Real-world data on microsatellite instability status in various unresectable or metastatic solid tumors

Microsatellite instability-high (MSI-H) is an important biomarker for predicting the effect of immune checkpoint inhibitors (ICIs) on advanced solid tumors. Microsatellite instability-high is detected in various cancers, but its frequency varies by cancer type and stage. Therefore, precise frequency is required to plan ICI therapy. In this study, the results of MSI tests actually carried out in clinical practice were investigated. In total, 26 469 samples of various cancers were examined between December 2018 and November 2019 to determine whether programmed cell death-1 blockade was indicated. The results of MSI tests were obtained for 26 237 (99.1%) of these samples. The male : female ratio was 51:49 and mean age was 64.3 years. In all samples, the overall frequency of MSI-H was 3.72%. By gender, the frequency of MSI-H was higher in female patients (4.75%) than in male patients (2.62%; P < .001). A comparison by age revealed that the frequency of MSI-H was significantly higher in patients younger than 40 years of age (6.12%) and 80 years or older (5.77%) than in patients aged between 60 and 79 years (3.09%; P < .001). Microsatellite instability-high was detected in 30 cancer types. Common cancer types were: endometrial cancer, 16.85%; small intestinal cancer, 8.63%; gastric cancer, 6.74%; duodenal cancer, 5.60%; and colorectal cancer, 3.78%. Microsatellite instability-high was detected in cancer derived from a wide variety of organs. The frequency of MSI-H varied by cancer type and onset age. These data should prove especially useful when considering ICI treatment.     

Relationship between protein biomarkers of chemotherapy response and microsatellite status,tumor mutational burden and PD-L1 expression in cancer patients

Chemotherapy and checkpoint inhibitor immunotherapies are increasingly used in combinations. We determined associations between the presence of anti-PD-1/PD-L1 therapeutic biomarkers and protein markers of potential chemotherapy response. Data were extracted from a clinical-grade testing database (Caris Life Sciences; February 2015 through November 2017): immunotherapy response markers (microsatellite instability-high [MSI-H], tumor mutational burden-high [TMB-H], and PD-L1 protein expression) and protein chemotherapy response markers (excision repair complementation group 1 [ERCC1], topoisomerase 1 [TOPO1], topoisomerase 2 [TOP2A], thymidylate synthase [TS], tubulin beta 3 [TUBB3], ribonucleotide reductase regulatory subunit M1 [RRM1] and O-6-methyl guanine DNA methyltransferase [MGMT]). Relationships were determined by the Mantel-Haenszel chi-squared test or Fischer's exact tests. Overall, 28,034 patients representing a total of 40 tumor types were assessed. MSI-H was found in 3.3% of patients (73% were also TMB-H), TMB-H, 8.4% (28.3% were also MSI-H) and PD-L1 expression in 11.0% of patients (5.1% were also MSI-H; 16.4% were also TMB-H). Based on concurrent biomarker expression, combinations of immunotherapy with platinum (ERCC1 negativity) or with doxorubicin, epirubicin or etoposide (TOP2A positivity) have a higher probability of response, whereas combinations with irinotecan or topotecan (TOPO1 positivity), with gemcitabine (RRM1 negativity), and fluorouracil, pemetrexed or capecitabine (TS negativity) may be of less benefit. The potential for immunotherapy and taxane (TUBB3 negativity) combinations is present for MSI-H but not TMB-H or PD-L1-expressing tumors; for temozolomide and dacarbazine (MGMT negative), PD-L1 is frequently coexpressed, but MSI-H and TMB-H are not associated. Protein markers of potential chemotherapy response along with next-generation sequencing for immunotherapy response markers can help support rational combinations as part of an individualized, precision oncology approach.     

Impact of high tumor mutational burden in solid tumors and challenges for biomarker application

Accurate identification of patients with solid tumors likely to respond to immunotherapy is crucial. Tumor mutational burden (TMB) measures the number of somatic mutations in a tumor and is an emerging prognostic and predictive biomarker for anti-programmed cell death (PD) 1/anti-PD-ligand 1 therapy and other immunotherapeutic agents. Tumor mutational burden is assessed optimally by whole exome sequencing, but next generation sequencing provides TMB estimates in a more timely and cost-effective manner. Blood-based measurement of TMB in plasma offers an alternative to the need for adequate tumor tissue for molecular testing, and has demonstrated the ability to identify patients who derive benefit from immunotherapy. Tumor mutational burden has diverse prognostic impact in different solid tumor types and also has a demonstrated role in predicting improved survival in patients receiving immunotherapy. There are challenges to TMB adoption into standard clinical practice, including variations in its definition, with the mutational number defining TMB-high appearing to vary across cancer types. The magnitude of TMB also varies across different tumor types, with the highest levels reported in melanoma and other skin cancers (where ultraviolet light is the dominant mutational process), followed by non-small cell lung cancer and other squamous carcinomas. Concerns regarding inter-laboratory and inter-platform variations in analysis methods have been raised, highlighting the need for standardization. Integration of other genomic or pathological biomarkers with TMB may increase its prognostic and predictive capabilities and validation of individual or combination models in prospective trials is warranted.     

Tumor mutation burden determined by a 645-cancer gene panel and compared with microsatellite instability and mismatch repair genes in colorectal cancer

BackgroundTumor mutation burden (TMB) assessed by tumor-related gene panels (CRGP), microsatellite instability (MSI), and mismatch repair (MMR) has been proven to be associated with prognosis, and these factors are prognostic indicators in predicting the benefits of immune checkpoint blockade (ICB) in solid tumors. However, whether the TMB calculated by CRGPs, MSI, and MMR is associated with overall survival (OS) in patients with colorectal cancer (CRC) remains to be explored.MethodsThe prognostic threshold of the panel-TMB was explored by a panel of 645 genes (GP645) from 41 CRC patients in Jiangsu Cancer Hospital (JCH dataset). The results were further validated using 531 CRC patients from The Cancer Genome Atlas (TCGA) database.ResultsMutations of the GP645 genes were distributed on 21 chromosomes. Spearman correlation analysis showed that the panel-TMB was positively correlated with TMB measured by whole-exome sequencing (WES) (wTMB) in the TCGA dataset (R=0.75, P<0.001). Kaplan-Meier survival analysis demonstrated that higher panel-TMB in CRC patients was significantly associated with a poor OS (P=0.0062). MSI and MMR status were determined using the GP645 by next-generation sequencing (NGS). The proportions of MSI-H and dMMR accounted for less than 10% in CRC, the vast majority of MSI-H/dMMR samples also had high TMB [positive predictive value (PPV) =66.6%], and only 13.3% of samples with high TMB were classified as MSI-high/dMMR. In addition, patients with low-TMB were associated with MSS/pMMR (96.2%), and these results are consistent with earlier studies.ConclusionsGP645 was constructed to evaluate OS in Chinese CRC patients. Panel-TMB and MSI/MMR might be potential prognostic predictors of CRC patients using the GP645.     

Genetic investigation of DNA-repair pathway genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1 in sporadic colon cancer

Mutations in DNA repair genes have previously been identified as causative factors for hereditary nonpolyposis colon cancer (HNPCC). Recent evidence also supports an association between DNA sequence variation in these genes and sporadic colorectal carcinoma (CRC). Genetic investigation of DNA repair genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1, as possible susceptibility factors for sporadic CRC, was done using both a haplotype tagging and a candidate (i.e. coding) single nucleotide polymorphism (SNP) approach. Some 1,068 patients with operated CRC (median age at diagnosis: 59 years) were compared to 738 sex-matched control individuals (median age: 67 years). Haplotype tagging SNPs, previously reported risk variants and all known coding SNPs with a minor allele frequency >0.005 were genotyped in PMS2 (N = 10), MLH1 (N = 11), MSH2 (N = 18), MSH6 (N = 15), MUTYH (N = 7), OGG1 (N = 11) and MTH1 (N = 3). No evidence for an association between CRC and any of the 7 genes was detected, neither with the tagging or coding SNPs nor in a sliding window haplotype analysis (all nominal p-values >0.05). The previously reported risk variants D132H in MLH1 and R154H in OGG1 were not even observed in the German population. Genetic CRC risk factors so far identified in DNA repair genes seem to be rare and population-specific. Their association with the disease could not be replicated in German CRC samples. It remains to be elucidated by more systematic, large-scale experiments whether common variants in the same genes, but present across populations, represent risk factors for sporadic CRC.     

Measurement of tumor mutational burden (TMB) in routine molecular diagnostics: in silico and real-life analysis of three larger gene panels

Assessment of Tumor Mutational Burden (TMB) for response stratification of cancer patients treated with immune checkpoint inhibitors is emerging as a new biomarker. Commonly defined as the total number of exonic somatic mutations, TMB approximates the amount of neoantigens that potentially are recognized by the immune system. While whole exome sequencing (WES) is an unbiased approach to quantify TMB, implementation in diagnostics is hampered by tissue availability as well as time and cost constrains. Conversely, panel-based targeted sequencing is nowadays widely used in routine molecular diagnostics, but only very limited data are available on its performance for TMB estimation. Here, we evaluated three commercially available larger gene panels with covered genomic regions of 0.39 Megabase pairs (Mbp), 0.53 Mbp and 1.7 Mbp using i) in silico analysis of TCGA (The Cancer Genome Atlas) data and ii) wet-lab sequencing of a total of 92 formalin-fixed and paraffin-embedded (FFPE) cancer samples grouped in three independent cohorts (non-small cell lung cancer, NSCLC; colorectal cancer, CRC; and mixed cancer types) for which matching WES data were available. We observed a strong correlation of the panel data with WES mutation counts especially for the gene panel >1Mbp. Sensitivity and specificity related to TMB cutpoints for checkpoint inhibitor response in NSCLC determined by wet-lab experiments well reflected the in silico data. Additionally, we highlight potential pitfalls in bioinformatics pipelines and provide recommendations for variant filtering. In summary, our study is a valuable data source for researchers working in the field of immuno-oncology as well as for diagnostic laboratories planning TMB testing.     

Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with a microsatellite stable status in Chinese gastric cancer patients

BackgroundGastric cancer (GC) is one of the most common cancers worldwide. However, little is known about the combination of HER2 amplification and microsatellite instability (MSI) status in GC. This study aimed to analyze the correlation of HER2 amplification with microsatellite instability (MSI) status, clinical characteristics, and the tumor mutational burden (TMB) of patients.MethodsA total of 192 gastric cancer (GC) patients were enrolled in this cohort. To analyze genomic alterations (GAs), deep sequencing was performed on 450 target cancer genes. TMB was measured by an in-house algorithm. MSI status was inferred based on the MANTIS (Microsatellite Analysis for Normal-Tumor InStability) score.ResultsThe most frequently amplified genes in the GC patients included cyclin E1 (CCNE1), human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 2 (FGFR2), cyclin D1 (CCND1), fibroblast growth factor 19 (FGF19), fibroblast growth factor 3 (FGF3), and fibroblast growth factor 4 (FGF4). The frequency of HER2 amplification was 9.38% (18/192). HER2 amplification was higher in females than in males (14.52% vs. 6.92%, respectively, P=0.091), however, MSI was higher in males compared to females (7.69% vs. 4.84%, respectively, P=0.46). HER2 amplification was higher in metastatic loci compared to primary lesions (23.08% vs. 8.38%, respectively, P=0.079) and was lower in patients with high TMB (TMB-H) compared to those with low TMB (TMB-L) (4.0% vs. 11.35%, respectively, P=0.12). While the frequency of MSI in metastatic foci was higher than that in primary lesions (15.38% vs. 6.15%, respectively, P=0.48), MSI status was highly associated with TMB-H (20% vs. 0%, respectively, P=3.66×10⁻⁷). Furthermore, HER2 amplification was negatively correlated with MSI status in Chinese GC patients.ConclusionsHER2 amplification was negatively correlated with TMB-H and MSI status, and MSI status was significantly associated with TMB-H in Chinese GC patients. These data suggested that HER2 amplification might be a negative indicator for GC immunotherapy.     

Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas

Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care.     

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.     

A tale of two pathways: Review of immune checkpoint inhibitors in DNA mismatch repair-deficient and microsatellite instability-high endometrial cancers

The DNA mismatch repair (MMR) pathway is critical for correcting DNA mismatches generated during DNA replication. MMR-deficiency (MMR-D) leads to microsatellite instability (MSI) associated with an increased mutation rate, driving cancer development. This is particularly relevant in endometrial cancer (EC) as 25%–30% of tumors are of MMR-D/MSI-high (MSI-H) phenotype. Comprehensive assessment using immunohistochemistry (IHC) and sequencing-based techniques are necessary to fully evaluate ECs given the importance of molecular subtyping in staging and prognosis. This also influences treatment selection as clinical trials have demonstrated survival benefits for immune checkpoint inhibitors (ICIs) alone and in combination with chemotherapy for MMR-D/MSI-H EC patients in various treatment settings. As a portion of MMR-D/MSI-H ECs are driven by Lynch syndrome, an inherited cancer predisposition syndrome that is also associated with colorectal cancer, this molecular subtype also prompts germline assessment that can affect at-risk family members. Additionally, heterogeneity in the tumor immune microenvironment and tumor mutation burden (TMB) have been described by MMR mechanism, meaning MLH1 promoter hypermethylation versus germline/somatic MMR gene mutation, and this may affect response to ICI therapies. Variations by ancestry in prevalence and mechanism of MMR-D/MSI-H tumors have also been reported and may influence health disparities given observed differences in tumors of Black compared to White patients which may affect ICI eligibility. These observations highlight the need for additional prospective studies to evaluate the nuances regarding MMR-D heterogeneity as well as markers of resistance to inform future trials of combination therapies to further improve outcomes for patients with EC.     

Associations between family history of colorectal cancer and genetic alterations in tumors

A family history of colorectal cancer has been consistently associated with an increased risk of developing colon cancer. However, there is limited information on the association between family history of colorectal cancer and genetic alterations that occur in colon tumors. In this study, we evaluate the association among genetic alterations of Ki-ras and p53, microsatellite instability and having a family history of colorectal cancer in a study of incident colon cancer cases (n = 1993) and population-based controls (n = 2,410). Although there was a slight nonsignificant increase in risk of having an unstable tumor among those with a family history of colorectal cancer, this increase in risk disappeared after excluding those people with a known mutation in either of the mismatch repair genes hMLH1 or hMSH2. A family history of colorectal cancer was not associated with Ki-ras mutations overall, although those with a G to T mutation of the second base of codon 12 were more likely to have a family history of colorectal cancer than were those without this specific type of Ki-ras mutation. Cases with p53 mutations were less likely to have a family history of colorectal cancer than were cases without a p53 mutation. We believe that, given the general lack of association between having a family history of colorectal cancer and genetic alterations in tumors, these alterations are acquired through disease pathways that involve exposure from diet, lifestyle or other environmental factors.     

Immune Checkpoint Inhibition as Primary Adjuvant Therapy for an IDH1-Mutant Anaplastic Astrocytoma in a Patient with CMMRD: A Case Report—Usage of Immune Checkpoint Inhibition in CMMRD

Constitutional mismatch repair deficiency (CMMRD) is a rare autosomal recessive hereditary cancer syndrome due to biallelic germline mutation involving one of the four DNA mismatch repair genes. Here we present a case of a young female with CMMRD, homozygous for the c.2002A>G mutation in the PMS2 gene. She developed an early stage adenocarcinoma of the colon at the age of 14. Surveillance MRI of the brain at age 18 resulted in the detection of an asymptomatic brain cancer. On resection, this was diagnosed as an anaplastic astrocytoma. Due to emerging literature suggesting benefit of immunotherapy in this patient population, she was treated with adjuvant dual immune checkpoint inhibition, avoiding radiation. The patient remains stable with no evidence of progression 20 months after resection. The patient’s clinical course, as well as the rational for considering adjuvant immunotherapy in patients with CMMRD are discussed in this report.     

Evaluation of 30 DNA damage response and 6 mismatch repair gene mutations as biomarkers for immunotherapy outcomes across multiple solid tumor types

Objective:DNA damage response (DDR) genes have low mutation rates, which may restrict their clinical applications in predicting the outcomes of immune checkpoint inhibitor (ICI) treatment. Thus, a systemic analysis of multiple DDR genes is needed to identify potential biomarkers of ICI efficacy.Methods:A total of 39,631 patients with mutation data were selected from the cBioPortal database. A total of 155 patients with mutation data were obtained from the Fudan University Shanghai Cancer Center (FUSCC). A total of 1,660 patients from the MSK-IMPACT cohort who underwent ICI treatment were selected for survival analysis. A total of 249 patients who underwent ICI treatment from the Dana-Farber Cancer Institute (DFCI) cohort were obtained from a published dataset. The Cancer Genome Atlas (TCGA) level 3 RNA-Seq version 2 RSEM data for gastric cancer were downloaded from cBioPortal.Results:Six MMR and 30 DDR genes were included in this study. Six MMR and 20 DDR gene mutations were found to predict the therapeutic efficacy of ICI, and most of them predicted the therapeutic efficacy of ICI, in a manner dependent on TMB, except for 4 combined DDR gene mutations, which were associated with the therapeutic efficacy of ICI independently of the TMB. Single MMR/DDR genes showed low mutation rates; however, the mutation rate of all the MMR/DDR genes associated with the therapeutic efficacy of ICI was relatively high, reaching 10%–30% in several cancer types.Conclusions:Coanalysis of multiple MMR/DDR mutations aids in selecting patients who are potential candidates for immunotherapy.     

Differential expression of hMLH1 and hMSH2 is related to bladder cancer grade,stage and prognosis but not microsatellite instability

Defects in the DNA mismatch repair proteins result in microsatellite instability and malignancy in hereditary non-polyposis colorectal carcinoma (HNPCC). However, the role of mismatch repair (MMR) proteins and microsatellite instability (MSI) in transitional cell carcinoma of the bladder is less clear. In our study, the expression of 2 MMR proteins and the frequency of MSI in Transitional cell carcinoma of the bladder (TCC) were investigated. One hundred eleven patients with TCC of the bladder were studied, with complete clinicopathological data (median follow up of 5 years, range 5-16 years). Immunohistochemistry was used to detect the expression levels of hMLH1 and hMSH2. Microsatellite analysis for 14 loci (10 loci from the Bethesda consensus panel and the repeats in the TGFbetaR2, BAX, hMSH3 and hMSH6 genes) was performed on 84 tumors. Reduced expression of either MMR protein was seen in 26 of 111 tumors (23%). Reduced expression was seen more commonly in muscle invasive (p<0.03) and high grade TCC (p<0.03) than in superficial, low grade tumors. By 5 years, reduced expression of either MMR protein was associated with fewer recurrences of superficial tumors (p=0.015) and fewer relapses in all tumors (p=0.03), compared to tumors with normal expression. Nine tumors had reduced expression of both MMR proteins, analysis which suggests a synergistic reduction in expression (p=0.001). MMR expression was related to patient age, younger patients being more likely to have reduced MMR expression than older patients (p<0.01). MSI was seen at multiple loci in 1 tumor (1%) and at a single locus in 6 tumors (7%). MSI was not associated with MMR expression. Our findings indicate that reduced expression of the MMR proteins may have an important contribution in the development of a subset of TCCs and suggest a potential role for MMR expression as prognostic indicators.     

Mutation analysis of the MLH1, MSH2 and MSH6 genes in patients with double primary cancers of the colorectum and the endometrium: a population-based study in northern Sweden

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder that predisposes to predominantly colorectal and endometrial cancers due to germline mutations in DNA mismatch repair genes, mainly MLH1, MSH2 and in families with excess endometrial cancer also MSH6. In this population-based study, we analysed the mutation spectrum of the MLH1, MSH2 and MSH6 genes in a cohort of patients with microsatellite unstable double primary tumours of the colorectum and the endometrium by PCR, DHPLC and sequencing. Fourteen of the 23 patients (61%) had sequence variants in MLH1, MSH2 or MSH6 that likely affect the protein function. A majority (10/14) of the mutations was found among probands diagnosed before age 50. Five of the mutations (36%) were located in MLH1, 3 (21%) in MSH2 and 6 (43%) in MSH6. MSH6 seem to have larger impact in our population than in other populations, due to a founder effect since all of the MSH6 families originate from the same geographical area. MSH6 mutation carriers have later age of onset of both colorectal cancer (62 vs. 51 years) and endometrial cancer (58 vs. 48 years) and a larger proportion of endometrial cancer than MLH1 or MSH2 mutation carriers. We can conclude that patients with microsatellite unstable double primary cancers of the colorectum and the endometrium have a very high risk of carrying a mutation not only in MLH1 or MSH2 but also in MSH6, especially if they get their first cancer diagnosis before the age of 50.     

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.