Gene mutations in the Ras pathway and the prognostic implication in Korean patients with juvenile myelomonocytic leukemia

2011 ◽  
Vol 91 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Hyung-Doo Park ◽  
Soo Hyun Lee ◽  
Ki Woong Sung ◽  
Hong Hoe Koo ◽  
Nak Gyun Jung ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Prognostic Implication ◽  
Ras Pathway ◽  
Korean Patients ◽  
Myelomonocytic Leukemia

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.

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 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.

Somatic mutations activating Wiskott-Aldrich syndrome protein concomitant with RAS pathway mutations in juvenile myelomonocytic leukemia patients

2018 ◽  
Vol 39 (4) ◽  
pp. 579-587 ◽  
Author(s):  
Alessandro Coppe ◽  
Leonardo Nogara ◽  
Matteo Samuele Pizzuto ◽  
Alice Cani ◽  
Simone Cesaro ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Pathway ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein ◽  
Myelomonocytic Leukemia

Genome-Wide Single Nucleotide Polymorphism Analysis in Juvenile Myelomonocytic Leukemia Uncovers Long-Range Uniparental Disomy Surrounding the NF1 Locus in Cases Associated with Type 1 Neurofibromatosis but Not in Cases with Mutant RAS or PTPN11.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1453-1453
Author(s):  
Christian Flotho ◽  
Doris Steinemann ◽  
Gudrun Göhring ◽  
Charles Mullighan ◽  
Geoffrey Neale ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mitotic Recombination ◽  
Chromosome 7 ◽  
Juvenile Myelomonocytic Leukemia ◽  
Single Nucleotide ◽  
Ras Pathway ◽  
Nf1 Gene ◽  
Myelomonocytic Leukemia ◽  
Pathway Deregulation

Abstract Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder of early childhood with myeloproliferative and myelodysplastic properties. The proliferative component is a result of RAS pathway deregulation caused by somatic mutation in the RAS or PTPN11 oncogenes (60% of cases) or by underlying neurofibromatosis type 1 (NF-1) with a germline NF1 gene defect (clinically 11% of cases). To search for potential collaborating genetic abnormalities, we used Affymetrix GeneChip Mapping 50K arrays to analyze over 116,000 single nucleotide polymorphisms (SNPs) across the genome using DNA from bone marrow or peripheral blood granulocytes from 16 children with JMML and normal karyotype (mutant RAS, n=4; mutant PTPN11, n=7; NF-1, n=5). Quantitative evaluation of hybridization intensities at each SNP locus failed to identify recurrent allelic gains or losses in the 16 cases. We were specifically interested in chromosome 7 because monosomy 7 or interstitial/terminal 7q deletions are found in about 30% of JMML cases and it is conceivable that submicroscopic 7q lesions occur in the other cases but remain undetected by standard techniques. However, at the resolution provided by the arrays used here, we saw no evidence for genomic copy number alterations on chromosome 7. Interestingly, evaluation of the SNP allelotypes identified large regions of copy-neutral loss of heterozygosity (LOH) on chromosome 17q, including the NF1 locus, in 4 of the 5 samples from patients with JMML and NF-1. The LOH region spanned a genomic range of approximately 55 Mbp in each case and included more than 1,400 contiguous SNPs. Allelic copy numbers were normal within the homozygous regions, indicating uniparental isodisomy (UPD). Compatible with isodisomy, 17q was normal in the corresponding conventional karyotypes. By contrast, the array data provided no evidence for 17q UPD in any of the 12 JMML cases without NF-1. In all four cases with 17q UPD, the recombination breakpoints appeared to be confined to a 400-kbp region on 17q11.1–17q11.2; however, lack of parental or nonleukemic DNA precluded definitive mapping of the breakpoints. Of note, the 17p chromosomal arm retained heterozygosity in all cases, indicating that the p53 tumor suppressor was not affected by the UPD event. We confirmed 17q disomy in the four NF-1 samples using matrix-based comparative genomic hybridization and are currently verifying homozygosity of multiple microsatellites spaced across chromosome 17. Furthermore, NF1 mutational analysis is under way to show that the individual lesion within this tumor suppressor gene is biallelic in leukemic cells from patients with JMML and NF-1. In summary, we assume that a mitotic recombination event in an early hematopoietic progenitor cell led to UPD involving the NF1 locus. Our observations provide strong confirmatory evidence that it is indeed the NF1 gene that is responsible for the increased incidence of JMML in NF-1 patients. In addition, our data support the emerging role of mitotic recombination as a second hit in leukemogenesis and corroborate the concept that RAS pathway deregulation is central to JMML pathogenesis.

scholarly journals Comprehensive Mutational Analysis of Juvenile Myelomonocytic Leukemia Using Whole-Genome Sequencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2974-2974
Author(s):  
Yusuke Okuno ◽  
Hideki Muramatsu ◽  
Norihiro Murakami ◽  
Nozomu Kawashima ◽  
Manabu Wakamatsu ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Driver Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Whole Genome ◽  
Ras Pathway ◽  
Myelomonocytic Leukemia

Background Juvenile myelomonocytic leukemia (JMML) is a rare and exclusively pediatric myelodysplastic/myeloproliferative neoplasm. This disease is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). It has been shown that causative somatic and/or germline mutations activating the RAS pathway are located in PTPN11, NF1, NRAS, KRAS, and CBL in 85% of patients with JMML. Furthermore, up to 20% of the patients have additional secondary mutations including SETBP1, and JAK3 mutations. In 2% of the patients, we identified, by RNA sequencing, activating kinase lesions affecting ALK or ROS1. Such findings suggest that other kinase fusions are present in JMML. There is an exceptional scarcity of somatic passenger mutations on the exome, suggesting that a small number of driver mutations drive JMML. However, to date, this hypothesis has not been investigated by whole-genome sequencing. Patients and Methods We performed a whole-genome sequencing (WGS) study in 48 patients with JMML. Bone marrow specimens and in vitro-cultured T cells were used as tumor and germline samples, respectively. Next-generation sequencing was performed using a HiSeq X platform (Illumina). Data analysis was performed by our in-house pipeline. Specifically, the pipeline detects single nucleotide variants (SNVs), copy number variants, somatic loss of heterozygosity (LOH), and chromosomal structural variations (SVs). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results In each patient we detected an average of 28 somatic mutations. These were primarily C-to-T transition in the CpG context, indicating that the mutations occurred by cell division. Besides RAS pathway and known secondary mutations, we observed no significant accumulation of somatic mutations in either coding or non-coding regions. Although we detected RAS pathway mutations in 90% of the patients, all mutations were on exome. However, we identified germline microdeletions affecting CBL and NF1, which had not been identified by exome sequencing. Additionally, we found two LOH events that affected NF1. Bi-allelic inactivation of NF1 is generally observed in patients with JMML; however, no pathogenic SNVs were identified in these two patients. We identified two chromosomal translocations that caused activating kinase lesions (i.e., RANBP2-ALK and TBL1XR1-ROS1). These had been pointed out in our previous RNA sequencing study. Another patient carried a complex SV that affected XPO1 (encoding exportin 1 or chromosome region maintenance 1 protein homolog). Although fusion genes involving XPO1 are reported to be present in lymphoid malignancies, the role of this SV in JMML remains unclear. Conclusions JMML is characterized by driver mutations that are largely present within the exome. However, WGS can still play a role in identifying both coding and non-coding mutations. LOH events without pathogenic SNVs suggest the presence of novel regulatory mechanisms of NF1. Conclusively, JMML is characterized by a paucity of somatic alterations and driver mutations. Hence, current research efforts should focus on RAS pathway mutations and known secondary mutations, many of which can be targeted. Disclosures No relevant conflicts of interest to declare.

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