Girish Venkataraman

Current status of prognostication in classical Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by a paucity of neoplastic Hodgkin/Reed Sternberg (HRS) cells within a complex cellular milieu that is rendered immunologically incapable of reacting against CD30+HRS cells due to a plethora of immune escape mechanisms initiated by the neoplastic cells. Accounting for 25% of all lymphomas and nearly 95% of all Hodgkin lymphomas, patients with cHL are typically young adults. Besides traditional prognostic factors, such as the International Prognostic Index (IPI), newer imaging and ancillary biomarkers (CD68, Galectin‐1 and plasma microRNA) have shown promise. Furthermore, the evolution of gene expression profiling (GEP) in recent years has enabled the development of several practically feasible GEP‐based predictors with prognostic relevance. This review discusses the current status of clinical prognostication in cHL, the critical role of histological evaluation in light of several mimicking entities, and the relevance of tissue as well as serum biomarkers pertaining to immune escape mechanisms and recent GEP studies.

Highly clonal regulatory T-cell population in follicular lymphoma - inverse correlation with the diversity of CD8(+) T cells.

The immune microenvironment in follicular lymphoma (FL) plays an important role in controlling disease characteristics. To characterize the T-cell receptor (TCR) repertoire in follicular lymphoma (FL) tissues, we applied a next-generation sequencing platform and deeply sequenced TCR cDNAs of T-cell subset populations present in pretreatment FL biopsy specimens. T regulatory cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who had not received anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both human leukocyte antigen (HLA) class I and II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T-cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease.

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patients clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

Clonal evolution underlying leukemia progression and Richter transformation in patients with ibrutinib-relapsed CLL

Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481 , we identified BTKT316A , a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

De novo CD5+ diffuse large B-cell lymphoma: Adverse outcomes with and without stem cell transplantation in a large, multicenter, rituximab treated cohort

De novo CD5+ diffuse large B‐cell lymphomas (DLBCL) are a distinct subgroup of DLBCL with poor prognosis. However the role of rituximab‐containing therapy and salvage stem cell transplantation in this patients’ population remain to be defined. We retrospectively reviewed clinical features and outcomes of 102 patients with de novo CD5+ DLBCL treated with rituximab‐containing therapy at nine different institutions. By Hans’ criteria, 64 patients had activated B‐cell (ABC) subtype, 24 germinal center B‐cell (GCB) subtype, and 14 were not evaluated. No patients had a myc translocation. Eighty‐three patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R‐CHOP), 7 with rituximab, etoposide, cyclophosphamide, doxorubicin, vincristine, prednisone (R‐EPOCH), and 6 with R‐CHOP with methotrexate, 3 g/m2. The overall response rate to front‐line therapy was 85%. The 3‐year progression free survival (PFS) and overall survival (OS) for all patients were 40 and 65%, respectively. The 3‐year PFS for ABC‐ and GCB‐subtypes was 34 and 45%, respectively. The 3‐year OS for ABC‐ and GCB‐subtypes was 62 and 67%, respectively. The median time to second treatment failure was 3 months and 1 month for ABC‐ and GCB‐subtypes, respectively. Twenty of 28 (71%) transplanted patients with autologous, allogeneic, or both, relapsed. This study confirms the poor prognosis of de novo CD5+ DLBCL in a large multi‐center cohort despite initial rituximab‐containing chemotherapy and suggests that stem cell transplantation fails to salvage the majority of these patients. Approaches to prevent recurrence and/or novel therapies for relapsed disease are needed for this subgroup of DLBCL patients. Am. J. Hematol. 91:395–399, 2016.

Predominance of CD4+ T Cells in T-Cell/Histiocyte-Rich Large B-Cell Lymphoma and Identification of a Subset of Patients With Peripheral B-Cell Lymphopenia

Objectives T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a morphologic variant of large B-cell lymphoma whose flow cytometry findings are not well characterized. Methods Nineteen cases with flow cytometric immunophenotyping were identified from the case records of four institutions between 2001 and 2016. Results In most cases, neoplastic B cells were not detected by flow cytometry. Overall, cases showed a predominance of CD4+ T cells, which in some cases was marked. Significant coexpression of CD57 was seen on CD4+ T cells where this marker was analyzed, which correlated with PD-1 expression. Two cases also showed a profound systemic B-cell lymphopenia, which was associated in one case with hypogammaglobulinemia. Conclusions Overall, our work challenges previous findings that cases of THRLBCL are rich in CD8+ T cells and highlights parallels between THRLBCL and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). Also, an association of THRLBCL with systemic B-cell lymphopenia has not been previously reported but may represent an underrecognized manifestation.

GATA3 Immunohistochemical Staining in Hodgkin Lymphoma: Diagnostic Utility in Differentiating Classic Hodgkin Lymphoma From Nodular Lymphocyte Predominant Hodgkin Lymphoma and Other Mimicking Entities

Background: Classic Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) are clinically distinct entities, with different prognostic and treatment implications. In addition, several large B-cell lymphomas and some T-cell lymphomas can mimic CHL. Differentiating these entities from CHL is crucial for ensuring appropriate therapy. GATA3 is a T-cell transcription factor involved in T-cell maturation and has been previously shown to be overexpressed in CHL cells via gene expression profiling. We investigated the utility of GATA3 immunostain in differentiating CHL from NLPHL and other mimicking entities. Materials and Methods: We accrued 17 NLPHLs, 49 CHLs [23 nodular sclerosis (NS), 3 syncytial variants, 3 lymphocyte rich and 13 mixed cellularity types], 4 primary mediastinal large B-cell lymphomas (PMBLs), 2 Epstein-Barr virus (EBV) positive diffuse large B-cell lymphomas (DLBCLs) (EBV+LBCLs), 2 T-cell/histiocyte-rich large B-cell lymphomas (TCHRBCLs), 1 gray zone lymphoma, and 2 tissue microarrays consisting of 72 DLBCLs. One slide from each was stained with GATA3 and percent positive tumor cells and intensity of nuclear expression was semiquantitatively graded independently by 2 board certified hematopathologists. Results: GATA3 was positive in 80% of CHLs. Both percent positivity and intensity of staining varied greatly. Syncytial variant of NS subtype showed the highest positivity rate (3/3; 100%), followed by NS (20/23; 87%), mixed cellularity (9/13; 70%), and lymphocyte rich (2/3; 67%). GATA3 was negative in all NLPHLs, EBV+LBCLs, TCRBCLs, and DLBCLs stained. The single gray zone lymphoma and 3/4 PMBLs were positive. Conclusions: Nuclear expression of GATA3 can be used to delineate CHL from NLPHL. GATA3 positivity effectively excludes NLPHL with 100% negative predictive value. However, as 20% of CHL can be negative for GATA3, CHL cannot be ruled out with negative GATA3. Additional findings include GATA3 positivity among PMBLs, whereas all 72 DLBCLs were negative for GATA3. This finding further highlights similarities between CHL and PMBL.

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3 −/dim CD4 + population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3−/dimCD4+ aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3−/dimCD4+ T-cell population constituting 29.3% (range 7.9–62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein–Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3−/dimCD4+ population (mean: 19.5%, range: 3–71.8%). Using a threshold of 3% for CD3−/dimCD4+ T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann–Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3−/dimCD4+ aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

Bone Marrow Lymphoma

Lymphomas are malignant hematopoietic neoplasms arising from mature lymphoid cells that typically reside within extramedullary lymphoid tissues, including the lymph nodes and spleen. However, lymphomas can frequently involve the extranodal tissues, including peripheral blood (leukemia) and/or the bone marrow. There are even rare instances in which the bone marrow is the sole location of involvement by lymphoma [1]. A variety of infiltration patterns may be observed (paratrabecular, interstitial, or sinusoidal) in the various B and T cell lymphomas, besides varying background microenvironment inflammatory cells (e.g., in Hodgkin lymphoma, T cell lymphomas). Both pattern and microenvironment are useful features that assist in characterizing marrow involvement by providing clues to the nature of the lymphoma in cases where marrow involvement is the presenting feature. Ancillary studies such as flow cytometry and immunohistochemistry are both complementary studies in evaluating for marrow involvement.

Morphology and Immunophenotype of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an indolent mature B-cell lymphoproliferative disorder that often presents with bone marrow and peripheral blood involvement. Lymphadenopathy and proliferation in extramedullary tissues are often seen. The key to the precise diagnosis of CLL begins with assessment of the cytomorphology of the neoplastic cells, often from peripheral blood, but also the architectural patterns of involvement from tissue sections in bone marrow and lymph node biopsies. Although not 100% sensitive and specific, these morphologic features are strong clues in the broad differential diagnosis. Immunophenotyping complements and aids the morphologic impressions. Although often an indolent disease, some patients tend to have progressive disease and an aggressive clinical course. Staining for CD38 and 70 kD zeta-associated protein (ZAP-70) helps to prognosticate the patient’s disease and predict the tumor’s mutational status for the immunoglobulin heavy-chain, variable region (IGHV). Since defining the disease in the mid-nineteenth century, the workup of CLL now combines multiple disciplines, from basic characterization of morphology to immunophenotyping, and now molecular genetics that all aid in arriving at a precise diagnosis and help to choose the proper therapies for affected patients. In this section, the morphology and immunophenotype of CLL are discussed, with special emphasis on differential diagnosis and transformation (Richter syndrome). The issue of monoclonal B-cell lymphocytosis is also addressed, the criteria and description of which have recently been modified in the new 2016 WHO revision.