RAS/RAF Mutations In Chinese Patients With Colorectal Serrated Adenocarcinoma And Prognostic Model For Colorectal Cancer

Part I An analysis of KRAS/NRAS/BRAF mutations and prognosis in Chinese patients with colorectal serrated adenocarcinomasObjectivesTo analyze clinicopathologic features,KRAS/NRAS/BRAF mutations and prognostic factors of Chinese patients with colorectal serrated adenocarcinomas,to provide a reference with prognostic information and therapeutic intervention for clinical management.MethodsAll the patients with colorectal cancers underwent surgical operations in the Second Affiliated Hospital Zhejiang University School of Medicine during the years from 2005 to 2011.Pathological sections were reviewed by two pathologists and reclassification was made in line with "the tumor classification criterion of the digestive system" by WHO published in 2010.The fluorescence PCR were used to detect KRAS/NRAS/BRAF mutations in specimens of 85 serrated adenocarcinomas,115 traditionally well-moderately differentiated adenocarcinomas and 36 adenomas.Immunohistochemical staining in all adenocarcinomas was performed to analyze the difference of the expression levels of 5 biomarkers(SOX2,MSH2,P53,CD44 and MAPK)in different subtypes of adenocarcinoma,and to compare the relationship between the expression levels of the5 biomarkers and KRAS/NRAS/BRAF mutations.All patients with colorectal carcinomas were followed-up to analyze the prognostic differences between the patients with serrated adenocarcinomas and the patients with well-moderately differentiated adenocarcinomas.Kaplan-Meier method was used to analyze prognostic differences between different KRAS/NRAS/BRAF status.Multivariate COX regression was used to screen prognostic indicators for patients with colorectal serrated adenocarcinomas.ResultsThe proportion of serrated adenocarcinoma was 14.1%among all adenocarcinoma,with an average age of 63.87 years and an average diameter of 4.03 cm.The significant differences of lymph nodes metastasis(χ2=19.382,P<0.001),pTNM stage(χ2=4.720,P=0.030),3-year and 5-year overall survival(χ2=9.898,14.287;P=0.002,<0.001,respectively)were found between the well-moderately differentiated adenocarcinoma and the serrated adenocarcinoma.The KRAS/NRAS/BRAF mutation rate was 72.2%in adenomas which was higher than that in adenocarcinomas(52.5%),with a significant difference(χ2 = 4.805,P =0.028).The mutation of KRAS exon 2 was the highest among all mutations,with a proportion of 61.1%in adenoma and 42.5%in adenocarcinoma respectively,which has a statistically significant difference between them(χ2 = 4.264,P = 0.039).However,there was no significant difference in KRAS exon 3,exon 4,NRAS exon 2 and exon 3 and BRAF exon 15 mutations between adenomas and adenocarcinomas.Unfortunately,NRAS exon 4 mutation was not detected neither in adenomas nor in adenocarcinomas.Two mutation sites of KRAS were detected in 3 adenomas and one adenocarcinoma.KRAS mutation was associated with gender and tumor diameter.The mutations in females were higher than that in males(χ2 = 7.572,P = 0.006).The mutation in tumors with more than 3 cm in diameters was higher than that in tumors with 3 cm or less(χ2=4.854,P=0.028).Whereas BRAF mutation was correlated with tumor site and histological subtypes.The BRAF mutation occurred in 4 right hemicolon,2 in rectal colon and none in left hemicolon(χ2=6.324,P=0.042).All 6 BRAF mutations occurred in serrated adenocarcinomas,compared with none in well-moderately differentiated adenocarcinomas(χ2 = 6.119,P = 0.013).KRAS/NRAS/BRAF mutations were associated to patients’ gender,tumor diameter,histological subtypes and 5-year overall survival.KRAS/NRAS/BRAF mutations in females,in more than 3 cm in diameter,in serrated adenocarcinoma and in the patients who died within five years were higher than that in male patients,in 3 cm or less in diameter,in well-moderately differentiated adenocarcinoma and in the patients who survived within five years,respectively,with significant differences(χ2=7.336,4.037,8.832,7.422;P=0.007,0.045,0.003,0.006,respectively).The expressions of all 5 biomarkers(SOX2,MSH2,P53,CD44 and MAPK)had no correlation with histological subtypes.But,MAPK expression correlated with both grass type and pTNM stage in the serrated adenocarcinoma.MAPK expression in fungoid type was higher than that in ulcerative type or in infiltrating type(χ2=6.864,P=0.033).MAPK expression in stage Ⅰ-Ⅱ was higher than that in stage Ⅲ-Ⅳ(χ2=5.207,P=0.022).The expression of both SOX2 and CD44 was associated with KRAS/NRAS/BRAF mutations.All 7 SOX2 positive cases were detected KRAS/NRAS/BRAF mutations,compared with only 50.4%of mutations in SOX2 negative cases,with a significant difference(χ2 = 6.556,P = 0.010).KRAS/NRAS/BRAF mutations were found in 61(59.2%)CD44 positive colorectal carcinomas,which were higher than that in CD44 negative cases(15,40.5%),and the difference between them was statistically significant(χ2＝ 3.829,P = 0.050).Kaplan-Meier analysis containing 200 patients with colorectal cancer indicated that the patients with serrated adenocarcinomas had worse prognoses than patients with well-moderately differentiated adenocarcinomas(χ2 = 13.231,P<0.001).The prognoses of patients with KRAS/NRAS/BRAF mutations of colorectal carcinomas were worse than that with wild type(χ2= 13.231,P<0.001).The prognoses of patients only with BRAF mutations were worse than patients with wild type,but the difference was not statistically significant(χ2 = 2.992,P = 0.084).According to histological subtypes and KRAS/NRAS/BRAF mutations,200 cases of colorectal cancers were reclassified into 4 groups:well-moderately differentiated adenocarcinomas with wild types,well-moderately differentiated adenocarcinomas with mutations,serrated adenocarcinomas with wild types and serrated adenocarcinomas with mutations.The prognostic results indicated that serrated adenocarcinomas with KRAS/NRAS/BRAF mutations had the worst prognoses,whereas no significantly prognostic difference was found between the other three groups(well-moderately differentiated adenocarcinomas with KRAS/NRAS/BRAF mutations,well-moderately differentiated adenocarcinomas wild types and serrated adenocarcinomas with wild types).A multivariate COX regression of the survival analysis containing 85 patients with colorectal serrated adenocarcinomas demonstrated that pTNM stage(P=0.015,HR=20.508,95%CI:1.796-234.151),KRAS mutation(P=0.026,HR=3.035,95%CI:1.14-8.076),NRAS mutation(P=0.001,HR=24.792,95%CI:3.792-155.447)and BRAF mutation(P=0.04,HR=7.319,95%CI:1.092-49.062)were independent prognostic factors.ConclusionsKRAS/NRAS/BRAF mutation in adenoma was higher than that in adenocarcinoma.KRAS/NRAS/BRAF mutation and BRAF mutation in the serrated adenocarcinoma was higher than that in well-moderately differentiated adenocarcinoma.BRAF mutation rate in Chinese patients with colorectal cancer was lower than that in European and American patients.Compared with well-moderately differentiated adenocarcinoma,serrated adenocarcinoma was more likely to occur lymph nodes metastasis,and to have a higher TNM stage,a lower 3-year and 5-year overall survival.The patients with serrated adenocarcinomas and KRAS/NRAS/BRAF mutations had worse prognoses than patients with serrated adenocarcinomas and KRAS/NRAS/BRAF wild types.The pTNM and KRAS/NRAS/BRAF were identified as independently prognostic predictors for patients with colorectal serrated adenocarcinomas.Part II Prognostic Model for Patients with Colorectal Cancer ObjectivesTo analyze the relationship between the expression levels of prognosis-related markers in colorectal cancer and clinical pathological parameters,to find the best combination of markers and to establish a stable model for predicting the prognoses of patients with colorectal cancers.MethodsThe clinical pathologic data of 1164 patients with colorectal cancers who underwent surgical treatment were collected from the Second Affiliated Hospital Zhejiang University School of Medicine during the years from 2001 to 2011.All patients were followed up.The paraffin blocks containing specimens of surgical resection were used to make tissue microarrays(TMAs).Fifteen markers(Lgr5,CA4,ANKRD12,PEDF,E-Cad,FOXE1,AZGP1,SOX2,MSH2,P53,CD44,SPARCL1,ADCY2,SHP2 and MAPK)were detected by immunohistochemistry for analyzing the relationships between clinical pathological parameters and prognoses of patients with colorectal cancers.Logistic regression was used to establish a prognostic prediction model for the 5-year overall survival of patients with colorectal cancer.ResultsA total of 35 TMAs was prepared,with 1164 tumor tissues and 954 tumor adjacent tissue sites.Each TMA contained from 48 to 62 sample sites.Both HE stainning and immunohistochemical staining showed that some samples were missing or not qualified in TMAs,due to no tumor tissues or no tumor adjacent mucosal tissues on the slides.The number of valid samples of cancer adjacent mucosa was significantly less than that of tumor tissues.The expression levels of 15 markers in colorectal cancers were different,with the lowest expression rate of SOX2 which is only 7.13%and the highest expression rate of ADCY2 which is up to 93.13%.Among these 15 markers,14 markers had differential expressions between tumors and tumor adjacent mucosa.The expression levels of all 15 markers had some associations with gender,age,tumor size,tumor location,tumor histological type,differentiation,depth of invasion,lymph node metastasis,distant metastasis and pTNM stage.The expression of Lgr5 is correlated with tumor location,tumor size,tumor differentiation,depth of invasion,lymph node metastasis,distant metastasis and pTNM stage.The expression of CA4 is associated with tumor differentiation,lymph node metastasis and pTNM stage.The expression of ANKRD12 is associated with lymph node metastasis.The expression of PEDF is correlated with tumor differentiation,depth of invasion,lymph node metastasis,distant metastasis and pTNM stage.The expression of E-Cadherin is correlated with histological type,differentiation,lymph node metastasis,distant metastasis and pTNM stage.The expression of FOXE1 is associated with tumor size,depth of invasion,lymph node metastasis,distant metastasis and pTNM stage.The expression level of AZGP1 is correlated with tumor invasion depth,lymph node metastasis,distant metastasis and pTNM stage.The expression of SOX2 was associated with age,tumor size,differentiation,depth of invasion,lymph node metastasis and distant metastasis.The expression of MSH2 is associated with age and tumor location.The expression of P53 is not correlated with any clinical pathological parameter.The expression of CD44 is associated with tumor differentiation,depth of invasion,lymph node metastasis and pTNM stage.The expression of SPARCL1 is associated with tumor location,differentiation,lymph node metastasis,distant metastasis and pTNM stage.The expression of ADCY2 is associated with gender,tumor differentiation,lymph node metastasis,distant metastasis and pTNM stage.The expression of SHP2 is associated with tumor lymph node metastasis and pTNM stage.The expression of MAPK is correlated with tumor differentiation,depth of invasion,lymph node metastasis,distant metastasis and pTNM stage.Univariate analysis demonstrated the correlations between the prognoses of patients with colorectal cancer and age,histological type,tumor differentiation,depth of invasion,lymph node metastasis,distant metastasis,pTNM stage and the expression level of markers Lgr5,CA4,ARKND12,PEDF,E-Cadherin,FOXE1,AZGP1,SOX2,MSH2,CD44,SPARCL1,ADCY2,SHP2 and MAPK.Multivariate analysis indicated that age,tumor histological type,tumor differentiation,depth of invasion,lymph node metastasis,distant metastasis,AZGP1,E-Cad,FOXE1,MAPK and SOX2 were independently prognostic factors for patients with colorectal cancers.The five-year overall survival prognosis model of colorectal cancer composed of clinical pathological parameters including age,tumor histological type,depth of invasion,lymph node metastasis and distant metastasis was superior to that composed biomarkers including Lgr5,E-CAD,FOXE1,AZGP1 and SOX2.The predictive effect of the five-year overall survival prognosis model was improved,when it composed of both clinical pathology parameters and markers,including age,histological type,depth of invasion,lymph node metastasis,distant metastasis,E-Cadherin,FOXE1,AZGP1 and SOX2(AUC=0.863,P<0.001,95%CI:0.820-0.905).ConclusionsThe markers could be used to establish a prognostic model for patients with colorectal cancer.The values of markers to predict prognosis of colorectal cancer didn’t exceed that of clinical pathological parameters.The ideal efforts will be obtained,if combination of clinical pathology parameters and markers is used to predict 5-year overall survival of patients with colorectal cancers.