Hanson, Curtis A.

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.

Abstract Abstract 3881 Background: Chronic lymphocytic leukemia (CLL) is largely incurable neoplasm of the blood and has one of the highest familial risks of disease among cancers. Monoclonal B-cell lymphocytosis (MBL) with a CLL immunophenotypic profile (i.e., CLL-like MBL) is a precursor state to CLL. In the general adult population, the prevalence rate of MBL is 5–9% and increases with age. This is in contrast to the 13–18% rate of MBL in first-degree relatives of CLL patients from high-risk families (defined here as families with at least two confirmed living cases of CLL). Few longitudinal studies of MBL incidence exists. One study evaluated MBL status over time in a cohort of individuals with MBL from a rural valley in Northern Italy. Our work herein will be the first to evaluate the status of MBL over time in relatives of CLL patients from high-risk families. Methods: Using a cohort of 41 high-risk CLL families, we evaluated the MBL status and clonal blood B-cell counts in 94 relatives of CLL patients at two time points at least 1 year a part. High sensitivity, multi-color flow cytometry was performed on fresh or previously frozen blood samples in three laboratories: Duke University, the US Food and Drug Administration, and Mayo Clinic. The number of cell events collected for flow cytometry evaluation varied from 1 to 5 x105 cells. To assure concordance across flow cytometry laboratories, 15 samples were evaluated at multiple labs. The concordance of MBL calling was a 100%. We defined three monotypic B-cell subsets as CLL-like MBL (CD5+ CD20dim), atypical MBL (CD5+ CD20+), and non-CLL like MBL (CD5−CD20+). Results: Of the 94 relatives, the median age at consent was 57 years (range 42–93) and 39% were male. At the initial MBL screening, 70 (74%) relatives had normal immunophenotype (NIPT) and 24 (26%) had MBL (1 atypical MBL, 3 non-CLL like MBL, and 20 CLL-like MBL). Seven of the 24 individuals with MBL had monoclonal B-cell counts available at baseline screening; median = 147.4 cells/uL (range 22.7–1663). The 94 relatives underwent repeat evaluation with a median follow up time of 1.8 years (range 1–6). At the second evaluation, we found an increase in the prevalence of MBL, with 11 of the 70 (12%) initially NIPT relatives developing a new MBL clone (10 CLL-like, 1 non-CLL like). These 11 incident MBL cases were from 9 families with 2 families each having 2 relatives changing MBL status. The median number of monoclonal B-cells in these 11 incident MBL cases was 0.58 cells/uL (range 0.38–60.3). The 23 out of the 24 relatives with baseline MBL (20 CLL-like MBL and 3 non-CLL like MBL) had a persistent MBL clone. Only 6 of these 23 individuals had monoclonal B-cell counts available at both time points, and among these 6, the clonal B-cell counts decreased at follow-up screening; median difference −64.1 cells/uL (range −1204.3– 10.5). The second evaluation of the initial atypical MBL relative had too few clonal cells for flow assessment. Conclusion: We found that our CLL-like MBL clones persisted over the period of observation in relatives from high-risk CLL families; this finding is similar to that previously reported using a population-based study conducted in Northern Italy. Notably, 12% of relatives from high-risk CLL families developed incident MBL during just 2 years of follow up. Although these MBL clones are all low count (< 1500 cells/uL), these data provide further evidence that MBL is a marker of inherited predisposition to CLL. It will be important to see how our rate of change of MBL status compares to that of the general population as we continue to follow our cohort of CLL families. Disclosures: No relevant conflicts of interest to declare.

Abstract Background U2AF1 mutations occur in approximately 16% of patients with primary myelofibrosis (PMF) and were significantly associated with anemia, thrombocytopenia, older age, JAK2V617F, ASXL1 mutations and normal karyotype (Leukemia 2014;28:431); furthermore, U2AF1 mutations were associated with inferior survival in univariate but not multivariable analysis. In the current study, we looked for the possibility of a differential effect from U2AF1 mutation variants on these observations in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of U2AF1 mutational status. Previously published methods (Leukemia 2014;28:431), including targeted next generation sequencing (Blood 2015 126:354), were used to screen for U2AF1 and other prognostically-relevant mutations. Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results Patient characteristics: U2AF1 mutational status was available for 457 patients and 72 (16%) harbored one of several mutation variants: these mutations affected residue Q157 in 44 (10%) patients, S34 in 24 (5%) and the remaining 1% displayed other variants. The 44 patients with U2AF1Q157 mutations included 23 with Q157P, 18 with Q157R and 3 with Q157-Y158insYE. The 24 patients with U2AF1S34 included 16 with S34F and 8 with S34Y. Only one patient harbored both Q157 and S34 mutations (Q157R, S34Y). Among all 457 study patients, median age was 63 years, 64% were males, dynamic international prognostic scoring system (DIPSS)-plus (JCO 2011;29:392) risk distributions were 13% low, 18% intermediate-1, 38% intermediate-2 and 32% high and driver mutation distributions were 54% JAK2, 22% CALR type 1/ type1-like, 4% CALR type 2/type 2-like, 7% MPL and 13% triple-negative. Cytogenetic studies were available in 449 patients: 39% abnormal and 10% unfavorable. All 457 patients were screened for ASXL1 mutations with 37% mutated, 450 for SRSF2 mutations with 15% mutated, 403 for SF3B1 with 8% mutated, 366 for IDH1/2 with 5% mutated and 369 for EZH2 with 4% mutated. Median follow-up was 4.4 years and during this period, 318 (70%) deaths and 53 (12%) leukemic transformations were documented. Phenotypic correlates of U2AF1 mutation variants: Because of the relatively small number of informative cases with specific mutations, we classified all patients into Q157 (n=44) and S34 (n=24) mutation variants and compared them with the 385 U2AF1 un-mutated cases. First, we confirmed our previous observations regarding the association between U2AF1 mutations in general and older age (p=0.0003), JAK2V617F (p<0.0001), ASXL1 mutations (p=0.0002), normal karyotype (p=0.03), hemoglobin <10 g/dL (p<0.0001) and platelet count <100 x 10(9)/L (p<0.0001); when the two U2AF1 mutation categories were analyzed separately, the corresponding p values for Q157 were 0.0005, 0.001, <0.0001, 0.04, <0.0001 and <0.0001 and for S34 were 0.12, 0.001, 0.41, 0.41, <0.0001 and 0.67. Phenotypic correlates of U2AF1 mutation variants: In univariate analysis, survival was adversely affected by U2AF1Q157 (p<0.0001; HR 2.2, 95% CI 1.6-3.1) but not by U2AF1S34 (p=0.8; HR 1.1, 95% CI 0.6-1.8) mutations (Figure 1a). Furthermore, the negative survival effect of U2AF1Q157 mutations was independent of anemia, thrombocytopenia, ASXL1, SRSF2, IDH1/2 and EZH2 mutations, as well as driver mutational status; multivariable analysis that included all molecular alterations identified U2AF1Q157 (HR 1.6, 95% CI 1.1-2.3), ASXL1 (HR 2.3, 95% CI 1.8-3.5), SRSF2 (HR 1.6, 95% CI 1.2-2.2) and absence of CALR type-1/like (HR 2.6, 95% CI 1.8-3.5) mutations as independent risk factors for survival. Finally, the survival effect of U2AF1Q157 mutations was independent of DIPSS-plus in patients with hemoglobin ≥10 g/dL (HR 2.6, 95% CI 1.3-5.3; p=0.007) (Figure 1c) but not in those with hemoglobin <10 g/dL (p=0.98) (Figure 1b). Conclusion Both U2AF1Q157 and U2AF1S34 mutation variants in PMF are associated with JAK2V617F and severe anemia whereas only the former is associated with ASXL1 mutations and thrombocytopenia. More importantly, U2AF1Q157, but not U2AF1S34, mutation variants in PMF are predictive of inferior survival, independent of other adverse mutations, and, in the absence of severe anemia, independent of DIPSS-plus. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3903 INTRODUCTION: Monoclonal B-Cell Lymphocytosis is an asymptomatic hematologic condition characterized by the presence of small populations of clonal B-cells in the blood or bone marrow occurring in 3–5% of the general population over age 40. Although MBL is considered the precursor state to CLL and other sub-types of indolent non-Hodgkin Lymphoma, most individuals with MBL will not develop a B-cell malignancy. It is unknown, however, whether individuals with MBL may be at risk for other adverse outcomes associated with CLL, such as increased risk of infection. To explore this aspect, we evaluated the risk of hospitalization for infection among patients with clinically recognized MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. METHODS: The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a 50 mile radius. To explore the natural history of infection in a local, community dwelling cohort of individuals with clinically identified, CLL-phenotype MBL, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed MBL seen in the MCR Hematology Division between January 1999 and December 2009. Analysis was limited to MBL cases who resided within 50 miles of MCR. The comparison cohort consisted of 689 adult patients also resided within 50 miles of MCR who were seen for a general medical examination between September 2002 and December 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. Rates of infection among individuals with MBL were also compared to a cohort of 174 patients with newly diagnosed CLL diagnosed in the same time interval and who resided within 50 miles of MCR. All hospitalizations in these 3 groups were audited to document hospitalization with infection including cross-referenced with the electronic Mayo Clinic infection database, which contains all culture results obtained from hospitalized patients at MCR. Serologies and viral assays (e.g. CMV viral load) were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotics were considered to have a culture negative infection. Among the cases, only hospitalizations after CLL or MBL diagnosis were considered. RESULTS: In May 2010, there were 524 individuals in the Mayo Clinic MBL/CLL Database with the diagnosis of CLL-phenotype MBL made between January 1999 – December 2009. Of these, 154 (29%) patients resided within a 50 mile radius of Mayo Clinic and were considered a local, community-based cohort of MBL patients. Median age at MBL diagnosis for these 154 patients was 70 years (range 45–94). After median follow-up of 4.2 years (range 0.3–10.6 years), 25 (16%) individuals with MBL were hospitalized with infection as compared to 20/689 (3%) individuals in the control population (odds ratio MBL relative to control=6.5; p<0.001) and 32/174 (18%) in the cohort of patients with newly diagnosed CLL (OR MBL relative to CLL=0.9; p=0.61) (Figure 1). The 25 MBL patients hospitalized for infection were hospitalized a total of 39 times (median 1 hospitalization for infection per individual). A specific infectious organism was identified by cultures or serologies in 8/39 (21%) hospitalizations among individuals with MBL, 10/27 (37%) hospitalizations for infection among controls, and 60 (63%) of the 89 hospitalizations for infection in CLL cases. Finally we performed a pooled multivariable analysis of all patients (n=1017) adjusting for age, sex, presence of MBL, diagnosis of CLL, and whether patients with CLL or MBL received treatment for CLL during follow-up. All factors were independently associated with risk of hospitalization for infection [Age (per year, OR=1.02, p=0.02), sex (male, OR=2.6, p=0.001), presence of MBL (OR=3.3, p<0.001), diagnosis of CLL (OR=3.0, p=0.002), and treatment for CLL (OR=3.3, p<0.001)]. CONCLUSIONS: In this cohort study, patients with newly diagnosed clinical-MBL had a 6.5 fold risk of hospitalization for infection relative to the control cohort. MBL was an independent risk factor for hospitalization for infection after controlling for age, sex, and treatment. Disclosures: Zent: GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: 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.