Methylation Profiling of Three Homogenous Cancers: Chronic Myelogenous Leukemia (CML), Acute Promyelocytic Leukemia (APL) and Gastrointestinal Stromal Tumors (GIST).

Aberrant promoter DNA methylation is found in many cancers and is associated with aberrant gene silencing. Some abnormal cancer related methylation changes have been associated with a number of clinical features including pathologic features, prognosis, and treatment response. However, other DNA methylation changes do not appear to have phenotypic consequences and may reflect a stochastic event or a downstream event of tumorogenesis. In order to obtain a better understanding of DNA methylation changes in cancer we chose to study three very homogenous cancers: CML, APL and GIST. CML is a leukemia characterized by the Philadelphia chromosome, t(9:22), and is very sensitive to imatinib mesylate. APL is another leukemia characterized by a specific chromosomal translocation, t(15:17), and a high response to all-trans-retinoic acid. Lastly, GIST is a sarcoma with a high frequency of c-kit mutations and sensitivity to imatinib mesylate. In this study, we used bisulfite PCR/Pyrosequencing to accurately quantitate the absolute amount of DNA methylation of 15 genes (MDR, ID4, PROX1, ER, Jun B, p73, p15, ABL1, THBS1, p16, RASSF1A, RUNX3, SOCS1, RARA, and PML). We analyzed the blood and bone marrow of 58 patients with CML (chronic phase n=32, accelerated phase n=11, blast crisis n=13), and 23 patients with APL. We also analyzed tumors from 9 GIST patients. We show clearly that DNA methylation changes are not random events, but related to the biology of the cancer. We have identified at least four specific types of aberrant DNA methylation.

1. Cancer specific hypermethylation of genes related to clinical phenotype. ID4 and PROX1 were hypermethylated in APL and CML, but not GIST. Methylation of these genes in CML was strongly associated with the development of blast crisis (P=0.0005), and the methylation of these two genes was also associated with an increase in the percentage of bone marrow blasts in APL (p<0.0001).

2. DNA methylation related to geographic changes that are related to recombinatorial translocation events. ABL hypermethylation was isolated to CML patients who have the bcr-abl translocation. In contrast, RARalpha hypermethylation was unique to APL patients, who have the PML-RARalpha translocation.

3. General DNA methylation changes associated with malignant transformation. Hypermethylation of MDR, ER, p16 and p15 was found in all three types of cancer. Interestingly, RUNX3 was hypermethylated in normal tissue, but became hypomethylated in all three cancers studied.

4. Genes that are resistant to hypermethylation (p73 and RASSF1A).

In conclusion, ID4 and PROX1 hypermethylation is a marker of disease progression in leukemia such as blast crisis in CML and higher percentage of bone marrow blasts in APL. Methylation of ABL and RARalpha are specific to CML and APL, respectively, and is likely related to specific chromosomal translocations. There are genes that are subject to small increases or decreases in DNA methylation that do not correlate with any obvious biology or clinical features. Finally, there are genes that are clearly resistant to DNA methylation changes. Our study shows that DNA methylation changes are clearly non-random events, and suggests there are several mechanisms that drive DNA methylation changes.