic lymphohistiocytosis-related T-cell lymphoproliferations.Immunodeficiencies predispose to T-cell hyperplasias, which must not be overdiagnosed as lymphoma. Many T-cell lymphomas in the immunodeficiency setting are likely coincidental, with specific exceptions. Systemic T- or NK-cell lymphomas are part of a spectrum of EBV+ T or NK lymphoproliferations and can present in the acquired immunodeficiency setting.

    View details for DOI 10.1093/ajcp/aqw213

    View details for Web of Science ID 000397108600005

    View details for PubMedID 28395105

  • HHV8/KSHV-Positive Lymphoproliferative Disorders and the Spectrum of Plasmablastic and Plasma Cell Neoplasms 2015 SH/EAHP Workshop Report-Part 3 AMERICAN JOURNAL OF CLINICAL PATHOLOGY Md, A. C., Said, J., Gratzinger, D., Chan, J. K., De Jong, D., Jaffe, E. S., Natkunam, Y., Goodlad, J. R. 2017; 147 (2): 171-187


    The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review immunodeficiency-related lymphoproliferative disorders with plasmablastic and plasma cell differentiation.The workshop panel reviewed human herpes virus 8 (HHV8)/Kaposi sarcoma herpesvirus (KSHV)-associated lesions and other lesions exhibiting plasma cell differentiation, including plasmablastic proliferations with features of myeloma/plasmacytoma, plasmablastic neoplasms presenting in extranodal sites and effusion-based lymphomas, and rendered a consensus diagnosis.The spectrum of HHV8/KSHV-associated proliferations ranged from multicentric Castleman disease (MCD) to MCD with plasmablastic aggregates to HHV8+ diffuse large B-cell lymphoma and germinotrophic lymphoproliferative disorder. Comparisons across effusion-based lymphomas with and without HHV8/KSHV and plasmablastic lymphomas in immunodeficient and immunocompetent patients were discussed.The presence or absence of HHV8/KSHV is a defining feature in disorders associated with Castleman disease, although their differential diagnosis and recognition of progression may be challenging. Plasmablastic proliferations overlap with myeloma/plasmacytoma as well as extranodal and effusion-based lymphomas. The involvement of Epstein-Barr virus is typically variable.

    View details for DOI 10.1093/ajcp/aqw218

    View details for Web of Science ID 000397108600004

    View details for PubMedID 28395104

  • Classical Hodgkin Lymphoma with Reduced ß2M/MHC Class I Expression Is Associated with Inferior Outcome Independent of 9p24.1 Status. Cancer immunology research Roemer, M. G., Advani, R. H., Redd, R. A., Pinkus, G. S., Natkunam, Y., Ligon, A. H., Connelly, C. F., Pak, C. J., Carey, C. D., Daadi, S. E., Chapuy, B., De Jong, D., Hoppe, R. T., Neuberg, D. S., Shipp, M. A., Rodig, S. J. 2016; 4 (11): 910-916


    In classical Hodgkin lymphoma (cHL), malignant Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple mechanisms, including perturbed antigen presentation and enhanced PD-1 signaling. HRS cell expression of the PD-1 ligands is attributable, in part, to copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) Amplification of PD-L1/PD-L2 is associated with advanced clinical stage and inferior progression-free survival (PFS) following first-line (induction) therapy. The relationships between altered expression of β2-microglobulin (β2M), MHC class I, and MHC class II by HRS cells, PD-L1/PD-L2 amplification, and clinical outcome in cHL are poorly defined. We assessed these variables in diagnostic biopsy specimens from 108 patients with cHL who received uniform treatment and had long-term follow-up and found decreased/absent expression of β2M/MHC class I in 79% (85/108) and decreased/absent expression of MHC class II in 67% (72/108) of cases. Patients with decreased/absent β2M/MHC class I had shorter PFS, independent of PD-L1/PD-L2 amplification and advanced stage. Decreased or absent MHC class II was unrelated to outcome. These results suggest that MHC class I-mediated antigen presentation by HRS cells is an important component of the biological response to standard chemo/radiotherapy. The paucity of β2M/MHC class I expression on HRS cells also prompts speculation regarding alternative mechanisms of action of PD-1 blockade in cHL. Cancer Immunol Res; 4(11); 910-6. ©2016 AACR.

    View details for PubMedID 27737878

    View details for PubMedCentralID PMC5210180

  • PD-L1 and PD-L2 Genetic Alterations Define Classical Hodgkin Lymphoma and Predict Outcome. Journal of clinical oncology Roemer, M. G., Advani, R. H., Ligon, A. H., Natkunam, Y., Redd, R. A., Homer, H., Connelly, C. F., Sun, H. H., Daadi, S. E., Freeman, G. J., Armand, P., Chapuy, B., De Jong, D., Hoppe, R. T., Neuberg, D. S., Rodig, S. J., Shipp, M. A. 2016; 34 (23): 2690-2697


    Classical Hodgkin lymphomas (cHLs) include small numbers of malignant Reed-Sternberg cells within an extensive but ineffective inflammatory/immune cell infiltrate. In cHL, chromosome 9p24.1/PD-L1/PD-L2 alterations increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and their further induction through Janus kinase 2-signal transducers and activators of transcription signaling. The unique composition of cHL limits its analysis with high-throughput genomic assays. Therefore, the precise incidence, nature, and prognostic significance of PD-L1/PD-L2 alterations in cHL remain undefined.We used a fluorescent in situ hybridization assay to evaluate CD274/PD-L1 and PDCD1LG2/PD-L2 alterations in 108 biopsy specimens from patients with newly diagnosed cHL who were treated with the Stanford V regimen and had long-term follow-up. In each case, the frequency and magnitude of 9p24.1 alterations-polysomy, copy gain, and amplification-were determined, and the expression of PD-L1 and PD-L2 was evaluated by immunohistochemistry. We also assessed the association of 9p24.1 alterations with clinical parameters, which included stage (early stage I/II favorable risk, early stage unfavorable risk, advanced stage [AS] III/IV) and progression-free survival (PFS).Ninety-seven percent of all evaluated cHLs had concordant alterations of the PD-L1 and PD-L2 loci (polysomy, 5% [five of 108]; copy gain, 56% [61 of 108]; amplification, 36% [39 of 108]). There was an association between PD-L1 protein expression and relative genetic alterations in this series. PFS was significantly shorter for patients with 9p24.1 amplification, and the incidence of 9p24.1 amplification was increased in patients with AS cHL.PD-L1/PD-L2 alterations are a defining feature of cHL. Amplification of 9p24.1 is more common in patients with AS disease and associated with shorter PFS in this series. Further analyses of 9p24.1 alterations in patients treated with standard cHL induction regimens or checkpoint blockade are warranted.

    View details for DOI 10.1200/JCO.2016.66.4482

    View details for PubMedID 27069084

  • Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma. Blood Matthews, J. M., Bhatt, S., Patricelli, M. P., Nomanbhoy, T. K., Jiang, X., Natkunam, Y., Gentles, A. J., Martinez, E., Zhu, D., Chapman, J. R., Cortizas, E., Shyam, R., Chinichian, S., Advani, R., Tan, L., Zhang, J., Choi, H. G., Tibshirani, R., Buhrlage, S. J., Gratzinger, D., Verdun, R., Gray, N. S., Lossos, I. S. 2016; 128 (2): 239-248


    Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), yet 40-50% of patients will eventually succumb to their disease demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNA's that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that Germinal Center Kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Since the majority of DLBCL tumors (~80%) exhibit activation of GCK, this therapy may be applicable to most patients.

    View details for DOI 10.1182/blood-2016-02-696856

    View details for PubMedID 27151888

  • Myeloid Cell Nuclear Differentiation Antigen (MNDA) Expression Distinguishes Extramedullary Presentations of Myeloid Leukemia From Blastic Plasmacytoid Dendritic Cell Neoplasm AMERICAN JOURNAL OF SURGICAL PATHOLOGY Johnson, R. C., Kim, J., Natkunam, Y., Sundram, U., Freud, A. G., Gammon, B., Cascio, M. J. 2016; 40 (4): 502-509


    Myeloid neoplasms constitute one of the most common malignancies in adults. In most cases these proliferations initially manifest in the blood and marrow; however, extramedullary involvement may precede blood or marrow involvement in a subset of cases, making a definitive diagnosis challenging by morphologic and immunohistochemical assessment alone. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive entity that frequently presents in extramedullary sites and can show morphologic and immunophenotypic overlap with myeloid neoplasms. Given that BPDCN and myeloid neoplasms may both initially present in extramedullary sites and that novel targeted therapies may be developed that exploit the unique molecular signature of BPDCN, new immunophenotypic markers that can reliably separate myeloid neoplasms from BPDCN are desirable. We evaluated the utility of myeloid cell nuclear differentiation antigen (MNDA) expression in a series of extramedullary myeloid leukemias (EMLs) and BPDCN. Forty biopsies containing EML and 19 biopsies containing BPDCN were studied by MNDA immunohistochemistry. The majority of myeloid neoplasms showed nuclear expression of MNDA (65%). In contrast, all cases of BPDCN lacked MNDA expression. These findings show that MNDA is expressed in the majority of EMLs and support the inclusion of MNDA immunohistochemistry in the diagnostic evaluation of blastic hematopoietic infiltrates, particularly when the differential diagnosis is between myeloid leukemia and BPDCN.

    View details for Web of Science ID 000374901700011

    View details for PubMedID 26796502

  • Isolated Follicles Enriched for Centroblasts and Lacking t(14;18)/BCL2 in Lymphoid Tissue: Diagnostic and Clinical Implications PLOS ONE Nybakken, G. E., Bala, R., Gratzinger, D., Jones, C. D., Zehnder, J. L., Bangs, C. D., Cherry, A., Warnke, R. A., Natkunam, Y. 2016; 11 (3)
  • A single-institution retrospective analysis of outcomes for stage I-II primary mediastinal large B-cell lymphoma treated with immunochemotherapy with or without radiotherapy LEUKEMIA & LYMPHOMA Binkley, M. S., Hiniker, S. M., Wu, S., Natkunam, Y., Mittra, E. S., Advani, R. H., Hoppe, R. T. 2016; 57 (3): 604-608
  • An analysis of MYC and EBV in diffuse large B-cell lymphomas associated with angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified HUMAN PATHOLOGY Hoffmann, J. C., Chisholm, K. M., Cherry, A., Chen, J., Arber, D. A., Natkunam, Y., Warnke, R. A., Ohgami, R. S. 2016; 48: 9-17
  • The Epstein-Barr Virus (EBV) in T Cell and NK Cell Lymphomas: Time for a Reassessment CURRENT HEMATOLOGIC MALIGNANCY REPORTS Gru, A. A., Haverkos, B. H., Freud, A. G., Hastings, J., Nowacki, N. B., Barrionuevo, C., Vigil, C. E., Rochford, R., Natkunam, Y., Baiocchi, R. A., Porcu, P. 2015; 10 (4): 456-467


    While Epstein-Barr virus (EBV) was initially discovered and characterized as an oncogenic virus in B cell neoplasms, it also plays a complex and multifaceted role in T/NK cell lymphomas. In B cell lymphomas, EBV-encoded proteins have been shown to directly promote immortalization and proliferation through stimulation of the NF-κB pathway and increased expression of anti-apoptotic genes. In the context of mature T/NK lymphomas (MTNKL), with the possible exception on extranodal NK/T cell lymphoma (ENKTL), the virus likely plays a more diverse and nuanced role. EBV has been shown to shape the tumor microenvironment by promoting Th2-skewed T cell responses and by increasing the expression of the immune checkpoint ligand PD-L1. The type of cell infected, the amount of plasma EBV DNA, and the degree of viral lytic replication have all been proposed to have prognostic value in T/NK cell lymphomas. Latency patterns of EBV infection have been defined using EBV-infected B cell models and have not been definitively established in T/NK cell lymphomas. Identifying the expression profile of EBV lytic proteins could allow for individualized therapy with the use of antiviral medications. More work needs to be done to determine whether EBV-associated MTNKL have distinct biological and clinical features, which can be leveraged for risk stratification, disease monitoring, and therapeutic purposes.

    View details for DOI 10.1007/s11899-015-0292-z

    View details for Web of Science ID 000366354900015

    View details for PubMedID 26449716

  • Immunoarchitectural patterns of progressive transformation of germinal centers with and without nodular lymphocyte-predominant Hodgkin lymphoma HUMAN PATHOLOGY Hartmann, S., Winkelmann, R., Metcalf, R. A., Treetipsatit, J., Warnke, R. A., Natkunam, Y., Hansmann, M. 2015; 46 (11): 1655-1661


    Progressive transformation of germinal centers (PTGC) has been frequently described in association with Hodgkin lymphoma, particularly nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). The aim of this study was to evaluate morphologic features of PTGC for better delineation of PTGC from early involvement by NLPHL. A total of 160 cases of PTGC were evaluated and included in the following 3 groups: 93 patients with PTGC who never developed a lymphoma, 23 patients with synchronous PTGC and NLPHL, and 44 patients with PTGC with antecedent or subsequent history of lymphoma. By histopathologic evaluation, 5 patterns of PTGC that reflected progressive dismantling of germinal centers were identified. There was no difference in the distribution of patterns 1 to 4 among the 3 groups of PTGC; however, in patients showing synchronous involvement of PTGC and NLPHL, pattern 5, which resembles a naïve B-cell follicle, was significantly more frequently observed (14/23) when compared with patients with PTGC who never developed a lymphoma (30/93; P = .0161). Furthermore, recognition of the spectrum of immunoarchitectural patterns of PTGC, including architectural and cytologic features, was helpful to better differentiate nodules involved by PTGC from NLPHL.

    View details for DOI 10.1016/j.humpath.2015.07.006

    View details for Web of Science ID 000364360600011

    View details for PubMedID 26410017

  • Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF-kappa B pathway INTERNATIONAL JOURNAL OF CANCER Ramachandiran, S., Adon, A., Guo, X., Wang, Y., Wang, H., Chen, Z., Kowalski, J., Sunay, U. R., Young, A. N., Brown, T., Mar, J. C., Du, Y., Fu, H., Mann, K. P., Natkunam, Y., Boise, L. H., Saavedra, H. I., Lossos, I. S., Bernal-Mizrachi, L. 2015; 136 (10): 2341-2351


    Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

    View details for DOI 10.1002/ijc.29301

    View details for Web of Science ID 000351208300010

    View details for PubMedID 25359525

  • Management of Nodular Lymphocyte Predominant Hodgkin Lymphoma in the Modern Era INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS King, M. T., Donaldson, S. S., Link, M. P., Natkunam, Y., Advani, R. H., Hoppe, R. T. 2015; 92 (1): 67-75


    To analyze treatment outcomes for nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) at a single institution.Patients with newly diagnosed NLPHL between 1996 and 2013 were reviewed retrospectively. Patients treated before 1996 were excluded because the majority received extended field radiation therapy (RT) alone.Fifty-five patients (22 ≤ 21 years old) were identified. The median follow-up time was 6.8 years. Among 37 patients with limited-stage (I-II) disease, treatments included involved field RT at a median dose of 36 Gy (n=9), rituximab monotherapy (n=9), observation (n=3), and response-adaptive therapy (n=16), in which the RT dose was reduced from 25.5 Gy to 15 Gy or was eliminated based on interim imaging after chemotherapy. The 5-year progression-free survival (PFS) was 76.4% (95% confidence interval [CI], 63.1-92.4). Nine patients experienced progression, including 5 receiving rituximab, 2 undergoing observation, and 2 receiving response-adaptive therapy. Rituximab was associated with an inferior PFS compared with RT alone (P=.02). The difference in PFS between response-adaptive therapy and RT alone was not statistically significant (P=.39). Among 18 patients with advanced-stage (III-IV) disease, treatments included chemotherapy alone (n=3), combined modality therapy (CMT) (n=2), response-adaptive therapy (n=2), rituximab (n=7), and observation (n=4). The 5-year PFS was 29.9% (CI, 13.3-67.4). Twelve patients experienced progression, including 1 receiving chemotherapy, 1 receiving CMT, 6 receiving rituximab, and 4 undergoing observation. There was no significant PFS difference between rituximab and non-rituximab therapies (P=.19) within the caveat of small sample sizes. In the entire cohort, 9 patients (3 with limited disease, 6 with advanced disease) experienced large cell transformation (LCT). Seven patients died; of those, 5 died with LCT.For limited disease, response-adaptive therapy demonstrated comparable outcomes with RT alone. Rituximab monotherapy resulted in inferior outcomes for limited disease and a high relapse rate for advanced disease.

    View details for DOI 10.1016/j.ijrobp.2015.02.001

    View details for Web of Science ID 000353988200011

    View details for PubMedID 25863755

  • Expression Profiles of MYC Protein and MYC Gene Rearrangement in Lymphomas AMERICAN JOURNAL OF SURGICAL PATHOLOGY Chisholm, K. M., Bangs, C. D., Bacchi, C. E., Molina-Kirsch, H., Cherry, A., Natkunam, Y. 2015; 39 (3): 294-303


    MYC translocations are a defining feature of Burkitt lymphoma and a group of diffuse large B-cell lymphoma (DLBCL) with inferior outcome. However, the clinical relevance of MYC gene rearrangement and its relationship with MYC protein expression has not been well characterized in lymphomas. Tissue microarrays containing 1214 lymphomas were successfully evaluated by immunohistochemistry using anti-MYC clone Y69 and a dual-color break-apart fluorescence in situ hybridization probe to detect MYC gene rearrangements. Aggressive B-cell lymphomas including Burkitt lymphoma and DLBCL showed the highest level of MYC protein staining defined as staining in >50% of lymphoma cells. A significant proportion of plasmablastic, B-lymphoblastic and T-lymphoblastic, and extranodal NK/T-cell lymphomas also showed staining in >50% of cells, whereas only occasional plasma cell myeloma, mantle cell lymphoma, and classical Hodgkin lymphoma showed a high level of staining. Small B-cell lymphomas, when positive, showed MYC protein in <50% of cells. In aggressive B-cell lymphomas, MYC rearrangement and MYC immunohistochemistry showed a high concordance rate; however, some DLBCL and all T-cell and NK-cell lymphomas with MYC protein expression lacked MYC gene rearrangements. Our results provide a baseline for MYC protein expression in lymphomas and indicate that its expression is not specific to lymphoma subtypes, cell lineage, or expected clinical behavior and is highly variable. In addition, MYC protein expression is not necessarily correlated with MYC gene rearrangements and suggests the need for caution in the interpretation of MYC immunohistochemistry in the differential diagnosis of lymphomas.

    View details for Web of Science ID 000349803400002

    View details for PubMedID 25581730

  • Large B-cell lymphoma with T-cell-rich background and nodules lacking follicular dendritic cell meshworks: description of an insufficiently recognized variant HUMAN PATHOLOGY Treetipsatit, J., Metcalf, R. A., Warnke, R. A., Natkunam, Y. 2015; 46 (1): 74-83


    Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is characterized by nodular or nodular and diffuse growth of scattered large neoplastic B cells associated with follicular dendritic cell (FDC) meshworks. Variant patterns, which at least focally show a T-cell-rich background, and rare cases lacking FDC meshworks that overlap with T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) are also recognized. We reviewed 195 cases spanning the diagnostic spectrum of NLPHL and THRLBCL and identified 5 cases with distinctive features that were difficult to classify according to the World Health Organization criteria or previously described variants. Clinically, they involved peripheral and central lymph node sites or the mediastinum, and the majority also had recurrent disease. Four cases showed large T-cell-rich nodules with fibrosis, and 1 showed diffuse THRLBCL-like pattern with a minor component of nodularity. All cases completely lacked FDC meshworks despite a prominent nodular growth pattern. Large atypical cells in all cases were CD20+ CD30- CD15- B cells, although a small subset (<10%) of CD30+ and CD15+ large cells were seen in 1 case. In 4 cases, the background mainly contained CD4+ PD-1+ or CD57+ T cells that ringed large atypical B cells. In 1 case, B-cell predominance and a ringing pattern of CD57+ T cells were noted in nodules, whereas they were lacking in the diffuse areas. Recognition of these variant cases expands the spectrum between NLPHL and THRLBCL and points to the need for further refinement of diagnostic criteria for appropriate classification and clinical management.

    View details for DOI 10.1016/j.humpath.2014.09.009

    View details for Web of Science ID 000346540100010

    View details for PubMedID 25456392

  • Variable Expression of B-cell Transcription Factors in Reactive Immunoblastic Proliferations A Potential Mimic of Classical Hodgkin Lymphoma AMERICAN JOURNAL OF SURGICAL PATHOLOGY Treetipsatit, J., Rimzsa, L., Grogan, T., Warnke, R. A., Natkunam, Y. 2014; 38 (12): 1655-1663


    Reactive immunoblastic proliferations can histologically mimic classical Hodgkin lymphoma (CHL), and show diffuse CD30 expression in large cells. The lack of expression of CD15 in a subset of CHL further complicates their separation from immunoblastic proliferations. Loss of expression of B-cell transcription factors is frequently exploited in making a diagnosis of CHL; however, the staining patterns of B-cell transcription factors in immunoblastic proliferations have not been extensively studied. Thirty-three cases of reactive immunoblastic proliferations were evaluated using a panel of immunohistochemistry for CD30, CD15, CD20, CD3, κ, λ, CD45RB, MUM1, PAX5, OCT2, and BOB.1, as well as Epstein-Barr virus (EBV)/EBV-encoded ribonucleic acid in situ hybridization. A newly developed dual-color chromogenic in situ hybridization technology for detection of κ/λ mRNAs was also used. The majority of immunoblasts expressed CD30 in 14 of 33 (42%) cases; none expressed CD15. Loss or weak expression of at least 1 transcription factor in B immunoblasts, most commonly PAX5, was noted in 24 of 29 (83%) cases. A polytypic light chain expression pattern was detected by immunohistochemistry in 14 of 22 (63.6%) cases and by dual-color chromogenic in situ hybridization in 9 of 10 (90%) cases studied. EBV-encoded ribonucleic acid was detected in 8 of 33 (24.2%) cases, 5 of which were clinically unrelated to infectious mononucleosis. We conclude that B-cell transcription factors can show loss or weak expression in a significant proportion of reactive immunoblastic proliferations, and, therefore, staining for B-cell transcription factors together with CD30 should be interpreted with caution before a diagnosis of CHL is made.

    View details for Web of Science ID 000345131700010

    View details for PubMedID 24921642

  • Indolent T-lymphoblastic Proliferation With Disseminated Multinodal Involvement and Partial CD33 Expression. American journal of surgical pathology Ohgami, R. S., Sendamarai, A. K., Atwater, S. K., Liedtke, M., Fleming, M. D., Natkunam, Y., Warnke, R. A. 2014; 38 (9): 1298-1304


    Although indolent T-lymphoblastic proliferations (iT-LBP) are rare, this diagnosis should be excluded in any patient with an extrathymic proliferation of immature TdT+T cells. Unlike T-lymphoblastic leukemia/lymphoma, patients with iT-LBP do not require chemotherapy. We report a case of iT-LBP with disseminated multinodal involvement in an otherwise healthy 49-year-old woman. Multiple lymph node biopsies were performed over the course of several months demonstrating persistent and anatomically diffuse involvement. Over 18 months, and without therapy, she has remained healthy, and her lymphadenopathy significantly improved. No bone marrow or peripheral blood involvement was ever identified. Atypical T cells showed an immunophenotypic spectrum of T-cell antigen expression with partial CD33 on a subset of T cells detected by both flow cytometry and immunohistochemistry. Both T-cell clonality and Human Androgen Receptor Assay (HUMARA) studies, performed on lymph node biopsy specimens, were negative. This case represents the first detailed clinical, morphologic, molecular, and immunophenotypic description of disseminated multinodal involvement by nonclonal iT-LBP with partial CD33 expression on T cells.

    View details for DOI 10.1097/PAS.0000000000000197

    View details for PubMedID 24618611

  • Large B-Cell Lymphomas Poor in B Cells and Rich in PD-1+ T Cells Can Mimic T-Cell Lymphomas. American journal of clinical pathology Ohgami, R. S., Zhao, S., Natkunam, Y. 2014; 142 (2): 150-156


    To characterize the clinicopathologic features of cases of large B-cell lymphomas, poor in B cells and densely rich in programmed cell death-1 (PD-1)+ reactive T cells, which can mimic T-cell lymphomas.A single-institute retrospective review of cases between 2010 and 2013 was performed.Of 178 cases of large B-cell lymphomas, eight cases of large B-cell lymphomas poor in B cells and diffusely rich in sheets of PD-1+ T cells were identified. These cases either were initially misdiagnosed as a T-cell lymphoma or substantiated a broader differential diagnosis including a T-cell lymphoma. Five cases were T-cell histiocyte-rich large B-cell lymphomas, and three cases were diagnosed as large B-cell lymphomas rich in T cells. In three of these cases, a subset of the PD-1+ T cells showed either morphologic nuclear atypia or atypical expression of T-cell antigens on flow cytometry and/or immunohistochemistry.Large B-cell lymphomas poor in B cells and rich in T cells can have diffuse sheets of reactive PD-1+ T cells, some with atypical morphologic and immunophenotypic features mimicking a T-cell lymphoma. Careful assessment of the immunoarchitecture and background inflammatory and stromal cells can prevent erroneous diagnoses in such cases.

    View details for DOI 10.1309/AJCPFJWKQ6GTVQE6

    View details for PubMedID 25015854

  • Myeloid cell nuclear differentiation antigen is expressed in a subset of marginal zone lymphomas and is useful in the differential diagnosis with follicular lymphoma HUMAN PATHOLOGY Metcalf, R. A., Monabati, A., Vyas, M., Roncador, G., Gualco, G., Bacchi, C. E., Younes, S. F., Natkunam, Y., Freud, A. G. 2014; 45 (8): 1730-1736


    The diagnosis of marginal zone lymphomas (MZL) is challenged by the lack of specific markers that distinguish them from other low-grade non-Hodgkin B-cell lymphomas. Myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein that labels myelomonocytic cells as well as B lymphocytes that localize to the marginal zone areas of splenic white pulp. We evaluated MNDA expression in a large series of B-cell lymphomas to assess the sensitivity and specificity of this antigen for the characterization of MZL. A total of 440 tissue sections containing extramedullary B-cell lymphomas and 216 bone marrow biopsies containing atypical or neoplastic lymphoid infiltrates were stained for MNDA by immunohistochemistry. Among the extramedullary lymphoma cases, approximately 67% of nodal MZL, 61% of extranodal MZL, and 24% of splenic MZL expressed MNDA. MNDA was also infrequently expressed in other B-cell neoplasms including mantle cell lymphoma (6%), chronic lymphocytic leukemia/small lymphocytic lymphoma (13%), follicular lymphoma (FL) (4%), lymphoplasmacytic lymphoma (25%), and diffuse large B-cell lymphoma (3%). In contrast, MNDA was only expressed in 2.3% of all bone marrow biopsies involved by lymphoid infiltrates, including 2 cases of FL and one case of MZL. Collectively, these data support the inclusion of MNDA in the diagnostic evaluation of extramedullary B-cell lymphomas, particularly those in which the differential diagnosis is between low-grade FL and MZL.

    View details for DOI 10.1016/j.humpath.2014.04.004

    View details for Web of Science ID 000339642100023

    View details for PubMedID 24925224

  • Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas DIAGNOSTIC PATHOLOGY Rimsza, L. M., Day, W. A., McGinn, S., Pedata, A., Natkunam, Y., Warnke, R., Cook, J. R., Marafioti, T., Grogan, T. M. 2014; 9


    Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.The virtual slide(s) for this article can be found here:

    View details for DOI 10.1186/1746-1596-9-144

    View details for Web of Science ID 000341387600001

    View details for PubMedID 25047073

  • LMO2 and BCL6 are associated with improved survival in primary central nervous system lymphoma BRITISH JOURNAL OF HAEMATOLOGY Lossos, C., Bayraktar, S., Weinzierl, E., Younes, S. F., Hosein, P. J., Tibshirani, R. J., Posthumus, J. S., DeAngelis, L. M., Raizer, J., Schiff, D., Abrey, L., Natkunam, Y., Lossos, I. S. 2014; 165 (5): 640-648


    Primary central nervous system lymphoma (PCNSL) is an aggressive sub-variant of non-Hodgkin lymphoma (NHL) with morphological similarities to diffuse large B-cell lymphoma (DLBCL). While methotrexate (MTX)-based therapies have improved patient survival, the disease remains incurable in most cases and its pathogenesis is poorly understood. We evaluated 69 cases of PCNSL for the expression of HGAL (also known as GCSAM), LMO2 and BCL6 - genes associated with DLBCL prognosis and pathobiology, and analysed their correlation to survival in 49 PCNSL patients receiving MTX-based therapy. We demonstrate that PCNSL expresses LMO2, HGAL(also known as GCSAM) and BCL6 proteins in 52%, 65% and 56% of tumours, respectively. BCL6 protein expression was associated with longer progression-free survival (P = 0·006) and overall survival (OS, P = 0·05), while expression of LMO2 protein was associated with longer OS (P = 0·027). Further research is needed to elucidate the function of BCL6 and LMO2 in PCNSL.

    View details for DOI 10.1111/bjh.12801

    View details for Web of Science ID 000335826500008

    View details for PubMedID 24571259

  • Use of CD137 ligand expression in the detection of small B-cell lymphomas involving the bone marrow HUMAN PATHOLOGY Zhao, S., Xing, Y., Natkunam, Y. 2014; 45 (5): 1024-1030


    Staging for small B-cell lymphomas is important for prognostic and therapeutic decision making; however, the detection of lymphoid infiltrates in the bone marrow is often hampered by the lack of specific diagnostic markers. We recently described the hematopoietic tissue distribution patterns of CD137 and CD137 ligand (CD137L), which have shown promise as immunotherapeutic targets. CD137 expression was primarily confined to cells in the microenvironment, whereas CD137L was expressed in neoplastic cells in most B-cell lymphomas. Here we evaluate the use of CD137L in the detection of small B-cell lymphomas involving the bone marrow. To test the potential efficacy of CD137L in detecting bone marrow lymphoid infiltrates, 166 small B-cell lymphomas were evaluated by immunohistochemistry and double-immunofluorescence labeling on formalin-fixed, paraffin-embedded bone marrow core biopsies. CD137L was highly expressed in bone marrows involved by small B-cell lymphomas and included hairy cell leukemia, mantle cell lymphoma, follicular lymphoma, B-lymphoblastic leukemia, and chronic lymphocytic leukemia. In addition, a small subset of marginal zone lymphoma and most of lymphoplasmacytic lymphoma showed staining. Normal bone marrow cells including myeloid, erythroid and megakaryocytic precursors, and reactive lymphoid aggregates lacked staining. Our findings show that immunohistochemistry for CD137L is capable of reliably distinguishing small B-cell lymphomas from reactive lymphoid aggregates. These data also suggest that CD137L is useful in providing staging information for clinical diagnosis and is likely to furnish a potential target for minimal residual disease assessment as well as immunotherapy in patients with stage 4 disease.

    View details for DOI 10.1016/j.humpath.2013.12.019

    View details for Web of Science ID 000335093800015

    View details for PubMedID 24746207

  • Bevacizumab and cyclosphosphamide, doxorubicin, vincristine and prednisone in combination for patients with peripheral T-cell or natural killer cell neoplasms: an Eastern Cooperative Oncology Group study (E2404). Leukemia & lymphoma Ganjoo, K., Hong, F., Horning, S. J., Gascoyne, R. D., Natkunam, Y., Swinnen, L. J., Habermann, T. M., Kahl, B. S., Advani, R. H. 2014; 55 (4): 768-772


    Peripheral T-cell lymphoma (PTCL) and natural killer (NK) cell lymphoma have poor survival with conventional cytotoxic chemotherapy. Because angiogenesis plays an important role in the biology of PTCL, a fully humanized anti-vascular endothelial growth factor (VEGF) antibody, bevacizumab (A), was studied in combination with standard cyclosphosphamide, doxorubicin, vincristine and prednisone (CHOP) chemotherapy (ACHOP) to evaluate its potential to improve outcome in these patients. Patients were treated with 6-8 cycles of ACHOP followed by eight doses of maintenance A (15 mg/kg every 21 days). Forty-six patients were enrolled on this phase 2 study from July 2006 through March 2009. Forty-four patients were evaluable for toxicity and 39 were evaluable for response, progression and survival. A total of 324 cycles (range: 2-16, median 7) were administered to 39 evaluable patients and only nine completed all planned treatment. The overall response rate was 90% with 19 (49%) complete response/complete response unconfirmed (CR/CRu) and 16 (41%) a partial response (PR). The 1-year progression-free survival (PFS) rate was 44% at a median follow-up of 3 years. The median PFS and overall survival (OS) rates were 7.7 and 22 months, respectively. Twenty-three patients died (21 from lymphoma, two while in remission). Grade 3 or 4 toxicities included febrile neutropenia (n = 8), anemia (n = 3), thrombocytopenia (n = 5), congestive heart failure (n = 4), venous thrombosis (n = 3), gastrointestinal hemorrhage/perforation (n = 2), infection (n = 8) and fatigue (n = 6). Despite a high overall response rate, the ACHOP regimen failed to result in durable remissions and was associated with significant toxicities. Studies of novel therapeutics are needed for this patient population, whose clinical outcome remains poor.

    View details for DOI 10.3109/10428194.2013.816700

    View details for PubMedID 23786456

  • Multiplexed ion beam imaging of human breast tumors. Nature medicine Angelo, M., Bendall, S. C., Finck, R., Hale, M. B., Hitzman, C., Borowsky, A. D., Levenson, R. M., Lowe, J. B., Liu, S. D., Zhao, S., Natkunam, Y., Nolan, G. P. 2014; 20 (4): 436-442

    View details for DOI 10.1038/nm.3488

    View details for PubMedID 24584119

  • FHIT, EGFR, and MSH2: Possible Etiopathologic, Prognostic, and Predictive Role in Non-Small Cell Lung Carcinoma in Egyptian Patients APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Younes, S. F., Aiad, H. A., Asaad, N. Y., Kandil, M. A., Natkunam, Y., Mokhtar, N. M. 2014; 22 (4): 275-283


    The high incidence and mortality of lung carcinoma in Egypt necessitates studying the factors that may be implicated in non-small cell lung carcinoma (NSCLC) pathogenesis and could affect patient management. The aim was to study FHIT, epidermal growth factor receptor (EGFR), and MSH2 protein expression in Egyptian patients with NSCLC. Immunohistochemical staining for FHIT, EGFR, and MSH2 was performed on 64 specimens from NSCLC patients and correlated with prognostic parameters, response to therapy, and overall survival. FHIT loss was observed in 64% of NSCLC patients and was significantly associated with SCC (P=0.003) and poor tumor grade (P=0.043). EGFR overexpression was observed in 47% of NSCLC patients and was significantly associated with SCC (P=0.002). MSH2 was reduced in 23.4% of NSCLC patients and was significantly associated with adenocarcinoma (P=0.024). In a univariate analysis, a significant relationship was seen between the poor overall survival in NSCLC patients and high T-stage (P=0.029), presence of metastasis (P=0.014), advanced-stage grouping (P=0.004), and FHIT loss (P=0.033). Further, FHIT loss was significantly related to disease progression in patients treated with chemotherapy (P=0.038). We conclude that all 3 markers play a role in the development of NSCLC in Egyptian patients. We suggest that FHIT loss be used as a predictor for progression in chemotherapy-treated NSCLC patients.

    View details for DOI 10.1097/PAI.0b013e3182988fa5

    View details for Web of Science ID 000334606700006

    View details for PubMedID 24185125

  • Mature results of a phase II study of rituximab therapy for nodular lymphocyte-predominant Hodgkin lymphoma. Journal of clinical oncology Advani, R. H., Horning, S. J., Hoppe, R. T., Daadi, S., Allen, J., Natkunam, Y., Bartlett, N. L. 2014; 32 (9): 912-918


    Universal expression of CD20 by malignant cells in nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) led us to evaluate rituximab (R) as a therapeutic option.Patients with previously treated or newly diagnosed NLPHL were treated with R (375 mg/m(2) once per week for 4 weeks) or, after a protocol amendment, with R plus R maintenance (MR; administered once every 6 months for 2 years). Primary and secondary outcome measures were progression-free survival (PFS) and overall response rate (ORR), respectively.A total of 39 patients were enrolled (R, n = 23; R + MR, n = 16). After four once-per-week treatments, ORR was 100% (complete response, 67%; partial response, 33%). At median follow-ups of 9.8 years for R and 5 years for R + MR, median PFS were 3 and 5.6 years (P = .26), respectively; median overall survival (OS) was not reached. Estimated 5-year PFS and OS for patients treated with R versus R + MR were 39.1% (95% CI, 23.5 to 65.1) and 95.7% (95% CI, 87.7 to 100) versus 58.9% (95% CI, 38.0 to 91.2) and 85.7% (95% CI, 69.2 to 100), respectively. Nine of 23 patients experiencing relapse had evidence of transformation to aggressive B-cell lymphoma; six of these patients had infradiaphragmatic involvement at study entry.R is an active agent in NLPHL. Although responses are not durable in most patients, a significant minority experience remissions lasting > 5 years. R + MR results in a nonsignificant increase in PFS compared with R. R may be considered in the relapsed setting for NLPHL. The potential for transformation of NLPHL to aggressive B-cell lymphoma underscores the importance of rebiopsy and long-term follow-up.

    View details for DOI 10.1200/JCO.2013.53.2069

    View details for PubMedID 24516013

  • LMO2 (LIM domain only 2) is expressed in a subset of acute myeloid leukaemia and correlates with normal karyotype HISTOPATHOLOGY Patel, J. L., Pournazari, P., Haggstrom, S., Kosari, F., Shabani-Rad, M., Natkunam, Y., Mansoor, A. 2014; 64 (2): 226-233


    LMO2 is a transcription factor that plays a key role in haematopoiesis. Expression of LMO2 has been demonstrated in germinal centre B cells, various B cell lymphomas and T lymphoblastic lymphoma/leukaemia (T-ALL), but has not been studied extensively in acute myeloid leukaemia (AML).We studied LMO2 expression by immunohistochemistry in biopsies from a cohort of AML patients (n = 196) and correlated it with established prognostic factors such as age, bone marrow morphology and cytogenetic findings.Forty per cent (79 of 196) of the samples from AML patients showed moderate/strong expression of LMO2 protein. LMO2 expression showed a significant positive correlation with normal cytogenetics (65% versus 24%, P < 0.0001) and a moderately negative correlation with complex karyotype [rs (98) = -0.218, P < 0.002]. AML associated with core binding factor [(t(8;21)/inv(16)/t(16;16)] had low LMO2 expression compared to diploid karyotype (29% versus 65%; P = 0.013). Expression of LMO2 protein exhibited an insignificant association with age (P = 0.197). Lower expression of LMO2 protein was noted in AML associated with myelodysplasia-related changes, compared to AML subtypes based on FAB classification (M0-M7) (21% versus 44%, P = 0.0187).LMO2 is expressed in a subset of AML patients and is associated with normal karyotype, which is different from T-ALL, where specific translocation (11p13) mediates protein expression.

    View details for DOI 10.1111/his.12242

    View details for Web of Science ID 000328347800005

    View details for PubMedID 24330148

  • Expression of the Activating Receptor, NKp46 (CD335), in Human Natural Killer and T-Cell Neoplasia. American journal of clinical pathology Freud, A. G., Zhao, S., Wei, S., Gitana, G. M., Molina-Kirsch, H. F., Atwater, S. K., Natkunam, Y. 2013; 140 (6): 853-866


    Objectives To evaluate the expression of CD335 (NKp46), an activation receptor that is selectively expressed on natural killer (NK) cells. Methods We assessed CD335's potential utility as a diagnostic marker in 657 cases by flow cytometry and 410 cases by immunohistochemistry. Results We observed that CD335 was highly specific for NK cells in nonneoplastic tissues. Moreover, 61 (90%) of 68 of NK cell neoplasms demonstrated CD335 expression, whereas B-cell, myelomonocytic, and plasma cell neoplasms lacked expression. Notably, 16 (20%) of 82 mature T-cell neoplasms, particularly T-cell large granular lymphocytic leukemia, mycosis fungoides, and ALK+ anaplastic large cell lymphoma, aberrantly expressed CD335. Conclusions Collectively, these data support the diagnostic utility of CD335 in evaluating hematopoietic malignancies and suggest that CD335 could be a useful target for selective immunotherapy in patients with mature NK and T-cell neoplasms.

    View details for DOI 10.1309/AJCPWGG69MCZOWMM

    View details for PubMedID 24225754

  • Molecular and genomic aberrations in Chlamydophila psittaci negative ocular adnexal marginal zone lymphomas AMERICAN JOURNAL OF HEMATOLOGY Zhu, D., Ikpatt, O. F., Dubovy, S. R., Lossos, C., Natkunam, Y., Chapman-Fredricks, J. R., Fan, Y., Lossos, I. S. 2013; 88 (9): 730-735


    The etiology and pathogenesis of ocular adnexal extranodal marginal zone lymphoma (OAEMZL) are still unknown and the association with Chlamydophila psittaci (C. psittaci) has been shown in only some geographic regions. Herein we comprehensively examined the frequency of chromosomal translocations as well as CARD11, MYD88 (L265P) and A20 mutations /deletions in 45 C. psittaci negative OAEMZLs. t(14;18)(q32;q21) IGH-MALT1 and t(11;18)(q21;q21) API2-MALT1 were not detected in any of the analyzed tumors while 3 tumors harbored IGH translocations to an unidentified partner. CARD11 mutations were not found in all the analyzed tumors while MYD88 L265P mutation was detected in 3 (6.7%) tumors. A20 mutations and deletions were each detected in 7(15.6%) and 6(13.3%) of the tumors, respectively. Therefore, the observed genetic aberrations could account for the activation of NF-kB signaling pathway in only a minority of the cases. Further studies are needed to identify the molecular mechanisms underlying the pathogenesis of OAEMZL.

    View details for DOI 10.1002/ajh.23490

    View details for Web of Science ID 000323313500002

    View details for PubMedID 23720088

  • LITAF, a BCL6 target gene, regulates autophagy in mature B-cell lymphomas BRITISH JOURNAL OF HAEMATOLOGY Bertolo, C., Roa, S., Sagardoy, A., Mena-Varas, M., Robles, E. F., Martinez-Ferrandis, J. I., Sagaert, X., Tousseyn, T., Orta, A., Lossos, I. S., Amar, S., Natkunam, Y., Briones, J., Melnick, A., Malumbres, R., Martinez-Climent, J. A. 2013; 162 (5): 621-630


    We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.

    View details for DOI 10.1111/bjh.12440

    View details for Web of Science ID 000323158400006

    View details for PubMedID 23795761

  • Follicular lymphoma in young adults: a clinicopathological and molecular study of 200 patients MODERN PATHOLOGY Duarte, I. X., Domeny-Duarte, P., Wludarski, S. C., Natkunam, Y., Bacchi, C. E. 2013; 26 (9): 1183-1196


    Follicular lymphoma is clinically heterogenous, and therefore necessitates the identification of prognostic markers to stratify risk groups and optimize clinical management. It is relatively rare in patients younger than 40 years, and the clinicopathologic characteristics and biological behavior in this age group are poorly understood. In the current study, samples from a cohort of 200 patients between 19 and 40 years were evaluated retrospectively with respect to clinical, histologic, and genetic features. These were then correlated with clinical outcome. The median age at presentation was 35 years with a slight female prepoderance (56%). Most of the cases are presented with nodal disease (90%). Concomitant follicular lymphoma and diffuse large B-cell lymphoma were observed in 7 (4%) patients. Immunohistologic studies showed the expression of CD10 (91%), BCL6 (97%), BCL2 (95%), MUM1/IRF4 (12%), MDM2 (17%), and CD23 (25%). BCL2 rearrangement was present in 74%, and BCL6 in 20%. The estimated overall survival of patients was 13 years (mean). The presence of anemia, elevated lactose dehydrogenase, bone marrow involvement, and high-risk follicular lymphoma international prognostic index correlated with adverse overall survival. Our findings revealed that follicular lymphoma in young adults demonstrate similarities with that of older adults, including the frequency of presentation at various anatomic sites, grade, and adverse prognostic factors.Modern Pathology advance online publication, 19 April 2013; doi:10.1038/modpathol.2013.50.

    View details for DOI 10.1038/modpathol.2013.50

    View details for Web of Science ID 000324011100004

    View details for PubMedID 23599146

  • CD30 targeting with brentuximab vedotin: a novel therapeutic approach to primary effusion lymphoma BLOOD Bhatt, S., Ashlock, B. M., Natkunam, Y., Sujoy, V., Chapman, J. R., Ramos, J. C., Mesri, E. A., Lossos, I. S. 2013; 122 (7): 1233-1242


    Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent monomethyl auristatin E (MMAE). Brentuximab vedotin is an effective treatment for relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized KSHV and EBV positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients.

    View details for DOI 10.1182/blood-2013-01-481713

    View details for Web of Science ID 000323392900023

    View details for PubMedID 23838350

  • Improvements in observed and relative survival in follicular grade 1-2 lymphoma during 4 decades: the Stanford University experience. Blood Tan, D., Horning, S. J., Hoppe, R. T., Levy, R., Rosenberg, S. A., Sigal, B. M., Warnke, R. A., Natkunam, Y., Han, S. S., Yuen, A., Plevritis, S. K., Advani, R. H. 2013; 122 (6): 981-987


    Recent studies report an improvement in overall survival (OS) of patients with follicular lymphoma (FL). Previously untreated patients with grade 1-2 FL referred from 1960-2003 and treated at Stanford were identified. Four eras were considered: era 1, pre-anthracycline (1960-1975, n=180); era 2, anthracycline (1976-1986, n=426), era 3, aggressive chemotherapy/purine analogs (1987-1996, n=471) and era 4, rituximab (1997-2003, n=257). Clinical characteristics, patterns of care and survival outcomes were assessed. Observed OS was compared with the expected OS calculated from Berkeley Mortality Database life tables derived from population matched by gender and age at time of diagnosis. The median OS was 13.6 years. Age, gender and stage did not differ across the eras. Although primary treatment varied, event free survival after the first treatment did not differ between eras (p=0.17). Median OS improved from approximately 11 years in eras 1 and 2 to 18.4 years in era 3 and has not yet been reached for era 4 (p<0.001) with no suggestion of a plateau in any era. These improvements in OS exceeded improvements in survival in the general population during the same time period. Several factors, including better supportive care and effective therapies for relapsed disease, are likely responsible for this improvement.

    View details for DOI 10.1182/blood-2013-03-491514

    View details for PubMedID 23777769

  • Clinicopathological features of aggressive B-cell lymphomas including B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell and Burkitt lymphomas: a study of 44 patients from Argentina. Annals of diagnostic pathology Bürgesser, M. V., Gualco, G., Diller, A., Natkunam, Y., Bacchi, C. E. 2013; 17 (3): 250-255


    Aggressive B-cell lymphomas incorporate a wide spectrum of lymphomas that pose challenges in diagnosis as well as treatment. We evaluated the clinicopathological features of 44 patients with aggressive B-cell lymphomas which were classified into 3 groups based on the World Health Organization 2008 classification as follows: including 30 cases of diffuse large B-cell lymphoma (DLBCL), 8 cases of Burkitt lymphoma (BL) and 6 cases of B-cell lymphoma, unclassifiable, with features intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma (BCLU). Male predominance was observed in BL and BCLU groups and the mean age varied from 29 years in BL, 61 years in DLBCL and 70 years in BCLU. Patients with BCLU presented at more advanced stages and had a higher international prognostic index. By immunohistochemistry, they shared characteristics of both BL (including more frequent expression of SOX11) and DLBCL. FISH analyses showed three cases with more than one rearrangement: one MYC/BCL2 and two BCL2/BCL6, in addition to which one case with BCL2/IGH translocation and another with MYC rearrangement were also detected. The mean follow-up survival time of BCLU was 6.6 months, which was significantly shorter in comparison to DLBCL (31 months) and BL (30 months), respectively. The importance of recognizing this BCLU group relies on its different clinical course, poor prognosis and shorter survival than DLBCL and BL. An accurate diagnosis is critical for risk stratification and to improve therapeutic approaches and outcomes.

    View details for DOI 10.1016/j.anndiagpath.2012.11.001

    View details for PubMedID 23246412

  • Indolent T-Lymphoblastic Proliferation (iT-LBP): A Review of Clinical and Pathologic Features and Distinction from Malignant T-Lymphoblastic Lymphoma ADVANCES IN ANATOMIC PATHOLOGY Ohgami, R. S., Arber, D. A., Zehnder, J. L., Natkunam, Y., Warnke, R. A. 2013; 20 (3): 137-140


    In recent years, a new pathologic entity has emerged: indolent T-lymphoblastic proliferation (iT-LBP). iT-LBPs share immunophenotypic similarities with T-lymphoblastic lymphoma; however, T-lymphoblastic proliferations are clinically indolent, and unlike the malignant counterpart, these expansions of nonclonal terminal deoxynucleotidyl transferase (TdT)+ T cells do not require treatment. Here we review the clinical and pathologic features, which are required for an accurate diagnosis of an iT-LBP. We demonstrate specific criteria can be used to accurately diagnose iT-LBP, notably: (1) confluent groups of TdT+ T cells in a biopsy specimen, (2) relative preservation of surrounding normal lymphoid architecture, (3) TdT+ T cells without morphologic atypia, (4) absence of thymic epithelium, (5) nonclonal TdT+ T cells, (6) immunophenotype of developmentally normal immature thymic T cells, and (7) clinical evidence of indolence (follow-up >6 mo without progression).

    View details for DOI 10.1097/PAP.0b013e31828d17ec

    View details for Web of Science ID 000317588700001

    View details for PubMedID 23574769

  • Usefulness of HGAL and LMO2 Immunohistochemistry in the Identification of Follicular Lymphomas of the Non-Gastric Gastrointestinal Tract APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Chapman-Fredricks, J., Younes, S. F., Fan, Y., Sandoval-Sus, J. D., Natkunam, Y., Lossos, I. S. 2013; 21 (3): 200-204


    We studied the sensitivity of 2 relatively new markers of germinal center B-cell origin, namely human germinal center-associated lymphoma (HGAL) and Lim-only transcription factor 2 (LMO2), in the identification of follicular lymphomas (FLs) of the nongastric gastrointestinal (GI) tract.We retrospectively reviewed cases of endoscopically derived primary, nongastric GI lymphomas including FL, grade 1 or 2, and extranodal marginal zone lymphoma (ENMZL) of mucosa-associated lymphoid tissue, classified based on morphologic features and immunohistochemical analysis. HGAL and LMO2 immunohistochemical stains were then prospectively performed in each case. When discrepant immunohistochemical results were obtained, fluorescent in situ hybridization was performed for t(14;18) IGH/BCL2 and IGH rearrangement using a dual color fusion and a dual color break-apart probe, respectively.All but one of the CD10-negative ENMZL cases were negative for both HGAL and LMO2. One case originally classified as ENMZL was positive for both HGAL and LMO2. Fluorescent in situ hybridization did not detect either t(14;18) IGH/BCL2 or IGH rearrangement in this case. It is likely, based on positivity of 2 established germinal center B-cell markers, that this represents a FL which was originally misclassified as an ENMZL based on CD10 negativity. Of the cases of FL (all CD10 and/or BCL-6 positive), 8 (80%) were positive for both HGAL and LMO2.Although HGAL and LMO2 did not demonstrate an increased sensitivity in the identification of FL of the nongastric GI tract in this series, they still were helpful in the reclassification of one of our cases, and may therefore be useful adjuncts in the identification of FL of the nongastric GI tract.

    View details for DOI 10.1097/PAI.0b013e31826399aa

    View details for Web of Science ID 000317961100004

    View details for PubMedID 22914613

  • Syk-Induced Phosphatidylinositol-3-Kinase Activation in EpsteinBarr Virus Posttransplant Lymphoproliferative Disorder AMERICAN JOURNAL OF TRANSPLANTATION Hatton, O., Lambert, S. L., Phillips, L. K., Vaysberg, M., Natkunam, Y., Esquivel, C. O., Krams, S. M., Martinez, O. M. 2013; 13 (4): 883-890


    Posttransplant lymphoproliferative disorder (PTLD)-associated Epstein-Barr virus (EBV)+ B cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein LMP2a, a B cell receptor (BCR) mimic, provides survival signals to virally infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV-associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol-3'-kinase (PI3K)/Akt signaling in EBV+ B cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV-infected B cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib-treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV-associated malignancies.

    View details for DOI 10.1111/ajt.12137

    View details for Web of Science ID 000316911900011

    View details for PubMedID 23398911

  • The spectrum of lymphoblastic, nodal and extranodal T-cell lymphomas: characteristic features and diagnostic dilemmas HUMAN PATHOLOGY Savage, N. M., Johnson, R. C., Natkunam, Y. 2013; 44 (4): 451-471


    T-cell lymphomas represent a heterogeneous group of neoplasms that encompass considerable clinical, morphologic, and immunophenotypic variation. The diagnosis of T-cell lymphoma is challenging because of its relative rarity, the lack of an immunophenotypic marker of clonality, and significant morphologic overlap with infectious/inflammatory processes and neoplasms, including Hodgkin and other non-Hodgkin lymphomas, and even mesenchymal or epithelial lesions. In the current World Health Organization classification of hematopoietic tumors, all except 1 subtype (ie, T-lymphoblastic lymphoma) are recognized as mature neoplasms derived from postthymic T cells. In addition to T-lymphoblastic lymphoma, this review will focus on nodal and extranodal T-cell lymphomas and exclude T-cell lymphomas presenting primarily in the skin. Extranodal natural-killer-cell/T-cell lymphoma, nasal type, will also be discussed because the derivation of this lymphoma from natural killer and natural killer-like T cells shows morphologic and immunophenotypic features that overlap with other T-cell lymphomas. In this review, we discuss the salient clinicopathologic, immunophenotypic, and genetic features, as well as our approaches to the diagnosis of lymphoblastic, nodal, and extranodal T-cell lymphomas.

    View details for DOI 10.1016/j.humpath.2012.02.007

    View details for Web of Science ID 000317246700001

    View details for PubMedID 22658223

  • Integration of Genomic Medicine into Pathology Residency Training The Stanford Open Curriculum JOURNAL OF MOLECULAR DIAGNOSTICS Schrijver, I., Natkunam, Y., Galli, S., Boyd, S. D. 2013; 15 (2): 141-148


    Next-generation sequencing methods provide an opportunity for molecular pathology laboratories to perform genomic testing that is far more comprehensive than single-gene analyses. Genome-based test results are expected to develop into an integral component of diagnostic clinical medicine and to provide the basis for individually tailored health care. To achieve these goals, rigorous interpretation of high-quality data must be informed by the medical history and the phenotype of the patient. The discipline of pathology is well positioned to implement genome-based testing and to interpret its results, but new knowledge and skills must be included in the training of pathologists to develop expertise in this area. Pathology residents should be trained in emerging technologies to integrate genomic test results appropriately with more traditional testing, to accelerate clinical studies using genomic data, and to help develop appropriate standards of data quality and evidence-based interpretation of these test results. We have created a genomic pathology curriculum as a first step in helping pathology residents build a foundation for the understanding of genomic medicine and its implications for clinical practice. This curriculum is freely accessible online.

    View details for DOI 10.1016/j.jmoldx.2012.11.003

    View details for Web of Science ID 000315841600001

    View details for PubMedID 23313248

  • Selective Immunophenotyping for Diagnosis of B-cell Neoplasms: Immunohistochemistry and Flow Cytometry Strategies and Results APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Boyd, S. D., Natkunam, Y., Allen, J. R., Warnke, R. A. 2013; 21 (2): 116-131


    Determining the immunophenotype of hematologic malignancies is now an indispensable part of diagnostic classification, and can help to guide therapy, or to predict clinical outcome. Diagnostic workup should be guided by morphologic findings and evaluate clinically important markers, but ideally should avoid the use of overly broad panels of immunostains that can reveal incidental findings of uncertain significance and give rise to increased costs. Here, we outline our approach to diagnosis of B-cell neoplasms, combining histologic and clinical data with tailored panels of immunophenotyping reagents, in the context of the 2008 World Health Organization classification. We present data from cases seen at our institution from 2004 through 2008 using this approach, to provide a practical reference for findings seen in daily diagnostic practice.

    View details for DOI 10.1097/PAI.0b013e31825d550a

    View details for Web of Science ID 000315464500004

    View details for PubMedID 22820658

  • CD137 Ligand Is Expressed in Primary and Secondary Lymphoid Follicles and in B-cell Lymphomas Diagnostic and Therapeutic Implications AMERICAN JOURNAL OF SURGICAL PATHOLOGY Zhao, S., Zhang, H., Xing, Y., Natkunam, Y. 2013; 37 (2): 250-258


    CD137 ligand (4-1BB ligand, TNFSF9, CD137L) is a member of the tumor necrosis factor family whose binding to its receptor, CD137 (4-1BB, TNFRSF9), mediates costimulatory and prosurvival signals necessary for T-cell activation and regulation of humoral immune responses. Recent studies have shown that anti-CD137 immunotherapy has promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. Here, we define the tissue expression profile of CD137L, which has not been previously explored. We characterized the expression of CD137L in normal and neoplastic human hematopoietic and nonhematopoietic tissue and found that CD137L is preferentially expressed in B cells of the primary follicles, mantle zones of the secondary follicles, germinal centers, and in normal endothelial cells. Double immunofluorescence labeling in tissue sections and flow cytometry analysis further showed that CD137L is a potential new marker of memory B cells. Evaluation of over 700 human hematopoietic tumors revealed that the majority of B-cell lymphomas expressed CD137L, which include mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. In contrast, CD137L expression was lacking in Hodgkin lymphoma and T-cell lymphoma. Our findings suggest that CD137L is a novel diagnostic marker of subtypes of non-Hodgkin B-cell lymphomas and raise the possibility that its expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and other malignancies.

    View details for DOI 10.1097/PAS.0b013e318268c6ea

    View details for Web of Science ID 000313576200012

    View details for PubMedID 23095505

  • Germinal centre protein HGAL promotes lymphoid hyperplasia and amyloidosis via BCR-mediated Syk activation NATURE COMMUNICATIONS Romero-Camarero, I., Jiang, X., Natkunam, Y., Lu, X., Vicente-Duenas, C., Gonzalez-Herrero, I., Flores, T., Luis Garcia, J., McNamara, G., Kunder, C., Zhao, S., Segura, V., Fontan, L., Martinez-Climent, J. A., Javier Garcia-Criado, F., Theis, J. D., Dogan, A., Campos-Sanchez, E., Green, M. R., Alizadeh, A. A., Cobaleda, C., Sanchez-Garcia, I., Lossos, I. S. 2013; 4

    View details for DOI 10.1038/ncomms2334

    View details for Web of Science ID 000316614600008

    View details for PubMedID 23299888

  • Examination of Genetic Aberrations in Chlamydophila Psittaci negative MALT Lymphomas of the Ocular Adnexa 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Zhu, D., Ikpatt, O. F., Dubovy, S. R., Natkunam, Y., Chapman-Fredricks, J. R., Fan, Y., Lossos, I. S. AMER SOC HEMATOLOGY. 2012
  • TdT(+) T-lymphoblastic Populations Are Increased in Castleman Disease, in Castleman Disease in Association With Follicular Dendritic Cell Tumors, and in Angioimmunoblastic T-cell Lymphoma AMERICAN JOURNAL OF SURGICAL PATHOLOGY Ohgami, R. S., Zhao, S., Ohgami, J. K., Leavitt, M. O., Zehnder, J. L., West, R. B., Arber, D. A., Natkunam, Y., Warnke, R. A. 2012; 36 (11): 1619-1628


    T-lymphoblastic lymphoma is an aggressive neoplasm requiring prompt clinical treatment. Conversely, indolent T-lymphoblastic proliferation mimics T-lymphoblastic lymphoma but consists of a proliferation of non-neoplastic TdT+ T cells, requiring no treatment. Recently, we identified several cases of indolent T-lymphoblastic proliferations in extrathymic lymphoid tissues: 1 in a patient suffering from Castleman disease (CD) associated with a follicular dendritic cell sarcoma/tumor, 1 in a patient with a history of angioimmunoblastic T-cell lymphoma (AITL), and 1 in association with acinic cell carcinoma. Interestingly, in the case of the patient with a history of AITL, these TdT+ T cells were seen in multiple anatomic sites over the span of 5 years. Here we review these 3 cases and extend our findings by demonstrating that TdT+ T-lymphoblastic populations are increased in lymph nodes of patients with CD (P=0.011), CD in association with follicular dendritic cell tumors, and AITL (P<0.01) compared with other T-cell or B-cell lymphomas or reactive lymph nodes. Finally, analysis of 352 nonhematolymphoid tumors including carcinomas, melanomas, and sarcomas demonstrates that TdT+ T cells are not increased in these tumors. Our studies not only present several detailed cases of indolent T-lymphoblastic proliferations, but also correlate these populations with specific hematologic diseases.

    View details for DOI 10.1097/PAS.0b013e318264e223

    View details for Web of Science ID 000310059600004

    View details for PubMedID 23060347

  • The contribution of HGAL/GCET2 in immunohistological algorithms: a comparative study in 424 cases of nodal diffuse large B-cell lymphoma MODERN PATHOLOGY Gualco, G., Bacchi, L. M., Domeny-Duarte, P., Natkunam, Y., Bacchi, C. E. 2012; 25 (11): 1439-1445


    Diffuse large B-cell lymphoma can be subclassified into at least two molecular subgroups by gene expression profiling: germinal center B-cell like and activated B-cell like diffuse large B-cell lymphoma. Several immunohistological algorithms have been proposed as surrogates to gene expression profiling at the level of protein expression, but their reliability has been an issue of controversy. Furthermore, the proportion of misclassified cases of germinal center B-cell subgroup by immunohistochemistry, in all reported algorithms, is higher compared with germinal center B-cell cases defined by gene expression profiling. We analyzed 424 cases of nodal diffuse large B-cell lymphoma with the panel of markers included in the three previously described algorithms: Hans, Choi, and Tally. To test whether the sensitivity of detecting germinal center B-cell cases could be improved, the germinal center B-cell marker HGAL/GCET2 was also added to all three algorithms. Our results show that the inclusion of HGAL/GCET2 significantly increased the detection of germinal center B-cell cases in all three algorithms (P<0.001). The proportions of germinal center B-cell cases in the original algorithms were 27%, 34%, and 19% for Hans, Choi, and Tally, respectively. In the modified algorithms, with the inclusion of HGAL/GCET2, the frequencies of germinal center B-cell cases were increased to 38%, 48%, and 35%, respectively. Therefore, HGAL/GCET2 protein expression may function as a marker for germinal center B-cell type diffuse large B-cell lymphoma. Consideration should be given to the inclusion of HGAL/GCET2 analysis in algorithms to better predict the cell of origin. These findings bear further validation, from comparison to gene expression profiles and from clinical/therapeutic data.

    View details for DOI 10.1038/modpathol.2012.119

    View details for Web of Science ID 000310795400001

    View details for PubMedID 22743653

  • Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number GENES & DEVELOPMENT Sankaran, V. G., Ludwig, L. S., Sicinska, E., Xu, J., Bauer, D. E., Eng, J. C., Patterson, H. C., Metcalf, R. A., Natkunam, Y., Orkin, S. H., Sicinski, P., Lander, E. S., Lodish, H. F. 2012; 26 (18): 2075-2087


    Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.

    View details for DOI 10.1101/gad.197020.112

    View details for Web of Science ID 000308975900009

    View details for PubMedID 22929040

  • CD137 Is Expressed in Follicular Dendritic Cell Tumors and in Classical Hodgkin and T-Cell Lymphomas Diagnostic and Therapeutic Implications AMERICAN JOURNAL OF PATHOLOGY Anderson, M. W., Zhao, S., Freud, A. G., Czerwinski, D. K., Kohrt, H., Alizadeh, A. A., Houot, R., Azambuja, D., Biasoli, I., Morais, J. C., Spector, N., Molina-Kirsch, H. F., Warnke, R. A., Levy, R., Natkunam, Y. 2012; 181 (3): 795-803


    CD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy.

    View details for DOI 10.1016/j.ajpath.2012.05.015

    View details for Web of Science ID 000309251100009

    View details for PubMedID 22901750

  • Identification of LMO2 transcriptome and interactome in diffuse large B-cell lymphoma BLOOD Cubedo, E., Gentles, A. J., Huang, C., Natkunam, Y., Bhatt, S., Lu, X., Jiang, X., Romero-Camarero, I., Freud, A., Zhao, S., Bacchi, C. E., Martinez-Climent, J. A., Sanchez-Garcia, I., Melnick, A., Lossos, I. S. 2012; 119 (23): 5478-5491


    LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis.

    View details for DOI 10.1182/blood-2012-01-403154

    View details for Web of Science ID 000307391400022

    View details for PubMedID 22517897

    View details for PubMedCentralID PMC3369683

  • LIM domain only 2 protein expression, LMO2 germline genetic variation, and overall survival in diffuse large B-cell lymphoma in the pre-rituximab era LEUKEMIA & LYMPHOMA Cerhan, J. R., Natkunam, Y., Morton, L. M., Maurer, M. J., Asmann, Y., Habermann, T. M., Vasef, M. A., Cozen, W., Lynch, C. F., Allmer, C., Slager, S. L., Lossos, I. S., Chanock, S. J., Rothman, N., Hartge, P., Dogan, A., Wang, S. S. 2012; 53 (6): 1105-1112


    Both LMO2 (LIM domain only 2) mRNA and protein expression in diffuse large B-cell lymphoma (DLBCL) have been associated with superior survival. However, a role for germline genetic variation in LMO2 has not been previously reported. Immunohistochemistry (IHC) for LMO2 was conducted on tumor tissue from diagnostic biopsies, and 20 tag single nucleotide polymorphisms (SNPs) from LMO2 were genotyped from germline DNA. LMO2 IHC positivity was associated with superior survival (hazard ratio [HR] = 0.55; 95% confidence interval [CI] 0.31-0.97). Four LMO2 SNPs (rs10836127, rs941940, rs750781, rs1885524) were associated with survival after adjusting for LMO2 IHC and clinical factors (p < 0.05), and one of these SNPs (rs941940) was also associated with IHC positivity (p = 0.02). Compared to a model with clinical factors only (c-statistic = 0.676), adding the four SNPs (c-statistic = 0.751) or LMO2 IHC (c-statistic = 0.691) increased the predictive ability of the model, while inclusion of all three factors (c-statistic = 0.754) did not meaningfully add predictive ability above a model with clinical factors and the four SNPs. In conclusion, germline genetic variation in LMO2 was associated with DLBCL prognosis and provided slightly stronger predictive ability relative to LMO2 IHC status.

    View details for DOI 10.3109/10428194.2011.638717

    View details for Web of Science ID 000304311500016

    View details for PubMedID 22066713

  • Aggressive EBV-associated Lymphoproliferative Disorder: A Prodrome to Diffuse Large B-cell Lymphoma? APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Batra, R., Medeiros, B. C., Zehnder, J. L., Warnke, R. A., Natkunam, Y. 2012; 20 (3): 325-330


    A 19-year-old male patient presented with intermittent high fever and left cervical lymphadenopathy. The lymph node biopsy findings were interpreted as "Epstein-Barr virus (EBV)-associated lymphoproliferative disorder consistent with infectious mononucleosis." No molecular studies were performed at that time. The patient was followed without treatment. Five months later, the patient again presented with fever, lymphadenopathy, and splenomegaly. The lymph node biopsy showed features of a diffuse large B-cell lymphoma. Molecular studies on this lymph node biopsy showed a clonal EBV population, although polymerase chain reaction studies failed to reveal a clonal B-cell or T-cell population. A concurrent bone marrow biopsy showed features consistent with hemophagocytic syndrome. He had elevated ferritin, soluble interleukin-2 receptors and persistent EBV viremia. The patient responded to Rituxan for a short period with undetectable EBV levels. Subsequent right cervical lymph node, liver, and jejunal biopsies showed involvement by diffuse large B-cell lymphoma and the patient expired soon thereafter.

    View details for Web of Science ID 000303140100012

    View details for PubMedID 22505014

  • The spectrum of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma: a description of 10 cases MODERN PATHOLOGY Gualco, G., Natkunam, Y., Bacchi, C. E. 2012; 25 (5): 661-674


    B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, is a diagnostic provisional category in the World Health Organization (WHO) 2008 classification of lymphomas. This category was designed as a measure to accommodate borderline cases that cannot be reliably classified into a single distinct disease entity after all available morphological, immunophenotypical and molecular studies have been performed. Typically, these cases share features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, or include characteristics of both lymphomas. The rarity of such cases poses a tremendous challenge to both pathologists and oncologists because its differential diagnosis has direct implications for management strategies. In this study, we present 10 cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma and have organized the criteria described by the WHO into four patterns along with detailed clinical, morphological and immunophenotypic characterization and outcome data. Our findings show a male preponderance, median age of 37 years and a mediastinal presentation in 80% of cases. All cases expressed at least two markers associated with B-cell lineage and good response to combination chemotherapy currently employed for non-Hodgkin lymphomas.

    View details for DOI 10.1038/modpathol.2011.200

    View details for Web of Science ID 000303617400003

    View details for PubMedID 22222636

  • IgG4-Related Systemic Sclerosing Disease of the Ocular Adnexa A Potential Mimic of Ocular Lymphoma AMERICAN JOURNAL OF CLINICAL PATHOLOGY Karamchandani, J. R., Younes, S. F., Warnke, R. A., Natkunam, Y. 2012; 137 (5): 699-711


    IgG4-related sclerosing disease has been described in the orbit and ocular adnexa. Of 164 biopsies of the ocular region for suspected lymphoma, we identified 6 cases of IgG4 disease, 4 of which were previously unrecognized. All 6 cases demonstrated increased plasma cells in a background of sclerosis and increased absolute numbers of IgG4-expressing cells. Our results confirm the difficulty in diagnosing IgG4-related sclerosing disease in the ocular region. Based on the findings, we suggest that specimens from biopsies of the eye and ocular adnexa for which a definitive diagnosis of lymphoma is not established undergo further workup for IgG and IgG4, particularly if increased plasma cells and sclerosis are present. When IgG4-expressing plasma cells account for greater than 50% of IgG-expressing plasma cells, a diagnosis of IgG4 disease should be considered. Timely recognition would benefit patients by allowing appropriate management with corticosteroid therapy and avoiding more aggressive or unnecessary therapeutic options.

    View details for DOI 10.1309/AJCPE1G8DRHXRPIH

    View details for Web of Science ID 000303141300002

    View details for PubMedID 22523207

  • Expression of the CD2AP adaptor molecule in normal, reactive and neoplastic human tissue. Pathologica Rizvi, H., Paterson, J. C., Tedoldi, S., Ramsay, A., Calaminici, M., Natkunam, Y., Lonardi, S., Tan, S., Campbell, L., Hansmann, M., Jones, D., Dikic, I., Shaw, A. S., Pileri, S. A., Stein, H., Mason, D. Y., Facchetti, F., Marafioti, T. 2012; 104 (2): 56-64


    To study the expression of CD2-associated protein (CD2AP), an adaptor protein involved in T-cell signalling and renal function, in normal, reactive and neoplastic human lymphoid tissues.We used immunohistochemical techniques to evaluate monoclonal antibodies against CD2AP on over 400 formalin fixed paraffin embedded tissue blocks retrieved from the host institutions of three authors. The samples tested included normal, reactive and neoplastic lymphoid tissue. In lymphoid tissues, strong CD2AP staining was observed in plasmacytoid dendritic cells (pDCs), weak and variable in mantle zone B cells and moderate in rare germinal center cells. CD2AP labeled cortical and rare medullary thymocytes and isolated mononuclear cells in bone marrow trephines. Furthermore, epithelial and endothelial cells expressed CD2AP. Among neoplasms, the greatest number of CD2AP-positive cases were found in diffuse large B cell (21/94), NK T-cell lymphomas (7/67), "blastic plasmacytoid dendritic cell neoplasms" (9/10) and some types of solid tumor.Our finding that mature peripheral T cells are CD2AP-negative but immature cortical thymocytes are positive may prove useful for diagnostic purposes. Moreover, our results demonstrate that CD2AP represents a useful marker of normal and neoplastic pDC and may be used in a diagnostic panel in reactive or neoplastic lymphoid proliferations.

    View details for PubMedID 22953501

  • Lack of association of tumor-associated macrophages with clinical outcome in patients with classical Hodgkin's lymphoma ANNALS OF ONCOLOGY Azambuja, D., Natkunam, Y., Biasoli, I., Lossos, I. S., ANDERSON, M. W., Morais, J. C., Spector, N. 2012; 23 (3): 736-742


    A recent study demonstrated that an increased number of CD68+ macrophages were correlated with primary treatment failure, shortened progression-free survival (PFS) and disease-specific survival (DSS) in patients with classical Hodgkin's lymphoma (cHL).The aim of the present study was to verify the relationship between the number of CD68+ and CD163+ macrophages with clinical outcomes in a cohort of 265 well-characterized patients with cHL treated uniformly with the standard doxorubicin, bleomycin, vinblastine and dacarbazine chemotherapy regimen. Two pairs of hematopathologists carried out independent pathological evaluations of tissue microarray slides.There were no associations between clinical characteristics and the expression of CD68 or CD163. However, higher levels of CD68 and CD163 expression were correlated with the presence of Epstein-Barr virus-positive Hodgkin tumor cells (P = 0.01 and 0.037, respectively). The expression of CD68 or CD163 was not associated with either the PFS or the DSS.CD68 and CD163 expression require further evaluation before their use can be recommended for prognostic stratification of patients with cHL.

    View details for DOI 10.1093/annonc/mdr157

    View details for Web of Science ID 000300733300029

    View details for PubMedID 21602260

  • Immature T-Cell Populations in Lymph Nodes of Castleman Disease and Angioimmunoblastic T-Cell Lymphoma Suggest Alternate Sites of T-Cell Development 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Ohgami, R. S., Chun, S., Ohgami, J., Zehnder, J. L., Van de Rijn, M., Arber, D. A., Natkunam, Y., Warnke, R. AMER SOC HEMATOLOGY. 2011: 1395–96
  • Identification of LMO2 Transcriptome and Interactome in Diffuse Large B-Cell Lymphoma by Integrated Experimental and Computational Approach 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Cubedo, E., Gentles, A. J., Huang, C., Natkunam, Y., Bhatt, S., Jiang, X., Lu, X., Romero-Camarero, I., Plevritis, S. K., Martinez-Climent, J. A., Sanchez-Garcia, I., Melnick, A., Lossos, I. S. AMER SOC HEMATOLOGY. 2011: 201–2
  • Immunostaining to identify molecular subtypes of diffuse large B-cell lymphoma in a population-based epidemiologic study in the pre-rituximab era. International journal of molecular epidemiology and genetics Morton, L. M., Cerhan, J. R., Hartge, P., Vasef, M. A., Neppalli, V. T., Natkunam, Y., Dogan, A., Dave, B. J., Jain, S., Levy, R., Lossos, I. S., Cozen, W., Davis, S., Schenk, M. J., Maurer, M. J., Lynch, C. F., Rothman, N., Chatterjee, N., Yu, K., Staudt, L. M., Weisenburger, D. D., Wang, S. S. 2011; 2 (3): 245-252


    Gene expression profiling studies have distinguished diffuse large B-cell lymphomas (DLBCLs) by cell of origin, with distinct pathogenetic mechanisms and prognosis. We attempted to identify DLBCL molecular subtypes in an epidemiologic study of 214 DLBCL patients diagnosed during 1998-2000 with archival tissues to investigate etiology. Immunohistochemical staining for CD10, BCL6, LMO2, MUM1/IRF4, and BCL2 and fluorescence in situ hybridization for t(14;18) were conducted, with ≥93% blinded duplicate agreement. CD10, LMO2, and BCL2 expression was similar to previous reports (32%, 44%, and 44% of DLBCLs, respectively), but BCL6 and MUM1/IRF4 expression was lower than expected (29% and 5%, respectively). We classified 112/214 (52%) cases as germinal center B-cell-like DLBCL (GCB-DLBCL; Hans et al., Blood 2004; CD10+ or CD10-/BCL6+/MUM1-), with no difference in prognosis compared with non-GCB-DLBCL (Cox regression, P=0.48). Comparing other GCB correlates, LMO2 expression and t(14;18) were more common but not exclusive to GCB-DLBCL as defined in our study, whereas BCL2 expression did not differ between DLBCL molecular subtypes. We could not confidently identify patients with GCB-DLBCL using these immunohistochemistry-based markers on archival tissues.

    View details for PubMedID 21915363

  • Prediction of survival in diffuse large B-cell lymphoma based on the expression of 2 genes reflecting tumor and microenvironment BLOOD Alizadeh, A. A., Gentles, A. J., Alencar, A. J., Liu, C. L., Kohrt, H. E., Houot, R., Goldstein, M. J., Zhao, S., Natkunam, Y., Advani, R. H., Gascoyne, R. D., Briones, J., Tibshirani, R. J., Myklebust, J. H., Plevritis, S. K., Lossos, I. S., Levy, R. 2011; 118 (5): 1350-1358


    Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.

    View details for DOI 10.1182/blood-2011-03-345272

    View details for Web of Science ID 000293510000028

    View details for PubMedID 21670469

    View details for PubMedCentralID PMC3152499

  • Clinicopathologic and Molecular Features of 122 Brazilian Cases of Nodal and Extranodal NK/T-Cell Lymphoma, Nasal Type, With EBV Subtyping Analysis AMERICAN JOURNAL OF SURGICAL PATHOLOGY Gualco, G., Domeny-Duarte, P., Chioato, L., Barber, G., Natkunam, Y., Bacchi, C. E. 2011; 35 (8): 1195-1203


    Extranodal natural killer/T-cell lymphoma, nasal type (NK/TCL) is more prevalent in Asia and in some areas of South and Central America, but it is rarely seen in the United States and Europe. In this study, a series of 122 cases of NK/TCL from Brazil was analyzed with respect to clinicopathologic features. Clinical characteristics and geographic distribution were evaluated in 97 cases of nasal/nasopharyngeal region and 23 cases in extranasal sites including 6 nodal cases. Clinical staging and follow-up information was available in a subset of 21 patients. All cases harbored Epstein-Barr virus (EBV), 95% and 85% expressed cytoplasmic CD3 and CD56, respectively, and all cases were positive for at least 1 marker for cytotoxic granules. The global distribution of EBV subtypes showed predominance of strain subtype A, 89%, and subtype B, 11%. No dual infections were detected. TCR-γ TCR-gene rearrangement was observed in 7 cases; all of them extranodal. Three of TCR-γ(+) cases showed EBV subtype A. Two TCR-γ(+)/CD56(+) cases showed EBV subtype B. Geographic distribution of NK/TCL showed higher frequency in the southeast and northeast regions of Brazil. Striking differences among geographic regions were seen with the vast majority of EBV subtype B (86%) occurring in the south and southeast regions.

    View details for DOI 10.1097/PAS.0b013e31821ec4b5

    View details for Web of Science ID 000292728200014

    View details for PubMedID 21716086

  • MicroRNAs Are Independent Predictors of Outcome in Diffuse Large B-Cell Lymphoma Patients Treated with R-CHOP CLINICAL CANCER RESEARCH Alencar, A. J., Malumbres, R., Kozloski, G. A., Advani, R., Talreja, N., Chinichian, S., Briones, J., Natkunam, Y., Sehn, L. H., Gascoyne, R. D., Tibshirani, R., Lossos, I. S. 2011; 17 (12): 4125-4135


    Diffuse large B-cell lymphoma (DLBCL) heterogeneity has prompted investigations for new biomarkers that can accurately predict survival. A previously reported 6-gene model combined with the International Prognostic Index (IPI) could predict patients' outcome. However, even these predictors are not capable of unambiguously identifying outcome, suggesting that additional biomarkers might improve their predictive power.We studied expression of 11 microRNAs (miRNA) that had previously been reported to have variable expression in DLBCL tumors. We measured the expression of each miRNA by quantitative real-time PCR analyses in 176 samples from uniformly treated DLBCL patients and correlated the results to survival.In a univariate analysis, the expression of miR-18a correlated with overall survival (OS), whereas the expression of miR-181a and miR-222 correlated with progression-free survival (PFS). A multivariate Cox regression analysis including the IPI, the 6-gene model-derived mortality predictor score and expression of the miR-18a, miR-181a, and miR-222, revealed that all variables were independent predictors of survival except the expression of miR-222 for OS and the expression of miR-18a for PFS.The expression of specific miRNAs may be useful for DLBCL survival prediction and their role in the pathogenesis of this disease should be examined further.

    View details for DOI 10.1158/1078-0432.CCR-11-0224

    View details for Web of Science ID 000291644700029

    View details for PubMedID 21525173

  • LMO2 protein expression predicts survival in patients with rheumatoid arthritis and diffuse large B-cell lymphoma LEUKEMIA & LYMPHOMA Baecklund, E., Backlin, C., Mansouri, M., Klareskog, L., Askling, J., Iliadou, A. N., Enblad, G., Lossos, I. S., Natkunam, Y., Rosenquist, R. 2011; 52 (6): 1146-1149

    View details for DOI 10.3109/10428194.2011.559801

    View details for Web of Science ID 000290869100033

    View details for PubMedID 21375434

  • HGAL Protein Expression Persists in Disorders of Germinal Center Dissolution Potential Role of HGAL in the Germinal Center Microenvironment APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Temmins, C., Zhao, S., Lossos, I. S., Natkunam, Y. 2011; 19 (3): 266-272


    Human Germinal Center-associated Lymphoma (HGAL) is a germinal center (GC) B-cell marker associated with a favorable outcome in diffuse large B-cell and classic Hodgkin lymphomas (CHL). To test its potential role in GC function, 75 cases involving GC disruption including 23 progressive transformation of germinal centers (PTGC), 25 follicle lysis and 27 Castleman disease (CD) were studied. HGAL protein expression uniformly correlated with GC B-cells in all except a subset of hyaline-vascular CD that showed severe regression of GCs. HGAL staining highlighted dismantled GCs in PTGC, in contrast to weak or absent CD10 and BCL6 staining. In follicle lysis, HGAL staining was comparable to that of CD10, BCL6, and CD21 in highlighting lysed follicles. Our findings show that HGAL protein expression effectively discriminates clusters of GC B-cells in disrupted follicles. Its persistence in disrupted GCs, suggests that it may be necessary for GC maintenance and supports its proposed role of confining B-cells to the GC microenvironment.

    View details for DOI 10.1097/PAI.0b013e3181f89a4d

    View details for Web of Science ID 000289279800011

    View details for PubMedID 21475040

  • The Efficacy of HGAL and LMO2 in the Separation of Lymphomas Derived From Small B Cells in Nodal and Extranodal Sites, Including the Bone Marrow AMERICAN JOURNAL OF CLINICAL PATHOLOGY Younes, S. F., Beck, A. H., Ohgami, R. S., Lossos, I. S., Levy, R., Warnke, R. A., Natkunam, Y. 2011; 135 (5): 697-708


    We studied the efficacy of 2 germinal center B-cell markers, HGAL and LMO2, in the separation of lymphomas derived from small B cells, particularly follicular lymphoma (FL) and marginal zone lymphoma occurring in nodal, extranodal, splenic, and bone marrow sites using immunohistochemical analysis for CD10, BCL6, BCL2, HGAL, and LMO2. Our results showed that HGAL and LMO2 are sensitive and specific markers for detecting FL in nodal and extranodal sites. In contrast, all markers were down-regulated in FL infiltrates in the bone marrow. CD10 and HGAL were expressed in a subset of FLs in the bone marrow and were highly correlated with each other and with CD21, a marker of follicular dendritic cells. We conclude that HGAL and LMO2 should be considered in immunohistochemical panels used for the routine workup of lymphomas derived from small B cells. In the bone marrow, staining for HGAL or CD10 can be helpful in making a diagnosis of FL, although they are absent in a subset of cases.

    View details for DOI 10.1309/AJCP7Z2BIBUNQPLZ

    View details for Web of Science ID 000289743400007

    View details for PubMedID 21502424

  • T-cell lymphomas: a tale of heterogeneity masking clarity LEUKEMIA & LYMPHOMA Natkunam, Y. 2011; 52 (1): 1-2

    View details for DOI 10.3109/10428194.2010.536602

    View details for Web of Science ID 000286901600001

    View details for PubMedID 21133725

  • Programmed death 1 expression in variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma: comparison with CD57 and lymphomas in the differential diagnosis HUMAN PATHOLOGY Churchill, H. R., Roncador, G., Warnke, R. A., Natkunam, Y. 2010; 41 (12): 1726-1734


    Recent studies have exploited an antibody directed against programmed death 1 expressed by follicular helper T-cells in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma. We had previously described clinically relevant, variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma and, in this study, sought to address the diagnostic utility of programmed death 1 in comparison with CD57 in variant nodular lymphocyte predominant Hodgkin lymphoma. Immunohistologic staining for programmed death 1 was carried out on biopsies of 67 patients with variant nodular lymphocyte predominant Hodgkin lymphoma. Thirty-four additional cases of nodular lymphocyte predominant Hodgkin lymphoma with associated diffuse areas, de novo T-cell and histiocyte-rich large B-cell lymphoma, and lymphocyte-rich classic Hodgkin lymphoma were also studied. Our results show that programmed death 1 positivity was found in the majority of nodular lymphocyte predominant Hodgkin lymphoma cases with a classic nodular architecture (87%) as compared with 50% for CD57 and was particularly helpful in identifying extranodular large atypical cells. Nodular lymphocyte predominant Hodgkin lymphoma with diffuse areas showed a gradual decrease in programmed death 1 reactivity from nodular to diffuse areas, although a significant proportion (40%-50%) of cases retained programmed death 1 positivity also in diffuse areas. In addition, T-cell and histiocyte-rich large B-cell lymphoma and lymphocyte-rich classic Hodgkin lymphoma displayed programmed death 1 positivity in a significant subset of cases (33%-40%). In conclusion, our study supports the utility of programmed death 1 in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma and shows greater sensitivity of staining of programmed death 1 as compared with CD57 across all variants of nodular lymphocyte predominant Hodgkin lymphoma. Loss of programmed death 1 reactivity did not correlate with diffuse areas, progression, or the ability to differentiate nodular lymphocyte predominant Hodgkin lymphoma from T-cell and histiocyte-rich large B-cell lymphoma. These findings suggest the need for continued vigilance in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma and its immunoarchitectural variants as well as related lymphomas in their differential diagnosis.

    View details for DOI 10.1016/j.humpath.2010.05.010

    View details for Web of Science ID 000284975800009

    View details for PubMedID 20825974

  • Clinical and Pathological Features of Non-Hodgkin Lymphomas Harboring Concurrent t(14;18) and 8q24 Anomalies 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Alizadeh, A. A., Anderson, M., Kohrt, H. E., Shyam, R. M., Bangs, C. D., Cherry, A. M., Advani, R., Natkunam, Y., Levy, R. AMER SOC HEMATOLOGY. 2010: 1291–92
  • In Situ Vaccination with TLR9 Agonist Combined with Local Radiation In Mycosis Fungoides: Analysis of Phase I/II Study 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Kim, Y. H., Gratzinger, D., Harrison, C., Brody, J., Czerwinski, D., Xing, L., Morales, A., Ai, W., Abdulla, F., Navi, D., Tibshirani, R. J., Advani, R., Natkunam, Y., Hoppe, R. T., Levy, R. AMER SOC HEMATOLOGY. 2010: 130–30
  • Oral and Extraoral Plasmablastic Lymphoma Similarities and Differences in Clinicopathologic Characteristics AMERICAN JOURNAL OF CLINICAL PATHOLOGY Hansra, D., Montague, N., Stefanovic, A., Akunyili, I., Harzand, A., Natkunam, Y., De La Ossa, M., Byrne, G. E., Lossos, I. S. 2010; 134 (5): 710-719


    Plasmablastic lymphoma (PBL), initially characterized as an aggressive lymphoma arising in the jaw and oral mucosa in HIV-infected patients, was recently reported to occur with extraoral manifestations, heterogeneous histologic findings, and variable association with immunodeficiency states. We reviewed clinical, morphologic, and immunophenotypic features of 13 cases of PBL to determine whether these different subtypes represent distinct morphologic and clinical entities. Two distinct subtypes of PBL were identified and classified as oral and extraoral PBL. The oral PBLs were strongly associated with HIV infection and commonly demonstrated plasmablastic morphologic features without plasmacytic differentiation. Extraoral PBLs tended to occur in patients with underlying non-HIV-related immunosuppression and universally demonstrated plasmacytic differentiation. The patients with oral PBL demonstrated better overall survival compared with patients with extraoral PBL (P = .02). Our findings suggest that PBL with oral and extraoral manifestation represent 2 distinct clinicopathologic entities.

    View details for DOI 10.1309/AJCPJH6KEUSECQLU

    View details for Web of Science ID 000283106600003

    View details for PubMedID 20959653

  • Immunoarchitectural Patterns in Follicular Lymphoma: Efficacy of HGAL and LMO2 in the Detection of the Interfollicular and Diffuse Components AMERICAN JOURNAL OF SURGICAL PATHOLOGY Younes, S. F., Beck, A. H., Lossos, I. S., Levy, R., Warnke, R. A., Natkunam, Y. 2010; 34 (9): 1266-1276


    Follicular lymphoma (FL) can exhibit variant histologic patterns that can lead to confusion with other B-cell lymphomas and reactive conditions. Diagnostic markers such as CD10 and BCL2 may be difficult to interpret in variant FL patterns, and are often diminished or absent in the interfollicular and diffuse components. We evaluated 2 recently characterized germinal center B-cell markers, human germinal center associated lymphoma (HGAL), and LIM-only transcription factor 2 (LMO2), in 127 FL patient biopsies (94 nodal, 33 extranodal), and correlated the findings with histologic pattern, cellular composition, grade, and additional immunostains (CD20, CD3, CD21, CD10, BCL2, and BCL6). Architectural patterns included predominantly follicular (75%) and follicular and diffuse components (25%); 10 cases showed marginal zone differentiation and 3 were floral variants. Eighty-nine cases were low grade (38 grade 1; 51 grade 2) and 38 were grade 3 (29 grade 3A and 9 grade 3B). HGAL had the highest overall sensitivity of detecting FL and was superior in detecting the interfollicular and diffuse components compared with BCL2, LMO2, CD10, and BCL6. All 28 cases that lacked CD10, expressed HGAL, and the majority also expressed LMO2. Our results show that HGAL and LMO2 are sensitive markers for FL diagnosis. The addition of HGAL and LMO2 to the immunohistologic panel is beneficial in the work-up of nodal and extranodal B-cell lymphomas and the efficacy of HGAL in detecting the follicular, interfollicular and diffuse components of FL is of particular value in the setting of variant immunoarchitectural patterns.

    View details for DOI 10.1097/PAS.0b013e3181e9343d

    View details for Web of Science ID 000281579800005

    View details for PubMedID 20697248

  • Expression of LMO2 Is Associated With t(14;18)/IGH-BCL2 Fusion but Not BCL6 Translocations in Diffuse Large B-Cell Lymphoma AMERICAN JOURNAL OF CLINICAL PATHOLOGY Durnick, D. K., Law, M. E., Maurer, M. J., Natkunam, Y., Levy, R., Lossos, I. S., Kurtin, P. J., McPhail, E. D. 2010; 134 (2): 278-281


    Diffuse large B-cell lymphoma (DLBCL) can be separated for prognostic purposes using gene expression profiling (GEP) into 2 subgroups: germinal center B-cell (GCB) and activated B-cell phenotypes. However, GEP is impractical for routine clinical use, and immunophenotyping is an imperfect surrogate. Therefore, we studied the relationship between expression of the purported germinal center marker LMO2 and the presence of IGH-BCL2 fusions, BCL6 translocations, and LMO2 translocations. In addition, we investigated the usefulness of LMO2 expression as a marker of GCB subtype in DLBCL. Immunohistochemical and fluorescence in situ hybridization studies were successfully performed on 101 cases of de novo DLBCL that had been incorporated into a tissue microarray. There was a statistically significant association between IGH-BCL2 fusion and LMO2 protein expression (P = .02) but not between BCL6 translocations and LMO2 expression. LMO2 translocations were not identified. Although uncommon, all cases that had both IGH-BCL2 fusion and BCL6 translocations expressed LMO2. The findings suggest LMO2 as a potential marker for the GCB phenotype.

    View details for DOI 10.1309/AJCPATUP1D0HGCUG

    View details for Web of Science ID 000280067600015

    View details for PubMedID 20660332

  • Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sarosiek, K. A., Cavallin, L. E., Bhatt, S., Toomey, N. L., Natkunam, Y., Blasini, W., Gentles, A. J., Ramos, J. C., Mesri, E. A., Lossos, I. S. 2010; 107 (29): 13069-13074


    Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-kappaB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.

    View details for DOI 10.1073/pnas.1002985107

    View details for Web of Science ID 000280144500066

    View details for PubMedID 20615981

    View details for PubMedCentralID PMC2919898

  • Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. 2010; 30 (5): 984-U224


    We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

    View details for DOI 10.1161/ATVBAHA.110.202796

    View details for Web of Science ID 000276677700015

    View details for PubMedID 20167654

    View details for PubMedCentralID PMC2874560

  • C-C Chemokine Receptor 1 Expression in Human Hematolymphoid Neoplasia AMERICAN JOURNAL OF CLINICAL PATHOLOGY Anderson, M. W., Zhao, S., Ai, W. Z., Tibshirani, R., Levy, R., Lossos, I. S., Natkunam, Y. 2010; 133 (3): 473-483


    Chemokine receptor 1 (CCR1) is a G protein-coupled receptor that binds to members of the C-C chemokine family. Recently, CCL3 (MIP-1alpha), a high-affinity CCR1 ligand, was identified as part of a model that independently predicts survival in patients with diffuse large B-cell lymphoma (DLBCL). However, the role of chemokine signaling in the pathogenesis of human lymphomas is unclear. In normal human hematopoietic tissues, we found CCR1 expression in intraepithelial B cells of human tonsil and granulocytic/monocytic cells in the bone marrow. Immunohistochemical analysis of 944 cases of hematolymphoid neoplasia identified CCR1 expression in a subset of B- and T-cell lymphomas, plasma cell myeloma, acute myeloid leukemia, and classical Hodgkin lymphoma. CCR1 expression correlated with the non-germinal center subtype of DLBCL but did not predict overall survival in follicular lymphoma. These data suggest that CCR1 may be useful for lymphoma classification and support a role for chemokine signaling in the pathogenesis of hematolymphoid neoplasia.

    View details for DOI 10.1309/AJCP1TA3FLOQTMHF

    View details for Web of Science ID 000274687800016

    View details for PubMedID 20154287

  • The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation HAEMATOLOGICA-THE HEMATOLOGY JOURNAL Marafioti, T., Paterson, J. C., Ballabio, E., Chott, A., Natkunam, Y., Rodriguez-Justo, M., Plonquet, A., Rodriguez-Pinilla, S. M., Klapper, W., Hansmann, M., Pileri, S. A., Isaacson, P. G., Stein, H., Piris, M. A., Mason, D. Y., Gaulardll, P. 2010; 95 (3): 432-439


    T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified.In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas.Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules.ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.

    View details for DOI 10.3324/haematol.2009.010991

    View details for Web of Science ID 000276292100015

    View details for PubMedID 20207847

  • Characterization of D-cyclin proteins in hematolymphoid neoplasms: lack of specificity of cyclin-D2 and D3 expression in lymphoma subtypes MODERN PATHOLOGY Metcalf, R. A., Zhao, S., Anderson, M. W., Lu, Z. S., Galperin, I., Marinelli, R. J., Cherry, A. M., Lossos, I. S., Natkunam, Y. 2010; 23 (3): 420-433


    D-cyclin proteins play a central role in cell-cycle regulation and are involved in the pathogenesis of lymphomas. In mantle-cell lymphoma, the t(11;14) translocation leads to overexpression of cyclin-D1, in addition to which cyclin-D1-negative mantle-cell lymphoma that overexpress cyclin-D2 or D3 have also been described. Although cyclin-D2 and D3 have been implicated in the prognosis of specific lymphoma subtypes, a thorough characterization of D-cyclin protein expression in human hematolymphoid neoplasia has not been reported. To evaluate the tissue expression patterns of D-cyclins, particularly D2 and D3, in normal and neoplastic hematolymphoid tissues, we optimized the commercially available antibodies for D-cyclins for use on paraffin-embedded tissue and stained tissue microarrays of over 700 patient samples. Our results show that cyclin-D2 and D3 proteins are expressed in many more lymphoma subtypes than cyclin-D1. Cyclin-D1, D2 and D3 were expressed in 100, 22 and 6% of mantle-cell lymphomas and 2, 49 and 20% of diffuse large B-cell lymphomas. Fluorescence in situ hybridization studies confirmed the presence of the CCND1/IGH translocation in the majority of mantle-cell lymphoma, but not in diffuse large B-cell lymphoma that expressed cyclin-D1 protein. In addition, a subset of follicular, marginal zone, lymphoplasmacytic, lymphoblastic, classical Hodgkin, mature T-cell and natural killer cell lymphomas and acute myeloid leukemias also expressed cyclin-D2 and D3. These data support the hypothesis that dysregulation of cell-cycle control by D-cyclins contribute to the pathogenesis of hematolymphoid neoplasia, and suggest a potential role for these proteins in the prognostic and therapeutic aspects of these diseases. For diagnostic purposes, however, the expression of D-cyclin proteins should be interpreted with caution in the subclassification of lymphoma types.

    View details for DOI 10.1038/modpathol.2009.173

    View details for Web of Science ID 000275108600009

    View details for PubMedID 20062012

  • PD-1 Expression in T-cell Lymphomas and Reactive Lymphoid Entities: Potential Overlap in Staining Patterns Between Lymphoma and Viral Lymphadenitis AMERICAN JOURNAL OF SURGICAL PATHOLOGY Krishnan, C., Warnke, R. A., Arber, D. A., Natkunam, Y. 2010; 34 (2): 178-189


    Peripheral T-cell lymphomas are a heterogeneous group that often requires the use of ancillary testing for accurate diagnosis. This is particularly applicable to the diagnosis of angiommunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, unclassified (PTCLU), because of their histologic and immunophenotypic overlap with reactive lymphoid proliferations. Recently, immunohistochemistry for programmed death-1 (PD-1), a marker of follicular helper T cells, was shown to be sensitive in the detection of AITL and PTCLU. The sensitivity of this marker in reactive entities, however, has not been adequately evaluated. We confirm that PD-1 staining is a highly sensitive marker in the diagnosis of peripheral T-cell lymphomas: increased extrafollicular PD-1-positive cells were seen in 93% (76/82) of AITL, 62% (16/26) of PTCLU, and 11% (2/18) of anaplastic-lymphoma-kinase (ALK)-negative anaplastic large-cell lymphomas. The majority of reactive lymphadenopathies including Cat-scratch disease, Kikuchi lymphadenitis, Castleman disease, and reactive follicular hyperplasia showed no PD-1 staining outside follicles. Some reactive lymph nodes, showed increased extrafollicular PD-1-positive cells in a pattern similar to AITL and PTCLU, and include progressive transformation of germinal centers, viral lymphadenitis (Epstein-Barr virusand human immunodeficiency virus) and Rosai-Dorfman disease. This study shows that PD-1-positive cells may be increased in a number of settings other than T-cell lymphomas. We conclude that staining for PD-1 in reactive and atypical lymphadenopathies should be interpreted with caution and in the context of other ancillary immunophenotypic and molecular studies before a diagnosis of AITL or PTCLU is entertained.

    View details for Web of Science ID 000274219800005

    View details for PubMedID 20087161

  • Immunoarchitectural Patterns in Follicular Lymphoma: Efficacy of HGAL and LMO2 in the Detection of the Interfollicular Component 99th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Younes, S. F., Beck, A., Warnke, R. A., Lossos, I. S., Levy, R., Natkunam, Y. NATURE PUBLISHING GROUP. 2010: 330A–330A
  • CD81 protein is expressed at high levels in normal germinal center B cells and in subtypes of human lymphomas HUMAN PATHOLOGY Luo, R. F., Zhao, S., Tibshirani, R., Myklebust, J. H., Sanyal, M., Fernandez, R., Gratzinger, D., Marinelli, R. J., Lu, Z. S., Wong, A., Levy, R., Levy, S., Natkunam, Y. 2010; 41 (2): 271-280


    CD81 is a tetraspanin cell surface protein that regulates CD19 expression in B lymphocytes and enables hepatitis C virus infection of human cells. Immunohistologic analysis in normal hematopoietic tissue showed strong staining for CD81 in normal germinal center B cells, a cell type in which its increased expression has not been previously recognized. High-dimensional flow cytometry analysis of normal hematopoietic tissue confirmed that among B- and T-cell subsets, germinal center B cells showed the highest level of CD81 expression. In more than 800 neoplastic tissue samples, its expression was also found in most non-Hodgkin lymphomas. Staining for CD81 was rarely seen in multiple myeloma, Hodgkin lymphoma, or myeloid leukemia. In hierarchical cluster analysis of diffuse large B-cell lymphoma, staining for CD81 was most similar to other germinal center B cell-associated markers, particularly LMO2. By flow cytometry, CD81 was expressed in diffuse large B-cell lymphoma cells independent of the presence or absence of CD10, another germinal center B-cell marker. The detection of CD81 in routine biopsy samples and its differential expression in lymphoma subtypes, particularly diffuse large B-cell lymphoma, warrant further study to assess CD81 expression and its role in the risk stratification of patients with diffuse large B-cell lymphoma.

    View details for DOI 10.1016/j.humpath.2009.07.022

    View details for Web of Science ID 000276493600015

    View details for PubMedID 20004001

  • Lymphoma cell VEGFR2 expression detected by immunohistochemistry predicts poor overall survival in diffuse large B cell lymphoma treated with immunochemotherapy (R-CHOP) BRITISH JOURNAL OF HAEMATOLOGY Gratzinger, D., Advani, R., Zhao, S., Talreja, N., Tibshirani, R. J., Shyam, R., Horning, S., Sehn, L. H., Farinha, P., Briones, J., Lossos, I. S., Gascoyne, R. D., Natkunam, Y. 2010; 148 (2): 235-244


    Diffuse large B cell lymphoma (DLBCL) is clinically and biologically heterogeneous. In most cases of DLBCL, lymphoma cells co-express vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, suggesting autocrine in addition to angiogenic effects. We enumerated microvessel density and scored lymphoma cell expression of VEGF, VEGFR1, VEGFR2 and phosphorylated VEGFR2 in 162 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone)-like regimens. VEGFR2 expression correlated with shorter overall survival (OS) independent of International Prognostic Index (IPI) (P = 0.0028). Phosphorylated VEGFR2 (detected in 13% of cases) correlated with shorter progression-free survival (PFS, P = 0.044) and trended toward shorter OS on univariate analysis. VEGFR1 was not predictive of survival on univariate analysis, but it did correlate with better OS on multivariate analysis with VEGF, VEGFR2 and IPI (P = 0.036); in patients with weak VEGFR2, lack of VEGFR1 coexpression was significantly correlated with poor OS independent of IPI (P = 0.01). These results are concordant with our prior finding of an association of VEGFR1 with longer OS in DLBCL treated with chemotherapy alone. We postulate that VEGFR1 may oppose autocrine VEGFR2 signalling in DLBCL by competing for VEGF binding. In contrast to our prior results with chemotherapy alone, microvessel density was not prognostic of PFS or OS with R-CHOP-like therapy.

    View details for DOI 10.1111/j.1365-2141.2009.07942.x

    View details for Web of Science ID 000272884100006

    View details for PubMedID 19821819

  • Building "tissue" microarrays from suspension cells. Methods in molecular biology (Clifton, N.J.) Zhao, S., Natkunam, Y. 2010; 664: 93-101


    Tissue microarray (TMA) is a highly efficient method that allows for large-scale measurement of -expression of RNA or protein in multiple tissue sections simultaneously. Most TMAs are made from paraffin--embedded tissues. In this chapter, we detail a method that enables construction of TMAs from small volumes of cells in suspension. A TMA is built using pellets of 1 x 10(6) to 5 x 10(7) spun cells after fixation, processing, and embedding. The entire procedure is carried out in a microcentrifuge tube and yields excellent preservation of cytomorphology and immunoreactivity from both fresh and frozen suspension cells. It is particularly useful for the study of hematopoietic neoplasms presenting in the blood and bone marrow, fine needle aspirates, and body fluids as well as cultured cells. In addition, this versatile method may facilitate the exploration of gene expression profiling and protein expression in clinical trials where regular tissue biopsies are not available.

    View details for DOI 10.1007/978-1-60761-806-5_10

    View details for PubMedID 20690056

  • Prediction of Survival in Diffuse Large B-Cell Lymphoma Based On the Expression of Two Genes: Integration of Tumor and Microenvironment Contributions 51st Annual Meeting and Exposition of the American-Society-of-Hematology Alizadeh, A. A., Gentles, A. J., Alencar, A. J., Kohrt, H. E., Houot, R., Talreja, N., Shyam, R., Natkunam, Y., Gascoyne, R. D., Briones, J., Advani, R., Lossos, I. S., Levy, R. AMER SOC HEMATOLOGY. 2009: 258–58
  • Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1152–S1152
  • Microtubule-associated Protein-2 is a Sensitive Marker of Primary and Metastatic Neuroblastoma AMERICAN JOURNAL OF SURGICAL PATHOLOGY Krishnan, C., Higgins, J. P., West, R. B., Natkunam, Y., Heerema-McKenney, A., Arber, D. A. 2009; 33 (11): 1695-1704


    Microtubule-associated protein-2 (MAP-2) is a protein expressed in high levels in cells derived from the neural crest. To the best of our knowledge, MAP-2 expression has not been thoroughly evaluated in tissues outside of the central nervous tissue. We examined the diagnostic utility of MAP-2 as a marker of neuroblastoma and attempted to characterize the expression of this protein in other tumors in the morphologic differential diagnosis of neuroblastoma.MAP-2 showed significant cytoplasmic reactivity in 95% of primary and 100% of metastatic neuroblastomas. Included within this set of tumors were 3 undifferentiated neuroblastomas, all of which showed strong staining. MAP-2 did not show significant staining in the majority of other small round blue cell tumors within the morphologic differential. Additionally, MAP-2 showed comparable sensitivity in staining primary neuroblastomas as compared with synaptophysin, chromogranin, CD56, and beta-catenin. In contrast to other markers of neuroblastoma, MAP-2 did not show significant cross reactivity to native bone marrow precursors, thus eliminating a potential source of confusion. In normal tissues, MAP-2 staining was essentially restricted to organs derived from the neural crest (adrenal medulla, endocrine organs). Variant patterns of staining were seen in exocrine organs, monocyte/macrophages and solitary fibrous tumor/hemangiopericytoma family of tumors. Rarely, high-grade adult sarcomas exhibiting strong cytoplasmic MAP-2 staining were seen.MAP-2 is a sensitive and specific marker of neuroblastoma, both in the primary tumor and bone marrow biopsy settings. We think that MAP-2, in conjunction with synaptophysin, is a very powerful immunohistochemical marker in differentiating neuroblastoma from its morphologic mimics.

    View details for Web of Science ID 000271795800016

    View details for PubMedID 19701075

  • Extracellular Signal-Regulated Kinase Positively Regulates the Oncogenic Activity of MCT-1 in Diffuse Large B-Cell Lymphoma CANCER RESEARCH Dai, B., Zhao, X. F., Hagner, P., Shapiro, P., Mazan-Mamczarz, K., Zhao, S., Natkunam, Y., Gartenhaus, R. B. 2009; 69 (19): 7835-7843


    The MCT-1 oncogene was originally identified from lymphoma cell lines. Herein we establish that MCT-1 is highly expressed in 85% of human diffuse large B-cell lymphomas (DLBCL) and that knocking down MCT-1 by a specific short hairpin RNA in DLBCL cells induces apoptosis, supporting a critical role for MCT-1 in DLBCL cell survival. However, the mechanism underlying MCT-1 regulation is largely unknown. We find that MCT-1 is phosphorylated and up-regulated by extracellular signal-regulated kinase (ERK). Furthermore, by using a small inhibitory molecule targeting ERK, we interrupted MCT-1 phosphorylation and stability. Significantly, cells with distinct levels of MCT-1 protein displayed differential sensitivity to ERK inhibitor-induced apoptosis. Treatment with the ERK inhibitor showed marked in vivo antitumor activity in a human DLBCL xenograft model. Our findings establish a functional molecular interaction between MCT-1 and the MEK/ERK signaling pathway and suggest that the activation of MCT-1 function by its upstream kinase ERK plays an important role in lymphomagenesis.

    View details for DOI 10.1158/0008-5472.CAN-09-1606

    View details for Web of Science ID 000270487600044

    View details for PubMedID 19789340

  • Characterization of c-Maf Transcription Factor in Normal and Neoplastic Hematolymphoid Tissue and Its Relevance in Plasma Cell Neoplasia AMERICAN JOURNAL OF CLINICAL PATHOLOGY Natkunam, Y., Tedoldi, S., Paterson, J. C., Zhao, S., Rodriguez-Justo, M., Beck, A. H., Siebert, R., Mason, D. Y., Marafloti, T. 2009; 132 (3): 361-371


    c-Maf, a leucine zipper-containing transcription factor, is involved in the t(14;16)(q32;q23) translocation found in 5% of myelomas. A causal role for c-Maf in myeloma pathogenesis has been proposed, but data on c-Maf protein expression are lacking. We therefore studied the expression of c-Maf protein by immunohistochemical analysis in myelomas and in a wide variety of hematopoietic tissue. c-Maf protein was detected in a small minority (4.3%) of myelomas, including a t(14;16)(q32;q22-23)/IgH-Maf+ case, suggesting that c-Maf protein is not expressed in the absence of c-Maf rearrangement. In contrast, c-Maf was strongly expressed in hairy cell leukemia (4/4) and in a significant proportion of T-cell (24/42 [57%]) and NK/T-cell (49/97 [51%]) lymphomas, which is in keeping with prior gene expression profiling and transgenic mouse studies. Up-regulation of c-Maf protein occurs in a small subset of myelomas, in hairy cell leukemia, and in T- and NK-cell neoplasms. Its detection may be of particular value in the differential diagnosis of small cell lymphomas.

    View details for DOI 10.1309/AJCPEAGDKLWDMB1O

    View details for Web of Science ID 000269157600006

    View details for PubMedID 19687312

  • Immunoarchitectural Patterns in Nodal Marginal Zone B-Cell Lymphoma A Study of 51 Cases AMERICAN JOURNAL OF CLINICAL PATHOLOGY Salama, M. E., Lossos, I. S., Warnke, R. A., Natkunam, Y. 2009; 132 (1): 39-49


    Nodal marginal zone lymphoma (NMZL) represents a rare and heterogeneous group that lacks markers specific for the diagnosis. We evaluated morphologic and immunoarchitectural features of 51 NMZLs, and the following immunostains were performed: CD20, CD21, CD23, CD5, CD3, CD43, CD10, Ki-67, BCL1, BCL2, BCL6, HGAL, and LMO2. Four immunoarchitectural patterns were evident: diffuse (38 [75%]), well-formed nodular/follicular (5 [10%]), interfollicular (7 [14%]), and perifollicular (1 [2%]). Additional features included a monocytoid component (36 [71%]), admixed large cells (20 [39%]), plasma cells (24 [47%]), compartmentalizing stromal sclerosis (13 [25%]), and prominent blood vessel sclerosis (10 [20%]). CD21 highlighted disrupted follicular dendritic cell meshwork in 35 (71%) of 49 cases, and CD43 coexpression was present in 10 (24%) of 42 cases. A panel of germinal center-associated markers was helpful in eliminating cases of diffuse follicle center lymphoma. Our results highlight the histologic and immunoarchitectural spectrum of NMZL and the usefulness of immunohistochemical analysis for CD43, CD23, CD21, BCL6, HGAL, and LMO2 in the diagnosis of NMZL.

    View details for DOI 10.1309/AJCPZQ1GXBBNG8OG

    View details for Web of Science ID 000267206400007

    View details for PubMedID 19864232

  • Prognostic significance of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR), and vascularity in diffuse large B-cell lymphoma treated with immunochemotherapy (R-CHOP) 45th Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO) Gratzinger, D., Advani, R., Zhao, S., Talreja, N., Tibshirani, R. J., Horning, S. J., Levy, R., Lossos, I. S., Gascoyne, R. D., Natkunam, Y. AMER SOC CLINICAL ONCOLOGY. 2009
  • Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens NATURE MEDICINE Fan, A. C., Deb-Basu, D., Orban, M. W., Gotlib, J. R., Natkunam, Y., O'Neill, R., Padua, R., Xu, L., Taketa, D., Shirer, A. E., Beer, S., Yee, A. X., Voehringer, D. W., Felsher, D. W. 2009; 15 (5): 566-571


    Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie key cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. Therefore, we have developed a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in nanoliter volumes. Our method can quantify amounts of MYC oncoprotein and B cell lymphoma protein-2 (BCL2) in Burkitt's and follicular lymphoma; identify changes in activation of extracellular signal-related kinases-1 (ERK1) and ERK2, mitogen-activated kinase-1 (MEK), signal transducer and activator of transcription protein-3 (STAT3) and STAT5, c-Jun N-terminal kinase (JNK) and caspase-3 in imatinib-treated chronic myelogeneous leukemia (CML) cells; measure an unanticipated change in the phosphorylation of an ERK2 isomer in individuals with CML who responded to imatinib; and detect a decrease in STAT3 and STAT5 phosphorylation in individuals with lymphoma who were treated with atorvastatin. Therefore, we have described a new and highly sensitive method for determining oncoprotein expression and phosphorylation in clinical specimens for the development of new therapeutics for cancer.

    View details for DOI 10.1038/nm.1903

    View details for Web of Science ID 000265889300036

    View details for PubMedID 19363496

  • Low Stage Follicular Lymphoma: Biologic and Clinical Characterization According to Nodal or Extranodal Primary Origin AMERICAN JOURNAL OF SURGICAL PATHOLOGY Weinberg, O. K., Ma, L., Seo, K., Beck, A. H., Pai, R. K., Morales, A., Kim, Y., Sundram, U., Tan, D., Horning, S. J., Hoppe, R. T., Natkunam, Y., Arber, D. A. 2009; 33 (4): 591-598


    Studies suggest that primary extranodal follicular lymphoma (FL) is not infrequent but it remains poorly characterized with variable histologic, molecular, and clinical outcome findings. We compared 27 extranodal FL to 44 nodal FL using morphologic, immunohistochemical, and molecular genetic techniques and evaluated the clinical outcome of these 2 similarly staged groups. Eight cases of primary cutaneous follicle center lymphoma were also studied. In comparison to nodal FL, a greater number of extranodal FL contained a diffuse growth pattern (P=0.004) and lacked CD10 expression (P=0.014). Fifty-four percent of extranodal and 42% of nodal FL cases showed evidence of t(14;18), with minor breakpoints (icr, 3'BCL2, 5'mcr) more commonly found in extranodal cases (P=0.003). Outcome data showed no significant differences in overall survival (P=0.565) and progression-free survival (P=0.627) among extranodal, nodal, and primary cutaneous follicle center lymphoma cases. Analysis of all cases by t(14;18) status indicate that the translocation-negative group is characterized by a diffuse growth pattern (P=0.043) and lower BCL2 expression (P=0.018). The t(14;18)-positive group showed significantly better overall survival (P=0.019) and disease-specific survival (P=0.006) in comparison with the t(14;18)-negative group. In low stage FL, the status of t(14;18) seems to be more predictive of outcome than origin from an extranodal versus nodal site.

    View details for Web of Science ID 000264818800014

    View details for PubMedID 19065102

    View details for PubMedCentralID PMC4847152

  • Lack of Utility of CD20 Immunohistochemistry in Staging Bone Marrow Biopsies for Diffuse Large B-cell Lymphoma APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Baiyee, D., Warnke, R., Natkunam, Y. 2009; 17 (2): 93-95


    The utility of CD20 immunohistochemistry in the evaluation of staging bone marrow biopsies of newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients has not been extensively studied. We used 113 routinely processed bone marrow biopsies to study the extent and pattern of involvement by lymphoma and CD20 staining. Twelve (10.6%) of 113 cases had involvement by morphology, and 5 (41.7%) of these showed histologic discordance between the primary site and the bone marrow. All cases with morphologic evidence of bone marrow involvement showed staining for CD20. Four (3.5%) of 113 cases had non-neoplastic aggregates that stained for CD20. One case (0.9%) showed a small benign lymphoid aggregate by immunohistochemistry that was not evident by morphology. Our results demonstrate that CD20 staining did not detect any examples of bone marrow involvement by DLBCL that were not evident by morphology. We conclude that immunohistochemistry for CD20 adds no increase in the sensitivity of detection of bone marrow infiltration by DLBCL.

    View details for Web of Science ID 000263734200001

    View details for PubMedID 19521275

  • The Transcription Factor LMO2 Is a Robust Marker of Vascular Endothelium and Vascular Neoplasms and Selected Other Entities AMERICAN JOURNAL OF CLINICAL PATHOLOGY Gratzinger, D., Zhao, S., West, R., Rouse, R. V., Vogel, H., Gil, E. C., Levy, R., Lossos, I. S., Natkunam, Y. 2009; 131 (2): 264-278


    The transcription factor LMO2 is involved in vascular and hematopoietic development and hematolymphoid neoplasia. We have demonstrated that LMO2 is expressed nearly ubiquitously in native and neoplastic vasculature, including lymphatics. LMO2 reactivity is otherwise virtually absent in nonhematolymphoid tissues except in breast myoepithelium, prostatic basal cells, and secretory phase endometrial glands. Vasculature is LMO2- in adult and fetal heart, brain of older adults, hepatic sinusoids, and hepatocellular carcinoma. LMO2 is uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms with the exception of epithelioid vascular neoplasms of pleura and bone. Among nonvascular neoplasms, LMO2 reactivity is present in giant cell tumor of tendon sheath, juvenile xanthogranuloma, a subset of gastrointestinal stromal tumors, small round blue cell tumors, and myoepithelial-derived neoplasms. The restricted expression pattern, nuclear localization, and crisp staining of LMO2 in paraffin blocks make it an attractive candidate for the diagnostic immunohistochemistry laboratory.

    View details for DOI 10.1309/AJCP5FP3NAXAXRJE

    View details for Web of Science ID 000262540200013

    View details for PubMedID 19141387

  • CD81 Protein Is Expressed in Normal Germinal Center B-Cells and in Subtypes of Human Non-Hodgkin Lymphomas 98th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Luo, R. F., Zhao, S., Tibshirani, R., Lossos, I. S., Advani, R., Gratzinger, D., Wong, A., Talrega, N., Levy, R., Levy, S., Natkunam, Y. NATURE PUBLISHING GROUP. 2009: 275A–275A
  • Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma LEUKEMIA & LYMPHOMA Azambuja, D., Lossos, I. S., Biasoli, I., Morais, J. C., Britto, L., Scheliga, A., Pulcheri, W., Natkunam, Y., Spector, N. 2009; 50 (11): 1830-1836


    The human germinal center-associated lymphoma (HGAL) gene has prognostic value in diffuse large B-cell lymphoma, and expression of its cognate protein is germinal center-specific. A previous study had suggested that HGAL protein expression might also be related to the outcome in patients with Hodgkin lymphoma (HL). The aim of this study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 patients with classic HL treated uniformly with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD). Tissue microarray analysis showed HGAL staining in 188 specimens (81%). Failure-free survival (FFS) was superior in patients with early-stage disease, low-risk IPS, and HGAL-positive patients. The estimated 5-year FFS for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (p = 0.03). In the multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse FFS. This study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical HL.

    View details for DOI 10.3109/10428190903242628

    View details for Web of Science ID 000272145000021

    View details for PubMedID 19883310

  • Gray zones around diffuse large B cell lymphoma. Conclusions based on the workshop of the XIV meeting of the European Association for Hematopathology and the Society of Hematopathology in Bordeaux, France. Journal of hematopathology Quintanilla-Martinez, L., De Jong, D., de Mascarel, A., Hsi, E. D., Kluin, P., Natkunam, Y., Parrens, M., Pileri, S., Ott, G. 2009; 2 (4): 211-236


    The term "gray-zone" lymphoma has been used to denote a group of lymphomas with overlapping histological, biological, and clinical features between various types of lymphomas. It has been used in the context of Hodgkin lymphomas (HL) and non-Hodgkin lymphomas (NHL), including classical HL (CHL), and primary mediastinal large B cell lymphoma, cases with overlapping features between nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B cell lymphoma, CHL, and Epstein-Barr-virus-positive lymphoproliferative disorders, and peripheral T cell lymphomas simulating CHL. A second group of gray-zone lymphomas includes B cell NHL with intermediate features between diffuse large B cell lymphoma and classical Burkitt lymphoma. In order to review controversial issues in gray-zone lymphomas, a joint Workshop of the European Association for Hematopathology and the Society for Hematopathology was held in Bordeaux, France, in September 2008. The panel members reviewed and discussed 145 submitted cases and reached consensus diagnoses. This Workshop summary is focused on the most controversial aspects of gray-zone lymphomas and describes the panel's proposals regarding diagnostic criteria, terminology, and new prognostic and diagnostic parameters.

    View details for DOI 10.1007/s12308-009-0053-9

    View details for PubMedID 20309430

  • LMO2 Protein Expression Predicts Survival in Patients with Diffuse Large B-Cell Lymphoma Treated with Immunochemotherapy (RCHOP): A Multicenter Validation Study. 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Advani, R., Talreja, N., Tibshirani, R., Zhao, S., Alizadeh, A., Briones, J., Bordes, R., Cohen, J., Horning, S., Levy, R., Lossos, I. S., Natkunam, Y. AMER SOC HEMATOLOGY. 2008: 1291–91
  • Lymphoma-Expressed VEGF-a,VEGFR-1, VEGFR-2, and Microvessel Density Are Not Predictive of Overall Survival in Follicular Lymphoma. 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Gratzinger, D., Zhao, S., Ai, W., Tibshirani, R., Levy, R., Natkunam, Y. AMER SOC HEMATOLOGY. 2008: 1290–90
  • Neither CD68+Nor CD163+Macrophages Are Associated with Decreased Survival in Follicular Lymphoma 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Gratzinger, D., Ai, W., Tibshirani, R., Levy, R., Natkunam, Y. AMER SOC HEMATOLOGY. 2008: 1284–84
  • PTP1B is a negative regulator of interleukin 4-induced STAT6 signaling BLOOD Lu, X., Malumbres, R., Shields, B., Jiang, X., Sarosiek, K. A., Natkunam, Y., Tiganis, T., Lossos, I. S. 2008; 112 (10): 4098-4108


    Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4-induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A "substrate-trapping" mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)-inducible manner. We delineate a new negative regulatory loop of IL-4-JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and enhances PTP1B protein stability to suppress IL-4-induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell-like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4-induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes.

    View details for DOI 10.1182/blood-2008-03-148726

    View details for Web of Science ID 000260691300030

    View details for PubMedID 18716132

  • Immunohistochemical characterization of nasal-type extranodal NK/T-Cell lymphoma using a tissue microarray AMERICAN JOURNAL OF CLINICAL PATHOLOGY Schwartz, E. J., Molina-Kirsch, H., Zhao, S., Marinelli, R. J., Warnke, R. A., Natkunam, Y. 2008; 130 (3): 343-351


    Nasal-type extranodal natural killer (NK)/T-cell lymphoma is an uncommon malignancy. By using a tissue microarray, we characterized 84 cases of extranodal NK/T-cell lymphoma with regard to expression of 18 immunohistochemical markers and the presence of Epstein-Barr virus (EBV) RNA. In our series, CD2 was positive in 69 (93%) of 74 cases, CD3 in 68 (84%) of 81, CD5 in 22 (27%) of 81, CD20 in 0 (0%) of 82, CD29 in 75 (91%) of 82, CD30 in 29 (35%) of 84, CD43 in 81 (96%) of 84, CD54 in 58 (72%) of 81, CD56 in 46 (58%) of 79, CD62L in 23 (28%) of 83, CD183 in 66 (80%) of 83, BCL2 in 33 (39%) of 84, cutaneous lymphocyte antigen in 21 (25%) of 84, granzyme B in 70 (83%) of 84, Ki-67 in 59 (71%) of 83, linker for activation of T cells in 60 (71%) of 84, perforin in 66 (86%) of 77, TIA1 in 76 (90%) of 84, and EBV in 73 (87%) of 84. Hierarchical cluster analysis separated primary cutaneous cases from cases manifesting in other sites based on lower expression of the cell adhesion molecule CD54.

    View details for DOI 10.1309/V561QTM6854W4WAV

    View details for Web of Science ID 000258538900003

    View details for PubMedID 18701406

  • Tissue microarrays from bone marrow aspirates for high-throughput assessment of immunohistologic markers in pediatric acute leukemia PEDIATRIC AND DEVELOPMENTAL PATHOLOGY Hazard, F. K., Zhao, S., Schiffman, J. D., Lacayo, N. J., Dahl, G. V., Natkunam, Y. 2008; 11 (4): 283-290


    Gene expression profiling studies have been employed to investigate prognostic subgroups in pediatric acute leukemia. Tissue microarrays (TMAs) are useful for high-throughput analysis of protein expression of target genes in acute leukemia samples and for validation of gene microarray analysis. Using cryopreserved samples of pediatric acute leukemia bone marrow aspirates, we constructed TMA from as few as 1 million cells. Bone marrow core biopsies from the same patients were included on the same TMA for comparison. A panel of 15 immunohistochemical markers typically used for diagnosis as well as those targeting recently characterized, prognostically relevant molecules of interest in pediatric acute leukemia was used to evaluate protein expression. Staining results confirm that suspension cells from bone marrow aspirates can be effectively used to derive protein expression data from multiple cases simultaneously with comparable efficacy to that of biopsy tissue. This method allows for new markers of diagnostic, prognostic, or therapeutic importance to be screened on large numbers of study patients. Furthermore, this technique may facilitate the inclusion of small samples, aspirates, and body fluids in large-scale studies of protein expression in clinical trials and protocols in which tissue biopsies are often unavailable.

    View details for DOI 10.2350/07-04-0253.1

    View details for Web of Science ID 000259353300005

    View details for PubMedID 17990919

  • Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP BLOOD Malumbres, R., Chen, J., Tibshirani, R., Johnson, N. A., Sehn, L. H., Natkunam, Y., Briones, J., Advani, R., Connors, J. M., Byrne, G. E., Levy, R., Gascoyne, R. D., Lossos, I. S. 2008; 111 (12): 5509-5514


    Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies. However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP-treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffin-embedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS; P = .002) and progression-free survival (PFS; P = .038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (P = .002 and .010, respectively) and was independent of the IPI for prediction of OS (P = .008). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies.

    View details for DOI 10.1182/blood-2008-02-136374

    View details for Web of Science ID 000256786500021

    View details for PubMedID 18445689

  • Expression of HGAL in primary cutaneous large B-cell lymphomas: evidence for germinal center derivation of primary cutaneous follicular lymphoma MODERN PATHOLOGY Xie, X., Sundram, U., Natkunam, Y., Kohler, S., Hoppe, R. T., Kim, Y. H., Cook, J. R., Hammel, J., Swerdlow, S. H., Guitart, J., Smith, M. D., Bosler, D., Listinsky, C., Lossos, I. S., Hsi, E. D. 2008; 21 (6): 653-659


    The classification of primary cutaneous large B-cell lymphoma (PCLBCL) is based on standard morphology, immunohistochemistry, and clinical presentation. There are two major subtypes in the current WHO-EORTC classification: follicle center lymphoma and diffuse large B-cell lymphoma, leg-type (DLBCL-LT). The goals of this study were to examine a series of DLBCLs to determine (1) whether the immunohistochemical paradigm of germinal center B-cell and non-germinal center B-cell types of systemic DLBCL could be applied to PCLBCL; (2) whether application of the newly described germinal center B-cell marker, human germinal center-associated lymphoma (HGAL) also discriminates between these types as a further support for germinal center B-cell origin for primary cutaneous center lymphoma; and (3) whether any of these biologic markers were of prognostic significance. To this end, 32 cases of diffuse PCLBCL (22 primary cutaneous follicular center lymphomas and 10 DLBCL-LT) were classified based on the WHO-EORTC criteria and studied for expression of CD20, BCL2, BCL6, CD10, MUM-1, and HGAL by immunohistochemistry. Results were correlated with clinical features. HGAL and BCL6 expression and germinal center B-cell phenotype were associated with primary cutaneous follicular center lymphoma. The combination of HGAL and BCL6 positivity had the highest sensitivity (88%) and specificity (100%) for predicting subtype compared to either marker alone. Both HGAL and BCL6 were associated with the germinal center B-cell phenotype. The correlation of HGAL expression with the germinal center B-cell phenotype demonstrates the role of this marker in the classification of cutaneous large B-cell lymphomas. BCL6 expression was the only immunohistochemical marker associated with overall survival. Characterizing PCLBCLs with markers of B-cell maturation stage is a useful framework for studying, classifying, and clinically stratifying these lymphomas.

    View details for DOI 10.1038/modpathol.2008.30

    View details for Web of Science ID 000256112900002

    View details for PubMedID 18264083

  • LMO2 protein expression, LMO2 germline genetic variation, and overall survival in diffuse large B-cell lymphoma (DLBCL) 10th International Conference on Malignant Lymphoma Cerhan, J., Natkunam, Y., Morton, L., Maurer, M., Habermann, T., Chanock, S., Cozen, W., Lynch, C., Severson, R., Allmer, C., Lossos, I., Levy, R., Rothman, N., Slager, S., Hartge, P., Dogan, A., Wang, S. OXFORD UNIV PRESS. 2008: 107–107
  • Closing the gap: A comparison of observed versus expected survival in follicular lymphoma (FL) at Stanford University from 1960-2003 Tan, D., Rosenberg, S. A., Lavori, P., Sigal, B. M., Levy, R., Hoppe, R. T., Warnke, R., Advani, R., Natkunam, Y., Plevritis, S. K., Horning, S. J. AMER SOC CLINICAL ONCOLOGY. 2008
  • The transcription factor LMO2 is a robust marker of vascular endothelium and vascular neoplasms with rare exceptions Gratzinger, D., Zhao, S., Vogel, H., Gil, E. C., Levy, R., Lossos, I., Natkunam, Y. FEDERATION AMER SOC EXP BIOL. 2008
  • Expression of the human germinal-centre-associated lymphoma protein in diffuse large B-cell lymphomas in patients with rheumatoid arthritis BRITISH JOURNAL OF HAEMATOLOGY Baecklund, E., Natkunam, Y., Backlin, C., Iliadou, A., Askling, J., Ekbom, A., Feltelius, N., Klareskog, L., Enblad, G., Lossos, I. S., Levy, R., Sundstroem, C., Rosenquist, R. 2008; 141 (1): 69-72


    Diffuse large B-cell lymphomas (DLBCL) can be subdivided into germinal centre (GC)-like and non-GC-like subtypes by CD10, BCL6 and MUM1/IRF4 status. We previously reported that patients with severe rheumatoid arthritis (RA) are at increased risk of non-GC DLBCL. This study examined a new GC-marker, human germinal-centre-associated lymphoma (HGAL) protein, in RA-DLBCL. Of 111, 38 (34%) DLBCL were HGAL-positive and showed less disseminated disease and a tendency toward improved overall survival compared to HGAL-negative cases. This supports that a majority of RA-DLBCL are of non-GC origin, indicating a specific role for activated peripheral B cells in the pathogenesis of RA-DLBCL.

    View details for DOI 10.1111/j.1365-2141.2008.07011.x

    View details for Web of Science ID 000253757400008

    View details for PubMedID 18324968

  • LMO2 protein expression predicts survival in patients with diffuse large B-Cell lymphoma treated with anthracycline-based chemotherapy with and without rituximab JOURNAL OF CLINICAL ONCOLOGY Natkunam, Y., Farinha, P., Hsi, E. D., Hans, C. P., Tibshirani, R., Sehn, L. H., Connors, J. M., Gratzinger, D., Rosado, M., Zhao, S., Pohlman, B., Wongchaowart, N., Bast, M., Avigdor, A., Schiby, G., Nagler, A., Byrne, G. E., Levy, R., Gascoyne, R. D., Lossos, I. S. 2008; 26 (3): 447-454


    The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab.DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome.In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis.We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

    View details for DOI 10.1200/JCO.2007.13.0690

    View details for Web of Science ID 000254177200020

    View details for PubMedID 18086797

  • The Stanford Tissue Microarray Database NUCLEIC ACIDS RESEARCH Marinelli, R. J., Montgomery, K., Liu, C. L., Shah, N. H., Prapong, W., Nitzberg, M., Zachariah, Z. K., Sherlock, G. J., Natkunam, Y., West, R. B., van de Rijn, M., Brown, P. O., Ball, C. A. 2008; 36: D871-D877


    The Stanford Tissue Microarray Database (TMAD; is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license.

    View details for DOI 10.1093/nar/gkm861

    View details for Web of Science ID 000252545400154

    View details for PubMedID 17989087

    View details for PubMedCentralID PMC2238948

  • Expression of the germinal center B cell-associated marker LMO2 versus BCL2 and BCL6 translocations in diffuse large B-cell lymphoma 97th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Durnick, D. K., Law, M. E., Natkunam, Y., Levy, R., Lossos, I. S., Renistein, E. D., Dogan, A. NATURE PUBLISHING GROUP. 2008: 252A–252A
  • LMO2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy with or without rituximab 97th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Natkunam, Y., Farinha, P., Hsi, E. D., Hans, C. P., Tibshirani, R., Sehn, L. H., Connors, J. M., Gratzinger, D., Zhan, S., Pohlman, B., Nagler, A., Levy, R., Gascoyne, R. D., Lossos, I. S. NATURE PUBLISHING GROUP. 2008: 267A–267A
  • Prognostic significance of VEGF, VEGF receptors, and microvessel density in diffuse large B cell lymphoma treated with anthracycline-based chemotherapy LABORATORY INVESTIGATION Gratzinger, D., Zhao, S., Tibshirani, R. J., Hsi, E. D., Hans, C. P., Pohlman, B., Bast, M., Avigdor, A., Schiby, G., Nagler, A., Byrne, G. E., Lossos, I. S., Natkunam, Y. 2008; 88 (1): 38-47


    Vascular endothelial growth factor-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of lymphoma cell proliferation and/or survival induced by autocrine vascular endothelial growth factor receptor-mediated signaling. We have recently shown that diffuse large B cell lymphomas expressing high levels of vascular endothelial growth factor protein also express high levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether tumor vascularity, or expression of vascular endothelial growth factor protein and its receptors, contribute to patient outcomes. Our results show that increased tumor vascularity is associated with poor overall survival (P=0.047), and is independent of the international prognostic index. High expression of vascular endothelial growth factor receptor-1 by lymphoma cells by contrast is associated with improved overall survival (P=0.044). The combination of high vascular endothelial growth factor and vascular endothelial growth factor receptor-1 protein expression by lymphoma cells identifies a subgroup of patients with improved overall (P=0.003) and progression-free (P=0.026) survival; these findings are also independent of the international prognostic index. The prognostic significance of overexpression of this ligand-receptor pair suggests that autocrine signaling via vascular endothelial growth factor receptor-1 may represent a survival or proliferation pathway in diffuse large B cell lymphoma. Dependence on autocrine vascular endothelial growth factor receptor-1-mediated signaling may render a subset of diffuse large B-cell lymphomas susceptible to anthracycline-based therapy.

    View details for DOI 10.1038/labinvest.3700697

    View details for Web of Science ID 000251820600004

    View details for PubMedID 17998899

  • Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma JOURNAL OF PATHOLOGY Tedoldi, S., Mottok, A., Ying, J., Paterson, J. C., Cui, Y., Facchetti, F., van Krieken, J. H., Ponzoni, M., Oezkal, S., Masir, N., Natkunam, Y., Pileri, S. A., Hansmann, M., Mason, D. Y., Tao, Q., Marafioti, T. 2007; 213 (4): 429-440


    The neoplastic Reed-Sternberg cells characteristic of classical Hodgkin's lymphoma (cHL) are of B-cell origin but they almost always show striking loss of a range of B-cell-associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkin's lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL-6 expression, 'ongoing' Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down-regulation of a number of markers associated with the B-cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down-regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL.

    View details for DOI 10.1002/path.2242

    View details for Web of Science ID 000251292800010

    View details for PubMedID 17935142

  • Survival in follicular lymphoma: The Stanford experience, 1960-2003. 49th Annual Meeting of the American-Society-of-Hematology Tan, D., Rosenberg, S. A., Levy, R., Lavori, P., Tibshirani, R., Hoppe, R. T., Warnke, R., Advani, R., Natkunam, Y., Yuen, A., Horning, S. J. AMER SOC HEMATOLOGY. 2007: 1005A–1005A
  • Major histocomplatibility class II (MHCII) and germinal center associated gene expression correlate with overall survival in ritiximab and CHOP-like treated diffuse large B.cell lymphoma (DLBCL) patients, using 49th Annual Meeting of the American-Society-of-Hematology Malumbres, R., Johnson, N. A., Sehn, L. H., Natkunam, Y., Tibshirani, R., Briones, J., Connors, J. M., Levy, R., Gascoyne, R. D., Lossos, I. S. AMER SOC HEMATOLOGY. 2007: 23A–23A
  • The utility of PAX5 immunohistochemistry in the diagnosis of undifferentiated malignant neoplasms MODERN PATHOLOGY Jensen, K. C., Higgins, J. P., Montgomery, K., Kaygusuz, G., van de Rijn, M., Natkunam, Y. 2007; 20 (8): 871-877


    PAX5 is a B-cell transcription factor whose expression at the protein level is reliably detected by immunohistochemistry in routine biopsies. The purpose of this study was to investigate whether PAX5 immunohistochemistry has diagnostic benefit as a B-cell marker in the work-up of undifferentiated malignant neoplasms. Twenty-five cases previously diagnosed as undifferentiated malignant neoplasms were selected. In addition, 59 hematolymphoid and 884 non-hematolymphoid malignancies were studied such that the specificity of PAX5 immunohistochemistry could be addressed. Two of the 25 (8%) undifferentiated neoplasms showed diffuse staining for PAX5, which indicated a B-cell derivation for these neoplasms that was not appreciated at the time of initial diagnosis. PAX5 staining was detected in the vast majority of hematolymphoid tumors of B-cell derivation but only in 5 of 884 (less than 1%) non-hematolymphoid tumors. Our results further show that PAX5 may be the only detectable marker of B lineage in lymphomas that lack or show equivocal CD45RB and CD20 expression. We conclude that the addition of PAX5 to a panel of immunohistologic markers used in the interrogation of undifferentiated neoplasms is of diagnostic benefit. Its expression can also facilitate the diagnosis of classical and nodular lymphocyte-predominant Hodgkin lymphoma with atypical morphologic and immunohistologic features. Lastly, we have shown that the lack of its expression at the protein level in many epithelial and mesenchymal neoplasms renders PAX5 expression an extremely specific marker of the B lineage.

    View details for Web of Science ID 000248219700008

    View details for PubMedID 17529924

  • International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia LEUKEMIA Rawstron, A. C., Villamor, N., Ritgen, M., Boettcher, S., Ghia, P., Zehnder, J. L., Lozanski, G., Colomer, D., Moreno, C., Geuna, M., Evans, P. A., Natkunam, Y., Coutre, S. E., Avery, E. D., Rassenti, L. Z., Kipps, T. J., Caligaris-Cappio, F., Kneba, M., Byrd, J. C., Hallek, M. J., Montserrat, E., Hillmen, P. 2007; 21 (5): 956-964


    The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

    View details for DOI 10.1038/sj.leu.2404584

    View details for Web of Science ID 000245999900014

    View details for PubMedID 17361231

  • Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors AMERICAN JOURNAL OF PATHOLOGY Lehman, N. L., Tibshirani, R., Hsu, J. Y., Natkunam, Y., Harris, B. T., West, R. B., Masek, M. A., Montgomery, K., van de Rijn, M., Jackson, P. K. 2007; 170 (5): 1793-1805


    The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.

    View details for DOI 10.2353/ajpath.2007.060767

    View details for Web of Science ID 000246050400033

    View details for PubMedID 17456782

    View details for PubMedCentralID PMC1854971

  • Mast cell tryptase and microphthalmia transcription factor effectively discriminate cutaneous mast cell disease from myeloid leukemia cutis JOURNAL OF CUTANEOUS PATHOLOGY Sundram, U. N., Natkunam, Y. 2007; 34 (4): 289-295


    Cutaneous mast cell disorders are uncommon, but a subset, especially mastocytoma and mast cell leukemia, can histologically mimic myeloid leukemia cutis. Our objective was to employ a panel of cytochemical and immunohistochemical markers to determine which ones would be most useful in separating these two entities.We stained 17 cases of cutaneous mast cell disease and 20 cases of myeloid leukemia cutis with Giemsa, toluidine blue, or pinacyanol erythrosinate (PE), as well as with antibodies against mast cell tryptase, microphthalmia transcription factor (MiTF), CD117 (c-kit), myeloperoxidase, CD43, CD25, CD2, and CD68.Mast cell tryptase and MiTF emerged as highly sensitive and specific markers for mast cell disease in this context, as both antibodies stained all cases of mast cell diseases but none of myeloid leukemia cutis. Although CD117 stained all cases of mast cell disease, it also stained 2 of 18 cases of myeloid leukemia cutis. PE appeared to be specific for mast cell disease, as 11 of 12 cases stained with this marker, compared with 0 of 18 cases of myeloid leukemia cutis.Our results show that mast cell tryptase and MiTF are equally effective in distinguishing mast cell disease from myeloid leukemia cutis.

    View details for DOI 10.1111/j.1600-0560.2006.00602.x

    View details for Web of Science ID 000245099700001

    View details for PubMedID 17381798

  • Microvessel density and expression of vascular endothelial growth factor and its receptors in diffuse large B-cell lymphoma subtypes AMERICAN JOURNAL OF PATHOLOGY Gratzinger, D., Zhao, S., Marinelli, R. J., Kapp, A. V., Tibshirani, R. J., Hammer, A. S., Hamilton-Dutoit, S., Natkunam, Y. 2007; 170 (4): 1362-1369


    Angiogenesis is known to play a major role in neoplasia, including hematolymphoid neoplasia. We assessed the relationships among angiogenesis and expression of vascular endothelial growth factor and its receptors in the context of clinically and biologically relevant subtypes of diffuse large B-cell lymphoma using immunohistochemical evaluation of tissue microarrays. We found that diffuse large B-cell lymphoma specimens showing higher local vascular endothelial growth factor expression showed correspondingly higher microvessel density, implying that lymphoma cells induce local tumor angiogenesis. In addition, local vascular endothelial growth factor expression was higher in those specimens showing higher expression of the receptors of the growth factor, suggesting an autocrine growth-promoting feedback loop. The germinal center-like and nongerminal center-like subtypes of diffuse large B-cell lymphoma were biologically and prognostically distinct. Interestingly, only in the more clinically aggressive nongerminal center-like subtype were microvessel densities significantly higher in specimens showing higher vascular endothelial growth factor expression; the same was true for the finding of higher vascular endothelial growth factor receptor-1 expression in conjunction with higher vascular endothelial growth factor expression. These differences may have important implications for the responsiveness of the two diffuse large B-cell lymphoma subtypes to anti-vascular endothelial growth factor and anti-angiogenic therapies.

    View details for DOI 10.2353/ajpath.2007.060901

    View details for Web of Science ID 000245233000022

    View details for PubMedID 17392174

  • The oncoprotein LMO2 is expressed in normal germinal-center B cells and in human B-cell lymphomas BLOOD Natkunam, Y., Zhao, S., Mason, D. Y., Chen, J., Taidi, B., Jones, M., Hammer, A. S., Dutoit, S. H., Lossos, I. S., Levy, R. 2007; 109 (4): 1636-1642


    We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

    View details for DOI 10.1182/blood-2006-08-039024

    View details for Web of Science ID 000244219400043

    View details for PubMedID 17038524

  • Expression of the RNA-binding protein VICKZ in normal hematopoietic tissues and neoplasms HAEMATOLOGICA-THE HEMATOLOGY JOURNAL Natkunam, Y., Vainer, G., Chen, J., Zhao, S., Marinelli, R. J., Hammer, A. S., Hamilton-Dutoit, S., Pikarsky, E., Amir, G., Levy, R., Yisraeli, J. K., Lossos, I. S. 2007; 92 (2): 176-183


    VICKZ family members are RNA-binding regulatory proteins expressed during embryogenesis but not usually found in normal adult tissue. The presence of VICKZ in normal germinal centers (GC) prompted us to characterize the expression pattern of this protein in lymphoid and hematopoietic tissues.We generated a pan-VICKZ antibody that recognized all three isoforms of VICKZ protein and screened 889 patients' samples by immunohistologic methods. We also analyzed the expression of VICKZ in normal hematopoiesis tissue by staining samples of tonsils, lymph nodesVICKZ protein expression was documented for the first time in normal human GC and in follicular (126/165), mediastinal large B-cell (9/10), Burkitt (2/2), diffuse large B-cell (DLBCL, 155/200), lymphocyte-predominant Hodgkin's (12/13), classical Hodgkin's (101/108), and anaplastic large cell (6/8) lymphomas and in lymphoid and myeloid leukemias. Since DLBCL may derive from GC or non-GC B cells we performed hierarchical cluster analysis for VICKZ, HGAL, BCL6, CD10, MUM1/IRF4 and BCL2 which showed that VICKZ is expressed in both subtypes. In addition, VICKZ mRNA isoforms were differentially expressed in lymphoma subtypes and over 40% of DLBCL expressed hVICKZ2, an isoform not usually present in normal GC B cells.We show that in normal lymphoid tissues VICKZ is expressed in GC lymphocytes but in lymphoid neoplasms its expression is not limited to GC-derived lymphoma subtypes. However, VICKZ exhibits differential expression in lymphoma subtypes and thus may be a marker of potential value in the diagnosis and study of hematopoietic neoplasia. The aberrant expression of its isoforms in DLBCL raises the possibility that these isoforms may be associated with different functions and suggests that further study of their role in normal and neoplastic lymphoid cells is warranted.

    View details for Web of Science ID 000244233600006

    View details for PubMedID 17296566

  • Expression of the human germinal center-associated lymphoma (HGAL) protein identifies a subset of classic Hodgkin lymphoma of germinal center derivation and improved survival BLOOD Natkunam, Y., Hsi, E. D., Aoun, P., Zhao, S., Elson, P., Pohlman, B., Naushad, H., Bast, M., Levy, R., Lossos, I. S. 2007; 109 (1): 298-305


    The human germinal-center-associated lymphoma (HGAL) gene and its cognate protein are expressed in a germinal center (GC)-specific manner. Its expression in classic Hodgkin lymphoma (cHL) prompted us to address whether HGAL expression could distinguish biologically distinct subgroups of cHL. Tissue microarrays from 145 patients treated with curative intent showed HGAL staining in 75% and was closely correlated with MUM1/IRF4 (92%) expression. BCL6 (26%), CD10 (0%), BCL2 (31%), Blimp1 (0.02%), and Epstein-Barr virus (EBV) (20%) showed no specific correlation; neither did phospho-STAT6, a key mediator of IL-4 and IL-13 signaling that induces HGAL and is implicated in cHL pathogenesis. In our study cohort, the 5-year overall survival (OS) correlated with young age (less than 45 years, P < .001), low stage (stage I and II, P = .04), and low International Prognostic Score (P = .002). In univariate analysis, HGAL expression was associated with improved OS (P = .01) and failure-free survival (FFS) (P = .05) but was not independent of other factors in multivariate analysis of OS or FFS. The expression of the GC-specific marker HGAL in a subset of cHL suggests that these cHLs retain characteristics of GC-derived lymphomas. The association with improved OS in univariate but not multivariate analysis suggests that HGAL expression is related to known clinical parameters of improved survival.

    View details for DOI 10.1182/blood-2006-04-014977

    View details for Web of Science ID 000243153900050

    View details for PubMedID 16954503

  • The biology of the germinal center. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Natkunam, Y. 2007: 210-215


    The immune system requires the production of high affinity antibodies of different subclasses to accomplish its many effector functions. Specific steps in B-cell ontogeny that occur within germinal centers of secondary lymphoid organs create much of the diversity in the immune system. This process also provides the raw material for the genesis of B-cell lymphomas as misdirection of the molecular machinery that regulate these steps can cause chromosomal translocations, prevent apoptosis and promote proliferation of abnormal clones. Many recent avenues of investigation have elucidated that the germinal center is a dynamic microenvironment where B-cells undergo repeated rounds of mutation and selection. Gene expression studies have further shown that malignancies derived from germinal center B-cells elaborate specific gene expression signatures that derive from neoplastic cells as well as elements of the host response such as T-cells and macrophages. This review will examine the current understanding of B-cell development in the germinal center and the key molecules involved in this process. Interactions between lymphoma cells and their cellular partners and models in the growth and development of follicular lymphoma will be presented.

    View details for PubMedID 18024632

  • The oncoprotein LMO2 is expressed in a germinal center B-cell-associated pattern and predicts survival in patients with diffuse large B-cell lymphoma. 48th Annual Meeting of the American-Society-of-Hematology Natkunam, Y., Hsi, E. D., Hans, C., Zhao, S., Taidi, B., Chen, J., Mason, D. Y., Marafioti, T., Jones, M., Pohlman, B., Hammer, A. S., Hamilton-Dutoit, S., Avigdor, A., Schiby, G., Nagler, A., Byrne, G. E., Levy, R., Lossos, I. S. AMER SOC HEMATOLOGY. 2006: 243A–243A
  • Low CD27 expression in plasma cell dyscrasias correlates with high-risk disease: An immunohistochemical analysis AMERICAN JOURNAL OF CLINICAL PATHOLOGY Morgan, T. K., Zhao, S., Chang, K. L., Haddix, T. L., Domanay, E., Cornbleet, P. J., Arber, D. A., Natkunam, Y. 2006; 126 (4): 545-551


    Genome-wide expression studies using complementary DNA microarrays recently suggested a number of intriguing candidate genes for distinguishing plasma cell dyscrasias. Our objective was to test select markers using immunohistochemical analysis and a tissue microarray from paraffin-embedded bone marrow core biopsy specimens obtained from 8 patients with monoclonal gammopathy of undetermined significance, 17 with plasmacytoma, 160 with multiple myeloma, and 15 with plasma cell leukemia (PCL). We immunostained serial sections for CD138, CD27, CD56, p27, Ki-67, CD3, and CD20. Each core was scored in duplicate by observers blinded to phenotype and reported as the average percentage of CD138+ cells. The Mann-Whitney U test was used to determine significance between groups. PCL showed significantly less immunostaining for CD27 (P < .01) and p27 (P < .05) compared with plasmacytoma and multiple myeloma. Low CD27 expression also was associated with plasmacytoma progression to multiple myeloma (P <.05). Our results support the hypothesis that low CD27 expression correlates with high-risk disease, including primary PCL and decreased progression-free survival in solitary plasmacytoma.

    View details for DOI 10.1309/ELGMGX81C2UTP55R

    View details for Web of Science ID 000240638900007

    View details for PubMedID 16938662

  • EBV can protect latently infected B cell lymphomas from death receptor-induced apoptosis JOURNAL OF IMMUNOLOGY Snow, A. L., Lambert, S. L., Natkunam, Y., Esquivel, C. O., Krams, S. M., Martinez, O. M. 2006; 177 (5): 3283-3293


    The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV- and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-kappaB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.

    View details for Web of Science ID 000240002800065

    View details for PubMedID 16920969

  • Jaw I/LRMP, a germinal centre-associated marker for the immunohistological study of B-cell lymphomas JOURNAL OF PATHOLOGY Tedoldi, S., Paterson, J. C., Cordell, J., Tan, S., Jones, M., Manek, S., Dei Tos, A. P., Roberton, H., Masir, N., Natkunam, Y., Pileri, S. A., Facchetti, F., Hansmann, M., Mason, D. Y., Marafioti, T. 2006; 209 (4): 454-463


    Jaw1, also known as lymphoid-restricted membrane protein (LRMP), is an endoplasmic reticulum-associated protein. High levels of Jaw1/LRMP mRNA have been found in germinal centre B-cells and in diffuse large B-cell lymphomas of 'germinal centre' subtype. This paper documents Jaw1/LRMP expression at the protein level in human tissues by immunohistochemical and western blotting analysis using an antibody reactive with paraffin-embedded tissues. Jaw1/LRMP was highly expressed in germinal centre B-cells (in keeping with gene expression data), in 'monocytoid B-cells', and in splenic marginal zone B-cells. It was absent, or present at only low levels, in mature T-cells, although cortical thymocytes were weakly positive. Among lymphoid neoplasms, Jaw1/LRMP was found in germinal centre-derived lymphomas (follicle centre lymphoma, Burkitt's lymphoma, lymphocyte-predominant Hodgkin's disease) but not in T-cell neoplasms (with the exception of a single T lymphoblastic lymphoma). Classical Hodgkin's disease and myeloma lacked Jaw1/LRMP but many cases of chronic lymphocytic leukaemia (but not mantle zone lymphoma) were Jaw1/LRMP-positive. Approximately half of the marginal zone lymphomas were Jaw1/LRMP-positive. In diffuse large B-cell lymphomas, Jaw1/LRMP was found in three-quarters (24/32) of the cases classified phenotypically as being of 'germinal centre' type, but it was also expressed in almost half (13/28) of the 'non-germinal centre' cases. A similar proportion of 'non-germinal centre' cases were positive for the protein products of two other genes expressed highly in germinal centre cells (HGAL/GCET2 and PAG). The fact that all three of these proteins are expressed in a significant proportion of diffuse large B-cell lymphomas assigned to the 'non-germinal centre' category indicates that the immunophenotypic categorization of diffuse large B-cell lymphoma according to cellular origin may be more complicated than currently understood. Finally, the expression of Jaw1/LRMP in other types of lymphoma and in non-lymphoid tissues/tumours may be of interest in differential diagnosis and research.

    View details for DOI 10.1002/path.2002

    View details for Web of Science ID 000239669600005

    View details for PubMedID 16739114

  • Prognostic immunohistologic markers in human tumors: why are so few used in clinical practice? LABORATORY INVESTIGATION Natkunam, Y., Mason, D. Y. 2006; 86 (8): 742-747


    Technological advances in gene cloning and genome-wide analyses have greatly increased the number of new tumor markers that can be detected by immunohistologic techniques. While many of these have been evaluated with respect to prognosis, there is a striking discrepancy between the number of markers reported to confer prognostic information and those that are used in clinical practice. We argue that lessons learned from epidemiological studies are applicable to studies of immunohistologic markers; in particular, advances in both fields can be vitiated by non-causal associations. We suggest that the most valuable immunohistologic markers are those that reflect genetic abnormalities, that are linked to the cell of origin, or that reflect tumor infiltrating cells or stromal reactions. It should also be appreciated that a marker that is genuinely predictive of prognosis may nevertheless not find any application in clinical practice if it becomes obsolete through the introduction of newer therapies or because there is no choice of alternative treatment strategies.

    View details for DOI 10.1038/labinvest.3700447

    View details for Web of Science ID 000239201300001

    View details for PubMedID 16855595

  • The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas HAEMATOLOGICA-THE HEMATOLOGY JOURNAL Paterson, J. C., Tedoldi, S., Craxton, A., Jones, M., Hansmann, M., Collins, G., Roberton, H., Natkunam, Y., Pileri, S., Campo, E., Clark, E. A., Mason, D. Y., Marafioti, T. 2006; 91 (6): 772-780


    We explored the expression of LCK and BAFF-R (B-cell activating factor receptor) both of which are known to play a role in signaling and apoptosis, in routine tissue biopsies. It was hypothesized that their expression patterns might yield information on apoptosis as it occurs in normal and reactive lymphoid cells, and also be of value for the detection of lymphoma subtypes.Both molecules were studied in paraffin-embedded tissue sections and cell lines by immunoperoxidase staining, and were also studied by western blotting. Human tonsillar B-cell subsets were analyzed by flow cytometry for LCK expression.LCK was detected for the first time in germinal centers and, at lower levels, in mantle zone B cells. The presence of LCK in B cells was confirmed by western blotting. Cross-linking surface IgM reduced LCK expression whereas cross-linking surface CD40 appeared to have the opposite effect. BAFF-R was present on mantle zone B cells but absent or weakly expressed in germinal center cells. Most lymphomas of germinal center origin (e.g. follicular lymphoma) and also many mantle cell lymphomas, chronic lymphocytic leukemia (CLL) and most T-cell neoplasms expressed LCK. In contrast, BAFF-R was expressed in a variety of B-cell lymphomas, but often absent in grade 3 follicular lymphomas and diffuse large B-cell lymphomas (DLBCL). Both LCK-positive and BAFF-R-positive DLBCL tended to be of germinal-center phenotype.The reciprocal expression pattern of LCK and BAFF-R in germinal center and mantle zone B cells may reflect their opposing roles in apoptosis. Their detection in lymphoma tissue biopsies may therefore be of clinical relevance in predicting response to treatment.

    View details for Web of Science ID 000238322400017

    View details for PubMedID 16769579

  • CD10 expression in peripheral T-cell lymphomas complicated by a proliferation of large B-cells MODERN PATHOLOGY Reichard, K. K., Schwartz, E. J., Higgins, J. P., Narasimhan, B., Warnke, R. A., Natkunam, Y. 2006; 19 (3): 337-343


    CD10 expression by the neoplastic T cells in angioimmunoblastic T-cell lymphoma was recently described. As cases of peripheral T-cell lymphoma, unspecified, fail to show similar CD10 expression, this feature helps discriminate between these two entities, particularly in cases exhibiting morphologic overlap. Given these findings, we studied CD10 expression in a subtype of peripheral T-cell lymphoma known as peripheral T-cell lymphoma complicated by a proliferation of large B cells and compared it with angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation. A total of 33 cases were identified including peripheral T-cell lymphoma complicated by a proliferation of large B cells (10), angioimmunoblastic T-cell lymphoma (10) and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation (13). Diagnoses were established by hematoxylin and eosin (H&E) stain, immunohistochemistry and/or molecular findings (polymerase chain reaction for T-cell receptor-gamma gene rearrangement). Two of 10 cases of peripheral T-cell lymphoma complicated by a proliferation of large B cells showed aberrant CD10 expression (20%) compared to 9/10 cases of angioimmunoblastic T-cell lymphoma (90%) and 8/13 of angioimmunoblastic T-cell lymphoma with a large B-cell proliferation (62%). One case each of angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation showed a rare, but not unequivocal, CD10+ atypical cell. Four cases of angioimmunoblastic T-cell lymphoma with a large B-cell proliferation were CD10 negative. Of the 2 CD10+ peripheral T-cell lymphoma complicated by a proliferation of large B cells, one had no H&E or IHC features of angioimmunoblastic T-cell lymphoma and showed only a rare positive cell. The second case, a lung biopsy, exhibited diffuse CD10 tumor cell positivity. The predominant staining pattern in the CD10+ cases was characterized by scattered, mostly individual, morphologically neoplastic cells. A rare case showed clusters of positive cells. Our data indicate that only 20% of cases of peripheral T-cell lymphoma complicated by a proliferation of large B cells show CD10 expression by the neoplastic T cells in contrast to angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation which exhibit CD10 staining in 90 and 62% of cases, respectively. This finding does not reach statistical significance with a P-value of 0.57 (Fisher's exact test). As these entities appear to be biologically distinct and may portend different overall survivals, CD10 expression may serve as an additional discriminating criterion.

    View details for DOI 10.1038/modpathol.3800536

    View details for Web of Science ID 000235592800001

    View details for PubMedID 16400325

  • Loss of CD19 expression in B-cell neoplasms HISTOPATHOLOGY Masir, N., Marafioti, T., Jones, M., Natkunam, Y., Rudiger, T., Hansmann, M. L., Mason, D. Y. 2006; 48 (3): 239-246


    To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas.A polyclonal antibody to the C terminus of CD19 was used to immunostain paraffin-embedded samples of normal and neoplastic lymphoid tissues. CD19 was widely expressed in normal B cells and in extramedullary plasma cells. It was found in most B-cell neoplasms, but expression in follicular lymphoma was weak (33/69) or negative (four cases). Similarly, CD19 expression in diffuse large B-cell lymphomas was weak (28/56) or negative (eight cases). In T-cell-rich B-cell lymphomas, CD19 was also weak (4/10) or negative (three cases). CD19 was often absent in post-transplant B lymphoproliferative disease, classical Hodgkin's disease and plasma cell neoplasms. An unexpected finding was the frequent absence of CD19 in the neoplastic cells in lymphocyte predominant Hodgkin's disease.CD19 can now be detected in routine biopsy specimens. In contrast to the classical pan-B marker CD20, CD19 is not always strongly expressed in B-cell neoplasms. Furthermore, the lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin's disease (which express most B-cell-associated markers) commonly lack CD19.

    View details for DOI 10.1111/j.1365-2559.2005.02317.x

    View details for Web of Science ID 000234799900003

    View details for PubMedID 16430470

  • BCL-6 negativity, female gender, and morphologic type, predict poor overall survival in primary cutaneous large B-Cell lymphomas with a diffuse architecture 95th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Xie, X., Sundram, U., Kohler, S., Lossos, I., Natkunam, Y., Cook, J. R., Swerdlow, S. H., Guitart, J., Smith, M., Bosler, D., Listinsky, C., Hammel, J., Hsi, E. D. NATURE PUBLISHING GROUP. 2006: 252A–252A
  • RNA-binding protein VICKZ is expressed in a germinal center associated pattern among lymphoma subtypes 95th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Natkunam, Y., Vainer, G., Zhao, S., Amir, G., Pikarsky, E., Hammer, A. S., Hamilton-Dutoit, S., Levy, R., Yisraeli, J., Lossos, I. S. NATURE PUBLISHING GROUP. 2006: 238A–239A
  • BCL-6 negativity, female gender, and morphologic type predict poor overall survival in primary cutaneous large B-cell lymphomas with a diffuse architecture 95th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Xie, X., Sundram, U., Kohler, S., Lossos, I., Natkunam, Y., Cook, J. R., Swerdlow, S. H., Guitart, J., Smith, M., Bosler, D., Listinsky, C., Hammel, J., Hsi, E. D. NATURE PUBLISHING GROUP. 2006: 252A–252A
  • Expression of the human germinal center associated lymphoma (HGAL) protein identifies a subset of classical Hodgkin lymphoma of germinal center derivation and improved outcome 95th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Natkunam, Y., Hsi, E. D., Zhao, S., Elson, P., Pohlman, B., Levy, R., Lossos, I. S. NATURE PUBLISHING GROUP. 2006: 239A–239A
  • The T-cell oncoprotein LMO2 is expressed in normal germinal center B-cells and in germinal center-derived B-cell lymphoma 95th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Natkunam, Y., Levy, R., Zhao, S., Taidi, B., Lossos, I. S. NATURE PUBLISHING GROUP. 2006: 239A–239A
  • Transmembrane adaptor molecules: a new category of lymphoid-cell markers BLOOD Tedoldi, S., Paterson, J. C., Hansmann, M. L., Natkunam, Y., Rudiger, T., Angelisova, P., Du, M. Q., Roberton, H., Roncador, G., Sanchez, L., Pozzobon, M., Masir, N., Barry, R., Pileri, S., Mason, D. Y., Marafioti, T., HOREJSI, V. 2006; 107 (1): 213-221


    Transmembrane adaptor proteins (of which 7 have been identified so far) are involved in receptor signaling in immune cells. They have only a short extracellular region, with most of the molecule comprising a substantial intracytoplasmic region carrying multiple tyrosine residues that can be phosphorylated by Src- or Syk-family kinases. In this paper, we report an immunohistologic study of 6 of these molecules in normal and neoplastic human tissue sections and show that they are restricted to subpopulations of lymphoid cells, being present in either T cells (LAT, LIME, and TRIM), B cells (NTAL), or subsets of both cell types (PAG and SIT). Their expression in neoplastic lymphoid cells broadly reflects that of normal lymphoid tissue, including the positivity of plasma cells and myeloma/plasmacytoma for LIME, NTAL, PAG, and SIT. However, this study also revealed some reactions that may be of diagnostic/prognostic value. For example, lymphocytic lymphoma and mantle-cell lymphoma showed similar profiles but differed clearly from follicle-center lymphoma, whereas PAG tended to be selectively expressed in germinal center-derived subsets of diffuse large B-cell lymphoma. These molecules represent a potentially important addition to the panel of immunophenotypic markers detectable in routine biopsies that can be used in hematopathologic studies.

    View details for DOI 10.1182/blood-2005-06-2273

    View details for Web of Science ID 000234235200040

    View details for PubMedID 16160011

  • Utility of syndecan-1 (CD138) expression in the diagnosis of undifferentiated malignant neoplasms - A tissue microarray study of 1,754 cases APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Kambham, N., Kong, C., Longacre, T. A., Natkunam, Y. 2005; 13 (4): 304-310


    Syndecan-1, a heparan sulfate-rich membrane glycoprotein, is expressed in plasma cells and is considered a reliable marker of plasmacytic differentiation. However, it has not been widely tested in non-hematolymphoid tissues, and thus its utility in the setting of an undifferentiated malignant neoplasm has not been evaluated. The authors conducted an extensive study of CD138 staining in over 1,700 normal, benign, and malignant non-hematolymphoid tissues, using five tissue microarrays. Immunohistochemical staining was performed with two commercially available CD138 monoclonal antibodies directed against syndecan-1 (Serotec, Oxford, UK, and DAKO, Carpenteria, CA). In addition to the specific membrane staining, many normal tissues and epithelial tumors showed strong cytoplasmic immunoreactivity. A small subset of mesenchymal neoplasms also showed membrane and cytoplasmic immunoreactivity. In squamous cell carcinoma of the head and neck, renal cell carcinoma, and prostate adenocarcinoma, the intensity of CD138 staining inversely correlated with the histologic grade of the carcinoma. However, statistically significant staining differences and their correlation with histologic grades differed depending on whether the Serotec or the DAKO antibody was used. These results indicate that CD138 immunoreactivity is widespread in normal and neoplastic epithelial tissues, as well as a variety of undifferentiated epithelial and mesenchymal processes. The authors conclude that the expression of syndecan-1, although relatively specific to plasma cells within the hematolymphoid system, should be interpreted with extreme caution in the setting of an undifferentiated neoplasm. Furthermore, the two commercially available monoclonal CD138 antibodies tested in this study showed significant differences in their immunoreactivity in different tumor types.

    View details for Web of Science ID 000233572700002

    View details for PubMedID 16280658

  • TMA-combiner, a simple software tool to permit analysis of replicate cores on tissue microarrays MODERN PATHOLOGY Liu, C. L., Montgomery, K. D., Natkunam, Y., West, R. B., Nielsen, T. O., Cheang, M. C., Turbin, D. A., Marinelli, R. J., van de Rijn, M., Higgins, J. P. 2005; 18 (12): 1641-1648


    We have previously published a suite of software tools that facilitates the reformulation of tissue microarray (TMA) data so that it may be analyzed using techniques originally devised for analysis of cDNA microarray data. However, current microarray data often feature multiple scores for a given tissue sample and antibody combination. Furthermore, an efficient and systematic method for combining scores that takes into account the differing staining properties of tissue epitopes has not been described. We thus present the TMA-Combiner, a new Microsoft Excel-based macro that permits analysis of data for which tissues may have two or more scores per antibody, and permits combination of data from multiple different tissue microarrays. It accomplishes this by rendering one score per tissue per antibody from two or more scores, using one of multiple user-selectable combination rules developed to account for the differing staining properties of tissue epitopes. This greatly facilitates analysis of tissue microarrays, particularly for users with large repositories of data, and may facilitate discovery of biological trends and help refine diagnostic accuracy of tissue markers in clinical samples.

    View details for DOI 10.1038/modpathol.3800491

    View details for Web of Science ID 000233372100016

    View details for PubMedID 16258508

  • Follicular dendritic cell immunohistochemical markers in angioimmunoblastic T-cell lymphoma APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Troxell, M. L., Schwartz, E. J., van de Ruin, M., Ross, D. T., Warnke, R. A., Higgins, J. P., Natkunam, Y. 2005; 13 (4): 297-303


    Angioimmunoblastic T-cell lymphoma is characterized by a paracortical proliferation of medium to large neoplastic T cells, often with clear cytoplasm, in a background of arborizing high endothelial venules, many surrounded by follicular dendritic cells (FDCs). IHC staining may be applied to highlight these extrafollicular FDCs, traditionally using CD21, or CD23. Several alternative FDC markers have been described, including CNA.42, cystatin A/acid cysteine proteinase inhibitor (ACPI, involved in antigen presentation), and fascin (an actin binding protein). The authors stained a collection of 45 angioimmunoblastic T-cell lymphomas with CD21, CD23, CNA.42, cystatin A, and fascin for direct comparison of FDC staining characteristics in this setting. CD21 highlighted the expected dendritic network of cell processes, within residual follicles and outside of follicles, often adjacent to proliferating vessels. CD23 exhibited similar staining quality but was less sensitive than CD21. CNA.42 showed only diffuse weak labeling of FDCs. Cystatin A stained the cytoplasm of follicular dendritic cells within and outside of follicles; however, staining was often not sharply localized to dendritic cell processes, and scoring was further complicated by reactivity with other cell types in over half of the cases. Likewise, fascin stained a variety of cell types, including strong staining of interdigitating dendritic-like cells, moderate staining of endothelial cells, and only weak staining of follicular dendritic cells within and outside of follicles. Thus, CD21 remains the most reliable marker of follicular dendritic cells in angioimmunoblastic T-cell lymphoma.

    View details for Web of Science ID 000233572700001

    View details for PubMedID 16280657

  • RNA-binding protein VICKZ is expressed in a germinal center associated pattern among lymphoma subtypes. 47th Annual Meeting of the American-Society-of-Hematology Natkunam, Y., Vainer, G., Zhao, S. C., Amir, G., Pikarsky, E., Hammer, A. S., Hamilton-Dutoit, S., Levy, R., Yisraeli, J. K., Lossos, I. S. AMER SOC HEMATOLOGY. 2005: 542A–542A
  • Expression of the human germinal center associated lymphoma (HGAL) protein identifies a subset of classical Hodgkin lymphoma of germinal center derivation and improved outcome. 47th Annual Meeting of the American-Society-of-Hematology Natkunam, Y., Hsi, E. D., Zhao, S. C., Elson, P., Pohlman, B., Levy, R., Lossos, I. S. AMER SOC HEMATOLOGY. 2005: 11A–12A
  • Cytologic diagnosis of Burkitt lymphoma. Cancer Troxell, M. L., Bangs, C. D., Cherry, A. M., Natkunam, Y., Kong, C. S. 2005; 105 (5): 310-318


    The diagnosis and classification of lymphoma require correlation of morphologic, immunophenotypic, and molecular-cytogenetic studies. Fine-needle aspiration biopsy (FNAB) is a valuable diagnostic technique that allows material to be collected for these ancillary studies, and for morphologic evaluation.The authors report a series of seven cases clinically or morphologically suspicious for Burkitt lymphoma. Fluorescence in situ hybridization studies (FISH) for c-myc were performed on FNAB material and correlated with cytologic and immunophenotypic data.Six of seven specimens were positive for c-myc rearrangement by FISH. However, only three of these cases represented Burkitt lymphoma, with one additional case of atypical Burkitt lymphoma. The other cases included diffuse large B-cell lymphoma, monomorphic posttransplant B-cell lymphoma, and an aggressive B-cell lymphoma, with the latter case negative for c-myc rearrangement by FISH. Of 2 non-Burkitt lymphoma specimens tested, 1 was positive for the immunoglobulin H/bcl-2 rearrangement, in addition to the c-myc rearrangement, suggesting transformation from a lower grade lymphoma.These cases illustrated the value of FNAB in the diagnosis of Burkitt lymphoma, as well as the importance of obtaining material for, and integrating results of, ancillary studies for the final diagnosis.

    View details for PubMedID 15986398

  • Cytologic diagnosis of Burkitt lymphoma - Role of ancillary studies 94th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Troxell, M. L., Bangs, C. D., Cherry, A. M., Natkunam, Y., Kong, C. S. JOHN WILEY & SONS INC. 2005: 310–18

    View details for DOI 10.1002/cncr.21307

    View details for Web of Science ID 000232474300009

  • Treatment of verruca vulgaris with topical cidofovir in an immunlocompromised patient: a case report and review of the literature TRANSPLANT INFECTIOUS DISEASE Cha, S., Johnston, L., Natkunam, Y., Brown, J. 2005; 7 (3-4): 158-161


    Lesions caused by verrucus vulgaris are commonly refractory to therapy and may become large, painful, or disfiguring in immunocompromised patients. Cidofovir is a potent nucleoside analog antiviral agent shown to have in vitro and in vivo activity against a broad spectrum of DNA viruses. We report a successful use of topical cidofovir to treat verruca vulgaris lesions in a highly immunocompromised patient, who was not considered a candidate for conventional therapy.

    View details for Web of Science ID 000236936200012

    View details for PubMedID 16390407

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


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

    View details for Web of Science ID 000229725900037

    View details for PubMedID 15958642

  • Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation BLOOD Natkunam, Y., Lossos, L. S., Taidi, B., Zhao, S. C., Lu, X. Q., Ding, F. Y., Hammer, A. S., Marafioti, T., Byrne, G. E., Levy, S., Warnke, R. A., Levy, R. 2005; 105 (10): 3979-3986


    We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

    View details for DOI 10.1182/blood-2004-08-3112

    View details for Web of Science ID 000229009000042

    View details for PubMedID 15677569

  • Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma LEUKEMIA Marafioti, T., Pozzobon, M., Hansmann, M. L., Gaulard, P., Barth, T. F., Copie-Bergman, C., Roberton, H., Ventura, R., Martin-Subero, J. I., Gascoyne, R. D., Pileri, S. A., Siebert, R., Hsi, E. D., Natkunam, Y., Moller, P., Mason, D. Y. 2005; 19 (5): 856-861


    Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

    View details for DOI 10.1038/sj.leu.2403702

    View details for Web of Science ID 000228692500024

    View details for PubMedID 15744341

  • Expression of CD163 (hemoglobin scavenger receptor) in normal tissues, lymphomas, carcinomas, and sarcomas is largely restricted to the monocyte/macrophage lineage AMERICAN JOURNAL OF SURGICAL PATHOLOGY Nguyen, T. D., Schwartz, E. J., West, R. B., Warnke, R. A., Arber, D. A., Natkunam, Y. 2005; 29 (5): 617-624


    CD163, a hemoglobin scavenger receptor, is expressed in monocytes and macrophages. We tested the expression of the CD163 protein in 1,105 human malignancies and normal tissues using tissue microarrays and conventional paraffin-embedded tissue sections. Besides staining nonneoplastic monocytes and histiocytes (tissue macrophages), membranous/cytoplasmic staining for CD163 was primarily limited to neoplasms with monocytic/histiocytic differentiation. CD163 reactivity was not observed in normal tissues, lymphomas, carcinomas, and in a majority of mesenchymal neoplasms, including follicular dendritic cell tumors (0 of 4), although it stained admixed histiocytes. Staining for CD163 was seen in Rosai-Dorfman disease (5 of 6), histiocytic sarcoma (3 of 4), littoral cell angioma (6 of 6), and Langerhans cell histiocytosis (3 of 5). A subset of atypical fibrous histiocytomas (9 of 16), benign fibrous histiocytomas (6 of 9), and atypical fibroxanthomas (1 of 3) also showed CD163 staining. Our studies also confirm earlier work showing that CD163 is expressed in acute myeloid leukemia with monocytic differentiation (AML, FAB subtype M5) (2 of 6), as well as a majority of giant cell tenosynovial tumors (7 of 8). Its limited range of expression and tissue specificity indicate that CD163 may have significant diagnostic utility in separating specific tumors with monocytic and histiocytic derivation from other entities in their differential diagnosis.

    View details for Web of Science ID 000228707200007

    View details for PubMedID 15832085

  • Expression of the bcl-6 and MUM1/IRF4 proteins correlate with overall and disease-specific survival in patients with primary cutaneous large B-cell lymphoma: a tissue microarray study JOURNAL OF CUTANEOUS PATHOLOGY Sundram, U., Kim, Y., Mraz-Gernhard, S., Hoppe, R., Natkunam, Y., Kohler, S. 2005; 32 (3): 227-234


    Systemic B-cell lymphomas have been studied using microarrays, which has led to a better understanding of their molecular characteristics. Initial microarray studies of these lymphomas have implicated several genes as important predictors of outcome. In this study, we used a tissue microarray (TMA) to characterize primary cutaneous large B-cell lymphomas (PCLBCL).We studied 14 patients for whom clinical follow up was available, including four patients whose lesions were limited to the leg on presentation. Immunohistochemical staining with CD20, CD44, CD21, CD5, CD10, bcl-2, bcl-6, Ki67, p53, and multiple myeloma 1 (MUM1) was examined.Our results identify two subgroups of lymphomas. The first group showed staining with bcl-6 and had an overall survival of 176 months (p = 0.003). The majority of this group was negative for MUM1. The second group lacked staining with bcl-6 and had an overall survival of 26 months, with a majority of these cases staining with MUM1. Three of four patients with PCLBCL of the leg showed no staining with bcl-6.Our study demonstrates the utility of TMAs in the analysis of PCLBCL and that expression of bcl-6 and MUM1 correlates with survival.

    View details for Web of Science ID 000226857100005

    View details for PubMedID 15701085

  • A novel method for making "tissue" microarrays from small numbers of suspension cells APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Montgomery, K., Zhao, S. C., van de Rijn, M., Natkunam, Y. 2005; 13 (1): 80-84


    Tissue microarrays (TMAs) are a highly efficient method for large-scale protein expression studies. To date most TMAs have been constructed using paraffin-embedded specimens. The authors developed a method that allows construction of TMAs from small numbers of cells in suspension. Spun pellets of 1x10 to 1x10 cells are directly processed and embedded in paraffin in an Eppendorf tube. Cylindrical cores of 0.6 mm are taken from these tubes and embedded in a recipient paraffin block to create a TMA. This relatively simple but versatile method enables very small numbers of cells in suspension to be analyzed using the TMA technology and allows for the study of hematolymphoid and related disorders of the blood and bone marrow for which solid tissue samples cannot be readily obtained. With the increasing trend toward obtaining small samples for screening and diagnostic purposes, this method provides a means to manipulate small volume samples for high-throughput immunohistochemical analysis. This method is also amenable for use for cultured cells.

    View details for Web of Science ID 000227276000013

    View details for PubMedID 15722798

  • The NFATc1 transcription factor is widely expressed in white cells and translocates from the cytoplasm to the nucleus in a subset of human lymphomas BRITISH JOURNAL OF HAEMATOLOGY Marafiot, T., Pozzobon, M., Hansmann, M. L., Ventura, R., Pileri, S. A., Roberton, H., Gesk, S., Gaulard, P., Barth, T. F., Du, M. Q., Leoncini, L., Moller, P., Natkunam, Y., Siebert, R., Mason, D. Y. 2005; 128 (3): 333-342


    Stimulation of lymphoid cells via their surface receptors triggers signalling pathways that terminate in the nucleus, where they induce alterations in gene transcription. Nuclear factor of activated T cells (NFAT) transcription factors, involved in a major Ca2+-dependent signalling pathway, normally reside in the cytoplasm but re-locate to the nucleus when activation of the pathway (e.g. following ligation of antigen receptors) leads to their dephosphorylation. This study found that one member of the NFAT family (NFATc1/NFAT2) can be detected in routine biopsy samples, where it is seen in essentially all lymphoid cells, but is absent from the great majority of non-haematopoietic cells. An immunohistological evaluation of NFATc1 in almost 300 lymphomas showed that most neoplastic lymphoid cells also express NFATc1 as a cytoplasmic constituent, although it is absent in classical Hodgkin's disease and plasma cell proliferations. Of particular interest was the finding that NFATc1 was relocated to the nucleus in a minority of lymphoid neoplasms (usually diffuse large B-cell lymphomas or Burkitt lymphoma), presumably reflecting activation of the NFAT pathway. It would be of interest to correlate this feature with patterns of gene expression and also with prognosis, since it may identify a subset of human lymphoma that is distinct in its molecular and clinical features.

    View details for DOI 10.1111/j.1365-2141.2004.05313.x

    View details for Web of Science ID 000226513100007

    View details for PubMedID 15667535

  • Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B cell derivation. 46th Annual Meeting of the American-Society-of-Hematology Natkunam, Y., Lossos, I. S., Taidi, B., Zhao, S. C., Lu, X. Q., Ding, F. Y., Hammer, A. S., Marafioti, T., Byrne, G. E., Levy, S., Warnke, R. A., Levy, R. AMER SOC HEMATOLOGY. 2004: 624A–624A
  • The number of CD25+tumor-infiltrating cells may predict clinical response to rituximab in follicular lymphoma patients. 46th Annual Meeting of the American-Society-of-Hematology Park, C. Y., Bohen, S. P., Levy, R., Warnke, R. A., Natkunam, Y. AMER SOC HEMATOLOGY. 2004: 214A–214A
  • Expression pattern of FCRL (FREB, FcRX) in normal and neoplastic human B cells BRITISH JOURNAL OF HAEMATOLOGY Masir, N., Jones, M., Pozzobon, M., Marafioti, T., Volkova, O. Y., Mechetina, L. V., Hansmann, M. L., Natkunam, Y., Taranin, A. V., Mason, D. Y. 2004; 127 (3): 335-343


    FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms.

    View details for DOI 10.1111/j.1365-2141.2004.05193.x

    View details for Web of Science ID 000224484800012

    View details for PubMedID 15491296

  • Immunophenotypic and genotypic characterization of progression in follicular lymphomas APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Natkunam, Y., Soslow, R., Matolcsy, A. A., Dolezal, M., Bhargava, V., Knowles, D. M., Warnke, R. 2004; 12 (2): 97-104


    Progression of follicular lymphomas (FLs) is often accompanied by a spectrum of histologic changes and an aggressive clinical course. Although molecular alterations have been implicated in this event, the underlying factors are largely unknown. We studied the expression of selected tumor suppressor genes (P53 and retinoblastoma [RB]), oncogenes (MYC and BCL2), and a transferrin-receptor related protein (Trump) in sequential biopsies in 16 patients. Eleven patients progressed from grade I or II FL to aggressive B-cell lymphomas with diffuse morphology, whereas 5 patients presented with diffuse aggressive lymphomas and recurred with indolent lymphomas. Immunoreactivity for P53 correlated with higher histologic grade in lymphomas progressing from indolent to aggressive; however, only 1 patient who presented with aggressive lymphoma demonstrated a P53 gene mutation. Neither P53 immunoreactivity nor genotypic alterations correlated with presentation with an aggressive histology and relapse with FL. Growth fraction, as assessed by Ki-67 staining, and Trump expression correlated with histologic grade. Immunoreactivity for RB, BCL2, and MYC was seldom associated with progression. Eight of 9 cases tested exhibited identical immunoglobulin heavy and light chain rearrangements or identical BCL2 gene rearrangements in the sequential lymphomas. We conclude that P53 and Trump protein expression and proliferation activity correlate with histologic grade, but not with recurrence or progression of FL. Our results further indicate that progression of FL to diffuse aggressive lymphomas and presentation of an aggressive B-cell lymphoma followed by FL are clonally related.

    View details for Web of Science ID 000221718400001

    View details for PubMedID 15354733

  • Intracellular signalling molecules as immunohistochemical markers of normal and neoplastic human leucocytes in routine biopsy samples BRITISH JOURNAL OF HAEMATOLOGY Pozzobon, M., Marafioti, T., Hansmann, M. L., Natkunam, Y., Mason, D. Y. 2004; 124 (4): 519-533


    We have investigated whether intracellular signal transduction molecules can be used as immunohistological markers of normal and neoplastic human leucocytes in routine tissue sections. We obtained selective labelling of white cells for eight such molecules (the 'linker' molecules SLP-76 and BLNK, the Src family kinases Lyn, Fyn, Syk and Hck, and the phospholipases PLC-gamma1 and PLC-gamma2). Antibodies to SLP-76 and PLC-gamma1 selectively labelled T cells, and antibodies to BLNK, Lyn, Fyn, Syk and PLC-gamma2 labelled B cells (although Fyn immunostaining was restricted to mantle zone B cells). Antibodies to the Syk and Hck kinases labelled probable thymocyte precursors at the periphery of the thymic cortex. In addition to lymphoid cells, several other leucocyte types were immunostained (e.g. SLP-76, Lyn, Syk and Hck were found in megakaryocytes, myeloid cells and/or macrophages, and PLC-gamma2 was detected in arterial endothelium). SLP-76 and PLC-gamma1 were found in most T-cell lymphomas studied, and some B-cell lymphomas were also positive for PLC-gamma1 (e.g. diffuse large cell and Burkitt's lymphoma). The five B cell-associated markers were found in most B-cell non-Hodgkin's lymphomas, although some diffuse large B-cell lymphomas were negative (e.g. for Lyn) and anti-Fyn tended not to stain small B-cell neoplasms. The observation that a range of leucocyte signalling molecules can be detected in routine biopsies offers new possibilities for studying normal and neoplastic human white cells in diagnostic tissue samples.

    View details for DOI 10.1111/j.1365-2141.2004.04802.x

    View details for Web of Science ID 000188539200014

    View details for PubMedID 14984504

  • Atypical cellular disorders. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program McClain, K. L., Natkunam, Y., Swerdlow, S. H. 2004: 283-296


    Some immunologic diseases are characterized by profound loss or primary dysfunction of a given population of cells. The atypical cellular disorders discussed here all bear some similarities in that abnormal proliferations of lymphocytes and macrophages or dendritic cells result in lymphadenopathy, skin rashes, bone lesions and infiltrations of nearly any other organ system. What are the similarities and the differences between Langerhans cell histiocytosis (LCH), sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease, and Castleman's disease (CD)? Studies on LCH have some advantages since it was described before the others, and organized clinical trials have been done since the 1980s. The understanding of SHML benefited from a registry maintained by Drs. Rosai and Dorfman. CD was described fifty years ago and for one subtype has the most clearly defined etiology (HHV-8 infection) of the three atypical cellular disorders discussed here. In Section I, Dr. Kenneth McClain examines the unanswered question of whether LCH is a malignant clonal disorder or an inflammatory response triggered by aberrant cytokine expression or a virus. Advocates of the malignant proliferation theory rest their case primarily on the following two points: Clonality of the CD1a+ Langerhans cells was demonstrated by analysis of the human androgen receptor in patients with single bone lesions (Low Risk) or multisystem disease including spleen, liver, bone marrow, or lung (High Risk). Although no consistent chromosomal abnormalities have been reported, loss of heterozygosity (LOH) has been defined by comparative genomic hybridization. Those in the "inflammatory response" camp note that non-clonal proliferation of Langerhans cells in adult pulmonary LCH also have LOH by the same method. The pathologic cells have not been successfully grown in culture or immune-deficient mice and don't have a "malignant" morphology. While the basic scientific arguments continue, important advances in the treatment of LCH have been made by international collaborations of the Histiocyte Society. Risk groups have been clearly defined and the response to therapy after the initial 6 weeks is known to be the strongest prognostic variable for outcome. In Section II, Dr. Yasodha Natkunam reviews the features of SHML, which most often presents as painless cervical lymphadenopathy, although many patients can have extranodal involvement as well. These sites include the skin, respiratory tract, bone, lung, gastrointestinal tract, and brain. The diagnosis rests on finding intact lymphocytes in the cytoplasm of activated macrophages as well as accumulation of mature plasma cells. Hemolytic or non-hemolytic anemias, hypergammaglobulinemia, and elevated erythrocyte sedimentatin rate (ESR) are often found with SHML. An intriguing finding of human herpesvirus (HHV)-6 viral proteins in SHML has been reported in several patients, but needs further study. SHML associated with lymphoproliferations triggered by defects in apoptosis are discussed since this mechanism may provide a clue to the etiology. Therapy for SHML varies greatly in reported case series. Many patients have spontaneous regression or resolution after surgical removal of isolated node groups. Others with systemic involvement may benefit from chemotherapy, but no clinical trials have been done. In Section III, Dr. Steven Swerdlow clarifies key features of the four types of CD. Localized cases are divided into the hyaline vascular type and plasma cell type. Both are usually cured by surgical excision and have symptoms mainly of a mass lesion, although the latter often also has constitutional symptoms. The two types are distinguished largely by the nature of the follicles and the number of interfollicular plasma cells. Interleukin (IL)-6 expression is increased in the plasma cell type. Multicentric CD of the plasmablastic type is most often found in HIV-positive patients with coincident HHV-8 infection. Many have lymphomas or Kaposi sarcomas. Other cases of multicentric CD are also most like the plasma cell type, however, with disseminated disease and constitutional symptoms. A wide variety of anti-neoplastic drugs, radiation therapy, anti-IL-6 and rituximab or atlizumab have been used with varying success in patients with multicentric CD. Clinical trials are needed for SHML and CD and registration of adult and pediatric patients on current LCH trials are encouraged.

    View details for PubMedID 15561688

  • Characterization of variant patterns of nodular lymphocyte predominant Hodgkin lymphoma with immunohistologic and clinical correlation AMERICAN JOURNAL OF SURGICAL PATHOLOGY Fan, Z., Natkunam, Y., Bair, E., Tibshirani, R., Warnke, R. A. 2003; 27 (10): 1346-1356


    Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) has traditionally been recognized as having two morphologic patterns, nodular and diffuse, and the current WHO definition of NLPHL requires at least a partial nodular pattern. Variant patterns have not been well documented. We analyzed retrospectively the morphologic and immunophenotypic patterns of NLPHL from 118 patients (total of 137 biopsy samples). Histology plus antibodies directed against CD20, CD3, and CD21 were used to evaluate the immunoarchitecture. We identified six distinct immunoarchitectural patterns in our cases of NLPHL: "classic" (B-cell-rich) nodular, serpiginous/interconnected nodular, nodular with prominent extranodular L&H cells, T-cell-rich nodular, diffuse with a T-cell-rich background (T-cell-rich B-cell lymphoma [TCRBCL]-like), and a (diffuse) B-cell-rich pattern. Small germinal centers within neoplastic nodules were found in approximately 15% of cases, a finding not previously emphasized in NLPHL. Prominent sclerosis was identified in approximately 20% of cases and was frequently seen in recurrent disease. Clinical follow-up was obtained on 56 patients, including 26 patients who had not had recurrence of disease and 30 patients who had recurrence. The follow-up period was 5 months to 16 years (median 2.5 years). The presence of a diffuse (TCRBCL-like) pattern was significantly more common in patients with recurrent disease than those without recurrence. Furthermore, the presence of a diffuse pattern (TCRBCL-like) was shown to be an independent predictor of recurrent disease (P = 0.00324). In addition, there is a tendency for progression to an increasingly more diffuse pattern over time. Analysis of sequential biopsies from patients with recurrent disease suggests that the presence of prominent extranodular L&H cells might represent early evolution to a diffuse (TCRBCL-like) pattern. We also report three patients who presented initially with diffuse large B-cell lymphoma and later developed NLPHL.

    View details for Web of Science ID 000185584800007

    View details for PubMedID 14508396

  • Plasmablastic lymphoma presenting in a human immunodeficiency virus-negative patient: a case report ANNALS OF HEMATOLOGY Nguyen, D. D., Loo, B. W., Tillman, G., Natkunam, Y., Cao, T. M., Vaughan, W., Dorfman, R. F., Goffinet, D. R., Jacobs, C. D., Advani, R. H. 2003; 82 (8): 521-525


    Plasmablastic lymphoma (PBL), an aggressive non-Hodgkin's lymphoma that carries a poor prognosis, previously has been identified almost exclusively in patients infected with the human immunodeficiency virus (HIV). We present a case of a 42-year-old HIV-negative patient presenting with an isolated nasal cavity mass, the typical presentation for PBL. The patient was given systemic chemotherapy, central nervous system prophylaxis, and consolidative locoregional radiotherapy and achieved a complete clinical response. This case suggests PBL should be considered in HIV-negative patients with characteristic findings.

    View details for DOI 10.1007/s00277-003-0684-3

    View details for Web of Science ID 000184757100013

    View details for PubMedID 12783213

  • Expression of the B-cell proliferation marker MUM1 by melanocytic lesions and comparison with S100, gp100 (HMB45), and MelanA MODERN PATHOLOGY Sundram, U., Harvell, J. D., Rouse, R. V., Natkunam, Y. 2003; 16 (8): 802-810


    The diagnosis of malignant melanoma remains one of the most difficult to render in surgical pathology, partially because of its extreme histologic variability. Limits in the sensitivity and/or specificity of the currently available melanocytic markers such as anti-S100, HMB45, and anti-MelanA further complicate this problem. Previous work has demonstrated that the B-cell proliferation/differentiation marker MUM1/IRF4 is detected in malignant melanoma and hematolymphoid malignancies, but not in any other neoplasm tested (including colonic, lung, breast, and ovarian carcinomas). In the current study, we have examined MUM1 protein expression in 61 melanocytic lesions and compared the diagnostic usefulness of this marker with that of anti-S100, HMB45, and anti-MelanA. The results indicate that MUM1 is positive in 33/36 (92%) cases of melanoma (21/22 [95%] conventional primary melanomas and 12/14 [86%] metastatic melanomas). In comparison, positivity was seen with anti-S100 in 36/36 cases (100%, 22 primary and 14 metastatic), HMB45 in 28 cases (78%, 17 primary and 11 metastatic), and anti-MelanA in 27 cases (75%, 19 primary and 8 metastatic). Although negative in schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors, MUM1 is detected in only one in eight cases of spindle cell and desmoplastic melanomas. With the exception of desmoplastic and spindle cell melanomas, MUM1 appears to be a sensitive and specific immunohistochemical stain for melanocytic lesions and may prove to be a useful addition to the current panel of melanoma markers.

    View details for DOI 10.1097/01.MP.0000081726.49886.CF

    View details for Web of Science ID 000185084000010

    View details for PubMedID 12920225

  • Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas CANCER RESEARCH Nepomuceno, R. R., Balatoni, C. E., Natkunam, Y., Snow, A. L., Krams, S. M., Martinez, O. M. 2003; 63 (15): 4472-4480


    EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.

    View details for Web of Science ID 000184562500028

    View details for PubMedID 12907620

  • Rituximab in lymphocyte-predominant Hodgkin disease: results of a phase 2 trial BLOOD Ekstrand, B. C., Lucas, J. B., Horwitz, S. M., Fan, Z., Breslin, S., Hoppe, R. T., Natkunam, Y., Bartlett, N. L., Horning, S. J. 2003; 101 (11): 4285-4289


    Lymphocyte-predominant Hodgkin disease (LPHD) is a unique clinical entity characterized by indolent nodal disease that tends to relapse after standard radiotherapy or chemotherapy. The malignant cells of LPHD are CD20+ and therefore rituximab may have activity with fewer late effects than standard therapy. In this phase 2 trial, 22 patients with CD20+ LPHD received 4 weekly doses of rituximab at 375 mg/m2. Ten patients had previously been treated for Hodgkin disease, while 12 patients had untreated disease. All 22 patients responded to rituximab (overall response rate, 100%) with complete response (CR) in 9 (41%), unconfirmed complete response in 1 (5%), and partial response in 12 (54%). Acute treatment-related adverse events were minimal. With a median follow-up of 13 months, 9 patients had relapsed, and estimated median freedom from progression was 10.2 months. Progressive disease was biopsied in 5 patients: 3 had recurrent LPHD, while 2 patients had transformation to large-cell non-Hodgkin lymphoma (LCL). All 3 patients with recurrent LPHD were retreated with rituximab, with a second CR seen in 1 patient and stable disease in 2. Rituximab induced prompt tumor reduction in each of 22 LPHD patients with minimal acute toxicity; however, based on the relatively short response duration seen in our trial and the concerns about transformation, rituximab should be considered investigational treatment for LPHD. Further clinical trials are warranted to determine the optimal dosing schedule of rituximab, the potential for combination treatment, and the possible relationship of rituximab treatment to the development of LCL.

    View details for DOI 10.1182/blood-2002-08-2644

    View details for Web of Science ID 000183072800018

    View details for PubMedID 12586628

  • Software tools for high-throughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays AMERICAN JOURNAL OF PATHOLOGY Liu, C. L., Prapong, W., Natkunam, Y., Alizadeh, A., Montgomery, K., Gilks, C. B., van de Rijn, M. 2002; 161 (5): 1557-1565


    The creation of tissue microarrays (TMAs) allows for the rapid immunohistochemical analysis of thousands of tissue samples, with numerous different antibodies per sample. This technical development has created a need for tools to aid in the analysis and archival storage of the large amounts of data generated. We have developed a comprehensive system for high-throughput analysis and storage of TMA immunostaining data, using a combination of commercially available systems and novel software applications developed in our laboratory specifically for this purpose. Staining results are recorded directly into an Excel worksheet and are reformatted by a novel program (TMA-Deconvoluter) into a format suitable for hierarchical clustering analysis or other statistical analysis. Hierarchical clustering analysis is a powerful means of assessing relatedness within groups of tumors, based on their immunostaining with a panel of antibodies. Other analyses, such as generation of survival curves, construction of Cox regression models, or assessment of intra- or interobserver variation, can also be done readily on the reformatted data. Finally, the immunoprofile of a specific case can be rapidly retrieved from the archives and reviewed through the use of Stainfinder, a novel web-based program that creates a direct link between the clustered data and a digital image database. An on-line demonstration of this system is available at

    View details for Web of Science ID 000179197000006

    View details for PubMedID 12414504

    View details for PubMedCentralID PMC1850765

  • The usefulness of immunohistochemistry in the diagnosis of follicular lymphoma in bone marrow biopsy specimens AMERICAN JOURNAL OF CLINICAL PATHOLOGY West, R. B., Warnke, R. A., Natkunam, Y. 2002; 117 (4): 636-643


    We used a panel of paraffin antibodies to determine whether neoplastic and nonneoplastic lymphoid aggregates in the bone marrow can be distinguished reliably. Formalin-fixed, paraffin-embedded bone marrow core biopsy specimens with lymphoid aggregates were stained using primary antibodies directed against bcl-2, bcl-6, CD5, CD10, CD20, and CD23. We studied 61 cases (26 follicular lymphoma and 35 benign or atypical aggregates). We found that no single stain is sufficient for identification of neoplastic lymphoid aggregates. However, this distinction was made possible by using a panel of antibodies. Under the conditions we tested, the most useful antibodies were CD10, bcl-2, CD5, and CD20. Most benign or atypical aggregates do not express CD10 and CD23. In addition, nonneoplastic aggregates had a large population of T cells. bcl-2 was useful in an architectural context for distinguishing neoplastic aggregates. bcl-6 often was expressed in both neoplastic and nonneoplastic aggregates and, thus, poorly discriminated between these processes. We studied the expression of CD10 and bcl-6 in selected lymph nodes in some cases.

    View details for Web of Science ID 000174715000018

    View details for PubMedID 11939740

  • Epstein-Barr virus infection is associated with endothelial bcl-2 expression in transplant liver allografts TRANSPLANTATION Millan, M. T., Natkunam, Y., Clarke-Katzenberg, R., Desai, D., Prapong, W., So, S. K., Esquivel, C. O., Sibley, R., Ferran, C., Martinez, O. M. 2002; 73 (3): 465-469


    In liver transplant recipients with Epstein-Barr virus (EBV) disease, we reported a low rate of acute rejection after stopping or markedly lowering immunosuppression. This observation led to the hypothesis that EBV, as a means of viral persistence, induces expression of antiapoptotic factors and these factors, in turn, confer protection to the transplanted organ. Bcl-2, an antiapoptotic factor induced by EBV in various host cells, is not normally expressed in the liver. We questioned whether bcl-2 is expressed in the transplanted liver and whether its expression is modified by EBV.Retrospective liver biopsy specimen from liver transplant patients diagnosed with EBV (n=12) were examined for the presence of bcl-2 by immunohistochemistry and compared with EBV (-) transplant (n=15), and nontransplant (n=13) livers.The most significant finding was the presence of endothelial bcl-2 expression in the majority of EBV (+) transplant samples examined (67%) and its relative absence in the other two groups (P<0.005). There was also bcl-2 expression in the hepatocytes and lymphocytes of the majority of transplant liver samples, irrespective of EBV status.We have identified a strong association between EBV infection and endothelial bcl-2 expression in transplant livers. We also found that transplantation, in itself, was associated with bcl-2 expression in the hepatocytes and lymphocytes of liver allografts.

    View details for Web of Science ID 000174115400023

    View details for PubMedID 11884946

  • Large atypical cells of lymphomatoid papulosis are CD56-negative: a study of 18 cases JOURNAL OF CUTANEOUS PATHOLOGY Harvell, J. D., Vaseghi, M., Natkunam, Y., Kohler, S., Kim, Y. 2002; 29 (2): 88-92


    Histologically, diffuse dermal infiltrates of large atypical lymphocytes can be seen in lesions as indolent as type C lymphomatoid papulosis (LyP) to ones as aggressive as NK/T-cell lymphoma. While lesions of lymphomatoid papulosis are definitionally positive for CD30, their ability to express CD56 has not been formally studied. The objective of the current study was to determine whether or not the large atypical cells of LyP express the natural killer cell marker, CD56.Biopsies from 18 patients with LyP were studied with monoclonal antibodies to CD30, CD56, CD8, and TIA-1. These included four type C LyP lesions. Clinical information was obtained by chart review and included extent of LyP lesions, presence/absence of disease at follow-up, and any associated hematologic malignancies,.None of the biopsies exhibited CD56 positivity within the large atypical cells of LyP. While some biopsies demonstrated CD56-positive, small, presumably reactive, lymphocytes within the infiltrate, their presence did not correlate with extent of disease, persistence of disease, or propensity for an associated non-LyP hematologic malignancy.The large atypical cells of types A and C LyP do not exhibit positivity for CD56, and thus a panel of antibodies that includes CD30 and CD56 can readily distinguish between the benign end of the spectrum of CD30-positive lymphoproliferations and aggressive NK/T-cell lymphoma.

    View details for Web of Science ID 000174804900004

    View details for PubMedID 12150138

  • Software tools for high-throughput analysis and image retrieval of immunohistochemistry stains obtained on tissue microarrays Lui, C. L., Natkunam, Y., Prapong, W., Montgomery, K., Botstein, D., Brown, P. O., van de Rijn, M. NATURE PUBLISHING GROUP. 2002: 341A–341A
  • Characterization of variant patterns of lymphocyte predominance Hodgkin's disease with immunohistochemical correlation Fan, Z., Natkunam, Y., Warnke, R. NATURE PUBLISHING GROUP. 2002: 240A–240A
  • Apoptosis stimulating protein of p53 (ASPP2) expression differs in diffuse large B-cell and follicular center lymphoma: Correlation with clinical outcome LEUKEMIA & LYMPHOMA Lossos, I. S., Natkunam, Y., Levy, R., Lopez, C. D. 2002; 43 (12): 2309-2317


    ASPP2 interacts with the tumor suppressor protein p53, promotes damage-induced apoptosis, and can specifically stimulate p53 apoptotic function. Thus, ASPP2 may function as a tumor suppressor and/or play a role in the cellular response to cytotoxic injury. To explore the role of ASPP2 in human cancer, we determined ASPP2 expression in two lymphoma subtypes with differing clinical outcomes: diffuse large B-cell lymphoma (DLBCL) and follicular center lymphoma (FCL). A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect ASPP2 mRNA. Sixty-one DLBCL and twenty-three FCL cases were analyzed and normalized ASPP2 levels were expressed relative to an mRNA standard. We found that ASPP2 mean expression strongly correlated with lymphoma subtype: DLBCL = 11.74 and FCL = 4.99 (p = 0.029, unpaired 2-tailed t-test). Importantly, ASPP2 expression was variable in DLBCL but not FCL (DLBCL-range, 0.04-94.6; FCL-range, 1.2-15.0). In these DLBCL cases, serum lactate dehydrogenase (LDH) was an independent predictor of survival with median survival in the high LDH group of 24 months and median survival not achieved in the normal-low LDH group (p = 0.014, Log-Rank Test). Mean ASPP2 levels trended toward an inverse correlation with LDH levels: High LDH, ASPP2 = 6.2; Normal-low LDH, ASPP2 = 18.2 (p = 0.074, unpaired 2-tailed t-test). In the DLBCL cases with ASPP2 levels > 7.8, only 10% (1/10) had a high LDH, in contrast to cases with ASPP2 levels < 7.8 in which 59% (26/44) had a high LDH (p = 0.011, Fisher Exact Test). Thus, low ASPP2 mRNA levels may correlate with poor clinical outcome in lymphoma which is consistent with the hypothesis that ASPP2 may play a role in tumor formation and/or sensitivity to cytotoxic agents. Larger studies as well as analysis of different tumor types are warranted.

    View details for DOI 10.1080/1042819021000040017

    View details for Web of Science ID 000178910400009

    View details for PubMedID 12613517

  • Durable remission in recurrent T-cell-rich B-cell lymphoma with the anti-CD20 antibody rituximab CLINICAL LYMPHOMA Natkunam, Y., Stanton, T. S., Warnke, R. A., Horning, S. J. 2001; 2 (3): 185-187


    A diagnostic continuum exists between lymphocyte-predominant Hodgkin's disease, T-cell-rich B-cell lymphoma (TCRBCL), and diffuse large B-cell lymphoma. While TCRBCLs are uncommon, their clinical and morphologic presentation can mimic other Hodgkin's and non-Hodgkin's lymphomas from which they must be distinguished for diagnosis and treatment. We present an unusual case of a 30-year-old man with recurrent TCRBCL arising from lymphocyte-predominant Hodgkin's disease with remarkable response to treatment with the anti-CD20 antibody, rituximab.

    View details for Web of Science ID 000173328300009

    View details for PubMedID 11779297

  • Modified cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone therapy for posttransplantation lymphoproliferative disease in pediatric patients undergoing solid organ transplantation JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY Suryanarayan, K., Natkunam, Y., Berry, G., Bangs, C. D., Cherry, A., Dahl, G. 2001; 23 (7): 452-455


    The authors report the use of a cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP)-based chemotherapy regimen in treating six children with posttransplantation lymphoproliferative disorder (PTLD) that developed after solid organ transplantation.The chemotherapy regimen consisted of a 29-day induction with CHOP and then as many as 15 cycles of maintenance therapy using methotrexate and cytarabine alternating with vincristine, adriamycin, mercaptopurine, and prednisone.All patients attained remission. One patient died of sepsis while in remission. Four of the five remaining patients have been followed-up in remission for as long as 8 years without losing the graft. One of the patients experienced relapse after completing therapy and subsequently died with disease.The authors conclude that pediatric patients with PTLD after solid organ transplantation that fails conservative management can be treated successfully with CHOP-based chemotherapy.

    View details for Web of Science ID 000171516000011

    View details for PubMedID 11878581

  • A unique AML1 (CBF2A) rearrangement, t(1;21)(p32;q22), observed in a patient with acute myelomonocytic leukemia CANCER GENETICS AND CYTOGENETICS Cherry, A. M., Bangs, C. D., Jones, P., Hall, S., Natkunam, Y. 2001; 129 (2): 155-160


    The AML1 (CBFA2) gene is the most frequent target of chromosomal rearrangements observed in human acute leukemia. These rearrangements include the commonly reported t(8;21)(q22;q22) or AML1/ETO fusion in AML-M2, the t(3;21)(q26;q22) or AML1 fusion with one of three genes, MDS1, EAP or EVI1, in therapy-related AML and MDS, as well as in blast crisis in CML and the t(12;21)(p13;q22) or TEL/AML1 fusion in B-cell ALL. In addition to the t(3;21), other AML1 translocations have also been reported in therapy-related MDS and AML, particularly after treatment with topoisomerase II inhibitors. AML1 gene rearrangements have also been observed less frequently with numerous other chromosomal partners. Here, we describe a patient with AML-M4 and a previously unreported rearrangement involving the AML1 locus and an unknown locus on the short arm of chromosome 1 at 1p32.

    View details for Web of Science ID 000171105500011

    View details for PubMedID 11566347

  • Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma BLOOD Lossos, I. S., Jones, C. D., Warnke, R., Natkunam, Y., Kaizer, H., Zehnder, J. L., Tibshirani, R., Levy, R. 2001; 98 (4): 945-951


    Diffuse large B-cell lymphoma (DLBCL) is characterized by a marked degree of morphologic and clinical heterogeneity. Establishment of parameters that can predict outcome could help to identify patients who may benefit from risk-adjusted therapies. BCL-6 is a proto-oncogene commonly implicated in DLBCL pathogenesis. A real-time reverse transcription-polymerase chain reaction assay was established for accurate and reproducible determination of BCL-6 mRNA expression. The method was applied to evaluate the prognostic significance of BCL-6 expression in DLBCL. BCL-6 mRNA expression was assessed in tumor specimens obtained at the time of diagnosis from 22 patients with primary DLBCL. All patients were subsequently treated with anthracycline-based chemotherapy regimens. These patients could be divided into 2 DLBCL subgroups, one with high BCL-6 gene expression whose median overall survival (OS) time was 171 months and the other with low BCL-6 gene expression whose median OS was 24 months (P =.007). BCL-6 gene expression also predicted OS in an independent validation set of 39 patients with primary DLBCL (P =.01). BCL-6 protein expression, assessed by immunohistochemistry, also predicted longer OS in patients with DLBCL. BCL-6 gene expression was an independent survival predicting factor in multivariate analysis together with the elements of the International Prognostic Index (IPI) (P =.038). By contrast, the aggregate IPI score did not add further prognostic information to the patients' stratification by BCL-6 gene expression. High BCL-6 mRNA expression should be considered a new favorable prognostic factor in DLBCL and should be used in the stratification and the design of risk-adjusted therapies for patients with DLBCL. (Blood. 2001;98:945-951)

    View details for Web of Science ID 000170364100008

    View details for PubMedID 11493437

  • Analysis of MUM1/IRF4 protein expression using tissue microarrays and immunohistochemistry MODERN PATHOLOGY Natkunam, Y., Warnke, R. A., Montgomery, K., Falini, B., van de Rijn, M. 2001; 14 (7): 686-694


    The gene encoding MUM1 was characterized as a possible translocation partner in chromosomal abnormalities involving a significant number of multiple myelomas. The overexpression of the MUM1 protein as a result of translocation t(6;14) (p25;q32) identified MUM1 as a putative regulatory molecule involved in B-cell differentiation and tumorigenesis. The expression of MUM1 protein in multiple myelomas supports this hypothesis. In the current study, using tissue microarray technology, we have tested the expression of the MUM1 protein in 1335 human malignancies and normal tissues. Our data show that the MUM1 protein is expressed in a wide spectrum of hematolymphoid neoplasms and in malignant melanomas but is absent in other human tumors. In addition, in tissue microarrays as well as in conventional paraffin sections, MUM1 staining was found to lack specificity in detecting plasmacytic differentiation as compared with two markers, CD138/Syndecan and VS38, commonly used in paraffin immunohistochemistry for detection of plasma cells.

    View details for Web of Science ID 000169927200008

    View details for PubMedID 11455001

  • Natural killer/natural killer-like T-Cell lymphoma, CD56+, presenting in the skin: An increasingly recognized entity with an aggressive course JOURNAL OF CLINICAL ONCOLOGY Mraz-Gernhard, S., Natkunam, Y., Hoppe, R. T., Leboit, P., Kohler, S., Kim, Y. H. 2001; 19 (8): 2179-2188


    To describe and identify the clinical and pathologic features of prognostic significance for natural killer (NK) and NK-like T-cell (NK/T-cell) lymphoma presenting in the skin.This study was a retrospective review of 30 patients with CD56+ lymphomas initially presenting with cutaneous lesions, with analysis of clinical and histopathologic parameters.The median survival for all patients was 15 months. Those with extracutaneous manifestations at presentation (11 patients) had a shorter median survival of 7.6 months as compared with those without extracutaneous involvement (17 patients), who had a more favorable median survival of 44.9 months (P =.0001). Age, gender, extent of cutaneous involvement, and initial response to therapy had no statistically significant effect on survival. Seven patients (24%) had detectable Epstein-Barr virus (EBV) within neoplastic cells. The patients with tumor cells that coexpress CD30 (seven patients) have not yet reached a median survival after 35 months of follow-up as compared with those with CD30- tumor cells (20 patients), who had a median survival of 9.6 months (P <.02). Routine histopathologic characteristics had no prognostic significance nor did the presence of CD3epsilon, EBV, or multidrug resistance.NK/T-cell lymphoma is an aggressive neoplasm; however, a subset with a more favorable outcome is identified in this study. The presence of extracutaneous disease at presentation is the most important clinical variable and portends a poor prognosis. The extent of initial skin involvement does not reliably predict outcome. Patients from the United States with NK/T-cell lymphoma presenting in the skin have a low incidence of demonstrable EBV in their tumor cells. Patients with coexpression of CD30 in CD56 lymphomas tend to have a more favorable outcome.

    View details for Web of Science ID 000168178300009

    View details for PubMedID 11304770

  • Natural killer cell precursor acute lymphoma/leukemia presenting in an infant ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Natkunam, Y., Cherry, A. M., Cornbleet, P. J. 2001; 125 (3): 413-418


    Lymphoma/leukemia derived from immature natural killer (NK) cells occur most commonly in adults and are characterized by blastic cytologic features and an aggressive outcome. Predilection for extranodal sites and absence of the Epstein-Barr virus associated with mature NK cell malignancies further distinguish this entity. We present a NK precursor acute lymphoma presenting with multiple masses in an infant without circulating blasts or marrow replacement by disease. The diagnostic difficulty arose from several factors, including young age, presentation with multiple masses, blastic cytologic features mistaken for a small, round, blue cell tumor, and the absence of lineage-specific markers. The CD56+, CD34+, CD33+, MPO-, cytoplasmic CD3+, CD45-, CD7-, HLA-DR-, and TdT- immunophenotype of this neoplasm overlaps with previously reported cases of myeloid/NK precursor acute leukemia and blastic NK cell lymphoma/leukemia. This case emphasizes the need for a strong index of suspicion to recognize this rare entity and to distinguish it from solid tumors and other hematolymphoid neoplasms that occur in infancy.

    View details for Web of Science ID 000167405000023

    View details for PubMedID 11231495

  • Angiocentric lymphomas (lymphomatous vasculitis) SEMINARS IN DIAGNOSTIC PATHOLOGY Natkunam, Y., Warnke, R. A. 2001; 18 (1): 67-77


    Angiocentric lymphomas are a heterogeneous spectrum of hematolymphoid malignancies that share a particular histologic characteristic, namely, an angiocentric or perivascular growth pattern. They include a variety of T-, B-, and natural killer-cell derived lymphomas that digress in many clinicopathologic features, immunophenotype, and prognosis. The term angiocentric lymphomas was initially used to refer to natural killer and natural killer-like T-cell lymphomas that show a prominent angiocentric growth pattern. With better immunophenotypic and molecular characterization together with evolving knowledge regarding their biology and pathogenesis, these lymphomas have now been reclassified. Apart from morphology, many features pertinent to the diagnosis of natural killer and natural killer-like T-cell lymphomas are shared by other peripheral T-cell and B-cell lymphomas, and by a subset of leukemias. The salient clinicopathologic features of natural killer and natural killer-like T-cell lymphomas together with the inherent difficulty of their identification and an integrated approach to their diagnosis are outlined in this article.

    View details for DOI 10.1053/sdia.2001.22141

    View details for Web of Science ID 000167281300008

    View details for PubMedID 11296995

  • Co-expression of CD56 and CD30 in lymphomas with primary presentation in the skin: clinicopathologic, immunohistochemical and molecular analyses of seven cases JOURNAL OF CUTANEOUS PATHOLOGY Natkunam, Y., Warnke, R. A., Haghighi, B., Su, L. D., Le Boit, P. E., Kim, Y. H., Kohler, S. 2000; 27 (8): 392-399


    Natural killer and natural killer-like T-cell lymphomas presenting in the skin usually demonstrate aggressive behavior, an angiocentric distribution and a characteristic immunophenotype. In contrast, primary cutaneous CD30+ lymphoproliferative disorders form a heterogeneous spectrum including anaplastic large cell lymphomas, the majority of which display a good prognosis. Lymphomas with co-expression of CD56 and CD30 are extremely rare and the significance of this co-expression is unknown.Seven retrospectively identified cases of lymphomas with co-expression of CD56 and CD30 presenting in the skin comprise this study. Immunohistochemistry, in situ hybridization for Epstein-Barr virus and T-cell receptor gene rearrangement studies were performed on paraffin sections.This subset of cutaneous lymphomas showed a variable clinical course that ranged from resolution without treatment, treatment-failure and recurrence, to death from disease. Histologic, immunophenotypic and molecular studies were of limited utility in predicting prognosis.Cutaneous lymphomas co-expressing CD56 and CD30 share many clinicopathologic features with natural killer and natural killer-like T-cell lymphomas or anaplastic large cell lymphomas, two entities with widely disparate clinical behavior. It is important to recognize that these lymphomas may behave more aggressively than primary cutaneous anaplastic large cell lymphomas do. Longer follow-up and further investigations on larger numbers of cases are necessary to fully characterize this rare subset of cutaneous lymphomas.

    View details for Web of Science ID 000088444100003

    View details for PubMedID 10955685

  • Blastic/blastoid transformation of follicular lymphoma - Immunohistologic and molecular analyses of five cases AMERICAN JOURNAL OF SURGICAL PATHOLOGY Natkunam, Y., Warnke, R. A., Zehnder, J. L., Jones, C. D., Milatovich-Cherry, A., Cornbleet, P. J. 2000; 24 (4): 525-534


    Progression of follicular lymphoma to a higher-grade malignancy frequently heralds a poor prognosis. Clinical transformation is variably accompanied by a spectrum of histologic changes characterized by alteration in growth and cytology. Although several cytogenetic events and potential oncogenes have been documented in this progression, the underlying molecular mechanisms are largely unknown. We present five patients with an unusual histologic transformation of follicular lymphoma manifested by blastic/blastoid morphology. This transformation is histologically distinct from other types of transformation of follicular lymphoma. All five cases exhibited the t(14;18) translocation and expressed the BCL-2 protein. In addition, two of the five patients showed increased levels of the p53 protein within neoplastic cells implicating a possible role for this oncogene in blastic/blastoid transformation. The lack of BCL-1 and myeloid antigens by immunohistochemistry and flow cytometry studies served to distinguish blastic/blastoid transformation of follicular lymphoma from its morphologic mimics. This distinction is clinically important because lymphoblastic and myeloid leukemias require significantly different therapeutic modalities and show better prognosis. Moreover, the lack of Epstein-Barr virus-specific mRNA suggests that this virus is unlikely to participate in blastic/blastoid transformation of follicular lymphoma.

    View details for Web of Science ID 000086211700006

    View details for PubMedID 10757399

  • Utility of paraffin section immunohistochemistry for C-KIT (CD117) in the differential diagnosis of systemic mast cell disease involving the bone marrow AMERICAN JOURNAL OF SURGICAL PATHOLOGY Natkunam, Y., Rouse, R. V. 2000; 24 (1): 81-91


    Systemic mast cell disease is characterized by an abnormal infiltration of mast cells involving several parenchymal organs and the bone marrow. Its spectrum of clinical and histologic presentation is highly variable and is not necessarily correlated with prognosis. Mast cell disorders presenting as atypical infiltrates in the bone marrow may simulate or be associated with other hematolymphoid malignancies, from which they must be distinguished. The paucity of reliable histochemical and immunohistochemical markers for the detection of mast cells in paraffin sections further confounds this diagnosis. The authors have employed immunohistochemistry for the C-KIT encoded tyrosine kinase receptor protein, CD117, for detection of mast cells on paraffin sections of 89 bone marrow specimens including systemic mast cell disease and other disorders. CD117 staining was found in all cases of mast cell disorders (seven of seven), and in one case of chronic myelogenous leukemia in blast crisis. None of the other myeloid disorders tested (0 of 16), or any of the cases of Hodgkin's disease (0 of 12), B-cell lymphomas (0 of 32), T-cell lymphomas (0 of 3), or histiocytic proliferations (0 of 3) showed staining for CD117. CD117 expression is effective in the separation of mast cell disease from disorders that may simulate it histologically.

    View details for Web of Science ID 000084643300010

    View details for PubMedID 10632491

  • Immunoblot analysis of CD34 expression in histologically diverse neoplasms AMERICAN JOURNAL OF PATHOLOGY Natkunam, Y., Rouse, R. V., Zhu, S., Fisher, C., van de Rijn, M. 2000; 156 (1): 21-27


    CD34 is a heavily glycosylated transmembrane protein of approximately 110 kd whose function is essentially uncharacterized. First identified in a myeloid leukemia cell line, immunohistological reactivity with anti-CD34 antibodies is also encountered in a histologically diverse subset of nonhematolymphoid neoplasms including angiosarcoma, solitary fibrous tumors, epithelioid sarcomas, spindle cell lipomas, dermatofibrosarcoma protuberans, and myofibroblastomas. Immunohistological reactivity for CD34 in hematopoietic stem cells and endothelial cells has been shown to correspond to the expression of the CD34 protein. With the exception of gastrointestinal stromal tumors, CD34 protein expression has not been investigated in other CD34 immunohistologically reactive nonhematolymphoid neoplasms. We undertook this study to examine whether the observed reactivity for anti-CD34 antibodies in apparently unrelated tumors is due to the expression of the same protein or whether shared epitopes elaborated by other proteins could account for this reactivity. Immunoblot analyses with anti-CD34 antibodies of six different CD34 immunohistologically reactive lesions show the same approximately 110-kd molecular weight protein. In addition, two cases of dermatofibrosarcoma protuberans show double bands at approximately 110 kd. Laser-capture microdissection of CD34 immunohistologically reactive epithelioid sarcoma and nonreactive epidermal cells illustrates that this reactivity is specific to tumor cells. These results show that the observed immunohistological reactivity with anti-CD34 antibodies is due to the expression of the CD34 protein and not to shared epitopes on unrelated proteins.

    View details for Web of Science ID 000084773300005

    View details for PubMedID 10623649

  • Aggressive cutaneous NK and NK-like T-cell lymphomas - Clinicopathologic, immunohistochemical, and molecular analyses of 12 cases AMERICAN JOURNAL OF SURGICAL PATHOLOGY Natkunam, Y., SMOLLER, B. R., Zehnder, J. L., Dorfman, R. F., Warnke, R. A. 1999; 23 (5): 571-581


    Natural killer (NK) and NK-like T-cell lymphomas are rare hematolymphoid malignancies that predominate in the upper aerodigestive system. They also involve other extranodal sites, including the skin. Primary cutaneous manifestations of NK and NK-like T-cell lymphomas are uncommon, and the clinicopathologic features are poorly understood. We have studied 12 patients of varied ethnic backgrounds with CD56-positive lymphomas in the skin. Six patients subsequently progressed to disseminated disease. These lymphomas showed the following immunophenotype: CD56+, CD43+, TCRb-, CD3-/+, CD20-, CD30-/+, CD4-, and CD8-. Two cases exhibited T-cell receptor gene rearrangements supporting a T-cell origin for these lymphomas, whereas the remaining 10 cases were likely derived from NK cells. Our results show inconsistent association of these lymphomas with Epstein-Barr virus (EBV), the multidrug resistance phenotype, and expression of P53. In addition, we found a previously unreported correlation between lymphomas harboring EBV mRNA and the expression of the multidrug resistance phenotype. These lymphomas were aggressive and were associated with rapid clinical progression, treatment failure, multiple relapses, and an average survival of 15 months from the time of diagnosis. Our results indicate the importance of recognizing this disease as a distinct subset of aggressive cutaneous lymphomas that may be diagnosed on the basis of morphology, immunophenotype, and gene rearrangement studies.

    View details for Web of Science ID 000080178200012

    View details for PubMedID 10328090

  • Aggressive natural killer-like T-cell malignancy with leukemic presentation following solid organ transplantation AMERICAN JOURNAL OF CLINICAL PATHOLOGY Natkunam, Y., Warnke, R. A., Zehnder, J. L., Cornbleet, P. J. 1999; 111 (5): 663-671


    NK-like T-cell malignancies are part of a spectrum of lymphoproliferative diseases that complicate immunosuppression associated with solid organ transplantation. We describe 2 patients with long-standing immunosuppression following solid organ transplantation. Both patients had systemic symptoms that included fever, myalgia, and weight loss. Organ involvement and lymphadenopathy were not initially observed. Unique to these 2 cases are the initial leukemic symptoms, which led to further characterization and identification of NK-like T-cell malignancies. Both patients exhibited an anomalous T/NK phenotype, CD56 positivity, and atypical blastic architecture of the large granular lymphocytes. Clonal rearrangement of T-cell receptor genes was detected in both patients. In 1 patient, a cytogenetic abnormality involving 8q24 was demonstrated. The disease course in both patients was aggressive, with involvement of multiple sites and rapid demise. This study emphasizes the importance of including NK-like T-cell malignancies in the differential diagnosis of lymphoproliferative disorders associated with immunosuppression and recognizing that an aggressive clinical course may follow leukemic presentation of disease.

    View details for Web of Science ID 000079920500011

    View details for PubMedID 10230357

  • Epstein-Barr virus strain type and latent membrane protein 1 gene deletions in lymphomas in patients with rheumatic diseases ARTHRITIS AND RHEUMATISM Natkunam, Y., ElenitobaJohnson, K. S., Kingma, D. W., Kamel, O. W. 1997; 40 (6): 1152-1156


    Recent studies have shown that immunomodulatory therapy for the treatment of rheumatic diseases can be associated with the development of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. The present study was undertaken to determine the strain type of EBV in lymphoproliferative disorders that occur in patients with rheumatic disease and to investigate EBV latent membrane protein 1 (LMP-1) gene deletions that occur in these lymphoproliferative disorders.Ten EBV-associated lymphoid neoplasms in patients with rheumatoid arthritis or dermatomyositis were analyzed by polymerase chain reaction to determine EBV strain type and to investigate for the presence of a previously characterized 30-basepair deletion in the LMP-1 gene.The results indicated that lymphoproliferative disorders in these patients can harbor EBV strain type A or B, with a predominance of type A infection (80%). It was also shown that both wild-type and mutated LMP-1 genes can be found in these neoplasms, with the deleted form of the LMP-1 gene occurring in one-third of cases in this series.LMP-1 deletions associated with certain aggressive lymphoid neoplasms are not required for the genesis of lymphoproliferative disorders in patients with rheumatic disease. The relative frequencies of type A and type B EBV strains in these lymphoproliferative disorders show similarities to the frequencies in patients with post-solid organ transplantation immunosuppression-associated lymphoproliferative disorders.

    View details for Web of Science ID A1997XC37600020

    View details for PubMedID 9182927



    Terminal differentiation of B cells to plasma cells in vivo is characterized by secretion of Ig and extinction of MHC class II expression on the cell surface. We show that IL-6 signaling leads to marked increases in the synthesis and secretion of Ig in clonal human B cell lines and newly isolated polyclonal B lymphocytes in vitro. The IL-6-induced cells resemble plasma cells in ultrastructure and in reduced expression of surface MHC class II. Enhanced Ig synthesis is a result of coordinated transcriptional activation of Ig genes without promoter or isotype specificity, and differential accumulation of the mRNA encoding the secreted form of Ig heavy chain. It is saturable and subject to negative control when IL-6 stimulation is prolonged. Coordinate with temporal changes in Ig synthesis, the DNA-binding activity and the synthesis of the B cell-enriched transcription factor Oct-2 are regulated. Thus, differentiation of B cells with IL-6 in vitro recapitulates the hallmarks of terminal B differentiation in vivo; Oct-2 may have a role in this process.

    View details for Web of Science ID A1994PM58100012

    View details for PubMedID 7930570



    The molecular analysis of the regulation of nuclear proteins induced by interleukin-6 has provided new insights into this largely unknown signal transduction pathway. Transcription factors of the CCAAT/enhancer-binding protein and AP-1 families, as well as the octamer-binding proteins and the tumor suppressor gene product pRB, are regulated by interleukin-6 in a cell type specific manner, suggesting that they may play a role in the nuclear signaling by interleukin-6.

    View details for Web of Science ID A1993KN29300020

    View details for PubMedID 8452668