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Background: Postoperative infection in tissue expander breast reconstruction causes increased morbidity, cost, and suboptimal patient outcomes. To improve outcomes, it is important to preoperatively identify factors that might predispose to infection and minimize them when possible. It is hypothesized that certain patient characteristics are associated with an increased infection rate. Methods: A retrospective, 6-year, single-institution review of patient records was performed from 413 tissue expanders placed in 300 women for postmastectomy breast reconstruction. Infection was defined as any case where antibiotics were given in response to clinical signs of infection. Fourteen potential risk factors were analyzed. A generalized estimation equations approach was used to perform univariable and multivariable analyses. Results: Antibiotics were given to treat clinical infection in 68 of 413 expanders (16.5 percent), with a median time to diagnosis of 6.5 weeks (range, 1 to 52 weeks). Univariable analysis showed significant association with breast size larger than C cup (p < 0.001), previous irradiation (p = 0.007), repeated implant (p = 0.008), and delayed reconstruction (p = 0.04). All variables except delayed reconstruction remained significant (p < 0.002 for all) in a multivariable model. Additional significant covariates in this model included one surgical oncologist (p = 0.003) and contralateral surgery (p = 0.046). Given infection, one surgical oncologist was associated with an increased rate of mastectomy flap necrosis (p = 0.01). Conclusions: Certain patient characteristics are associated with increased infection in tissue expansion breast reconstruction. Understanding how these predispose to infection requires additional study. Patients identified with these characteristics should be educated about these risks and other reconstructive options to optimize the success of their breast reconstruction.

Purpose Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated. Patients and Methods Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy. Results Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P ≤ .01), resulted in improved tolerability and treatment delivery. Conclusion Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.

Purpose: The precise molecular targets of IFN-α therapy in the context of malignant melanoma are unknown but seem to involve signal transducers and activators of transcription 1 signal transduction within host immune effector cells. We hypothesized that the in vitro transcriptional response of patient peripheral blood mononuclear cells (PBMC) to IFN-α would be similar to the in vivo response to treatment with high-dose IFN-α.Experimental Design: The gene expression profiles of PBMCs and immune cell subsets treated in vitro with IFN-α were evaluated, as were PBMCs obtained from melanoma patients receiving adjuvant IFN-α.Results: Twenty-seven genes were up-regulated in PBMCs from normal donors after treatment with IFN-α in vitro for 18 hours (>2-fold, P < 0.001). A subset of these genes (in addition to others) was significantly expressed in IFN-α–treated T cells, natural killer cells, and monocytes. Analysis of gene expression within PBMCs from melanoma patients (n = 13) receiving high-dose IFN-α-2b (20 MU/m2 i.v.) revealed significant up-regulation (>2-fold) of 21 genes (P < 0.001). Also, the gene expression profile of in vitro IFN-α–stimulated patient PBMCs was similar to that of PBMCs obtained from the same patient after IFN-α therapy.Conclusions: This report is the first to describe the transcriptional response of T cells, natural killer cells, and monocytes to IFN-α and characterize the transcriptional profiles of PBMCs from melanoma patients undergoing IFN-α immunotherapy. In addition, it was determined that microarray analysis of patient PBMCs after in vitro stimulation with IFN-α may be a useful predictor of the in vivo response of immune cells to IFN-α immunotherapy.

Purpose To analyze the prognostic impact of Wilms’ tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML). Patients and Methods We studied 196 adults younger than 60 years with newly diagnosed primary CN-AML, who were treated similarly on Cancer and Leukemia Group B (CALGB) protocols 9621 and 19808, for WT1 mutations in exons 7 and 9. The patients also were assessed for the presence of FLT3 internal tandem duplications (FLT3-ITD), FLT3 tyrosine kinase domain mutations (FLT3-TKD), MLL partial tandem duplications (MLL-PTD), NPM1 and CEBPA mutations, and for the expression levels of ERG and BAALC. Results Twenty-one patients (10.7%) harbored WT1 mutations. Complete remission rates were not significantly different between patients with WT1 mutations and those with unmutated WT1 (P = .36; 76% v 84%). Patients with WT1 mutations had worse disease-free survival (DFS; P < .001; 3-year rates, 13% v 50%) and overall survival (OS; P < .001; 3-year rates, 10% v 56%) than patients with unmutated WT1. In multivariable analyses, WT1 mutations independently predicted worse DFS (P = .009; hazard ratio [HR] = 2.7) when controlling for CEBPA mutational status, ERG expression level, and FLT3-ITD/NPM1 molecular-risk group (ie, FLT3-ITDnegative/NPM1mutated as low risk v FLT3-ITDpositive and/or NPM1wild-type as high risk). WT1 mutations also independently predicted worse OS (P < .001; HR = 3.2) when controlling for CEBPA mutational status, FLT3-ITD/NPM1 molecular-risk group, and white blood cell count. Conclusion We report the first evidence that WT1 mutations independently predict extremely poor outcome in intensively treated, younger patients with CN-AML. Future trials should include testing for WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile.

Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and times at which tumor lysis is observed in treated patients. Mitochondrial membrane depolarization was detected in flavopiridol-treated CLL cells by 6 hours, well before the onset of cell death. Flavopiridol-induced mitochondrial depolarization was not blocked by caspase inhibitors or by the calcium chelator EGTA, but was reduced by Bcl-2 overexpression. Intracellular calcium mobilization was noted at early time points using fluorescence microscopy. Furthermore, electron paramagnetic resonance oximetry showed a gradual but significant reduction in cellular oxygen consumption rate by 6 hours, corresponding with ultrastructural mitochondrial damage detected by electron microscopy. These observations suggest that in CLL and 697 cells, flavopiridol mediates its cytotoxic effects via induction of the mitochondrial permeability transition and changes in intracellular calcium.

A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML).Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features--that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature.Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1beta.A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways.

The prognostic relevance of FLT3 D835/I836 mutations (FLT3-TKD) in cytogenetically normal acute myeloid leukemia (CN-AML) remains to be established. After excluding patients with FLT3 internal tandem duplications, we compared treatment outcome of 16 de novo CN-AML patients with FLT3-TKD with that of 123 patients with wild-type FLT3 (FLT3-WT), less than 60 years of age and similarly treated on Cancer and Leukemia Group B protocols. All FLT3-TKD+ patients and 85% of FLT3-WT patients achieved a complete remission (P = .13). Disease-free survival (DFS) of FLT3-TKD+ patients was worse than DFS of FLT3-WT patients (P = .01; estimated 3-year DFS rates, 31% vs 60%, respectively). In a multivariable analysis, FLT3-TKD was associated with worse DFS (P = .02) independent of NPM1 status and percentage of bone marrow blasts. To gain further biologic insights, a gene-expression signature differentiating FLT3-TKD+ from FLT3-WT patients was identified. The signature (333 probe sets) included overexpression of VNN1, C3AR1, PTPN6, and multiple other genes involved in monocarboxylate transport activity, and underexpression of genes involved in signal transduction regulation. These associations with outcome, other prognostic markers, and the elucidated expression signature enhance our understanding of FLT3-TKD–associated biology and may lead to development of novel therapies that improve clinical outcome of CN-AML patients with FLT3-TKD.

The clinical impact of MLL partial tandem duplication (MLL-PTD) was evaluated in 238 adults aged 18 to 59 years with cytogenetically normal (CN) de novo acute myeloid leukemia (AML) who were treated intensively on similar Cancer and Leukemia Group B protocols 9621 and 19808. Twenty-four (10.1%) patients harbored an MLL-PTD. Of those, 92% achieved complete remission (CR) compared with 83% of patients without MLL-PTD (P = .39). Neither overall survival nor disease-free survival significantly differed between the 2 groups (P = .67 and P = .55, respectively). Thirteen MLL-PTD+ patients relapsed within 1.4 years of achieving CR. MLL-PTD+ patients who relapsed more often had other adverse CN-AML–associated molecular markers. In contrast with previously reported studies, 9 (41%) MLL-PTD+ patients continue in long-term first remission (CR1; range, 2.5-7.7 years). Intensive consolidation therapy that included autologous peripheral stem-cell transplantation during CR1 may have contributed to the better outcome of this historically poor-prognosis group of CN-AML patients with MLL-PTD.

The precise molecular targets of interferon-alpha (IFN-alpha) therapy of melanoma are unknown but likely involve signal transducer and activator of transcription 1 (STAT1) signal transduction within host immune effector cells. We hypothesized that microarray analysis could be utilized to identify candidate molecular targets important for mediating the anti-tumor effect of exogenously administered IFN-alpha.To identify the STAT1-dependent genes regulated by IFN-alpha, the gene expression profile of splenocytes from wild type (WT) and STAT1(-/-) mice was characterized.This analysis identified 30 genes that required STAT1 signal transduction for optimal expression in response to IFN-alpha (p0.001). These genes include granzyme b (Gzmb), interferon regulatory factor 7 (Irf7), Fas death domain-associated protein (Daxx), and lymphocyte antigen 6 complex, locus C (Ly6c). The expression of 20 genes was found to be suppressed in the presence of STAT1 including chemokine ligand 2 (Ccl2), Ccl5, and Ccl7. Nineteen genes were significantly upregulated in murine splenocytes following treatment with IFN-alpha regardless of the presence of STAT1 including CD86, lymphocyte antigen 6 complex, locus A (Ly6a), and Tap binding protein (Tapbp). The expression of representative IFN-responsive genes was confirmed at the transcriptional level by Real Time PCR.This report is the first to demonstrate that STAT1-mediated signal transduction plays a major role in the transcriptional response of murine immune cells to IFNalpha.

The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner. It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes. Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL. Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.

Background: Richter's transformation (RT), the occurrence of an aggressive lymphoma in patients with prior chronic lymphocytic leukemia (CLL), occurs in up to 10% of CLL patients. Anthracycline-based chemoimmunotherapy remains standard, but outcomes are poor (median survival approximately 12 months). R-EPOCH (rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) has demonstrated superior efficacy to R-CHOP in HIV-associated diffuse large B-cell lymphoma, Burkitt's lymphoma, and primary mediastinal B-cell lymphoma. We conducted a single-institution retrospective cohort study to characterize efficacy and toxicity of R-EPOCH as first-line treatment for RT. Methods: The study included patients treated with R-EPOCH as first-line therapy for histologically confirmed RT at the Ohio State University between January 1st 2006 and May 31st 2014. Characteristics of the CLL and RT, ECOG performance status, and laboratory assessment of bone marrow and organ function were abstracted from medical records. Toxicity of R-EPOCH was assessed by reviewing the frequency of adverse events (AE). R-EPOCH treatment outcomes were assessed as documented by the treating physician and not secondarily verified. Progression free survival (PFS) and overall survival (OS) durations were calculated from date of Richter's biopsy until date of event (PFS: relapse/death; OS: death), censoring patients without event at last follow-up. PFS and OS estimates were obtained using the Kaplan-Meier method. Univariable proportional hazards models were used to model PFS and OS as a function of each clinical variable. Results: The study included 46 patients. Median age at RT diagnosis was 67 (range 38-83). Median number of months from CLL to RT diagnosis was 53 (range 0.4-198). A median of 3 (range 0-13) prior CLL treatments had been given with 10 (22%) patients receiving ibrutinib as the most recent. The majority of patients where CLL risk was evaluable demonstrated poor-risk disease: 24/43 (56%) complex karyotype, 20/41 (49%) del(17)(p13.1), 27/32 (84%) unmutated IGHV. The majority of evaluable patients had an ECOG performance status of 0 or 1 (18/28, 64%). Table 1 shows RT characteristics for these patients. Treatment with R-EPOCH was started a median of 5 days after RT diagnosis (range 0-110). The majority of patients did not complete 6 cycles: 16 (35%) completed 1 cycle, 10 (22%) 2 cycles, 5 (11%) 3 cycles, 4 (9%) 4 cycles, 2 (4%) 5 cycles, and 9 (19%) 6 cycles. Of 131 cycles given, 114 (87%) were fully evaluable for AE with results in table 2. Outcome of R-EPOCH treatment was known for 44 patients: 9 (20%) achieved complete response, 8 (18%) clinical response documented by the treating physician, 14 (32%) progressive disease, and 13 (30%) died without (known) lymphoma progression. With a median follow up of 39 (range 13-54) months, 9 (20%) patients were alive without lymphoma progression. All patients with lymphoma progression died. Median PFS was 3.5 months (95% CI: 2.0-7.6) and median OS was 5.9 months (95% CI: 3.2-10.3) (Figure 1). In univariable analysis, risk of death was higher for patients with complex CLL karyotype (HR 4.38, p=0.0002), del(17)(p13.1) (HR 3.04, p=0.003), higher number of CLL treatments (HR 1.16, p=0.004), higher bilirubin (HR 1.68, p=0.04), and higher serum creatinine (HR 1.65, p=0.05). Conclusions: Patients with RT treated with first-line R-EPOCH had poor PFS (median 3.5 months) and OS (median 5.9 months) with only 20% of patients alive at last follow up. Characteristics of underlying CLL influenced outcomes of R-EPOCH with worse PFS and OS in deletion 17p and complex karyotype patients. Better therapies for RT are urgently needed, especially in patients with poor risk CLL. Table 1. Characteristics of Aggressive Lymphoma n=46 Histology, no. (%)- Large Cell- Early large-cell/Prolymphocytic- Plasmablastic (EBV+)- High-grade B-cell 42 (91) 2 (4) 1 (2) 1 (2) Extranodal Disease- Yes- No 20 (43) 26 (57) Bulky Disease, no. (%)- Yes ≥5, Table 2. Adverse Events by Cycle Cycle 1 (n=40) Cycle 2 (n=29) Cycle 3 (n=15) Cycle 4 (n=12) Cycle 5 (n=11) Cycle 6 (n=7) Any AE* Infection Neutropenic Fever Hospitalization ICU Stay 29 (72%) 14 14 14 4 17 (59%) 7 4 7 0 7 (47%) 1 0 1 0 4 (33%) 1 0 1 0 2 (18%) 1 1 1 0 1 (14%) 0 0 1 0 *Hospitalization, infection, neutropenic fever, non-neutropenic fever, ICU stay, transfusion, mucositis, fatigue requiring treatment delay, and ileus. Disclosures Stephens: Immunomedics: Research Funding; Acerta Pharma BV: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Maddocks:Janssen: Research Funding; Novartis: Research Funding; Pharmacyclics: Consultancy, Research Funding. Jones:AbbVie: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding.

MOR208 is an Fc engineered CD19 monoclonal antibody which has been shown in a Phase I trial in patients with relapsed and refractory CLL to be generally well tolerated and have preliminary efficacy, with an overall response rate (ORR) of 30% by IWCLL 2008 guidelines (Woyach et al, Blood 2014). Compared to non-engineered CD19 monoclonal antibodies, MOR208 has significantly enhanced antibody dependent cellular cytotoxicity (ADCC), which can be further augmented in vitro with the addition of lenalidomide. Given the in vitro synergy of these agents, acceptable individual safety profiles, and efficacy of each as a single agent, we chose to combine MOR208 and lenalidomide in patients with both previously treated and previously untreated CLL. This study is a single institution phase II trial of MOR208 in combination with lenalidomide with an initial safety run-in as part of each cohort. MOR208 was given at a dose of 1 mg/kg on cycle 1 day 1, then 9 mg/kg on days 2, 8, 15, and 22 of cycle 1, and then on day 1 of cycles 2-12. Lenalidomide was started at a dose of 2.5 mg daily on cycle 1 day 8 and given continuously. The dose of lenalidomide could be escalated up to 10 mg daily in patients without toxicity. After 12 cycles, lenalidomide could be continued indefinitely in responding patients. Toxicity was assessed using the National Cancer Institute's Common Criteria for Adverse Events v4.0 for non-hematologic toxicity, and IWCLL 2008 guidelines for hematologic toxicity. Disease response by IWCLL 2008 guidelines was assessed at cycle 7 day 1 and at the end of cycle 12. This study will enroll 20 patients with treatment-naïve CLL and 20 patients with relapsed/refractory CLL. At this time, 7 patients with relapsed/refractory disease and 5 patients with treatment-naïve disease have been enrolled and evaluated. The most common toxicities observed related to protocol therapy have been infusion related reactions, fatigue, thrombocytopenia, and neutropenia. In patients with relapsed disease, all toxicities except neutropenia have been grade 1 or 2, and 2 patients experienced grade 3 neutropenia. Of the 5 patients with treatment-naïve CLL, two experienced significant infusion reactions on cycle 1 day 1 that prevented further administration of MOR208. After a protocol amendment escalating steroid premedication, no further grade 3 infusion reactions have been observed. While the majority of patients were able to escalate lenalidomide to either 5 or 10 mg, all patients had lenalidomide eventually dose reduced to 2.5 mg daily due to cytopenias, rash, or fatigue. This combination has shown preliminary efficacy. In the cohort of patients with relapsed disease, two experienced progressive disease during cycle 2 and cycle 5 respectively. The remaining 5 patients achieved stable disease (SD, n=3) or a partial response (PR, n=2) at cycle 7 day 1, with one patient converting to PR by cycle 12. Three patients completed 12 cycles of therapy, and the remaining two completed 12 cycles and now remain on lenalidomide alone at cycle 18 and cycle 19 respectively. In the cohort of patients with treatment-naïve disease, three patients completed more than 1 day of therapy. All of these patients achieved a PR at cycle 7 day 1, and are now in cycle 10 (n=1) or cycle 11 (n=2). In conclusion, this Phase II trial in progress demonstrates preliminary safety and activity of the combination of MOR208 and lenalidomide in patients with CLL. This combination also has the potential to positively modulate the immune system, and detailed correlative studies are evaluating T cell and NK cell function in these patients. Trial accrual is ongoing and updated results will be presented at the meeting. Disclosures Jones: AbbVie: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Byrd:Acerta Pharma BV: Research Funding.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression, and dysregulated expression of lncRNAs has been reported in many cancers. One of the most documented ways by which lncRNAs can contribute to cancer aggressiveness is by altering gene expression through associations with chromatin modifying proteins. However, the vast majority of lncRNAs dysregulated in cancer remain to be functionally characterized. Specifically, there have been few studies investigating the role of lncRNAs in chronic lymphocytic leukemia (CLL). Numerous studies have been published documenting the role of micro RNAs (miRs) in CLL, indicating that non-coding RNAs can play a significant role in this disease. Therefore we hypothesize that dysregulated lncRNA expression in CLL contributes to aggressive disease. We performed microarray analysis using Arraystar Human LncRNA Array v2.0, a platform that analyzes over 30,000 lncRNA transcripts in addition to 30,000 coding transcripts. We found that many lncRNAs are aberrantly expressed in CLL compared to healthy donor B cells. We identified the lncRNA treRNA (TRERNA1) as overexpressed in CLL cells (p = .0014). treRNA has been previously described to have enhancer-like function (Ørom et al., 2010) as well as translational regulatory functions (Gummireddy et al., 2013). It has been reported as overexpressed in breast cancer lymph-node metastases and colon cancer (Gummireddy et al., 2013). In addition to expressing spliced treRNA, CLL cells contain a transcript that retains the intron between the two coding exons due to insufficient splicing. Therefore we investigated the prognostic significance of both spliced and retained intron treRNA (ri-treRNA) in subsequent studies. We obtained 144 well-characterized CLL patient samples from the CLL Research Consortium (CRC) and measured transcript expression by quantitative reverse transcription PCR (qRT-PCR). We separated patients into high or low expressers of treRNA relative to the overall median expression and found that patients with higher expression of treRNA have significantly shorter time to treatment (p = .04). High expression of treRNA also correlates with the poor prognostic indicators unmutated IGHV (p < .0001) and low ZAP70 methylation (p Disclosures Flinn: Celgene Corporation: Research Funding.

Introduction: Lenalidomide, an immunomodulatory agent, and ibrutinib, a selective and covalent inhibitor of Bruton's tyrosine kinase, are orally bioavailable agents with single-agent activity in several histologic subtypes of relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). We are conducting a phase I study of the combination of ibrutinib and lenalidomide to determine the maximum tolerated dose, dose limiting toxicities (DLT), and preliminary efficacy in pts with relapsed/refractory NHL and updated results are presented. Methods: Patients (pts) with relapsed/refractory B-cell NHL including diffuse large B-cell (DLBCL), transformed, mantle cell (MCL), marginal zone (MZL), lymphoplasmacytic (LPL), and follicular (FL) lymphoma who have received at least one prior therapy were eligible. Prior autologous but not allogeneic stem cell transplant and prior lenalidomide were permitted. Prior ibrutinib, CNS involvement, and pts requiring anticoagulation were not permitted. ANC > 1000/mm3, platelets > 50,000/mm3, and creatinine < 2.0 mg/dL, ALT/AST ≤ 2.5 x ULN, bilirubin ≤ 1.5 x ULN, and ECOG PS 0-2 were required at study entry. Treatment consisted of escalating doses of lenalidomide days 1-21 and ibrutinib days 1-28 of a 28 day cycle. A standard 3+3 dose escalation schema was followed. DLTs included: treatment delays > 14 days for toxicity; grade 5 toxicity; tumor lysis syndrome requiring dialysis; tumor flare reaction nonresponsive to corticosteroids; ANC < 500/mm3 or platelets 7 days; grade 3 or 4 febrile neutropenia or infection; and any grade 3 or 4 non-hematologic toxicity with the following exceptions: DVT, diarrhea, nausea or vomiting amenable to medical therapy, correctable electrolyte abnormalities; grade 3 fatigue, or grade 3 maculopapular rash that resolved within 7 days. Pts without significant toxicity could continue treatment until disease progression. Response was assessed every 3 months for 12 months and then every 6 months until disease progression. Results: Twenty-five pts have been treated. Median age is 67 years (range 45-85) with 16 males. Histologies include DLBCL/transformed lymphoma (n=9), MCL (n=7), FL (n=4), MZL (n=2), and LPL (n=3). Four pts were treated at dose level (DL) 1 (lenalidomide 15 mg/ibrutinib 420 mg). One pt was replaced for rapid disease progression and 1 pt experienced DLT consisting of a grade 2 ischemic stroke. As a result of this DLT, DL 1 was expanded to 6 evaluable pts. A second DLT was observed, a grade 3 rash that resolved within 7 days but recurred on day 22. A total of 6 pts were then treated at DL-1 (lenalidomide 10 mg/ibrutinib 280 mg), and no DLTs were encountered. The protocol was amended to include additional dose levels. Pts enrolled on dose level -1A with lenalidomide 10 mg and ibrutinib 420 mg. One DLT occurred at this dose level, a grade 3 rash that failed to resolve within 7 days. The dose level was expanded to 6 pts without further DLT. DL-1B includes an intra-pt dose escalation of the lenalidomide from 10 mg in cycle 1 to 15 mg in cycle 2 with ibrutinib 420 mg. Six pts have been enrolled on this dose level. Three pts have been replaced including 2 with cytopenias not meeting DLT criteria but precluding dose escalation and one with progressive disease. Three pts at DL-1B remain on treatment. Related grade 3-4 toxicities occurred in 16/24 currently evaluated pts (67%), including primarily hematologic toxicity, rash, increased LFTs, pneumonia, hypokalemia, and syncope. Pts have received a median of 3 cycles of therapy to date (range 1-19) and 9 remain on therapy. At DL 1, a pt with DLBCL achieved a complete response (CR) and a pt with transformed follicular achieved a partial response (PR). At DL-1, a pt with DLBCL achieved a CR and 1 pt each with MCL and FL achieved PR. At DL -1A, 1 pt each with MCL and MZL achieved a PR. Overall response rate for 18 assessable pts is 39%. Five pts had best response of stable disease. Sixteen pts have discontinued the study including 3 pts with DLTs, 2 for alternative treatment, 2 for toxicity, and 9 pts with progression. Conclusions: Combined therapy with lenalidomide and ibrutinib in pts with relapsed NHL is well-tolerated, although DLTs of recurrent rash and stroke were encountered. Lenalidomide 10 mg and ibrutinib 420 mg was tolerated and pts are currently enrolling in an intra-pt dose escalation cohort. Preliminary efficacy has been observed in pts with relapsed/refractory DLBCL, MCL, FL, MZL, and transformed NHL. Disclosures Christian: Pharmacyclics: Research Funding; Acerta: Research Funding; Immunomedics: Research Funding; Celgene: Consultancy; Novartis: Other: IDSM; Seattle Genetics: Consultancy, Research Funding; Janssen: Research Funding. Off Label Use: The use of ibrutnib and lenalidomide in combination in relapsed/refractory non-Hodgkin's lymphoma is off-label. Kuruvilla:Karyopharm: Honoraria, Research Funding; Roche Canada: Honoraria; Seattle Genetics: Honoraria, Research Funding. Smith:Celgene: Consultancy; Pharmacyclics: Consultancy. Porcu:Cell Medica: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; Shape: Research Funding. Maddocks:Acerta: Research Funding; Pharamcyclics: Research Funding; Novartis: Research Funding. Byrd:Pharmacyclics: Research Funding. Blum:Celgene: Research Funding; cephalon: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding.

Introduction: The appropriate dose of antithymocyte globulin (ATG) to be used in reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT) is yet to be determined. In our previous retrospective analysis we compared two dosing schedules of ATG (7.5 mg/kg vs 6 mg/kg) in 136 patients who had undergone RIC allo-HSCT, and found no significant differences in the incidence of acute or chronic graft-versus-host disease (GVHD), non-relapse mortality (NRM), relapse, progression-free survival (PFS) or overall survival (OS). As of October 2013, our ATG dosing was lowered from 6.5 to 4.5 mg/kg. We now compare the outcomes of patients who received ATG at a dose of 4.5 mg/kg (r-ATG) to 6 mg/kg (R-ATG) Methods: We retrospectively analyzed the outcomes of patients who underwent unrelated or mismatch related RIC allo HSCT for hematologic malignancies and received r-ATG (40 patients) vs R-ATG (216 patients) at The Ohio State University Comprehensive Cancer Center between October 2007 and September 2014. Cumulative incidences of GVHD, infection, NRM, and relapse were analyzed using Gray's test, accounting for competing risks. PFS and OS were analyzed using the log-rank test. Results: Patients with hematologic malignancies included AML/MDS (53%), NHL/Hodgkin's (19%), CLL (12%), and other hematologic malignancies including ALL (16%). There was a significant association between disease group and ATG dose (p Conclusions While there was an increase in grade II-IV acute GVHD with r-ATG, there was no significant difference in acute GVHD grade III-IV, relapse, NRM, PFS and OS between the two cohorts. r-ATG resulted in a lower proportion of CMV reactivation, but in a higher proportion of other competing risks. Given the increasing use of RIC allo-HSCT for treating malignant hematologic conditions, the correct dose and schedule of ATG administration should be defined by prospective randomized controlled trials. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Knowledge of the cell-of-origin is essential for the full understanding of the causes of a malignant disease and for the rational design of targeted therapies. The B cell compartment is composed of a highly complex mixture of subtypes, each with distinct phenotypes and roles within the immune system. In chronic lymphocytic leukemia (CLL), heterogeneity in the biology and clinical course of the disease is thought to be linked to divergent cellular origins. We and others have previously shown that the epigenome of CLL, as measured by the global pattering of DNA methylation, is highly clonal and remarkably stable over time and thus represents a powerful approach to trace founder subtype populations. Here we combine epigenomic and transcriptomic analysis using next-generation sequencing approaches to compare CLL cells to highly purified and specific B cell subpopulations at various stages of maturation. We find that B cell maturation involves substantial unidirectional epigenetic programming that occurs as a continuum throughout the transition between naïve to fully-mature memory B cell subpopulations. Combining 258 CLL cases using Illumina 450K analysis reveals that all CLLs arise from a discrete window within the spectrum of B cell maturation that is more similar to mature B cells, with the majority of cases clustering at two distinct points correlating with unmutated IGHV versus highly mutated ( Citation Format: Christopher C. Oakes, Marc Seifert, Assenov Yassen, Lei Gu, Martina Przekopowitz, Amy Ruppert, Andrius Serva, Sandra Koser, David Brocks, Daniel Lipka, Olga Bogatyrova, Daniel Mertens, Marc Zapatka, Peter Lichter, Hartmut Doehner, Ralf Kueppers, Thorsten Zenz, Stephan Stilgenbauer, John Byrd, Christoph Plass. Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B25.

Background Pretreatment cytogenetics are not routinely used to predict patient outcomes in mantle cell lymphoma (MCL). Based on the prognostic utility of cytogenetics in other diseases, we reviewed the effect of a complex karyotype (CK) in MCL. Patients and Methods We included patients evaluated between November, 2002, and May, 2011. Those with ≥ 3 chromosomal abnormalities on a pre-treatment cytogenetic evaluation were defined as CK. Demographic, clinical, and survival differences between patients with CK and non-CK (NCK) were assessed. Results Of 80 patients, 32 (40%) had CK, which was associated with high-risk clinical risk factors. Therapy did not differ between the groups, nor did rate of autologous stem cell transplant (ASCT). The 2-year progression-free survival (PFS) estimates were 70% and 48% for patients with NCK and CK, respectively ( P = .02). Two-year overall survival (OS) estimates were also greater in those with NCK versus CK (85% vs. 58%; P = .02). When controlling for high-risk Mantle Cell Lymphoma International Prognostic Index (MIPI) score ( P = .006), bulky disease ( P = .01), and ASCT in first remission ( P = .01), CK was not significantly associated with PFS ( P = .18). Conclusion CK is associated with shortened PFS and OS in MCL but has not been demonstrated to be prognostic independent of other variables in this series.

Importance The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuing therapy with this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up. Objective To determine features associated with discontinuation of ibrutinib therapy and outcomes. Design, Setting, and Participants A total of 308 patients participating in 4 sequential trials of ibrutinib at The Ohio State University Comprehensive Cancer Center were included. These clinical trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014. Main Outcomes and Measures Patients were evaluated for time to therapy discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued therapy because of disease progression, targeted deep sequencing was performed in samples at baseline and time of relapse. Results With a median follow-up of 20 months, 232 patients remained on therapy, 31 had discontinued because of disease progression, and 45 had discontinued for other reasons. Disease progression includes Richter’s transformation (RT) or progressive CLL. Richter’s transformation appeared to occur early and CLL progressions later (cumulative incidence at 12 months, 4.5% [95% CI, 2.0%-7.0%] and 0.3% [95% CI, 0%-1.0%], respectively). Median survival following RT was 3.5 months (95% CI, 0.3-6.0 months) and 17.6 months (95% CI, 4.7 months–“not reached”) following CLL progression. Sequencing on peripheral blood from 8 patients with RT revealed 2 with mutations in BTK , and a lymph node sample showed no mutations in BTK or PLCG2 . Deep sequencing on 11 patients with CLL progression revealed BTK or PLCG2 mutations in all. These mutations were not identified before treatment in any patient. Conclusions and Relevance This single-institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib therapy discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with RT. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCG2 with progression and clearly show that CLL progressions are associated with these mutations, while RT is likely not. Trial Registrations clinicaltrials.gov Identifiers:NCT01105247,NCT01217749,NCT01589302, andNCT01578707

Purpose To analyze the prognostic impact of mutated KIT (mutKIT) in core-binding factor acute myeloid leukemia (AML) with inv(16)(p13q22) and t(8;21)(q22;q22). Patients and Methods Sixty-one adults with inv(16) and 49 adults with t(8;21), assigned to postremission therapy with repetitive cycles of higher dose cytarabine were analyzed for mutKIT in exon 17 (mutKIT17) and 8 (mutKIT8) by denaturing high-performance liquid chromatography and direct sequencing at diagnosis. The median follow-up was 5.3 years. Results Among patients with inv(16), 29.5% had mutKIT (16% with mutKIT17 and 13% with sole mutKIT8). Among patients with t(8;21), 22% had mutKIT (18% with mutKIT17 and 4% with sole mutKIT8). Complete remission rates of patients with mutKIT and wild-type KIT (wtKIT) were similar in both cytogenetic groups. In inv(16), the cumulative incidence of relapse (CIR) was higher for patients with mutKIT (P = .05; 5-year CIR, 56% v 29%) and those with mutKIT17 (P = .002; 5-year CIR, 80% v 29%) compared with wtKIT patients. Once data were adjusted for sex, mutKIT predicted worse overall survival (OS). In t(8;21), mutKIT predicted higher CIR (P = .017; 5-year CIR, 70% v 36%), but did not influence OS. Conclusion We report for the first time that mutKIT, and particularly mutKIT17, confer higher relapse risk, and both mutKIT17 and mutKIT8 appear to adversely affect OS in AML with inv(16). We also confirm the adverse impact of mutKIT on relapse risk in t(8;21) AML. We suggest that patients with core-binding factor AML should be screened for mutKIT at diagnosis for both prognostic and therapeutic purposes, given that activated KIT potentially can be targeted with novel tyrosine kinase inhibitors.

We investigated the relative prognostic significance of cytogenetics in 635 adult acute myeloid leukemia (AML) patients 60 years of age or older treated on front-line protocols. Classification trees and tree-structured survival analysis (TSSA) were used to identify important cytogenetic groups, and their prognostic significance was then assessed in multivariable analysis (MVA). Overall, 48.5% achieved complete remission (CR); 6.6% survived at 5 years. Complex karyotypes with at least 3 abnormalities (complex ≥ 3) and a group including “rare aberrations” predicted lower CR rates (25% and 30%) versus other patients (56%). Compared with complex ≥ 3, the odds of CR were significantly higher for noncomplex karyotypes without rare aberrations on MVA. Cytogenetically, complex ≥ 5 predicted inferior disease-free survival on TSSA, remaining significant on MVA together with white blood cell count (WBC), sex, and age. For survival, complex ≥ 5, rare aberrations, and core-binding factor (CBF) abnormalities were prognostic (P < .001), with 5-year survivals of 0%, 0%, and 19.4%, respectively, and 7.5% for remaining patients. Together with WBC, marrow blasts, sex, and age, the cytogenetic groups remained significant on MVA. In conclusion, pretreatment cytogenetics adds to other prognostic factors in older AML patients. Patients with complex ≥ 5 appear to benefit minimally from current treatment and are better suited for investigational therapy or supportive care. (Blood. 2006;108:63-73)

Purpose To test the prognostic significance of ETS-related gene (ERG) expression in cytogenetically normal primary acute myeloid leukemia (AML). Patients and Methods Pretreatment blood samples from 84 cytogenetically normal AML patients aged less than 60 years, who were characterized for BAALC expression, FLT3 internal tandem duplication (ITD), and MLL partial tandem duplication (PTD) and uniformly treated on Cancer and Leukemia Group B 9621 protocol, were analyzed for ERG expression by real-time reverse transcriptase polymerase chain reaction. Patients were divided into quartiles according to ERG levels and were compared for clinical outcome. High-density oligonucleotide arrays were used to identify genes differentially expressed between high and low ERG expressers. Results With a median follow-up of 5.7 years, patients with the upper 25% of ERG expression values had a worse cumulative incidence of relapse (CIR; P < .001) and overall survival (OS; P = .011) than the remaining patients. In a multivariable analysis, high ERG expression (P < .001) and the presence of MLL PTD (P = .027) predicted worse CIR. With regard to OS, an interaction was observed between expression of ERG and BAALC (P = .013), with ERG overexpression predicting shorter survival only in low BAALC expressers (P = .002). ERG overexpression was an independent prognostic factor even when the unfavorable group of FLT3 ITD patients lacking an FLT3 wild-type allele was included. High ERG expression was associated with upregulation of 112 expressed-sequenced tags and named genes, many of which are involved in cell proliferation, differentiation, and apoptosis. Conclusion ERG overexpression in AML patients with normal cytogenetics predicts an adverse clinical outcome and seems to be associated with a specific molecular signature.

Purpose Evaluate the outcome of induction and postremission therapy in adults younger than 60 years with normal cytogenetics acute myeloid leukemia (AML). Patients and Methods In 490 patients, induction included cytarabine and daunorubicin (AD) or cytarabine and escalated doses of daunorubicin and etoposide ± PSC-833 (ADE/ADEP). Intensification included one cycle of high-dose cytarabine (HDAC) followed by etoposide/cyclophosphamide and mitoxantrone/diaziquone (group I), three HDAC cycles (group II), four intermediate-dose cytarabine (IDAC) or HDAC cycles (group III), or one HDAC/etoposide cycle and autologous stem-cell transplantation (ASCT; group IV). Results Of 350 patients receiving AD, 73% achieved complete remission (CR), compared with 82% of 140 receiving ADE/ADEP (P = .04). Splenomegaly was associated with a lower CR rate (P < .001), and ADE/ADEP, with a higher CR rate in younger patients (P = .005). The 5-year disease-free survival (DFS) rate was 28% each for intensification groups I and II, compared with 41% and 45% for groups III and IV, respectively (P = .02). The 5-year cumulative incidence of relapse (CIR) was 62% and 67% for groups I and II, respectively, compared with 54% and 44% for groups III and IV, respectively (P = .049). The type of postremission intensification remained significant for DFS and CIR in multivariable analysis. Conclusion In younger adults with normal cytogenetics AML, splenomegaly predicts a lower CR rate, and the postremission strategies of either four cycles of I/HDAC or one cycle of HDAC/etoposide followed by ASCT are associated with improved DFS and reduced relapse compared with therapies that include fewer cycles of cytarabine or no transplantation.

Secondary cytogenetic abnormalities at diagnosis of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) have been associated with an inferior outcome in reported series of largely chemotherapy-treated patients. To date, no study has specifically focused on the prognostic significance of secondary karyotypic abnormalities, detected at the time of diagnosis, in interferon (IFN)-alpha treated patients. We compared the outcome of 29 newly diagnosed Ph+ CML patients with additional abnormalities to that of 234 sole Ph+ patients, treated on CALGB protocols with IFN-alpha alone or together with IFN-gamma or low-dose cytarabine. Complete and partial cytogenetic responses were achieved in 20 and 19% of sole Ph+ patients, compared to 23 and 18%, respectively, of patients with additional abnormalities (P=1.00). None of 4 patients with 'high-risk' secondary abnormalities [+8, +Ph and i(17)(q10)], for whom follow-up cytogenetic samples were available, achieved a cytogenetic response. With a median follow-up of 11.3 years, the median overall survival (OS) was 6.0 years for sole Ph+ patients compared to 7.5 years for patients with additional abnormalities (P=0.70), with corresponding 8-year OS of 36 and 38%, respectively. On multivariable analysis, only age (P

Secondary cytogenetic abnormalities at diagnosis of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) have been associated with an inferior outcome in reported series of largely chemotherapy-treated patients. To date, no study has specifically focused on the prognostic significance of secondary karyotypic abnormalities, detected at the time of diagnosis, in interferon (IFN)-alpha treated patients. We compared the outcome of 29 newly diagnosed Ph+ CML patients with additional abnormalities to that of 234 sole Ph+ patients, treated on CALGB protocols with IFN-alpha alone or together with IFN-gamma or low-dose cytarabine. Complete and partial cytogenetic responses were achieved in 20 and 19% of sole Ph+ patients, compared to 23 and 18%, respectively, of patients with additional abnormalities (P=1.00). None of 4 patients with 'high-risk' secondary abnormalities [+8, +Ph and i(17)(q10)], for whom follow-up cytogenetic samples were available, achieved a cytogenetic response. With a median follow-up of 11.3 years, the median overall survival (OS) was 6.0 years for sole Ph+ patients compared to 7.5 years for patients with additional abnormalities (P=0.70), with corresponding 8-year OS of 36 and 38%, respectively. On multivariable analysis, only age (P0.001) and white blood cell count (P=0.02) were associated with outcome. We conclude that, with the possible exception of +8, +Ph and i(17)(q10), additional chromosomal abnormalities at diagnosis are not associated with inferior outcome in Ph+ CML patients treated with IFN-alpha-based therapy.

Cytogenetic aberrations are important prognostic factors in acute myeloid leukemia (AML). Of adults with de novo AML, 45% lack cytogenetic abnormalities, and identification of predictive molecular markers might improve therapy. We studied the prognostic impact of BAALC (Brain And Acute Leukemia, Cytoplasmic), a novel gene involved in leukemia, in 86 de novo AML patients with normal cytogenetics who were uniformly treated on Cancer and Leukemia Group B 9621. BAALC expression was determined by comparative real-time reverse transcriptase–polymerase chain reaction in pretreatment blood samples, and patients were dichotomized at BAALC's median expression into low and high expressers. Low expressers had higher white counts (P = .03) and more frequent French-American-British M5 morphology (P = .007). Compared to low expressers, high BAALC expressers showed significantly inferior overall survival (OS; median, 1.7 vs 5.8 years, P = .02), event-free survival (EFS; median, 0.8 vs 4.9 years, P = .03), and disease-free survival (DFS; median, 1.4 vs 7.3 years, P = .03). Multivariable analysis confirmed high BAALC expression as an independent risk factor. For high BAALC expressers the hazard ratio of an event for OS, EFS, and DFS was respectively 2.7, 2.6, and 2.2. We conclude that high BAALC expression predicts an adverse prognosis and may define an important risk factor in AML with normal cytogenetics.

Overexpression of the oncomiR, miR-155, is known to be predictive of poor outcome in chronic lymphocytic leukemia (CLL) patients. Using NanoString Technologies’ nCounter platform, we interrogated the miR-155 expression levels in 109 previously untreated CLL patients receiving chemoimmunotherapy on CALGB 9712 or CALGB 10101. The data, dichotomized around median expression, showed that high expressers of miR-155 had shorter progression-free survival (p=0.005) and a higher risk of death after 4 years on study (p=0.004). The expression of miR-155 was not significantly associated with the majority of baseline demographic, clinical and cytogenetic characteristics, including age, Rai stage and high-risk cytogenetics [del(17p)/del(11q)] (p>0.15). Association of high miR-155 expression with IGHV un-mutated disease(p=0.03) and ZAP70 methylation Previously, associations have been made between IGHV unmutated status/high ZAP70 levels and a B cell receptor (BCR) activated phenotype. Therefore, to investigate the close association between BCR activation and miR-155 in CLL, we explored regulation of BCR pathways through ibrutinib-mediated inhibition of Bruton’s tyrosine kinase (BTK) and its ability to modulate miR-155. Ibrutinib is an irreversible inhibitor of BTK, an integral kinase in the BCR pathway, and ibrutinib treatment has been shown to decrease pro-survival signaling via the AKT, ERK and NFκB pathways. Serial samples from ibrutinib-treated (420 mg/day) patients were obtained pre-treatment and on days 8 and 29 of therapy on the Phase Ib/II trial OSU-10053 (n=12). miR-155 expression, interrogated using real time PCR (RT-PCR), was significantly lower pre-treatment versus day 8 (p Disclosures Jaglowski: Pharmacyclics: Research Funding. Lin:GlaxoSmithKline: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding; Genentech: Research Funding.

Introduction: Lenalidomide, an immunomodulatory agent, and ibrutinib, a selective and covalent inhibitor of Bruton’s tyrosine kinase, are orally bioavailable agents with promising single agent activity in relapsed/refractory B-cell NHL. We are conducting a phase I study of the combination of ibrutinib and lenalidomide to determine the maximum tolerated dose, dose limiting toxicities (DLT), and preliminary efficacy in patients with relapsed/refractory NHL. Methods: Patients with relapsed/refractory B-cell NHL including diffuse large B-cell (DLBCL), transformed, mantle cell, marginal zone, lymphoplasmacytic, and follicular lymphoma who have received at least one prior therapy were eligible. Prior autologous but not allogeneic stem cell transplant and prior lenalidomide were permitted. Prior ibrutinib, CNS involvement, and patients requiring anticoagulation were not permitted. ANC > 1000/mm3, platelets > 50,000/mm3, and creatinine < 2.0 mg/dL, ALT/AST ≤ 2.5 x ULN, bilirubin ≤ 1.5 x ULN, and ECOG PS 0-2 were required at study entry. Treatment consisted of escalating doses of lenalidomide days 1-21 and ibrutinib days 1-28 of a 28 day cycle. A standard 3+3 dose escalation schema was followed. DLTs were assessed during cycle 1 and included: treatment delays > 14 days for toxicity; grade 5 toxicity; tumor lysis syndrome requiring dialysis; tumor flare reaction nonresponsive to corticosteroids; ANC < 500 / mm3 or platelets 7 days; grade 3 or 4 febrile neutropenia or infection; and any grade 3 or 4 non-hematologic toxicity with the exception of DVT, diarrhea, nausea, or vomiting amenable to medical therapy, correctable electrolyte abnormalities; grade 3 fatigue, or grade 3 maculopapular rash that resolved within 7 days. Patients without significant toxicity could continue treatment until disease progression. Response was assessed every 3 months for 12 months and then every 6 months until disease progression. Results: From September 2013 until July 2014, 13 patients have been treated. Median age is 68 years (range 45-85) with 9 males. Histologies include DLBCL (4), transformed (4), follicular (2), mantle cell (2), and lymphoplasmacyctic (1) lymphoma. Median number of prior therapies is 3 (range 2-9) with 4 patients having undergone prior autologous transplant. Four patients were treated at dose level (DL) 1 (lenalidomide 15 mg and ibrutinib 420 mg). One patient was replaced for rapid disease progression and 1 patient experienced DLT consisting of a grade 2 ischemic stroke while on aspirin with no history of cardiovascular disease that resulted in discontinuation of study therapy. As a result of this DLT, dose level 1 was expanded to 6 patients. A second DLT was observed, a grade 3 rash that resolved within 7 days with interruption of therapy but recurred with diffuse erythroderma within 4 hours of ibrutinib resumption on day 22 at 280 mg. A total of 6 patients were then treated at DL -1 (lenalidomide 10 mg and ibrutinib 280 mg), and no DLTs were encountered. Related grade 3-4 toxicities occurred in 4/13 patients (31%), including one patient with hypokalemia, neutropenia, thrombocytopenia, and pneumonia, two others with neutropenia and one with maculapapular rash. Patients have received a median of 3 cycles of therapy to date (range 1-5) and 5 remain on therapy. At DL 1, a patient with DLBCL achieved a complete response (CR) and patient with transformed follicular achieved a partial response (PR). At DL -1, a patient with DLBCL achieved a CR and a patient with mantle cell lymphoma achieved a PR. Four patients achieved stable disease. Reasons for study discontinuation include two patients with DLTs who were not evaluated for response, one patient with stable disease who went off study for alternative treatment, and five patients who have progressed. Conclusions: Combined therapy with lenalidomide and ibrutinib in patients with relapsed NHL has been well tolerated at doses of 10 mg and 280 mg respectively, although DLTs of recurrent rash and stroke were encountered with 15 mg lenalidomide and 420 mg ibrutinib. Dose escalation to an intermediate dose level with lenalidomide 10 mg and ibrutinib 420 mg is planned. If this dose level is deemed safe, intra-patient dose escalation of the lenalidomide from 10 mg in cycle 1 to 15 mg in cycle 2 with ibrutinib 420 mg continuously will be tested. Preliminary efficacy has been observed in patients with relapsed/refractory DLCL, MCL, and transformed NHL. Disclosures Christian: Janssen Research & Development, LLC: Research Funding; Celgene: Consultancy. Off Label Use: Use of lenalidomide and ibrutinib in B-cell non-Hodgkin's lymphoma. Smith:Celgene: Consultancy, Research Funding; Pharmacyclics/Janssen: Consultancy, Speakers Bureau. Porcu:Celgene: Honoraria. Byrd:Phamacyclics: Research Funding. Blum:Janssen, Pharmacyclics: Research Funding.

7010 Background: The Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib (I) is very effective in chronic lymphocytic leukemia (CLL), with progression free survival of 76% at 26 months (mo) for patients (pts) with relapsed disease (Byrd J et al, NEJM 2013). We evaluated pts treated with I to explore features associated with progressive disease (PD) and subsequent outcomes. Methods: 267 pts from The Ohio State University Comprehensive Cancer Center participating in 3 previously reported Institutional Review Board approved trials of I were included; 196 pts received single agent I and 71 received I plus ofatumumab. A subset of pts with PD had Ion Torrent deep sequencing (DS) performed on peripheral blood at baseline and relapse. Results: With a median follow-up of 16 mo (

Complexity has been associated with an adverse outcome in CLL (Kujawski et al. Blood 2008, 112:1993), and cytogenetic complexity often evolves as the patient progresses (Blood 2013, 121:1403). We sought to determine whether the degree of complexity as determined by cytogenetic analyses correlated with time-to-treatment (TTT) or with overall survival (OS) and its relationship with del(17p). Patients (pts) with CLL who had cytogenetic analyses completed between December 2006 and May 2011 were identified from The Ohio State University Wexner Medical Center (OSU) databases. The first cytogenetic analysis evaluated within one year of diagnosis and prior to treatment was considered the diagnostic sample. All but one cytogenetic analyses were performed using CpG +/- pokeweed mitogen and phorbol 12-myristate 13-acetate stimulation. Cytogenetic methods and analyses were by standard laboratory protocols. Fluorescence in situ hybridization (FISH) was performed on all cases. For these analyses, probes for ATM (11q22.3) and TP53 (17p13.1) (Abbott Molecular, Des Plaines, IL) were used according to the manufacturer's recommendations. Complex karyotype (CK) was defined as ≥3 independent aberrations. Associations between CK and clinical or molecular characteristics were tested using Fisher's exact or Wilcoxon rank sum tests. TTT was calculated from the date of diagnosis until the date of treatment, censoring pts who had not started treatment at the date last seen. OS was calculated from the date of diagnosis until the date of death or last follow-up. Estimates of TTT and OS were calculated using the Kaplan-Meier method, and differences in survival distributions were compared using the log-rank test. Diagnostic samples meeting the above criteria were identified in 143 pts. Median age was 58 (range 33-85), 38% were female, and 44% had Rai stage 0. Twenty pts (14%) had del(11q) and 16 (11%) had del(17p), 10 of which were identified by both FISH and karyotype, 4 by FISH alone, and 2 by karyotype alone. CK was detected in 22 pts (15%, 95% CI: 0.10-0.22). CK tended to be more prevalent in males (20% vs 7% in females, p=0.05) and in those with Rai stage ≥ I (20% vs 8% with Rai stage 0, p=0.06), but there was no significant association with age (p=0.98). Only 5 CK pts (23%) had del(11q) compared to 12% without CK (NCK; p=0.20). CK was strongly associated with del(17p), where 41% of CK pts harbored this typically high-risk aberration versus only 6% in NCK pts (p TTT was significantly shorter in pts with CK compared to NCK (p=0.0005), with 12 mos estimates of 55% (95%CI=0.31-0.74) and 83% (95%CI=0.75-0.89), respectively (Figure 1A). In the subsets of pts with and without del(17p), the negative impact of CK persisted as shown in Figures 2A-B. In a proportional hazards model for TTT, CK remained significantly associated with shorter TTT independent of del(17p) and Rai stage (hazard ratio=2.20, 95%CI=1.03-4.71; p=0.04). With a median follow-up of 32 months for OS, there have been only 14 events. Still, early deaths tended to occur more often in those with CK than NCK; five deaths occurred in the first 12 mos following diagnosis, 3 in the CK group (13.6%) and 2 in the NCK group (1.7%), and hence there was a significant difference in OS (p=0.015; Figure 1B). The limited information prevented meaningful comparisons between CK and NCK by del(17p) subgroups. In summary, CK is indicative of both shorter TTT and OS. Although there is a strong correlation of del(17p) with CK, presence of CK appears to predict TTT independent of del(17p). Complexity should be evaluated in a larger, prospective series to further clarify its role in clinical outcome. Disclosures: No relevant conflicts of interest to declare.

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient's CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient's normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.

Abstract 3865 ZAP-70 expression is a prognostic marker differentiating chronic lymphocytic leukemia (CLL) patients with indolent disease from those rapidly progressing after diagnosis and requiring treatment. We previously demonstrated in four independent clinical cohorts that DNA methylation at a single CpG dinucleotide in the ZAP-70 promoter region impacts on ZAP-70 transcriptional regulation and is prognostic in CLL (Claus et al. J Clin Oncol. 2012;30(20):2483–91). Here, we confirm and extend our previous findings by investigating the significance of ZAP-70 methylation relative to established prognostic biomarkers in CLL: IGHV mutation status or expression of ZAP-70 protein or CD38. DNA methylation analysis of the ZAP-70 core promoter region was conducted in mononuclear cells of 295 untreated CLL patients, previously reported by the CRC (Rassenti et al. Blood 2008;112:1923–30) using MALDI-TOF mass spectrometry (MassARRAY, Sequenom). The 295 patients had a median age of 55 years (range 26–82), 69% were male, and 46% were Rai stage 0, 47% stage I/II, and 7% stage III/IV at diagnosis. ZAP-70 protein and CD38 expression were measured by four-color flow cytometry and IGHV mutational status was assessed, as previously described. By hierarchical clustering, the previously identified single CpG dinucleotide (“CpG unit 1”, position +223 relative to the transcription start) separated patients with high methylation levels across the entire region from those with lower methylation levels at this specific site. With a median follow-up of 3.9 years in 50 patients who had not started treatment and 5.6 years among those still alive (n=196), patients with lower methylation levels at CpG unit 1 had shortened time to first treatment (TTT) and overall survival (OS) (both p Hence, the prognostic value of ZAP-70 CpG unit 1 methylation was confirmed in a large patient cohort with extensive follow-up. We defined a clinically applicable threshold for ZAP-70 methylation assessment and determined its relative value to current prognostic markers in CLL. ZAP-70 methylation predicted longer TTT among patients with CLL cells lacking ZAP-70 expression, but not among those with ZAP-70+ CLL cells, although this could reflect sampling bias due to the small number of patients (n=16) in our cohort with both high methylation and expression of ZAP-70. Lastly, methylation was the strongest risk factor for OS, and neither IGHV mutation status nor expression of ZAP-70 or CD38 significantly improved the ability to predict prognosis. In conclusion, ZAP-70 DNA methylation represents a clinically valuable biomarker for predicting TTT and OS in previously untreated patients with CLL. Disclosures: Gribben: Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Pharmacyclics: Honoraria; Roche: Honoraria; Celgene: Honoraria. Kay:Celgene: Research Funding.

Abstract 183 CLL is a common leukemia which demonstrates variable reactivity of the B cell receptor (BCR) to antigen ligation, but constitutive activation of this pathway. Bruton's Tyrosine Kinase (BTK) is a member of this pathway which shows transcriptional upregulation and constitutive activity in CLL. Ongoing trials of ibrutinib, an orally bioavailable inhibitor of BTK, have shown outstanding preliminary activity in CLL. However results with other known reversible and more selective irreversible BTK inhibitors have had variable results. As ibrutinib potentially has other targets in addition to BTK, this raises the question of whether BTK is an important target in CLL. To answer this question and determine the role of BTK in the development and expansion of CLL, we have used Eμ-TCL1 (TCL1) transgenic mouse model of CLL, where the TCL1 oncogene is under the control of the VH promoter-IgH-Eμ enhancer and adult transgenic mice spontaneously develop CLL. To determine whether BTK is an important target in CLL as opposed to an alternative target of ibrutinib, we crossed the B6/TCL1 mouse with the XID mouse, which harbors a mutation in the PH domain of BTK, rendering it kinase-inactive. In B-cells derived from these mice we observed continued TCL1 expression and diminished BCR signaling with BCR ligation. XID/TCL1 mice (n=65) had a significantly prolonged overall survival (OS) compared to those with wild-type (WT) BTK (n=79) (Median not reached versus (vs) 13.4 months respectively, p In conclusion, our data show that active BTK is critical for the development and expansion of CLL in the TCL1 mouse model of CLL. Continuous global inhibition of this kinase through mutation or pharmacologic inhibition delays the development of CLL, confirming that BTK is an important target for drug development. Disclosures: Chang: Pharmacyclics, Inc.: Employment. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Abstract 2691 Background: Pre-treatment cytogenetics are utilized for risk-stratification in many hematologic malignancies but not in MCL, where improved risk-stratification is needed due to the heterogeneity of outcomes. A review at our institution found a significant association of CK with median time to relapse (1005 days for CK vs. not reached in those without CK (NCK), p=0.008) in pts undergoing autologous transplantation in first remission (Cohen, BMT Tandem Mtgs 2012). To evaluate the impact of CK at diagnosis on PFS and overall survival (OS) regardless of initial therapy or eligibility for transplant, we reviewed all pts with newly diagnosed MCL at the Ohio State University (OSU) from 2000–2011. Methods: We included all pts with confirmed MCL and available bone marrow (BM, n = 68) or peripheral blood (n = 12) pretreatment cytogenetics. Unstimulated cytogenetic analysis, standardly performed at OSU from 2000–2008, was performed in 48 pts, and stimulated analysis utilizing CpG oligonucleotides was performed in 32 pts beginning in Jan 2008. CK was defined as ≥3 unrelated chromosomal abnormalities, including t(11;14). Associations between CK and clinical variables were performed using Fisher's Exact and Wilcoxon rank sum tests. PFS and OS curves were constructed from date of pathologic diagnosis until date of relapse or death by the Kaplan-Meier method and evaluated using the log-rank test. A multivariable proportional hazards model for PFS was constructed using a limited backwards selection procedure. Results: In 80 untreated pts, median age was 63 (range 37–81), 70% were male, 28% had bulky disease ≥5cm, and 95% had stage IV disease. Sixty-five of 77 evaluable pts (84%) had BM involvement. Median time from diagnosis to pretreatment cytogenetic analysis was 21 days (range:−20 to 1094). CK was detected in 32 pts (40%, 95% CI: 0.29 to 0.52), and CK rate was higher in stimulated versus unstimulated analyses (56% vs 29%, p=0.02). CK was associated with several adverse markers, including a higher MCL international prognostic index (MIPI; median 6.3 vs 5.9, p=0.003) and simplified MIPI scores (median 6 vs 4, p=0.0006), increased percent involvement of BM cellularity (68% vs 10%, p=0.005), splenomegaly (73% vs 38%, p=0.005), and B-symptoms (57% vs 26%, p=0.01). CK was not associated with age (p=0.98) or bulky disease ≥5cm (p=0.80). Thirty-six pts (45%) underwent transplant in first remission, 35 autologous and 1 allogeneic. Intensive induction included R-HyperCVAD (n=10), methotrexate with augmented R-CHOP (n=31), and R-CHOP (n=17); less intensive induction included R-Bendamustine (n=4) and other non-anthracycline containing therapies. There were no large differences between CK and NCK patients with respect to receipt of intensive induction (76% vs 75%) or transplant in first remission (44% vs. 46%). With a median follow-up of 24 months for PFS and 32 months for OS, the estimated PFS at 2 years for CK and NCK pts was 47% and 71%, respectively (p=0.04; Figure 1A), and the estimated OS was 57% and 84% (p=0.02). For pts with unstimulated evaluation, the association of CK with shortened PFS and OS remained significant (PFS, p=0.02 and OS, p=0.01), with no association of method of analysis with PFS (p=0.35). In a multivariable analysis, high-risk (≥6.2) vs. low-risk ( Conclusions: Pre-treatment detection of a CK, even without stimulation, is prognostic for shortened PFS and OS in MCL and should be evaluated in larger, prospective series with uniformly treated patient populations. The presence of del(17p), detected in only 8% of pts, appears to predict a particularly poor PFS and should also be prospectively evaluated. Disclosures: No relevant conflicts of interest to declare.

e18508 Background: ORR of 97-99% and 5-yr PFS of 75-79% are reported with front-line DA-REPOCH in pts age 19-85. In these prospective series, 72% of pts escalated ≥ 1 dose level (DL), febrile neutropenia (FN) occurred in 36-51% pts, and 3-4% died of toxicity. Methods: We performed retrospective analysis of pts ≥ 65 treated with DA-REPOCH front-line for DLBCL at Ohio State University from 2002-2011. PFS estimates were calculated by the Kaplan-Meier method. Logistic regression and proportional hazards models were fit to identify variables associated with ORR and PFS, respectively. Results: 69 pts ≥ 65 yrs (range, 65-92) received DA-REPOCH for de novo (n=42) or transformed (prior CLL=16, FL=9, HL=2) DLBCL, including 28 pts ≥ 75. Performance status (PS) was ≥ 2 in 43%, 86% were stage III-IV, 77% had an aaIPI ≥ 2, 19% had bulky disease ≥ 10 cm, 83% had extranodal disease, and median creatinine clearance (CrCl) was 64 ml/min. Median number of cycles was 5 (range 1-7). Max DL was 1, 2, and 3 in 72, 13, and 6% of pts, respectively, and 9% received all cycles below DL 1. Max DL and number of cycles were similar between pts 65-74 and > 75. Dose reductions (DR), delays, FN, and hospitalization occurred in 48, 39, 38, and 42% of pts, respectively. DR were associated with age ≥ 75 (p=0.001) or reduced CrCl (p=0.02) and delays strongly associated with PS ≥ 2 (p=0.001). FN was associated with bulky disease (p=0.03); 31% of pts without bulky disease developed FN compared to 67% with bulk. Nine pts (13%, 3 pts ≥ 75) died of toxicity (7 pts at DL 1 and 2 at DL < 1). ORR was 67% (95% CI 52-75%). Median PFS was 17 mos (95% CI 6-43). Increased LDH (p=0.008 and p

Abstract 5189 Background: Somatic hypermutation (SH) of the immunoglobulin heavy chain gene is a well-described prognostic factor in chronic lymphocytic leukemia (CLL), with patients (pts) exhibiting < 98% homology having an improved prognosis compared to their unmutated counterparts. Prior series have identified loci VH 1–69, 3–23, and 4–34, as the most frequently utilized loci in CLL (Donisi et al, Diagn Mol Pathol, 15: 206–215, 2006). The impact of SH remains unclear in mantle cell lymphoma (MCL). While one retrospective series found an incidence of 60% of SH with an improved overall survival (OS) in pts with SH (Lai et al, Modern Pathology, 19: 1498–1505, 2006), a prospective study reported an incidence of 28% and no association with OS (Camacho et al, Blood, 101: 4042–4046, 2003). The immunoglobulin heavy chain gene loci most commonly utilized in the two series also differed, VH 1–69, 4–59, and 3–74 in the retrospective review, and VH 3–21, 3–23, 4–34, and 4–59 in the prospective study. Given the variable rates of SH and unclear association with prognosis, we evaluated SH in a series of pts with MCL with available tissue treated at the Ohio State University. Methods: Non-bone marrow tissue samples for all pts with MCL were evaluated for the presence of SH. The sequences of the IgVH results were compared to the most homologous germline V sequences in IMGT/Gene-DB. Sequences that differed by more than 2% from their corresponding germline sequences were considered hypermutated whereas sequences that differed by less than 2% were considered unmutated. Summary statistics are presented and survival estimates were calculated by the Kaplan-Meier method. Results: In 11 pts, the median age was 63 years (range 43–80), 82% were male, all pts had stage IV disease, and the median MIPI score was 5.7 (range 5.3–8.1). Five of 10 pts with available data had splenomegaly, 6 pts had BM involvement, and 6 pts had extranodal disease (GI tract n=2, upper airway n=2, lung n=1, and kidney n=1). Median lactate dehydrogenase (LDH) and median white blood cell (WBC) count at diagnosis were 156 U/L (range 102–274) and 6.6 × 109/L (range 5.3–235.0), respectively. 9 patients received 1 therapy while one patient received 2 and one received 3 therapies. Five pts underwent autologous transplant in first remission, and 1 pt underwent allogeneic transplant at first relapse. Median time from initial diagnosis to first treatment was 1.7 months (range 0.5–10.2 months). With a median follow up of 2.1 years (range 0.6–3.7) median progression-free survival (PFS) from diagnosis was 3.2 years (95% CI 1.1–5.0) and median OS was 7.8 years (95% CI 1.5–7.8). There have been 4 deaths due to disease progression (n=3) and pneumonia (n=1). The most frequently utilized IgVH gene loci were VH1-8 (n=3), with VH 4-34 (n=2). Although VH 4-34 is implicated in CLL, both patients had immunophenotyping consistent with MCL, a t(11;14) translocation, and no lymphocytosis. 2 pts demonstrated SH, representing 18% of the sample (95% CI 2–52%). One of these 2 pts with SH was a 63 year old male, with nodal and bone marrow involvement and a MIPI score of 5.5. He was treated with RMCHOP for two cycles followed by autologous transplant according to Damon et al (J Clin Oncol, 27: 6101–6108, 2009), and died 3 years after transplant due to pneumonia without evidence of relapse. The second pt was a 66 year old female treated with 6 cycles of RCHOP who relapsed 4.8 years later. Salvage regimens included R-Bortezomib, R-Bendamustine, FCR, and R-GemOx, and she died due to disease progression 7.8 years after diagnosis. These two pts utilized loci VH 3-9 and VH 4-59 respectively. Conclusions: In our series, the estimated incidence of SH is lower than what has previously been reported, and the utilized IgVH loci are different. While VH 3-9 and VH 4-59 were observed in the 2 pts with SH, in prior series, both were associated with a decreased rate of SH. The duration of PFS and OS appears longer for these two hypermutated pts in our review; however, further evaluation is needed due to the small numbers of pts with evaluable tissue and the limited follow-up. In addition, the most commonly utilized Ig locus VH1-8 in this study does not appear to be utilized as frequently in other series, which may reflect regional differences in antigen exposure or other environmental factors. Multicenter prospective evaluations should be undertaken to determine the incidence of SH as well as associations of SH and selective IgVH loci with pt prognosis in MCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 289 Introduction: Chronic lymphocytic leukemia (CLL) is a disease mainly of older adults, with a median age of 72 at diagnosis, yet landmark trials that have established chemoimmunotherapy as standard initial therapy for this disease include patients (pts) with median ages between 58–64 years. Outcomes in other types of leukemia have been shown to be influenced by age, and one randomized phase III study (German CLL5) demonstrated a lack of benefit of fludarabine (F) over chlorambucil (Ch) in CLL pts over age 64. To help determine the current optimal standard therapy for older pts with CLL we reviewed the data on all pts enrolled on successive phase II and III CALGB CLL trials for previously untreated pts to determine if the efficacy varied by age. Particular interest was paid to ideal chemotherapy choice and the benefit of rituximab or alemtuzumab in older pts. Methods: 663 pts with untreated CLL enrolled on CALGB first-line studies were evaluated (515 pts 70 vs Results: Median follow-up was 91 months (range, 16–236). Overall response rate (ORR) was significantly different among the treatment regimens (p Discussion: These data support chlorambucil as an acceptable treatment for CLL pts age 70 or older, and an appropriate chemotherapeutic backbone for future trials. The addition of rituximab to fludarabine-containing regimens significantly improves both PFS and OS in younger and older pts, confirming the importance of this agent in current front-line CLL regimens. The addition of consolidation alemtuzumab did not improve response rates, PFS, or OS regardless of age, compared to similar regimens that did not include alemtuzumab consolidation, although these data are less mature compared to those derived from the older studies. Collectively, these data suggest that future clinical trials for older CLL pts should incorporate rituximab or other anti-CD20 antibodies with similar or improved efficacy, but not fludarabine or alemtuzumab. Disclosures: No relevant conflicts of interest to declare.

Abstract 1739 Background: Triple negative breast cancers (TNBC) are sensitive to agents that cause DNA crosslinking and strand breaks and have high epidermal growth factor receptor (EGFR) expression and amplification. Therefore, we sought to determine the safety and efficacy of sequential combination therapy with bendamustine (B) and erlotinib (E). While lymphopenia was an expected toxicity, ≥ grade (gr) 3 infections were not anticipated and overall hematologic toxicity and infection rates were expected to be lower than that observed in treatment of hematologic malignancies. Methods: A phase I/II trial of B and E was performed in patients (pts) with metastatic TNBC with ECOG performance status ≤ 2 and ≤ 1 prior chemotherapy (CTX) for metastasis. Pts were required to have adequate bone marrow function and could not have active infection or history of HIV on anti-retroviral therapy. Pts received B on days 1, 2 and E on days 5 – 21 of a 28 day cycle. Phase I consisted of dose escalation of 2 sequential 3 patient cohorts: dose level 1, B 120 mg/m2 intravenous (IV) and E 100 mg oral (po), and dose level 2, B 120 mg/m2 IV and E 150 mg po. Dose limiting toxicity (DLT) was defined on toxicities related to the study therapy observed during cycle 1, including: absolute neutrophil count < 1 × 109/L for > 7 days despite use of white blood cell growth factor support, platelet count < 25 × 109/L for > 7 days, associated with bleeding, or < 10 × 109/L at any time, gr 4 rash, gr 3 diarrhea uncontrolled medically, gr 4 diarrhea, or other non-hematologic toxicity ≥ gr 3. Response was assessed every 2 cycles. Results: 11 pts were treated, 5 on dose level 1 and 6 on dose level 2. One pt had DLT on dose level 2. However, infection-related serious adverse events (SAE) were observed in 4/11 (36%) pts during or after treatment was completed and possibly associated with prolonged lymphopenia (see Table). Within 1 month (mo) of completing 6 cycles Pt 1 was hospitalized for Pneumocystis carinii (PCP) pneumonia (PNA). Pt 5 completed 4 cycles, but received no further CTX; 2 mo later, she developed brain metastases treated with whole brain radiation and dexamethasone. At 3 mos after treatment she was hospitalized for dyspnea, hypoxia and PNA and received empiric PCP treatment, broad spectrum antibiotics and anti-fungal therapy. No diagnostic procedure was done. Within 1 mo of completion of 6 cycles, Pt 6 was hospitalized with PNA concerning for opportunistic pathogen. Pt 8 received 1 cycle, stopped study therapy for DLT (gr 3 fatigue, gr 3 dyspnea), and was hospitalized for progressive disease. She had persistent gr 4 lymphopenia when transitioned to hospice. Conclusion: Combination therapy with B and E causes excessive toxicity with severe and prolonged lymphopenia resulting in clinically significant depressed CD4 counts and opportunistic infections. The extent of lymphopenia is more severe than that observed in prior published clinical trials of B. We hypothesize that E may potentiate B-related lymphopenia. Since toxicity observed is potentially life threatening this combination should not be pursued further. Pts treated on the trial with persistent lymphopenia will have serial lymphocyte and CD4 counts performed and will be placed on prophylaxis for opportunistic infections. We suggest that future trials of B combinations in TNBC patients should take into account the potential for cumulative toxicity to lymphocytes and the need for monitoring patients during and after treatment. Updated results will be presented. This study was approved and funded by the National Comprehensive Cancer Network (NCCN) from general research support provided by Cephalon, Inc. Disclosures: No relevant conflicts of interest to declare.

Abstract 1386 Background: Hyperacute TLS, defined as a rise in uric acid, potassium, lactate dehydrogenase (LDH), and/or phosphate that occurs within 4.5 hours and peaks 24–48 hours after flavopiridol, has been described in up to 40% of pts with CLL treated with this agent. Methods: A retrospective analysis of 116 pts with relapsed or refractory CLL treated with single agent flavopiridol at the Ohio State University on phase I or II protocols was conducted to determine predictive factors for TLS. Pts received flavopiridol 30–50 mg/m2 bolus + 30–50 mg/m2 4-hour continuous IV infusion (CIVI) on days 1, 8, 15, and 22 every 35 days or days 1, 8, and 15 every 28 days. Results: In 116 pts, the median age was 60 (range, 31–84), median number of prior therapies was 4 (range, 1–14), 69% pts were male, 79% were Rai stage III-IV, 53% had bulky disease ≥ 10 cm, 52% had splenomegaly, and 69% had del(17p) and/or del(11q). Median pre-treatment laboratory values included B2-microglobulin (B2M) 4.4 (range, 0.8–14.9), absolute lymphocyte count (ALC) 7134/mm3 (range, 0–266,310), white blood cell count (WBC) 13,950/mm3 (range, 1,300-314,500), and LDH 199 U/L (range, 102–654). The incidence of TLS was 46% (95% CI: 36%-55%), with 14 of 53 pts (26%) with TLS requiring dialysis. In univariable analyses using logistic regression, variables associated with the occurrence of TLS were female gender (p 150,000/mm3, 75% pts with WBC 100–150,000, and 38% pts with WBC < 100,000/mm3. TLS rates were 74% and 19% in pts with B2M above and below the median (4.4), respectively. In a secondary analysis, we examined if peak flavopiridol and its glucoronide metabolite (flavo-G) levels correlated with TLS or gender. In a subset of 85 pts with available data, flavo-G levels were associated with TLS (p=0.001), but this was independent of pt gender. When peak flavo-G levels were distributed into quartiles, 50% women in the lowest quartile developed TLS compared to 93% in the highest quartile. Likewise, 14% of men with the lowest flavo-G levels developed TLS as opposed to 57% in the highest quartile. With respect to pt outcomes, 49% with TLS and 44% without TLS responded to flavopiridol. In a multivariable model controlling for number of prior treatments, cytogenetic risk group, Rai stage, age, and gender, response rates were not significantly different (p=0.34) in patients with and without TLS. However, overall survival (OS) and progression-free survival (PFS) were inferior in pts with TLS (p=0.03 and p=0.04, respectively). Eighty-six pts have died, including 13 of 14 pts with TLS who required dialysis. For pts with TLS that did not require dialysis, OS was not significantly different (p=0.88), although PFS was still worse in this subgroup (p=0.03, Figure 1). Conclusions: Female pts and pts with B2M ≥ 4.4, WBC ≥ 100,000/mm3, or bulky adenopathy ≥ 10 cm were at highest risk and should be monitored for hyperacute TLS with flavopiridol. TLS does not appear to be predictive of response or improved PFS in pts receiving flavopiridol. Supported by NCI K23 CA109004, NCI U01 CA076576, NCI N01 CM62207, LLS SCOR 7080-06, and the D. Warren Brown Foundation. Disclosures: Off Label Use: The efficacy of the cyclin dependent kinase inhibitor, flavopiridol is under investigation in CLL.

Abstract 1378 Although the most rapidly growing portion of the United States population is the elderly (Yancik R and Ries L, Semin Oncol 2004), they are consistently underrepresented in cancer clinical trials (Hutchins L et al, N Engl J Med 1999). The incidence of chronic lymphocytic leukemia (CLL) is markedly increased in older people, with a median age at diagnosis of 72 years (Ries L et al, SEER Clinical Statistics Review 2008). In contrast, an age range of between 58 and 66 years has been noted in patients (pts) enrolled in key trials to evaluate the first-line treatment for CLL (Eichhorst B et al, Leuk Lymphoma 2009). Both fludarabine and chemoimmunotherapy with rituximab have not demonstrated much promise in the older subset of patients. Developing new therapeutics that have clinical benefit and also demonstrate feasibility of administration to this patient population is of great interest. Flavopiridol, is a pan-cyclin-dependent kinase inhibitor, that has demonstrated significant clinical activity in relapsed and refractory CLL pts including those with del(17p13.1)(Christian B et al, Clin Lymphoma Myeloma 2009). We sought to determine the feasibility and impact of treating patients over the age of 70 with flavopiridol by reviewing outcomes of two clinical trials using single-agent flavopiridol in patients with relapsed or refractory CLL at our institution (Byrd J et al, Blood 2007; Lin T et al, J Clin Oncol 2009). Pts were divided into categories based on age [≥70 years old (n = 21) and Disclosures: Off Label Use: The efficacy of the cyclin dependent kinase inhibitor, flavopiridol is under investigation in CLL. Jones:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbott Laboratories: Research Funding.

Abstract 2456 The cyclin-dependent kinase inhibitor flavopiridol has been shown to be a highly effective therapy in patients (pts) with relapsed or refractory CLL, and we have shown in vitro that flavopiridol induces apoptosis independent of the p53 pathway. Here we analyze the results of two early single-agent flavopiridol trials at our institution, OSU 0055 and OSU 0491, to study the effect of poor risk cytogenetic abnormalities on overall response rate (ORR), progression-free survival (PFS) and overall survival (OS). 112 pts are included in this analysis; 49 from the phase I and 63 from the phase II study. Data from the two trials were combined for all analyses. Specific genetic abnormalities were identified by fluorescence in situ hybridization, and complexity was determined by stimulated karyotype analysis. Dohner's hierarchical classification was used to group the pts into those with del (17p), those with del (11q) and those with other cytogenetic abnormalities or normal cytogenetics. Using this classification, 40 pts (37%) had del (17p), 37 (33%) had del (11q), and 35 (31%) had neither of these abnormalities. These groups were not significantly different in terms of age, sex, Rai stage, or percent fludarabine-refractory. The median number of prior treatments was 4 (range 1–11) and did not differ among cytogenetic groups (p=0.21). The presence of bulky adenopathy (≥5 cm) was significantly different among the groups (p=0.01). 89% of pts with del (11q) had bulky disease, while only 60% with del (17p) and 77% of pts without these abnormalities had bulky disease. Complex cytogenetics (≥3 abnormalities) were much more likely in pts with del (17p), where 63% possessed a complex karyotype, as opposed to 32% of pts with del (11q) and 26% of pts with other abnormalities (p=0.003). The ORR was 47%, and was not significantly different among the cytogenetic groups (p=0.17). ORR for pts with del (17p) was 48%, for pts with del (11q) was 57%, and for those without these cytogenetic abnormalities was 34%. The overall median PFS was 10 months (mo.). While the estimated medians were slightly higher in pts with poor risk cytogenetics, 10 mo. for both del (17p) and del (11q) groups, and 8 mo. for pts without these abnormalities, the risk of progression changed significantly over time and by 24 mo., the estimated PFS for pts with del (17p), del (11q), and other cytogenetics was respectively, 4%, 5%, and 24%. Overall median OS was 28 mo. and was not significantly different among the groups (20 mo. for del (17p), 36 mo. for del (11q), and 26 mo. for pts without these abnormalities; p=0.13). Multivariable models were fit for ORR, PFS, and OS, which included cytogenetic group, treatment schedule, age, Rai stage, number of prior treatments, bulky adenopathy and presence of complex karyotype. With respect to ORR, odds of response were not significantly different among the cytogenetic groups (p=0.21), and notably, complex karyotype was not a significant predictor of ORR (p=0.15). Since similar patterns of PFS were observed for pts with del (17p) and del (11q), these groups were combined and the risk of progression relative to pts without these abnormalities were allowed to change over time. Although cytogenetic group was not statistically significant (p=0.07), it should be noted that the risk of progression tended to be lower for pts with del (17p)/del (11q) in the first six months and then increased with follow-up as compared to pts without these abnormalities (hazard ratio (HR) 0.7 at 3 mo., 1.1 at 6 mo., 1.5 at 12 mo. and 2.2 at 24 mo.). The only independent predictor of shorter PFS was the presence of bulky adenopathy (HR=2.0, 95% confidence interval (CI) 1.1–3.7). While some differences were observed in PFS, OS was not significantly different among the cytogenetic groups when controlling for other variables (p=0.24) but was impacted (p=0.005) by the number of prior treatments (HR=1.2, 95% CI 1.1–1.3). Collectively, these data show that pts with cytogenetically high-risk disease who are treated with flavopiridol have a high ORR that is not significantly different from those without these cytogenetic abnormalities. While there may be differences with respect to PFS that are being evaluated further, OS did not differ among cytogenetic groups. Flavopiridol is thus an attractive agent to study in the first-line setting for pts with poor-risk cytogenetic abnormalities, as well as to use in further single agent and combination studies in relapsed or refractory disease. Disclosures: Grever: Sanofi Aventis: Dr. Grever is on the use patent for flavopiridol. This patent has not been awarded and has no monetary value at this time. Byrd: Sanofi Aventis: Dr. Byrd is on the use patent for flavopiridol. This patent has not been awarded and has no monetary value at this time.

Background: Breast circulating tumor cells (CTCs) are commonly isolated by positive selection enrichment technology, which targets the epithelial cell adhesion activating molecule, EpCAM. However, CTCs with low or no EpCAM expression, such as those from basal and normal-like subtypes, are more likely to be missed by this method. We developed a novel negative enrichment technology to detect CTCs with mesenchymal and stem cell markers in localized and metastatic breast cancer (BC). Patients and Methods: Twenty nine patients with localized and metastatic BC initiating chemotherapy were enrolled. CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points: pretreatment, after one cycle of treatment (TP1) and at end of treatment or at disease progression in metastatic patients (TP2). Immunocytochemical staining for nucleus, cytokeratin (8,18,19), vimentin, and CD44 was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. Enrollment is ongoing. Results: Median age was 57 yrs (range 28-78 yrs); stage distributions were I-2 (7%), II-10 (35%), III-3 (10%), and IV-14 (48%); 19 (66%) were estrogen receptor positive (ER+), 4 (14%) estrogen and progesterone receptor negative (ER-PR-HER2 non-overexpressing) and 6 (21%) HER2 overexpressing. Negative enrichment yielded an average log10 depletion of nucleated cells of 2.74 and an overall, average log10 depletion of 5.2 (>100,000 enrichment). No CTCs were identified in 5 healthy volunteers or in buffy coats purchased from the Red Cross. CTCs were identified in all stages at pretreatment but decreased to 0 in three patients by TP1. Baseline median CTC level was 373/mL in localized BC (range 2.2-1975/mL) and 761/mL in metastatic BC (range 9.9-47513/mL). In localized BC, the median percent (%) change in CTCs was +2% at TP1 (n= 13; range −100% to +14945%) and −95% at TP2 (n=5; range −100% to −13%). In metastatic BC, median CTC numbers decreased by 41% at TP1 (n=11; range −100% to +4222%) and increased at TP2 in 2 patients by 231% and 730%. Baseline CTC levels and changes at TP1 were not significantly different between localized and metastatic groups by the Wilcoxen Rank Sum test. All tested CTCs expressed vimentin and CD44. In addition to CTCs, a population of cells without detectable cytokeratin expression but positive for the other markers was identified in some samples. Further characterization of these cells for epithelial mesenchymal transition markers is underway. Conclusions: CTCs identified with this novel negative selection method have both mesenchymal and stem cell markers in localized and metastatic BC. Higher CTC numbers with epithelial characteristics are detected with this method relative to what is reported with positive selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3284.

Abstract 539 Introduction: The addition of rituximab to fludarabine-based regimens in CLL has been shown to produce high response rates with extended remissions. The long-term follow-up of these regimens with respect to progression, survival, and development of secondary malignancies has been limited. Patients and Methods: We report the long-term follow up of the chemoimmunotherapy trial CALGB 9712 (Blood 2003;101:6-14). This trial randomized 104 untreated, symptomatic patients to receive either 6 monthly cycles of fludarabine plus rituximab (FR) followed 2 months later by 4 weekly doses of rituximab (concurrent arm) or 6 monthly cycles of single agent fludarabine followed by rituximab consolidation using 4 weekly doses (sequential arm). With a median follow up of 92 months (range: 60-107), we analyzed the updated CALGB database and flow sheets submitted by treating physicians. Results: The overall response rate (ORR) was 84% (95% CI: 77%-91%), with a 90% ORR in the concurrent group (95% CI: 82%-98%) and a 77% ORR in the sequential group (95% CI: 66%-89%). Complete response (CR) was seen in 38% of patients (95% CI: 30%-45%), and partial response (PR) in 46% (95% CI: 38%-54%). The median OS was 85 months (95% CI: 71-95), with 71% of patients alive at 5 years (95% CI: 61%-79%). The median PFS was 37 months (95% CI: 27-45), with 27% progression-free at 5 years (95% CI: 19%-36%). With long-term follow up, the estimated median OS and PFS for the concurrent group were 84 months (95% CI: 57-100) and 32 months (95% CI: 23-55), respectively; the median OS and PFS for the sequential group were 91 months (95% CI: 71-110) and 40 months (95% CI: 23-50), respectively. Patients with del(17p13.1)/del(11q22.3)(18 patients) and unmutated IgVH(43 patients) continue to have an inferior OS (P=0.01 and P=0.04, respectively) and PFS (P=0.03 and P=0.04, respectively) compared to those without these abnormalities. We next assessed the frequency of therapy-related myeloid neoplasms (t-MN) and other cancers occurring after this chemoimmunotherapy regimen. No patient has developed MDS or AML prior to relapse. One patient (1%) developed t-MDS following relapse and receipt of FCR 41 months after completing trial therapy; t-MDS was diagnosed 9 months later. Richter's transformation was noted in three (3%) of the CALGB 9712 patients with large cell (n=2) or Hodgkin lymphoma (n=1). Second malignancies have included localized basal cell or squamous cell skin cancer in 12 (12%) patients whereas 11 (11%) have developed other epithelial malignancies including 4 GI, 3 lung, 3 melanomas, and 1 prostate cancer. Conclusions: Long-term follow up of patients enrolled on CALGB 9712 demonstrates extended OS and PFS with fludarabine plus rituximab, given either concurrently or sequentially, with an estimated 17%(95% CI: 9%-27%) of responders still in remission 8 years later. Looking at other published data, patients treated with FR administered concurrently or sequentially do not appear to have an increased risk of t-MN or second cancers. These long-term data reaffirm that FR is one of several acceptable frontline treatments for symptomatic patients with CLL. Disclosures: Morrison: Genentech: Speakers Bureau.

We appreciate the comments of Mead et al regarding our recent article,[1][1] and agree it is important to discern the reason(s) for the apparent differences in outcomes of patients with cytogenetically normal acute myeloid leukemia (CN-AML) and point mutations in the FLT3 -tyrosine kinase domain (

The WT1 gene on chromosome 11p13 encodes a zinc-finger protein regulating gene transcription. A recent study conducted on 70 CN-AML patients (pts) found that 7 (10%) harbored WT1 mutations (WT1mut) and suggested that WT1mut are associated with failure to achieve complete remission (CR) (Summers et al., Leukemia2007;21:550). However, the prognostic impact of WT1mut remains to be elucidated in a larger set of homogeneously treated adults with de novo CN-AML and in the context of established prognostic molecular markers. We studied the prevalence and prognostic impact of WT1mut in 196 younger [ Figure Figure

To test if small deletions or amplifications (ie. below the resolution of cytogenetics) exist in bone marrow-derived tumor DNA from acute myeloid leukemia (AML) patients (pts), we used a dense tiling path array comparative genomic hybridization (aCGH) platform consisting of 386,165 unique oligomers spaced evenly at ∼6Kb intervals across the genome. We analyzed 144 adult de novo AML pts; 64 had normal karyotypes, and 80 had 1 or 2 clonal aberrations. Similar numbers of FAB M0/1, M2, M3, and M4 pts were included, and all samples had >30% blasts (median=72%). To generate a cancer-free control set of data, we also analyzed 23 DNA samples from normal individuals matched for age and ethnicity, and with no history of cancer. Both the tumor and cancer-free control DNA samples were co-hybridized with a pool of control DNAs from blood of 4 healthy young males. To define the sensitivity and specificity of the aCGH platform, we examined its ability to detect cytogenetically defined chromosome gains and losses. Of the 33 gains and losses present in >20% of metaphases, 29 (88%) were detected by aCGH. Of the 20 gains and losses present in ≤20% of metaphases, aCGH detected only 5 (25%). Three of 63 (4.8%) balanced translocations [t(15;17), t(8;21), t(9;11)] were detected using aCGH, indicating that breakpoints of some translocations contained small deletions. Further, we identified many previously described germline copy number variants (CNVs) in both the AML pts and cancer-free controls. To improve our ability to define even smaller somatic microdeletions and amplifications, we tested 20 AML pts using CGH arrays containing 1.5 million probes per genome (average probe spacing 1.5 Kb). To preclude detection of germline CNVs, the higher resolution CGH experiments were performed comparing tumor and skin-derived DNA from the same patient. These same sample pairs were also analyzed individually with the Affymetrix 500K SNP arrays. Using stringent criteria to define abnormal segments, we identified 64 altered loci in the 20 AML pts that were not apparent cytogenetically, and that contained ≥1 gene. SNP arrays confirmed aCGH findings in 7/9 loci >100 Kb, and in 1/55 loci

Although CEBPA mutations (CEBPA+) have been reported to predict favorable outcome in CN-AML, their prognostic value has not been evaluated in the context of such established prognostic molecular markers in CN-AML as the combination of FLT3-ITD and NPM1 mutational status and BAALC and ERG expression. 169 adults aged

Background: In a previous study we showed that AML with deletion 9q (9q-) occurring in the context of a non-complex karyotype is associated with CEBPA loss-of-function mutations; their prevalence was 41% (Frohling et al., Genes Chromosomes Cancer2005;42:427). We hypothesized that disruption of CEBPA function and loss of a critical segment of 9q cooperate in leukemogenesis. This is consistent with the model of leukemogenesis, in which mutations from different complementation groups cooperate, e.g., CEBPA mutations, representing the class II mutation impairing differentiation, occur simultaneously with 9q- associated with loss and/or mutation of an as yet unidentified gene that confers the proliferative advantage (class I mutation). 9q- is also associated with t(8;21)/RUNX1/RUNX1T1, another class II mutation. Importantly, in initial studies screening for NPM1 mutations, a few patients (pts) with 9q- were reported to harbor NPM1 mutations. Objective: To evaluate the incidence and clinical significance of mutations in the CEBPA and NPM1 genes in a large series of AML with 9q aberrations. Methods: Fifty-seven pts exhibiting a 9q aberration on chromosomal banding analysis were screened for CEBPA and NPM1 mutations. Pts were classified into three groups: those with 9q- occurring as a sole aberration or together with one additional abnormality other than t(8;21) (n=35); pts with 9q- occurring within a complex karyotype, defined as ≥3 abnormalities (n=10); and pts with 9q- secondary to t(8;21) (n=12). Results: The frequencies of CEBPA and NPM1 mutations in group 1 were 49% and 29%, respectively. Strikingly, either a CEBPA or NPM1 mutation was identified in 27 of the 35 (77%) pts within this subgroup. CEBPA and NPM1 mutations did not occur concurrently. In contrast, only one CEBPA and no NPM1 mutations were detected in group 2; no pt in group 3 had mutations in CEBPA or NPM1. Although the number of pts is still limited, within group 1, pts with CEBPA mutations and those with NPM1 mutations had higher CR rates, 86% and 80%, respectively, than pts with wild-type CEBPA and NPM1, whose CR rate was 57%. Overall survival of 9q- pts with CEBPA mutations was significantly better than that of the remaining pts comprising those with NPM1 mutations and pts with wild-type CEBPA and NPM1 (p=0.03). Conclusions: Abnormalities of 9q occurring in AML pts with a non-complex karyotype and without t(8;21) are highly associated with CEBPA or NPM1 gene mutations. CEBPA and NPM1 mutations are mutually exclusive, a finding that further supports the hypothesis that both CEBPA and NPM1 mutations act as class II mutations, which cooperate with a class I mutation affecting a thus far unknown gene on 9q. Currently, additional pts with 9q- are under investigation.