Characterization Of Genotype-phenotype And Clinico-pathological Features In Chinese Hereditary Nonpolyposis Colorectal Cancer Families

Colorectal cancer represents a major public health problem accounting for over 1 million cases of new cancers and about half a million deaths worldwide. Despite curative surgery in those presenting early, the risk of recurrence is significantly high. The worldwide increase in incidence of colorectal cancer, according to WHO recommendations ,supposes a number of measures and interventions that are applied for prevention, early diagnosis, treament, posttherapeutical rehabilitation and patient care.Inheritance plays a special role in colorectal cancer development, but most patients do not have apparent evidence of genetic disorders. About 25% of all CRC are estimated to be hereditary. Familial colorectal cancer accounts for about 15% of all. Hereditary nonpolyposis colorectal cancer for about 5%-10%, and familial adenomatous polyposis for about 1%. The first step in a patient'sevaluation and follow-up is a detailed review of the family history, to determine the number of relatives of the family history, to determine the number of relatives affected, their relationship to each other.Hereditary nonpolyposis colorectal cancer syndrome is the most common form of hereditary colorectal cancers. Predisposed individuals have increased lifetime riskof developing colorectal, endometrial and other cancers. The syndrome is primarily due to heterozygous gennline mutations in one of the mismatch repair genes;mainly hMLHl and hMSH2(80%-90%) among hMLHK hMSH2. hMSH6> hPSML hPSM2> hMSH3 > hMSH5. hMLH3 #1 EX01. The resulting mismatch repair deficiency leads to microsatellite instability which is the hallmark of tumors arising within this syndrome, as well as a variable proportion of sporadic tumors.With the development of genetic investigation in hereditary nonpolyposis colorectal cancer syndrome, only the study of individuals genetic investigation is less than going on probe mechanism of leading to hereditary nonpolyposis colorectal cancer lucubrately long time. So, it would be ideal to have sufficient amount of carefully characterized cells with same phenotype and function of human native about hereditary nonpolyposis colorectal cancer. At present, application of CyA along with establishment of immortalized EBV-transformed human lymphoblastoid cell lines is a new breakthrough.Assessing on genotype-phenotype and clinico-phaenotype, features may represent the ethnic and geographical characteristics that may have some diagnostic significance in China and/or Asia compared with Europe. Clinically, hereditary nonpolyposis colorectal cancer families were diagnosed based on the Amsterdam criteria. Although the Amsterdam criteria unified the diagnosis of hereditary nonpolyposis colorectal cancer worldwide, it was too rigid for small families and it excluded extracolonic cancers associated with hereditary nonpolyposis colorectal cancer. A lot of new, modified or suggested criteria and guidelines were proposed, such as the Japanese criteria, suspected HNPCC criteria, etc. Based on the suspected HNPCC criteria, considering the specific features of the tumor spectrum in Chinese population, the Chinese Hereditary Colorectal Cancer Collaboration established the criteria for Chinese hereditary nonpolyposis colorectal cancer families.The aim of our study is to analyze the clinical features of Chinese hereditary nonpolyposis colorectal cancer families and to screen the germline mutations of human mismatch repair gene hMLHl and hMSH2, as well as to establish immortalized human cell line under this condition Thirty-one independent Chinesehereditary nonpolyposis colorectal cancer families collected in Zhejiang province and part others. All of them met Chinese hereditary nonpolyposis colorectal cancer criteria.Part one Characterization of Oinico-pathological features inChinese hereditary nonpolyposis colorectal cancer families.Objective: To evaluate the Clinico-pathological features of Chinese hereditary nonpolyposis colorectal cancer families according to the number of fulfilling Amsterdam criteria, Japanese criteria, Bethsda Guideline and Chinese criteria in thirty-one families.Methods: Thirty-one independent Chinese hereditary nonpolyposis colorectal cancer families were collected in Zhejiang province and other provinces. All of them met Chinese hereditary nonpolyposis colorectal cancer criteria. Clinical data were recorded including gender, site of colorectal cancer, age of onset, history of multiple colorectal cancers, associated extracolonic cancers, clinical stage, tumor pathological features etc. There are five groups including AC group, JC group, sHNPCC group, BG group, CHNPCC group according to Amsterdam criteria, Japanese criteria, sHNPCC, Bethsda Guideline and Chinese criteria, and control group was arranged by 35 sporadic colon cancer cases in the same time period.Results: One hundred and thirty-six malignant neoplasm were found in 107 patients including 14 multiple cancers. One hundred and six of the 136 neoplasm (77.9%) were diagnosed as colorectal cancer, with an average age of onset at 48.6+ 29.0 years old. Gastric cancer was the most common extracolonic cancer (10.3%) in these families. There were 9 multiple colorectal tumors and 5 colorectal tumors associating with extracolonic cancer, accounting for 8.5% and 4.5% of the total CRCs, respectively. The average age of malignant neoplasm onset in all the patients was 48.8±13.3 years old and the ratio of males to females was 1.7:1. Among five groups with intact pathological data available tumor tissues were compared with 35 SCRC cases, the tumor of Chinese hereditary colorectal cancer showed more frequentexophytic growth pattern, higher ratio of poorly differentiated carcinoma, A/B Duke's stage, more Crohn's like lymphoid reactionCPO.05).Conclutions: (1) Onset age of Chinese hereditary nonpolyposis colorectal cancer proband is earlier than sporadic colon cancer almostly twenty years. (2) Chinese hereditary nonpolyposis colorectal cancer families had some particular clinical features, such as a left-sided predominance, less synchronous or metachronous colorectal cancers, and frequent occurrence of gastric cancer. (3) Chinese hereditary nonpolyposis colorectal cancer tumors usually have a favourable prognosis.Part two Characterization of genetic features in Chinese hereditary nonpolyposis colorectal cancer families by denaturinghigh-performance liquid chromatography.Objective: to build up and evaluate the method of detecting mutations of mismatch repair gene hMLHl and hMSH2 by denaturing high-performance liquid chromatography and to analyze the genetic features of Chinese hereditary nonpolyposis colorectal cancer families. And to evaluate Chinese hereditary colorectal cancer criteria by difference criteria groups.Methods: 31 peripheral blood DNA samples extracted from 31probands in Chinese hereditary nonpolHNPCC families. Polymerase chain reaction and denaturing high performance liquid chromatoaphy methods were employed to screen the mutations. Sequencing analysis was followed to find out the exact sites and character of mutation in samples showing abnormal denaturing high performance liquid chromatoaphy profiles. There are four groups including ACJC,BG,CHNPCC according to Amsterdam criteria, Japanese criteria, Bethsda Guideline and Chinese criteria, as well as, gene mutation group and no gene mutation group.Results: Twenty-three different sequence variations in hMLHl and hMSH2 genes were found in 17 families. Fifteen sequence variations were located in theexons, including 5 SNPs, 3 silent mutations, 3 missense mutations, 2 nonsense mutation and 2 frameshift mutations. The latter seven mutations were seemed to be pathogenic, and in the introns, 5 SNPs ,the significance of the other three remained uncertain. 4 of five sequence variations are mutation carriers, the other developed colorectal cancer in one family. Anther kindred have three mutation carriers. There is no significance difference among four groups(p>0.05).Conclutions: (1) Denaturing high performance liquid chromatoaphy was confirmed to be highly effective, convenient technique with consistent result for the detection of MSH2 and MLH1 gene mutations.(2) Germline mutations of hMLHl and hMSH2 genes were identified in about one-third hereditary nonpolyposis colorectal cancer kindreds fulfilling Chinese hereditary nonpolyposis colorectal cancer criteria. Missense mutation and silent mutation were often seen. (3) Chinese hereditary colorectal cancer criteria not only presents the clinical features of hereditary colorectal cancer pedigree, but also reflected the features of Chinese cancer pedigree.Part three Collection and trimming of Chinese hereditary nonpolyposis colorectal cancer families and establishment of humanlymphoblastoid cell linesObjective: to collect and trim all 31 families involved in this study fulfilled the Chinese hereditary nonpolyposis colorectal cancer criteria. And to establish human lymphoblastoid cell linesMethods: 31families were collected in Zhejiang Province and others partly, all of whom fulfilled the Chinese hereditary nonpolyposis colorectal cancer criteria. Peripheral blood samples (5-8ml) were collected from 251 of three hundred and twenty-nine participants: in all families after formal written consents were signed. EBV-transformed human lymphoblastoid cell lines were established by using CyA.Results: 251 Peripheral blood samples of three hundred and twenty-nine in families (76.3%)were collected, 218 EBV-transformed lymphoblastoid cell lines (93.2%)were established, 208 permantent lymphoblastoid cell lines(82.9%) were formed with average 18days.Conclusions: Establishment of Chinese hereditary nonpolyposis colorectal cancer kindreds permanent lymphoblastoid cell lines leading in China could promote the development of hman genetics study. The method by using CyA can be routinely used for the efficient banking of large number of lymphoblastoid cell lines.