Analysis of the K-ras/B-raf/Erk signal cascade, p53 and CMAP as markers for tumor progression in colorectal cancer patients.
Colorectal cancer patients may succumb to their disease because of local recurrence or formation of metastasis. To develop a prognostic tool for these fatal types of progression, 23 patients with colorectal carcinoma were included in this study for the detection at the time of surgery of the incidence of K-ras, B-raf and p53 mutations, the phosphorylation status of Erk and the expression of cystatin-like metastasis-associated protein (CMAP) in tumor, mucosa and liver samples. Polymerase chain reaction-restriction fragment length polymorphism and PCR-SSCP were used to detect the respective mutations. The results of these assays were complemented by sequencing the K-ras, B-raf and p53 mutations. A multiplex RT-PCR assay was used to detect the CMAP mRNA levels and the phosphorylation status of Erk in tumor samples was assessed by Western blot using a phosphospecific Erk antibody. The carcinomas were classified as stages T4 (70%), T3 (17%), T2 (9%) and T1 (4%) and thus represent a group of advanced colorectal carcinomas. The carcinomas (8 out of 23, 39.1%) were mutated in K-ras codons 12 or 13 and two patients had a B-raf (V599) mutation in their tumor. Of 22 tumors, 11 (50%) were positive for pErk, indicating the activation of the RAS/RAF/ERK signaling pathway. Of the 23 tumors, 13 (65.5%) showed an increased CMAP RNA level. Notably, 10 of these 13 patients have already died and two developed liver metastasis. Mutations in p53 were found in only 6 patients (26%), with 6 being detected in carcinoma, 1 in mucosa and 1 in liver tissue. These alterations were classified as non-sense (n=1), mis-sense (n=2) and frame-shift mutations (n=1) as well as intron polymorphisms (n=5). There was a significant correlation between Erk activation and K-ras codon 12 mutation (p=0.016), but not between K-ras codon 13 or B-raf mutations and Erk activation. Furthermore, there was a significant correlation of each positive marker with tumor stage (p=0.001).
Laboratory of Molecular Diagnostics, National Oncology Centre, 1756 Sofia, Bulgaria.
Milka
M
Georgieva
Maria
M
Krasteva
Elka
E
Angelova
Krassimir
K
Ralchev
Valentin
V
Dimitrov
Stanislav
S
Bozhimirov
Elena
E
Georgieva
Martin R
MR
Berger
D014408:Biomarkers, Tumor / Q000032:analysis*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015891:Cystatins / Q000032:analysis*; D018450:Disease Progression; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D016158:Genes, p53; D011905:Genes, ras; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
D014408:Biomarkers, Tumor; C116891:CST7 protein, human; D015891:Cystatins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases