Mutational signature profiling classifies subtypes of clinically different mismatch-repair-deficient tumours with a differential immunogenic response potential.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

Clinical and Pathological Characterization of Lynch-Like Syndrome.

BACKGROUND & AIMS: Lynch syndrome is characterized by DNA mismatch repair (MMR) deficiency. Some patients with suspected Lynch syndrome have DNA MMR deficiencies but no detectable mutations in genes that encode MMR proteins-this is called Lynch-like syndrome (LLS). There is no consensus on management of patients with LLS. We collected data from a large series of patients with LLS to identify clinical and pathology features. METHODS: We collected data from a nationwide-registry of patients with colorectal cancer (CRC) in Spain. We identified patients whose colorectal tumors had loss of MSH2, MSH6, PMS2, or MLH1 (based on immunohistochemistry), without the mutation encoding V600E in BRAF (detected by real-time PCR), and/or no methylation at MLH1 (determined by methylation-specific multiplex ligation-dependent probe amplification), and no pathogenic mutations in MMR genes, BRAF, or EPCAM (determined by DNA sequencing). These patients were considered to have LLS. We collected data on demographic, clinical, and pathology features and family history of neoplasms. The χ2 test was used to analyze the association between qualitative variables, followed by the Fisher exact test and the Student t test or the Mann-Whitney test for quantitative variables. RESULTS: We identified 160 patients with LLS; their mean age at diagnosis of CRC was 55 years and 66 patients were female (41%). The Amsterdam I and II criteria for Lynch syndrome were fulfilled by 11% of cases and the revised Bethesda guideline criteria by 65% of cases. Of the patients with LLS, 24% were identified in universal screening. There were no proportional differences in sex, indication for colonoscopy, immunohistochemistry, pathology findings, or personal history of CRC or other Lynch syndrome-related tumors between patients who met the Amsterdam and/or Bethesda criteria for Lynch syndrome and patients identified in universal screening for Lynch syndrome, without a family history of CRC. CONCLUSIONS: Patients with LLS have homogeneous clinical, demographic, and pathology characteristics, regardless of family history of CRC.

A novel non-invasive colorectal cancer diagnostic method: Volatile organic compounds as biomarkers.

INTRODUCTION: Population-based fecal tests for colorectal cancer (CRC) screening have shown to reduce mortality thanks to the early detection of the disease. However, currently available fecal tests are limited in their sensitivity and specificity. Our aim is to look for volatile organic compounds in fecal samples as biomarkers for CRC detection. MATERIAL AND METHODS: Eighty participants were included; 24 had adenocarcinoma, 24 had adenomatous polyps and 32 presented no neoplasms. Fecal samples were collected 48 h preceding the colonoscopy from all participants, except CRC patient samples that were collected after 3-4 weeks from the colonoscopy. Magnetic headspace adsorptive extraction (Mag-HSAE) followed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) was performed on stool samples to identify volatile organic compounds as biomarkers. RESULTS: p-Cresol was significantly more abundant in the cancer samples (P < 0.001) with an area under the curve (AUC) of 0.85 (CI 95%; 0.737-0.953), having a sensitivity and specificity of 83% and 82%, respectively. In addition, 3(4H)-dibenzofuranone,4a,9b-dihydro-8,9b-dimethyl- (3(4H)-DBZ) was also more abundant in the cancer samples (P < 0.001) with an AUC of 0.77 (CI 95%; 0.635-0.905), sensitivity of 78% and specificity of 75%. When combined (p-cresol and 3(4H)-DBZ), the AUC was 0.86, sensitivity 87% and specificity 79%. p-Cresol also appeared to be promising as a biomarker for pre-malignant lesions with an AUC of 0.69 (CI 95%; 0.534-0.862), sensitivity 83% and specificity 63%, P = 0.045. CONCLUSIONS: Volatile organic compounds emitted from feces and determined by a sensitive analytical methodology (Mag-HSAE-TD-GC-MS), employing a magnetic graphene oxide as extractant phase, could be used as a potential screening technology for CRC and pre-malignant lesions.

Lynch-like Syndrome: Potential Mechanisms and Management.

Lynch syndrome is an autosomal dominant disorder caused by germline mutations in DNA mismatch repair (MMR) system genes, such as MLH1, MSH2, MSH6, or PMS2. It is the most common hereditary colorectal cancer syndrome. Screening is regularly performed by using microsatellite instability (MSI) or immunohistochemistry for the MMR proteins in tumor samples. However, in a proportion of cases, MSI is found or MMR immunohistochemistry is impaired in the absence of a germline mutation in MMR genes, BRAF mutation, or MLH1 hypermethylation. These cases are defined as Lynch-like syndrome. Patients with Lynch-like syndrome represent a mixture of truly hereditary and sporadic cases, with a risk of colorectal cancer in first-degree relatives that is between the risk of Lynch syndrome in families and relatives of sporadic colon cancer cases. Although multiple approaches have been suggested to distinguish between hereditary and sporadic cases, a homogeneous testing protocol and consensus on the adequate classification of these patients is still lacking. For this reason, management of Lynch-like syndrome and prevention of cancer in these families is clinically challenging. This review explains the concept of Lynch-like syndrome, potential mechanisms for its development, and methods for adequately distinguishing between sporadic and hereditary cases of this entity.

Effects of Somatic Methylation in Colonic Polyps on Risk of Developing Metachronous Advanced Colorectal Lesions.

The utility of molecular markers for predicting the risk of metachronous advanced colorectal lesions (MACLs) remains poorly investigated. We examined the relationship between somatic hypermethylation in polyps at baseline and the risk of developing MACL. This retrospective cohort study included 281 consecutive patients with colonic polyps who were enrolled between 2007 and 2009 and followed-up until 2014. MACLs were defined as adenomas of ≥10 mm, high-grade dysplasia, or with a villous component; and serrated lesions of ≥10 mm or with dysplasia. In total, 595 polyps were removed at baseline colonoscopy and analyzed for pathological characteristics and CpG island methylator phenotype (CIMP) using the MS-MLPA (Methylation-Specific -- Multiplex Ligation-dependent Probe Amplification) technique. Forty-five patients (16.0%) showed at least one CIMP+ polyp. MACL risk was higher in patients with CIMP+ polyps (odds ratio (OR), 4.50; 95% CI, 1.78-11.4; p = 0.002). Patients with CIMP+ polyps also exhibited shorter time to MACL development (33.8 months vs. 50.1 months; p < 0.001), even with adjustment for polyp size and number (OR, 2.40; 95% CI, 1.33-4.34). Adding CIMP analysis improved the sensitivity (57.0% to 70.9%), negative predictive value (71.1% to 77.3%), and overall accuracy (49.8% to 52.0%) for MACL risk estimation. These results highlight that CIMP may be a useful marker for endoscopic surveillance.

Increased Th17-Related Cytokine Serum Levels in Patients With Multiple Polyps of Unexplained Origin.

OBJECTIVES: Most patients with multiple colonic polyps do not have a known genetic or hereditary origin. Our aim was to analyze the presence of inflammatory cytokines and levels of glucose, insulin, and C-reactive protein (CRP) in patients with multiple colonic polyps. METHODS: Eighty-three patients with 10 or more adenomatous or serrated polyps and 53 control people with normal colonoscopy were included. Smoking habits were registered, and glucose, CRP, and basal insulin in the serum/blood were measured. Quantification of IL-2, IL-4, IL-6, IL-10, IL-11, IL-17A, and IL-23 cytokine levels in the serum was performed by a high-sensitivity enzyme-linked immunosorbent assay. RESULTS: Smoking and diabetes were more prevalent in those with colonic polyps than in the control people (67% vs 16%, P = 0.001; 11% vs 2%, P = 0.048). In addition, the cytokine serum levels were higher, i.e., IL-2 (P = 0.001), IL-4 (P = 0.001), IL-6 (P = 0.001), IL-17A (P = 0.001), IL-23 (P = 0.014), and CRP (P = 0.003). Adjusting for sex, smoking, and diabetes in a multivariate analysis, IL-2, IL-4, IL-6, IL-17A, and IL-23 remained independently elevated in cases with multiple polyps. DISCUSSION: These results indicate that immune responses mediated by Th17 cells may be involved in the pathogenesis of multiple colonic polyps.

Risk of Cancer in Family Members of Patients with Lynch-Like Syndrome.

Lynch syndrome (LS) is a common cause of hereditary colorectal cancer (CRC). Some CRC patients develop mismatch repair deficiency without germline pathogenic mutation, known as Lynch-like syndrome (LLS). We compared the risk of CRC in first-degree relatives (FDRs) in LLS and LS patients. LLS was diagnosed when tumors showed immunohistochemical loss of MSH2, MSH6, and PMS2; or loss of MLH1 with BRAF wild type; and/or no MLH1 methylation and absence of pathogenic mutation in these genes. CRC and other LS-related neoplasms were followed in patients diagnosed with LS and LLS and among their FDRs. Standardized incidence ratios (SIRs) were calculated for CRC and other neoplasms associated with LS among FDRs of LS and LLS patients. In total, 205 LS (1205 FDRs) and 131 LLS families (698 FDRs) had complete pedigrees. FDRs of patients with LLS had a high incidence of CRC (SIR, 2.08; 95% confidence interval (CI), 1.56-2.71), which was significantly lower than that in FDRs of patients with LS (SIR, 4.25; 95% CI, 3.67-4.90; p < 0.001). The risk of developing other neoplasms associated with LS also increased among FDR of LLS patients (SIR, 2.04; 95% CI, 1.44-2.80) but was lower than that among FDR of patients with LS (SIR, 5.01, 95% CI, 4.26-5.84; p < 0.001). FDRs with LLS have an increased risk of developing CRC as well as LS-related neoplasms, although this risk is lower than that of families with LS. Thus, their management should take into account this increased risk.

Colorectal cancer molecular classification using BRAF, KRAS, microsatellite instability and CIMP status: Prognostic implications and response to chemotherapy.

OBJECTIVE: The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. DESIGN: This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). RESULTS: Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P<0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P<0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005). CONCLUSION: We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy.

KRAS and BRAF somatic mutations in colonic polyps and the risk of metachronous neoplasia.

BACKGROUND & AIMS: High-risk features of colonic polyps are based on size, number, and pathologic characteristics. Surveillance colonoscopy is often recommended according to these findings. This study aimed to determine whether the molecular characteristics of polyps might provide information about the risk of metachronous advanced neoplasia. METHODOLOGY: We retrospectively included 308 patients with colonic polyps. A total of 995 polyps were collected and tested for somatic BRAF and KRAS mutations. Patients were classified into 3 subgroups, based on the polyp mutational profile at baseline, as follows: non-mutated polyps (Wild-type), at least one BRAF-mutated polyp, or at least one KRAS-mutated polyp. At surveillance, advanced adenomas were defined as adenomas ≥ 10 mm and/or with high grade dysplasia or a villous component. In contrast, advanced serrated polyps were defined as serrated polyps ≥ 10 mm in any location, located proximal to the splenic flexure with any size or with dysplasia. RESULTS: At baseline, 289 patients could be classified as wild-type (62.3%), BRAF mutated (14.9%), or KRAS mutated (22.8%). In the univariate analysis, KRAS mutations were associated with the development of metachronous advanced polyps (OR: 2.36, 95% CI: 1.22-4.58; P = 0.011), and specifically, advanced adenomas (OR: 2.42, 95% CI: 1.13-5.21; P = 0.023). The multivariate analysis, adjusted for age and sex, also showed associations with the development of metachronous advanced polyps (OR: 2.27, 95% CI: 1.15-4.46) and advanced adenomas (OR: 2.23, 95% CI: 1.02-4.85). CONCLUSIONS: Our results suggested that somatic KRAS mutations in polyps represent a potential molecular marker for the risk of developing advanced neoplasia.

Serrated colorectal cancer: Molecular classification, prognosis, and response to chemotherapy.

Molecular advances support the existence of an alternative pathway of colorectal carcinogenesis that is based on the hypermethylation of specific DNA regions that silences tumor suppressor genes. This alternative pathway has been called the serrated pathway due to the serrated appearance of tumors in histological analysis. New classifications for colorectal cancer (CRC) were proposed recently based on genetic profiles that show four types of molecular alterations: BRAF gene mutations, KRAS gene mutations, microsatellite instability, and hypermethylation of CpG islands. This review summarizes what is known about the serrated pathway of CRC, including CRC molecular and clinical features, prognosis, and response to chemotherapy.