Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer

Abstract

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation‐specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell‐free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer‐specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer‐free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.

Epithelial ovarian cancer and the use of circulating tumor DNA: A systematic review

“…The methylation status of RASSF1A and ESR1 had the lowest sensitivities of around 25% [15,16]. Another eight studies used MSP to assess the methylation status of two to seven genes, which improved the sensitivity to between 58% and 93% [18][19][20][21][40][41][42][43]. One of the studies with the highest sensitivity also had the lowest specificity (56%).…”

Methylation in HOX Clusters and Its Applications in Cancer Therapy

“…The methylation profile of most HOX genes has been investigated in a variety of cancer types, and is considered a valuable biomarker for their diagnosis and prognosis (Table 1) [22,[24][25][26][27]. The HOX gene hypermethylation is often linked to the silencing of HOX gene targets working as tumor-suppressor and/or apoptotic genes ( Figure 1) [3,28].…”

“…The aberrant methylation of HOXA9 is characteristic of a wide variety of cancers, and is used as a biomarker for diagnoses and prognoses in distinct sample types. In serum, for example, the hypermethylation of HOXA9 was recently proposed as a marker to detect early epithelial ovarian cancer [27]. Moreover, the methylation profile of this gene was considered, in combination with other genes, to be potentially applicable for prostate cancer clinical staging based on urine collection [91].…”

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

“…MethyLight is a high-throughput quantitative real time PCR that utilizes a Taq-Man-based fluorescent probe to measure DNAm with a detection limit of 0.01% [91]. A multiplex MethyLight assay was applied to the serum cfDNA for the early detection of epithelial ovarian cancer [92]. A multiplex quantitative PCR, ligase reaction detection reaction quantitative PCR (LDRqPCR), has been set up to improve MSP on cfDNA and was shown to be able to evaluate seven CpG markers relevant in colon cancer [93,94].…”