A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in eighty percent of early (UICC I) colon cancer stages in humans

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/ or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed. 2009 Elsevier Ltd. All rights reserved. * Corresponding author at: Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. Tel. : +49 511 856 7545; fax: +49 511 856 7409. E-mail address: Pablo.Steinberg@tiho-hannover.de (P. Steinberg). Sparks et al. [12] suggested that mutations in CTNNB1, which, as APC, is involved in the Wnt signalling pathway and encodes the protein b-catenin, could uniquely substitute for APC mutations at early stages of CRC development. Later studies reported that CTNNB1 mutations can be observed in CRC, although at a significantly lower frequency than that originally reported by Sparks et al. [12] and that CTNNB1 mutations are associated with CRC showing MSI [13–16]. Subsequently Johnson et al. [17] showed that CTNNB1 mutations are strongly associated with HNPCC. In recent years an alternative concept to the above-mentioned adenoma–carcinoma sequence, the so-called serrated pathway, in which hyperplastic polyps give rise to serrated adenomas, these then leading to the development of serrated carcinomas, has been postulated [18–20]. At the molecular level the serrated carcinomas are characterized by MSI, mutation of B-RAF/K-RAS (mutually exclusive) as well as extensive DNA methylation [20], whereby BRAF/K-RAS mutations represent early steps in tumour development along the serrated pathway [21,22]. In a recent study Jass et al. [23] explored the possibility that the early evolution of CRC is not limited to the adenoma–carcinoma sequence and the serrated adenoma pathway, but often combines components of both pathways in a so-called fusion pathway. Specifically, it was suggested that methylation of the DNA repair gene O-6-methylguanine DNA methyltransferase, mutation of K-RAS and inactivation of the tumour suppressor gene p53 provide critical combinations of molecular ‘‘cross-over’’ between the two pathways at the stage of precancerous polyps [23]. In the last two decades research groups worldwide screened thousands of tissue samples from CRC patients with varying panels of molecular gene markers that had been reported to be mutated during CRC development. However, due to the diversity of the CRC types analyzed (in some cases coupled with insensitive screening methods) the sensitivity of the marker panels was rather low or high sensitivities reported in small cohorts were not reproducible if applied to a large number of tissues. Very recently a new panel of primers that allows the analysis of the mutational hotspots within the genes coding for APC, CTNNB1, B-RAF and K-RAS was conceived [24]. The four above-mentioned genes were selected because they are involved in the Wnt and the Ras-Raf-MEK-MAPK signalling cascades (MAPK, mitogen-activated protein kinase; MEK, MAPK/ ERK kinase) and therefore play a substantial role in the adenoma– carcinoma and in the serrated adenoma pathways. The long-term goal is to make use of this new primer panel to develop a non-invasive method to detect colon cancer at a very early stage by stool DNA analysis. In a first step towards this longterm goal the percentage of CRC carrying at least one of the four above-mentioned genes in a mutated form alone and in combination with MSI was determined in 50 sporadic CRC samples by using the new primer panel. Furthermore, the results of this study were compared with those obtained with gene marker combinations previously reported in the scientific literature. 2. Material and methods