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Abstract Background and Significance : Chronic lymphocytic leukemia (CLL) is the most heritable hematologic malignancy; however, no common CLL predisposition genes are known. Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by small accumulations of B lymphocytes in the peripheral blood. MBL has a CLL-like immunophenotype, may progress to overt CLL, and is over represented in CLL families. Therefore, MBL observed in the context of familial CLL may be a marker of inherited risk for development of CLL. Detailed characterization of family-associated MBL may also provide mechanistic insights into the pathogenesis of familial CLL. Our strategy was to detail the biologic characteristics of CLL in family-associated MBL. Methods : Persons with MBL were identified by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. Flow cytometry was used to determine the surface immunophenotype including CD38 and intracellular ZAP-70. We defined MBL as populations of CD19+, CD5+, CD20lo, CD23+ B cells that comprised at least 2% of the CD19+ peripheral B cell compartment and did not exceed 5.0 × 109 MBL cells/L. RNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences. MBL cells were sorted in bulk for FISH for loci associated with clinical CLL. Results : Twenty-two out of 190 (12%) unaffected family members were found to have MBL. We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 32%; range 2%–97%). Nonetheless, the absolute size of the MBL clone was small, 15 of 17 individuals had < 200 × 106 MBL cells/L. CD38 expression (defined as CD38 surface expression in ≥30% of MBL cells) was observed in 8 of 18 subjects tested. ZAP-70 (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 4 of 12 participants. Among 12 subjects tested, 5 MBL expressed both surface IgD and IgM, 3 expressed IgD only, 2 expressed IgM only, and 2 did not express IgD or IgM. Analysis of immunoglobulin heavy and light chains has been completed in 8 individuals. Both immunoglobulin heavy chain variable (IgVH) region mutated (n = 12) and unmutated (n = 4) sequences were observed. Four of 8 individuals had 2 or more unrelated MBL clones (range 2–5), including one individual with both unmutated and mutated clones. Among the 16 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IgVH genes were 3–07 (3 MBL clones), 3–15 (3), and 4–34 (3). No VH1 family gene rearrangements were observed. In one individual, a VH3–07 MBL clone showed intraclonal diversification suggestive of antigen driven immunoglobulin sequence changes. Twenty productive light chain rearrangements were identified among the 16 MBL clones, with 11 Vλ and 9 Vκ genes used. We observed 6 productive rearrangements of Vλ1–51. MBL cells were bulk sorted for FISH from 9 subjects. Mono or biallelic deletion of 13q14.3 was observed in 5 subjects, the other 4 were normal. Conclusions : Our findings confirm that MBL is commonly observed among the unaffected family members from CLL kindreds. We found that some MBL clones express ZAP-70, CD38 or have unmutated IgVH genes and thus are similar to clinical CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. Small MBL clones are commonly oligoclonal. Importantly, although the immunoglobulin heavy and light chain genes rearranged in these MBL clones are all commonly used in CLL, the absence of VH1 and abundance of Vλ1–51 rearrangements do suggests differences in BCR usage between CLL and MBL. Further investigation of family associated MBL may clarify the genetics and immunobiology of familial CLL.

Abstract Background: Recent literature suggests that statins may have anticancer potential. This effect is thought to be mediated through 2 primary mechanisms - impairment of protein prenylation and interference with the formation of cholesterol-rich lipid microdomains, or “lipid rafts” within the cell membrane. Both of these processes are critical for signaling activity of numerous proteins important for lymphomagenesis and tumor survival. Recent data, however, suggest that statin use may directly inhibit rituximab binding to CD20 and therefore rituximab efficacy. These findings raised significant concerns about statin use during rituximab treatment. Here we report on statin use and clinical outcome in a cohort of FL and DLBCL treated patients, most of whom were treated with rituximab containing regimens. Methods: 293 newly diagnosed FL patients and 228 newly diagnosed DLBCL patients were prospectively enrolled in our Lymphoma SPORE registry from 9/2002 through 6/2007. Pathology was centrally reviewed. All patients were followed for retreatment, progression-free and overall survival. Statin use at the time of diagnosis and time of initial treatment was abstracted from the medical record. An event was defined by disease progression, retreatment, or death due to any cause. Results: 19% of FL patients and 22% of DLBCL patients were on statins at diagnosis; 16% and 19% were on statins during treatment, respectively. Initial therapy for the FL patients was observation (40%), R-CHOP (19%), R-CVP (12%), rituximab alone (8%), CVP (7%), RT (6%), and other (8%). All DLBCL patients received rituximab with CHOP or a CHOP-like regimen. At a median follow-up of 36 months (range 3–73), 109 (37%) and 65 (29%) of FL and DLBCL patients had an event; 19 (6%) and 46 (20%) of FL and DLBCL patients died, respectively. After adjusting for FLIPI, grade, and initial therapy type, statin use at diagnosis was associated with better event-free survival (HR = 0.57, 95% CI: 0.34–0.95, p=0.03) in FL patients. Statin use during treatment in FL patients was also associated with better event-free survival, though not statistically significant (HR = 0.67, 95% CI: 0.39–1.16, p=0.15). The improvement in EFS for FL patients was consistent across initial therapies, including observation. Statin use was not associated with IPI-adjusted overall survival or event-free survival in DLBCL (all p > 0.50). Conclusions: Statin therapy does not appear to be associated with inferior clinical outcome in DLBCL treated with rituximab and CHOP or CHOP-like therapy. Therefore direct inhibition of rituximab binding to CD20 may have limited clinical significance or/and may be overcome by inhibitory impact of statins on cell signaling. The latter possibility is supported by our observation that statin use is associated with improved event-free survival in follicular lymphoma. Figure Figure Figure Figure

Abstract Background: Inhibitors of HMG-CoA reductase (“statins”) are effective in reducing the risk of cardiovascular disease. Preclinical data from rodents suggested that they may increase the incidence of some cancers, including lymphoma. However, monitoring of randomized trials in humans have generally found decreased cancer risks, and these findings have been supported by a growing number of observational studies, including three recent reports of a protective association in NHL. The suggested anti-carcinogenic mechanisms for statins include their impact on inflammatory (including a shift from a Th1 to Th2 profile) and angiogenesis pathways, as well as the induction of apoptosis by modulation of signaling pathways, all of which are of biologic relevance in NHL. Methods: We evaluated the history of statin use and risk of NHL in a clinic-based study of 243 newly diagnosed NHL cases and 499 frequency matched controls enrolled at the Mayo Clinic from 2002-2005. Risk factor data were collected using self-administered questionnaires, and included history of use of cholesterol lowering drugs two or more years before diagnosis (for cases) or enrollment (for controls); specific drug names were not available. Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI), adjusting for age, gender, and residence. NHL subtypes were centrally reviewed, and subtype-specific risks were estimated using polychotomous logistic regression. Results. The mean age at diagnosis was 59.5 years for cases and 55% were men; among controls, the mean age at enrollment was 61.8 years and 54% were men. Twenty-one percent of the cases reported ever using statins compared to 30% of the controls, supporting a lower risk of NHL for ever users (OR=0.69; 95% CI 0.47–1.00). The lowest risk was seen for the longest users; i.e., compared to never users, person who had 10 or more years of use had the greatest reduction in risk (OR=0.52; 95% CI 0.24–1.10). A history of high cholesterol was also inversely associated with risk of NHL (OR=0.72; 95% CI 0.55–0.93). However, the association for the type of treatment for cholesterol lowering varied. Those who were medically treated had inverse association with risk of NHL (OR=0.63; 95% CI 0.46–0.86), while the no treatment and dietary change only groups were found to have no association with risk. The reduction in NHL risk with medical treatment was specific to statin use, as the use of other cholesterol lowering drugs was not associated with risk (OR=1.13; 95% CI 0.50–2.57). These results were not confounded by education, family history of NHL, body mass index, cigarette smoking or alcohol use. In subtype analysis, the inverse association was seen for diffuse large B-cell lymphoma (OR=0.27; 95% CI 0.08–0.90), but not for CLL/SLL or follicular NHL. Conclusions. Statin use was associated with a lower risk of developing NHL, particular diffuse large B-Cell lymphoma.

Abstract Background: A significant percentage of DLBCL patients present with a composite histology, often seen as a node containing both follicular lymphoma and DLBCL or DLBCL in the node and discordant indolent lymphoma in the bone marrow. Literature from the pre-rituximab era suggests DLBCL patients with transformed lymphoma or composite histology have worse outcome than de novo DLBCL. Here we report on early events in a cohort of R-CHOP treated patients. Goal: To determine whether patients with composite lymphoma have an inferior event free survival (EFS) and overall survival (OS) compared to de novo diffuse large B-cell lymphoma when treated with R-CHOP. Methods: Newly diagnosed patients treated with an R-CHOP containing regimen were prospectively enrolled in our Lymphoma SPORE registry from 9/2002 through 6/2007. Pathology was centrally reviewed. All patients were followed for retreatment, disease progression, and death. Results: 401 DLBCL patients were enrolled; 14% (57/401) had a composite histology. 33 patients had DLBCL and another histology, predominantly follicular lymphoma (n=29), in the same node. 20 patients had a non-DLBCL histology in a distinct location from the DLBCL; this was primarily indolent lymphoma in the bone marrow (n=15). 4 patients had both. 19% (75/401) of patients died during follow-up and 30% (121/401) had an event (death due to any cause, progression, or retreatment). Median follow-up for living patients was 34 months (range, 5–73). Composite DLBCL patients had higher event-free (3 year EFS = 79%) and overall (3 year OS = 93%) survival then de novo DLBCL (3 year OS = 66%, 3 year EFS 79%), p=0.05 and p=0.005 respectively. These differences remained statistically significant after adjusting for the International Prognostic Index (IPI): EFS HR = 0.53, 95% CI: 0.29–0.97, p=0.02; OS HR=0.28, 95% CI: 0.10–0.76, p=0.002. OS and EFS for composite DLBCL more closely resembled R-CHOP treated grade III follicular lymphoma (A,B) from the same cohort (3 year EFS = 81%, 3 year OS = 93%). Improved outcome for composite DLBCL was consistent whether the additional histology was in the same node or distinct from the DLBCL. Conclusions: R-CHOP treated DLBCL patients with indolent discordant bone marrow involvement or other composite histology have improved early OS and EFS compared to de novo DLBCL. Further follow-up is needed to assess the long-term prognosis of composite DLBCL in the rituximab era. Histology N Age > 60 Stage III/IV LDH > ULN PS > 1 >2 EN Sites 3 YR EFS 3 YR OS * Denotes statistically significant difference at p=0.05 de novo DLBCL 344 58% 56% 56% 17% 22% 66% 78% Composite DLBCL 57 65% 77%* 34%* 18% 32% 79%* 93%* Figure Figure

Abstract B cell non-Hodgkin lymphoma (NHL) is a serious and frequently fatal illness. Some of the genetic changes that lead to lymphoma have been discovered, but many of the other contributing mechanistic details underlying transformation events are not yet known. Members of the TNF super-family have been shown to be key mediators in the formation and regulation of both normal and malignant cell growth. APRIL (A Proliferation Inducing-ligand), a TNF family member expressed by monocytes, macrophages, and tumor cells, stimulates B-cell NHL growth and transgenic over-expression of APRIL in mice results in B cell hyperplasia and lymphoma. The ability of APRIL to bind to TNFR related receptors, BCMA (B Cell Maturation Antigen) and TACI (Transmembrane Activator and CAML Interactor), and with lower affinity to heparan sulfate proteoglycans (HSPG) has complicated studies to better understand APRIL mediated signaling. It has been shown that NHL cells variably express TACI and BCMA, with TACI being most predominant, however, the specific mechanisms of how APRIL induces NHL cell growth remain to be fully defined. Therefore, we sought to specifically characterize APRIL-mediated signaling events through TACI to better understand the role of this receptor-ligand interaction in B cell NHL biology. Using a TACI-expressing NHL cell line (Karpas) we found that APRIL consistently increased NHL cell proliferation, but had very little effect on cell survival. Since the PI-3K/Akt pathway has been shown to be important in regulating cell cycle progression, we explored the possibility that APRIL-TACI interactions resulted in activation of this pathway. We found that stimulation of TACI-expressing NHL cells with APRIL results in phosphorylation of Akt but had no effect on NF-kB classical pathway activation. Because APRIL can also bind to cells via proteoglycans, we also tested the ability of APRIL to activate Akt in the presence of heparin and found that APRIL-mediated Akt phosphorylation was still detectable. These results suggest that APRIL-mediated signaling occurs independent of proteoglycan binding. In addition to activating Akt, we also found that stimulation of NHL cells with APRIL resulted in up-regulation of cyclin D1 and p21, but not c-myc and p27. Inhibition of the PI3K/AKT signal transduction pathway with the pharmacological inhibitor wortmannin prevented APRIL-induced proliferation as well as up-regulation of p21 and cyclin D1 levels suggesting that PI-3K kinase is required. To further substantiate the contribution of TACI in APRIL-mediated signaling, we selectively targeted TACI expression using a siRNA approach. When TACI expression was inhibited, APRIL no longer induced NHL-B cell proliferation, Akt phosphorylation or up-regulation of p21 and cyclin D1. Taken together, these studies suggest that APRIL-TACI interactions result in activation of the PI3K/Akt pathway and up-regulation of cyclin D1 and p21, and help define the mechanism by which APRIL enhances B cell NHL growth. The inhibition of APRIL by blocking APRIL/receptor or interruption of downstream signaling may provide potential therapeutic option for patients with B-cell non-Hodgkin’s Lymphoma.

Abstract Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin lymphoma. MCL is characterized by the chromosomal translocation t(11;14) leading to overexpression of cyclin D1 (CCND1), a regulator of the cell cycle. NF-κB plays a central role in key cellular processes, including cell cycle regulation and apoptosis, and it has been shown to be constitutively activated in MCL cell lines and in patient biopsy samples and to play a key role in the growth and survival of MCL cells. In an exploratory study, we evaluated the hypothesis that germline variation in candidate genes from the cell cycle and NF-κB pathways predict overall survival in MCL. Methods: We genotyped 235 SNPs from 29 cell cycle genes and 447 SNPs from 55 NF-κB genes in 39 MCL patients aged 38–64 years who participated in a population-based study conducted from 1998–2000 through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles county. Tagging single nucleotide polymorphisms (SNPs) were selected from HapMap. Stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site. To assess the statistical significance of a gene, we first used principal components analysis to capture the genetic variation of the SNPs within a gene. Cox proportional hazards analysis was then used to assess the association between the top principal components from a gene with overall survival. Results: Through early 2005, there were 22 deaths in 39 patients (56%). The median follow-up of the 17 surviving patients was 56 months (range 36–70). Eleven of the 55 genes (TNFSF14 p=0.0007, NF-κBIA p=0.003, IL1R2 p=0.01, TNFRSF25 p=0.02, TNFSF13B p=0.02, FAS p=0.03, TLR2 p=0.04, TNFSF10D p=0.04, TNFSF10 p=0.06, MAP3K2 p=0.06) in the NF-κB pathway were associated with overall survival. In the cell cycle pathway, only 3 of the 29 genes (CASP5 p=0.06, CASP9 p=0.09, BCL2 p=0.09) were associated with overall survival, and a previously reported SNP in CCND1 (rs603965) showed no association (p=0.5). Permutation p-values for observing at least this number of associated tests in each pathway due to chance (from multiple testing) was p=0.8 for the cell cycle pathway and p=0.03 for NF-κB pathway. The small sample size precluded multi-gene modeling. Conclusion: In a population series of MCL patients, we found suggestive evidence that host genetic variation in candidate NF-κB pathway genes affects overall survival. These results complement tumor expression data implicating this pathway in the pathogenesis of MCL. In addition, such findings further support the value of developing therapeutic targets from this pathway.

Abstract Recent studies have reported an increased risk of breast, endometrial, ovarian, and gall bladder cancer associated with ERT. One proposed mechanism by which ERT increases risk is through the binding of estrogen to receptors at the tissue site. Estrogen receptors are also present on certain hematopoietic cells as well as on myeloid leukemia cells. Further, downregulation of the estrogen receptor through DNA methylation has been associated with increased acute myeloid leukemia (AML) survival. We evaluated whether ERT is associated with an increased risk of leukemia, particularly AML, in the Iowa Women’s Health Study. A cohort of 37,172 post-menopausal Iowa women ages 55 to 69 years with no history of prior cancer was linked annually to the population-based state cancer registry through 2001. In addition to other self-reported cancer risk factors, participants were asked about current and former use of ERT in 1986 and on four subsequent follow-up questionnaires. A total of 201 cases of leukemia were identified over 16 years of follow-up including 64 AMLs and 87 chronic lymphocytic leukemias (CLLs). Compared to never users of ERT at study baseline, current [multivariate relative risk (RR), 1.09; 95% Confidence Interval (CI) 0.70–1.72) and former users (RR=0.81, 95% CI=0.58–1.14) were at no increased risk of developing leukemia. For AML, current users also had no increased risk (RR=0.86, 95% CI=0.39–1.92) and there was a suggestion that former users had a slight decreased risk (RR=0.68, 95% CI=0.38–1.22). For CLL, all results were around unity. Duration of ERT or time-dependent use had no appreciable effect on the RRs. We conclude that ERT use is unlikely to be a risk factor for leukemia, including AML. Supported by NCI R01 CA39741.

Abstract Background. Intracellular one-carbon transfer reactions are essential for nucleotide synthesis and methylation of biologic compounds including DNA. Previous studies have linked genetic variants in one-carbon metabolism genes with risk of developing NHL, but little is known regarding the impact of these variants on disease outcome. We evaluated the hypothesis that inherited genetic variation in genes involved in one-carbon metabolism is associated with overall survival in DLBCL. Methods. We genotyped 30 single nucleotide polymorphisms (SNPs) from 18 candidate one-carbon metabolism genes in 215 DLBCL patients who participated in a population-based case-control study conducted from 1998–2000 using the SEER (Surveillance, Epidemiology and End Results) cancer registries in the Detroit, Iowa, Los Angeles and Seattle. Stage, B-symptoms, first course of therapy, date of last follow-up and vital status through early 2005 were obtained from cancer registry files. Cox proportional hazards analysis was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals for the association between individual SNPs and overall survival, adjusting for age, demographic and clinical factors. We also used parallel modeling strategies to identify the best summary multi-SNP risk score to predict survival. Results. The median age at diagnosis was 57 years (range, 20–74), and 50 (23%) of the patients died during follow-up, with a median follow-up of 57 months (range, 31–78 months) for surviving patients. After adjusting for demographic and clinical variables, SNPs in SHMT1 (rs1979276; HRCT/TT=2.47, 1.31–4.67), BHMT (rs585800; HRAT/TT=2.02, 1.16–3.54), and TCN1 (rs526934; HRTT=1.86, 1.04–3.33) were the strongest and most robust predictors of survival. A summary score of the number of deleterious genotypes (0–3) from these three genes was strongly associated with survival (p=7.9 x 10−5) after accounting for demographic and clinical variables (HR=2.58 per deleterious genotype, 95% CI 1.75–3.80). A risk score combining the three SNPs with clinical and demographic variables (score of 0 to 5) was even more strongly associated with survival (p=1.4 x 10−13); the Kaplan Meier survival curves are shown in the Figure. In a time-dependent ROC analysis, the combined risk score had a concordance index of 0.75 at 5 years of follow-up (95% CI 0.69–0.81). Conclusion: Host genetic variation in the one-carbon metabolism genes SHMT1, BHMT, and TCN1, individually and particularly in combination, was associated with overall DLBCL survival after accounting for clinical and demographic factors, supporting a role for this pathway in disease progression. Future work should evaluate interactions of genes from this pathway with dietary nutrients and therapeutic agents in DLBCL prognosis. Figure Figure

Abstract Next‐generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL‐IPI) in newly diagnosed CLL and high‐count monoclonal B‐cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi‐stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time‐to‐first treatment (TTT), adjusted for CLL‐IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17‐1.39, P = 2.6 × 10 −8 ; c ‐statistic = 0.76). When stratified by CLL‐IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37‐1.72, P = 7.0 × 10 −14 ). Overall, 80% of low/intermediate CLL‐IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12‐2.07, P = .007; c ‐statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL‐IPI, especially among low/intermediate CLL‐IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.

7536 Background: While most patients (pts) with follicular lymphoma (FL) achieve an initial response to treatment, the majority relapse. Recently, Goldman et al. reported that surveillance imaging (SI) in FL is not associated with improved post-relapse outcomes and is frequently associated with false positive scans (ASH 2017). The goal of this study was to validate these findings. Methods: Pts were enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), a prospective cohort of newly diagnosed lymphoma pts. All pts were followed for events including relapse, re-treatment, and death, with events verified by medical records. Pts eligible for this study had biopsy proven FL grade 1, 2, or 3a who had achieved a response after induction therapy and later relapsed. Initial and post-treatment management was per treating physician. Medical records were re-reviewed in pts with events for clinical details at relapse in relationship to planned follow-up visits and SI. Relapse detection was categorized either by clinical suspicion (CS) via history, exam, and/or labs; or by SI in an asymptomatic pt. Univariate survival analysis was conducted for each variable. The hazard ratio with 95% confidence interval based on Cox Proportional Hazard models was presented along with the log rank test p-value. Kaplan-Meier plots were produced to evaluate the difference in overall survival (OS) between groups. Results: 1121 FL pts were enrolled in MER from 2002-2015, of which 117 were eligible. Median age at diagnosis was 60 years (range 35-83), and 61% were men. Median time to relapse of the 117 pts was 26 months (range 9-125). At a median follow-up from relapse of 69 months (range 0.23-179), 26 pts died. Pts completed a median of 3 imaging studies (range 0-15) during post-induction surveillance. 63 relapses (56%) were detected based on CS; 50 (44%) were detected by SI; and 4 were unknown. There was no difference in OS from relapse for those with relapse detected via CS vs. SI (HR = 0.98 [0.45,2.12], p = 0.96). Conclusions: Routine SI does not appear to improve survival outcomes in FL after achieving first remission, and its common practice should be questioned. Further investigation of prognostic markers to identify high-risk FL pts who may benefit from SI is needed.