Low prevalence of biliary tract cancer with defective mismatch repair genes in a Japanese hospital-based population

Recent studies have reported that immune checkpoint inhibitors are effective against various defective mismatch repair (dMMR)/microsatellite instability-high (MSI-H) cancers. A limited number of reports are available on the frequency of dMMR/MSI-H carcinoma in biliary tract cancer (BTC), describing its clinicopathological characteristics and prognosis. The latter carcinoma is also associated with Lynch syndrome (LS). The present study was performed to investigate the frequency of patients with dMMR/MSI-H in BTC and the clinical characteristics of BTC with dMMR/MSI-H in a single institution in Japan. A total of 116 patients with BTC who underwent curative surgical resection at Kagawa University Hospital between January 2008 and December 2017 were included. The protein expression levels of the mismatch repair (MMR) genes [mutL homolog 1 (MLH1), mismatch repair endonuclease PMS2 (PMS2), MutS homolog (MSH)2 and MSH6] were assessed by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissue specimens. Subsequently, MSI testing was performed on patients who exhibited loss of MMR protein expression. Loss of expression of one or more proteins was detected in five cases (4.3%). Loss of MLH1/PMS2 expression was observed in one case of intrahepatic cholangiocarcinoma, whereas loss of PMS2 expression was noted in one case of perihilar cholangiocarcinoma. Loss of MSH2/MSH6 and MSH6 expression was noted in two cases of distal cholangiocarcinoma and loss of PMS2 expression in one case of ampullary carcinoma. Out of the five patients, two demonstrated MSI-H. Microsatellite stability was observed in two cases and for one case, no data were available. Two MSI-H cases were patients with loss of expression of MLH1/PMS2 and MSH2/MSH6. None of the five patients exhibited a past medical history or family history of suspected LS. The frequency of dMMR in BTC was ~5%, which was similar to that reported by similar studies performed in other countries. In the present study, IHC appeared to be more useful than MSI testing for detecting MMR abnormalities with regards to the detection rate. Furthermore, there may only be a limited number of patients with BTCs who are likely to benefit from the therapeutic effects of treatment with immune checkpoint inhibitors.     

DNA错配修复蛋白表达与胃腺癌临床病理特征的关系及意义

目的:检测DNA错配修复（mismatch repair，MMR）主要蛋白（MLH1、MSH2、MSH6和PMS2)在人胃腺癌组织中的表达，并分析错配修复缺陷（defective mismatch repair，dMMR）与胃腺癌临床病理因素及预后的关系。方法:采用免疫组织化学染色法检测4种MMR蛋白（MLH1、MSH2、MSH6和PMS2)在120例人胃腺癌组织中的表达，并从癌症基因组图谱（The Cancer Genome Atlas，TCGA）公共数据库下载432例胃腺癌患者的临床病理资料和微卫星不稳定性（microsatellite instability，MSI）的检测结果，分析MSI与胃腺癌临床病理特征的关系，利用TCGA的数据分析高频度微卫星不稳定（MSI-H）与胃腺癌患者预后的关系。结果:免疫组化染色结果显示在120例胃腺癌组织中，MMR蛋白表达正常（pMMR）组106例（88.3%），MMR蛋白表达缺失（dMMR)组14例（11.7%），其中MLH1缺失2例（1.7%）、PMS2缺失13例（10.8%）、MLH1和PMS2共同缺失2例（1.7%）、MSH2和MSH6共同缺失1例（0.8%）。统计分析结果显示，dMMR与胃腺癌淋巴结转移相关（P=0.022），而与其他临床病理因素无关。TCGA数据统计分析结果显示，MSI-H与胃腺癌患者年龄（P=0.001)、性别（P=0.000)、原发肿瘤部位（P=0.000)、Lauren分型（P=0.011）、肿瘤浸润深度T分期（P=0.024)、淋巴结有无转移（P=0.008)有关。Kaplan-Meier生存分析结果显示MSI-H型胃腺癌患者有预后更好的趋势，但差异不具有统计学意义（P=0.070）。结论:120例中国胃腺癌患者中MSI/dMMR型胃腺癌占比为11.7%，且dMMR状态与胃腺癌的淋巴结转移呈负相关；MSI-H型胃腺癌患者具有年龄大、多为女性、肿瘤多位于胃远端、肿瘤浸润深度T分期低、无淋巴结转移的特征，且MSI-H型胃腺癌具有预后更好的趋势，但不具有统计学意义。MSI状态与胃癌预后的关系尚需进一步深入研究和大样本数据的验证。     

MMR蛋白在515例子宫内膜样腺癌中表达及其与临床病理学特征的相关性分析

背景与目的：林奇综合征（Lynch syndrome,LS）相关的子宫内膜癌有着独特的临床病理学特征及治疗手段。对新发子宫内膜癌患者采用免疫组织化学（immunohistochemistry,IHC）染色的方法检测错配修复（mismatch repair,MMR）蛋白表达情况,可以有效地筛查LS相关的癌症患者。本研究探讨MMR蛋白（MLH1、MSH2、MSH6及PMS2）在子宫内膜样腺癌中的表达情况及其与患者临床病理学特征的关系。方法：收集中国医科大学盛京医院2018年1月—2020年8月共515例子宫内膜样腺癌连续性病例为研究对象,年龄范围为28 ~ 81（57.73±8.41）岁。采用IHC染色的方法检测癌组织中MLH1、MSH2、MSH6和PMS2蛋白表达情况,应用聚合酶链式反应（polymerase chain reaction,PCR）方法对MLH1蛋白表达缺失的标本进行基因的甲基化检测,并且分析MMR蛋白表达缺失情况与子宫内膜样腺癌临床病理学特征的关系。只要有一种MMR蛋白表达缺失即判定为MMR蛋白错配修复缺陷（deficient mismatch repair,dMMR）,蛋白全部阳性则判定为MMR表达完整（proficient mismatch repair,pMMR）。结果：515例子宫内膜样腺癌中有138例（26.8%）发生MMR蛋白表达缺失,MLH1、PMS2、MSH2及MSH6蛋白表达缺失率分别是16.3%（84/515）、19.0%（98/515）、5.4%（28/515）、8.0%（41/515）。MMR蛋白的缺失以MLH1和PMS2联合表达缺失（60.9%,84/138）为主;其次为MSH2和MSH6联合表达缺失（18.8%,26/138）;MSH2、MSH6和PMS2联合表达缺失有2例（1.4%,2/138）;PMS2、MSH2和MSH6蛋白单独表达缺失比例分别为8.0%（11/138）、1.4%（2/138）、10.1%（14/138）。对27例MLH1蛋白表达缺失标本进行甲基化检测,结果显示,阳性率为85.2%（23/27）。515例子宫内膜样腺癌组织中的MMR蛋白表达缺失与患者发病年龄、国际妇产科联合会（The International Federation of Gynecology and Obstetrics,FIGO）分期、组织学分化程度、浸润深度、脉管转移、神经侵犯、淋巴结转移、p53异常表达、肿瘤浸润淋巴细胞（tumor infiltrating lymphocyte,TIL）及肿瘤伴瘤周淋巴细胞有相关性,而与是否累及子宫下段无关。与pMMR的患者相比,dMMR的患者发病年龄更小,FIGO临床分期多为Ⅲ ~ Ⅳ期,组织学分化程度多为低分化,肿瘤多无肌层浸润,肿瘤多出现脉管神经侵犯及淋巴结转移,肿瘤浸润淋巴细胞增多,且肿瘤伴瘤周淋巴细胞更显著,MSH6蛋白缺失患者多无p53异常表达。结论：dMMR的子宫内膜样腺癌患者具有独特的临床病理学特征。应用免疫组织化学染色方法检测MMR蛋白表达情况,并对MLH1表达缺失的标本进行基因甲基化检测,能初步筛查LS患者,对肿瘤患者免疫治疗具有一定指导意义。     

Mismatch repair markers in preoperative and operative endometrial cancer samples; expression concordance and prognostic value

Background The endometrial cancer mismatch repair (MMR) deficient subgroup is defined by loss of MSH6, MSH2, PMS2 or MLH1. We compare MMR status in paired preoperative and operative samples and investigate the prognostic impact of differential MMR protein expression levels.Methods Tumour lesions from 1058 endometrial cancer patients were immunohistochemically stained for MSH6, MSH2, PMS2 and MLH1. MMR protein expression was evaluated as loss or intact to determine MMR status, or by staining index to evaluate the prognostic potential of differential expression. Gene expression data from a local (n = 235) and the TCGA (n = 524) endometrial cancer cohorts was used for validation.Results We identified a substantial agreement in MMR status between paired curettage and hysterectomy samples. Individual high expression of all four MMR markers associated with non-endometrioid subtype, and high MSH6 or MSH2 strongly associated with several aggressive disease characteristics including high tumour grade and FIGO stage, and for MSH6, with lymph node metastasis. In multivariate Cox analysis, MSH6 remained an independent prognostic marker, also within the endometrioid low-grade subgroup (P < 0.001).Conclusion We demonstrate that in addition to determine MMR status, MMR protein expression levels, particularly MSH6, may add prognostic information in endometrial cancer.Subject terms: Endometrial cancer, DNA mismatch repair, Endometrial cancer, Prognostic markers, Gynaecological cancer     

筛查结直肠癌DNA错配修复基因缺失的方法分析

目的:通过对筛查结直肠癌DNA错配修复(mismatch repair,MMR)基因缺失两种最常用的检测方法的分析,寻找更为经济有效的检测策略。方法:分析新疆医科大学第一附属医院2018年9月至2019年9月收治并行手术的结直肠癌患者的肿瘤组织223例,采用免疫组织化学法检测平台检测MLH1、MSH2、PMS2、MSH6的表达缺失情况,PCR-毛细管电泳法检测肿瘤微卫星不稳定(microstatellites instability,MSI)状态。结果:在223例结直肠癌中,27例(12.1%)MMR蛋白表达缺失(MMR deficiency,dMMR),196例(87.9%)MMR蛋白表达完整(MMR proficient,pMMR)。MLH1、MSH2、MSH6和PMS2的缺失率分别为9.0%(20/223)、1.8%(4/223)、2.7%(6/223)和9.4%(21/223)。包含PMS2和MSH6的2种抗体试验筛查dMMR结直肠癌的灵敏度和特异度与4种抗体试验(MLH1、MSH2、PMS2、MSH6)的灵敏度和特异度均相同。微卫星高度不稳定(MSI-high,MSI-H)2...     

Impact of Institutional Universal Microsatellite-Instability (MSI) Reflex Testing on Molecular Profiling Differences Between Younger and Older Patients with Colorectal Cancer

IntroductionDNA mismatch repair deficient (dMMR) or microsatellite instability-high (MSI-H) colorectal cancer (CRC) is found in about 15% of early-stage diseases and 5% of metastatic diseases. We reviewed a large, single-institutional database after implementation of universal reflex dMMR/MSI-H testing in CRC to compare profiles of younger (≤50) and older (>50) patients.Patients and MethodsBetween 2009 and 2017, all patients diagnosed with CRC at the University of Florida underwent reflex somatic tumor testing for dMMR by immunohistochemistry (MLH1, PMS2, MSH2, MSH6), MSI by PCR, and Next-Generation Sequencing. Statistical analysis was conducted with 2-sample comparison tests and logistic regression models.ResultsThere were 375 patients included in the final analysis. Patients were grouped as younger (ages ≤50 years-old; n = 80) or older (>50 years-old; n = 295). Compared to tumors from older patients, tumors from younger patients were less likely to be dMMR/MSI-H (12.5% vs. 21.4%, P = .013) and less likely to have a BRAF mutation (1.5% vs. 16.1%, P = .002). BRAF mutation status was highly associated with MMR status; BRAF-mutated tumors were 29.7 times more likely than BRAF-WT tumors to be dMMR/MSI-H (P = < .001, 95% CI 11.3-78.3).ConclusionsTumors of younger patients were less likely than tumors of older patients to have a dMMR/MSI-H or BRAF mutation. Universal MMR/MSI testing in our dataset identified a relatively large population of older patients with sporadic CRC who were eligible for immunotherapy.     

Relationship between MLH1, PMS2, MSH2 and MSH6 gene-specific alterations and tumor mutational burden in 1057 microsatellite instability-high solid tumors

Microsatellite instability-high (MSI-H) and tumor mutational burden (TMB) are predictive biomarkers for immune-checkpoint inhibitors (ICIs). Still, the relationship between the underlying cause(s) of MSI and TMB in tumors remains poorly defined. We investigated associations of TMB to mismatch repair (MMR) protein expression patterns by immunohistochemistry (IHC) and MMR mutations in a diverse sample of tumors. Hypothesized differences were identified by the protein/gene affected/mutated and the tumor histology/primary site. Overall, 1057 MSI-H tumors were identified from the 32 932 tested. MSI was examined by NGS using 7000+ target microsatellite loci. TMB was calculated using only nonsynonymous missense mutations sequenced with a 592-gene panel; a subset of MSI-H tumors also had MMR IHC performed. Analyses examined TMB by MMR protein heterodimer impacted (loss of MLH1/PMS2 vs. MSH2/MSH6 expression) and gene-specific mutations. The sample was 54.6% female; mean age was 63.5 years. Among IHC tested tumors, loss of co-expression of MLH1/PMS2 was more common (n = 544/705, 77.2%) than loss of MSH2/MSH6 (n = 81/705, 11.5%; P < .0001), and was associated with lower mean TMB (MLH1/PMS2: 25.03 mut/Mb vs MSH2/MSH6 46.83 mut/Mb; P < .0001). TMB also varied by tumor histology: colorectal cancers demonstrating MLH1/PMS2 loss had higher TMBs (33.14 mut/Mb) than endometrial cancers (20.60 mut/Mb) and other tumors (25.59 mut/Mb; P < .0001). MMR gene mutations were detected in 42.0% of tumors; among these, MSH6 mutations were most common (25.7%). MSH6 mutation patterns showed variability by tumor histology and TMB. TMB varies by underlying cause(s) of MSI and tumor histology; this heterogeneity may contribute to differences in response to ICI.     

Association between deficient mismatch repair system and efficacy to irinotecan-containing chemotherapy in metastatic colon cancer

The present study investigated the association between deficient mismatch repair (dMMR) and efficacy outcomes of irinotecan-based first-line chemotherapy in patients with metastatic colorectal cancer (mCRC). Among 297 patients with sporadic mCRC receiving an irinotecan-containing regimen as first-line chemotherapy, 197 with available paraffin-embedded tissues were included in the current analysis. Tumors displaying loss of MMR protein (MLH1 or MSH2) and/or a microsatellite instability-high (MSI-H) genotype by PCR were classified as dMMR. Deficient mismatch repair was found in 23 evaluable tumors, among which eight displayed negativity for MLH1 expression, 11 for MSH2 expression, and four for both. The overall response rate was 47.2% (46.0% in proficient MMR (pMMR) and 56.5% in dMMR), with no significant difference between the two groups (P = 0.569). Median progression-free survival was 8.85 months in patients with dMMR tumors and 6.82 months in patients with pMMR tumors, but this difference did not reach statistical significance (P = 0.089). Median overall survival was not different between the two groups (P = 0.413). Efficacy outcomes of first-line irinotecan-based chemotherapy did not differ significantly between mCRC patients with pMMR and those with dMMR. Our data collectively indicate that MMR status is not effective as a single predictive marker for response to irinotecan-based chemotherapy in mCRC patients.     

Colorectal Carcinoma in Malaysians: DNA Mismatch Repair Pattern in a Multiethnic Population

Background: The interesting preponderance of Chinese with colorectal carcinoma (CRC) amongst the threemajor ethnic groups in Malaysia prompted a study to determine DNA mismatch repair (MMR) status in ourCRC and attempt correlation with patient age, gender and ethnicity as well as location, grade, histological typeand stage of tumour. Histologically re-confirmed CRC, diagnosed between 1st January 2005 and 31st December2007 at the Department of Pathology, University of Malaya Medical Centre, were immunohistochemically stainedwith monoclonal antibodies to MMR proteins, MLH1, MSH2, MSH6 and PMS2 on the Ventana BenchmarkXT autostainer. Of the 142 CRC cases entered into the study, there were 82 males and 60 females (M:F=1.4:1).Ethnically, 81 (57.0%) were Chinese, 32 (22.5%) Malays and 29 (20.4%) Indians. The patient ages rangedbetween 15-87 years (mean=62.4 years) with 21 cases <50-years and 121 ≥50-years of age. 14 (9.9%) CRCshowed deficient MMR (dMMR). Concurrent loss of MLH1 and PMS2 occurred in 10, MSH2 and MSH6 in 2with isolated loss of MSH6 in 1 and PMS2 in 1. dMMR was noted less frequently amongst the Chinese (6.2%)in comparison with their combined Malay and Indian counterparts (14.8%), and was associated with rightsided and poorly differentiated tumours (p<0.05). 3 of the 5 (60.0%) dMMR CRC cases amongst the Chineseand 1 of 9 cases (11.1%) amongst the combined Malay and Indian group were <50-years of age. No significantassociation of dMMR was noted with patient age and gender, tumour stage or mucinous type.     

Isolated loss of PMS2 expression in colorectal cancers: frequency, patient age, and familial aggregation.

PURPOSE: Most colorectal cancers that have high levels of microsatellite instability (MSI-H) show loss of immunohistochemical expression of proteins that participate in the DNA mismatch repair process, most often involving MLH1 and MSH2. Less commonly, a third DNA mismatch repair protein, MSH6, may also be lost as the primary event. Rarely, tumors with MSI-H show normal expression of these three proteins. The genetic deficiency leading to the MSI-H phenotype in such cases is unknown. PMS2 is another member of the DNA mismatch repair complex. Its expression is generally lost in tumors with MLH1 loss of expression. Rarely, there is selective loss of PMS2 expression. We sought to describe the frequency and clinical correlates of selective loss of expression of PMS2 with the MSI-H tumor phenotype. EXPERIMENTAL DESIGN: Two thousand seven hundred nineteen colorectal cancers from both clinic- and research-based ascertainment were studied. Tumor MSI testing and immunohistochemistry for MLH1, MSH2, MSH6, and PMS2 were conducted. Medical records were abstracted for age at diagnosis, gender, colorectal cancer site, and family history. RESULTS: Five hundred thirty-five of the 2,719 tumors were MSI-H. Of these, 93% showed loss of expression of MLH1, MSH2, and/or MSH6. Thirty-eight showed normal expression for these proteins. PMS2 immunohistochemical staining was successful in 32 of 38 of these tumors. Of the 32, 23 showed selective loss of expression of PMS2. This was associated with young age of diagnosis and right-sided location but not with a striking family history of cancer. CONCLUSIONS: Overall, 97% of the MSI-H tumors showed loss of expression for one or more of these four mismatch repair proteins. Selective loss of expression of PMS2 was present in 72% of cases in which colorectal cancers had an MSI-H phenotype but no alteration of expression of MLH1, MSH2, and MSH6. The underlying mechanism involved cannot be determined from this study but could involve point mutations in other DNA mismatch repair genes with retention of immunohistochemical expression, somatic inactivation of PMS2, or germ line mutation of PMS2.     

Mismatch repair deficiency concordance between primary colorectal cancer and corresponding metastasis

Universal screening for mismatch repair deficiency (dMMR) in cancer is increasingly being implemented to detect Lynch syndrome and aid in treatment decisions. The mismatch repair (MMR) immunohistochemistry (IHC) concordance rate between primary colorectal cancer (CRC) and metastasis is unknown. At times, only metastatic tumor is available for screening (lymph node, liver, lung etc.) rather than the primary tumor. Therefore, it is important to confirm that tissue from metastases can be used for screening for dMMR. We tested dMMR primary and metastatic tumor to assess concordance between the two. We identified dMMR CRC resected at Ohio State University from 1999 to 2013 and stained a corresponding metastasis for all four MMR proteins (MLH1, MSH2, MSH6, PMS2) with IHC. A total of 50 primary CRC with dMMR and available regional lymph nodes (LN; 26 cases) or other metastatic tissue (24 cases) were identified. Thirteen cases were explained by MLH1 hypermethylation and 10 cases had Lynch syndrome. Two cases had somatic MMR mutations and the etiology for dMMR was unknown in 25 cases. All cases showed concordance in IHC staining between the primary tumor and corresponding metastatic tissue. In 36 cases, metastatic LN/other site was resected at the same time as the primary tumor. In 14 cases, time lapsed [median 16.5 months; quartile (Q)1 8.0; Q3 25; range 3–69] from the primary resection until metastatic resection. Metastatic tissue can be used to screen for Lynch syndrome and dMMR.     

The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer

Identification and characterization of the genetic background in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is important since control programmes can in a cost-effective manner prevent cancer development in high-risk individuals. HNPCC is caused by germline mismatch repair (MMR) gene mutations and the genetic analysis of HNPCC therefore includes assessment of microsatellite instability (MSI) and immunohistochemical MMR protein expression in the tumor tissue. MSI is found in >95% of the HNPCC-associated tumors and immunostaining using antibodies against the MMR proteins MLH1, MSH2, and MSH6 has been found to correctly pinpoint the affected gene in about 90% of the cases. The PMS2 antibody was the most recently developed and we have in a clinical material assessed the added value of PMS2 immunostaining in 213 patients with suspected hereditary colorectal cancer. All 119 MSS tumors showed retained expression for all four antibodies and PMS2 did thus not identify any underlying MMR defect in these cases. However, PMS2 immunostaining contributed to the characterization of the MMR defect in a subset of the MSI tumors. Concomitant loss of MLH1 and PMS2, which functionally interact in the MutLα complex, was found in 98% of the tumors from patients with germline MLH1 mutations. Among the 12 MSI-high tumors with retained expression of MLH1, MSH2 and MSH6, 8 tumors showed loss of PMS2 staining, and mutations in MLH1 were identified in 2 and mutations in PMS2 in 3 of these individuals. In summary, isolated loss of PMS2 was found in 8% of the MSI-high tumors in our series, including 8/12 previously unexplained MSI-high tumors, in which mutations either in MLH1 or in PMS2 were identified in five cases.     

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.     

甲状腺滤泡癌组织中PD-L1与dMMR相关蛋白表达的关系及其临床意义

目的:探讨甲状腺滤泡癌组织中PD-L1与dMMR相关蛋白表达的关系及其临床意义.方法:收集2015年1月至2020年6月福建医科大学附属第二医院60例甲状腺滤泡癌患者的癌组织蜡块,再次经H-E染色确诊,采用免疫组化法检测癌组织和癌旁组织中PD-Ll的表达以及MMR系统4个基因(MLH1、MSH2、MSH6、PMS2)编...     

The role of MLH1, MSH2 and MSH6 in the development of multiple colorectal cancers

There is increased incidence of microsatellite instability (MSI) in patients who develop multiple primary colorectal cancers (CRC), although the association with hereditary nonpolyposis colon cancer (HNPCC) is unclear. This study aims to evaluate the underlying genetic cause of MSI in these patients. Microsatellite instability was investigated in 111 paraffin-embedded CRCs obtained from 78 patients with metachronous and synchronous cancers, and a control group consisting of 74 cancers from patients with a single CRC. Tumours were classified as high level (MSI-H), low level (MSI-L) or stable (MSS). MLH1, MSH2 and MSH6 gene expression was measured by immunohistochemistry. Methylation of the MLH1 promoter region was evaluated in MSI-H cancers that failed to express MLH1, and mutational analysis performed in MSI-H samples that expressed MLH1, MSH2 and MSH6 proteins. The frequency of MSI-H was significantly greater in the multiple, 58 out of 111 (52%), compared to the single cancers, 10 out of 74 (13.5%), P < 0.01. Of the 32 patients from whom two or more cancers were analysed, eight (25%) demonstrated MSI-H in both cancers, 13 (41%) demonstrated MSI-H in one cancer and 11 (34%) failed to demonstrate any MSI-H. MSI-H single cancers failed to express MLH1 or MSH2 in seven out of nine (78%) cases and MSI-L/MSS cancers failed to express MLH1 or MSH2 in one out of 45 (2.2%) cases, all cancers expressed MSH6. MSI-H multiple cancers failed to express MLH1 or MSH2 in 21 out of 43 (48%) cases and MSI-L/MSS cancers failed to express MLH1 or MSH2 in four out of 32 (12.5%) cases. MSH6 expression was lost in five MSI-H multiple cancers, four of which also failed to express MLH1 or MSH2. Loss of expression of the same mismatch repair (MMR) gene was identified in both cancers from six out of 19 (31%) patients. Methylation was identified in 11 out of 17 (65%) multiple and three out of six (50%) single MSI-H cancers that failed to express MLH1. Mutational analysis of 10 MSI-H multiple cancers that expressed MLH1, MSH2 and MSH6 failed to demonstrate mutations in the MLH1 or MSH2 genes. We suggest that, although MSI-H is more commonly identified in those with multiple colorectal cancers, this does not commonly arise from a classical HNPCC pathway.     

肠癌微卫星状态检测方法的比较

目的:探索PCR法与IHC法对微卫星状态判断的差异性与一致性。方法:收集南京鼓楼医院病理科2014年6月至2015年12月病理诊断明确的大肠癌手术切除标本485例,所有标本的微卫星状态通过检测癌组织及正常组织中BAT25、BAT26、D5S346、D2S123及D17S250等5个标准位点的突变状态确定,并同时采用IHC法检测标本癌组织中MLH1、MSH2、MSH6和PMS2等DNA错配修复基因蛋白的表达,对比两种检测结果之间的一致性。结果:PCR法共检测到MSI状态39例,包括30例MSI-H和9例MSI-L,MSS状态446例,MSI占所有病例的8.04%,其中MSI-H的比率为6.19%。485例患者的免疫组化检测结果显示蛋白缺失（dMMR）者61例,蛋白不缺失（pMMR）者424例,dMMR占所有病例的12.58%。IHC法检测的灵敏度和特异度分别为86.67%和92.31%,两种方法检测微卫星状态的符合率为91.96%。结论:IHC法和PCR法检测微卫星状态具有较高的一致性,IHC法经济、便捷,更便于临床推广,当IHC法检测4种修复蛋白均无缺失时,则可以诊断为MSS/MSI-L,无需进一步行PCR,而当发现有任一修复蛋白有缺失时,则需行PCR检测进一步判定是否存在微卫星不稳定状态。我们的这一结论,将为临床工作节省大量的时间与成本。     

Microsatellite instability and DNA mismatch repair protein deficiency in Lynch syndrome colorectal polyps

Colorectal cancers associated with Lynch syndrome are characterized by deficient DNA mismatch repair (MMR) function. Our aim was to evaluate the prevalence of microsatellite instability (MSI) and loss of MMR protein expression in Lynch syndrome-associated polyps. Sixty-two colorectal polyps--37 adenomatous polyps, 23 hyperplastic polyps, and 2 sessile serrated polyps (SSP)--from 34 subjects with germline MMR gene mutations were tested for MSI using a single pentaplex PCR for five mononucleotide repeat microsatellite markers, and also for expression of MLH1, MSH2, MSH6, and PMS2 proteins by immunohistochemistry. High-level MSI (MSI-H) was seen in 15 of 37 (41%) adenomatous polyps, one of 23 (4%) hyperplastic polyps, and one of two (50%) SSPs. Loss of MMR protein expression was seen in 18 of 36 (50%) adenomatous polyps, zero of 21 hyperplastic polyps, and zero of two SSPs. Adenomatous polyps 8 mm or larger in size were significantly more likely to show MSI-H [OR, 9.98; 95% confidence interval (CI), 1.52-65.65; P = 0.02] and deficient MMR protein expression (OR, 3.17; 95% CI, 1.20-8.37; P = 0.02) compared with those less than 8 mm in size. All (six of six) adenomatous polyps 10 mm or larger in size showed both MSI-H and loss of MMR protein expression by immunohistochemistry. Our finding that the prevalence of MMR deficiency increases with the size of adenomatous polyps suggests that loss of MMR function is a late event in Lynch syndrome-associated colorectal neoplasia. Although testing large adenomatous polyps may be of value in the diagnostic evaluation of patients with suspected Lynch syndrome, the absence of an MMR-deficient phenotype in an adenoma cannot be considered as a strong evidence against Lynch syndrome, as it is with colorectal carcinomas.     

Genome-wide copy neutral LOH is infrequent in familial and sporadic microsatellite unstable carcinomas

Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.     

Immunohistochemical expression pattern of MMR protein can specifically identify patients with colorectal cancer microsatellite instability

The microsatellite instability (MSI) pathway is found in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in 12 % of sporadic colorectal cancer (CRC). It involves inactivation of deoxyribonucleic acid mismatch repair (MMR) genes MLH1, MSH2, PMS2, and MSH6. MMR germline mutation detections are an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC. In this study, we analyzed for the first time in Tunisia the potential value of immunohistochemical assessment of MMR protein to identify microsatellite instability in CRC. We evaluate by immunohistochemistry MMR protein expression loss in tumoral tissue compared to positive expression in normal mucosa. Immunohistochemistry revealed loss of expression for MLH1, MSH2, MSH6, and PMS2 in 15, 21, 13, and 15 % of cases, respectively. Here, we report a more elevated frequency of MSI compared to data of the literature. In fact, by immunohistochemistry, 70 % of cases were shown to be MSS phenotype, whereas 30 % of cases, in our set, were instable. Moreover, according to molecular investigation, 71 % of cases were instable (MSI-H) and remaining cases were stable (29 %). Thus, we found a perfect association between MMR immunohistochemical analyses and MSI molecular investigation. Immunohistochemical analysis of MMR gene product expression may allow one to specifically identify MSI phenotype of patients with colorectal carcinomas.     

结直肠癌组织MSI状态及其与临床病理参数之间的相关性

目的:分析结直肠癌组织微卫星不稳定（MSI）状态及其与临床病理参数之间的相关性。方法:利用免疫组化法检测MLH1、MSH2、MSH6、PMS2错配修复蛋白的表达,分析441例结直肠癌组织的MSI状态。结果:免疫组化检测发现,441例结直肠癌中,微卫星稳定（MSS）为375例,MLH1、MSH2、MSH6、PMS2错配修复蛋白任一表达缺失共66例,占14.97%（66/441）;其中MLH1、MSH2、MSH6、PMS2单一表达缺失率分别为1.4%（6/441）、0.2%（1/441）、0.7%（3/441）、2.3%（10/441）;MLH1和PMS2同时表达缺失率9.1%（40/441）,MSH2和MSH6同时表达缺失率1.1%（5/441）,MSH6和PMS2同时表达缺失率0.2%（1/441）。结直肠癌患者MSI与MSS在民族、肿瘤部位、分化程度、T分期、N分期、肿瘤大小等临床病理特征方面存在差异,而在性别、年龄、大体类型、病理类型、M分期、临床分期、神经和脉管侵犯方面均无明显差异。结论:新疆少数民族、右半结肠、低分化、T4、N0、肿瘤＞5 cm的结直肠癌患者更易发生MSI。