Genome-wide DNA methylation is predictive of outcome in juvenile 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.

Download Full-text

Genome-Wide DNA Methylation Profiling Reveals Distinct Molecular Subgroups of Juvenile Myelomonocytic Leukemia with Clinical Significance

Leukemia Research ◽

10.1016/s0145-2126(17)30147-9 ◽

2017 ◽

Vol 55 ◽

pp. S22

Author(s):

D. Lipka ◽

T. Witte ◽

R. Toth ◽

J. Yang ◽

A. Baude ◽

...

Keyword(s):

Dna Methylation ◽

Clinical Significance ◽

Juvenile Myelomonocytic Leukemia ◽

Molecular Subgroups ◽

Genome Wide ◽

Dna Methylation Profiling ◽

Myelomonocytic Leukemia ◽

Methylation Profiling

Download Full-text

Aberrant DNA Methylation Is Associated With Poor Outcomes In Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v122.21.517.517 ◽

2013 ◽

Vol 122 (21) ◽

pp. 517-517

Author(s):

Hirotoshi Sakaguchi ◽

Hideki Muramatsu ◽

Xinan Wang ◽

Yinyan Xu ◽

Yoko Hibi ◽

...

Keyword(s):

Dna Methylation ◽

Chromosomal Aberration ◽

Research Funding ◽

Methylation Pattern ◽

World Health ◽

Juvenile Myelomonocytic Leukemia ◽

Aberrant Methylation ◽

Aberrant Dna Methylation ◽

Poor Outcomes ◽

Myelomonocytic Leukemia

Abstract Recent studies suggest that aberrant methylation plays a fundamental role in the development of a variety of cancers, including myeloid malignancies. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is characterized by both excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor. It is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) according to the World Health Organization classification. We recently reported that somatic mutations in SETBP1 and JAK3 were identified in JMML patients and were associated with poor outcomes (Nat Genet 2013;45:937–41). The goal of this study was to clarify the clinical significance of aberrant DNA methylation in JMML. We studied 92 children (61 boys and 31 girls) who were diagnosed with JMML in institutions throughout Japan. A diagnosis of JMML was made based on internationally accepted criteria. We quantitatively evaluated the CpG methylation pattern in the promoter regions of 16 candidate genes (APC, BMP4, CALCA, CDH13, CDKN2A, CDKN2B, CHFR, DAPK, DMR-H19, ER, IGF2, MGMT, MLH1, RARB, RASSF1, TP73) from genomic DNA derived from bone marrow specimens at the time of diagnosis. This was accomplished by bisulfite conversion and the pryosequencing technique. We defined aberrant methylation as >3 standard deviations from the mean methylation level derived from 8 healthy individuals. The median age at diagnosis was 16 months (range, 0.3–160). By genetic analysis, PTPN11, NF1, NRAS, KRAS, and CBL mutations were found in 39 (42%), 7 (8%), 12 (13%), 13 (14%), and 11 (12%) patients, respectively. In addition, 16 patients had SETBP1 or JAK3 mutations. Karyotypic abnormalities were detected in 15 patients, including 8 with monosomy 7. The median monocyte count, percentage of hemoglobin F, and platelet count at the time of diagnosis were 4.6x109/L (range, 0.2–31.6), 21% (range, 0–68), and 61.0x109/L (range, 1.4–483), respectively. The median observation period was 18 months (range, 1–287). During observation, 56 of the 92 patients received allogeneic hematopoietic stem cell transplantation (HSCT), and 30 of 92 patients died. Outcomes were assessed according to transplantation-free survival (TFS), in which HSCT and death were censored, and overall survival (OS) by the Kaplan-Meier method. Aberrant methylation of BMP4, CALCA, CDKN2A, CDKN2B, DMR-H19 and RARB were detected, of which hypermethylation of BMP4, CALCA, CDKN2A, and RARB were associated with poor TFS according to univariate analyses (P<0.10). We integrated the number of aberrant methylation of these four genes to arrive at an aberrant methylation score (AMS). An AMS of 0, 1, 2, 3, and 4 was seen in 36, 29, 19, 7, and 1 of the 92 patients, respectively. The AMS was significantly higher in patients with SETBP1 or JAK3 mutations than in other patients (P=0.03): 1, 8, 3, 3, and 1 of the 16 patients showed an AMS of 0, 1, 2, 3, and 4, respectively. The probability of 5-year TFS was 42% in the AMS = 0 cohort and 4% in the AMS = 1 to 4 cohort, respectively (log-rank, P<0.001). Moreover, the probability of 5-year OS was 65% in the AMS = 0 to 2 cohort and 8% in the AMS = 3 and 4 cohort, respectively (log-rank, P=0.004). In multivariable analysis using the Cox-proportional hazard model, AMS = 1 to 4 (hazard ratio [HR], 2.6; 95% confidential interval [CI], 1.2–5.5; P=0.013), mutations of PTPN11 or NF1 (HR, 2.7; 95% CI, 1.3–5.5; P=0.010), and chromosomal aberration (HR, 3.3; 95% CI, 1.7–6.5; P=0.001) were independent predictors of poor TFS. Further, chromosomal aberration (HR, 4.4; 95% CI, 1.6–11.8; P=0.004) and platelet counts <33x109/L (HR, 2.8; 95% CI, 1.3–6.4; P=0.013) were independent predictors of poor OS. The present study shows that aberrant methylation of four genes (BMP4, CALCA, CDKN2A, and RARB) is associated with poor outcomes in JMML patients. Patients with SETBP1/JAK3 mutations frequently show the hypermethylation of these genes. Further, allogeneic HSCT is associated with improved outcomes for patients with AMS = 1 and 2. Therefore, these results suggest that examination of the methylation pattern of these four genes may help guide clinical decisions for the management of patients with JMML. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Download Full-text

Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome

Blood ◽

10.1182/blood-2010-08-298968 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4871-4880 ◽

Cited By ~ 60

Author(s):

Christiane Olk-Batz ◽

Anna R. Poetsch ◽

Peter Nöllke ◽

Rainer Claus ◽

Manuela Zucknick ◽

...

Keyword(s):

Dna Methylation ◽

Cox Regression ◽

Cpg Island ◽

Cpg Islands ◽

Prognostic Scores ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Prognostic Features ◽

Aberrant Dna Methylation ◽

Myelomonocytic Leukemia

Abstract Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

Download Full-text

International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-20-3184 ◽

2020 ◽

Vol 27 (1) ◽

pp. 158-168

Author(s):

Maximilian Schönung ◽

Julia Meyer ◽

Peter Nöllke ◽

Adam B. Olshen ◽

Mark Hartmann ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

International Consensus ◽

Consensus Definition ◽

Definition Of ◽

Myelomonocytic Leukemia

Download Full-text

Abnormal promoter DNA methylation in juvenile myelomonocytic leukemia is not caused by mutation in DNMT3A

Blood ◽

10.1182/blood-2011-07-370684 ◽

2011 ◽

Vol 118 (16) ◽

pp. 4490-4491 ◽

Cited By ~ 7

Author(s):

Marcin W. Wlodarski ◽

Jessica Mötter ◽

Thomas A. Gorr ◽

Christiane Olk-Batz ◽

Henrik Hasle ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

Promoter Dna Methylation ◽

Promoter Dna ◽

Myelomonocytic Leukemia

Download Full-text

Genome-Wide Methylation Analysis Using the Digital Restriction Enzyme Analysis of Methylation for Stratification of Patients with Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-127792 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2973-2973

Author(s):

Hironobu Kitazawa ◽

Hideki Muramatsu ◽

Norihiro Murakami ◽

Yusuke Okuno ◽

Manabu Wakamatsu ◽

...

Keyword(s):

Restriction Enzyme ◽

Methylation Level ◽

Enzyme Analysis ◽

Juvenile Myelomonocytic Leukemia ◽

Restriction Enzyme Analysis ◽

Consensus Clustering ◽

Cpg Sites ◽

Genome Wide ◽

Svm Model ◽

Myelomonocytic Leukemia

Background Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/ myeloproliferative neoplasm that occurs during infancy and early childhood. The clinical course of the disease varies widely. The majority of children require allogenic hematopoietic stem cell transplantation (HSCT) for long term survival, but the disease will eventually resolve spontaneously in ~15% of patients. Previous studies have identified clinical and molecular risk factors in JMML. More recently, three groups independently discovered that genome-wide methylation profiling using 450K Illumina array revealed that the high methylation (HM) subgroup was significantly associated with poor survival compared to the low methylation (LM) subgroup (Murakami 2018 Blood, Stieglitz 2017 Nat. Commun., Lipka 2017 Nat. Commun.). 450K could be a standard assay for stratification of JMML. However, it is now unavailable because the manufacture replaced it with EPIC array. Here, we developed a next-generation sequencing-based clinical test recapitulate 450K clustering results using the digital restriction enzyme analysis of methylation (DREAM) method (Jelinek 2012 Epigenetics). Patients and Methods We studied 99 children (67 boys and 32 girls) with JMML. All the patients were included in our previous publications. First, we assessed JMML samples with DREAM. Briefly, genomic DNA was sequentially cut with two enzymes SmaI and XmaI recognizing the same sequence, CCCGGG sites in DNA. Enzyme-treated DNA was then used to generate sequencing libraries according to the Illumina protocols, and run on an Illumina Hiseq 2500. We assessed 10 JMML samples with reduced representation bisulfite sequencing (RRBS) (Meissner 2005 Nucleic Acids Res.). In brief, purified genomic DNA was digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. The CpG-rich DNA fragments (40-220 bp) were size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome analyzer. Results We analyzed 99 samples using the DREAM with 8.87 (4.09-16.35) million reads (median, [range]), and determined methylation level in 62,525 (52,356-75,185) CpG sites (median [range]). We observed a strong correlation between DREAM methylation ratio and 450K beta-value of overlapping CpG sites (Pearson r2 = 0.95 [0.913-0.962], median [range]). We performed unsupervised consensus clustering with DREAM methylation data of 7,704 CpG sites within ±1 kb from TSS on autosomal chromosomes detected in ≥95% of the samples with imputation of the missing data using the median of each CpG site methylation level. Clustering identified two distinct subgroups, the HM subgroup (n = 35) and the LM subgroup (n = 64), matching 95% (94 of 99) with the 450K clustering results. The HM subgroup patients showed significantly poorer 5-year OS than the LM subgroup patients (41.9% [95% confidence interval {CI}], 25.3%-57.6%) vs. 71.4% [95% CI, 56.2%-82.1%]; P = 0.00345). Discrepancies in the clustering results between DREAM and 450K were observed in only 5 patients (2 survived and 3 died); all 5 patients were reclassified as those with LM with DREAM from being HM with 450K. We also performed RRBS methylation analysis on 10 patients. Unsupervised consensus clustering using promoter-associated 4,971 CpG sites measured with RRBS identified HM (n = 5) and LM (n = 5) subgroups and completely matched with the classification made using DREAM and 450K. Then, we developed a prediction model of the methylation subgroups using a machine-learning program. We selected 85 CpG sites from 7,704 CpG sites used for unsupervised clustering of the DREAM assay that showed a distinct difference in the average methylation level (>0.3) between the HM and LM subgroups of the learning cohort (n = 70) and developed a support vector machine (SVM) model. As a validation cohort, we analyzed the remaining 29 JMML samples with a SVM model and confirmed a high matching rate with 450K clustering results (100%, 29 of 29). Conclusions We could develop a methylation test for JMML using the DREAM assay. Both the unsupervised clustering analysis and SVM model could repeat the result of 450K-based methylation classification, i.e., the HM and LM subgroups. The relatively lower cost of the DREAM assay (US$200/sample) enabled us to incorporate methylation classification in JMML in most settings. Disclosures No relevant conflicts of interest to declare.

Download Full-text

MEK Inhibition Demonstrates Activity in Relapsed, Refractory Patients with Juvenile Myelomonocytic Leukemia: Results from COG Study ADVL1521

Blood ◽

10.1182/blood-2021-144850 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3679-3679

Author(s):

Elliot Stieglitz ◽

Mignon L. Loh ◽

Julia Meyer ◽

Chujing Zhang ◽

Donald A Barkauskas ◽

...

Keyword(s):

United States ◽

Clinical Trial ◽

Dna Methylation ◽

Objective Response ◽

The United States ◽

Juvenile Myelomonocytic Leukemia ◽

International Consensus ◽

Molecular Responses ◽

Mek Inhibition ◽

Myelomonocytic Leukemia

Abstract Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response. Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling. Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

Download Full-text