Prognosis Significance Of SOX9/SOX8 And Podoplanin/CD34 Expression In Colorectal Cancer

Colorectal cancers (CRC) is one of the most common malignant tumors, the incidence of which ranks 2^{nd 3rd} worldwide and 2nd In developed countries only next to lung cancer. The death of CRC in our country ranks 4^th to 6^th among 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 meet the challenge of how to prolong the patient's survivals. Therefore, more sensitive, specific and more effective markers are pursued to shed a light on the diagnosis, treatment and prognosis of CRC as well. Two sorts of bio-molecular markers: SOX9, SOX8 and podoplanin, CD34 have been investigated with the intention to find their expression patterns and prognostic significance in CRC.226 specimens of patients were selected who had been undergone CRC surgical operation during 1991-2002 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 Consecutive sections and then performed HE staining and EnVision immunohistochemistry staining.Statistical analysis was performed by software SPSS13.0 for windows. Analysisof univariate significance and correlation test was done for all data. X2 test or Fisher exact test, T test were exclusively used to analyze interclass numerical data while Spearman analysis were used for the correlation. 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 ascertainsurvival rate difference. All data are presented here in the form of (X ±S), PO.05 isused as an indication of statistical significance.Immunohistochemistry EnVision method was used to detect SOX9 and SOX8 expression in colorectal normal mucosa and tumors to indicate the significance of SOX9 and SOX8 in the prognosis of carcinomas. SOX9, SOX8 expression were all detected in colorectal normal and cancer-neighboring mucosa, adenoma as well as adencarcinoma of 226 samples with paired normal and cancer-neighboring mucosa and 41 sporadic colorectal adenoma samples. The expression rates of SOX9 were 9.09%(10/110), 14.04% (16/114), 48.78%(20/41), 60.11%(113/188) respectively, while SOX8 gene expression rates were 0%(0/117), 0%(0/125), 29.97% (12/41), 15.20%(31/204), respectively, both of which showed a tendency of increasing, SOX9 in particular. SOX9 gene expression could be mainly detected in 1/3 sections at the base of crypt in normal mucosa, while the expression remained almost similar between the cancer-neighboring and normal mucosa, with a slight high in cancer-neighboring mucosa. But in adenoma and adencarcinoma, SOX9 gene expressed stronger than that of nomal and cancer-neighboring mucosa, especially in invisive front of adencarcinoma and tumor buddings. SOX8 gene was faintly expressed in normal and cancer-neighboring mucosa with a comparison with SOX9. According to twice scoring methods, its total score was less than 1, taken as negative. SOX8 gene expressed more intense in adenoma than in adencarcinoma but both weaker than SOX9. Interestingly, in adencarcinoma, both SOX9 and SOX8 gene expression were extraordinarily correlative to the histology type, that was SOX9 express highly in well or moderately differentiated adencarcinoma whilemuch weaker or even not express in mucinous adenocarcinoma, signet-ring cell carcinoma and poor or undifferentiated carcinoma. On the contrary, SOX8 gene expression could be detected extremely strong in mucous adenocarcinoma, signet-ring cell carcinoma as well as poor or differentiated carcinoma, however, it could only be detected weakly or not at all in mild- well-differentiated adencarcinoma.Dependence analysis between SOX8, SOX9 expression and clinical pathological parameters was made in order to establish the relationship between SOX9> SOX8 expression and the prognosis of CRC. There was no statistical significance between SOX9^ SOX8 expression and clinical pathological parameters. Nevertheless, when the Univariate COX proportional hazard model was used to find the relationship of SOX9 > SOX8 expression and the prognosis of CRC, we find that over-expression of SOX9, TNM stagingm/IV, tumor buddings (Si5) , which served as an indicator of well-differentiated adencarcinoma were notably correlated to adverse prognosis of colorectal cancer (P<0.01,log-rank test). Further multivariate COX analysis showed that SOX9 over-expression( a-f), TNM staginglll/IV, tumor buddings (Si 5) , could be an independent indicator of in CRC contrasted with SOX8, which had nothing to do with the prognosis of CRC. All those statistic data have shown us that there must be a close correlation between SOX9 gene expression and CRC, which implies SOX9 is an independent indicator of survival of CRC.Immunohistochemistry En Vision was also used to detect the second group of specific bio-molecular makers: podoplanin and CD34 maker of tumor microlymphatic vessel and microvacular vessel, respectively. Therefore, microvascular density (MLD) and microvessel density (MVD)fin the different area of tumors could be measured to detect the distribution of microlymphatic vessel and microvacular vessel and find out the relationship of migration and CRC prognosis. We find that Podoplanin and CD34 can either be detected in normal tumor neighboring mucosa or colorectal cancer, but in total, podoplanin (MLD) expression was much lower than CD34 (MVD). Similaritiesexist in the distribution pattern of Podoplanin and CD34 in colorectal carcinoma, that is, both of them are most distributed in tumor invasive front, less in superficial zone, least in center part, Podoplanin especially, which sometimes couldn't be detected at all. Podoplanin (MLD) distribution in colorectal cancer, normal cancer neighboring mucosa is (13.03±4.03) , (13.30±3.06) respectively, andCD34 (MVD) is(31.77±8.57)and (29.32±11.69) respectively. Podoplanin(MLD) distribution in tumor invasive front, central region and superficial zone is (19.64±7.21) , (5.64±2.87) , (13.86±4.61) respectively, and CD34 (MVD) is (40.93±11.71) , (21.07±8.38) , (34.67±12.38) respectively. It is infered that MLD distribution of tumor invasive front is more intense compared with central region, superficial zone as well as cancer-neighboring mucosa MLD (P <0.01 or P <0.05) by correlation analyses, however, there are no statistic difference between total colorectal cancer MLD and cancer-neighboring mucosa MLD (P X).O5) . In addition, statistics refered to tumor's anterior field MVD distribution shows the similar pattern to MLD, which distributed much more than in central region, superficial region and normal cancer neighboring mucosa (P <0.01 or P <0.05) . There are no difference between total colorectal cancer MVD and normal cancer neighboring mucosa MVD either (P X).O5) . It also can inferred from those statistics that tumor invasive front MLD was mainly correlative with tumor invasive depth, TNM Stage, lymphatic/vascular invasion, lymphatic node metastasis and distant organ metastasis (P <0.05) which means carcinoma with deeper infiltration, late TNM stage, lymphatic invasion, lymphatic node metastasis and distant organ metastasis have more MLD. The MVD of CRC front is highly correlative with age, tissue grade, tumor budding, blood vessel invasion, TNM stage and invasive depth (P <0.05). However, MVD in superficial and central region, as well as total MVD, have no statistical significance with clinical pathological parameters (P >0.05), all of which infers that there must be a close relation between invasive front MLD, MVD and tumor invasion as well as tumor metastasis.To establish a more distinct relationship between the MLD/MVD and CRCprognosis, the univariate COX proportional hazard model was used Lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) as well as MLD/ MVD of different tumor sections were included in this model. Statistics data present here showed that MLD/MVD and LVI/BVI in tumor invasive front were significantly related with poor prognosis of CRC iP <0.05 ).However All of them were not independent prognosticators in colorectal cancer by multivariate COX proportional hazard mo6e\(P >0.05). Univariate analysis by Kaplan-Meier curve revealed that CRC 5-year survival is much lower in over- MLD/MVD group with LVI/BVI than poor- MLD/MVD group with no LVI/BVI. The data above reveals that in CRC cases, MLD/MVD of tumor invasive fornt, lymphatic invasion and blood vessel invasion are credible indicators of adverse prognosis for CRC.Conclusion:1 > SOX9 hyper-expression was an independent indicator of adverse prognosis of CRC.SOX9 expresses highly in non-mucous adenocarcinoma/signet-ring cell carcinoma, While SOX8 is mainly detected in mucous adenocarcinoma/signet-ringcell carcinoma.2. Podoplanin(MLD) and CD34(MVD) in CRC are both distributed in the frontier and highly correlative with lymphatic invasion(LVI) and blood vessel invasion (BVI).MLD/MVD of anterior field and LVI/BVL were credible indicator of adverse prognosis for CRC .