Novak, Anne J.

Abstract Background:The pathological behavior of Waldenströms macroglobulinemia (WM) cells relies on aberrant B-cell receptor (BCR), and Toll-like receptor (TLR) signaling and is intricately supported by malfunctions in initiation of apoptosis. In this dynamic, a complex shift between functionally active pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family occurs and bestows the malignant cells with a significant survival advantage. This defective apoptotic signaling is associated with aggressive clinical behavior, chemoresistance, and a poor prognosis in many B-cell cancers, including WM. Thus, BCR/TLR induced proliferation and abnormal apoptotic signaling due to dysregulation of Bcl-2 signaling, act in concert for the assurance of the malignant WM cells survival. Clinically, mitigation of BCR signaling with the first in class BTK inhibitor, ibrutinib, has shown promising results in WM and other B-cell cancers. However, clinical responses to the BTK inhibitor alone are less than optimal and development of ibrutinib-resistant disease is being recognized as an important and impending dilemma. We have previously shown the utility of disrupting pro-apoptotic Bcl-2 protein interactions in WM, chronic lymphocytic leukemia and multiple myeloma and that this functional interference results in re-sensitization (or heightened sensitivity) of tumor cells towards chemoimmunotherapy. As such, we examined the effects of ABT-199, a Bcl-2 specific small molecule inhibitor, in preclinical models of WM. Importantly; we conducted this investigation in proprietarily developed WM cells that have acquired resistance mechanisms towards either ibrutinib (IR WM subclones) or bortezomib (BR WM subclones). Materials:WM cell lines (BCWM.1, MWCL.1 and RPCI-WM1; WT cells) including their bortezomib resistant (BR) or ibrutinib resistant (IR) subclones were used in this study. Results: An initial comparison of Bcl-2 family members, Bcl-2, Mcl-1, BCL-xL, A1 (anti-apoptotic members), Noxa, Puma, Bim (pro-apoptotic initiators) and Bak and Bax (pro-apoptotic effectors), revealed that BR WM cells express increased levels of pro-apoptotic Bcl-2 family members, particularly Bcl-2, relative to WT cells. Similarly, A1 and Bcl-2 levels were up regulated in IR cell lines suggesting that the Bcl-2 mediated survival pathway is involved in resistance to both bortezomib and ibrutinib. Cell growth (MTS) assays using ABT-199 indicated that both BR and IR cells were equally sensitivity to Bcl-2 inhibition, with an IC50 ranging between 2 – 4mM. ABT-199 induced a dose dependent increase in MOMP with concomitant induction of apoptotic cell death, the latter confirmed by observation of PARP-1 cleavage. ABT-199 induced an intrinsic apoptotic program with the activation of caspase 9 and caspase 3 in all the cells tested. This data indicated the Bcl-2 pathway as being relevant and essential in WM cell lines even under drug resistant conditions. To evaluate the priming effect of ABT-199 on bortezomib and ibrutinib induced cell death, MTS assays were performed with these drugs in presence of IC25 concentrations of ABT-199. The addition of ABT-199 significantly reduced the IC50of both bortezomib and ibrutinib in WT, BR and IR cells. This suggested that re-sensitization of BR and IR cells towards either bortezomib or ibrutinib, respectively, was possible in presence of ABT-199. Further, combined addition of ABT-199 with bortezomib or ibrutinib showed synergistic cell death (combination index range: 0.004 – 0.6, CalcuSyn, Biosoft UK) in BR or IR WM cells, respectively. Conclusion: Dysregulation of Bcl-2 signaling is associated with the pathogenesis and promotion of drug resistance in B-cell cancers, including WM. Our report is first to highlight in WM preclinically, the antiproliferative effect of disrupting Bcl-2, through use of ABT-199. Altogether, our data suggests that therapeutic interference of Bcl-2 function with ABT-199 complements the anti-WM activity of ibrutinib or bortezomib in a synergistic manner and can in fact restore sensitivity of either drug in WM cells that are resistant to proteasome or BTK inhibitors. Focused mRNA and protein profiling analyses are currently underway to delineate the mechanisms accounting for synergy observed between concomitant Bcl-2/BTK inhibition as well Bcl-2/proteasome inhibition. Disclosures Ansell: Biothera: Research Funding. Martin:Janssen: Honoraria. Coleman:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Speakers Bureau.

Abstract To date, whole genome and exome sequencing studies of follicular lymphoma (FL) have primarily focused on identification of small site mutations that are recurrent in FL tumorigenesis or involved in tumor clonal evolution. A comprehensive genomic and transcriptomic survey of various mutation types including large structural variants (SVs) in FL cases with detailed clinical annotations and long-term follow-up has not been accomplished. To gain insight into genetic biomarkers that may predict clinical features, we performed exome and whole genome mate-pair sequencing of fresh frozen tumor and paired peripheral blood DNAs, and transcriptome sequencing of tumor RNAs, from 8 FL patients. The patients we selected were either below the median age of FL onset (n=7, median 54.5 yrs) or had a family history of lymphoma (n=1). These patients were clinically diverse, and included patients who had Grade 1 or 2 disease (n=4), classified as “indolent”; and patients with Grade 3a disease (n=2) or who subsequently had pathologic transformation (n=2), classified as “aggressive”. The coding regions of the genome (exome) were captured using Agilent SureSelect Target Capture Kit V2.0, and sequenced at 100-bp paired-end. The single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using The Genome Analysis Toolkit (GATK), and the exon level copy number variants (CNVs) we identified using patternCNV. The whole-genome mate-pair libraries of both normal and tumor DNAs with 3kb insert sizes were sequenced paired end at 50-bp. The large SVs of CNVs, large INDELs, translocations, and inversions were identified using the SnowShoes-SV algorithm. The RNA sequencing libraries of 8 FL tumors were constructed using Illumina TruSeq protocol, and sequenced at 50-bp paired end. The RNA-Seq data were analyzed using TopHat and the fusion transcripts were identified using SnowShoes-FTD. Our analysis of SNVs and INDELs revealed mutations in previously reported genes including MLL2, CREBBP, TNFRSF14, and histone cluster genes (HIST1H2AM, HIST1H2BD). In addition, we identified novel recurrent mutations in cysteine-rich PAK1 inhibitor (CRIPAK) in 25% of the tumors. In a secondary analysis performed by Sanger sequencing or re-analysis of publically available RNA sequencing data, we identified CRIPAK mutations in 44% of FL (n=32) and 28% of DLBCL tumors (n=102). Bioinformatics analysis shows that the coding region of CRIPAK is highly enriched with the protein functional domain, post-SET, which is usually found in histone lysine methyltransferases (HMTase) genes including MLL2 and EZH2 that are known to be important in lymphomagenesis. Interestingly, CRIPAK is part of the same regulatory network consisted of previously identified lymphoma genes including MLL2, EZH2, CREBBP, and EP300, according to the shortest path algorithm by MetaCore (Philadelphia, Pennsylvania). Recurrent SVs identified in the FL tumors included the well-known IGH-BCL2 translocation, or t(14;18), in 6 out of 8 cases (75%) and the chr1q amplifications in 4 out of 8 (50%) tumors. Other non-recurrent large CNVs involving entire chromosomes or chromosome arms, as well as other inter- and intra-chromosomal structural variants were detected in individual tumors. In addition, we identified and validated 6 fusion transcripts from the transcriptome sequencing data in 3 out of 8 cases (38%). While our sample size is small, we found that SNVs and INDELs in MLL2, CREBBP, TNFRSF14, CRIPAK, and histone cluster genes, as well as t(14;18), did not distinguish indolent or aggressive tumors. However, all aggressive (4/4, 100%) and none of the indolent tumors had gains in chr1q; and the presence of RNA fusion transcripts were observed in aggressive tumors only (3/4, 75%). Additionally, we found that aggressive tumors had higher numbers of genes with point mutations (SNVs and short INDELs) (40 ± 7.6 vs. 26 ± 2.6; aggressive vs. indolent), higher numbers of genes impacted by copy number aberrations (1060 ± 263.7 vs. 233 ± 133.9), and higher numbers of large SVs (24 ± 10 vs. 6 ± 1.6). Taken together, our comprehensive analysis of 8 FL tumors reveals genetic diversity among newly diagnosed FL patients, identifies novel and recurrent mutations in CRIPAK, and finds that high tumor complexity and DNA instability may be indicators of aggressive disease. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 925 Diffuse large B-cell lymphomas (DLBCL) consists of biologically distinct subtypes: Germinal Center B-cell (GCB)–like and Activated B-cell (ABC)-like. Studies to date suggest that patients with tumors that have ABC features have an inferior survival; therefore, it is important to develop new therapies specifically for ABC-type DLBCL based on mechanistic differences. It has been shown that the ABC-DLBCL has high levels of total and phosphorylated STAT3 (pSTAT3) protein and this STAT3 activity may play a role in the survival of ABC-DLBCL cells. Indeed, when ABC and GCB cell were incubated with the novel JAK2 inhibitor TG101348 (TargeGen, San Diego, CA) we found potent cytotoxicity (LD50 2μM) in ABC DLBCL but not in GCB DLBCL. This provides further rationale to study the regulation of STAT3 in the survival of ABC type DLBCL cells. In studies of HDAC expression in ABC DLBCL cells, we found over-expression of the class 1 histone deacetylases, specifically HDAC1 and HDAC3. We then performed co-immunoprecipitation experiments to learn the functional interaction between STAT3 and HDACs. We found that STAT3 formed complexes with HDAC1 to a greater extent than HDAC3. We also showed that STAT3 did co-precipitate with HDAC1 suggesting a reciprocal relationship between these two proteins. These results suggested that HDAC1 (but not HDAC3) might be involved in the regulation of STAT3. The HDAC inhibitor LBH589 (LBH, Novartis Pharm) was more cytotoxic to ABC (LD50, 10-20nM) than GCB (LD50, 50-100nM) and was shown to dephosphorylate STAT3. These results led to experiments designed to determine the effect of LBH on STAT3 acetylation. When ABC cells were incubated with LBH, co-immunoprecipitation data showed a dose-dependent increased in acetylation of STAT3 without significant change on the levels of STAT3 immunoprecipitated. Next, we asked that how LBH is dephosphorylating STAT3. One potential mechanism is activation of a protein phosphatase. Immunoprecipitation of protein phosphatase 2A (PP2A) in the lysates of LBH-treated cells followed by western blotting of STAT3 demonstrated a positive correlation between LBH dose and amount of STAT3 co-immunoprecipitated with PP2A. Our data show that when LBH is incubated with ABC cells, the LBH increases binding of PP2A to STAT3 and the increased phosphatase activity turns off STAT3 signaling. Since both HDAC inhibitor (LBH) and the JAK2 inhibitor (TG) had selective cytotoxic effects on ABC cells, and since both agents dephosphorylate STAT3, we combined the two drugs. We found that the combination exhibited enhanced inhibitory effects on the growth of ABC cells and was able to alter the association of STAT3 with HDAC1 and subsequently dephosphorylate STAT3 to greater extent than either agent alone. Furthermore, we knocked down HDAC1 expression (through siRNA) in OCILy10 (ABC) cells and showed that inhibition of HDAC1 inhibits constitutively activated STAT3. This data suggests that inhibiting HDAC1 expression results in STAT3 dephosphorylation. In summary, we have demonstrated that a key consequence of HDAC expression in ABC cells is continuous activation of STAT3. These data indicate that HDACs activate STAT3 and that PP2A may be important in maintaining STAT3 in the activated state in the ABC cells. This study describes a new mechanism by which STAT3 mediates ABC cell survival and provides the rationale for targeting ABC type DLBCL by simultaneously inhibiting HDAC activity and STAT3 activation by pharmacologic grade inhibitors such as LBH589 and TG101348, which are currently in clinical trial. A phase 1 and 2 trial of JAK2 inhibitor, TG101348 for ABC/GCB type DLBCL is planned. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2668 Poster Board II-644 Interleukin-12 (IL-12) has been demonstrated to induce IFN-g production by T and NK cells and thereby contribute to anti-tumor immunity. However, the administration of IL-12 to boost anti-tumor immunity in B-cell lymphoma has shown no clinical benefit. In fact, clinical trials of IL-12 in combination with rituximab in follicular B-cell lymphoma (FL) showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res. 2006 15; 12:6056-63). The goal of this study was therefore to determine the role of IL-12 in the antitumor response in B-cell NHL. First, we measured serum levels of IL-12 in patients with untreated FL before treatment with rituximab and normal healthy controls. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). To determine the mechanism by which IL-12 may contribute to a poor prognosis, we investigated the role of IL-12 on induction of immune tolerance. First, we found that TIM-3, a member of the T cell immunoglobulin and mucin domain-containing protein (TIM) family that functions to terminate TH1-mediated immunity and promote tolerance, was constitutively expressed on a subset of intratumoral T cells accounting for approximately 15% and 25% of the intratumoral CD4+ and CD8+ T cells, respectively. In contrast, less than 2% of T cells from peripheral blood of normal individuals expressed TIM-3. TIM-3-expressing T cells were distinct from regulatory T cells since CD25+ and Foxp3+ T cells lacked TIM-3 expression. Secondly, we found that TIM-3-expressing CD4+ cells were unable to produce cytokines such as IL-2, IFN-g or IL-17 and that TIM-3-expressing CD8+ T cells failed to produce Granzyme B, IFN-g or IL-2. We also observed that TIM-3-expressing T cells lost the capacity to proliferate in response to TCR activation. These results suggest that TIM-3 expressing CD4+ and CD8+ T cells are functionally exhausted. Thirdly, we observed that TIM-3 expression on T cells could be induced by activation and that IL-12 was the strongest stimulus to induce TIM-3 expression on CD4+ and CD8+ T cells. Finally, we found by immunohistochemistry (IHC) that Galectin-9 (Gal-9), a ligand for TIM-3, was abundantly expressed on lymphoma B cells. In vitro incubation with a stable form of Gal-9 induced apoptosis of CD4+ and CD8+ T cells in a dose dependent fashion. Gal-9-mediated apoptosis of T cells was attenuated by a TIM-3 Fc protein and isolated TIM-3+ T cells exhibited a significantly higher apoptosis rate than TIM-3− T cells in response to Gal-9. These results indicate that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in lymphoma patients. Given the findings that IL-12 strongly induces TIM-3 expression on effector T cells and that the TIM-3/Gal-9 pathway impairs the immune response, we conclude that increased serum levels of IL-12 suppress anti-tumor immunity in follicular lymphoma patients and is associated with a poor prognosis. Disclosures: Witzig: Novartis: Research Funding.

Abstract Abstract 143 IL-12 induces IFN-g production and contributes to anti-tumor immunity. However, administration of IL-12 in cancer patients has resulted in a limited clinical benefit. In follicular B-cell lymphoma (FL), a clinical trial of IL-12 in combination with rituximab showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res 2006), suggesting that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in FL patients. Recently, a type of immune response, termed “ T cell exhaustion” describing a condition in which T cells exhibit reduced differentiation, proliferation, and effector function, has been shown to impair anti-tumor immunity and result in disease progression. TIM-3, a family member of T-cell immunoglobulin and mucin domian-containing proteins, inhibits TH1-mediated immune response and promotes immunological tolerance. A recent study has suggested that TIM-3 may play an important role in mediating T cell exhaustion. However, the biological and clinical relevance of TIM-3 in cancers remains completely unknown. In this study, we determined whether T cell exhaustion exists in the tumor microenvironment, whether IL-12 contributes to T cell exhaustion, and whether TIM-3-mediated T cell exhaustion impacts patient outcome in FL. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). Both lymphoma B cells and monocytes were able to produce IL-12 and contributed to elevated serum levels of IL-12 in FL. Importantly, we found that IL-12 strongly induced TIM-3 expression in a dose-dependent manner. Endogenous production of IL-12 by lymphoma B cells and monocytes was capable of regulating TIM-3 expression on T cells from FL. As a consequence, TIM-3 was highly expressed on a subset of T cells from PBMCs or lymph nodes from FL patients while its expression was negligible or moderate on T cells from normal PBMCs or benign lymph nodes, respectively. The number of TIM-3-expressing T cells accounted for approximately 32% and 39% of CD4+ or CD8+ T cells in biopsy specimens and 6.2% and 6.7% in peripheral blood of FL patients. TIM-3+ T cells, co-expressed with PD-1, exhibited a reduced ability to proliferate and decreased cytokine production compared to TIM-3- T cells. Similarly, IL-12-induced TIM-3+ T cells gradually lost the capacity to produce cytokines over a period of time. These results suggest that TIM-3-expressing T cells are functionally exhausted. In addition, TIM-3+ T cells were prone to apoptotic induction by its ligand galectin (Gal) -9. The phosphorylation of p38 was higher in TIM-3+ T cells compared to TIM-3- T cells when exposed to Gal-9, suggesting MAPK pathway was involved in Gal-9-mediated apoptosis of TIM-3+ T cells. Finally, increased numbers of intratumoral TIM-3-expressing cells were associated with a higher histological grade, higher LDH levels and a poor survival in FL patients. Taken together, these results indicate that IL-12, in contrast to its role in augmenting immune response through IFN-g, induces T cell exhaustion by upregulating TIM-3 expression. We further demonstrated that lymphoma B cells produce IL-12 thereby contributing to T cell exhaustion by promoting TIM-3 expression on intratumoral T cells. Impairment of anti-tumor immunity due to T cell exhaustion induced by the IL-12-TIM-3 pathway may account for the observation that high levels of serum IL-12 and increased number of TIM-3+CD4+ T cells correlate with a worse outcome in FL patients. These findings not only reveal a novel IL-12-TIM-3 pathway that plays an important role in impairing tumor immunity and detrimentally affecting prognosis in FL patients, but may have therapeutic potential for cancer patients. Disclosures: No relevant conflicts of interest to declare.

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