Correlation Analysis of DNA Methylation Level and Clinical Characteristics in JMML Patients

Abstract Objective: As a rare, aggressive pediatric myeloproliferative disease, juvenile myelomonocytic leukemia (JMML) encompassed both biological features of myelodysplastic syndrome and myeloproliferative neoplasm. Studies have shown that the methylation level in JMML patients is closely related to prognosis, and patients with high methylation level have poor prognosis. This study aimed to find clinical indicators that were associated with different methylation levels and prognosis. Methods: The clinical information of 24 JMML patients with DNA samples admitted to our center from December 2013 to May 2020 was retrospectively analyzed, and the DNA methylation level of their whole genome was detected. Results: The median age of onset was 14.5 months (0.1-153 months) among the 24 cases, including 17 males and 7 females. At diagnosis, the median WBC count was 27.1×10 9/L (6.2-98.1×10 9/L), and the median platelet count was 38×10 9/L (10-277×10 9/L). Chromosome karyotype abnormalities were found in 12.5% (3/24) of patients. Next-generation sequencing results showed that 79.2% (19/24) patients had at least one Ras pathway-related classical gene mutation, and 41.7% (10/24) patients had two or more somatic mutations. Genomic DNA methylation levels were divided into three groups: 10 cases in the hypomethylation group, 4 cases in the moderate methylation group, and 10 cases in the hypermethylation group. There were significant differences in age, platelets, PTPN11 gene mutation and the number of somatic mutations ≥2 in different methylation groups (P<0.05). The age of hypomethylated group was significantly lower than that of hypermethylated group (P<0.05), and the platelets of hypomethylated group was significantly higher than that of hypermethylated group (P<0.05). Patients ≤12m and platelets＞32×10 9/L had lower DNA methylation level (P<0.0001). The number of patients with PTPN11 gene mutation in the hypomethylated group was significantly lower than that in the hypermethylated group (P<0.05), and the number of patients with ≥2 mutations in the low and medium methylated groups was significantly lower than that in the hypermethylated group (P<0.05). Correlation analysis showed that hypermethylation level was significantly correlated with PTPN11 gene mutation and ≥2 somatic mutations (P<0.001). Conclusions: JMML patients with high methylation level in the DNA genome at diagnosis were older and with lower platelet levels, and hypermethylation were significantly correlated with high-risk prognostic factors such as PTPN11 gene mutation and ≥2 somatic mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Decitabine for treatment of children with JMML

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Low Mutation Rate and No Significant Prognostic Implication of SF3B1, U2AF1 and SRSF2 Genes in Myelodysplastic Syndrome without Harboring Ring Sideroblast

Blood ◽

10.1182/blood.v120.21.4950.4950 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4950-4950

Author(s):

Hye-Ran Kim ◽

Trang Nguyen Thi Dai ◽

Min-Gu Kang ◽

Stephanie J. Won ◽

Hwan-Young Kim ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Gene Mutation ◽

Mutation Rate ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Study Cohort ◽

Prognostic Implication ◽

Hematopoietic Stem ◽

Exon 2 ◽

Number Of Patients

Abstract Abstract 4950 Background: Myelodysplastic syndrome (MDS) is an hematopoietic stem cells (HSCs) disorder, leading to malignant cells that ultimately grow uncontrollably. Somatic mutations of spliceosomal gene such as SF3B1, U2AF1 and SRSF2 has been widely described in MDS and other hematological malignancies. Many reports state that hematopoietic malignancies mostly result from somatic mutations in HSCs in the bone marrow. Some functional studies have been performed and have shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, such kinds of mutation are hallmarks of dominantly acting and growing specific cancer. Many efforts have been made to unravel the mutation considered disease background of MDS. Genotype–phenotype associations have been demonstrated for somatic spliceosomal gene mutation in MDS with ring sideroblasts but there is not much research regarding aberrant splicing pathways in MDS without harboring ring sideroblast. Therefore, this study investigated the prevalence and prognostic implication of the SF3B1, U2AF1 and SRSF2 splice gene mutation in these patients in Korea. Materials and Methods: The study cohort of 92 MDS patients was examined for somatic mutations in SF3B1, U2AF1 and SRSF2 splicing gene using direct sequencing method. The clinical and hematologic data were also recorded. The collected data was analyzed by SPSS for Windows version 18. 0. Pearson's chi-square tests, one way ANOVA analysis, and Student t-test were performed. Survival rates of multiple myeloma patients according to the mutation result of SF3B1, U2AF1 and SRSF2 splicing gene were analyzed using Kaplan-Meier log-rank test. Results: Our 92 MDS patients showed recurrent mutation and polymorphisms. Mutations in K666N, H662Q and K700E of SF3B1; S34T, S34P and Q157P of U2AF1; P95H, P95R and P95L of SRSF2 were found in 8 (8. 7%), 6 (6. 5%), and 11 (11. 9%) patients, respectively. The patients displayed 39198T>T/C polymorphism (88. 0%) in exon 18 of SF3B1, 8345T>T/G polymorphism (7. 6%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 12%, 80. 4% and 7. 6%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82. 0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10. 0% polymorphism and 90. 0% non polymorphism. The patient with either polymorphisms or mutations in both SF3B1, U2AF1 and SRSF2 had no effect on overall survival and disease-free survival. Conclusion: Our results show that mutation rate of SF3B1, U2AF1 and SRSF2 splice gene in Korean MDS patients without harboring ring sideroblast displayed relatively rare and infrequent molecular event. Moreover, alteration of SF3B1, U2AF1 and SRSF2 splice gene was not significantly implicated in the clinical outcomes and prognosis. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v114.22.2604.2604 ◽

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.

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Identification of GBA Gene Mutations in 19 Pakistani Unrelated Patients of Gaucher Disease

Blood ◽

10.1182/blood-2018-99-120385 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4952-4952 ◽

Cited By ~ 1

Author(s):

Arshi Naz ◽

Qurat Abedin ◽

Shariq Ahmed ◽

Saima Siddiqui ◽

Tahir Shamsi

Keyword(s):

Bone Marrow ◽

Genetic Analysis ◽

Gene Mutation ◽

Gaucher Disease ◽

Conflicts Of Interest ◽

Demographic Data ◽

White Blood Cells ◽

Bone Marrow Morphology ◽

Number Of Patients ◽

Gba Gene

Abstract Introduction: Gaucher disease (GD) is one of the lysosomal storage diseases that is rare and inherited autosomal recessively. There is insufficiency of glucocerebrosidase enzyme that leads to the build up of un-degraded substrates in white blood cells causing anemia, hepatosplenomegaly and skeletal disease. This enzyme deficiency is linked with the defect of its gene (GBA) that codes for this enzyme. Initial diagnosis is made by the estimation of glucocerebrosidase level in blood and confirmed by genetic analysis of GBA gene. To identify the mutations of GBA gene in Pakistani patients with GD from different regions of Pakistan. Sampling & methodology: The sample and demographic data was collected in National Institute of Blood Disease and Bone marrow Transplantation after approval of IRB and written informed consent of patients. We collected total 19 blood samples, out of which 5 had Gaucher's disease, 10 samples were parents of the index cases and 4 were control. The methodology consisted of DNA extraction and quantification from peripheral blood. Genetic analysis of coding regions of GBA gene was done via gene amplification, gel electrophoresis and sequencing. Result: Mutation was found in two out of five families that makes the prevalence of GBA gene mutation 40%. These were diagnosed on reduced enzyme levels and found to have L444P (c.1448T>C) mutation in homozygous form in 10th exon of GBA gene. The parents of that patient carried the same mutation in one allele. Rest of the patients who were diagnosed on bone marrow morphology showed no mutation in GBA gene. Conclusion: Our results illustrate that GBA gene mutation was found in those patients who were diagnosed by the estimation of β-glucosidase enzyme levels rather than on bone marrow morphology. In our population, the mutation L444P was found, which is the most frequent gene mutation found in the world. Since this study is conducted in a small number of patients therefore it is recommended that large cohorts of patients should be evaluated in future for genetic mutations among Gaucher's patients in Pakistan Key words: Gaucher disease, storage disorder, GBA gene Disclosures No relevant conflicts of interest to declare.

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Calr Mutational Status in Egyptian Patients with Myeloproliferative Neoplams

Blood ◽

10.1182/blood-2019-128194 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5388-5388

Author(s):

Eman A. Soliman ◽

Samah I. El-Ghlban ◽

Abdelaleem H. Abdelaleem ◽

Sherin Abdel-Aziz ◽

Sameh Shamaa ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Therapy ◽

Hcv Infection ◽

Number Of Patients ◽

Significant Difference ◽

Egyptian Patients ◽

Calr Mutations

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

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No Recurrent Mutation of SF3B1, U2AF1 and SRSF2 Spliceosomal Gene in Multiple Myeloma but Polymorphisms of These Genes Are Associated with the Prediction of Worse Prognosis

Blood ◽

10.1182/blood.v120.21.3958.3958 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3958-3958

Author(s):

Trang Nguyen Thi Dai ◽

Hye-Ran Kim ◽

Min-Gu Kang ◽

Stephanie J. Won ◽

Hwan-Young Kim ◽

...

Keyword(s):

Multiple Myeloma ◽

Light Chain ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Bone Lesion ◽

Disease Free Survival ◽

Free Light Chain ◽

Immunoglobulin Level ◽

Exon 2 ◽

Number Of Patients

Abstract Abstract 3958 Background: Recently, a striking example of the effects on acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The sequencing of the DNA from abnormal blood cells from patients with several types of leukemia such as AML, CLL, CMML, pre-leukemic syndromes, and MDS, has shown that a high proportion of these cases are associated with somatic mutations in spliceosomal proteins. Also, evidence of cancer-specific alternative splicing and oncogenic somatic mutations in spliceosome subunits has been steadily growing. However, there is not much research regarding aberrant splicing pathways in Multiple myeloma (MM) patients. Therefore, we tried to investigate the presence and prognostic implication of mutation of the SF3B1 and U2AF1 protein in these patients in South Korea. Materials and Methods: We examined a cohort of 87 MM patients and 100 healthy controls for somatic mutations in SF3B1, U2AF1 and SRSF2 by using direct sequencing method. The medical records were reviewed for age, sex, plasma cell percentage, serum M protein, immunoglobulin level, free light chain ratio, calcium, creatinine, hemoglobin, bone lesion, albumin, beta 2 microglobulin, lactate dehydrogenase, treatment outcome, and so on. The collected data was analyzed by SPSS for Windows version 18.0. We performed Pearson's chi-square tests, one way ANOVA analysis, and Student t-test. Survival rates of myeloma patients according to the result of SF3B1, U2AF1 and SRSF2 sequencing were analyzed using Kaplan-Meier log-rank test. Results: Our 87 MM patients showed no mutation including known recurrent ones in SF3B1, U2AF1 and SRSF2 genes. However, the patients displayed 39198T>T/C polymorphism (70.1%) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8%) in exon 2 of U2AF1 and 5399C/T polymorphism (100%) in exon 1 of SRSF2. In the entire cohort, the number of patients with no polymorphism, one polymorphism, two polymorphisms and three polymorphisms was counted up to 0%, 24.1%, 67.8% and 8.0%, respectively. The T/C polymorphism at position 39198 of SF3B1 exon 18 and the T/G polymorphism at position 8345 of U2AF1 exon 2 were analyzed by allele-specific PCR using normal control. Results in 100 normal controls, polymorphism of SF3B1 exon 18 was taken into account of 82.0%, the remaining is non polymorphism while U2AF1 exon 2 showed 10.0% polymorphism and 90.0% non polymorphism. Sex (p=0.048) and free light chain ratio (p=0.002) showed significant results according to polymorphism status while other clinical characteristics were not associated. The patient with polymorphisms in both SF3B1 and U2AF1 had worse overall survival (P=0.042) and disease-free survival (P<0.01), compared to patients without polymorphism. Conclusion: Our results show no recurrent SF3B1, U2AF1 and SRSF2 mutations in MM patients rather polymorphisms in SF3B1 and U2AF1 gene were significantly implicated in the prediction of poor prognosis. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2019-121681 ◽

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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Response to upfront azacitidine in juvenile myelomonocytic leukemia in the AZA-JMML-001 trial

Blood Advances ◽

10.1182/bloodadvances.2020004144 ◽

2021 ◽

Vol 5 (14) ◽

pp. 2901-2908

Author(s):

Charlotte M. Niemeyer ◽

Christian Flotho ◽

Daniel B. Lipka ◽

Jan Starý ◽

Claudia Rössig ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

Newly Diagnosed ◽

Open Label ◽

Hematopoietic Stem ◽

Curative Therapy ◽

Time Profiles ◽

Genome Wide ◽

Number Of Patients ◽

Myelomonocytic Leukemia

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most children with juvenile myelomonocytic leukemia (JMML). Novel therapies controlling the disorder prior to HSCT are needed. We conducted a phase 2, multicenter, open-label study to evaluate the safety and antileukemic activity of azacitidine monotherapy prior to HSCT in newly diagnosed JMML patients. Eighteen patients enrolled from September 2015 to November 2017 were treated with azacitidine (75 mg/m2) administered IV once daily on days 1 to 7 of a 28-day cycle. The primary end point was the number of patients with clinical complete remission (cCR) or clinical partial remission (cPR) after 3 cycles of therapy. Pharmacokinetics, genome-wide DNA-methylation levels, and variant allele frequencies of leukemia-specific index mutations were also analyzed. Sixteen patients completed 3 cycles and 5 patients completed 6 cycles. After 3 cycles, 11 patients (61%) were in cPR and 7 (39%) had progressive disease. Six of 16 patients (38%) who needed platelet transfusions were transfusion-free after 3 cycles. All 7 patients with intermediate- or low-methylation signatures in genome-wide DNA-methylation studies achieved cPR. Seventeen patients received HSCT; 14 (82%) were leukemia-free at a median follow-up of 23.8 months (range, 7.0-39.3 months) after HSCT. Azacitidine was well tolerated and plasma concentration-–time profiles were similar to observed profiles in adults. In conclusion, azacitidine monotherapy is a suitable option for children with newly diagnosed JMML. Although long-term safety and efficacy remain to be fully elucidated in this population, these data demonstrate that azacitidine provides valuable clinical benefit to JMML patients prior to HSCT. This trial was registered at www.clinicaltrials.gov as #NCT02447666.

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Germline Mutations in CBL Cause a Predisposition to Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.310.310 ◽

2009 ◽

Vol 114 (22) ◽

pp. 310-310 ◽

Cited By ~ 1

Author(s):

Charlotte M Niemeyer ◽

Michelle Kang ◽

Ingrid Furlan ◽

Danielle Shin ◽

Debbie S Sakai ◽

...

Keyword(s):

Autosomal Dominant ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Familial Cancer ◽

Myeloproliferative Neoplasm ◽

Germline Mutations ◽

Clinical Syndrome ◽

Juvenile Myelomonocytic Leukemia ◽

Published Data ◽

Myelomonocytic Leukemia

Abstract Abstract 310 Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm (MPN) of early childhood. Up to 60% of patients harbor activating mutations in either NRAS, KRAS, or PTPN11, while another 15% of children have neurofibromatosis type 1 (NF-1) and demonstrate loss of the wildtype NF1 allele in their hematopoietic cells at diagnosis. We recently described that an additional 10–15% of children with JMML harbor missense homozygous mutations in exons 8 and 9 of CBL (Loh, Blood, 2009). Cbl is a complex protein that functions primarily as an E3 ubiquitin ligase but also serves numerous important adaptor functions. Mutations in CBL have recently been reported in adults with MPNs and the available evidence in adults indicates that these lesions are somatically acquired. We noted that a number of children with JMML and CBL mutations had neurological conditions including developmental delay and dysmorphic stigmata, although these features were not 100% penetrant. Based on these observations, we performed mutational studies in fibroblasts and buccal epithelial cells, which were available from 13 JMML patients with homozygous CBL mutations in their bone marrow at diagnosis. We now show that in all 13 patients the initial CBL mutation occurred as a heterozygous germline event (Table 1). Interestingly, a child with the 1222 T>C later developed a brain tumor with a homozygous CBL lesion. Mutational studies on parental DNA were informative in 11 cases and indicated autosomal inheritance in 6 families. Furthermore, two of these children had extensive family histories in which several young family members died of JMML. There were no known features of NF-1 in either pedigree. Subsequent analysis of these two pedigrees revealed a pattern of autosomal dominant inheritance that spanned 4 generations in 1 family and 3 generations in the other. To rule out normal genetic variation, a cohort of 240 healthy individuals were screened without detection of a CBL abnormality. One hallmark feature of JMML myeloid progenitor cells is their sensitivity to granulocyte-macrophage colony stimulating factor (GM-CSF) in colony-forming assays. Retroviral transduction of the 371 Tyr>His and 384 Cys>Arg mutations into wildtype murine fetal liver cells failed to induce a hypersensitive phenotype. However, recently published data suggest that a full oncogenic phenotype is conferred when the wildtype allele is deleted (Sanada, Nature, 2009), as occurs in the human diseases. Transduction of the most common human JMML mutations into BaF3-EpoR cells that have had murine Cbl knocked down is ongoing, as is further biochemical analysis. In summary, germline mutations in CBL cause a clinical syndrome with a predisposition to JMML, and importantly, can be inherited in an autosomal dominant fashion, thus establishing CBL as a new familial cancer predisposition gene. Disclosures: No relevant conflicts of interest to declare.

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Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Blood ◽

10.1182/blood.v122.21.45.45 ◽

2013 ◽

Vol 122 (21) ◽

pp. 45-45 ◽

Cited By ~ 1

Author(s):

Hui Huang ◽

Daniel E. Bauer ◽

Mignon L. Loh ◽

Govind Bhagat ◽

Alan B. Cantor ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Phosphorylation ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Brdu Incorporation ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

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DNA Methylation Differences in Twins Discordant for Adolescent/Young Adult Hodgkin Lymphoma

Blood ◽

10.1182/blood.v126.23.179.179 ◽

2015 ◽

Vol 126 (23) ◽

pp. 179-179

Author(s):

Jun Wang ◽

Amie Hwang ◽

Dan Weisenberger ◽

David J. Van Den Berg ◽

Victoria K. Cortessis ◽

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Keyword(s):

Dna Methylation ◽

Hodgkin Lymphoma ◽

Methylation Level ◽

Conflicts Of Interest ◽

Developed Countries ◽

T Test ◽

Blood Draw ◽

Blood Samples ◽

Dna Methylation Age ◽

Paired T Test

Abstract Hodgkin lymphoma (HL) is derived from germinal center B-lymphocytes, has a peak incidence in adolescents and young adults (AYA) in developed countries and results in substantial treatment-related late effects. We previously identified a SNP (rs1860661G) in the TCF3 gene, inversely associated with HL risk, that is also mutated in tumor cells. The protein encoded by TCF3 is responsible for maintaining the B-cell program, which is downregulated in HRS cells; therefore higher expression levels may be protective.1 DNA methylation is associated with repression of gene expression and may thereby play a role in HL pathogenesis. We extracted DNA from blood samples obtained from HL survivors and their unaffected monozygotic MZ twins (4 female and 5 male twin pairs discordant for AYA HL). Average age at blood draw was 62 years, and average time since diagnosis of the case was 34 years. We also extracted DNA from blood samples from 25 unaffected MZ twin pairs as control pairs. DNAs were bisulfite converted and DNA methylation quantified using the Illumina Infinium HumanMethylation27 BeadChip array, which scores methylation levels as β values. We compared average DNA methylation β value differences at the TCF3 locus cg16524139 (near rs1860661) in HL survivors and their unaffected co-twins using a paired t-test. We also examined the TCF3 germline genotype by DNA methylation level rank in HL-discordant pairs. We also computed DNA methylation age, a global measure of physiological aging, using a validated algorithm2 and compared the result in the HL survivor to that of each corresponding unaffected co-twin using a paired t-test. We found a small but significantly higher DNA methylation level at the TCF3 locus in HL survivors compared to their unaffected co-twins (p=0.05), but no difference between the members of healthy MZ twin pairs (p=0.81). The protective allele (G) was more prevalent in the 6 pairs with higher DNA methylation level in HL survivors (33%), compared to the 3 pairs with higher levels in unaffected co-twins (17%). This pattern would be expected if methylation at the cg16524139 were to diminish TCF3 expression related to a protective rs1860661G variant. HL survivors had an average DNA methylation age of 64.1 years compared to 61.3 years in their unaffected MZ co-twins (p=0.04). Differential DNA methylation in HL survivors and their unaffected twins suggests a role for DNA methylation in HL, possibly as a result of treatment or disease. 1. Cozen W, Timofeeva MN, Li D, Diepstra A, Hazelett D, Delahaye-Sourdeix M, et al.: A meta-analysis of Hodgkin lymphoma reveals 19p13.3 TCF3 as a novel susceptibility locus. Nat Comm 25:3856. doi: 10.1038/ ncomms 4856, 2014 2. Horvath S: DNA methylation age of human tissues and cell types. Genome Biol, 14:R115 doi:10.1186/gb-2013-14-10-r115, 2013. Disclosures No relevant conflicts of interest to declare.

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