Kar-Tong Tan

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD)

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Mutational profiling of a MonoMAC syndrome family with GATA2 deficiency

Ling-Wen Ding1,+, Takayuki Ikezoe2,+, Kar-Tong Tan1,+, Masakazu Mori3, Anand Mayakonda1, Wenwen Chien1, De-Chen Lin1,4, Yan-Yi Jiang1, Michael Lill4, Henry Yang1, Qiao-Yang Sun1,*, and H. Phillip Koeffler1,4 1Cancer Science Institute of Singapore, National University of Singapore, Singapore 2Department of Hematology, Fukushima Medical University, 1 Hikari-ga-oka Fukushima, Fukushima 960-1295, Japan

Corrigendum: Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia (Leukemia (2016) 30 (1672-1681) DOI: 10.1038/leu.2016.69)

V Madan, P Shyamsunder, L Han, A Mayakonda, Y Nagata, J Sundaresan, D Kanojia, K Yoshida, S Ganesan, N Hattori, N Fulton, KT Tan, T Alpermann, MC Kuo, S Rostami, J Matthews, M Sanada, L-Z Liu, Y Shiraishi, S Miyano, E Chendamarai, HA Hou, G Malnassy, T Ma, M Garg, LW Ding, QY Sun, W Chien, T Ikezoe, M Lill, A Biondi, RA Larson, BL Powell, M Lübbert, WJ Chng, HF Tien, M Heuser, A Ganser, M Koren-Michowitz, SM Kornblau, HM Kantarjian, D Nowak, WK Hofmann, H Yang, W Stock, A Ghavamzadeh, K Alimoghaddam, T Haferlach, S Ogawa, LY Shih, V Mathews and HP Koeffler

Mutational profiling of acute lymphoblastic leukemia with testicular relapse

Relapsed acute lymphoblastic leukemia (ALL) is the leading cause of deaths of childhood cancer. Although relapse usually happens in the bone marrow, extramedullary relapse occasionally occurs including either the central nervous system or testis (<1–2%). We selected two pediatric ALL patients who experienced testicular relapse and interrogated their leukemic cells with exome sequencing. The sequencing results and clonality analyses suggest that relapse of patient D483 directly evolved from the leukemic clone at diagnosis which survived chemotherapy. In contrast, relapse leukemia cells (both bone marrow and testis) of patient D727 were likely derived from a common ancestral clone, and testicular relapse likely arose independently from the bone marrow relapsed leukemia. Our findings decipher the mutational spectra and shed light on the clonal evolution of two cases of pediatric ALL with testicular relapse. Presence of CREBBP/NT5C2 mutations suggests that a personalized therapeutic approach should be applied to these two patients.

Clonality and clonal evolution analysis of paediatric ALL based on B-cell receptor/T-cell receptor rearrangement

The prognosis of paediatric acute lymphoblastic leukaemia (ALL) has greatly improved in recent decades, with currently 85–90% of the patients cured with routine chemotherapy. However, once the disease relapses, the leukaemic cells often became refractory to therapy, leading to cancer death. Malignant B/T lymphocytes of ALL cells have a unique variable, diversity and joining (VDJ) junction sequence of B-cell/T-cell receptor genes (BCR/TCR), which can be used as a ‘molecular fingerprint’ to trace the clonality. To gain a better insight into ALL clonal dynamic and evolution, we initially investigated the clonality of 34 diagnosis-relapse matched paediatric ALL samples by analysing the IGH rearrangement repertoire. Genomic DNA was extracted and the VDJ junction at the IGH loci was amplified from each leukaemic sample using primer pools designed based on the FR1 region (van Dongen et al, 2003) (Table S1). Polymerase chain reaction products were gel purified, ligated with adaptors and sequenced with Hiseq4000 (PE250, Illumina, San Diego, CA, USA). Sequencing reads were mapped to the IGH database using MiXCR (Bolotin et al, 2015) and IgBLAST (https://www.ncbi.nlm. nih.gov/igblast/). Several clonal evolutionary patterns were observed: Pattern I, the dominant clone (defined as the clone having the highest clonal fractions of BCR/TCR in each case) observed at diagnosis survived chemotherapy and persisted as a dominant clone at relapse (top, Fig 1A); Pattern II, multiple clones present at diagnosis and relapse (middle, Fige 1A. IIa, subdominant clones emerged or increased at relapse; IIb, subdominant clones eliminated or decreased at relapse; IIc, persistent subdominant clones at relapse); Pattern III, minor subclone at diagnosis became dominant clone at relapse or minor subclones at diagnosis emerged as oligoclones at relapse (bottom, Fig 1A). In line with a recent finding (Bashford-Rogers et al, 2016), a large number of potential minor subclonal rearrangements (<2% of the clonal fraction) was detected at diagnosis, most of which persisted at relapse. In total, 170 index rearrangements (defined as rearrangement with clonotype fraction >2%) were detected in these 34 ALL patients. Among these index rearrangements, IGHV4-34 was the most frequently used heavy chain variable gene segment, followed by IGHV4-39 and IGHV6-1 (Fig 1B); and a highly diversified complementarity-determining region 3 (CDR3) was observed (Fig 1C). Notably, more than 45% of these index rearrangements encoding nonproductive BCR, containing either out-of-frame CDR3 or CDR3 disrupted by inside stop-codon (Fig 1D, Figure S1). The prevalence of nonproductive IGH rearrangements support the recent hypothesis that BCR may act as a tumour suppressor in most cases of precursor ALL (Muschen, 2015). Alternatively, these cells may have bypassed the BCR checkpoint by gaining a functional oncogenic driver that mimics the BCR signalling. The study was subsequently extended to another cohort of 219 paediatric ALL patients at diagnosis (Qian et al, 2017). BCR/TCR reads were extracted from the RNA sequencing data and mapped to the BCR/TCR sequence database using MiXCR. A total of 201 samples had a dominant rearrangement of either IGH/IGK/IGL or TRA/TRB (supported by more than 30 reads, Fig 2A,B, Table S2). Eighteen patients lacked expression of clonal BCR/TCR. Forty-eight patients (21%) had multiple clonal rearrangements (either ≥ 3 IGH/ or ≥ 3 IGL/or ≥ 3 TRA/or >4 IGK/or > 4 TRB (Langerak & van Dongen, 2012)), indicating a potential bi/oligoclonality of these samples. Table S3). Remarkably, 91 ALL samples (41%) carried nonproductive BCR/TCR on both alleles or on the only expressed allele. The stop-codon interrupted/frameshift mRNA had often undergone nonsense-mediated mRNA decay and, the vast majority of the non-productive BCR/TCR transcripts were only barely expressed (Fig 2D). Potential VH replacement events were detected in 12 patients (Table S4), suggesting a potential ongoing clonal diversification in these patients. Notably, unusual VJ rearrangement of IGH was detected in the dominant clone of 5 samples (Table S5). The mechanism of this VJ rearrangement, which may be attributed to the aberrant expression of RAG1/2 and AID in ALL cells, requires further study. Patients were grouped based on the expression of their haematopoietic lineage marker genes and BCR/TCR rearrangements. Most of the patients showed a good correlation between their B/T cell lineage gene expressions and their BCR/TCR rearrangement pattern (Fig 2A). However, 24 cases of B-ALL (defined by expression of CD19/CD79 etc.) surprisingly expressed a dominant TCR rearrangement (Table S6); 12 were supported by more than 100 TCR reads and thus considered as high confident rearrangements. For example, one patient (Immunophenotype: HLA-DR+/CD34 + / CD38 + /CD10-/CD19 + /CD20-/CD22 + /cyCD79a+/cyIgM/cyCD3-/CD2-/CD5-/CD7-/CD13dim/CD15dim/CD33 + / MPO-) expressed a productive TCR rearrangement in the dominant clone (TRBV28-TRBD-TRBJ2-7, 2,939 reads). In Correspondence

Diagnosis and relapse: cytogenetically normal acute myelogenous leukemia without FLT3 -ITD or MLL -PTD

Diagnosis and relapse: cytogenetically normal acute myelogenous leukemia without FLT3 -ITD or MLL -PTD

Profiling the B/T cell receptor repertoire of lymphocyte derived cell lines

BackgroundClonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations.MethodsWe systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data.ResultsRearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution.ConclusionsOur analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.

Abstract 806: MGA is a potential tumor suppressor in acute myeloid leukemia

MGA is an incompletely studied gene with a high mutation frequency in MLL-PTD AML (9%) and in core bind factor AML (8%). This gene encodes a MAX-interacting protein and is believed to act as a transcription factor that suppresses MYC binding to its target. By in silico analysis, we found that MGA is expressed in normal myeloid hematopoietic cells and AML, and the expression level is comparable with TET2 or DNMT3A. Further data mining of TCGA revealed a high frequency of inactivating mutations of the MGA gene in a variety of cancers such as various adenocarcinomas. To interrogate functionally its role in leukemogenesis, lentiviral constructs containing either shRNA or CRISPR-sgRNA targeted to different regions of the MGA gene were generated. MGA expressing AML cell line EOL-1 was silenced by shRNA or CRISPER system. Silencing was confirmed by western blot (shRNA) and Sanger Sequencing (sgRNA). An increase of methylcellulose colony number (~30%) was observed in MGA silenced cell lines. Control EOL-1 cells or EOL-1 cells silenced with MGA CRISPR sgRNAs were injected into both flanks of NSG mice, and tumor masses were harvested 21 days after injection. Silencing of MGA by CRISPR-sgRNA consistently enhanced in vivo xenograft cell growth. In addition, western blot analyses revealed silencing of MGA in EOL-1 cells increased protein levels of Cyclin E1 and phos-RB (S807 phosphorylation inhibits the ability of RB to target protein allowing cell cycle progression), indicative of a proliferative advantage conferred by the silencing of MGA. MGA may be a potential regulator of the MYC pathway. We, therefore, examined whether silencing of MGA alters MYC transcriptional activity. Luciferase reporter assay was carried out in 293FT cells stabilized with either scramble or shRNA- targeting MGA. Luciferase activities were measured 48 h after transfection of cells with MYC activity reporter pMyc4ElbLuc and normalized to the corresponding co-transfected Renilla luciferase activity. A fourfold increase in luciferase activity was observed in MGA silenced cells when compared with non- targeting shRNA controls. Furthermore, Kaplan–Meier survival analysis was performed in the TCGA-AML patients by comparison of cases with highest versus lowest expression of MGA. P-values were calculated by log-rank test. MGA expression data and patient survival data were retrieved from TCGA-AML patients RNA seq, or microarray (70 AML patients). The MGA expression ‘high’ and ‘low’ groups were defined by 15% higher than the median or 15% lower than the median, respectively. AML patients with lower levels of MGA in their leukemic samples had a worse outcome compared with those whose leukemic cells expressed higher levels of MGA. Collectively, our results suggest that MGA may function as a potential tumor-suppressor in AML. Citation Format: Qiaoyang Sun, Lingwen Ding, Kar-Tong Tan, Wenwen Chien, Xinyi Loh, Jinfen Xiao, Anand Mayakonda, Dechen Lin, Yanyi Jiang, Henry Yang, Sigal Gery, H. Phillip Koeffler. MGA is a potential tumor suppressor in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2017-806

Abstract 2450: Mutational profiling of MLL-PTD acute myeloid leukemia

In this study, we performed whole-exome and targeted sequencing on 85 MLL-PTD AML patients. These AMLs have oncogenic tandem duplication of the MLL gene. At least one well-known oncogenic driver mutation was identified in over 90% of the MLL-PTD patients. In line with earlier sequencing studies of other AML subtypes and the TCGA-AML-sequencing project, DNMT3A was the most often mutated epigenetic regulator (25%); IDH1/2 hotspot mutations were identified in 31% of patients. TET family was the third most prominently mutated epigenetic regulator (TET1 (5%), TET2 (16.3%). Mutations of epigenetic regulators also occurred in polycomb-associated proteins (EZH2, ASXL family members), chromatin remodelers (ARID2, ARID1A), genes associated with histone acetylation (CREBBP, EP300, KAT6A, KAT6B) and histone methylation (MLL2, MLL3). Proliferation-related pathway was extensively mutated, with 54 of 80 MLL-PTD patients (67.5%) carrying at least one mutation of proliferative genes. Specifically, FLT3 mutations were found in 46% of patient samples. Notably, some FLT3-ITD patients had more than one type of internal tandem duplication (ITD) insertion, probably reflecting existence of multiple subclones in these leukemias. We found highly prevalent mutations of cohesin genes: STAG2 (16%), SMC1A (6%), SMC3 (1%), RAD21 (1%) and CTCF (6%). Cohesin pathway is more frequently mutated in MLL-PTD patients (26%) than the AML samples from either TCGA (13%) or a meta-analysis of 1000 AML (9.1%). Remarkably, an extremely high proportion of the mutations had a strong tendency to disrupt the coding sequence in STAG2, emphasizing their crucial tumor-suppressor role in this AML subtype (16% in MLL-PTD vs 3% in TCGA-AML. RNA processing pathway was also strikingly altered in MLL-PTD patients. The most prominently mutated genes within this category were the splicing factors. They included U2AF1 (13%, S34F/Y), SRSF2 (3%), SF3A1 (5%), ZRSR2 (3%), DHX15 (1%) and CWC22 (1%). Multiple mutations co-occur with MLL-PTD which are usually acquired in a sequential manner. A potential ordering for acquisition of many mutations include IDH2/DNMT3A/U2AF1/TET2→MLL-PTD→RAS-receptor tyrosine kinase based on the following reasons: #1, real-time-PCR showed that MLL-PTD was absent in remission while mutations of IDH2, DNMT3A, TET2 and U2AF1 were still retained with a high VAF. This suggests that MLL-PTD was acquired after mutations of IDH2, DNMT3A, TET2 and U2AF1; #2, MLL-PTD is highly stable during disease progression as compared with mutations of the RAS-RTK. On the other hand, RAS-RTK mutations frequently exist as subclonal mutations and tend to be unstable during disease progression. These observations support a notion that MLL-PTD was acquired prior to RAS-RTK. Taken together, MLL-PTD is acquired after those remission-persisting, initiating mutations (IDH2, DNMT3A, TET2 and U2AF1), but prior to lesions of the proliferation-related drivers. Citation Format: Lingwen Ding, Qiaoyang Sun, Kar-Tong Tan, Wenwen Chien, Anand Mayakonda, Dechen Lin, Xinyi Loh, Jinfen Xiao, Manja Meggendorfer, Tamara Alpermann, Manoj Garg, Su-Lin Lim, Vikas Madan, Norimichi Hattori, Yasunobu Nagata, Satoru Miyano, Allen Yeoh Eng Juh, Hsin-An Hou, Yan-Yi Jiang, Yan-Yi Jiang, Sumiko Takao, Li-Zhen Liu, Siew-Zhuan Tan, Siew-Zhuan Tan, Michael Lill, Mutsumi Hayashi, Akitoshi Kinoshita, Hagop M. Kantarjian, Steven M. Kornblau, Seishi Ogawa, Torsten Haferlach, Henry Yang, H. Phillip Koeffler. Mutational profiling of MLL-PTD acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2017-2450