Hanson, Curtis A.

Abstract Background JAK inhibitors (JAKi) currently represent a frontline treatment modality in patients with symptomatic intermediate- or high-risk myelofibrosis (MF). While the efficacy of JAKi in decreasing spleen size and improving disease-related symptoms is well documented, the effect of this drug class on overall survival and risk of leukemic transformation in MF patients is less well understood, primarily due to short follow up in individual patient cohorts. Furthermore, there is scant information on baseline predictors of overall and leukemia-free survival, and also spleen and/or anemia responses, at the onset of JAKi therapy. We conducted a sponsor-independent single-center retrospective review of patients treated with JAKi with long follow up to address the aforementioned issues. Here, we report our findings regarding baseline predictors of clinical outcome and treatment response in MF patients treated with JAKi. Methods Clinical, cytogenetic and molecular data were retrospectively collected on patients treated with one or more JAKi on a clinical trial at the Mayo Clinic, Rochester, MN. Baseline clinical data pertained to the onset of treatment with the first JAKi. Karyotype information was available for all patients. Screening for JAK2V617F, MPLW515, ASXL1, and SRSF2 mutations was performed as previously described (Leukemia. 2013 Apr 26. doi: 10.1038/leu.2013.119). Spleen size was measured by palpation by clinical investigators. Anemia and spleen responses were adjudicated as per IWG criteria (Tefferi, Blood, 2006). Information on survival and leukemic transformation was updated in July 2013. Results A total of 157 MF patients were studied (60% male; median age 65 years, range 34-89 years). Ninety seven patients (62%) had primary MF, 42 (27%) post-polycythemia vera MF, and 18 (11%) post-essential thrombocythemia MF. The DIPSS-plus distribution was: Low/Int-1 20 (13%), Int-2 66 (42%), and High 71 (45%). Other characteristics were: JAK2V617F positive 78%, unfavorable karyotype 17%, hemoglobin <10 g/dL 58%, leukocyte count >25 x 109/L 31%, platelet count <100 x 109/L 15%, peripheral blasts ≥1% 69%, red blood cell transfusion-dependent 37%, and constitutional symptoms 56%. Forty one patients (38%) were ASXL1 mutated (n=108) and 20 (18%) SRSF2 mutated (n=110). One hundred and thirty nine patients (89%) were evaluable for spleen response (median spleen size by palpation 19 cm, range 6-32 cm) and 91 (58%) for anemia response by IWG criteria. The first JAKi was momelotinib (CYT387) in 79 patients (50%), ruxolitinib in 51 (33%) and fedratinib (SAR302503) in 27 (17%). Median follow up from first JAKi treatment was 30 months (range 1-67 months); during this period, 72 deaths (46%) and 14 (9%) leukemic transformations have been documented. The median overall survival of the entire cohort was 41 months. Multivariable analysis identified the following DIPSS-plus independent predictors for survival: palpable spleen size >20 cm (HR 2.5), ASXL1 mutated status (HR 3.1) and SRSF2 mutated status (HR 3.8) (all p<0.01) (Figure). In contrast, there was no difference in survival with regards to the first JAKi used for MF treatment (p=0.3). Multivariable analysis identified the following predictors for leukemia-free survival: SRSF2 mutated (HR 9.7), unfavorable karyotype (HR 8.7), and palpable spleen size >20 cm (HR 9.1) (all p<0.01). Predictors of spleen response included baseline spleen size (≤20 cm 56%, >20 cm 34%; p=0.01), with borderline significance for ASXL1 mutation status (unmutated 51%, mutated 31%; p=0.06). Predictors of anemia response included RBC transfusion status at baseline (transfusion dependent 38%, transfusion independent 12%; p<0.01) and JAK2V617F status (unmutated 46%, mutated 22%; p=0.02). Conclusions Larger baseline spleen size and ASXL1 and SRSF2 mutated status predicted for inferior overall survival, independent of DIPSS-plus, at the onset of first JAKi treatment in MF patients. Similarly, multivariable analysis identified larger baseline spleen size and presence of unfavorable karyotype and SRSF2 mutations as being predictive for inferior leukemia-free survival. Spleen response rates were significantly superior in patients with smaller baseline spleen size and possibly ASXL1 unmutated status. Anemia response rates were significantly higher in patients who were transfusion-dependent at baseline and had JAK2V617F unmutated status. Disclosures: Pardanani: Bristol Myers Squibb: Clinical trial support Other; Sanofi: Clinical trial support, Publication support services, Clinical trial support, Publication support services Other; PharmaMar: Clinical trial support, Clinical trial support Other; JW Pharmaceutical Corp.: Clinical trial support, Clinical trial support Other. Off Label Use: Use of Ruxolitinib, Momelotinib, Fedratinib and Pomalidomide for treatment of Myelofibrosis in Clinical Trial setting.

Abstract Background In myelodysplastic syndromes (MDS), we have previously shown the adverse prognostic effect of ALC <1.2 x 10(9)/L (Nisha et al. Am J Hematol 2010;85:160). In the current larger study of 889 patients with MDS, we sought to validate and expand on our previous observations and also examine the effect of lymphocyte-to-monocyte ratio (LMR). Methods We retrospectively recruited 889 consecutive patients with primary MDS who were untreated at the time of referral to our institution, and in whom ALC and absolute monocyte count (AMC) at time of referral was documented. The diagnosis of MDS and leukemic transformation (LT) was according to WHO criteria (Blood. 2009;114:937). Complete follow-up information was updated in January 2015. Comparison of laboratory and clinical features was made between patients with subnormal and normal ALC; patients with above normal ALC were considered separately. Overall and leukemia-free survivals of patients with normal ALC was compared to those with either subnormal ALC or, in order to validate our previous observation, with ALC <1.2 x 10(9)/L. Results Patient characteristics: Median (range) values for the 889 study patients (69% males) included: age 72 (18-98), hemoglobin 9.6 g/dL (5.4-15.7), leukocyte count 3.4 x 10(9)/L (0.4-35), AMC 0.2 x 10(9)/L (0-1.7), platelet count 106 x 10(9)/L (2-1804), circulating blasts 0% (0-18) and bone marrow blasts 3% (0-19). Transfusion need was documented in 33% of patients and abnormal karyotype in 49%. Risk stratification by the revised international prognostic scoring system (IPSS-R) was very high in 10%, high in 16%, intermediate in 20%, low in 36% and very low in 16%. The median (range) ALC for the entire study population of 889 patients was 1.16 x 10(9)/L (0.02-8.9). The normal range of ALC at our institution was 0.9-2.9 x 10(9)/L; the number of patients with subnormal, normal and above normal ALC was 261 (29%), 598 (67%) and 30 (4%), respectively. In addition, 442 (50%) and 417 (47%) patients had ALC below 1.2 x 10(9)/L and ALC between 1.2 and the upper limit of normal, respectively. After a median follow-up of 27 months, 712 (80%) deaths and 116 (13%) leukemic transformations were documented. Comparison of patients stratified by absolute lymphocyte count Compared to patients with normal ALC, patients with subnormal ALC displayed lower hemoglobin (p=0.002), higher red blood cell transfusion need (p=0.0003), lower leukocyte count (p<0.0001), lower monocyte count (p=0.002) and lower platelet count (p<0.0001), whereas borderline association was also apparent for older age (p=0.06). In univariate analysis, survival was adversely affected by lower ALC, treated as a continuous variable (p=0.01). Similarly, when compared to patients with normal ALC, patients with subnormal ALC (p=0.001; HR 1.3, 95% CI 1.1-1.5) and those with ALC <1.2 x 10(9)/L (p=0.0002; HR 1.3, 95% CI 1.2-1.6) experienced significantly shortened survival. There was no difference in survival between patients with above normal ALC and those with either subnormal (p=0.58) or normal (p=0.41) ALC; accordingly, patients with above normal ALC were excluded from subsequent analysis. In multivariable analysis, the adverse effect of subnormal ALC was shown to be independent of age, gender, anemia, thrombocytopenia, neutropenia , circulating blast percentage, bone marrow blast percentage or cytogenetic risk stratification by and IPSS; the same held true for ALC <1.2 x 10(9)/L. The association with IPSS-R was of borderline significance for both ALC <1.2 x 10(9)/L (p=0.06) and subnormal ALC (p=0.1). The adverse effect of ALC was most apparent in patients with very low or low risk disease per IPSS-R (p=0.0007 for subnormal ALC and 0.0003 for ALC <1.2 x 10(9)/L), compared to those with higher risk status (p=0.67 and 0.7, respectively). Neither subnormal ALC (p=0.4) nor ALC <1.2 x 10(9)/L (p=0.1) affected leukemia free survival. Analysis using LMR in place of ALC showed similar but not necessarily superior results. Conclusions Compared to those with normal ALC, MDS patients with either subnormal ALC or with ALC below 1.2 x 10(9)/L experienced shorter overall, but not leukemia-free, survival; this adverse effect was most apparent in lower risk patients and was independent of most of the currently established risk factors for survival in MDS. The observations from the current study suggest the possible therapeutic value of augmenting host immunity in lower risk MDS. Disclosures Al-Kali: Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding.

Abstract Background Spliceosome pathway gene mutations are recurrent in myeloid malignancies with the highest frequencies reported for myelodysplastic syndromes associated with ring sideroblasts (MDS-RS; 85%), MDS without RS (44%) and chronic myelomonocytic leukemia (CMML; 55%) (Nature 2011;478:64). SF3B1 mutations were the most frequent (75%) in MDS-RS and SRSF2 mutations in CMML (28%); U2AF1 mutational frequencies were 12% in MDS without RS and 8% in CMML. We have previously described SRSF2 (Blood. 2012;120:4168) and SF3B1 (Leukemia. 2012;26:1135) mutations in 17% and 6.5% of patients with primary myelofibrosis (PMF); prognostic relevance was shown for the former but not the latter. Objectives The objectives of the current study were to i) describe the incidence of U2AF1 mutations in PMF and their correlation with clinical features, karyotype and other mutations and ii) examine the prognostic significance of U2AF1 mutations in PMF, in the context of both conventional prognostic models and other prognostically-relevant mutations. Methods Information on clinical and laboratory parameters and karyotype was available in all study patients, at time of referral, which coincided with time of sample collection for mutation screening. Risk stratification was according to the Dynamic International Prognostic Scoring System (DIPSS)-plus system. U2AF1 and other mutations were analyzed using standard PCR techniques and bidirectional sequencing; for U2AF1, two hot spots that included residues S34 and Q157 were amplified. Results A total of 251 PMF patients (median age 63 years; 160 males) were studied. DIPSS-plus risk distribution was high in 32%, intermediate-2 in 38%, intermediate-1 in 17% and low in 13% of the patients. The frequency of each DIPSS-plus adverse feature was as follows: age >65 years (42%), transfusion need (32%), hemoglobin <10 g/dL (47%), leukocyte count >25 x 10(9)/L, (16%), platelet count <100 x 10(9)/L (22%), ≥1% blasts (57%), constitutional symptoms (35%), and unfavorable karyotype (9%). Karyotype was normal in 156 (63%) patients. At a median follow-up of 48 months, 158 (63%) deaths and 27 (11%) leukemic transformations were recorded. Mutational frequencies Forty-one (16.3%) patients harbored U2AF1 mutations: 16 (39%) Q157P, 10 (24%) Q157R, 8 (20%) S34F, 4 (10%) S34Y and one each for Q157P/E159A, Q157R/S34Y and Q157-Y158insYE. Frequencies for other mutations were 11% for SRSF2, 7.3% for SF3B1, 31% for ASXL1, 5.5% for EZH2, 5% for IDH1/2 and 58% for JAK2V617F. U2AF1 mutations were usually, but not always, exclusive of other spliceosomal mutations: one patient expressed all three spliceosomal mutations. The frequency of any one of the three spliceosomal mutations was 34%. Clinical correlates U2AF1 mutations were significantly associated with older age (p=0.02), JAK2V617F (p=0.002), mutant ASXL1 (p=0.04), transfusion need (p<0.0001), hemoglobin <10 g/dL (p<0.0001), platelets <100 x 10(9)/L (p<0.0001) and normal karyotype (p=0.006). The associations with anemia, thrombocytopenia, JAK2V617F and normal karyotype were inter-independent; the frequency of U2AF1 mutations in the presence of anemia was 29%, transfusion need 36%, thrombocytopenia 35%, JAK2V617F 23% and normal karyotype 21%. Prognostic interactions U2AF1 mutations were associated with inferior overall (p=0.004) but not leukemia-free (p=0.6) survival. However, the survival association was fully accounted for by the above-mentioned clustering of U2AF1 mutations with anemia and thrombocytopenia. Similarly, although multivariable analysis of U2AF1, SRSF2, ASXL1, EZH2 and IDH mutations identified the first three as being independently predictive of poor survival, only ASXL1 and SRSF2 remained significant when either anemia or thrombocytopenia was included as a co-variate. Conclusions U2AF1 mutations are the most frequent spliceosome pathway mutations in PMF, cluster with normal karyotype and JAK2V617F, and are strongly associated with anemia and thrombocytopenia; the latter suggests a pathogenetic contribution to ineffective hematopoiesis in PMF. The current study also suggests that more than one third of patients with PMF carry a spliceosome mutation. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Chronic lymphocytic leukemia (CLL) can effect renal function in a variety of ways including direct infiltration of the kidney, ureteral obstruction by lymphadenopathy, and treatment related tumor lysis syndrome (uric acid nephropathy). Rarely, CLL has also been reported to be associated with light chain nephropathy, renal amyloidosis, membranoproliferative glomerulonephritis (MPGN), granulomatous interstitial nephritis (GIN), and minimal change disease (MCD). Nearly all the data on the effects of CLL on renal function is at the case report level. We systematically evaluated the prevalence of renal insufficiency at diagnosis as well the incidence of acquired renal insufficiency during follow-up in a large cohort of patients with newly diagnosed CLL to more accurately define the effects of CLL on the kidney and its impact on clinical outcomes. Methods Between January 1995 -February 2013, previously untreated CLL patients seen in the Division of Hematology at Mayo Clinic at diagnosis (<12 months) and who had baseline assessment of serum creatinine were included in this analysis. Patients with serum creatinine (Cr) ≥1.5 mg/dL at baseline were classified as having renal insufficiency at diagnosis. Patients who initially had baseline creatinine <1.5 mg/dL but who developed a Cr≥1.5 mg/dL during the course of their disease were considered to have acquired renal insufficiency. Results Existing renal insufficiency at the time of CLL diagnosis: Of 2047 patients who met the eligibility criteria, 153 (7.5%) patients had renal insufficiency (Cr≥1.5 mg/dL) at the time of CLL diagnosis including 15 (0.7%) with a Cr≥3 mg/dL. Renal insufficiency was also more common among men (9.3% vs. 3.9%; p<0.00001), those with advanced stage disease (Rai 0=7.0%; Rai I-II=6.4%, Rai III-IV=20.2%; p<0.0001), and CD49d positive patients (6.8% vs. 3.8%; p<0.038). Patients with renal insufficiency at diagnosis were also older (median age 72.2 vs. 63.9; p<0.0001). No difference in the prevalence of renal insufficiency at diagnosis was observed based on cytogenetic abnormalities detected by FISH or CD38, ZAP-70 or IGHV gene mutation status. Although renal insufficiency at diagnosis was strongly associated with OS on univariate analysis (p<0.001), no association was observed between renal insufficiency and TTT or OS on multi-variate analysis adjusting for age, sex, and Rai stage. Acquired renal insufficiency during CLL disease course: Among the 1894 patients with normal renal function at diagnosis, 304 (16.1%) acquired renal insufficiency (Cr≥1.5 mg/dL) during the course of their CLL disease course including 43 (2.3%) with peak Cr≥3 mg/dL. In addition to age (older) and male sex, a number of CLL disease characteristics were associated with a higher likelihood of acquired renal insufficiency including: IGHV UM (OR=2.0; p=0.0001), unfavorable FISH (del17p- or 11q-; OR=2.0; p=0.001), and being CD49d+ (OR=1.8; p=0.002), ZAP-70+ (OR=1.6; p=0.004), or CD38+ (OR=1.4; p=0.0.032),. Shorter TTT (p<0.001) and OS (P<0.001) was observed among patients with initially normal creatinine who acquired renal insufficiency (Figure 1A and 1B). On MV analysis adjusting for age, sex, and stage at diagnosis, acquired renal insufficiency remained an independent predictor of TTT (OR=1.77; p=0.001) and OS (OR=2.67; p<0.001). Renal insufficiency and therapy selection After median follow-up of 4.5 years (range 0-18.0), 620 of 2047 (30.3%) patients have progressed to require treatment. Patients with renal insufficiency prior to treatment were less likely to receive purine nucleoside analogue based therapy and more likely to receive single agent alkylator based treatment. Conclusions Approximately 1 in every 13 patients (7.5%) with CLL has renal insufficiency at the time of diagnosis and an additional 16.1% acquire renal insufficiency during the course of the disease. The risk of developing renal insufficiency is associated with a variety of CLL B-cell characteristics and is associated with TTT and OS. Data on causes of acquired renal insufficiency is being abstracted and will be presented at the meeting. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Off Label Use: MK2206 in a phase 1 trial of CLL.

Abstract Activating mutations in colony stimulating factor 3 receptor gene (CSF3R) have recently been identified as specific driver mutations in chronic neutrophilic leukemia (CNL)(NEJM 2013;368:1781). We sought to outline the clinical and laboratory characteristics and natural history of this molecularly defined entity. Methods : An IRB- approved retrospective analysis of all cases of CSF3R-mutated WHO-defined CNL was undertaken. Methods of mutation analysis have been previously published. (Leukemia 2013; 27: 1870) Results : From 10/1995 to 3/2013, fourteen patients (median age 67 years; 57% male) with CSF3R-mutated CNL were evaluated at the Mayo Clinic. Most of the CSF3R mutations identified occurred in isolation: T618I (n=11), M696T (n=2) and I598I SYN (n=1), although one T618I mutant patient also carried a cytoplasmic domain mutation (2341_2342insC ). All patients were BCR/ABL negative. Five carried a SETBP1 mutation: G870D (n=2), D868N (n=2) and G872R (n=1), all of whom carried the T618I mutation. Only one of 10 tested carried a CALR mutation (E398D) and none of 13 the JAK2 V617F mutation. Apart from fatigue, most patients were asymptomatic at diagnosis and leukocytosis had been mostly an incidental laboratory finding. Ten (71%) patients had preceding chronic leukocytosis, median duration 12.5 months (range 5-84), 5 reported weight loss, which was accompanied by fevers/ sweats in 3. Five (36%) patients had prior exposures to adjuvant chest wall irradiation for breast cancer (n=2), radioiodine for thyroid disease (n=2) and methotrexate/cyclophosphamide for autoimmune disease (n=2). Five (36%) patients had palpable splenomegaly. Peripheral blood (PB) parameters, median (range) at diagnosis were: Hb 10.8 g/dl (8.5-16.0), WBC: 54 x 10(9)/L (21.7-176.2), platelets: 201 x 10(9)/L (25-476), neutrophils: 85% (78-94), immature cells (myelocytes + metamyelocytes): 6% (0-11) and monocytes: 3% (0-10). Neutrophils had toxic granulation and Dohle Bodies in 71% of cases. LDH was elevated in all patients: 354 U/L (225-496). None had a monoclonal protein. The bone marrow (BM) was hypercellular in all patients: 95% (80-100%) and granulocytic precursors were markedly increased with left-shifted (n=11) or normal maturation. BM blasts were < 5%. Reticulin fibrosis was absent (n=5), 1+ (n=5) or 1-2+ (n=1) in those with staining. Cytogenetics were normal in all but one at diagnosis. One patient was lost to follow up after diagnosis. Hydroxyurea (HU) was the most frequently used therapy (initial therapy in 10 patients) and was ultimately received by all. Although initially effective at controlling leukocytosis, clinical course of most was defined by progressive refractory mature neutrophilia with the development of red cell (n=6) and platelet transfusion (n=3) dependancy. Other therapies included IFN or PEG/IFN (n=4), hypomethylating agents (n=4), ruxolitinib (n=2), thalidomide (n=2), cladribine (n=2) and imatinib (n=1). None of these additional drugs, induced remission and they provided primarily palliative and transient benefit. One patient transformed into AML at 17 months, one received induction chemotherapy for suspected AML (PB and BM blasts at 11-13%) at 19 months and two evolved into WHO-defined CMML, at 20 and 27 months, respectively. Both patients with documented/suspected AML were both T618I and SETBP1 positive. Of the cases progressing to CMML (one with T618I, one with M696T) neither was SETBP1 positive. At last follow up, eight patients had died and median survival was 22.5 months. Progressive refractory neutrophilic leukocytosis often preceded and contributed to death. Four (29%) patients are currently alive after a median follow-up of 61 months (14.3- 69.4). Conclusion : CSF3R-mutated CNL is an aggressive disease (median survival <2 years) characterized by progressive neutrophilic leukocytosis which is only partially controlled with current drugs, including the putative targeted therapy ruxolitinib (both ruxolitinib-treated patients progressed on treatment). Patients with CNL could progress into AML or CMML; however, most succumb from complications associated with progressive treatment-refractory leukocytosis. The observation that both patients with documented/suspected AML were SETBP1-mutation positive suggests potential pathogenetic link. Disclosures Off Label Use: Ruxolitinib, cladribine, thalidomideand inteferon. These drugs were used in a historical account of CNL and I report on these in a retrospective chart review only..

Abstract Introduction Approximately 2-8% chronic lymphocytic leukemia (CLL) patients develop diffuse large B-cell lymphoma (DLBCL, so-called Richter syndrome [RS]) over long-term follow-up. The outcomes of patients with RS are variable and poorly understood. Methods CLL patients seen at Mayo Clinic from January 1995 to April 2013 who developed biopsy-proven DLBCL during follow-up were included in this analysis. Clinical characteristics, therapy and overall survival (OS) of all patients with RS were analyzed. Results One hundred and twenty patients with biopsy-proven RS were identified in the Mayo Clinic database. The median age at diagnosis of RS was 67 years (range, 29-88 years), 87 (73%) were male, and 83 (69%) had received prior CLL therapy. The median duration between diagnosis of CLL and development of RS was 3.8 years (range, 0-34.5 years). Stereotyped B-cell receptors were found in 9/30 (30%) RS patients compared to 219/1294 (17%) non-RS patients (OR=2.1, p=0.07). IGHV4-39 gene usage was present in 2/26 (8%) RS patients compared to 57/1354 (4%) non-RS patients (p=0.3). Of the 30 RS biopsy samples in which Epstein Barr virus (EBV) in-situ hybridization studies were performed, EBV was detected in 7 (23%) patients. After a median follow-up of 5.2 years, 86 (72%) patients have died. Causes of death were as follows: progressive DLBCL (n=46, 53%), progressive CLL (n=2, 2%), infection (n=8, 9%), unknown (n=20, 23%) and other (n=10, 12%). The median OS of the entire cohort was 0.9 years (95% CI, 0.5-1.3 years). Tsimberidou and colleagues proposed a prognosis score (RS score) based on the following variables at the time of RS diagnosis: Eastern Co-operative Group performance status of 2-4, lactate dehydrogenase >1.5 x normal, platelets<100 x 109/L, tumour size >5 cm and >1 prior therapy for CLL. Each parameter was assigned one point, and patients were then assigned to one of four risk groups based on their calculated RS score: 0 or 1, low risk; 2, low-intermediate risk; 3, high-intermediate risk; 4 or 5, high risk. The OS according to the RS score for our group of patients is shown in Figure 1. Median survival in the low, intermediate-1, intermediate-2 and high-risk groups were 2.4 years, 0.7 years, 0.3 years and 0.2 years, respectively (p<0.001). Fifteen (12%) patients received cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) as initial therapy for RS, and 68 (57%) received rituximab in addition to CHOP (R-CHOP). There was no difference in survival among patients who received CHOP compared to R-CHOP (median, 1.8 years. vs. 1.2 years, p=0.5). Twenty-four patients (20%) received non-CHOP-based chemotherapy, and treatment was unknown in 4 (3%) patients. Nine patients (7%) died before definitive therapy could be instituted. Thirteen (11%) patients were able to proceed to stem cell transplantation (SCT): 10 (8%) autologous SCT, 2 (2%) allogeneic SCT and 1 (1%) autologous SCT followed by an allogeneic SCT. Although RS patients who underwent SCT were younger compared to those who did not undergo SCT (median, 59 years. vs. 69 years, p=0.001), no differences in prior CLL therapy or receipt of R-CHOP therapy for DLBCL was noted between the two groups. The median survival of patients who proceeded to SCT was superior compared to those who did not (5.2 years vs. 0.7 years; p=0.005). Conclusion Stereotyped B-cell receptors but not VH4-39 had a suggested evidence of an increased risk of RS in our cohort. Although 90% RS patients received intensive chemotherapy, only 11% patients were able to proceed to SCT. Median survival of RS patients remains dismal at 1 year, and newer therapies are needed to improve outcomes. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Background Increased serum levels of lactate dehydrogenase (LDH) typically accompany primary myelofibrosis (PMF) and might be linked to increased cell turnover from clonal myeloproliferation, including leukocytosis, low grade hemolysis and extra-medullary hematopoiesis in the liver. Despite the fact that serum LDH has been extensively utilized as a prognostic marker in both lymphoid neoplasms and solid tumors, there are limited studies in PMF that have systematically examined the clinical significance of the degree of serum LDH elevation in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF, including both pre-PMF and overt PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of serum LDH at time of referral. Marked elevation of serum LDH was defined as a value of ≥1000 U/L (i.e. over 4-fold increase from the upper limit of the normal range for our institution, which was 122-222 U/L), based on preliminary analysis of the threshold for prognostic effect. Targeted next generation sequencing was used to screen for prognostically-relevant mutations (Blood 2015126:354). Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results LDH values in overt PMF versus pre-PMF: The entire study population consisted of 357 patients, including 311 with overt PMF and 46 with pre-PMF. The median serum LDH level was 514 U/L (range 136-2263): overt PMF 532 U/L (range 136-2263); pre-PMF 401 U/L (range 180-1237) (p=0.0003). Accordingly, in order to avoid the confounding effect of the difference in serum LDH level between overt PMF and pre-PMF, and considering the small number of patients with pre-PMF, further analysis was limited to the 311 patients with overt PMF. Patient characteristics: The 311 patients (median age 64 years; 66% males) with overt PMF included 205 (66%) JAK2, 49 (16%) type 1/like CALR, 13 (4%) type 2/like CALR, 16 (5%) MPL mutated cases and 28 (9%) were "triple-negative". DIPSS-plus risk distribution was 31% high, 43% intermediate-2, 15% intermediate-1 and 12% low; 30% displayed red cell transfusion-dependency and 37% abnormal karyotype, including 14% with unfavorable karyotype. 184 patients were screened for ASXL1 mutations with 42% mutated and 205 for SRSF2 mutations with 16% mutated. Clinical correlates of markedly elevated LDH (≥1000 U/L): Among all 311 study patients with overt PMF, 37 (12%) displayed marked elevation of LDH (≥1000/L). Patients with marked elevation of LDH displayed significantly higher leukocyte count (p=0.005; R-squared = 0.05), circulating blast percentage (p=0.03; R-squared = 0.07) and SRSF2 mutational frequency (44% vs 12%; p<0.0001). Survival analysis: After a median follow up of 3 years, 199 (64%) deaths and 31 (10%) leukemic transformations were documented. In univariate analysis, increased serum LDH level was associated with inferior survival, both as a continuous variable (p=0.002) and as a categorical variable with the cutoff level of 1000 U/L (HR 2.02, 95% CI 1.3-3.1; p=0.001); the survival effect LDH ≥1000/L was independent of DIPSS-plus (HR 1.6, 95% CI 1.1-2.5). Other variables that were significantly associated with shortened survival, on univariate analysis, included all 8 DIPSS-plus variables (p≤0.01 in all instances), absence of CALR type 1/type 1-like (p<0.0001) and presence of ASXL1 (p<0.0001) or SRSF2 (p=0.0006) mutations. In multivariable analysis that included only genetic risk factors, serum LDH retained its significance (HR 2.2, 95% CI 1.3-3.6), along with absence of CALR type 1/type 1-like, or presence of ASXL1 or SRSF2 mutations or unfavorable karyotype. In multivariable analysis that included only clinical variables, serum LDH ≥1000/L was again independently predictive of poor survival (HR 1.7, 95% CI 1.1-2.6), along with age >65 years, hemoglobin <10 g/dL, platelets <100 x 10(9)/L, leukocyte count >25 x 10(9)/L and constitutional symptoms. Patients with marked LDH elevation were also more likely to undergo leukemic transformation (HR 3.1, 95% CI 1.2-7.6). Conclusion The current study suggests that marked elevation of serum LDH in PMF indicates aggressive tumor biology that is currently not accounted for by established clinical or genetic risk factors; the low R-squared values seen in relation to leukocyte count and circulating blasts are consistent with this assumption. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND: Although chemoimmunotherapy (CIT) has improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 40-50% of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays. Interactions with nurturing environments can enhance CLL B-cell resistance to apoptosis. These interactions include cytokine mediated prosurvival signals by angiogenic molecules, such as VEGF and bFGF that nurture CLL B-cells in an autocrine fashion and promote CLL cell survival partly through up regulation of anti-apoptotic proteins. These findings provide a strong rationale for testing anti-VEGF therapy in combination with a purine nucleoside analogue CIT regimen for upfront treatment. We conducted a randomized phase 2 CIT trial using pentostatin, cyclophosphamide, and rituximab with (PCR-B) or without (PCR) bevacizumab (B), an anti-VEGF monoclonal anti-body. METHODS: Eligible patients were previously untreated and had CLL in need of treatment by NCI-WG criteria (Blood 111:5446). Patients were randomized using a dynamic allocation procedure stratifying for stage (0-II vs. III-IV) and FISH (17p or 11q deletion vs. other) to receive either 6 cycles of rituximab (100 mg on day 1 of cycle 1; 375 mg/m2on day 2 of cycle 1 and day 1 of cycles 2-6) followed by pentostatin (2 mg/m2) and cyclophosphamide (600 mg/m2) (PCR) administered every 21 days. Patients in the PCR-B cohort also received bevacizumab 15mg/kg on day 1 of cycles 1-5 and days 1, 22, & 43 of cycle 6. All patients underwent complete response evaluation 3 months after day 1 of cycle 6 (or last cycle of treatment for those completing < 6 cycles). MRD was assessed using 6-color flow cytometry (Leukemia 21:956) at the completion of treatment. RESULTS: 68 patients were enrolled through the Mayo Clinic Cancer Research Consortium between 1/2009 and 1/2013. Three patients were excluded from analysis: 1 patient canceled prior to treatment, 1 was dosed incorrectly, and 1 was ineligible due to immunophenotyping inconsistent with CLL. Median age of eligible patients was 63 years (range 43-81) and 43 (66%) were men. With respect to disease stage, 3 (5%), 38 (58%), and 24 (37%) had low, intermediate and high Rai stage disease. Eleven (17%) patients had deletion 17p or 11q & 29 (45%) had unmutated IGHV. No statistically significant differences were observed in these variables by treatment arm. All 65 evaluable patients have completed active treatment, with 54 (83.1%) completing the intended 6 cycles (PCR group 27/32 [84.4%] and PCR-B 27/33 [81.8%]). Hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 10 (31.3%) patients on PCR and 12 (36.4%) on PCR-B (p=0.79). Non-hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 9 (28.1%) patients on PCR and 18 (54.4%) on PCR-B (p=0.04). The most common such events were hypertension (PCR: 3.1% vs. PCR-B: 21.2%), proteinuria (0% vs. 6.1%) and creatinine increase (3.1% vs. 6.1%). Across both arms, 64/65 (98.5%) patients achieved a response including 31/32 (96.9%) treated with PCR and 33/33 (100%) treated with PCR-B (p=0.49). CR/CRi was achieved in 10/32 (31.3%) patients treated with PCR & 18/33 (54.5%) treated with PCR-B (p=0.08). Of the 28 who achieved a CR/CRi, MRD analysis was completed on 26, of whom 12 (46%) were MRD negative. With respect to treatment arm, 5/32 (16%) patients on PCR and 7/33 (21%) on PCR-B achieved an MRD negative CR. Median time to retreatment for all 65 patients was 44.8 (95% CI: 34.6 – NA) months. Median overall survival has not yet been reached. With current follow-up no differences between treatment-free survival (p=0.38), progression-free survival (p=0.23), or overall survival (p=0.45) are observed by treatment arm. Plasma levels of angiogenic cytokines VEGF, bFGF, thrombospondin (TSP) and the chemokines CCL3 and CCL4 were measured prior to treatment and at the time of the post treatment response evaluation. Correlations of these cytokines with clinical outcome will be presented. CONCLUSION: The addition of bevacizumab to purine analogue-based CIT was generally well-tolerated and may increase complete remission rates in patients with CLL. No clear improvement in treatment free survival has been observed to date. Disclosures Shanafelt: Hospiria: Research Funding; Pharmacyclics/Jannsen: Research Funding; Cephalon: Research Funding; Celgene: Research Funding; glaxoSmithKline: Research Funding; Genetech: Research Funding; Polyphenon E Int'l: Research Funding. Off Label Use: Off label use of pentostatin for treatment of CLL. Off label use of bevacizumab for treatment of CLL. . Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract Background : Polycythemia Vera (PV) is associated with JAK2 mutations. In essential thrombocythemia (ET), ̴ 85% of patients harbor one of 3 "driver" mutations, with frequencies of ̴ 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are referred to as "triple negative". We applied next-generation sequencing (NGS) with a 27-gene panel of myeloid malignancy-relevant genes, in order to describe the prevalence of "non-driver" mutations and their prognostic relevance in PV and ET. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1.Paired-end indexed libraries were prepared using the NEBNext Ultra Library prep protocol (NEB, Ipswich, MA /Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software (PerkinElmer, Danvers, Massachusetts) was utilized to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK -Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (COSMIC database), or if they have not been associated with a dbSNP. Results: 314 patients with PV (n =133; median age 64 years, 53% females) or ET (n =181; median age 58 years, 59% females) were evaluated. Median follow-up was 9.8 years for PV and 9.2 years for ET. During this time 59 (44%) deaths, 15 (11%) fibrotic and 5 (4%) leukemic transformations were documented for PV and the corresponding percentages for ET were approximately 33%, 8% and 2%. Driver mutation distribution was 98% JAK2 for PV and 52% JAK2, 24% CALR, 20% triple-negative and 4% MPL for ET. Polycythemia Vera Mutations other than JAK2, CALR or MPL were seen in 58 (44%) patients; 29% harbored one, 14% two and one 3 mutations. None of 3 JAK2 -unmutated cases expressed non-driver mutations. Mutational frequencies were 18% TET2, 11% ASXL1, 5% SH2B3, 3% SF3B1 and ≤2% SETBP1, IDH2, DNMT3A, CEBPA, CSF3R and SUZ12, SRSF2, ZRSR2, TP53, CBL, NRAS, RUNX1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected overall (OS; Figure 1) and myelofibrosis-free (MFS) survival; respective HR (95% CI) were 2.6 (1.3-5.2) and 1.7 (0.97-3.1) and 13.7 (2.9-63.4) and 5.1 (1.5-17.6) for ≥2 mutations and one mutation, respectively. OS was also adversely affected by SRSF2 (p=0.006) and RUNX1 (p=0.04) and borderline affected by TET2 (p=0.06), IDH2 (p=0.08), ASXL1 (p=0.19) and SF3B1 (p=0.19) mutations. In multivariable analysis, SRSF2 and RUNX1 retained significance whereas the others remained borderline significant. In both univariate and multivariable analyses, leukemia-free survival (LFS) was adversely affected by IDH2 and RUNX1 mutations. In univariate analysis, ASXL1, IDH2, RUNX1 and KIT mutations predicted fibrotic progression, whereas SETBP1 was of borderline significance (p=0.07); all, including SETBP1, were significant during multivariable analysis. Essential thrombocythemia Mutations other than JAK2, CALR or MPL were seen in 83 (46%) patients; 35% harbored one, 7% two and 4% three mutations; prevalence in JAK2, CALR, MPL mutated and triple-negative cases was 52%, 43%, 43% and 35%, respectively (p=0.52). Mutational frequencies were: 13% TET2, 11% ASXL1, 6% DNMT3A, 5% SF3B1, 4% CEBPA, 2% TP53, SH2B3, EZH2 and CSF3R and <2% for SETBP1, IDH2, SRSF2, ZRSR2, CBL, NRAS, RUNX1, U2AF1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected OS (Figure 2) but not MFS or LFS; HR (95% CI) for OS were 6.6 (2.5-17.8) for 3 mutations and 2.2 (1.3-3.9) for one or two mutations. In univariate analysis, survival was adversely affected by CBL, EZH2, SF3B1, SRSF2 and IDH2 mutations. In multivariable analysis, EZH2 and SF3B1 remained significant. Univariate analysis identified SETBP1 and SF3B1 mutations as risk factors for fibrotic progression and EZH2, TP53 and CSF3R mutations for leukemic transformation. Conclusions: "Non-driver" mutations occur in more than 40% of patients with PV or ET; the number of such mutations and presence of certain specific ones likely predict OS, MFS or LFS. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

Abstract Background : In primary myelofibrosis (PMF), ̴ 88% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 60%, 22% and 6% for JAK2, CALR and MPL, respectively. Other "non-driver" mutations have also been described in PMF and some of them and their number have been associated with inferior survival (Leukemia. 2014;28:1804). We applied next generation sequencing (NGS) with a broader panel of MPN-relevant genes, in order to identify additional mutations of prognostic relevance as well as obtain additional information regarding the prognostic value of 'number of mutations'. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA specimens obtained at time of referral for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. Paired-end indexed libraries were prepared from individual patient DNA using the NEB Next Ultra Library prep protocol on the Agilent Bravo liquid handler (NEB, Ipswich, MA/Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software was utilized (PerkinElmer, Danvers, Massachusetts) to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK-Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP. Results: 180 PMF patients were evaluated (median age 63 years; 65% males). DIPSS-plus risk distribution was 32% high, 38% intermediate-2, 17% intermediate-1 and 13% low. Driver mutation distribution was 62% JAK2, 22% CALR, 9% triple-negative and 7% MPL. Karyotype was abnormal in 41% of patients and unfavorable in 12%. Mutations other than JAK2, CALR or MPL (i.e. "non-driver" mutations) were seen in 150 (83%) patients including 88% of "triple-negative" cases. 62 (34%) patients harbored one, 55 (31%) two, 16 (9%) three and 17 (10%) four or more. Mutational frequencies were: ASXL1 36%, TET2 18%, SRSF2 17%, U2AF1 17%, ZRSR2 11%, SF3B1 10%, DNMT3A 9%, CEBPA (9%), Tp53 7%, SETBP1 6%, CBL 5%, IDH1/2 5%, SH2B3 4%, CSF3R 4%, NRAS 4%, RUNX1 3% and ≤2% for SUZ12, KIT, PTPN11, NPM1 and EZH2. DIPSS-plus high/intermediate-2 risk patients displayed higher number of mutations (p=0.0004) and higher mutational frequencies for ASXL1 (p=0.02), SRSF2 (p=0.004) and CBL (p=0.02). Associations noted included JAK2 with U2AF1 (p=0.03), unfavorable karyotype with CBL (p=0.01) and normal karyotype with ZRSR2 mutations (p=0.04). At a median follow-up of 4 years, 111 (62%) deaths were documented. For examination of impact on survival, we considered 'number of mutations' and specific mutations with >2% frequency. Accordingly, in univariate analysis, survival was adversely affected by 'number of mutations' (Figure 1) and presence of ASXL1, SRSF2, IDH1/2, U2AF1, RUNX1 and CBL mutations. For multivariable analysis, we considered three categories (zero, 1-3 and ≥4) for number of mutations based on the results from univariate analysis (Figure 1); the results showed ≥4 mutations, 1-3 mutations, RUNX1, CBL, ASXL1 and SRSF2 mutations were independently associated with shortened survival; the respective HR (95% CI) were 4 (1.4-11.1), 3 (1.3-6.8), 2.9 (1.1-8.1), 2.8 (1.3-6.3), 1.8 (1.2-2.7) AND 1.7 (1.03-2.7). When the multivariable analysis was repeated including only the 150 patients with at least one non-driver mutation, the 'number of mutations' was no longer significant (p=0.35) but ASXL1, CBL, RUNX1 and SRSF2 mutations retained their significance. The prognostic relevance of ASXL1 and CBL continued to be apparent even after the addition of DIPSS-plus and driver mutation profile to the multivariable model. Conclusions: Mutations other than JAK2, CALR or MPL occur in more than 80% of patients with PMF, including those with "triple-negative" driver mutational status. The absence of such mutations is independently favorable for survival while the prognostic effect of their presence is influenced by ASXL1, CBL, RUNX1 and SRSF2 mutations. Figure 1. Figure 1. Disclosures Pardanani: Stemline: Research Funding.