| Background & ObjectiveColorectal cancer (CRC), includes neoplasms of colon and rectum, is a much common malignant tumor which do great harm to peoples'health. The mobidity of CRC is rising year by year in China along with the improvement of peoples'living standard and alteration of lifestyle. Although there were some progress in diagnosis and therapy, the total treatment effectiveness of CRC did not show any significant improvement and the 5-year survival rate after radical operation was still about 50%. When CRC was detected early, more than 90% of the patients would live at least 5 years. Unfortunately, early-stage tumors amenable to curative therapies were the most difficult to detect. One-thirds of the CRC patients had advanced stage of cancer or metastasis when diagnosed with symptoms, and their relative survival rate was not more than 40%. Additionally, more than 20% of the CRC patients presented with local recurrence and/or metastasis after radical operation, which also affected the survival rate seriously. There may be some differences both in tumor biological behaviors and in local recurrence, metastasis and prognosis after curative resection between the cancers of colon and rectum. So far few studies had evolved, let alone the identical results.Currently, there are some difficulties in early diagnosis, accurate prognosis estimation of CRC, as well as monitoring of local recurrence and/or metastasis after radical surgery. All the non-invasive detection techniques used for clinical diagnosis and screening of CRC are short of adequate sensitivity and specificity. The most reliable prognostic factor identified to date in CRC is the staging of disease at the time that therapy is initiated. Neither the modified Dukes staging system, nor the TNM staging system, is commonly accepted as an ideal prognostic factor. For example, near 30% of the patients of Dukes'B stage died of local recurrence and/or metastasis in 5 years after radical treatment, although they had no lymph node metastases. On the other hand, about 70% of the CRC local recurrence and/or metastasis appeared in 2 years after radical operation, but the optimized therapy opportunity often missed when they were diagnosed by symptom and routine medical examination. Furthermore, the high false positive rate and false negative rate also existed in the aspect of clinical iconography, detection techniques of relevant oncogene or tumour markers.By now, CRC is proved not a single desease, but an accumulation of sophisticated biological evolutions include polygene, multi-procedure and multistage under the role of environment and heredity. Also in the procedure existed the function lost of cancer suppressor gene and activation of oncogene. Therefore, the attempt of early diagnosis, prognosis evaluation and monitoring for local recurrence and/or metastasis by the detection of single or several factors has unavoidablely the conflict between the sensitivity and specificity. Protein is the production of gene expression and tumor is believed to be a disease of proteins'pitfall. Numerous of proteins changed during the formation of tumor, included different modifications after protein translation as well as the expression level, resulted in the change of protein expression profiles of tumor tissue or body fluid. Therefore there is much need in the proteome level for further research of the development of malignant tumor. Proteomic methods detect the functional units of expressed genes, through biochemical analysis of cellular proteins, to provide a protein fingerprint. The proteomic reflects both the intrinsic genetic programme of the cell and the impact of its immediate environment and is therefore valuable in biomarker discovery. Surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) is a new method of proteomics developed in recent years. Not only because of its high sensitivity and high throughput, but also its ability of reflecting the full protein profiles of tested sample and eventually find the protein biomarkers, it is a promising method for early diagnosis, prognosis evaluation and monitoring of local recurrence and/or metastasis of tumorous diseases.The objectives of this research were, firstly, to investigate the different tumorous biological behaviors between the cancer of colon and rectum through comparative analysis of clinical and pathological characteristics of local recurrence and/or metastasis after radical operation, then to find the most effective factors which influence the survival time in order that provide the evidence on how to choose the treatments protocols for clinic. Secondly, to creat a differential diagnostic model for colorectal cancer from other malignant tumors after screening the different protein expression between the two kinds of tumor using the technique of SELDI-TOF-MS. And thirdly, through the comparative analysis of serum different protein expression profiles before and after operation between the group of local recurrence and/or metastasis and the group of tumor-free survivors after CRC radical treatment, to find the special protein biomarkers relevant to recurrence and metastasis. Then to explore the potential role in monitoring CRC recurrence and/or metastases of the special proteins.Methods1. Follow-up was executed for the patients of CRC local recurrence and/or metastasis after radical operation for CRC. A total of 132 CRC patients with a complete preoperative medical history and follow-up data were retrospectively analyzed with respect of their clinic and pathological factors.2. Three hundred and twenty-five serum samples were collected, including 68 cases of CRC, 150 cases of other malignant tumors (breast, gastric, esophagus, liver, lung and kidney, each of them 25 cases), 40 cases of colorectal benign diseases and 67 healthy people. Their sera protein fingerprintings were read by SELDI-TOF-MS. Using Biomarker Wizard and Biomarker Patterns software to analyze the distinct proteins between colorectal cancer and other malignant tumors and create a best decision classification tree model for differential diagnosing colorectal cancer.3. Three hundred and forty-six serum samples were collected, including 141 cases of pre- and post-operation CRC local recurrence and/or metastasis, 109 cases of pre- and post-operation CRC tumor-free survivors, 44 cases of colorectal benign diseases and 52 healthy people. Follow-up was executed for at least 2 years. Their sera protein fingerprintings were read by SELDI-TOF-MS. The special protein biomarkers relevant to recurrence and metastasis were screened and their potential roles in monitoring recurrence and/or metastasis of CRC after curative resection were evaluated.4. Statistical analysis. The data was imported into SPSS 13.0 for statistical analysis. Contingency table chi-square test and Students't test or one-way ANOVA were utilized for comparative analysis of nominal data and measurement data, respectively. Survival rate was evaluated by life table method. Kaplan-Meier method and Log-rank statistics were used for estamiation of median survival time and comparative analysis, respectively. The Cox proportional hazards model was used to define the relationship between a number of covariates and survival time. The power of diagnosis test was evaluated by receiver operating characteristic curve. Differences between groups were considered significant if the P values were less than 0.05 in a 2-sided test. Results1. Of the patients with CRC local recurrence and/or metastasis after curative resection, the main primary cancer located in rectum, which occupied 72.7% (96/132), especially the middle and lower rectum cancer accounted for 53.0% (70/132). There were some differences in primary tumor gross type, lymph node metastasis, tissue type, cell differentiation and the number of recurrence and/or metastasis between the patients of colon cancer and rectal cancer with local recurrence and/or metastasis after curative resection. The survival rate of rectal cancer with recurrence or metastasis was much better than that of colon cancer, and the survival rate of single recurrence or metastasis was much better than that of multiple recurrences and/or metastases. Multiple factors analysis of Cox regression model suggested that treatment mode, number of recurrence and/or metastasis, primary tumor invasion and lymph node metastasis correlated with the survival time of patients. Among them, treatment mode was the most important. Re-operation was better than conservative treatment and radical operation was better than palliative surgery.2. The protocol of serum protein profiling with IMAC30 chips had a high sensitivity and was reliable and repeatable. The inner- and intra-assay coefficient of the same protein peak location was 0.3% and 0.5%, and the inner- and intra-assay coefficient of the same protein intensity was 9% and 14%, respectively.3. A best decision classification tree model for differential diagnose colorectal cancer from other malignant tumors using the serum protein fingerprinting was successfully established. In the testing mode the accuracy of diagnosis for CRC and other cancer was 86.2% and 82.0%, respectively. Blinded validation suggested that the total accuracy for prediction was 74.4%. ROC curve analysis suggested that six proteins with M/Z of 8911, 8919, 8964, 11726, 14049 and 14139 were significantly differential expressed between the two groups.4. There were 13 proteins of high expression in the pre-operation sera from the patients with recurrence or metastasis compared with that from the patients with tumor-free, among which 9 proteins with M/Z 4527, 7501, 7763, 7799, 8739, 9099, 9314, 15620 and 15821 AUC>0.7 and the lower of 95% CI>0.5. There existed no significant difference between serum proteins from patients of tumor free for more than 3 months after operation and that from normal control. While the protein M/Z 7763 was still highly expressed in the patients of recurrence or metastasis and presented a tendency of elevation except for a transit down regulation. The sensitivity, specificity and Youden index for prediction of CRC recurrence or metastasis after radical operation utilizing the protein M/Z 7763 were 92.0%, 83.9% and 0.759, respectively. And the values of CEA were 52.0%, 82.3% and 0.343, respectively.Conclusion1. There are some differences in tumor biological behaviors between colon cancer and rectal cancer. The prognosis of rectal cancer recurrence and/or metastasis after curative surgery is better than that of colon cancer. Over all, re-operation, especially radical treatment is suggested for the patients with CRC recurrence and/or metastasis. That is helpful to prolong the survival time and to improve the quality of living.2. The modified protocol of serum protein profiling was stable, repeatable, and was applicable to other malignant tumor.3. The serum protein fingerprinting differential diagnostic model for colorectal cancer yields rather a high sensitivity and specificity.4. Six proteins with M/Z of 8911, 8919, 8964, 11726, 14049 and 14139 were significantly differential expressed in the sera between the groups of CRC and other malignant tumors.5. The protein of M/Z 7763, which has a prediction of poor prognosis, exists in the serum from patients of pre-operation.6. The protein of M/Z 7763 has a high sensitivity and specificity in the diagnosis of CRC recurrence and/or metastasis, which is much better than CEA and has a potential role in therapy monitoring.
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