Clinicopathological Study On Biologic Markers For Colorectal Carcinoma

ObjectiveIn order to search for new molecular markers as prognostic factors in colorectal carcinoma and to provide more information for clinical practice, guiding treatment and reducing recrudescence or metastasis, the following biologic markers as P16, NF-kB/P65, FHIT, DNA-PKcs, Id2, EGFR and Her-2 were investigated. Furthermore, their relevance to tumor biological characteristics, five-year survival rate, diseases free survival (DFS) and overall survival (OS) of patients were analyzed.Materials and methodsOur study is a retrospective investigation. Five hundred and fifty-eight cases of colorectal carcinoma from 1993-2006, in the Department of Chinese PLA General Hospital were collected and reviewed. According to the criteria of Colorectal Carcinoma Histologic Typing of WHO, a total of 359 cases of colorectal carcinoma with complete clinical data were enrolled in the study and of which, 198 were successfully followed up with inquisition.Facture of tissue chip: According to the principle of tissue chip, 359 cases of paraffin-embedded colorectal carcinoma and 35 cases of normal mucosa beside the carcinomas were made to seven tissue chips by a simple self-made mould and feasible method with repetitive experiments.Immunohistochemistry: The seven biologic markers—P16,NF-kB/P65, FHIT, DNA-PKcs,Id2, EGFR and Her-2 expression were detected in 359 cases of colorectal carcinoma and 35 cases of normal mucosa beside the carcinomas by PV6000 immunohistochemical method. Their relationships to clinical pathology and prognosis were also analysed.Somatic Mutations of EGFR gene: EGFR mutation status was detected by DNA extraction and PCR-sequences in 25 cases of paraffin-embedded colorectalcarcinomas.Western Blot: The differential expression of DNA-PKcs was detected in 16 cases of colorectal carcinoma and normal mucosa beside the carcinomas by protein extract and Western Blot.Cell culture and fluorescent immunohistochemistry of Colo205 cell: According to the technique of cell culture, dyeing of Hoechst33342 and PI, cell counting and fluorescent immunocytochemistry, the expressions of P16, NF-KB/P65, FHIT, DNA-PKcs and Id2 in side population (SP) cancer stem-like cells were detected.ResultsThe positive expression rates of biologic markers were as follows: 53.2%(191/359) was for P16, 67.7%(243/359) for NF-kB/P65, 46.0%(165/359) for FHIT, 63.5%(228/359) for DNA-PKcs, 68.0%(244/359) for Id2, 64.9%(233/359) for EGFR, and 1.7%(6/359) for Her-2. Among them, DNA-PKcs, NF-kB/P65, P16, FHIT and Id2 were related to clinical stage. The expressions of DNA-PKcs, NF-KB/P65, P16 and Id2 were increased, positively corresponding to clinical stage (TNM) from I to IV (P< 0.05) while FHIT, presented a negative correlation with the stage (P=0.000) . The expression of FHIT showed a negative correlation with the invasion while DNA-PKcs and Id2 were on the contrary (P< 0.05). The expressions of DNA-PKcs, P16, NF-kB/P65, FHIT and Id2 were also related to abdominal and distant metastasis. The positive expression ratios of DNA-PKcs, P16, NF-KB/P65 and Id2 in patients with abdominal and distant metastasis were higher than in those without (P<0.05) . FHIT presented a negative correlation with abdominal and distant metastasis, in which the positive ratio of FHIT expression in patients with abdominal and distant metastasis was only 10.6%, whereas in those without, was up to 62.1%. The expressions of DNA-PKcs, NF-KB/P65, P16, FHIT and Id2 also showed a correlation with lymphatic invasion.The expression of DNA-PKcs, NF-kB/P65, EGFR and Id2 in carcinomawere significantly higher, but of FHIT was statistically lower, than that in normal mucosa beside tumors (P<0.05) .By univariant survival analysis, all P16, NF-kB/P65, FHIT, DNA-PKcs, Id2, age of patients, TNM stage, lymphatic invasion, infiltration, abdominal and distant metastases and therapeutic method but EGFR and Her-2 showed significant correlation with the prognosis of colorectal adenocarcinoma.In Cox multivariant survival analysis, NF-kB/P65, abdominal and distant metastases and therapeutic method were independent prognostic indicators for DFS. The negative expression of NF-kB/P65, nonmetastases and chemotherapy after operation presented a longer survival time without tumor and better prognosis. NF-kB/P65, abdominal and distant metastases, TNM stage and therapeutic method showed significant effect on OS of colorectal carcinoma. The negative expression of NF-kB/P65, lower clinical stage, nonmetastasis and chemotherapy after operation indicated as a better prognosis.Somatic EGFR mutations were identified in 1 of 25 colorectal carcinoma patients and 5 of the patients existed silent mutations.The protein where extracted from colorectal carcinoma and normal mucosa beside the carcinomas were identified by Western Blot with DNA-PKcs binding activity. The expression of DNA-PKcs in colorectal carcinoma was higher than normal mucosa beside the carcinomas by Western Blot (P=0.013) .The side population (SP) cancer stem-like cells were counted from 4% to 10% in Colo205 colon carcinoma cell line by dyeing of Hoechst33342 and PI. The expression of DNA-PKcs in SP cancer stem-like cell was observed obviously stronger than in other carcinomatous cell. The expressions of P16, NF-kB/P65 and FHIT in SP cancer stem-like cell were much weaker than other non-SP carcinomatous cell while that of Id2 showed no difference.Conclusions:1 NF-kB/P65, abdominal and distant metastasis and therapeutic method were independent prognostic indicators for DFS and NF-kB/P65, abdominal and distantmetastasis, TNM stage and therapeutic method showed significant effect on OS of colorectal carcinoma.2 The negative expression of NF-kB/P65 represented a longer survival time without tumor and better prognosis.3 The expressions of DNA-PKcs, P16, NF-KB/P65, FHIT and Id2 were related to distant metastasis. It is suggested that the expressions of above-mentioned biologic markers may play an important role in the development and progression of the tumor and they may become new prognostic markers for colorectal carcinoma.4 The positive expression rates of DNA-PKcs, NF-kB/P65, EGFR and Id2 were significantly higher than normal mucosa beside the carcinomas while FHIT was on the contrary.5 Somatic mutations on EGFR may be related to metastasis of colorectal carcinoma.6 The side population (SP) cancer stem-like cell was counted from 4% to 10% of Colo205 colon cancer cell by dyeing of Hoechst33342 and PI. The expression of DNA-PKcs in SP cancer stem-like cell was stronger than other carcinomatous cell. The expressions of P16, NF-kB/P65 and FHIT in SP cancer stem-like cell were much weaker than other carcinomatous cell while that of Id2 had no difference.