ALBERT

Description: Introduction: Allogeneic hematopoietic stem cell transplantation (Allo-HCT) is the only curative treatment for myelodysplastic syndrome (MDS). The portion of patients referred for transplant and the proportion who are subsequently transplanted is unknown. Aim: Tostudy the frequency of allo-HCT in pts with MDS and identify factors associated with transplant referral and barriers to transplant. Methods: Pts were included if they were under the age of 75 and seen at our center by a leukemia physician between 2008-2015 and within 6 months of MDS diagnosis. Pts were eligible for allo-HCT if they did not have major organ dysfunction, i.e. left ventricular ejection fraction 2mg/dL, FEV1

Description: Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio

Description: Key Points KMT2C mutations occur in 15% and 25% of patients with cHCL and vHCL, respectively, along with CCND3 and U2AF1 mutations each in 13% of vHCLs. NF1, NF2, N/KRAS, and IRS1 alterations contribute to clinical resistance to vemurafenib treatment in patients with cHCL.

Description: The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are characterized by mutations, which drive JAK-STAT pathway activation. Several studies have demonstrated the presence of recurrent somatic mutations outside of the JAK-STAT pathway, which accumulate over time, and may impact disease phenotype and outcome. We sought to determine the influence of somatic mutations on clinical phenotype and prognosis. We sequenced a total of 30 genes recurrently mutated in myeloid malignancies in a cohort of 162 MPN patients (pts) using a next generation sequencing platform. The cohort included 49 pts with ET, 26 PV, 38 Primary Myelofibrosis (MF), 11 Post ET MF, 14 Post PV MF, 12 with leukemic transformations of MPN (LT), 7 with MPN-unclassified (MPN-U) and 5 others. Median age was 59 years and 79 were men. A Total of 288 gene mutations were identified with the most commonly mutated genes being JAK2 (n=121, 74%), TET2 (n=31, 19%), DNMT3A (n=18, 11%), ASXL1 (n=16, 10%), IDH2 (n=10, 6%), RAS (n=12, 7%), TYK2 (n=8, 5%) and TP53 (n=7, 4%). We did not find any mutations in NPM1, CBL, SRSF2 and no FLT3 -ITD. CALR was not assessed in 20 pts and these were excluded from mutation number analysis. Importantly, we identified a relationship between the absolute number of mutations found per pt, disease phenotype, and age (table 1). Pts with/without prior chemotherapy or radiotherapy exposure did not have a difference in mutation number (1.5 vs. 1.9). Cases of ET or PV with fibrotic transformation had more mutations in ASXL1, RAS, EZH2, PHF6 and MPL than pre fibrotic ET or PV suggesting these may be relevant in disease progression and development of fibrosis. Mutations in TET2, RAS and PHF6 were more frequent in cases with LT compared to those with chronic phase MPN. Pts over 40 were more likely to have mutations in TET2 (p=0.026) and JAK2 (p=0.019) and ASXL1 mutations were more common in pts with abnormal cytogenetics than in those with normal cytogenetics (p=0.003). Thrombotic events, which are an important cause of morbidity in MPN patients, negatively correlated with mutations in ASXL1 (p=0.044). Prognosis as measured by DIPPS and DIPSS-Plus scores appeared to correlate with the average number of mutations found in MF patients (table 2). We examined several cases for which serial samples were available, and noted the acquisition of new mutational events despite ongoing therapy. We noted that the most commonly acquired mutations occurred in epigenetic modifying (DNMT3A, TET, IDH, ASXL1) and in growth signaling pathway (RAS, CBL) genes. These occurred despite active therapy and often without an overt change in clinical phenotype. Further details of these serial samples will be presented. We conclude that the number and spectrum of somatic mutations correlate with disease phenotype of MPN. Younger pts have fewer mutations, as do pts with normal cytogenetics. JAK2 and TET2 mutations were more common in older pts. We show that a subset of pts acquire mutations in epigenetic modifiers and in genes involved in growth signaling pathways during disease course, and that mutations in TET2, RAS and PHF6 were enriched at the time of leukemic transformation. Taken together, these results indicate that mutations outside the JAK-STAT pathway influence disease phenotype, and that the acquisition of mutations over time may predict for disease progression. Serial evaluation of mutational burden over time therefore warrants exploration in the clinical setting. Table 1. Average number of mutations appeared to correlate with disease phenotype, age and abnormal cytogenetics. Average Number of Mutations N Mean (SD) P-value Age 〈 40 years 13 1.4 (0.9) 0.026 Age 〉 40 years 12 2 (1) No Thrombosis 113 2 (1) 0.712 Thrombosis 28 1.9 (1) Normal Cytogenetics 64 1.8 (0.9) 0.016 Abnormal Cytogenetics 40 2.3 (1.2) ET/PV/PMF 99 1.8 (0.8) 0.029 LT 10 3 (1.5) ET/PV 66 1.6 (0.7) 0.01 Post ET/PV MF 22 2.3 (1.1) ET 44 1.6 (0.7) 〈 0.001 PV 22 1.5 (0.9) PMF 33 2.2 (0.9) Post ET/PV MF 22 2.3 (1.1) LT 10 3 (1.5) Table 2. Disease prognostic scores in MF appear to correlate with the average number of mutations found per patient. Risk category N Average number mutations DIPSS Low 8 1.5 Intermediate-1 19 2.4 Intermediate-2 9 1.8 High 1 5 DIPSS-Plus Low 6 1.5 Intermediate-1 12 2 Intermediate-2 14 2.2 High 1 5 Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Description: Few studies have compared treatment outcomes and disease complications between classical and variant hairy cell leukemia (HCL). We reviewed records of patients (pts) with HCL treated at Memorial Sloan Kettering Cancer Center between 1983 and 2013 and identified 331 pts. To reduce bias we limited analysis to the 183 pts who were reviewed and treated at MSKCC within 3 months of diagnosis (table 1). The median follow-up was 46.8 months. Median overall survival (OS) for the entire cohort was not reached and 5 and 10 year OS was 94% and 83% respectively. Median OS for classical and variant HCL was not reached in either group (Fig 1) while 5 and 10 year OS appeared equal. The time to next treatment (TNT) following initial therapy was longer for classical HCL (Fig 2). Pts with classical HCL were also more likely to achieve remission with first therapy and required fewer individual lines of therapy (Table 1). Cladribine was used first line in 122 pts and resulted in a median TNT of 138 months (97.1-NA), pentostatin was used in 9 pts and resulted in similar remission duration as cladribine with TNT of 81 months (80.5-NA) (p=0.82). 5 pts had abnormal cytogenetics at diagnosis and this did not influence OS when compared to the 62 pts who had a normal karyotype with estimated 5 year OS of 100% and 97%, respectively. 31 pts required treatment for disease relapse. The median time to 3rd therapy was not reached however 72% and 52% of all pts were estimated to require a 3rd treatment at 5 and 10 years, respectively. Cladribine was used to treat 1st relapse in 18 pts while 5 were treated with the combination of cladribine and rituximab. This resulted in a median TNT of 66.3 (37.2-NA) months in the cladribine group, median TNT was not reached for the combination group. We found that initial treatment of HCL with cladribine appeared to result in a longer disease remission when compared with the second treatment with a median TNT of 138 and 66 months respectively. 22 pts died during follow up, with 2 deaths due to HCL, 6 due to secondary malignancy, the remainder were unknown. 27 Secondary cancers were identified the most common were non-melanoma skin cancer (5), prostate cancer (5), melanoma (4) and other lymphoproliferative disorders (4). The most common reasons for needing retreatment varied depending on disease type with the recurrence of cytopenias accounting for 23/25 pts with classical HCL and only 1 with variant HCL. Symptomatic splenomegaly prompted re-treatment in 4/5 with variant and 3/25 classical HCL. B symptoms were uncommon occurring in 1 pt with classical and 1 with variant disease at relapse. The major complication of 1st therapy was febrile neutropenia necessitating admission for intravenous antibiotics, which occurred in 40/139 pts. There were no mortalities due to bacterial sepsis following 1st therapy. We conclude that OS of pts with HCL variant is equal to that of classical disease; however patients with classical HCL have a far longer duration of first remission and a greater chance of complete remission. Patients with variant HCL have different clinical features at relapse and appear to require more lines of therapy to maintain disease control. We found that responses to cladribine following first treatment appear longer than for second treatment. The combination of cladribine and rituximab may result in a longer second remission. We did not find a higher incidence of secondary malignancy in patients with variant HCL. Table 1. Comparison of patients with classical and variant HCL. Classical HCL (N=146) Variant HCL (N=10) Median age (range) 52 (27-84) 67 (39-78) P=0.005 Men (%) 114 (78%) 6 (60%) Median WBC at diagnosis 3.7x10^9/L 9.7x10^9/L CD25 expression 146/146 0/10 BRAF* (V600E mutated/ assessed) 3/6 1/2 Number needing therapy 109 10 Indication for re-treatment 25 5 Cytopenia 23/25 1/5 splenomegaly 3/25 4/25 Median OS Not reached Not reached 5 year OS 94% 100% 10 year OS 84% 67% TNT following 1st treatment (months) 120 20 P=0.002 CR to 1st therapy 87/109 5/10 Median number of therapies (range) 1 (0-7) 4 (2-7) Splenectomy 1 3 Secondary cancers 27 0 Deaths 15 1 *BRAF mutation was infrequently assessed as we analyzed patients up to 2013. Figure 1. OS for classical and variant HCL is equal despite the shorter remission duration and more frequent need for re-treatment in HCL variant. Figure 1. OS for classical and variant HCL is equal despite the shorter remission duration and more frequent need for re-treatment in HCL variant. Figure 2. Remission duration following first therapy is significantly longer in patients with classical than variant HCL. Figure 2. Remission duration following first therapy is significantly longer in patients with classical than variant HCL. Disclosures Park: Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy.

Description: Background: We previously reported potent anti-tumor activity of the oral BRAF inhibitor vemurafenib in patients with relapsed or refractory BRAF mutant hairy cell leukemia (HCL) (Park et al. ASH 2014). According to the study design, patients whose disease relapsed following the initial treatment were allowed to be re-treated with vemurafenib. Here we report the clinical outcome of patients who were retreated with vemurafenib at relapse following initial treatment, as well as the result of genomic analysis that provided an insight into mechanisms of resistance to BRAF inhibition in HCL. Patients and Methods: Patients with BRAF mutant HCL who were refractory or resistant to purine analogs, or who had ≥2 relapses with an indication for treatment (ANC ≤1.0, HGB ≤10, or PLT ≤100K) were enrolled. Eligible patients received vemurafenib 960mg twice daily for 3 months. Bone marrow (BM) evaluations were performed after 3 months to assess response. Patients with partial (PR) or complete response (CR) with detectable minimal residual disease were allowed to receive vemurafenib for up to 3 additional months. After a maximum of 6 months of therapy, patients were observed with monthly CBC. At disease relapse with peripheral blood (PB) counts low enough to meet the initial eligibility criteria, re-treatment with vemurafenib was allowed until disease progression or unacceptable toxicity. Serial PB and/or BM samples were collected for targeted next-generation sequencing analysis of a 300-gene panel to detect contributors to resistance and genes collaborating with BRAF mutations in HCL. Results: 26 patients have been enrolled. 2 patients discontinued treatment before response assessment: 1 patient due to primary refractory disease to vemurafenib and 1 patient due to grade 3 photosensitivity. 24 patients completed at least 3 months of treatment, and therefore are available for efficacy evaluations. Of the 24 evaluable patients, all patients achieved response (10 CR and 14 PR) with the overall response rate of 100% when assessed after 3 months of vemurafenib. With the median follow up of 11.7 months (range, 1.3 - 25.4 months), 7 patients experienced disease relapse (3 previous CR and 4 PR). Of the 7 relapse patients, 6 met re-treatment criteria and restarted vemurafenib. 4 of the 6 patients regained response (all PR) with complete hematologic recovery and remain on therapy. 2 patients discontinued re-treatment before response assessment: 1 patient due to grade 2 photosensitivity and fatigue, and 1 patient due to resistant disease with refractory cytopenia and a rapid increase in splenomegaly. Targeted genomic analysis in 20 patients pre-vemurafenib revealed at least 1 somatic alteration coexisting with the BRAF V600E mutation in every patient, including deletion of 7q in more than half of patients and recurrent mutations in MLL3 and MED12 (Figure). Genomic analysis of the patient with de novo resistance to vemurafenib identified a missense mutation in IRS1 (Insulin Receptor Substrate 1; IRS1 P1201S) in addition to the BRAF V600E mutation. Functional characterization of the IRS1 P1201S mutation in vitro revealed potent induction of MAP kinase and PI3K-AKT signaling by the IRS1 mutant relative to wildtype, consistent with prior knowledge that IRS1 activates both MAP kinase and PI3K-AKT signaling. These data suggest that bypass activation of ERK and parallel activation of the AKT pathway contributed to de novo vemurafenib resistance. In the patient with acquired resistance to vemurafenib, genetic analysis of pretreatment, remission and relapse PB mononuclear cells revealed emergence of 2 separate, activating subclonal KRAS mutations at relapse. The mutations in KRAS were not seen at pretreatment or at remission. Activating RAS mutations are well known mediators of vemurafenib resistance in BRAF V600E-mutant malignancies, and, in this case, the detection of KRAS mutations coincided with clinical relapse and insensitivity to vemurafenib. Conclusions: Despite high response rates after a short course of vemurafenib in most patients, we observed de novo and acquired resistance to vemurafenib. Serial genomic analysis revealed ERK-dependent and independent mechanisms of BRAF inhibitor resistance in HCL. Our data provide the first insights into genetic mechanisms of RAF inhibitor resistance in HCL and suggest combinatorial therapeutic strategies that may have a role in the therapy of HCL. Figure 1. Figure 1. Disclosures Park: Amgen: Consultancy; Juno Therapeutics: Other: Advisory Board, Research Funding; Genentech: Research Funding. Off Label Use: Vemurafenib in HCL. Stone:Agios: Consultancy; AROG: Consultancy; Juno: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Celator: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding. Rai:Nash Family Foundation: Research Funding; Karches Family Foundation: Research Funding; Nancy Marks Family Foundation: Research Funding; Leon Levy Foundation: Research Funding. Altman:Seattle Genetics: Other: Advisory board; Ariad: Other: Advisory board; Spectrum: Other: Advisory board; Novartis: Other: Advisory board; BMS: Other: Advisory board; Astellas: Other: Advisory board; assistance with abstract preparation. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: MF is a Philadelphia-negative myeloproliferative neoplasm (Ph-negative MPN) associated with driver mutations in the JAK-STAT pathway (e.g. JAK2, CALR, MPL) and other mutations in genes that lead to epigenetic changes and altered RNA splicing (e.g. TET2, SRSF2, ASXL1, EZH2). The RAS-signaling pathway is frequently altered in acute myeloid leukemia (AML) and other myeloid malignancies, but few studies have evaluated the prevalence of such mutations in patients with MF. We sought to describe the frequency and clinical features of RAS mutations in patients with MF. Methods: We analyzed next-generation sequencing data from 723 patients with a diagnosis of primary MF (N=520), post-PV MF (N=119) and post-ET MF (N=84). Sequencing was performed with either paired tumor-normal whole exome sequencing (WES; N=56) or selected gene panel for genes associated with myeloid malignancies (N=667). The following 16 genes were analyzed in all 723 patients and were considered as the common denominator for analysis: ASXL1, CALR, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, RUNX1, TET2, TP53, WT1. RAS mutations were considered as oncogenic mutations in NRAS and/or KRAS. Molecular high risk (MHR) mutations were considered as mutations in any one of the 4 genes: ASXL1, EZH2, IDH1, IDH2 (SRSF2 mutations were not included since they were not evaluated in all cases). Odds ratio (OR) and P-values were estimated using Fisher's exact test in pairwise comparisons among genetic features, and P-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure, with significant Q-values considered as those

Description: Introduction: MF is a Philadelphia-negative myeloproliferative neoplasm (Ph-negative MPN) with an heterogeneous outcome. In 2009, Cervantes et al. published the International Prognostic Score System (IPSS) to better determine outcomes in this disease. In the last decade, several recurrently mutated genes have been described in MF, some of them associated with prognostic impact in survival. We propose a novel prognostic score that incorporates molecular and cytogenetic data in patients with MF. Methods: We analyzed clinical, cytogenetic and molecular data from 623 patients with a diagnosis of primary MF (N=445), post-PV MF (N=109) and post-ET MF (N=69). Data was extracted from medical records at time of sample collection for analysis. Mutation data was obtained by next-generation sequencing analysis, performed with either paired tumor-normal whole exome sequencing (N=46) or selected gene panel for genes associated with myeloid malignancies (N=577). The following 16 genes were analyzed in all 623 patients and were considered as the common denominator for analysis: ASXL1, CALR, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, RUNX1, TET2, TP53, WT1. RAS mutations were considered as oncogenic mutations in NRAS and/or KRAS. Molecular high risk (MHR) mutations were considered as mutations in any one of the 4 genes: ASXL1, EZH2, IDH1, IDH2 (SRSF2 mutations were not included since they were not evaluated in all cases). Cytogenetic data was stratified into 4 risk categories (based on Tam et al, Blood 2009): (1) Diploid; (2) Del(13q)/Del(20q)/Trisomy 9; (3) Abnormalities of chromosomes 5, 7, 17 and complex karyotype; (4) Other abnormalities. To develop the model, the data was split into a training dataset (N=434) and a test dataset (N=189). Variables initially included in the initial training model were those with a p-value65 years), hemoglobin (25x109/L), peripheral blood blasts (〉1%), presence of constitutional symptoms, sex (male vs female), platelet count (

Description: Despite significant progress in elucidating the predictive value of somatic mutations in AML following conventional chemotherapy, the implications of extended genomic profiling in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT) are poorly understood. Moreover, little work has been performed correlating somatic mutations identified on an extended gene panel with AML relapse and survival following allo-HSCT, and comparison of the prognostic significance of somatic mutations, transplant conditioning intensity, stem cell source, AML disease types and cytogenetics in an unselected cohort of AML patients undergoing allo-HSCT has not been described. We performed mutation profiling on diagnostic bone marrow samples from 153 patients with AML who underwent allo-HSCT. Median age was 58 years (19-78), and median follow-up after allo-HSCT was 8 months (1-33). Three sequencing platforms were used (Foundation Medicine Hematology NGS panel, N=32; Institutional 28 gene myeloid panel, N=51; 6 gene PCR panel, N=70). Hazard ratios (HR) for relapse and OS were calculated for genes mutated in 5 or more patients (IDH 1/2, JAK2, DNMT3A, FLT3 ITD & TKD, NPM1, TET2, RUNX1, ASXL1 and TP53). Secondary AML was defined as AML arising from a prior myeloid disorder or therapy related AML. Categorical variables were compared with Fischers exact test and continuous variables using Kruskal-Wallis test. Cox regression estimated hazard ratios (HR) for factors associated with relapse and OS post-allo-HSCT. Significant factors were included in multivariate analysis. Factors associated with significantly reduced OS following allo-HSCT included blasts over 5% prior to allo-HSCT, secondary AML, therapy-related AML, monosomal karyotype, mutant RAS and mutant TP53. Blasts over 5% before allo-HSCT, complex or monosomal karyotype and TP53 mutation were associated with risk of relapse (Table 1). No other somatic mutations predicted relapse. Median OS from diagnosis for TP53-mutated AML patients undergoing allo-HSCT was 6 months (Fig 1). Allo-HSCT characteristics were not associated with relapse or OS in this cohort (Table 1). Given its association with OS and relapse, we focused subsequent analyses on patients with mutant TP53. No significant difference in conditioning or donor source was noted between TP53-mutated and wild-type patients. Mutations in TP53 were associated with NCCN poor risk AML, complex karyotype and monosomal karyotype (Table 2). Three patients (33%) with TP53 mutations did not have a complex or monosomal karyotype but did have either MLL rearrangement (N=1) or adverse cytogenetics with del(5q) (N=2). While the number of patients with TP53 mutations was small in this unselected cohort (9/83, 11%), multivariate analysis found that mutations in TP53 and presence of over 5% blasts before allo-HSCT were both predictive of relapse after transplant (table 3). In the TP53-mutant group, 4/9 patients relapsed and 5/9 patients died. 3 died due to non-relapse mortality, and 2 died due to AML. 2 relapsed patients remained alive at last follow-up; one received azacitidine/DLI and attained a flow-cytometry-based MRD-positive CR before progressing, and the other had progressive disease following Decitabine. 3/4 relapsed patients had sequencing at the time of relapse. TP53 mutations were identified at relapse in all 3 patients, indicating that the TP53 mutated clone was involved in relapse. In summary, we found that TP53 mutations and pre-transplant bone marrow blasts were associated with relapse after allo-HSCT in this unselected cohort of AML patients. TP53 mutations were associated with NCCN high-risk AML, complex and monosomal karyotype and therapy-related AML. We did not find other somatic mutations to be associated with relapse. Further study in a larger cohort is necessary to confirm the prognostic implications of TP53 mutations and to assess the value of other mutations in predicting outcomes after allo-HSCT. The poor outcomes observed for patients with TP53-mutated AML highlight the need to assess TP53 mutation status to identify patients who may benefit from maintenance strategies or other pharmacologic or cellular approaches after transplant to reduce the risk of relapse in these patients. Disclosures Levine: Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Description: Aim: Evaluation of tandem minimal residual disease (MRD) assessment using multi-gene next generation sequencing (NGS) and multi-parameter flow cytometry (MFC) in acute myeloid leukemia (AML) pts undergoing allogeneic hematopoietic stem cell transplant (allo-HCT). Methods: MRD was measured on the same bone marrow aspirate using 10-color MFC and a targeted myeloid specific 28-gene NGS panel pre and post allo-HCT in available samples from 122 consecutive pts with AML transplanted between 2014 and 2015. Any level of MRD measured by MFC in the blast compartment was regarded as positive, while somatic mutations detected above a pre-defined variant allele frequency (VAF) threshold on bulk marrow by multi-gene NGS were regarded as positive. Mutations identified on diagnostic or relapse samples were tracked throughout the disease course. FLT3-ITD and TKD mutations were detected in a stand-alone PCR based assay and VAF was not quantified. Results: NGS (HR: 2.37 (95% CI 1.06-5.28) and HR: 3.23 (95% CI 1.21-8.62)) and MFC (HR: 2.44 (95% CI 1-5.97) and HR: 4.62 (95% CI 1.32-16.09)) predicted overall survival (OS) and time-to-relapse respectively with median observation time of 12 months among survivors. MRD detection using both assays was associated with relapse, with MRD detected by MFC being the most predictive (table 1). NGS was applicable to 85% of tested pts with probable pathogenic mutations seen at diagnosis, while all pts tested at diagnosis had abnormal blasts detected by MFC. Transplant factors including donor source, conditioning intensity, stem cell source and GVHD prophylaxis were not associated with transplant outcomes while complex and monosomal karyotype were associated with OS and time-to-relapse (table 1). Pre allo-HCT concordance rate of MRD detection using the two assays was 70% (table 2). 12 (20%) pts had detectable MRD by MFC and not NGS. Five of these patients had NGS assessment at diagnosis and on manual review of NGS results 3 of these 5 had diagnostic mutations detected on pre allo-HCT samples at VAF below threshold to call mutations. Six (10%) were MFC negative but had detectable mutations by NGS, which were typically clonal hematopoiesis (CH) type mutations with VAF ranging between 3-20%, only 1/6 of these has relapsed post allo-HSCT. MRD pre allo-HCT using both MFC and NGS was associated with relapse; however, the risk was highest in pts who had pre transplant MRD detected concurrently using both techniques and cumulative incidence of relapse was lowest in those who were MRD negative using both techniques (Figure 1A & B). No significant change in mutant DNMT3A, TET2 and JAK2 variant allele frequency (VAF) was seen between assessment at diagnosis and pre transplant, while a significant reduction in NPM1 and IDH VAF was noted. For pts in CR or CRi pre allo-HCT MRD burden quantified using MFC (on blasts) was markedly lower than the corresponding VAF of residual mutations (on bulk marrow) measured in the same sample (Figure 1C) with VAF of residual mutation ranging between 10-20% suggesting that a large bulk of cells at the time of CR were derived from the abnormal clone. We tracked pathogenic mutations identified on diagnostic samples in pts who had marrow MFC and NGS MRD assessment after transplant. In pts who relapsed, multi-gene NGS detected mutations earlier than MFC although at very low allele burden (