High incidence of advanced colorectal neoplasia during endoscopic surveillance in serrated polyposis syndrome.

BACKGROUND: Serrated polyposis syndrome (SPS) has been associated with an increased risk of colorectal cancer (CRC). Accordingly, intensive surveillance with annual colonoscopy is advised. The aim of this multicenter study was to describe the risk of advanced lesions in SPS patients undergoing surveillance, and to identify risk factors that could guide the prevention strategy. METHODS: From March 2013 to April 2015, 296 patients who fulfilled criteria I and/or III for SPS were retrospectively recruited at 18 centers. We selected patients in whom successful clearing colonoscopy had been performed and who underwent subsequent endoscopic surveillance. Advanced neoplasia was defined as CRC, advanced adenoma, or advanced serrated lesion that were ≥ 10 mm and/or with dysplasia. Cumulative incidence of advanced neoplasia was calculated and independent predictors of advanced neoplasia development were identified. RESULTS: In 152 SPS patients a total of 315 surveillance colonoscopies were performed (median 2, range 1 - 7). The 3-year cumulative incidence of CRC and advanced neoplasia were 3.1 % (95 % confidence interval [CI] 0 - 6.9) and 42.0 % (95 %CI 32.4 - 51.7), respectively. Fulfilling both I + III criteria and the presence of advanced serrated lesions at baseline colonoscopy were independent predictors of advanced neoplasia development (odds ratio [OR] 1.85, 95 %CI 1.03 - 3.33, P  = 0.04 and OR 2.62, 95 %CI 1.18 - 5.81, P  = 0.02, respectively). During follow-up, nine patients (5.9 %) were referred for surgery for invasive CRC (n = 4, 2.6 %) or because of polyp burden (n = 5, 3.3 %). After total colectomy, 17.9 % patients developed advanced neoplasia in the retained rectum. CONCLUSIONS: Patients with SPS have a substantial risk of developing advanced neoplasia under endoscopic surveillance, whereas CRC incidence is low. Personalized endoscopic surveillance based on polyp burden and advanced serrated histology could help to optimize prevention in patients with SPS.

Low efficacy of metronidazole in the eradication of Blastocystis hominis in symptomatic patients: Case series and systematic literature review. / Escasa eficacia de metronidazol en la erradicación de Blastocystis hominis en pacientes sintomáticos: serie de casos y revisión sistemática de la literatura.

INTRODUCTION: Blastocystis hominis (B. hominis) is a protozoan commonly found in the gastrointestinal tract. There are doubts about its clinical significance. Metronidazole (MTZ) is the recommended first-line treatment. MATERIALS AND METHODS: A retrospective review was carried out between 2011 and 2012. A total of 151 samples were randomly selected from 383 samples positive for B. hominis. Inclusion criteria were: suggestive symptoms, treatment indication and microbiological follow-up. A systematic review was performed of all studies that evaluated the effect of MTZ on B. hominis infection. RESULTS: Forty-six patients met the inclusion criteria (64% women; age, 44.2±2 years). MTZ was used in 39 patients, 31 of whom obtained a clinical response (79.5%) but only 15 a microbiological response (48.4%). No dose-effect relationship was observed. Twenty patients with no initial microbiological response received a second round of treatment (MTZ, cotrimoxazole, paramomycin, others), with a microbiological response in 70%. Overall, B. hominis was cured in 72% (95% CI: 57%-83%). Of 54 treatments associated with a clinical response, a microbiological response occurred in 31 (57%), while in the remaining 12 with no clinical response, microbiological cure was observed in only 2 (17%) (P=.022). The eradication rate in the systematic review varied between 0% and 100%. CONCLUSIONS: There seems to be a relationship between the clinical and microbiological response to B. hominis treatment. The microbiological response to MTZ treatment is insufficient in our geographical setting. The systematic review shows that the response to MTZ is very variable.

Colorectal cancer risk factors in patients with serrated polyposis syndrome: a large multicentre study.

OBJECTIVE: Serrated polyposis syndrome (SPS) is associated with an increased colorectal cancer (CRC) risk, although the magnitude of the risk remains uncertain. Whereas intensive endoscopic surveillance for CRC prevention is advised, predictors that identify patients who have high CRC risk remain unknown. We performed a multicentre nationwide study aimed at describing the CRC risk in patients with SPS and identifying clinicopathological predictors independently associated with CRC. DESIGN: From March 2013 through September 2014, patients with SPS were retrospectively recruited at 18 Spanish centres. Data were collected from medical, endoscopy and histopathology reports. Multivariate logistic regression was performed to identify CRC risk factors. RESULTS: In 296 patients with SPS with a median follow-up time of 45 months (IQR 26-79.7), a median of 26 (IQR 18.2-40.7) serrated polyps and 3 (IQR 1-6) adenomas per patient were detected. Forty-seven patients (15.8%) developed CRC at a mean age of 53.9±12.8, and 4 out of 47 (8.5%) tumours were detected during surveillance (cumulative CRC incidence 1.9%). Patients with >2 sessile serrated adenomas/polyps (SSA/Ps) proximal to splenic flexure and ≥1 proximal SSA/P with high-grade dysplasia were independent CRC risk factors (incremental OR=2, 95% CI 1.22 to 3.24, p=0.006). Patients with no risk factors showed a 55% decrease in CRC risk (OR=0.45, 95% CI 0.24 to 0.86, p=0.01). CONCLUSIONS: Patients with SPS have an increased risk of CRC, although lower than previously published. Close colonoscopy surveillance in experienced centres show a low risk of developing CRC (1.9% in 5 years). Specific polyp features (SSA/P histology, proximal location and presence of high-grade dysplasia) should be used to guide clinical management.

Susceptibility genetic variants associated with colorectal cancer risk correlate with cancer phenotype.

BACKGROUND & AIMS: Ten common low-penetrant genetic variants have been consistently associated with colorectal cancer (CRC) risk; little is known about the correlation between these variants and CRC phenotype. Characterization of such a correlation would improve CRC management and prevention programs. We assessed the association between these genetic variants and CRC phenotype in patients and modeled pairwise combinations to detect epistasis. METHODS: The validation population corresponded to a prospective, multicenter, population-based cohort (EPICOLON I) of 1096 patients with newly diagnosed CRC. The replication set was an independent, prospective, multicenter Spanish cohort (EPICOLON II) of 895 patients with newly diagnosed CRC. For individual single nucleotide polymorphism (SNP) association analyses, a multivariate method using logistic regression was applied in EPICOLON I and subsequently prospectively validated in EPICOLON II. Interactions between SNPs were assessed using the likelihood ratio test. RESULTS: Validated results confirmed that the C allele on 8q23.3 (rs16892766) was significantly associated with advanced-stage tumors (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.15-1.90; P value = 4.9 x 10(-3)). The G allele on 8q24.21 (rs6983267) was more common in patients with a familial history of CRC (OR, 2.02; 95% CI, 1.35-3.03; P value = 3.9 x 10(-4)). The combination of rs6983267 on 8q24.21 and rs9929218 on 16q22.2 was associated with a history of colorectal adenoma (carriers of GG and AA, respectively; OR, 2.28; 95% CI, 1.32-3.93; P = 5.0 x 10(-4)). CONCLUSIONS: CRC susceptibility variants at 8q23.3, 8q24.21, and 16q22.2 appear to be associated with cancer phenotype. These findings might be used to develop screening and surveillance strategies.

Case-control study for colorectal cancer genetic susceptibility in EPICOLON: previously identified variants and mucins.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family. RESULTS: None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles). CONCLUSIONS: ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.

Long-Term Experience with Hyperthermic Chemotherapy (HIVEC) Using Mitomycin-C in Patients with Non-Muscle Invasive Bladder Cancer in Spain.

(1) Background: Intravesical mitomycin-C (MMC) combined with hyperthermia is increasingly used in non-muscle invasive bladder cancer (NMIBC), especially in the context of a relative BCG shortage. We aim to determine real-world data on the long-term treatment outcomes of adjunct hyperthermic intravesical chemotherapy (HIVEC) with MMC and a COMBAT® bladder recirculation system (BRS); (2) Methods: A prospective observational trial was performed on patients with NMIBC treated with HIVEC using BRS in nine academic institutions in Spain between 2012-2020 (HIVEC-E). Treatment effectiveness (recurrence, progression and overall mortality) was evaluated in patients treated with HIVEC MMC 40mg in the adjuvant setting, with baseline data and a clinical follow-up, that comprise the Full Analysis Set (FAS). Safety, according to the number and severity of adverse effects (AEs), was evaluated in the safety (SAF) population, composed by patients with at least one adjunct HIVEC MMC instillation; (3) Results: The FAS population (n = 502) received a median number of 8.78 ± 3.28 (range 1-20) HIVEC MMC instillations. The median follow-up duration was 24.5 ± 16.5 (range 1-81) months. Its distribution, based on EAU risk stratification, was 297 (59.2%) for intermediate and 205 (40.8%) for high-risk. The figures for five-year recurrence-free and progression-free survival were 50.37% (53.3% for intermediate and 47.14% for high-risk) and 89.83% (94.02% for intermediate and 84.23% for high-risk), respectively. A multivariate analysis identified recurrent tumors (HR 1.83), the duration of adjuvant HIVEC therapy <4 months (HR 1.72) and that high-risk group (HR 1.47) were at an increased risk of recurrence. Independent factors of progression were high-risk (HR 3.89), recurrent tumors (HR 3.32) and the induction of HIVEC therapy without maintenance (HR 2.37). The overall survival was determined by patient age at diagnosis (HR 3.36) and the treatment duration (HR 1.82). The SAF population (n = 592) revealed 406 (68.58%) patients without AEs and 186 (31.42%) with at least one AE: 170 (28.72%) of grade 1-2 and 16 (2.7%) of grade 3-4. The most frequent AEs were dysuria (10%), pain (7.1%), urgency (5.7%), skin rash (4.9%), spasms (3.7%) and hematuria (3.6%); (4) Conclusions: HIVEC using BRS is efficacious and well tolerated. A longer treatment duration, its use in naïve patients and the intermediate-risk disease are independent determinants of success. Furthermore, a monthly maintenance of adjunct MMC HIVEC diminishes the progression rate of NMIBC.

Telomerase mRNA expression and immunohistochemical detection as a biomarker of malignant transformation in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease is a premalignant condition for developing colorectal cancer. Since a correlation has been suggested between telomere length, chromosomal instability and neoplastic transformation in this setting, we sought to investigate whether telomerase expression in colorectal mucosa may constitute a biomarker for malignant transformation in patients with inflammatory bowel disease. PATIENTS AND METHODS: Forty-seven patients with inflammatory bowel disease with and without cancer or dysplasia were evaluated for human telomerase reverse transcriptase hTERT immunostaining in paraffin-embedded, formalin-fixed colorectal tissues. In addition, hTERT mRNA expression was assessed in fresh frozen specimens from a second set of 35 patients with inflammatory bowel disease at high or low risk for neoplastic transformation. RESULTS: Five out of 10 patients (50%) with colorectal cancer or high-grade dysplasia exhibited hTERT immunochemical detection in adjacent, non-transformed colonic mucosa. However, this phenomenon was also observed in non-affected mucosa of patients with either long-standing (13 out of 19 patients; 68%) or short duration (13 out of 18 patients; 72%) disease without cancer or dysplasia. On the other hand, hTERT mRNA expression in non-affected colorectal mucosa from patients at high risk for neoplastic transformation due to long-standing disease was higher than in those at low risk (7.42+/-6.43 vs. 2.87+/-1.47, respectively; p=0.006). CONCLUSIONS: Whereas hTERT immunostaining provides equivocal results, the observation that patients at high risk for colorectal cancer because of long-standing inflammatory bowel disease overexpress hTERT mRNA in non-affected colorectal mucosa suggests its potential usefulness as a biomarker of the risk of malignant transformation.

[Epigenetics of cancer]. / Epigenética del cáncer.

The pathogenesis of cancer includes both genetic and epigenetic mechanisms. The term <> refers to any alteration of gene expression, potentially hereditary, which is not accompanied by a modification in the DNA sequence. The effects of epigenetic changes include alteration of DNA transcription, aberrant activation of specific genes, predisposition of genetic instability through alteration of the control of chromosome replication and silencing of the genes implicated in cancer initiation and progression. Among the various epigenetic alterations that lead to altered gene expression, methylation is the main epigenomic mechanism implicated in cancer, whether through a phenomenon of overall hypomethylation or hypermethylation localized in the promoters of specific genes.

Endoscopic requirements of colorectal cancer screening programs in average-risk population. Estimation according to a Markov model.

BACKGROUND: Although colorectal cancer (CRC) screening strategies are quite common in the United States, their systematic introduction in Europe has been delayed until the year 2008. To estimate endoscopic requirements of four different CRC screening strategies (annual and biennial fecal occult blood testing (FOBT), flexible sigmoidoscopy every 5 years, and colonoscopy every 10 years) in an average-risk population. METHODS: A long-term Markov process model was designed combining three adherence rates for the four above-mentioned screening strategies in individuals aged from 50 to 74. Estimations included endoscopic procedures performed for both screening and surveillance purposes. Models were adjusted for age-related adenoma and CRC incidence rates, life expectancy, and cancer-related survival. RESULTS: The mean number of annual colonoscopies per 100,000 individuals aged 50-74 ranged from 100 to 271 for annual FOBT, from 75 to 203 for biennial FOBT, from 222 to 601 for sigmoidoscopy, and from 903 to 2,449 for colonoscopy-based strategies, depending on the adherence rate. According to these estimations, annual and biennial FOBT strategies would generate a slight decrease of current endoscopic activity (1.4-3.8% and 2.7-7.2%, respectively), whereas sigmoidoscopy and colonoscopy-based strategies would induce a 4.7-12.8% and 32-87% increase, respectively, with respect to a non-screening scenario. The model confirmed a 3-16% mean reduction of CRC incidence depending on the strategy and adherence rate. CONCLUSION: Whereas endoscopic capacity exists for widespread CRC screening with annual or biennial FOBT, implementation of potentially more effective strategies, such as flexible sigmoidoscopy or colonoscopy, would result in a significant increase of current endoscopic resources.

Identification of MYH mutation carriers in colorectal cancer: a multicenter, case-control, population-based study.

BACKGROUND & AIMS: Whereas it has conclusively been demonstrated that biallelic MutY human homolog (MYH) mutations confer a significant risk for colorectal cancer (CRC), the influence of monoallelic mutations remains controversial. Characterization of MYH-associated CRC is critical to identify individuals who might benefit from preventive strategies. This prospective, multicenter, case-control, population-based study was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) devising a set of clinical criteria to identify MYH carriers among newly diagnosed CRC. METHODS: Genotyping for Y165C and G382D was performed by TaqMan technology. Single-stranded conformation polymorphism analysis was performed in heterozygotes to screen for mutations in the entire gene. All individuals were re-screened for any additional pathogenic variant. RESULTS: Biallelic and monoallelic MYH mutations were found in 8 (0.7%) and 19 (1.7%) of 1116 CRC patients, respectively. None of the 934 control subjects carried biallelic mutations, whereas 22 (2.3%) of them were monoallelic carriers. In a meta-analysis including all previous case-control studies, monoallelic MYH carriers were not at increased risk for CRC (odds ratio, 1.11; 95% confidence interval, 0.90-1.37), although a significant association was found with the Y165C mutation in either homozygotes or heterozygotes (odds ratio, 1.67; 95% confidence interval, 1.17-2.40). Furthermore, presence of more than 15 synchronous colorectal adenomas or CRC diagnosed before the age of 50 years was the most effective set of criteria for the identification of biallelic MYH mutation carriers. CONCLUSIONS: This study proposes the first set of clinical criteria designed to identify CRC patients with biallelic MYH mutations, and it argues against an increased risk for monoallelic carriers.

Identification of Lynch syndrome: how should we proceed in the 21st century?

Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is the most common form of hereditary colorectal cancer. Although great advances in the understanding of its molecular basis have taken place in the last decade, optimal selection of individuals for HNPCC genetic testing remains controversial. This is especially relevant since colonoscopy has been proven effective for reducing colorectal cancer incidence and mortality in individuals at-risk for this disorder. In this manuscript, we summarize the most significant contributions to this important issue that have appeared in the last few years.

[Cyclooxygenase 2 and colorectal cancer: therapeutic implications]. / Ciclooxigenasa 2 y cáncer colorrectal: implicaciones terapéuticas.

There is epidemiological evidence that suggests an inverse association between the consumption of non-steroidal anti-inflammatories (NSAIDs) and the risk of developing certain neoplasms. This association led to the identification of the therapeutic target of these drugs, cyclooxygenase type 2 (COX-2). Later studies have demonstrated that COX-2 is over-expressed in many malignant and pre-malignant lesions of different origins, among which are included colorectal neoplasms. This factor explains the beneficial effect observed with the use of classic NSAIDs and more recently, with selective COX-2 inhibitors (coxibs), in the treatment and/or prevention of several neoplasms.

[High-grade dysplasia as a risk factor of metachronous advanced colorectal neoplasms in patients with advanced adenomas]. / Displasia de alto grado como factor de riesgo de neoplasia colorrectal avanzada metacrónica, en pacientes con adenomas avanzados.

BACKGROUND: Patients with advanced adenomas (AA) have a high risk of developing advanced colorectal neoplasms. Therefore, shorter monitoring intervals have been recommended in this patient subgroup. High grade dysplasia (HGD) is the main marker of cancer transformation. However, its predictive value for developing advanced neoplams in patients with advanced adenoma is unknown. AIM: To investigate if HGD increases the risk for developing advanced neoplasms in patients with AA. METHODS: Between January 1996 and December 1997 every patient with an AA endoscopically resected were considered for inclusion. Patients with a history of colorectal cancer (CRC), inflammatory bowel disease, familial adenomatous polyposis or patients who met the Amsterdam criteria, and those without colonoscopic monitoring were excluded. We assessed the development of advanced neoplasms during the study period. RESULTS: 71 patients were included and classified into 2 groups, depending on the presence (n = 49) or lack (n = 22) of HGD in the initial colonoscopy. The probability of developing advanced neoplasms (log rank, p = 0.47; Breslow, p = 0.58) or AA with HGD (log rank, p = 0.47; Breslow, p = 0.53) in the study period was similar between both groups. The number of metachronic polyps (p = 0.67), adenomas (p = 0.73), AA (p = 0.93) and AA with HGD (p = 0.88) was also similar. CONCLUSION: The risk of developing advanced neoplasms is not different between AA with HGD and those with other characteristics of AA (villous pattern and larger than 1 cm). Therefore, changes in monitoring intervals are not warranted.

POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer.

Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.

Multiple sporadic colorectal cancers display a unique methylation phenotype.

Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC). Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP); KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in ß value ≥0.1). Methylight assays validated the results for 4 selected genes (p = 0.0002). Eight out of 12(66.6%) multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2%) solitary tumors (p = 0.004). Interestingly, 76 out of the 102 (74.5%) hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

MSH6 and MUTYH deficiency is a frequent event in early-onset colorectal cancer.

PURPOSE: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. EXPERIMENTAL DESIGN: We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. RESULTS: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. CONCLUSIONS: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives.