Abstract A13: DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA
Authors: Singh Alka , Badarukhiya Jaydeep Aravindbhai , Gupta Sameer , Sachan Manisha 
Published: June 2020

Abstract
Background: Accumulated evidence revealed that aberrant CpG island hypermethylation plays significant roles in carcinogenesis that can serve as a promising target for molecular detection in body fluids. Ovarian cancer remains the most lethal gynecologic malignancy and is characterized by few early symptoms, late stage presentation, and resulting poor survival, which thereby renders challenge to its prompt diagnosis. Despite a myriad of attempts at early diagnosis of ovarian cancer, this clinical aim still remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant circulating DNA methylation patterns provide highly specific cancer signals.
Methods: To develop a DNA methylation-based screening assay for early diagnosis of ovarian cancer, we quantitatively assessed the promoter methylation of DAPK1, HOXA9, RASSF1A, and SOX1 genes in 105 ovarian cancer and 30 non-neoplastic ovarian specimens by means of a high-throughput quantitative, real-time PCR-based technique (MethyLight) and clonal bisulfite sequencing. The best-performing gene cassette was further evaluated for their methylation status in cell-free DNA from serum of matched 35 ovarian cancer patients and 20 normal controls. Area under the ROC (Receiver-operator characteristic) curve was used to evaluate the discriminatory performance of both individual and combined gene panel.
Results: RASSF1A, HOXA9, DAPK1, and SOX1 displayed a methylation frequency of 72.72, 82.67, 68.0, and 80.0%, respectively, in tissue samples. We identified DNA methylation of HOXA9 and SOX1 as the best discriminator between cancer and non-neoplastic tissue. In the multiplex assay, the combined panel of HOXA9 and SOX1 achieved a sensitivity of 88.0% with a specificity of 86.7%, when either or both of the gene promoters showed methylation. These genes appear to have great potential to be evaluated for their methylation level in cell-free DNA as a noninvasive diagnostic marker for early diagnosis of ovarian cancer. In cell-free DNA, this novel panel achieved a sensitivity of 62.86.0% and a specificity of 95% with an area under the ROC curve of 0.81 for discriminating OC samples from normal control. The results of MethyLight were concordant with those of clonal bisulfite sequencing.
Conclusion: Our findings revealed the potential of epigenetic biomarker for early detection of OC in cell-free DNA as a minimally invasive tool. Here, we have established that promoter hypermethylation can be detected in serum cell-free DNA from patients with early as well as advanced-stage ovarian cancer. Based on the multiplex MethyLight assay, study will focus on analyzing the clinical utility of HOXA9 and SOX1 as methylation biomarkers. These findings further underline the potential of multiplexing individual markers into a panel that helps to achieve higher sensitivity and specificity than individual marker and provides robustness to a noninvasive early screening test.
Citation Format: Alka Singh, Jaydeep Aravindbhai Badarukhiya, Sameer Gupta, Manisha Sachan. DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A13.