| Background and ObjectivesHereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal and dominantly inherited cancer predisposition caused by a constitutional defect in a mismatch repair (MMR) gene, with a disease penetrance that approaches 80-90%. It is estimated to cause 2-15% of colorectal cancers. HNPCC patients usually develop tumors at a young age (≤40) and have a tendency for synchronous or metachronous tumors. HNPCC patients are also at risk of developing CRC (68-75%), endometrial cancer (25-50% for women), gastric cancer (15%) and ovarian cancer (8-12% for women). 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. The international and clinical diagnostic criteria (the Amsterdam criteria) for HNPCC have been established and put into general use in clinical diagnosis. However, not all the HNPCC families can meet the Amsterdam criteria, usually because of a small family size, a weak family history of cancer, or a high age at onset. 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, hMSH3,hMSH6, hPMS1 and hPMS2. hMLH1 and hMSH2 genes account for more than 90% of the HNPCC families with identified germline mutations. Genomic instability results from mutations of MMR genes and the loss of DNA repair function. High microsatellite instability (MSI-H) is a hallmark for deficient MMR. Inmore than 90% of HNPCC patients meeting the Amsterdam criteria, microsatellite instability has been found in the tumor tissue. Since many pathogenic mutations of MMR genes cause protein truncation, a negative or less intense nuclear staining is observed in such cases with MMR gene 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 very difficult to be detected. Well established techniques for mutation detection range from relatively simple methods, e.g., single-strand conformation analysis (SSCP) and heteroduplex analysis, to more complex procedures, e.g., direct sequencing, protein truncation test, denaturing gradient gel electrophoresis. SSCP and heteroduplex analyses tend to lack sensitivity, whereas the more sensitive methods are often very laborious, expensive and slow. Ideally, the methods used in mutation analysis of large numbers of DNA fragments should be sensitive, non-hazardous, relatively inexpensive and fully automated or at least semiautomated to minimize time and labor costs. Rapid turnover time is also of fundamental importance to clinicians and their patients. A new method called denaturing high-performance liquid chromatography (DHPLC) has been developed by this study and can fulfill most of the above-mentioned requirements. In this study, the research subjects were young patients with colorectal cancer. First, immunohistochemical staining for the hMSH2 and hMLHl proteins in tumor tissue and MSI analysis were done in a part of the young patients with colorectal cancer (younger than 40 years of age). Next we selected the patients with absence of hMSH2/ hMLHl expression and MSI-H as highly suspected HNPCC. Second, DHPLC analysis and DNA sequencing were used to detect the mutations of hMSH2 and hMLHlgenes in highly suspected HNPCC. The patients with identified mutations were diagnosed as HNPCC. Third, all germline mutations in HNPCC that had been reported in China or other countries were collected to establish a mutation database and to fabricate oligochips which were to be used in diagnosis of HNPCC. Methods1. 2037 cases of colorectal cancer were collected and then analyzed with respect of gender, age, involved region, histology and attack rate. The objective of this study is to explore the clinicolpathological characteristics and attack rate of young patients withcolorectal cancer in Guangdong Province.2. Immunohistochemical staining was used to detect expression of hMSH2 and hMLHl genes in young patients with colorectal cancer. The patients with absence of hMSH2/ hMLHl expression were highly suspected HNPCC.3. Microsection and PCR-SSCP were used to evaluate the frequency of microsatellite instability in young patients with colorectal cancer. The patients with MSI-H were highly suspected HNPCC.4. DHPLC analysis for the detection of mutation: PCR amplification for DHPLC analysis was performed using primers designed for each exon of both hMLHl and hMSH2. Heteroduplex double peaks were found by DHPLC analysis in the exons with mutations.5. DNA sequencing: all exons with heteroduplex double peaks were sequenced to identify mutations that occurred.6. All germline mutations in HNPCC reported in China or other countries were collected for preliminary construction of mutation database.Results1. 2037 cases of colorectal cancer were treated in Nanfang Hospital from 1986 to 2002 and their average age was 53 years. The patient cohort cosisted of 1157 males (56.80%) and 880 females (43.20%), and the rectum was still the most frequently involved site (55.87%). Of the 2037 patients with colorectal cancer, 381 (18.70%) were younger than 40 of age. Of the 381 young patients with colorectal cancer, 194 (50.92%) were men and 187 (49.08%) were women, and their average age was 34 years. In the 118 (30.97%) young patients, the initial cancer was located in the right colon. Clinical stage was advanced (Dukes C and D) in 201 (52.75%) of the 381 patients. Acordding to Bethesda guideline, 381 young patients with colorectal cancer were chosen to be suspected HNPCC, and 20 HNPCC kindreds fulfilling Amsterdam criteria I or II were collected after reexamination of medical records.2. Of the 166 suspected HNPCC patients, 37 (22.29%) showed absence of hMLHl expression by 1HC, 124 (74.70%) showed normal fflC expression of hMLHl, 49 (29.52%) showed absence of hMSH2 expression by IHC, and 112 (67.47%) showed normal IHC expression of hMSH2. Loss of immunohistochemical expression for at least one of these MMR proteins was found in 80 of the 166 (48.19%). Thenegative rates of hMLHl protein in right colon, left colon and rectum cancer were 26.67% (12/45), 5.13% (2/39) and 14.29% (11/77), respectively, while the negative rates of hMSH2 protein in right colon, left colon and rectum cancer were 33.33% (15/45), 15.38% (6/39) and 18.18% (4/77), respectively. 80 highly suspected HNPCC patients were screened by IHC detection.3. Of the 73 suspected HNPCC patients, 41 (56.16%) were MSI-H, 21 (28.77%) were MSI-L, and 11 (15.07%) were MSS. The incidence of MSI-H had a striking association with the age of the patients at diagnosis. Of tumors in patients whose ages at diagnosis were younger than 30 years, 81.25% (n=13) showed MSI-H; of those whose ages at diagnosis were from 31 to 35,66.67% (n=12) showed MSI-H; however, of those whose ages at diagnosis were from 36 to 40,41.02% (n=16) showed MSI-H (P=0.014; x2 test). Of the 73 suspected HNPCC patients, 11 (15.07%) showed absence of hMLHl expression by IHC, and 26 (35.62%) showed absence of hMSH2 expression by IHC, and 1 showed absense of both. The detection rate of absence of MMR expression also had a striking association with the age of the patients at diagnosis. Of tumors in patients whose ages at diagnosis were younger than 30 years, 68.75% (n=ll) showed absence of MMR expression; of those whose age at diagnosis were from 31 to 35, 66.67% (n=12) showed absence of MMR expression; however, of those whose ages at diagnosis were from 36 to 40, 33.33% (n=13) showed absence of MMR expression (P=0.014; x2 test). 41 highly suspected HNPCC patients were screened by MSI analysis.4. Of 90 highly suspected HNPCC patients, 54 had heteroduplex double peaks found in DHPLC screening in 16 exons of hMSH2, including 9 in exon 1, 11 in exon 3, 2 in exon 11, 4 in exon 12 and 28 in exon 13; 11 had heteroduplex double peaks found in DHPLC screening in 19 exons of hMLHl, including 2 in exon 6 and 9 in exon 13. Of 90 patients detected with DHPLC, 72.22% (65/90) were found to show heteroduplex double peaks.5. Sequencing results showed that 62 alterations were found in 39 amplicons of hMSH2, including 1 point mutation in the promoter, 1 frameshift mutation, 14 nonsense mutations, 9 missense mutations, 2 synonymous mutations and 35 alterations in introns; and that 7 alterations were found in 6 amplicons of hMLHl, including 1 frameshift mutation, 1 nonsense mutation, 1 missense mutation, 1synonymous mutation and 3 alterations in introns. of 90 patients analysised by sequencing, 50% (45/90) were found to have alterations.6. Of 90 highly suspected HNPCC patients, 45 had at least one alteration in hMSH2 and/or hMLHl genes, and 33 with pathogenic mutations were daignosed as HNPCC patients. Of the 45, 87% were affected by hMSH2, and 13% by hMLHl. The mutations were scattered throughout the genes, with some hot spot areas in exons 13, 1, 3 and 12 for hMSH2, and exon 13 for hMLHl.7. The 466 germline alterations reported in different parts of the world were likely to be pathogenic and were referred to as mutation. Of them, 51% (236/466) affected hMLHl, including 45% frameshift mutations, 11% nonsense mutations and 31% missense mutations; 39% (181/466) affected hMSH2, including 50% frameshift mutations, 19% nonsense mutations and 18% missense mutations. Of the 56 pathogenic mutations reported in China (including 16 germline mutations we found), 33 affected hMSH2, including 37% missense mutations, 36% frameshift mutations and 24% nonsense mutations; 23 affected hMLHl, including 48% missense mutations, 35% frameshift mutations and 13% nonsense mutations.Conclusion1. The incidence of colorectal cancer has been rising rapidly in China, and the average age of the patients is 10 years younger than that in the western developed countries. There have been more male victims than female ones. The left-sided colorectum is still the most frequently involved site. The incidence of young colorectal cancer in China is much higher than that in the western developed countries, which suggests the incidence of HNPCC is at a dangerously high level in our country. Compared with patients older than 40 years of age, young patients are more prone to right colonic cancer. The clinical stage usually reaches the advanced (Dukes C and D) in more than half of the young patients, which suggests it is important to identify HNPCC patients and its kindreds with routine detection and effective treatment as early as possible.2. IHC can be used to detect the expression of hMLHl and hMSH2 in young patients with colorectal cancer, because of the 80 highly suspected HNPCC were indentified, 37 showed absence of hMLHl expression, and 49 showed absence of hMSH2 expression.3. The frequency of MSI is significantly high among patients younger than 40 years of age, and MSI rate in these tumors increases significantly as the age at cancer diagnosis decreases, because 41 highly suspected HNPCC were indentified by MSI analysis, with a detection rate being 56.16% (41/73). 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 young patients with colorectal cancer.4. The DHPLC technique is a good mdthod used for mutation analysis of a large number of DNA fragments, because 65 cases with MMR gene alteratons were indentified by DHPLC, with a detection rate being 72.22% (65/90). The data show that a high proportion of the young colorectal cancer patients have a germline mutation in one of the MMR genes (hMSH2, or hMLHl).5. There are some differences of HNPCC germline mutation between Chinese and other peoples, because 65 cases indentified by DHPLC were sequenced and 69 alterations were found. Of those 69 alterations, 39 were reported in relative literature and 30 were found by our research. Our study has provided new detection targets and important evidence for study of HNPCC in China, and provided new mutations for international HNPCC mutation database.6. Of the 90 highly suspected HNPCC patients, 45 had at least one alteration in hMSH2 and/or hMLHl genes, and 33 patients with pathogenic mutations were daignosed as HNPCC patients. Our data indicate that our study population has a much higher mutation rate in hMSH2 than in hMLHl (6.5:1), which suggests that the emphasis of mutation detection is on hMSH2 gene in Guangdong Province.7. 466 germline mutations that were reported in other countries and 56 germline mutations that were reported in China (including 16 germline mutations we found) have been collected for preliminary construction of mutation database of HNPCC. This work has provided a solid foundation for fabrication of oligochips that can be used in diagnosis of HNPCC.
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