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MYCderegulation occurs in 67% of multiple myeloma (MM) cases and associates with progression and worse prognosis in MM. EnhancedMYCexpression is known to be driven by translocation or amplification events, but it only occurs in 40% of MM patients. Here, we describe a new mechanism ofMYCregulation, whereby epigenetic regulation ofMYCby increased accessibility of a cell-type specific enhancer leads to increasedMYCexpression. We found enhancer activity does not associate with enhancer hijacking events. We identified specific binding of c-MAF, IRF4, and SPIB transcription factors to the enhancer can activateMYC. In addition, we discovered focal amplification of this specific enhancer in approximately 4% of MM patients. Together, our findings define a new epigenetic mechanism ofMYCderegulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

High expression ofMYCand its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between theMYClocus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding genePVT1are enriched inMYC-intact cases. To identify genomic drivers ofMYCactivation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in theMYClocus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements betweenMYCand immunoglobulin (Ig) loci. Rearrangements betweenMYCand non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within theBCL6super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrentMYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines withoutMYCrearrangement were highly dependent on a previously uncharacterized 3’ enhancer within theMYClocus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that thePVT1promoter limitsMYCactivation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3’ rearrangements that removePVT1from its position inciswith the rearrangedMYCgene.Key pointsCRISPR-interference screens identify a conserved germinal center B cellMYCenhancer that is essential for GCB-DLBCL lackingMYCrearrangements.Functional profiling ofMYCpartner loci reveals principles ofMYCenhancer-hijacking activation by non-immunoglobulin rearrangements.

Supplementary Table 1 lists oligonucleotide sequences and cell line information

Supplementary Table 2 details known and de novo motif analysis for distal acetylation clusters

Supplementary table 4 lists ETV6-regulated gene-enhancer linkages

Supplementary Table 6 details GGAA repeat overlap analysis of new and previously published ETS factor datasets.

Contains Supplementary Figures 1-12 and additional information about Supplementary tables

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6–RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6–RUNX1+/− like B-ALL “signature” genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6–RUNX1+/− like B-ALL.Significance:We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease.See related commentary by Mercher, p. 2.This article is highlighted in the In This Issue feature, p. 1

Supplementary Table 5 annotates genome-wide candidate ETV6-repressed enhancers

Supplementary Table 3 summarizes TF ChIP-Seq results and motif enrichment statistics

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6–RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6–RUNX1+/− like B-ALL “signature” genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6–RUNX1+/− like B-ALL. Significance: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1

Distal enhancers play critical roles in sustaining oncogenic gene expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+ B-ALL “signature” genes, including likely oncogenic drivers. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of many repeat enhancer-activated genes. Together, our findings reveal a novel epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene expression program of ETV6-RUNX1+ B-ALL.SignificanceWe show that the oncogenic gene expression program of a common pediatric leukemia relies on repetitive noncoding elements that are not conserved between humans and rodents, placing important limitations on animal models for this disease. Our findings may present new opportunities for targeting cancer-specific chromatin dysregulation in leukemia.

Background Breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) is a rare malignancy originating from the periprosthetic capsule of a textured, most often macrotextured, breast implant. Identified in women whose indications for breast implants can be either aesthetic or reconstructive, the genomic underpinnings of this disease are only beginning to be elucidated. Objectives The aim of this study was to evaluate the exomes, and in some cases the entire genome, of patients with BIA-ALCL. Specific attention was paid to copy number alterations, chromosomal translocations, and other genomic abnormalities overrepresented in patients with BIA-ALCL. Methods Whole-exome sequencing was performed on 6 patients, and whole-genome sequencing on 3 patients, with the Illumina NovaSeq 6000 sequencer. Data were analyzed with the Illumina DRAGEN Bio-IT Platform and the ChromoSeq pipeline. The Pathseq Genome Analysis Toolkit pipeline was used to detect the presence of microbial genomes in the sequenced samples. Results Two cases with STAT3 mutations and 2 cases with NRAS mutations were noted. A critically deleted 7-Mb region was identified at the 11q22.3 region of chromosome 11, and multiple nonrecurrent chromosomal rearrangements were identified by whole-genome sequencing. Recurrent gene-level rearrangements, however, were not identified. None of the samples showed evidence of potential microbial pathogens. Conclusions Although no recurrent mutations were identified, this study identified mutations in genes not previously reported with BIA-ALCL or other forms of ALCL. Furthermore, not previously reported with BIA-ALCL, 11q22.3 deletions were consistent across whole-genome sequencing cases and present in some exomes. Level of Evidence: 5

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.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to cobind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and point to Ets1-mediated enucleation of Notch–Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition. Significance: Notch signaling controls developmentally important and tissue-specific activities, raising barriers for developing anti-Notch therapies. Pivoting away from pan-Notch inhibitors, we show antileukemic but less toxic effects of targeting ETS1, a T-cell NOTCH1 cofactor. These results demonstrate the feasibility of context-dependent suppression of NOTCH1 programs for the treatment of T-ALL. This article is highlighted in the In This Issue feature, p. 127

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a subtype of T-cell lymphoma with much of its pathogenesis poorly understood. Mutations in the JAK/STAT pathway have been identified in a substantial proportion of cases, as well as copy number aberrations (CNAs). However, a more discrete characterization is vital to identify those with a germline predisposition to develop BIA-ALCL when exposed to a textured implant, and to identify targets for precision anti-BIA-ALCL therapeutics.To determine if there are recurrent gene mutations in BIA-ALCL patients in a cohort at a single institution.Ten patients were included that were diagnosed with BIA-ALCL between April 2015 and May 2020. Whole exome sequencing (WES) was performed on 8 tumor and normal BIA-ALCL samples and whole genome sequencing (WGS) was performed on 3 BIA-ALCL fluid samples. Samples were sequenced on an Illumina NovaSeq 6000. Data was analyzed using the Illumina DRAGEN Bio-IT Platform v3.8 workflow.This study was carried out at Barnes Jewish Hospital/Siteman Cancer Center.Patients were included if they have a pathology proven diagnosis of BIA-ALCL.Incidence and quantification of recurrent mutations in tumor samples of patients with BIA-ALCL evaluated via WES and WGS.Nearly all BIA-ALCLs showed substantial CNAs. Most patients had missense mutations in large genes with predominantly C to T transitions. Two cases had STAT3 mutations, consistent with previously reported data. Whole genome sequencing showed a critically deleted 7Mb region on chromosome 11 at 11q22.3 region in all 3 samples evaluated. This region contains the ATM gene which upon deletion in mouse models has shown an increased number of tumors. Clinically, the overall prognosis of BIA-ALCL was favorable; one patient passed from complications.There are no highly recurrent mutations in BIA-ALCL, however, 11q22.3 deletions were consistent across WGS cases and present in some exomes. Given the small sample size due to the rarity of the disease, genomic evaluation of a larger cohort is necessary to confirm this finding. Financial support for this study was provided by the Aesthetic Surgery Education Research Foundation and Barnes Hospital Foundation.

Subtypes of B-lymphoblastic leukemia (B-ALL) are defined by unique genetic aberrations and gene expression programs. ETV6-RUNX1+ (E-R+) B-ALL, a common pediatric subtype, shares a gene activation signature with “ETV6-RUNX1-like” B-ALL that lacks the E-R fusion gene. Both E-R+ and E-R-like B-ALL show frequent inactivating mutations or deletions of ETV6, which encodes an ETS family transcriptional repressor (core binding motif GGAA/T). Signature genes overexpressed in E-R+ B-ALL, such as EPOR and PIK3C3, were shown to sustain the malignant phenotype. We performed H3K27ac ChIP-Seq and ATAC-Seq on 26 B-cell cancer cell lines to identify subtype-specific active enhancer-like elements. Unbiased K-means clustering identified distal elements that were selectively acetylated in four E-R+ B-ALL cell lines (all with inactivation of the second ETV6 allele) and in a fifth B-ALL cell line with biallelic ETV6 deletion. Motif analysis of this cluster showed strong enrichment for tandem repeats of the sequence “GGAA” (p Doxycycline-inducible expression of ETV6 in ETV6-deficient, E-R+ B-ALL cells led to significant growth inhibition. Restored ETV6 bound extensively to GGAA repeats (80% of ETV6 binding sites), repressed repeat enhancer acetylation, and repressed expression of adjacent E-R+ signature genes (n=40). Repression of a published E-R+ signature gene set was significant by GSEA analysis (p GGAA repeat enhancers are a known driver of Ewing sarcoma, where they are activated by oncogenic fusions of the ETS activators FLI1 or ERG. Active GGAA repeat enhancers and repeat-regulated genes in E-R+ B-ALL show only partial overlap with repeat-regulated genes in Ewings, possibly due to tissue-specific factors. Wild-type ERG and FLI1 are highly expressed in B-ALL. We found that knockdown of ERG, but not FLI1, led to significant downregulation of repeat enhancer-regulated E-R+ signature genes in B-ALL. ChIP-Seq showed that the ERG binds nearly all active GGAA repeat enhancers in three ETV6-deficient B-ALL cell lines, but not in ETV6-intact B-ALL. We identify GGAA repeat enhancers, sustained by ETV6 deficiency and strong ERG expression, as a unifying mechanism for a subtype-specific oncogenic gene expression program in B-ALL. Our findings may have crucial implications for E-R+ B-ALL modeling, as most GGAA repeats are not conserved between mice and humans. Mechanisms that activate leukemia-specific repeat enhancers may represent appealing targets for future therapeutic development. Citation Format: Joshua W. Goldman, Rohan Kodgule, Alexander C. Monovich, Travis Saari, Cody N. Hall, Niharika Rajesh, Juhi Gupta, Athalee Aguilar, Noah Brown, Mark Y. Chiang, Marcin P. Cieslik, Russell J. Ryan. ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1461.

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: Copy number alterations (CNA) have been described in childhood precursor B-lineage acute lymphoblastic leukemia (B-ALL) which in conjunction with chromosomal abnormalities drive leukemogenesis. There is no consensus on the clinical incorporation of CNA in B-ALL. An integrated genomic classification (IGC) has been proposed which includes CNA and cytogenetics. Methods: We correlated this IGC with immunophenotypic minimal residual disease (MRD) as well as other standard criteria for 245 patients of B-ALL such as National Cancer Institute (NCI) risk, D+8 prednisolone response, cytogenetics, and ploidy status. Results: MRD was detectable in 81 patients (33.1%). The most common abnormalities were seen in CDKN2A/B (25.7%) followed by PAX5(20%), ETV6(16.7%), IKZF1(15.5%), Rb1(5.3%), BTG (3.3%), EBF1(2.0%), and PAR1(0.8%). On integrating CNA into the IGC, 170 patients (69.4%) were classified into good genomic risk (GEN-GR) whereas 75 (30.6%) belonged to the poor genomic risk (GEN-PR) category. The IGC showed a significant correlation with MRD and NCI risk. The presence of CNA predicted MRD clearance in intermediate cytogenetics group. Conclusion: These data seem to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The IGC is of prognostic relevance and offers an additional avenue for prognostication and risk-adapted therapy.

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.

Introduction: Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. Materials and Methods: One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. Results: Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. Discussion and Conclusion: We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance.

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

Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in NPM1 mutated AML (NPM1mut 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 NPM1mut 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 NPM1mut AML.

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.