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 Protein Expression and Microsatellite Instability in Cutaneous Squamous Cell Carcinoma

There exist relatively sparse and conflicting data on high-level microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) in cutaneous malignancies. We aimed to determine the expression profiles of MMR proteins (MSH2, MSH6, MLH1, and PMS2) in different progression stages of cutaneous squamous cell carcinoma (cSCC, 102 patients in total) by immunohistochemistry, and search for MSI-H in patients with low-level MMR or dMMR using multiplex-PCR. Low-level MMR protein expression was observed in five patients: One patient with primary cSCC < 2 mm thickness and low-level MLH1, three patients with primary cSCC > 6 mm (including one with low-level MSH2, as well as MSH6 expression, and two with low-level PMS2), and one patient with a cSCC metastasis showing low-level MSH2 as well as MSH6. Intergroup protein expression analysis revealed that MLH1 and MSH2 expression in actinic keratosis was significantly decreased when compared to Bowen’s disease, cSCC < 2 mm, cSCC > 6 mm, and cSCC metastasis. In cases with low-level MMR, we performed MSI-H tests revealing three cases with MSI-H and one with low-level MSI-L. We found low-level MMR expression in a small subset of patients with invasive or metastatic cSCC. Hence, loss of MMR expression may be associated with tumour progression in a small subgroup of patients with non-melanoma skin cancer.     

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.     

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.     

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。     

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。     

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)27例(12.1%),微卫星稳定(microsatellite stable,MSS)196例(87.9%),无微卫星低度不稳定(MSI-low,MSI-L)。BAT-25、BAT-26、NR-21、NR-24、NR-27、MONO-27共6个位点的灵敏度分别为88.9%、92.6%、96.3%、70.4%、92.6%、77.8%。将MSI定义为3种标记(NR-21、NR-27、BAT-26)至少有1处不稳定时,得出的结果与6种标记组(BAT-25、BAT-26、NR-21、NR-24、NR-27、MONO-27)完全相同。结论:简化的两种检测方案为结直肠癌dMMR/MSI的鉴定提供了更为简便、可靠、价格低廉的方法。     

Evaluation of yield and experiences of age-related molecular investigation for heritable and nonheritable causes of mismatch repair deficient colorectal cancer to identify Lynch syndrome

Universal mismatch repair deficiency (dMMR) testing of colorectal cancer (CRC) is promoted as routine diagnostics to prescreen for Lynch syndrome. We evaluated the yield and experience of age-related molecular investigation for heritable and nonheritable causes of dMMR in CRC below age 70 to identify Lynch Syndrome. In a prospective cohort of 3602 newly diagnosed CRCs below age 70 from 19 hospitals, dMMR, MLH1 promoter hypermethylation, germline MMR gene and somatic MMR gene testing was assessed in daily practice. Yield was evaluated using data from the Dutch Pathology Registry (PALGA) and two regional genetic centers. Experiences of clinicians were evaluated through questionnaires. Participating clinicians were overwhelmingly positive about the clinical workflow. Pathologists routinely applied dMMR-testing in 84% CRCs and determined 10% was dMMR, largely due to somatic MLH1 hypermethylation (66%). Of those, 69% with dMMR CRC below age 70 without hypermethylation were referred for genetic testing, of which 55% was due to Lynch syndrome (hereditary) and 43% to somatic biallelic pathogenic MMR (nonhereditary). The prevalence of Lynch syndrome was 18% in CRC < 40, 1.7% in CRC age 40-64 and 0.7% in CRC age 65-69. Age 65-69 represents most cases with dMMR, in which dMMR due to somatic causes (13%) is 20 times more prevalent than Lynch syndrome. In conclusion, up to age 65 routine diagnostics of (non-)heritable causes of dMMR CRCs effectively identifies Lynch syndrome and reduces Lynch-like diagnoses. Above age 64, the effort to detect one Lynch syndrome patient in dMMR CRC is high and germline testing rarely needed.     

Microsatellite instability and mutation analysis among southern Italian patients with colorectal carcinoma: detection of different alterations accounting for MLH1 and MSH2 inactivation in familial cases.

BACKGROUND: Microsatellite instability (MSI) is due to defective DNA mismatch repair (MMR) and has been detected at various rates in colorectal carcinoma (CRC). The role of MSI in colorectal tumorigenesis was assessed further in this study by both microsatellite analysis of two CRC subsets [unselected patients (n = 215) and patients <50 years of age (n = 95)], and mutation screening of the two major MMR genes MLH1 and MSH2 among familial CRC cases. PATIENTS AND METHODS: PCR-based microsatellite analysis was performed on paraffin-embedded tissues. In CRC families, MLH1/MSH2 mutation analysis and MLH1/MSH2 immunostaining were performed on germline DNA and MSI+ tumour tissues, respectively. RESULTS: The MSI+ phenotype was detected in 75 (24%) patients, with higher incidence in early-onset or proximally located tumours. Among 220 patients investigated for family cancer history, MSI frequency was markedly higher in familial [18/27 (67%)] than in sporadic [32/193 (17%)] cases. Three MLH1 and six MSH2 germline mutations were identified in 14 out of 36 (39%) CRC families. Prevalence of MLH1/MSH2 mutations in CRC families was significantly increased by the presence of: (i) fulfilled Amsterdam criteria; (ii) four or more CRCs; or (iii) one or more endometrial cancer. While MSH2 was found mostly mutated, almost all [8/9 (89%)] familial MSI+ cases with loss of the MLH1 protein were negative for MLH1 germline mutations. CONCLUSIONS: Both genetic (for MSH2) and gene-silencing (for MLH1) alterations seem to be involved in CRC pathogenesis.     

Common variants in mismatch repair genes and risk of invasive ovarian cancer

Mismatch repair (MMR) is important for repairing of nucleotide mismatches during DNA replication. Germline mutations in MMR genes are associated with hereditary non-polyposis colorectal cancer (HNPCC). Ovarian cancer occurs as part of the HNPCC phenotype, and so common variants in MMR genes are candidates for ovarian cancer susceptibility. We performed a large multicentre case-control study to investigate associations of common variations in MMR genes and ovarian cancer using a single nucleotide polymorphism (SNP) tagging approach. A total of 2570 controls and 1531 cases from three separate studies were genotyped for 44 tagging SNPs (stSNP) in seven MMR genes (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2). Genotype frequencies were marginally different between cases and controls for PMS2 rs7797466 (P(2df) = 0.046) with a 1.17-fold (95% CI 1.03-1.33) increase in risk for each 'a' allele carried (P-trend = 0.013). Haplotype analysis of PMS2 also showed significant differences in frequencies between cases and controls (P(7df) = 0.005), with one haplotype accounting for most of the effect. There was also marginal evidence for a recessive protective effect with common homozygote as the baseline comparator for two SNPs--MSH6 rs3136245 (OR 0.67; 95% CI 0.46-0.98) and MSH3 rs6151662 (OR 0.28; 95% CI 0.08-0.91)--but the comparisons of genotype frequencies for these variants were not significant (P = 0.10 and 0.054). In conclusion, it is unlikely that common variants in MLH1, MLH3, PMS1, MSH2, MSH3 and MSH6 contribute significantly to ovarian cancer susceptibility. The observed association of PMS2 rs7797466 with ovarian cancer warrants confirmation in an independent study.     

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.     

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.     

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.     

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.     

Gene-related cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)

The family histories of 130 individuals with documented hereditary non-polyposis colorectal cancer (HNPCC) (caused by mutations in mismatch-repair (MMR) genes MSH2 (n = 64), MLH1 (n = 62) or MSH6 (n = 4)) were obtained, and incidence of cancers in those families was compared to that in the general population. There were a total of 982 cancers in 723 individuals. Colorectal cancer (CRC) was the commonest type (64% and 55% in individuals from families with germline MLH1 and MSH2 mutations respectively). Median age at diagnosis of first CRC in MSH6 mutation families was 59 years compared to 45 years in both MLH1 and MSH2 mutation families. The relative risk (RR) of endometrial cancer was 55 in MSH2 mutation families, compared with 27 in MLH1 mutation families, and 37 in MSH6 mutation families; median age at diagnosis 49 years. Even within MSH2 families, endometrial cancer tended to cluster, with 28 of the 58 cases coming from families with three or more cases (P < 0.001). Absolute risk of endometrial cancer in MLH1 families was still greater than any other cancer (other than CRC). 5% of cancers in both MLH1 and MSH2 mutation families were gastric (RR = 12); 53% of these were diagnosed before 50 years. Seven cases of small intestinal cancer occurred in MSH2 and MLH1 mutation families (RR = 26). There were 13 cases of cancer of the ureter; all were in MSH2 families. These cancers tended to cluster within families (P < 0.001); three of seven families with urothelial cancer had such cases in two or more individuals; two others had kidney cancer. Nineteen of 27 ovarian cancers (70%) were in MSH2 mutation families and 70% of these were diagnosed before age 50 years. There were 9 cases of sebaceous skin cancer, 3 in two MLH1 and 6 in four MSH2 mutation families. Of 22 pancreatic cancers, 14 were known to be diagnosed before 60 years. Breast cancer RR was 1.7 overall. The type of mutation (truncating or other type, and site of mutation) showed no obvious correlation with the presence or absence of extra-colonic cancers in families.     

Comparison of Tissue Mismatch Repair Protein Deficiency between Early- and Advanced-Stage Endometrial Cancer

Objective: ESGO/ESTRO/ESP guidelines recommend that DNA mismatch repair (MMR) proteins or microsatellite instability tests should be performed in all cases of endometrial cancer. This study aims to clarify the relationship of MMR protein deficiency (dMMR) between early and advanced stages of endometrial cancer. Secondary objective is to identify dMMR affecting factors in endometrial cancer. Methods: This cross-sectional study was conducted on endometrial cancer patients who underwent surgery at HRH Princess Maha Chakri Sirindhorn Medical Center, Srinakharinwirot University, between May 2013 and April 2021. Patients with endometrial cancer whose tumor tissue was available for analysis were identified. The expression of MMR proteins was assessed by immunohistochemistry, including MLH1, MSH2, MSH6, and PMS2. Then, the pathological specimens were reviewed. Results: A total of 207 patients with endometrial cancer were assessed for data analysis. MMR deficiency was observed in 92 cases (44.4%). We found patients with dMMR in both the early and advanced stages of endometrial cancer-68/155 cases (43.9%) and 24/52 cases (46.2%), respectively (P = 0.774).  Statistically significant differences were found only in myometrial invasion (adjusted prevalence odds ratio 2.35, 95% CI 1.21 to 4.57, P = 0.012). Conclusion: Our study showed no difference in tissue dMMR between early- and advanced-stage endometrial cancer. The dMMR was not associated with improved outcomes in patients with endometrial cancer. Even though ESGO/ESTRO/ESP guidelines recommend the performance of MMR IHC or MSI tests in all endometrial cancer cases, we can select the appropriate patients those categorized as “advanced stage” or “recurrent”-who may gain the most benefits from the immunotherapy modality of treatment.     

Constitutional mismatch repair deficiency and Lynch syndrome among consecutive Arab Bedouins with colorectal cancer in Israel

We assessed the molecular characteristics and the frequency of mutations in mismatch-repair genes among Bedouin patients with colorectal cancer (CRC) in Israel. Bedouin patients with a diagnosis of CRC at a major hospital in the southern part of Israel were deemed eligible for this study. The primary screening method was immunohistochemical staining for mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2). For subjects with abnormal immunohistochemical staining, we performed microsatellite instability (MSI) analyses, and for tumors with a loss of MLH1 expression we also performed BRAF testing. In MSI high cases we searched further for germline mutations. Of the 24 patients enrolled, four subjects (16.7%) had MSI high tumors: one subject was found to harbor a biallelic PMS2 mutation, one subject had Lynch syndrome (LS) with MSH6 mutation and two subjects had a loss of MLH1/PMS2 proteins/BRAF ^{wild type}/normal MLH1 sequence. Ten patients (41.7%) were younger than 50 at the time of diagnosis and none had first degree relatives with CRC. In conclusion, in this cohort of 24 consecutive Arab Bedouins with CRC, one patient was found to harbor a constitutional mismatch repair deficiency, one patient had LS with MSH6 mutation, and two patients had unresolved loss of MLH1/PMS2 proteins/BRAF ^{wild type} phenotype.     

Identification of Lynch syndrome-associated DNA mismatch repair-deficient bladder cancer in a Japanese hospital-based population

Background

The prevalence of Lynch syndrome (LS)-associated DNA mismatch repair (MMR)-deficient bladder cancer (BC) has scarcely been investigated.

Methods

Immunohistochemistry for four MMR proteins (MLH1, MSH2, MSH6, and PMS2) was performed in formalin-fixed paraffin-embedded (FFPE) sections prepared from the resected specimens of 618 consecutive newly diagnosed BC cases. Genetic/epigenetic analyses were performed in patients displaying the loss of any MMR proteins in the tumor.

Results

Of the 618 patients, 9 (1.5%) showed the loss of MMR protein expression via immunohistochemistry; specifically, 3, 3, 2, and 1 patients displayed the loss of MLH1/PMS2, PMS2, MSH6, and MSH2/MSH6, respectively. All nine patients were male with a median age of 68 years (63–79 years). One had been previously diagnosed as having LS with an MSH2 variant. Genetic testing demonstrated the presence of a pathogenic PMS2 variant (n?=?1), a variant of uncertain significance in MSH2 (n?=?1), and no pathogenic germline variants of the MMR genes (n?=?1). One patient with MSH6-deficient BC did not complete the genetic testing because of severe degradation of DNA extracted from the FFPE specimen, but the patient was strongly suspected to have LS because of their history of colon cancer and MSH6-deficient upper urinary tract cancer. There remained a possibility that the remaining four patients who refused genetic testing had LS.

Conclusions

The prevalence of LS-associated MMR-deficient BC was estimated to be 0.6–1.1% among unselected BC cases.

     

Screening for germline mutations of MLH1, MSH2, MSH6 and PMS2 genes in Slovenian colorectal cancer patients: implications for a population specific detection strategy of Lynch syndrome

Microsatellite instability (MSI) is present in more than 90% of colorectal cancers of patients with Lynch syndrome, and is therefore a feasible marker for the disease. Mutations in MLH1, MSH2, MSH6 and PMS2, which are one of the main causes of deficient mismatch repair and subsequent MSI, have been linked to the disease. In order to establish the role of each of the 4 genes in Slovenian Lynch syndrome patients, we performed MSI analysis on 593 unselected CRC patients and subsequently searched for the presence of point mutations, larger genomic rearrangements and MLH1 promoter hypermethylation in patients with MSI-high tumours. We detected 43 (7.3%) patients with MSI-H tumours, of which 7 patients (1.3%) harboured germline defects: 2 in MLH1, 4 in MSH2, 1 in PMS2 and none in MSH6. Twenty-nine germline sequence variations of unknown significance and 17 deleterious somatic mutations were found. MLH1 promoter methylation was detected in 56% of patients without detected germline defects and in 1 (14%) suspected Lynch syndrome. Due to the minor role of germline MSH6 mutations, we adapted the Lynch syndrome detection strategy for the Slovenian population of CRC patients, whereby germline alterations should be first sought in MLH1 and MSH2 followed by a search for larger genomic rearrangements in these two genes. When no germline mutations are found tumors should be further tested for the presence of germline defects in PMS2 and MSH6. The choice about which gene should be tested first can be guided more accurately by the immunohistochemical analysis. Our study demonstrates that the incidence of MMR mutations in a population should be known prior to the application of one of several suggested strategies for detection of Lynch syndrome.     

Little evidence for involvement of MLH3 in colorectal cancer predisposition

Mutations in the DNA MMR genes MSH2, MLH1, MSH6 and PMS2 underlie a large subset of HNPCC cases, and a hallmark of the tumors is MSI. In many HNPCC families, however, a causative mutation has not been found. Therefore, the involvement of additional, thus far unknown, genes in MSI as well as MSS colorectal tumor predisposition is possible. The role of a relatively recently cloned MMR gene, MLH3, in familial CRC has been studied; but the results appear somewhat conflicting. To further evaluate the role of MLH3 in CRC predisposition, we analyzed 30 Finnish CRC cases for germline mutations by sequencing. These cases were selected from a large series of Finnish CRC patients, to match features previously proposed to associate with MLH3 germline defects. We found 5 missense variants, 4 of which were also found in Finnish cancer-free controls. The only remaining variant does not appear to be an attractive candidate for a disease-associated mutation because the amino acid change is located outside the conserved residues. We also screened for the previously reported variants, including a frameshift change, the most likely pathogenic MLH3 mutation observed so far. The frameshift was not present in the 30 CRC cases or in 700 cancer-free controls. While it is a difficult task to exclude a role of MLH3 in HNPCC, our study could not confirm a role for MLH3 in CRC predisposition.