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We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p − patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD− patients had a significantly improved survival as compared to patients who became NGS-MRD− subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD− but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission and evaluated MRD using sensitive error corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients harbored PI NGS-MRD and 40.9% harbored PC NGS-MRD (median VAF 0.76%). Patients harboring NGS-MRD had a significantly higher cumulative incidence of relapse (p=0.003), inferior overall survival (p=0.001) and relapse free survival (p

// Nikhil Patkar 1, * , Rohan Kodgule 1, 5, * , Chinmayee Kakirde 1 , Goutham Raval 1 , Prasanna Bhanshe 1 , Swapnali Joshi 1 , Shruti Chaudhary 1 , Y. Badrinath 1 , Sitaram Ghoghale 1 , Shraddha Kadechkar 1 , Syed Hasan Khizer 2 , Sadhana Kannan 3 , Dhanalaxmi Shetty 4 , Anant Gokarn 2 , Sachin Punatkar 2 , Hasmukh Jain 2 , Bhausaheb Bagal 2 , Hari Menon 6 , Manju Sengar 2 , Navin Khattry 2 , Prashant Tembhare 1 , Papagudi Subramanian 1 and Sumeet Gujral 1 1 Haematopathology Laboratory, ACTREC, Tata Memorial Centre, Navi Mumbai, India 2 Adult Haematolymphoid Disease Management Group, Tata Memorial Centre, Mumbai, India 3 Biostatistics, ACTREC, Tata Memorial Centre, Navi Mumbai, India 4 Dept of Cytogenetics, ACTREC, Tata Memorial Centre, Navi Mumbai, India 5 Homi Bhabha National Institute, Training School Complex, Mumbai, India 6 Haemato-Oncology, CyteCare Cancer Hospital, Bangalore, India * These authors contributed equally to this work Correspondence to: Nikhil Patkar, email: nvpatkar@gmail.com Keywords: acute myeloid leukemia; NPM1; measurable residual disease; next-generation sequencing; multiparameter flow cytometry Received: April 16, 2018 Accepted: November 16, 2018 Published: November 27, 2018 ABSTRACT Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in NPM1 mutated AML ( NPM1 mut AML). However, the clinical utility of next generation sequencing (NGS) to detect MRD in AML remains unproven. We analysed the clinical significance of monitoring MRD using ultradeep NGS (NGS-MRD) and flow cytometry (FCM-MRD) in 137 samples obtained from 83 patients of NPM1 mut AML at the end of induction (PI) and consolidation (PC). We could monitor 12 different types of NPM1 mutations at a sensitivity of 0.001% using NGS-MRD. We demonstrated a significant correlation between NGS-MRD and real time quantitative PCR (RQ-PCR). Based upon a one log reduction between PI and PC time points we could classify patients as NGS-MRD positive ( 1log reduction). NGS-MRD, FCM-MRD as well as DNMT3A mutations were predictive of inferior overall survival (OS) and relapse free survival (RFS). On a multivariate analysis NGS-MRD emerged as an independent, most important prognostic factor predictive of inferior OS (hazard ratio, 3.64; 95% confidence interval [CI] 1.58 to 8.37) and RFS (hazard ratio, 4.8; 95% CI:2.24 to 10.28). We establish that DNA based NPM1 NGS MRD is a highly useful test for prediction of relapse and survival in NPM1 mut AML.

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM. To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9. To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such as IRF4 and MAF, that play key roles in MM pathogenesis. ChIP-sequencing for these enhancer-associated TFs and luciferase reporter assays targeting their binding sites confirmed the binding and involvement of IRF4 and MAF in activating enhancer elements in MM cells with intact MYC loci. MYC abnormalities are well-known secondary genetic events that trigger the progression of precursor diseases to MM. To define the genetic status of the identified enhancer elements in malignant cells, we examined whole-genome sequencing (WGS) data of 906 MM patients from the MMRF CoMMpass cohort. We found focal amplification of the enhancer region in 2.8% (n = 26) of patients. Transcriptional analysis on the same patient cohort revealed a significant increase in MYC mRNA levels in enhancer-amplified patients compared to MM cases with no MYC aberrations. These results indicate that enhancer activity is required to induce MYC expression and progression of patients with intact MYC loci. Collectively, our findings reveal a novel mechanism of MYC deregulation in malignant plasma cells: selective gain of chromatin accessibility at the enhancer elements and amplification of the enhancer region allow for binding of regulatory factors IRF4 and MAF, which increase the transcription of MYC in the absence of the known MYC chromosomal abnormalities. Our results point to the importance of non-coding regulatory elements and their associated TF networks as drivers of MM progression and suggest a new approach to identify predictive biomarkers and therapeutic targets that could improve patient outcomes in MM and other cancers. Disclosures Fulco: Bristol Myers Squibb: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Introduction The monitoring of a patient's response to chemotherapy, called, measurable residual disease (MRD) is one of the most important predictors of outcome in Acute Myeloid Leukemia (AML). Although universally applicable, FCM-MRD for AML suffers from low sensitivity as compared to precursor B lineage acute lymphoblastic leukemia. Here, we evaluated the clinical utility of error corrected next generation sequencing (NGS) to detect MRD (NGS-MRD) in AML using single molecule molecular inversion probes (smMIPS). We compare NGS-MRD and FCM-MRD and determine their impact on patient outcome. We demonstrate that error corrected NGS-MRD at early timepoints in therapy is an independent and significant predictor of outcome in patients of AML treated with conventional therapies. Methods We created a 35 gene "hotspot" panel comprising of a pool of 302 smMIPS. In brief, this panel covers regions of 35 commonly mutated genes in AML.FLT3-ITD were detected using a novel one-step PCR based NGS assay. Post mapping, singleton reads (originating from one UMI) were discarded and consensus family based variant calling was performed. We then created a site and mutation specific error model to ascertain the relevance of an observed variant at each site. A limit of detection (LOD) experiment demonstrated a lower detection limit of 0.05%. For FLT3-ITD the LOD was 0.002%. A total of 393 adult patients of AMLwere accrued over a period of six years.Patients were treated with standard 3+7 induction followed by 3 doses of HiDAC. Allogeneic bone marrow transplantation was offered where feasible. Somatic mutations at diagnosis were evaluated using a smMIPS based 50 gene myeloid panel which was applicable to 327 patients [83.2% of AMLs, median 2 mutations per case (range 1 - 6 trackable mutations)].MRD assessment could be performed in 201 adult patients of AML in morphological remission (not performed in the rest because of suboptimal quality DNA at MRD time points or missing sample).Samples were sequenced on multiple S4 flow cells of a NovaSeq 6000 using 150PE chemistry.FCM-MRD was obtained from the bone marrow at end of induction (PI, n=200) and end of first consolidation cycle (PC, n=98). NGS-MRD sample also obtained at the same time points (PI, n=196& PC, n=127) from the bone marrow (n=266) or peripheral blood (n=45). Results The interaction of mutations that were trackable at diagnosis can be seen in Figure 1A. A total of 345 mutations could be detected in 196 patients (Figure 1B) with a median VAF of 1.01% [0.82% after exclusion of mutations in DNMT3A, TET2, ASXL1 (DTA) genes; (median of 2 mutations for PI and one for PC timepoint)]. The median consensus read coverage was 11,127 for the smMIPS assay, whereas for the FLT3-ITD assay it was 13,96,366.The median follow-up of the cohort was 42.3 months. The presence of NGS-MRD (70.9%) was associated with inferior overall survival (OS; p=0.001) [hazard ratio(HR)- 2.24; 95% confidence interval (CI)- 1.47 to 3.43] and relapse free survival (RFS; p=0.0002) [HR- 2.28; 95% CI- 1.58 to 3.31] at PI time point as well as PC time points [40.94% positive; OS (p=0.008)(HR- 1.92; 95% CI- 1.14 to 3.22) and RFS (p=0.004)(HR- 1.90; 95% CI- 1.18 to 3.05)].Similarly, FCM-MRD (44%) was predictive of inferior OS (p=0.0002)(HR- 2.08; 95% CI- 1.38 to 3.13)and RFS (p=0.0008)(HR- 1.81; 95% CI- 1.26 to 2.60) at PI as well as PC time points [21.4% positive, OS (p=0.04)(HR- 1.87; 95% CI- 0.89 to 3.91) and RFS (p=0.001)(HR- 2.38; 95% CI- 1.17 to 4.81)]. On multivariate analysis post induction NGS MRD emerged as the most important independent prognostic factor predictive of inferior outcome for OS [HR- 1.94; 95% CI-1.15 to 3.27; (p Conclusion In conclusion, we demonstrate that error corrected panel-based sequencing is feasible for MRD monitoring in AML and may offer an advantage over existing techniques. Maximum clinical utility may be leveraged by combining FCM and NGS modalities. Figure Disclosures No relevant conflicts of interest to declare.

Atypical breakpoints and variant APL cases involving alternative chromosomal aberrations are seen in a small subset of acute promyelocytic leukemia (APL) patients. Over 7 different partner genes for RARA have been described. Although rare, these variants prove to be a diagnostic challenge and require combination of advanced cytogenetic and molecular techniques for accurate characterization. Heterogeneity occurs not only at the molecular level but also at clinico-pathological level influencing treatment response and outcome. In this case series we describe the molecular heterogeneity of APL seen in a single tertiary referral centre with a focus on seven variant APL cases from a single tertiary cancer center in India over a period of two and a half years. We discuss five cases with PLZF-RARA fusion and two novel PML-RARA variants, including a Bcr3 variant involving fusion of PML exon4 and RARA exon3 with an additional 40 nucleotides originating from RARA intron2, another involving exon 6 of PML and exon 3 of RARA with addition of 126 nucleotides, which mapped to the central portion of RARA intron 2 To the best of our knowledge this is the first of kind case series from India

Introduction: Mainstay of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR) as evident by less than 5% blasts. Post remission strategies then focus on either consolidation chemotherapy or allogeneic bone marrow transplantation (aBMT). However, not all patients by this remission criterion achieve long term remission and a subset of patients' relapse. This relapse originates from further expansion chemoresistant clones. Detection of minimal residual disease (MRD) following chemotherapy, early in the course of treatment, is highly predictive of outcome and offers a window of opportunity to intensify treatment and prevent relapse as seen in ALL. Here, we describe assessment of immunophenotypic MRD using a 10-colour two tube assay and a combination of difference from normal and leukemia associated immunophenotype (LAIP) approach in patients of adult AML. Methods: We accrued 310 patients of adult (>18 years) AML, other than with t(15;17), over a 66-month period (1st July 2012- 31st December 2017) after obtaining informed consent. All patients received standard induction chemotherapy. MRD testing was done post induction and after first consolidation with high dose cytarabine. Patients accrued from 1st July 2012 to 28th February 2015 (85 patients) were processed using a three tube 8 colour MRD assay. Subsequently samples of 225 patients were processed using a two tube 10 colour MRD assay. Identical panel was used for diagnostic sample, post induction and post consolidation. 500,000 events were acquired per tube with the 3-tube assay and 1.6 million events per tube obtained per tube with the 2 tube, 10 colour assay. Analysis of MRD was done using Kaluza 1.3 by a combination of difference from normal approach that focused on the development of Myeloid progenitors to mature cells and LAIP approaches. Cytogenetic studies by conventional karyotyping and FISH was done as per NCCN version 2 2014 recommendations. Patients were classified into favorable, intermediate and poor cytogenetic risk. The presence of FLT3-ITD, NPM1 and CEBPA mutations were detected by a fragment length analysis-based assay. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the MRD assays, cytogenetic and molecular risk groups were analyzed for their impact on OS and RFS using log rank test. Results: The median age of entire cohort was 33 years (M : F = 1.6 : 1). Based on cytogenetics, 35.1% were classified as favorable risk whereas 52.5% and 12.2% were intermediate and poor risk respectively. FLT3-ITD, NPM1 and CEBPA mutations were detected in 21.9%, 29.0% and 7.7% of patients respectively. Post induction MRD was assessed in 298 patients of which 114 (38.3%) had detectable residual disease (range 0.004-22.6%, median:0.9%). Post consolidation MRD was assessed in 186 patients of which 49 (26.4%) were MRD positive (range 0.002-19.8%, median: 0.37%).The 3 year OS, RFS and median RFS of the entire cohort was 59.0%, 44.8% and 18.7 months respectively. Poor risk cytogenetics as expected was predictive of inferior OS (median OS = 14.6 months vs not reached, p=0.05) as well as RFS (median RFS = 9.3 months vs 18.6 months, p=0.003 respectively). FLT3, NPM1 and CEBPA mutations were not informative as predictors of outcome.The presence of MRD at post induction time point predicted an inferior OS [(p=0.08), HR:1.53; 95% CI (0.93-2.51)] & RFS [(p=0.0002), HR:2.14; 95% CI (1.4-3.3)] as compared to the MRD negative group. Similarly, patients harboring MRD at the end of consolidation had an inferior OS [(p=0.04), HR:1.83; 95% CI (0.94-3.6)] & RFS [(p=0.02), HR:1.79; 95% CI (1.04-3.09)] as compared to the MRD negative group. On multivariate analysis post induction FCM MRD emerged as the most important independent prognostic factor predictive of inferior outcome for RFS [(p=0.01); HR:2.14; 95% CI (1.17 - 3.4)] followed by poor cytogenetic risk [(p=0.05); HR:1.8; 95% CI (1.0 to 3.26)]. Conclusion: Our data demonstrates that MRD by flow cytometry at end of induction as well as consolidation are important prognostic factors together with known factors such as high risk cytogenetics. AML MRD is a very useful guide for post remission strategies in AML and should be incorporated into routine treatment algorithms. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Introduction: One of the mainstays of chemotherapy in acute myeloid leukemia (AML), other than acute promyelocytic leukemia, is induction with a goal to achieve morphological complete remission (CR) as evident by less than 5% blasts. Post remission strategies then focus on either consolidation chemotherapy or allogeneic bone marrow transplantation (aBMT). However, not all patients by this remission criterion achieve long term remission and a subset of patients relapse. This relapse originates from further expansion chemoresistant clones. Detection of minimal residual disease (MRD) following chemotherapy, early in the course of treatment, is highly predictive of outcome and offers a window of opportunity to intensify treatment and prevent relapse. Here, we describe assessment of immunophenotypic MRD using a 10-colour two tube assay and a "difference from normal" approach. Methods: We accrued 100 consecutive patients of adult (>18 years) AML, other than with t(15;17), over a 14 month period after obtaining informed consent. All patients received "3+7" induction therapy with daunorubicin and cytarabine. If patients were in morphological CR at end of induction, they either received 3 courses of 12-18 gm/m2 high dose cytarabine (HiDAC) or aBMT if feasible.MRD testing was done at two time points, post induction and post 1st Cycle HiDac using a two tube 10 colour assay. (CD15, CD13, CD19, CD34, CD56, CD7, CD45, CD11b, HLA-DR, CD117, CD14, CD123, CD64, CD33, CD36 & CD38). A minimum of one million events were acquired per tube on a Navios flow cytometer. Identical panel was used at MRD time points as well as on the diagnostic sample. Analysis of MRD was done using Kaluza 1.3 by a difference from normal approach that focused on the development of progenitors to monocytes. Conventional karyotyping and FISH was done as per standard recommendations and patients were classified into favorable, intermediate and poor cytogenetic risk. The presence of FLT3-ITD, NPM1 and CEBPAmutations was detected by a fragment length analysis based assay. Overall survival (OS) was calculated from start of induction therapy to time of last follow up or death. Relapse free survival (RFS) was calculated after achieving of 1st remission (CR) till relapse or death or last follow up if in CR. Results of the MRD assays, cytogenetic and molecular risk groups were analyzed for their impact on OS and DFS. Results: A total of 100 AML patients were treated and followed up over a 14 month period. Based on cytogenetics, 36.7% were classified as favorable risk whereas 54.1% and 9.2% were intermediate and poor risk respectively. FLT3-ITD, NPM1 and CEBPA mutations were harbored by 9%, 19% and 8% of patients respectively. The OS was 63% and RFS was 50% with a median follow up of 6 months. Of these, 24 had induction death and 17 had refractory disease. Post induction MRD was assessed in 70 patients of which 25 (35.7%) had detectable residual disease (range 0.02-55%, median:1.5%). Post consolidation MRD was assessed in 49 patients of which 14 (28.6%) were MRD positive (range 0.002-7.7%, median: 0.03%). Favorable risk cytogenetics was predictive of better RFS (p=0.007) but not OS. FLT3-ITD positive status was associated with worse OS (p=0.01) but not RFS. Patients harboring MRD at the end of induction were associated with worse OS (p=0.08) & RFS (p=0.04), whereas post consolidation positive MRD status was strongly associated with inferior RFS (p=0.04). Conclusion: Our data is in agreement with other studies that determination of immunophenotypic MRD is extremely important in predicting outcome. AML MRD is a very useful guide for guiding post remission strategies in AML and should be incorporated into routine treatment algorithms. Acknowledgments: Dr Nikhil Patkar is supported by the Wellcome Trust - DBT / India Alliance through an Intermediate Fellowship for Clinicians and Public Health Researchers. This research is supported through an India Alliance grant (IA/CPHI/14/1/501485). Disclosures Patkar: Wellcome Trust-DBT India Alliance: Research Funding.