HRS Cell Research Articles

Circulating tumor DNA for monitoring classic Hodgkin lymphoma patients: Correlation with FDG-PET/CT.

The value of circulating tumor DNA (ctDNA) as a biomarker of disease activity in classic Hodgkin lymphoma (cHL) patients has not yet been well established. By profiling primary tumors and ctDNA, we identified common variants between primary tumors and longitudinal plasma samples in most of the cases, confirming high spatial and temporal heterogeneity. Although ctDNA analyses mirrored HRS cell genetics overall, the prevalence of variants shows that none of them can be used as a single biomarker. Conversely, the estimation of hGE/mL, based on measures of total ctDNA, reflects disease activity and is almost perfectly correlated with standard parameters such as PET/CT that are associated with refractoriness.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

The microenvironmental niche in classic Hodgkin lymphoma is enriched for CTLA-4–positive T cells that are PD-1–negative

AbstractClassic Hodgkin lymphoma (cHL) is a tumor composed of rare, atypical, germinal center–derived B cells (Hodgkin Reed-Sternberg [HRS] cells) embedded within a robust but ineffective inflammatory milieu. The cHL tumor microenvironment (TME) is compartmentalized into “niches” rich in programmed cell death-1 ligand (PD-L1)–positive HRS cells and tumor-associated macrophages (TAMs), which associate with PD-1–positive T cells to suppress antitumor immunity via PD-L1/PD-1 signaling. Despite the exquisite sensitivity of cHL to PD-1 checkpoint blockade, most patients eventually relapse and need therapeutic alternatives. Using multiplex immunofluorescence microscopy with digital image analysis, we found that cHL is highly enriched for non–T-regulatory, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)–positive T cells (compared with reactive lymphoid tissues) that outnumber PD-1–positive and lymphocyte-activating gene-3 (LAG-3)–positive T cells. In addition, T cells touching HRS cells are more frequently positive for CTLA-4 than for PD-1 or LAG-3. We further found that HRS cells, and a subset of TAMs, are positive for the CTLA-4 ligand CD86 and that the fractions of T cells and TAMs that are CTLA-4–positive and CD86-positive, respectively, are greater within a 75 μm HRS cell niche relative to areas outside this region (CTLA-4, 38% vs 18% [P = .0001]; CD86, 38% vs 24% [P = .0007]). Importantly, CTLA-4–positive cells are present, and focally contact HRS cells, in recurrent cHL tumors following a variety of therapies, including PD-1 blockade. These results implicate CTLA-4:CD86 interactions as a component of the immunologically privileged niche surrounding HRS cells and raise the possibility that patients with cHL refractory to PD-1 blockade may benefit from CTLA-4 blockade.

Single Cell Transcriptome Analysis Reveals Disease-Defining T Cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Abstract 5053: Epstein-Barr virus prevalence in classical Hodgkin lymphoma tumors is explained by histologic subtype, not race/ethnicity in a multiethnic US population

Abstract Epstein-Barr virus (EBV) is present in a varying proportion of classical Hodgkin lymphoma (cHL) tumors reportedly associated with age, sex, race/ethnicity and histologic subtype. As part of a study to examine the tumor microenvironment and survival in a multiethnic set of US cHL cases, we examined the distribution of EBV expression in tumor blocks from a subset of 269 of the available cases. We confirmed cHL diagnosis and histological subtype in H&E sections of FFPE tumor blocks of excisions and core biopsies from cases provided by Southern California Kaiser, Winship Cancer Center at Emory University, City of Hope National Medical Center, Grady Memorial Hosiptal, University of California Los Angeles and University of Southern California hospitals diagnosed from 1996-2016. Immunostain results were available for most cases. Tissue microarrays were constructed with two 2 mm cores from each case with 29 cases on each array. EBER was assessed using in situhybridization and scored as negative, positive in HRS cells or positive in the surrounding normal infiltrate. Demographic and clinical information, including the biopsy anatomic site, subtype, race/ethnicity (Hispanic, African American, Asian, non-Hispanic white), age at diagnosis and gender. Multiple logistic regression was conducted to assess the relationship between age, race/ethnicity, gender, subtype and EBV tumor status. Race/ethnicity data was available at this time for 237 cases. Of these, 186 were nodular sclerosis (NS), 53 were mixed cellularity (MC), 8 were lymphocyte depleted (LD), 3 were lymphocyte rich (LR), and 10 were not otherwise specified (NOS). Fifty-seven cases were African American, 48 were non-Hispanic white, 115 were Hispanic, 16 were Asian and one was other race. 106 were female (42%). Age at diagnosis ranged from 5-84 years, with 118 (50%) in the adolescent/young adult (AYA) range (15-35). EBV prevalence in HRS cells by subtype was 0% for LD, 47% for MC, 33% for LR, 23% for NS and 50% for NOS. When restricted to the two most common subtypes, NS and MC, histologic subtype alone was a statistically significant predictor of EBV tumor status (p=.0008) and when adjusting for age, sex, site and race/ethnicity (p=.0011). In NS cases, EBV HRS cell positivity was highest in non-Hispanic whites (31%), followed by Hispanics (23%) and African Americans (18%). Among MC cases, it was very high among African Americans (80%) compared to Hispanics and non-Hispanic whites (33-39%). The patterns were similar when restricted to the AYA age group. 6.5%, 4% and 20% of EBV-negative NS, MC and NOS cases, respectively, had EBV present in non-malignant lymphocytes but not in HRS cells. Histologic subtype was the strongest predictor of EBV HRS cell positivity in this set of multiethnic cHL patients. Unlike previous reports, EBV-positive tumors were not more common among non-whites, except for African American MC cases. Citation Format: Rachel Bolanos, Amie Hwang, Chun Chao, Christopher Flowers, Sheeja Pullarkat, Jose Aparicio, Sophia Wang, Karen Mann, Leon Bernal-Mizrachi, Joo Song, Christian Steidl, Christine Lee, Wendy Cozen, Iran Siddiqi. Epstein-Barr virus prevalence in classical Hodgkin lymphoma tumors is explained by histologic subtype, not race/ethnicity in a multiethnic US population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5053.

Immune targeting of the microenvironment in classical Hodgkin's lymphoma: insights for the hematologist.

While up to 80% of patients with Hodgkin’s lymphoma (HL) are cured with first-line therapy, relapsed/refractory (R/R) disease remains a clinical challenge and is fatal for many young patients. HL is unique in that the tumor cells (Hodgkin Reed–Sternberg; HRS cells) are a small fraction (<1%) of the tumor bulk, with the remaining tumor composed of the cells of the tumor microenvironment (TME). The support and integrity of the TME is necessary for HRS cell growth and survival. Targeting the programmed death 1 pathway has shown exciting activity in relapsed HL and led to United States Food and Drug Administration approval of the checkpoint inhibitors, nivolumab and pembrolizumab, for R/R HL. Novel combinations with checkpoint blockade therapy (CBT), targeted approaches such as combinations of CBT with brentuximab vedotin or chemotherapy, chimeric antigen receptor T-cells, and the use of CBT to potentially sensitize to subsequent therapy are being investigated as treatment approaches. As understanding of the HL TME grows, hopefully this will increase the number of rational therapeutic targets.

The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G1

Classical Hodgkin Lymphoma (cHL) is primarily a B cell lymphoid neoplasm and a member of the CD30–positive lymphomas. cHL and the other CD30–positive lymphomas are characterized by the elevated expression and/or constitutive activation of the activator protein-1 (AP-1) family transcription factors, c-Jun and JunB; however, the specific roles they play in the pathobiology of cHL are unclear. In this report we show that reducing either c-Jun or JunB expression with short-hairpin RNAs (shRNAs) reduced the growth of cHL cell lines in vitro and in vivo, primarily through impairing cell cycle transition through G1. We further investigated the effect of c-Jun and JunB knock-down on proliferation in another CD30–positive lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). We found that JunB knock-down in most ALK+ ALCL cell lines examined also resulted in reduced proliferation that was associated with a G0/G1 cell cycle defect. In contrast, c-Jun knock-down in multiple ALK+ ALCL cell lines had no effect on proliferation. In summary, this study directly establishes that both c-Jun and JunB play roles in promoting HRS cell proliferation. Furthermore, we demonstrate there are similarities and differences in c-Jun and JunB function between cHL and ALK+ ALCL.

New Recurrently Mutated Genes in Classical Hodgkin Lymphoma Revealed By Whole-Exome Sequencing of Microdissected Tumor Cells

Dissecting the genetics of classical Hodgkin lymphoma (cHL), a common lymphoma derived from germinal center B (GCB) cells, has remained a difficult task compared to other lymphomas, due to the rarity (usually <5%) of tumor cells (the Hodgkin and Reed/Sternberg - HRS - cells) in the lymph node.Thus, we microdissected 1200-1800 single HRS cells per case from frozen biopsies of 36 patients, along with a similar number of adjacent non-neoplastic cells, and performed in duplicate whole-genome DNA amplification (WGA) followed by independent whole-exome sequencing, thus allowing to control for possible biases introduced by the WGA step. The putative somatic protein-changing mutations identified in a major tumor clone (i.e., ≥20% variant allele frequency) were then validated by targeted deeper sequencing of the same WGA DNA, as well as by ultra-deep sequencing and digital PCR of unamplified whole-biopsy DNA.We found highly recurrent (11/36 patients; 31%), heterozygous, STAT6 missense mutations in exons encoding for the DNA binding domain, identical to the activating STAT6 variants known in primary mediastinal B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. ShRNA-induced STAT6 knockdown caused marked apoptosis in the STAT6-mutated cHL cell line L1236, which expresses phosphorylated STAT6 (pSTAT6), while it showed little or no effect in the STAT6-unmutated/pSTAT6+ cHL lines L428 and HDLM2, and in the STAT6-unmutated/STAT6-unphosphorylated cHL line L540. Thus, STAT6 mutations in cHL appear to confer a survival advantage distinct from (and beyond that of) STAT6 phosphorylation, possibly due to their aberrant DNA binding activity following pSTAT6-dependent entry into the nucleus. Interestingly, STAT6 mutations were often associated with disruptive somatic mutations of the JAK-STAT signaling inhibitor SOCS1 (frequency in the evaluable STAT6-mutated versus unmutated patients: 6/7, i.e. 87%, versus 4/14, i.e. 29%; p=0.02), suggesting a genetic interaction. Indeed, transduction of wild-type SOCS1 induced apoptosis of STAT6-mutated L1236 cells to a much greater extent than STAT6-unmutated L428 cells, despite both carry a SOCS1disruptive mutation and express pSTAT6. Furthermore, the presence in additional cases of genetic lesions of other JAK-STAT pathway members (mutations activating STAT3, STA5B, JAK1 or inactivating the inhibitor PTPN1; JAK2 copy number gains) attests to the pervasive role of JAK-STAT signaling in the genetics of cHL (up to ~70% of patients affected).GNA13, encoding Gα13, carried mutations, mostly heterozygous, in 8/36 (22%) patients. By transmitting signals from the S1PR2 and P2RY8 receptors, Gα13 ensures the confinement of proliferating GCB cells within secondary lymphoid follicles, while constraining at the same time their expansion by facilitating apoptosis in this potentially dangerous niche (Nature 2014;516:254). GNA13 mutations in cHL included disruptive (frame-shifting and non-sense) variants and were similar to those identified in GC B cell-derived Burkitt lymphoma and DLBCL of the GCB-type. The apparent histogenetic selectivity of inactivating GNA13mutations, almost never found in post-germinal center ABC-DLBCL, suggests they may facilitate cHL lymphomagenesis by rescuing from apoptosis crippled GC B cells (the proposed HRS cell precursors) and by promoting their dissemination outside the follicles, where HRS cells are indeed observed in lymph nodes with early, partial involvement by cHL (Am J Surg Pathol 1983;7:145).Heterozygous missense mutations of XPO1 occurred in 6/36 patients (17%) at the hot-spot amino acid residue E571, which is recurrently targeted also in chronic lymphocytic leukemia and PMBCL. XPO1encodes exportin-1, which shuttles outside the nucleus more than 200 proteins, including tumor suppressors involved in cHL pathogenesis and functioning in the nucleus (e.g., NFKBIA, FOXOs, TP53) (Blood 2012;120:4621). XPO1 inhibition by the clinical compound selinexor caused growth inhibition and apoptosis of cHL cell lines in a mutation-dependent manner.By providing a global characterization of the cHL coding genome, this works uncovered several new genes implicated in the genetics of this lymphoma, and most prominently in the JAK-STAT pathway, that likely play a significant role in its biology.#Shared first authorship°Shared last authorship DisclosuresNo relevant conflicts of interest to declare.

Expression of PIM Kinases in Reed-Sternberg Cells Fosters Immune Privilege and Tumor Cell Survival in Classical Hodgkin Lymphoma

The classical Hodgkin lymphoma (cHL) is characterized by a presence of rare malignant Reed-Sternberg (RS) cells surrounded by abundant reactive infiltrate. Tumor RS cells express multiple cytokines, chemokines and immunoregulatory proteins, such as PD-L1 or galectin-1, that enhance recruitment of the infiltrating cells and allow malignant cells to escape from host immune surveillance. Since targeting these immunomodulatory molecules is a highly promising therapeutic strategy in cHL, identification of pathways and mechanisms orchestrating tumor immune evasion and supporting RS cell survival can further reveal targetable vulnerabilities of cHL. Expression of immunoregulatory proteins in cHL is modulated by tumor-specific activity of certain pro-survival transcription factors, such as NFκB and STATs. Since the activity of these transcription factors is modulated by oncogenic PIM1/2/3 serine/threonine kinases, we hypothesized that PIMs may support RS cell survival and foster their immune privilege. For these reasons, we investigated PIM1/2/3 expression in cHL and determined mechanisms underlying their expression. Furthermore, we assessed impact of PIM inhibition on expression of key factors engaged in development of the immunosuppressive microenvironment and HRS cell survival.Our analyses revealed that PIM1/2/3 are ubiquitously expressed in primary and cultured RS cell lines. At least one PIM isoform was expressed in each cell line and in 97% of 67 primary cHL biopsies. RS cells treated with JAK1/2/3 inhibitor exhibited reduced PIM1 and PIM2 levels. Genetic inhibition of canonical NFkB activity with IkB super-repressor or shRNA-mediated ablation of alternative NFkB pathway led to decrease of PIM2 and PIM3, suggesting that PIM-1/2/3 expresion in cHL depends at least in part on JAK-STAT and NFkB activity.To assess the role of PIM kinases in cell viability, we silenced expression of each PIM isoform (individually or simultaneously) in HDLM-2 cells. Knockdowns of individual PIM isoforms were associated with marked increased in remaining isoforms expression and were not associated with toxicity. In marked contrast, downregulation of all three isoforms increased cellular apoptosis by 17%. For this reason, for subsequent studies we used a newly developed pan-PIM inhibitor (SEL24-B489). The inhibitor was toxic to all cells with IC50 ranging from 3-5 µM. To determine mechanisms underlying toxicity, we assessed the activities of specific PIM substrates: 4EBP1, S6, and p65 (RelA). SEL24-B489 rapidly decreased PIM-dependent phosphorylation of these molecules in all tested cell lines. Furthermore, it reduced DNA binding activity of the NFκB-p65 complexes, indicating that PIM kinases modulate NFκB activity in cHL. For this reason, we next assessed the consequences of PIM inhibition on NFκB-dependent transcription. SEL24-B489 significantly downregulated mRNA of NFkB target genes associated with HRS cell survival and proliferation, such as Bfl-1, RelB and CD40. In cells treated with PIM inhibitor SEL24-B489 we also found markedly decreased expression NFkB-dependent cytokines and chemokines specifically shaping pro-tumoral microenvironment, such as IL-8, CCL5 and IL-13. In addition, cell lines exposed to SEL24-B489 treatment exhibited decreased expression of immunomodulatory PD-L1 and Gal-1 proteins. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using HDLM-2 cells. In contrast to animals exposed to vehicle alone, we observed inhibition of tumor growth by 95,8% in SEL24-B489-treated animals (p=0,0002). Consistent with the in vitro data, we observed strong downregulation of phospho-S6, GAL-1 and PD-L1 proteins in tumor sections from PIM inhibitor-treated animals.Taken together, we demonstrated that the oncogenic PIM-1/2/3 kinases are expressed in RS cells and their activity can be specifically blocked using a pan-PIM inhibitor SEL24-B489. PIM inhibition significantly reduced activity of specific PIM substrates and decreased the expression of NFκB-dependent pro-survival genes and key immunomodulatory proteins. These results highlight the pleiotropic activity of SEL24-B489 and indicate that PIM kinases are promising therapeutic targets in cHL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment.

M2 Macrophages but not M1 Macrophages Are Associated with Worst Outcome in Classical Hodgkin Lymphoma

We have shown recently that in EBV+ paediatric classical Hodgkin lymphoma (cHL) the tumour microenvironment is characterized by a cytotoxic/Th1 profile and higher numbers of CD14+, CD68+ and CD163+ cells when compared to EBV- cases. Our results suggested also that CD163+ cells represent a heterogeneous cell population. The objectives of this study were to evaluate the macrophage polarization in the tumour microenvironment of paediatric cHL and its impact on survival. The tumour microenvironment was analysed by double-immunohistochemistry based on the combination of CD68 or CD163 with pSTAT1 (M1 macrophages), as well as CD68 or CD163 with CMAF markers (M2 macrophages). Gene expression of STAT1 and LYZ were also evaluated by RT-qPCR. Results were analysed in context of age, histological characteristics, EBV-status, clinical follow-up and our previous study of T-cell populations in these cases. Results: 100 cHL cases were studied, including 69% nodular sclerosis (NS) and 23% mixed cellularity (MC) cases; 44.8% of cases were EBV-positive. Patients <= 14 years showed higher numbers of CD8+ lymphocytes (P = 0.002). EBV+ cases exhibited higher numbers of CD68+pSTAT1+ macrophages (P = 0.02). Higher gene expression of LYZ and STAT1 was observed in EBV+ cases and cases with higher numbers of CD68+pSTAT1+ macrophages. Better overall-survival was observed in cases with high numbers of CD163+pSTAT1+ macrophages (P = 0.02); while high numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P = 0.02). Furthermore, cases showing high numbers of both CD163+CMAF+ and Granzyme B+ cells were associated with worse PFS (P = 0.009). Our results confirm that CD163 is not a M2-specific marker and suggest that in paediatric cHL the polarization of macrophages may depend on EBV status of HRS cell, and high numbers of M2 macrophages, but not M1 macrophages, is associated with worse PFS.

Microenvironment Assessment, Disease Characteristics and Prognosis In HIV-Associated (HIV-cHL) and HIV-Negative Classical Hodgkin Lymphoma: The Influence Of Epstein Barr Virus (EBV)

IntroductionStudies of EBV positive (+) and negative (-) classical Hodgkin lymphoma (cHL) have shown the importance of the immune microenvironment in affecting Reed-Sternberg (hRS) cell survival, proliferation, and biologic behavior. For example, macrophage infiltrates may correlate with inferior disease outcome and survival and proliferation of the hRS cells depends on trophic signals from various inflammatory cells, including CD4+ T cells. The latter finding may explain why HIV-cHL patients (pts) usually present with higher circulating CD4+ T-cell counts (cCD4) compared to HIV-related non-Hodgkin lymphoma. Pathobiologically, HIV-cHL differs from HIV negative cHL (cHL) in that it is nearly always EBV+, has higher numbers of hRS cells, presents with more advanced median stage, exhibits more commonly the mixed cellularity (MC) pattern, and some studies suggest it is more clinically aggressive. To investigate the microenvironment of HIV-cHL and its influence on cHL biology, we assessed the immune cell composition and clinical characteristics of HIV-cHL and compared the findings to those of EBV+ and EBV- cHLs. Methods31 HIV-cHL and 40 cHL (8 EBV+/32 EBV-) cases were identified and corresponding tissue microarrays (TMAs) created. TMAs were evaluated for EBV (EBER), CD30, and microenvironment-associated antigens: PAX5, CD3, CD4, CD8, CD68, CD163 (% positive), TIA1, FOXP3 (relative number 0-4+); the hRS-macrophage microenvironment was evaluated by assessing the number of hRS where >50% of the circumference of the neoplastic cell was associated with CD68+ cells. Results were compared based on HIV status, EBV status (in HIV negative pts), demographics, cCD4 and histology; each was correlated with overall survival. Analyses were performed using non-parametric Fisher's exact test, Kaplan-Meier method and Cox Proportional Hazards model. ResultsM F ratio was 9:1 in the HIV group vs. 1.3:1 in the HIV negative pts (p <0.001). The median cCD4 in the HIV+ pts at HIV-cHL diagnosis was 248 cells/mm3. No differences in age, stage, B symptoms, bulky disease, IPS score, and hRS concentration were found between HIV+ and HIV negative cHLs pts. 7% of cHLs and 33% of HIV-cHLs were classified as MC (p<0.01). 90% of HIV-cHLs were EBV+ compared to 20% of the cHL cases. Statistically significant differences were seen in the number of CD4+ and CD8+ cells between the EBV+ and EBV- cHLs irrespective of HIV status. The only difference among all the immune microenvironment markers observed between EBV+ HIV-cHL and EBV+ cHL cases was that of peri-hRS CD68 cells, which were more abundant in the EBV+ HIV-cHL (Table 1; 51% vs. 30% p<0.005). Similar differences in peri-hRS CD68 staining were observed between the HIV-cHL and all cHL pts. The only deaths in HIV-cHL pts occurred when CD68 was >15% (p<0.05). No statistical differences were noted for OS with respect to cCD4, histologic subtype, race, gender, or HIV status. ConclusionsNo significant differences were observed between HIV-cHL and cHL pts with respect to stage, B symptoms, bulky disease, IPS score, or overall survival, but there were more MC cases in the HIV-cHL cohort. The microenvironment of HIV-cHL and EBV+ cHL is similar, but different from EBV- cHL with respect to percentages of CD163+, PAX5+, CD4+ and CD8+ cells. The location of CD68+ macrophages was the only discordant result between the EBV+ HIV-cHL and EBV+ cHL cohorts. The differences in location of the CD68+ cells, which may be dependent on HIV status, suggests greater influence of these cells on the biologic behavior of the neoplastic process, correlating with poor survival. However, the similarity in the microenvironments in HIV-cHL and EBV+ cHL with respect to CD4, CD8, and CD163 staining implies an important role for EBV on disease biology, as well. Thus, these data suggest that EBV as well as HIV, play prominent roles in determining the immune response and disease behavior in HIV-cHL, warranting further study Disclosures:No relevant conflicts of interest to declare.

Targeted Therapy in Relapsed Classical Hodgkin Lymphoma

Although frontline treatment of advanced Hodgkin lymphoma (HL) produces high cure rates, disease either will not respond to or will relapse after initial therapy in approximately a quarter of patients. Many patients with disease relapse can be successfully salvaged with second-line chemotherapy followed by autologous stem cell transplantation (ASCT). Patients whose disease relapses after ASCT are rarely cured. A unique pathophysiologic feature of HL is that the malignant Reed-Sternberg (HRS) cell is rare and resides within a microenvironment of inflammatory and immune-related cells. The recent FDA approval of the anti-CD30 antibody-drug conjugate brentuximab vedotin (BV) for patients with either primary refractory HL or those whose disease relapses after ASCT represents a major advance in therapy. This article focuses on BV and other novel agents that target the HRS cell surface, intracellular signaling pathways, and tumor microenvironment.

Induction of Endoplasmic Reticulum Stress by Sorafenib and Activation of NF-κB by Lestaurtinib as a Novel Resistance Mechanism in Hodgkin Lymphoma Cell Lines

Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoma show aberrant expression and activation of several receptor tyrosine kinases (RTK) in the majority of cases. Therefore, we tested whether tyrosine kinase inhibitors (TKI) already in clinical use or late stages of clinical trials have antiproliferative effects on HRS cell lines and evaluated the targets, affected signaling pathways, and mechanisms of cell death and resistance. Sorafenib and lestaurtinib had antiproliferative effects on HRS cell lines at concentrations achievable in patients. Sorafenib inhibited platelet-derived growth factor receptor (PDGFR) α, TRKA and RON, caused decreases in total and phosphorylated amounts of several signaling molecules, and provoked caspase-3-independent cell death, most likely due to endoplasmic reticulum stress as indicated by upregulation of GADD34 and GADD153 and phosphorylation of PERK. Lestaurtinib inhibited TRKA, PDGFRα, RON, and JAK2 and had only a cytostatic effect. Besides deactivation, lestaurtinib also caused activation of signaling pathways. It caused increases in CD30L and TRAIL expression, and CD30L/CD30 signaling likely led to the observed concomitant activation of extracellular signal-regulated kinase 1/2 and the alternative NF-κB pathway. These data disclose the possible use of sorafenib for the treatment of Hodgkin lymphoma and highlight NF-κB activation as a potential novel mechanism of resistance toward TKIs.

Immunohistochemical expression of angiogenic cytokines in childhood Hodgkin lymphoma

This immunohistochemical study was carried out to evaluate the role of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), their inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1), and microvessel density (MVD) in the clinicopathologic behavior of childhood Hodgkin's lymphoma (HL). Paraffin-embedded histologic sections from 15 children with HL were immunohistochemically stained with MMP-2, MMP-9, TIMP-1, VEGF, and CD31 antibodies to investigate the correlation between the expression of these markers and the clinicopathologic characteristics of HL. Expression of MMP-2 and VEGF in Hodgkin and Reed–Sternberg cells (HRS) was more frequent in nodular sclerosis than in other subtypes ( p=0.07 and 0.08, respectively). None of the study parameters in HRS cell were associated with age, sex, disease stage, extranodal disease, and the occurrence of bulky tumor. There was a trend toward advanced stage in negative TIMP-1 staining in HRS cells ( p=0.06). In reactive lymphocytes, MMP-2 expression was correlated with MVD ( r=0.68, p=0.005), and MMP-9 expression was correlated with B symptoms ( p=0.003). Also, low TIMP-1 expression in reactive lymphocytes was frequently found in patients with advanced stage ( p=0.048). There was a positive correlation with the ratio of MMP-2 expression in reactive lymphocytes and MVD ( r=0.68, p=0.005). Expression of MMP-9 in reactive lymphocytes was correlated with MVD without statistical significance ( r=0.487, p=0.06). Our results suggest that, as in many solid tumors, angiogenesis and angiogenic factors may play an important role in childhood HL. Larger series of patients are needed to determine the prognostic value of angiogenesis in childhood HL.

The Geldanamycin Analogue Heat Shock Protein 90 (HSP90) Inhibitor 17-AAG in Lymphomas: Molecular Mechanisms of 17-AAG-Induced Cell Death in Hodgkin Disease and Non- Hodgkin Lymphoma Cell Lines.

Heat Shock Proteins (HSP) are stress-induced proteins that act as molecular chaperones in all benign and malignant cells. HSPs are important for maintaining the function of several client proteins involved in transcriptional regulation, signal transduction, and cell-cycle control. HSP90 has been recently identified as a potential therapeutic target in several types of tumors. However, the effect HSP90 inhibition in Hodgkin disease (HD) and non-Hodgkin lymphomas (NHL) remains unknown. The geldanamycin analogue 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a small molecule inhibitor of HSP90, which interferes with ATP-dependent chaperones activity, with subsequent degredation of client proteins by the proteasome. We studied the activity of 17-AAG in a panel of cell lines of HD (HD-MyZ, HD-LM2, L-428, KM-H2) and NHL (SUDHL1, and Jurkat) origin. HSP90 was expressed in all HD cell lines but not in normal peripheral blood lymphocytes of healthy donors. 17-AAG demonstrated a significant antiproliferative effect in a dose (01 micromolar - 5 micromolar) and time (24 hours to 72 hours) dependent manner in all cells lines, but was most prominant in the HD-LM2 cells. In all HD-derived cell lines, 17-AAG had more than 50% inhibitory effect over control cells. This effect was most prominant after 72 hours of incubation. Subsequently, we examined the molecular mechanisms responsible for this antiproliferative effect. Both cell cycle arrest and induction of apoptosis were observed. In the HD-LM2 cell line, 17-AAG predominantly induced a G0/G1 cell cycle phase arrest, while in the L-428 cell line there was a G2/M phase arrest (as determined by propidium iodide staining and flow-cytometric analysis). This effect was associated with alterations in MDM2 and cyclin D1 proteins. Cell cycle arrest was followed by apoptosis as determined by Annexin-V binding assay, which was predominantly mediated by caspase 9 activation. 17-AAG also reduced the cellular content of Bcl-2, cFLIP, and AKT, but had no significant effect on XIAP or cIAP2. Furthermore, 17-AAG decreased the total ERK1/2 which has been shown to play an important role in HRS cell survival. 17-AAG-induced apoptosis was partially reversed by the pancaspase inhibitor ZVAD-FMK. Preliminary data suggested that 17-AAG did not enhance the effect of doxoorubicin or gemcitabine chemotherapy. These data suggest a first proof-of-principle that inhibition of HSP90 might be of therapeutic value in HD and NHL.

First hyperpolarizability measurements via hyper-Rayleigh scattering at 1500 nm

A hyper-Rayleigh scattering, HRS, system constructed around an optical parametric power oscillator and allowing wide fundamental wavelength tunability is described in detail. The design of the HRS cell, which may overcome problems inherent with other cell designs, is discussed. The potential of this HRS system for non-resonant first hyperpolarizability, β, determination is highlighted via measurement of a dipolar chromophore at 1500 nm. The magnitude of the non-linearity may be explained by either increased conjugation in the charge-transfer state or by increased magnitude in non-diagonal β-tensor elements.