Novak, Anne J.

Background:With an expanding array of novel treatment options in follicular lymphoma (FL), we largely lack effective biomarkers to predict response in this molecularly heterogeneous disease. Activating mutations focused on components of the nutrient‐sensing arm of the mTORC1 pathway (RRAGC, ATP6V1B2, ATP6AP1), are particularly enriched in FL, occurring in ∼30% of cases. Meanwhile, clinical mTOR inhibitors (mTORi) have shown some promise, with response rates of 50–60% in relapsed/refractory FL.Aims:We therefore sought to determine whether gene mutation status ‐ particularly in genes regulating the mTOR pathway – may represent biomarkers of mTORi‐response in FL.Methods:We retrospectively analyzed pre‐treatment tumor samples from relapsed/refractory FL patients treated on two phase II clinical trials with single agent mTOR inhibitors, everolimus (NCT00436618) or temsirolimus (NCT00290472), with well characterized clinical outcomes. In total, 21 patients had available biopsies, consisting of 15 everolimus‐ and 6 temsirolimus‐treated cases.Tumor DNA extracted from formalin fixed paraffin embedded (FFPE) tissue was subjected to targeted sequencing using a 22 gene panel comprising genes recurrently mutated in FL, and PI3K/AKT/mTOR pathway genes mutated in germinal center lymphomas. Libraries were prepared using capture‐based target enrichment (Nonacus), and sequenced on the NextSeq550 (Illumina) with a mean coverage depth of x700. A stringent bioinformatic pipeline was adopted with variants called with VarScan2, and a 10% variant allele frequency (VAF) threshold was employed to filter out sequencing artefacts due to the FFPE material. 100% (15/15) of a selection of variants were validated by Sanger sequencing. Staining for pS6 was undertaken on tissue slides to examine downstream mTOR activation by immunohistochemistry.Results:Overall response rate (CR and PR) was 48% (10/21). Mutational analysis showed no significant association between clinical response and mTORC1‐pathway mutations (mut) taken individually or collectively; although, absolute numbers of mutations in these genes were low, including RRAGC (responder: 0/10 mut vs non‐responder: 1/11 mut), ATP6AP1 (responder: 1/10 vs non‐responder: 1/11), and ATP6V1B2 (responder: 1/10 vs non‐responder 3/11). Notably, no difference in pS6 staining was seen between responders and non‐responders.However, we found mutations in the epigenetic regulator CREBBP to be enriched in the responder group (100% (10/10) responders vs 54.5% (6/11) non‐responders, p = 0.04). This effect was more pronounced when considering only mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP, with HAT mutations seen in 100% (10/10) responders, and 27.3% (3/11) non‐responders (p = 0.001).Summary/Conclusion:Mutations in the mTOR pathway did not predict for response to mTOR inhibition in our study. The enrichment of CREBBP mutations in responders, and particularly those affecting the catalytic HAT domain, points to CREBBP as a potential biomarker of response which warrants validation in trials of drugs modulating the PI3K/AKT/mTOR axis. More broadly our data may lend support to a distinct clinical and biological behavior of CREBBP HAT domain‐mutant disease that also warrants further evaluation. Taken together, our findings highlight the potential opportunities afforded by undertaking correlative studies, and underlines the need to incorporate prospective biomarker discovery into clinical trials in order to help focus future therapeutic strategies more rationally.

Background:Recent large next generation sequencing studies have redefined the genomic landscape of diffuse large B‐cell lymphoma (DLBCL). While many of the more common genetic variants, primarily mutations, have been biologically characterized, little is known about newly emerging genetic aberrations. In particular, somatic copy number alterations remain poorly studied due to the large genomic regions they cover and the number of genes involved. This, in combination with lack of remission or early relapse in a portion of DLBCL, opens new opportunities to explore yet unidentified genetic contributions to lymphomagenesis.Aims:The goal of this study was to identify variants associated with failure to achieve event free survival at 24 months (EFS24) and define their biologic significance in DLBCL.Methods:Newly diagnosed DLBCL treated with standard immunochemotherapy were included in this study (n = 246). Copy number data was generated by using the molecular inversion probe OncoScan™ FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA). OncoScan OSCHP files were analyzed using Nexus Copy Number 9.0 software (Biodiscovery, El Segundo, USA). Data interpretation and copy number calling was done using the human reference genome GRCh37/hg19. Files were analyzed using the Nexus FASST2‐Segmentation algorithm, which is based on a Hidden Markov Model approach for calling genetic events. For functional analysis, SOCS6 was overexpressed and studied in GCB‐DLBCL cell lines.Results:Upon review of genomic copy number data from DLBCL cases (n = 246), loss of 18q22.1‐q23 was a significant event in our cohort (p = 0.018), is found in 11% (n = 27) of DLBCL, and is enriched in germinal center DLBCL (GCB‐DLBCL) patients (18/107, 17%, p < 0.05) compared to activated B‐cell (ABC)/non‐GCB DLBCL patients (1/93, 1%). Furthermore, GCB‐DLBCL patients with a loss of 18q22.1‐q23 were significantly more likely to fail EFS24 (50%, p < 0.05) compared to GCB‐DLBCL patients without the loss (29%). Genes located at 18q22.1‐q23 include, TMX3, DOK6 and SOCS6. SOCS family proteins have been shown to play an important role in tumor biology, although little is known about SOCS6. SOCS6 gene expression is downregulated in a variety of solid tumors and is hypermethylated in some hematological malignancies. RNASeq data from DLBCL suggest variable expression across tumors. Using a panel of GCB cell lines we found that SOCS6 expression was low to negative, and publically available cell line data suggests this region is hypermethylated. Expression of SOCS6 was higher in a panel of mantle cell lymphoma and ABC‐DLBCL cell lines and this again correlated with their cell line methylation data, where no hypermethylation was found in those cell lines. Together, this data suggests that downregulation of SOCS6 through genomic loss or hypermethylation may have biologic importance. To explore the role of SOCS6, we restored expression in GCB cell lines and immuno‐fluorescence staining suggest localization to the nucleus and cytoplasm, as previously reported in other cell types. Studies to explore the role of SOCS6 in cytokine signaling and STAT family protein regulation are ongoing.Summary/Conclusion:Using genome copy number data from 246 DLBCL, we have found that loss of 18q22.1‐q23 (p = 0.018) is associated with GCB‐DLBCL and failure to achieve EFS24. Furthermore, we have identified that SOCS6 may have clinical and biologic significance in GCB‐DLBCL.

Abstract Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

AbstractIndolent B‐cell lymphomas other than follicular lymphoma account for up to 10% of all B‐cell neoplasms. While they typically follow a slowly progressive course, some patients experience rapid progression and early mortality. Prognostic scoring systems have not been adopted, hindering the ability of clinicians or researchers to predict outcomes, or risk‐stratify patients during clinical trials. To address this, we evaluated the utility of existing prognostic indices and novel, early disease‐related outcomes, to predict subsequent long term survival. Baseline characteristics and outcomes data were generated from a longitudinal cohort study that prospectively enrolled 632 patients newly diagnosed with marginal zone lymphoma, lymphoplasmacytic lymphomas, or B‐cell lymphomas not otherwise specified, beginning in 2002. The International Prognostic Index (IPI), Follicular Lymphoma International Prognostic Index (FLIPI), and MALT International prognostic index (MALT‐IPI) demonstrated c‐statistics that ranged from 0.593‐0.612 for event‐free survival (EFS), and 0.683‐0.714 for overall survival (OS). Patients who attained event‐free survival at 12 months (EFS12) experienced similar mortality to the US general population (standardized mortality ratio [SMR] 1.19; 95% CI 0.95‐1.46). Patients who did not attain EFS12 had subsequent worse morality (SMR 3.14 (95% CI 2.05‐4.59). The MALT‐IPI demonstrated utility in predicting subsequent long‐term outcomes among patients with non‐follicular indolent B‐cell lymphomas. This index should be used by clinicians giving guidance to patients at the time of initial diagnosis, and risk stratification during clinical studies. The divergent long‐term outcomes experienced by patients who do or do not attain EFS12 suggest there exists a subset of patients who harbor high‐risk disease. Future research efforts should focus on methods to identify these patients at the time of diagnosis, in order to enable risk‐tailored therapy.

Key Points Cytokines in the BM microenvironment regulate PD-1 ligand expression and secretion in WM. Secreted PD-1 ligands modulate T-cell function in WM.

SummaryWe evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co‐infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency‐matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT‐PCR), and those with RNA concentrations <5000 genome equivalents/ml were confirmed using nested RT‐PCR methods. To assess the role of HPgV in lymphoma prognosis, we used 2948 cases from a cohort study of newly diagnosed lymphoma patients (included all cases from the case‐control study). There was a positive association of HPgV viraemia with risk of lymphoma overall (Odds ratio = 2·14; 95% confidence interval [CI] 1·63–2·80; P < 0·0001), and for all major subtypes except Hodgkin lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma, and this was not confounded by other lymphoma risk factors. In contrast, there was no association of HPgV viraemia with event‐free survival (Hazard ratio [HR] = 1·00; 95% CI 0·85–1·18) or overall survival (HR = 0·97; 95% CI 0·79–1·20) for lymphoma overall, or any of the subtypes. These data support the hypothesis for a role of HPgV in the aetiology of multiple lymphoma subtypes.

Objective Determinants of the increased risk of diffuse large B-cell lymphoma (DLBCL) in SLE are unclear. Using data from a recent lymphoma genome-wide association study (GWAS), we assessed whether certain lupus-related single nucleotide polymorphisms (SNPs) were also associated with DLBCL. Methods GWAS data on European Caucasians from the International Lymphoma Epidemiology Consortium (InterLymph) provided a total of 3857 DLBCL cases and 7666 general-population controls. Data were pooled in a random-effects meta-analysis. Results Among the 28 SLE-related SNPs investigated, the two most convincingly associated with risk of DLBCL included the CD40 SLE risk allele rs4810485 on chromosome 20q13 (OR per risk allele=1.09, 95% CI 1.02 to 1.16, p=0.0134), and the HLA SLE risk allele rs1270942 on chromosome 6p21.33 (OR per risk allele=1.17, 95% CI 1.01 to 1.36, p=0.0362). Of additional possible interest were rs2205960 and rs12537284. The rs2205960 SNP, related to a cytokine of the tumour necrosis factor superfamily TNFSF4, was associated with an OR per risk allele of 1.07, 95% CI 1.00 to 1.16, p=0.0549. The OR for the rs12537284 (chromosome 7q32, IRF5 gene) risk allele was 1.08, 95% CI 0.99 to 1.18, p=0.0765. Conclusions These data suggest several plausible genetic links between DLBCL and SLE.