Dyke, Daniel L. Van

Abstract Abstract 3538 Background: Cytogenetics detection of common recurring genetic defects by FISH is an important clinical prognostication tool in CLL and is frequently used by clinicians for counseling and risk stratification. Thus, any improvement in FISH technology is a welcome advance in our care for CLL patients. Mayo Clinic CLL FISH analyses are prepared directly from buffy coat samples rather than mononuclear cells or lymphocytes. FISH probes (deletion 6q, 11q, 13q, 17p, +12 & IGH/CCND1) are scored among consecutive nuclei without selection for nuclear morphology (so called clinical scoring). CLL B-cells are observed, microscopically, primarily as round lymphocytes with round nuclei, and we found (ASH 2010, #3606) that the % abnormal nuclei in the CLL round cell population is significantly higher than that of the non-round nuclei population. In 79 of 87 (91%) patients, the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 – 57%). Round nuclei (p = 0.004) correlated better with % B-cells by flow cytometry than either consecutive unselected nuclei (p = 0.046) or the lobed (non-round) nuclei (p = 0.40). Utilizing this “Poor Man's” sorting technique, we proposed that scoring consecutive round nuclei would improve sensitivity of detection of CLL FISH defects in patients, especially those with low B-cell counts. In a retrospective study of Mayo Clinic patients with normal baseline FISH who had a FISH defect in follow-up, we showed that by re-scoring 28 baseline cases for round nuclei exclusively, 12 originally normal samples remained normal but 16 (57%) were abnormal using the “Poor Man's” scoring technique. In the present study, we prospectively compared FISH scoring on unsorted samples using the “Poor Man's” technique vs the clinical technique. Methods: After IRB approval, we collected 63 consecutive unsorted Mayo Clinic patient blood samples referred for CLL FISH testing with an absolute lymphocyte count (ALC)< 30K × 109/L, and scored each sample using both the “Poor Man's” round nuclei method and the standard clinical method. The kappa statistic was used to adjust for chance agreement. Bland Altman plots of difference (“Poor Man's” result minus clinical result) vs the average of the two methods were generated. Results: Median age of the 40 males (64%) and 23 females (36%) was 66 years (range 36–87), and median ALC was 6.803 (range 0.980–29.750). The most common FISH defects were 11q-, trisomy 12 & 13q- by clinical technique (14%, 22% & 46% respectively) as well as by “Poor Man's” method (16%, 24% & 49% respectively). A high degree of agreement was observed between the two methods when positive/negative comparisons were made (p<0.0001). Additional FISH defects were observed in 12 of 63 (19%) patients (one 11q-, one +12; six 13q-, and four 17p-) when we utilized the “Poor Man's” method that were not observed by the clinical method. Discussion: The “Poor Man's” sorting method compares well with the clinical method for the presence and absence of FISH defects. The abnormal CLL FISH patterns are truly confined to round nuclei. The “Poor Man's” method is more sensitive than the clinical method, resulting in higher reported abnormal cells and detection of more defects. Importantly, additional FISH defects were identified in 12 patients via “Poor Man's” method that were not observed by clinical technique and 6 of these involved a change in the patients' hierarchical (Döhner) classification due to detection of the more unfavorable prognostic markers of trisomy 12 or deletion 11q or 17p. Thus, selectively scoring FISH in the round nuclei increases the sensitivity for detection of a genetic defect in CLL and provides a practical framework for prognostication in CLL patients with low lymphocyte counts and, we would speculate, including those in clinical remission or with minimal residual disease. 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.

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

Abstract Abstract 4104 Background: The International Prognostic Scoring System (IPSS) for primary myelofibrosis (PMF) uses five predictors of inferior survival: age > 65 years, hemoglobin < 10 g/dL, leukocytes > 25 × 109/L, circulating blasts ≥ 1% and constitutional symptoms (Cervantes F et al. Blood 2009; 113: 2895). The dynamic IPSS (DIPSS) utilizes the same prognostic variables but can be applied at any time during the disease course (Passamonti F et al. Blood 2010; 115: 1703). IPSS-independent risk factors for survival have since been described and include unfavorable karyotype, red cell transfusion need and platelets < 100 × 109/L. Objectives: i) To determine if the aforementioned IPSS-independent risk factors for survival in PMF (i.e. unfavorable karyotype, red cell transfusion need and platelets < 100 × 109/L) are also DIPSS-independent. ii) To develop and validate a refined DIPSS model that incorporates DIPSS-independent prognostic factors for survival. iii) To establish a prognostic model for leukemia-free survival in PMF. Methods: The Mayo Clinic database for PMF was used to identify patients in whom bone marrow histologic and cytogenetic information was obtained at the time of their referral. WHO criteria were used for PMF diagnosis and leukemic transformation (Vardiman JW et al. Blood 2009; 114: 937). None of the patients in the current study were included in the original group of patients used to describe DIPSS (Passamonti F et al. Blood 2010; 115: 1703). Results: A total of 793 patients met the above-stipulated criteria. The study population was divided into two groups based on whether or not patients were seen at the Mayo Clinic within (n=428; training set) or beyond (n=365; test set) their first year of diagnosis. i) Objective 1: Multivariable analysis that included DIPSS risk category (low, intermediate-1, intermediate-2 and high risk), karyotype (favorable or unfavorable), platelet count (≥ or <100 × 109/L) and transfusion status (independent or dependent) was performed using the training set and all four variables were found to be significant as independent predictors of survival (p<0.0001, <0.0001, 0.0008 and 0.01, respectively). ii) Objective 2: Using the respective hazard ratios (HR) obtained during the multivariable prognostic analysis in the training set, HR-weighted adverse points were assigned: 3 points for high-risk DIPSS, 2 points for intermediate-2 risk DIPSS, and 1 point each for intermediate-1 risk DIPSS, unfavorable karyotype, platelets < 100 × 109/L or transfusion need. Accordingly, a refined DIPSS model (i.e. DIPSS-plus) was devised using the training set (Figure 1) and subsequently validated in the test set (Figure 2): low-risk (0 adverse points), intermediate-1 risk (1 adverse point), intermediate-2 risk (2-3 adverse points) and high-risk (4-6 adverse points). Considering the entire group of 793 study patients, the respective median survivals were 185, 78, 35 and 16 months (p<0.0001). ii) Objective 3: Multivariable analysis identified platelet count (p=0.0007) and karyotype (p=0.04) but not DIPSS or transfusion status as independent predictors of leukemia-free survival. We used these two variables to classify patients into a low (favorable karyotype and platelets ≥ 100 × 109/L) or high (unfavorable karyotype or platelets < 100 × 109/L) risk category for leukemic transformation; the respective 5- and 10-year risk of leukemic transformation were 6% and 12% vs. 18% and 31%, respectively (p<0.0001; HR 3.3, 95% CI 1.9–5.6). Conclusions: DIPSS-plus effectively incorporates prognostic information from karyotype, platelet count and transfusion status into the DIPSS prognostic model for PMF. Unfavorable karyotype or platelets <100 × 109/L is associated with a significantly higher risk of leukemic transformation in PMF. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2486 In patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL), deletion of 17p13 (17p-) resulting in loss of one allele of TP53 and 11q22 (11q-) resulting in loss of one allele of ATM predicts a significantly poorer prognosis. These lesions are nearly always monoallelic in patients with CLL and their consequences are considerably influenced by the integrity of the remaining allele. A dysfunctional mutation in the remaining allele, resulting in more aggressive disease, ultimately occurs in most patients with 17p- (> 80%) but fewer patients with 11q- (30%). Because ATM and TP53 are integral components of the p53 pathway, we hypothesize that patients with clonal 17p- and 11q- in the same CLL cell would have a worse prognosis than patients with only one of these deletions. To test this hypothesis, we compared the clinical outcomes (overall survival (OS) and time to treatment (TTT)) of patients with 17p- and 11q- in the same cell to patients with either 17p- or 11q- or neither defect. Methods: We used the Mayo Clinic CLL Database and the Cytogenetics laboratory database to identify all CLL patients seen in the Division of Hematology from 1/1/1995-3/29/2012 who had FISH analysis. Data was extracted on demographics, date of first (sentinel) occurrence of 11q- and 17p-, treatment, and vital status. FISH studies were performed on uncultured blood or bone marrow by standard methods using the Mayo Clinic CLL FISH panel of centromere specific control probes and probes for 6q (MYB), 11q (ATM), 13q14, and 17p (TP53) deletions, trisomy 12 and IGH. Patients with both 11q- and 17p- were further tested with the Abbott Molecular (Des Plaines, IL) ATM/TP53 combination probe set, to determine if the 11q and 17p deletions were present in the same cells. Patients were grouped into 4 cohorts: 1) clonal 17p- and 11q-; 2) 17p- only; 3) 11q- only; and 4) neither 17p- nor 11q-. OS and TTT were calculated and Kaplan-Meier curves were drawn from sentinel FISH date until their last known alive date or death date for OS and until treatment or last known untreated date for TTT. We censored all data on 3/29/2012 and analyzed using log-rank tests from date of sentinel FISH. Sanger sequencing of exons 4–9 of TP53 was done in samples from 15 patients with clonal 17p- and 11q- with an available sample collected within 1 year of the sentinel FISH test and without treatment in that interval. All mutations were confirmed by a repeat assay and analyzed for their predicted effect using the UMD p53 database. Results: We studied 2234 CLL patients with a median follow-up of 4.8 years (range 0 – 29.6 years). Twenty (1%) patients had clonal 17p- and 11q-, 159 (7%) had 17p-, 251 (11%) had 11q-, and 1804 (81%) had neither 17p- nor 11q-. Median OS from sentinel FISH study (Figure 1) was significantly shorter in the clonal 17p- and 11q- group (1.9 years), 17p- group (3.2 y), and 11q- group (4.7 y) compared to the patients with neither 17p- nor 11q- (9.2 y) (p < 0.0001). OS was significantly shorter in the patients with clonal 17p- and 11q- cells vs those with 17p- (p=0.048). TTT was also significantly different among the 4 groups (p<0.0001) with median time to treatment of 0.07 y in the clonal 17p- and 11q-, 0.2 y in 17p-, 0.7 y in 11q-, and 5.6 y in the neither 17p- nor 11q- group. TP53 analysis performed in samples from 15 patients with clonal 17p- and 11q- showed that 10 patients had mutations in the remaining TP3 allele predicted to be deleterious to normal p53 protein function. Median OS was 2.1 years for those with TP53 mutations and 0.9 years for those without mutations (p = 0.03) and median TTT was 0.25 years for those with TP53 mutations and 0.05 years for those without mutations (p = 0.049). Conclusion: “Double hit” clonal deletions of 17p and 11q occurred in 1% of this large cohort of CLL patients and were associated with a significantly poorer prognosis. We recommend that testing for this “double hit” phenomenon, by use of an ATM/TP53 combination probe set, should thus be performed in all patients with 17p- and 11q- on FISH analysis because these patients have a poorer prognosis than those with either 17p- or 11q- and could be candidates for novel therapies. The finding that dysfunctional TP53 mutations in the remaining allele did not worsen prognosis in patients with clonal 17p- and 11q- suggests that monoallelic deletion of both ATM and TP53 in a clone of CLL cells is sufficient to disable p53 pathway function in these patients. Future study of clonal 17p- and 11q- CLL could thus be informative in this regard. Disclosures: Kay: Celgene: 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. Zent:Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding.

Abstract Abstract 4122 Background: We have previously identified sole +9, 13q- or 20q- as “favorable” and sole +8 or complex karyotype as “unfavorable” cytogenetic abnormalities in primary myelofibrosis (PMF) (Blood 2010; 115: 496). The purpose of the current study, which includes more than twice the number of patients included in previous studies, was to identify additional prognostically-relevant cytogenetic abnormalities in PMF and refine cytogenetic risk categorization for overall and leukemia-free survival. Methods: Clinical and laboratory data were collected from consecutive patients with PMF seen at our institution and in whom cytogenetic information at or within 1 year of diagnosis was available. Diagnosis of PMF and acute myeloid leukemia were according to the World Health organization (WHO) criteria. Results: A total of 433 patients with PMF were included in the current study. Median age at diagnosis was 65 years. IPSS risk distributions were low in 12% of patients, intermediate-1 in 25%, intermediate-2 in 24% and high in 39%. JAK2V617F mutational frequency was 60%. Cytogenetic findings were normal in 275 (64%) patients. Among the 158 (36%) patients with abnormal karyotype, 109 (69% of abnormal cases) represented sole abnormalities, 23 (15%) two abnormalities and 26 (17%) three or more (i.e. complex) abnormalities. In an effort to identify cytogenetic categories of similar prognosis, each one of 12 operational cytogenetic categories was separately compared with both normal and complex karyotype. Accordingly, we were able to devise a two-tired cytogenetic risk stratification with highly significant differences in overall and leukemia-free survival (Figures 1 and 2): unfavorable (complex karyotype or sole or two abnormalities that include +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p- or 11q23 rearrangement) and favorable (all other cytogenetic findings including normal karyotype). Median survivals of patients with favorable and unfavorable karyotype were 5.2 and 2.0 years, respectively (p<0.0001). The corresponding 5-year survival rates were 51% and 8% (HR 3.1, 95% CI 2.2–4.3; p<0.0001). Multivariable analysis confirmed the IPSS-independent prognostic value of cytogenetic risk categorization (p<0.0001; HR 2.1, 95% CI 1.5–3.1) and also identified thrombocytopenia (platelet count < 100 × 109/L) as another independent predictor of inferior survival (p<0.0001; HR 1.9; 95% CI 1.4–2.6). A similar multivariable analysis showed that cytogenetic risk profile (p=0.001; HR 4.1, 95% CI 1.7–9.6) and platelet count (p=0.04; HR 2.3, 95% CI 1.0–5.0), but not IPSS (p=0.27), predicted leukemia-free survival; the 5-year leukemic transformation rates for unfavorable and favorable karyotype were 46% and 7%, respectively (HR 5.5, 95% CI 2.5–12.0; p<0.0001). These results, in terms of both overall and leukemia-free survival analysis, did not change when patients receiving allo-SCT were censored at the time of their transplant. Among patients with favorable karyotype; the incidences of leukopenia and thrombocytopenia were highest in patients with sole 20q- (41% and 38%, respectively) and lowest in those with sole 13q- (0% and 0%, respectively). Conclusions: The current study provides the rationale and necessary details for incorporating cytogenetic findings and platelet count in future prognostic models for PMF. The study also revealed a highly significant association between sole 20q- and both leukopenia and thrombocytopenia, which suggests a 20q- haploinsufficient gene effect. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3606 Background: Cytogenetic abnormalities are important prognostic indicators in CLL. At Mayo Clinic, CLL FISH analyses are derived from buffy coat samples not purified for lymphocytes. Scoring is therefore performed on consecutive qualifying nuclei regardless of cell size, shape or morphology. This scoring may not reflect the percent of abnormal nuclei of the affected cell type, but rather the entire population of cells present in the sample. We sought to determine whether the percent abnormal nuclei in only the affected cell type (e.g. lymphocytes) of patients with CLL differed from that of the general cell population. Scoring only B-lymphocytes could increase the sensitivity of the test in patients with low B-cell counts, either early in their disease or after treatment. Cell sorting techniques could be used to reach this goal but can be expensive, labor intensive and add to completion time. We propose a “Poor Man's Cell Sorting” technique based on cell morphology when stained with 4',6-diamidino-2-phenylindole (DAPI). In CLL samples, both round and so called “lobed” cells are seen by DAPI staining but we hypothesize that the CLL B-cells typically present as perfectly round in shape (Fig. 1). Thus our hypothesis: if only the round cells are scored for a genetic defect, would this more accurately represent the malignant leukemic B-cell population and allow for enhanced disease status by FISH? Methods: After IRB approval, we identified 87 CLL patients (75% male; 25% female, mean age 62.4 y, range 43–89 y) in the Mayo Clinic CLL Database, who were seen at Mayo Clinic between March, 2002 - July, 2010. We selected patients known to have low level FISH abnormalities and who therefore should have a significant population of both lymphocytes and neutrophils. Selection criteria included <20×106 B-cells per microliter (by flow cytometry) of peripheral blood and ≤40% of interphase nuclei expressing a specific FISH abnormality. Although most of the 87 patients exhibited more than one FISH abnormality, we focused on only one FISH defect per patient for this study. The original specimen (slide or fixed cell pellet) was rescored for % abnormal nuclei among 100 consecutive round and 100 consecutive lobed (or multilobular) nuclei, using DAPI to identify nuclear architecture. These scores were compared to each other and to the original clinical FISH analysis (scored for 200 consecutive nuclei and not selected for nuclear morphology). Results: Among 87 cases, FISH signals were scored for 6q-(1), 11q-(9), +12(15), 13q-(58), 17p-(2) and 2 IGH rearrangements. In 86/87 cases, the abnormal lobed nuclei did not have FISH defects. One patient exhibited 35% +12 by clinical assay, 52% +12 in the round nuclei, and 11% by lobed nuclei. For all cases, the mean percentage of abnormal nuclei was greater in the round cells (46%) vs original scoring (23.6%). In 79/87 patients (91%), the % of abnormal nuclei was greater in the round cells vs original FISH (mean increase 24.3%; range 1.5–57%). One patient's score was the same in round cells vs original, and 7 patients exhibited fewer abnormal nuclei in the round cells vs original. For these 7 cases, the mean % abnormal nuclei was 5.6% for round nuclei vs 8.4% for original score (score differences ranged from 1 – 5.5%). Overall, by univariate regression analysis, round cells (p=0.0043) have a better correlation with % B-cells, ascertained by flow cytometry, than either the current clinical approach (p=0.0462) or the lobed-cell approach (p=0.4058). Discussion: In virtually all cases (99%), the abnormal FISH patterns were confined to the round nuclei, and the lobed nuclei were virtually always normal by FISH. For patients with <20×106 lymphocytes, selecting for round nuclei uniformly resulted in identification of a higher percentage of abnormal cells. Further studies, including comparison of our round nucleus approach to CLL FISH analysis to actual sorted B-lymphocyte cell selection and the association of this leukemic restricted estimation of abnormal FISH levels to clinical outcome are necessary. Given our findings, we believe that estimating FISH defects restricted to the leukemic B-cell population will become an important adjunct to cytogenetic analysis for patients with low lymphocyte counts, including those in clinical remission and those with minimal residual disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: The Food and Drug Administration (FDA) approved ibrutinib in February 2014 based on results from a pivotal phase 2 clinical trial that enrolled 85 patients with relapsed/refractory CLL. A subsequent phase 3 trial confirmed the superior progression-free survival (PFS) and overall survival (OS) of ibrutinib compared to ofatumumab in 391 CLL patients. However, there is no information on the efficacy and safety of ibrutinib in CLL patients treated outside the context of clinical trials. Additionally, it is unclear if the reasons for discontinuation of ibrutinib differ in routine clinical practice from those described in patients treated on clinical trials. We conducted a retrospective study to evaluate these aspects in a large cohort of CLL patients treated with ibrutinib off-protocol. Methods: After IRB approval, CLL patients seen at Mayo Clinic, Rochester, MN, and treated with commercial ibrutinib from November 2013 through July 2015 were evaluated. The baseline characteristics, time to therapy discontinuation, reasons for discontinuation and survival following discontinuation were abstracted. Results: One hundred thirty-five patients with CLL were treated during the study interval; 124 received ibrutinib for relapsed/refractory CLL and 11 received ibrutinib for treatment-naïve CLL with TP53 disruption (either del17p on FISH or TP53 mutation). Patients with relapsed/refractory CLL (n=124) Patients were high-risk, with a median of 3 prior therapies (range, 1-15), 81% had unmutated IGHV, and 38% had either del11q or del17p on FISH testing. After a median follow-up of 6.4 months, 101 remain on ibrutinib therapy. The estimated proportion of patients continuing ibrutinib at 6 months was 84% (95% CI: 77-92%) and at 12 months was 70% (95% CI: 59-83%). Of the 23 who discontinued ibrutinib therapy, 8 discontinued for progressive disease (2 progressive CLL; 6 Richter's transformation). The remaining 15 discontinued therapy for reasons other than progression, including: infection (n=3), bleeding (n=3), diarrhea (n=1), atrial fibrillation (n=1), rash (n=1), immune thrombocytopenia (n=1), renal dysfunction (n=1), severe edema (n=1), MDS (n=1) and physician preference (n=2). Table 1 shows the reasons for discontinuation of ibrutinib in patients treated on clinical trials (Maddocks, JAMA Oncology, 2015) compared to the current cohort of patients. The estimated cumulative incidence of discontinuation of ibrutinib therapy due to CLL progression/Richter's transformation at 6 and 12 months was 9% (95% CI: 4-17%) and 12% (95% CI: 5-23%), respectively. The estimated cumulative incidence of ibrutinib discontinuation due to reasons other than progression at 6 and 12 months was 10% (95% CI: 5-17%) and 22% (12-34%), respectively (Figure). The median survival after discontinuation of ibrutinib for patients who had CLL progression was 3.5 months, not reached in patients who developed Richter's transformation and not reached in those patients who discontinued ibrutinib due to reasons other than progression. Patients with treatment-naïve CLL (n=11) All patients had evidence of TP53 disruption (9 had del17p, 3 had TP53 mutation, and 1 had both). After a median follow-up of 2.8 months, no patient has had progression of CLL, and 2 patients have discontinued ibrutinib therapy for reasons other than CLL progression (atrial fibrillation, n=2). The estimated proportion of patients continuing ibrutinib at 6-months was 71% (95% CI: 45-100%). All 11 are still alive, and no patient has undergone allogeneic stem cell transplantation. Conclusion: This is the first study that describes the outcomes of a large cohort of CLL patients treated with ibrutinib outside the context of clinical trials. Patients treated in routine practice appear more likely to discontinue ibrutinib for reasons other than progression of disease, compared to those participating in clinical trials. Our results confirm the poor survival of CLL patients who progress on ibrutinib. Table 1. Comparative analysis of reasons for discontinuation of ibrutinib in patients with relapsed/refractory CLL: Characteristic Current Study (%) Maddocks (JAMA Oncology 2015) (%) No. of patients 124 308 Median follow-up (months) 6.4 20 Reasons for Discontinuation Progression of disease CLL 2 (2) 13 (4) Richter's transformation 6 (5) 15 (5) Non-progression of disease Infection 3 (2) 28 (9) Other 12 (10) 17 (5) Figure 1. Figure 1. Disclosures Ding: Merek: Research Funding. Kay:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Tolero Pharm: Research Funding. Shanafelt:Cephalon: Research Funding; Celgene: Research Funding; Glaxo-Smith_Kline: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Genentech: Research Funding.

Abstract Abstract 3069 Background: Monosomal karyotype (MK) is defined as the presence of two or more distinct autosomal chromosome monosomies or a single autosomal monosomy associated with at least one structural abnormality (Breems DA et al. J Clin Oncol 2008; 26: 4791). In acute myeloid leukemia (AML), MK has been shown to be prognostically worse than complex or other unfavorable karyotype (Breems DA et al. J Clin Oncol 2008; 26: 4791). In primary myelofibrosis (PMF), complex karyotype or isolated trisomy 8 predicts inferior survival (Hussein K et al. Blood 2010; 115: 496). Objective: To determine if MK in PMF is prognostically distinct from previously defined poor cytogenetic risk categories including complex karyotype and isolated trisomy 8. Methods: The Mayo Clinic database for PMF was used to identify consecutive patients with unfavorable karyotype including complex karyotype and sole trisomy 8. WHO criteria were used for PMF diagnosis and leukemic transformation (Vardiman JW et al. Blood 2009; 114: 937). Results: Among 793 PMF patients with cytogenetic information at the time of their first time referral to the Mayo Clinic, 452 displayed a normal karyotype and 341 (43%) an abnormal karyotype. Of the latter, 41 (12%) displayed complex karyotype and 21 (6%) sole trisomy 8. Among the 41 patients with complex karyotype, 17 (42%) met the criteria for MK and 24 (58%) displayed complex karyotype without monosomies. Overall survival was significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 2.3, 95% CI 1.1–4.8) or trisomy 8 (p=0.02; HR 2.4, 95% CI 1.2–5.1) (Fig. 1). Prognosis among all three groups was significantly worse than patients with normal karyotype (Fig. 1). Leukemia-free survival was also significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 6.9, 95% CI 1.3–37.3) or trisomy 8 (p=0.02; HR 14.8, 95% CI 1.7–130.8) (Fig. 2). LFS in patients with normal karyotype was similar to those with either complex karyotype without monosomies (p=0.31) or trisomy 8 (p=0.86) (Figure 2). Conclusions: Monosomal karyotype in PMF is distinctly associated with extremely poor overall and leukemia-free survivals that are significantly worse than those seen in PMF patients with other unfavorable karyotype including complex karyotype without monosomies and sole trisomy 8 abnormalities. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2922 The myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by ineffective hematopoiesis and a highly variable clinical course, for which novel treatments are beginning to emerge. Conventional cytogenetic studies (CCS) of bone marrow (BM) are routinely used in clinical practice to detect abnormal clones in proliferating (metaphase) cells, identifying clonal aberrations in ∼50% of de novo MDS cases. Cytogenetics is also one of the key International Prognostic Scoring System (IPSS) components used to estimate overall survival and leukemia-free survival in MDS. Chromosome abnormalities may be quantified at presentation and during treatment by the use of fluorescence in situ hybridization (FISH) with DNA probes for specific chromosome loci (e.g., chromosomes 5, 7, 8 and 20) in non-proliferating (interphase) nuclei. However, it is not clear whether or not peripheral blood CCS will yield the same diagnostic and prognostic data as bone marrow CCS at disease presentation or whether patients without apparent chromosome abnormalities by CCS have “hidden” abnormalities that can be identified by interphase FISH. To answer these questions, 15 members of the International Working Group on MDS Cytogenetics agreed to perform CCS and FISH in parallel on both peripheral blood and bone marrow samples collected from MDS patients. To be certain that all participating sites scored and interpreted their individual FISH data in a similar fashion, a quality assurance (QA) study was completed with each site studying two identical test (proficiency) samples. Concordance among sites was very good to excellent allowing for the establishment of a standardized protocol with clear scoring criteria before patient samples were processed. In the second phase of the study, a total of 77 MDS patients were accrued to the study with 61% showing an abnormal karyotype. A FISH panel consisting of eight probe sets [-5/5q-, -7/7q-/der(1;7), +8/8q-, -11/+11/11q-/add(11q), 12p-/+21/t(12;21), -13/13q-, 17p- and 20q-/i(20q)/i(20p), Abbott Molecular, Inc.] was performed on both specimen types. While CCS was frequently unsuccessful (57.5%) in the PB specimens, FISH was informative (concordant with BM/PB CCS) in 92% of cases, with 49% of PB FISH demonstrating an abnormal clone. FISH was discordant in 4 of 77 BM and PB samples (5%), while CCS and FISH on BM and PB were discordant in 6 of 77 BM specimens (8%) and 6 of 73 PB specimens (8%). The data suggest that evaluation of interphase nuclei from PB on follow-up (non-diagnostic) FISH studies on MDS patients will be equally informative (and less costly and stressful) as a BM sample. Disclosures: Slovak: PerkinElmer: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Abstract Abstract 1246 Poster Board I-268 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a variable clinical course. Genomic aberrations as detected by FISH analysis and the mutational status of the IgVH genes have been found to have prognostic clinical significance with unmutated IgVH genes associated with clinically more aggressive disease. Previous studies have suggested that unmutated IgVH B-CLL is associated with increased chromosomal instability and increased chromosomal aberrations leading to accelerated disease progression. We recently performed a high-throughput, high-resolution array-based comparative genomic hybridization (aCGH) in a cohort of B-CLL patients. Fifty-eight patients have been analyzed by using the Sureprint G3 microarray (one million probes, Agilent). Our working hypothesis was that we would see increased genomic aberrations and instability in the unmutated IgVH group. The total number of copy number aberrations and total megabases (Mb) deleted or gained per genome were used as a surrogate marker for genomic instability. Variations in chromosomal aberrations in various VH families were also evaluated. B-CLL with unmutated IgVH genes utilized members of the VH1, VH3, VH4 and VH5 families with VH1-69 the most prevalent. B-CLL with mutated IgVH genes utilized VH families VH1, VH2, VH3, VH4, and VH6. Considerable variation was seen in the number of genomic abnormalities between cases. Overall, 315 abnormalities have been identified (236 deletions and 79 gains), with a median of 4 copy-number abnormalities per patient (range 0-32). In patients with IgVH mutational status available the median number of chromosomal aberrations in the mutated group was 5 (range 1-17) and unmutated group was 4 (range 0-20). Copy number deletions were significantly more common than chromosomal gains for the total group and in both the unmutated and mutated IgVH gene groups. Mean number of megabases (Mb) of DNA deleted in the unmutated IgVH group was 17.80Mb (standard deviation 24.17 Mb, median 6.63 Mb, range 0 - 90.55Mb) and 40.65 Mb in the mutated IgVH group (standard deviation 86.04 Mb, median 15.35Mb, range 0 – 307.57Mb). Mean number of Mb gained in the unmutated group was 37.74Mb (standard deviation 57.58Mb, median 0.44Mb, range 0 – 132.47Mb) and 32.99Mb (standard deviation 59.76Mb, median 0.14Mb, range 0 – 172.50Mb) in the mutated group. Nine patients had B-CLL with VH1-69 genes and 8 of them were unmutated. Median number of chromosomal deletions was 2 and gains also 2 (ranges 0 – 8 deletions and 0 – 4 gains) for this group. Similar findings were seen for the other VH families. These results initially suggest that total copy number variations do not account for the observed clinical differences between mutated and unmutated IgVH B-CLL. The catalogue of potentially clinically relevant chromosomal abnormalities found in this aCGH study is being currently evaluated. Disclosures Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Fonseca:BMS: Consultancy; Amgen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.