International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia

PurposeKnown clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation.Experimental designPublished DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients).ResultsAnalysis of pooled, published data established three DNA methylation subgroups as a de facto standard. Unfavorable prognostic parameters (PTPN11 mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS.ConclusionsThis study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML

RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML

Additional file 1: of CREBBP is a target of epigenetic, but not genetic, modification in juvenile myelomonocytic leukemia

This file contains additional details on methods and Tables S1–S4. (PDF 218 kb

Additional file 1: of CREBBP is a target of epigenetic, but not genetic, modification in juvenile myelomonocytic leukemia

This file contains additional details on methods and Tables S1–S4. (PDF 218 kb

Epigenetic silencing of AKAP12 in juvenile myelomonocytic leukemia

<p>A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of <i>AKAP12</i> has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three <i>AKAP12</i> promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The <i>AKAP12</i>α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased <i>AKAP12</i>α expression. Promoter methylation of <i>AKAP12</i>α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. <i>In silico</i> screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the <i>AKAP12</i>α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of <i>AKAP12</i> promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the <i>AKAP12</i> promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates <i>AKAP12</i>α expression <i>in vitro</i>, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of <i>AKAP12</i>α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.</p

RASA4 undergoes DNA hypermethylation in resistant juvenile myelomonocytic leukemia

Aberrant DNA methylation at specific genetic loci is a key molecular feature of juvenile myelomonocytic leukemia (JMML) with poor prognosis. Using quantitative high-resolution mass spectrometry, we identified RASA4 isoform 2, which maps to chromosome 7 and encodes a member of the GAP1 family of GTPase-activating proteins for small G proteins, as a recurrent target of isoform-specific DNA hypermethylation in JMML (51% of 125 patients analyzed). RASA4 isoform 2 promoter methylation correlated with clinical parameters predicting poor prognosis (older age, elevated fetal hemoglobin), with higher risk of relapse after hematopoietic stem cell transplantation, and with PTPN11 mutation. The level of isoform 2 methylation increased in relapsed cases after transplantation. Interestingly, most JMML cases with monosomy 7 exhibited hypermethylation on the remaining RASA4 allele. The results corroborate the significance of epigenetic modifications in the phenotype of aggressive JMML

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

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