Yinyan Xu

Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations

Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML‐related mutations.

Correlation of Clinical Features With the Mutational Status of GM-CSF Signaling Pathway-Related Genes in Juvenile Myelomonocytic Leukemia

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

Downregulation of GATA-2 and overexpression of adipogenic gene-PPARγ in mesenchymal stem cells from patients with aplastic anemia

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells and fatty replacement in the bone marrow. Transcriptional factor GATA-2 plays several important roles in both hematopoiesis and adipogenesis. Decreased levels of GATA-2 compromise the proliferation and survival of hematopoietic stem cells. GATA-2 suppresses adipocyte differentiation through direct inhibition of adipogenic factors, including peroxisome proliferator-activated receptor-gamma (PPARgamma). Previous studies have shown that expression of GATA-2 is decreased in marrow CD34-positive cells in AA. To elucidate the mechanisms of fatty marrow replacement, we evaluated the mRNA expression for GATA-2 and PPARgamma in mesenchymal stem cells (MSCs) from patients with AA by quantitative real-time polymerase chain reaction. GATA-2 expression by MSCs from AA patients was significantly lower than in normal subjects. Conversely, expression of PPARgamma was significantly higher in AA patients. Western blot analysis demonstrated that protein levels of GATA-2 were lower in AA patients than those in normal subjects. Moreover, incubation with interferon-gamma induced downregulation of GATA-2 levels in MSCs from normal subjects. These findings indicate that fatty marrow replacement in AA patients can be explained by downregulation of GATA-2 and overexpression of PPARgamma in MSCs. Decreased expression of GATA-2 might be responsible for the pathogenesis and development of the clinical features of the disease.

Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was −0.99 standard deviation (SD) (range −4.01–+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P<0.001). The combined absence of a minor paroxysmal nocturnal hemoglobinuria population and a short telomere length is an efficient predictor of poor immunosuppressive therapy response, which should be considered while deciding treatment options: immunosuppressive therapy or first-line hematopoietic stem cell transplantation. The trial was registered in www.umin.ac.jp with number UMIN000017972.

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia

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.

GATA2 and secondary mutations in familial myelodysplastic syndromes and pediatric myeloid malignancies

GATA2, a member of the GATA transcription factor family, plays a critical role in hematopoiesis,1 vascular2 and neural development. Mutations in the exons and intron 5 of this gene have been identified as the cause of several hematologic disorders.3 GATA2-related disorders include familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML);4,5 chronic myeloid leukemia (CML);6 monocytopenia and mycobacterial infection (MonoMAC) syndrome;7 and dendritic cell, monocyte, B and NK lymphoid (DCML) deficiency.8 Patients with MonoMAC syndrome or DCML deficiency exhibit increased susceptibility to infection and often progress to MDS and AML.2,8 Because GATA2 is associated with the development of vascular and lymphatic systems, patients with GATA2 deficiency may present with lymphedema, monosomy 7, and MDS, known as Emberger syndrome.2,9

Peripheral blood lymphocyte telomere length as a predictor of response to immunosuppressive therapy in childhood aplastic anemia

Predicting the response to immunosuppressive therapy could provide useful information to help the clinician define treatment strategies for patients with aplastic anemia. In our current study, we evaluated the relationship between telomere length of lymphocytes at diagnosis and the response to immunosuppressive therapy in 64 children with aplastic anemia, using flow fluorescence in situ hybridization. Median age of patients was ten years (range 1.5–16.2 years). Severity of the disease was classified as very severe in 23, severe in 21, and moderate in 20 patients. All patients were enrolled in multicenter studies using antithymocyte globulin and cyclosporine. The response rate to immunosuppressive therapy at six months was 52% (33 of 64). The probability of 5-year failure-free survival and overall survival were 56% (95% confidence interval (CI): 41–69%) and 97% (95%CI: 87–99%), respectively. Median telomere length in responders was −0.4 standard deviation (SD) (−2.7 to +3.0 SD) and −1.5 SD (−4.0 to +1.6 (SD)) in non-responders (P<0.001). Multivariate analysis showed that telomere length shorter than −1.0 SD (hazard ratio (HR): 22.0; 95%CI: 4.19–115; P<0.001), platelet count at diagnosis less than 25×109/L (HR: 13.9; 95%CI: 2.00–96.1; P=0.008), and interval from diagnosis to immunosuppressive therapy longer than 25 days (HR: 4.81; 95%CI: 1.15–20.1; P=0.031) were the significant variables for poor response to immunosuppressive therapy. Conversely to what has been found in adult patients, measurement of the telomere length of lymphocytes at diagnosis is a promising assay in predicting the response to immunosuppressive therapy in children with aplastic anemia.

Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes

Purpose:Precise genetic diagnosis of inherited bone marrow failure syndromes (IBMFS), a heterogeneous group of genetic disorders, is challenging but essential for precise clinical decision making.Methods:We analyzed 121 IBMFS patients using a targeted sequencing covering 184 associated genes and 250 IBMFS patients using whole-exome sequencing (WES).Results:We achieved successful genetic diagnoses for 53 of 121 patients (44%) using targeted sequencing and for 68 of 250 patients (27%) using WES. In the majority of cases (targeted sequencing: 45/53, 85%; WES: 63/68, 93%), the detected variants were concordant with, and therefore supported, the clinical diagnoses. However, in the remaining 13 cases (8 patients by target sequencing and 5 patients by WES), the clinical diagnoses were incompatible with the detected variants.Conclusion:Our approach utilizing targeted sequencing and WES achieved satisfactory diagnostic rates and supported the efficacy of massive parallel sequencing as a diagnostic tool for IBMFS.Genet Med advance online publication 19 January 2017