The comparison of DNA methylation patterns across cancer types (pan-cancer methylome analyses) has revealed distinct subgroups of tumors that share similar methylation patterns. Integration of these data with the wealth of information derived from cancer genome profiling studies performed by large international consortia has provided novel insights into the cellular aberrations that contribute to cancer development. There is evidence that genetic mutations in epigenetic regulators (such as DNMT3, IDH1/2 or H3.3) mediate or contribute to these patterns, although a unifying molecular mechanism underlying the global alterations of DNA methylation has largely been elusive. Knowledge gained from pan-cancer methylome analyses will aid the development of diagnostic and prognostic biomarkers, improve patient stratification and the discovery of novel druggable targets for therapy, and will generate hypotheses for innovative clinical trial designs based on methylation subgroups rather than on cancer subtypes. In this review, we discuss recent advances in the global profiling of tumor genomes for aberrant DNA methylation and the integration of these data with cancer genome profiling data, highlight potential mechanisms leading to different methylation subgroups, and show how this information can be used in basic research and for translational applications. A remaining challenge is to experimentally prove the functional link between observed pan-cancer methylation patterns, the associated genetic aberrations, and their relevance for the development of cancer.

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.

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.

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.

In the last decade, circulating nucleic acids such as microRNAs (miRNAs) and cell-free DNA (cfDNA) have become increasingly important in serving as potential novel biomarkers for a variety of human diseases. If cell-free nucleic acids are to become routinely used in diagnostics, the difference in plasma miRNA and cfDNA levels between healthy and diseased subjects must exceed pre-analytical and analytical variability. Until now, few studies have addressed the time limitations of pre-processing or explored the potential use of long-term blood storage tubes, which might need to be implemented in real-life diagnostics. In this study, we analyzed the stability of four breast cancer-associated miRNAs and two cancer-associated genes under various storage conditions, to test their limitations for potential application in clinical diagnostics. In two consecutive experiments, we tested the limits of conventional EDTA tubes, as well as long-term storage blood collection tubes (BCTs) from four different manufacturers. We found that circulating miRNAs are relatively stable when stored in EDTA monovettes for up to 12 h before processing. When stored in BCTs, circulating miRNAs and cfDNA are stable for up to 7 days, depending on the manufacturer. Norgen tubes were superior for cfDNA yield, while Streck tubes performed the worst in our study with hemolysis induction. In conclusion, plasma prepared from whole blood is suitable for the quantification of both cf-miRNAs and cfDNA simultaneously.

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BackgroundAberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown.MethodsWe screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors.ResultsWe identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML.ConclusionsOur study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.

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 AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative highresolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., a, b, and g) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12a promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12a expression. Promoter methylation of AKAP12a correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12a 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 AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT-and HDAC-inhibitors reactivates AKAP12a expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12a in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.