Risk of colorectal polyps and of malignancies in asymptomatic carriers of mutations in the main DNA mismatch repair genes.

OBJECTIVE: Mutation carriers (Mut+) in DNA mismatch repair genes are predisposed to cancer of various organs and to adenomatous polyps; however, they may remain asymptomatic and cancer or polyp-free for several years. We purposed to analyse the clinical follow-up of individuals carrying constitutional mutations in the MLH1, MSH2 or MSH6 genes who were unaffected by benign polyps or malignant tumours at diagnosis. MATERIAL AND METHODS: Mut + subjects (n.81) were members of Lynch syndromes in whom mutations were detected between 1993 and 2015; all were asymptomatic at diagnosis. They were informed of the cancer risk and surveillance was suggested. As controls, 113 nongene carriers (Mut-) in the same Lynch families were identified. RESULTS: About one-fourth of the mutation carriers developed polyps, mostly adenomas; polyps were less (12%, p < .05) in Mut - subjects, and hyperplastic lesions were the prevalent histology. More polyps were detected in MLH1 vs. MSH2 mutation carriers. In Mut+, 21 malignant tumours developed in 14 carriers vs. 4 tumours in 3 patients among Mut- (p < .001). Tumours were mostly of the Lynch spectrum; however, three glioblastomas were developed, together with neoplasms of various organs (duodenum, thyroid, skin, lung and cervix). Mean age of tumour occurrence was 43.0 years in Mut + vs. 53.0 among Mut-. CONCLUSIONS: Cancer developed more often in Mut+, with no consistent difference between MLH1 and MSH2 carriers. More polyps (mostly adenomas) were detected in MLH1 carriers. The majority (13 of 21) of malignant tumours occurred in organs for which there is no recommended surveillance, and were lethal in three patients.

Disease presentation, treatment and survival for Italian colorectal cancer patients: a EUROCARE high resolution study.

We analysed presentation, treatment and survival in a representative population-based sample of 3753 Italian colorectal cancer cases, diagnosed 2003-05: 70% were >65 years, 44% stage I-II, 27% stage IV and 92% received surgery. Chemotherapy was given to 58% of stage III colon cases, radiotherapy to 25% of rectal cases. Four percent of surgical cases underwent endoscopic polypectomy, and in 57% ≥11 lymph nodes were examined. Five-year relative survival was good (60%), independent of sex and site. Adherence to treatment guidelines was satisfactory, but wider use of faecal blood testing and colonoscopy will anticipate stage at diagnosis and likely improve survival.

Revised guidelines for the clinical management of Lynch syndrome (HNPCC): recommendations by a group of European experts.

Lynch syndrome (LS) is characterised by the development of colorectal cancer, endometrial cancer and various other cancers, and is caused by a mutation in one of the mismatch repair genes: MLH1, MSH2, MSH6 or PMS2. In 2007, a group of European experts (the Mallorca group) published guidelines for the clinical management of LS. Since then substantial new information has become available necessitating an update of the guidelines. In 2011 and 2012 workshops were organised in Palma de Mallorca. A total of 35 specialists from 13 countries participated in the meetings. The first step was to formulate important clinical questions. Then a systematic literature search was performed using the Pubmed database and manual searches of relevant articles. During the workshops the outcome of the literature search was discussed in detail. The guidelines described in this paper may be helpful for the appropriate management of families with LS. Prospective controlled studies should be undertaken to improve further the care of these families.

Incidence trend of malignant polyps through the data of a specialized colorectal cancer registry: clinical features and effect of screening.

OBJECTIVES: The purposes of the study are to describe the incidence trend of malignant polyp of large bowel over a 25-year period in the District of Modena and to assess the effect of an organized colorectal cancer screening program. MATERIAL AND METHODS: Through the data of a specialized colorectal cancer Registry, we evaluate the clinical and pathological features of the polyps. Trend analysis was assessed with the Joinpoint Regression Program. RESULTS: A total of 172 patients with malignant polyps were diagnosed throughout the study (3.5% of 4.835 registered patients); their overall frequency during the registration period increased from zero cases in the initial years (1984-85) to 57 cases in the past 3 years (2006-2008). Crude incidence rate passed from 0.37 in 1986-89 to 10.2 in 2006. Joinpoint trend analysis of crude rates showed a significant increase of incidence during the study period, with percent of annual variation ranging between 38.6% (95% CI 12.5-70.7) and 7.3% (95% CI 2.6-12.1). During the screening period (2005-2008, the past 4 years of registration) there was a significant increase of sessile polyps (p < 0.001), while other clinical and morphological features, including the number of low- and high-risk malignant polyps, remained unchanged. The surgery (after polypectomy) tended to raise both in low- and high-risk subgroups. CONCLUSION: The incidence of malignant polyps increased significantly from the initial to the most recent periods of colorectal cancer registration. Screening was associated with changes in gross morphology of polyps and with an increased use of the surgery after endoscopic polypectomy.

Lymph node evaluation in stage IIA colorectal cancer and its impact on patient prognosis: a population-based study.

BACKGROUND: The analysis of regional lymph nodes is particularly relevant in patients with stage II colorectal cancer, in whom the role of adjuvant chemotherapy remains unclear. The aim of this study was to assess the relationship between number of examined lymph nodes and survival in patients with stage IIA (pT3N0M0) colorectal cancer, and to determine the optimal number of lymph nodes that should be examined. METHODS: The study group included all the surgically-treated colorectal cancer patients in stage IIA (n = 657) who were identified through the population-based Cancer Registry of the Province of Modena (Northern Italy), during the period 2002-2006. RESULTS: The median number of harvested lymph nodes was 19 (range 1-68). Considering, as a reference point, patients with 12 or less lymph nodes, subjects with n ≥ 20 lymph nodes examined showed, in univariate analysis, a significantly higher cancer specific (p = 0.01) and relapse-free survival (p = 0.003). The results were confirmed by multivariate analysis (Cox model). CONCLUSION: The result suggests that colorectal cancer patients in stage IIA with n ≥ 20 lymph nodes examined exhibit better survival when compared with subjects in whom fewer lymph nodes were examined. The number of 20 lymph nodes is the essential requirement for an oncologic resection of the large bowel.

PLZF expression during colorectal cancer development and in normal colorectal mucosa according to body size, as marker of colorectal cancer risk.

Promyelocytic leukemia zinc finger protein (PLZF) is a protein involved in various signaling, growth regulatory, and differentiation pathways, including development/function of some T cells. Here, we aimed at the detection of PLZF during colorectal carcinogenesis, using immunofluorescence, and at the evaluation of the colocalization of PLZF with CD2 and CD56 positive cells (T, γ δ , NK, and NKT cells), using confocal-microscopy, along colorectal carcinogenesis, since its earliest stages, that is, dysplastic aberrant crypt foci (ACF). Furthermore, we analyzed PLZF in the normal colonic mucosa (NM) according to anthropometric parameters of the subject. NM exhibited strong CD56 fluorescent staining. This infiltration was lost in both ACF and colorectal carcinoma (CRC), while PLZF presence increased from NM to ACF and CRC. Strong association was found between CD56+ colonic mucosa cell infiltration and body mass index. Interestingly, an increased stromal PLZF-reactivity was present in NM of obese subjects. This study shows that overexpression of PLZF and exclusion of NK cells in dysplastic microenvironment are very early events in the stepwise sequence leading to CRC and that lower levels of CD56+ cells in NM, together with increased levels of PLZF+ cells, can be a reflection of colon cancer risk due to obesity.

Increased expression of CD133 is a strong predictor of poor outcome in stage I colorectal cancer patients.

OBJECTIVE: Stage I colorectal carcinomas display a highly variable behavior which is not accurately predicted by the available prognostic markers. CD133 is considered a useful marker to identify the so-called cancer stem cells in colorectal cancers (CRCs) and its expression has been shown to have prognostic significance in CRC patients. This study aimed to verify whether immunohistochemical evaluation of CD133 might correlate with the progression risk of stage I CRC patients. MATERIAL AND METHODS: Expression levels of the CD133 molecule were analyzed and compared in two series of stage I surgically resected CRC patients showing disease progression and death for the disease and patients with no evidence of disease progression after at least 6 years after surgery. RESULTS: A positive staining for CD133 was detected in 52% of the cases with poor prognosis and only in 9% of the group with good prognosis, and this difference was highly significant (p < 0.001). A significant correlation was detected between CD133 expression and histological parameters, such as tumor budding, vascular invasion, and presence of lymph node micrometastases but not tumor grading, gender, and age. Disease-free survival and cancer-specific survival of CD133 negative tumors were significantly longer compared to positive cases. In multivariate analyses, CD133 staining confirmed to be a predictor of shorter survival independent from vascular invasion but not from lymph nodes micrometastases. CONCLUSIONS: These findings demonstrate that CD133 immunostaining is a useful predictor of high risk progression in stage I CRC patients and might help to identify patients eligible for adjuvant chemotherapy.

Identification of Lynch syndrome among patients with colorectal cancer.

CONTEXT: Lynch syndrome is the most common form of hereditary colorectal cancer (CRC) and is caused by germline mutations in DNA mismatch repair (MMR) genes. Identification of gene carriers currently relies on germline analysis in patients with MMR-deficient tumors, but criteria to select individuals in whom tumor MMR testing should be performed are unclear. OBJECTIVE: To establish a highly sensitive and efficient strategy for the identification of MMR gene mutation carriers among CRC probands. DESIGN, SETTING, AND PATIENTS: Pooled-data analysis of 4 large cohorts of newly diagnosed CRC probands recruited between 1994 and 2010 (n = 10,206) from the Colon Cancer Family Registry, the EPICOLON project, the Ohio State University, and the University of Helsinki examining personal, tumor-related, and family characteristics, as well as microsatellite instability, tumor MMR immunostaining, and germline MMR mutational status data. MAIN OUTCOME: Performance characteristics of selected strategies (Bethesda guidelines, Jerusalem recommendations, and those derived from a bivariate/multivariate analysis of variables associated with Lynch syndrome) were compared with tumor MMR testing of all CRC patients (universal screening). RESULTS: Of 10,206 informative, unrelated CRC probands, 312 (3.1%) were MMR gene mutation carriers. In the population-based cohorts (n = 3671 probands), the universal screening approach (sensitivity, 100%; 95% CI, 99.3%-100%; specificity, 93.0%; 95% CI, 92.0%-93.7%; diagnostic yield, 2.2%; 95% CI, 1.7%-2.7%) was superior to the use of Bethesda guidelines (sensitivity, 87.8%; 95% CI, 78.9%-93.2%; specificity, 97.5%; 95% CI, 96.9%-98.0%; diagnostic yield, 2.0%; 95% CI, 1.5%-2.4%; P < .001), Jerusalem recommendations (sensitivity, 85.4%; 95% CI, 77.1%-93.6%; specificity, 96.7%; 95% CI, 96.0%-97.2%; diagnostic yield, 1.9%; 95% CI, 1.4%-2.3%; P < .001), and a selective strategy based on tumor MMR testing of cases with CRC diagnosed at age 70 years or younger and in older patients fulfilling the Bethesda guidelines (sensitivity, 95.1%; 95% CI, 89.8%-99.0%; specificity, 95.5%; 95% CI, 94.7%-96.1%; diagnostic yield, 2.1%; 95% CI, 1.6%-2.6%; P < .001). This selective strategy missed 4.9% of Lynch syndrome cases but resulted in 34.8% fewer cases requiring tumor MMR testing and 28.6% fewer cases undergoing germline mutational analysis than the universal approach. CONCLUSION: Universal tumor MMR testing among CRC probands had a greater sensitivity for the identification of Lynch syndrome compared with multiple alternative strategies, although the increase in the diagnostic yield was modest.

Biallelic PMS2 Mutations in a Family with Uncommon Clinical and Molecular Features.

We describe a patient with constitutional mismatch repair-deficiency (CMMR-D) in whom the syndrome started at age 10 with the development of multiple adenomas in the large bowel. In the successive 25 years, four malignancies developed in different organs (rectum, ileum, duodenum, and lymphoid tissue). The patient had biallelic constitutional pathogenic variants in the PMS2 gene. We speculate that besides the PMS2 genotype, alterations of other genes might have contributed to the development of the complex phenotype. In the nuclear family, both parents carried different PMS2 germline mutations. They appeared in good clinical condition and did not develop polyps or cancer. The index case had a brother who died at age three of lymphoblastic leukemia, and a sister who was affected by sarcoidosis. Tumor tissue showed diffuse DNA microsatellite instability. A complete absence of immunoreactivity was observed for the PMS2 protein both in the tumors and normal tissues. Next-generation sequencing and multiple ligation-dependent probe amplification analyses revealed biallelic PMS2 germline pathogenic variants in the proband (genotype c.[137G>T];[(2174+1_2175-1)_(*160_?)del]), and one of the two variants was present in both parents-c.137G>T in the father and c.(2174+1-2175-1)_(*160_?)del in the mother-as well as c.137G>T in the sister. Moreover, Class 3 variants of MSH2 (c.1787A>G), APC (c.1589T>C), and CHEK2 (c.331G>T) genes were also detected in the proband. In conclusion, the recognition of CMMR-D may sometimes be difficult; however, the possible role of constitutional alterations of other genes in the development of the full-blown phenotype should be investigated in more detail.

Analysis of telomere dynamics in peripheral blood cells from patients with Lynch syndrome.

BACKGROUND: In patients with Lynch syndrome, germline mutations in DNA mismatch repair (MMR) genes cause a high risk of developing a broad spectrum of cancers. To date, the management of patients with Lynch syndrome has represented a major challenge because of large variations in age at cancer onset. Several factors, including genetic anticipation, have been proposed to explain this phenotypic heterogeneity, but the molecular mechanisms remain unknown. Telomere shortening is a common event in tumorigenesis and also has been observed in different familial cancers. In this study, the authors investigated the possibility of a relation between telomere length and cancer onset in patients with Lynch syndrome. METHODS: The mean telomere length was measured using quantitative polymerase chain reaction in peripheral blood samples from a control group of 50 individuals, from 31 unaffected mutation carriers, and from 43 affected patients, and the results were correlated with both gene mutation and cancer occurrence. In affected patients, telomere attrition was correlated with age at cancer onset. In all patients, a t test was used to assess the linearity of the regression. RESULTS: A significant correlation between telomere length and age was observed in both affected and unaffected mutation carriers (P = .0016 and P = .004, respectively) and in mutS homolog 2 (MSH2) mutation carriers (P = .0002) but not in mutL homolog 1 (MLH1) mutation carriers. Telomere attrition was correlated significantly with age at onset in MSH2 carriers (P = .004), whereas an opposite trend toward longer telomeres in patients with delayed onset was observed in MLH1 carriers. CONCLUSIONS: The current data suggested that telomere dynamics differ between MLH1 and MSH2 mutation carriers. It is possible that subtle, gene-specific mechanisms can be linked to cancer onset and anticipation in patients with Lynch syndrome.

Integrated analysis of unclassified variants in mismatch repair genes.

PURPOSE: Lynch syndrome is a genetic disease that predisposes to colorectal tumors, caused by mutation in mismatch repair genes. The use of genetic tests to identify mutation carriers does not always give perfectly clear results, as happens when an unclassified variant is found. This study aimed to define the pathogenic role of 35 variants present in MSH2, MLH1, MSH6, and PMS2 genes identified in our 15-year case study. METHODS: We collected clinical and molecular data of all carriers, and then we analyzed the variants pathogenic role with web tools and molecular analyses. Using a Bayesian approach, we derived a posterior probability of pathogenicity and classified each variant according to a standardized five-class system. RESULTS: The MSH2 p.Pro349Arg, p.Met688Arg, the MLH1 p.Gly67Arg, p.Thr82Ala, p.Lys618Ala, the MSH6 p.Ala1236Pro, and the PMS2 p.Arg20Gln were classified as pathogenic, and the MSH2 p.Cys697Arg and the PMS2 p.Ser46Ile were classified as likely pathogenic. Seven variants were likely nonpathogenic, 3 were nonpathogenic, and 16 remained uncertain. CONCLUSION: Quantitative assessment of several parameters and their integration in a multifactorial likelihood model is the method of choice for classifying the variants. As such classifications can be associated with surveillance and testing recommendations, the results and the method developed in our study can be useful for helping laboratory geneticists in evaluation of genetic tests and clinicians in the management of carriers.

Lymph node micrometastasis and survival of patients with Stage I (Dukes' A) colorectal carcinoma.

OBJECTIVE: Although patients with Stage I colorectal cancer show an excellent prognosis, a few of them die of metastatic disease. In this subgroup of individuals, the search of occult metastasis might reveal that early dissemination of tumor cells could be the cause of cancer progression. MATERIAL AND METHODS: Through a Cancer Registry, we selected all patients with Stage I disease who died of metastatic tumor; a total of 32 patients were identified and in 25 of them paraffin-embedded material was available. The group was matched to 70 Stage I patients with favorable prognosis (controls). In cases and controls resected lymph nodes were cut, and micrometastases were searched using pan-cytokeratin antibodies. RESULTS: Micrometastases were detected in 18 of 25 (72%) Stage I patients who died of the disease, while they were almost absent among controls (1 of 70, p < 0.001 by χ(2) test). Vascular invasion and tumor budding were more frequent among Stage I patients with an unfavorable prognosis than in controls. By regression analyses, micrometastases (HR 12.3, CI 4.8-32) and vascular invasion (HR 3.5, CI 1.4-8.5) maintained an independent association with prognosis (cancer-specific survival). CONCLUSION: Micrometastasis in the lymph nodes can be revealed in the majority of patients with early colorectal cancer who die of tumor progression, while they appear extremely rare in Stage I individuals with good prognosis. The selection of patients through histology (vascular invasion) and search of occult metastatic cells might represent a way to identify individuals who might benefit from adjuvant chemotherapy.

Definition and management of colorectal polyposis not associated with APC/MUTYH germline pathogenic variants: AIFEG consensus statement.

An expert consensus panel convened by the Italian Association for Inherited and Familial Gastrointestinal Tumors (Associazione Italiana per lo Studio della Familiarità ed Ereditarietà dei Tumori Gastrointestinali, AIFEG) reviewed the literature and agreed on a number of position statements regarding the definition and management of polyposis coli without an identified pathogenic mutation on the APC or MUTYH genes, defined in the document as NAMP (non-APC/MUTYH polyposis).

Endoscopic Papillectomy for Ampullary Adenomas: Different Outcomes in Sporadic Tumors and Those Associated with Familial Adenomatous Polyposis.

BACKGROUND: Ampullary adenomas are rare and potentially malignant. Surgery was the standard treatment but endoscopic papillectomy (EP) is a possible alternative. AIM: We retrospectively evaluated the principal clinical outcomes of EP in all patients referred to our unit also dividing sporadic ampullary adenoma (SAA) from familial adenomatous polyposis (FAP)-associated adenomas. METHODS: All consecutive patients who underwent endoscopic papillectomy because of ampullary adenoma were considered. The primary outcome was the technical success of EP. Secondary outcomes included the number of procedures, the adverse event rate, the recurrence rate, the concordance of histology pre- and post-EP, and the evaluation of factors related to technical success. RESULTS: Between January 2001 and December 2015, sixty-two patients were included (21 FAP and 41 SAA). Technical success was achieved in 75.8% and was different in the two groups (FAP 95.2%, SAA 65.8%, p 0.025). Intraductal invasion was negatively associated with technical success (41.7% vs. 84.0%; p 0.005). The intestinal subtype was predictive of success (79.7% vs. 0%; p 0.012) as well as en bloc resection (90.3% vs. 61.3%; p 0.016). Adverse events were reported in 14 patients (22.6%). CONCLUSIONS: EP is an effective and safe procedure and is a viable alternative to surgery. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03494543.

Relative role of APC and MUTYH mutations in the pathogenesis of familial adenomatous polyposis.

OBJECTIVE: Familial adenomatous polyposis (FAP) is an interesting model for the study of colorectal tumour. Two genes contribute to the FAP phenotype - APC and MUTYH - but their relative role is still undefined. The objective of this study was to evaluate the contribution of the two genes to the pathogenesis of FAP by means of a series of FAP families. MATERIAL AND METHODS: Sixty-one unrelated families with a diagnosis of FAP and a total of 187 affected individuals were evaluated. After extracting DNA, APC and MUTYH genes were sequenced. RESULTS: In the whole series of patients, colectomy with ileorectal anastomosis was the most frequent surgery, although the number of patients treated by total proctocolectomy and ileoanal anastomosis was increasing. Duodenal and jejunal-ileal adenomas were present in more than half of the patients. Constitutional mutations were detected in 37 of the 45 families (82.2%); there were 33 families with APC and 4 with MUTYH alterations. Age at onset of polyposis and age at surgery were 10-15 years delayed for carriers of MUTYH mutations; cancer at diagnosis was frequent, and extracolonic manifestations were diagnosed in the majority of MUTYH-positive families. MUTYH-associated polyposis showed the horizontal transmission expected for recessive inheritance (at variance with the dominant pattern seen with APC mutations). CONCLUSIONS: At least two genes are associated with the FAP phenotype. APC mutations account for the majority of cases, while MUTYH mutations can be observed in 10% of patients. There are few but definite differences between APC- and MUTYH-associated FAP, such as age at diagnosis and pattern of transmission.

O6-methylguanine-DNA methyltransferase promoter hypermethylation in colorectal carcinogenesis.

Epigenetic alterations have been reported in colorectal neoplasia which can either complement or in some cases be predisposed to genetic alterations such as K-ras mutations. We examined the promoter methylation status of the CDKN2A and O6-methylguanine-DNA methyltransferase (MGMT) genes, after sodium bisulfite conversion and DNA amplification with methylation specific PCR. Moreover, we searched for G to A transitions in codons 12 and 13 of the K-ras oncogene in normal colorectal mucosae, aberrant crypt foci (ACF, early premalignant lesions) and carcinomas. CDKN2A hypermethylation was an infrequent event in ACF (2 of 26, 7.7%). On the contrary, MGMT hypermethylation was found in the normal mucosae (3 of the 12 samples, 25%), in 14 of the 26 ACF (53.8%) and in 7 of the 9 (77.8%) carcinomas examined. K-ras mutations were evident in 6 ACF (23%) and in 3 carcinomas (33.3%), mostly associated with MGMT promoter hypermethylation. These findings strongly support the hypothesis that epigenetic mechanisms play an important role in the early steps of colorectal carcinogenesis.

Microsatellite instability and colorectal cancer prognosis.

PURPOSE: Many studies have evaluated the role of high levels of microsatellite instability (MSI) as a prognostic marker and predictor of the response to chemotherapy in colorectal cancer (CRC); however, the results are not conclusive. The aim of this study was to analyze the prognostic significance of high levels of MSI (MSI-H) in CRC patients in relation to fluorouracil-based chemotherapy. EXPERIMENTAL DESIGN: In three different institutions, 1,263 patients with CRC were tested for the presence of MSI, and CRC-specific survival was then analyzed in relation to MSI status, chemotherapy, and other clinical and pathologic variables. RESULTS: Two hundred and fifty-six tumors were MSI-H (20.3%): these were more frequently at a less advanced stage, right-sided, poorly differentiated, with mucinous phenotype, and expansive growth pattern than microsatellite stable carcinomas. Univariate and multivariate analyses of 5-year-specific survival revealed stage, tumor location, grade of differentiation, MSI, gender, and age as significant prognostic factors. The prognostic advantage of MSI tumors was particularly evident in stages II and III in which chemotherapy did not significantly affect the survival of MSI-H patients. Finally, we analyzed survival in MSI-H patients in relation to the presence of mismatch repair gene mutations. MSI-H patients with hereditary non-polyposis colorectal cancer showed a better prognosis as compared with sporadic MSI-H; however, in multivariate analysis, this difference disappeared. CONCLUSIONS: The type of genomic instability could influence the prognosis of CRC, in particular in stages II and III. Fluorouracil-based chemotherapy does not seem to improve survival among MSI-H patients. The survival benefit for patients with hereditary non-polyposis colorectal cancer is mainly determined by younger age and less advanced stage as compared with sporadic MSI-H counterpart.

Aberrant DNA methylation profiles of inherited and sporadic colorectal cancer.

BACKGROUND: Aberrant DNA methylation has been widely investigated in sporadic colorectal carcinomas (CRCs), and extensive work has been performed to characterize different methylation profiles of CRC. Less information is available about the role of epigenetics in hereditary CRC and about the possible clinical use of epigenetic biomarkers in CRC, regardless of the etiopathogenesis. Long interspersed nucleotide element 1 (LINE-1) hypomethylation and gene-specific hypermethylation of 38 promoters were analyzed in multicenter series of 220 CRCs including 71 Lynch (Lynch colorectal cancer with microsatellite instability (LS-MSI)), 23 CRCs of patients under 40 years in which the main inherited CRC syndromes had been excluded (early-onset colorectal cancer with microsatellite stability (EO-MSS)), and 126 sporadic CRCs, comprising 28 cases with microsatellite instability (S-MSI) and 98 that were microsatellite stable (S-MSS). All tumor methylation patterns were integrated with clinico-pathological and genetic characteristics, namely chromosomal instability (CIN), TP53 loss, BRAF, and KRAS mutations. RESULTS: LS-MSI mainly showed absence of extensive DNA hypo- and hypermethylation. LINE-1 hypomethylation was observed in a subset of LS-MSI that were associated with the worse prognosis. Genetically, they commonly displayed G:A transition in the KRAS gene and absence of a CIN phenotype and of TP53 loss. S-MSI exhibited a specific epigenetic profile showing low rates of LINE-1 hypomethylation and extensive gene hypermethylation. S-MSI were mainly characterized by MLH1 methylation, BRAF mutation, and absence of a CIN phenotype and of TP53 loss. By contrast, S-MSS showed a high frequency of LINE-1 hypomethylation and of CIN, and they were associated with a worse prognosis. EO-MSS were a genetically and epigenetically heterogeneous group of CRCs. Like LS-MSI, some EO-MSS displayed low rates of DNA hypo- or hypermethylation and frequent G:A transitions in the KRAS gene, suggesting that a genetic syndrome might still be unrevealed in these patients. By contrast, some EO-MSS showed similar features to those observed in S-MSS, such as LINE-1 hypomethylation, CIN, and TP53 deletion. In all four classes, hypermethylation of ESR1, GATA5, and WT1 was very common. CONCLUSIONS: Aberrant DNA methylation analysis allows the identification of different subsets of CRCs. This study confirms the potential utility of methylation tests for early detection of CRC and suggests that LINE-1 hypomethylation may be a useful prognostic marker in both sporadic and inherited CRCs.