ALBERT

Description: Abstract 173 One of the most common karyotypic abnormalities identified in myelodysplastic syndromes (MDS) is monosomy 7 (del7) or deletion of the long arm of chromosome 7 (del7q). The presence of del7/del7q carries a poor prognosis in MDS, MDS/myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML); the impact of these defects appears similar. Recently, a copy-neutral type of loss of heterozygozity (LOH also referred to as a somatic UPD) has been identified on 7q. Microdeletion on 7q corresponding to the EZH2 locus led to identification of inactivating mutations in this gene, though hemizygous EZH2 mutations are only rarely found and do not fully explain del7/7q pathogenesis. We performed a comprehensive analysis of myeloid neoplasms (N=189), using next generation whole exome sequencing technology, including MDS (N=34), MDS/MPN (N=26) or MPN (N=4) and 124 with AML (both primary and secondary). Among them, LOH7, involving del7/del7q were observed in 17% of cases (N=33). To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalyitic filtering approach, whereby paired DNA (tumor/CD3 lymphocyte) were sequenced and results aligned using Burrows-Wheeler Aligner and variants detected using GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on searching for del7/7q linked somatic mutational events involved comparisons of mutations in the area of del7q to cases diploid for this locus. We hypothesized that there may be heterozygous mutations of 7q, which could lead to functional haploinsufficiency that is also a result of del7q (haploinsuffcient theory, heterozygous mutations). Conversely, mutations may be either unique to del7q hemizygous inactivation, or shared between 7q diploid and haploid cases. In total, we found alterations in 12 genes located on chromosome 7 (6% of all alterations found). Using filtering strategies we narrowed the focus to “tier 1” mutations to avoid false positives; 11 mutated genes were found in cases with del7/7q and 2 in UPD7q. For example, novel hemzygous (but not heterozygous mutations) of an E3 ubiquitin ligase CUL1 gene were detected only in cases with del7/7q, suggesting that the wild type allele is protective. In cases with diploid 7q, 24 heterozygous alterations were observed (10 genes shared with del7/7q). The previously described EZH2 mutations were seen in heterozygous, homozygous and hemizygous configurations, but were most common in UPD7q (100%), while only 7% of del7/7q cases were positive. Notably, 5/12 mutant genes were located in commonly deleted regions (CDRs) either 7q22, 7q34 or 7q35–36. These CDRs also contain recurrently mutated lesions, including 7q22 (CUX1:n=4; STAG3:n=2), 7q34 (a splicing factor; LUC7L2: n=3) and 7q35–36 (EZH2: n=10). When we investigated the association between haploinsufficiency and heterozygous mutations, among those on del7/7q, cases with wild type forms of corresponding genes showed decreased expression. Similarly, such mutations were occasionally present in diploid configuration; here again the wild type cases showed a decreased expression. These findings suggest that mutated genes located in CDRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. EZH2 is a good example of such a gene. We also searched accessory genetic events observed on other chromosomes along with del7/7q and UPD7. By SNP-A, there were clear differences among 3 LOH7 groups, in which del7 was more associated with accessory chromosomal defects than cases with UPD7q or del7. Similarly, mutational patterns were specific to each LOH cohort. For example, while well known frequently mutated genes, such as U2AF1, TET2 and TP53, were commonly found in all 3 LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in monosomy 7, not in del7q or UPD7. Similarly, LOH7q was associated with somatic mutations in SETBP1 and RUNX1. In conclusion, we detected several candidate genes that could be associated with del7/7q and UPD7. Some mutations were heterozygous in cases with diploid 7q and correlated with CDRs on del7/7q without mutation. Certain mutations are specifically observed with del7, while others are commonly observed in all categories of LOH7, including EZH2. Moreover, some genes outside of the chromosome 7 were coincidently mutated with LOH7. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Description: Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1698 Hypomethylating agents decitabine and azacitidine are standard treatments for myelodysplastic syndromes (MDS). In their use, one hopes to rectify cytopenias and prolong survival by retarding further disease progression. However, individual treatment responses vary from complete remission (CR) to complete refractoriness. In general, at least 4 cycles of therapy are administered prior to assessing response. Thus, patients may have prolonged exposure to ineffective therapy, suffering toxicities without clinical benefit, while alternative and potentially more effective treatments are delayed. Currently, there are no reliable phenotypic or mutational markers for predicting response to hypomethylating agents. Once whole exome sequencing (WES) became available for more routine analysis, we theorized that somatic mutational patterns may help identify patients who would most benefit from these drugs, thereby maximizing response rate by rational patient selection. To pursue this hypothesis, we screened a cohort of 168 patients with MDS who received either azacitidine or decitabine for the presence of somatic mutations. Only those who received sufficient therapy, i.e., completed at least 4 cycles, were selected for outcome analysis. Targeted Sanger sequencing, including a panel of up to 19 genes frequently affected by somatic mutations was performed. For a representative subset of 26 patients (this subset is expanding) of whom there were 15 responders and 11 non-responders, mutational analysis was performed by WES to select target genes for further analysis. WES utilizes paired DNA (tumor vs. CD3+ lymphocytes) to produce raw sequence reads aligned using Burrows-Wheeler Aligner (BWA). Variants are detected using the Broad Institute's Best Practice Variant Detection GATK toolkit. Median age was 68 years (range, 55–85), 50% were female, and MDS subtypes were as follows: RA/RCUD/RARS 13%, RCMD 16%, RAEB-1/2 20%, MDS/MPN & CMML-1/2 31%, and sAML 20%. Response was assessed using IWG 2006 criteria at 4 and 7 months after therapy initiation. Overall response was 48%; rate of CR (including marrow/cytogenetic CR) was 28%, any HI 20%, SD 22%, and no response 29%. The cohort was then dichotomized into “responders” and “non-responders,” with responders classified as those achieving CR or any HI. Baseline patient characteristics were similar between both groups, including average age at treatment initiation, disease subtypes, proportion of abnormal/complex karyotypes, and presence of common cytogenetic aberrations. Overall, the most frequently mutated genes include TET2/IDH1/IDH2, SRSF2, ASXL1, SF3B1, RUNX1, EZH2/EED/SUZ12, SETBP1, CBL, and PPIAF2. The highest rate of refractoriness was noted in mutants of TET2/IDH1/IDH2 (67%), SF3B1 (67%), U2AF1/2 (67%). We also identified several genes whose mutants were few but associated exclusively with refractory disease (100%), including KIT, ZRSR2, PRPF8, LUC7L2. We next applied a recursive partitioning algorithm to construct a decision tree for identifying the most pivotal mutations associated with response: we found mutant CBL and PPFIA2 to be strongly associated with response, whereas mutant U2AF1/2, SF3B1 and PRPF8 were strongly associated with refractoriness. Our final approach was to dichotomize the cohort by the presence/absence of each mutation/group of mutations, and response within mutant vs. wild type cases was compared. Among refractory cases, TET2/IDH1/IDH2 (26%) and SF3B1 (17%) were most frequently mutated; among responders, mutations in RUNX1 (19% vs. 4%]), CBL (14% vs. 0%), SRSF2 (23% vs. 9%), and SETBP1 (18% vs. 4%) were most frequent. When multiple genes were combined in “either-or” fashion, mutation in TET2, SF3B1, PRPF8, or LUCL71 was significantly associated with refractoriness (52%, p=.0287), whereas mutations of RUNX1, CBL, SRSF2, SETBP1, or PPFIA2 mutation was significantly associated with response (86%, p=.0001). Mutational patterns appear to predict response to standard hypomethylating agents. Identification of the most predictive genes could guide development of molecular maker-based selection of patients for hypomethylating agent therapy, but will require ongoing analysis and additional prospective testing for validation. Disclosures: Advani: Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Description: Introduction Intensive consolidation chemotherapy in acute myeloid leukemia (AML) patients induces important hematologic toxicity with potential life-threatening infections that can lead to delays in further treatment or death in complete remission (CR). Recent single and multi-center studies (Guo et al. Blood 2011, JCO 2012, ASH 2015) have shown that the infusion of HLA-mismatched peripheral stem cell without immunosuppressive prophylaxis can accelerate hematologic recovery after chemotherapy, without developing graft versus host disease (GVHD). Objectives The primary objective of this phase I-II trial is to confirm the safety (absence of GVHD) and efficacy (reduction of neutropenia duration) of HLA-mismatched stem cells infusion after consolidation chemotherapy with idarubicine and cytarabine (3+7) in 50 younger patients with intermediate/high-risk AML. Herein we present the preliminary results of the phase I trial with 9 adult patients (safety cohort). Methods Patients younger than 65 years old with AML in CR after induction therapy that were assigned to receive consolidation course with idarubicin (12 mg/m2/day IV 1-3) and cytarabine (200 mg/m2/day IV 1-7) according to PETHEMA protocol were enrolled in this study in a single Spanish institution. To determine safety of HLA-mismatched stem cells infusion a standard 3+3 design was used in this preliminary study: cohorts of 3 patients were established with decreasing immunosuppressive prophylaxis, 3 additional patients would be enrolled with the same immunosuppression if limiting toxicity (LT) was observed in 1 out of 3 patients. LT was defined as global GVHD〉grade 2 or grade 4 infusion related reactions. The first cohort of 3 patients was assigned to receive immunosuppression with cyclosporine (1-1.5 mg/kg/bid) and prednisone (0.5 mg/kg/qid), the second cohort to receive only prednisone (0.5 mg/kg/qid), and the third would not receive immunosuppressive treatment. The immunosuppression resulting of this phase would be used in an expansion cohort. Stem cells were obtained after mobilization with G-CSF and apheresis from HLA-mismatched family-related donors and were infused the day 9 of the consolidation course. The donor and recipient HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 alleles were genotyped using a PCR-SSP method. Hematologic recovery was defined as days from start of chemotherapy to neutrophil count 〉0.5x109/L and to platelet count 〉50x109/L. G-CSF was administered only in case of severe infection. This study was approved by the Ethical Committee, and inform consent was obtained from all patients and donors. Results From March 2015 to June 2016, 9 patients were enrolled in this study. Median age was 46 (28-64) and 6 were male. All were in CR after induction therapy, 6 had intermediate risk cytogenetic, and 3 high risk cytogenetic/molecular AML. All the donors were family-related and HLA compatibility was 3/6 for 8 patients and 5/6 in one patient. HLA-mismatched stem cells infusion characteristics were: median number of mononuclear cells, CD34+ and CD3+ T cells infused per course was 4.5 (2.1-6.6)x108/kg, 3.3 (0.7-4.9)x106/kg and 1.7 (0.8-2.3)x108/kg, respectively. LT was not reached and no diagnosis of GVHD was made. Two patients developed cytarabine related rash and other 2 patients infectious diarrhea. No patient needed further immunosuppressive treatment. Median duration of neutropenia and thrombocytopenia was 30 (27-50) and 44 (22-51) days, respectively. 3 patients received G-CSF and 2 developed severe infections. Median blood cell unit and platelet units transfused was 4 (2-20) and 8 (2-32). These results are similar to those observed in a historical cohort (non-matched) of 59 patients with AML who received consolidation with 3+7 at the same institution between January 2010 to January 2015 (median duration of neutropenia and thrombocytopenia was 29 (17-57) and 36 (18-206) days, respectively). Conclusion The infusion of HLA-mismatched stem cell is safe after consolidation with idarubicin and cytarabine in younger patients. The methodology and in consequence the results of our safety cohort (with immunosuppressive prophylaxis) are not comparable to the previous experience reported by other groups. The reduction of hematologic recovery remains to be confirmed with this schedule in a larger cohort without immunosuppressive prophylaxis. Research granted by IIS La Fe (2014/0368) Disclosures Boluda: Instituto de Investigación Sanitaria La Fe: Employment.

Description: Abstract 307 Whole-exome (WES) sequencing revealed tremendous mutational heterogeneity in leukemia. While WES can be applied for discovery, it also has potential as a diagnostic tool that can overcome the shortcomings of current methods. We theorized that, in addition to mutation discovery, systematic application of WES in MDS may reveal distinct mutational patterns allowing for new molecular classification. We performed WES in 116 paired exomes, including MDS (n=57), MDS/MPN (n=36), and sAML (n=23). We also included comparative analysis with pAML (N=202; TCGA), and other publicly available data for a total of 333 exomes; 10 patients were studied serially. Paired DNA (marrow/CD3+ cells) was subjected to WES, sequence-aligned by BW Aligner, and variants detected via GATK pipeline (Broad Institute). We used defined criteria to minimize false-positives: P

Description: Abstract 1405 Genes involved in congenital genetic cancer susceptibility syndromes are also targets of somatic mutations in various tumors. Examples include WT1, NF1, CBL, TP53 and MLL2 affected both in germ line as well as somatic mutations present in malignant disorders. To apply this approach to investigation of pathogenic mutations in myeloid malignancies, we selected 183 congenital disorders in which germline mutations of disease specific genes are reported to be pathogenic. Their main clinical presentations are skeletal abnormalities (N=54 disorders), skin abnormality (N=24), mental retardation (N=17) and hematological disorders (N=12). In total, we searched for mutations in 204 genes associated with these congenital disorders. We analyzed whole exome of various myeloid malignancies, including 60 cases with myelodysplastic syndromes (MDS), 29 MDS/MPN, 5 with MPN and 122 with acute myeloid leukemia (AML) and found somatic mutations in 62 genes, which also mutated in germ line in various congenital syndromes. Of those, the most frequently mutated genes were TP53 (25 cases) and WT1 (16 cases), associated with germline mutation of Li-Fraumeni syndrome and Wilms tumor, respectively. Some somatic mutations, for example, NF1 (R1276Q) and PTPN11 (D61N), were exactly the same as observed in corresponding congenital disorders (Neurofibromatosis or Noonan syndrome). One of the novel findings is that somatic SET binding protein 1 (SETBP1) mutations (D868N, G870S and I871T) were commonly observed in sAML and CMML, and were identical to germline mutations in Schinzel-Giedion syndrome (see designated abstract). We found recurrent somatic SETBP1 mutations in 15% of each CMML and sAML. Moreover, multiple genes pathogenic in Usher syndrome (congenital hearing and vision loss, complicated by vasoproliferative retinal tumor), were somatically mutated in various myeloid neoplasms. Out of 9 genes which are causative for this syndrome, 15 mutations of 6 genes (MYO7A, USH1C, CDH23, PCDH15, USH2A, and GPR98) were observed in 13 cases, including 2 frameshift and 13 missense mutations. These genes coordinate with each other to form a functional network. CDH23 and PCDH15 are cadherins and act as cell adhesion molecules. MYO7A are actin-based motor molecules with a variety of functions. USH1C serves as an anchor and codes for a scaffolding protein to form a complex with all the other proteins. Through the PDZ binding site, USH1C forms a complex with CDH23, which was the most frequently mutated gene in this family (1 frame shift and 3 misssense mutations). CDH23 mutations were observed in 2 cases with primary AML, sAML and MDS. Specifically, a somatic missense mutation G2771S of CDH23 in a secondary AML case was identical to germline of Usher syndrome. The second most frequently mutated gene, GPR98, is located in 5q14.3 locus; a small hemizygous clone found in del5q of an MDS case. In a serial sample analysis, this mutation increased to become the larger main clone during AML evolution. Moreover, in this case, an additional CDH23 mutation was acquired in the course of leukemic expansion. In such cases with Usher syndrome gene mutations, U2AF1, ZRSR2, EZH2, IDH2 and ETV6 mutations were also observed, suggesting pathogenic cooperation with these well-known tumor suppressor genes and oncogenes. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

Description: Abstract 2 MDS and other chronic myeloid malignancies such as MDS/MPN are characterized by a frequent progression to secondary AML (sAML), a likely multistep process of acquisition of genetic abnormalities. Genes involved in congenital genetic cancer susceptibility syndromes are often targets of somatic mutations in various tumors. For instance, germ-line mutations of SETBP1 are associated with Schinzel-Giedion syndrome (SGS), which is characterized by skeletal malformations, mental retardation and frequent neuroepithelial tumors. While SETBP1 overexpression in myeloid malignancies links to poor prognosis, somatic mutations of SETBP1 were not previously identified in leukemias. When we performed whole exome sequencing of 20 cases with myeloid malignancies, in addition to detecting previously described lesions, such as TET2, CBL and ASXL1, we identified a somatic SETBP1 mutation (D868N) in 2 cases with RAEB. Analysis of DNA from CD3+ cells from these patients confirmed its somatic nature. Sanger sequencing was applied to all coding exons in an additional 48 cases, leading to detection of 2 additional somatic mutations (G870S and I871T) in 2 patients with CMML and sAML, respectively. These findings prompted us to further expand our screening cohort: targeted SETBP1 sequencing was performed in a total of 734 patients (283 with MDS, 106 with sAML, 167 with MDS/MPN, 138 MPN and 146 with primary AML): 52 mutations were detected in 52 patients (7.1%); D868N, G870S and I871T alterations were more frequently observed (N=27, N=16 and N=5, respectively), while D868Y, S869N, D880E and D880N were less prevalent. These mutations, of which 92% (48 out of 52) were identical to those in the SGS germ line, were detected in 15% with CMML (24/156), 15% with sAML (16/106) and 7% CML blast phase (2/28). Clinically, mutant cases were associated with higher age (p=.014), deletion of chromosome 7q (p=.0005) and shorter median survival (28 vs. 13 months, p