Clinical and Genetic Characterization Of Patients With C-CBL Mutated Juvenile Myelomonocytic Leukemia By Whole-Exome/Deep Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1565-1565 ◽  
Author(s):  
Hideki Muramatsu ◽  
Hirotoshi Sakaguchi ◽  
Xinan Wang ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Point Mutations ◽  
Allele Frequencies ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Pathway ◽  
Gm Csf ◽  
School Of Medicine ◽  
Whole Exome ◽  
Buccal Smear

Abstract Introduction Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in a mutually exclusive manner in >80% affected children. Patients and Methods We examined 108 children (71 boys and 37 girls) diagnosed with JMML in institutions throughout Japan. Written informed consent for sample collection was obtained from the parents of the patients. Molecular analysis, including whole-exome sequencing, was approved by the Ethics Committees of Nagoya University Graduate School of Medicine and Graduate School of Medicine, the University of Tokyo, in accordance with the Declaration of Helsinki. JMML diagnoses were based on internationally accepted criteria. Using exome sequencing and target deep sequencing, we identified 17 (7 boys and 10 girls) patients with c-CBLmutations. The median age at diagnosis was 11 months (range, 1-67 months). Seven of these 17 (41%) patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). Results Comparison of the clinical characteristics between patients with c-CBL mutations (n = 17) and those without c-CBL mutations (n = 91) revealed a statistically significant male predominance restricted to the patients without c-CBL mutations [7/17 (41%) vs. 64/91 (70%); p = 0.02]. The genetic alterations of c-CBLobserved in the 17 patients were as follows: 12 point mutations, 1 splice site mutation, and 4 deletions [c.1105del66: p.Glu369_Asp390del (n = 1); c.1217del22: p.Thr406fs (n = 1); c.1096-110del643: p.Glu366_Phe468del (n = 1); and c.1177_1227+86del: p.Ile393_Gln409del (n = 1)]. We could confirm heterozygous germline mutations in all the 5 patients (100%) whose germline sample was available [buccal smear and nail (n = 2), buccal smear (n = 1), and CD3+T cell (n = 2)]. Deep sequencing using a next generation sequencing platform enabled precise estimation of the mutated allele frequencies of each mutation, and we categorized the patients with c-CBL mutations into 3 distinctive groups: (A) homozygous (allele frequencies >86%; n = 10), (B) heterozygous (35%–42%; n = 5), and (C)small clone mutations (19% and 7%; n = 2). While the majority of point mutations and splice site mutations are homozygous [11/13 (85%)], which is consistent with previous reports, all 4 deletion mutations showed heterozygosity (p = 0.0037). Surprisingly, both patients with small clone mutations harbored other RAS pathway mutations simultaneously with higher mutated allele frequencies (%) [PTPN11 (c.181G>T, p.Asp61Tyr; 46%; n = 1) and KRAS (c.38G>A, p.Gly13Asp; 42%; n = 1)], suggesting that c-CBL mutations occurred as secondary genetic events in these patients. Any patient with JMML with a c-CBL mutation does not have SETBP1 and JAK3 mutations, which we recently identified as secondary mutations in JMML (Sakaguchi et.al., Nature Genetics2013). Unexpectedly, central nervous system complications were observed in 3 of the 17 (18%) patients (Moyamoya disease, n = 2, and acute disseminated encephalomyelitis, n = 1). Although 4 patients survived without post HSCT event, late graft rejection (n = 1), relapse as acute myeloid leukemia (n = 1), and death due to unknown reasons (n = 1) was observed among the 7 patients who underwent HSCT. Nine of 10 patients with JMML survived without HSCT. Consequently, the probability of 5-year transplantation-free survival [95% confidence interval (CI)] of the 17 patients with c-CBL mutations was significantly superior to that of the other 91 patients without c-CBL mutations [38% (9%–69%) vs. 14% (5%–26%), p < 0.001]. Conclusion JMML patients with c-CBL mutations have genetic and clinical heterogeneity. However, this subgroup of JMML showed better survival outcome compared with c-CBL wild-type JMML, although with several characteristic clinical events precluding patients’ quality of life. Further clinical research is warranted to elucidate determining factors for clinical heterogeneity of c-CBL mutated JMML patients. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Clinical and Genetic Characterization Of Patients With C-CBL Mutated Juvenile Myelomonocytic Leukemia By Whole-Exome/Deep Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1564-1564
Author(s):  
Hideki Muramatsu ◽  
Hirotoshi Sakaguchi ◽  
Xinan Wang ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Point Mutations ◽  
Allele Frequencies ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Pathway ◽  
Gm Csf ◽  
School Of Medicine ◽  
Whole Exome ◽  
Buccal Smear

Abstract Introduction Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in a mutually exclusive manner in >80% affected children. Patients and Methods We examined 108 children (71 boys and 37 girls) diagnosed with JMML in institutions throughout Japan. Written informed consent for sample collection was obtained from the parents of the patients. Molecular analysis, including whole-exome sequencing, was approved by the Ethics Committees of Nagoya University Graduate School of Medicine and Graduate School of Medicine, the University of Tokyo, in accordance with the Declaration of Helsinki. JMML diagnoses were based on internationally accepted criteria. Using exome sequencing and target deep sequencing, we identified 17 (7 boys and 10 girls) patients with c-CBLmutations. The median age at diagnosis was 11 months (range, 1-67 months). Seven of these 17 (41%) patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). Results Comparison of the clinical characteristics between patients with c-CBL mutations (n = 17) and those without c-CBL mutations (n = 91) revealed a statistically significant male predominance restricted to the patients without c-CBL mutations [7/17 (41%) vs. 64/91 (70%); p = 0.02]. The genetic alterations of c-CBLobserved in the 17 patients were as follows: 12 point mutations, 1 splice site mutation, and 4 deletions [c.1105del66: p.Glu369_Asp390del (n = 1); c.1217del22: p.Thr406fs (n = 1); c.1096-110del643: p.Glu366_Phe468del (n = 1); and c.1177_1227+86del: p.Ile393_Gln409del (n = 1)]. We could confirm heterozygous germline mutations in all the 5 patients (100%) whose germline sample was available [buccal smear and nail (n = 2), buccal smear (n = 1), and CD3+T cell (n = 2)]. Deep sequencing using a next generation sequencing platform enabled precise estimation of the mutated allele frequencies of each mutation, and we categorized the patients with c-CBL mutations into 3 distinctive groups: (A) homozygous (allele frequencies >86%; n = 10), (B) heterozygous (35%–42%; n = 5), and (C)small clone mutations (19% and 7%; n = 2). While the majority of point mutations and splice site mutations are homozygous [11/13 (85%)], which is consistent with previous reports, all 4 deletion mutations showed heterozygosity (p = 0.0037). Surprisingly, both patients with small clone mutations harbored other RAS pathway mutations simultaneously with higher mutated allele frequencies (%) [PTPN11 (c.181G>T, p.Asp61Tyr; 46%; n = 1) and KRAS (c.38G>A, p.Gly13Asp; 42%; n = 1)], suggesting that c-CBL mutations occurred as secondary genetic events in these patients. Any patient with JMML with a c-CBL mutation does not have SETBP1 and JAK3 mutations, which we recently identified as secondary mutations in JMML (Sakaguchi et.al., Nature Genetics2013). Unexpectedly, central nervous system complications were observed in 3 of the 17 (18%) patients (Moyamoya disease, n = 2, and acute disseminated encephalomyelitis, n = 1). Although 4 patients survived without post HSCT event, late graft rejection (n = 1), relapse as acute myeloid leukemia (n = 1), and death due to unknown reasons (n = 1) was observed among the 7 patients who underwent HSCT. Nine of 10 patients with JMML survived without HSCT. Consequently, the probability of 5-year transplantation-free survival [95% confidence interval (CI)] of the 17 patients with c-CBL mutations was significantly superior to that of the other 91 patients without c-CBL mutations [38% (9%–69%) vs. 14% (5%–26%), p < 0.001]. Conclusion JMML patients with c-CBL mutations have genetic and clinical heterogeneity. However, this subgroup of JMML showed better survival outcome compared with c-CBL wild-type JMML, although with several characteristic clinical events precluding patients’ quality of life. Further clinical research is warranted to elucidate determining factors for clinical heterogeneity of c-CBL mutated JMML patients. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Whole Exome Analysis Reveals Spectrum of Gene Mutations in Juvenile Myelomonocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 170-170
Author(s):  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Hirotoshi Sakaguchi ◽  
Kenichi Yoshida ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Deep Sequencing ◽  
Somatic Mutations ◽  
Gene Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Pathway ◽  
Gm Csf ◽  
Whole Exome ◽  
Myelomonocytic Leukemia

Abstract Abstract 170 Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods: We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation. Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers' protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results: For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases. Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion: Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

Whole-Exome Sequencing and Targeted Deep Sequencing of cfDNA Enables a Comprehensive Mutational Profiling of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 197-197 ◽  
Author(s):  
Salomon Manier ◽  
Jihye Park ◽  
Samuel Freeman ◽  
Gavin Ha ◽  
Marzia Capelletti ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Clonal Evolution ◽  
Target Coverage ◽  
Whole Genome ◽  
Driver Genes ◽  
Whole Exome ◽  
Low Pass ◽  
Targeted Deep Sequencing ◽  
Tumor Dna

Abstract Background . Cell-free DNA (cfDNA) sequencing enables serial temporal sampling, which offers the possibility of following the dynamics of biomarkers and clonal evolution in Multiple Myeloma (MM) over time. The use of cfDNA in clinical practice as a molecular biomarker and for monitoring response/resistance is dependent on a comprehensive profile of matched cfDNA and tumor DNA (tDNA) samples. Here we performed Ultra-Low Pass Whole Genome Sequencing (ULP-WGS) followed by whole-exome sequencing (WES) and targeted deep sequencing of matched cfDNA/tDNA samples from MM patients. Methods. We performed next generation sequencing of matched cfDNA/tDNA samples for 63 patients with newly diagnosed or relapsed MM, SMM, or MGUS. Libraries were constructed using the Kappa Hyper kit and sequenced by ultra-low-pass whole-genome sequencing (ULP-WGS, 0.1x coverage) to quantify tumor fraction within cfDNA. WES was performed on 30 matched samples cfDNA/tDNA/germline DNA from 10 patients with more than 5% of tumor fraction. Libraries were hybridized to the Nextera Rapid Capture Exome kit (Illumina) and then sequenced on HiSeq 4000 (Illumina). Targeted deep sequencing was performed on 32 matched cfDNA/tDNA samples from 16 patients using the HaloPlex HS technology (Agilent), allowing for molecular barcoding. Libraries were constructed according to the manufacturer's instructions and sequenced on HiSeq 2500 (Illumina). Sequencing data were analyzed using the Firehose pipelines, including MuTect, ABSOLUTE, ReCapSeg, GISTIC and MutSig. Results. We first used a cost-effective approach to establish the tumor content of cfDNA in a large-scale manner by ULP-WGS. Among 63 tested samples (53 MM, 6 SMM and 4 MGUS patient samples), the tumor fraction within cfDNA ranged from 0 to 81% with a mean of 10%. About 43% of these samples had tumor fraction greater than 5% within cfDNA. To assess whether cfDNA can capture the genetic diversity of MM and inform clinical management, we performed WES of matched cfDNA/tDNA/germline DNA samples for 10 patients (mean target coverage 194x). Copy number alterations (CNAs) assessed by WES (ReCapSeg) were consistent between cfDNA and tumor DNA. Similarly, focal CNAs assessed by GISTIC were consistent between tDNA and cfDNA. We then examined the overlap of somatic single nucleotide variants (SSNVs) between WES of cfDNA and matched tDNA. We found, on average, 100% of the clonal and 96% of the subclonal (range 54-100%) SSNVs that were detected in the tumor were confirmed to be present in cfDNA. Similarly, for mutations detected in the cfDNA, we found, on average, 100% of the clonal and 99% of the subclonal (range 98-100%) SSNVs were confirmed in the tumor. To assess whether targeted deep sequencing of cfDNA could be a good proxy for tumor biopsy we used a targeted deep sequencing approach of known MM driver genes. Libraries were prepared using unique molecular barcodes to avoid duplication rates, for 32 matched cfDNA/tDNA samples from 16 patients with MM. The mean target coverage was 596x. We found similar frequencies of altered MM driver genes in both cfDNA and tDNA, including KRAS, NRAS, and TP53, indicating that cfDNA can be used for precision medicine. Conclusions. Our study demonstrates that both WES and targeted deep sequencing of cfDNA are consistently representative of tumor DNA alterations in terms of CNAs, focal CNAs and SSNVs. This approach could therefore be used to longitudinally follow clonal evolution across the course of the disease and precision medicine in patients with MM. Disclosures Palumbo: Takeda: Employment, Honoraria; Janssen Cilag: Honoraria. Kumar:Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; Onyx: Consultancy, Research Funding. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria. Facon:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy; Janssen: Consultancy, Speakers Bureau; Bristol: Consultancy; Millenium/Takeda: Consultancy; Celgene: Consultancy, Speakers Bureau; Karyopharm: Consultancy. Ghobrial:Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Noxxon: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

scholarly journals Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia

Blood ◽  
2012 ◽  
Vol 120 (7) ◽  
pp. 1485-1488 ◽  
Author(s):  
Sayoko Doisaki ◽  
Hideki Muramatsu ◽  
Akira Shimada ◽  
Yoshiyuki Takahashi ◽  
Makiko Mori-Ezaki ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Somatic Mosaicism ◽  
Juvenile Myelomonocytic Leukemia ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Mild Disease ◽  
Myeloid Neoplasm ◽  
Buccal Smear ◽  
Spontaneous Improvement ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

scholarly journals Immunophenotypic and Genetic Overlap between JMML and CMML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1803-1803
Author(s):  
Cody E. Cotner ◽  
Mitul Modi ◽  
Gerald Wertheim ◽  
Michele Paessler ◽  
Sarah K. Tasian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Histone Modification ◽  
Research Funding ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cell Transplant ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Bone Marrow Biopsies ◽  
Ras Pathway ◽  
Myelomonocytic Leukemia

Abstract Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution. Methods: The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel. CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis. Results: We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance. We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining. Conclusions: Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

scholarly journals Comprehensive Genetic Analysis in Cases of Juvenile Myelomonocytic Leukemia for Prognostic Estimation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3159-3159
Author(s):  
Norihiro Murakami ◽  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Hirotoshi Sakaguchi ◽  
Kenichi Yoshida ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Research Funding ◽  
Survival Rates ◽  
Gene Mutations ◽  
Genetic Alterations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Genetic Mutations ◽  
Ras Pathway ◽  
Pathway Gene ◽  
Myelomonocytic Leukemia

Abstract Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm (MPN) that occurs during childhood and has a poor prognosis. Somatic or germline mutations in canonical RAS pathway genes, i.e., PTPN11, NF1, NRAS, KRAS, and CBL, are reported be detected in approximately 85% patients. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for JMML. Although spontaneous remission is occasionally observed in others with supportive therapy, some patients show aggressive disease progression despite HSCT. Recent studies have identified several additional genetic events in an array of genes, including SETBP1 and JAK3, but the relationship between genetic alterations and clinical outcomes remains unclear. Patients and Methods: A total of 131 patients (88 boys, 43 girls) with JMML were enrolled in the study. The median age was 15 months (range, 1-160 months). Eighty-two of 131 patients underwent HSCT, and 36 patients died (disease related, n = 27, transplantation-related complications, n = 16, infection, n = 5, unknown, n = 3). We performed comprehensive genetic analyses of the 131 JMML patients using whole-exome sequencing (n = 68, 52%) or targeted deep sequencing (n = 92, 70%), and assessed the impact of genetic alterations on clinical outcomes in 119 patients, excluding 12 patients with Noonan syndrome-related myeloproliferative disorder (NS/MPD). Results: We identified canonical RAS pathway gene mutations in 115 of 131 patients (88%). Although most RAS pathway mutations were mutually exclusive, coexisting secondary RAS pathway mutations were found in nine patients (8%). In addition, 28 patients harbored secondary genetic alterations in other genes, including SETBP1 (n = 10), JAK3 (n = 12), ASXL1 (n = 6), SH3BP1 (n = 1), RRAS2 (n = 2), and SOS1 (n = 3). In total, 34 of 131 patients harbored secondary genetic mutations. In univariate analysis, patients with secondary genetic mutations showed poorer survival rates than patients without these mutations [5-year transplantation-free survival (TFS) (95% CI) = 8.8% (2.3%-21.1%) vs. 24.1% (15.2%-34.1%), p = 0.007; 5-year overall survival (OS) (95% CI) = 49.6% (32.0%-65.0%) vs. 62.3% (50.8%-71.8%), p = 0.135]. On the basis of the dominant canonical RAS pathway mutations classification, patients with PTPN11 and NF1 mutations were significantly associated with the presence of secondary genetic mutations compared to patients with other RAS pathway gene mutations (PTPN11 (20 of 43, 47%), NF1 (5 of 7, 71%), NRAS (2 of 18, 11%), KRAS (4 of 20, 20%), CBL (1 of 17, 6%), p < 0.001). Consistent with previous reports, patients with PTPN11 and NF1 mutations had inferior survival rates than other JMML patients [5-year TFS (95% CI) = 0% vs. 32.7% (21.5%-44.3%), p < 0.001; 5-year OS (95% CI) = 45.3% (31.1%-58.5%) vs. 68.1% (55.2%-78.0%), p = 0.006]. Multivariate survival analysis identified the RAS pathway mutations (i.e., patients with PTPN11 and NF1 mutations vs. others) [TFS: HR (95% CI) = 3.732 (2.382-5.847), p < 0.001; OS: HR (95% CI) = 1.983 (1.117-3.521), p < 0.019] and low platelet count (<33 × 109/L vs. ≥ 33 × 109/L) [TFS: HR (95% CI) = 1.816 (1.160-2.843), p < 0.001] as independent risk factors for TFS and OS. In subgroup analysis of 50 patients with PTPN11 and NF1 mutations, there were no significant survival differences between patients with (n = 25) or without (n = 25) secondary genetic mutations [5-year TFS (95% CI) = 0% vs. 0%, p = 0.753; 5-year OS (95% CI) = 39.6% (20.8%-57.9%) vs. 51.7% (30.9%-69.1%), p = 0.589]. Discussion: Consistent with previous studies, secondary genetic mutations were associated with inferior survival rates, but high correlations were observed in JMML patientswith PTPN11 and NF1 mutations. Our results suggest that comprehensive genetic mutational profiling is essential to estimate prognosis and to stratify JMML patients who require HSCT and/or novel treatment modalities. Disclosures Ogawa: Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Kojima:SANOFI: Honoraria, Research Funding.

scholarly journals A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Takumi Miura ◽  
Satoshi Yasuda ◽  
Yoji Sato
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Allele Frequencies ◽  
Genetic Mutations ◽  
Sequencing Data ◽  
Simple Method ◽  
Whole Exome ◽  
Generation Sequencing

Abstract Background Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. Results Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. Conclusions For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

scholarly journals PCNT point mutations and familial intracranial aneurysms

Neurology ◽  
2018 ◽  
Vol 91 (23) ◽  
pp. e2170-e2181 ◽  
Author(s):  
Oswaldo Lorenzo-Betancor ◽  
Patrick R. Blackburn ◽  
Emily Edwards ◽  
Rocío Vázquez-do-Campo ◽  
Eric W. Klee ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Intracranial Aneurysms ◽  
Point Mutations ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Interaction Domain ◽  
Knockout Mouse Model ◽  
Whole Exome ◽  
Primordial Dwarfism

ObjectiveTo identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing.MethodsWe performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants.ResultsWe identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases.ConclusionThe PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt−/−) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease.

scholarly journals Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia

2021 ◽  
Vol 218 (2) ◽  
Author(s):  
Eleni Louka ◽  
Benjamin Povinelli ◽  
Alba Rodriguez-Meira ◽  
Gemma Buck ◽  
Wei Xiong Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Childhood Leukemia ◽  
Clonal Evolution ◽  
Juvenile Myelomonocytic Leukemia ◽  
Hematopoietic Stem ◽  
Ras Pathway ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin−CD34+CD38−CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a “first hit,” (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

scholarly journals Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4309-4309
Author(s):  
María Abáigar ◽  
Jesús M Hernández-Sánchez ◽  
David Tamborero ◽  
Marta Martín-Izquierdo ◽  
María Díez-Campelo ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Clonal Evolution ◽  
Clonal Expansion ◽  
Driver Mutations ◽  
Advisory Committees ◽  
Disease Evolution ◽  
Mutational Burden ◽  
Whole Exome ◽  
Leukemic Phase

Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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