ALBERT

Description: Erythropoietin (EPO) is a plasma glycoprotein that binds erythroid progenitors in the bone marrow and stimulates their proliferation and differentiation. EPO is secreted into the circulation by specialized kidney peritubular fibroblasts. Though the transcriptional regulation of EPO production has been well studied, the intracellular regulation of EPO trafficking remains poorly understood. In an effort to identify genes involved in EPO secretion, we developed a genome-wide functional screen that provides a quantifiable and selectable readout of intracellular EPO accumulation. In order to perform such a screen, we generated a reporter HEK293T cell line stably expressing EPO fused to GFP and as an internal control, alpha-1-antitrypsin (A1AT) fused to mCherry. We showed that both EPO and A1AT are efficiently secreted from the cell and that treatment with Brefeldin A (which disrupts endoplasmic reticulum [ER] to Golgi transport) results in intracellular accumulation of EPO and A1AT. These findings demonstrate that the machinery required for the efficient secretion of EPO via the classical secretory pathway is intact in this cell line. To identify genes that affect EPO secretion, we mutagenized the reporter cell line with a CRISPR/Cas9 knock-out library (GeCKO-v2), which delivers SpCas9, a puromycin resistance cassette, and a pooled collection of 123,411 single guide RNAs (sgRNAs) that include six independent sgRNAs targeting nearly every coding gene in the human genome. Transduction was performed at low multiplicity of infection (MOI ~0.3), such that most infected cells receive 1 sgRNA to mutate 1 gene in the genome. Puromycin selection was applied from days 1-4 post-transduction. After 14 days, cells with normal mCherry but increased (top 7%) or decreased (bottom 7%) GFP fluorescence were isolated. Integrated sgRNAs sequences were quantified by deep sequencing and analyzed for their enrichment in the GFP high compared to the GFP low population. This strategy allows the identification of genes that affect EPO but not A1AT levels, therefore ruling out genes that affect global secretion. This screen, performed in triplicates, identified that the sgRNAs targeting surfeit locus protein 4 (SURF4) are the mostly enriched sgRNAs (at the gene level) in the GFP high population: 5 out of 6 sgRNAs targeting SURF4 were enriched in the GFP high population, at a genome-wide statistical level. To validate these results, we generated a sgRNA targeting SURF4 and demonstrated that SURF4 deletion results in intra-cellular accumulation of EPO with no effect on A1AT. We confirmed these results in several independent reporter cell line clones, excluding an artifact unique to the original reporter clone used in the screen. Additionally, the intracellular EPO accumulation in SURF4 deficient cells was rescued by SURF4 cDNA, ruling out an off-target sgRNA effect. We next showed that SURF4 interacts with EPO (by co-immunoprecipitation) and that EPO accumulates in the ER of SURF4 deleted cells (using endo-H and fluorescent confocal microscopy). In contrast to EPO, we found that SURF4 deletion does not result in the intracellular accumulation of a related glycoprotein, thrombopoietin. To examine if SURF4 facilitates the secretion of EPO when expressed at a more physiological level, we deleted SURF4 in HEP3B cells induced to express EPO from its endogenous locus and found that SURF4 also mediates the secretion of EPO under these conditions. Taken together, the studies summarized above demonstrate that SURF4 is the ER cargo receptor that promotes the efficient secretion of EPO. Additional work is currently ongoing to further characterize the role of SURF4 in the secretion of EPO. Disclosures No relevant conflicts of interest to declare.

Description: Introduction von Willebrand disease (VWD) is a common inherited bleeding disorder with a reported incidence ranging from 0.01% to 1%. VWD is classified as quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). Classical VWD type 2A, the most common qualitative defect of VWD, is characterized by decreased VWF-dependent platelet adhesion and a selective deficiency of high molecular weight VWF multimers. The majority of type 2A VWD is inherited in an autosomal dominant fashion. This subtype is caused by missense variants acting through a variety of mechanisms including defects in multimer assembly or an intrinsically increased sensitivity to cleavage by ADAMTS-13. The majority of pathogenic mutations are located in exon 28, predominantly affecting the A2 domain. Missense mutations have also been reported in the D3 and A1 domains (exons 22 and 25-28). Less common missense mutations include those responsible for dimerization (CK domain, exon 52) and multimerization defects (D2 domain, exons 11-17). Our objective was to determine the genomic distribution of mutations underlying type 2A VWD in patients referred to a USA-based clinical reference laboratory. Methods In a clinical reference laboratory, BloodCenter of Wisconsin, VWF gene sequence analysis results from January 2009 to July 2018 were reviewed. Molecular testing was performed by polymerase chain reaction (PCR) amplification and bi-directional Sanger sequencing or capture-based enrichment and next-generation sequencing (NGS). Complete coding region and splice junction of VWF gene was compared to the reference sequence (NM_00552.3). Variant interpretation was performed according to American College of Medical Genetics (ACMG) standards and guidelines by utilizing genome databases, population frequency data, variant specific literature search and predictive computational tools. Variant interpretation was reviewed by a team comprising of genetic counselors, molecular experts and experts in coagulation disorders correlating these findings with the patient's clinical phenotype provided. Results Pathogenic variants were identified in 46 cases received. Of these, 56.5 % (26 of 46) were in exon 28 of the VWF gene.(Figure 1) 21.7% of mutations (10 out of 46) were identified in exon 26, 4.3% were identified in exon 25 (2 of 46) and 6.5% (3 out of 46) were identified in exon 52 of VWF gene. The remaining 10.7% were identified in exons 6,11,12,22 and 27 (1 each). The distribution of these mutations according to VWF domains was also studied. 39% of these mutations were found in the A2 domain, 30.4% were identified in the D3 domain, and 13% were found in the A1 domain. A small number of these mutations were identified in the D1 domain (1/46), D2 domain (2/46) and CK domain (2/46). Phenotypic information was available for most patients evaluated. Conclusions In a predominantly Caucasian population with others of mixed ethnicities, the distribution of VWD type 2A mutations was studied in patients evaluated in a clinical reference laboratory. Most mutations were identified in VWF Exon 28 and in the A2 domain. Next generation sequencing platform enables sequencing of large amounts of DNA in parallel thereby allowing all VWF exons to be evaluated at one time. However, in smaller laboratories, without access to a NGS platform, initial evaluation of the VWF Exon 28 for evaluation of VWD type 2A variants is a cost effective measure. Subsequently, sequencing of other less implicated exons may be performed utilizing a reflexive algorithm. Genetic testing can be very useful in diagnosis of type 2 variants, where mutations in specific regions correspond to the defect in VWF function. Disclosures Friedman: Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Description: Background Elotuzumab is an approved monoclonal antibody targeting SLAMF7 on plasma and NK cells that enhances the activity of lenalidomide, pomalidomide, and bortezomib in multiple myeloma (MM). A recent study showed improved outcomes with the combination of pomalidomide, bortezomib, and dexamethasone vs. bortezomib and dexamethasone in relapsed or refractory MM (Richardson PG et al., Lancet Oncol 2019). We therefore studied elotuzumab with pomalidomide, bortezomib, and dexamethasone (elo-PVD) in relapsed and refractory MM. Methods The primary objective was to determine the overall response rate (ORR). Patients with relapsed and refractory disease and ≥1 prior lines of treatment (including lenalidomide and a proteasome inhibitor) were eligible to participate. Prior treatment with pomalidomide was permitted. Elotuzumab was weekly for the first 2 cycles and then every other week. Pomalidomide was given on days 1-21; bortezomib was on days 1, 8, 15; and dexamethasone was weekly. Each cycle was 28 days. Results The trial has completed accrual in September 2018 with 48 patients receiving treatment. The median age was 64 (range 40-80), and median number of prior regimens was 3 (range 1-9); 25% had high risk FISH. All patients had prior lenalidomide and proteasome inhibitor (bortezomib 96%, 29% carfilzomib) and were refractory to their last line of therapy. Other prior therapies included: autologous stem cell transplant (48%), pomalidomide (33%), daratumumab (25%), and isatuximab (4%). 46 patients were assessable for response (2 patients did not complete cycle 1 and were not evaluable for response: 1 due to rapid disease progression; 1 stroke. The median length of follow up was 18.8 months (range 0.5-23.4): 16 patients continue on study; 27 patients discontinued for progressive disease; 3 patients discontinued for adverse events (AEs) (sepsis, pneumonia, stroke); 1 patient underwent auto SCT; and 1 patient was lost to follow up. Best ORR was 61% (PR = 16, VGPR = 10, CR = 2). ORR for patients with prior anti-CD38 antibody, 46%; carfilzomib, 46%; pomalidomide, 43%. Median PFS was 9.8 months (95% CI 6.8-Inf). In patients with 1 prior line of therapy, ORR was 74% and median PFS was not reached (95% CI 12-Inf); 18 month PFS was 68%. Grade ≥ 3 hematologic AEs included anemia (10%), neutropenia (29%), and thrombocytopenia (15%). Additional common grade ≥ 3 AEs included lung infection (27%) and hypophosphatemia (15%). Common non-hematologic AEs all grades included fatigue (grade 1-2 only, 70%), upper respiratory infection (grade 1-2, 56%; grade 3, 2%), diarrhea (grade 1-2 only, 42%), constipation (grade 1-2 only, 35%), hyperglycemia (grade 1-2, 46%; grade 3, 4%), and sensory neuropathy (grade 1-2 only, 31%), with 2 possibly related deaths (sepsis, pneumonia). Conclusions Elo-PVD is one of the first trials of a quadruplet regimen in relapsed and refractory MM incorporating a monoclonal antibody. In patients with refractory disease, elo-PVD shows encouraging responses. With the limitations of cross trial comparisons and small patient numbers, for patients with 1 prior line of treatment and refractory disease, a PFS at 18 months of 68% with elo-PVD compares favorably with a median PFS of 17.8 months in a similar subgroup of PVD in the OPTIMISMM trial (Dimopoulos MA et al., ASH 2018). Patients who received prior pomalidomide, carfilzomib, and/or anti-CD38 monoclonal antibody also benefited. Treatment was well-tolerated with manageable toxicity and with attention to infectious AEs. Disclosures Yee: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Karyopharm: Consultancy; Adaptive: Consultancy; Amgen: Consultancy, Honoraria. Lipe:Celgene: Consultancy; amgen: Research Funding; amgen: Consultancy. Nadeem:Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy. O'Donnell:Celgene: Consultancy; Amgen: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Takeda: Consultancy. Branagan:Pharmacyclics: Consultancy; Janssen: Consultancy; Surface Oncology: Consultancy. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Richardson:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Raje:Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy. OffLabel Disclosure: The combination of elotuzumab, pomalidomide, bortezomib, and dexamethasone is an off-label use in relapsed and refractory multiple myeloma.

Description: Key Points Overall, no benefit of granulocyte transfusion therapy was observed, but the power of the study was reduced due to low accrual. Post hoc secondary analysis suggested that patients receiving higher doses tended to have better outcomes than those receiving lower ones.

Description: Key Points RH genotyping of red cells may improve matching of patients and donors and reduce Rh alloimmunization. RH genotype matching may improve use of an African American blood donor inventory.

Description: Background: At Hofstra-North Shore LIJ School of Medicine, we have opportunities for our medical students to participate in experiential quality improvement projects. As part of this initiative, some students participate on health-care improvement teams using a Clinical Microsystems approach. This approach is a conceptual framework that has been applied to various departments in the North Shore-LIJ Health System in order to improve quality and patient safety. The basis of Clinical Microsystems is to identify a need in the hospital that may be targeted in order to improve the efficacy of healthcare quality and delivery. Once the objective for healthcare improvement is determined, a multidisciplinary team is created spanning one or more microsystems to target the specific barrier. One of the multidisciplinary teams focuses on the care of patients with sickle cell disease. LIJ Hospital admits between 150 and 200 patients with sickle cell disease a year primarily due to recurrent pain crises. The initial sickle cell multidisciplinary improvement team included physicians, patients, nurses, social workers and two medical students. The role of the students on this team included literature searches and survey administration. The team's initial analysis found that a lack of consistent outpatient follow up was resulting in frequent readmissions for this population to acute care facilities for pain control. To address this, the team was instrumental in establishing a primary care outpatient clinic focused exclusively on caring for patients with sickle cell disease, run by a primary care physician, in 2012. The current study was conducted by a medical student, and her mentor, who were original members of the sickle cell improvement team. The study tested the hypothesis that enrolling patients with sickle cell disease in an outpatient clinic with a dedicated physician focused on sickle cell disease management and pain control would decrease the number of admissions this population has for acute care at LIJ. The study also highlights the opportunity to involve medical students in a meaningful way in hands-on quality improvement projects in the early stages of training. Methods: This was a retrospective study of all adults with sickle cell disease, 21 years or older, who were seen at the primary care outpatient sickle cell clinic. We compared the rates of hospital admissions and length of stay in the one-year prior and one-year after their establishment of care at the clinic. All data was identified through manual and automated searches of the Electronic Health Record. Results: Since the opening of the clinic in 2012, 107 adults established care at the clinic. Within the first year that a patient began being cared for by the clinic, their admission rate dropped 27%, from an average of 3.775 to 2.75 admissions per year (p=0.0003). There were a total of 151 admissions one-year pre-intervention and 110 admissions one-year post-intervention. Conclusion: This project supports the value of a dedicated primary care outpatient sickle cell clinic on decreasing the admission rates for patients with sickle cell disease. Within one year of establishing care at the clinic, admissions rates decreased significantly, emphasizing the role of dedicated outpatient primary care in the management of patients with sickle cell disease. This study also highlights the importance and feasibility of integrating medical students into a quality improvement project early on during medical school. There are opportunities for students to have meaningful roles on hospital based improvement projects and learn quality improvement methodologies. An important component to this success is faculty mentoring to support a student's involvement in the project. The student involved in this study experientially participated in multidisciplinary team-based rapid cycles of change, process mapping, data collection and analysis. Similar projects could offer students an opportunity to participate during medical school in a longitudinal quality improvement project to develop skills they will need as physicians to identify, participate and measure the effects of improvement efforts. Disclosures No relevant conflicts of interest to declare.

Description: Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Description: Introduction: Hodgkin lymphoma (HL) is a common malignancy of children and adolescents and is highly curable with a 5-year overall survival (OS) rate of 〉 97%, yet dose-intensified chemotherapy regimens in combination with radiation therapy come with a high cost in form of long-term toxicity and morbidity (Castellino et al., Blood 2011). This major clinical challenge has resulted in the evaluation of risk-adapted treatment regimens in clinical trials aiming to achieve the optimal equilibrium between high survival rates and prevention of treatment-related toxicity. However, risk stratification is currently limited to the use of clinical factors as there are no validated integral biomarkers that can be employed to either improve risk stratification or as surrogate markers of treatment outcome in pediatric HL. The aim of our study was to perform gene expression profiling (GEP) to uncover disease biology underlying treatment response and develop a prognostic model to tailor first-line therapy in pediatric HL. Methods: We selected 203 formalin-fixed, paraffin-embedded tissue (FFPET) specimens from patients enrolled in a randomized phase 3 clinical trial (AHOD0031) of the Children's Oncology Group (COG) based on the availability of archived FFPET blocks. That trial was designed to assess the value of early chemotherapy response for tailoring subsequent therapy in intermediate-risk pediatric HL. We performed GEP on RNA extracted from pre-treatment FFPET biopsies using NanoString technology and a customized codeset encompassing probes for 784 genes. These genes were either previously reported to be associated with prognosis and outcome in HL or represent the cellular diversity of the tumor microenvironment. Event free survival (EFS) and OS were estimated using the Kaplan-Meier method. Gene expression data were used to develop a predictive model for EFS using penalized Cox regression with parameters trained using leave-one-out cross-validation. Results: Of the 203 tissue samples obtained from the Biopathology Center at the Cooperative Human Tissue Network, 182 (89.7%) passed quality assurance testing, similar to the pass rate achieved for adult HL samples obtained from the Eastern Cooperative Oncology Group trial E2496 (Scott et al., JCO 2013). We applied our previously published 23-gene predictor for OS (Scott et al., JCO 2013), developed using biopsies from adult HL patients to our pediatric cohort. After calibrating the new codeset, 53 patients were classified as "high-risk" and 129 as "low-risk". Importantly, the model failed to predict inferior outcomes in the "high-risk" group (5-year OS 100% vs 95%, log-rank P = 0.125; 5-year EFS 82% vs 70%, log-rank P = 0.159), with patients in the "high risk" group trending to have superior outcomes than the "low risk" patients. Moreover, only 2 genes from this model, IFNG and CXCL11, were significantly associated with EFS in univariate Cox regression analysis (P = 0.003 and 0.048, respectively) but with inverse hazard ratios in the pediatric group compared to adult patients. Therefore, we sought to develop a novel EFS predictive model for pediatric patients treated in the AHOD0031 trial. Using univariate Cox regression we identified 79 genes significantly associated with EFS (raw P 〈 0.05). Using the expression of these 79 genes as the input to penalized Cox regression, we developed a 16-gene model to predict EFS in our training cohort. Using an optimized cut-off for the model score, 31% of patients were designated high-risk and had significantly inferior post-treatment outcome (5-year EFS 38% vs 89%, log-rank P 〈 0.0001). When multivariate analyses were performed including our EFS-model score, disease stage and initial treatment response as variables, only the model score was significantly associated with EFS (P 〈 0.0001, HR 11.3 (95% CI 5.5-23.4)). Conclusions: Failure of the GEP-based model developed in adult HL suggests distinct biology underlies treatment failure in the pediatric age group. We describe the development of a novel predictive model for EFS in intermediate-risk pediatric HL patients that will be validated in an independent cohort of patients treated in the AHOD0031 trial. Successful validation of the model may provide a clinically relevant biomarker for pediatric and adolescent HL patients allowing refinement of risk stratification and the combination of molecular and clinical risk factors at diagnosis. Disclosures Scott: Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Description: Background: Recent advancements in comprehensive sequencing technologies has enabled researchers to uncover numerous somatic variants within many disease types, but their respective contributions to disease pathogenesis and potential roles as outcome biomarkers are less well-defined. Even fewer studies have integrated somatic mutation events with genomic gain and loss events of respective genes, mostly due to the need to utilize a second platform to robustly assess genomic copy number. To further understand the roles of these genomic aberrations in the disease biology of mature B-cell neoplasms, we developed a next-generation sequencing (NGS) panel of 220 genes for profiling of Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), and secondarily for Chronic Lymphocytic Leukemia (CLL). Methods: The 220 genes represented in the panel included genes based on reported frequency of mutations, targets of therapeutic agents, involvement in disease-enriched pathways, prognostic value, and associated with targeted therapy resistance. Also included were genes that mapped to sites of genomic gain and loss commonly observed in these neoplasms. Of all the genes, 163 were enriched for DLBCL, 144 for FL, 44 for MCL, and 107 for CLL. Probes were designed to cover the coding exons of the 220 genes using a hybrid-capture approach (Nimbledesign, Roche, Inc.), with a total capture size of 0.92 Mb comprising 4086 target regions. Optimization of DNA fragmentation and library preparation was performed for DNA extracted from formalin-fixed paraffin-embedded (FFPE) biopsy material, being the most common intended tissue type and also for which tumor burden could be assessed. Sequencing was performed using a Miseq sequencer (Illumina, Inc.) with the goal to enable detection of somatic variants down to an allele variant frequency (AVF) of 5%. CLC Genomics Workbench (Qiagen, Inc.) was used for alignment and variant calling. Filtering was performed to exclude synonymous, intronic, and untranslated region variants, variants within homopolymer regions (〉6), and variants detected below 5% AVF. Results: The average depth of coverage achieved across an initial sample set of over 50 FFPE DLBCL specimens was approximately 350X, with only one sample having more than 10% of the target regions not achieving a coverage of 60X or greater (within 95% of the target region). As part of a proof-of-concept study, we sequenced a cohort of 85 DLBCL FFPE samples from a single institution from patients undergoing biopsy as part of their routine clinical care. Clinical outcome, cell-of-origin subtype (by the Hans method) and genomic gain/loss data by array-CGH were available. All studies were performed with IRB approval. Of the 85 cases, 80 had also been submitted to bi-directional Sanger's sequencing for Exons 5-8 of TP53 to establish accuracy of variant detection. Of cases completed to date, additional TP53 mutations have been detected by NGS due to an AVF below that detectable by Sangers or only detected in one direction. In addition, of the 14 cases completely sequenced and filtered to date, EZH2 variants in four samples have been detected in the GCB subtype while four CD79B variants were in samples of the non-GCB subtype, consistent with previously reported associations. Furthermore, six of the eight samples which harbored an EZH2 or CD79B variants had mutations within the catalytic domain at Y646 (EZH2) or the ITAM residue Y196 (CD79B), both of which have clinical implications. Initial assessment of the ability of the panel to infer copy number showed concordance with aCGH in detecting large chromosomal aberrations. The sensitivity and specificity of this approach is currently being optimized. Conclusion: Overall then, a hybrid-capture NGS panel has been developed, targeting genes enriched for involvement in the most frequent subtypes of mature B-cell neoplasms and optimized for application to FFPE biopsy specimens. While emerging pathogenomic patterns observed in the cases sequenced to date have confirmed previously observed patterns, completion of the NGS analysis of the 85 DLBCL cohort will permit more complex integrated analysis of somatic variants and genomic gain/loss with pathologic and clinical outcome data. Disclosures Friedman: Cancer Genetics Inc.,: Employment, Equity Ownership. Guttapalli:Cancer Genetics, Inc.: Employment, Equity Ownership. Ma:Cancer Genetics, Inc.: Employment, Equity Ownership. Thodima:Cancer Genetics, Inc.: Employment, Equity Ownership. Kamalakaran:Cancer Genetics, Inc.: Employment, Equity Ownership. Houldsworth:Cancer Genetics, Inc.: Employment, Equity Ownership.

Description: Introduction: Randomized studies have demonstrated that compared to chemotherapy alone (ChemoTx), combined modality therapy (CMT) improves early event-free survival in HL patients with early stage disease. However, long-term follow up from randomized trials suggests that overall survival (OS) when receiving ChemoTx alone is equivalent or superior to OS compared with CMT. In addition, many studies have described late effects in HL survivors. While the negative impact of late-effects on LE have been estimated for pediatric HL patients (Yeh, Blood, 2012), these estimates have limited generalizability to adult HL patients due to differences in treatment regimens and exposure-related late-effects risks. To address this gap, we sought to develop an adjustable Markov model to predict LE for adult HL patients treated with contemporary therapy. Methods: We created a Markov "state transition model" in which a cohort of patients moves through different health states. The patient cohort (base case, 18 years old) starts with initial diagnosis, and upfront treatment with 12-year OS modeled from the CCG 5942 (Wolden, JCO, 2012; COG, updated data, 2015). Following the first 12 years, the probabilities of dying were modeled by summing background mortality rates and the mortality rate associated with late effects. Background mortality rate estimates came from the 2010 CDC gender-specific LE data. Late effects mortality rates were estimated from excess absolute risk (EAR) estimates due to late effects from the Childhood Cancer Study (CCSS) Cohort 1 subjects across all disease stages who were treated with extended field RT (EFRT), higher alkylating agent therapy, and less anthracycline compared to contemporary cohorts. (Castellino, Blood, 2011) Recognizing that recent comparisons of RT doses and fields from CCSS survivors to those treated with involved field radiotherapy (IFRT) have demonstrated a reduction in RT to healthy tissues of approximately 50% (Koh, Radiation Oncology, 2007), we assumed that this RT reduction would reduce incremental mortality risk attributable to therapy by 50%. Thus, for patients treated with CMT containing IFRT, we reduced the reported EAR estimate for the CCSS-1 HL patients by 50%. Furthermore, for HL patients treated with ChemoTx alone, we assumed incremental mortality risk would be reduced by 75% (i.e., EAR reduced by 75% for this group). Because late effects mortality rates were based on pediatric data, we conducted extensive sensitivity analyses on EAR estimates to portray the scope of uncertainty surrounding LE estimates. Results: We built on previous work on this topic by utilizing 12-year OS from CCG 5942 and by adapting data from the CCSS-1 cohort to reflect the impact of late effects on LE with more modern therapy (e.g. IFRT). 12-year OS for early stage, favorable risk HL patients treated on CCG 5942 was 98.9% and 100% for patients treated with ChemoTx and CMT, respectively. LE for an 18 year old without HL was 60.9 years. Without consideration of the burden of late effects (i.e., EAR=0), a patient with early stage, favorable risk HL had a LE similar to a healthy 18 year old without HL. For HL patients, LE with ChemoTx alone (base case, COPP/ABV) was 58.0 years and the LE for treatment with CMT (i.e., COPP/ABV + IFRT) was 55.7 years. Additionally, reduced LE was also apparent for HL patients who received ChemoTx alone (see Figure). Finally, in order to apply these data to individual HL patients, we created an adjustable model with variables including age, gender, risk group (favorable/unfavorable), and gender- and treatment-specific EAR that may potentially be applied to an individual HL patient. Conclusion: We created an adjustable Markov model that predicts LE for adult HL patients treated with contemporary therapy. This model, including longer term OS data, demonstrated that contemporary therapy reduces the late effects burden. However, for survivors of early stage HL, we found that LE loss due to late effects substantially exceeds LE loss due to HL. To further enhance this model for the potential application in adults with HL, further synthesis of available pediatric and adult data (accounting for contemporary therapy) is needed to account for differences in EAR by age and gender over a life span. Altogether, models that synthesize clinical trial data provide valuable information to providers and may help guide them and HL patients towards individualized therapeutic decisions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.