Hanson, Curtis A.

Abstract Abstract 2409 Background: Serum free light chain (FLC) quantitation has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and it has been incorporated in response criteria for both MM and AL amyloidosis. Two recent studies have shown FLC abnormalities are present in 39–54% of CLL patients and are associated with shorter time to treatment (TTT) and inferior overall survival (OS) in CLL. Here we report the largest study to date of FLC abnormalities in a prospective cohort of CLL patients and explore the effects of both monoclonal and polyclonal FLC elevations. Methods: Newly diagnosed CLL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from stored research serum samples using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the reference range (k>19.4 mg/L, l>26.3 mg/L). Monoclonal FLC elevation was defined as elevated FLC with an abnormal FLC ratio. Polyclonal FLC elevation was defined as elevated FLC with a normal FLC ratio (0.26–1.65). Patients with normal FLC but abnormal FLC ratio were evaluated as a distinct group (Abnormal FLC Ratio). Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for TTT and OS. Cox proportional hazards models were used to assess the association of FLC with TTT and OS. Results: 385 CLL patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a median follow-up of 47 months (range 1–94), 135 patients (35%) have been treated and 43 patients have died (11%). Abnormalities in FLC were present in nearly half of the CLL patients (48.8%). Although 32.4% had an elevated kappa or lambda, only 14.0% had a monoclonal FLC elevation while the remaining 18.4% had a polyclonal FLC elevation. An additional 16.4% had an abnormal ratio without elevated FLC. Monoclonal FLC elevation was associated with high risk features of CD38+, CD49D+, ZAP70+, IGHV unmutated and having high risk FISH (del 17p13; del 11q22). Monoclonal FLC elevation was highly associated with shorter TTT (HR=3.24, p=0.0007) and poor overall survival (HR=4.14, p=0.0006). These associations remained statistically significant after adjusting for stage and prognostic factors. Abnormal FLC ratio without elevated FLC was also associated with shorter TTT (HR=1.74, p=0.03), though this association did not hold after adjustment for stage and prognostic factors. Polyclonal FLC elevations were associated high risk FISH and poor overall survival (HR=6.41, p<0.0001, adjusted HR = 4.82, p=0.0002) but not with TTT. Conclusions: In the largest series of newly diagnosed CLL patients reported to date, nearly half of CLL patients have abnormal FLC concentrations. Monoclonal FLC elevation is an independent predictor of shorter TTT and inferior overall survival in CLL, is associated with high risk markers, and may be a marker of an aggressive tumor. Polyclonal FLC elevation without abnormal FLC ratio is independently associated with poor overall survival but not TTT suggesting it may be a non-specific marker of host frailty/comorbidity rather than disease specific outcomes. Regardless of cause, CLL patients with elevated FLC have poor outcome and FLC may have utility as a biomarker in CLL. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 2443 Background: There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association (GWA) study of CLL identified genetic variants located on chromosomes 2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13 that increased the risk of CLL within a European population. We replicated 5 of these 6 loci in an independent sample of CLL cases and controls from the United States. We now investigate whether these loci also influences MBL, a reported precursor condition of CLL. In addition, a follow-up analysis of the initial GWA study identified four more CLL-susceptibility loci on 2q37.3, 8q24.21, 15q21.3, and 16q24.1. Herein, we also evaluate the association of these four loci with risk of CLL. Methods: Peripheral blood samples were obtained from three ongoing studies: the Genetic Epidemiology CLL (GEC) Consortium, the Mayo Clinic non-Hodgkin lymphoma (NHL)/ CLL study, and the Mayo Clinic Biobank. We implemented rigorous genotyping quality-control measures, and successfully genotyped a total of 407 CLL patients, 965 controls, and 60 MBLs from these studies. Within each locus, the previously reported single nucleotide variants (SNPs) or variants in high linkage disequilibrium (LD) with the previously reported SNPs were evaluated with risk of MBL or CLL. Tests for association was done using the Cochran-Armitage trend test, and unconditional logistic regression was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CLL or MBL risk Results: In our evaluation of the six initially reported CLL-susceptibility loci (2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13) with MBL risk, we found three of the six had suggestive associations (p-value < 0.20) with ORs comparable to and in the same direction as those observed from CLL risk. Our strongest finding was with rs13397985 at locus 2q37.1 (OR= 1.56; 95% CI: 1.05, 2.31; p-trend = 0.041), followed by rs17483466 at locus 2q13 (OR= 1.49; 95% CI: 0.99, 2.24; p-trend = 0.074). As expected given our previously reported findings with CLL risk, the association between rs11083846 on chromosome 19q13 and MBL risk was not significant (p-trend = 0.70). Of the four recently reported CLL-susceptibility loci SNPs located on 2q37.3, 8q24.21, 15q21.3, and 16q24.1, we found all to be associated with CLL risk but one. Specifically, the strongest association was seen for locus 8q24.21 (best tagged SNP rs1021955; OR = 1.37; 95% CI: 1.10, 1.70; p-trend = 0.005), followed by locus 16q24.1 (best tagged SNP rs305065; OR= 0.77; 95% CI: 0.61, 0.97; p-trend = 0.024). However, we found no associations for locus 15q21.3 for the previously reported SNP nor for any SNPs in LD with the previously reported SNP. Conclusions: Our MBL results provide additional robust genetic evidence that MBL is a precursor to CLL and that it shares similar underlying genetic predisposition. Also our results confirm three of the four recently reported CLL-susceptibility loci and further support the role of a genetic basis in the etiology of CLL. More research is needed to elucidate the potential manner in which these genetic loci function in CLL or MBL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 460 Background: SF3B1 mutations were recently reported to occur in myelodysplastic syndromes (MDS), especially in the presence of ring sideroblasts (RS) (Papaemmanuil et al. Leukemia Research 2011, Volume 35, Supplement 1, page S18). We sought to accurately define the interaction between SF3B1 mutations, bone marrow morphology, karyotype and prognosis in MDS with ≥15% RS (MDS-RS). Methods: Mayo Clinic databases and cell banks were queried to identify patients with MDS and ≥15% RS (MDS-RS). All study patients were required to have undergone bone marrow examination and cytogenetic evaluation at diagnosis. Pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow RS percentage and confirm World Health Organization (WHO) morphologic categories. Detailed analysis of clinical and cytogenetic parameters was performed to risk stratify patients according to the revised International Prognostic Scoring System (IPSS-R) (Greenberg et al. Leukemia Research 2011, Volume 35, Supplement 1, page S6). All patients were annotated for their mutational status involving JAK2, MPL and IDH. PCR sequencing was used to identify SF3B1 mutations. Results I: Baseline patient information: The study is currently ongoing; to date, results are availab1e in 88 patients (median age 72 years, 69% males) including 36 (41%) patients with refractory anemia with RS (RARS), 36 (41%) with refractory cytopenia with multilineage dysplasia and ≥15% RS (RCMD-RS), 11 (12%) with refractory anemia with excess blasts (RAEB)-1 and ≥15% RS (RAEB1-RS) and 5 (6%) with RAEB2-RS. Median (range) values were 9.1 g/dL (5.8–15.7) for hemoglobin, 4.1 × 109/L (1.2–35.2) for leukocytes and 164 × 109/L (6–585) for platelets. IPSS-R risk categories were 20% very good, 49% good, 11% intermediate, 3% poor and 16% very poor. Karyotype was normal in 60%, other very good/good risk 16%, intermediate-risk 2%, and poor/very poor in 22%; 14% had monosomal karyotype (MK). Six (7%), four (5%) and zero (0%) patients displayed IDH, JAK2 or MPL mutations. Red cell transfusion need was documented in 27 (31%) patients. Results II: Prevalence and distribution of SF3B1 mutations and clinical correlates: SF3B1 mutations (all heterozygous) were detected in 40 (∼46%) of all 88 study patients: 25 (63%) K700E, 6 (15%) K666N/Q/R, 3 (8%) E622D, 2 (5%) H662D/Q, 2 (5%) Y623C, 1 (3%) R625C, and 1 (3%) T663I. One patient each displayed both SF3B1K700E and IDH2R140Q or SF3B1K700E and JAK2V617F. SF3B1 mutational frequencies were 69% for RARS, 36% for RCMD-RS, 18% for RAEB1-RS and 0% for RAEB2-RS (p=0.0007). SF3B1 mutations clustered with normal (59%) and other very good/good risk (57%) karyotype and were infrequent in poor/very poor karyotype (5%; p=0.0004). Additional significant correlations were noted between SF3B1 mutations and lower IPSS-R risk category (p=0.0004), advanced age (p=0.006), higher platelet count (p=0.0003). Results II: SF3B1 mutations and prognostic relevance in MDS-RS: To date, 68 (∼77%) deaths and 10 (∼11%) leukemic transformations have been documented. Median follow-up for living patients was 82 months. Univariate survival analysis considering all 88 study patients identified the following as strongly significant risk factors: IPSS-R (p<0.0001), WHO morphologic categories (p<0.0001), cytogenetic risk categories (p<0.0001), and transfusion need (p=0.0003). In multivariable analysis, only IPSS-R and transfusion need remained significant. In univariate analysis, the presence of SF3B1 mutations was significantly associated with better overall (p=0.04) and leukemia-free (p=0.03) survival; however, in both instances, significance was completely accounted for by WHO morphologic risk categorization and the morphology-adjusted p values were 0.6 and 0.2, respectively. In other words, when RARS and RCMD were analyzed separately, there was no additional prognostic value from the presence or absence of SF3B1 mutations (Figures 1 and 2; p=0.67 and 0.44, respectively). Conclusions: SF3B1 mutations are prevalent in RARS and RCMD-RS but do not provide additional, WHO morphology-independent or IPSS-R-independent, prognostic information. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4610 Introduction Infections are a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Up to 50% of CLL patients develop infectious complications, and 30–50% of all deaths in CLL patients are attributed to infections. Despite these data, the available research on infectious complications in CLL is primarily limited to patients participating in clinical trials of chemotherapy, obscuring the distinction between the risk of infection due to the disease’s natural history relative to the risk associated with therapy related immune suppression. 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 community-based cohort of CLL patients, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed CLL who resided within 50 miles of MCR and diagnosed between January, 1999 and December, 2009. The comparison cohort consisted of 689 adult patients who 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. All hospitalizations at MCR among both cohorts were audited to document hospitalization with infection and cross-referenced with the Mayo Clinic infection database, which includes all culture results obtained from hospitalized MCR patients. Serologies and viral assays 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 antibiotic therapy were considered to have a culture negative infection. RESULTS: In May 2010, there were 2022 CLL patients in the Mayo Clinic CLL Database diagnosed between 1999 and 2009. Of these, 174 (9%) were local CLL patients residing within 50 miles of MCR. Median age at diagnosis for these non-referred CLL patients was 69 years (range 27–97). Most had early stage disease (60% stage 0; 32% stage I-II). On prognostic evaluation, 121 (75%) patients were CD38 negative, 66 (61%) ZAP-70 negative, 47 (50%) IGHV mutated, and 56 (62%) had either no abnormality or 13q14- as a sole abnormality on FISH analysis. 64 (37%) patients progressed to require treatment for CLL during follow-up. After median follow-up of 4.0 years (range 0–11), 32 (18.4%) CLL patients were hospitalized with infection at least once as compared to 20/689 (2.9%) individuals in the control cohort (odds ratio=7.5; p<0.0001) Figure 1A. Even prior to receiving treatment for CLL, the rate of hospitalization for infection remained higher than the control cohort (p=0.002; Figure 1B). The 32 CLL patients hospitalized with infection were admitted with infection a total of 89 times (median 1 hospitalization for infection per patient). A specific infectious organism was identified by cultures or serologies in 60 (63%) of the 89 infections in CLL cases and 10/27 (37%) infections among controls. Hospitalization for an opportunistic infection (e.g. cytomegalovirus, cryptococcus, filamentous fungi, mycobacteria, Pneumocystis) occurred in 5/174 (3%) CLL patients during follow-up compared to 0/689 controls (p<0.001). Finally, we performed a pooled multivariable analysis of all patients (n=863) to identify factors (age, sex, CLL diagnosis, CLL treatment) associated with infection risk. Most factors were independently associated with hospitalization for infection [Age (per year, OR=1.03, p=0.02), sex (male, OR=4.0, p<0.001), diagnosis of CLL (OR=2.1, p=0.09), and treatment for CLL (OR=5.9, p<0.001)]. In univariate analysis of CLL patients [sex (OR=4.2, p=0.01), higher Rai stage (p=0.046), unfavorable (del 17p13; del 11q23) FISH [OR=4.6, p=0.02] and unmutated IGHV [OR=2.8, p=0.07]) were associated with risk of infection. CONCLUSIONS: In this cohort study, patients with newly diagnosed CLL had a 7.5-fold risk of hospitalization for infection relative to the control cohort. After 4 year follow-up, ∼1 in 5 CLL patients required hospitalization for infection. Although CLL treatment was a substantial risk factor for infection, the risk of infection among untreated patients remained higher than controls. Disclosures: 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. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract Background In routine myelodysplastic syndromes (MDS) practice, physicians do not submit bone marrow (BM) slides for central review. Instead, they rely on available BM reports to assess prognosis and make therapeutic decisions. Such practice is likely to introduce inaccuracies, especially in the diagnosis of MDS-unclassifiable (MDS-U). The objectives of the current study were to describe the natural history of MDS-U that is diagnosed based on routine BM and peripheral blood (PB) smear reports and compare its prognosis to refractory anemia (RA) or refractory cytopenia with multi-lineage dysplasia (RCMD), which were diagnosed in the same manner. Methods Mayo Clinic databases were reviewed to identify consecutive patients who meet current WHO criteria for MDS-U, RA and RCMD (Swerdlow et al. WHO classification of Tumors, Lyon 2008). No central review was required and diagnosis was based on routine Mayo Clinic pathology reports. Included in the current WHO-defined MDS-U are patients with findings of i) refractory cytopenia with unlineage dysplasia (RCUD) or RCMD with 1% blasts in the PB, 2) MDS with unilineage dysplasia and pancytopenia and 3) persistent cytopenia with ≤1% PB blasts, <5% BM blasts, <10% BM myeloid cell dysplasia and cytogenetic abnormalities considered presumptive evidence of MDS. Patients who had exposure to chemotherapy and/or radiotherapy were excluded. Results A total of 382 patients met WHO diagnostic criteria for MDS-U (n=146; 38%), RA (n=26; 7%), and RCMD (n=210; 55%). Presenting clinical characteristics for patients belonging to each one of these three categories are listed and compared in table 1. There was no difference between the three subgroups in age or gender distribution. Similarly, with the exception of thrombocytopenia that was more prevalent in RCMD, complete blood count and transfusion need at diagnosis were not significantly different between the groups. Risk distribution using conventional prognostic models for survival did not reveal significant differences between MDS-U, RCMD and RA. At the time of this analysis, 265 deaths and 25 leukemic transformations were documented. Overall and leukemia-free survival was similar between the three groups (Figure 1). IPSS and IPSS-R karyotypes (Figure 2&3), however delineate patients into distinct category (P=0.001 and P= 0.01) Conclusions Distinguishing between MDS-U, RCMD and RA, in daily practice, has limited clinical relevance. It might be simpler and more practical to consider MDS without excess blasts under one common morphologic category and utilize instead cytogenetic and molecular information for further prognostic stratification and identify patient subgroups with specific therapeutic needs. Myelodysplastic syndrome-unclassifiable (MDS-U) (n=146) compared with refractory anemia (RA) (n=26) and refractory cytopenia with multilineage dysplasia (RCMD) (n=210) Disclosures: No relevant conflicts of interest to declare.

Abstract Background Although the clinical importance of del 17p13 in patients with chronic lymphocytic leukemia (CLL) is well recognized, the implications of when this defect is identified are less clearly defined. In particular, the distinction between the identification of del 17p13 at the time of diagnosis and secondary acquisition of del 17p13 during the course of the disease are poorly characterized. Methods We identified all patients with CLL cared for at the Mayo Clinic between 1/1/2000 and 12/31/2012 who underwent baseline fluorescence in-situ hybridization (FISH) testing prior to receiving treatment. The Results of repeat FISH testing were reviewed to identify cases with clinically ascertained clonal evolution. Results A total of 1757 patients with newly diagnosed CLL were seen at Mayo Clinic prior to receiving treatment. Among these, 1243 had FISH testing performed prior to treatment and within 36 months of diagnosis. The median time from diagnosis to initial FISH was 0.8 months (11.5 to 35.4 months). The Results of baseline FISH testing among these patients is as follows: 486 (39%) had del 13q14, 234 (19%) had trisomy 12, 115 (9%) had del 11q23, 59 (5%) had del 17p13, 15 (1%) had other (e.g., del6q) and 334 (37%) had no abnormalities detected. Among these patients, 344 (28%) underwent repeat FISH testing during the course of their disease. Repeat FISH testing was typically performed for clinical suspicion of karyotype evolution or prior to therapy selection in patients with a long time interval between first prognostic FISH and initiation of treatment. Among these 344 patients, 41 (12%) acquired new cytogenetic abnormalities on repeat FISH. Classification of these 344 patients by the Dohner classification at diagnosis and time of follow-up FISH is shown in Table 1. Among the 41 pts who acquired a new FISH detectable genetic abnormality, the newly acquired defect resulted in a change in Dohner FISH risk category for 39 patients including 15/41 (37%) who acquired a del 17p13. Baseline clinical and prognostic characteristics of patients who developed clonal evolution to those who did not are shown in Table 2. Patients with unmutated immunoglobulin heavy chain (IGHV) gene mutation status (p<0.0001) and CD49d (p=0.003) expression were more likely to experience clonal evolution. Among the 319 patients who did not have del 17p13 on baseline FISH, 15 (5%) acquired del 17p13 during the course of their disease. The overall survival from the date del 17p13 was identified is shown for those with del 17p13 at diagnosis (n=59) compared to those who acquired del 17p13 later in the course of the disease (n=15) in Figure 1A. Similarly, among 339 patients who did not have del 6q23 on baseline FISH, 9 (3%) acquired del 6q23 during the course of their disease. The overall survival from the date del 6q23 was identified is shown for those with del 6q23 at diagnosis (n=11) and those who acquired del 6q23 later in the course of the disease (n=9) in Figure 1B. Conclusion Acquired cytogenetic evolution is clinically ascertained by FISH during the course of the disease in approximately 12% of patients. The newly acquired defects in these patients result in a change in Dohner classification for in >90% of these patients including ∼37% who acquire del 17p13. The clinical implications of del 17p13 is influenced by the timing of ascertainment with markedly shorter survival for those who acquire del 17p13 during the course of the disease relative to those with this defect at diagnosis. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Recent observations have underscored the biologic relevance of intratumoral angiogenesis and its potential impact on prognosis. Increased bone marrow angiogenesis has been demonstrated in a variety of hematologic disorders, including multiple myeloma. The extent and prognostic significance of bone marrow angiogenesis in 114 patients with myelofibrosis with myeloid metaplasia (MMM) was investigated. A control group of 44 patients without bone marrow disease, 15 patients with polycythemia vera, and 17 patients with essential thrombocythemia was also studied. Bone marrow microvessel density was assessed by a semiquantitative method, visual microvessel grading, and 2 separate quantitative methods, visual count and computerized image analysis. Angiogenesis estimation by all 3 methods was highly comparable. On visual microvessel grading, a grade 3 or 4 increase in bone marrow angiogenesis was demonstrated in 70% of patients with MMM, 33% of patients with polycythemia vera, 12% of patients with essential thrombocythemia, and 0% of normal controls. In a multivariate analysis, increased angiogenesis in MMM correlated significantly with increased spleen size and was found to be a significant and independent risk factor for overall survival. Increases in marrow angiogenesis correlated with hypercellularity and megakaryocyte clumping. In contrast, these 2 features were inversely proportional to reticulin fibrosis, whereas increases in marrow angiogenesis were independent of reticulin fibrosis. These preliminary findings suggest that neo-angiogenesis is an integral component of the bone marrow stromal reaction in MMM and may provide useful prognostic information and a rationale for the therapeutic investigation of anti-angiogenic agents.

Abstract Abstract 1711 Background: Mutations in genes of the splicing machinery, such as SF3B1, SRSF2 and U2AF35 are common in patients with myelodysplastic syndromes [MDS] (Nature 2011;478:64) and chronic myelomonocytic leukemia [CMML] (Haematologica 2012;Epub). In MDS, SRSF2 gene mutations are an independent risk factor for shortened over-all (OS) and leukemia-free survival (LFS) (Blood 2012;119:3578). In MDS with ring sideroblasts (RS), SF3B1 mutations have a high prevalence (∼50%), but do not influence either, the OS or the LFS (Blood 2012;119:569). We carried out this study to evaluate the prevalence, clinical correlates and prognosis of the aforementioned spliceosome mutations in CMML. Methods: The study included 227 patients with WHO defined CMML who were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SRSF2, SF3B1 and U2AF35. Results I: Prevalence and clinical correlates Among the 227 study patients, 153 (67%) were male, median age was 71 years (range, 17–90 years) and 192 (85%) met the WHO criteria for CMML-1. Ninety (40%) patients had SRSF2 mutations (86% CMML-1), 13 (6%) had SF3B1 mutations (75% CMML-1) and 20 (9%) had U2AF35 mutations (95% CMML-1). One-hundred and twenty three (54%) patients had at least one of three spliceosome mutations (86% CMML-1). Mutational hot spots were P95 for SRSF2 (P95L-n=36/H-n=32/R-n=13/A-n=1), K700E (n=7) and H662Q (n=2) for SF3B1, and Q157 (Q157R-n=5/P-n=5/G-n=1) and S34F (n=7) for U2AF35. Seven patients (54%) with SF3B1 mutations had ≥1% RS, with 5 (38%) showing ≥15% RS. Mutations involving all three spliceosome genes were mutually exclusive. The cytogenetic distribution based on the Spanish risk stratification system (Haematologica 2011;96:375) was; SRSF2 mutations: 69 (77%) low risk, 11 (12%) intermediate risk, and 10 (11%) high risk (+8-n=3, del/monosomy 7-n=2, monosomal karyotype-n=5); SF3B1 mutations: 8 (62%) low risk and 5 (38%) intermediate risk; U2AF35 mutations: 15 (75%) low risk, 3 (15%) intermediate risk and 2 (10%) high risk (p=0.89). The distribution of mutations according to the MD Anderson prognostic scoring system [MDAPS] (Blood 2002;99:840) was; SRSF2 - low-n=41, intermediate-1-n=26, intermediate-2-n=18, high-n=5, SF3B1- low-n=7, intermediate-1-n=3, intermediate-2-n=2, high-n=1, and U2AF35- low-n=11, intermediate-1-n=5, intermediate-2-n=3, high-n=1 (p=0.73). There was no statistically significant difference, among the three mutation groups, in prognostically relevant parameters, including gender distribution, median age, hemoglobin values, platelet counts, peripheral blood (PB) and BM blast counts, absolute neutrophil counts (ANC) and absolute monocyte counts (AMC). The only notable difference was that patients with the SF3B1 mutation had a lower median white blood cell count (p=0.04) and a lower absolute lymphocyte count (p=0.045). Results II: Prognostic impact of spliceosome mutations At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. Median survivals for patients with mutations involving SRSF2, SF3B1 and U2AF35 were 24, 17 and 12 months, respectively. In univariate analysis, the presence of SRSF2 (p=0.67), SF3B1 (p=0.96) or U2AF35 (p=0.49) mutations had no prognostic impact on OS. Similarly, none of the three spliceosome mutations affected LFS; corresponding p values were 0.55 for SRSF2, 0.9 for SF3B1 and 0.38 for U2AF35 mutations respectively. We then examined possible prognostic value of having none of these mutations (n=104) vs otherwise (n=123) and the results were once again negative (p=0.87). Conclusions: SRSF2 is the most frequently mutated spliceosome gene in CMML, but neither it nor SF3B1 or U2AF35 mutations affect overall or leukemia-free survival in CMML. Furthermore, the current study suggests limited genotype-phenotype association, save for the already established association between SF3B1 mutations and RS. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1742 Background Karyotype is one of the most potent and reproducible risk factors for both overall (OS) and leukemia-free (LFS) survival in primary myelofibrosis (PMF) (Blood 2011;118:4595). It is currently not clear if the number of metaphases studied or the abnormal metaphase percentage alters this prognostic impact. Methods: An updated Mayo Clinic database of karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify a consecutive series of patients and their cytogenetic information obtained at time of referral was centrally re-reviewed. Cytogenetic results were interpreted and reported according to the International System for Human Cytogenetic Nomenclature; abnormal karyotype was defined by the presence of at least 2 metaphases with structural abnormalities or monosomy or 3 metaphases with polysomy, regardless of number of metaphases examined. For this particular study, the presence of less than 20 evaluable metaphases did not disqualify patients. “Very high risk” karyotype included monosomal karyotype, inv(3) or i(17q) abnormalities (Blood 2011;118:4595). “unfavorable” karyotype included complex or any sole or two abnormalities that included +8, −7/7q-, -5/5q-, inv(3), i(17q), 12p-, or 11q23 rearrangement (Blood 2011;118:4595). All other cytogenetic abnormalities were considered “favorable” Results: A total of 590 patients (median age 65 years; range 19–89 years) including 424 (72%) males. The DIPSS-plus (JCO 2011;29:392) risk distribution was 40% high, 39% intermediate-2, 12% intermediate-1 and 9% low. Cytogenetic findings included 17 (3%) very high risk, 69 (12%) unfavorable, 165 (28%) favorable and 339 (57%) normal karyotypes. The number of bone marrow metaphases studied to report these cytogenetic findings were ≥20 in 468 (79%) patients, 11 to 19 in 71 (12%) patients and ≤10 in 51 (9%) patients; the proportion of cases studied with ≥20 metaphases were 53% for very high risk, 74% for unfavorable, 83% for favorable and 80% for normal karyotype (p=0.006). Among patients with abnormal karyotype, the abnormal metaphase percentage was ≥75% in 148 (59%) patients, 50 to 74% in 36 (15%) patients, 26 to 49% in 27 (11%) patients and ≤25% in 38 (15%) patients; the proportion of patients with ≥75% was 59% for very high risk, 67% for unfavorable and 56% for favorable karyotypes (p=0.70). As expected, OS was significantly different among very high risk, unfavorable, favorable and normal karyotype patients with respective median survivals of 8, 23, 41 and 57 months (p<0.0001). The number of metaphases studied (p=0.62) or the abnormal metaphase percentage (p=0.12), by themselves, did not affect survival. Similarly, the survival difference among the aforementioned cytogenetic risk groups was equally apparent when patients with ≥20 metaphases studied (n=468; P<0.0001) and those with <20 metaphases studied (n=122; p<0.0001) were separately analyzed. Analysis of patients with very high risk or unfavorable karyotype (n=86) revealed no significant effect of abnormal metaphase percentage on survival (Figure; p=0.80). A similar scenario was demonstrated for patients with favorable karyotype (Figure; p=0.24). Conclusions: Neither the number of metaphases examined nor the abnormal metaphase percentage appear to influence the currently recognized cytogenetic risk stratification in PMF. The current study has implications for both clinical practice and clinical research involving cytogenetic prognostication in hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2902 Background: Although chronic lymphocytic leukemia (CLL) is a low grade B-cell malignancy, 2–8% of patients (pts) are reported to undergo transformation to diffuse large B-cell lymphoma (DLBCL; Richter's syndrome [RS]). Whether the risk of transformation is related to the underlying biology of CLL or is a consequence of treatment for CLL is uncertain. Also, the role of prognostic markers such as immunoglobulin heavy chain (IGHV) gene mutation status, ZAP-70 and CD38 expression, and genetic abnormalities detected by fluorescence in situ hybridization (FISH) in predicting the development of RS is not well defined. The available data on stem cell transplant (SCT) in pts with RS is based on retrospective case series of selected RS pts and is of uncertain relevance to the majority of CLL pts experiencing RS. Therefore, we conducted a cohort study of newly diagnosed CLL pts to explore the incidence and natural history of RS. Methods: The Mayo Clinic CLL database includes all consenting pts with a pathologic diagnosis of CLL. Information on date of diagnosis, treatment history, and disease-related complications is abstracted from clinical records on all pts and then maintained prospectively. We used this database to identify newly diagnosed pts with CLL from January 2000 through July 2011, who were seen at our institution within 12 months of diagnosis. Pts with biopsy-confirmed RS were included in this analysis. For pts referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included in this study. The Mayo Clinic Institutional Review Board approved the study. Results: Between January 2000 and July 2011, 1641 pts with newly diagnosed CLL were seen at Mayo Clinic within 12 months of diagnosis, and this cohort had a median follow-up of 4.0 yrs (range, 0–12.3 yrs). Of these pts, 37 (2.3%) were diagnosed with RS. The median time to development of RS after a diagnosis of CLL was 1.8 yrs (range, 0–11.7 yrs). The incidence of RS at 2 and 5 yrs post CLL diagnosis were 1.3% and 2.1% respectively (Figure). Pts who developed RS had more advanced (Rai III/IV) stage disease (22% vs. 6%; p<0.001) and higher beta 2-microglobulin levels (median 3.1 mg/dL vs. 2.3 mg/dL; p=0.003) at diagnosis of their CLL compared to pts without RS. Pts who developed RS were also more likely to be positive for ZAP-70 (58% vs. 33%; p=0.03), CD49d (77% vs. 31%; p=0.0004), and CD38 (57% vs. 29%; p=0.001), have unmutated IGHV (76% vs. 44%; p=0.003) and have either del(11q22.3) or del(17p13) on FISH analysis (46% vs. 13%; p<0.0001). RS transformation occurred prior to treatment of CLL in 17 (46%) pts and after treatment of CLL in 20 pts (54%). Treatment for CLL was associated with an increased risk of RS (92% vs 34%; p<0.0001). The odds ratio (OR) of RS was increased for pts who received purine nucleoside analogues (OR=3.0; 95%CI=[1.6, 5.9]; p=0.001), alkylating agents (OR=2.5; 95% CI=[1.3, 4.9]; p=0.004), or both (OR=3.3; 95% CI=[1.7, 6.4]; p=0.0003) for treatment of their CLL. Median overall survival after development of RS was 2.1 yrs. Most pts (29/37, 78%) received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Two (5%) pts underwent an autologous SCT and 5 (14%) underwent an allogeneic SCT. Additionally, 1 (3%) patient received an autologous SCT followed by an allogeneic SCT. By the end of the study period, 17 (46%) pts with RS had died. Conclusion: The overall rate of biopsy-proven RS in this large cohort of pts with newly diagnosed CLL was 2.3% (5 year incidence: 2.1%). Clinical characteristics (Rai stage III/IV) and biologic characteristics of the CLL B-cell (positive expression of ZAP-70, CD38 and CD49d; unmutated IGHV; and either del(11q22.3) or del(17p13) on FISH) at the time of diagnosis were associated with future risk of RS. Exposure to purine nucleoside analogues and/or alkylating agents for treatment of CLL was also associated with increased risk of RS. Disclosures: Zent: GlaxoSmithKline: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Genzyme: Research Funding; Biothera: Research Funding. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.