Tumor Heterogeneity Revealed byKRAS,BRAF, andPIK3CAPyrosequencing:KRASandPIK3CAIntratumor Mutation Profile Differences and Their Therapeutic Implications

Abstract

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.

Pulmonary metastasectomy for colorectal cancer: analysis of prognostic factors affecting survival

“…Moreover, BRAF mutation status, as well as KRAS, have been shown to be predictive for whether a patient may benefit to EGFR-inhibitors therapy, such as cetuximab (13). Therefore, detection of these mutations is crucial in the clinical scenario of CRC.…”

The molecular characteristics of colorectal cancer: Implications for diagnosis and therapy (Review)

“…The mutation rates of NRAS, in contrast, are lower (1-3%) and activating mutations of HRAS has not been detected in CRC (40,91,92). Previously, pyrosequencing of KRAS, BRAF and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α revealed that 53.8% of patients exhibit a KRAS mutation in codons 12 or 13, of which 57.9% were c.38G>A (pG13D), and 22.2% were c35G>T (p.G12V) mutations (93).…”

Updated guidelines for biomarker testing in colorectal carcinoma: a national consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology

“…They also detected tumours harbouring two concomitant KRAS mutations. Other authors have subsequently confirmed these same findings [26][27][28]31]. Based on these studies, it is recommended that tests for RAS status be conducted on metastatic tissue.…”

“…However, still a significant proportion of patients selected this way do not respond to therapy. Potential reasons for this, besides alternative resistance mechanisms, may include intratumour heterogeneity [26], differences between the primary tumour and its metastases [27], and the sensitivity of methods used to identify mutations [28].…”