Citations

Background: NGS of myeloid mutations is an integral part of AML clinical decision-making. There is currently no information regarding concordance between NGS panels in AML using samples from the same patient across various platforms in different diagnostic laboratories. To study this important question, we analyzed NGS of myeloid mutations in diagnostic samples from The Beat AML Master Trial (BAMT) for newly diagnosed older AML patients, and compared variant calls made between institutional laboratories enrolling the study subject with those made by Foundation Medicine (FM), the central laboratory used for treatment assignment in this precision medicine trial. Methods: We identified newly diagnosed AML patient samples (peripheral blood (PB) and/or bone marrow (BM)) from 2 lead institutions in the BAMT(Ohio State, OSU and Oregon Health and Sciences University, OHSU) that were analyzed by both the institutional and by FM from Nov 15, 2016 to Apr 15, 2019. Samples sent to both laboratories >3 days apart were excluded. Samples were analyzed at the institutional laboratories using their respective NGS mutational panels and by FM using the FoundationOne®Heme(FMH) NGS panel which utilizes capture based sequencing. The OSU NGS assay utilizes sequencing on Illumina MiSeq. The OHSU NGS assay employs semiconductor-based sequencing (Ion Torrent PGM platform). The variant allele frequency (VAF) sensitivity for detection for all 3 laboratories range from 1-2%. We evaluated the ability to identify mutations in 8 genes : FLT3, IDH1/ 2, NPM1, TET2, DNMT3A, WT1 and TP53 used in treatment assignment in theBAMT. A detection cutoff of 2% was used to define the presence or absence of a mutation. Overall, agreement was defined as the number of times the local and central laboratories made the same call divided by the total number of patients. Sensitivity was defined as the number of present calls made locally divided by the number of present calls made centrally, and specificity as the number of absent calls made locally divided by the number of absent calls made centrally. The overall kappa statistic, controlling for institution, provided another measure of agreement between local and central calls, where a value of 1 indicates perfect agreement. Results: 194 patient samples were identified using methods above and analyzed locally at the screening institution (125 at OSU, 69 at OHSU) and centrally at FM. Type of tissue analyzed for variants between local site and FM were 59 PB, 129 BM, and 6 with BM/PB mismatch. Overall agreement in presence/absence calls between local and central results for each of the 8 genes was over 95% (Table 1). There was perfect agreement for NPM1. The sensitivity was above 94% for all genes except TP53 (88.6%) and WT1 (63.6%). Failure to detect a mutation locally was primarily due to reporting of all TP53 variants, including variants of unknown significance (VUS) (5) by FM as agreed upon in the study protocol, detection at low levels below local site sensitivity cutoff (1), detection of variants in a portion of gene not covered at the local site(1)and possible artifact (1). For the WT1 gene, discordance in 5 samples included VUS (3) reported by FM ,a variant detected in a portion of the gene not covered at the local site(1).and difference in leukemic tissue analyzed with mutation not detected by the central laboratory on a PB sample, and present at the institutional lab on a BM sample; affecting the overall agreement and specificity but not sensitivity. Specificity was at least 98% for each of the 8 genes. Finally, most discrepancies in reported mutations in FLT3 (n=2), IDH1 (n=1), IDH2 (n=2), DNMT3A (n=4) and TET2 (n=5) were due to reporting of VUS in one laboratory and not by another. Conclusion: Detection of pathogenic myeloid mutations using orthogonal assays showed a high degree of concordance for genes used in therapeutic assignment on the BAMT.The small number of discordant results, in TP53 and WT1, were attributed to the reporting of VUS. This study illustrates the importance of quality control and standardization as NGS continues to be widely utilized in AML for clinical decision making, with a variety of platforms across multiple laboratories. Our next steps involve evaluating the differences in VAFs reported between local and central laboratories when a given mutation is identified, as well as the potential reasons for observed differences and clinical implications of known pathogenic mutations vs putative VUS. Disclosures Borate: Daiichi Sankyo: Consultancy; AbbVie: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Pfizer: Consultancy. Vergilio:Foundation Medicine: Employment; Roche Holding AG: Equity Ownership. Stein:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Patel:France Foundation: Honoraria; Dava Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Astellas: Research Funding; Abbvie: Research Funding; AI Therapeutics: Research Funding; Forma: Research Funding; Incyte: Research Funding; Kite: Research Funding; Takeda: Research Funding. Stock:Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Schiller:Amgen: Other, Research Funding; Astellas: Research Funding; Biomed Valley Discoveries: Research Funding; Bristol Myer Squibb: Research Funding; Celgene: Research Funding, Speakers Bureau; Constellation Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J&J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding; Agios: Research Funding, Speakers Bureau. Blum:AmerisourceBergen: Consultancy; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Astellas,: Research Funding; Xencor: Research Funding; Forma: Research Funding. Kovacsovics:Pfizer: Research Funding; Jazz: Consultancy; Novartis: Research Funding; Abbvie: Research Funding; Amgen: Consultancy, Research Funding. Foran:Agios: Honoraria, Research Funding. Druker:Pfizer: Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding. Byrd:Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Prelude Therapeutics: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees. Mims:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

AbstractCurrent published guidelines for perioperative pharmacy services have limited information on the development and implementation of a robust clinical pharmacy program across the surgical continuum of care. This publication defines the responsibilities and scope of practice of the Perioperative Clinical Pharmacist (PCPh), supporting the PCPh as a critical member of the interprofessional surgical patient care team. Opportunities for pharmacist role integration into perioperative medication management processes are described along with published examples of successful PCPh practice models, including those with interventions targeted toward Enhanced Recovery Pathways (ERP). Recommended training and competencies for future and practicing perioperative pharmacists, in addition to considerations for precepting and scholarly activities, are also outlined. Finally, future developments in perioperative pharmacy practice are discussed, including technological advancements, improved predictive models, and expansion of collaborative practice agreements.

Abstract Background: Blasts and leukemic stem cells of acute myeloid leukemia (AML) as well as several other hematologic malignancies express CD123, potentially providing a target for novel therapies. XmAb14045 (also known as SQZ622) is a potent bispecific antibody targeting both CD123 and CD3 that stimulates targeted T cell-mediated killing of CD123-expressing cells, regardless of T cell antigen specificity. It is a full-length immunoglobulin molecule designed to be dosed intermittently, in contrast to smaller constructs that are referred to as "DART" or "BiTE" bispecific antibodies that require a continuous infusion. Methods: Patients with relapsed or refractory AML, B cell acute lymphoblastic leukemia (B-ALL), blast phase chronic myelogenous leukemia, or blastic plasmacytoid dendritic cell neoplasm were eligible to enroll on this first-in-human, multicenter, open-label phase 1 dose-escalation study (XmAb14045-01) with standard 3+3 design. The primary objective was to estimate the maximum tolerated dose (MTD) or recommended dose and schedule of XmAb14045. Secondary objectives included safety, preliminary antileukemic activity, and pharmacokinetics/pharmacodynamics of XmAb14045. Treatment was administered weekly in 28 day cycles, using a weight-based dose with a single dose level in Part A and, in Part B, an initial priming dose on Day 1 followed by an escalated dose on subsequent weeks. Premedication to prevent cytokine release syndrome (CRS) was instituted as needed and included a steroid, acetaminophen, and diphenhydramine. Patients were premedicated at all XmAb14045 doses ≥0.075 µg/kg. Therapy was continued for as long as tolerated and there was continuing evidence of therapeutic benefit in the opinion of the investigator. Treatment response was assessed by the 2017 European LeukemiaNet (ELN) criteria after Cycle 1 and after each odd-numbered cycle. CRS was graded using the CRS revised grading system (Lee et al., Blood, 2014). Results: At data cut-off, 64 patients have been treated to date, 63 with relapsed/refractory AML and 1 with B-ALL. Patients had a median age of 61 years and were heavily pretreated (median of 3 prior therapies [range 1-8], including 19 [30%] who had undergone prior allogeneic stem cell transplantation). The recommended dose for Part A was 1.3 µg/kg after a single dose-limiting toxicity of Grade 4 CRS at 2.3 µg/kg; no MTD has been identified. CRS or its component symptoms was the most common treatment-emergent adverse event (TEAE). CRS episodes began within approximately 1-4 hours of the start of drug infusion and occurred in 49 of 64 patients (77%). Seven patients (11%) developed Grade ≥3 CRS, the majority of these on the first dose. There were no CRS-related deaths. Excluding CRS-related events, additional TEAEs occurring in >10% of patients included fatigue (31%), febrile neutropenia (30%), peripheral edema (30%), cough (23%), elevated hepatic transaminases (19%; all recovered without sequelae), pneumonia (17%), stomatitis (14%), hyperglycemia (13%), and sepsis (11%). No myelosuppression requiring dose modification or evidence of tumor lysis syndrome was seen. In Part A, single agent antileukemic activity was documented with a best response of CR (2) or CRi (1) in 3/13 AML patients (CR/CRi rate 23%) treated at the two highest dose levels studied to date (1.3 and 2.3 µg/kg weekly); no CR, CRi, or morphologic leukemia-free state (MLFS) responses were seen at lower doses. Antileukemic activity occurred quickly; all responders had achieved at least an MLFS response after 4 doses (1 cycle). Two responders were bridged to stem cell transplantation, and the third was ineligible for medical reasons but remains in remission at 14+ weeks after initiating therapy. Conclusions: XmAb14045 demonstrated evidence of antileukemic activity in heavily pretreated patients with relapsed/refractory AML treated in Part A at the 1.3 and 2.3 µg/kg doses administered once weekly, with a 23% CR/CRi rate. CRS was the most common toxicity but was generally manageable with premedications. The study is ongoing with further optimization of dose, schedule, and premedication regimen for CRS anticipated during accrual to dose escalation cohorts in Part B. ClinicalTrials.gov Identifier: NCT02730312 Disclosures Ravandi: Xencor: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; Seattle Genetics: Research Funding; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria; Sunesis: Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Orsenix: Honoraria; Xencor: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding. Foran:Xencor, Inc.: Research Funding; Agios: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Mawad:Swedish Cancer Institute: Employment. Blum:Astellas: Consultancy; Pfizer: Consultancy; Boehringer Ingelheim: Research Funding; Forma: Research Funding; Xencor: Research Funding; Tolero: Research Funding. Saville:Xencor, Inc.: Employment, Equity Ownership. Johnson:Xencor, Inc.: Employment, Equity Ownership. Vanasse:Novartis: Employment, Equity Ownership. Ly:Xencor, Inc.: Employment, Equity Ownership. Mims:Novartis: Consultancy; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Abstract Background High frequency mutations in both the colony-stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1) have recently been described in World Health Organization (WHO)-defined chronic neutrophilic leukemia (CNL) (NEJM 2013;368:1781; Leukemia. 2013. Prepublished on 2013/04/23). Response to treatment with ruxolitinib (10-15 mg twice-daily) was also described in one patient with CSF3RT618I mutation (NEJM 2013;368:1781). Primary cells from this patient were reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). In a report of 12 patients with WHO-defined CNL, CSF3RT618I was seen in 10 (83%) patients, four (40%) of whom also expressed SETBP1 mutations (Leukemia. 2013. Prepublished on 2013/04/23). In the current study, we investigated the clonal distribution of mutant CSF3R and SETBP1in a CNL patient expressing both mutations and describe our observations regarding the effect of JAK inhibitors, both in vitro and in vivo. Methods Under institutional review board approved protocol, peripheral blood and buccal cells were collected and density gradient centrifugation was applied to enrich for mononuclear and granulocyte cell fractions. Antibody-labeled magnetic bead separation was used to obtain CD3+ and CD34+ cell fractions. DNA sequencing was used to screen for CSF3R and SETBP1 mutations. Single colonies were obtained by plating mononuclear cells in duplicate with cytokine enriched methylcellulose with and without the addition of fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for both SETBP1 and CSF3Rmutations. Results The study patient was a 66-year-old lady with history of radiation therapy after lumpectomy for breast cancer in 1997. In October, 2012 the discovery of three synchronous lesions in the left breast necessitated mastectomy. White blood cell count (WBC) was approximately 13 x 10(9)/L at the time. Subsequently, her WBC gradually increased to 180 x 10(9)/L. Bone marrow examination on March 15, 2013 showed predominantly granulocytic proliferation with over 95% cellularity and no dysplastic features or monocytosis. Cytogenetic studies and mutation screening for JAK2V617F and BCR-ABL1were negative. A diagnosis of CNL was made and the patient was placed on hydroxyurea therapy. i) In vitro studies The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34+ myeloid progenitors; neither mutation was present in buccal or CD3+ cells. Colony forming unit (CFU) assay-derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I. Fedratinib and the JAK1 inhibitor revealed activity in suppressing colony formation by >50% at a drug concentration of ≥600 nM. Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations. However, CSF3R unmutated colonies were more likely to emerge than SETBP1unmutated colonies after in vitro drug exposure, which resulted in the appearance of occasional colonies that were negative for both mutations. ii) Treatment with ruxolitinib Ruxolitinib treatment at 10 mg twice-daily was started on 6/10/13, in addition to 500 mg/day of hydroxyurea. At the time, WBC was 86.5 x 10(9)/L, Hgb 11.8 g/dL and platelets 122 x 10(9)/L. On 6/17/13, WBC decreased to 43.9 and hydroxyurea was held. On 6/25/13, WBC increased to 89.6 x 10(9)/L and ruxolitinib was increased to 15 mg twice-daily. On 7/15/13, WBC had further increased to 190 x 10(9)/L and hydroxyurea was added to the treatment regimen at 500 mg/day. On 8/2/13, WBC was recorded at 91.3 x 10(9)/L. Conclusions In a double mutated CNL patient, we found CSF3R and SETBP1 mutations to be myeloid cell-restricted and co-expressed in erythroid and granulocytic cells; the latter antedated the former in order of acquisition. In vitro JAK inhibition had non-selective activity in suppressing myeloid colony formation. Treatment of the study patient with single agent ruxolitinib was ineffective. 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.

Abstract Background: Reactive oxygen species cause DNA damage and altered immunologic responses, and have been linked to the development of NHL. Diets rich in fruits and vegetables are excellent sources of antioxidants, and vitamins C, E, polyphenols, and carotenoids, along with selected micronutrients such as zinc are thought to be responsible for most of the antioxidant activity in foods. We evaluated the hypothesis that vegetables, fruits and nutrients involved in antioxidant activity would protect against developing NHL. In a secondary analysis, we also assessed heterogeneity of this hypothesis for the most common subtypes of NHL: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma, and diffuse large B cell lymphoma (DLBCL). Methods: We evaluated dietary antioxidant intake and NHL risk in a clinic-based study of 416 newly diagnosed NHL cases and 926 frequency-matched controls enrolled at the Mayo Clinic from 2002–2007. Usual diet two years before diagnosis/enrollment was assessed using a self-administered, 128-item food frequency questionnaire. Dietary intake of antioxidants was estimated using the Food Processor SQL system, further supplemented with data from US Department of Agriculture nutrient databases. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusted for age, sex, residence, and total energy. NHL subtypes were centrally reviewed, and subtype-specific risks were estimated using polychotomous logistic regression. Results: The mean age at diagnosis was 60.8 years for cases and 57% were male; for controls, the mean age at enrollment was 60.8 years and 54% were men. NHL risk was inversely associated with intake of total vegetables (OR for highest compared to lowest quartile, 0.46; 95% CI 0.32–0.68; p-trend<0.001), and specifically green leafy (OR=0.54; 95% CI 0.39–0.76; p-trend<0.001) and cruciferous (OR=0.62; 95% CI 0.43–0.88; p-trend=0.02) vegetables, but not legumes or red/yellow/orange vegetables. There was no association with total fruit intake or intake of citrus fruits in particular. When modeled together, the association for green leafy vegetables was unchanged while the association for cruciferous vegetables attenuated (OR=0.75; 95% CI 0.50–1.13; p-trend=0.3). Higher intake of total vitamin A (OR=0.60; 95% CI 0.41–0.88; p-trend<0.001) and vitamin E (OR=0.53; 95% CI 0.34–0.82; p-trend<0.001), but not vitamin C, were associated with lower NHL risk. For vitamin A, there was no association with preformed Vitamin A, but there was an inverse association with beta-carotene (OR=0.56; 95% CI 0.39–0.81; p-trend<0.001); only weak inverse associations were observed for other carotenoids (lutein/zeaxanthin, lycopene, and cryptoxanthin). When modeled together, the inverse association for vitamin E was unchanged while the association for beta-carotene attenuated. For micronutrients, there were inverse associations for manganese (OR=0.60; 95% CI 0.40–0.90; p-trend=0.02), selenium (OR=0.47; 95% CI 0.27–0.80; p-trend=0.01), and zinc (OR=0.46; 95% CI 0.27–0.80; p-trend<0.001) but not copper; when modeled simultaneously, both manganese (p-trend=0.03) and zinc (p-trend=0.02) remained significantly and inversely associated with NHL risk. Further adjustment for education, family history of NHL, smoking, alcohol use, and body mass index did not alter these results. There was no NHL subtype heterogeneity for the associations with total vegetables, green leafy vegetables or vitamin E, while inverse associations were not observed for manganese and follicular lymphoma or zinc and CLL/SLL. Conclusion: Higher intakes of vegetables, particularly green leafy vegetables, vitamin E, manganese, and zinc were associated with lower risk of NHL. These finding extend a growing literature from case-control and cohort studies that support a protective role of foods and nutrients that function in antioxidant related pathways against development of NHL, and therefore may represent one of the few known modifiable risk factors for this cancer.

Abstract Background: Elevated serum BLyS levels have been found in patients with both malignant and autoimmune diseases when compared to those of healthy donors, suggesting that BLyS may play a pathogenic role. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the mechanistic details underlying control of BLyS expression remain to be defined. In previous work we found that a single nucleotide polymorphisms (SNP) in the BLyS promoter (−871C→T) resulted in increased BLyS transcription suggesting that genetic variation in the BLyS gene may influence its expression. Because elevated BLyS levels have been detected in patients with NHL we wanted to determine if additional SNPs in the BLyS gene were associated with high serum BLyS levels and risk of developing of NHL. Methods: We genotyped 9 tagSNPs within the BLyS gene in a clinic-based study of 441 incident Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. We evaluated the association of individual SNPs as well as haplotypes from the BLyS gene with risk of NHL. We also jointly tested the main effects for all independent (r2<0.25) SNPs using a multivariate logistic regression (MLR) model. As a secondary analysis, for those SNPs showing significant results (< 5% significance), we evaluated associations between those who had all high risk alleles at the SNPs of interest compared to those who had all low risk alleles at the SNPs of interest. Additionally, we evaluated serum BLyS levels by ELISA in these same subjects. Serum BLyS levels were detemined by ELISA. Results: In the individual single SNP logistic regression analysis, 3 of the 9 tagSNPs were significant at p<0.05. Haplotype and MLR results were nonsignificant. However, when we categorized participants into low and high risk groups based on risk alleles at the three statistically significant SNPs, we found the high risk variant had an odds ratio (OR) of 2.086 (p=0.0001) for risk of B-cell NHL. When the analysis was restricted by histologic subtype we found that diffuse large B-cell lymphoma or follilcular lymphoma grade 3 had an OR of 3.163 (p=0.01), follicular lymphoma grade I/II had an OR= 2.547 (p=0.046), and chronic lymphocytic leukemia (CLL) had an OR=1.238 (p=0.13). Because there was not a significant correlation of the high risk variant with CLL, we performed an additional analysis in which we included all B cell NHL cases excluding CLL and the OR was 2.799 (p=0.0001). We next wanted to determine if serum BLyS levels correlated with either the high or low risk variant. The mean serum BLyS level in those individuals (untreated cases and controls) who carried the low risk variant at all three SNPs was significantly lower (p=0.0061) at 1.3 ng/ml (n=25, range: undetectable-4.4 ng/ml) compared to 4.3 ng/ml in those with the high risk variant (n=74, range: undetectable- 66.8 ng/ml). Conclusions: In summary, we have found that genetic variation in the BLyS gene is significantly associated with an increased risk of developing B-cell NHL, particularly follicular and large B-cell lymphoma. Further, we found that genetic variation is also associated with an increase in serum BLyS levels.Taken together, our data further highlight the role of the BLyS gene in B cell malignancies and characterization of the precise genetic and environmental mechanisms that regulate BLyS may have significant clinical impact.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Rearrangements of the MLL (mixed lineage leukemia) gene on chromosome 11q23 are present in 5-10% of either acute myeloid (AML) or acute lymphoblastic leukemia (ALL), and are associated with a poor prognosis. Fusion proteins involving the MLL histone methyltransferase (HMT) result in recruitment of another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of Histone H3 lysine 79 (H3K79) at MLL target genes and enhanced expression of leukemogenic genes such as HOXA9 and MEIS1. EPZ-5676 is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against additional HMTs. EPZ-5676 treatment of cultured cells reduced histone H3K79 methylation, decreased MLL target gene expression and resulted in selective killing of MLL-rearranged (MLL-r) leukemia cells by inducing apoptosis and differentiation. EPZ-5676 administration led to tumor regression in an MLL-r leukemia xenograft model. We report preliminary results of an ongoing Phase 1 trial of EPZ-5676 in patients with advanced hematologic malignancies, including acute leukemia with MLL-r. Methods: This is a 2-part phase 1 open label dose escalation study investigating the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and anti-leukemic activity of EPZ-5676 in adult patients (pts) with relapsed/refractory leukemia (CT.gov: NCT01684150). In the dose-escalation phase, patients with AML, ALL, acute mixed lineage leukemia, myelodysplastic syndrome, myeloproliferative neoplasia (MPN) or chronic myeloid leukemia were eligible. Eligibility in the expansion phase of the study was restricted to pts with MLL-r or partial tandem duplication of MLL (MLL-PTD). EPZ-5676 was administered via continuous intravenous infusion (CIV) for 21 days of a 28 day cycle in the dose escalation phase and CIV for all 28 days of a 28 day cycle in the expansion phase, until disease progression or unacceptable toxicity. Results: Between October 2012 and July 2014, 37 pts have enrolled; 36 are evaluable for safety (received at least 1 dose of EPZ-5676) and 28 pts (19 MLL-r, 4 MLL-PTD) are evaluable for anti-leukemia activity (completed at least one post baseline marrow evaluation). The median age at time of enrollment was 53 years (range: 20 to 81 years); the median number of prior systemic therapies was 2 (range: 1 to 6) and 14 pts had a prior allogeneic stem cell transplant. Of the treated pts, disease characteristics were: 31 pts with AML (21 MLL-r, 5 MLL-PTD), 4 pts with ALL (3 MLL-r) and 1 pt with MPN (MLL-r). Table 1: Number of evaluable pts per cohort Dose (mg/m2/day) 12 24 36 54 80 90 21 day CIV 1 5 4 6 3 - 28 day CIV - - - - - 17 The median time on study was 29 days (range: 5-196 days). Adverse events (AEs) reported in >15% of pts were: anemia, fever/neutropenia, thrombocytopenia, constipation, diarrhea, nausea, chills, fatigue, mucosal inflammation, peripheral edema, hypomagnesemia, dyspnea and sepsis; of which leukocytosis, nausea and hypomagnesemia were assessed by the investigator as drug related. Grade I PR interval prolongation, without associated QTc changes, was observed in 2 pts. There were no deaths attributed to study drug treatment, and there were 2 pts with treatment discontinuation for potentially drug-related AEs. Dose proportional PK was observed with rapid attainment of steady-state plasma concentrations on Day 1 of treatment. Preliminary PD data demonstrated inhibition of H3K79 methylation from baseline in marrow (median: 52%, range 0-81%) and peripheral blood mononuclear cells (median: 43%, range 25-67%) at doses above 36 mg/m2/day. PD effects were observed by day 8. The 21 day CIV pts recovered H3K79 methylation towards baseline by day 28, and the 28 day CIV pts maintained methyl-mark inhibition throughout cycle 1. To date, best responses for pts with MLL-r are morphologic CR (1 pt.), cytogenetic CR (1 pt), and resolution of leukemia cutis (2 pts). In addition, a treatment-related increase in neutrophils (PMN) and/or monocytes was observed in 6 pts with a median day of onset of 15 days (range 8-28 days). The identification of the MLL-r by split signal FISH in mature PMN suggests a differentiation effect on the leukemic clone. Conclusions: EPZ-5676 is well tolerated and exhibits anti-leukemic activity in MLL-rearranged acute leukemia. These data provide rationale for continued clinical investigation of potential utility of EPZ-5676 in patients with rearrangements of the MLL gene. Disclosures Stein: Janssen Pharmaceuticals: Consultancy. Garcia-Manero:Epizyme, Inc: Research Funding. Rizzieri:Novartis, Inc: Consultancy, Speakers Bureau; Ariad, Inc: Speakers Bureau. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Altman:Astellas: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy. Pollock:Epizyme: Employment, Equity Ownership. Waters:Epizyme, Inc: Employment, Equity Ownership. Legler:Epizyme, Inc: Employment. Thomson:Epizyme, Inc: Employment. Daigle:Epizyme, Inc: Employment, Equity Ownership. McDonald:Epizyme, Inc: Employment. Campbell:Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme, Inc: Employment. Hedrick:Epizyme, Inc: Employment; Pharmacyclics, Inc: Equity Ownership. Copeland:New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other; Mersana: Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Employment, Equity Ownership.

Abstract Abstract 3634 PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7. Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T.Dose levelVeliparib Oral Daily Days 1–8Topotecan Continuous IV Days 3–7Carboplatin Continuous IV Days 3–7Clinical Responses/#Pts1A10 mg p.o. BID1.0 mg/m2/day—1/3(CRi)1B10 mg p.o. BID1.3 mg/m2/day—1/3(CR)2A10 mg p.o. BID1.3 mg/m2/day150 mg/m2 IV0/42C10 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV2/6(CRi, PR)2D10 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV1/6(PR)2E10 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CRi)3C20 mg p.o. BID1.0 mg/m2 IV120 mg/m2 IV0/63D20 mg p.o BID1.2 mg/m2 IV120 mg/m2 IV2/6(CR, PR)3E20 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)440 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/6(CR, CR)5§80 mg p.o. BID1.2 mg/m2 IV150 mg/m2 IV2/4(CR, CRi) Of 56 pts treated at doses of V 10–80 mg orally BID Days1–8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 25 (45%) have experienced > grade 3 non-hematologic toxicity, 6(11%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 27% (8CR, 4 CRi, 3 PR), with 7 in relapsed AML, 7 in refractory AML and 1 refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V is detectable in marrow supernatant and cells, does not accumulate with multiple dose administrations in plasma but does in marrow, and is not altered by T or C administration. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to <50% of pretreatment values in 30/41 (73%) blood and 3/4 patients at V 40mg per day dose level had greater than 80% suppression of PAR. The MTD has not been reached and dose escalation is ongoing. Disclosures: No relevant conflicts of interest to declare.