Prognostic Significance Of Stroma Proportion And α-SMA Expressing Fibroblasts In Colorectal Cancer

Colorectal cancers(CRC)is one of the most common malignant tumors,the incidence of which ranks 2^nd～3^rdworldwide and 2^ndin developed countries only next to lung cancer. The death of CRC in our country ranks about 4^thamong malignant tumors and keeps increasing.The incidence and mortality of CRC is increasing by year as the result of people's changing way of life and diet.Despite of the great progress made in the study of molecular biology of colorectal cancer,the prognosis for CRC can't be precisely predicted yet.Many should be done to improve the veracity of evaluation about patient's survivals.Epithelial mesenchymal transition in the growth and development of carcinoma is a hot study direction.More and more research has shown the tumor microenvironment has important function in carcinoma development.Tumor microenvironment is composed of extracellular matrix(ECM)and mesenchymal cells including fibroblast,vascular endothelial cell,smooth muscle cell and inflammatory cells among which the fibroblast is the major type.The interaction between tumor cells and mesenchymal cells plays an important role in carcinogenesis.The tumor microenvironment in tumors tissue,called "reactive stroma" is not the same as in normal tissue.The "reactive stroma" is characterized by:firstly,changes in ECM elements;secondly,increased microvessel density;lastly,more inflammatory cells infiltration as well as increased and activated fibroblasts.The fibroblasts in tumors ECM are activated,marked by features of both fibroblast and smooth muscle cell,which was thereby called as myofibroblast.Profuse rough endoplasmic reticulum and stress fiber in cytoplasm and fibrous joint on cell memberance constitute of the ultramicrostructure of myofibroblast.Myofibroblast displays the eukaryotic cytoskeleton system,which can be carectorized and recognized by immunohistochemistry staining ofα-SMA,vimentin,desmin etc.α-SMA is a generally received marker for myofibroblast,which partly possesses function of fibroblast and smooth muscle cell and is involoved in embryonic development,damage/repair,immunity and carcinogenesis etc.by secreting kinds of factors.Myofibroblasts of peri-tumor induce malignant trasmition,change of matrix elements and angiogenesis etc.by paracrine (matrix-epithelium interaction)to either promote or inhibit cancer development.Despite the clear knowledge of contribution of stroma to tumorogenesis,its mechanism is so complicated and hasn't been fully understood up to now,hence,people are paying effort to study and explore it.Based on above,we selected parenchyma-stromal ratio,stroma area,α-SMA positive area and propotion ofα-SMA positive area as indexes and expected to find important information in regard to colorectal carcinoma metastasis and prognosis by retrospective study and statistics method.18 colorectal normal mucosa and 160 specimens of patients were selected who had been undergone CRC surgical operation during 1990-2000 in Zhejiang province Xiaoshan Public Hospital,The First Affiliated Hospital of Zhejiang University and Zhejiang Tumor Hospital.All the samples were performed with 10%neutro-formalin fixing,paraffin embedding,4μm sections and then performed EnVision immunohistochemistry staining.After all the data was got through the image-analysis by computer,statistical analysis was performed by software SPSS12.0 for windows.Analysis of univariate significance and correlation test was done for all data.One-way ANOVA,T test and x² test were exclusively used to analyze interclass numerical data.Univariate and multivariate COX proportional hazard model were used to analyze the patient's prognosis,Kaplan -Meier methods were used to portrait the survival curve,Log-rank test were used to ascertain survival rate difference.All data are presented here in the form of((?)±S),P＜0.05 is used as an indication of statistical significance.Stromal area and parenchyma-stromal ratio of the tumors specimens were targeted as an index in the first part.EnVision methord of immunohistochemistry were used to analyze theα-SMA expression.Then the stromal area and parenchyma-stromal ratio of the specimens were detected by image analysis.Among all the sample,the stromal area in colorectal cancer,normal mucosa is(4739991.14±1234931.84)and(3334058.83±708003.98)respectively,and parenchyma-stromal ratio is(1.0558±0.8452)and(1.7266±0.6428)respectively.The stromal area of colorectal cancer is larger than that of normal mucosa,while the parenchyma-stromal ratio is decreased evidently,both of which are of statistical significance.The relationship between the stromal area/parenchyma-stromal ratio and the clinical pathological parameters of CRC was analyzed and we found no statistically significant difference between the stromal area/parenchyma-stromal ratio and age,sex,location,tumor budding number(P＞0.05);while clear statistically significant difference was found between the stromal area/parenchyma-stromal ratio and tumor invasive depth,TNM stage, lymphatic node metastasis,and histological type(P＜0.05).To establish a more distinct relationship between the stromal area or parenchyma-stromal ratio and CRC prognosis,the univariate COX proportional hazard model was used.Statistics data present here showed that stromal area or parenchyma-stromal ratio,tumor invasive depth,TNM stage,lymphatic node metastasis,distant organ metastasis,tissue grade were significantly related with poor prognosis of CRC(P＜0.05).Univariate analysis by Kaplan-Meier curve revealed that CRC 5-year survival is much lower in large stromal area or low parenchyma-stromal ratio group than small stromal area or high parenchyma-stromal ratio group.The data above reveals that in CRC cases,the stromal area or parenchyma-stromal ratio of tumor is an independent indicator of survival of CRC.Immunohistochemistry EnVision method were used in the second part to detectα-SMA expression in colorectal normal mucosa and tumors.In order to find the association of distribution of myofibroblast in colorectal cancer stroma and cancer metastasis and prognosis,α-SMA positive area and positive area ratio are calculated,by image analysis mentioned in the preceding part.As a result,α-SMA expression were detected in all colorectal normal mucosa as well as colorectal carcinoma of 160 samples.In the colorectal normal mucosa,α-SMA was detected at the 2/3 lower part of crypt,mostly surrounding glands in lamina propria and connecting to muscularis mucosa at the bottom of the glands.However,in colorectal cancerα-SMA expression was irregular,α-SMA positive area distribution in colorectal cancer and normal mucosa is(864481.18±496634.39)and(283663.15±153710.86)respectively;positive area ratio is(0.1901±0.1056)and(0.0874±0.0503) respectively.There is statistically significant difference between them(P＜0.01).In an analysis of relation betweenα-SMA positive area,positive area ratio and clinical parameters,we found no significant association ofα-SMA positive area,positive area ratio with age,gender,tumor location,infiltration depth,TNM stage,lypmatic metastasis and distant metastasis(P＞0.05)except tumor histological type(P＜0.05 or P＜0.01).Significant difference among tumor histological type also was found by x² test, that the ratio of stageâ… /â…¡and no lymphatic metastasis was high in the group of highα-SMA positive area ratio(P＜0.05).At the same time,the Univariate COX proportional hazard model,life table, Kaplan-Meier curve were used to find the relationship ofα-SMA positive area,positive area ratio and the prognosis of CRC.No distinct relationship was found(P＞0.05).In conclusion:1,There are significant difference between colorectal cancer and normal mucosa of the stromal area and parenchyma-stromal ratio,while the stromal area of colorectal cancer was increased. 2,The stromal area and parenchyma-stromal ratio of CRC were related with infiltration depth,TNM stage,lymphatic metastasis and tumor histological type.They are two independent indicator of survival of CRC while the stromal area is more powerful than the latter.3,There are significant difference between colorectal cancer and normal mucosa of the distribution of myofibroblasts.Myofibroblasts in the stromal area of CRC was increased.4,The distribution of myofibroblasts in the stromal area of CRC was related with the tumor histological type and didn't show any relation to the prognosis of CRC.