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Objective. SPIRIT 2 is the largest phase 3 prospective randomized open-label trial comparing imatinib (I) 400mg with dasatinib (D) 100mg daily in newly diagnosed chronic phase CML. The primary endpoint was 5 year event-free survival. Methods. 812 (406 in each arm) of 814 patients recruited started study medication (median age 53.2, 275/812 (33.8%) were over 60 years old). Patients were recruited at 144 hospitals between August 2008 and March 2013 and randomized to receive either imatinib 400mg or dasatinib 100mg daily. Secondary endpoints included overall survival, rates of treatment failure, cytogenetic/molecular response - RT-PCR BCR-ABL/ABL ratio of Disclosures O'Brien: Bristol Myers Squibb: Research Funding; CTI: Other: Chair of Independent Data Monitoring Committee; National Institute for Health and Care Excellence (NICE): Other: Chair of Technology Appraisal Committee. Osborne:Servier: Honoraria; Pfizer: Honoraria; Takeda: Honoraria; MSD: Honoraria; Roche: Honoraria; Novartis: Honoraria. Bell-Gorrod:Bristol-Myers Squibb: Consultancy; Merck EDM Serono: Consultancy; PharmaMar: Consultancy; Novartis: Consultancy; GlaxoSmithKline: Consultancy. Latimer:Pfizer: Consultancy; BMS: Consultancy; Merck: Consultancy; Astra Zeneca: Consultancy; Bluebirdbio: Consultancy; Janssen: Consultancy. Apperley:Incyte: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Byrne:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Pocock:Kent & Canterbury Hospital: Employment. Copland:Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Clark:Ariad/Incyte: Consultancy; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, frequent mutations (RRAGC, V-ATPase) in the amino acid sensing pathway connected to MTOR signaling have been described in FL. The RRAGC mutations activate MTOR. The V-ATPase is a multi-protein complex primarily involved in proton pumping and acidification of cellular compartments. Here, we report initial results from functional analyses of the common hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2. Methods: We introduced the ATP6V1B2 hotspot mutations by site-directed mutagenesis. The cDNAs were cloned into lentiviral or retroviral vectors and used to generate stable lymphoma cells lines and HEK293T cells. We also generated recombinant S. cerevisiae expressing a mutation homologous to human ATP6V1B2 p.R400Q at the endogenous VMA2locus (R381Q). We modeled the location of the mutations onto the existing 3D structures. Results: Some of the stable cell lines lost the expression of the ATP6V1B2 mutants over time, while others were more permissive. Given the critical role of V-ATPase in lysosomal acidification and autophagy we measured LC3-II levels in various recombinant cell lines and detected very elevated LC3-II levels in the V-ATPase mutant lines. Highly elevated LC3-II levels at steady state could indicate impairments in LC3-II metabolism as seen with chemical V-ATPase inhibition (bafilomycin A1 treatment) or elevated autophagic initiation and flux. We resorted to multiple experimental approaches to resolve this question: i) chemical MTOR inhibition strongly activates autophagy. Using rapamycin and Torin 2, we induced LC3-II to levels comparable to those seen with the V-ATPase mutants; ii) using inhibitors of lysosomal LC3-II degradation, we further increased already elevated LC3-II levels in V-ATPase mutant HEK293T cell lines; iii) in S. cerevisiae Vma2R381Qcells we found elevated autophagy activity based on analysis of the LC3 homolog Atg8, vacuolar/lysosomal processing of a GFP-Atg8 chimera, and enzymatic analysis of an autophagy marker, Pho8∆60. Together, these findings currently allow for the working hypothesis that V-ATPase mutants strongly increase autophagic flux. We performed studies of the effects of V-ATPase mutants on MTOR activity as measured through RPS6KB/S6-kinase phosphorylation. In transient transfections in HEK293T cells and in stable HEK293T cells lines achieving expression slightly above endogenous V-ATPase levels, performed under conditions of leucine starvation or sufficiency, we detected MTOR activation. We mapped the mutated p.Y371C and p.R400Q residues onto the published electron microscopy-derived structures of yeast V-ATPase. We found that both residues are closely spaced and are located at the interface of the V1B2 and V1A subunits; the latter with potential effects on subunit interactions, the conformational state of the V1 complex (open, loose or tight) and possibly effects on ATPase activity. To gain insights into the physiological consequences of these findings, we measured growth and viability of a recombinant lymphoma cell line (SUDHL4) expressing wild type and mutant V-ATPase in the presence of lowered leucine concentration. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. In a parallel experiment, we induced cellular stress by intentionally overgrowing these lines and measured survival over time. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. Complementary measurements of the biochemical and functional consequences of V-ATPase mutations in isolated primary FL B cells are ongoing and will be updated at the meeting. Conclusion: Our initial efforts at functional characterization of the common FL-associated hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2 uncovered substantially elevated autophagic flux. To our knowledge, this is the first report of mutational activation of autophagy in follicular lymphoma. Our current working model is that this elevated flux promotes lysosomal amino acid generation that subsequently activates MTOR through previously proposed inside-out signaling pathways. Combined, these changes may allow for improved survival of FL cells under conditions of nutrient stress. These findings may allow for future therapeutic targeting of this pathway in FL. Disclosures No relevant conflicts of interest to declare.

Cytogenetic studies (CG) of 475 chronic lymphocytic leukemia (CLL) cases showed trisomy 12 in 6.1% or 26% of patients with abnormal karyotypes. Fluorescence in situ hybridization (FISH) detected trisomy 12 in 35% of 117 CLL patients. Only 34.6% of cases detected by FISH were detected by CG. Twelve patients had low levels of trisomic cells (4% to 11%) relative to clonal B cells (47.5% to 86%), suggestive of clonal evolution. Untreated patients with trisomy 12 were predominantly male (P < .05) and had an increased incidence of splenomegaly (P < .03). Patients with trisomy 12 were more likely to be previously treated and had advanced Binet stage compared with those without trisomy 12. The median survival was shorter in patients with trisomy 12 (7.8 years) and patients with other chromosomal abnormalities without trisomy 12 by FISH (5.5 years) than in patients with diploid karyotypes (14.4 years). The response to fludarabine was similar to that of patients with diploid karyotypes, but there was a trend for earlier disease progression. FISH detected residual disease in all patients with trisomy 12 in complete (n = 6) or partial remission (n = 4). As few as 1 trisomic cell in 5,000 was detected by performing FISH on fluorescence-activated cell sorter-sorted cells. Trisomy 12 was absent in T cells in patients with trisomy 12. We conclude that FISH identifies trisomy 12 approximately 2.6 times more often than CG, readily identifies minimal residual disease, and predicts for a shorter median survival.

The chromosomal translocation t(15;17)(q22:21) of acute promyelocytic leukemia (APL) fuses PML, a novel gene, with RAR alpha, a retinoic acid receptor gene. PML-RAR hybrid transcripts were studied in 18 cases of APL using RNA-PCR. Two forms were noted: one designated 5′, producing a 439-bp chimeric fragment, and a 3′ form, producing a pair of fragments of 765 bp and 909 bp. 5′ forms were found in 7 of the 18 cases while the other 11 patients expressed the 3′ forms. The chromosome 15 specific probes K3 and K2 were used to study genomic breakpoints in 12 APL patients. Comparison of these results with RNA PCR in 11 patients for whom both were available yielded a rearrangement pattern predictive of whether the hybrid transcript was 5′ or 3′. In this way, an additional three patients in whom DNA but not RNA was available were identified as having 3′ (downstream) breakpoints and, therefore, 3′ hybrid forms. Thus, 21 cases categorized as having 5′ or 3′ PML-RAR transcripts were analyzed for various phenotypic differences. Surface phenotyping of leukemic promyelocytes demonstrated expression of the CD2 antigen in all cases with the 5′ splice variant. Only 1 of 11 cases with the 3′ form showed CD2 expression. This difference is significant at P = .001.

The chromosomal translocation t(15;17)(q22:21) of acute promyelocytic leukemia (APL) fuses PML, a novel gene, with RAR alpha, a retinoic acid receptor gene. PML-RAR hybrid transcripts were studied in 18 cases of APL using RNA-PCR. Two forms were noted: one designated 5′, producing a 439-bp chimeric fragment, and a 3′ form, producing a pair of fragments of 765 bp and 909 bp. 5′ forms were found in 7 of the 18 cases while the other 11 patients expressed the 3′ forms. The chromosome 15 specific probes K3 and K2 were used to study genomic breakpoints in 12 APL patients. Comparison of these results with RNA PCR in 11 patients for whom both were available yielded a rearrangement pattern predictive of whether the hybrid transcript was 5′ or 3′. In this way, an additional three patients in whom DNA but not RNA was available were identified as having 3′ (downstream) breakpoints and, therefore, 3′ hybrid forms. Thus, 21 cases categorized as having 5′ or 3′ PML-RAR transcripts were analyzed for various phenotypic differences. Surface phenotyping of leukemic promyelocytes demonstrated expression of the CD2 antigen in all cases with the 5′ splice variant. Only 1 of 11 cases with the 3′ form showed CD2 expression. This difference is significant at P = .001.

The retinoic acid receptor alpha (RAR alpha) and the myl gene are involved in the translocation breakpoint t(15;17)(q22;q21) in acute promyelocytic leukemia (APL). The majority of the breakpoint sites have been mapped within the second intron of the RAR alpha gene; however, the breakpoint sites on the myl gene are variable. Using primer sets derived from exon 2 or exon 3 of the RAR alpha gene and a primer derived from the myl cDNA, we were able to amplify the breakpoint sites of the fusion transcripts of all six APL RNA samples by the reverse transcriptase-polymerase chain reaction (RT-PCR). A DNA fragment of 290 bp (breakpoint A) was amplified using RNA samples from three patients, whereas two DNA fragments of 630 and 774 bp (breakpoint B) were amplified using RNA samples from the other three APL patients. DNA sequence analysis of the amplified fragments suggests that the APL breakpoints clustered within two different introns of the myl gene. Northern blot analysis demonstrated that fusion transcripts RAR alpha/myl and myl/RAR alpha of varying sizes were detected in patients with different breakpoint sites on the myl gene. In addition, we analyzed five APL samples in complete remission and detected t(15;17)- positive cells. We conclude that the t(15;17) breakpoints in APL can be amplified by PCR using a single primer set and that minimal residual disease can be demonstrated in APL using RT-PCR.

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome- positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

Cytoplasmic protein extracts from chronic myelogenous leukemia (CML) cells contained an activity that altered the electrophoretic mobility of complexes formed between nuclear proteins and the transcriptional enhancers of interferon (IFN)-inducible genes. Exposure of CML cells to IFN-alpha diminished the effect of the CML cytoplasmic proteins on these nuclear protein-DNA complexes. The presence of clinical responsiveness to IFN-alpha correlated with the sensitivity to the IFN- induced change in the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that the action of IFN-alpha in CML may be linked to a pathway that can result in posttranslational modification of nuclear proteins.

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome- positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

Introduction: The landscape of gene mutations in CLL prior to therapy is well-characterized. Comparatively less is known about gene mutations and their frequency in CLL patients that have relapsed after potent chemo-immunotherapy. Further, despite knowledge of subclonal TP53 mutations that enrich and likely drive CLL relapse in a fraction of cases, a comprehensive profile of gene mutations and their variant allele frequencies (VAFs) and clonal dynamics before and after chemo-immunotherapy in CLL is lacking. Methods: We have procured paired pre-treatment and post-treatment samples from 53 CLL cases that had relapsed after chemo-immunotherapy and purified CLL CD19+ cells and CD3+ T-cells to purity with FACS. DNA from relapsed CLL was subjected to exome capture and whole exome sequencing (WES) at a mean coverage of 72-fold (range 52-102) and sequence data analyzed using three variant callers: MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Somatically acquired gene mutations occurring in 2 or more rCLL cases were confirmed by Sanger sequencing in relapsed CLL samples and also re-sequenced in pre-treatment samples. Genes with mutation frequencies ≥5% in rCLL underwent custom gene panel-based deep coverage re-sequencing in paired pre-treatment and post-treatment samples. Analysis of deep re-sequencing data was done using the Broad GATK HaplotypeCaller v3.3.0 in parallel with VarScan2. Selected low-level variants were measured using droplet digital PCR (ddPCR) that was adapted to detection of VAFs as low as 1/10,000. Results: In CLL relapsed from potent chemo-immunotherapy, we detected mutated TP53, NOTCH1, SF3B1, XPO1, BIRC3, MYD88, NXF1, POT1, CACNA1E, CHD2, EGR2, FAM50A, FAT3, FBXW7, MGA, SAMHD1 and ZMYM3 with frequencies ≥5%. An additional 64 genes were mutated in 2/53 rCLL cases each. We performed ultra-deep panel-based re-sequencing of the 17 genes with frequencies ≥5% in 53 paired diagnosis and relapse samples, complementing selected variants with ddPCR validation to determine VAFs. TP53 mutations constituted the most frequently enriched gene at relapse (7/53=13%) and the VAFs of all TP53 mutations substantially increased at relapse often from very minor subclones at diagnosis. Importantly, none of the clonal TP53 mutations in rCLL appeared directly induced by chemotherapy, but instead all were selected from pre-existing subclones. Similarly, subclonal mutations in SAMHD1 substantially enriched in four cases at relapse (4/53=8%) suggesting a role in resistance to chemotherapy. The majority of NOTCH1 mutations (8/13) were already fully clonal at diagnosis without further enrichment at relapse. Three (3/13) subclonal NOTCH1 mutations substantially enriched at relapse, while two (2/13) clonal NOTCH1 mutations substantially decreased. The VAFs for SF3B1 mutations similarly demonstrated three patterns: i) clonal that remained clonal (4/10), ii) clonal that substantial declined and became subclonal at relapse (4/10), and, iii) subclonal that enriched but remained subclonal (2/10) at relapse. Of the 13 remaining genes, most demonstrated no consistent enrichment or depletion or remained subclonal at relapse. Of biological interest, the genes FBXW7, MYD88, NOTCH1, NXF1, ZMYM3, XPO1, SF3B1 and POT1, were often already fully clonal in the pre-treatment samples, suggesting an early role in CLL pathogenesis rather than a later role in the development of CLL relapse. Conclusion: In this large WES study focused on gene mutations in relapsed CLL paired with analysis of subclone dynamics using deep panel re-sequencing and ddPCR, we identify the genes TP53 and likely SAMHD1 as drivers of CLL relapse in 20% of cases. Multiple other genes previously implicated as CLL drivers did not consistently enrich at relapse. Further, a subset of the mutated genes was often already fully clonal pre-treatment; these genes likely serve an important role early in CLL pathogenesis that is independent of therapy. The majority of relapsed CLL in this cohort were not associated with the recurrent clonal emergence of known CLL driver mutations and based on the gene mutations frequencies reported here, much larger rCLL cohorts would need analysis to confirm possible additional low frequency gene drivers of rCLL. Disclosures Malek: Gilead Sciences: Equity Ownership; Abbvie: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving. Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL. Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB. To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC. We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells. We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis. We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples. Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL. Disclosures Malek: Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

CML patients should be screened for Abl kinase domain (KD) mutations before changing tyrosine kinase inhibitor (TKI) therapy to ensure that the second-line TKI will be effective against mutations that arose on the first drug. Sanger sequencing (SS) can confidently detect mutations present in >20% of BCR-ABL molecules but will fail to detect minor resistant sub-clones. If missed, these minor sub-clones may be further selected by the second-line TKI and cause treatment failure. The T315I mutation can be particularly problematic as it is resistant to most TKIs except ponatinib. It seems reasonable to detect all mutations if possible, and avoid second line drugs that are known to be ineffective in their presence. We have developed a next generation sequencing strategy (Illumina MiSeq, 2 x 300 bp) that enables confident detection of all Abl KD mutations present at a level of at least 1% in the BCR-ABL cDNA, this level being 3-5x above the background calling-error rate. Two 500 bp PCR products are sufficient to cover the entire kinase domain (aa M237 to E505). With 300 bp paired-end sequencing of the products, a 65 bp region containing the 315 codon (codons 306-326) is sequenced on both strands. By excluding base changes that are not corroborated on both strands, mutations in this region can be detected with a 10-fold higher accuracy (in 0.1% of BCR-ABL molecules). Samples with low disease burden (1% BCR-ABL/ABL positivity) can also be amplified sufficiently with 50 cycles of PCR, minimising artefactual DNA polymerase-induced mutations. Indexing allows the simultaneous analysis of 80 PCR's in a single MiSeq run (Abl KD of 40 patients). Important aspects of the method are: 50% PhiX DNA is added to the library to increase complexity, and the flow cell is seeded at low density (300,000 clusters per mm2) to reduce sequencing errors.Overlapping paired reads are combined to produce a single FASTQ sequence (modified FLASH source code). Any bases in the overlapping region that do not agree with their counterpart on the other strand are labelled "N" and given a quality score (Q score) of 20.Combined sequences are quality parsed (FASTX Tool Kit) to exclude sequences that do not have a Q score of at least 20 at all basesParsed high quality sequences are compared to the reference sequence. We have sequenced the BCR-ABL KD of patients who were sub-optimal responders (BCR-ABL/ABL ratio of >1% at >= 11 months on therapy) in the NCRI SPIRIT 2 trial of first-line imatinib vs dasatinib. Of 60 sub-optimally responding imatinib patients, 6 (10%) had high level mutations (in >20% of BCR-ABL molecules): T315I, L387F, G250E, N331D, M244V x 2. The patients with L387F and G250E were switched to dasatinib and proceeded to respond well. The patient with T315I was also initially switched to dasatinib but failed to respond (BCR-ABL/ABL 29% after 1 year). This patient eventually received ponatinib with good response (BCR-ABL/ABL One patient with M244V was switched to nilotinib. Initially this caused relative selection of a pre-existing M387F mutated clone, this mutation increasing from 11 months). Our study demonstrates the value of using 2 x 300 bp paired-end sequencing to detect high and low level mutations, even in patients with low-level disease burden, to guide the choice of an appropriate second-line TKI. Disclosures Dickson: Ariad: Research Funding. Kennedy:Ariad: Research Funding. Cork:Roche: Research Funding; Ariad: Research Funding; BMS: Research Funding; Novartis: Research Funding. Hedgley:Roche: Research Funding; BMS: Research Funding; Ariad: Research Funding; Novartis: Research Funding. Copland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Holyoake:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. O'Brien:BMS: Consultancy, Honoraria, Research Funding; Pzifer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ramashoye:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Diabetes negatively influences cancer-related outcomes in several malignancies. Previous studies have demonstrated key drivers in the pathogenesis of chronic lymphocytic leukemia (CLL) involving the insulin and insulin-like receptor pathways. We aimed to investigate whether hyperinsulinemic states such as diabetes impact clinical outcomes in CLL patients. Methods: This is a retrospective cohort study of 291 patients enrolled into a CLL translational clinical trial from 2004-2011 at a single university. Clinical outcomes were evaluated through exhaustive medical record review. Diabetes was classified as Type 1, Type 2, or steroid-induced diabetes requiring therapy. Covariates included Rai stage and intensity of CLL treatment, determined on whether patients had low- or high-intensity regimens. The primary end points were disease-specific and overall survival. Overall survival and disease-specific survival was measured from date of diagnosis to date of death, or date of censoring, which was February 2015. Disease-specific death was defined as death due to infection, CLL progression and/or complication from CLL therapy. Causes of death were verified by 2 independent physician reviewers with expertise in CLL, and dates of death were confirmed by the national death index database. Disease-specific and overall survival was calculated using Kaplan-Meier analysis. Proportional hazards (Cox) regression models were fit to the data using overall or disease-specific survival as endpoints. We also performed a separate parallel survival analysis including only patients who were untreated prior to enrollment. Multivariate analyses were performed incorporating known high-risk CLL features including Rai stage, IgVH mutation status, del11q, del17p, and TP53 mutation status. Cox proportional hazards were used in the multivariate analysis. Results: Over ten years, 291 CLL patients were enrolled into our study. Of these, 19% had diabetes (Type 2 DM, n = 46, Type 1 DM, n = 2, steroid-induced DM, n = 8). Only 5 patients were treated with low-intensity regimens. Disease-specific survival was significantly shorter in diabetics compared to non-diabetics. The median disease-specific survival in diabetic patients was 128.4 months, compared to non-diabetics, which was not reached (p=0.0068). Overall survival was also significantly shorter in the diabetic patients. The median overall survival in diabetic patients was 124.5 months, compared to 260.0 months in non-diabetic patients (p=0.0002). The majority of deaths in the diabetic cohort (71%) and the non-diabetic cohort (77%) were due to CLL-specific deaths. There was a substantial amount of infection-related deaths in the diabetic cohort (46%) and the non-diabetic cohort (32%). The presence of diabetes retained its significance as a prognostic variable of survival when analyzed against high-risk CLL features. Table 1. Multivariate analysis of 236 CLL patients with available data points Patient variable Hazard Ratio p -value 95% CI Diabetes 2.19 0.001 1.36-3.53 Rai stage 1.29 0.309 0.79-2.08 IgVH unmutated 2.81 0.000 1.74-4.54 Deletion 17p/mTP53 2.30 0.001 1.41-3.76 Deletion 11q 1.29 0.429 0.68-2.45 When analyzing only CLL patients untreated prior to enrollment, diabetes maintained a significantly poorer disease-specific and overall survival- as well as independent prognostic variable status against high-risk CLL features. Conclusion: Diabetes is associated with significantly worse disease-specific and overall survival among patients with CLL. The 11 year difference in median overall survival is impressive, as the life expectancy of an average diabetic patient at the median age of our cohort is estimated to be 19 to 22 years based on recent national studies. CLL-related deaths comprised the majority of both diabetic and non-diabetic deaths. Diabetes is an independent poor prognostic variable when analyzed against known high-risk features of CLL. Future investigations into other clinical variables, including glycemic control and benefits of therapies with less hematologic toxicity and/or infectious risk, are of interest. Disclosures Malek: Janssen Pharmaceuticals: Research Funding; Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership.

Introduction: Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p (all lines). Overall response rates to ibrutinib in treatment naïve or relapsed CLL are high and most patients experience prolonged remission durations, although cases of acquired ibrutinib resistance have been reported. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment cell interactions. Weakening of these interactions underlies some of the mechanisms by which ibrutinib is working in vivo. However, comparatively little is known about CLL cell-intrinsic determinants of apoptotic responses induced by ibrutinib. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance and ultimately clinical outcome are warranted. Methods: We first characterized 50 paired CLL samples procured before and after standard CLL therapies (predominantly chemo immunotherapies) for IGVH status, ZAP70 positivity, CD38 expression, TP53 mutations and FISH and analyzed the relapsed samples by whole exome sequencing (WES). Preliminary ibrutinib treatment studies using CD40-expressing fibroblasts or IL-4 or both in CLL co-culture resulted in low ibrutinib activity, precluding measurements of cell-intrinsic response determinants. Therefore, to assay ibrutinib-mediated CLL cell kill, CLL samples were purified using negative selection and incubated in RPMI medium supplemented with 10% FCS for 72 hours with ibrutinib at varying doses (0.25 µM - 5 µM). Cell death and viability was measured using AnnexinV-PI staining and flow cytometry. IC50 values were calculated using the program XLfit. Results: CLL cells demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.37 µM - 9.69 µM in pre-treatment samples and 0.56 µM - >10 µM in post treatment samples (mean IC50 values for pre-treatment and relapsed samples were 2.7 µM and 3.7 µM, respectively). Multiple cell-intrinsic determinants of IC50 values to ibrutinib were identified; CLL samples from IGVH unmutated samples were more sensitive than samples from IGVH mutated samples (mean IC50 values for pre-treatment samples were (IGVH -UM) 2.1 µM and (IGVH -M) 3.5 µM (p=0.01), and relapsed samples of (IGVH -UM) 2.8 µM and (IGVH -M) 4.0 µM (p=0.03), respectively, consistent with a greater dependency/signaling capacity of the BCR in IGVH-UM CLL. Similar findings were derived for ZAP70 status, with CLL samples with IC50 results for paired samples demonstrated a strong concordance within paired samples (Pearson correlation coefficient 0.66). However, ~10% of CLL samples were substantially more resistant to ibrutinib at relapse when compared with their paired diagnosis samples. Of these 5 samples, three had acquired mutations in TP53 following prior chemotherapy; a hypothesis-generating finding that acquired TP53 mutations reduce ibrutinib sensitivities. An expansion cohort of 7 additional ex vivo ibrutinib-treated TP53-mutated CLL samples confirmed the novel and clinically relevant finding that TP53 mutated CLL cells are substantially less sensitive to ibrutinib than TP53 wild type cells (mean IC50 values of 6.1 µM and 2.7 µM, respectively; p Conclusion: Ibrutinib ex-vivo treatment of 50 paired CLL samples procured before and after standard CLL therapies identified cell-intrinsic determinants of ibrutinib sensitivity, including IGVH unmutated CLL and trisomy 12 and implicates prior chemotherapy-selected mutations in TP53 as an intrinsic cause of reduced response. Expansion data identified TP53 mutations as a cell-intrinsic ibrutinib resistance factor in CLL. This novel finding may explain observed shorter remission durations for CLL patients with del17p/mTP53 undergoing ibrutinib therapy. Ex vivo data overall appear concordant with findings in patients treated with ibrutinib thus externally validating our approach and may allow for further refinements in ibrutinib therapy and response prediction. Disclosures Balasubramanian: Pharmacyclics LLC, an AbbVie Company: Equity Ownership; Janssen: Employment, Equity Ownership. Malek:Janssen Pharmaceuticals: Research Funding; Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership.

Background: Imatinib and dasatinib are established drugs in the first-line treatment of chronic myeloid leukemia (CML). Several studies, including SPIRIT2 have shown that first-line dasatinib (100mg once daily) has a superior complete cytogenetic and major molecular response rate compared to imatinib (400mg once daily), but no significant differences in progression-free or overall survival have been shown in any study. To date, there has been no direct comparison of quality of life (QoL) using generic and cancer-specific instruments for first-line treatment of chronic-phase CML with imatinib and dasatinib. SPIRIT2 (STI571 Prospective International Randomised Trial 2) is the first randomized clinical trial to incorporates generic and cancer-specific QoL measurement for first-line therapy. Methods: Quality of life is a secondary endpoint in the SPIRIT2 trial and has been assessed at baseline, and at 1, 2, 3, 6 and 12 months post trial entry and thereafter annually. The EQ-5D, FACT-G, FACT-BRM and the FACT-TOI have been used as QoL measures in this trial. The FACT-G covers cancer-specific QoL measure dimensions such as physical well-being, functional well-being, social and family well-being, emotional well-being and the FACT-BRM and the FACT-TOI different subsets of them. The QoL scores (EQ-5D, FACT-G, FACT-BRM, FACT-TOI) were calculated at different time points and comparison of the mean scores for both treatment groups was made. Results: A comparison between imatinib and dasatinib shows no significant difference in QoL in generic instruments and also in cancer-specific instruments. EQ-5D was 0.77 and 0.79 at baseline and 0.80 and 0.82 at one year for dasatinib and imatinib, respectively (2-3 basis points increase over 1 year). Similar results were obtained for the FACT-G, FACT-BRM and the FACT-TOI. There was a slight increase for the FACT-G (4-5 basis points), FACT-TOI (3-4 basis points) and FACT-BRM (8-10 basis points) after 1 year for both treatments, but this difference was not significant. The effects on the well-being and the emotional dimensions have been analysed for both drugs and there was no change over time, demonstrating results similar to the imatinib arm of the IRIS trial. Conclusions: Standard dose imatinib and dasatinib are both used as first-line treatments for CML and, despite different side effect profiles, there is no significant difference in QoL using the instruments described here between these two drugs over time. These data will allow the derivation of utility values to contribute to future health economic/technology appraisals. Additional analyses of how generic and cancer-specific measures of different QoL instruments change in CML patients over time in those patients that develop side effects, e.g. fluid retention with imatinib or pleural effusion with dasatinib will be presented. Disclosures Copland: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cork:BMS: Research Funding; Novartis: Research Funding; Roche: Research Funding; Ariad: Research Funding. Hedgley:Ariad: Research Funding; Roche: Research Funding; BMS: Research Funding; Novartis: Research Funding. Gills:Novartis: Research Funding; Ariad: Research Funding; BMS: Research Funding; Roche: Research Funding. Holyoake:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Bescoby:Roche: Research Funding; Ariad: Research Funding; BMS: Research Funding; Novartis: Research Funding. Pocock:Janssen: Honoraria. Clark:Novartis: Honoraria, Research Funding, Speakers Bureau; Pzifer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding. O'Brien:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Pzifer: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding.

Introduction Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete. Methods To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis. Results The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D>D/G or p.419D>D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL). Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models. Conclusion We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D>D/G or p.419D>D/H). Disclosures: Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.

Introduction Genomic complexity as measured through SNP array-based (SNP-A) genomic profiling is a strong negative predictor of survival outcome in adult acute myelogenous leukemia (AML). The recent discovery of multiple novel recurrently mutated genes in AML has led to the development of several prognostic models based on various combinations of genes along with the well-established risk factors of age and karyotype, but these models do not account for the strong effect of SNP-A-based genomic complexity. In this study, we seek to determine the relative importance of AML genomic complexity and gene mutation status on overall survival (OS) in AML. Methods We employed SNP-A genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of 13 recurrently mutated genes to determine aCNA/cnLOH lesion load and gene mutational status for 156 consecutively collected previously untreated AML patients. AML cell samples were processed with a Ficoll gradient, negatively selected using Miltenyi microbead columns, and then further purified with flow cytometric cell sorting. Processed DNA isolated from highly purified AML blasts and paired buccal DNA was subsequently hybridized to Affymetrix SNP 6.0 arrays. aCNAs were visually identified using the dChip program in paired data displays and corroborated by algorithmic lesion scoring, and cnLOH was detected using internally developed software. Using the same DNA, we resequenced the recurrently mutated exons of NPM1, FLT3, CEBPA, IDH1, IDH2, NRAS, KRAS, and TP53, and all coding exons of RUNX1, ASXL1, TET2, DNMT3A, and BCORL1. Clinical data including age, overall survival, and cytogenetics were ascertained. Cytogenetic risk categories were assigned based on the SWOG criteria. Univariate analyses were performed using Kaplan-Meier estimates of survivor functions, and Cox proportional hazards models were used for multivariate analyses. Results At the time of analysis, 119 (76%) patients had died. aCNA/cnLOH were common with ≥1, ≥2, and ≥3 lesions detected in 62%, 38%, and 26% of cases, respectively. Univariate overall survival analysis of all clinical and molecular variables demonstrated a significantly increased OS for mutations of NPM1 (p=0.01) and decreased OS for mutations of TP53 (p60 (p Within the total cohort, 103 patients (66%) were diagnosed with de novo AML. In this group of de novo AML, aCNA/cnLOH were again common with ≥1, ≥2, and ≥3 lesions in 56%, 29%, and 18% of cases, respectively. Statistically significant univariate variables adversely affecting OS included mutations of TET2 (p=0.023) and TP53 (p60 (p Conclusion Genomic complexity is common in adult AML and constitutes a dominant independent predictor of short survival outcomes. Through use of multivariate analysis incorporating genomic complexity and the mutation status of 13 recurrently mutated genes and controlling for age- and cytogenetic-based risk, only genomic complexity and mutations of TP53 emerge as significant independent predictors of short overall survival. Disclosures: No relevant conflicts of interest to declare.

Introduction Chronic lymphocytic leukemia with elevated genomic complexity (CLL-HGC) is clinically aggressive and is characterized by shortened survival. While it is known that CLL-HGC is enriched for specific aCNAs and that a subset of CLL-HGC carries TP53 mutations, it is currently unclear what other molecular aberrations cause or contribute to genomic instability in CLL with wild type TP53. Methods Within a cohort of 255 CLL cases previously analyzed on SNP 6.0 arrays, we identified 50 CLL-HGC cases with ≥ 3 acquired genomic copy number aberrations (aCNA) and/or acquired uniparental disomy (aUPD). Of these 50 CLL-HGC cases, 26 cases were wild type for TP53, and of these, 23 were subjected to whole exome sequence analysis (WES). Exome capture and WES was performed on DNA isolated from FACS-purified CD19+ and CD3+ cells, and massively parallel sequencing was performed using 96 bp paired-end sequencing on a HiSeq2000 sequencer. Bioinformatics analysis followed validated in-house pipelines. The following genes and exons were re-sequenced in all samples using Sanger sequencing: TP53 (exons 2-10), SF3B1 (exons 13-17), NOTCH1 (exon 34), and POT1 (all coding exons). Genetic data were complemented with assays for p53 protein expression and inducibility (following Nutlin 3 treatment of purified CLL cells), radiation-induced CLL cell apoptosis and ATM Ser-1981 auto-phosphorylation following CLL cell irradiation. Results Sanger sequence validation of all nominated candidate gene mutations in paired samples (T+N) confirmed 192 mutated genes in 23 CLL cases, a mutational load per case that is comparable to published unselected CLL cohorts. A gene mutated at high frequency, akin to TP53, was not identified. Recurrently mutated genes (N=2 or 3 out of 23 cases) in CLL-HGC included MYD88, NCKAP5, EGR2 and NXF1. Mutations in other genes previously suggested to contribute to genomic instability or CLL clinical aggressiveness were largely absent (ATM: N=1; NOTCH1: N=0; SF3B1: N=1 and POT1: N=0). A detailed gene-by-gene review of the mutated genes revealed that some of the genes can be grouped into functional classes that may have relevance to the observed genomic phenotype: nuclear export (XPO, NXF1), apoptosis regulation (BAX, KHDC1, PACS2, FBXW7), RB-E2F-p53 axis (DYRK1A, TRIM16, RB1CC1, E2F7), signaling (MYD88, EGR2), chromosome segregation (BSDC1, DDX46, PDIA4, BOD1L, ZW10) and p53 network (IRF2, SERTAD4, SYVN1, BAX). Notably absent were mutations in other DNA-ds-break response and repair or DNA maintenance pathway genes. Functional data uncovered 3 CLL-HGC cases with impaired p53 protein induction following chemical p53 activation and two cases with impaired ATM-Ser-1981 auto-phosphorylation. Conclusion We describe the results of WES in 23 CLL samples with high genomic complexity and wild type TP53. Overall, the following conclusions can be supported from this work: i) CLL-HGC with wild type TP53 is not associated with a high-frequency mutated gene; ii) the gene mutation load in CLL-HGC with wild type TP53 is similar to unselected CLL cases; iii) most CLL-HGC with wild type TP53 carry mutations in genes that fall into functional classes that may have a role in CLL genome destabilization or a permissive role in the survival of CLL cells with spontaneously occurring DNA lesions. Additional functional studies are in progress testing specific CLL-HGC-associated mutated alleles for effects on chromosomal stability and the p53 network. Overall, the results point to a multi-factorial origin of genomic instability in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 147 Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and currently incomplete. Methods: To further our understanding of the genetic basis of Follicular Lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from eleven cases of FL and one case of DLBCL transformed from prior FL followed by paired-end (96 base pair read length per side) massively parallel sequencing. Data from three independent HiSeq2000-based runs were pooled to maximize coverage. The sequence data were characterized by excellent technical QC parameters including a depth of coverage range of 43–73 and with 90% of bases in the target region covered by at least 10 reads. Data were subsequently analyzed using a validated bioinformatics pipelines (Personal Genome Diagnostics Inc., Baltimore, Maryland) serving as the primary data source to nominate candidate mutated genes. To complement the next gen sequencing data, we analyzed the 12 FL cases that constituted the discovery cohort for next generation sequencing for acquired genomic copy number aberrations (aCNA/LOH) and acquired uniparental disomy (aUPD) using SNP 6.0 array profiling. Data for paired DNA samples (sorted FL B-cells versus CD3 cells) were analyzed using dChip-based visual and algorithmic data analysis methods. Results: The bioinformatics pipeline nominated between 13 and 86 (mean of 47) somatically mutated genes per FL case; of these, 480 represented distinct genes. Importantly, 32 genes were nominated to be mutated in ≥2 out of 12 cases and all these candidate recurrent gene mutations and various other genes for a total of N=122 genes were subjected to Sanger sequence validation. Overall, we validated 68 genes as mutated in at least 1/12 FL discovery set cases. These included frequent mutations in MLL2 (8/12), CREBBP (7/12) and BCL2 (5/12). In addition, we identified 19 novel recurrently mutated genes (≥2 out of 12 FL cases with mutations). From these 19 genes and functionally related genes we selected 10 genes for a complete mutation analysis in a total 57 FL cases. Within the group of recurrently mutated genes we have identified a gene involved in apoptosis threshold regulation as well as multiple novel genes involved in B-cell transcriptional control. Further, we identify frequent mutations in the linker Histone genes HIST1H1 B-E. Finally, through incorporation of SNP array profiling data, we identify multiple candidate target genes for frequent aUPD in FL and further a list of single mutated genes (PTEN, A20, ARID1A, VAV1, TLR2 and TLR8 and others) that can be directly implicated in the pathogenesis of afflicted FL cases. Conclusion: This large genomic profiling study identifies 19 novel recurrently mutated genes in FL, including an apoptosis regulator, transcription factors and linker histones, thereby substantially broadening our understanding of the genetic basis of FL. Disclosures: Lebovic: Genentech: Speakers Bureau.

Abstract 2324 Poster Board II-301 The presence of genomic aberrations in CLL, including del17p and del11q, affects CLL patient survival, and thus an in-depth understanding of the genes influenced through these deletions is important. Further, genes affecting CLL disease progression that is observed in patients with recurrent genomic deletions, including del11q, remain incompletely characterized. Focusing therefore on del11q in CLL, we employed comparative expression array profiling (Affymetrix 133 2.0 plus arrays) of RNA from sorted CD19+ cells from 10 cases with and nine cases without del11q to identify stable transcriptome differences associated with del11q. Through these efforts we have identified for the first time differential expression of the insulin receptor (INSR) in CLL and predominant INSR expression in CLL with del11q. We have validated this finding in a cohort of 219 CLL cases. Our aggregate data suggest biologically and clinically meaningful INSR expression in a substantial subset of all CLL cases. INSR stimulation by insulin in CLL cells expressing the INSR resulted in the activation of canonical INSR signaling pathways, including the AKT-mTOR and Ras/Raf/Erk pathways, and INSR activation partially abrogated CLL cell apoptosis ex vivo. Clinically, CLL cases with INSR expression displayed larger lymphnodes and higher Rai stage at study enrollment and the disease progressed faster requiring repeat therapy earlier than CLL cases with low or absent INSR expression. This novel data provides the first description of a pathobiological role of the insulin receptor in CLL biology and disease progression, thus identifying a potential mediator and mechanism for del11q-associated CLL phenotypes. The identification of a transmembrane tyrosine kinase receptor with direct effects on CLL biology and clinical behavior offers opportunities for CLL prognostication and targeted therapy development and further strengthens the case for involvement of the INSR in cancer biology. Disclosures: Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

Chronic lymphocytic leukemia (CLL) is associated with characteristic genomic aberrations as defined by FISH and SNP arrays. CLL with del11q is characterized by a relatively aggressive clinical course, and about one third of cases carry mutations in ATM on the retained allele. Little information is available on additional molecular aberrations associated with del11q in CLL. We have analyzed del11q in a subset of 178 CLL cases using DNA from FACS-sorted CD19+ cells compared with buccal DNA using the Affymetrix 50k SNP array platform. Genomic copy number analysis using dChipSNP identified two non-overlapping minimal deleted regions on 11q, one of which spans ATM. We proceeded to use comparative expression array profiling of RNA derived from sorted CD19+ cells from 10 CLL cases with and nine cases without del11q using the Affymetrix U133 2.0 Plus platform. Analysis of normalized data identified ~85 probe sets with differential expression (del11q versus reference) that displayed a false discovery rate of 90% pure CLL cells from 10 cases with del11q and with high INSR expression versus cases without del11q and absent expression confirmed the accuracy of Q-PCR-based estimates of INSR expression. Work is in progress to measure effects of INSR expression on CLL survival and insulin-induced signaling in CLL in an effort to dissect functional consequences of INSR expression in CLL.

Inhibition of either coagulation factor Xa (FXa) or thrombin (FIIa) alters ex vivo biomarkers of coagulation and decreases thrombus size in animal models of experimental thrombosis. The objective of this study was to determine if combining FXa and FIIa inhibitors would synergistically reduce the magnitude of IIa generated without elevating markers of bleeding. A synergistic combination may translate into lower doses, more effective anticoagulation and better safety. To predict the FXa and FIIa inhibitor combinations with the maximum potential for synergy, PD 0313052, a potent, selective FXa inhibitor, and argatroban, a potent FIIa inhibitor, were each tested independently and in combination using an in vitro thrombin generation assay. Individually, PD 0313052 and argatroban reduced total thrombin generation (TG) in a concentration dependent manner with IC50’s of 497±148 and 882±193 nM, respectively. Subsequently, PD 0313052 and argatroban were combined in 96 well plates at concentrations ranging from 0.125x to 8x their respective IC50 concentrations. Analysis using the Bliss Independence Model identified statistically significant synergistic activity, with the greatest increase (33%) over simple additivity at 249 and 441 nM FXa to FIIa, respectively, both below their respective IC50 concentrations. Furthermore, combinations of PD 0313052 and argatroban were evaluated in an assay measuring activated clotting time (ACT). Although both PD 0313052 and argatroban dose-dependently elongated the ACT, the combination of 0.5x the TG IC50 concentrations, which demonstrated the greatest synergy in the TG assay, showed the smallest increase in the ACT, prolonging clotting time by only 15%. These data demonstrate that the combination of a specific factor Xa inhibitor and a specific IIa inhibitor can synergistically reduce thrombin generation without appreciable elevation of ACT, suggesting that dual inhibition of FXa and FIIa, using relatively low doses of each, may provide efficacious and safe treatment for thromboembolic diseases.

A complete hematologic remission was achieved in a patient with therapy- related preleukemia and transfusion-dependent pancytopenia after treatment with recombinant human granulocyte-macrophage colony- stimulating factor (GM-CSF). The patient remained in remission for nearly 1 year despite the discontinuation of GM-CSF treatment. Several lines of evidence suggest that normal hematopoiesis was restored after GM-CSF treatment. First, the cytogenetic anomaly, which was present before GM-CSF, completely disappeared after three cycles of treatment. Cytogenetic conversion was documented by conventional karyotypic evaluation of mitotic bone marrow cell preparations as well as by premature chromosome condensation analysis of the nonmitotic cells of bone marrow and peripheral blood. Second, the growth pattern and cycle status of bone marrow granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells were found to be normal during remission. Third, X chromosome-linked restriction fragment length polymorphism- methylation analysis of DNA from mononuclear cells (greater than 80% lymphocytes) and mature myeloid elements showed a polyclonal pattern. These findings suggest that restoration of hematopoiesis in this patient after GM-CSF treatment may have resulted from suppression of the abnormal clone and a selective growth advantage of normal elements.

Cytogenetic studies were performed in 546 patients with acute leukemia between 1968 and 1975. Two hundred thirty-four patients were aneuploid (42.9%), and 312 patients were diploid (57.1%). Among these, 32 patients were found to exhibit similar chromosomal alterations that appeared to involve specifically chromosomes 8 and 21. Banding studies in at least 15 of these patients confirmed the presence of a translocation between these two chromosomes. The cytogenetic findings were correlated with the hematologic and clinical data. It was found that each of these individuals had a typical picture of acute granulocytic leukemia with Auer rod-positive and peroxidase-positive cells. Ultrastructurally, the patients in this group also consistently demonstrated the presence of a nuclear bleb that has been positively associated with aneuploidy in acute leukemia. Clinically, they seemed to respond better to therapy than other adult patients with acute granulocytic leukemia. It is proposed that the 8/21 translocation acute leukemia represents a definite subgroup within the general category of acute granulocytic leukemia, with an incidence of approximately 7.3%.