Microsatellite Instability And KRAS Gene Mutations In Sporadic Colorectal Cancer

Background Mismatch repair genes(MMR) mutations which are the main reason of Lynch syndrome(LS) in colorectal cancer(CRC). The MMR gene mutations can result in the missing expression of correspond repair protein(MLH1, MSH2, MSH6 and PMS2). Tumors with MMR deficiency exhibit microsatellite instability(MSI) because these regions of the genomes are particularly unstable and susceptible to errors that do not get corrected resulting from the defective MMR system. MSI is related with LS, and that patients who were detected with high-level microsatellite instability(MSI-H) is an important tip for LS. Since then, a large number of studies have showed that MSI is present in 90% of LS and 10% of sporadic CRC, and MSI-H CRC has an unique clinical and pathological features. MSI-H CRC is not only an useful prognostic indicator, but also has a guiding significance for CRC adjuvant chemotherapy. Studies have demonstrated that MSI-H CRC is not sensitive to 5-fluorouracil(5-FU) based adjuvant chemotherapy. Studies have shown that the prognosis of KRAS mutations of microsatellite stable(MSS) CRC patients was worse than KRAS wild type. KRAS protein is signaling molecules which located downstream of epidermal growth factor receptor(EGFR), when KRAS gene mutations can lead to RAS / RAF / MAPK pathway is active and sustained, then loss of normal regulation of cell growth. KRAS gene mutations frequency of codon 12 and 13 is the higher, and the mutation rate is 30%-45% in CRC.Cetuximab / panitumumab which are used for anti-EGFR targeted therapy in metastatic CRCs and the treatment effect is significant. when KRAS gene mutations, CRC patients are unable to benefit from the anti-EGFR. KRAS gene mutations should be detected for CRC patients before receiving anti-EGFR therapy.Objective In this study, we analyzed the clinical and pathological features of MSI-H CRC and low-frequency microsatellite instability(MSI-L) /MSS CRC by detecting the distribution of MSI in sporadic CRC. And we used the both method fluorescence polymerase chain reactions(PCR) and immunohistochemical(IHC) method to detect MRI types of CRC patients, then compared the consistency of the two methods about MSI and analyzed the sensitivity and specificity of the immunohistochemical method to help us establish routinely reasonable testing methods and procedures for the detection of MSI. Under detecting MSI types, we used quantitative real-time PCR(q PCR) to detect KRAS gene mutations in the CRC, and analysis the correlation between the MSI and KRAS gene mutationsMethods 319 surgery paraffin tissue samples which were randomly selected from January 2014 to January 2016 in newly diagnosed patients with CRC who underwent surgical treatment surgical were used for this study. The paraffin tissues would be cut volume for DNA extraction and the DNA was used to detect MSI types and KRAS gene mutations. Besides, the paraffin tissues were cut into pieces to detect MMR proteins(MLH1, MSH2, MSH6 and PMS2) in CRC tumor tissues by IHC. Statistical analysis was performed using SPSS 20 software.Results 1. The distribution of MSI types in sporadic CRC: the incidence of MSI-H, MSI-L and MSS colorectal cancer were respectively 11.9%, 0.31% and 87.8%. Among the five bethesda markers, Bat26 and Bat25 which were found simultaneously in 34 cases of MSI-H CRC, and the changed sites of D2S123, D17S250 and D5S346 were 11 cases, 11 cases and 6 cases, respectively. And five sites all changed had only 3 cases. 2. MSI-H CRC has the unique clinical and pathological features: MSI-H was higher among younger than 50 years old of CRC patients(P < 0.001), which tended to occur in the right colon(P < 0.001) and a lower degree of differentiation features(P = 0.034) and had less lymph node metastasis(P = 0.001). In addition, MSI-H had the higher incidence of mucinous adenocarcinoma(P = 0.044) and more concentrated depth of invasion in the T3/T4(P = 0.010), and the higher incidence in the phase I/II than stage III/IV(P = 0.004). Multivariate analysis showed that MSI-H was associated with age, lymph node metastasis, differentiation and tumor location. 3. MSI typs and MMR protein expression had a high consistency in sporadic CRC. The concordance rate of both methods was 97. 8%, Kappa = 0.894. And the sensitivity and specificity of IHC were 87.1% and 99.3%, respectively. The positive predictive value and negative predictive value of IHC method were 94.4% and 98.2%, respectively. 4. KRAS gene mutation rate was 43.9% in CRC and codon 12 and 13 mutation rate was 34.8% and 9.1%, respectively. The C.34 G > A was the most common mutation site(19.4%). 5. The relationship between the KRAS gene mutations and clinical and pathological features in CRC: KRAS gene had a higher mutation rate in female patients(P = 0.001), distant metastasis(P = 0.004) and pathological stage III/IV(P = 0.002). Multivariate analysis showed that the KRAS gene mutations were associated with gender and pathological stage. 6. There was no significant difference between KRAS gene mutation rate of MSI CRC and MSS CRC(P > 0.05). But the mutation rate of codon 12 in MSS CRC was more than MSI CRC and the mutation rate of codon 13 was higher than codon 12 in MSI CRC(P < 0.001).Conclusions 1. The incidence of MSI-H was 11.9% in sporadic CRC. MSI-H CRC had an unique clinical and pathological features. 2. There was a high consistency between MSI types and protein expression of MMR, and the IHC method to detect MSI types had high sensitivity and specificity. 3. The mutation rate of KRAS gene was 43.9% in sporadic CRC. KRAS gene mutations had a relationship with female, distant metastasis and pathological stage III/IV of CRC. 4. There was no significant difference between KRAS gene mutation rate of MSI CRC with MSS CRC. And the mutation rate of codon 12 in MSS CRC was more than MSI CRC.