Citations

Abstract Introduction Approximately 2-8% chronic lymphocytic leukemia (CLL) patients develop diffuse large B-cell lymphoma (DLBCL, so-called Richter syndrome [RS]) over long-term follow-up. The outcomes of patients with RS are variable and poorly understood. Methods CLL patients seen at Mayo Clinic from January 1995 to April 2013 who developed biopsy-proven DLBCL during follow-up were included in this analysis. Clinical characteristics, therapy and overall survival (OS) of all patients with RS were analyzed. Results One hundred and twenty patients with biopsy-proven RS were identified in the Mayo Clinic database. The median age at diagnosis of RS was 67 years (range, 29-88 years), 87 (73%) were male, and 83 (69%) had received prior CLL therapy. The median duration between diagnosis of CLL and development of RS was 3.8 years (range, 0-34.5 years). Stereotyped B-cell receptors were found in 9/30 (30%) RS patients compared to 219/1294 (17%) non-RS patients (OR=2.1, p=0.07). IGHV4-39 gene usage was present in 2/26 (8%) RS patients compared to 57/1354 (4%) non-RS patients (p=0.3). Of the 30 RS biopsy samples in which Epstein Barr virus (EBV) in-situ hybridization studies were performed, EBV was detected in 7 (23%) patients. After a median follow-up of 5.2 years, 86 (72%) patients have died. Causes of death were as follows: progressive DLBCL (n=46, 53%), progressive CLL (n=2, 2%), infection (n=8, 9%), unknown (n=20, 23%) and other (n=10, 12%). The median OS of the entire cohort was 0.9 years (95% CI, 0.5-1.3 years). Tsimberidou and colleagues proposed a prognosis score (RS score) based on the following variables at the time of RS diagnosis: Eastern Co-operative Group performance status of 2-4, lactate dehydrogenase >1.5 x normal, platelets<100 x 109/L, tumour size >5 cm and >1 prior therapy for CLL. Each parameter was assigned one point, and patients were then assigned to one of four risk groups based on their calculated RS score: 0 or 1, low risk; 2, low-intermediate risk; 3, high-intermediate risk; 4 or 5, high risk. The OS according to the RS score for our group of patients is shown in Figure 1. Median survival in the low, intermediate-1, intermediate-2 and high-risk groups were 2.4 years, 0.7 years, 0.3 years and 0.2 years, respectively (p<0.001). Fifteen (12%) patients received cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) as initial therapy for RS, and 68 (57%) received rituximab in addition to CHOP (R-CHOP). There was no difference in survival among patients who received CHOP compared to R-CHOP (median, 1.8 years. vs. 1.2 years, p=0.5). Twenty-four patients (20%) received non-CHOP-based chemotherapy, and treatment was unknown in 4 (3%) patients. Nine patients (7%) died before definitive therapy could be instituted. Thirteen (11%) patients were able to proceed to stem cell transplantation (SCT): 10 (8%) autologous SCT, 2 (2%) allogeneic SCT and 1 (1%) autologous SCT followed by an allogeneic SCT. Although RS patients who underwent SCT were younger compared to those who did not undergo SCT (median, 59 years. vs. 69 years, p=0.001), no differences in prior CLL therapy or receipt of R-CHOP therapy for DLBCL was noted between the two groups. The median survival of patients who proceeded to SCT was superior compared to those who did not (5.2 years vs. 0.7 years; p=0.005). Conclusion Stereotyped B-cell receptors but not VH4-39 had a suggested evidence of an increased risk of RS in our cohort. Although 90% RS patients received intensive chemotherapy, only 11% patients were able to proceed to SCT. Median survival of RS patients remains dismal at 1 year, and newer therapies are needed to improve outcomes. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Introduction We have previously developed a primary human bone marrow model in CLL which allows us to study marrow derived mesenchymal stroma cells (MSC) and mimic in vivoMSC-leukemic interactions in CLL (Ding, Blood, 2010) These interactions proved to be bidirectional with functional implications for both cell types. However the biologic and clinical implications of these bilateral cellular interactions in relation to MSC function in CLL patients have not been completely studied. MSC are clearly established as an essential component in forming supportive marrow niches for hematopoiesis. Our focus in this study thus was to further investigate on the status of CLL MSC function in comparison with normal MSC and the impact of the leukemic CLL B cell on MSC function and its role in forming marrow niches in CLL. Methods Bone marrow (BM) aspirates of CLL patients or normal age-matched subjects were used to generate MSC and these MSC were then assessed for various functions. Specifically we assessed the growth, differentiation capacity, gene expression, cytokine secretion profile and transcriptome analysis of CLL and normal MSC using colony forming unit (CFU) assay, MSC differentiation assay, Affymetrix U133 2.0 plus array and RNA sequencing respectively. To determine MSC CFU frequency, fixed number of nucleated cells (5 or 10 million) from BM aspirates were added in MSC growth medium for 10 days to form CFU. CFU frequency was then calculated by dividing the number of MSC colonies by the fixed number of inputted nucleated cells. In order to test the specific gene changes that occurred in MSC after their interaction with CLL leukemic cells, normal MSC were co-cultured with CLL cells for 1 week and subsequently followed by cell growth, gene expression analysis with RNA sequencing and cytokine profiles using a multiplex assay. Results CLL MSC generated significantly lower CFU colonies compared to normal subjects (CLL [n=24] vs normal [n=6] mean: 2.2 vs. 22 CFU per million nucleated cells, p=0.01). In order to test if the lower number of CLL MSC CFU was due to their reduced growth capacity or lower frequency, we then performed the CFU assay using identical numbers of CLL and normal MSC at the start of culture. CLL MSC was found to have significantly lower CFU capacity compared to normal MSC (mean: 2.1 [CLL] vs 4.5 [normal] CFU/10 input cell, n=7 in each group, p=0.03). To investigate the mechanism(s) for the reduced growth capacity of CLL MSC, we compared the gene expression profiles of non-stimulated CLL vs. normal MSC (n=10 for each group) with the following outcomes: CLL MSC had increased expression of cell cycle inhibitor: p16(Ink4a) and p57, both key markers of cell senescence (Baker, Nature, 2011); CLL MSC overexpressed homeobox B cluster of transcription factors, key regulator for cell development and differentiation. Wnt inhibitors DKK1/DKK2 and non-canonical Wnt 5b known to be associated with aging were found to be 10 and 2 fold more elevated in CLL MSC. In addition, MSC abundantly produced a milieu of the cytokines MCP-1/IL-8/IL-6/IL-1Ra after interaction with CLL leukemic cells. These cytokines are associated with a senescent associated secretory phenotype (SASP). Co-culture experiments of normal MSC with CLL Leukemic cells revealed significant impact of leukemic cells on normal MSC functions. Co-culture with a low density of CLL leukemic cells (CLL to MSC ratio: ≤10,000/1) increased MSC growth (1.2 fold). However, high density co-culture of CLL cells with MSC (CLL to MSC ratio of 100,000/1) inhibited MSC growth (0.8 fold). Transcriptome analysis of normal MSC before and after one week of co-culture with CLL leukemic cells showed that normal MSC had alteration of ~1500 genes. Many of the modified genes (CDKN2B, DKK2, LIF, HGF, FOXQ1 etc) are known to be involved in regulation of cell growth and senescence. Conclusion Collectively, these results demonstrated that bone marrow derived CLL MSC have decreased proliferative potential and possess senescent phenotypes. Importantly these senescent features were inducible in normal MSC after their interaction with CLL leukemic cells implying that leukemic B cells can educate MSC to become senescent. The implications of the CLL MSC senescence status are multiple and include: defective hematopoietic niches due to dysfunctional MSC, immune dysregulation and the enhanced nurturing of CLL B cell by these MSC. We are now investigating several of these latter features in our current work. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract BACKGROUND: Although chemoimmunotherapy (CIT) has improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 40-50% of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays. Interactions with nurturing environments can enhance CLL B-cell resistance to apoptosis. These interactions include cytokine mediated prosurvival signals by angiogenic molecules, such as VEGF and bFGF that nurture CLL B-cells in an autocrine fashion and promote CLL cell survival partly through up regulation of anti-apoptotic proteins. These findings provide a strong rationale for testing anti-VEGF therapy in combination with a purine nucleoside analogue CIT regimen for upfront treatment. We conducted a randomized phase 2 CIT trial using pentostatin, cyclophosphamide, and rituximab with (PCR-B) or without (PCR) bevacizumab (B), an anti-VEGF monoclonal anti-body. METHODS: Eligible patients were previously untreated and had CLL in need of treatment by NCI-WG criteria (Blood 111:5446). Patients were randomized using a dynamic allocation procedure stratifying for stage (0-II vs. III-IV) and FISH (17p or 11q deletion vs. other) to receive either 6 cycles of rituximab (100 mg on day 1 of cycle 1; 375 mg/m2on day 2 of cycle 1 and day 1 of cycles 2-6) followed by pentostatin (2 mg/m2) and cyclophosphamide (600 mg/m2) (PCR) administered every 21 days. Patients in the PCR-B cohort also received bevacizumab 15mg/kg on day 1 of cycles 1-5 and days 1, 22, & 43 of cycle 6. All patients underwent complete response evaluation 3 months after day 1 of cycle 6 (or last cycle of treatment for those completing < 6 cycles). MRD was assessed using 6-color flow cytometry (Leukemia 21:956) at the completion of treatment. RESULTS: 68 patients were enrolled through the Mayo Clinic Cancer Research Consortium between 1/2009 and 1/2013. Three patients were excluded from analysis: 1 patient canceled prior to treatment, 1 was dosed incorrectly, and 1 was ineligible due to immunophenotyping inconsistent with CLL. Median age of eligible patients was 63 years (range 43-81) and 43 (66%) were men. With respect to disease stage, 3 (5%), 38 (58%), and 24 (37%) had low, intermediate and high Rai stage disease. Eleven (17%) patients had deletion 17p or 11q & 29 (45%) had unmutated IGHV. No statistically significant differences were observed in these variables by treatment arm. All 65 evaluable patients have completed active treatment, with 54 (83.1%) completing the intended 6 cycles (PCR group 27/32 [84.4%] and PCR-B 27/33 [81.8%]). Hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 10 (31.3%) patients on PCR and 12 (36.4%) on PCR-B (p=0.79). Non-hematologic grade 3+ adverse events deemed at least possibly related to treatment were observed in 9 (28.1%) patients on PCR and 18 (54.4%) on PCR-B (p=0.04). The most common such events were hypertension (PCR: 3.1% vs. PCR-B: 21.2%), proteinuria (0% vs. 6.1%) and creatinine increase (3.1% vs. 6.1%). Across both arms, 64/65 (98.5%) patients achieved a response including 31/32 (96.9%) treated with PCR and 33/33 (100%) treated with PCR-B (p=0.49). CR/CRi was achieved in 10/32 (31.3%) patients treated with PCR & 18/33 (54.5%) treated with PCR-B (p=0.08). Of the 28 who achieved a CR/CRi, MRD analysis was completed on 26, of whom 12 (46%) were MRD negative. With respect to treatment arm, 5/32 (16%) patients on PCR and 7/33 (21%) on PCR-B achieved an MRD negative CR. Median time to retreatment for all 65 patients was 44.8 (95% CI: 34.6 – NA) months. Median overall survival has not yet been reached. With current follow-up no differences between treatment-free survival (p=0.38), progression-free survival (p=0.23), or overall survival (p=0.45) are observed by treatment arm. Plasma levels of angiogenic cytokines VEGF, bFGF, thrombospondin (TSP) and the chemokines CCL3 and CCL4 were measured prior to treatment and at the time of the post treatment response evaluation. Correlations of these cytokines with clinical outcome will be presented. CONCLUSION: The addition of bevacizumab to purine analogue-based CIT was generally well-tolerated and may increase complete remission rates in patients with CLL. No clear improvement in treatment free survival has been observed to date. Disclosures Shanafelt: Hospiria: Research Funding; Pharmacyclics/Jannsen: Research Funding; Cephalon: Research Funding; Celgene: Research Funding; glaxoSmithKline: Research Funding; Genetech: Research Funding; Polyphenon E Int'l: Research Funding. Off Label Use: Off label use of pentostatin for treatment of CLL. Off label use of bevacizumab for treatment of CLL. . Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients. Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping. To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases. Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort. Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status. Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract Multiple myeloma (MM) is 2-3 times more common among African-Americans compared to non-Hispanic whites. The 2-3-fold increased risk among family members of cases suggests a genetic contribution to risk. Genome-wide association studies (GWAS) in populations of European ancestry have identified seven novel risk loci at 2p23.3 (rs6746082), 3q26.2 (rs10936599), 3p22.1 (rs1052501), 6p21.32 (rs2285803), 7p15.3 (rs4487645), 17p11.2 (rs4273077) and 22q13.1 (rs877529) (Broderick, et al. Nat Genet, 2011, Chubb, et al. Nat Genet, 2013), three of which were replicated in another European series (Martino et al., Br J Haematol, 2012). Here we examined the index signals and conducted fine-mapping for each locus in a case-control study of 1,049 multiple myeloma cases and 7,084 controls of African ancestry to identify better markers of risk and novel independent loci in seven previously reported regions in this high risk population. Incident cases were recruited from 10 clinical centers and SEER cancer registries from 2011 to 2013 and genotyped using the Illumina HumanCore GWAS array. Control data were obtained from previous genome-wide studies of breast and prostate cancer, genotyped using the Illumina 1M-Duo in 4425 male controls from the African Ancestry Prostate Cancer Consortium (consisting of 14 independent studies) and 2632 female controls from a breast cancer GWAS of African-American women (consisting of 9 independent studies). Imputation to 1000 Genomes (March 2012 release) was conducted for regions around six of the previously identified single nucleotide polymorphisms [SNPs] (the HLA region harboring rs2285803 is still being imputed, results will be presented). A case-control analysis of SNPs/indels >1% frequency within 250 kb of each index variant was conducted using unconditional multivariable logistic regression adjusting for age, sex and five leading principal components. Region-specific alpha levels were determined through permutation tests. The minimum alpha level across the six regions was α=0.002. All previously reported risk variants were common in African-Americans (minor allele frequency [MAF] >0.05). For five of the six SNPs, we had ≥94% power to detect the same effect observed in non-Hispanic whites, and 64% power for the less common variant rs10936599 (MAF=0.07). We observed directionally consistent effects (odds ratio [OR]>1) for the six risk variants tested, with three replicating at p≤0.05 (7p15.3, p=1.4x10-7; 17p11.2, p=0.05; 22q13.1, p=0.02). For three of the six regions, we observed better markers of risk in African-Americans that were correlated with the index SNP in Europeans (7p15.3, rs56333627, p=1.5x10-5, r2=0.89; 17p11.2, rs34562254, p=2.9x10-3, r2=0.90; 22q13.1, rs2092410, p=1.1x10-4 r2=.71). The missense variant identified in the 17p11.2 region (rs34562254, Pro251Leu) is located in TNFRSF13B, which encodes the protein TACI, a B cell surface receptor which plays a role in B cell maturation, apoptosis and antibody production by inducing activation of transcription factors including NFAT and NFκβ. In addition, there is evidence suggesting that TACI is involved in MM pathogenesis. Our results demonstrate that many of the risk loci for MM found in European ancestry populations are also risk loci in men and women of African ancestry and that by fine-mapping, we are able to identify variants that better capture risk in populations of African ancestry. Disclosures Terebelo: Celgene Corp: Membership on an entity's Board of Directors or advisory committees. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Abstract Abstract 4252 Background: Multiple recent studies have demonstrated the survival benefits of reduced intensity allogeneic stem cell transplant for high risk CLL patients. These transplant series, however, represent highly selected cases where age, co-morbidities and performance status preclude transplant in many patients with CLL. The portion of newly diagnosed CLL patients that are eventually considered for transplant is unknown, as well as the proportion that are referred for transplant evaluation; the proportion that actually receive stem cell transplant, and the reasons for not pursuing transplant in referred patients. We conducted a cohort study of newly diagnosed CLL patients to address this gap in knowledge. Methods: We used the Mayo Clinic database to identify all patients with newly diagnosed CLL seen at Mayo Clinic within 1 year of diagnosis between July of 2003 and May of 2011 (time interval during which reduced intensity allogeneic stem cell transplantation was part of routine care at Mayo Clinic). Electronic medical record and laboratory studies were reviewed to identify all patients referred for stem cell transplant evaluation (either allogeneic transplant for progressive CLL or autologous stem cell transplant for Richter's transformation). Patients referred for transplant were analyzed to determine what proportion completed transplant and, for those who were not transplanted, the reason they were not transplanted (e.g. lack of insurance approval, co-morbidity, lack of a donor, inadequate disease control, death prior to transplant or patient preference). Results: A total of 1250 patients with newly diagnosed CLL between July 2003 and May 2011 who were seen at Mayo Clinic within 1 year of diagnosis were identified. The mean age at diagnosis was 63.6 yrs and 34.2% were women. With respect to disease stage, 48.7% were Rai stage 0, 45.5% Rai stage I/II, and 5.8% Rai stage III/IV. CLL FISH analysis revealed 126 (13.6%) patients who had either del (17p) or del (11q) and 801 (86.4%) patients who had other FISH abnormalities or normal FISH results. After a median follow-up of 41 months, 425 (34%) of patients have received treatment. A total of 54 patients (4.3%) were referred for a transplant evaluation during follow-up. Of these 54 patients, 48 (3.8% overall cohort) were referred for consideration of allogenic transplant for treatment of progressive CLL and 6 (0.5%) were referred for autologous transplant for treatment of Richter's transformation. Baseline characteristics associated with subsequent transplant referral included age at diagnosis (median: 56 yrs) (p &lt; 0.0001, Odds Ratio (OR) for each year older = 0.91, 95% Confidence Interval (CI)=(0.88, 0.95)), Rai stage (III/IV vs. 0) (p = 0.0002, OR = 22.7, 95% CI=(5.1, 102.4)) and FISH (del(11q), del(17p) vs Normal, del(13q) or Trisomy 12) (p &lt; 0.0001, OR = 7.3, 95% CI=(3.3, 16.1)). Gender, Rai stage (I/II vs. 0), and patient residence (local vs. not local) were not significant. Among the 54 patients referred for transplant, 28 (51.9%) completed a transplant. This included 22 (45.8%) patients referred for allogenic transplant and 6 (100%) patients referred for autologous transplant. Among the 26 patients referred who did not undergo transplant, the principal reasons were patient preference (n = 6, 23.1%), patient still being evaluated for transplant as of the study end date (n = 6, 23.1%), alternative therapy chosen (n = 5, 19.2%), inadequate disease control (n = 3, 11.5%), death prior to transplant (n = 3, 11.5%), lack of insurance approval (n = 2, 7.7%), and lack of a donor (n = 1, 3.8%). Conclusion: 4.3% of the newly diagnosed CLL patients in this series were considered for stem cell transplant at some point during follow-up. Young age, high Rai stage and high risk FISH abnormalities are principal reasons for transplant referral. Only 51.9% of those considered for transplant completed transplant. The principal reasons for referral patients not completing transplant who had adequate time to follow were patient preference, alternative therapy, inadequate disease control and death prior to transplant. The remaining 6 patients (23%) are in process of transplant evaluation with longer follow required. These analyses provide first incidence data for CLL transplant referral in a cohort of newly diagnosed patients. Disclosures: Zent: Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKlime: Research Funding. Shanafelt:Celgene: Research Funding; Teva/Cephalon: Research Funding; GlaxoSmith Klein: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Polyphenon E International: Research Funding.

Abstract Abstract 724 Background: Chromosome content identifies two pathogenic pathways, each occurring in about half of patients with MGUS and multiple myeloma (MM). Hyperdiploid MM (HRD) has 48–75 chromosomes with multiple trisomies selectively involving chromosomes 3, 5, 7, 9, 11, 15, 19 and 21; only 10% of these HRD tumors have primary IgH translocations and no frequent focal genetic mutations have been identified. In contrast primary IgH translocations are identified in over 70% of non-hyperdiploid MM (NHRD). Rearrangements of MYC have been detected by FISH in only 16% of untreated MM, but over 90% of MM cell lines, identifying a late role for MYC in the progression of MM. The introduction of a MYC transgene into a mouse strain predisposed to MGUS results in mice that uniformly develop MM, suggesting a distinct early role of MYC in the progression of MGUS to MM. We report here that rearrangements in a 4Mb region surrounding MYC are present in 70% of HRD, representing the most frequent focal genetic mutation in this genetic subtype of MM. Results: We analyzed the MMRC reference collection of gene expression (Affymetrix Hu133Plus2) and copy number (Agilent 244k CGH) data and performed FISH to identify MYC rearrangements with IgH or IgL loci in 218 patients with untreated and relapsed MM. We found MYC rearrangements in 48% of MM (identified only by FISH in 5%, only by aCGH in 33%, and by both FISH and aCGH in 10%), including 43% of untreated, and 51% of relapsed MM. Using a hyperdiploid index calculated from the median copy number of the chromosomes involved in trisomies we determined that rearrangements of MYC were present in 70% of the top third, 35% of the middle third, and 25% of the bottom third. Using the paired gene expression data we found that the expression of MYC was approximately two-fold higher in the samples with rearrangements compared to those without rearrangements (p<0.001) and about three-fold higher in MM tumors without rearrangements compared to MGUS (p<0001). Using paired RNA and DNA from the MMRC reference collection we determined in 22 informative patients that MYC rearrangements are associated with monoallelic expression of MYC (p<0.01), consistent with cis-dysregulation of MYC. Analysis of the various changes on aCGH, and fine mapping of the genetic architecture of the rearrangements using next generation sequencing identifies a promiscuous array of rearrangements that often result in the introduction of an enhancer within the MYC locus, resulting in its cis-dysregulation. Since they cannot be comprehensively identified by either CGH or FISH alone, more sensitive techniques, such as next generation sequencing approaches, will be required to comprehensively identify all MYC rearrangements in MM. Conclusions: Rearrangements of MYC are the most frequent focal genetic mutation in untreated MM and are particularly prevalent in hyperdiploid MM. While only one third involve an immunoglobulin locus, they all result in cis-dysregulated expression of MYC, and may be one mechanism responsible for the progression of MGUS to MM. Tumors lacking MYC rearrangements bi-allelically over-express MYC by a trans mechanism including potentially inactivating mutations of BLIMP1/PRDM1, or activating mutations of IRF4. We propose two largely non-overlapping pathogenic pathways in MM: HRD associated with frequent MYC rearrangements, and NHRD associated with frequent primary IgH translocations. The prevalence of MYC rearrangements increases with tumor progression, identifying a role for MYC both early and late in tumorigenesis. As therapies that have been reported to target MYC (e.g., IMiDs®, bortezomib, bromodomain inhibitors) are used in the clinic, it will be important to associate their effect with the presence or absence of MYC rearrangements. Disclosures: Bergsagel: Constellation Pharmaceuticals: Consultancy. Keats:Tgen: Employment.

Abstract Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-Cell malignancy typically associated with aggressive clinical course. Leukemic T-cells demonstrate post-thymic T-cell phenotypes (Tdt-, CD1a-, CD5+, CD2+ and CD7+) and commonly are CD4+/CD8- T-cells, but co-expression of CD4+/CD8+ or CD8+/CD4- have also been observed. Rearrangement of chromosome 14 involving TCL1 locus is the cornerstone for T-PLL diagnosis which leads to overexpression of oncogene TCL1. As a binding partner for TCL1, AKT expression correlates with worse prognosis. Despite advances in identification of novel mutations, therapeutic option is limited with most patients having very short survival. Herein, we performed a detailed analysis of specific T-cell subsets affected in T-PLL and a comprehensive genomic analysis by whole exome sequencing (WES) and RNA sequencing. We also explored the preclinical efficacy of targeting AKT activation by AKT inhibitor MK2206 on leukemic T-cells isolated from T-PLL patients. Methods T-PLL leukemic cells were isolated from blood or marrow samples of T-PLL patients (n=9) and were tested for involved T-cell subsets by multicolor flow cytometric analysis. Leukemic T-cells were treated with escalating doses of MK2206 (0 to 10 µM) for about 80 hours and were assessed for their apoptosis levels using Annexin V flow cytometric analysis. Whole exome sequencing (WES) and RNA sequencing were conducted using genomic DNA and RNA isolated from purified leukemic T cells from T-PLL patients. Results Phenotypic analysis of T-cell subsets in T-PLL patients showed that leukemic T-cells in 4 untreated T-PLL patients are consistent with naïve T-cell phenotype (CD45RA+, CD45RO-, CCR7+). Leukemic T-cells in 3 of the untreated patients and in 2 of the treated patients have central memory T (CD45RA-, CD45RO+, CCR7+) and effector memory T phenotypes (CD45RA-, CD45RO+,CCR7-) respectively. MK2206 treatment was able to induce dose-dependent apoptosis on the isolated PBMC (containing > 90% leukemic T-cells) of T-PLL patients (n=4) (IC50: 5 µM). FISH analysis found a rearrangement of TCL1 locus on chromosome 14 in all T-PLL cases (n=9). Three cases have also been detected to have del (11q) involving ATM locus and one patient has both del(11q) and del(17p). Subsequent WES (n=7) and RNA sequencing (n=6) analysis revealed recently reported mutations in ATM (frame shift mutation and early stop mutation W579*) in 2 cases, JAK3 (M511I) in 2 cases and STAT5B (T628S and N642H) in 2 cases. Importantly, novel somatic mutations in gene IKZF1 (N159S), NTRK1 (R33W), AP2A2 (P514L), HDAC8 (I115R), RARB (G90W) and TNIP2 (K104Q) were detected by WES. Mutations in EML4 (L548W and F304S) and VAV3 (C282Y and splice site mutation) were also identified in 2 different T-PLL cases. RNA sequencing revealed several fusion transcripts resulting in early stop of several different genes including PTPRT, L3MBTL1, UCKL1 in one T-PLL case. Conclusion T-PLL leukemic T-cells are consistent with either naïve or central memory T-cell subsets in untreated patients. The AKT inhibitor MK-2206 was capable of inducing apoptosis and could be a potential therapeutic agent for T-PLL patients. In addition, we detected known mutations in ATM and JAK-STAT pathways. Novel mutations in genes involving DNA binding and chromatin remodeling (IKZF1, HDAC8) or kinase signal pathway (NTRK1, TNIP2, VAV3, EML4) were uncovered. These results suggest that further therapeutic approaches could be developed to target DNA binding factors or JAK-STAT or AKT-TCL1 signal pathway with an ultimate goal to improve survival of T-PLL patients. Disclosures Off Label Use: Off label use of MK2206: MK-2206 is an AKT inhibitor obtained from Selleck Chemicals and is used to treat leukemic cells in vitro to test the leukemic sensitivity to AKT inhibition. .

Abstract Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3927 PURPOSE: CLL B-cells interact with their microenvironment in a variety of ways that influence leukemia cell survival. Contact mediated interactions are the most widely studied. More recently, bidirectional cytokine mediated interactions have been found to modulate spontaneous and drug induced CLL B-cell death. Specifically, upregulation of CCL2, CCL3, and CCL4 are found when CLL B-cells are co-cultured with various accessory cells. We set out to more comprehensively profile the effect of CLL-stromal cell co-culture on immune cytokine regulation and to evaluate the effect of novel therapeutic agents in clinical testing for patients with CLL on these interactions. METHODS: Freshly isolated CLL B-cells (n=24) were cultured in AIM V media for 24 hours alone or in direct contact with HS-5 stromal cells. HS-5 cells were cultured alone in AIM V to determine basal level of immune cytokine secretion. Immune cytokine levels in the media under these 3 conditions (CLL alone; HS5 alone; co-culture) were assessed using multiplex cytokine arrays (Invitrogen). The protective effects of the 6 most highly upregulated cytokines on spontaneous apoptosis of CLL B-cells after 7 day culture were then examined by culturing leukemic cells with and without recombinant cytokines. The effect of sub-lethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206 and the Bruton's Tyrosine Kinase inhibitor PCI-32765 were examined. To compare the effect of these drugs to a more conventional chemotherapeutic agent, we evaluated fludarabine (F-Ara-A) under the same conditions. To explore the ability of these novel agents to overcome stromal rescue, CLL B-cells pre-exposed to bendamustine (BEN) were cultured in media alone, or in direct contact with stroma with and without sub-lethal doses of CAL-101, MK2206, or PCI-32756. Leukemic cell viability was assessed by flow cytometry using annexin/PI staining. RESULTS: Six cytokines increased ≥4 fold (e.g. >400%, all p<0.0001) on coculture relative to the expected additive value of individual cultures. Consistent with previous reports, (Schulz 2011; Burger 2009) CCL3 and CCL4 were found to have the greatest upregulation (both >20 fold the additive value). The other highly upregulated cytokines detected have not been previously reported to be influenced by coculture: IL12 (15 fold additive value), IL1β (11 fold additive value), TNF-α (11 fold additive value), and IL-1RA (4 fold additive value). Although transwell experiments (n=6) suggested upregulation of these cytokines was largely contact dependent, substantial upregulation of several cytokines still occurred without direct contact. Each of the 6 most upregulated cytokines reduced spontaneous apoptosis when CLL B-cells were cultured for 7 days in media plus recombinant cytokine. When CLL B-cells and HS-5 cells were co-cultured with CAL-101, MK-2206, or PCI-32765, the effects on cytokine secreted upregulation were cytokine and drug specific (e.g. one drug affected individual cytokines differently; different drugs affected different cytokines). For example, PCI-3276 and CAL-101 had similar affects on the upregulation of CCL3 and CCL4 on co-culture but opposite effects on TNF-α and IL-1β. In contrast, F-Ara-A had minimal effect on upregulation of any of these 6 cytokines. Finally, we evaluated the ability of these novel agents to overcome stromal rescue of CLL B-cells treated with conventional chemotherapeutic agents. At sublethal doses CAL-101, MK2206, PCI-32765 each significantly overcame stromal rescue of BEN exposed CLL B-cells. (Fig.1) CONCLUSIONS: Direct physical interaction of CLL B-cells with bone marrow stromal cells resulted in profound upregulation of multiple immune cytokines. The 6 most upregulated cytokines identified in these experiments all reduced spontaneous apoptosis of CLL B-cells on extended culture. Sublethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206, and the Bruton's Tyrosine Kinase inhibitor PCI-32765 had distinct effects on stromal cell mediated cytokine upregulation. Importantly, each of these 3 drugs also overcame stromal mediated rescue of BEN exposed CLL B-cells. These in vitro findings support the ongoing clinical trials of BEN in combination with CAL-101 (ASH 2011), PCI-32765 (ASCO 2012) or MK2206 (Ding et. al., unpublished observation). Disclosures: Kay: Celgene: Research Funding. Shanafelt:Celgene: Research Funding; Hospira: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Polyphenon E International: Research Funding.