Classical Hodgkin Lymphoma Cell Lines Research Articles

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.

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.

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.

Bruton Tyrosine Kinase Inhibition in Classical Hodgkin Lymphoma Cell Lines and Animal Models

Introduction: Hodgkin's Lymphoma (HL) is a malignancy of mature (post germinal center) B-lymphocytes that afflicts almost 20,000 individuals annually in North America and Europe. While the majority of patients diagnosed with HL are cured with multi-agent chemotherapy, 15 % of patients are refractory to conventional chemotherapy and almost half of patients with high-risk disease relapse. For these patients few effective therapeutic options exist. Only 3 drugs have been approved in the last 30 years for relapsed HL and novel therapies are needed. Bruton's Tyrosine Kinase (BTK) is a member of the TEC family and plays a central role in B-cell signaling, activation, proliferation and differentiation. Several studies have shown that BTK has been associated with PI3K, mTOR, FAS and NF-κB signaling. Furthermore, BTK can be activated by CD40 ligand binding, chemokines such as IL-6 and activation of toll like receptor (TLR) pathways suggesting a possible clinical role for BTK inhibition in HL.Methods: Classical HL cell lines (KM-H2, L1236, L-428 and HDLML) were exposed to Ibrutinib (0-25 micro M concentrations for 0-72 hrs) followed by cell death analysis using multiple cytotoxicity assays including trypan blue viability analysis, Annexin V FITC and Histone DNA ELISA apoptosis assays and molecular assays including western blotting and RT-PCR. Female ICR SCID mice were inoculated with KH-H2 by subcutaneous injection (5x106 cells). Once tumors formed ~100 mg tumor fragments were inoculated in 12 mice. Mice were administered either vehicle (N=6) or Ibrutinib (50 mg/kg i.v.) (N=6) daily for 4 weeks. Residual tumor tissue was analyzed for BTK suppression, inhibition of downstream signaling and activation of pro-apoptotic pathways.Results: Ibrutinib suppressed viability and induced apoptosis in HL cell lines in a dose and time dependent manner (IC50s ~5-7.5 µM in KM-H2, L1236 and L-428). Western blotting and RT-PCR revealed significant induction of apoptotic markers and down-regulation of pro-survival markers. Ibrutinib treatment resulted in suppression of known BTK downstream targets including PI3K, mTOR, FAS and NF-kB. Ibrutinib given at 50 mg/kg i.v. daily for three weeks caused statistically significant inhibition of KM-H2 sub-cutaneous xenograft (Figure 1). Molecular analysis of residual tumor tissue by RT-PCR revealed statistically significant down-regulation of BTK, PI3K, Akt, Rictor, 4EBP1 and related markers. Immunohistochemical analysis of residual tumors for H&E, Ki67, BTK and related downstream signaling is ongoing and will be presented in detail during the meeting.Conclusion: Our present studies are the first showing in vitro and ex vivo action of BTK inhibition in classical HL. These findings suggest in vitro activity and merit further examination. A phase II study examining the activity of ibrutinib in relapsed or refractory HL is currently enrolling (NCT02824029). [Display omitted] DisclosuresMohammad:karyopharm therapeutics: Research Funding. Azmi:Janssen: Research Funding; karyopharm therapeutics: Research Funding. Ramchandren:Janssen: Research Funding; pharmacyclics: Research Funding.

Diminished expression of 5hmc in Reed-Sternberg cells in classical Hodgkin lymphoma is a common epigenetic marker

Loss of the epigenetic marker 5-hydroxymethylcytosine (5hmC) has been demonstrated in a variety of neoplasms. Several recent studies have shown epigenetic alteration in Classical Hodgkin lymphoma (CHL), which may impact treatment. We demonstrate near universal depletion of 5hmC in the neoplastic Hodgkin Reed-Sternberg (H/RS) cells in all cases of CHL (49/49). We hypothesized that the addition of vitamin C–a cofactor for the ten-eleven translocation (TET) enzymes which oxidize 5-methylcytosine (5mC) to 5hmC - may replenish levels of 5hmC. The CHL cell line L428 was grown in optimal conditions and then subjected to vitamin C treatment, which demonstrated reduced cell viability as well as caspase activation and increased concentration of 5hmC. A more detailed understanding of the epigenetic landscape of CHL may help guide future therapies.

Beyond Exhaustion: The PDL1-PD1 Axis Shapes the Classical Hodgkin Lymphoma Microenvironment

Background: PD1 inhibitor (PD1i) efficacy is taken as evidence of T cell exhaustion in Classical Hodgkin Lymphoma (CHL), however, direct evidence for exhaustion is limited. PD1 expression does not predict PD1i response in CHL. Additionally, PDL1 and PD1 expression are not correlated and are independently prognostic. This suggests a more complex picture than PD1 expression simply reflecting exhaustion induced by PDL1. Aim: To investigate PD1/PDL1 interaction in CHL Methods: 149 CHL cases were compared to 27 reactive lymph node controls (RLN) by immunohistochemistry (IHC). PD1 expression was validated with two clones. Multiplex IHC (mIHC) was performed in 47 CHL and 27 RLN by stripping, reprobing and image alignment. Imaging Mass Cytometry (IMC) was analysed by t-SNE clustering and spatial point pattern modelling (SPPM). SPPM is a powerful technique adopted from other disciplines and its use in multiplex image analysis is novel. Flow-based CFSE proliferation and cytokine assays were performed in 15 CHL and 5 RLN single cell suspensions (SCS) with CD3/28 and PMA/Ionomycin stimulation. Co-cultures were with KMH2 and L428 cell lines and healthy donor naïve T helper cells (TH). Results: We evaluated PDL1 expression by IHC and found that PDL1hi CHL cells were surrounded by PDL1+ macrophages (Mφ), consistent with the literature. Mφ PDL1 intensity correlated inversely with distance from CHL cells consistent with induction by a CHL-secreted factor (p <0.001), a finding we validated in vitro (p <0.001). Deep phenotyping using IMC identified CD14+CD16-CD163-PDL1+-, CD14-CD68+CD163-PDL1+ and CD14+CD16+CD163+PDL1- myeloid subsets by t-SNE. SPPM revealed that tumor and vessel location predicted their distribution with subsets one and two localising to tumor whilst subset three localised to vessel. Our data supports the hypothesis that CHL recruits and induces a PDL1+ myeloid environment. We next sought evidence of T cell exhaustion. PD1 expression by IHC was lower in CHL than RLN (p<0.001). Cells co-expressing PD1 with TIM3 or LAG3 were significantly reduced in CHL compared to RLN by mIHC. Cells co-expressing PD1 with TBET or EOMES were reduced in or showed no significant difference between CHL and RLN. No correlation was seen between PD1 and PDL1 expression. We performed functional validation, evaluating proliferative and IL2/IFNγ production capacity, and identified no significant difference between CHL and RLN SCS with or without the addition of Pembrolizumab or isotype control. This constitutes a more detailed assessment of exhaustion with higher case numbers than publications to date. Our data does not support the presence of an exhausted T population above that seen in reactive controls and finds no relationship between PD1 and PDL1 expression. The absence of exhaustion does not exclude a role for the PD1-PDL1 axis, which regulates multiple facets of T cell homeostasis including motility, differentiation (skewing towards T regulatory cells (TReg)), and TReg effector functions. Many of these are characterised by transient PD1 expression unlike the sustained expression seen in exhaustion. In contrast to PD1, CHL PDL1 and MHC class 2 expression do predict PD1i response. CHL MHC2 expression suggests antigen presentation and a role for TH cells. This led us to examine connections between PDL1 and the TH compartment. Co-culture of CHL cell lines with naïve TH led to enrichment of CD25hiCD127loFOXP3+ TReg which upregulated PD1 compared to TReg from paired CD3/28 controls (p<0.001). FOXP3+ cells were enriched by IHC compared to RLN (p<0.001). Unlike PD1, FOXP3 associated with CHL MHC2 expression (p=0.012), which in turn associated with PDL1 expression (p=0.02). SPPM of TC and TH subsets revealed that PDL1 expression predicted the location of FOXP3+ TReg and negatively predicted the location of TC and TH17. PDL1 was a stronger predictor than CHL cell density. These data support a role for PDL1 in shaping T differentiation and trafficking irrespective of observed PD1 expression. Conclusion: We find that CHL cells are PDL1hi and induce a PDL1+ myeloid environment, but detect little evidence of T exhaustion. Instead we find that PDL1 expression shapes T (particularly TReg) differentiation and trafficking. Our data challenges the narrative that PD1i in CHL act by reversing exhaustion and highlights novel mechanisms with the potential to provide insight into CHL biology and inform the hunt for biomarkers of response. Figure Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

OP-38 - Initial presentation of prostate cancer may hint different clinical characteristics along with serving predictive biomarker in castration-resistant prostate cancer patients treated with abiraterone acetate

Loss of the epigenetic marker 5-hydroxymethylcytosine (5hmC) has been demonstrated in a variety of neoplasms. Several recent studies have shown epigenetic alteration in Classical Hodgkin lymphoma (CHL), which may impact treatment. We demonstrate near universal depletion of 5hmC in the neoplastic Hodgkin Reed-Sternberg (H/RS) cells in all cases of CHL (49/49). We hypothesized that the addition of vitamin C–a cofactor for the ten-eleven translocation (TET) enzymes which oxidize 5-methylcytosine (5mC) to 5hmC - may replenish levels of 5hmC. The CHL cell line L428 was grown in optimal conditions and then subjected to vitamin C treatment, which demonstrated reduced cell viability as well as caspase activation and increased concentration of 5hmC. A more detailed understanding of the epigenetic landscape of CHL may help guide future therapies.

The Treg/Th17 Axis Is Skewed in Classical Hodgkin Lymphoma By PDL1+Ve but Not PDL1-Ve Lymphoma Cells and By Lymphoma MHC Class 2 Expression.

Introduction: Classical Hodgkin Lymphoma (CHL) is responsive to Programmed Death 1 inhibition (PD1i). Recognition that 9p24 amplifications promote PDL1 upregulation in lymphoma cells provides insight into CHL's particular sensitivity to PD1 inhibitors, but their mechanism of action remains unclear. Although it is widely believed that PD1i act by reversing CD8 T cell exhaustion, neither CD8 infiltration, PD1 expression nor lymphoma MHC class 1 (MHC1) expression predict PD1i response in CHL. Additionally, while most CHL cells adopt CD8 evasion mechanisms such as MHC1 loss, MHC2 expression is frequently retained suggesting a vulnerability to CD4-mediated cytotoxicity. CD4 cells dominate the CHL microenvironment and recent data demonstrates that MHC2 alongside PDL1 expression on CHL cells predict PD1i response suggesting an important role for CD4 cells in PD1i action.Aim: To evaluate the impact of the PDL1 axis and MHC2 expression on the CD4 compartment in CHL and their implications for PD1i activity.Methods: We evaluated 146 CHL patients by immunohistochemistry (IHC), with comparison to 24 reactive lymph node (RLN) controls. We designed a novel spatial analysis method utilising intra-tumor cell heterogeneity to test the influence of lymphoma PDL1 expression upon the T cell infiltrate. By combining IHC dual staining with stripping and reprobing we stained sections for CD30/PDL1/CD3 and serial sections on either side for GATA3/CD8, FoxP3/CD8, RORgT/CD8 and Tbet/CD8. Images were virtually overlaid and spatial analysis was performed using Visiopharm software. Distances from T cell subtypes were measured to either CD30+PDL1- CHL cells, CD30+PDL1+ CHL cells or CD30-PDL1+ microenvironmental cells. Skewing was tested statistically by comparison to parent cell distribution (e.g. CD8+ to CD3+). Spatial analysis was performed in 10 blindly selected cases.Results/Discussion: Despite high PDL1 expression our data showed limited evidence for an exhausted PD1+CD8 population; CD8 tumor-infiltrating lymphocytes (TIL) were frequent, but PD1+TIL were rare (median 33% vs 0.08% nucleated cells respectively). No correlation was seen between PD1+TIL and CD8+TIL or PDL1. Stripping and reprobing of PD1+ IHC sections demonstrated that PD1+ cells were CD8 negative. This led us to consider alternative mechanisms of action for PD1i.Since PD1i has a profound impact on naïve CD4 differentiation and influences the type of effector generated, we next quantified CD4 effector subtypes. We found that Tbet+TIL (Th1, antitumor) and FoxP3+TIL (Treg, protumor) were enriched in CHL, whereas RORgT+TIL (Th17, antitumor) were suppressed compared to RLN (p=<0.0001, 0.0004 and <0.0001 respectively). No significant difference was seen for GATA3+TIL (Th2, protumor). We further evaluated Treg and Th17 due to their shared developmental pathway which is known to be influenced by PDL1. FoxP3:Th17 ratios were heavily skewed towards Treg in CHL compared to RLN (p=<0.0001). Spatial analysis demonstrated enrichment of FoxP3+TIL and a paucity of RORgT+TIL in proximity to CD30+ lymphoma cells and with greater enrichment around PDL1+ lymphoma cells than PDL1- lymphoma cells. This supports a hypothesis of skewing of the Treg/Th17 axis by the lymphoma cell by the PD1-PDL1 axis. Finally, we evaluated the relationship between effector subtypes and CHL MHC2 expression across our cohort and found a positive association between FoxP3+TIL numbers and lymphoma cell MHC2 expression (p=0.0014).Conclusion: The skewing of Th17/Treg via the PD1-PDL1 axis and positive association between Treg numbers and lymphoma MHC2 expression are consistent with CHL cells promoting Treg differentiation. This is supported in the literature by reports of MHC2-dependent induction of Treg by CHL cell lines. Paradoxically, Treg are associated with improved response to therapy despite evidence that they are induced by CHL cells. It is therefore hypothesised that Treg directly suppress lymphoma cell expansion. However, these data support our alternative hypothesis that PDL1 and MHC2 expression contribute to CHL cells establishing a Treg-rich and Th17-poor immunosuppressive microenvironment. Intervention with PD1i therapy or chemotherapy causing immunogenic cell death then shifts the infiltrate to a less suppressive environment leaving MHC2+ cells vulnerable to CD4-mediated cytotoxicity. We are currently modelling this in vitro. [Display omitted] DisclosuresGribben:Abbvie: Honoraria; Medical Research Council: Research Funding; Novartis: Honoraria; Cancer Research UK: Research Funding; NIH: Research Funding; Acerta Pharma: Honoraria, Research Funding; Pharmacyclics: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Honoraria; TG Therapeutics: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Wellcome Trust: Research Funding; Kite: Honoraria.

PO-379 Novel tumour specific miRNA expressed in classical hodgkin lymphoma cell lines

IntroductionMiRNAs are short non coding RNAs involved in post-transcriptional regulation of gene expression that can acquire oncogenic properties in consequence of overexpression or mutations. Tumour genomes undergo global demethylation that triggers genetic instability and activates transcription of sequences normally silenced by methylation. These include the overexpression of tumour specific miRNAs that can deregulate various processes in the malignant cell. Therefore, the aim of the study was to identify aberrantly activated miRNAs in classical Hodgkin lymphoma (cHL).Material and methodsWe have screened NGS small RNA reads (HiSeq 4000 (Illumina); single-end 50 bp seq) of cHL cell lines (n=7) and compared them to non-HL cell lines (n=10) and germinal centre B cell pools (GCB)(n=10) to find yet undescribed miRNAs. High quality reads validated using FastQC and CAP-miRSeq software mapping to the reference genome (GRCh37) (using Bowtie software) not reported as miRNAs before were identified. In order to find miRNAs expressed exclusively in cHL we have moreover compared the sequences in respect to sequence-derived gene expression data from 143 malignant lymphomas generated by the ICGC MMML-Seq consortium (www.icgc.org). Last, the expression of novel miRNAs seemingly specific for cHL was validated by real-time qPCR using custom designed molecular probes (Thermo Fisher Scientific).Results and discussionsSeven novel miRNAs expressed in at least 3/7 cHL cell lines but neither in the non-HL cell lines nor in the GCB cells and observed with a frequency <1% in the ICGC MMML-Seq cohort were identified. As a proof of principle we intended to confirm the expression of miRNA #1 and #7 using real-time qPCR. In concordance with the NGS data expression of miRNA #7 (chr2:213543512–213543573) was observed in 4/7 cHL cell lines exclusively. In silico analyses of potential target mRNAs has shown enrichment in gene ontologies including regulation of signalling (p<0.001) (GO:0023051) or regulation of actin cytoskeleton organisation (p<0.05) (GO:0032956) - processes deregulated in human neoplasms. The expression of miRNA #1 was not confirmed and the validation of the further 5 novel miRNAs is ongoing.ConclusionOur data provide evidence for aberrant activation of miRNAs in neoplastic cells probably not expressed under physiologic conditions in the same cell type. The identified miRNA #7 and potentially the currently tested 5 further candidates contribute to the unique cHL miRNAome.

Recurrent somatic loss of TNFRSF14 in classical Hodgkin lymphoma.

Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed-Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole-genome sequencing-mediated copy number analysis of purified HRS cells. We performed low-coverage sequencing of small numbers of intact HRS cells and paired non-neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS-derived cell lines (KM-H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD-1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236-4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases.

Synthesis, characterization and anticancer activity of gold(III) complexes with (1R,2R)-(−)-1,2-diaminocyclohexane

Two gold(III) complexes, [(DACH)AuCl2]Cl (1) and [(DACH)2Au]Cl3 (2), derived from (1R,2R)-(−)-1,2-diaminocyclohexane and sodium tetrachloroaurate dihydrate, NaAuCl4·2H2O, were synthesized and fully characterized using elemental analysis and different physical methods, such as FTIR, 1H NMR, 13C NMR, solid-state NMR and X-ray crystallography. The data confirm the coordination of the DACH ligand to the gold(III) metal centre through its nitrogen atoms. According to in vitro cytotoxic studies, complex 2 was more effective against the three human tumor cell lines evaluated (the ovarian cancer cell line A2780, its cisplatin-resistant clone A2780cis and the classical Hodgkin lymphoma cell line L-540). The fold-resistance of the A2780 and A2780cis cells to cisplatin was about twofold lower for both gold(III) complexes.

Potential role of hypoxia in early stages of Hodgkin lymphoma pathogenesis.

A unique feature of the germinal center B cell-derived Hodgkin and Reed/Sternberg cells of classical Hodgkin lymphoma is their lost B cell phenotype and the aberrant expression of factors of other hematopoietic cell types, including ID2 and NOTCH1. As cellular dedifferentiation and upregulation of ID2 and NOTCH1 are typical consequences of a hypoxic response, we wondered whether hypoxia may impose an HRS cell-like phenotype in B cells. Culturing normal B cells or cell lines of germinal center-type diffuse large B-cell lymphoma under hypoxic conditions caused partial downregulation of several B cell markers, ID2 upregulation, and increased NOTCH1 activity. The hypoxic cells acquired further features of Hodgkin and Reed/Sternberg cells, including increased JUN expression, and enhanced NFκB activity. The Hodgkin and Reed/Sternberg cell-expressed epigenetic regulators KDM4C and PCGF2, as well as the phosphatase DUSP1 were partially induced in hypoxic B cells. Inhibition of DUSP1 was toxic for classical Hodgkin lymphoma cell lines. Thus, hypoxia induces key Hodgkin and Reed/Sternberg cell characteristics in mature B cells. We speculate that hypoxic conditions in the germinal center may impose phenotypic changes in germinal center B cells, promoting their survival and initiating their differentiation towards a Hodgkin and Reed/Sternberg cell-like phenotype. These may then be stabilized by transforming events in the Hodgkin and Reed/Sternberg precursor cells.

Brefeldin A exerts differential effects on anaplastic lymphoma kinase positive anaplastic large cell lymphoma and classical Hodgkin lymphoma cell lines.

To obtain further insights into the biological differences of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) and classical Hodgkin lymphoma (HL), we screened microbial culture filtrates to search for compounds that would exert a significantly greater effect on the viability of ALK+ ALCL cell lines compared to HL cell lines and identified Brefeldin A (BFA) as a suitable candidate. BFA inhibited phosphorylation of ALK and its downstream molecule, signal transducer and activator of transcription 3 (STAT3), one of the central pathways for the survival of ALK+ ALCL cells. In HL cell lines BFA did not affect CD30 expression orconstitutive nuclear factor (NF)-κB activity, both of which are critical forHL cell survival. BFA induced disruption of the Golgi apparatus in ALK+ ALCL cell lines, which was accompanied by a decrease in active ADP-ribosylation factor 1 (ARF1), whereas BFA had no significant effect on these parameters in HL cell lines. These results add extra insights into the biological distinction between ALK+ ALCL and HL cells and highlight the Golgi apparatus as a target for the treatment of ALK+ ALCL.

Exome Sequencing Of Purified Hodgkin Reed-Sternberg Cells Reveals Recurrent Somatic Mutations In Genes Responsible For Antigen Presentation, Chromosome Integrity, Transcriptional Regulation and Protein Ubiquitination

IntroductionMost genomic studies of classical Hodgkin lymphoma (CHL) have been confined to cell lines due to the difficulty of isolating sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. One approach to evaluate primary cases has been to microdissect HRS cells from fresh-frozen tissue biopsies, which has been used for gene expression profiling and array comparative genomic hybridization to assess copy number alterations (Hartmann et al., Haematologica 93, 2006; Brune et al, J Exp Med 205, 2008; Steidl et al., Blood 120, 2012; Steidl et al., Blood 116, 2010; Tiacci et al., Blood 120, 2012). However, laser capture microdissection is technically challenging, does not provide a pure tumor cell population, and yields very small amounts of nucleic acids that may not be adequate for deep sequencing. Typically, generating whole-exome sequence data from fewer than 104 cells, including single cells, requires whole genome amplification (WGA), and is of a quality suitable to detect large scale copy-number alterations, but not nucleotide level information to identify point mutations. MethodsWe used a flow cytometric cell isolation method, which has enabled rapid isolation of thousands of viable HRS cells from primary CHL tumors (Fromm, et al., Am J Clin Pathol 126, 2006). Here we developed a new ultra-low-input DNA exome sequencing protocol which we combined with flow cytometry using CD64, CD95, CD30, CD5, CD20, CD15, CD40, and CD45, to produce what is to our knowledge the first full exome deep sequencing study of primary cases of Hodgkin lymphoma. We obtained a sequence depth of over 10X with >90% coverage of the target exome in HRS, as well as tumor-infiltrating T cells (also sorted and used as somatic control), in ten primary cases of CHL and performed mutation, copy number variation, and loss-of-heterozygosity (LOH) analysis. ResultsWe have identified 61 recurrent mutations, and 12 genes had somatic mutations in over 30% of cases. Non-synonymous or splice site mutations were seen in genes involved in antigen presentation (B2M), chromosome integrity (BCL7A), NF-kB activation (A20/TNFAIP3) and protein ubiquitination (HECW2 and UBE2A). We also obtained high-resolution copy number variation data indicating specific regions of gains and losses and intragenic chromosomal breakpoints. The most common genetic alterations from the combined analyses were in A20, present either as mutations or LOH, and alterations in b2-microglobulin (B2M), which were found in 80% of the cases sequenced. Alterations of B2M were inactivating and bi-allelic, leading to a lack of expression of MHC class I protein complex on the cell surface. The alterations include start codon mutations, exon-one splice donor site mutations, out of frame first-exon deletions and gene loss through chromosome-level deletion (LOH). Where possible, mutations were confirmed at the RNA level and resulted in lack of B2M protein expression documented by immunohistochemistry in an expanded cohort, where a total of 20 of 27 cases (74%) lacked B2M expression. Ectopic expression of B2M in a CHL cell line induced MHC class 1 expression, indicating that this genetic alteration is singly responsible for this defect in antigen presentation. In addition, B2M inactivation was highly prevalent in nodular sclerosis CHL, but was not found in any of the five cases of mixed cellularity CHL examined, indicating that these two types of CHL may belong to two different genetic categories. ConclusionsWe report the first exome deep sequencing of purified HRS cells from CHL tumor specimens, and reveal consistent alterations in important biological processes. The methodology developed allows exome sequencing from very low DNA input (10 ng), which has broader applications such as using FNA specimens from multiple tumor types. The genomic landscape of CHL revealed commonalities and differences with other lymphoma subtypes, like the consistent presence of A20 alterations that can lead to NF-kB activation. Inactivating mutations in B2M explain the complete lack of MHC class I expression, which likely affect the microenvironment. Since B2M expression is normal in all the cases evaluated that were histologically classified as mixed cellularity CHL, it is possible that this genetic alteration and resulting lack of B2M protein expression is a more accurate biomarker of CHL subtype than histological distinction. Disclosures:No relevant conflicts of interest to declare.

Sphingosine-1-phosphate receptor 1 in classical Hodgkin lymphoma: assessment of expression and role in cell migration

Classical Hodgkin lymphoma (CHL), a neoplasm of abnormal B lymphocytes (Hodgkin–Reed–Sternberg (HRS) cells), has been described to have a typical pattern of clinical presentation and dissemination often involving functionally contiguous lymph nodes. Despite the progress made in understanding CHL pathophysiology, the factors that regulate the spread of lymphoma cells in CHL are poorly understood. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid present at high concentrations in the plasma and lymphatic fluid, is known to have a critical role in regulating lymphocyte trafficking mainly through sphingosine-1-phosphate receptor 1 (S1PR1). In this study, we explore the role of the S1P–S1PR1 axis in Hodgkin lymphoma cell migration and the expression of S1PR1 in CHL cell lines and clinical cases. We found that S1PR1 is present in the KM-H2 and SUP-HD1 Hodgkin lymphoma cell lines at the mRNA and protein level. In addition, functionally, S1P potently stimulated migration of both cell lines. S1P-induced migration was inhibited by the S1PR1 antagonist, VPC44116, and the S1PR1 functional antagonist, FTY720-P, but was potentiated by the S1PR2-specific antagonist, JTE013. We also determined that S1PR1 induced migration in the KM-H2 and SUP-HD1 cells via the heterotrimeric G-protein Gi and the phosphatidylinositol-3-kinase pathway. Immunohistochemical assessment of the tissue from CHL samples revealed that a subset of cases (7/57; 12%) show strong, membranous staining for S1PR1 in HRS cells. Altogether, our data indicate that S1PR1 is a functional receptor on HRS cells, which governs tumor cell migration and is expressed in a subset of CHL cases. Given the availability of S1PR1 antagonists, some of which are used clinically for modulation of the immune system, these results suggest that S1PR1 could be a future therapeutic target in the treatment of those cases of S1PR1-positive, refractory/recurrent CHL.

Aptamer‐Drug Conjugates for Targeted Therapy of CD30‐expressing Lymphomas

CD30 is a biomarker for diagnosis and targeted therapy of Anaplastic Large cell lymphoma (ALCL) and Classical Hodgkin Lymphoma (CHL). Our previous studies have demonstrated specific binding of RNA aptamer to CD30 positive tumor cells. However, for in vivo targeted therapy there is a concern about biostability of the RNA aptamers.To overcome this, an ssDNA aptamer was developed via a hybrid SELEX approach using CD30‐expresing lymphoma cells and purified CD30 protein to yield the aptamer C2NP which specifically bound to CD30 expressing ALCL and CHL cells at concentrations ≤10 nm and did not react to control tumor cells. Importantly, C2NP is highly stable and more than 50% of them remained after incubation in human serum at 37°C for 24 hours, indicating the suitability for in vivo use.For targeted therapy study, C2NP was conjugated with a chemotherapeutic agent and a fluorescent reporter to form an aptamer‐drug conjugate (ADC). The specificity of ADC was confirmed by flow cytometry and intracellular delivery of chemo‐drug was observed by microscopy. Chemical conjugation of drug had no effect on aptamer binding specificity and affinity to lymphoma cells. In addition, potential therapeutic effects of the aptamer‐drug conjugate are under investigation by using cultured ALCL and CHL cell‐lines currently. This is the first study to use an aptamer‐drug conjugate for targeted therapy of CD30‐exprssing lymphomas.

PU.1 is a tumor suppressor for B cell malignancies

PU.1 is a tumor suppressor for B cell malignancies

Detection of ABCC1 expression in classical Hodgkin lymphoma is associated with increased risk of treatment failure using standard chemotherapy protocols

BackgroundThe mechanisms responsible for chemoresistance in patients with refractory classical Hodgkin lymphoma (CHL) are unknown. ATP-binding cassette (ABC) transporters confer multidrug resistance in various cancers and ABCC1 overexpression has been shown to contribute to drug resistance in the CHL cell line, KMH2.FindingsWe analyzed for expression of five ABC transporters ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2 using immunohistochemistry in 103 pre-treatment tumor specimens obtained from patients with CHL. All patients received first-line standard chemotherapy with doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens. ABCC1 was expressed in Hodgkin and Reed-Sternberg (HRS) cells in 16 of 82 cases (19.5%) and ABCG2 was expressed by HRS cells in 25 of 77 cases (32.5%). All tumors were negative for ABCB1, ABCC2 and ABCC3. ABCC1 expression was associated with refractory disease (p = 0.01) and was marginally associated with poorer failure-free survival (p = 0.06). Multivariate analysis after adjusting for hemoglobin and albumin levels and age showed that patients with CHL with HRS cells positive for ABCC1 had a higher risk of not responding to treatment (HR = 2.84, 95%, CI: 1.12-7.19 p = 0.028).ConclusionsExpression of ABCC1 by HRS cells in CHL patients predicts a higher risk of treatment failure and is marginally associated with poorer failure-free survival using standard frontline chemotherapy regimens.

N-terminal PAX8 polyclonal antibody shows cross-reactivity with N-terminal region of PAX5 and is responsible for reports of PAX8 positivity in malignant lymphomas

Recently, reports using immunohistochemistry and a polyclonal antibody directed against the N-terminal region of PAX8 describe PAX8 expression in malignant lymphomas. As the N-terminal regions of PAX family members, including the B-cell transcription factor PAX5, have high sequence homology, we investigated PAX8 positivity in malignant lymphomas. Comparative sequence analysis between the N- and C-terminal regions of PAX8 and PAX5 proteins confirmed homologies of 70% and 39%, respectively. We then compared the results using N-terminal (high homology) and C-terminal (lower homology) anti-PAX8 antibodies to assess PAX8 expression in reactive tissues, diffuse large B-cell lymphoma and classical Hodgkin lymphoma, using routine immunohistochemical methods. Expression of PAX8 was also assessed in diffuse large B-cell lymphoma and classical Hodgkin lymphoma cell lines using real-time qRT-PCR methods. Our results show that reactive and neoplastic B-cells are positive for PAX8 using the N-terminal antibody, but negative for PAX8 when the C-terminal antibody was used. PAX8 mRNA levels were not detected in any of the B-cell lymphoma cell lines studied. These results indicate that benign and malignant B-cells do not express PAX8. We conclude that positivity for PAX8 reported by others in B-cell lymphomas is likely due to cross-reactivity between the N-terminal regions of PAX8 and PAX5, due to the high sequence homology of these two regions.

PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Abstract 2628PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth.In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo.We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.