Cerhan, James R.

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.

7021 Background: Case-control studies (CCS) have identified germline variants (GVs) & genes associated with AML risk and/or outcome. However, whether these genes also harbor somatic mutations (SMs) in AML and whether these SMs are associated with clinical phenotypes and outcome is unknown. We systemically interrogated PubMed (terms "AML genetic polymorphisms"), yielding 612 reports (1981-2018), from which we identified unique variants/single nucleotide polymorphisms (SNPs) in 100 genes associated with AML risk and/or outcome in CCS. To determine the prevalence and relevance of SMs in these genes in an unselected AML population, we performed an analysis using WES of cases in the highly-annotated MCAEC (Finn, Ca Epidemiol 2015). Methods: We performed WES using remnant cytogenetic pellets from AML diagnosis in 98 MCAEC patients with available samples, sequenced at a depth of ~100 million paired-end 100bp reads using Agilent SureSelectXT Human All Exon V5 + UTRs target enrichment kit. Sequencing reads were aligned to human reference genome, and SMs including non-synonymous (NS) and truncating single nucleotide variants (SNVs) and small INDELs were identified and filtered using Exome Sequencing Project, 1000 genome, HapMap, & Mayo Clinic (MC) internal Biobank GV database. Copy number aberrations (CNAs) were identified & filtered using public CN polymorphism databases. For this analysis we evaluated the prevalence of somatic SNVs, INDELs, & CNAs in the 100 nominated genetic risk genes in collaboration with MC Cancer Center to determine their association with clinical phenotype and outcome. Results: Of the 100 unique genes, there were 40 with SM in our dataset, with 126 total mutations. Most were mutated in a very small number of patients, allowing insufficient statistical power to perform association analyses. However, an NS mutation in CYP2B6 (c.785A > G, p.Lys262Arg) had a minor allele frequency of 12.0%; this variant has been reported in association with breast cancer risk, but not with AML. Conclusions: We studied SM’s in AML genetic risk genes using WES of tumor samples. While representing a smaller sample size, this suggests their contribution to AML development may be limited in a broader clinical population. These results & prevalence of the novel CYP2B6 rs2279343 variant in AML must be confirmed in larger cohorts.

8563 Background: Optimal initial treatment of follicular lymphoma (FL) is unknown. Rituximab as monotherapy (R) or as a component of immunochemotherapy (R+Chemo) is established as effective and it is now reasonable to re-examine the role of chemotherapy dosing. We explored clinical features, systemic treatment and chemotherapy delivery with comparative effectiveness of delivered dose intensity (DDI) on outcomes. Methods: We reviewed the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource database along with medical records on newly diagnosed grade I-IIIa FL who received systemic therapy from 2002 to 2009. Presenting clinicopathologic factors, outcomes and systemic therapy details including doses of chemotherapy were collected. The event-free (EFS) and overall survival (OS) effects of systemic therapy and chemotherapy DDI were analyzed with multivariate Cox regression. Confounding effects of FLIPI, grade, stage, and age were considered in the analysis. Results: From 2002 to 2009, 631 newly diagnosed FL were enrolled. Median follow up duration was 52.7 months. We identified 322 grade I-IIIa FL treated with systemic therapy including 93 R and 229 R+Chemo. Age and stage were similarly distributed between the R and R+Chemo groups; however, patients in the R group had lower grade (p<0.01) and FLIPI (p=0.03). Multivariate analysis showed no significant differences in EFS (HR=1.24, p=0.28) or OS (HR=0.55, p=0.13) for R compared to R+Chemo. Among R-CVP or R-CHOP treated FL, DDI data were collected for 73 doxorubicin (dox) and 137 cyclophosphamide (cyc) patients. Eighty-five percent of patients received 90% or more pre-planned DDI. After controlling for confounding factors, higher cycDDI was associated with improved EFS (HR 0.55, P=0.04) and OS (HR 0.74, P=0.03). No significant OS or EFS effects of doxDDI were observed. Conclusions: Addition of chemotherapy to rituximab was not associated with a detectable difference in survival outcomes in grade I-IIIa FL at a median follow-up of 52.7 months. Among R+Chemo treated FL, chemotherapy was delivered completely in most patients and more completed delivery of cyclophosphamide was associated with improved EFS and OS.

8504 Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma. The optimal follow-up strategy for patients (pts) in remission is not clear. The goal of this study is to determine the utility of surveillance scans in a large, prospective, multi-institutional cohort of DLBCL pts. Methods: Patients 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. Patients eligible for this study had biopsy proven DLBCL and were treated with anthracycline based immunochemotherapy (IC). Initial and post-treatment management was per treating physician. Medical records were re-reviewed in pts with events for clinical details at relapse and relationship to planned follow-up visits and surveillance scans. Results: 644 pts with DLBCL treated with IC were enrolled in MER from 2002-2009. Median age was 63 years (range 18-92), 54% were men, and median f/u was 59 months (range 8-116). 537 pts entered post-treatment observation; 109 (20%) of the 537 pts relapsed and 41 died from other causes. 42% of relapses were in the first 12 months following diagnosis, 27% between 12-24 months, and 31% >24 months. In the 109 who relapsed, 62% of pts (62/100, 9 unknown) presented to their physician earlier than a planned follow-up visit due to symptoms. At the time of relapse, 68% were symptomatic, 42% of pts had abnormal physical exam, and 55% had elevated LDH; 87% of pts had ≥1 of these features. Of the 38 pts with relapse detected at a planned visit, 26 had clinical features of relapse and 12 pts had relapse detected solely by planned surveillance scan; 4 pts had relapse of low-grade or other subtype and 8 had DLBCL relapse (4 of whom had equivocal/positive PET at the end of IC). Thus, surveillance scanning detected DLBCL relapse prior to clinical manifestations in only 8/537 pts (1.5%) observed post DLBCL therapy. Conclusions: The vast majority of DLBCL relapses occur outside of planned follow-up visits and are accompanied by symptoms, physical exam, or laboratory abnormalities. Routine surveillance scans post-therapy add little to detection of DLBCL relapse.

5082 Background: We evaluated the association of germ-line variation in 10 candidate genes important in prostate cancer biology (NKX3-1, JAK2, SLCO2B1, STAT3, CYP19A1, HSD17B4, TRMT11, PRMT3, HSD17B12, NCOA4) with failure of AA in advanced prostate cancer patients. Methods: Patients (N=619) were enrolled for genotyping at the time of AA failure. Candidate genes were selected from the literature and previous pilot studies in smaller cohorts which had identified variation in TRMT11 and HSD17B12 to be associated with AA failure. Genes were tagged using single nucleotide polymorphisms (SNPs) from HapMap with minor allele frequency of >5% and r2≥0.8. DNA was extracted from peripheral blood and genotyped using Illumina Veracode platform. The primary endpoint was time to failure on AA, defined as time from initiating continuous AA to two serial PSA increases over the nadir or clinical or imaging based progression, whichever came first. Principal component analysis was used for gene-levels tests. Association with the primary endpoint was assessed using proportional hazards regression models at the gene-level and at the SNP level. For SNP level results we estimated per allele (ordinal model) hazard ratios (HR) and 95% confidence intervals (CI) using Cox regression, adjusted for Gleason score (GS). Results: We successfully genotyped 60 SNPs (from 10 genes) in 519 subjects. The median age of the cohort was 71 years (range 43, 92). The GS distribution was 51% subjects with GS≥8; 33% with GS=7 and 17% with GS<7. Median time to AA failure for the cohort was 2.3 years (IQ range: 1.0-4.5). In gene level analyses adjusting for GS, TRMT11 (p=0.004), HSD17B12 (p=0.016), and JAK2 (p=0.044) genes were associated with time to AA failure. Four of the 18 genotyped SNPs in JAK2 were associated with AA failure after adjustment for GS (Table). Conclusions: Variation in the JAK2 gene is significantly associated with time to AA failure. Validation is needed to develop these as predictive biomarkers for AA in advanced prostate cancer. [Table: see text]