TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K-ras mutations

CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.     

TP53 and mutations in human cancer

TP53 is the most frequently mutated gene in human cancer, with a predominance of missense mutations scattered over 200 codons. In many cancers, specific mutation patterns can be identified, which are shaped by site-specific mutagenesis and by biological selection. In tobacco-related cancers (lung, head and neck), organ-specific patterns are observed, with many mutations compatible with the ones experimentally induced by tobacco carcinogens. In several other cancers, such as squamous cell carcinoma of the oesophagus or hepatocellular carcinoma (HCC), mutation patterns show geographic variations between regions of high and low incidence, suggesting a role for region-specific risk factors. HCC from high-incidence regions showing also a high prevalence of a specific Ser-249 TP53 mutation is one of the most striking examples of a mutagen fingerprint. All such assessments are useful to generate clues on the mutagenic mechanisms involved in human cancer. Moreover, it has been shown that DNA retrieved from plasma can be successfully used for detection of TP53 mutations, which gives hope for earlier more accurate detection of human cancers.     

Polymorphisms in insulin-related genes predispose to specific KRAS2 and TP53 mutations in colon cancer

Risk factors for colon cancer may not only influence the overall risk of cancer but also the risk for specific types of mutations. We evaluated the effect of polymorphisms in four insulin-related genes (G972R in IRS1, G1057D in IRS2, a CA repeat in IGFI and an A/C polymorphism at -202 of IGFBP3) on the risk of microsatellite instability and KRAS2 and TP53 mutations in a population-based set of 1788 cases of colon cancer and 1981 controls. The GR/RR IRS1 genotypes were associated with an increased risk of colon cancers with the KRAS2 G12D mutation (OR 2.3, 95% CI 1.5, 3.5 versus controls, OR 1.7, 95% CI 1.1, 2.6 versus KRAS2 wild type), the "no 192" IGFI genotype increased the risk of the KRAS2 G13D mutation (OR 2.3, 95% CI 1.2, 4.2 versus controls, OR 2.1, 95% CI 1.1, 4.0 versus wild type), and the DD IRS2 genotype increased the risk of the G12V KRAS2 mutation (OR 1.8, 95% CI 0.9, 3.5 versus controls, OR 2.0, 95% CI 1.0, 4.0 versus wild type). Polymorphisms in IRS1 and IGF1 were also associated with an approximately two-fold increased risk of specific TP53 mutations relative to controls without cancer. We conclude that polymorphisms in some insulin-related genes are associated with an increased risk of colon cancer with specific KRAS2 and TP53 mutations, implying a link between these genetic changes and specific mutational pathways in carcinogenesis.     

Clinicopathologic features in colorectal cancer patients with microsatellite instability

The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.     

Assessment of microsatellite instability in colorectal cancer patients from Brazil

The replication error status analysis of DNA, through microsatellite instability detection, has become an indispensable tool for hereditary non-polyposis colorectal cancer screening. This study investigated the microsatellite instability in Brazilian individuals presenting colorectal cancer. In this study, 66 patients were clinically analyzed according to Amsterdam II and Bethesda guidelines. Normal and tumour tissues were collected and analyzed for MSI degree according to molecular markers BAT25, BAT26, BAT40, APC–D5S346, D2S123, and D17S250. Eight patients (12.1%) fulfilled the Amsterdam II guidelines, and 15 (22.7%) met the Bethesda guidelines. BAT25 was the most sensitive marker (86.7%), while BAT26 was the least sensitive (66.7%). The specificity of both markers was 100%, but all of the markers must be used since the contribution of each marker to the sensitivity and specificity of the test is complementary. Proximal tumours were significantly predominant among RER⁺ patients. Conclusions: Patients with a family history of colorectal cancer with the tumour in the proximal colon must be screened to replication error status as early as possible in order to avoid the progression of the disease.     

Thymidylate synthase polymorphism and microsatellite instability: association in colorectal cancer

5-Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5' and 3'untraslated region (5'-3' UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5'UTR) is more frequently associated with MSI+ and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5'UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5' UTR) genotypes with the MSI+ and MSI-, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study

Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.     

Prognostic value of circulating KRAS2 gene mutations in colorectal cancer with distant metastases

While tissue KRAS2 mutations have been extensively investigated, the role of circulating mutant KRAS2 gene in patients with colorectal carcinoma remains obscure. The aim of the present study was to explore the prognostic significance of circulating KRAS2 gene mutational status in subjects undergoing primary treatment for colorectal cancer. Codon 12 KRAS2 mutations were examined in DNA samples extracted from the serum of 86 patients with colorectal cancer and were compared with the KRAS2 status of their primary tumors. Tissue and serum KRAS2 status was compared with other clinicopathological variables (including CEA and CA 19-9 levels) and with cancer-related survival. KRAS2 mutations were found in tissue samples of 28 patients (33%); serum KRAS2 mutations were detected in 10 of them (36%). Serum KRAS2 status was significantly associated with Dukes' stage D (p=0.001) and with preoperative CA 19-9 levels (p=0.01). At multivariate analysis, cancer-related survival was associated with Dukes' stage (p<0.0001), CEA level (p=0.02), and mutant circulating KRAS2 (p=0.01). All 7 stage D patients with serum KRAS2 mutations died of the disease within 24 months of primary treatment; cancer-related survival was significantly better in 9 stage D patients without serum KRAS2 mutations, with 5 patients (56%) alive after 24 months and 1 patient (13%) alive after 44 months. Residual disease after surgery was evident in all 7 stage D patients with mutant circulating KRAS2, and in 5 out of 9 stage D patients without serum mutations. Serum KRAS2 status may impact substantially on the management of stage D colorectal carcinoma, since it appears to cor-relate with prognosis in this patient subgroup.     

TP53 in gastric cancer: mutations in the l3 loop and LSH motif DNA-binding domains of TP53 predict poor outcome

The aim of this study was to clarify whether specific p53 mutations may have biological relevance in terms of disease relapse or death in gastric carcinomas (GC). Resected specimens from a consecutive series of 62 patients with GC undergoing potentially curative surgery were prospectively studied. The mutational status of exons 5-8 of the p53 gene was investigated in 62 cases using the PCR-SSCP and sequencing. Presence of microsatellite instability (MSI) was evaluated in 56 cases by analyzing loci highly sensitive of MSI. Twenty mutations of p53 were detected in 17 of the 62 cases analyzed (27%). Ten mutations (50%) occurred in highly conserved domains. According to the p53 specific functional domains: 4/20 mutations (20%) were in the L3 loop and 3/20 (15%) in LSH motif. Eight of the 56 GC resulted MSI-H, 5 (9%) MSI-L, and 43 (77%) MSI stable (MSS). None of the 8 (14%) MSI-H GC showed p53 mutations. p53 mutations were associated with intestinal histotype. Moreover, specific mutations in functional domain (L3 and LSH), together with advanced TNM stage, node involvement, depth of invasion, diffuse histotype, proved to be significantly related to quicker relapse and to shorter overall survival. Specific mutations in p53 functional domains, rather than any mutations in this gene, may be biologically more significant in terms of patients outcome, indicating that these mutations might have biological relevance to identify subgroups of patients at higher risk of relapse or death who might benefit from a more aggressive therapeutic approach.     

Prognostic value of mutations in TP53 and RAS genes in breast cancer

The identification of molecular indicators of higher risk for specific subgroups of cancer patients may allow to develop more aggressive therapeutic strategies aimed at cases with the highest likelihood of response. This would avoid unnecessary toxicity to patients and alleviate the burden of cancer care for healthcare systems. Activated oncogenes and mutated tumor suppressor genes are causal determinants of the appearance and progression of tumors in man. They therefore represent potential indicators of prognosis and/or response to therapy. However, even in cases of well-studied oncogenes and tumor suppressor genes such as TP53 and RAS, their attributed prognostic and predictive value is often based on studies of insufficient statistical power that often lead to conflicting conclusions. Findings in favor or against the use of TP53 and RAS as prognostic and predictive indicators in breast cancer are reviewed and discussed here.     

MiRNA genes constitute new targets for microsatellite instability in colorectal cancer

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥ 80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis.