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.     

微卫星不稳定性在结直肠癌中的意义

 微卫星不稳定性（MSI）是结直肠癌发生发展的重要分子机制,对结直肠癌具有重要临床意义。MSI是林奇综合征的诊断标志物。同时由于MSI阳性的结直肠癌比阴性者表现出更好的预后,且对5-氟尿嘧啶类化疗药物不敏感,MSI同样成为结直肠癌的预后和预测化疗疗效的标志物。     

微卫星不稳定状态对Ⅳ期结直肠癌患者化疗反应性和预后的影响

背景与目的：错配修复缺陷导致的微卫星不稳(microsatellite inistability,MSI)状态对Ⅱ、Ⅲ期结直肠癌患者的预后及化疗敏感性有重要作用,但在晚期肠癌中研究较少。该研究探讨微卫星不稳对晚期结直肠癌患者化疗敏感性及预后的影响。方法：收集采用XELOX或FOLFOX为一线化疗方案的Ⅳ期肠癌患者的原发肿瘤组织,免疫组化方法检测肿瘤组织中错配修复基因hMLH1、hMSH2、hMSH6和hPMS2的蛋白表达,分析微卫星状态与患者临床特征、预后及化疗反应的相关性。结果：共收集113例晚期结直肠癌患者,未发现MSI与患者总体生存时间(overall survival,OS)以及化疗敏感性存在相关。亚组分析中我们发现79例原发灶姑息性切除的患者中,MSI 患者(22例)较MSS患者(57例)的中位无疾病进展时间(progression-free survival,PFS)明显延长(19.9个月vs 7个月,P=0.005),但MSI与OS无关(P=0.07)。对该79例患者预后行Cox多因素分析显示,MSI是影响患者PFS的独立危险因素(P=0.043,MSS/MSI,HR=2.079)。此外,该群患者的MSI状态与疾病控制率(59.1%vs 31.6%,P=0.025)相关。结论：在原发灶姑息性切除的Ⅳ期结直肠癌患者中,微卫星不稳定状态与无疾病进展时间和化疗的疾病控制率呈正相关,因而有必要对该群患者进行微卫星检测。     

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.     

结直肠癌的微卫星不稳定与染色体不稳定

结直肠癌中存在微卫星不稳定（MSI）和染色体不稳定（CIN）。微卫星不稳定指微卫星序列中重复单位的获得或丢失，多由于DNA错配修复系统改变所致。染色体不稳定包括整条染色体的获得或缺失，染色体易位、重排等，结直肠癌中常见有1p和8p的删除、17p和18q的杂合性缺失以及20q的扩增。两种类型的结直肠癌具有不同的临床病理特征，在基因表达、疗效和预后等方面的差异性有待进一步研究。     

大肠癌微卫星不稳定性及其临床意义

目的 了解大肠癌微卫星不稳定性（ＭＩ）情况。方法 用６个微卫星位点检测ＰＣＲ扩增所选位点ＰＣＲ产物用８％变性聚丙烯酰胺凝胶电泳、银染。结果 ６０例大肠癌中,２０例表现ＭＩ,其中１１例为复制错误（ＲＥＲ）阳性。ＲＥＲ阳性与ＲＥＲ阴性大肠癌患者相比,发病年龄较年轻,一级亲属有恶性肿瘤病史者明显高于ＲＥＲ阴性,倾向位于结肠,呈浸润性生长,Ⅲ￣Ⅳ期比例高。５例大肠癌伴大肠腺瘤病例中,４例腺瘤有ＭＩ。结论     

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.     

大肠癌细胞周期调控因子表达与微卫星不稳定性的关系

目的　探讨大肠癌组织中细胞周期调控因子表达与微卫星不稳定性 (MSI)的关系。方法　采用免疫组化、PCR SSLP法对 5 6例大肠癌分别进行细胞周期调控因子CyclinD1、P16、P2 1WAF1/CIP1、P5 3、Rb以及PCNA的观测和D18S34、D17S799、D5S40 9、TCF 2、P5 3和Rb 6个碱基序列位点MSI检测。结果　大肠癌MSI总阳性率 44 .6 % ,其中 1个位点阳性 7例 ,≥ 2个位点阳性 18例 ,占32 .1%。MSI阳性 (MSI )大肠癌CyclinD1、P5 3蛋白阳性表达率显著低于MSI阴性 (MSI-)大肠癌 ,而P2 1蛋白阳性表达率显著高于MSI-肿瘤组 (P <0 .0 5 ) ,MSI 大肠癌P16阳性率也较MSI-组略增高 ,MSI 和MSI-大肠癌Rb蛋白阳性表达率较接近。结论　P2 1蛋白过度表达对大肠癌MSI的发生可能具有较重要的促进作用 ,CyclinD1、P5 3蛋白表达减少或P16蛋白过度表达与大肠癌MSI的发生可能也有关。     

Evidence of tumor microsatellite instability in gastric cancer with familial aggregation

About 90% of gastric cancer (GC) cases appear in a sporadic setting. Nonetheless, in high incidence areas high familial aggregation rates have been recently described. Microsatellite instability (MSI) is thought to be an important molecular phenotype both in sporadic GC and in tumors of the HNPCC spectrum. The aim of this study was to assess the frequency of MSI in GC with familial aggregation. Five quasimonomorphic mononucleotide repeats (BAT-26, BAT-25, NR-24, NR-21 and NR-27) were analyzed in 250 GC patients. Seventy-five patients (30%) had at least one-first-degree family member affected by GC and 63 patients (25.2%) showed MSI. The frequency of MSI was significantly higher in patients with a positive family history of GC (38.7%) compared to patients with other tumor types within the family (15.7%) or with a negative oncological familial history (21.9%, P = 0.004). Within cases with a positive familial oncological history, the MSI frequency in families with GC only was similar to the one observed in families with GC and colon cancer (P = 0.96). Nonetheless, in families with GC and lung cancer, the frequency of MSI was significantly lower (5.6%, P = 0.007). MSI occurs in GCs with familial aggregation. Similar MSI rates have been observed in GC patients with other family members affected by GC or colon cancer. The same does not occur in families with other members affected by lung cancer. Our data seem to suggest that familial aggregation for either GC alone or gastric and colon cancer share common etiological factors in contrast to families with gastric and lung cancers. C. Pedrazzani and G. Corso are contributed equally.     

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.     

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1, resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation’s effect upon the DNA MMR system.     

Cytotoxic CD8+ T cells and tissue resident memory cells in colorectal cancer based on microsatellite instability and BRAF status

BACKGROUNDRecent studies in non-colorectal malignancy have associated T resident memory (T_RM) cells with improved patient survival. It is unknown if T_RM plays a role in colorectal cancer (CRC).AIMTo examine the potential role of T_RM cells in providing immunogenicity in CRC stratified by microsatellite instability (MSI) and BRAF status.METHODSPatients with known MSI and BRAF mutation status were eligible for inclusion in this study. CRC tumour sections stained with haematoxylin and eosin were microscopically reviewed and the images scanned prior to assessment for location of invading edge and core of tumour. Sequential sections were prepared for quantitative multiplex immunohistochemistry (IHC) staining. Opal Multiplex IHC staining was performed with appropriate positive and negative controls and imaged using a standard fluorescent microscope fitted with a spectral scanning camera (Mantra) in conjunction with Mantra snap software. Images were unmixed and annotated in inForm 2.2.0. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15.RESULTSSeventy-two patients with known MSI and BRAF status were included in the study. All patients were assessed for MSI by IHC and high resolution capillary electrophoresis testing and 44 of these patients successfully underwent quantitative multiplex IHC staining. Overall, there was a statistically significant increase in CD8+ T_RM cells in the MSI (BRAF mutant and wild type) group over the microsatellite stable (MSS) group. There was a statistically significant difference in CD8+ T_RM between high level MSI (MSI-H):BRAF mutant [22.57, 95% confidence interval (CI): 14.31-30.84] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0076 andMSI-H:BRAF wild type [16.18 (95%CI: 10.44-21.93)] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0279. There was no statistically significant difference in CD8 T cells (both CD8+CD103- and CD8+CD103+T_RM) between MSI-H: BRAF mutant and wild type CRC.CONCLUSIONThis study has shown that CD8+ T_RM are found in greater abundance in MSI-H CRC, both BRAF mutant and MSI-H:BRAF wild type, when compared with their MSS counterpart. CD8+ T_RM may play a role in the immunogenicity in MSI-H CRC (BRAF mutant and BRAF wild type). Further studies should focus on the potential immunogenic qualities of T_RM cells and investigate potential immunotherapeutic approaches to improve treatment and survival associated with CRC.     

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。     

Mitochondrial microsatellite instability of colorectal cancer stroma

Mitochondrial microsatellite instability (mtMSI) and mutations of mitochondrial DNA has been reported in cancer epithelia of carcinomas. However, mtMSI in cancer stroma has not yet been identified in human cancers. In this study, we attempted to determine if mtMSI occurs in the cancer stroma of sporadic colorectal cancers, and if the stromal mtMSI has any correlations with stromal nuclear MSI (nMSI) and cancer epithelial mtMSI. Nine microsatellite sequences within the D-loop and 5 coding genes for mtMSI, and 9 microsatellites for nMSI were analyzed in the microdissected cancer epithelia and adjacent stromas of 48 sporadic colorectal cancers. Overall, 23 somatic mitochondrial DNA alterations were detected in 15 cancer epithelia (31.2%) and 5 stromas (10.4%). The mutations consisted of 19 D-loop mtMSI alterations, and 1 missense and 3 framshift mutations of repeat sequences within the coding genes. All of the 5 stromal genetic alterations showed D-loop mtMSI. In regards to other MSI status, the stromal mtMSI had no association with stromal nMSI or epithelial mtMSI, either. These findings indicate that in addition to the cancer epithelia the cancer stroma harbor mtMSI, and suggest a possible role of stromal mtMSI in the pathogenesis of colorectal cancers. Furthermore, the data suggest that stromal mtMSI may occur independently of stromal nMSI and epithelial mtMSI in sporadic colorectal cancers.     

Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency

BackgroundPatients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs.Patients and methodsMSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation.ResultsAmong 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44).ConclusionsLS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.     

Genetic predisposition to colorectal cancer: Implications for treatment and prevention

Colorectal cancer (CRC) is the third most common cancer diagnosed in men and women and approximately 5% of cases are associated with identifiable germline mutations associated with hereditary cancer syndromes. Lifetime risks for CRC can approach 50%–80% for mutation carriers in the absence of endoscopic and/or surgical intervention, and early identification of at-risk individuals can guide clinical interventions for cancer prevention and treatment. Personal and family history and molecular phenotype of CRC tumors are used in determining which patients should be referred for clinical genetic evaluation. Outcomes of genetic testing performed using next-generation sequencing (NGS) multigene panels suggest there can be significant overlap in clinical features among the various hereditary cancer syndromes. This review summarizes new developments in diagnosis and management of patients with genetic predisposition to CRC.     

大肠重复癌微卫星不稳定性研究

目的　探讨微卫星不稳定性 (MSI)在大肠重复癌与单发大肠癌中的变化规律。方法　PCR SSLP法对 38例大肠重复癌患者的 5 1例癌灶及对照 35例单发大肠癌分别进行 5个碱基序列位点MSI检测。结果　大肠重复癌中复制错误 (RER)阳性率为 5 2 .9% (2 7/5 1) ,对照组为 17.1% (6 /35 ) ,两组差异有非常显著意义 (χ2 =11.2 5 ,P <0 .0 1)。大肠重复癌组中 ,RER阳性与低分化、近端大肠密切关联。RER阳性组与阴性组在年龄、性别、是否伴有转移、Dukes分期上未见异常。结论　MSI在大肠重复癌的发生上起着重要作用 ,MSI可作为预测大肠重复癌发生的有用标志 ,对MSI大肠癌患者应警惕多重癌发生的可能性。     

An overview of colorectal cancer survival rates and prognosis in Asia

Colorectal cancer is a rapidly rising trend in Asia.The incidence in many Asian countries is on par with the West.Several studies have provided data regarding the survival of patients with colorectal cancer.In Asia,the overall cure rate of colorectal cancer has not improved dramatically in the last decade,5-year survival remaining at approximately 60%.Colorectal cancer survival time has increased in recent years,but mortality rate remains high.Although studies have determined a number of factors that can predict survival of patients after diagnosis,life expectancy has not been increased dramatically.It seems that among the prognostic factors explored so far,the most important are those that relate to early diagnosis of cancer.Primary detection is feasible since efficient screening modalities are available.Colonoscopic surveillance is needed,especially in subjects at higher risk.