基于中国人Lynch综合征临床标准的家族性结直肠癌胚系突变谱多中心研究

  目的  Lynch综合征作为一种常染色体显性遗传的恶性肿瘤综合征,基于肿瘤家族史对患者进行初筛是目前Lynch综合征筛查的主要手段。本研究旨在明确符合中国人Lynch综合征临床标准的结直肠癌患者最终确诊为Lynch综合征的比例,以及二代测序在Lynch综合征临床诊断中的应用。  方法  2017年2月至2019年10月纳入85例就诊于国内7家医院（浙江大学医学院附属第二医院、北京协和医院、江苏省人民医院、北京大学第一医院、辽宁省肿瘤医院、四川大学华西医院和温州市中心医院）无血缘关系的符合中国人Lynch综合征临床标准的结直肠癌患者,利用含61个已报道与遗传性肿瘤相关基因的二代测序平台检测入组先证者的胚系突变。对于检出的临床意义不明的基因变异,通过一代测序、多重荧光PCR毛细管电泳检测肿瘤组织微卫星状态等手段判读该变异的致病意义。  结果  符合中国人Lynch综合征临床标准的结直肠癌患者中,28.2%检测出Lynch综合征关键基因已知致病性或疑似致病性胚系突变,另外15.3%患者携带上述基因的临床意义不明的变异。通过对这些变异进行分析,本研究认为MLH1基因c.2240_2255 delCTGATCTATACAAAGT通读突变为遗传性结直肠癌的致病性突变;然而目前尚缺乏充足证据证明MLH3 基因第2～11号外显子大片段重复与遗传性结直肠癌的发生存在密切联系。  结论  本研究发现同样基于符合中国人Lynch综合征临床标准的结直肠癌患者,与既往利用一代测序的研究报道相比,二代测序并未提高Lynch综合征的确诊率。但是,二代测序会带来大量临床意义不明的突变。需要谨慎对待尚未明确的基因突变检测结果,必要时应该进行更深入的家系研究和更全面的分子检测以明确这些突变的致病意义。      

二代测序在家族遗传性高危胃肠肿瘤筛查中的应用

  目的  探索二代测序技术对家族遗传性高危胃肠肿瘤患者进行遗传筛查的意义及高危因素在筛选患者中的价值。  方法  选取2016年3月至2016年4月收治于北京大学肿瘤医院的322例结直肠癌及胃癌患者,筛选出25例遗传性胃肠肿瘤高危患者,运用二代测序技术对患者的外周血白细胞DNA进行42个遗传性肿瘤综合征相关基因的胚系检测。  结果  24%（6/25）患者检测出遗传性肿瘤相关基因的病理性胚系突变,其中50%（3/6）患者肿瘤组织的免疫组织化学检测表现为错配修复蛋白表达缺失,83%（5/6）患者发病年龄≤50岁且具有恶性肿瘤家族史。发生胚系突变的6例遗传性肿瘤相关基因分别为MYH基因错义突变1例,APC基因缺失突变1例和遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer,HNPCC）相关基因的突变4例（包括MLH1、MLH3、TGFBR2的错义突变和MSH6的无义突变各1例）,且提供了MLH3的胚系致病突变的家系验证。  结论  通过二代测序技术对本研究入组的25例患者进行家族遗传性肿瘤综合征的筛查,检测出遗传性肿瘤相关基因的胚系致病突变6例,提示运用二代测序技术对家族遗传性高危消化道肿瘤患者进行遗传筛查具有提高检测阳性率的临床应用价值。      

错配修复基因蛋白在结直肠癌诊治中的临床应用

  目的  探讨错配修复基因（mismatch repair gene,MMR）蛋白MLH1、MSH2、MSH6、PMS2在结直肠癌中的表达及在临床中的应用。  方法  选取四川省人民医院2015年1月至2016年9月收治的607例结直肠癌患者,采用免疫组织化学法检测手术标本中MMR蛋白的表达情况,研究其与临床病理学的关系,并评价其在Lynch综合征和散发性结直肠癌筛查中的价值。  结果  607例患者中MMR表达缺失率为35.58%。MMR蛋白表达缺失的阴性组与表达正常的阳性组,在年龄、性别、肿瘤大小、P53、CD34、D2-40的比较,差异均无统计学意义（P>0.05）;两组患者在肿瘤位置、分化程度、TNM分期、淋巴结转移、VEGF、Ki-67的比较,差异均有统计学意义（P < 0.05）。联合检测MLH1、MSH2、PSM2、MSH6蛋白可以作为初步筛选Lynch综合征患者的方法。  结论  对结直肠癌患者的手术标本进行MMR检测,筛查Lynch综合征患者和家族成员,进行管理及干预,可降低部分人群患结直肠癌的风险。      

MLH1基因突变在Lynch综合征中的研究进展

结直肠癌是常见的消化系统恶性肿瘤,有些类型具有家族聚集性。Lynch综合征作为最常见的遗传性结直肠癌综合征,主要因相关基因突变致错配修复蛋白表达异常、减少或缺失而致病,其主要包括MLH1、MSH2、MSH6和PMS2基因。考虑到MLH1和MSH2基因的胚系突变占Lynch综合征总突变近90%,因此本文将着重综述近年来错配修复蛋白基因MLH1的突变,整理在不同国家和地区的重要创始人突变。通过对公共数据库已有报道突变及单中心收录数据比较,从而对其诊断,基因筛查,为了解不同人种和地域的Lynch综合征创始人突变提供一定的指导意义。     

Lynch综合征筛查及治疗研究进展

家族遗传性结直肠癌是一类由于基因胚系变异导致的疾病,其中最常见的是Lynch综合征,也被称为遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer,HNPCC）。近年来随着对基因组认识的加深,研究显示Lynch综合征临床表型和治疗靶点均有别于散发型结直肠癌。本文将从Lynch综合征筛查策略、类Lynch综合征再定义,以及Lynch综合征免疫治疗和化学预防等方面综述其研究进展。      

国人卵巢癌DNA损伤修复基因突变图谱分析

  目的  同源重组修复（homologous recombination repair,HRR）基因BRCA1/2在卵巢癌的发生发展中起到了重要作用,相关研究比较深入,而对于其他DNA损伤修复（DNA-damage repair,DDR）基因突变在中国卵巢癌人群中的分布及其与患者临床特征之间的关系仍缺乏详细的探讨。  方法  本研究纳入2019年6月至2020年6月在天津医科大学肿瘤医院接受手术治疗的卵巢癌患者122例,收集其外周血样本和67例对应肿瘤组织标本,利用二代测序技术检测19个DDR基因的变异情况、分布特点和临床病理相关性。  结果  胚系DDR基因突变主要集中在HRR基因,占80.37%,致病性和可能致病性突变全部集中在HRR基因。携带胚系HRR（germline HRR,gHRR）突变的患者较BRCA突变的更多（31.15% vs. 23.77%）,且呈现家族聚集现象,病理类型以浆液性腺癌为主,并与铂类药物敏感性相关。肿瘤组织层面DDR基因突变中HRR基因突变率仅次于TP53,占39.39%。检出肿瘤组织HRR（tumor HRR,tHRR）突变的患者较gHRR突变的患者多5.97%。tHRR突变患者对铂类药物治疗敏感,且复发风险更低。  结论  揭示中国卵巢癌DDR基因突变特征,提示基于二代测序的DDR多基因检测可指导卵巢癌患者的精准诊疗。      

Lynch 综合征的诊治进展和家系管理*

Lynch 综合征（Lynch syndrome,LS）是遗传性结直肠癌中最常见（约占5%）的一类常染色体显性遗传病,错配修复基因的种系突变和微卫星不稳定是其区别于其他遗传性结直肠癌的两大特点。近年来,研究发现在诊断和治疗上,LS与散发性结直肠癌有一定的区别;此外除了患者本人的诊断和治疗,整个家系的管理也至关重要。此类疾病应当引起临床高度重视。本文对LS的最初定义、诊断标准和筛查标准的变迁、最新治疗进展和家系管理进行综述,旨在帮助临床了解LS,给予患者合理的治疗,以及对其家系成员适当的干预和监控,尽可能降低患癌风险。      

云南省7个Lynch综合征家系中致癌基因突变研究

目的 探讨云南省7个Lynch综合征（Lynch syndrome,LS）家系中常见致病基因突变情况。方法 选取昆明医科大学第一附属医院肿瘤科2008年3月—2013年12月收治的7个经过免疫组织化学检测和微卫星不稳定检测确定为MSI-H和dMMR的家系先证者,并且这7个家系都符合AmsterdamⅡ诊断标准。采用目标区域捕获结合高深度测序技术,对家系首发患者的遗传性结直肠癌相关5个基因外显子及其邻近±2 bp内含子区变异进行分析。结果 7个家系（2个白族家系、2个彝族家系、3个汉族家系）中,2个白族家系和2个彝族家系均未发现已知突变位点。结论 Lynch综合征家系致病突变可能存在民族差异。     

Lynch综合征研究进展

[摘要] Lynch 综合征（Lynch syndrome,LS）是一种常染色体显性遗传病,是由于几种DNA 错配修复（mismatch repair,MMR）基因（MLH1、MSH2、MSH6、PMS2）中的一种出现种系突变,或由于EPCAM基因缺失导致MSH2 表达丢失引起。LS是遗传性结直肠癌（colorectal cancer,CRC）最常见的原因,其特征为患CRC和子宫内膜癌（endometrial cancer,EC）的风险显著增加,且存在发生其他几种恶性肿瘤的风险。对于LS 的诊断,目前几种临床病理学标准（如阿姆斯特丹标准等）已被用于识别存在Lynch 综合征风险的个体。然而,这些标准的敏感性及特异性有限,仍有赖于临床医生对LS的警惕并关注其家族史。伴有MMR基因变异的LS相关肿瘤通常具有微卫星高度不稳定的特征,由于移码突变新抗原的存在,可以激发强大而持久的免疫反应和肿瘤浸润淋巴细胞浸润,所以对于LS患者,免疫检查点抑制剂将会是一种很有前景的治疗方法。由于LS是一种基因遗传病,与DNA错配修复缺陷具有独特关系,对其的充分理解对相关肿瘤的诊断、预防和治疗具有重要的临床意义。     

高通量测序分析PIK3CA突变型与野生型结直肠癌的肠道菌群特征

  目的  采用16S rDNA二代高通量测序技术,研究PIK3CA突变型与野生型结直肠癌患者中的肠道菌群特征。  方法  选取2016年12月至2017年12月华中科技大学同济医学院附属协和医院结直肠癌患者19例,采用 16S rDNA高通量测序技术分析PIK3CA突变型（n=4）和PIK3CA野生型（n=15）之间肿瘤组织中微生物多样性和组成差异。  结果  在门水平上,结直肠癌患者的肠道菌群主要由变形菌门、栖热菌门、放线菌门、厚壁菌门和拟杆菌门组成,占总群落的98%以上。Alpha多样性分析显示,PIK3CA突变型与野生型结直肠癌患者之间微生物多样性有显著性差异,且PIK3CA突变型患者中微生物多样性明显高于野生型。Beta多样性分析显示PIK3CA突变型与野生型结直肠癌患者的肿瘤微生物特征有显著性差异。Spearman关联网络分析结果显示,在PIK3CA野生型结直肠癌肿瘤组织中,双歧杆菌属与假单胞菌呈正相关。  结论  PIK3CA突变型与野生型结直肠癌患者肿瘤组织中的微生物群落结构有显著性差异,且PIK3CA突变患者中的肠道微生物多样性更丰富。      

Constitutional MLH1 methylation presenting with colonic polyposis syndrome and not Lynch syndrome

At least one-third of patients meeting clinical criteria for Lynch syndrome will have no germline mutation and constitutional epimutations leading to promoter methylation of MLH1 have been identified in a subset of these patients. We report the first case of constitutional MLH1 promoter methylation associated with a colonic polyposis syndrome in a 39 year-old man with a family history of colorectal cancer (CRC) and a personal history of 21 polyps identified over 8 years as well as the development of two synchronous CRCs over 16 months who was evaluated for a hereditary cancer syndrome. Immunohistochemistry (IHC) of multiple tumors showed absent MLH1 and PMS2 expression, though germline testing with Sanger sequencing and multiplex ligation-dependent probe amplification of these mismatch repair genes (MMR) genes was negative. A next generation sequencing panel of 29 genes also failed to identify a pathogenic mutation. Hypermethylation was identified in MLH1 intron 1 in tumor specimens along with buccal cells and peripheral white blood cells, confirming the diagnosis of constitutional MLH1 promoter methylation. This case highlights that constitutional MLH1 methylation should be considered in the differential diagnosis for a polyposis syndrome if IHC staining shows absent MMR gene expression.     

Screening for Lynch syndrome in young Saudi colorectal cancer patients using microsatellite instability testing and next generation sequencing

Individuals with Lynch syndrome (LS) have germline variants in DNA mismatch repair (MMR) genes that confer a greatly increased risk of colorectal cancer (CRC), often at a young age. Identification of these individuals has been shown to increase their survival through improved surveillance. We previously identified 33 high risk cases for LS in the Saudi population by screening for microsatellite instability (MSI) in the tumor DNA of 284 young CRC patients. The aim of the present study was to identify MMR gene variants in this cohort of patients. Peripheral blood DNA was obtained from 13 individuals who were at high risk of LS due to positive MSI status and young age (<60 years at diagnosis). Next generation sequencing, Sanger sequencing and Multiplex Ligation-dependent Probe Amplification were used to screen for germline variants in the MLH1, MSH2, MSH6 and PMS2 MMR genes. These were cross-referenced against several variant databases, including the International Society for Gastrointestinal Hereditary Tumors Incorporated database. Variants with pathogenic or likely pathogenic significance were identified in 8 of the 13 high risk cases (62%), comprising 4 in MLH1 and 4 in MSH2. All carriers had a positive family history for CRC or endometrial cancer. Next generation sequencing is an effective strategy for identifying young CRC patients who are at high risk of LS because of positive MSI status. We estimate that 7% of CRC patients aged <60 years in Saudi Arabia are due to LS, potentially involving around 50 new cases per year.     

Women with synchronous primary cancers of the endometrium and ovary: do they have Lynch syndrome?

PURPOSE: Lynch syndrome (hereditary nonpolyposis colorectal cancer; HNPCC) is an autosomal-dominant cancer predisposition syndrome that increases risk for multiple cancers, including colon, endometrial, and ovarian cancer. Revised Bethesda Criteria recommend that patients with two HNPCC-associated cancers undergo molecular evaluation to determine whether they have a mismatch repair (MMR) defect associated with HNPCC. The purpose of our study was to determine the likelihood of MMR defects (MSH2, MSH6, MLH1) in women with synchronous endometrial and ovarian cancer. PATIENTS AND METHODS: Between 1989 and 2004, 102 women with synchronous endometrial and ovarian cancers were identified; 59 patients had tumor blocks available for analysis. Patients were divided into risk groups based on family history: high (met Amsterdam criteria), medium (personal history or first-degree relative with an HNPCC-associated cancer), and low (all others). Protein expression for MSH2, MSH6, and MLH1 was evaluated by immunohistochemistry. Microsatellite instability and MLH1 promoter methylation analyses were performed on a subset of cases. RESULTS: Median age was 50 years. Two patients met Amsterdam criteria for HNPCC. Five additional patients, all medium-risk, had molecular findings consistent with a germline mutation of either MSH2 or MLH1. None of the low-risk patients had molecular results consistent with a germline mutation. CONCLUSION: Overall, 7% of women in our cohort met either clinical or molecular criteria for Lynch syndrome. All of these women had a prior history or a first-degree relative with an HNPCC-associated cancer. Limiting genetic evaluation to women with synchronous endometrial and ovarian cancer who have a family history suggestive of HNPCC may appropriately identify women with Lynch syndrome.     

Comprehensive screening for mutations associated with colorectal cancer in unselected cases reveals penetrant and nonpenetrant mutations

Germline mutation testing in patients with colorectal cancer (CRC) is offered only to a subset of patients with a clinical presentation or tumor histology suggestive of familial CRC syndromes, probably underestimating familial CRC predisposition. The aim of our study was to determine whether unbiased screening of newly diagnosed CRC cases with next generation sequencing (NGS) increases the overall detection rate of germline mutations. We analyzed 152 consecutive CRC patients for germline mutations in 18 CRC‐associated genes using NGS. All patients were also evaluated for Bethesda criteria and all tumors were investigated for microsatellite instability, immunohistochemistry for mismatch repair proteins and the BRAF*V600E somatic mutation. NGS based sequencing identified 27 variants in 9 genes in 23 out of 152 patients studied (18%). Three of them were already reported as pathogenic and 12 were class 3 germline variants with an uncertain prediction of pathogenicity. Only 1 of these patients fulfilled Bethesda criteria and had a microsatellite instable tumor and an MLH1 germline mutation. The others would have been missed with current approaches: 2 with a MSH6 premature termination mutation and 12 uncertain, potentially pathogenic class 3 variants in APC, MLH1, MSH2, MSH6, MSH3 and MLH3. The higher NGS mutation detection rate compared with current testing strategies based on clinicopathological criteria is probably due to the large genetic heterogeneity and overlapping clinical presentation of the various CRC syndromes. It can also identify apparently nonpenetrant germline mutations complicating the clinical management of the patients and their families.     

Mutation and association analyses of the candidate genes ESR1, ESR2, MAX, PCNA, and KAT2A in patients with unexplained MSH2-deficient tumors

Lynch syndrome (Hereditary non-polyposis colorectal cancer/HNPCC) is a cancer susceptibility syndrome which is caused by germline mutations in DNA mismatch repair (MMR) genes, in particular MLH1 and MSH2. A pathogenic germline mutation in the respective MMR gene is suggested by the finding of a loss of a mismatch repair protein in tumor tissue on immunohistochemical staining combined with an early age of onset and/or the familial occurrence of colorectal cancer. Pathogenic germline mutations are identifiable in around 60% of patients suspected of Lynch syndrome, depending on the familial occurrence. The aim of the present study was to identify novel susceptibility genes for Lynch syndrome. 64 Healthy controls and 64 Lynch syndrome patients with no pathogenic MSH2 mutation but a loss of MSH2 expression in their tumor tissue were screened for rare and disease causing germline mutations in the functional candidate genes ESR1, ESR2, MAX, PCNA, and KAT2A. Thirty variants were identified, and these were then genotyped in an independent sample of 36 mutation negative Lynch syndrome patients and 234 controls. Since a trend towards association was observed for KAT2A, an additional set of 21 tagging SNPs was analyzed at this locus in a final case-control sample of 142 mutation negative Lynch syndrome patients and 298 controls. The mutation analysis failed to reveal any rare disease-causing mutations. No association was found at the single-marker or haplotypic level for any common disease-modifying variant. The present results suggest that neither rare nor common genetic variants in ESR1, ESR2, MAX, PCNA, or KAT2A contribute to the development of Lynch syndrome.     

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred’s. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.