Citations

Abstract Abstract 1746 Diagnosis of essential thrombocythemia (ET) has been updated in the last World Health Organization (WHO) classification, that provides a guideline to distinguish ET from early/prefibrotic primary myelofibrosis. These two conditions differ in term of disease evolution and survival (Barbui et al, J. Clin. Oncol. 2011). To design a prognostic model for survival in WHO-defined ET (International Prognostic Score for Essential Thrombocythemia, IPSET), we studied 891 patients with strict WHO diagnostic criteria. Demographics have been presented elsewhere (Barbui et al, J. Clin. Oncol. 2011). We tested the prognostic impact on survival of age, prior thrombosis, leukocyte count, platelet count, hemoglobin level, JAK mutational status, splenomegaly. Harrell's C concordance statistics were used to set the best cutoff levels of continuous variables in order to predict survival. Univariate Cox proportional hazard regression showed that age over 60 years (P<.0001), leukocyte count greater than 11 x109/L (P<.0001), anemia (P=.04), platelet count greater than 1000 x109/L (P<.004) and prior history of thrombosis (P< 0001) were significant risk factors for survival. Age over 60 years (hazard ratio-HR- 6.5, 95% CI: 3.9–10.7; P<.0001), leukocyte count greater than 11 × 109/L (HR: 3.1, 95% CI: 2–4.7; P<.0001) and prior history of thrombosis (HR: 2.9, 95% CI: 1.9–4.5; P< 0001) retained statistical significance on survival by multivariable analysis and were included in the new prognostic model. We assigned each factor an integer weight close to the corresponding HR in the multivariable Cox regression: weight 2 for age over 60 years; weight 1 for leukocyte count greater than 11 x109/L and for prior thrombosis. Finally, we defined the IPSET score by allocating patients into three risk categories with significantly different survival according to the sum of weights: low risk (sum of weights=0; n= 342; median survival not reached), intermediate risk (sum=1-2; N=374; median survival 24.5 years), and high risk (sum=3-4, n=158; median survival 13.8 years). Fisher exact test did not reveal a difference in the distribution of the causes of death among IPSET categories. We validated the model in two independent cohorts: one from Germany (132 WHO-defined ET) and another from France (234 ET patients not strictly diagnosed according to WHO). Log rank test and Cox regression showed significant differences in term of survival among IPSET categories in both series. Finally, we designed an age-specific IPSET model for patients younger than 60 years (IPSET-y). Using the same methodology, we found that age over 45 years (weight 2), leukocyte count greater than 11 × 109/L (weight 1) and prior history of thrombosis (weight 1) were independent risk factors for survival. IPSET-y defined three risk categories (LR, sum=0; IR, sum=1-2; HR, sum=3–4) with a median survival not reached in LR and IR, and of 22.3 years in HR. In conclusion, IPSET, based on age over 60 years, leukocyte count greater than 11 x109/L and history of thrombosis allows prognostic assessment of WHO-defined ET. The validation process makes IPSET applicable in all patients phenotypically appearing as ET. IPSET models may be useful for treatment decision-making. Disclosures: Vannucchi: Italfarmaco: Consultancy; Novartis: Honoraria. Gisslinger:AOP Orphan Pharmaceuticals AG: Research Funding.

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 3803 Background: Ring sideroblasts (RS) represent abnormal mitochondrial iron deposits that are commonly present in myelodysplastic syndromes (MDS). The presence of ≥15% RS, by definition (World Health Organization-WHO) is necessary for a diagnosis of a MDS-RS; with refractory anemia with ring sideroblasts (RARS) being a specific morphologic category. RS can also be seen with other morphologic categories such as; refractory cytopenias with multilineage dysplasia (RCMD), MDS-unclassifiable (MDS-U) and refractory anemia with excess blasts (RAEB-1/2). Amongst these categories, RARS is generally believed to have the best survival rate with the lowest risk of leukemic transformation and this has been attributed to the absence of associated dysplasia in more than one lineage. However, it has not been systematically shown whether or not the exact percentage of RS provides additional prognostic information in the context of an accurate morphologic categorization and risk stratification by karyotype or the revised IPSS (IPSS-R). Methods: 200 patients with primary MDS without excess blasts and ≥1% RS were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis and the pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow (BM) RS percentage and to confirm WHO morphologic categories. Molecular profiling included analysis for IDH1, IDH2, JAK2 and MPL mutations. For the purposes of this study patients were divided into 4 categories based on the RS percentage; <5%, 5–14%, 15–50% and >50%. In addition, each patient was assigned IPSS and IPSS-R prognostic scores at diagnosis and risk stratified by karyotype according to the IPSS-R cytogenetic risk categories. Cox proportional regression method was used for multivariable analysis. Results: Of the overall 200 study patients, 140 (70%) were male and median age was 71 years (range, 17–90 years). At presentation, 34 (17%) patients were red cell transfusion-dependent. There were 56 (28%) patients with <5% RS (RCMD-52, MDS-U-14), 32 (16%) with 5–14% RS (RCMD-29, MDS-U-3), 79 (39%) with 15–50% RS (RARS-43, RCMD-29, MDS-U-2) and 33 (16%) with >50% RS (RARS-13, RCMD-17, MDS-U-3). Three patients (2%) were IDH1 mutant (RARS-1, RCMD-2), 17 (8%) IDH2 mutant (RARS-1, RCMD-14, MDS-U-2), 5 JAK2 V617F (3%) mutant (RARS-2, RCMD-3) and one patient with RCMD had the MPL W515L mutation. At a median follow-up of 33 months, 156 (73%) deaths and 24 (12%) leukemic transformations were documented. Median survivals were 63 months for MDS with >50% RS, 43 months for MDS with 15–50% RS, 35 months for MDS with 5–14% RS, and 14 months for MDS with <5% RS (p=0.005). In univariate analysis, additional risk factors included decreased hemoglobin, decreased platelet count, increased circulating blasts, WHO morphologic categories, red cell transfusion need, IPSS, IPSS-R and cytogenetic risk categories per IPSS-R. On a multivariable analysis only the IPSS-R prognostic score (p<0.0001) and the WHO morphologic categories (p=0.02 for RARS) retained significance. Univariate analysis disclosed significantly inferior leukemia-free survival (LFS) in patients with RCMD, thrombocytopenia, increased circulating blasts, and poor IPSS and IPSS-R prognostic scores. On multivariable analysis once again only the IPSS-R prognostic scores (p=0.02) and WHO morphologic categories (p=0.02) retained their significance. In other words, RS percentage, independent of WHO classification, as a continuous or categorical variable (i.e.: <5%, 5–14%, 15–50% & >50%) did not affect either overall survival or LFS. Conclusions: In MDS without excess blasts, once an accurate WHO morphologic categorization is made based on the presence or absence of multi-lineage dysplasia, there is no additional prognostic value for quantifying bone marrow ring sideroblasts. WHO classification and IPSS-R prognostic scores remain the most important factors in assessing patients with MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: The prognostic significance of cytogenetic abnormalities in chronic myelomonocytic leukemia (CMML) was recently revisited (AJH, 89; 813-818, 2014 and Blood April, 2013). Using a large Mayo Clinic-French Consortium database, we analyzed the molecular and prognostic correlates of cytogenetic abnormalities in CMML. Methods: CMML diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Results: Spectrum and frequency of cytogenetic abnormalities: A total of 409 patients participated in this study including, 268 (66%) from the Mayo Clinic and 141 (34%) from the French CMML consortium. Of these, 396 (97%) had ≥20 metaphases and 13 (3%) had ten to 19, analyzed. One hundred and fifteen (30%) patients displayed an abnormal karyotype, including 82 (71%) sole, 20 (17%) two and 13 (11%) complex abnormalities. The most common abnormalities were; +8 (23%), -Y (20%), -7/7q- (14%), 20q- (8%), +21 (8%) and der (3q) (8%). Other cytogenetic abnormalities included 5q-, 12p-, 13q- and i(17q), present at a much lower frequency (0.9-4%). Phenotypic correlates: Abnormal vs normal karyotype was associated with older age (p=0.03), hemoglobin<10 g/dL (p=0.0009), white blood cell count (WBC) >15 x 109/L (p=0.02), absolute neutrophil count (ANC) >10 x 109/L (p=0.03), absolute lymphocyte count (ALC) >2.5 x109/L ( p=0.04), peripheral blood (PB) blast ≥1% (p<0.0001), bone marrow (BM) blast ≥10% (p<0.0001) and circulating immature myeloid cells (IMC) (p=0.0003). +8 (p=0.01), +21 (p=0.03) and der (3q) (p=0.03) were associated with hemoglobin <10 g/dL. -Y was associated with older age (p=0.04), lower PB (p=0.04) and BM (p=0.02) blasts. -7/7q was associated with leukocytosis (p=0.005), neutrophilia (p=0.04), and higher PB blasts (p=0.004). 20q- was associated with thrombocytopenia (p=0.04). Molecular correlates: ASXL1 mutations were associated with abnormal karyotype (p=0.04) and SRSF2 with normal karyotype (p=0.02). In comparison to other abnormal karyotypes, the incidence of ASXL1 mutations was lower in –Y (P=0.04) and der(3q) (p=0.03). U2AF1 mutations were associated with monosomal karyotype (p=0.03) and SF3B1 with der (3q) (p<0.0001). Prognostic relevance : Median follow-up was 1.8 years with 244 (60%) deaths and 79 leukemic transformations (19%). A step-wise survival analysis resulted in three distinct cytogenetic risk categories (Figure 1): high (complex and monosomal karyotype), intermediate (all abnormalities not in high or low risk) and low (normal, sole -Y and sole der (3q)); the corresponding median survivals were 0.2 (HR 8.1, 95% CI 4.6-14.2), 1.7 (HR 1.7, 95% CI 1.2-2.3). In multivariable analysis, the particular cytogenetic risk stratification remained significant in the context of Mayo molecular model (p<0.0001), MDAPS (p<0.0001), and the GFM risk model (P<0.0001). The Mayo-French cytogenetic risk model was also effective in predicting leukemic transformation with HR of 10.9 (95% CI 4.2-27.8) for high and 2.2 (95% CI 1.3-3.7) for intermediate risk groups. Conclusion: Cytogenetic abnormalities are seen in approximately 30% of patients with CMML and display significant associations with certain molecular and phenotypic characteristics. We describe a novel cytogenetic prognostic model for both over-all and leukemia free survival in CMML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background : Recent studies have suggested significant associations between karyotype and certain molecular or phenotypic features in primary myelofibrosis (PMF). In the current study of 835 consecutive patients, we examined the spectrum and prevalence of cytogenetic abnormalities in PMF and their molecular and phenotypic correlates. Methods : PMF diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Spectrum and frequency of cytogenetic abnormalities : Analyzable metaphases were obtained in 826 (99%) of 835 patients studied; 681(82%) had ≥20 metaphases analyzed. 352 (42.6%) patients had abnormal karyotype, including 240 (68.2%) sole, 64 (18.2%) two and 48 (13.6%) complex; comparison of these groups revealed lower platelet count (p<0.01), higher DIPSS-plus score (p=0.03) and higher percentage of younger patients (p=0.04) with complex abnormalities. Monosomal karyotype was noted in 20 (5.7%) patients. Approximately 150 individual abnormalities were identified; most frequent were 20q- (23.3%), 13q- (18.2%), +8 (11.1%), +9 (9.9%), duplication of chromosome 1q (9.7%) and -7/7q- (7.1%). Other notable abnormalities including i(17q) (1.4%), 12p- (1.1%) and inv(3) (0.6%) were much less frequent. Trisomy 8 was the most frequent in the context of complex abnormality (25%). Among the 500 patients seen within one year of initial diagnosis, 179 (35.8%) had abnormal karyotype, which included 121 (67.6%) sole, 31 (17.3%) two and 27 (15.1%) complex abnormalities; the most common abnormalities were 20q- (24.6%), 13q- (15.1%), +8 (14%) and +9 (10%) whereas 11q- (1.7%), i(17q) (1.1%), inv(3) (0.6%), and 12p- (0.6%) were infrequent. Molecular correlates : 476 patients were annotated for JAK2, CALR and MPL mutations; abnormal karyotype frequencies were 43% in JAK2, 42% CALR, 33% MPL mutated and 34% triple-negative cases (p=0.3). 13q- was associated with mutant CALR (p=0.03) and +9 with mutant JAK2 (p=0.02). Subsets of patients were also screened for ASXL1, EZH2, IDH, SRSF2, U2AF1, and SF3B1 mutations; in all instances, mutational frequencies were higher in patients with normal karyotype, reaching significance with ASXL1 (p=0.02) and U2AF1 (p=0.01). Mutant SRSF2 was associated with 20q- (p=0.02). Phenotypic correlates : Phenotypic correlates included abnormal karyotype with anemia (p=0.02), leukopenia (p<0.01) and thrombocytopenia (p<0.01); complex karyotype with younger age (p=0.04) and thrombocytopenia (p<0.01); leukopenia with 20q-, +8 and -7/7q-; and thrombocytopenia with 20q- and -7/7q-. Cytopenias were less likely to occur with 13q- (p<0.01), which was instead associated with thrombocytosis (p<0.01). 20q- was associated with lower incidence of marked leukocytosis (p=0.02). Trisomy 8 was associated with lower incidences of constitutional symptoms (p<0.01) and marked splenomegaly (p<0.01). Conclusions : The association of 13q- with CALR mutations in PMF might underlie its association with both thrombocytosis and favorable prognosis. The association of +9 with JAK2 mutations might reflect selective clonal advantage through JAK2V617F dosage enhancement or mutation-induced chromosomal instability. The association of 20q- with mutant SRSF2 and thrombocytopenia warrant further clarification of its reported association with favorable prognosis. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3833 Background: Survival in primary myelofibrosis (PMF) is assessed by the Dynamic International Prognostic Scoring System (DIPSS) (Blood 2010;115:1703) and DIPSS-plus (JCO 2011;29:392). The latter is based on eight risk factors including age>65 years, constitutional symptoms, hemoglobin <10 g/dL, red blood cell transfusion need, leukocyte count >25 × 109/L, platelet count <100 × 109/L, peripheral blood (PB) blasts ≥1% and unfavorable karyotype. Recent studies have highlighted the prognostic significance of monosomal karyotype (MK) (Blood 2011;117:5612), unfavorable karyotype (Leukemia 2011;25:82) and platelet count <100 × 109/L ((JCO 2011;29:392), for leukemic transformation (LT) in PMF. Methods: A combination of multivariable and receiver operating characteristic (ROC) analyses were applied to a Mayo Clinic database of 884 karyotypically-annotated patients with PMF, in order to define karyotype-dependent and karyotype-independent risk models for LT. The karyotype-independent model was further validated in a separate cohort of 525 PMF patients from an international database (Blood 2010;115:1703). Patients in the Mayo cohort were evaluated at time of referral and those of the international database at time of initial diagnosis. Results I: Sixty cases of LT were documented. Karyotype (categorized into MK, inv(3)/i(17q) abnormalities, other unfavorable, and favorable), PB blast percentage (as a continuous variable), and platelet count (as a continuous variable) were the most reliable (p<0.01) in predicting leukemia-free survival (LFS). The best discriminant levels for inferior LFS were MK (HR 6.2, 95% CI 2.2–17.1), inv(3)/i(17q) abnormalities (HR 7.6, 95% CI 1.8–33.3), PB blast ≥2% (HR 2.4, 95% CI 1.4–4.2) and platelet count ≤41 × 109/L (HR 2.6, 95% CI 1.3–5.1). MK and inv(3)/i(17q) abnormalities were subsequently consolidated into one “very high risk” cytogenetic category (n =27), with a corresponding HR of 6.6 (95% CI 2.7–16.0). HR-based risk scores were assigned to “very high risk” karyotype (2 points), PB blast ≥2% (one point) and platelet count ≤50 × 109/L (one point) to classify patients into low (no adverse points; n =522), intermediate (one adverse point; n =290) and high risk (two or more adverse points; n =72) groups; the corresponding 3-year LT rates were 3%, 10% (HR 2.6; 95% CI 1.4–4.6) and 35% (HR 9.4, 95% CI 4.7–18.7) (Figure 1). Results II: The karyotype-independent model was based on PB blast percentage and platelet count only; low-risk (no adverse points; n =525), intermediate-risk (one adverse point; n =311) and high-risk (two adverse points; n =48); the corresponding 3-year LT rates were 3%, 12% (HR 3.1, 95% CI 1.8–5.4; p<0.0001) and 25% (HR 6.0, 95% CI 2.6–14.1). The karyotype-independent model was validated in a separate cohort of 525 PMF patients with 70 documented cases of LT, from an international database (Blood 2010;115:1703). Conclusions: The current leukemia risk models are applicable at diagnosis or during follow-up, in the presence or absence of cytogenetic information, and complement DIPSS-plus in the comprehensive prognostication of patients with PMF. Disclosures: Vannucchi: Novartis: Honoraria.

Abstract Background: KIT D816V is considered the driver mutation in systemic mastocytosis (SM) however this mutation alone does not explain the diverse clinical manifestations of SM. While mutations in other myeloid-relevant genes have been reported in SM, their pattern(s) of co-segregation and prognostic value, independent of age and SM subtype, have not been fully defined in a mature dataset. Objectives: In this pilot study of advanced SM patients, we sequenced a panel of myeloid-relevant genes for known pathogenic mutations, to ascertain: (i) the frequency of individual mutations; (ii) the pattern(s) of mutation co-segregation; and (iii) the prognostic value of mutations, both individually and in combination, on overall- (OS) and leukemia-free survival (LFS). Methods: The current study was approved by the Mayo Clinic institutional review board. SM was classified as per World Health Organization criteria. DNA was isolated from archived bone marrow (BM) cell pellets. Next-generation deep sequencing by HiSeq SBS chemistry (Illumina) was performed to investigate the coding regions of 22 genes: KIT, JAK2, CALR, SRSF2, U2AF1, SF3B1, ASXL1, TET2, EZH2, SETBP1, DNMT3A, IDH-1/2, SUZ12, CBL, NRAS, FLT3, PTPN11, RUNX1, CEBPA, TP53 and NPM1. Results: We studied 19 patients with advanced SM; 6 had aggressive SM (ASM) and 13 had SM with an associated hematological disease (SM-AHD). Of the latter, 5 patients (38%) had myeloproliferative neoplasm-unclassified (MPN-u), 3 (23%) chronic myelomonocytic leukemia (CMML), 2 (15%) acute myeloid leukemia (AML), and 1 (8%) each polycythemia vera, myelodysplastic syndrome, and multiple myeloma. The median follow up from diagnosis was 20 months (range 2 to 84). Over this period, 5 patients (26%) developed leukemic transformation and 12 patients (63%) have died. KIT D816V was identified in 9 (47%) patients; 9 of 10 patients who tested negative by deep sequencing were screened by allele-specific PCR (sensitivity ~0.01%), with 8 additional patients testing positive. Thus, overall KIT D816V frequency was 89%. Fourteen (74%) patients harbored at least one additional non-KIT mutation; of these 7 patients had one additional mutation (ASXL1/TET2/SF3B1 = 2 each and JAK2 =1), 3 patients each had two and three additional mutations, and 1 patient had 4 additional mutations. The most frequently mutated genes (other than KIT) were ASXL1/TET2 (5 patients each, 26%), CBL (4 patients, 21%), SETBP1/SF3B1/DNMT3A (2 patients each, 11%) and JAK2/CALR/FLT3/IDH-1/RUNX1/TP53 (1 patient each, 5%). There was no overlap between patients harboring TET2 and ASXL1 mutations however 2 and 1 CBL-mutated patients concurrently harbored TET2 and ASXL1 mutations, respectively. SETBP1 and SF3B1 but not DNMT3A mutations occurred independently of TET2 and ASXL1 mutations. The distribution of mutations was similar between ASM and SM-AHD subgroups. When considered individually, the following non-KIT mutated genes (present in at least 2 patients) were associated with significantly inferior OS: ASXL1, CBL and SETBP1 (p<0.05). When the 3 mutated genes were considered together, only ASXL1 and CBL maintained their significance, independent of age and SM subtype, for inferior OS (Hazard ratio (95% confidence interval)=34.8 (3.1-393.9) and 59.7 (4.1-861.5), respectively). Patients harboring ASXL1 and/or CBL mutations (n=8, 8 deaths, median OS=11 months) had a significantly worse OS as compared to those without either mutation (n=11, 4 deaths, median OS=84 months) (p=0.0002) (Fig 1). When considering the number of non-KIT mutations (zero, one, two, three and four mutations in 5, 7, 3, 3 and 1 patients, respectively), the group with ≥3 mutations (n=4, 4 deaths, median OS=7 months) had a significantly inferior OS as compared to those with <3 mutations (n=15, 8 deaths, median OS=48 months) (p=0.01) (Fig 2). Presence of mutations was not correlated with LFS in this analysis. Conclusions: In this cohort of advanced SM patients, non-KIT mutations were frequent (prevalence 74%). TET2, ASXL1, SETBP1 and SF3B1, but not CBL or DNMT3A, mutations were mutually exclusive. Presence of ASXL1 and CBL mutations was associated with significantly inferior OS (HR=35 and 60, respectively), independent of known prognostic factors. An increasing number of non-KIT mutations (≥3, HR=5.9) was also associated with a significantly worse survival outcome. Sequencing results from a larger cohort will be presented at the meeting. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

Abstract Abstract 3898 Background: Although chemoimmunotherapy (CIT) has substantially improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 4050 of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays. There remains interest in identifying new approaches to improve the effectiveness of CIT. Ofatumumab is a humanized anti-CD20 monoclonal antibody that is approved for patients with relapsed CLL refractory to fludarabine and alemtuzumab. The single agent activity of ofatumumab in patients with refractory CLL suggests it may have greater efficacy than rituximab in patients with CLL. To explore the efficacy of ofatumumab based CIT, we initiated a clinical trial studying the effect of ofatumumab in combination with pentostatin and cyclophosphamide (PCO) for patients with previously untreated CLL. METHODS: Eligible patients were previously untreated and had CLL in need of treatment according to the NCI-WG criteria (Blood 111:5446). The treatment schema included 6 cycles of ofatumuamb (300 mg on day 1 of cycle 1;1000 mg on day 2 of cycle 1 and day 1 of cycles 26) followed by pentostatin (2 mg/m2) and cyclophosphamide (600 mg/m2) administered every 21 days. All patients underwent complete response evaluation including evaluation for MRD using sensitive flow cytometry (Leukemia 21:956) at the completion of treatment. RESULTS: 33 patients were enrolled at Mayo Clinic and Duke University between 3/2010 and 9/2010. All patients were eligible for treatment and have completed active treatment. Clinical characteristics included: 76 male, median age 63 (range: 5083); intermediate Rai risk 27; and high Rai risk 73. On prognostic testing 33 were CD38, 48 Zap-70 , 58 IGHV unmutated, and 6 had high risk FISH (del 17p13; del 11q22). 26 of 33 eligible patients (79) completed all 6 cycles of PCO induction. Seven patients went off treatment early (toxicity: n5; refusal: n1; progression: n1). Adverse events deemed at least possibly related to PCO induction included 8 (24) patients with grade 3 hematologic toxicity and 5 (15) with grade 3 non-hematologic toxicity. The overall response rate to PCO induction was 94 (31/33) with 15 (45) CR/CRi, 5 (15) CCR, 9 (27) nPR, and 2 (6) PR. 2/10 (20) patients with CR's tested for MRD were also MRD negative by 6 color flow cytometry analysis. Finally, we compared this cohort of patients treated with ofatumuamb-based CIT to our prior 108 patient cohort of patients treated with rituximab-based CIT both of which used an identical chemotherapy backbone (pentostatin and cyclophosphamide). Although the age, gender, ALC, and prognostic profile (CD38, ZAP-70, and IGHV mutation status) of treated patients were similar (all p0.3), the ofatumumab cohort included more advanced stage patients (Rai stage III/IV: 73 vs. 41 p0.002). Ofatumumab-based CIT appeared to be better tolerated than our previous experience with rituximab-based CIT (grade 3 hematologic toxicity ofatumumab-CIT 24 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{8$}\!\left/ \!\raisebox{-1ex}{$33$}\right.}\]\) \end{document} vs. rituximab-CIT51 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{55$}\!\left/ \!\raisebox{-1ex}{$108$}\right.}\]\) \end{document}, p0.009; grade 3 non-hematologic toxicity ofatumumab-CIT 15 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{5$}\!\left/ \!\raisebox{-1ex}{$33$}\right.}\]\) \end{document} vs. rituximab-CIT 24 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{26$}\!\left/ \!\raisebox{-1ex}{$108$}\right.}\]\) \end{document}, p0.34). Despite a higher proportion of Rai Stage III/IV patients in the ofatumumab cohort, the efficacy of ofatumumab-based CIT also compared favorably to our previous experience with rituximab based CIT (Table). CONCLUSION: Front-line ofatumumab-based CIT appears to be well-tolerated in patients with CLL. Compared to our historic experience with rituximab based CIT, ofatumumab-based CIT appeared to have less hematologic toxicity and improved efficacy. This trial has been expanded to provide a larger experience with ofatumumab-based CIT. Disclosures: Shanafelt: Hospira: Research Funding; Glaxo-Smith-Kine: Research Funding; Genentech: Research Funding. Off Label Use: Off label use of pentostatin and Ofatumumab. Lanasa:GlaxoSmithKline: Consultancy, Speakers Bureau. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding. Tun:Celgene: Research Funding. Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Abstract Background : TET2, located on chromosome 4q24, is frequently mutated in the majority of patients with chronic myelomonocytic leukemia (CMML). TET2 has 11 exons, and variations, especially in exon 3 have been described as a part of age related clonal hematopoiesis (Jaiswal NEJM 2015). In CMML, somatic TET2 mutations in the absence of ASXL1 mutations (ASXL1wt/TET2mt) were previously shown to impart a favorable outcome on survival (Patnaik BCJ 2015). In the current larger CMML patient cohort, we describe the number and type of TET2 mutations and examine their phenotypic and prognostic effects. Methods : 261 patients with CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. TET2 (NM_001127208.2) coverage extended from exons 3-11, with frame shift, non-sense, and missense variations considered pathogenic. Previously annotated single nucleotide polymorphisms (http//www.hapmap.org) were considered non-pathogenic. Results: Among the 261 study patients, 65% were males and median age was 70 years (range, 28-91). 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations were documented. Mutational frequencies ≥4% were encountered in; ASXL1 45%, TET2 42%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4% and IDH2 4%. i) TET2 mutations type, number and phenotypic correlates: TET2 mutations were seen in 109 (42%) patients; these included frameshift 33 (30%), nonsense 29 (27%) and missense 11 (10%) variants whereas 36 (33%) had more than one type of mutation. Overall, 57 (52%) patients had more than 1 TET2 mutations: 52 (48%) patients had 1, 46 (42%) 2 and 11 (10%) ≥3 TET2 mutations. Among all 109 TET2 mutated patients, 65% were male, and median age was 71 years with no significant difference in age and gender distribution between mutated and un-mutated cases, or type of TET2 mutations; however, older patients were more likely to carry multiple TET2 mutations (p=0.01). Compared to their un-mutated counterparts, TET2 mutated cases were less likely to have a low hemoglobin (p<0.001), circulating immature myeloid cells (IMC) (p=0.001), peripheral blood (PB) (p=0.009) and BM blasts (p=0.009), and have high-risk stratification per Mayo Molecular Model (p<0.001); these differences were not affected by the type or number of TET2 mutations. TET2 mutated cases were more likely to have a higher frequency of SRSF2 (p=0.004) and lower frequency of ASXL1 (p=0.03), Tp53 (p=0.04) and IDH1/2 mutations (p<0.001); these associations were also not affected by the type or number of TET2 mutations. ii) Impact on survival: Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was superior in TET2 mutated (median 33 months) versus wild-type (median 21 months) patients [p=0.03; HR 1.3 95% CI 1.12-1.86] (figure 1A). This survival difference remained significant after adjustment for age (p=0.04), leukocyte count (p=0.017), absolute monocyte count (p=0.02), absolute lymphocyte count (p=0.02), platelet count (p=0.015), circulating IMC (p=0.03), DNMT3A (p=0.02) and ASXL1 (p=0.045) mutations; however, significance was lost after adjustment for abnormal karyotype (p=0.32) and the Mayo Molecular Model (p=0.0003). These observations were not affected by the type or number of TET2 mutations. Finally, our previous observation regarding the survival advantage of ASXL1wt/TET2mt versus other genotypes was most apparent for patients with multiple TET2 mutations (p=0.02) (figure 1B). Conclusions: TET2 mutations are common in CMML and constitute approximately equal proportions of frameshift and non-sense mutations, while missense mutations are less frequent. Majority of TET2 mutated CMML cases harbor more than one mutant variant. Regardless, the relevance of type and number of TET2 mutations in CMML was limited to an association between older age and number of mutations and the latter with possibly improved survival in the absence of clonal ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 700 Studies conducted over the last 10 years suggest that 3–5% of the general population over age 40 harbor a clonal population of lymphocytes of CLL phenotype when evaluated using sensitive flow cytometry assays (Rawstron Blood 100:635, 2002; Ghia Blood 103:2337, 2003). This discovery led to creation of the new diagnostic entity, monoclonal B-cell lymphocytosis (MBL; Marti BJH 130:325, 2005), which is considered the precursor state to chronic lymphocytic leukemia (CLL) (Landgren NEJM 360:659, 2009). The current diagnostic criteria for CLL-like MBL specify patients have a clonal B-cell population with appropriate phenotype, an absolute B-cell count <5 × 10^9/L, and no characteristics of a lymphoproliferative disorder (e.g. lymphadenopathy, organomegaly, B-symptoms) while individuals with a clone of CLL phenotype and B-cell count >5 × 10^9/L or who have lymphadenopathy are classified as having CLL (Hallek Blood 111:5446, 2008). The natural history of MBL appears to differ based on the absolute B-cell count. From an epidemiologic perspective, the majority of individuals with MBL have very small clonal lymphocyte populations, B-cell counts within the normal range, and appear to be at low likelihood of developing CLL (Rawstron ASH 2009). Such individuals with “low count” MBL do not appear to be at increased risk of death (Rawstron ASH 2009). From a clinical perspective, most patients with MBL are identified due to lymphocytosis and have B-cell counts between 3–5 × 10^9/L. While such individuals with “high count” MBL progress to require treatment for CLL at a rate of ~1.5% per year (Shanafelt JCO 27:3959, 2009), whether their survival differs from that of age- and sex-matched individuals is unknown. Conceptually, patients with the precursor state to malignancy should have a risk of death similar to those without the precursor state unless they progress to develop malignancy. To determine the impact of clinically “high count” MBL on survival, we compared the survival of 300 patients with clinically recognized MBL to the age- and sex-matched population. In addition, to explore whether the current B-cell threshold used to segregate MBL from CLL optimally classifies risk, we also evaluated the survival of 94 patients with newly diagnosed Rai 0 CLL and a B-cell count between 5–10 × 10^9/L and 218 newly diagnosed Rai 0 CLL patients with an ALC > 10 × 10^9/L to the age- and sex-matched population. Although it appeared slightly lower, the survival of patients with clinical recognized MBL (n=300, median age 68.6) was not statistically different than age- and sex-matched individuals (p=0.14; Figure 1A). In contrast, the survival of patients with newly diagnosed Rai 0 CLL and a B-cell count between 5–10 × 10^9/L (p=0.03) and newly diagnosed Rai 0 CLL patients with an ALC > 10 × 10^9/L (p=0.00018) were both significantly shorter than expected for the age- and sex-matched population. While these data are consistent with the designation of clinically recognized MBL as a premalignant condition, a more nuanced story emerged when the individuals with clinically recognized MBL were stratified by the CD38 status of the B-cell clone. Although the survival of individuals with a CD38- clone (n=213; 78%) was not different than age- and sex-matched individuals (p=0.14), the survival for those with a CD38+ clone (n=59; 22%) was substantially shorter than the age- and sex-matched population (p=0.0002; Figure 1B). Collectively, these data provide additional clinical evidence in support of the 5 × 10^9/L B-cell threshold to distinguish between MBL and CLL as patients with a B-cell count between 5–10 × 10^9/L or higher are at increased risk of death compared to the age- and sex-matched population. However they also suggest that, in addition to absolute B-cell count, biologic characteristics of the B-cell clone influence survival in patients with clinically recognized MBL. Additional studies are needed to determine whether such characteristics should be considered when classifying individuals with B-cell clones <5 × 10^9/L as having a premalignant or malignant condition. Figure 1A: Survival of patients with clinically recognized MBL (n=300) compared to the age- and sex-matched general population Figure 1A:. Survival of patients with clinically recognized MBL (n=300) compared to the age- and sex-matched general population Figure 1B: Survival of patients with clinically recognized MBL by CD38 status compared to the age- and sex-matched general population. CD38- p=0.14, CD38+ p=0.0002 Figure 1B:. Survival of patients with clinically recognized MBL by CD38 status compared to the age- and sex-matched general population. CD38- p=0.14, CD38+ p=0.0002 Disclosures: Shanafelt: Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding. Off Label Use: Lenalidomide. Kay:Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.