Citations

Abstract The targeted therapy ibrutinib inhibits B cell receptor signaling (BTK inhibitor) and has yielded high response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL). However, it is widely believed that the addition of immune therapies to targeted drugs will be required to activate anti-tumor immunity and work towards curative therapy. Identifying effective combinations of targeted drugs and/or standard chemotherapy with immunotherapy is a priority research area and particularly relevant for CLL, as patients' T cells have been shown to exhibit profound tolerance/exhaustion and notably, no activity was reported in a recent trial of anti-PD-1 immunotherapy for relapsed disease. Ibrutinib has shown beneficial immunomodulatory activity in CLL by inhibiting IL-2-inducible T cell kinase (ITK) as well BTK that is associated with increased effector CD4+and CD8+ T cell numbers and decreased expression of inhibitory checkpoint receptors such as PD-1 on patient T cells. Here we have performed comparative immune bioassays from a randomized phase III trial comparing ibrutinib-based therapy to traditional FCR chemoimmunotherapy to assess the effects of treatments on anti-tumor T cell function. Viable peripheral blood mononuclear cell samples were collected serially (baseline, 6 months and 12 months) from CLL patients on the randomized phase III E1912 trial of ibrutinib and rituximab versus FCR for previously untreated disease to allow longitudinal batched immune analysis. Cytotoxicity assays revealed that highly purified CD3+ T cells from the FCR treated patients at 6 and 12-month time-points did not change their activated killing function against autologous baseline CD19+ CLL tumor B cells (acting as target antigen-presenting cells pulsed with superantigen, sAg) compared to pre-treatment/baseline exhausted T cells (n=22). In contrast, patients treated with ibrutinib-based therapy had a significant increase in activated anti-tumor T cell killing function (P<.01, n=22) at both 6-month (66% increase) and 12-month (89% increase) time-points. Flow cytometric analysis of circulating immune subsets revealed that the percentage of PD-1 and PD-L1 positive cells among CD8+ and CD4+ T cells (particularly effector compartments) were reduced with ibrutinib-based therapy, whereas only a partial reduction was detected following FCR treatment. However, patients' T cells from both treatment arms responded normally to T cell receptor engagement by upregulating these checkpoint molecules. This led us to explore ex vivo treatment of highly purified CD3+ T cells and CD19+ CLL B cells from both treatment arms with anti-PD-L1 or anti-PD-1 immunotherapy prior to cytotoxicity assays. Our functional data revealed that the T cells from both FCR time-points (6 and 12-months) were not sensitive to either anti-PD-L1 (n=14) or anti-PD-1 (n=14) treatment. In contrast, ibrutinib-based treatment sensitized anti-tumor T function (23% increase in killing) following anti-PD-L1 treatment (n=14) at the 6-month time-point only (P<.01) but not with anti-PD-1. To investigate the mechanism underlying these effector function differences, we compared the ability of highly purified CD3+ T cells from each treatment arm (n=45) time-point to form F-actin immunological synapses with baseline autologous CLL tumor B cells. Quantitative confocal image analysis revealed that ibrutinib-based therapy significantly (P<.01) enhanced polarization of F-actin, tyrosine-phosphorylated proteins and granzyme B at immune synapses with tumor cells at both 6 and 12-month time-points, whereas FCR treated patient T cells failed to mobilize these lytic synapse molecules. Importantly, our assays have revealed that T cells from both FCR treatment time-points formed "non-polarized" immune synapses with tumor cells, in keeping with cytotoxic dysfunction and insensitivity to additional checkpoint immunotherapy. In contrast, our functional correlative bioassays have revealed that ibrutinib-based therapy can reactivate exhausted cytolytic T cell function and suggest to us, a potential therapeutic window for anti-PD-L1 immunotherapy at the earlier 6-month time-point. We believe this data supports the concept of incorporating functional bioassays to immune-monitoring assays associated to clinical trials that should aid knowledge-led design of future combination immunotherapy. Disclosures Kay: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding; MedImmune: Research Funding.

Abstract Background Predictors of outcomes after reduced intensity conditioning (RIC) stem cell transplantation (SCT) in chronic lymphocytic leukemia (CLL) are not well established. The Dana Farber (DF) investigators (Brown et al, leukemia 2013) developed a prognostic scoring model for overall survival (OS) and disease free survival (DFS) that assigned one point to each of comorbidity index, absolute lymphocyte count, LDH and disease status. The goals of the present study were to 1) validate the DF model 2) identify additional predictors of outcomes after SCT. Methods This study is a retrospective review of the Mayo Clinic CLL database and was approved by the Institutional Review Board. All CLL patients that underwent RIC-SCT were included. The prognostic significance of prognostic factors at time of RIC-SCT were analyzed, including the DF model. Survival was estimated and compared using the Kaplan Meier and Log Rank tests. Results From 2006 to 2012, 50 patients with CLL underwent RIC-SCT at the Mayo Clinic. Of these, 33 were male (66%) and the median age was 56 (29-69) years. The median time from diagnosis to RIC-SCT was 4.7 years (0.6-22.9). Indications for RIC-SCT were 17p deletion in 24 (48%) patients and purine nucleoside analogue (PNA) refractory disease in 26 (52%) patients. Transplant related mortality was 6% and non-relapse mortality at 5 years was 14%. Acute graft versus host disease (GVHD) developed in 30 (60%) patients and chronic (c) GVHD was noted in 32 patients (64%). The 5-year relapse rate was 38%. The DF prognostic score stratified three categories for OS (score of 0, 1-2, ≥ 3), but was not associated with DFS in this cohort. We next evaluated the impact of other patient, disease, and transplant characteristics on clinical outcome. Development of cGVHD post-transplant was the dominant predictor of both DFS (HR 0.26, 95% CI=0.10-0.69, P=0.006) and OS (HR 0.04, 95% CI=0.01-0.19, P<0.0001, figure 1). The presence of 17p deletion at time of RIC-SCT was associated with shorter DFS (HR 3.99, 95% CI=1.43-11.18, P=0.008), but did not impact OS. Developing cGVH but not DF score remained an independent predictor of OS on a multi-variate analysis including both factors. Given these findings, we evaluated what pre-treatment patient, disease, and transplant characteristics predicted for subsequent development of cGVHD. Patients with higher scores on DF prognostic model were less likely to develop cGVHD (score 0=66%, 1-2=76%, ≥3=31%; P=0.01). Three CLL characteristics (complete or partial remission at time of RIC-SCT, absence of 17p deletion, and absence of ZAP70 overexpression) were predictors for development of cGVHD while alemtuzumab exposure ≤3 months prior to RIC-SCT decreased likelihood of cGVHD. On multivariate modeling using these four predictors, only ZAP70 overexpression was an independent negative predictor of developing cGVHD (Table 1). Rates of cGVHD post RIC-SCT in ZAP 70 positive and ZAP70 negative patients were 50% and 92%, respectively (p=0.03). Conclusion This study found that while the DF prognostic score is an effective way to predict outcome prior to RIC-SCT, development of cGVHD post-transplant is ultimately the most significant predictor for both OS and DFS after transplant in patients with CLL. Pretransplant characteristics that predict subsequent cGVHD include DF prognostic score and ZAP70 status. This is the first report to identify biologic characteristics of CLL B-cells associated with development of cGVHD post-transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract Despite recent advances and improvement in treatment options, chronic lymphocytic leukemia (CLL) is still an incurable disease. Recent advancement in parallel sequencing technology has provided better understanding of genetic landscape and clonal architecture underlying disease course. Furthermore, mutation status of TP53, SF3B1, NOTCH1, and BIRC3 has improved current CLL risk stratification. In order to better understand the genetic basis and clonal evolution underlying disease progression we analyzed a homogeneously treated cohort of CLL cases at multiple time points before and after therapy. We performed WES in 30 sequential samples from 12 CLL cases. All 12 cases received PCR (pentostatin, cyclophosphamide and rituximab) based chemoimmunotherapy as initial treatment. Of these 12 cases, two had samples available both >6 months prior to enrollment and at treatment initiation, seven patients who experienced relapse had samples available at both treatment initiation and at and the time of relapse, and three patients had samples >6 months prior to enrollment, at treatment initiation, and at relapse. These cases were previously characterized by aCGH/FISH. R function Mclust and kernel density estimate (KDE) plots were used to identify subclonal heterogeneity. An average of 102-fold depth coverage was obtained with an average of 80% of the targeted regions being covered by at least 30X. Overall, we detected a total of 219 nonsynonymous Single Nucleotide Variants (SNV) and indels (average 19, range 11-33). Del13q (42%) was identified as most prevalent copy number abnormality followed by trisomy 12 (33%), del11q and del17p (17% each). Recurrent mutations were identified in NOTCH1 (33%), DDX3X (25%), TP53, SF3B1 XPO1 and MED12 (17% each). Other tumor implicated genes such as CARD11, NOTCH2, NOTCH4, DIS3, TRAF2, NFKBID, CHK2 and RB1 were found mutated in individual cases. In all 12 cases we identified at least one relevant cytogenetic abnormalities (del11q32, del13q14, del17p13, trisomy 12) and/or a mutated driver gene. Interestingly we found NOTCH1, DDX3X and XPO1 mutated at a significantly higher prevalence than previous studies (10%, 2% and 4%, respectively). Since all cases in our cohort correspond to individuals with progressive disease, a plausible explanation for the higher prevalence of mutations in our cohort is that these mutations are associated with disease progression. By WES we identified multiple subclones in 58% of cases (7 of 12 cases), as compared to previously identified 33% (4 of 12 cases) by CGH/FISH. In 5 chemotherapy naïve cases, no significant increase in genomic complexity was detected with disease progression. In contrast, 5 out of 10 cases with samples analyzed before CIT and at the time of relapse had changes in clonal dynamics, with initially dominant subclones diminishing in response to therapy and the available empty niche being occupied by expansion of another “fitter” subclone at the time of relapse. No acquisition of tumor associated gene mutations was seen post therapy or associated with relapse in 9 of 10 cases. In the remaining case we identified an interesting example of convergent evolution. The pre-therapy sample was comprised of a unique clone characterized with del11q13, mutations in SF3B1 (G742D) and DDX3X (pE196fs). After therapy that clone was either eradicated or reduced to below detection level. Interestingly another clone emerged after relapse that harbored an independent del11q22 and mutations affecting different aminoacids of SF3B1 (K700E) and DDX3X (pL21fs). The strong selection of impairment in these genes during disease progression suggests a key role of these pathways/genes in the pathogenesis of this patient. In conclusion, these studies provide additional insight into the genomic landscape and clonal architecture in a cohort of homogeneously treated CLL cases before and after therapy. Stable clonal architecture was seen in prior to treatment, whereas clonal evolution leading to increased tumor heterogeneity occurred after the under selection selective pressure of therapy in relapsed cases. Early identification of tumoral heterogeneity and underlying genetic abnormalities might provide better targets for therapy to decrease the likelihood of relapse. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract The characteristic clinical heterogeneity observed in chronic lymphocytic leukemia (CLL) reflects its biological diversity and as a consequence, better understanding of the biology of this disease has led to the identification of a number of useful prognostic tools. Advances in massively parallel sequencing technologies have unearthed genetic and epigenetic heterogeneity and reinforced the concept that specific genetic lesions and the development of genomic complexity can modulate the pathology of CLL. In addition, DNA methylation profiling has shown that CLL patients can be grouped into at least three clinically relevant epigenetic subgroups with distinct outcomes. However, despite this explosion in knowledge, defining the prognosis of individual CLL patients remains a significant clinical challenge. Consequently, there is a need to identify prognostic tests that can provide reliable personalized risk assessments. We have recently shown that a subset of CLL patients manifest telomere dysfunction, which drives genomic instability and clonal evolution. We went on to show an association between short, dysfunctional telomeres and 'high-risk' genetic lesions1,2. Importantly, longitudinal analysis confirmed that tumor cell telomere length remained remarkably stable throughout the course of the disease suggesting that the telomere length is fixed at an early point in disease pathogenesis making it a reliable predictor of both clinical progression and overall survival (OS) regardless of when the measurement is made. In this study, we generated telomere length profiles in 224 newly diagnosed, early stage CLL patients seen at the Mayo clinic in Rochester using high-throughput single telomere length analysis (HT-STELA). We then tested the ability of telomere length to predict time to treatment (TTT) and OS and compared its predictive power with DNA methylation profiling and CLL-IPI scores. Patients with a mean telomere length inside the fusogenic range (IFR) had a significantly shorter time to treatment (P<0.0001; HR = 3.2) and OS (P = 0.0027; HR = 2.3) when compared to patients with a mean telomere length outside the fusogenic range (OFR) (Figure 1A and B). When considering epigenetic subgroups, we found a strong association between telomere length and 'epitype'; high programmed (HP) CLL cells had significantly longer telomeres than intermediate programmed (IP), and low programmed (LP) CLL cells (P = 0.0002 and P<0.0001 respectively). Furthermore, bifurcation of the HP and IP groups into telomere length OFR and IFR subsets identified patients with distinctly different TTT (P = 0.0008; HR = 2.74) and OS (P = 0.004; HR = 2.4). 79/81 (97.5%) of the LP group had short telomere length IFR, but when we divided the LP group above and below the mean telomere length of the fusogenic range (≤/>2.17kb), patients with the shortest telomeres had a significantly worse OS (P = 0.034; HR = 2.83). Telomere length was also strongly associated with CLL-IPI score; the CLL-IPI low-risk group (CLL-IPI 1) had the longest telomeres and the CLL-IPI high-risk group (CLL-IPI 4) had the shortest telomeres (P<0.0001). More heterogeneity in telomere length was observed in the CLL-IPI 1 and 2 groups and patients with telomere length IFR in these 'low-risk' groups showed significantly shorter TTT (P<0.0001; HR = 4.24) and overall survival (P<0.0001; HR = 10.1) (Figure 1C and 1D). Finally, multivariate modelling, using Cox proportional hazards with forward selection, revealed that telomere length retained independent prognostic significance when CLL-IPI score was entered in the model for time to treatment (HR = 1.42; P = 0.023) and OS (HR = 1.68; P = 0.008). In conclusion, telomere length analysis provides more accurate prognostic information about the progression and outcome of CLL patients and can augment the prognostic power of the CLL-IPI for early stage CLL. References Lin TT et al. Blood. 2010;116(11):1899-907. Britt-Compton et al. Leukemia. 2012; 26(4):826-30. Disclosures Norris: Cardiff University: Patents & Royalties: Telomere measurement patent. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Jansen: Research Funding. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Fegan:Napp: Honoraria; Gilead Sciences, Inc.: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

Abstract Background Approximately 2-8% patients with chronic lymphocytic leukemia (CLL) will transform to diffuse large B-cell lymphoma (DLBCL, Richter syndrome [RS]). Clinical characteristics and molecular markers at the time of CLL diagnosis are associated with the risk of RS; however, there are no data regarding germline genetic variations and the risk of RS. Genomewide association studies (GWAS) have shown several single-nucleotide polymorphisms (SNPs) that are associated with a higher risk of familial CLL. It is not known whether any of these polymorphisms also predispose to RS. Methods Since 2002, all consecutive patients with newly diagnosed (<9 months diagnosis) CLL at Mayo Clinic were offered enrollment into a prospective genetic epidemiology study. Patients completed extensive epidemiologic questionnaires and baseline clinical, laboratory, and biologic data were abstracted using a standard protocol. Genotyping of germline tissue at diagnosis was performed using an Illumina iSelect panel and Affymetrix 6.0 SNP chip. All patients were prospectively and longitudinally followed at defined time-points with systematic collection of data on treatments, second cancers, and RS. All patients with biopsy-proven DLBCL during follow-up were considered to have undergone transformation into RS. Time to RS was calculated from CLL diagnosis date until RS or until last follow-up date for those with no RS. SNPs were modeled in two ways: ordinal and dominant. Cox regression was used to estimate hazard ratio (HR) for individual SNPs with time to transformation. Results Thirteen of the GWAS-discovered SNPs associated with risk of developing CLL were available and genotyped on 620 CLL patients. Median age at diagnosis of CLL was 62 years (range 27-88), and 428 (69%) were male. Three hundred and ten (51%) patients were low (0) Rai stage, 271 (45%) were intermediate (I-II) Rai stage, and 22 (4%) were advanced (III-IV) Rai stage. The immunoglobulin heavy chain gene was unmutated in 189 (40%) patients; 157 (32%) patients expressed ZAP-70, 163 (29%) expressed CD38 and 104 (31%) expressed CD49d. Fluorescence in-situ hybridization (FISH) revealed that 210 (41%) patients had del13q, 90 (18%) patients had trisomy 12, 37 (7%) had del11q, 23 (5%) had del17p and 141 (28%) had no detectable FISH abnormalities. As of last follow-up, 239 (39%) patients received therapy for CLL. After a median follow-up of 5.9 years (range 0-11), 15 (2.4%) patients developed biopsy-proven RS. The median time to RS in these 15 patients was 4.5 years (range 1.0-8.7 years). The ordinal HR for the 13 SNPs tested, their corresponding genes, and p-values are shown in Table 1. Germline polymorphisms in a single SNP, rs4987852, encoding for BCL2 (chromosome 18), was significantly associated with an increased risk of RS (ordinal HR=3.9; 95% CI=1.6-9.8; p-value=0.004). This allele was present in 48/605 (8%) non-transformed CLL patients compared to 4/15 (27%) of patients with RS. Time to RS according to the Kaplan-Meier analysis for rs4987852 is shown in Figure 1. This SNP is located in a region in which t(14;18) translocation breakpoints commonly occur in follicular lymphoma and overexpression of BCL2 leads to an increased incidence of B-cell lymphomas in mice. Conclusion Our results suggest that inherited genetic polymorphisms predispose CLL patients to develop RS. Specifically, SNP (rs4987852) present on the BCL2 gene on chromosome 18 in CLL is associated with an increased risk of transformation to RS. These observations require replication in other CLL cohorts. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is defined as somatic mutations in clonal blood cells of individuals without any other hematological abnormalities and have been identified using whole exome sequencing (WES) from pre-specified genes. CHIP is reportedly associated with aging, reduced overall survival, cardiovascular disease, and hematologic disease, mainly myeloid cancers. Additionally, whole genome sequencing and WES studies in CLL cases have identified putative CLL driver genes with recurrent somatic variants or copy number alterations. Here, we investigate the associations of CHIP and mutations in CLL somatic driver genes with risk of CLL and MBL, a precursor to CLL. Using WES, 571 individuals from 132 CLL families were sequenced from two different sites, Mayo Clinic (92 families) and National Cancer Institute (NCI, 40 families), including 251 CLLs, 74 MBLs, and 246 unaffected relatives. DNA was extracted from buccal cells (Mayo Clinic) and peripheral blood (NCI). Allele counts from gVCF files were extracted for specific genes and analyzed. Variants were annotated using the Variant Effect Predictor in Ensembl, genome build GRCh37 (http://useast.ensembl.org/index.html). Highly-sensitive flow cytometry of the blood was used to screen for MBL. CHIP was defined as in Genovese et al., 2014 as the presence of any of the following: A) disruptive mutations in ASXL1, TET2, or PPM1D; B) missense mutation p.617F in JAK2; C) disruptive or missense mutations in DNMT3Alocalized to exons 7 to 23D) any mutations reported at least 7 times in hematological malignancies in the Catalogue of Somatic Mutations in Cancer (COSMIC); and (E) No variant allelic fraction (VAF) threshold. CLL driver genes included disruptive or missense mutations in ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, or FBXW7with VAF greater than 0.1. Mutations from COSMIC and CLL driver genes were excluded if the minor allele frequency in 1000 Genomes was greater than 0.5%. Data was analyzed using logistic regression adjusting for age, sex, and site. Odds ratios (OR) and 95% confidence intervals (CI) were estimated. Median age for CLL, MBL, and unaffected was 63, 61.5, and 54, respectively. The unaffected relatives were significantly younger than both the CLL and MBL groups (p<0.0001), with no difference between CLL and MBL (p=0.46). A total of 70 (12%) individuals from 54 different families had CHIP. CHIP was characterized in the majority of samples as mutations in DNMT3A, ASXL1, and TET2with 43, 9 and 8 individuals, respectively. As expected, CHIP was associated with age (p<0.001) but not sex (p=0.87). CHIP was present in 42 (17%) CLLs, 9 (12%) MBLs, and 19 (8%) unaffected relatives. There was a significant association between the presence of CHIP and CLL risk compared to unaffected relatives (OR= 2.19, 95% CI: 1.20 - 4.13, p=0.013) but not with MBL risk (OR=1.34, 95% CI: 0.54 - 3.11, p=0.51). Using the CLL driver genes, we found mutations in 119 (47%) CLL, 20 (27%) MBL, and 70 (29%) unaffected relatives. Mutations in CLL driver genes were associated with CLL risk compared to unaffected relatives (OR = 2.51, 95% CI: 1.64 - 3.87, p<0.0001), but there was no evidence for increased MBL risk (OR = 1.00). These results held when we subset the analysis to the Mayo only data where the DNA was extracted from buccal cells. This study further supports the association between mutations in CHIP related genes and risk of hematologic malignancies, in particular CLL. This is the first report of the prevalence of CHIP in MBLs. Although MBL and CHIP represent clonal population of blood cells, our data suggest the clones are different. Mutations in CLL driver genes are also associated with CLL risk but show little to no evidence with increased MBL risk. Interestingly, our unaffected relatives of CLL cases also had a high frequency of CLL driver mutations. Future studies are needed to understand these acquired mutations (both in CHIP- and CLL driver- related genes) in the unaffected to MBL to CLL continuum. Disclosures Parikh: Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Research Funding. Ding:Merck: Research Funding. Cerhan:Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding; Nanostring: Research Funding. Shanafelt:Jansen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract Background MBL is a precursor state to CLL, with a prevalence of ~5-8% in the general population and 15-18% in CLL families. To date, 41 single nucleotide polymorphisms (SNPs) have been found to be associated with CLL. We previously showed that a PRS of the weighted average of the number of risk alleles of these SNPs is associated with CLL risk using cases and controls from the International Lymphoma Epidemiology (InterLymph) Consortium. We validated this score in an independent sample of CLL cases and controls from the Genetic Epidemiology of CLL (GEC) Consortium, a cohort of families with ≥2 members with CLL. In the CLL families we also reported an association between the PRS and MBL risk, where 93% of MBLs were low-count MBL. Here we evaluate the PRS in an independent sample ascertained from the Mayo Clinic Biobank. Methods The Mayo Clinic Biobank is a large-scale bio-repository of adult patients assembled to provide a wide array of health-related research studies. Biobank participants (N=2530) were screened for MBL using a highly-sensitive, 8-color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow-cytometry assay on stored cryopreserved peripheral blood mononucleotide cells. Low-count and high-count MBL were defined as those individuals who had <85% and ≥85 clonal B-cells out of total B-cells, respectively. Individuals without MBL ("controls") were frequency matched to MBLs based on age and sex, after excluding non-Caucasian and non-interpretable flow results. Participants were genotyped using the Illumina OmniExpress array. Standard genotyping quality-control metrics were applied. We computed the PRS as previously published and categorized it by quintiles based on values previously used with InterLymph controls. The middle quintile served as the reference category. We used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age at consent, sex, and socioeconomic status. In addition, we compared the median PRS by low-count MBL and high count MBL using the Mann-Whitney U test. Results We identified 276 MBLs overall and 2109 controls; 97% (N=267) of MBLs had low-count MBL and only 3% (N=9) had high-count MBL. We then selected 844 controls frequency matched to the MBL cases on age and sex. Family history of leukemia/lymphoma was 11.3% among MBLs overall and 8.8% among controls. The median PRS was 7.88 and 7.46 (Figure) among MBLs overall and controls, respectively. The continuous PRS had a 1.75-fold increased risk for MBL overall (CI:1.5-2.1, P=2.65x10-12). Compared to the middle quintile, the highest quintile had 2.2-fold increased risk for MBL overall (CI:1.5-3.3, P=1.46x10-4) and the lowest quintile had 0.6-fold decreased risk (CI:0.4-0.96, P=0.03). The median PRS for low-count and high count MBL were 7.86 and 8.04, respectively. The continuous PRS had a 1.75-fold increased risk for low-count MBL (CI:1.5-2.1, P=4.81x10-12) and a 1.89-fold increased risk for high-count MBL (CI:0.8-4.2, P=0.13), however, the effect is not statistically significant between high-count and low-count MBL (P=0.89). Conclusion In this independent sample, we validated our previous findings that the CLL PRS is associated with MBL risk. Although the sample size of high-count MBL was limited (N=9), there is evidence of a weak trend towards a higher PRS compared to low-count MBL and controls Larger sample size of high-count MBL is warranted in order to stratify MBL risk by low-count and high-count MBL. These results may help identify individuals at higher risk of MBL including low-count and high-count MBL, beyond the risk associated with age and family history of CLL. Figure: Polygenic risk score distribution by control and MBL status Figure. Figure. Disclosures Parikh: Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding. Cerhan:Nanostring: Research Funding; Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board. Kay:Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding.

Abstract Abstract 4607 Background: Extramedullary Chronic Lymphocytic Leukemia (EM-CLL) is reported in the literature as case reports and series. The prognosis, patient characteristics, and treatment approaches to EM-CLL have not been fully analyzed in a comprehensive fashion. We therefore conducted a systematic review to better understand EM-CLL and how it might affect patients' clinical course and overall survival. Methods: A comprehensive PubMed search for English-language articles published between 1975 and 2010 in peer-reviewed journals was performed. EM-CLL was defined as CLL identified pathologically in these reports outside the bone marrow. Search terms included “EM-CLL, CLL outside the bone marrow, and CLL with each system [central nervous system (CNS), genitourinary (GU/GYN), gastrointestinal (GI), cardiac, pulmonary, head and neck (H+N), skin, and vascular]. Data regarding location, sex, age at diagnosis, systemic CLL disease setting (relapsed vs. initial diagnosis), laboratory studies/prognostic factors (when available), treatment for CLL (yes or no), use of chemotherapy (yes or no), use of radiation (yes or no), use of rituximab (R), use of purine analogues, outcome (alive or deceased), and follow-up were collected. A z-test was used for assessing proportion differences for gender, stage (Rai 0, 1, 2 vs. Rai 3, 4), and chemotherapy (yes or no). Univariate analyses were performed via the Chi-square test. A backward stepwise logistic regression analysis was performed to identify whether disease location, disease setting, age, and sex influence overall survival (OS). No other multivariate analyses were performed due to large percent of missing data for different variables of interest. A two-tailed P level of 0.05 was considered statistically significant in all analyses; which were performed using SPSS for windows, version 19.0 (SPSS Inc.) and StarStat (DataStar, Inc.). Results: A total of 192 cases were identified [63 (33%) skin, 51 (27%) CNS, 26 (14%) GI, 20 (10%) GU/GYN, 10 (5%) lung, 10 (5%) ocular, 6 (3%) heart, 5 (3%) H+N, and 1(0.5%) vascular]. Median age at detection of EM-CLL was 62 (range, 25–89); significantly more men were diagnosed with EM-CLL versus women (61% vs. 35%; z = 3.51, P <.001); more patients were at early stage (Rai 0, 1, 2) versus advanced (Rai 3 and 4) at time of EM-CLL but this difference was not statistically significant (22% vs. 18%; z = .43, P =.665). More patients with EM-CLL were treated with chemotherapy for their underlying systemic CLL than not (65% vs. 15%, z = 6.25, P <.001); very few patients received radiotherapy which was delivered for their EM-CLL (n=35; 18%) but treatment information was missing from approximately 20% of cases. Our analysis showed that there was no significant difference with regard to number of patients treated with chemotherapy before and after the contemporary chemoimmunotherapy era (defined here as before and after the year 2000) (87% vs. 78%, p =.171). As far as systemic therapy for CLL, 12% of pts received R and 14% received purine analogues. Tissue sites such as CNS (45%), ocular (33%), H+N (25%), and skin (19%) were significantly more likely to receive XRT (p <.001) but no significant differences between the sites were reported for chemotherapy (p =.106), R (p =.237), and purine analogues (p =.090) usage. OS data was missing in 56 cases (29%), 79 were alive (41%), and 54 (28%) were dead at the time of case reporting. Backward stepwise logistic regression analysis, which included data for 102 (53%) patients showed that EM-CLL detected in patients with systemic relapse had worse OS compared to those with EM-CLL at time of initial CLL diagnosis (p =.027), while no significant effect on OS was found for tissue site (p =.085), age (above or below 70, p =.796), and sex (p =.791). All other variables were not significant. Conclusions: To our knowledge, this analysis represents the largest collection of EM-CLL series examined comprehensively. EM-CLL is not an uncommon presentation in the spectrum of CLL/SLL with skin and CNS being the most commonly affected organs (33% and 27% respectively). Patients who develop EM-CLL in the relapsed setting have significantly worse OS compared to those who manifest EM-CLL at the time of initial diagnosis. We propose that this subset of CLL needs a prospective and robust accumulation of prognostic and clinical data as it will be critical in defining how extramedullary presentation affects the biology and natural course of CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: B-cell non-Hodgkin lymphomas (NHL) are common lymphoid malignancies in which malignant B-cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. The microenvironment that supports the growth and survival of NHL B cells consists of a complex network of immune cells and cytokines and its specific composition has been shown to have significant clinical implications. The intratumoral immune cells include effector and regulatory T (Treg) lymphocytes that are more than simple residual elements from the normal lymph node structure. We previously explored whether the extent of T cell infiltration played a role in the clinical outcome of patients with B-cell NHL and found that an increased percentage of CD4+ T cells in the diagnostic biopsy of lymphoma patients significantly correlated with an improved 5-year overall survival. We have also shown that intratumoral Treg cells significantly suppress the anti-tumor response. Based on the fact that intratumoral T-cells are important in B-cell NHL, we evaluated whether genetic variation in genes that regulate the T-cell response may be associated with lymphoma risk. Methods: We genotyped 257 single nucleotide polymorphisms (SNPs) from 50 candidate genes related to T-cell differentiation and function in a clinic-based study of 441 Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. Tagging and nsSNPs were selected from HapMap. The most prevalent homozygous genotype was used as the reference group and each SNP was modeled individually as having a log-additive effect, expressed as ordinal odds ratios (OR) per variant allele and 95% confidence intervals, in an age- and sex-adjusted logistic regression analysis. We also evaluated the consistency of the findings for the most common types of B-cell NHL - diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and CLL/SLL. For gene-level analyses, we used principal components (PC) analysis using all SNPs from a gene; those PCs that explained > 90% of the variability were then modeled in a logistic regression analysis and significance was determined using a likelihood ratio test. We also evaluated the association of haplotypes from each gene with risk of NHL using the score test implemented from HAPLO.SCORE. Results. The mean age at diagnosis was 60.1 years for cases and 58% were male; among controls, the mean age at enrollment was 61.7 years and 55% were men. In the PC gene analyses, PRF1 (perforin gene - involved in cytotoxicity; p=0.004), CD276 (B7-H3 gene - involved in co-stimulation; p=0.01), TBX21 (T-bet gene – regulates Th1 cell development; p=0.02), and IL6 (p=0.02) were significant at p<0.05; haplotype analysis showed similar results, with the addition of CARD15 (NOD2 gene – regulates Th1 responses; p=0.02). There were 2 SNPs in PRF1, and both the intronic SNP rs3758562 (OR=1.38; 1.13–1.69) and the synonymous coding SNP rs885821 (OR=0.80; 0.63–1.02) were associated with NHL risk overall and for each subtype. There were 2 SNPs in CD276, and both the intronic SNP rs7176654 (OR=1.29; 1.07–1.56) and the mrna-utr SNP rs3816661 (OR=0.82; 0.68–0.99) were associated with NHL risk overall and with the CLL/SLL and follicular NHL subtypes specifically. There were 2 SNPs in TBX21, and only the mrna-utr SNP rs7502875 (OR=0.73; 0.59–0.92) was associated with NHL risk overall and for each subtype. There were 11 SNPs in IL6, and the intronic SNP rs2069835 (OR=1.88; 1.24–2.84) and the locus-region SNP rs2069824 (OR=1.73; 1.20–2.50) were each associated with NHL risk overall and each subtype. Finally, there were 9 SNPs in CARD15, with only the nonsynonymous coding SNP rs5743291 (OR=1.43; 1.02–1.98) being associated with overall NHL risk, and risk was specific to FL. Conclusions. Genetic variation in several genes that play critical roles in T-cell maturation and function was associated with lymphoma risk. These genes appeared to be associated with the differentiation and function of effector T-cells particularly Th1 and Th17 cells. These results provide potentially important insights into the role of T-cells in lymphomagenesis.