Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1.

Mandy Schneider,Pablo Steinberg,Bettina Scholtka,Kornelia Berghof-Jäger,Uwe Gottschalk,Daniela Schatz,Siegbert Faiss

doi:10.3390/cancers3010091

Abstract

In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243–1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

Highlights

Sporadic colorectal cancer (CRC) is one of the leading cancer diseases in the Western world
In a previous study on CRC (UICC stages I–IV) [20] B-Raf and CTNNB1 mutations were successfully detected by single strand conformation polymorphism (SSCP) analysis, K-Ras mutations by restriction fragment length polymorphism (RFLP) analysis and APC mutations by direct sequencing of the mutation cluster region
All mutations in K-Ras, B-Raf and CTNNB1 were single base substitutions, which led to the exchange of an amino acid in the corresponding gene product

Summary

Introduction

Sporadic colorectal cancer (CRC) is one of the leading cancer diseases in the Western world. The ―traditional‖ pathway, the so-called adenoma-carcinoma-sequence, described by Vogelstein and colleagues in the early 1990s, is characterized by an early bi-allelic inactivation of APC caused by alterations in the mutation cluster region (codons 1243–1567) of this gene followed by an oncogenic. K-Ras codon 12 or 13 mutation, inactivation of the tumor suppressor gene Tp53 at the transition from adenoma to carcinoma [1,2,3] and chromosomal instability [4]. In accordance with this model, adenomas and even single aberrant crypt foci, the earliest putative CRC precursors, were shown to harbour mutations in the APC gene [3,5]. Sparks et al [6] suggested that mutations in CTNNB1, which, like APC, is involved in the Wnt signaling pathway and encodes the protein

Results

Discussion

Conclusion