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Australian Researchers Use Epigenetics to Stratify Triple Negative Breast Cancers

NEW YORK (GenomeWeb) – A group of researchers in Australia has used a methylation-specific sequencing technique to uncover certain methylation differences in triple-negative breast cancers that may have prognostic value.

Breast cancers are considered "triple negative" if they lack estrogen and progesterone receptors and do not over-express or amplify the HER2 gene. Patients with triple-negative breast cancer, or TNBC, seem to stratify into two groups, with cancers that progress either very rapidly, or very slowly. These cancers are clinically challenging to manage, and better stratification of patients might help in tailoring companion diagnostics or specific treatments. 

In a study published yesterday in Nature Communications, researchers at the Garvan Institute described how they identified differentially methylated regions using affinity capture of methylated DNA with the recombinant methyl-CpG binding domain of MBD2 protein followed by next-generation sequencing, a technique also known as MBDCap-Seq. This analysis was carried out on formalin-fixed, paraffin-embedded tissue biopsies.

More specifically, the researchers purified FFPE DNA using the Gentra Puregene kit from Qiagen, and then isolated methylated DNA with Thermo Fisher Scientific's MethylMiner kit. MBDCap-enriched DNA was prepared for sequencing with the Illumina ChIP-Seq DNA sample prep kit, with library preparation analyzed on an Agilent High Sensitivity DNA 1000 chip. The team validated sample-specific differential methylation using Sequenom MassARRAY methylation analysis.

Comparing coverage with the Illumina HumanMethylation450K array, the group found the MBDCap-seq method interrogated more than four million additional CpG sites, and was better at profiling certain blocks of hypermethylation, such as CpG shores and enhancers.

The researchers then used this method to identify differentially methylated DNA in a cohort of 19 TNBC biopsies and six controls. They detected 822 hypermethylated and 43 hypomethylated regions, which were then validated in a slightly larger independent cohort. These sites corresponded to 308 genes.

The authors zeroed in on regions that were differentially methylated specifically in TNBCs and not other breast cancers. They discovered 282 TNBC-specific probes could be used to classify tumor samples in The Cancer Genome Atlas HM450K cohort into TNBCs and non-TNBCs with 72 percent sensitivity and 94 percent specificity. Further, they found that 36 larger genomic regions contained three or more of these sites, and that these overlapped spans of DNA containing gene bodies or promoters.

Finally, the researchers performed cluster analysis of the sites and demonstrated that TNBC-specific methylation signatures could retrospectively stratify patients, with a cluster of largely hypomethylated sites correlating to better prognosis. Querying genomic regions with three or more signature sites revealed 14 regions linked to poor survival and three associated with good survival.

Recent research has suggested screening TNBC patients for BRCA mutations could be another viable avenue of intervention, as reported by GenomeWeb. Myriad plans a 2015 launch of a homologous recombination deficiency test to help guide platinum-based therapies for TNBC, and at the American Association for Molecular Pathology annual meeting Cepheid described a TNBC test cartridge in its oncology pipeline that would run on the Xpert platform.