Classical Hodgkin Lymphoma Cell Research Articles

Human leukocyte antigen (HLA) class I expression on Hodgkin-Reed-Sternberg cells is an EBV-independent major determinant of microenvironment composition in classic Hodgkin lymphoma.

Hodgkin-Reed-Sternberg cells (HRSCs) in classic Hodgkin Lymphoma (HL) frequently lack expression of human leukocyte antigen class I (HLA-I), considered to hamper activation of cytotoxic T cells in the tumor microenvironment (TME). Here, we demonstrate HLA-I expression on HRSCs to be a strong determinant of TME composition whereas expression of HLA-II was associated with only minor differential gene expression in the TME. In HLA-I-positive HL the HRSC content and expression of CCL17/TARC in HRSCs are low, independent of the presence of Epstein-Barr virus in HRSCs. Additionally, HLA-I-positive HL shows a high content of CD8+ cytotoxic T cells. However, an increased expression of the inhibitory immune checkpoint LAG3 on CD8+ T cells in close proximity to HRSCs is observed. Suggesting interference with cytotoxic activity, we observed an absence of clonally expanded T cells in the TME. While HLA-I-positive HL is not associated with an unfavorable clinical course in our cohorts, they share features with the recently described H2 subtype of HL. Given the major differences in TME composition, immune checkpoint inhibitors may differ in their mechanism of action in HLA-I-positive compared to HLA-I-negative HL.

Expression Patterns of GATA3 in Classical Hodgkin Lymphoma: A Clinico-Pathological Study.

GATA3 is a transcription factor involved in T-cell maturation and has been previously shown to be aberrantly overexpressed in malignant Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). However, the immunophenotypes of the cell types expressing GATA3 have not been precisely characterized so far in cHL tissues. In this single-center retrospective cohort study we analyzed the expression patterns of GATA3 alone and in combination with B, T, NK or macrophage-associated markers in 73 cases with newly diagnosed cHL and investigated for a possible correlation with clinical and laboratory parameters. Immunohistochemistry (single and double) was performed using GATA3 alone and in combination with CD20, CD3, CD56, CD68, CD30 or CD15. Clinical and laboratory parameters were collected and correlated with the expression of GATA 3. GATA3 nuclear expression was found in HRS cells in 39/73 (54%) cases of cHL. The Nodular Sclerosis (NS) subtype showed the highest positivity rate (35/56, 63%), followed by mixed cellularity (MC; 4/14, 29%) and lymphocyte rich (LR; 0/3). Double immunostainings showed that GATA3 was expressed by CD30+ or CD15+ HRS cells and a few CD3+ T-cells, whereas GATA3 expression was not detected in CD20, CD56 or CD68+ cells. GATA3-negative cHL was significantly associated with unfavorable prognostic factors such as older age at diagnosis and increased levels of serum β2-microglobulin. The heterogenous expression patterns of GATA3 in HRS cells that were observed in a substantial proportion of cHL, mainly in the NS subtype, further support the biological heterogeneity of cHL.

A Subset of Classical Hodgkin Lymphoma Cases Demonstrate CD19 Expression on Hodgkin Reed-Sternberg Cells By Immunohistochemistry

Background: The neoplastic Hodgkin Reed-Sternberg (HRS) cells in Classical Hodgkin Lymphoma (CHL) are thought to lose their B-cell phenotype and lack expression of most B-cell markers. Improvements in antibodies and detection methods have revealed that a subset of CHL cases may express some B-cell markers, including CD19 and CD20, with currently unclear significance. Introduction of CD19-directed therapies has changed the landscape of salvage treatment in CD19 expressing Non-Hodgkin Lymphomas. As such, we sought to characterize CD19 expression in HRS cells and its clinical implications and potential utility as a therapeutic target. Methods: We conducted an Institutional Review Board-approved retrospective review of records for CHL cases from 01/2015 to 07/2020 at a tertiary care center. 42 cases met the criteria and had sufficient tissue for analysis: 39 CHL, 2 CHL mixed with other B-cell lymphomas, and 1 Epstein Barr Virus Post-Transplant Lymphoproliferative Disease (EBV-PTLD)-CHL type. Cases were assessed for CD19 immunohistochemical (IHC) staining using the EP169 Rabbit Monoclonal Antibody. HRS cells were identified via morphology and characteristic immunoprofile, with all HRS cells expressing strong CD30 and dim PAX-5. CD19 staining intensity (0-3+) and pattern of expression (cytoplasmic, Golgi body, membranous) in HRS cells was independently evaluated by three hematopathologists. Based on their consensus, CD19 staining was categorized as negative, weak (variable across HRS cells), or positive. CD19 staining was compared with histologic subtype, other B-cell marker staining, EBV association (via EBER-ISH), and clinical data and outcomes, including, age, stage, treatment, response, overall survival, and demise. Results: Histologically, cases were composed of the following subtypes: 26 nodular sclerosis, 8 mixed cellularity, 3 lymphocyte-rich, 1 lymphocyte-depleted, 2 other (EBV-PTLD and malignant B-cell lymphoma) and 2 unclassifiable (due to treatment effect and biopsy size). Nine of 42 cases (21%) were EBV-associated by EBER-ISH. CD19 staining of any kind in HRS cells was found in 13/42 cases (31%), with 10/42 (24%) demonstrating positive staining and 3/42 (7%) demonstrating weak staining. Staining intensity and pattern varied across positive cases (Table 1 and Figure 1). CD19 staining was seen in 4/26 nodular sclerosis (15%), 6/8 mixed cellularity (75%), 1/3 lymphocyte-rich (33%), and 1/1 lymphocyte-depleted (100%) cases. The strongest correlation with other markers was seen with EBV, with 7/9 (78%) EBV+ cases staining with CD19. Five cases with CD19 staining also demonstrated predominantly patchy/dim CD20 staining. The clinical characteristics of CHL cases with CD19 expression varied, with an age range of 10-78 years old and variable clinical stage at time of tissue sampling. Following initial treatment, 4/13 patients failed to achieve remissions after frontline chemotherapy (mostly ABVD), with 3 of these patients now deceased due to disease. Conclusions: A significant subset of CHL cases demonstrate CD19 expression on HRS cells, and EBV-associated CHL has a high rate of CD19 expression, which may reflect its different underlying disease mechanism. Larger studies are necessary to characterize the staining pattern and intensity that may be most amenable to specific therapies and to better elucidate the prognostic implications of CD19 expression. Given the accessibility of CD19 IHC and the growing list of effective CD19-directed therapies, it is worthwhile assessing CD19 expression, particularly in EBV associated CHL, and further research into the utility of CD19 expression on HRS as a potential therapeutic target is warranted.

Whole slide imaging of tumour microenvironment in classical Hodgkin’s lymphoma: development of a clinical prediction model based on programmed death-ligand 1 and tumorous Reed-Sternberg cells

AimsThe prognostic impact of programmed death-ligand 1 (PD-L1) cells in classic Hodgkin lymphoma (cHL) tumour microenvironment remains undefined.MethodsModel development via Transparent Reporting of a multivariable prediction model for Individual Prognosis...

Elevated serum TARC levels precede classic Hodgkin lymphoma diagnosis by several years

AbstractTumor cells in classic Hodgkin lymphoma produce high quantities of the thymus- and activation-related chemokine (TARC). We measured TARC levels in prediagnostic serum samples and found strikingly increased values in the vast majority of patients, as early as 6 years before diagnosis.

Clonal composition and differentiation stage of human CD30+ B cells in reactive lymph nodes.

Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.

Digital spatial profiling of CD4+ T cells in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (CHL) harbors a small number of Hodgkin-Reed-Sternberg (HRS) cells scattered among numerous lymphocytes. HRS cells are surrounded by distinct CD4+ T cells in a rosette-like manner. These CD4+ T cell rosettes play an important role in the tumor microenvironment (TME) of CHL. To elucidate the interaction between HRS cells and CD4+ T cell rosettes, we completed digital spatial profiling to compare the gene expression profiles of CD4+ T cell rosettes and other CD4+ T cells separated from the HRS cells. Immune checkpoint molecules including OX40, programed cell death-1 (PD-1), and cytotoxic T lymphocyte associated protein 4 (CTLA-4) expression was higher in CD4+ T cell rosettes compared to other CD4+ T cells. Immunohistochemistry confirmed variable PD-1, CTLA-4, and OX40 expression in the CD4+ T cell rosettes. This study introduced a new pathological approach to study the CHL TME, and provided deeper insight into CD4+ T cells in CHL.

Diagnostic Utility of STAT6 and pSTAT6 Immunohistochemistry for Distinguishing Classic Hodgkin Lymphoma and Peripheral T-Cell Lymphoma With Hodgkin and Reed-Sternberg-like Cells.

Peripheral T-cell lymphomas (PTCLs), particularly nodal lymphomas of T-follicular helper cell origin, may include Hodgkin/Reed-Sternberg (HRS)-like cells in their microenvironment. These HRS-like cells are morphologically indistinguishable from HRS cells of classic Hodgkin lymphoma (CHL). Therefore, PTCLs with HRS-like cells pose a differential diagnosis vis-à-vis CHL. A previous study reported that, in contrast to HRS cells, programmed death-ligand 1 (PD-L1) expression is rare in HRS-like cells of PTCLs and suggested that PD-L1 immunohistochemistry is useful to differentiate HRS cells and HRS-like cells. In this study, we analyzed 21 patients with PTCL with HRS-like cells and 34 patients with CHL and assessed the diagnostic utility of STAT6, pSTAT6, and pSTAT3 immunohistochemistry in distinguishing HRS cells from HRS-like cells. In addition, we also performed PD-L1 immunohistochemistry to reconfirm its utility in distinguishing the 2 diseases. Compared with HRS cells in CHLs, HRS-like cells in PTCLs showed significantly less positivity for STAT6 (9.6% vs. 70%, P <0.001), pSTAT6 (9.6% vs. 70%, P <0.001), and PD-L1 (9.6% vs. 85%, P <0.001). Thus, we reconfirmed the diagnostic utility of PD-L1 immunohistochemistry in distinguishing CHLs from PTCLs with HRS-like cells. In contrast, both HRS-like and HRS cells were highly associated with pSTAT3 expression, with no significant difference in positive cell frequency (86% vs. 91%, P =0.66). On the basis of these findings, we conclude that, in addition to PD-L1, STAT6 and pSTAT6 immunohistochemistry are helpful diagnostic tools to distinguish CHLs from PTCLs with HRS-like cells.

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease.

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest.

"Coexistence of Classical Hodgkin Lymphoma and Langerhans Cell Proliferation Presenting as a Tumor in Tumor Pattern"

"Coexistence of Classical Hodgkin Lymphoma and Langerhans Cell Proliferation Presenting as a Tumor in Tumor Pattern"

A Unusual Case of Classic Hodgkin Lymphoma (cHL) with Aberrant T-Antigens Expression

Abstract Introduction/Objective T-cell-associated antigens (TCA) are rarely expressed by Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) and is associated with uncertain clinical significance. Methods/Case Report We present a very recent case of a 68 year-old female with no significant past medical history presented with a large right groin mass for 1 month. She denied any fever, night sweats, fatigue or weight loss. CT abdomen/pelvis showed inguinal/pelvic lymphadenopathy. Resection showed effaced nodal architecture with a vague nodular appearance and with large, atypical nucleolated cells in a background of small, mature lymphocytes and histiocytes. Eosinophils are conspicuously absent in the background. The large cells were polylobated lymphocyte and histiocyte rich (L &amp; H) like, classic HRS cells as well as multinucleated in appearance. The morphologic features overlapped between Classic Hodgkin Lymphoma, and Nodular Lymphocyte Predominant Hodgkin Lymphoma. Given the immunohistochemical (IHC) features of the neoplastic cells [CD45 negative, PAX5+ (weak), CD30+ (strong), CD15+ (majority) and MUM1 +] the diagnosis was most compatible with CHL. Interestingly, a subset of the Hodgkin cells (10-20%) aberrantly expressed a number of T-cell markers (CD2, CD3, CD4, and CD8). However, the background smaller T-cells did not show cytologic atypia or aberrancies of T-cell antigen markers. The large cells were negative for CD43 and ALK1. TCR gamma gene rearrangement was also negative for clonal T-lymphocytes, thus excluding the possibility of peripheral T-cell lymphoma with Hodgkin-like cells. Based on the morphology and IHC profile, this case proves to be CHL with multiple TCA expression including CD2, CD3, CD4 and CD8. She was planned for chemotherapy. Four months after the initial diagnosis and after completion of 2 cycles of chemotherapy, her PET CT scan showed improvement with right inguinal adenopathy being smaller in size and decreased avidity, indicating a response to treatment. She is due for 2 more cycles. Till todate she is doing well with no further symptoms. Results (if a Case Study enter NA) NA. Conclusion So far reported cases of classic Hodgkin lymphoma (CHL) with aberrant T-cell antigen (TCA) expression were associated with decreased event-free survival and overall survival when compared with TCA-negative CHLs. Our finding contributes to the literature and improves our knowledge of the disease.

P035: CRISPR/Cas9-mediated knockout reveals an important role of CD30 in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

P035: CRISPR/Cas9-mediated knockout reveals an important role of CD30 in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

NFkB Pathway and Hodgkin Lymphoma.

The tumor cells that drive classical Hodgkin lymphoma (cHL), namely, Hodgkin and Reed-Sternberg (HRS) cells, display hallmark features that include their rareness in contrast with an extensive and rich reactive microenvironment, their loss of B-cell phenotype markers, their immune escape capacity, and the activation of several key biological pathways, including the constitutive activation of the NFkB pathway. Both canonical and alternative pathways are deregulated by genetic alterations of their components or regulators, EBV infection and interaction with the microenvironment through multiple receptors, including CD30, CD40, BAFF, RANK and BCMA. Therefore, NFkB target genes are involved in apoptosis, cell proliferation, JAK/STAT pathway activation, B-cell marker expression loss, cellular interaction and a positive NFkB feedback loop. Targeting this complex pathway directly (NIK inhibitors) or indirectly (PIM, BTK or NOTCH) remains a challenge with potential therapeutic relevance. Nodular predominant HL (NLPHL), a distinct and rare HL subtype, shows a strong NFkB activity signature because of mechanisms that differ from those observed in cHL, which is discussed in this review.

Immune phenotypes and checkpoint molecule expression of clonally expanded lymph node-infiltrating T cells in classical Hodgkin lymphoma

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αβ sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4+ and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4+ and CD8+ T cells. Degrees of clonal T cell expansion varied between patients (range: 1–18 expanded clones per patient) and was almost exclusively restricted to CD8+ T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells.

PDL1 shapes the classical Hodgkin lymphoma microenvironment without inducing T-cell exhaustion.

Classical Hodgkin lymphoma (CHL) is unusually sensitive to PD1 inhibition and PDL1 is highly expressed on CHL cells and in the tumor microenvironment. This could be interpreted as evidence of exhaustion, but paradoxically, PD1+ lymphocyte infiltration does not predict response to PD1 inhibitors and no increase in cytotoxic markers is seen after PD1 therapy as might be expected with reversal of exhaustion. In contrast to PD1, elevated PDL1 does predict response to PD1 inhibitors and recent data associate both retained CHL MHC-II expression and increased T helper (TH) T-cell receptor diversity with response, suggesting a connection to the TH compartment. We performed a phenotypic, spatial and functional assessment of T-cell exhaustion in CHL and found co-expression of an exhaustion marker and lower PD1 expression in CHL than in reactive nodes whereas the proliferative and cytokine production capacity were similar in CHL and the reactive nodes. We found no correlation between PDL1 expression and exhaustion signatures. Instead, we identified a strong association between PDL1 expression and CHL MHC-II expression, TH recruitment, and enrichment of TH1 regulatory cells. These data suggest that a dominant effect of PDL1 expression in CHL may be TH engagement and promotion of a regulatory microenvironment rather than maintenance of exhaustion.

Distinct Gene Expression Patterns of Calcium Channels and Related Signaling Pathways Discovered in Lymphomas.

Cell surface calcium (Ca2+) channels permit Ca2+ ion influx, with Ca2+ taking part in cellular functions such as proliferation, survival, and activation. The expression of voltage-dependent Ca2+ (CaV) channels may modulate the growth of hematologic cancers. Profile analysis of Ca2+ channels, with a focus on the Ca2+ release-activated Ca2+ (CRAC) and L-type CaV channels, was performed on RNA sequencing data from lymphoma cell lines and samples derived from patients with diffuse large B cell lymphoma (DLBCL). CaV1.2 expression was found to be elevated in classical Hodgkin lymphoma (CHL) cell lines when compared to other B cell lymphoma cell lines. In contrast, CHL exhibited reduced expression of ORAI2 and STIM2. In our differential expression analysis comparing activated B cell-like DLBCL (ABC-DLBCL) and germinal centre B cell-like DLBCL (GCB-DLBCL) patient samples, ABC-DLBCL revealed stronger expression of CaV1.3, whereas CaV1.1, CaV1.2, and CaV1.4 showed greater expression levels in GCB-DLBCL. Interestingly, no differences in ORAI/STIM expression were noted in the patient samples. As Ca2+ is known to bind to calmodulin, leading to calcineurin activation and the passage of nuclear factor of activated T cells (NFAT) to the cell nucleus, pathways for calcineurin, calmodulin, NFAT, and Ca2+ signaling were also analyzed by gene set enrichment analysis. The NFAT and Ca2+ signaling pathways were found to be upregulated in the CHL cell lines relative to other B cell lymphoma cell lines. Furthermore, the calmodulin and Ca2+ signaling pathways were shown to be downregulated in the ABC-DLBCL patient samples. The findings of this study suggest that L-type CaV channels and Ca2+-related pathways could serve as differentiating components for biologic therapies in targeted lymphoma treatments.

Nodular Lymphocyte-predominant Hodgkin Lymphoma With Nodular Sclerosis: An Underrecognized Feature Associated With Pattern D.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) with unusual features, including some that can overlap morphologically with classic Hodgkin lymphoma (CHL), have been described. Herein, we describe 12 cases of NLPHL with fibrous bands and capsular fibrosis resembling, in part, nodular sclerosis (NS) CHL. Seven of 12 cases harbored Reed-Sternberg-like cells, further suggestive of CHL, but all cases lacked associated eosinophils and/or plasma cells in the background. In this cohort, all cases had areas of so-called pattern D (nodular T-cell rich) as a sole component in 7 (58%) cases or as a hybrid pattern along with pattern E (diffuse T-cell/histiocyte-rich) in 5 (42%) cases. The immunophenotype of the large neoplastic cells in these cases supported their being lymphocyte predominant cells of NLPHL, positive for CD20, CD79a, and OCT2, and negative for CD15 and CD30. However, PAX5 was weak in 9 of 11 cases similar to Hodgkin/Reed-Sternberg cells in CHL. We conclude that some cases of NLPHL are associated with fibrous bands and capsular fibrosis and resemble, in part, NS CHL. In our experience, NLPHL with NS-like features occurs in 10% to 15% of cases of NLPHL and is associated with a variant pattern (D and/or E). In addition, all patients in this cohort were not treated before biopsy, suggesting that the prominent sclerosis in these cases is inherent to disease biology. Recognition of NLPHL with NS-like features further expands the morphologic spectrum of NLPHL and helps avoid potential misdiagnosis as CHL.

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.

CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2

ABSTRACT In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Phase II single-arm study of brentuximab vedotin in Chinese patients with relapsed/refractory classical Hodgkin lymphoma or systemic anaplastic large cell lymphoma

Background Relapsed/refractory (R/R) classical HL (cHL) and systemic anaplastic large-cell lymphoma (sALCL) treatment options are limited in China and there is a need for new therapies. Research design and methods This single-arm, open-label, multicenter, Phase II study assessed efficacy, safety, and pharmacokinetics (PK) of single-agent brentuximab vedotin in Chinese patients with R/R cHL or sALCL. Patients received brentuximab vedotin as a 1.8 mg/kg intravenous infusion on Day 1 of a 3-week cycle (maximum 16 cycles). Results Patients (N=39) received a median of 10 cycles (range: 2–16) of brentuximab vedotin. The objective response rate was 69% (95% CI: 52–83%). Median duration of response, progression-free survival and overall survival were 12.1 months, 13.5 months (95% CI: 6.8 months–not estimable) and not reached after a median follow-up of 16.6 months. Twenty seven (69%) patients achieved an objective response (complete response: n=11 [28%]; partial response: n=16 [41%]). Brentuximab vedotin was well-tolerated with no on-study deaths. AEs were generally manageable and reversible. No new safety signals were identified. PK were consistent with those previously described in Western populations. Conclusion Brentuximab vedotin had a positive benefit-risk profile for Chinese patients with R/R cHL or sALCL confirming it as a potential treatment option. Clinical trial registration www.clinicaltrials.gov identifier is NCT02939014.