Molecular Detection of APC, K‐ras, and p53 Mutations in the Serum of Colorectal Cancer Patients as Circulating Biomarkers

Abstract

Early detection of tumor DNA in serum/plasma prior to the development of recurrence or metastases could help improve the outcome of patients with colorectal cancer (CRC) after tumor resection. Recent advances in the detection of tumor DNA in the serum/plasma has opened up numerous new areas for investigation and new possibilities for molecular diagnosis. APC and K- ras mutations are considered to be early-stage developments of CRCs, whereas p53 mutations are thought to be relatively late events in the tumorigenesis of CRCs. The aim of this study was to search for the presence of genetic mutations in the DNA extracted from the serum of CRC patients and healthy subjects. We simultaneously evaluate the significance of APC, K- ras, and p53 gene mutations in cancer tissues and their paired serum samples of 104 CRC patients by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) followed by direct sequencing. Additionally, analysis was carried out to detect the serum carcinoembryonic antigen (CEA) levels in CRC patients. Overall, we found at least one of the gene mutations in tumor tissues from 75% (78/104) of the CRC patients. Comparison of the three molecular markers showed that the detection rates in the serum were 30.4%, 34.0%, and 34.2% for APC, K- ras, and p53 genes, respectively. Of these patients, 46.2% (36/78) were identified as having positive serum results, whereas all healthy controls remained negative. The overall positive tumor DNA detection rates in the serum were 0% (0/7) for Dukes' A classification, 22.4% (11/49) for Dukes' B, 48.7% (19/39) for Dukes' C, and 66.7% (6/9) for Dukes' D. The detection rate increased as the tumor stage progressed ( p = 0.012). Concurrently, a significant difference was observed between lymph node metastases and positive serum tumor DNA detection ( p < 0.001). A significantly higher postoperative metastasis/recurrence rate in patients harboring gene mutations with serum tumor DNA than those without serum tumor DNA was also demonstrated ( p < 0.001). However, no significant correlation between the postoperative metastasis/recurrence and serum CEA levels was observed ( p = 0.247). These data suggest that the identification of circulating tumor DNA using the molecular detection of APC, K- ras, and p53 gene mutations is a potential tool for early detection of postoperative recurrence/metastases. Moreover, these genes may be potential molecular markers of poor clinical outcome in CRC patients.

Circulating Tumor DNA as a Liquid Biopsy for Cancer

“…(14 ). These findings were then confirmed by several other groups, and in the following years tumorspecific aberrations, including mutations in tumor suppressors and oncogenes (15 ), microsatellite instability (MSI) (16 ), and DNA methylation (17 ), were identified and provided concrete evidence that cfDNA is released into the circulation by tumors (Fig. 3).…”

The role of plasma cell-free DNA detection in predicting preoperative chemoradiotherapy response in rectal cancer patients

“…KRAS is a part of the epidermal growth factor (EGF) system, as a downstream mediator following EGF binding to its receptor EGFR (35). Thus, EGF is considered an important target of chemoradiotherapy.…”

Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours

“…Although there is a wide variation in techniques employed, earlier studies have demonstrated; (1) cfDNA can be detected in plasma of CRC patients and its level increases with stage of disease (Diehl et al 2005), (2) APC, p53 and KRAS mutations can all be detected in cfDNA and could serve as circulating biomarkers in CRC (Wang et al 2004) and (3) there is a potentially prognostic value of the peresence of cfDNA in resected CRC during postoperative follow up (Ryan et al 2003). More recently, Diehl et al (2008) demonstrated that circulating mutant DNA can be used to assess tumour dynamics in patients undergoing multimodality therapies for CRC.…”