ALBERT

Description: Abstract 2526 Introduction: Single nucleotide polymorphism (SNP) is an inter-individual genetic variation which could explain inter-individual differences of response/survival to chemotherapy. The present study was attempted to build up risk model of survival for acute myeloid leukemia (AML) patients with normal karyotype (AML-NK). Methods and materials: A total of 247 patients with AML-NK was included into the study. Genome-wide SNP array (Affymetrix SNP-array 6.0) was performed in the discovery set (n=118), and genotypes were analyzed for overall survival (OS). After identifying significant SNPs for OS in single SNP analyses, risk model was constructed. Replication was performed in an independent validation cohort (n=129). Results: Out of 632,957 autosomal SNPs meeting genotype data filtration criteria, a total of 82 SNPs were selected and passed into the next step of validation in an independent cohort. In the risk model generation step, finally 4 SNPs (rs2826063, rs12791420, rs11623492 and rs2575369) were meeting stringent criteria for SNP selection as follows: 1) p-value 〈 0.10 from Cox proportional hazards regression model in adjustment with age and WBC counts at diagnosis; 2) minor allele frequency 〉 0.05; 3) call rate 〉 95.0%; 4) high linkage disequilibrium r2 〈 0.8. These 4 SNPs were introduced into the risk model, and patients was grouped into 2 groups according to the number of deleterious variables including 4 SNPs and 2 clinical variables (i.e. age and WBC counts at presentation): risk score 0–2 as a low risk (n=80) and 3–6 as a high risk (n=38). The risk model could stratify the patients according to their OS in discovery (p=1.053656•10−4) and in validation set (p=5.38206•10−3). The risk model showed a higher AUC than those being incorporated only clinical or only 4 SNPs, suggesting improved prognostic stratification power of combined model. Conclusion: Genome-wide SNP based risk model obtained from 247 patients with AML-NK can identify high risk group of patients with poor survival using genome wide SNP data. (Clinicaltrials.govIdentifier:NCT01066338) Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Recent studies utilizing NGS demonstrated that residual allelic burden at complete remission (CR) is associated with worse overall survival (OS) and relapse incidence in AML. Quantitative PCR (qPCR) based disease monitoring is current practice in CBF-AML. However, qPCR requires standardization and the result for the same sample may vary depending on several factors. Also, other known prognostic factors such as cKIT mutation require an additional test. As RNA-seq can detect gene rearrangement as well as somatic mutations, we hypothesized that RNA-seq on samples taken at diagnosis and at remission can be used to monitor these genetic alterations simultaneously and can be utilized for minimal residual disease (MRD) monitoring in CBF-AML. Patients and Methods This study included 42 CBF-AML patients (23 RUNX1-RUNX1T1 and 19 CBFB-MYH11 AML). Overall, 84 bone marrow samples (42 diagnosis-CR pairs) were subjected to targeted RNA-seq using Illumina TruSight Pan-Cancer panel. After read mapping, gene count was measured using HTSeq followed by DEseq2 for gene expression quantification. Average number of sequenced reads was 3.5M reads with 87% overall mapping rate. Gene fusions in diagnostic samples were detected using EricScript. All 84 samples as well as 42 samples from T-cell fraction (CD3+, as a control) were also subjected to DNA sequencing, targeting a panel of 84 genes (Agilent SureSelect custom gene panel). Average on-target coverage was 1,606x. All other computational analyses were done using R and python. Results In diagnostic samples, class-defining gene fusion events were detected in all 42 patients. In CR samples, we tracked identical junctions identified in corresponding diagnostic samples. As expected, both CBFB-MYH11 and RUNX1-RUNX1T1 showed significant reduction in all CR samples compared to their corresponding diagnostic samples (p 〈 2.2e-13 and p 〈 6.3e-05, Fig A and B). CBFB-MYH11 was detectable in 6/19 CR samples (32%) and RUNX1-RUNX1T1 was detectable in 15/23 CR samples (65%). Reduction level of RUNX1-RUNX1T1 measured by RNA-seq showed positive correlation with the reduction level measured by qPCR (Pearson's Rho = 0.74, p 〈 5.4e-05, Fig C). As per mutational profile at diagnosis, we detected 74 mutations in 38 samples (n=38/42, 90%). NRAS (36%), KIT (36%), KRAS (17%) and, ASXL2 (17%) were commonly mutated. Survival analyses on each gene and each protein locus identified cKIT-D816 mutation as an adverse prognostic factor (HR = 3.57, [1.15 - 11.11], p = 0.028). We were able to detect all cKIT-D816 mutations in RNA-seq. Using information from NGS, we built a prognostic model for RUNX1-RUNX1T1 AML (n = 23). Decision tree analysis identified three distinct subgroups of RUNX1-RUNX1T1 AML on the basis of reduction level of RUNX1-RUNX1T1 and mutation profile (Fig D). Consistent with previous studies, 3-log or deeper reduction of RUNX1-RUNX1T1 transcript level was the most significant prognostic factor (low risk group). The algorithm further divided the patients who failed to achieve 3-log reduction according to the presence of cKIT-D816 mutation at diagnosis (intermediate and high risk group). For three defined groups, 2-year OS rates were 87%, 74%, and 33% (p = 0.08, Fig E) and 2-year relapse incidence rates were 13%, 42%, and 67% (p = 0.048, Fig F). Conclusion RNA-seq can be utilized to quantify RUNX1-RUNX1T1 and CBFB-MYH11 transcripts on diagnostic and CR samples in CBF-AML. We also showed that RNA-seq can stratify RUNX1-RUNX1T1 AML patients into three risk groups according to their long-term prognosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Predicting treatment outcome of acute leukemia has been an important issue. Many factors have been elucidated. We evaluated the impact of donor's availability on treatment outcome including mortality in patients with acute lymphoblastic leukemia (ALL). Methods A total of 294 patients with ALL were evaluated after receiving chemotherapy between year 2001 and July 2014 at our center. Patients were assessed for the need of hematopoietic stem cell transplants (HSCT), availability of HLA sibling match donor and the impact on overall outcome. Indications for transplantation were defined as (1) WBC-〉100,000 for T-ALL, 〉30 for B-ALL with, (2) cytogenetic abnormalities of t(9:22), (4:11) or (1:19), (3) relapse or primary refractory disease or (4) MRD positivity. Patients were divided into 3 categories, group A with an indication for HSCT and available donor (HSCT+/D+), group B with HSCT indication but no available donor (HSCT+/D-) and group C with no indication for HSCT regardless of donor status (HSCT-). Results The median age was 20 (14-63 years). 95 (32%) were female while 198 (68%) were male. 276 (86%) patients were newly diagnosed while 18 (14%) were relapsed. Immuno-phenotype was B for 191 (65%), T for 91 (31%) versus mixed lineage for 12 (4%). 33 (11%) patients were positive for Philadelphia chromosome. Median WBC at diagnosis was 23.2X109/L CNS involvement was positive in 26 (8%) patients. With a median follow-up of 60 months for survivors (range 2 to 116.5 months), 148 (50%) patients showed HSCT+/D+, HSCT+/D- in 79 (27%) and HSCT- in 67 (23%) patients. the 5-year OS for HSCT+/D-, HSCT+/D+ and HSCT- were 29%, 57% and 55% (p value

Description: Background: Azathioprine (AZA) has been used as a steroid sparing agent in allogeneic BMT program at the Princess Margaret Cancer Centre, Toronto, Canada for last two decades especially for cGVHD treatment. A previous clinical trial (Sullivan, Blood 1998) compared prednisone (PRD) alone vs PRD plus AZA for the treatment of extensive chronic GVHD (cGVHD) suggesting that PRD alone showed a better survival than PRD+AZA. However, the NIH consensus criteria (NCC, 2005) for cGVHD and new statistic endpoint to evaluate efficacy of cGVHD, failure free survival (FFS), have been recently introduced and increasingly used. Therefore, we conducted retrospective study attempted to evaluate the efficacy of PRD+AZA regimen compared to PRD alone regimen with respect to failure free survival (FFS) as well as overall survival (OS), non-relapse mortality (NRM)and relapse incidence. In order to adjust for the risk factors which affect the choice of treatment between different treatment options, propensity score matching (PSM) analysis was adopted in the present study. Methods: The patients diagnosed with late onset acute GVHD was excluded. A total of 240 patients were included in the analysis, transplanted at the Princess Margret Cancer Center between 2009 and 2013, diagnosed with cGVHD by NCC, and treated with PRD+AZA (n=98) or PRD alone (n=142) as first line treatment. Failure free survival (FFS), OS, NRM and relapse were compared between the 2 groups. A case-control study was performed with well-balanced pairs of PRD+AZA vs PRD patients. For the PSM analysis, propensity score (PS) was calculated. Clinical variables included in PS calculation were global score (GS) by NCC, subtype of cGVHD (classical vs overlapping), age, gender, duration from HCT to cGVHD initial treatment, performance status (PS), progressive type onset (PTO) of cGVHD, thrombocytopenia (TP) and each organ involvement of cGVHD per skin, gastrointestinal tract, liver, lung and musculoskeletal system. A total of 74 case-control pairs were selected within 0.1 of a difference in propensity score. RESULTS: With a follow-up of 43. 6 months, the 2-year FFS, OS, NRM and relapse incidence was 24.7 %, 75.6 %, 16.6% and 7.7%, respectively. The median FFS was 7.9 months (95% CI, 6.1-9.6 months). PRD+AZA group showed a longer FFS duration compared to PRD group (13.2 vs 5.6 months, p

Description: Abstract 2024 Introduction: Follicular lymphoma (FL) remains an incurable disease despite the use of immunochemotherapy or autologous stem cell transplant (ASCT). Allogeneic stem cell transplantation (Allo-SCT) remains a viable treatment option in FL because of its associated graft versus lymphoma effect and potential to achieve long term disease control. There is limited data in literature regarding long term outcome of follicular lymphoma post allo-SCT. Patients and Methods: We identified a total of 52 patients with chemosensitive FL underwent allo-SCT at our centre between 1989 and 2009. 89% were in a chemosensitive remission at time of transplantation. The conditioning regimens included BUCY in 48 (92%) patients. GVHD prophylaxis was cyclosporine and methotrexate until 2009 and subsequently cyclosporine and mycophenolate mofetil. Patients undergoing matched unrelated donor (MUD) transplant received alemtuzumab. Results: There were 30 (58%) males and 22 (42%) females. The median age at BMT was 45 years (range:24–64) and the median number of prior chemotherapy regimens was 3 (range: 1–6). Fifteen (29%) of patients had only received

Description: BACKGROUND: Tyrosine kinase inhibitor (TKI) resistance is the most relevant event during the treatment of chronic myeloid leukemia (CML), which correlates with high risk of treatment failure, disease progression and death, explaining half of treatment failed CML patients. However, the remaining half with TKI resistance does not show any ABL1 tyrosine kinase domain (TKD) mutation indicating the presence of alternative pathogenic pathways behind TKI resistance. Thus we hypothesized that the novel mutation besides ABL1-TKD mutation occurs during the development of TKI resistance. Using whole exome sequencing, we screened 13 pairs of CML cases with TKI resistance, but without ABL1-TKD mutation. The present study attempts to: 1) explore novel mutation(s) developing TKI resistance to CML treatment and 2) validate the somatic variants in an independent cohort of CML patients (n=100). METHODS: Thirteen CML cases with TKI resistance but not having ABL1 TKD mutation were included prospectively. Reason for TKI resistance includes Loss of MCyR (n=7) with (n=2) or without additional cytogenetic abnormality (ACA; n=5), progression to blastic crisis (n=3), development of ACA (n=1), development of clonal evolution in Ph neg clone (n=1), primary cytogenetic resistance (n=1). TKI resistance were demonstrated to imatinib (n=12), dasatinib (n=5), nilotinib (n=4) or ponatinib (n=2). The latest treatment includes ponatinib (n=3), dasatinib (n=8) alone (n=4), with smoothen inhibitor (n=2), or with after systemic chemotherapy (n=2), omacetaxine (n=1), and nilotinib (n=1). Disease stage at the time of exome sequencing was chronic phase (n=10) or blastic crisis (n=3). Germline and tumor samples at the time of TKI resistance were compared using whole exome sequencing (Illumina TruSeq kit, HiSeq 2000). Targeted sequencing for selected variants was performed to validate the result. All patients were confirmed the absence of ABL1-TKD mutations using Sanger sequencing. RESULTS: 1) Exome sequencing (Illumina Truseq kit) was performed as per the manufacturer's protocol using an Illumina HiSeq 2000 sequencer. DNA from buccal mucosa was used as a control for variant calling. Exome sequencing reads processing includes mapping to human genome hg19, marking PCR duplicates, realignment of indels, fixing mate information, and discard the reads with more than 2 mismatches to increase the true positive rate. In the end, we have on-target-coverage of 57x. Lastly, 72% of target positions are mapped more than 30x. 2) One hundred nineteen somatic variants were identified in 13 patients in 108 genes. Among them 5 genes have variants in multiple patients including DNMT3A (n=3), ASXL1 (n=2), NPIPB5 (n=2), ATXN3 (n=2) and EFEMP1 (n=2) . We also found at least 1 mutation in well-known driver genes in 6 patients (6/13 = 46%). 3) Three out of 4 patients with ACA carry variants at least one of DNMT3A (n=2), ASXL1 (n=2), and SETBP1 (n=1). Also, 2 out of 3 cases progressed to blastic crisis demonstrate variants in DNMT3A (n=1) and IDH1 (n=1). 4) Interestingly, in one patient, exome sequencing reveals ABL1-TKD mutation (T315I), which was not detected at the initial screening by Sanger sequencing. 5) The result of targeted sequencing in an independent cohort of CML patients (n=100) will be presented in the annual meeting of American Society of Hematology in Dec 2015. CONCLUSION: Our study suggest that DNMT3A and ASXL1 mutations seem to be the driver mutations involved in the development of TKI resistance/progression, independent of ABL1-TKD mutation. Also, exome sequencing can detect ABL1-TKD mutations including T315I prior to be detected by initial Sanger sequencing. Disclosures Lipton: Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding. Kim:Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Description: Abstract 2534 Background: Acute myeloid leukemia with normal karyotype (AML-NK) is known to be heterogeneous in the molecular level. Accordingly, it has become more critical to dissect this group of patients according to their prognosis using a molecular genetic technology. We attempted to analyze the incidence and prognostic implication of genetic abnormalities on survival in 426 adult patients with AML-NK. Methods: A total of 67 AML-NK patients achieved complete remission (CR), candidate mutations in 21 genes were identified by whole exome sequencing which has 41–89× coverage and by single-nucleotide polymorphism array analysis using marrow mononuclear cells at diagnosis of AML-NK. Subsequently, mutation analysis of 11 genes (i.e. FLT3/ITD, NPM1, DNMT3a, IDH1, IDH2, TET2, NRAS, WT1, DNAH11, SF3B1, and PHF6) which are known to be involved in the pathogenesis of hematologic diseases, were performed using Sanger sequencing in another subset of 359 AML-NK patients as a validation cohort. Results: Of 426 patients in total (median age: 51, ranges: 15–85), FLT3/ITD, NPM1, and DNMT3a mutations were associated with higher leukocytes counts at presentation of AML-NK. In 284 patients who received standard remission induction (RI) chemotherapy (excluding 119 patients with conservative treatment and 22 early death/1 follow-up loss after RI chemotherapy), those with FLT3/ITD mutation were significantly associated with a higher risk of relapse (p=0.02), a shorter leukemic-free survival duration (LFS)(p

Description: Introduction: The discoveries of JAK2-V617F as well as MPL and CARL mutations have greatly clarified the underlying genetics of Philadelphia negative myeloproliferative disease (MPN). Mutation status on these three genes, especially JAK2-V617F, can characterize over 90% of MPN patients. However, the heterogeneity of MPN in terms of its AML transformation and treatment response remains unclear. To assess the difference in mutational status between MPN patients who progress to secondary AML and those who do not, we aim to examine longitudinal samples taken from multiple time points using next generation sequencing. Patients and Methods: Bone-marrow (BM) samples were collected from 19 MPNpatientsfrom2003 to 2012atChonnamNational UniversityHwasunHospital.The diagnosis of MPN was established according to the revised criteria of the World Health Organization.Longitudinal samples were taken at the time of diagnosis and at a follow-up as well as its T-cell fractions (CD+3) isolated from the peripheral blood using MAC separation column.Targeted sequencing was performed using an Agilent custom probe set of a panel of 84 myeloid genes. We multiplexed and sequenced the samples using an IlluminaHiseq2000. Results: The mean on-target coverage for the 57 sequenced samples was 861.4x. We detected a total of 48 somatic mutations in 25 genes in 17 patients (89%) throughout the course of the disease. Five of the 25 genes were recurrently mutated (JAK2, IDH2, ASXL1, SRSF2 and, TP53). As expected, JAK2-V617F was the most commonly observed (15/17 patients). One of the patients without JAK2-V617F carried a MPL mutation and another was triple negative.At the time of follow-up, 12 patients had chronic MPN (11 stable disease and 1 spleen response) withRuxolitinib treatment for a median duration of 373 days (range 255 - 729). Among 7 progressed patients, 5 patients had additional mutation at diagnosis of MPN other than JAK2 or MPL: MPN-13 (DNMT3A and ASXL1), MPN-14 (SRSF2 and IDH2), MPN-15 (IDH2), MPN16 (U2AF1), MPN-17 (IDH2 and SRSF2) as shown in figures. On the other hand, 4/12 non-progressed patients carried additional mutations: MPN-01 (ZRSR2), MPN-05 (ASXL1, CBL, FGR, KMT2D and TET2), MPN-10 (ASXL1, CDH13, EED, EZH2, MN1, NF1 and TRRAP), MPN-11 (FOXP1). In summary, only ASXL1 and JAK2-V617F were recurrently mutated among the non-progressed group. At the time of leukemic transformation, 6 out of 7 patients acquired new mutations: MPN-13 (SETBP1 and TP53), MPN-14 (RUNX1, ASXL1, and IDH1), MPN-15 (TP53 and CASP8), MPN-16 (TP53), MPN-18 (CEBPA), and MPN-20 (ASXL1). In the remaining case (MPN-17), increase of JAK2-V617F VAF was also observed. In summary, TP53 mutation was the most common mutation to acquire by the time of leukemic transformation (n=3). Other mutations acquired by this stage were in SETBP1, RUNX1, IDH1, CEBPA, and ASXL1. We did not observe any significant difference in the allelic burden increase of JAK2-V617F from diagnosis to follow-up between the non-progressed and progressed groups (Figure A). Conclusion: There is no significant difference in mutation burden increase of JAK2-V617F between patients who progressed to secondary AML and patients that did not. Acquisition of mutations other than JAK2-V617F at both diagnosis and at follow-up is associated with the risk of transformation to secondary AML. Mutation profiling using a myeloid gene panel at timed follow-up after MPN diagnosis can be more helpful than monitoring JAK2-V617F status in these patients. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Large granular lymphocytes (LGL) are a morphologically recognizable subpopulation of lymphocytes comprising an immunophenotypically heterogeneous population of activated CD3+ T cells and CD3- natural killer (NK) cells that mediate non-MHC-restricted cytotoxicity. Increased number of circulating LGL can be found as a response to viral infections, autoimmune disease or malignant neoplasms, as a result of chronic antigenic stimulation. Of interest, LGL lymphocytosis has been reported to occur following hematopoietic cell transplantation (HCT), with a variable incidence of up to 20%. This population display improved transplant outcomes with a lower incidence of non-relapse mortality and relapse (Kim, BMT, 2013; Nann-Rütti, BBMT, 2012). The aim of the present study is to determine the risk factors associated with the development of LGL lymphocytosis after allogeneic HCT. Methods A total of 826 patients who underwent an allogeneic HCT at Princess Margaret Cancer Centre, Toronto, Canada from 2000 to 2012 were retrospectively analyzed. LGL lymphocytosis was defined as the presence of at least two of the followings: 1) Sustained peripheral blood lymphocyte count ≥3.0 x 109/L observed in at least three consecutive determinations over a period of 2-3 months; 2) Predominance (≥30%) of LGL lymphocytes in the peripheral blood, as assessed by morphologic or immunophenotypic criteria; 3) T-cell receptor monoclonality assessed by PCR. The patient population was divided into discovery and replication sets using 2 different methods: stratified randomization and propensity score matching, using relevant baseline variables such as donor type, CMV serostatus, conditioning, T-cell depletion. Results No significant imbalances were found between the discovery and replication sets in terms of relevant baseline characteristics and clinical outcomes, for both the randomly divided patients and the propensity score matched groups. The overall incidence of LGL lymphocytosis was 14.5% at 3 years. The incidence of LGL lymphocytosis was similar across all subgroups of patients, both for the randomly divided groups and the propensity score matching (P-value not significant). A multivariable analysis of the risk factors for the development of LGL lymphocytosis was performed, including the following variables: grades 3-4 acute graft-versus-host-disease (GVHD), chronic GVHD, CMV viremia, CMV serostatus of the recipient, donor type, transplant year and T-cell depletion (TCD) for GVHD prophylaxis. In the stratified randomization analysis, the following risk factors were identified: 1) Discovery set: chronic GVHD (Hazard Ratio: 8.3, 95% CI: 3.1-22.6, P