Genetic Basis of Myeloid Proliferation Related to Down Syndrome

Abstract Abstract 535 Background Transient abnormal myelopoiesis (TAM) represents a self-limited proliferation exclusively affecting perinatal infants with Down syndrome (DS), morphologically and immunologically characterized by immature blasts indistinguishable from acute megakaryoblastic leukemia (AMKL). Although spontaneous regression is as a rule in most cases, about 20–30% of the survived infants develop non-self-limited AMKL (DS-AMKL) 3 to 4 years after the remission. As for the molecular pathogenesis of these DS-related myeloid proliferations, it has been well established that GATA1 mutations are detected in virtually all TAM cases as well as DS-AMKL. However, it is still open to question whether a GATA1 mutation is sufficient for the development of TAM, what is the cellular origin of the subsequent AMKL, whether additional gene mutations are required for the progression to AMKL, and if so, what are their gene targets, although several genes have been reported to be mutated in occasional cases with AMKL, including JAK2/3, TP53 and FLT3. Methods To answer these questions, we identify a comprehensive spectrum of gene mutations in TAM/AMKL cases using whole genome sequencing of three trio samples sequentially obtained at initial presentation of TAM, during remission and at the subsequent relapse phase of AMKL. Whole exome sequencing was also performed for TAM (N=16) and AMKL (N=15) samples, using SureSelect (Agilent) enrichment of 50M exomes followed by high-throughput sequencing. The recurrent mutations in the discovery cohort were further screened in an extended cohort of DS-AMKL (N = 35) as well as TAM (N = 26) and other AMKL cases (N = 19) using target deep sequencing. Results TAM samples had significantly fewer numbers of somatic mutations compared to AMKL samples with the mean numbers of all mutations of 30 (1.0/Mb) and 180 (6.0/Mb) per samples in whole genome sequencing or non-silent somatic mutations of 1.73 and 5.71 per sample in whole exome sequencing in TAM and AMKL cases, respectively (p=0.001). Comprehensive detections of the full spectrum of mutations together with subsequent deep sequencing of the individual mutations allowed to reveal more complicated clonological pictures of clonal evolutions leading to AMKL. In every patient, the major AMKL clones did not represent the direct offspring from the dominant TAM clone. Instead, the direct ancestor of the AMKL clones could be back-traced to a more upstream branch-point of the evolution before the major TAM clone had appeared or, as previously reported, to an earlier founder having an independent GATA1 mutation. Intratumoral heterogeneity was evident at the time of diagnosis as the presence of major subpopulations in both TAM and AMKL populations, which were more often than not characterized by RAS pathway mutations. While GATA1 was the only recurrent mutational target in the TAM phase, 8 genes were recurrently mutated in AMKL samples in whole genome/exome sequencing, including NRAS, TP53 and other novel gene targets that had not been previously reported to be mutated in other neoplasms. The recurrent mutations found in the discovery cohort, in addition to known mutational targets in myeloid malignancies, were screened in an extended cohort of DS-associated myeloid disorders (N=61) as well as other AMKL cases, using high-throughput sequencing of SureSelect-captured and/or PCR amplified targets. Secondary mutations other than GATA1 mutations were found in 3 out of 26 TAM, 20 out of 35 DS-AMKL and 4 out of 19 other AMKL cases. Conclusion TAM is characterized by a paucity of somatic mutations and thought to be virtually caused by a GATA1 mutation in combination with constitutive trisomy 21. Subsequent AMKL evolved from a minor independent subclone acquiring additional mutations. Secondary genetic hits other than GATA1 mutations were common, where deregulated epigenetic controls as well as abnormal signaling pathway mutations play a major role. Disclosures: No relevant conflicts of interest to declare.

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Whole-exome and whole-genome sequencing in chronic lymphocytic leukemia: new biomarkers to target

Pharmacogenomics ◽

10.2217/pgs-2020-0022 ◽

2020 ◽

Vol 21 (13) ◽

pp. 957-962

Author(s):

Charbel Hobeika ◽

Gaelle Rached ◽

Alain Chebly ◽

Eliane Chouery ◽

Hampig Raphael Kourie

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput Sequencing ◽

Gene Mutations ◽

Lymphocytic Leukemia ◽

Whole Genome ◽

Mutational Status ◽

Whole Exome ◽

Worse Prognosis

Many biomarkers indicate prognosis in chronic lymphocytic leukemia; such as fluorescence in situ hybridization testing: 17p or 11q deletions have a worse prognosis than trisomy 12, 13q deletion or normal result, or the mutational status of the immunoglobulin heavy chain (IGHV): unmutated IGHV have a worse prognosis than mutated IGHV. Recently, many gene mutations ( TP53, NOTCH1 etc.,) have been linked to a worse prognosis. With the new era of high-throughput sequencing, it has become easier to study gene mutations and their implication in predicting prognosis. In this review, we aim to review all the studies that performed whole-exome sequencing or whole-genome sequencing on chronic lymphocytic leukemia cells and explore the implication of various genes in disease prognosis.

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Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽

10.1182/blood.v120.21.124.124 ◽

2012 ◽

Vol 120 (21) ◽

pp. 124-124

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Yuichi Shiraishi ◽

Yasunobu Nagata ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Conflicts Of Interest ◽

Massively Parallel Sequencing ◽

Gene Mutations ◽

Pediatric Leukemia ◽

Protein Coding ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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Whole Exome Sequencing of Acute Myeloid Leukaemia Patients Identifies Somatic and Germline Mutations in Fanconi Anaemia Genes

Blood ◽

10.1182/blood.v124.21.698.698 ◽

2014 ◽

Vol 124 (21) ◽

pp. 698-698

Author(s):

Kyaw Zeya Maung ◽

James X Gray ◽

Paul J Leo ◽

Mahmoud Bassal ◽

Anna L Brown ◽

...

Keyword(s):

Dna Repair ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Research Network ◽

Fanconi Anaemia ◽

Molecular Characteristics ◽

Whole Exome ◽

Recurrent Mutations

Abstract Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

Blood ◽

10.1182/blood.v122.21.3708.3708 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3708-3708

Author(s):

Atsushi Narita ◽

Hideki Muramatsu ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Asahito Hama ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Bone Marrow Failure Syndrome ◽

Cell Lineages ◽

Bone Marrow Failure Syndromes ◽

Whole Exome

Abstract Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

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Whole-Exome Resequencing Identifies Somatic Mutations Of BCOR and BCORL1 Transcriptional Corepressor Genes and Major Cohesin Complex Component Genes In Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.834.834 ◽

2013 ◽

Vol 122 (21) ◽

pp. 834-834

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yasunobu Nagata ◽

Ayana Kon ◽

Yusuke Okuno ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Transcriptional Corepressor ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML. Methods To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases. Results and Discussion A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%). Conclusion Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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Leukemogenesis in Seven Donor Cell Derived Myeloid Neoplasms Patients. Whole Exome Sequencing Reveals Clonal Dynamics

Blood ◽

10.1182/blood-2018-99-118894 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2752-2752

Author(s):

Julia Suárez González ◽

Juan Carlos Triviño ◽

Mi Kwon ◽

Angela Figuera Alvarez ◽

Guiomar Bautista ◽

...

Keyword(s):

Exome Sequencing ◽

Somatic Mutations ◽

Gene Mutations ◽

Donor Cell ◽

Hematopoietic Stem ◽

Time Points ◽

Post Transplant ◽

Myeloid Neoplasm ◽

Whole Exome ◽

The Moment

Abstract Introduction Donor cell derived myeloid neoplasm (DCMN), defined as the development of de novohematological malignancies from cells of donor origin,is a late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We report on seven cases of DCMN in which whole-exome sequencing (WES) at different time-points after allo-HSCT, as well as in a sample from each donor, was performed. The ultimate objective was to accurately described the clonal architecture, spatial heterogeneity and identify somatic mutations that are induced in the process of leukemogenesis and clonal evolution of myeloid neoplasm. Donors were also analyzed to detect underlying condition predisposing to the development of DCMN. Patient and Methods Seven patients with a confirmed diagnosis of DCMN and their donors were recruited from different Spanish institutions. This cohort included a total of 32 BM samples at different time points after allo-HSCTand one PB sample from each donor (one case received dual allo-HSCT, CB and PB from both donors were obtained), which provided a total of 40 samples. Genomic DNA samples were prepared according to Agilent SureSelect-XT Human exon 50Mb enrichment kit (Agilent Technlogies, Santa Clara, CA) preparation guide and libraries were sequenced on Illumina HiSeq platform (Illumina, San Diego, CA). DNA sequencing data from recipient post-transplant BM samples, were matched against their donor PB sample and previous post-transplant BM samples to identify the acquisition of mutations along the post allo-HSCT period.Germline variants in donors were studied in order to detect mutations that predisposed to the development of a myeloid neoplasm.The research protocol was approved by the Ethic Committee of Gregorio Marañón General University Hospital. Patients´ and donors´ information was collected from their medical records. Results Clinical and biological characteristics of the 7 patients with DCMN and their donors are shown in Table 1. Mutational profiles obtained from the follow-up samples at different time-points post-HSCT demonstrated high intra-tumor genetic heterogeneity and clonal dynamic for all cases. The number of variants are increased over time and at the moment of DCMN diagnosis, the median number of variants was 28, ranging from 18 to 92 variants (Figure 1). WES identified in DCMN patients gene mutations commonly seen in adult AML or MDS, such as in SETBP1, DNMT3A, TET2, RUNX1, CSF3R, EP300and IDH2.In addition, others non-silent variants were acquired in all cases. Among the additional novel alterated genes, we found 23 strong candidateswith oncogenic potential. LUC7L2, NOP14, LAMA5, SKOR2, EML1, SNX13, RHPN2, IRS1, MTG2, TENM2, MEFV, GSE1, NOTCH4, DTX1, CNOT4, PNKP, GRB7,SENP7,TAF1L, ZKSCAN2, ZBTB20, ZNF461 and MEGF10. Analysis of CNVs revealed numerical alterations across the post allo-HSCT samples in patients 1, 2, 3, 4, 5 and 7. The most common chromosomal alterations in DCMN were monosomy 7 and other chromosome 7 abnormalities, which detected in the 86% (6/7) of the patients. Although none of the donors developed a myeloid neoplasm at the moment of diagnosis of DCMN in recipient, donor 1 revealed an abnormal karyotype (45,XY,-7) at the moment of the allo-HSCT. All other donors harbored at least one pathogenic or probably-pathogenic variants, most probably of germline origin, in genes involved in hematological or solid tumor predisposition. Conclusions The development of DCMN involves dynamic genomic processes that begin months before the clinical onset. In this study, our integrated multi-step analysis revealed the intra-tumor heterogeneity and evolutionary history of seven DCMN. The present study reveals a process of sequential clonal expansions promoted by the acquisition of somatic mutations in donor hematopoietic cells. Detection of heritable or acquired gene mutations in donors associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-HSCT. Leukemic transformation ofdonor hematopoietic stem cells provides a useful in vivomodel to study the mechanisms involved in leukemogenesis. Novel approaches based on high-depth next generation sequencing to study consecutive samples from post-transplant period in these patients, appear promising to discover new genes involved in myeloid neoplasm and to decipher the mechanisms of leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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Whole-Exome Sequencing Reveals a Paucity of Somatic Gene Mutations in Aplastic Anemia and Refractory Cytopenia of Childhood

Blood ◽

10.1182/blood.v124.21.4388.4388 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4388-4388

Author(s):

Yusuke Okuno ◽

Atsushi Narita ◽

Hideki Muramatsu ◽

Kenichi Yoshida ◽

Asahito Hama ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Who Classification ◽

Somatic Mutations ◽

Gene Mutations ◽

Confidential Interval ◽

Cell Lineages ◽

Whole Exome ◽

Somatic Gene

Abstract Introduction: The appropriate classification of bone marrow (BM) failure syndromes in children is challenging, particularly in relation to histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study is to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 children with three diseases using whole-exome sequencing. Patients and Methods: Wediagnosed AA, RCC, or RCMD on the basis of morphology and histological findings of bone marrow (BM) according to the 2008 World Health Organization (WHO) classification criteria. Patients with AA exhibited hypocellular BM and no morphologically dysplastic changes in any of three hematopoietic cell lineages, while patients with RCC had <10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients with RCMD exhibited >10% dysplastic changes in two or more cell lineages. We obtained peripheral blood and BM samples from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). We performed exome capture from paired DNA (non-T cells/CD3+ lymphocytes) using SureSelect® Human All Exon V4 kit (Agilent Technologies, Santa Clara, CA), which covered all part of the coding exons, followed by massively-parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations and germline variants were detected through our pipeline for whole-exome sequencing (Genomon-exome). All candidate somatic nucleotide changes were validated by Sanger sequencing. The ethics committee of Nagoya University Graduate School of Medicine approved this study. Results: Whole-exome sequencing pipeline identified a total of 14 non-synonymous somatic (one nonsense, 11 missense, and two frameshift) changes among the 29 patients, which resulted in only 0.48 mutations per patient. The average numbers of somatic mutations per sample were not significantly different among these groups (0.50 in AA, 0.36 in RCC, and 0.60 in RCMD). As a whole, childhood AA, RCC, and RCMD were characterized by a paucity of somatic mutations compared with adult myelodysplastic syndromes (MDS) in which 10 or more mutations per exome were detected on average. Among the mutated genes, BCOR-inactivating mutations in two patients (p.S158fs in AA and p.E1286X in RCMD) were considered significant genetic events based on previous reports that it is a driver gene in MDS. With regard to germline events, we did not detect any germline mutations of inherited BM failure syndromes. Moreover, we did not identifiy significantly frequent germline events in the entire cohort or any genetic hallmarks to be able to discriminate between these three diseases. When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0%) vs. 6% (0%–26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100%–100%) vs. 95% (70%–99%)] were not significantly different. Conclusion: We usedwhole-exome sequencing analysis for gene mutational profiling of children with AA, RCC, and RCMD. Idiopathic bone marrow failure syndromes in children are characterized by a paucity of somatic gene mutations, irrespective of histological diagnosis. These findings suggest that histological diagnosis based on the WHO classification system does not discriminate the mutational profile of idiopathic BM failure syndromes in children. Disclosures No relevant conflicts of interest to declare.

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Whole-exome sequencing identifies somatic mutations and intratumor heterogeneity in inflammatory breast cancer

npj Breast Cancer ◽

10.1038/s41523-021-00278-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Rui Luo ◽

Weelic Chong ◽

Qiang Wei ◽

Zhenchao Zhang ◽

Chun Wang ◽

...

Keyword(s):

Breast Cancer ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Inflammatory Breast Cancer ◽

Somatic Mutations ◽

Gene Mutations ◽

Intratumor Heterogeneity ◽

Driver Gene ◽

Tissue Samples ◽

Whole Exome

AbstractInflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1, LAMA3, FLG2, UGT2B4, STK33, BRCA2, ACP4, PIK3CA, and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3, IL12RB2, and PDE4B may potentially generate ITH in IBC.

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Whole-Exome Sequencing Of Adult Philadephia-Negative Acute Lymphoblastic Leukemia From Diagnosis To Relapse After Allogeneic Hematopoietic Stem Cell Transplantation Reveals Clonal Evolution and Relapse-Associated Mutated Genes

Blood ◽

10.1182/blood.v122.21.156.156 ◽

2013 ◽

Vol 122 (21) ◽

pp. 156-156

Author(s):

Haowen Xiao ◽

Yi Luo ◽

Xiaoyu Lai ◽

Jimin Shi ◽

Yamin Tan ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Lymphoblastic Leukemia ◽

Gene Mutations ◽

Genetic Alterations ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Whole Exome ◽

Matched Samples

Abstract Introduction Although steady progress of effective chemotherapy in childhood acute lymphoblastic leukemia (ALL) carried with exceeding 80% of individuals now cured, the majority of adult patients with ALL are not cured by chemotherapy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option. However, relapse remains the most leading cause of death after allo-HSCT. Adverse genetic alterations are generally accepted to be responsible for treatment failure and relapse. Several structural chromosomal alterations including rearrangement of the myeloid-lymphoid or mixed-lineage leukemia gene (MLL) and Philadelphia chromosome (Ph), have been mostly found in relapsed ALL. However, many Ph-negative (Ph-) ALL patients with normal karyotype , lacking known risk factors, also experienced relapse. The underlying pathologic determinants leading to relapse and prognostic markers in these cases remain poorly understood. More importantly, allo-HSCT is a distinct treatment option from tradtional chemotherapy and has 2 important forms to eliminate and select on malignant cells. The malignant cells that go on to causing relapse must initially survive ablation of chemotherapy before allo-HSCT and conditioning regimen in allo-HSCT. Then, after allo-HSCT, they must survive the effect of graft-versus- leukemia (GVL) reaction. Following this rationale, we hypothesized that there may be pivotal genetic causes confer leukemic cells a fitness advantage to undergo huge selective pressures and expand after allo-HSCT. To elucidate the genomic basis underlying relapse after allo-HSCT to aid to discover novel predictive biomarkers and identify therapeutic targets, we carried out the first whole-exome sequencing analysis in longitudinal matched samples from diagnosis to relapse after allo-HSCT in adult patients with the most common subtype of ALL, Ph- B-cell ALL (B-ALL). Methods Whole-exome sequencing was conducted for 9 genomic DNA samples from 3 relapsed cases with Ph- B-ALL (discovery cohort) at 3 specific time points including: diagnosis, complete remission (CR) after induction chemotherapy before allo-HSCT, relapse after allo-HSCT to discover candidate relapse-associated mutated genes. We identified putative somatic mutations by comparing each tumor ( diagnostic samples or relapsed samples) to normal (CR samples) from the same patient. To confirm candidate somatic gene mutations, screen relapse-associated gene mutations and define the frequency of somatic mutations identified by whole-exome sequencing analysis, we further carried out target genes whole coding regions sequencing in an ALL extended validation cohort including 58 adult Ph- B-ALL cases, where 27 patients experienced relapse at a median time of 6.5 (range 2-33) months after allo-HSCT and 31 patients did not relapse after allo-HSCT at a median follow-up for 34 (range 12–56) months. Results (1) We discovered novel associations of recurrently mutated genes (CREBBP, KRAS, PTPN21) with the pathogenesis of adult Ph- B-ALL relapse after allo-HSCT, which were mutated in at least two relapsed cases, but were not mutated in non- relapsed patients. (2) The generation of high-depth whole-exome sequencing data in longitudinal matched samples from diagnosis to relapse after allo-HSCT in initial 3 patients allowed us to directly assessed the evolution of somatic mutations. Our data suggested that in the progression of leukemia relapse after allo-HSCT, the relapse clone had a clear relationship to the diagnosis clone, either arising from a subclone already exsiting in the diagnostic tumor, or originating from a common preleukemic progenitor with the diagnosis clone. In the latter pattern, the relapse clone acquires new genetic alterations while retaining some but not all of the alterations found in the diagnostic tumor. In contrast, in some cases, leukemia recurrences afer allo-HSCT may be composed of second malignancies with completely distinct sets of mutations from the primary tumor. Conclusions Our study is the first to explore genetic basis of adult Ph- B-ALL from diagnosis to relapse after allo-HSCT over time, which will provide novel genetic biomarkers on risk “index” to improve individualized treatment intensification and intervention strategies, and potential therapeutic targets for Ph--ALL relapse after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

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Whole exome sequencing of thymic neuroendocrine tumor with ectopic ACTH syndrome

Acta Endocrinologica ◽

10.1530/eje-16-0546 ◽

2017 ◽

Vol 176 (2) ◽

pp. 187-194 ◽

Cited By ~ 3

Author(s):

Yanli Li ◽

Ying Peng ◽

Xiuli Jiang ◽

Yulong Cheng ◽

Weiwei Zhou ◽

...

Keyword(s):

Neuroendocrine Tumor ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Neuroendocrine Tumors ◽

Somatic Mutations ◽

Ectopic Acth Syndrome ◽

Ectopic Acth ◽

Whole Exome ◽

Genetic Abnormalities ◽

Recurrent Mutations

Objective Thymic neuroendocrine tumor is the second-most prevalent cause of ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS), which is a rare disease characterized by ectopic ACTH oversecretion from nonpituitary tumors. However, the genetic abnormalities of thymic neuroendocrine tumors with EAS remain largely unknown. We aim to elucidate the genetic abnormalities and identify the somatic mutations of potential tumor-related genes of thymic neuroendocrine tumors with EAS by whole exome sequencing. Design and methods Nine patients with thymic neuroendocrine tumors with EAS who were diagnosed at Shanghai Clinical Center for Endocrine and Metabolic Diseases in Ruijin Hospital between 2002 and 2014 were enrolled. We performed whole exome sequencing on the DNA obtained from thymic neuroendocrine tumors and matched peripheral blood using the Hiseq2000 platform. Results We identified a total of 137 somatic mutations (median of 15.2 per tumor; range, 1–24) with 129 single-nucleotide mutations (SNVs). The predominant substitution in these mutations was C:G > T:A transition. Approximately 80% of detected mutations resulted in amino acid changes. However, we failed to discover any recurrent mutations in these nine patients. By functional predictions, HRAS, PAK1 and MEN1, previously reported in neuroendocrine tumors, were identified as candidate tumor-related genes associated with thymic neuroendocrine tumors. Conclusions Using whole exome sequencing, we identified genetic abnormalities in thymic neuroendocrine tumors with EAS. Thereby, this study acts as a further supplement of the genetic features of neuroendocrine tumors. Somatic mutations of three potential tumor-related genes (HRAS, PAK1 and MEN1) might contribute to the tumorigenesis of thymic neuroendocrine tumors with EAS.

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