The Study Of Molecular Markers In Colorectal Neoplasms And Accurate Stratification On Colorectal Cancer

BackgroundColorectal cancer (CRC) is one of the leading causes of cancer death in the world. In America, the incidence and mortality of colorectal cancer continue to trend downward, which benefit from the strategy of screening. In China, Based on limited resources and current screening technology, the quantitative high risk factors sequential screening program has been massively implemented for CRC screening. Combination of iFOBT twice by one-week interval for the first screening and follow up once annually and investigation of HRFQ (high risk factor Questionnaire) was used as primary screening methods in the first phase. If either the iFOBT or HRFQ was positive, as a high risk population, a colonoscopy was recommended in the second phase. This two-phase strategy was the most popular strategy for colorectal cancer screening in China currently. The strategy achieved a certain effect, but it was still needs to be improved.Colonoscopy was the golden standard for colorectal cancer screening, it had limited patient compliance. Fecal occult-blood testing was the only available noninvasive screening method that reduces the risk of death from colorectal cancer, but it had limited sensitivity.In Chinese, the detection rate of colonoscopy in "high risk" groups who undergo the preliminary screening was as low as 19.3%-27.2%.The compliance rate of colonoscopy was 34.9%. The poor detection rates suggested the specificity of preliminary screening was too low. Our team attempted to develop a convenient, noninvasive and tumor-specific screening way which will improve the specificity of preliminary screening and enhance compliance and detection rate of colonoscopy ultimatelyCancer came with gene mutation, chromosome instability, epigenetic changes and metabolic changes and so on. The fecal pyruvate kinase isoenzyme type M2 (fecal PK-M2) recognized as a key enzyme controlling the metabolism of cells with a high proliferation rate, such as tumor cells. It reflected the change of metabolism in cancer cells. The dimeric form of PK-M2 was released from tumors into the blood and can be quantified. So it was a potential ideal molecular marker in colorectal cancer screening.The mutant KRAS and BRAF would activate the MAPK (mitogen-activated protein kinase) pathway and promote the malignant transformation of CRC precurcor lesions. It was an important molecular event in "adenoma-carcinoma" sequence. Study on KRAS and BRAF mutation in CRC precurcor lesions can help us get new insights into the nature of colorectal cancer, discover new screening methods and establish new prevention strategy for colorectal cancer.Accurate stratification on colorectal cancer can guide the selection of clinical treatment and help judge prognosis. Our team analyzed the survival rate of 1368 colorectal cancer from 1985 to 2005. The date showed the patients in stage IIC had a poorer survival rate compared with stage ⅢA and stageⅢB. The observation cannot reflect accurate stratification. Our team proposed a new TNM stage model which comprehensive measured the ratio of T and N in colorectal cancer stratification. The model was considered as a reasonable stage system after the verification of the SEER data. But the model whether universal adapted to different populations of colorectal cancer will need to be verified. We attempted to analyze all colorectal cancer from 1985 to 2011 in the Second Affiliated Hospital, School of Medicine, Zhejiang University to verify the revised stage system. Patients with stage 0 and IV stage colorectal cancer were excluded from the study.The first chapter the study of molecular markers in colorectal neoplasms screeningThe applied study of fecal PK-M2 in colorectal cancer screeningMethod:425 subjects completed all test and be analyzed. All subjects took a high risk questionnaire surveys (HRFQ), FOB testing, fecal PK-M2 testing and colonoscopy. Subjects were classified according to the most advanced lesion identified. Advanced adenoma was defined as any lesion containing high-grade dysplasia, a polyp containing clinically significant villous architecture, or a tubular adenoma that was≥1cm in diameter. Minor polyps included tubular adenomas<1cm in diameter and hyperplastic polyps.Results:The fecal PK-M2 detected 3 of 3 invasive cancers, the occult-blood test detected 3 of 3 invasive cancers, too. The fecal PK-M2 detected 7 of 9 invasive cancers plus adenomas with high-grade dysplasia, whereas occult-blood test identified 8 of 9. Among 22 subjects with advanced neoplasia (defined as a tubular adenoma≥1cm in diameter, a polyp with a villous histologic appearance, a polyp with high-grade dysplasia, or cancer), the fecal PK-M2 was positive in 17, whereas occult-blood test is positive in 16. Specificity in subjects with negative findings on colonoscopy was 77.8 percent for the fecal pk-m2 and 73.9 percent for occult-blood test. The result of Chi-square test suggested fecal PK-M2 had higher sensitivity and specificity than FOB, but this difference did not reach statistical significance.Conclusion:The fecal PK-M2 had higher sensitivity and specificity than FOB, but this difference did not reach statistical significance. We needed more data to prove the value of PK-M2 in CRC screening in the following work.The second chapter Pathological distributions and implications of KRAS and BRAF mutations in a Chinese population with colorectal polypsMethod:The patients were recruited from a population-based colorectal cancer screening program. A total of 87254 subjects between 40 to 84 years old were screened by questionnaire based risk assessment and fecal immunochemical test from May 2011 toApril 2014. Among them, a total of 4302 subjects with at least one CRC precursor lesions were accepted endoscopic resection. Hematoxylin and Eosin (H&E)-stained slides from formalin-fixed paraffin-embedded (FFPE) lesion samples (n=4302) were reviewed by pathologists. In the 4302 lesions,495 lesions were selected and analyzed the genotype of KRAS and BRAF. This study was approved by the Committee for the Protection of Human Subjects at the Second Affiliated Hospital, School of Medicine, Zhejiang University.Results:Among the 495 colorectal polyps and adenomas,143 were identified as carrying KRAS codon12 or 13 mutations, which was 28.9% of the total polyps and adenomas. In the 143 subjects with mutant KRAS,93 (93/311,29.9%) were male,50 (50/184,27.2%) were female.46(46/149,30.9%) were located at right colon,97(97/346,28.0%) were located at left colon.20 (20/65,30.8%) were hyperplastic polyps,85 (85/337,25.2%) were low grade intraepithelial neoplasia,38 (38/93,40.9%) were high grade intraepithelial neoplasia. In low grade intraepithelial neoplasia with mutant KRAS,46 (46/123,37.4%) were tubuvillous adenoma,24 (24/155,15.5%) were tubular adenoma, 12 (12/52,23.1%) were serrated adenoma,3 (3/7) were villous adenoma.A total of 77 colorectal cancer precursor lesions were identified with BRAFV600E mutations,45 from male subjects, and 32 from female subjects.18(18/149,12.1%) were located at right colon,59 (59/346,17.1%) were located at left colon.24 were hyperplastic polyps,48 were low grade intraepithelial neoplasia, and 5 were high grade intraepithelial neoplasia. In low grade intraepithelial neoplasia with mutant BRAF,10 were tubuvillous adenoma,8 were tubular adenoma,26 (26/52,50%) were serrated adenoma,4 (4/7) were villous adenoma.Of the 77 BRAFV600E mutations,64.9%(50/77) presented in serrated adenoma (50%) and hyperplastic polyps (36.9%). There were only 7.1%(27/378) BRAF V600E mutations present in other types of colorectal adenoma (tubular adenoma, tubuvillous adenoma and villous adenoma).Conclusion:KRAS mutations were more common in adenomas with villous type and adenomas with high-grade dysplasia. BRAF mutations were associated with serrated adenoma and hyperplastic polyps. We found 20 KRAS mutations and 24 BRAF mutations in 65 hyperplastic polyps. Although morphological dysplasia in hyperplastic polyps was not found, we still cannot decide whether they could progress to adenomas in molecular level or not.The third chapter accurate stratification on colorectal cancer 11368 colorectal cancer prognosis and TNM stage analysisMethod:A total of 1,368 cases of colorectal cancer operated and pathologically confirmed at the Second Affiliated Hospital of Zhejiang University School of Medicine from 1985 to 2005 were collected. All cancer was pathologically staged according to AJCC TNM classification (7th edition). Univariate and multivariate analysis were applied to evaluate the prognostic factors. For univariate analysis, the overall survival rate was calculated using the Kaplan-Meier method, and the significant difference was evaluated by the log-rank test. Cox multivariate regression analysis was performed to identify the predictors of survival. All these analysis were conducted using SPSS software (version 19.0).Results:The 3-,5-, and 10-year overall survival rates were 67.5%,60.2%, and 53%, respectively. The survival rates for 3,5, and 10 years after radical resection were 77.6%, 69.9%, and 62.4% respectively. According to the TNM classification, the 5-year survival rates of the stage-Ⅰ,-Ⅱ,-Ⅲ, and -Ⅳ patients were 90.1%,72.6%,53.8%, and 10.4%, respectively. Multivariate analysis by Cox regression revealed that the following factors were independently associated with the cumulative survival rate of colorectal cancer:depth of invasion, lymph node metastasis, and lymph nodes harvested, adjacent organ involvement, metastasis, radical resection, and post-operative infection. The 5-year overall survival rate was lower in stage-ⅡC patients than in stage-ⅢA or -ⅢB patients. The survival curves of the above factors were significantly different (P< 0.05).Conclusion:The depth of invasion, lymph node metastasis, harvested lymph nodes, adjacent organ involvement, metastasis, radical resection and postoperative infection were the independent prognostic factors of colorectal cancer. The depth of invasion may be a more precise prognostic factor for stage- Ⅱ and -Ⅲ patients.2. TNM staging of colorectal cancer should be reconsidered according to weighting of the T stageMethodsRecords of 2,080 nonmetastatic, advanced cancer patients undergoing colorectal cancer surgery from 1985 through 2011 were reviewed for T, N stage pathology and follow-up information. Using overall and disease-specific survival data, the 7th edition TNM staging system and the T-plus staging system were compared for stage homogeneity and discrimination and gradient monotonicity.ResultsFor gradient monotonicity, the T-plus staging system was superior for both colon and rectal cancer. With Kaplan-Meier survival curves, the T-plus staging system discriminated among different stages, and the corresponding survival was inversely associated with the stage. However, for the 7th edition TNM staging system, stage Ⅲa had a better prognosis than stage Ⅱ for rectal cancer and stage I for colon cancer. For homogeneity within the same stage and discrimination between different stages, the two staging systems were similar for rectal cancer, but the T-plus system was clearly better for colon cancer.ConclusionsThe T-plus staging system provided good gradient monotonicity. For future colorectal cancer staging systems, we proposed replacement of lymph node status as the criterion to discriminate colorectal cancer stage II and stage III with greater weighting of the T stage.