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// 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.

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.