Hypermethylation in novel DNA biomarkers for prostate cancer predicted the cancer’s return after radical prostatectomy, according to a study published online Aug. 5 in the Journal of Clinical Oncology.
"To the best of our knowledge, this is the first report of a validated prognostic multigene methylation signature for [prostate cancer]," said Christa Haldrup, Ph.D., of Aarhus (Denmark) University Hospital and her colleagues (J. Clin. Oncol. 2013 Aug. 5 [doi: 10.1200/JCO.2012.47.1847]).
The study investigators identified six DNA methylation markers – hypermethylation of AOX1, C1orf114, GAS6, HAPLN3, KLF8, and MOB3B – as highly cancer specific. One of the potential markers, C1orf114 hypermethylation, and a methylation signature including the three genes AOX1, C1orf114, and HAPLN3 were independent predictors of time to biochemical recurrence after radical prostatectomy.
The researchers identified the markers using microarray-based screening and bisulfate sequencing of 20 nonmalignant tissue specimens and 29 prostate cancer (PC) tissue specimens. They then evaluated the markers’ diagnostic potential in a training cohort of 293 radical prostatectomy (RP) samples and a validation cohort of 114 RP samples.
High methylation of C1orf114 was significantly associated with biochemical recurrence of disease, according to multivariate analysis of the training cohort (hazard ratio, 3.10). This finding was confirmed in the validation cohort (HR, 3.27).
Similarly, a two-gene prognostic methylation signature (C1orf114 and HAPLN3) and the three-gene prognostic methylation signature also were significantly associated with biochemical recurrence in the training and validation cohorts.
When researchers analyzed the three-gene signature in high- and low-methylation subgroups, hypermethylation was significantly associated with recurrence in the training cohort (HR, 1.91) and validation cohort (HR, 2.33), the investigators said.
The two- and three-gene signatures performed markedly better as recurrence predictors than C10rf114 alone, and the three-gene signature performed slightly better than the two-gene signature, "suggesting that incorporation of a few genes into a simple dichotomized methylation-based test can improve robustness compared with a single-marker test," the investigators said.
"Three years after surgery, 52% and 41% of patients in the high-methylation group had experienced biochemical recurrence, compared with only 19% and 14% of patients in the low-methylation group in cohorts 1 and 2, respectively," they said.
Tissue samples for the study were collected in Denmark, Switzerland, Germany, and Finland. The training cohort consisted of "consecutive curatively intended RP of histologically verified, clinically localized PC" collected between 1993 and 2005 and previously used for tissue microarray construction. The validation cohort consisted of such samples collected from 1992 to 2003.
The six candidate genes were the most promising of eight that were initially selected for validation.
"Gene selection was based on difference in methylation levels between ADJ-N [adjacent normal] and PC samples, fold change in methylation levels between ADJ-N and PC samples, and, when possible, the presence of more than one CpG site indicating cancer-associated hypermethylation," the investigators wrote. "We preferably included genes not previously investigated in relation to PC."
For all candidate biomarkers, RP samples were significantly hypermethylated, compared with nonmalignant samples, they said.
"C1orf114 methylation as a continuous variable, and models based on dichotomized C1orf114, HAPLN3, and AOX1 methylation had significant independent prognostic value for prediction of biochemical recurrence in two RP patient cohorts from four different countries," the study authors said.
"The novel candidate methylation biomarkers were highly cancer specific" and were at level with or exceeded the known candidate PC methylation marker GSTP1 in this sample set, they added.
"The precise clinical utility of these new candidate methylation markers for PC diagnosis should be further investigated in studies including prostate biopsy, urine, or blood samples," the investigators said.
Future studies also should evaluate the prognostic potential of the markers in diagnostic biopsies. "Because only preoperative clinicopathologic parameters are available at this time, molecular markers may contribute relatively more independent prognostic information than after RP and, importantly, could be used to guide treatment decisions," the investigators said.
The Lundbeck Foundation, the John and Birthe Meyer Foundation, the Danish Cancer Society, and the Danish Council for Strategic Research supported the study. The authors reported having no disclosures.