Gene Detection And Analysis Of Hereditary Nonpolyposis Colorectal Cancer

Background and ObjectivesIncidence of colorectal cancer increased gradually in recent years, colorectal cancer has become one of the major diseases led to death in developed countries and regions. Revealed by the different mechanism of carcinogenesis, colorectal cancer can be divided into two groups: One is known as chromosome instability colorectal cancer (CIN), which covers more than 80% of sporadic colorectal cancer, the initial factors are APC gene defects, after a multi-stage, multi-gene mutations gradually develop cacinoma, while another is called with DNA microsatellite instability (MSI) of colorectal cancer, which covers hereditary nonpolyposis colorectal cancer (HNPCC), and 10%-15% of sporadic colorectal cancer, which defects mainly in mismatch repair genes (MMR), resulting MSI, develop colorectal cancer, also known as MSI colorectal cancer. HNPCC is an autosomal and dominantly inherited cancer predisposition caused by a constitutional defect in a MMR gene, with a disease pentrance that approaches 80-90%. It is estimated to cause 5-15% of colorectal cancer. HNPCC patients usually develop rumors at a young age and have a tendency for synchronous or metachronous tumors. The phenotype of HNPCC features an excess of early onset CRC with a propensity to involve the proximal (right-sided) colon, and a variety of extracolonic cancers,particularly carcinomas of the endometrium, ovary, stomach, small bowel, pancreas, hepatobiliary tract, brain and upper uroepithelium of ureter and renal pelvis, in addition to skin lesions .So it is important to identify HNPCC patients and its kindreds so that routine detection and effective treatment can be conducted as early as possible. Because no macroscopic or microscopic features are exclusively associated with HNPCC,the identification of mutations in MMR genes serves as the gold standard in diagnosing HNPCC. There are at least six genes associated with this predisposition: hMLH1, hMSH2, hPMS1, hPMS2, hMSH3 and h MSH6. hMLH1 and hMSH2 genes account for more than 90% of the HNPCC families with identified germline mutations. Therefore microsatellite analysis and immunohistochemical staining are commonly used as the first diagnostic screening test for HNPCC. The mutations are scattered throughout the genes without hot spots and consequently difficult to be detected. A new method called denaturing high-performance liquid chromatography(DHPLC) has been developed in this study and can fulfill most of the above-mentioned requirements. This study was based on the preliminary studies ,collected recent 504 colorectal cancer and 95 endometrial cancer patients to investigate and analyze,selected a group of suspected HNPCC patients and a group of HNPCC-associated endometrial cancer patients(≤50 years old) to immunohistochemical staining for hMLH1 and hMSH2 proteins, while the fresh collected samples of suspected HNPCC(≤50 years old) were used to microsatellite analysis, screening for highly suspected patients, then finnally DHPLC analysis and gene sequencing to identify mutations and diagnose HNPCC patients.Methods1. 504 cases of colorectal cancer were collected and then analyzed with respect of gender, age, involved region, histology and family history,and explored the clinicopathologic characteristics of young colorectal cancer patients.2. Immunohistochemical staining was used to detect expression of hMLH1 and hMSH2 genes in suspected HNPCC and HNPCC-associated endometrium cancer patients. With abnormal expression of hMLH1/MSH2 protein was considered as highly suspected HNPCC patients.3. MSI was used to evaluate the frequency of microsatellite analysis in suspected HNPCC patients. The patients with MSI-H were highly suspected HNPCC.4. DHPLC analysis for detection of hMLH1 and hMSH2 mutations:PCR amplification was performed using primers designed for each exon of both hMLH1 and hMSH2, DHPLC analyzed to predict mutations.5. DNA sequencing: all exons with heteroduplex double peaks were sequenced to identify mutations that occurred.Results1. Median age of 504 cases with colorectal cancer was 60. The high-risk age ranged from 41 to 70.75 cases among them were young patients (14.88%).The ratio between male and female was 1.48:1.226(44.84%) cases located in the rectum, 130(25.79%) in the lefthemi colon, 130(25.79%) in the righthemi colon, others were 18(3.57%). Among them 486 cases presented with a single lesion, 18 cases presented with multiple lesions. Histological types in all the cases were grouped as follows: well differentiated adenocarinoma 109 (21.63%), moderately differentiated adenocarinoma 275(54.56%), low differentiated adenocarinoma 38(7.54%), mucinous adenocarinoma 74(14.68%), and miscellaneous types 8(1.593%).Young colorectal cancer patients with poor differentiation occurring were 49.33%, while in the elderly were 19.35%. Difference between two groups showed statistical significance. The cases with confirmed stage A, B, C and D were 49 (9.72%), 256 (50.79%), 177 (35.12% ) and 22 (4.37% ), respectively, according to Dukes'staging system. The cases with the progressing stages (B, C, D stages) were 455 (90.28%) among all the cases. 33 (6.55%) have family history among 504 patients, thereinto 13 according with FAP, 8 HNPCC patients met with Amsterdam criteriaⅡ,their families were total 31 tumor patients, including 25 colorectal cancers, 6 other rumors (endometrial cancer, liver cancer, pancreatic cancer, nasopharyngeal carcinoma was one case respectively, lung cancer was two cases); another 12 patients ,their family members were only two colorectal cancer or HNPCC related tumor patients.2. To obtain feasible experiments and reliable results. We selected 157 suspected HNPCC patients from 504 cases, and 30 old colorectal cancer patients as control. Abnormality of immunohistochemical expression for at least one of these MMR proteins was found in 50 of the 157(31.85%)cases. The abnormal rate of hMLH1 and hMSH2 protein in right colon cancer was significantly higher than in left colon and rectum cancer. The abnormal rate of hMLH1 /hMSH2 protein in different age group were as follow: younger than 30 years old was 28.57%(2/7), 31 to 40 years old was 30.43%(21/69), 41 to 50years old was 33.33%(27/81).The abnormal rate of hMLH1/hMSH2 protein in family history patients was 70.59% (12/17) .The abnormal rate of hMLH1 and hMSH2 protein was 32.58% (29/89) in male and 30.88% (21/68) in female. The abnormal rate of hMLH1 and hMSH2 protein in 30 cases of control patients was 6.67%(2/30).The abnormal rate of hMLH1/hMSH2 protein in right colon, left colon and rectum cancer were 53.85%, 28.89% and 21.92%, respectively. In addition we selected 65 HNPCC-associated endometrium cancer patients, abnormality of hMLH1/hMSH2 protein was 30.77% (20/65) . The abnormal rate of hMLH1/hMSH2 protein in different age group were as follow: younger than 30 years old was 0%(0/2), 31 to 40 years old was 37.5%(9/24), 41 to 50years old was 28.21 %(11/39), The abnormal rate of hMLH1 and hMSH2 protein in 30 old endometrium cancer patients was 6.67%(2/30).3. To obtain MSI incidence in suspected HNPCC patients, we selected 71 suspected HNPCC patients, and 40 cases old CRC patients as control. The rate of MSI-H was 60.56%(43/71)in suspected HNPCC patients .Of patients whose age at diagnosis were younger than 30 years old, 66.67% (4/6) showed MSI-H;of those whose age at diagnosis were from 31 to 40, 62.16% (23/37) showed MSI-H. Of those whose age at diagnosis were from 41 to 50, 57.14% (16/28) showed MSI-H .Among 40 control patients 37.5%(15/40) showed MSI-H. Difference in each age groups(≤50 years old) was not significant, however difference was significant between suspected HNPCC and old group. To compare IHC and MSI methods,52 patients were analyzed by IHC and MSI simultaneously, 16 showed the abnormality of MMR protein and MSI-H, 24 showed normal MMR protein and MSS. The detection rate of two methods was similar to each other.4. Of 20 highly suspected HNPCC patients, 13 had heteroduplex double peaks in DHPLC . hMLH1 had 5 heteroduplex double peaks: including 2 in exon 1 and 2 in exon 8,1 in exon 15; hMSH2 had 8 heteroduplex double peaks: including 3 in exon 1,1 in exon 9,3 in exon 13,1 in exonl6B. Of 20 patients detected with DHPLC, 65.00%(13/20) were found to show heteroduplex double peaks.5. Sequencing results showed that 9 mutations were found in hMLH1 and hMSH2,3 mutations were new (Intron15 1731+15delT; hMSH2 Exon 13 2196T>C and Exon 16 2963C>G) , 6 mutations had existed (hMSH2 Intronl 218+8C>G, Exon 9 1452-1455delAATG and Intron 13 2006-5 A>G) ,among them 2 were hot mutations (hMSH2 Intronl 218+8OG and Exon 9 1452-1455delAATG).Conclusions 1. This study showed incidence of young colorectal cancer in China was far higher than Europe and USA. The trend of colorectal cancer incidence was gradually young,Genetic factors may play a important role, we need to increase awareness of young colorectal cancer and strengthen early detection and prevention to hereditary colorectal cancer patients.2. IHC can be used to detect expression of hMLH1/hMSH2 in suspected HNPCC and HNPCC-associated endometrium cancer. Identified highly suspected patients 50 and 20 cases respectively, and found mainly abnormal MMR protein was hMLH1 in these two tumors. MMR protein expression was closely related to age and family history, abnormal expression of MMR genes was an important factor to result in incidence of young colorectal cancer and endometrial cancer.3. Amplying MSI analyzed the state of suspected HNPCC patients. The frequency of MSI is not significant in patients younger than 50 years old,however diffenence was significant between suspected HNPCC patients and old patients .MSI analysis combined with MMR protein immunostaining can maximize the detection of MMR deficient tumor and may be a most useful tool for identifying highly suspected HNPCC from suspected patients. DHPLC is an effective and high-flowing technique to detect MSI under non-denaturing conditions.4. The data show that young patients have a high proportion of germline mutation in one of the MMR genes (hMSH2 or hMLH1). 20 highly suspected HNPCC detected by DHPLC,found 13 mutations, DNA sequencing and sequence comparing found 9 mutations, including 3 new mutations, 6 known mutations (including 2 hot spot mutations).