散发性大肠癌的错配修复缺陷研究

目的探讨错配修复缺陷的散发性大肠癌的临床病理特征及错配修复缺陷检测手段的应用。方法对71例散发性大肠癌行hMLH1启动子甲基化检测、微卫星不稳定检测以及hMLH1和hMSH2的免疫组化检测,分析错配修复缺陷的散发性大肠癌的临床病理特征,探讨三种检测方法的应用价值。结果hMLH1基因启动子甲基化、微卫星不稳定和错配修复蛋白表达的阳性率分别为9.9%,9.9%和71.0%,三者密切相关。hMLH1启动子甲基化和微卫星不稳定的散发性大肠癌均具有结肠癌多发和低分化腺癌相对多见的特征。错配修复蛋白表达阴性的散发性大肠癌仅具有低分化腺癌相对多见的特征。结论错配修复缺陷的散发性大肠癌具有结肠癌和低分化腺癌多发的倾向,hMLH1启动子甲基化和微卫星不稳定以及错配修复蛋白的失表达三者密切相关。     

Era of universal testing of microsatellite instability in colorectal cancer

Colorectal cancer (CRC) incidence and mortality are constantly decreasing, but CRC still remains the third most prevalent cancer and the third most common cause of cancer death in both males and females in the United States. Recent rapid declines in CRC incidence rates have largely been attributed to increases in screening that can detect and remove precancerous polyps, and the decrease in death rates for CRC largely reflects improvements in early detection, treatment and the understanding of molecular/genetic basis of CRC. One of the important molecular/genetic findings is the presence of microsatellite instability (MSI) in CRCs. Many studies have shown the importance of MSI testing in diagnosing Lynch syndrome and predicting prognosis and response to chemotherapeutic agents in CRCs. Increased emphasis has been placed on the importance of MSI testing for all newly diagnosed individuals with CRCs. Both immunohistochemical staining (IHC) and polymerase chain reaction (PCR)-based MSI testing show high sensitivity and specificity in detecting MSI. The current clinical guidelines and histopathology features are indicative of, but not reliable in diagnosing Lynch syndrome and CRCs with MSI. Currently, there are evidences that universal testing for MSI starting with either IHC or PCR-based MSI testing is cost effective, sensitive, specific and is getting widely accepted.     

肺癌微卫星不稳定性与错配修复基因缺陷的相关性研究

 目的　通过对肺癌微卫星不稳定性（MSI）的分析与错配修复基因蛋白表达的检测,探讨肺癌发病的分子机制。方法　从50例肺癌患者的正常肺组织、癌组织中提取DNA;SSCP法检测标本中MSI发生情况;免疫组织化学法检测错配修复基因hMLH1及hMSH2在肺癌中的表达情况。结果　50例肺癌中微卫星高度不稳定（MSI-H）14例,低度不稳定（MSI-I）21 例,稳定（MSS）15例,正常组织中未出现MSI,两者之间差异有统计学意义（P＝0.000）;免疫组化结果显示hMLH1在MSI肺癌组织中常为缺失表达,表达率为74 %（37／50）;hMSH2在MSI肺癌组织中也呈缺失表达,表达率为32 %（16／50）;而在MSS肺癌组织中均显示hMLH1、 hMSH2基因蛋白表达阳性。结论　肺癌的发生可能存在MSI途径,而hMLH1、hMSH2的表达失活则可能导致MSI的发生,因此,MSI可作为肺癌诊断的指标之一。     

全自动IdyllaTM系统在中国结直肠癌微卫星不稳定性检测中的可行性研究

背景与目的：微卫星不稳定性（microsatellite instability,MSI）与错配修复（mismatch repair,MMR）基因缺陷,作为结直肠癌（colorectal cancer,CRC）等泛肿瘤免疫治疗的重要生物标志物之一,其检测近年来备受关注。目前临床上常用的多重荧光聚合酶链反应（polymerase chain reaction,PCR）检测MSI的传统方法（如Promega等）,通常需要非肿瘤组织对照及较长的检测周期。Idylla^TM系统作为一种全自动定量PCR装置,检测MSI无需非肿瘤组织对照,且运行时间仅为2.5 h,具有较好的临床应用前景。但在国内临床应用前,应在中国CRC患者中验证该方法的可靠性。方法：收集2017年3月—2019年3月复旦大学附属肿瘤医院的87例CRC患者用40%甲醛溶液固定石蜡包埋（formalin-fixed paraffin-embedded,FFPE）的组织样本,分别采用免疫组织化学（immunochemistry,IHC）、传统PCR（Promega）和Idylla^TM 3种方法进行MMR/MSI检测并比较分析,评估Idylla^TM系统临床检测的灵敏度和特异度以及在不同肿瘤含量样本中的可重复性。结果：87例CRC患者中,IHC和Promega的检测结果完全一致,其中56例被判定为MMR缺陷（deficient MMR,dMMR）/高度MSI（MSI-high,MSI-H）,31例为MMR完整（proficient MMR,pMMR）/微卫星稳定（microsatellite stable,MSS）。然而,其中4例dMMR/MSI-H患者被Idylla^TM MSI系统诊断为MSS,1例pMMR/MSS患者被诊断为MSI-H。若以IHC/Promega为参考标准,Idylla^TM MSI系统的诊断灵敏度为92.9%（52/56）,特异度为96.8%（30/31）,总体一致率达94.3%（82/87）。随后,应用Idylla^TM系统对上述不一致的5例患者进行重复检测,其中4例肿瘤含量≥20%的病例检测结果与IHC/Promega MSI检测一致,但1例肿瘤含量仅为5%的病例仍与IHC/Promega不一致,被误诊为MSS。最后,针对该病例,通过激光捕获显微切割技术对肿瘤细胞进行富集后,应用Idylla^TM MSI系统再次检测,结果显示为MSI-H。此外,选取5例具有不同肿瘤含量（20% ~ 60%）的病例,分别进行3次重复性验证,结果显示,Idylla^TM系统具有高度可重复性。结论：对于肿瘤含量≥20%的CRC患者,Idylla^TM系统可以为临床MSI检测提供一种快速、可靠、全自动的解决方案,并具有较高的灵敏度、特异度和可重复性。     

Chk1 frameshift mutation in sporadic and hereditary non-polyposis colorectal cancers with microsatellite instability.

AIM: Protein kinase Chk1 (hChk1) is essential in human cells for cell cycle arrest in response to DNA damage, and has been shown to play an important role in the G2/M checkpoint. The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal cancers. The aim of this study was to investigate whether a frameshift mutation within the Chk1 gene contribute to the development or progression of eastern sporadic and hereditary non-polyposis colorectal cancer (HNPCC) with microsatellite instability (MSI). METHODS: We analyzed MSI using the 6 microsatellite markers and a frameshift mutation in the BRAF gene and in poly(A)9 within the Chk1 gene in 51 sporadic colorectal cancer and 14 HNPCC specimens. RESULTS: Eleven of the 51 sporadic colorectal cancers and all of the 14 HNPCCs were MSI-positive. Chk1 frameshift mutations were observed in 2 and 3 sporadic colon cancers and HNPCC, respectively, whereas no BRAF mutations were detected in these samples. Interestingly, all cases with the Chk1 frameshift mutation had high-frequency MSI. CONCLUSION: These results suggest that the Chk1 gene is a target of genomic instability in MSI-positive colorectal cancers and that the Chk1 framshift mutations might be involved in colorectal tumourigenesis through a defect in response to DNA damage in a subset of sporadic colorectal cancers and HNPCCs.     

肺癌微卫星不稳定性与错配修复基因缺陷的相关性研究

目的 通过对肺癌微卫星不稳定性(MSI)的分析与错配修复基因蛋白表达的检测,探讨肺癌发病的分子机制.方法 从50例肺癌患者的正常肺组织、癌组织中提取DNA;SSCP法检测标本中MSI发生情况;免疫组织化学法检测错配修复基因hMLH1及hMSH2在肺癌中的表达情况.结果 50例肺癌中微卫星高度不稳定(MSI-H)14例,低度不稳定(MSI-I)21例,稳定(MSS)15例,正常组织中未出现MSI,两者之间差异有统计学意义(P=0.000);免疫组化结果显示hMLH1在MSI肺癌组织中常为缺失表达,表达率为74%(37/50);hMSH2在MSI肺癌组织中也呈缺失表达,表达率为32%(16/50);而在MSS肺癌组织中均显示hMLH1、hMSH2基因蛋白表达阳性.结论 肺癌的发生可能存在MSI途径,而hMLH1、hMSH2的表达失活则可能导致MSI的发生,因此,MSI可作为肺癌诊断的指标之一.     

散发性结直肠癌微卫星不稳定状态与错配修复蛋白表达缺失及临床病理特征的相关性

目的： 探讨散发性结直肠癌微卫星不稳定(microsatellite instability,MSI)情况及其与错配修复 (mismatch repair,MMR) 蛋白MLH1、MSH2、MSH6、PMS2表达缺失的相关性,并总结MSI散发性结直肠癌的临床病理学特征。方法：多重荧光PCR法检测散发性结直肠癌肿瘤组织DNA的微卫星不稳定性,免疫组化(Immunohistochemistry,IHC)S-P法检测散发性结直肠癌肿瘤组织MLH1、MSH2、MSH6、PMS2蛋白的表达缺失,分析MSI发生与MMR蛋白表达缺失及临床病理特征的相关性。结果：75 例散发性结直肠癌检出MSI 21例(28%),包括 MSI-H 19例、MSI-L 2例,其他54例(72%)为MSS。检出MMR蛋白表达缺失16例(21.33%),其中15例(93.75%)为MSI-H、1例(6.25%)为MSS;MMR蛋白表达59例(78.67%),其中4例(6.78%)为MSI-H、2例(3.39%)MSI-L,其他53例为MSS。MSI组MMR蛋白缺失率(15/21,71.43%)显著高于MSS组(1/54,1.9%)（P<0.01）。MSI与患者年龄,是否黏液腺癌,肿瘤有无远处转移有关（P<0.01）,其中MSI-H好发于年龄>50岁、肿瘤无远处转移、MMR蛋白缺失人群,且类型以黏液腺癌为主。结论： 散发性结直肠癌肿瘤组织中MSI发生率高于MMR蛋白缺失率,并且MSI-H的散发性结直肠癌转移风险较低、预后较好。检测MSI 状态对提高结直肠癌的预防、诊断和治疗水平,降低结直肠癌的发病率和病死率有着重要意义。     

肺癌微卫星不稳定性与错配修复基因缺陷的相关性研究

目的 通过对肺癌微卫星不稳定性(MSI)的分析与错配修复基因蛋白表达的检测,探讨肺癌发病的分子机制.方法 从50例肺癌患者的正常肺组织、癌组织中提取DNA;SSCP法检测标本中MSI发生情况;免疫组织化学法检测错配修复基因hMLH1及hMSH2在肺癌中的表达情况.结果 50例肺癌中微卫星高度不稳定(MSI-H)14例,低度不稳定(MSI-I)21例,稳定(MSS)15例,正常组织中未出现MSI,两者之间差异有统计学意义(P=0.000);免疫组化结果显示hMLH1在MSI肺癌组织中常为缺失表达,表达率为74%(37/50);hMSH2在MSI肺癌组织中也呈缺失表达,表达率为32%(16/50);而在MSS肺癌组织中均显示hMLH1、hMSH2基因蛋白表达阳性.结论 肺癌的发生可能存在MSI途径,而hMLH1、hMSH2的表达失活则可能导致MSI的发生,因此,MSI可作为肺癌诊断的指标之一.     

Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: Report from the constitutional mismatch repair deficiency consortium

BackgroundConstitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome for which data regarding clinical manifestations, molecular screening tools and management are limited.MethodsWe established an international CMMRD consortium and collected comprehensive clinical and genetic data. Molecular diagnosis of tumour and germline biospecimens was performed. A surveillance protocol was developed and implemented.ResultsOverall, 22/23 (96%) of children with CMMRD developed 40 different tumours. While childhood CMMRD related tumours were observed in all families, Lynch related tumours in adults were observed in only 2/14 families (p = 0.0007). All children with CMMRD had café-au-lait spots and 11/14 came from consanguineous families. Brain tumours were the most common cancers reported (48%) followed by gastrointestinal (32%) and haematological malignancies (15%). Importantly, 12 (30%) of these were low grade and resectable cancers. Tumour immunohistochemistry was 100% sensitive and specific in diagnosing mismatch repair (MMR) deficiency of the corresponding gene while microsatellite instability was neither sensitive nor specific as a diagnostic tool (p < 0.0001). Furthermore, screening of normal tissue by immunohistochemistry correlated with genetic confirmation of CMMRD. The surveillance protocol detected 39 lesions which included asymptomatic malignant gliomas and gastrointestinal carcinomas. All tumours were amenable to complete resection and all patients undergoing surveillance are alive.DiscussionCMMRD is a highly penetrant syndrome where family history of cancer may not be contributory. Screening tumours and normal tissues using immunohistochemistry for abnormal expression of MMR gene products may help in diagnosis and early implementation of surveillance for these children.     

中国人遗传性非息肉病性结直肠癌错配修复缺陷表型分析

目的：对中国人遗传性非息肉病性结直肠癌的肿瘤组织进行hMSH2和hMLH1,蛋白表达监测及微卫星不稳定性检测。方法：共收集58个符合不同临床诊断标准的家系,对符合Amsterdam标准24个家系(AC组)的38个肿瘤(来自22个家系)、符合日本标准15个家系(JC组)的16个肿瘤(来自12个家系)、符合Bethesda指导纲要的19例患者(BG组)中12例的13个肿瘤组织进行研究。选取5个微卫星位点BAT26、BAT25、D2S123、D5S346和D17S250及单克隆抗体hMSH2和hMLH1用于分析。结果：AC组家系100％表现为高度微卫星不稳定性(MSI-H),其中81．8％(18／22)表现为hMSH2和hMLH1表达异常;JC组家系中,93．3％(14／15)和1／1个腺瘤表现为MSI-H,45．5％(5／11)表现为hMSH2或hMLH1表达异常;BG组家系中,53．8％(7／13)患者肿瘤表现为MSI-H,其中4／7表现hMSH1表达异常。结论：不同临床诊断标准的家系,其肿瘤组织MSI-H阳性和错配修复蛋白表达异常的频率不同,Amsterdam标准和日本标准可较准确地反映肿瘤组织中错配修复缺陷情况,但Bethesda指导纲要也不可或缺。在临床诊断的基础上,合用免疫组化和微卫星不稳定性检测,可以较全面地检测到错配修复缺陷肿瘤。错配修复蛋白表达异常和微卫星不稳定性密切相关。     

MutL homolog 1 methylation and microsatellite instability in sporadic colorectal tumors among Filipinos

BACKGROUNDColorectal cancer (CRC) ranks third in terms of incidence and second in mortality worldwide. In CRC, the silencing of mismatch repair genes, including the mutL homolog 1 (hMLH1) has been linked to microsatellite instability (MSI), the lengthening or shortening of microsatellite repeats. Very limited data have been presented so far on the link of hMLH1 methylation and MSI in Southeast Asia populations with sporadic CRC, and on its clinical significance.AIMTo investigate the significance of the MSI status and hMLH1 methylation in CRC Filipino patients.METHODSFifty-four sporadic CRC patients with complete clinical data were included in this study. Genomic DNA from CRC tumor biopsies and their normal tissue counterparts were profiled for MSI by high resolution melting (HRM) analysis using the Bethesda Panel of Markers (BAT25, BAT26, D2S123, D5S346, and D17S250). hMLH1 methylation screening was performed using bisulfite conversion and methylation specific polymerase chain reaction. Statistical analysis was conducted to calculate their associations to clinicopathological characteristics and survival relevance (Kaplan-Meier curves and the log-rank test).RESULTS hMLH1 methylation was observed in 9% and 35% of CRC and normal samples, respectively. Higher incidence of consistently methylated hMLH1 found in both normal and CRC was noticed for relation to location of tumor (P < 0.05). As for MSI status, D2S123 the most common unstable microsatellite and MSI-high (MSI-H) was the most common MSI profile, counted for 46% and 50% of normal and CRC tissues, respectively. The presence of MSI-low (MSI-L) and microsatellite stable (MSS) was 43% and 11% for normal, and 31% and 19% for CRC samples. The mean month of patients’ survival was shorter in patients whose normal and tumor tissues had methylated compared to those with unmethylated hMLH1 and with MSI-H compared to those with MSI-L/MSS (P < 0.05). This was supported by significant difference in Kaplan-Meier with log-rank analysis. This data indicated that hMLH1 methylation and high MSI status have prognostic value.CONCLUSIONThis study showed the clinical significance of hMLH1 methylation and MSI status in sporadic CRC Filipino patients, especially in the normal part of the tumor.     

微卫星不稳定散发性大肠癌的临床病理特征和DNA倍体研究

目的:探讨微卫星不稳定的散发性大肠癌的临床病理特征及微卫星不稳定表型和DNA倍体类型的关系。方法:对71例散发性大肠癌行BAT25和BAT26两个位点的微卫星不稳定检测和流式细胞术倍体分析,探讨微卫星不稳定状态和临床病理特征及DNA倍体类型的关系。结果:微卫星不稳定的阳性率为9.86%(7/71),微卫星不稳定表型和发病部位、组织学类型及分化程度相关(P<0.05),而与性别、年龄、淋巴结转移和分期无关。微卫星不稳定的散发性大肠癌中右半结肠癌和低分化腺癌的比例高于微卫星稳定者。68例患者检出二倍体和异倍体分别为18和50例,微卫星不稳定表型者5例为二倍体,因此和DNA倍体类型显著相关(P=0.012)。结论:微卫星不稳定的散发性大肠癌好发于右半结肠,具有低分化腺癌的倾向,多为二倍体。     

Value of the identification of microsatellite instability in colorectal cancer

Objective  Recent studies defend a possible prognostic and therapeutic value of the identification of microsatellite instability (MSI) in colorectal cancer. This work tries to assess the impact that the identification of MSI tumours can have in clinical practice. Material and methods  We recovered tumour samples from 92 of the 143 patients operated on for colorectal cancer in our institution between 1995 and 2000. Five MSI markers (BAT 25, BAT 26, D2S123, D5S346 and D17S250) were studied on them. The rate and clinicopathologic characteristics of MSI tumours were investigated along with their impact on the global and disease-free survival as compared with microsatellite stable (MSS) tumours. Results  All 5 microsatellite markers’ status were established in 73 patients (79.3% of the samples). Among them, 7 tumours showed instability in just one marker (low microsatellite instability [MSI-L]) whereas 5 tumours had mutations in 2 or more markers (high microsatellite instability [MSI-H]), for a total 15.4% rate of MSI tumours. All MSI-H tumours were located in the right colon. We could not find any impact from MSI detection on global or disease-free survival. Conclusions  MSI determination did not identify groups of patients with a different prognosis. Moreover, with such low incidence its determination can only be justified in those cases that fulfill Bethesda’s criteria to identify families with Lynch’s syndrome.     

修饰型微卫星不稳定性与散发结直肠癌p53基因点突变的相关性

目的 高频度微卫星不稳定性被认定为DNA错配修复缺陷的标志,但既往研究发现一个显著矛盾,即在高频度微卫星不稳定结直肠癌中,p53突变率较一般结直肠癌低.研究旨在确认该矛盾的存在并试图阐明其机制.方法 对180例散发结直肠癌采用高分辨率荧光标记微卫星分析法检测微卫星位点稳定性,PCR扩增直接测序检测p53突变.结果 微卫星不稳定性呈现修饰型和跳跃型两种变化.低频度微卫星不稳定性均呈现修饰型而无跳跃型变化;高频度微卫星不稳定性均检出了跳跃型变化,一部分也并存修饰型变化.微卫星不稳定与肿瘤部位及分化程度明显相关,p53突变与肿瘤分化明显相关.高频度微卫星不稳定肿瘤未检出p53突变,而低频度微卫星不稳定肿瘤p53突变率较高.结论 低频度微卫星不稳定性呈现的修饰型微卫星位点长度变化可能是DNA错配修复缺陷的表型;此表型与提高的碱基置换突变率有关.单纯DNA错配修复缺陷可能不足以导致微卫星不稳定性的跳跃型变化,高频度微卫星不稳定的真正原因仍有待阐明.     

Clinical implications of mismatch repair deficiency screening in patients with mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN)

IntroductionMixed neuroendocrine non-neuroendocrine neoplasms (MiNEN) are rare tumors, mainly encountered in the gastroenteropancreatic tract. Based on the limited available data, MiNEN is usually a highly aggressive neoplasm combining a high-grade neuroendocrine and a non-neuroendocrine component, associated with a poor prognostic outlook. Deficient DNA mismatch repair (MMR) results in microsatellite instability, which is a useful screening marker for identifying patients with Lynch syndrome and a prognostic factor for chemotherapeutic interventions. Little information on MMR status in MiNEN is available in published studies. Therefore, the purpose of this study was to explore the status and putative role of MMR on MiNEN.MethodsWe investigated the MMR status in 44 cases and characterized their clinicopathological features and prognoses. Immunohistochemistry was performed for four mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2).ResultsMean age at diagnosis was 61 years, and 75% of the patients were male. Lymph node metastases were observed in 14 (35.9%) patients. The most common tumor localizations were gastric (28 patients, 63.6%). Lack of immunohistochemical expression of MMR proteins was shown in 38.6% of cases. The common deletion rates of one or more proteins were 29.4% (5/17) for MLH1/PMS2 and 23.5% (4/17) for MLH1. Correlation between clinicopathological parameters showed that MMR deficiency was significantly associated with early TNM stage and better prognoses in patients with MiNEN.ConclusionMiNENs showed frequent losses of MMR protein expression, which contributes to the knowledge of the pathological and clinical aspects of MiNEN tumors.     

散发性结直肠癌中微卫星不稳定性及临床病理意义

目的通过微卫星位点BAT-25和BAT-26的分析,观察散发性结直肠癌原发和转移灶中微卫星不稳定性（MSI）的阳性率,并探讨其与临床病理参数的关系。方法收集73例结直肠癌原发灶和53例转移灶石蜡标本,分离基因组DNA,通过荧光标记多重PCR法扩增微卫星位点BAT-25和BAT-26;应用全自动DNA测序仪和GeneScan 3．1软件进行片段分析,观察这2个位点重复序列长度的变化。以1例己知有MSI-H的遗传性非息肉病性结直肠癌（HNPCC）病例为阳性对照。结果73例散发性结直肠癌中,MSI的阳性率为15．1％,MSI与患者的性别、肿瘤发生部位、分化程度和预后有关（P〈0．05）;53例转移患者中,转移灶的MSI阳性率（17．0％）略高于原发灶（13．2％）,差异无统计学意义（P〉0．05）,但有2例原发灶MSI阴性,转移灶MSI阳性。结论散发性大肠癌中MSI是一个常见的分子事件;MSI可作为临床判断大肠癌恶性程度、预后等的重要参考指标,根据MSI对散发性结直肠癌进行分类有重要的理论和实际意义;MSI在部分散发性大肠癌的转移中可能起一定的作用。