遗传性非息肉病性大肠癌的基因检测及分析
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摘要
研究背景与目的
大肠癌的发生率逐年上升,近年大肠癌已成为发达国家和地区导致死亡的主要疾病之一。根据癌变发生机理的不同,将大肠癌分为两类:一类称为染色体不稳定性大肠癌(CIN),它涵盖了80%以上的散发性大肠癌,其发生的始动因素主要是APC基因缺陷,经过多阶段、多基因突变而致癌;另一类称为微卫星不稳定(MSI)性大肠癌,它涵盖了遗传性非息肉病性大肠癌(HNPCC)和10%-15%散发性大肠癌,其发生主要是错配修复(MMR)基因缺陷导致MSI,引起大肠癌,因此又称为MSI性大肠癌。HNPCC是一种单基因显性遗传性疾病,占所有结直肠癌的5%-15%,外显率达80%-90%,多见于青年患者。临床特点主要为发病年龄早、好发于近端结肠,常伴发肠外肿瘤,尤其是子宫内膜癌、胃癌、小肠癌、前列腺癌及肝癌、泌尿系统肿瘤等。由于该肿瘤无明显形态学特征及特异的生化指标,家系调查和年青发病不足以建立最终诊断,因此证实MMR基因的种系突变是目前诊断HNPCC的金标准。已知与发生相关的基因包括hMLH1、hMSH2、hPMS1、hPMS2、hMSH3和hMSH6等,90%以上的突变发生于hMLH1和hMSH2。微卫星分析和免疫组织化学(IHC)染色常用于HNPCC最初的诊断性筛查。由于突变分布于整个MMR基因,并无突变热点。变性高效液相色谱技术(DHPLC)具有敏感、高效、经济的特点,是目前大规模筛查未知突变的一种实用方法。因此,本研究在前期研究的基础上,对新收集的504例大肠癌和95例子宫内膜癌进行调查分析,选择一组可疑HNPCC和HNPCC相关子宫内膜癌(均≤50岁)病例进行免疫组化检测,同时对新近收集的可疑HNPCC(≤50岁)新鲜组织标本进行微卫星分析,筛查高度可疑患者,然后通过DHPLC和基因测序分析确定突变位点并确定HNPCC患者。
方法
1.收集504例大肠癌病例,从性别、年龄、肿瘤好发部位、组织学类型及家族遗传史等方面进行分析,并探讨青年大肠癌的临床及病理特点。
2.运用IHC检测可疑HNPCC及HNPCC相关的子宫内膜癌hMLH1和hMSH2蛋白表达情况,将蛋白表达异常的病例确定为高度可疑HNPCC病例。
3.运用DHPLC在非变性条件下检测可疑HNPCC的微卫星情况,将MSI-H的病例确定为高度可疑HNPCC病例。
4.运用DHPLC在部分变性条件下检测hMLH1和hMSH2基因突变:PCR扩增待测DNA片段,DHPLC对PCR产物进行检测,确定突变的基因片段。
5.DNA测序:对DHPLC预测突变的DNA片段进行测序,确定突变种类,分析基因功能改变。
结果
1.504例大肠癌中位年龄为60岁,其中青年大肠癌(年龄≤40岁)75例,占14.88%。发病部位分别位于直肠226(44.84%)例;左半结肠130(25.79%)例,其中乙状结肠108(21.43%)例,降结肠18(3.57%)例,结肠脾曲4(0.79%)例;右半结肠130(25.79%)例,其中升结肠77(15.28%)例,横结肠30(5.95%)例,结肠肝曲8(1.59%)例,盲肠15(2.98%)例;其他18例(3.57%);其中单病灶486例,多发病灶18例。组织学分型为:腺癌496例(98.41%),其中高分化腺癌109例(21.63%),中分化腺癌275例(54.56%),低分化腺癌38例(7.54%),黏液腺癌74例(14.68%);其他类型8例(1.59%)。青年大肠癌分化不良者占49.33%,而中老年大肠癌分化不良者只占19.35%,二者差异有统计学意义。Dukes分期,进展期癌(B、C、D期)共455例,占90.28%。504例大肠癌患者中有家族遗传史的为33例(6.55%),其中13例为家族性腺瘤性息肉病(FAP);8例为符合Amsterdam标准Ⅱ的HNPCC,8个HNPCC家系总发病人数为31例,其中结直肠癌患者为25例,肠外肿瘤6例(子宫内膜癌1例、肺癌2例、肝癌1例、胰腺癌1例、鼻咽癌1例);另外12例患者家庭成员中只有2例患结直肠癌或相关肿瘤,为高度可疑HNPCC患者。
2.为了实验的可行和结果的可靠,从504例大肠癌中选取157例可疑HNPCC(≤50岁)为筛查对象,30例老年大肠癌(＞50岁)为对照;另选取65例HNPCC相关的子宫内膜癌(≤50岁)为筛查对象,30例老年子宫内膜癌(＞50岁)为对照;运用IHC方法对hMLH1和hMSH2蛋白表达进行分析。157例可疑HNPCC的hMLH1/hMSH2蛋白表达异常的总检出率为31.85%(50/157):不同年龄段hMLH1/hMSH2表达异常检出率依次:≤30岁为28.57%(2/7)、31-40岁为30.43%(21/69)、41-50岁为33.33%(27/81);有家族史的病例hMLH1/hMSH2表达异常的总检出率为70.59%(12/17);hMLH1/hMSH2蛋白在右半结肠、左半结肠和直肠表达异常的检出率分别为53.85%(21/39)、28.89%(13/45)和21.92%(16/73);男性患者hMLH1/hMSH2表达异常的检出率为32.58%(29/89),女性患者为30.88%(21/68);对照组30例老年大肠癌hMLH1/hMSH2表达异常的检出率为6.67%(2/30)。65例HNPCC相关的子宫内膜癌hMLH1/hMSH2蛋白表达异常的总检出率为30.77%(20/65);不同年龄段hMLH1/hMSH2表达异常的检出率依次:≤30岁为0%(0/2),31-40岁为37.5%(9/24),41-50岁为28.21%(11/39);对照组30例老年子宫内膜癌hMLH1/hMSH2表达异常的检出率为6.67%(2/30)。
3.为了获得MSI-H在可疑HNPCC病例中的发生情况,我们选取可行PCR扩增的71例可疑HNPCC为筛查对象,40例老年大肠癌作为对照,应用DHPLC在非变性的条件下检测微卫星情况。71例可疑HNPCC中,检出43例MSI-H怖?检出率为60.56%(43/71);各年龄段的检出率分别为:≤30岁:66.67%(4/6);31-40岁:62.16%(23/37);41-50岁:57.14%(16/28);40例老年大肠癌MSI-H的检出率为37.5%(15/40)。为了比较和分析IHC和MSI两种方法的优劣,对其中52例可疑HNPCC同时进行IHC和MS1分析,两种方法分析检出均为阳性的病例16例,二者均为阴性的病例24例。两种方法比较,高度可疑HNPCC的检出率相似。
4.根据IHC和MSI分析结果,从中选取20例高度可疑HNPCC进行DHPLC检测,hMLH1出现杂合双峰的有5例,其中Exon1有2例,Exon8有2例,Exon15有1例;hMSH2出现杂合双峰的有8例,其中Exon1有3例,Exon 9有1例,Exon13有3例,Exon16B有1例;DHPLC杂合双峰的检出率为65.00%(13/20)。经DNA测序和序列比对,发现了9个突变,其中新突变3个(Intron151731+15delT;hMSH2 Exon 13 2196T＞C和Exon 16 2963C＞G);已知突变6个(hMSH2 Intronl 218+8C＞GExon 9 1452-1455delAATG和Intron 132006-5A＞G),包括2个热点突变(hMSH2 Intronl 218+8C＞G和Exon 91452-1455delAATG.)。
结论
1.本组资料表明我国青年大肠癌的发生率较高,发病趋势正逐步年青化,遗传因素可能起一定作用,须提高对青年大肠癌的认识和加强对遗传性大肠癌的早期检测及预防。
2.通过IHC方法检测可疑HNPCC和HNPCC相关的子宫内膜癌hMLH1和hMSH2蛋白表达,分别确定高度可疑患者50例和20例,两种肿瘤MMR蛋白均以hMLH1表达异常为主,MMR蛋白异常表达与年龄及家族史密切相关,表明MMR蛋白异常表达是诱发低龄大肠癌和低龄子宫内膜癌的重要因素。
3.运用微卫星分析检出43例高度可疑HNPCC患者,检出率为60.56%,50岁以下患者MSI检出率较高且各年龄段检出率相似,而老年患者MSI检出率显著降低,50岁以下患者都应纳入筛查范围;DHPLC在非变性的条件下进行微卫星检测,具有敏感和高通量的优点。
4.MMR基因突变在低龄大肠癌中属于频发事件,20例高度可疑HNPCC经DHPLC检出13例突变,经DNA测序和序列比对,发现了9个突变,其中3个新突变,6个已知突变(其中热点突变2个)。
Background and Objectives
Incidence 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.
Methods
1. 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.
Results
1. 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).
大肠癌的发生率逐年上升,近年大肠癌已成为发达国家和地区导致死亡的主要疾病之一。根据癌变发生机理的不同,将大肠癌分为两类:一类称为染色体不稳定性大肠癌(CIN),它涵盖了80%以上的散发性大肠癌,其发生的始动因素主要是APC基因缺陷,经过多阶段、多基因突变而致癌;另一类称为微卫星不稳定(MSI)性大肠癌,它涵盖了遗传性非息肉病性大肠癌(HNPCC)和10%-15%散发性大肠癌,其发生主要是错配修复(MMR)基因缺陷导致MSI,引起大肠癌,因此又称为MSI性大肠癌。HNPCC是一种单基因显性遗传性疾病,占所有结直肠癌的5%-15%,外显率达80%-90%,多见于青年患者。临床特点主要为发病年龄早、好发于近端结肠,常伴发肠外肿瘤,尤其是子宫内膜癌、胃癌、小肠癌、前列腺癌及肝癌、泌尿系统肿瘤等。由于该肿瘤无明显形态学特征及特异的生化指标,家系调查和年青发病不足以建立最终诊断,因此证实MMR基因的种系突变是目前诊断HNPCC的金标准。已知与发生相关的基因包括hMLH1、hMSH2、hPMS1、hPMS2、hMSH3和hMSH6等,90%以上的突变发生于hMLH1和hMSH2。微卫星分析和免疫组织化学(IHC)染色常用于HNPCC最初的诊断性筛查。由于突变分布于整个MMR基因,并无突变热点。变性高效液相色谱技术(DHPLC)具有敏感、高效、经济的特点,是目前大规模筛查未知突变的一种实用方法。因此,本研究在前期研究的基础上,对新收集的504例大肠癌和95例子宫内膜癌进行调查分析,选择一组可疑HNPCC和HNPCC相关子宫内膜癌(均≤50岁)病例进行免疫组化检测,同时对新近收集的可疑HNPCC(≤50岁)新鲜组织标本进行微卫星分析,筛查高度可疑患者,然后通过DHPLC和基因测序分析确定突变位点并确定HNPCC患者。
方法
1.收集504例大肠癌病例,从性别、年龄、肿瘤好发部位、组织学类型及家族遗传史等方面进行分析,并探讨青年大肠癌的临床及病理特点。
2.运用IHC检测可疑HNPCC及HNPCC相关的子宫内膜癌hMLH1和hMSH2蛋白表达情况,将蛋白表达异常的病例确定为高度可疑HNPCC病例。
3.运用DHPLC在非变性条件下检测可疑HNPCC的微卫星情况,将MSI-H的病例确定为高度可疑HNPCC病例。
4.运用DHPLC在部分变性条件下检测hMLH1和hMSH2基因突变:PCR扩增待测DNA片段,DHPLC对PCR产物进行检测,确定突变的基因片段。
5.DNA测序:对DHPLC预测突变的DNA片段进行测序,确定突变种类,分析基因功能改变。
结果
1.504例大肠癌中位年龄为60岁,其中青年大肠癌(年龄≤40岁)75例,占14.88%。发病部位分别位于直肠226(44.84%)例;左半结肠130(25.79%)例,其中乙状结肠108(21.43%)例,降结肠18(3.57%)例,结肠脾曲4(0.79%)例;右半结肠130(25.79%)例,其中升结肠77(15.28%)例,横结肠30(5.95%)例,结肠肝曲8(1.59%)例,盲肠15(2.98%)例;其他18例(3.57%);其中单病灶486例,多发病灶18例。组织学分型为:腺癌496例(98.41%),其中高分化腺癌109例(21.63%),中分化腺癌275例(54.56%),低分化腺癌38例(7.54%),黏液腺癌74例(14.68%);其他类型8例(1.59%)。青年大肠癌分化不良者占49.33%,而中老年大肠癌分化不良者只占19.35%,二者差异有统计学意义。Dukes分期,进展期癌(B、C、D期)共455例,占90.28%。504例大肠癌患者中有家族遗传史的为33例(6.55%),其中13例为家族性腺瘤性息肉病(FAP);8例为符合Amsterdam标准Ⅱ的HNPCC,8个HNPCC家系总发病人数为31例,其中结直肠癌患者为25例,肠外肿瘤6例(子宫内膜癌1例、肺癌2例、肝癌1例、胰腺癌1例、鼻咽癌1例);另外12例患者家庭成员中只有2例患结直肠癌或相关肿瘤,为高度可疑HNPCC患者。
2.为了实验的可行和结果的可靠,从504例大肠癌中选取157例可疑HNPCC(≤50岁)为筛查对象,30例老年大肠癌(＞50岁)为对照;另选取65例HNPCC相关的子宫内膜癌(≤50岁)为筛查对象,30例老年子宫内膜癌(＞50岁)为对照;运用IHC方法对hMLH1和hMSH2蛋白表达进行分析。157例可疑HNPCC的hMLH1/hMSH2蛋白表达异常的总检出率为31.85%(50/157):不同年龄段hMLH1/hMSH2表达异常检出率依次:≤30岁为28.57%(2/7)、31-40岁为30.43%(21/69)、41-50岁为33.33%(27/81);有家族史的病例hMLH1/hMSH2表达异常的总检出率为70.59%(12/17);hMLH1/hMSH2蛋白在右半结肠、左半结肠和直肠表达异常的检出率分别为53.85%(21/39)、28.89%(13/45)和21.92%(16/73);男性患者hMLH1/hMSH2表达异常的检出率为32.58%(29/89),女性患者为30.88%(21/68);对照组30例老年大肠癌hMLH1/hMSH2表达异常的检出率为6.67%(2/30)。65例HNPCC相关的子宫内膜癌hMLH1/hMSH2蛋白表达异常的总检出率为30.77%(20/65);不同年龄段hMLH1/hMSH2表达异常的检出率依次:≤30岁为0%(0/2),31-40岁为37.5%(9/24),41-50岁为28.21%(11/39);对照组30例老年子宫内膜癌hMLH1/hMSH2表达异常的检出率为6.67%(2/30)。
3.为了获得MSI-H在可疑HNPCC病例中的发生情况,我们选取可行PCR扩增的71例可疑HNPCC为筛查对象,40例老年大肠癌作为对照,应用DHPLC在非变性的条件下检测微卫星情况。71例可疑HNPCC中,检出43例MSI-H怖?检出率为60.56%(43/71);各年龄段的检出率分别为:≤30岁:66.67%(4/6);31-40岁:62.16%(23/37);41-50岁:57.14%(16/28);40例老年大肠癌MSI-H的检出率为37.5%(15/40)。为了比较和分析IHC和MSI两种方法的优劣,对其中52例可疑HNPCC同时进行IHC和MS1分析,两种方法分析检出均为阳性的病例16例,二者均为阴性的病例24例。两种方法比较,高度可疑HNPCC的检出率相似。
4.根据IHC和MSI分析结果,从中选取20例高度可疑HNPCC进行DHPLC检测,hMLH1出现杂合双峰的有5例,其中Exon1有2例,Exon8有2例,Exon15有1例;hMSH2出现杂合双峰的有8例,其中Exon1有3例,Exon 9有1例,Exon13有3例,Exon16B有1例;DHPLC杂合双峰的检出率为65.00%(13/20)。经DNA测序和序列比对,发现了9个突变,其中新突变3个(Intron151731+15delT;hMSH2 Exon 13 2196T＞C和Exon 16 2963C＞G);已知突变6个(hMSH2 Intronl 218+8C＞GExon 9 1452-1455delAATG和Intron 132006-5A＞G),包括2个热点突变(hMSH2 Intronl 218+8C＞G和Exon 91452-1455delAATG.)。
结论
1.本组资料表明我国青年大肠癌的发生率较高,发病趋势正逐步年青化,遗传因素可能起一定作用,须提高对青年大肠癌的认识和加强对遗传性大肠癌的早期检测及预防。
2.通过IHC方法检测可疑HNPCC和HNPCC相关的子宫内膜癌hMLH1和hMSH2蛋白表达,分别确定高度可疑患者50例和20例,两种肿瘤MMR蛋白均以hMLH1表达异常为主,MMR蛋白异常表达与年龄及家族史密切相关,表明MMR蛋白异常表达是诱发低龄大肠癌和低龄子宫内膜癌的重要因素。
3.运用微卫星分析检出43例高度可疑HNPCC患者,检出率为60.56%,50岁以下患者MSI检出率较高且各年龄段检出率相似,而老年患者MSI检出率显著降低,50岁以下患者都应纳入筛查范围;DHPLC在非变性的条件下进行微卫星检测,具有敏感和高通量的优点。
4.MMR基因突变在低龄大肠癌中属于频发事件,20例高度可疑HNPCC经DHPLC检出13例突变,经DNA测序和序列比对,发现了9个突变,其中3个新突变,6个已知突变(其中热点突变2个)。
Background and Objectives
Incidence 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.
Methods
1. 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.
Results
1. 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).
引文
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[6]Saletti P,Edwin D,Pack K,et al.Microsatellite instability:application in hereditary nonpolyposis colorectal cancer[J].Ann Oncol,2001,12(2):151-160.
[7]Lamberti C,Kruse R,Ruelfs C,et al.Microsatellite instability—a useful diagno stic tool to select patients at high risk for hereditary nonpolyposis colorectal cancer:a study in different groups of patients with colorectal cancer[J].Gut,1999,(44):839-843.
[8]Thibodeau SN,Bren G,Schaid D.M icrosatellite instability in cancer of the proximal colon[J].Science,1993,260:816-819.
[9]Aaltonen LA,Salovaara R,Kristo P,et al.Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease[J].Engl J Med,1998,338:1481-1487.
[10]Walhberg SS,Schmeits J,Thomas G,et al.Evaluation of microsatellite instability and immunohistochemistry for the prediction of germline MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families[J].Cance Res,2002,62(12):3485-3492.
[11]刘晓蓉,王亚平,张军妮等.应用离子对反相高效液相色谱法检测微卫星DNA 不稳定[J].中华医学遗传学杂志,2004,21(1):56-60.
[12]Dietmaier W,Hofstadter F.Detection of microsatellite instability by realtime PCR and hybridization probe melting point analysis[J].Lab Invest,2001,81:1453-1456.
[13]Hirst GL,Illand M.Automated fluorescent detection of microsatellite instability[J].Mol Biotechno 1,2001,17:239-247.
[14]Dieumegard B,Grandjouan S,Sabourin JC,et al.Extensive screening for hereditary non-polyposis colorectal cancer[J].Cancer,2000,82(4):871-80.
[15]Terdiman JP,Gum JR,Conrad PG,et al.Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing [J] .Gastroenterology, 2001, 120(1): 21-30.
[16]Ward R, Meagher A, Tomlinson I, et al: Microsatellite instability and the clinicopathological features of sporadic colorectal cancer [J]. Gut, 2001, 48(6): 821-9.
[1]Holinski Feder E,Muller Koch Y,Friedl W,et al.DHPLC mutation analysis of the hereditary nonpolyposis colon cancer(HNPCC)genes hMLH1 and hMSH2[J].Biochem Biophys Methods,2001,47(1-2):21-32
[2]Skopek TP,Glaab WE,Monroe JJ,et al.Analysis of sequence alterations in a defined DNA region:comparison of temperature-modulated heteroduplex analysis and denaturing gradient electrophoresis[J].Mutat Res,1999,430(1):13-21.
[3]周明奎,李文哲,朱文斯.变性高效液相色谱在微生物基因检测中的应用研究进展[J].生命科学研究,2006,10(2):76-79.
[4]Ziqiang Yuan,Benjamin Legender Jr,et al.A novel mutation detection approach of hMLH1 and hMSH2 genes for screening of colorectal cancer[J].Cancer Detection and Prevention,2006,30:333-340.
[5]Vasen HF,Watson P,Mecklin JP,et al.New clinical criteria for hereditary nonpolyposis colorectal cancer(HNPCC,Lynch syndrome) proposed by the International Collaborative group on HNPCC[J].Gastroenterology.1999,116(6):1453-1456.
[6]Cruz Correa M,Giardiello FM.Diagnosis and management of hereditary colon cancer[J]. Gastroenterol Clin North Am, 2002, 31(2):537-549.
[7] Bubb VJ,Curtis LJ,Cunningham C,et al.Microsatellite instability and the role of hMSH2 in sporadic colorectal cancer[J].Oncogene. 1996,12(12):2641-2649.
[8] Chan TL,Yuen ST,Chung LP,et al.Frequent Microsatellite Instability and Mismatch Repair Gene Mutation In Young Chinese Patients With Colorectal Cancer[J]. Cancer, 1999, 91:1221-1226.
[9] Liu B,Farrington SM,Petersen GM,et al.Genetic instability occurs in the majority of young patients with colorectal cancer [J]. Nat Med, 1995, 1(4):348-352.
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[2]冯波,郑民华,马君俊,等.结直肠癌组织中激肽释放酶10基因的表达及其与临床及病理的关系[J].中华外科杂志,2006,44(9):623-627.
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[4]Hu ZB,Huo X,Lu DR,et al.Functional polymorphisms of Matrix Metalloproteinase-9 are associatedwith risk of occurrence and metastasis of lung cancer[J].Clin Cancer Res,2005,11(15):5433-5439.
[5]方淑梅,金霞,李琰,等.基质金属蛋白酶3基因多态性与遗传易感性及淋巴结转移的关系[J].癌症,2005,24(3):305-310.
[6]Sun T,Gao Y,Tan W,et al.Haplotypes in matrix metalloproteinase gene cluster on chromosome 11q22 contribute to the risk of lung cancer development and progression[J].Clin Cancer Res,2006,12(23):7009-7017.
[7]Lou Y,Fang CQ,Li JH.A study on the expression of CASP9 gene and its polymorphism distribution in non-small cell lung cancer[J].Zhonghua Yi Xue Yi Chuan Xue Za ZhI,2007,24(1):59-62.
[8]Hamai Y,Matsumura S,Matsusaki K,et al.A single nucleotide polymorphism in the 5' untranslated region of the EGF gene is associated with occurrence and malignant progression of gastric cancer[J].Pathobiology,2005,72(3):133-138.
[9]Matsumura S,Oue N,Nakayama H,et al.A single nucleotide polymorphism in the MMP-9 promoter affects tumor progression and invasive phenotype of gastric cancer[J].Cancer Res Clin Oncol,2005,131(1):19-25.
[10]Crawford NP,Ziogas A,Peel DJ,et al.Germline polymorphisms in SIPA1are associated with metastasis and other indicators of poor prognosis in breast cancer[J].Breast Cancer Res,2006,8(2):R16.
[11]Jin X,Kuang G,Wei LZ,et al.No association of the matrix metalloproteinase 1promoter polymorphism with susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in northern China[J].World J Gastroenterol,2005,11(16):2385-2389.
[12]董稚明,崔雅静,邝刚,等.胸苷酸合成酶基因多态性及其蛋白表达与食管鳞状细胞癌淋巴结转移的关系[J].癌症,2005,24(10):1225-1229.
[13]Cao ZG,Li CZ.A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances oral squamous cell carcinoma susceptibility in a Chinese population[J].Oral Oncol,2006,42(1):32-38.
[14]Kohaar I,Thakur N,Salhan S,et al.TNFalpha-308G/A polymorphism as a risk factor for HPV associated Cervical Cancer in Indian population[J].Cell Oncol,2007,29(3):249-256.
[15]Purdie KJ,Lambert SR,Teh MT,et al.Allelic imbalances and microdeletions affecting the PTPRD gene in cutaneous squamous cell carcinomas detected using single nucleotide polymorphism microarray analysis[J].Genes Chromosomes Cancer,2007,46(7):661-669.
[16]Ostrovsky O,Korostishevsky M,Levite I,et al.Characterization of HPSE gene single nucleotide polymorphisms in Jewish populations of Israel[J].Acta Haematologica,2007,117(1):57-64.
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[1]Lamberti C,Kruse R,Ruelfs C,et al.Microsatellite instability a useful diagnosis tool to select patients at high risk for hereditary nonpolyposis colorectal cancer:a study in different group s of patientswith colorectal cancer[J].Gut,1999,44(6):839-843.
[2]Parc YR,Halling KC,Burgart LJ,et al.Microsatellite instability and hMLH1/hMSH2 expression in young endometrial carcinoma patients:associations with family history and histopathology[J].Cancer,2000,86(1):60-66.
[3]Lynch HT,Lynch J.Lynch syndrome:genetics,natural history,genetic counseling,and prevention[J].Clin Oncol,2000,18(21 Supp 1):19-31.
[4]LindorNM,BurgartLJ,Leontovich O,et al.immunohistochemistry versus microsatellite instability testing in phenotyp ing colorectal tumors[J].Clin Oncol,2002,20(4):1043-1048.
[5]Loukola A,Eklin K,Laiho P,et al.Microsatellite Marker analysis in screening for hereditary nonpolyposis colorectal cancer(HNPCC)[J].Cancer Res,2001,61 (11):4545-4549.
[6]Saletti P,Edwin D,Pack K,et al.Microsatellite instability:application in hereditary nonpolyposis colorectal cancer[J].Ann Oncol,2001,12(2):151-160.
[7]Lamberti C,Kruse R,Ruelfs C,et al.Microsatellite instability—a useful diagno stic tool to select patients at high risk for hereditary nonpolyposis colorectal cancer:a study in different groups of patients with colorectal cancer[J].Gut,1999,(44):839-843.
[8]Thibodeau SN,Bren G,Schaid D.M icrosatellite instability in cancer of the proximal colon[J].Science,1993,260:816-819.
[9]Aaltonen LA,Salovaara R,Kristo P,et al.Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease[J].Engl J Med,1998,338:1481-1487.
[10]Walhberg SS,Schmeits J,Thomas G,et al.Evaluation of microsatellite instability and immunohistochemistry for the prediction of germline MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families[J].Cance Res,2002,62(12):3485-3492.
[11]刘晓蓉,王亚平,张军妮等.应用离子对反相高效液相色谱法检测微卫星DNA 不稳定[J].中华医学遗传学杂志,2004,21(1):56-60.
[12]Dietmaier W,Hofstadter F.Detection of microsatellite instability by realtime PCR and hybridization probe melting point analysis[J].Lab Invest,2001,81:1453-1456.
[13]Hirst GL,Illand M.Automated fluorescent detection of microsatellite instability[J].Mol Biotechno 1,2001,17:239-247.
[14]Dieumegard B,Grandjouan S,Sabourin JC,et al.Extensive screening for hereditary non-polyposis colorectal cancer[J].Cancer,2000,82(4):871-80.
[15]Terdiman JP,Gum JR,Conrad PG,et al.Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing [J] .Gastroenterology, 2001, 120(1): 21-30.
[16]Ward R, Meagher A, Tomlinson I, et al: Microsatellite instability and the clinicopathological features of sporadic colorectal cancer [J]. Gut, 2001, 48(6): 821-9.
[1]Holinski Feder E,Muller Koch Y,Friedl W,et al.DHPLC mutation analysis of the hereditary nonpolyposis colon cancer(HNPCC)genes hMLH1 and hMSH2[J].Biochem Biophys Methods,2001,47(1-2):21-32
[2]Skopek TP,Glaab WE,Monroe JJ,et al.Analysis of sequence alterations in a defined DNA region:comparison of temperature-modulated heteroduplex analysis and denaturing gradient electrophoresis[J].Mutat Res,1999,430(1):13-21.
[3]周明奎,李文哲,朱文斯.变性高效液相色谱在微生物基因检测中的应用研究进展[J].生命科学研究,2006,10(2):76-79.
[4]Ziqiang Yuan,Benjamin Legender Jr,et al.A novel mutation detection approach of hMLH1 and hMSH2 genes for screening of colorectal cancer[J].Cancer Detection and Prevention,2006,30:333-340.
[5]Vasen HF,Watson P,Mecklin JP,et al.New clinical criteria for hereditary nonpolyposis colorectal cancer(HNPCC,Lynch syndrome) proposed by the International Collaborative group on HNPCC[J].Gastroenterology.1999,116(6):1453-1456.
[6]Cruz Correa M,Giardiello FM.Diagnosis and management of hereditary colon cancer[J]. Gastroenterol Clin North Am, 2002, 31(2):537-549.
[7] Bubb VJ,Curtis LJ,Cunningham C,et al.Microsatellite instability and the role of hMSH2 in sporadic colorectal cancer[J].Oncogene. 1996,12(12):2641-2649.
[8] Chan TL,Yuen ST,Chung LP,et al.Frequent Microsatellite Instability and Mismatch Repair Gene Mutation In Young Chinese Patients With Colorectal Cancer[J]. Cancer, 1999, 91:1221-1226.
[9] Liu B,Farrington SM,Petersen GM,et al.Genetic instability occurs in the majority of young patients with colorectal cancer [J]. Nat Med, 1995, 1(4):348-352.
[1]Kuwai T,Kitadai Y,et al.Single nucleotide polymorphism in the hypoxiainducible factor-lalpha gene in colorectal carcinoma[J].Oncol Rep,2004,12(5):1033-1037.
[2]冯波,郑民华,马君俊,等.结直肠癌组织中激肽释放酶10基因的表达及其与临床及病理的关系[J].中华外科杂志,2006,44(9):623-627.
[3]Andersen CL,Wiuf C,Kruhoffer M,Korsgaard M,Laurberg S,Orntoft TF.Frequent occurrence of uniparental disomy in colorectal cancer[J].Carcinogenesis,2007,28(1):38-48.
[4]Hu ZB,Huo X,Lu DR,et al.Functional polymorphisms of Matrix Metalloproteinase-9 are associatedwith risk of occurrence and metastasis of lung cancer[J].Clin Cancer Res,2005,11(15):5433-5439.
[5]方淑梅,金霞,李琰,等.基质金属蛋白酶3基因多态性与遗传易感性及淋巴结转移的关系[J].癌症,2005,24(3):305-310.
[6]Sun T,Gao Y,Tan W,et al.Haplotypes in matrix metalloproteinase gene cluster on chromosome 11q22 contribute to the risk of lung cancer development and progression[J].Clin Cancer Res,2006,12(23):7009-7017.
[7]Lou Y,Fang CQ,Li JH.A study on the expression of CASP9 gene and its polymorphism distribution in non-small cell lung cancer[J].Zhonghua Yi Xue Yi Chuan Xue Za ZhI,2007,24(1):59-62.
[8]Hamai Y,Matsumura S,Matsusaki K,et al.A single nucleotide polymorphism in the 5' untranslated region of the EGF gene is associated with occurrence and malignant progression of gastric cancer[J].Pathobiology,2005,72(3):133-138.
[9]Matsumura S,Oue N,Nakayama H,et al.A single nucleotide polymorphism in the MMP-9 promoter affects tumor progression and invasive phenotype of gastric cancer[J].Cancer Res Clin Oncol,2005,131(1):19-25.
[10]Crawford NP,Ziogas A,Peel DJ,et al.Germline polymorphisms in SIPA1are associated with metastasis and other indicators of poor prognosis in breast cancer[J].Breast Cancer Res,2006,8(2):R16.
[11]Jin X,Kuang G,Wei LZ,et al.No association of the matrix metalloproteinase 1promoter polymorphism with susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in northern China[J].World J Gastroenterol,2005,11(16):2385-2389.
[12]董稚明,崔雅静,邝刚,等.胸苷酸合成酶基因多态性及其蛋白表达与食管鳞状细胞癌淋巴结转移的关系[J].癌症,2005,24(10):1225-1229.
[13]Cao ZG,Li CZ.A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances oral squamous cell carcinoma susceptibility in a Chinese population[J].Oral Oncol,2006,42(1):32-38.
[14]Kohaar I,Thakur N,Salhan S,et al.TNFalpha-308G/A polymorphism as a risk factor for HPV associated Cervical Cancer in Indian population[J].Cell Oncol,2007,29(3):249-256.
[15]Purdie KJ,Lambert SR,Teh MT,et al.Allelic imbalances and microdeletions affecting the PTPRD gene in cutaneous squamous cell carcinomas detected using single nucleotide polymorphism microarray analysis[J].Genes Chromosomes Cancer,2007,46(7):661-669.
[16]Ostrovsky O,Korostishevsky M,Levite I,et al.Characterization of HPSE gene single nucleotide polymorphisms in Jewish populations of Israel[J].Acta Haematologica,2007,117(1):57-64.