Mutations of the Spliceosome Complex Genes Occur In Adult Patients but Are Very Rare In Children with Myeloid Neoplasia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2797-2797
Author(s):  
Shinsuke Hirabayashi ◽  
Jessica Moetter ◽  
Kenichi Yoshida ◽  
Michael Heuser ◽  
Henrik Hasle ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Adult Patients ◽  
Refractory Anemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Buccal Swab ◽  
New Genes ◽  
Whole Exome ◽  
Normal Cytogenetics ◽  
Myeloid Neoplasia ◽  
Myelomonocytic Leukemia

Abstract Abstract 2797 Somatic point mutations occur frequently in adult patients with myelodysplastic syndromes (MDS) and are associated with distinct clinical features such as overall survival, genomic aberrations and clonal transformation. Recently a number of new genes had been identified which are involved in methylation and chromatin regulation (TET2, DNMT3A, ASXL1). Although mutations of these genes are present at high frequency in adult cohorts, they are very rare events in children with myeloid disease. Based on the results from a large-scale whole exome sequencing study of adult patients with myeloid neoplasia, a new group of genes involved in the formation of spliceosome complex was discovered to be recurrently somatically mutated in approximately 40% of patients with MDS and 55% of chronic myelomonocytic leukemia (CMML) cases (Yoshida, et al., unpublished data). Spliceosomes are macromolecular RNA-protein complexes that remove noncoding introns from precursor mRNA. We hypothesized that the disruption of the spliceosome complex might play a driving role in the leukemogenesis in pediatric MDS. Using targeted re-sequencing we investigated the 3 exclusive hotspots of 2 spliceosome genes that were found to be mutated in adult MDS: U2AF1 (S34), U2AF1 (Q157) and SFRS2 (P95). We analyzed a total of 339 WHO-defined pediatric cases: juvenile myelomonocytic leukemia (JMML), n=112; secondary CMML, n=12; Down syndrome-associated MDS, n=6; de novo acute myeloid leukemia (AML), n=12, MDS-related AML, n=50; refractory anemia with excess blasts (RAEB), n=52; RAEB in transformation (RAEB-T), n=11; and refractory cytopenia (RC), n=84. We found two heterozygous missense mutations: SFRS2 c.284C>T/P95L in a patient with JMML, and U2AF1 c.101C>A/S34Y in a patient with refractory cytopenia. The first patient presented with non-syndromic JMML with somatic mutation of PTPN11 c.226G>C/E76Q, normal cytogenetics, increased HbF(80%), splenomegaly, monocytosis, and was 4.5 years old at diagnosis. The patient underwent successful SCT. The analysis of buccal-swab DNA confirmed the somatic status of the SFRS2 mutation. The second patient was diagnosed at age of 17 years with systemic mastocytosis positive for c-kit D816V mutation, and with associated clonal hematological non-mast cell lineage disease: refractory cytopenia with normal cytogenetics. SCT was also performed with success on this patient. To ensure that the low rate of mutations found in our pediatric cohort was not due to technical issues, we also sequenced major mutation sites of U2AF1 and SFRS2 genes in 19 adult patients with MDS: refractory anemia (RA), n=8; RC with multilineage dysplasia (RCMD), n=3; RAEBI/II, n=5; CMML, n=3. We found 4 heterozygous changes: SFRS2 missense mutation c.284C>T/P95L in two patients with RA and RAEBII; SFRS2 deletion c.284_307/P95RfsX120 in one RA patient, and U2AF1 c.470A>C/Q157P in one RA patient. Interestingly, 3 out of 4 patients with a mutated spliceosome gene harbored a mutation of the ASXL1 gene, but none was mutated for DNMT3A. In summary, re-sequencing of 3 spliceosome gene hotspots revealed the presence of heterozygous mutations in 2/339 children and 4/19 adults with myeloid disease. The analysis of another gene-hotspot implicated in the MDS whole exome study, SF3B1 K700 is ongoing, but all pediatric cases analyzed to date were negative. The drastically reduced frequency of spliceosome mutations in pediatric compared to adult myeloid malignancies suggests a different pathogenetic mechanism in childhood disease, and fits well with previous reports that somatic mutations of non-Ras-pathway genes, such as DNMT3A, are less prevalent in pediatric cohorts. The functional impact of spliceosome mutations on leukemogenesis warrants further study. Disclosures: No relevant conflicts of interest to declare.

Myelodysplastic/Myeloproliferative Neoplasms

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 264-272 ◽  
Author(s):  
Mario Cazzola ◽  
Luca Malcovati ◽  
Rosangela Invernizzi
Keyword(s):  
Clinical Laboratory ◽  
Myeloproliferative Neoplasms ◽  
Chronic Myelomonocytic Leukemia ◽  
World Health ◽  
Refractory Anemia ◽  
Molecular Diagnostic ◽  
Juvenile Myelomonocytic Leukemia ◽  
Lymphoid Tissues ◽  
Ring Sideroblasts ◽  
Myelomonocytic Leukemia

Abstract According to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, myelodysplastic/myeloproliferative neoplasms are clonal myeloid neoplasms that have some clinical, laboratory, or morphologic findings that support a diagnosis of myelodysplastic syndrome, and other findings that are more consistent with myeloproliferative neoplasms. These disorders include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable. The best characterized of these latter unclassifiable conditions is the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis. This article focuses on myelodysplastic/myeloproliferative neoplasms of adulthood, with particular emphasis on chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts associated with marked thrombocytosis. Recent studies have partly clarified the molecular basis of these disorders, laying the groundwork for the development of molecular diagnostic and prognostic tools. It is hoped that these advances will soon translate into improved therapeutic approaches.

CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2604-2604
Author(s):  
Y. Lucy Liu ◽  
Priyangi A Malaviarachchi ◽  
Shelly Y. Lensing ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Protein ◽  
Gm Csf ◽  
Myelomonocytic Leukemia ◽  
Normal Controls

Abstract Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of Stable Cell Lines Representing Juvenile Myelomonocytic Leukemia with SHP2 Mutations

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4353-4353
Author(s):  
Yuming Zhao ◽  
Yao Guo ◽  
Chunxiao He ◽  
Dengyang Zhang ◽  
Han Zhong Pei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cytokine Signaling ◽  
Ras Signaling ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Myelomonocytic Leukemia

Abstract Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

Prospective Study of 90 Children Requiring Treatment for Juvenile Myelomonocytic Leukemia or Myelodysplastic Syndrome: A Report From the Children’s Cancer Group

2002 ◽  
Vol 20 (2) ◽  
pp. 434-440 ◽  
Author(s):  
William G. Woods ◽  
Dorothy R. Barnard ◽  
Todd A. Alonzo ◽  
Jonathan D. Buckley ◽  
Nathan Kobrinsky ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Prospective Study ◽  
De Novo ◽  
Refractory Anemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Allogeneic Bone ◽  
Monosomy 7 ◽  
Cancer Group ◽  
De Novo Aml ◽  
Myelomonocytic Leukemia

PURPOSE: We report the first large prospective study of children with myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML) treated in a uniform fashion on Children’s Cancer Group protocol 2891. PATIENTS AND METHODS: Ninety children with JMML, various forms of MDS, or acute myeloid leukemia (AML) with antecedent MDS were treated with a five-drug induction regimen (standard or intensive timing). Patients achieving remission were allocated to allogeneic bone marrow transplantation (BMT) if a matched family donor was available. All other patients were randomized between autologous BMT and aggressive nonmyeloablative chemotherapy. Results were compared with patients with de novo AML. RESULTS: Patients with JMML and refractory anemia (RA) or RA-excess blasts (RAEB) exhibited high induction failure rates and overall remission of 58% and 48%, respectively. Remission rates for patients with RAEB in transformation (RAEB-T) (69%) or antecedent MDS (81%) were similar to de novo AML (77%). Actuarial survival rates at 6 years were as follows: JMML, 31% ± 26%; RA and RAEB, 29% ± 16%; RAEB-T, 30% ± 18%; antecedent MDS, 50% ± 25%; and de novo AML, 45% ± 3%. For patients achieving remission, long-term survivors were found in those receiving either allogeneic BMT or chemotherapy. The presence of monosomy 7 had no additional adverse effect on MDS and JMML. CONCLUSION: Childhood subtypes of MDS and JMML represent distinct entities with distinct clinical outcomes. Children with a history of MDS who present with AML do well with AML-type therapy. Patients with RA or RAEB respond poorly to AML induction therapy. The optimum treatment for JMML remains unknown.

Myelodysplastic syndrome (MDS) and autoimmune diseases (AD): Hypomethylating agents to treat both.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e18018-e18018
Author(s):  
Omar Al Ustwani ◽  
Laurie Ann Ford ◽  
Sheila N. J. Sait ◽  
Anne Marie W. Block ◽  
Maurice P. Barcos ◽  
...  
Keyword(s):  
Treatment Plan ◽  
Myeloproliferative Neoplasm ◽  
Relapsing Polychondritis ◽  
Refractory Anemia ◽  
Hypomethylating Agents ◽  
Monosomy 7 ◽  
Double Stranded Dna ◽  
Clinical Presentations ◽  
Normal Cytogenetics ◽  
Myelomonocytic Leukemia

e18018 Background: AD occurring in the setting of MDS is challenging to recognize and incorporate into the treatment plan. We assessed the clinical presentations, laboratory abnormalities and outcome of patients with MDS and AD. Methods: Records of MDS patients treated at Roswell Park Cancer Institute between 2007 and 2010 were reviewed (n=123). Results: AD was identified in 10 MDS patients (8.1%): 70% were males, median age was 60.6 years (41-75). AD manifested as seronegative polyarthritis in 2, bronchiolitis obliterans in 2, Hashimoto’s thyroiditis in 2, and 1 (10%) for each of rheumatoid arthritis, systemic lupus, polymyalgia rheumatica, Sjogren syndrome and relapsing polychondritis. Laboratory autoimmune markers were: anti nuclear antibodies in 2, rheumatoid factor in 2, anti-double stranded DNA in 1 and anti phospholipid syndrome with thrombosis in 1. Corticosteroids were the most common used treatment for AD (60%). Regarding the MDS diagnosis; 50% had refractory anemia with excess of blasts-1 and -2, 20% refractory anemia with ring sideroblasts, 10% for each of chronic myelomonocytic leukemia, refractory anemia and MDS/myeloproliferative neoplasm. Normal cytogenetics were noted in 70% of patients, 20% with complex karyotype and 10% had monosomy 7. Hypomethylating agents were used to treat MDS in 80% of patients and in one case were associated with concomitant improvement of AD. Overall survival from diagnosis with MDS was 54 months (6-127). Conclusions: Our experience with one patient and review of the literature suggest that both AD and MDS could benefit from treatment with hypomethylating agents. This warrants a prospective clinical trial.

Cbl and TET2 Mutations Are Present in Refractory Ph+ Disorders Including Accelerated and Blast Crisis CML and ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2173-2173
Author(s):  
Hideki Makishima ◽  
Anna M. Jankowska ◽  
Heather Cazzolli ◽  
Bartlomiej P Przychodzen ◽  
Courtney Prince ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Blast Crisis ◽  
Uniparental Disomy ◽  
Ring Finger ◽  
Chronic Phase ◽  
Homozygous Deletion ◽  
Juvenile Myelomonocytic Leukemia ◽  
Tet2 Mutation ◽  
Myelomonocytic Leukemia

Abstract Abstract 2173 Poster Board II-150 Loss of heterozygosity (LOH) due to acquired uniparental disomy (UPD) is a commonly observed chromosomal lesion in myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML). Most recurrent areas of LOH point towards genes harboring mutations. For example, UPD11q23.3 and UPD4q24 were found to be associated with c-Cbl and TET2 mutations, respectively. Cbl family mutations (c-Cbl and Cbl-b) have been associated with atypical MDS/MPN including CMML and juvenile myelomonocytic leukemia (JMML) as well as more advanced forms of MDS and secondary AML (sAML). Ring finger mutants of Cbl abrogate ubiquitination and thereby tumor suppressor function related to inactivation of phosphorylated receptor tyrosine kinases, Src and other phosphoproteins. TET2 mutations are present in a similar disease spectrum. The TET family of proteins is involved in conversion of methylcytosine to methylhydroxycytosine which cannot be recognized by DNMT1. Thereby, the proteins seem to counteract maintenance hypermethylation. In our screen of MDS/MPN, we found c-Cbl and Cbl-b ring finger mutations in 5/58 (9%) of CMML and AML derived from CMML, 2/39 (5%) MDS/MPNu, 4/21 (19%) JMML and 14/148 (9%) RAEB/sAML. In the same cohort, TET2 mutations were present in 37% and 14% of patients with MDS/MPN and MDS, respectively. Of note we did not find any TET2 mutations in JMML. We and others have also noted that TET2 and c-Cbl mutations were also detected in atypical chronic myeloid leukemia. While translocations resulting in BCR/ABL fusion characterize CML, we stipulated that in analogy to other chronic myeloproliferative diseases, TET2 and c-Cbl mutations may be also present in CML and contribute to phenotypic heterogeneity within BCR/ABL associated disorders. In particular, progression of CML to accelerated phase (AP) or blast crisis (BC) could be associated with acquisition of additional lesions. When 22 patients with CML chronic phase (CP) were screened, no TET2 and c-Cbl mutations were found. However, we identified 1 c-Cbl, 2 Cbl-b (6%) and 6 TET2 (12%) mutations in 51 patients with CML-AP (N=18) and CML-BC (N=33) with myeloid and lymphoid/mix 24 and 9 phenotype, respectively. These mutations were mutually exclusive. We also noted that TET2 mutations were present in 1/9 CML in BC with lymphoid phenotype. We subsequently screened Ph+ ALL cases (N=9) and found a TET2 mutation in 1 case but no Cbl family mutations. In contrast when 9 Ph- ALL cases were screened as controls, neither TET2 or Cbl mutations were found. SNP-A analysis revealed 2 cases of LOH involving chromosome 4 (UPD4q24 and del4) in a patient with lymphoid blast crisis and Ph+ ALL, respectively. However, UPD was not found in Cbl family gene regions (11q23.3 or 3q13.11). A homozygous deletion of Cbl-b region was seen in a CP patient. Cbl family mutations were associated with a more complex karyotype than TET2 mutations (67% vs. 17% cases with abnormal phenotype). Patients with Cbl family mutations were resistant to imatinib which was effective in only 2 out of 6 patients with TET2mutations. Dasatinib was effective in 2 patients with TET2 mutation. Median over all survival of progressed CML was 47, 49 and 48 months in patients with Cbl, TET2 or no mutations, respectively. In conclusion, our results indicate that Cbl family mutations can occur as secondary lesions in myeloid type aggressive CML (AP and myeloid BC), but not in lymphoid types. TET2 mutations were identified in both lymphoid BC and Ph1+ALL, as well as myeloid BC and AP. In contrast to CMML or JMML in which a vast majority of mutations are homozygous, all Cbl family mutations were heterozygous (no LOH). Similarly, all but two TET2 mutations were heterozygous (1 hemizygous in del4 and 1 homozygous case in UPD4q), suggesting that additional cooperating lesions affecting corresponding pathways may be present. These mutations likely represent secondary lesions which contribute to more either progression (CML) or more aggressive features (Ph+ ALL) and characterize disease refractory to therapy with imatinib. Disclosures: No relevant conflicts of interest to declare.

Mir-183 Over-Expression: A Potential Biomarker for Juvenile Myelomonocytic Leukemia (JMML).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2558-2558
Author(s):  
Y. Lucy Liu ◽  
Yan Yan ◽  
Shelly Y. Lensing ◽  
Todd Cooper ◽  
Peter D. Emanuel
Keyword(s):  
Peripheral Blood ◽  
Robust Regression ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Potential Biomarker ◽  
Median Level ◽  
Myelomonocytic Leukemia

Abstract Abstract 2558 Juvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p<0.001); but the median level of miR-34b was only slightly higher in JMML subjects, and not significantly so, compared to normal individuals (median=1.4 vs 1.0, p>0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p<0.05). Based on a robust regression analysis, for every unit increase in the square root of RQ miR-183, the monocyte percentage significantly increased by 0.73% (SE=0.32%, p=0.023). This is the first evidence suggesting that microRNAs may contribute to the pathogenesis of JMML. miR-183 may also serve as an important biomarker that can be directly and quantitatively linked to significant clinical parameters in JMML. It also may ultimately provide a target for JMML therapy. Disclosures: No relevant conflicts of interest to declare.

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.

Decitabine, a Potential Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2516-2516 ◽  
Author(s):  
Y. Lucy Liu ◽  
Peter Dean Emanuel ◽  
Robert P. Castleberry
Keyword(s):  
Mononuclear Cells ◽  
Plasma Concentrations ◽  
Philadelphia Chromosome ◽  
Disease Free Survival ◽  
Adult Patients ◽  
Myelogenous Leukemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Pharmacokinetic Studies ◽  
Gm Csf ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS, NF1, and PTPN11, have been linked to be responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult for JMML. Only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28–55%), with 5-year disease-free survival rates of 25–40%. Decitabine, as one of the second generation of hypomethylating agents, has been demonstrated to produce encouraging responses in adult patients with chronic myelogenous leukemia or other hematopoietic disorders. Our recent studies have demonstrated that PTEN deficiency is detected in 67% of JMML patients. We hypothesize that hypermethylation of the PTEN promoter is one of the causes that lead to PTEN deficiency in JMML, and that a hypomethylating agent may improve PTEN expression in JMML cells, and thus inhibit hypersensitivity to GM-CSF. In order to test our hypothesis, we conducted an in vitro pilot study with JMML cells. Hypermethylation of the PTEN promoter was detected in 23/30 (77%) of JMML patients using Methylation-specific PCR. Sequencing confirmed that the CpG islands of the PTEN promoter were hypermethylated. A CFU-GM formation assay was used to evaluate the therapeutic sensitivity of Decitabine to JMML cells. Frozen mononuclear cells from peripheral blood samples of 5 JMML patients were plated in 0.3% agar medium with Decitabine ranging in concentration from 1nM to 1000nM. Significant inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion. The effective Decitabine concentrations in the cultures were lower or equivalent to the safe and tolerable plasma concentrations achievable in adult patients in clinical settings (30 mg/m2/day). Our data suggests that hypermethylation of the PTEN promoter is a common event in JMML, and Decitabine may be a potentially safe and effective reagent to treat JMML. Further pharmacokinetic studies should be conducted in the clinic to clarify the plasma concentration in pediatric patients, and the mechanism of Decitabine in inhibiting hypersensitivity of JMML cells to GM-CSF should be further explored since multiple genes are hypermethylated in cancers.

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