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.     

Tumours with loss of MSH6 expression are MSI-H when screened with a pentaplex of five mononucleotide repeats

Background:

Microsatellite instability (MSI) is commonly screened using a panel of two mononucleotide and three dinucleotide repeats as recommended by a consensus meeting on MSI tumours held at the National Cancer Institute (Bethesda, MD, USA). According to these recommendations, tumours are classified as MSI-H when at least two of the five microsatellite markers show instability, MSI-L when only one marker shows instability and MSS when none of the markers show instability. Almost all MSI-H tumours are characterised by alterations in one of the four major proteins of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 or PMS2) that renders them MMR deficient, whereas MSI-L and MSS tumours are generally MMR proficient. However, tumours from patients with a pathogenic germline mutation in MSH6 can sometimes present an MSI-L phenotype with the NCI panel. The MSH6 protein is not involved in the repair of mismatches of two nucleotides in length and consequently the three dinucleotide repeats of the NCI panel often show stability in MSH6-deficient tumours.

Methods:

A pentaplex panel comprising five mononucleotide repeats has been recommended as an alternative to the NCI panel to determine tumour MSI status. Several studies have confirmed the sensitivity, specificity and ease of use of the pentaplex panel; however, its sensitivity for the detection of MSH6-deficient tumours is so far unknown. Here, we used the pentaplex panel to evaluate MSI status in 29 tumours known to harbour an MSH6 defect.

Results:

MSI-H status was confirmed in 15 out of 15 (100%) cases where matching normal DNA was available and in 28 out of 29 (97%) cases where matching DNA was not available or was not analysed.

Conclusion:

These results show that the pentaplex assay efficiently discriminates the MSI status of tumours with an MSH6 defect.     

Distinct patterns of microsatellite instability are seen in tumours of the urinary tract

To date, two forms of microsatellite instability (MSI) have been described in human cancer. MSI typical of hereditary nonpolyposis colon cancer (HNPCC), is due to deficient DNA mismatch repair (MMR) and is defined with mono- and dinucleotide repeat microsatellites. A second variety of instability is best seen at selective tetranucleotide repeats (EMAST; elevated microsatellite alterations at select tetranucleotides). While MSI occurs infrequently in bladder cancers, EMAST is common. Sporadic tumours with the largest proportion showing MSI are those found most frequently in HNPCC kindreds. While bladder cancer is not frequently seen in HNPCC, upper urinary tract tumours (UTTs) are. Having previously found a low frequency of MSI in bladder cancer, we sought to determine the relative levels of MSI and EMAST in transitional cell carcinoma (TCC) of the upper and lower urinary tracts. Microsatellite analysis was performed at 10 mono- and dinucleotide and eight tetranucleotide loci, in 89 bladder and 71 UTT TCC. Contrasting patterns of instability were seen in urinary tumours. In bladder cancer, MSI was rare and EMAST was common. The presence of EMAST was not related to tumour grade, stage, subsequent outcome or immunohistochemical expression of the MMR proteins. In UTT, while MSI occurred frequently, EMAST was seen less frequently than in bladder cancer. When TCC of the upper and lower urinary tracts are compared, MSI-H is more frequent in UTT and EMAST more frequent in bladder cancer. Our findings show that, as for colorectal cancer, the pattern of MSI varies with location in the urinary tract. In addition, we have confirmed that MSI and EMAST are discrete forms of MSI, and that the presence of EMAST does not affect tumour phenotype.     

Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumors.

PURPOSE: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients. EXPERIMENTAL DESIGN: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6. RESULTS: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors. CONCLUSIONS: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.     

Different types of microsatellite instability in ovarian carcinoma

Microsatellite instability at mono- and dinucleotide repeats is the hallmark of the hereditary non-polyposis cancer syndrome (HNPCC) and is related to deficient DNA mismatch repair. In contrast, a distinct form of microsatellite instability at selective tetranucleotide repeats (EMAST or elevated microsatellite alterations at selected tetranucleotides) was described in several non-HNPCC cancer types. EMAST is probably unrelated to mismatch repair defects. We investigated the frequency of microsatellite instability at mononucleotide, dinucleotide and tetranucleotide repeats in a series of 75 ovarian carcinomas (53 serous and 22 non-serous). Microsatellite analysis was carried out using 5 mono- and dinucleotide markers from the National Cancer Institute Consensus Panel and 6 tetranucleotide markers, which have been reported as frequently unstable in the literature. High frequency of microsatellite instability (MSI-H) at mono- and dinucleotide repeats was observed in 9% and a low frequency (MSI-L) in 21% of serous carcinomas. MSI-H was detected in 4% and MSI-L in 18% of non-serous carcinomas. Nine percent of serous carcinomas showed instability at multiple and 9% at single tetranucleotide loci. All non-serous carcinomas were stable at tetranucleotide loci. In summary, EMAST (e.g., tumors with tetranucleotide instability without concomitant MSI-H) was observed in 13% of ovarian serous carcinomas. All EMAST positive tumors were of advanced stage. We conclude that EMAST occurs as a distinct form of microsatellite instability in ovarian cancer. EMAST seems to be particularly frequent in advanced serous carcinomas. Its clinical significance needs to be investigated.     

HMSH6 alterations in patients with microsatellite instability-low colorectal cancer

Two microsatellite instability (MSI) phenotypes have been described in colorectal cancer (CRC): MSI-H (instability at >30% of the loci examined) and MSI-L (MSI at 1-30% of the loci examined). The MSI-H phenotype, observed in both hereditary nonpolyposis colon cancer-associated CRC and approximately 15% of sporadic CRC, generally results from mutational or epigenetic inactivation of the DNA mismatch repair (MMR) genes hMSH2 or hMLH1. The genetic basis for the MSI-L phenotype, however, is unknown. Several other proteins, including hMSH3 and hMSH6, also participate in DNA MMR. Inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair single-base mispairs and small insertion-deletion loops but not large insertion-deletion loops. This suggests that hMSH6 mutations are more likely to be associated with a MSI-L phenotype than a MSI-H phenotype in CRC. To explore this possibility, we screened tumors from 41 patients with MSI-L CRC for hMSH6 mutations with conformation-sensitive gel electrophoresis (CSGE) and for hMSH6 protein expression by immunohistochemistry. Alterations found with CSGE were confirmed by DNA sequencing of normal and tumor tissue. One somatic (Asp389Asn) and 15 germ-line changes were found. Of the 15 germ-line changes, 9 were found in an intron (none involving splice junctions), and 6 were found in an exon (Gly39Glu, Leu395Val, and 4 silent alterations). Immunohistochemical staining for hMSH6 performed on 34 of the 41 tumors revealed strong nuclear hMSH6 expression in all of the cases. Overall, our results suggest that hMSH6 mutations do not play a major role in the development of sporadic CRC with a MSI-L phenotype.     

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.     

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。     

Mutations of the 'minor' mismatch repair gene MSH6 in typical and atypical hereditary nonpolyposis colorectal cancer

Mutations of the mismatch repair (MMR) genes MLH1 and MSH2 are associated with hereditary nonpolyposis colorectal cancer (HNPCC), a highly penetrant autosomal dominant condition characterized by hypermutability of short tandemly repeated sequences in tumor DNA. Mutations of another MMR gene, MSH6, seem to be less common than MLH1 and MSH2 defects, and have been mostly observed in atypical HNPCC families, characterized by a weaker tumor family history, higher age at disease onset, and low degrees of microsatellite instability (MSI), predominantly involving mononucleotide runs. We have investigated the MSH6 gene sequence in the peripheral blood of 4 HNPCC and 20 atypical HNPCC probands. Two frameshift mutations within exon 4 were detected in 2 patients. One mutation was found in a proband from a typical HNPCC family, who had developed a colorectal cancer (CRC), a gastric cancer and a rectal adenoma. The CRC and the adenoma showed mild MSI limited to mononucleotide tracts, while the gastric carcinoma was microsatellite stable. The other mutation was detected in an atypical HNPCC proband, whose CRC showed widespread MSI involving both mono- and dinucleotide repeats. The phenotypic variability associated with MSH6 constitutional mutations represents a complicating factor for the optimization of strategies aimed at identifying candidates to MSH6 genetic testing.     

Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications

Background

Microsatellite instability (MSI) constitutes an important oncogenic molecular pathway in colorectal cancer (CRC), representing approximately 15% of all colorectal malignant tumours. In roughly one third of the cases, the underlying DNA mismatch repair (MMR) defect is inherited through the transmission of a mutation in one of the genes involved in MMR, predominantly MSH2 and MLH1, or less frequently, MSH6 or PMS2. In the overwhelming number of sporadic cases, MSI results from epigenetic MLH1 silencing through hypermethylation of its promoter. MMR deficiency promotes colorectal oncogenesis through the accumulation of numerous mutations in crucial target genes harbouring mononucleotide repeats, notably in those involved in the control of cell proliferation and differentiation, as well as DNA damage signalling and repair.

Design

In this review, we describe the molecular aspects of the MMR system and the biological consequences of its defect on the oncogenic process, and we discuss the various experimental systems used to evaluate the efficacy of cytotoxic drugs on MSI colorectal cells lines. There is increasing evidence showing that MSI CRCs differ from all CRCs in terms of prognosis and response to the treatment. We report the clinical studies that have evaluated the prognostic and predictive value of MSI status on clinical outcome in patients treated with various chemotherapy regimens used in the adjuvant setting or for advanced CRCs.

Conclusion

In view of this, the opportunity of a systematic MSI phenotyping in the clinical management of patients with CRC is further discussed.     

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。     

Sporadic Early Onset Colorectal Cancer in Pakistan: a Case- Control Analysis of Microsatellite Instability

Background: Early onset sporadic colorectal cancer (CRC) is a biologically and clinically distinct entity hypothesized to exhibit differences in histological features and microsatellite instability (MSI) as compared to typical onset CRC. This study compared the MSI status, mismatch repair enzyme deficiency and clinicopathological features of early onset (aged 45 years) with controls (>45 years). Materials and Methods: A total of 30 cases and 30 controls were analyzed for MSI status using the Bethesda marker panel. Using antibodies against hMLH1, hMSH2 and hMSH6, mismatch repair protein expression was assessed by immunohistochemistry. Molecular characteristics were correlated with clinicopathological features. Results: The early onset sporadic CRCs were significantly more poorly differentiated tumors, with higher N2 nodal involvement and greater frequency of signet ring phenotype than the typical onset cases. MSI was observed in 18/30 cases, with 12/18 designated as MSI-high (MSI-H) and 6/18 designated as MSI-low (MSI-L). In the control group, 14 patients exhibited MSI, with 7 MSI-H and 7 MSI-L. MSI tumors in both cases and controls exhibited loss of hMLH1, hMSH2 and hMSH6. MSS tumors did not exhibit loss of expression of MMR proteins, except hMLH1 protein in 3 controls. No statistically significant difference was noted in MSI status or expression of MMR proteins in cases versus controls. Conclusions: Microsatellite status is comparable between early and typical onset sporadic CRC patients in Pakistan suggesting that differences in clinicopathological features between these two subsets are attributable to other molecular mechanisms.     

Immunohistochemical analysis of expression and allelotype of mismatch repair genes (hMLH1 and hMSH2) in bladder cancer

Mutation of human homologues of DNA mismatch repair (MMR) genes in tumours has been shown to be associated with the phenomenon of microsatellite instability (MSI). Several studies have reported the occurrence of MSI in bladder cancer, but evidence of involvement of MMR genes in the pathogenesis of this cancer is still unclear. We therefore utilized quantitative immunohistochemical (IHC) image analysis and PCR-based allelotype analysis to determine hMLH1 and hMSH2 genes alteration in a cohort of Egyptian bladder cancer samples. IHC analysis of 24 TCC and 12 SCC revealed marked- intra and intertumour heterogeneity in the levels of expression of the two MMR proteins. One TCC lost MLH1 expression and one lost MSH2, (1/24, 4%), and one SCC lost MSH2 (1/12, 8%). A large proportion of analysed tumours revealed a percentage positivity of less than 50% for MLH1 and MSH2 expression (44% and 69%, respectively). Complete loss of heterozygosity in three dinucleotide repeats lying within, or in close proximity to, hMLH1 and hMSH2 was rare (2/57, (4%) for MLH1; and 1/55, (2%) for MSH2), however allelic imbalance was detected in 11/57 (hMLH1) and 10/55 (hMSH2) at any of the informative microsatellite loci. These alterations in structure and expression of DNA MMR genes suggest their possible involvement in the tumorigenesis and/or progression of bladder cancer.     

Mutations of the 'minor' mismatch repair gene MSH6 in typical and atypical hereditary nonpolyposis colorectal cancer

Mutations of the mismatch repair (MMR) genes MLH1 and MSH2 are associated with hereditary nonpolyposis colorectal cancer (HNPCC), a highly penetrant autosomal dominant condition characterized by hypermutability of short tandemly repeated sequences in tumor DNA. Mutations of another MMR gene, MSH6, seem to be less common than MLH1 and MSH2 defects, and have been mostly observed in atypical HNPCC families, characterized by a weaker tumor family history, higher age at disease onset, and low degrees of microsatellite instability (MSI), predominantly involving mononucleotide runs. We have investigated the MSH6 gene sequence in the peripheral blood of 4 HNPCC and 20 atypical HNPCC probands. Two frameshift mutations within exon 4 were detected in 2 patients. One mutation was found in a proband from a typical HNPCC family, who had developed a colorectal cancer (CRC), a gastric cancer and a rectal adenoma. The CRC and the adenoma showed mild MSI limited to mononucleotide tracts, while the gastric carcinoma was microsatellite stable. The other mutation was detected in an atypical HNPCC proband, whose CRC showed widespread MSI involving both mono- and dinucleotide repeats. The phenotypic variability associated with MSH6 constitutional mutations represents a complicating factor for the optimization of strategies aimed at identifying candidates to MSH6 genetic testing.     

Mismatch repair gene expression and genetic instability in testicular germ cell tumor

Human mismatch repair (MMR) genes encode highly conserved interacting proteins that correct replication errors predisposing to hereditary gastrointestinal and genitourinary malignancies. A subset of sporadic genitourinary tumors also exhibits MMR deficiency and can be identified by measuring the frequency of microsatellite instability (MSI) in cancer cell DNA. We investigated expression of the two most commonly mutated MMR genes, MSH2 and MLH1, in sporadic testicular germ cell tumor (GCT) in order to: (1) determine the expression pattern of MSH2 and MLH1 proteins in normal seminiferous tubules and histologically distinct GCT subtypes, (2) correlate MMR gene expression with genetic instability in GCT and (3) develop a panel of molecular markers that can identify genetically distinct subsets of GCT for prognostic assessment. MSH2 and MLH1 had differential staining patterns in normal seminiferous tubules and malignant tissues. MSH2 was expressed in all stages of spermatogenesis up to but excluding mature sperm whereas MLH1 was predominantly expressed in premeiotic germ cells. All histological GCT subtypes showed differential immunostaining for MSH2 and MLH1 however pure seminoma had statistically significant fewer low MSH2 staining tumors than other subtypes (p = 0.046). Twenty-five percent of GCT exhibited increased frequency of MSI (MSI+ tumors) with 73, 70 and 43% of MSI+ tumors exhibiting low MSH2, low MLH1 or low MSH2 and low MLH1 staining respectively. Fifteen percent of testicular GCT exhibited loss of heterozygosity (LOH) but no MSI (LOH only tumors). Only 28, 17 or 6% of LOH only tumors exhibited low MSH2, low MLH1 or low MSH2 and low MLH1 staining respectively.     

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.     

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.     

Low-level microsatellite instability occurs in most colorectal cancers and is a nonrandomly distributed quantitative trait.

About 10-15% of colorectal cancers show high-level microsatellite instability. The characteristics and very existence of low-level instability (MSI-L) are unclear, although some studies have found associations between MSI-L and molecular characteristics, notably more frequent K-ras mutations and a low level of allele loss near APC. We have attempted to define a MSI-L group of tumors by analyzing 107 sporadic colorectal carcinomas at 44 microsatellites. Ten (9.7%) MSI-H cancers were identified, but there was no evidence for a discrete MSI-L group. However, the 97 non-MSI-H cancers showed greater variation in the frequency of MSI than was expected by chance. Most cancers (68%) in the non-MSI-H group showed some MSI and could therefore be classed as nominally MSI-L. No association was found between MSI-L (or the level of MSI) and any clinicopathological or molecular variable, including K-ras mutation and loss of heterozygosity at APC. The causes of variation in level of the MSI in non-MSI-H colorectal cancers are unknown, but the differences are quantitative and probably reflect the evolutionary histories of the cancers rather than qualitatively different genetic pathways of tumorigenesis.     

Lynch‐like syndrome: Characterization and comparison with EPCAM deletion carriers

Colorectal cancers (CRCs) with microsatellite instability‐high (MSI+) but without detectable germline mutation or hypermethylation in DNA mismatch repair (MMR) genes can be classified as Lynch‐like syndrome (LLS). The underlying mechanism and clinical significances of LLS are largely unknown. We measured MSI and MMR protein expression in 4,765 consecutive CRC cases. Among these, MSI+ cases were further classified based on clinical parameters, germline sequencing of MMR genes or polymerase ε (POLE) and δ (POLD1) and promoter methylation analysis of MLH1 and MSH2. We found that MSI+ and MMR protein‐deficient CRCs comprised 6.3% (N = 302) of this cohort. On the basis of germline sequencing of 124 cases, we identified 54 LS with MMR germline mutation (LS‐MMR), 15 LS with EPCAM deletions (LS‐EPCAM) and 55 LLS patients. Of the 55 LLS patients, six (10.9%) had variants of unknown significance in the genes tested, and one patient had a novel somatic mutation (p.S459P) in POLE. In patients with biallelic deletions of EPCAM, all tumors and their matched normal mucosa showed promoter hypermethylation of MSH2. Finally, we found that patients with LLS and LS‐EPCAM shared clinical features that differed from LS‐MMR patients, including lower frequency of fulfillment of the revised Bethesda guidelines (83.6 and 86.7% vs. 98.1% for LS‐MMR) and older mean age at CRC diagnosis (52.6 and 52.7 years vs. 43.9 years for LS‐MMR). We identified somatic mutation in POLE as a rare underlying cause for MMR deficiency in LLS. The similarity between LLS and LS‐EPCAM suggests LLS as a subset of familial MSI+ CRC.