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Abstract Background Diffuse large B cell lymphoma post-transplant lymphoproliferative disorder (DLBCL-PTLD) represents a fatal complication after a solid organ transplant (SOT). Approximately 50% of DLBCL-PTLD can be related to Epstein Barr Virus (EBV) infection. However, the EBV status is not taken into account in the DLBCL-PTLD categorization, although increasing evidence suggests its critical role in biological, clinical and prognostic aspects of the disease. In this single center retrospective study, we compared clinical and histological features, as well as outcomes of DLBCL-PTLDs by EBV status. Methods This study focused on patients with SOT who were diagnosed with DLBCL-PTLD at Mayo Clinic (Rochester, MN). Patients were identified through the Mayo Clinic Lymphoma Data base and the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), between 1989 and 2017. The histology was re-reviewed (RLK) and classified according to the WHO Classification or Tumours of Haematopoietic and Lymphoid Tissues 2017. Cell of origin (COO) was performed by Hans algorithm. EBV RNA in situ hybridization (EBER) was performed. Cox proportional hazards models were used to assess the association of clinical factors in overall survival (OS). Results 148 DLBCL-PTLD were identified. The median age at SOT and at the time of PTLD diagnosis were 50 years (range: 15-71) and 56 years (range: 18-82), respectively. The median time to PTLD diagnosis from SOT was 38 months (range: 1-499). The types of SOT included renal (44%), liver (36%), cardiac (9%), renal/pancreas (5%), pancreas (8%), lung (7%) and multiorgan (4%). 62% were stage III-IV, 30% had elevated LDH and 67% had an IPI >1. 88% of patients presented with extranodal disease, and 16% had involvement of the grafted organ. Eighty-three (56%) cases were EBV positive. The COO information was available on 35 cases. The non-GCB subtype was identified in 24 cases and GCB subtype in 11 cases. The initial treatment was as follows: reduction in immunosuppression (12%), immunosuppression reduction with rituximab (26%), rituximab alone (5%), chemotherapy with or without rituximab (35%). Patients with EBV positive PTLD were more likely to have grafted organ involvement (20% vs. 8%, p=0.047) and late PTLD (occurring after 12 months from SOT - 60% vs. 8%, p<0.001) than patients with EBV negative PTLD. There was no association between EBV status and age, sex, stage, LDH, and extranodal site involvement. At a median follow-up of 80 months (range 9-269), 83 patients (56%) had died. The 5-year survival rate from PTLD diagnosis was 56% (95% CI: 48-65%). The median OS was 85 months (95% CI: 37-148). There was no difference in OS in patients with EBV positive PTLD (median OS=84.7 months, HR=1.21, 95% CI: 0.77-1.92) versus patients with EBV negative DLBCL-PTLD (median OS=85.4 months, p=0.41). Conclusion Our study assessed the impact of EBV status on the presentation and clinical outcome of DLBCL-PTLD in a 148-patient single-institution cohort. 56% of cases were EBV positive. EBV positivity was associated with grafted organ involvement and late PTLD diagnosis. Patients with EBV-negative and EBV-positive disease had similar survival from PTLD diagnosis. Disclosures Maurer: Celgene: Research Funding; Morphosys: Research Funding; Nanostring: Research Funding. Cerhan:Nanostring: Research Funding; Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding.

Abstract Introduction: The development of immune checkpoint inhibitors (ICPis) has ushered in a new paradigm for the treatment of both solid and liquid malignancies. However, these therapies, which promote an activated T cell phenotype, can cause a unique spectrum of autoimmune toxicities known as immune-related adverse events (IRAEs), which can affect a variety of organ systems. Autoimmune hemolytic anemia (AIHA) has been described as a rare complication of ICPis, but existing data are mostly limited to isolated case reports. Further, guidelines for the diagnosis and treatment of these patients are lacking. We describe the clinical and laboratory features of 14 patients who developed ICPi-associated AIHA. Methods: We contacted hematology departments at 18 academic medical centers across the U.S. to inquire about potential cases of ICPi-associated AIHA. Nine centers contributed a total of 14 cases to the current series. We defined AIHA as an abrupt decrease in hemoglobin ≥2g/dL, the presence of at least two laboratory features of hemolysis (elevated serum lactate dehydrogenase [LDH], elevated reticulocyte percentage, low or absent serum haptoglobin, and spherocytes), and the exclusion of other causes of anemia. We collected data on demographics and comorbidities, features of AIHA, and response to treatment. We defined complete recovery (CR) and partial recovery (PR) of hemoglobin as an increase from the nadir hemoglobin to within 1 and 2 g/dL of the baseline value, respectively, in the absence of recent transfusion. Results: The clinical features of the 14 patients in our cohort with ICPi-associated AIHA are summarized in the Figure (panel A). Underlying malignancies included melanoma (n = 9), non-small cell lung cancer (n = 3), colorectal cancer (n = 1), and acute myelogenous leukemia (n = 1). Four patients had a pre-existing diagnosis of a lymphoproliferative disorder, and two of these patients were known to have a positive direct antiglobulin test (DAT) prior to initiation of ICPi. ICPis used included ipilimumab, nivolumab, a combination of ipilimumab and nivolumab, and pembrolizumab. Representative hemoglobin and LDH trends are shown in the Figure (panels B and C). The median (interquartile range [IQR]) interval from initiation of ICPis to AIHA was 55 (22-110) days. Eight patients (57%) were DAT positive: 3 were positive for both IgG and C3; 3 were positive for IgG alone; 2 were positive for C3 alone; and 1 was weakly positive on polyspecific testing. Five patients (36%) were DAT negative, and in one patient a DAT was not performed. The median (IQR) hemoglobin nadir was 6.3 (6.1-8.0) g/dL,and 11 patients (79%) required red blood cell transfusion. All patients were treated with glucocorticoids, with 3 requiring additional immunosuppressive therapy. All patients achieved a CR or PR of hemoglobin in response to treatment (CR, n = 11; PR, n =3). The median (IQR) time from the hemoglobin nadir to CR or PR was 47 (30-62) days. Seven patients (50%) experienced at least one other IRAE. Seven patients (50%) were re-challenged with ICPis, and one patient developed recurrent AIHA with re-challenge. Conclusion: ICPi-associated AIHA is a recently-described phenomenon, which will be encountered more frequently with the widespread use of ICPis. ICPi-associated AIHA shares many clinical features with other forms of AIHA; however, a unique aspect of ICPi-associated AIHA appears to be a relatively high incidence of DAT negativity (36% in our series, compared with 3-11% in other forms of AIHA). It is possible that more advanced DAT testing - i.e., "super-Coombs" - which was not performed in any of the patients in this series, would have yielded different results. Glucocorticoids appear to be an effective first-line regimen in most patients with ICPi-associated AIHA. Furthermore, we found that AIHA does not necessarily recur with ICPi re-challenge. Additional studies are needed to better understand the mechanisms underlying ICPi-associated AIHA and optimal treatment strategies. Disclosures Anagnostou: ASH Research Training Award for Fellows 2018: Research Funding; Mayo Clinic SPORE: Research Funding; Mayo Clinic Immune Monitoring Core: Research Funding; Mayo Clinic Hematology Research Division: Research Funding. Salama:BMS: Research Funding; Celldex: Research Funding; Dynavax: Research Funding; Genentech: Research Funding; Merck: Research Funding; Reata: Research Funding; Incyte: Research Funding; Immunocore: Research Funding; Array: Consultancy; Merck: Consultancy; Biocryst: Consultancy; BMS: Speakers Bureau.

Abstract Background Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with poor overall survival rates. The genetics of PTCLs are poorly understood. We have described t(6;14)(p25.3;q11.2) translocations in PTCLs involving the T-cell receptor-alpha (TRA@) and interferon regulatory factor-4 (IRF4) genes and overexpressing IRF4 protein (also called MUM1). IRF4/MUM1 is a lymphoid transcription factor involved in differentiation and growth, the expression of which is tightly regulated in normal T cells. However, nearly half of PTCLs constitutively express IRF4/MUM1 and this expression is associated with adverse overall survival in several PTCL subtypes. Because IRF4/TRA@ translocations are rare among PTCLs (<1%), we investigated the presence of other genetic abnormalities of IRF4 in PTCLs and examined their association with IRF4/MUM1 protein expression. Methods Studies were performed on paraffin tissue sections of PTCL specimens diagnosed by standard WHO criteria. IRF4/MUM1 immunohistochemical staining (IHC) was performed using the MUM1p antibody and considered positive if nuclear staining was present in >30% of tumor cell nuclei. Fluorescence in situ hybridization (FISH) was performed using breakapart probes constructed from bacterial artificial chromosome DNA that hybridized to loci flanking the IRF4 locus on 6p25.3. Cases with 3 or more fusion signals were considered to have extra copies of the IRF4 gene. In a subset of cases, PCR and Sanger sequencing was performed on DNA extracted from frozen PTCL tumor tissue with primer sets that amplified exons 1 to 10 of the IRF4 gene. Data were analyzed using the chi-square test or t test. Results PTCLs from 277 patients (175 M:102 F; median age, 61 y) were examined by both IHC and FISH. The subtype distribution was: PTCL, not otherwise specified (NOS), 93; anaplastic large cell lymphoma (ALCL), 89; angioimmunoblastic T-cell lymphoma (AITL), 27; cutaneous T-cell lymphoma (CTCL), 43; and cytotoxic PTCL, 25. IRF4/MUM1 was positive in 116 cases (42%). Extra copies of IRF4 were identified in 18 PTCLs (7%), 83% of which were IRF4/MUM1 protein-positive. In contrast, 39% of cases without extra copies of IRF4 were IRF4/MUM1 protein-positive (p=0.0002). The mean percentage of tumor cells with IRF4/MUM1 staining was 69% in cases with extra copies of IRF4, compared to 30% in cases without extra copies (p=0.0001). Extra copies of IRF4 were seen in 13% of ALCLs, 4% each of PTCLs, NOS and cytotoxic PTCLs, 2% of CTCLs, and 0% of AITLs (p=0.02). Two IRF4/MUM1-positive cases had an IRF4/TRA@ translocation (1%). We selected 30 IRF4/MUM1-positive PTCLs for Sanger sequencing. In this subset, we identified a previously undescribed non-synonymous variant in exon 5 (S309R) in 1 case (3%) without an extra copy of IRF4. Conclusions Extra copies of IRF4 are seen in ∼7% of PTCLs, are significantly associated with IRF4/MUM1 expression, and are preferentially observed in ALCLs. Both IRF4/TRA@ translocations and IRF4 mutations are rare in PTCLs. These data suggest that genetic abnormalities of the IRF4 gene – specifically extra copies of IRF4 – may contribute to IRF4/MUM1 expression in some PTCLs. Since IRF4/MUM1 expression is seen in ∼42% of PTCLs, these data also imply the existence of other regulatory mechanisms. Characterizing these mechanisms may be important clinically, since IRF4/MUM1 has been proposed as a therapeutic target in human cancers but drugs that directly inhibit its function are currently not available. Disclosures: No relevant conflicts of interest to declare.

Abstract Background : Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor was approved for the treatment of Waldenstrom Macroglobulinemia (WM) in January, 2015 based on the results of a clinical trial of 63 previously-treated patients with WM. Data on outcomes with ibrutinib outside of clinical trials are limited. Methods : Medical records of patients with WM seen consecutively between 02/01/2015 and 06/01/2018 at Mayo Clinic, MN, FL and AZ were reviewed and those treated with ibrutinib at any point during their disease were included. Refractoriness to prior therapy was defined as disease progression on therapy or within 2 months of stopping treatment. The response rates were assessed by WM VI International Workshop Criteria. Adverse events were graded using National Cancer Institute Common Toxicity Criteria for Adverse Events (AEs) v4.03. All time-to-event analyses were performed from the time of initiation of ibrutinib. Results : Eighty patients [previously treated, n=67 (84%), treatment-naïve, n=13 (16%)] received ibrutinib for active WM. Median follow-up was 18 months (95% CI: 12-19) and the median overall survival (OS) was not reached (NR) (95% CI: 27-NR) for the entire cohort at the date of last follow-up. The median age was 67 years (range: 50-88) and the median ECOG performance score was 1 (range: 0-3) at the time of ibrutinib initiation. Median lines of therapies prior to ibrutinib for the previously-treated patients was 2 (range: 1-9) and 35 patients (44%) had received ≥3 prior lines of therapy. Of the 80 patients, 52 patients (43 previously treated, 9 treatment-naïve) had data for response assessment. The baseline characteristics and laboratory parameters at initiation of ibrutinib are outlined in Table 1a and prior therapies are mentioned in Table 1b. Overall response rate (ORR) was 90% (100% in patients with ibrutinib as primary therapy and 88% in salvage therapy setting); major-response rate (MRR) was 77%. Median time to first and best response was 2.1 months (95% CI: 1.5-3.5) and 5.4 months (95% CI: 3.5-13), respectively. Other clinically relevant endpoints and AEs related to ibrutinib therapy are outlined in Table 2 and 3, respectively. The median time-to-next therapy was 33 months (95% CI: 28-NR). At last follow-up, 25 (32%) patients had discontinued ibrutinib therapy. The reasons for discontinuation was progression (n=8), elevated liver enzymes (n=4), cognitive changes (n=1), inability to achieve better than MR (n=1), atrial fibrillation (n=3), consolidation with autologous stem cell transplantation after PR with ibrutinib (n=1), transition to hospice care due to progressive head and neck cancer (n=1), infection in lower extremity (n=1), transformation to DLBCL (n=1), blistering rash which resolved after ibrutinib discontinuation (n=1), obscure GI bleed that resolved after ibrutinib discontinuation (n=1), fatigue (n=1) and for unclear reasons recorded as treatment intolerance (n=1). Following discontinuation of ibrutinib, 21 patients received salvage therapy. Of these, data regarding the response to salvage therapy was available in 9 (43%) patients (VGPR=2, PR=3, SD=3 and progressive disease=1); median PFS from the salvage therapy following ibrutinib discontinuation was 18 months (95% CI: 2-25). Of the 4 patients who were not treated following ibrutinib discontinuation; 1 elected hospice care due to concurrent head and neck cancer progression, and the other 3 patients elected to be monitored off therapy (one had achieved SD and two had PR from ibrutinib). Fourteen patients were evaluable for IgM rebound following ibrutinib discontinuation. Of those, 5 (36%) had sudden increase in IgM level [median: 158% from the IgM level after ibrutinib discontinuation (range: 22%-1368%)] within a median of 2 months (range: 1-3 months) and 1 patient required plasmapheresis for symptomatic hyperviscosity. Four of 5 patients received salvage therapy between 0-15 months. At last follow-up, 11 patients (14%) had died of whom 3 were on ibrutinib at the time of death [CNS involvement, (n=1), causes unrelated to WM, (n=2)]. Conclusions : Ibrutinib is an effective agent in WM patients with MYD88L265Pmutation. Our data regarding outcomes in non-trial setting appear comparable to the results observed in the pivotal clinical trial. Hematologists should be aware of unique ibrutinib associated toxicities and clinically relevant IgM rebound phenomenon following abrupt discontinuation of ibrutinib. Disclosures Ailawadhi: Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Pharmacyclics: Research Funding; Amgen: Consultancy. Gertz:janssen: Consultancy; Prothena: Honoraria; Medscape: Consultancy; Teva: Consultancy; celgene: Consultancy; annexon: Consultancy; Alnylam: Honoraria; Physicians Education Resource: Consultancy; spectrum: Consultancy, Honoraria; Apellis: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Ionis: Honoraria; Research to Practice: Consultancy. Kumar:Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; AbbVie: 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, Research Funding; KITE: 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, Research Funding; Janssen: 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; Takeda: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lacy:Celgene: Research Funding. Dispenzieri:Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dingli:Millennium Takeda: Research Funding; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Millennium Takeda: Research Funding. Kapoor:Celgene: Research Funding; Takeda: Research Funding.

Abstract Introduction: Lenalidomide (Len) in association with standard Rituximab-CHOP (R2CHOP21) has been shown to be safe and effective in newly diagnosed Diffuse Large B-cell Lymphomas (DLBCL) [Nowakowski et al. JCO 2014, Vitolo et al. Lancet Oncol 2014]. The addition of lenalidomide appears to benefit primarily patients with non-Germinal Center B-cell (non-GCB) phenotype as determined by immunohistochemistry (IHC). These early results led to currently ongoing randomized trial in ABC subtype of DLBCL, however efficacy of R2CHOP in ABC DLBCL as defined by Gene Expression Prophyling (GEP) has not been reported. In the present combined analysis of two independent phase 2 studies, we report the long-term follow-up (FU) outcome in DLBCL patients treated with R2CHOP, comparing GCB and ABC according to Nanostring Platform. Methods: We included all newly diagnosed histologically-confirmed de-novo DLBCL patients enrolled in two R2CHOP21 phase 2 trials, conducted by Mayo Clinic (MC) and Italian Lymphoma Foundation (FIL). Inclusion criteria in the two trials were similar, main differences included: all age and all International Prognostic Index (IPI) vs age between 60 and 80 years old and IPI >1, in MC vs FIL study, respectively. All pts received R-CHOP21 plus Len at 25 mg/day for 10 days/cycle and 15 mg/day for 14 days/cycle in MC and FIL trial, respectively. Cell of origin (COO) was determined by IHC, according to Hans algorithm, and retrospectively, in patients with available pathological material, by Nanostring, performed according to Scott algorithm and Masque-Soler signature in MC and FIL study, respectively. We analyzed the long-term FU outcome in terms of progression-free survival (PFS), time to progression (TTP), overall survival (OS), comparing between GCB and ABC phenotypes according to GEP. Results: A total of 112 DLBCL pts (63 MC, 49 FIL) were included. Main characteristics were: median age 69 years (y) (range 22-87), male 65 (58%) pts, advanced stage III-IV in 94 (84%), B symptoms in 38 (34%), International Prognostic Index (IPI) intermediate-high/high in 71 (63%). At a median follow-up of 5.1 years (y), 5y-PFS was 63.5%, 5y-TTP 70.1% and 5y-OS 75.4%. A total of 32 relapses were observed, with only 2 cases of Central Nervous System (CNS) relapse. Late relapse occurring beyond 3 years was observed in 4 cases (3 cases with GCB phenotype and one case with missing COO data). In a subgroup analysis by IPI 0-2 vs 3-5 were: 5y-PFS, 5y-TTP and 5y-OS were: 69.0% vs 59.0% (p=0.100), 73.2% vs 67.4% (p=0.285) and 82.3% vs 70.2% (p=0.059), respectively. Regarding for COO defined by IHC, GCB phenotype vs non-GCB were 45 (40%) vs 41 (37%) patients respectively; 26 (23%) patients were not evaluable. 5y-PFS, 5y-TTP and 5y-OS were: 52.8% vs 64.5% (p=0.198), 61.6% vs 69.6% (p=0.444) and 68.6% vs 74.1% (p=0.238) in GCB vs non-GCB, defined by IHC, respectively. Regarding Nanostring analysis, GCB vs ABC vs Unclassified (Uncl) were 31 (46%) vs 22 (32%) vs 15 (22%) respectively; 44 pts were not evaluable. 5y-PFS, 5y-TTP and 5y-OS were: 62.3% vs 70.8% vs 64.2% (p=0.645), 68.1% vs 79.8% vs 64.2% (p=0.662) and 76.0% vs 74.8% vs 79.0% (p=0.658) in GCB vs ABC vs Uncl, defined by Nanostring, respectively (Table1, Fig1). Conclusions: The association of Len with R-CHOP21 appears to overcome the negative prognostic impact of ABC phenotype determined by GEP, with high PFS, TTP and OS rate, maintained at a long-term FU analysis. Figure 1. Figure 1. Disclosures Chiappella: Teva: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Nanostring: Other: lecture fees; Roche: Other: lecture fees; Amgen: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees. Gaidano:Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria. Witzig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vitolo:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau.

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 Abstract 378 The need for new and more effective long-term treatments for Multiple Myeloma (MM) has led to the utilization and engineering of replication competent (oncolytic) viruses as potential therapies. Vesicular stomatitis virus (VSV) is a potent oncolytic agent with several features that make it a favorable choice as a potential myeloma therapy. Specifically, (i) VSV replicates rapidly and can be grown to high titers (for effective clinical use) (ii) VSV undergoes transcription and replication exclusively in the cytoplasm avoiding host genome integration (iii) there is low/absent pre-existing immunity against VSV among the general population, (iv) naturally occurring human VSV infections are generally asymptomatic or result in minimal flu-like symptoms, (v) VSV is not easily transmitted between individuals (natural transmission is by hematophagous insects). Previously we showed weak oncolytic efficacy with an attenuated strain of VSV coding for the sodium iodide symporter (NIS) gene, VSV-D51-NIS, in the immune competent 5TGM1 syngeneic MM mouse model (C57Bl/KalwRijHsd) (Goel at. al. Blood. 2007 Oct 1;110(7):2342-50). Since VSV replication is strongly inhibited by IFN-induced innate immune responses in normal cells, but not in myeloma cells and IFN has known anti-myeloma activity, we hypothesized that the IFN-coding VSVs would be safer and more potent than previously tested VSV recombinants. We therefore constructed and tested the efficacy of VSVs coding for b-Interferon (VSV-IFN) or b-IFN and NIS (VSV-IFN-NIS). Interestingly, all of the newly constructed viruses, including VSV-IFN-NIS, showed greatly superior replication kinetics compared to the previously reported VSV-D51-NIS virus. Furthermore, compared to VSV-D51-NIS, VSV-IFN-NIS vectors induced higher NIS expression in vitro. In vivo therapy studies showed that a single intravenous dose of each of the IFN-coding VSVs promoted tumor regression and significantly prolonged survival of immunocompetent mice bearing subcutaneous or orthotopic 5TGM1 myeloma tumors. Tc-99m imaging studies conducted in mice treated with VSV-IFN-NIS, showed tumor specific virus mediated NIS expression and radio-isotope uptake that increased concurrently with intratumoral viral spread. Most importantly, there was no evidence of neurotoxicity following treatment with the IFN-coding VSVs. These studies indicate that VSV-IFN-NIS has potential as a novel therapeutic agent for multiple myeloma that can be combined with radio-isotopes for both non-invasive imaging of viral biodistribution and radiovirotherapy. A phase I clinical study is currently planned. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: In both polycythemia vera (PV) and essential thrombocythemia (ET), aspirin and/or hydroxyurea therapy provide adequate protection from thrombosis but not from disease transformation into acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) or myelofibrosis (MF). In designing clinical trials with new drugs, including the use of small molecule JAK2 inhibitors, in either ET or PV, it is important to identify patients who are at a higher risk of early transformation into AML/MDS/MF as well as those in whom the long-term risk of these complications is too low to justify exposure to experimental drugs with unknown short-term and long-term toxicity profile. Methods: Information on an institutional database of 1061 patients with either PV (n=458) or ET (n=603) was updated in July of 2007. The World Health Organization criteria were used for diagnosis of PV, ET, AML, MDS, and MF. Two different approaches were used to assess both early and overall risk of AML/MDS/MF development in both ET and PV. In the first approach, three distinct groups were delineated and their presenting features compared; group A was comprised of patients who have remained AML/MDS/MF free after a minimum follow-up of 20 years whereas groups B and C included patients who developed either AML/MDS or MF, respectively, in the first decade of their disease. In the second approach, prognostic factors for AML/MDS/MF-free survival were examined among the entire cohort of 1061 patients. Results : In ET, the number of patients that fulfilled the above-mentioned criteria for inclusion in groups A, B, and C were 40, 12, and 8; the three groups displayed significant differences in the prevalence, at diagnosis, of lower than normal hemoglobin level (8% vs. 58% vs. 38%; p=0.0004), male sex (18% vs. 58% vs. 50%; p=0.01), and arterial thrombosis at diagnosis (p=0.03), respectively. However, only the former two remained significant during multivariable analysis that included age as a covariate. In PV, the number of patients in groups A, B, and C were 23, 18, and 12; the only difference between the three groups was the association between group B and leukocytosis (p=0.02). Cox regression analysis of the entire 1061 ET/PV patients cohort, including 65 AML/MDS/MF events in ET and 36 AML/MDS events in PV, confirmed the inferior AML/MDS/MF-free survival in ET associated with lower than normal hemoglobin and AML/MDS-free survival in PV associated with leukocytosis (Figure). Conclusion : The current study identifies PV patients with leukocytosis as being the most appropriate and ET patients without anemia as the least appropriate for consideration of participation in experimental drug treatment trials. Figure Figure Figure Figure

Abstract Background JAK inhibitors, including ruxolitinib, are to date incapable of inducing complete (CR) or partial (PR) remissions, reversal of bone marrow (BM) fibrosis or molecular responses in myelofibrosis (MF). This is consistent with the fact that JAK2 mutations are neither specific nor pathogenetically essential for the disease. Other currently available drugs in MF are equally ineffective in terms of disease-modifying activity. Methods In an investigator-sponsored single-center study (ClinicalTrials.gov Identifier: NCT01731951), imetelstat, a lipid-conjugated oligonucleotide inhibitor of human telomerase, was administered to patients with high or intermediate-2 risk MF (JCO 2011). Adverse events were monitored by common terminology criteria (Version 4.03) and responses by the International Working Group criteria (Blood 2013). Eligibility criteria included platelets ≥50 x 10(9)/L. Study drug and funding were provided by Geron Corporation (Menlo Park, CA, USA). Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A) or weekly x 3 followed by every three weeks (cohort B). Mutations with prognostic (ASXL1 and SRSF2) or phenotypic (SF3B1 and U2AF1) relevance were screened by DNA sequencing. Quantitative PCR was used to measure JAK2V617F burden (assay sensitivity 0.01%). Laboratory correlative studies included analyses of granulocyte telomere length, mononuclear cell telomerase activity and human telomerase reverse transcriptase (hTERT) isoforms. Results Thirty-three patients were accrued; the first 18 patients enrolled and followed for a minimum of 3 months or discontinued are presented in this abstract: 11 cohort A and 7 cohort B; 44% PMF, 33% post-PV MF and 22% post-ET MF. Median age was 68 years and baseline risk was high in 56% and intermediate-2 in 44%. Seven patients were transfusion-dependent. Median spleen size was 13 cm and 11 patients had constitutional symptoms. Karyotype was abnormal in 7 patients and 89% were JAK2-mutated. Fifteen (83%) patients were previously treated including 7 with a JAK inhibitor and 3 with pomalidomide. i) Toxicity At a median f/u of 3.2 months, 16 (89%) patients remain on treatment; the two discontinuations were from unrelated death and disease progression. In cohort A, there were no grade-4 treatment-related adverse events; grade-3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, two (29%) patients experienced grade-4 thrombocytopenia; grade-3 events were limited to thrombocytopenia, neutropenia and anemia in one patient each. Dose reduction was necessary in only two (11%) patients because of grade 3 or 4 myelosuppression. ii) Efficacy Overall response rate was 44%. This included five (28%) patients who met the BM and peripheral blood morphologic criteria for CR (n=4) or PR (n=1) and 3 patients with clinical improvement, pending validation of response duration and resolution of drug-induced grade-1 thrombocytopenia. The four (22%) CR patients experienced reversal of BM fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline and became transfusion-independent. Complete molecular responses were documented in 2 CR patients: one had U2AF1Q157P and 10% JAK2V617F and the other SF3B1K666E and 50% JAK2V617F. A third CR patient had a >50% reduction in U2AF1 469_insAGTATG mutation. Among 13 patients with leukocytosis, 10 (77%) normalized their count or had >50% reduction. Eleven (61%) patients had complete or partial resolution of leukoerythroblastosis. iii) Laboratory correlative studies Three (50%) of 6 spliceosome-mutated vs. 1 (8%) of 12 unmutated (p=0.045) achieved CR. Spliceosome-mutated patients were also more likely to experience grade-3/4 myelosuppression (67% vs. 25% ; p=0.09). Treatment was associated with suppression of telomerase activity, shortening of telomere length and alteration of hTERT isoform pattern. Conclusions The current study signifies the potential value of telomerase-based treatment strategies in MF and identifies imetelstat as an active drug in that regard. The observed morphologic and molecular remissions confirm selective anti-clonal activity, which has thus far eluded other drugs in MF, including JAK inhibitors. The association between response and spliceosome mutations suggests a broader application for the drug in myeloid malignancies.</abstract> Disclosures: Stuart: Geron: Consultancy.

Abstract Abstract 1751 Because IDH mutations are frequent in blast-phase myeloproliferative neoplasms (MPN), they might contribute to leukemic transformation. We examined this possibility in 301 consecutive patients with chronic-phase primary myelofibrosis (PMF). All study patients were fully characterized for karyotype, JAK2 and MPL mutational status, and Dynamic International Prognostic Scoring System-plus (DIPSS-plus) risk status. DNA from bone marrow or peripheral blood was used to screen for IDH1 and IDH2 mutations, by direct sequencing and/or high resolution melting (HRM) assay. Mutant IDH was detected in 12 patients (4%): seven IDH2 (five R140Q, one R140W and one R172G) and five IDH1 (three R132S and two R132C). MPL exon 10 was mutated in 18 patients (6.3%) and constituted W515L in 14 patients, W515K in 3 and a frameshift mutation in 1 patient. JAK2V617F was detected in 169 (56%) patients. Six patients displayed both JAK2V617F and IDH mutations (IDH2R140Q in 2 patients, IDH2R140W in 1 and IDH1R132S in 3); JAK2V617F allele burden was 1%, 7%, 22%, 27%, 30% and 96%, respectively. One patient displayed both IDHR140Q and MPLW515R. One-hundred and seven (36%) patients were negative for all three mutations. The 12 IDH-mutated patients were clinically compared to patients belonging to the three other molecular subgroups: mutated for JAK2 only (n=164), mutated for MPL only (n=18) and unmutated for all three (n=107). The four molecular subgroups were remarkably similar in their phenotype with few exceptions; IDH-mutated patients were significantly older than those with no mutations (p=0.04) whereas age distribution was similar between patients with mutant IDH, MPL or JAK2. In univariate analysis, overall survival (OS) for IDH-mutated patients was significantly shorter than those for JAK2-mutated (p=0.03), MPL-mutated (p=0.047) or unmutated (p=0.0009) patients. IDH-mutated patients also showed significantly shorter leukemia-free survival (LFS), compared to those with mutant JAK2 (p=0.0008), mutant MPL (p=0.02) or no mutations (p=0.001). After accounting for age, the presence of mutant IDH remained a significant disadvantage for both OS (p=0.04) and LFS (p=0.005). Multivariable analysis of OS that included risk categorization per DIPSS-plus confirmed the independent prognostic relevance of mutant IDH (p=0.03): HR for patients with no mutations =0.39, 95% CI 0.2–0.75; HR for JAK2-mutated patients =0.50, 95% CI 0.27–0.95; HR for MPL-mutated patients =0.53, 95% CI 0.23–1.2. A similar analysis for LFS that included risk factors for LT (i.e. unfavorable karyotype and platelet count <100 × 109/L) as covariates also confirmed the prognostic relevance of mutant IDH (p=0.003): HR for patients with no mutations =0.16; 95% CI 0.06–0.46; HR for JAK2-mutated patients =0.18; 95% CI 0.06–0.48; HR for MPL-mutated patients =0.09; 95% CI 0.01–0.76). Further analysis disclosed that the negative OS and LFS effect of mutant IDH was most pronounced in the presence (p=0.0002 and <0.0001, respectively) as opposed to the absence (p=0.34 and 0.64, respectively) of concomitant JAK2V617F expression. Analysis of paired samples obtained during the chronic and blast phases of the disease was possible in 2 IDH-mutated patients and showed the presence of both IDH and JAK2 mutations at both time points. Our observations suggest that IDH mutations in PMF are independent predictors of leukemic transformation and raise the possibility of leukemogenic collaboration with JAK2V617F. Disclosures: No relevant conflicts of interest to declare.