Viswanatha, David S.

Abstract Abstract 3840 Background: Patients with myeloproliferative neoplasms (MPN) harbor a multitude of somatic mutations involving JAK2, MPL, LNK, CBL, TET2, ASXL1, IDH1, IDH2, EZH2, DNMT3A, IKZF1 and TP53. None of these mutations have been consistently traced back to the ancestral clone and most of them are currently believed to represent secondary genetic events. The higher prevalence of IDH, LNK, IKZF1 and TP53 mutations in blast-phase MPN, as opposed to chronic-phase disease, suggests their pathogenetic contribution to leukemic transformation (Harutyunyan A, et al. N Engl J Med. 2011;364:488). We examined this possibility for TP53 mutations or its SNPs in chronic-phase PMF; TP53 SNPs involving codon 72 have been associated with increased risk in the development of several cancers. Methods: Mayo Clinic MPN database was utilized to identify adult patients (>18 years) with chronic-phase PMF. Study eligibility criteria included availability of bone marrow histology and cytogenetic information at time of referral. In addition, all patients were annotated for IDH, MPL and JAK2 mutational status. Patients with blast-phase disease at the time of their referral were excluded from the study. DNA from bone marrow or peripheral blood was extracted using conventional methods and amplified with one set of primers covering TP53 exons 4 through 9. The amplification products were then sequenced with eight primers using the Big Dye Terminator sequencing kit v1.1 (Applied Biosystems, Foster City, CA) and the ABI 3130xl Genetic Analyzer. Sequences were examined for mutations by the Sequencher software (Gene Codes, Ann Arbor, MI). Any mutation found was confirmed with a second PCR and sequencing reaction. A mutation was considered to be any sequence other than genomic reference sequence NC_000017.10 or a well documented polymorphism such as the rs1042522 in exon 4. Mutations were compared with the IARC p53 Database (http://www-p53.iarc.fr/) and/or the TP53 Mutation Database (http://p53.free.fr/index.html) to determine if the mutation had been reported before. Results I: Baseline characteristics and clinical course A total of 107 patients with chronic-phase PMF were studied. Median age at time of referral was 64 years (range 32–81) and 71% were males. DIPPS-plus risk assignment was low in 13%, intermediate-1 in 15%, intermediate-2 in 38% and high in 34%. Karyotype was normal in 70 (65%) patients, favorable in 26 (24%) and unfavorable in 11 (10%). JAK2, MPL and IDH mutations were detected in 62 (58%), 7 (7%) and 6 (6%) cases, respectively. To date, 74% of patients had died and 15% had documented leukemic transformation. Results II: TP53 mutations/polymorphisms and their clinical relevance TP53 mutations were discovered in only 4 (4%) cases: i) E204E; GAG>GAA (silent exon 6); ii) G245D; GGC>GAC (exon 7); iii) R175H; CGC>CAC (exon 5); and iv) six base insert (GGCGAG) after bp13767 (exon 6). There was no significant difference in presenting features or survival between TP53 mutated and unmutated cases. None of the four patients with TP53 mutation incurred leukemic transformation. TP53 exon 4 SNPs data for codon 72 was available on 104 patients and included 58 (56%) patients with homozygous Arg72Arg, 34 (33%) patients with heterozygous Pro72Arg and 12 (11%) patients with homozygous Pro72Pro. Allele frequencies were 0.7 for G (corresponding to arginine) and 0.3 for C (corresponding to proline), which is similar to the general population (0.65 and 0.35 respectively). There were no significant differences among the three codon 72 genotypes in terms of presenting characteristics or survival. Conclusions: TP53 mutations are infrequent in chronic phase PMF. A higher number of informative cases are needed to accurately define their phenotypic or prognostic effects. We are currently analyzing more cases in this regard. On the other hand, TP53 polymorphisms at codon 72 do not appear to influence phenotype or prognosis in chronic-phase PMF. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: High risk CLL patients treated with novel targeted therapies continue to experience disease progression (PD), and more than 50% of these PD patients evolve into Richter's syndrome (RS), (Maddocks, Jama Oncol 2015). Standard chemotherapy for large cell lymphoma has limited efficacy (median overall survival <12 months) in RS. Novel immunotherapy using a PD-1 antibody to block immune checkpoint signals to activate the cytotoxic T -cell has significant efficacy in relapsed Hodgkin lymphoma (Ansell, NEJM 2015). We conducted a phase 2 trial to test the safety and clinical efficacy of the anti-PD-1 antibody pembrolizumab in patients with relapsed/refractory CLL and relapsed low grade B non-Hodgkin lymphoma (B-NHL) (MC1485 trial). Methods: MC1485includes two arms: patients with relapsed/refractory CLL (including RS) and patients with relapsed B-NHL. The main end points of this study are safety and overall response rate. The first 6 patients enrolled in either arm were included in a designed safety interim analysis. Here we report the initial interim analysis from the CLL arm. Relapsed/refractory CLL including RS who had ECOG performance 0-2 and normal organ function were included. Patients with active autoimmune disease or interstitial lung disease were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks until progression, excessive toxicity, or completion of 2 years of therapy. Soluble plasma PD-L1 levels were measured in these 6 patients using an established ELISA assay (Frigola, Clin Cancer Res, 2011). CD8+ T cell subsets were assessed for their functional status using an established analysis (Liu, Oncoimmunology. 2013) at baseline and after 2 cycles of therapies. Results: As of July 27th, 2015, 16 relapsed/refractory CLL patients including 5 RS patients were enrolled. The median age was 71 years (58-81). The median number of prior therapies was 3 (1-6). All CLL patients had received chemoimmunotherapy (FCR/PCR). 4 out of 5 RS patients had received anthracycline-containing chemotherapy. 5 out of 8 patients who had received prior ibrutinib therapy progressed on ibrutinib. 8 patients had 17p-/TP53 mutation. Herein, we report preliminary results from the interim analysis of CLL arm including 4 RS and 2 CLL as well as the 5th RS who had response evaluation by PET. The median cycles of therapy administered was 4 (3-7). One patient experienced grade 2 cytokine release syndrome with transient febrile neutropenia, grade 3 headache and skin rashes. These symptoms resolved with steroid therapy. One patient experienced grade 3 myalgia and was also treated with steroids. The other patients in this safety cohort tolerated pembrolizumab well. The most common drug-related adverse events were grade 1 dyspnea (33%, 2/6) and grade 1 anemia (33%, 2/6). Based on investigator assessment using the Cheson 2007 International Working Group Criteria, 4 out of 5 RS patients had responded to therapy. One patient had a complete remission after 2 cycles and remains in remission. One patient had almost complete PET response after two cycles and is classified as PR as her bone marrow have not been re-evaluated yet. Two patients had notable responses with nodal and skin lymphoma improvements before they showed potential evidence of progressive diseases. The remaining 1 RS and 2 CLL had stable disease and continued on therapy at the last follow-up. Soluble PD-L1 levels (sPD-L1, n=6) were measured at baseline and after 2 cycles of therapies. 3 RS patients who had responded to therapy had decreased or stable sPD-L1 levels. The 4th RS and two CLL had increased sPD-L1 levels and had not demonstrated clinical response. CD8 T-cell subset analyses performed blindly (n=4) showed increased CD8 T cell function in one of the 4 patients tested. This patient who demonstrated CD8 T cell functional enhancement is the only patient in the 4 tested patients who had notable response to pembrolizumab. Conclusion: Pembrolizumab is tolerated in relapsed CLL and RS patients. Early efficacy observed in heavily pretreated RS patients including patients progressed on both anthracycline-based treatment and ibrutinib indicate PD-1 inhibition is a promising novel approach in patients with RS. Our CD8 T cell subset analysis may be used as a biomarker assay to predict clinical response in the era of novel immunotherapy. This trial and further correlative analysis is ongoing and will be presented at the ASH meeting. Disclosures Ding: Merek: Research Funding. Shanafelt:Cephalon: Research Funding; Pharmactckucs: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding; Celgene: Research Funding; Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding. Kay:Tolero Pharma: Research Funding; Hospira: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding.

Abstract Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision. Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision. Results : 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified. Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1). Conclusion : The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding.

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

Abstract Abstract 1245 Poster Board I-267 Introduction Chronic lymphocytic leukemia (CLL) patients with del(11q) by fluorescence in situ hybridization (FISH) have a poor prognosis. These patients often have younger age of onset, bulky lymphadenopathy, and short clinical response to treatment with purine analogues. Deletion of 11q in CLL is thought to involve loss-of-function of ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. As part of a larger study of early-intermediate stage, high risk CLL patients, we applied whole genome copy number variation (CNV) analysis to characterize the genomic alterations in CLL patients with and without del(11q) by FISH. Patients and Methods We studied 17 patients aged 29-67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on evaluation of molecular and immunophenotypic markers. Of these patients, six had del(11q) by FISH analysis. No patients in this study had del(11q) as a sole FISH abnormality. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 106 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a single nucleotide polymorphism (SNP)-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results CNV analysis reveals hemizygous deletions of 11q in all 6 patients positive for del(11q) by FISH. The size of the deletion varies from ∼980 Kb to ∼44 Mb. All deletions include the region of the ATM gene at 11q22.3. No large homozygous deletions of 11q were detected. The three largest interstitial deletions (∼39 Mb, ∼41 Mb, ∼44 Mb) span 11q14.1-q23.3. A slightly smaller deletion of ∼33 Mb spans 11q14.2-q23.3. One deletion of ∼8.6 Mb covers 11q22.1-q22.3. The smallest deletion of ∼980 Kb is found within 11q22.3, centered on the ATM gene. Three out of 6 patients show complex interstitial deletions of 11q that consist of two or more discontinuous segments of loss of heterozygosity (LOH) separated by short undeleted regions. Five out of 6 patients with 11q deletions also show 13q hemizygous deletion by CNV analysis and by FISH. In 4 of these patients, the 11q and 13q interstitial deletions are the only acquired CNV events detected in the CLL genome. In one patient with 11q and 13q interstitial deletions, CNV analysis also shows chromosome 12 copy number (CN)=3 (also detected by FISH), CN=3 at 3q24-telomere (tel), and LOH events consistent with hemizygous deletion at 11p13 and 7p15.2-tel. Most noteworthy are 2 patients negative for any FISH abnormality who show copy-neutral LOH of most of 11q. The region of copy-neutral LOH includes 11q12-tel in one patient and 11q13.1-tel in the other patient. The copy-neutral LOH event involves close to 100% of the CLL cells in one patient, but only about 50% of the CLL cells in the other patient. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells and provides a powerful tool for CLL clinical research. Applying this new genomic technology to CLL patients with 11q deletions reveals marked complexity in the size and structure of hemizygous deletion events, which include the region of the ATM gene at 11q22.3 and can be discontinuous, indicative of complex genomic events. We identified copy-neutral LOH of 11q in 2 patients, a finding not previously described in CLL cells. Copy-neutral LOH of 11q is a genetic mechanism by which biallelic mutations of ATM, in cooperation with the loss of other tumor suppressor genes on 11q, may contribute to poor outcomes in some CLL patients without detectable FISH abnormalities. Additional studies, including sequencing of the ATM gene in 11q copy-neutral LOH patients, are required to confirm this hypothesis. Disclosures Shanafelt: Genentech, Hospira, Polyphenon E International, Celgene, Cephalon, Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genetech, Genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech: Research Funding; Bayer: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Abstract Abstract 2420 Introduction: Interstitial deletion of chromosome 11q (11q-) detected by fluorescence in situ hybridization (FISH) is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL). CLL patients with 11q- often have younger age of onset, bulky lymphadenopathy, and poorer overall survival. The ataxia-telangiectasia mutated (ATM) gene, which encodes a protein kinase central to DNA damage response, is thought to be the critical gene in CLL 11q- through loss of ATM function. A subgroup of CLL patients with 11q- has been shown by other investigators to have a mutation in the remaining ATM allele, supporting a loss-of-function hypothesis. In an ongoing study, we are using SNP-based whole genome copy number variation (CNV) analysis to characterize in detail the 11q deletions in CLL patients. In addition, we have reported uniparental disomy (UPD) of 11q in CLL patients negative for FISH abnormalities. Here we report direct DNA sequence analysis of the ATM gene in CLL patients with 11q- and with UPD of 11q. Patients and Methods: We performed CNV analysis and ATM sequencing on 10 patients aged 29–67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on molecular and immunophenotypic markers. Eight patients had 11q- and 2 patients had UPD of 11q. CLL cells and normal cells were separated from patient peripheral blood samples by immunomagnetic beads. CNV analysis was performed on purified genomic DNA from CLL and normal cells for each patient to distinguish acquired copy number changes in malignant cells from polymorphic CNVs in the human genome. We used the Illumina 660w-quad beadchip, a SNP-based microarray for CNV analysis. CNV data was analyzed by CNV partition and PennCNV software. DNA sequence analysis of the ATM gene was performed by the dideoxy chain termination method using an ABI 3730 DNA Analyzer. All coding regions of the ATM gene (exons 4–65) and associated splice site junctions were amplified by PCR using purified genomic DNA from CLL cells. DNA sequence of the PCR products was determined on both strands. Sequence data was analyzed by Sequencher software. Results: In the 8 CLL patients with 11q−, CNV analysis demonstrates hemizygous interstitial deletions that vary from 980 Kb to 43.2 Mb. All deletions include the region of the ATM gene at 11q22.3. Two out of these 8 patients have a mutation in the residual ATM allele. One patient (CLL010) has a point mutation in exon 61 of ATM (c.8600G>A (gly2867glu)) that likely involves the substrate-binding region of the PI3-kinase domain. One patient (CLL029) has a frameshift mutation in exon 12 of ATM (c.1401_1405delCAAGA; asp467fs) that causes a premature stop signal at codon 484, presumably resulting in a truncated protein. In the 2 CLL patients with UPD of 11q, CNV analysis demonstrates copy-neutral loss of heterozygosity that extends from either 11q12.1 (CLL008) or 11q13.1 (CLL009) to the terminus of 11q. Both patients with UPD of 11q display homozygous mutation of the ATM gene. One patient (CLL008) has a point mutation in exon 58 of ATM (c.8161G>A (asp2721asn)) that likely involves the ATP binding site in the catalytic domain. One patient (CLL009) has a frameshift mutation in exon 24 of ATM (c.3238_3247del10bp/ins11bp; asp1080fs) that creates a premature stop signal at codon 1095, presumably resulting in a truncated protein. Conclusions: We report a new finding that ATM gene mutation occurs in CLL patients with UPD of 11q. Our study further expands observations reported by others concerning the role of ATM mutations in CLL. ATM mutations that we characterized include point mutations that may interfere with kinase activity and frameshift mutations that may result in a truncated ATM protein. Our sequence analysis supports a loss-of-function mechanism for ATM in a subgroup of high-risk, untreated CLL patients. The absence of detectable ATM mutations in the majority of CLL patients with 11q- may imply the existence of hidden ATM mutations not found by our targeted exon sequencing approach or epigenetic mechanisms that result in loss of gene expression. The finding that untreated CLL patients with UPD of 11q have homozygous ATM mutation supports a clonal selection hypothesis for identical alleles of mutated ATM in the absence of mutagenic chemotherapy. Selection of a CLL subclone with mutated ATM may confer a proliferative advantage through increased cell division and/or resistance to apoptosis following DNA damage. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 1265 Poster Board I-287 Introduction Fluorescence in situ hybridization (FISH), in combination with other markers, is used as a prognostic tool for CLL patients at diagnosis. The presence or absence of trisomy 12 and deletions at 13q, 11q, and 17p helps to predict disease progression and to stratify patients for therapeutic decisions. We hypothesized that whole genome single nucleotide polymorphism (SNP)-based copy number variation (CNV) analysis would capture all of the information in current CLL FISH panels and would reveal new CNV features in the CLL genome. Patients and Methods Nineteen early-intermediate clinical stage, untreated CLL patients aged 29 to 77 were determined to be at high risk for disease progression by FISH, IgVH mutation status, ZAP-70, and CD38 prognostic markers. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 162 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a SNP-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results 100% concordance is found between del(13q), del(11q), and del(17p) FISH abnormalities and loss of heterozygosity (LOH) at 13q, 11q, and 17p by CNV analysis. All three patients with trisomy 12 by FISH show copy number(CN)=3 of chromosome 12 by CNV analysis. Of 15 patients with del(13q) by FISH, 12 out of 15 have regions of hemizygous deletion on 13q that vary from ∼830 Kb to ∼38 Mb. The smallest region of LOH is located within 13q14.3. Three out of 15 patients show homozygous deletion within 13q14.3. One of these 3 patients has copy-neutral LOH of the entire 13q arm with an embedded 835 Kb segment of homozygous deletion at 13q14.3. Two patients have large discontinuous segments of LOH on 13q, indicating complex interstitial deletion events. Two out of 5 patients with del(13q) as a sole FISH abnormality show additional CNV events in the CLL genome. One of these patients has copy neutral LOH at 2q33.1-telomere(tel). One other patient with sole del(13q) FISH shows LOH events at 10q23.31-23.33 and at 15q15.1. Five out of six patients with del(11q) by FISH have either 13q LOH (n=4) or chromosome 12 CN=3 (n=1) without any other CNV events detected in the CLL genome. One patient with trisomy 12 and del(11q) by FISH has three additional acquired CNV abnormalities in the CLL genome: LOH at 7p15.2-tel, LOH at 11p13, and CN=3 at 3q24-tel. In contrast to patients with del(11q), del(13q), and trisomy 12, patients with del(17p) by FISH have numerous acquired CNV abnormalities in the CLL genome. These include LOH events at 1p34.3-p34.2, 2q34-q36.3, 3p21.31-tel, 4p13, 4p15.1-tel, 15q11.2-q14 and 15q14-q15.3, 16p13.3-tel, 16p13.11, 16p13.2, 18p11.21-tel, 20p11.21-tel, and 20q13.2-q13.31. CN=3 at 2p12-tel is detected in 2 out of 5 patients with 17p hemizygous deletion. One out of 5 patients with 17p hemizygous deletion shows CN=3 at 10q22.2-tel. One other patient also with 17p hemizygous deletion shows CN=3 at 22q12.2-tel. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells. These events include hemizygous deletion, homozygous deletion, copy-neutral LOH, and CN=3 duplication. Detection of copy-neutral LOH is not possible by FISH or array comparative genomic hybridization technology. The current study reveals a high number of acquired CNV events in earlier stage, untreated CLL patients with 17p hemizygous deletion. This observation, indicative of genomic instability, is consistent with the known poor prognosis of del(17p) patients. The new somatic CNV abnormalities detected in CLL cells may help to discover additional genes or signaling pathways involved in CLL initiation and progression. In addition, the new CNV markers may be used in larger clinical studies to improve CLL prognosis and patient stratification for therapy. Disclosures Shanafelt: Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

Abstract Early response to therapy, including assessment of MRD is a powerful prognostic factor in childhood ALL. However, there are many other known prognostic factors, and the precise relationship among these and MRD has been incompletely explored. In COG protocol P9900, patients were assigned to one of 3 risk-directed therapies based on NCI risk group, as well as TEL-AML1 translocations (TEL) and simultaneous trisomies of chromosomes 4 and 10 (DT). Infants and Ph+ patients were excluded. Good risk patients (9904) comprised NCI SR patients with either TEL or DT without CNS disease. Poor risk patients (9906) included a subset of NCI HR patients with particularly unfavorable risk factors based largely on age and white count, while 9905 had both NCI SR and HR patients not eligible for 9904 or 9906, including NCI HR patients with TEL or DT. MRD was satisfactorily measured with at least .01% sensitivity by 4-color flow cytometry in the marrow at end of induction (D29) or in blood at Day 8 (D8). D29 MRD was associated with poor outcome in all studies, and in both HR and SR patients on 9905 as shown: Study 4 y EFS ± SE (n) p value MRD <.01% MRD >.01% 9904 Overall 94±1%(668) 75±6%(105) <.0001 TEL-AML1 94±2%(367) 68±12%(38) <.0001 DT 95±1%(316) 80±7%(69) <.0001 9905 NCI-Standard Risk 89±2%(462) 63±6%(119) <.0001 9905 NCI-High Risk 83±4%(292) 49±9%(73) <.0001 9906 74±6%(156) 39±8%(84) <.0001 The proportion of D29 MRD positive DT(17.9%) patients was higher than that of TEL patients (9.4%) but MRD was also prognostic when each group was considered separately; similar findings were seen in HR TEL and DT patients. TEL or DT was associated with a significantly better prognosis in both HR and SR patients who were MRD negative, but were not favorable factors in MRD positive patients. D8 blood MRD was also prognostic in all studies, with higher MRD levels generally associated with poorer outcome. Patients who were both D29 and D8 MRD negative had a good outcome overall (93% 4 y EFS); combining all favorable risk factors, including TEL, DT, NCI risk group, D8 and D29 MRD identified a group of patients (about 13% of B-precursor ALL patients) with 98± 1% 4 y EFS. In multivariate analysis, D29 MRD was the most important prognostic factor. NCI Risk group, DT and D8 blood were also significant, but TEL and day 8 morphologic assessment of marrow were not. We conclude that MRD is a major prognostic factor in childhood ALL even accounting for other risk factors. The prognostic significance of TEL and DT depends on MRD status. The combination of good risk clinical and genetic factors, and absence of MRD in both D8 blood and D29 marrow identifies a group of patients with outstanding outcome on limited therapy.

Abstract Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders. Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data. Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested). Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case. Table 1. Clinically significant high oxygen affinity Hb variants at Mayo Clinic n Beta Alpha >30 Malmo Tarrant 10 - 20 Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, Abruzzo Dallas 5 - 10 Ty Gard, Ypsilanti, Alberta, Coimbra, North Chicago J-Cape Town 2 - 4 Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, Regina Chesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano 1 Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South Milwaukee Linwood, Longview*, Milledgeville, Sarasota Springs, Voorhees * novel Disclosures Pardanani: Stemline: Research Funding.

Abstract Background: Myelodysplastic syndromes (MDS) are rare hematological neoplasms that are more typically seen in elderly patients. Young patients (< 50 years old) have been reported to comprise between 3-6% in the Surveillance, Epidemiology and End Results (SEER) (Ma X et al, Cancer 2007; Rollison R et al, Blood 2008) with a better overall survival (OS). We hereby report the characteristics of young MDS patients with long survival follow-up. Methods: A total of 1012 MDS patients' data from Oct 1993 to Dec 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. Patients youngers than 50 years old (yMDS) were included as cohort 1, while the rest was included as control group (cohort 2). Prognostic factors were analyzed by univariate and multivariate analyses. Survival estimates were calculated using Kaplan-Meier curves and univariate and multivariate analyses was based on log-rank testing using JMP software version 10. Results: Characteristics: We identified 68 (7%) yMDS patients with a median age of 42 years (range, 18-49). Female gender was more common in yMDS (43% vs 31%, p= 0.05). Upon comparison between cohort 1 and 2, only platelets were significantly lower in yMDS (61 vs 102, p <0.0001), but not white blood cells, hemoglobin or degree of marrow fibrosis. MDS subtyping according to World Health Organization 2016 showed single lineage dysplasia in 15% vs 2%, multilineage dysplasia 24% vs 36% , ring sideroblasts 9% vs 15%, isolated del (5q) 1% vs 3%, excess blasts 44% vs 31%, and unclassifiable in 6% vs 5%. As expected, therapy related MDS (t-MDS) was more frequent in yMDS (33% vs 17%, p= 0.005). Transformation to acute myeloid leukemia (AML) was also more frequent in yMDS (28 % vs 11%, p= 0.0004). Compared to cohort 2, yMDS IPSS-R scores were very high, high, intermediate, low, and very low in 24% vs 13%, 29% vs 16%, 21% vs 20%, 19% vs 35%, and 7% vs 17%, respectively. Allogenic hematopoietic cell transplantation (HCT) was more frequent in yMDS (42% vs 4%, p< 0.0001). Survival outcome: Median OS was longer for cohort 1 vs cohort 2 but did not reach statistical significance (43 vs 21 months, p= 0.1). Median progression free survival (PFS) was shorter for cohort 1 vs cohort 2 but did not reach statistical significance (8 vs 12 months, p= 0.3). Median OS for cohort 1 based on R-IPSS was 44, 105, 40, 18, and 12 months for very low, low, intermediate, high and high risk groups, respectively (p= 0.09). Median OS was shorter in t-MDS vs de novo MDS in cohort 1 (13 vs 47 months, p = 0.04). Young patients who had transformed to AML had a worse median OS (18 vs 93 months, p=0.001). On multivariate analysis neither t-MDS nor R-IPSS had a statistically significant impact on OS. Conclusions: MDS is rarely diagnosed under the age of 50. IPSS-R was less powerful in detecting differences between its risk groups for this patient population, although more than half of the patients with yMDS had either high or very high risk. Among this cohort of yMDS patients, there was a significantly higher proportion with therapy-related myeloid neoplasms compared to older patients (one third of patients), with subsequent higher rates of transformation to AML and higher allogeneic HCT. In our study, we did not find an improved OS for yMDS patients compared to older patients. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.