Abstract
BackgroundInterrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR.MethodsIn this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood.ResultsOur results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27).ConclusionThis report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.
Highlights
Interrogation of site-specific CpG methylation in circulating tumor DNAs has been employed in a number of studies for early detection of breast cancer (BrCa)
A total of 229 CpG sites passed these filters. Among these are located in the loci of CPXM1, RASSF1A, and SC3BGA1, which have been reported in the literature to be indicative of BrCa [3]
Multiplex bisulfite PCR-ligase detection reaction (LDR)-qPCR assay to validate the 3 breast cancer methylation markers In essence, the analyses described above rationalized that the methylation level at 3 CpG sites (m_ncR1, mNR5A2, and m_PRKCB) can serve as serum markers for progressing BrCa
Summary
Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. The primary diagnostic screening method, mammography, has high rates of false positive and false negatives, can result in over-diagnosis,, uses harmful radiation, and is an uncomfortable process for patients [6, 7] This necessitates the pursuit of molecular markers more indicative of a tumor’s biological characteristics translatable to a reliable, non-invasive diagnostic assay. These include: a) Epi proColon, ColoVantage, Realtime mS9, all of which detect methylation in the SEPT9 gene for colon cancer detection [19]; b) Epi proLung which detects methylation of SHOX2 for lung cancer detection [20], and c) Colvera, which detects methylation at BCAT1 and IKZF1 for colon cancer recurrence [21]
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