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Abstract Abstract 1691 Poster Board I-717 Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein and a lymphoplasmacytic infiltrate in the bone marrow. The aberrant production of the monoclonal IgM can result in serum hyperviscosity that can cause significant morbidity in patients with this disease. In previous work, we have shown that IL-6 significantly upregulates IgM secretion by WM cells and that IL-6 secretion is regulated by CCL5 (Rantes). We have also shown that IL-6 mediated IgM secretion in WM requires phosphorylation of Stat1 and Stat3. Because IL-6 induced signaling involves the Jak/Stat pathway, we tested whether the use of a Jak/Stat inhibitor, TG101348, would result in down regulation of CCL5, IL-6 and IgM production and inhibit cell proliferation and viability in WM. First, we determined whether TG101348 could inhibit the production of CCL5 because other Jak inhibitors have been shown to inhibit cytokine production. Using the BCWM.1 cell line as well CD19+ malignant cells from bone marrow specimens from WM patients, we measured CCL5 by ELISA in the culture supernatant 24 hours after treatment with increasing concentrations of the inhibitor. We found that CCL5 secretion was decreased by 50% at a concentration of TG101348 of 250nM and was completely inhibited at 2μM. Next, we measured IL-6 production after treatment with TG101348. We had previously shown that stromal cells are the primary source of IL-6 and therefore used the stromal cell line HS-5 to measure IL-6 by ELISA after treatment with the inhibitor. Our previous work had also shown that IL-6 secretion was mediated by GLI (a member of the Hedgehog pathway) rather than the Jak/Stat pathway. Interestingly, we found that IL-6 secretion was inhibited in a dose dependent fashion but required higher doses for complete suppression (8μM). We then measured IgM production by malignant B-cells 24 hours after treatment with TG101348. Our previous work had shown that IL-6 mediated IgM secretion was dependent on the Jak/Stat pathway. We found that IgM production was inhibited by 50% at 500nM and completely suppressed at 2μM. Finally, we measured the effect of TG101348 on cell proliferation and survival. Using the BCWM.1 cell line, we found that cell proliferation as determined by tritiated thymidine uptake was inhibited in a dose dependent fashion with 50% inhibition at 1μM. Inhibition of cell viability as measured by Annexin V/propidium iodide staining, however, required higher concentrations and cell viability was inhibited with an IC50 of 8μM. These data confirm the role of Jak/Stat signaling in the CCL5-IL-6-IgM axis in WM. We found that TG101348 generally suppressed the signaling and growth of WM cells but that pathways that were known to be Jak/Stat dependent required significantly lower doses to be completely inhibited. These data provide a strong rationale for the use of inhibitors of this pathway, such as TG101348, in the treatment of patients with WM. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 440 Background: Complement plays a central role in innate immunity, and also functions as a regulator of the overall immune response, including T-cell responses. We recently reported that germline genetic variation in C2, C5, C7 and C9 was associated with risk of developing non-Hodgkin lymphoma (Br J Haematol 2009;145:614). Here we evaluate whether complement genes are associated with event-free survival (EFS) in follicular lymphoma. Methods: We genotyped 166 single nucleotide polymorphisms (SNPs) from 32 complement pathway genes in a prospective cohort study of 115 newly diagnosed follicular lymphoma patients enrolled at the Mayo Clinic from 2002-2005 as part of the Lymphoma SPORE. All patients were systematically followed through 2008 for EFS (defined as disease progression, retreatment, or death due to any cause). Tagging and nsSNPs were selected from HapMap for the genes encoding complement proteins and their key regulators (C1QB, C1QG, C1QR1, C1QTNF7, C1RL, C1S, C2, C3, C3AR1, C4BPA, C4BPB, C5, C5R1, C6, C7, C8B, C9, BF, CFH, CFHL5, CFHR1, CGHR4, CLU, CR1 (CD35), CR2 (CD21), DF, MASP2, MBL2, MCP (CD46), DAF (CD55), CD59 and SERPING1). Cox regression was used to estimate Hazard Ratios (HRs) for individual SNPs with EFS. The most prevalent homozygous genotype was used as the reference group, and each polymorphism was modeled individually as having a log-additive effect in the regression model. For gene-level analyses, we used a principal components based gene-level test. All models were adjusted for sex, FLIPI, follicular grade III, and initial treatment. Results. The median age at diagnosis was 61 years (range, 25-85). 57 (50%) of the patients had an event, at a median follow-up of 59 months (range, 33-73) for living patients. In gene-level analyses, CFH (p=0.004), CD55 (p=0.02), CFHR5 (p=0.02) and CFHR1 (p=0.02) were significant at p<0.05, and these genes had noteworthy q-values after consideration of multiple testing (q=0.04 for CFH and q=0.08 for CD55, CFHR5, and CFHR1). For CFH, 2 of the 11 tagSNPs (rs1329423, HR=0.46, p=0.001; and rs3766404, HR=2.0, p=0.003) were significantly associated with EFS, as was the nsSNP rs1065489 (amino acid change E936D: HR=0.39, p=0.004). For CD55, both tagSNPs (rs4844591, HR=0.58, p=0.03; and rs2564978, HR=0.58, p=0.03) were associated with EFS, as were 3 of 5 tagSNPs from CFHR5 (rs3748557, HR=0.52, p=0.03; rs12092294, HR=1.9, p=0.03; and rs6694672, HR=1.9, p=0.03) and the only tagSNP for CFHR1 (rs436719, HR=0.54, p=0.03). Conclusions. Genetic variation in CFH, CD55, CFHR5, and CFHR1 was associated with EFS in follicular lymphoma after adjustment for clinical variables. These genes are part of a gene cluster at 1q32-q32.1 involved in the regulation of C3. CD55 encodes a membrane complement regulatory protein which can allow tumor cells to evade complement attack. These results are being validated in a second set of SPORE patients. Support: P50 CA97274, R01 CA92153. Disclosures: No relevant conflicts of interest to declare.

SummaryIbrutinib‐related data in Waldenström macroglobulinaemia (WM) remain sparse, particularly outside of trials. We report on 80 patients [previously treated, n = 67 (84%), treatment‐naïve, n = 13 (16%)] with WM, evaluated consecutively at Mayo Clinic, who received ibrutinib off‐study after its approval in 2015 for WM. Overall response rate (ORR) was 91%; major‐response rate (MRR) was 78%. The median time to first response and best response was 2·9 [95% confidence interval (CI): 2–4] and 5·7 (95% CI: 4–12) months, respectively. The median follow‐up was 19 (95% CI: 14–21) months; 18‐month progression‐free survival (PFS) was 82%. The median time on therapy was 12·5 (95% CI: 9·3–16·7) months, and the median duration‐of‐response was 32 (range: 23–32) months. Twenty‐five patients (31%) had discontinued therapy at last follow‐up (68% due to treatment‐related toxicities) and 18% of patients required dose reduction. Fatigue (12%) and atrial‐fibrillation (11%) were common non‐haematological toxicities. IgM rebound occurred in 36% of patients who abruptly discontinued ibrutinib. Following ibrutinib discontinuation, 84% of patients received subsequent treatment, achieving an ORR of 57% and MRR of 50%. The median PFS from commencement of subsequent salvage therapy was 18 months. Ibrutinib therapy, outside of clinical trials, is effective in WM, but is associated with toxicities and challenges, including IgM rebound and a high drug discontinuation rate for reasons other than disease progression.

AbstractEpidemiologic studies show an increased risk of non‐Hodgkin lymphoma (NHL) in patients with autoimmune disease (AD), due to a combination of shared environmental factors and/or genetic factors, or a causative cascade: chronic inflammation/antigen‐stimulation in one disease leads to another. Here we assess shared genetic risk in genome‐wide‐association‐studies (GWAS).Secondary analysis of GWAS of NHL subtypes (chronic lymphocytic leukemia, diffuse large B‐cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and ADs (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis). Shared genetic risk was assessed by (a) description of regional genetic of overlap, (b) polygenic risk score (PRS), (c)"diseasome", (d)meta‐analysis.Descriptive analysis revealed few shared genetic factors between each AD and each NHL subtype. The PRS of ADs were not increased in NHL patients (nor vice versa). In the diseasome, NHLs shared more genetic etiology with ADs than solid cancers (p = .0041). A meta‐analysis (combing AD with NHL) implicated genes of apoptosis and telomere length.This GWAS‐based analysis four NHL subtypes and three ADs revealed few weakly‐associated shared loci, explaining little total risk. This suggests common genetic variation, as assessed by GWAS in these sample sizes, may not be the primary explanation for the link between these ADs and NHLs.

Background:Diffuse Large B Cell Lymphoma (DLBCL) is characterized by both biologic and genetic heterogeneity. While significant therapeutic advances have been made, nearly 30% of cases fail frontline treatment. Recent data suggest that 5–10% of DLBCL can be categorized as double/triple hit lymphoma (DH/TH), defined as having concurrent MYC, BCL2 and/or BCL6 gene rearrangements, identified by fluorescence in‐situ hybridization (FISH) probes. According to the 2017 revision of the World Health Organization classification of lymphoid neoplasms, DH/TH DLBCL is now considered a high grade B‐cell lymphoma (HGBL). While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, often through genomic amplification of oncogenes or loss of tumor suppressors. Despite their similarities, the contributions of CNAs in MYC, BCL2, and BCL6, alone, or in combination with gene rearrangements, are largely unknown in DLBCL and could identify a group of previously uncharacterized patients that may have high expression of MYC, BCL2 and/or BCL6 driven by alternative mechanisms.Aims:The goal of the study is to define the landscape and prognostic significance of gene rearrangements and copy gains in MYC, BCL2, and BCL6 in a cohort of newly diagnosed DLBCL.Methods:A total of 176 cases of DLBCL with both FISH and CNA data were available for the study. FISH was performed on formalin‐fixed paraffin‐embedded sections using break‐apart probes to MYC, BCL2, and BCL6 in the Mayo Clinic Department of Lab Medicine and Pathology. Somatic copy number alterations were identified using whole exome sequencing and the Affymetrix OncoScan® FFPE assay on patient tumor FFPE tissue, and were interrogated using Nexus Copy Number 9.0 software. All patients were treated with immunochemotherapy and prospectively followed for event‐free and overall survival.Results:FISH identified 3 groups of previously described patients, DH/TH or HGBL (n = 11), translocation positive (single MYC, BCL2, BLC6, or double BCL2/BLC6, n = 86), and translocation negative (n = 79). Expanding upon the translocation positive subgroup, copy number analysis identified patients (19 of 86, 22%) with copy number gains in a second or third gene resulting in genomic hits at both MYC and BCL2/6; we classified these as alternative‐double/triple‐hit (A‐DH/TH). An example of this would be a MYC translocation plus a BCL2 and/or BCL6 gain (n = 5), or a BCL2 and/or BCL6 translocation plus a copy gain in MYC (n = 14). Similar interrogation of a publically available dataset (n = 250), revealed similar proportions of these patient groups in an independent cohort of DLBCL, validating our findings. Cell of origin classification of the A‐DH/TH cases revealed that they were 53% GCB. A‐DH/TH patients, in comparison to the translocation‐positive/copy‐gain‐negative cases, were at greater risk of an event (Cox HR = 1.5, 95% CI = 0.71–3.0) and the cases trended towards a worse overall survival (log‐rank P = 0.065; Cox HR = 2.0, 95% CI = 0.94–4.3). Patients with copy‐gains alone had similar outcomes as those with no copy‐gain or translocations, suggesting a central role for at least one translocation event in predicting outcome.Summary/Conclusion:Our data suggest that use of CNA data in combination with traditional FISH can identify a unique subset of DLBCL, A‐DH/TH, that are missed by standard clinical practice. More importantly, we find that these patients do poorly and may benefit from more aggressive treatment, although we do acknowledge that our population size was small and further validation is needed.image

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.

SummaryBackgroundThe appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown.AimTo explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells.MethodsFifty‐one patients with Crohn's disease and ulcers >0.5 cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0 mm, 7 to 8 mm and 21 to 24 mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins‐6, ‐8 and ‐23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase‐positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient.ResultsHistological disease activity scores (P < 0.0001), proinflammatory gene expression levels (P < 0.005) and numbers of myeloperoxidase‐positive cells (P < 0.0001) were highest in biopsies from the ulcer edge in the colon and ileum, with decreasing gradients observed with distance from the edge (P < 0.05). No differences between colonic and ileal samples were detected for the parameters measured at any location.ConclusionsBiopsies from the ulcer edge in patients with Crohn's disease yielded the greatest histological disease activity and mRNA levels and had similar readouts in the colon and ileum. Research is needed to confirm this conclusion for other measures.