Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.Peer reviewe

Differential roles of EPS8 in carcinogenesis: Loss of protein expression in a subset of colorectal carcinoma and adenoma

Peer reviewe

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Instituto Multidisciplinario de Biología Celula

Body Weight, Physical Activity, and Risk of Cancer in Lynch Syndrome

Lynch syndrome (LS) increases cancer risk. There is considerable individual variation in LS cancer occurrence, which may be moderated by lifestyle factors, such as body weight and physical activity (PA). The potential associations of lifestyle and cancer risk in LS are understudied. We conducted a retrospective study with cancer register data to investigate associations between body weight, PA, and cancer risk among Finnish LS carriers. The participants (n = 465, 54% women) self-reported their adulthood body weight and PA at 10-year intervals. Overall cancer risk and colorectal cancer (CRC) risk was analyzed separately for men and women with respect to longitudinal and near-term changes in body weight and PA using extended Cox regression models. The longitudinal weight change was associated with an increased risk of all cancers (HR 1.02, 95% CI 1.00–1.04) and CRC (HR 1.03, 1.01–1.05) in men. The near-term weight change was associated with a lower CRC risk in women (HR 0.96, 0.92–0.99). Furthermore, 77.6% of the participants retained their PA category over time. Men in the high-activity group had a reduced longitudinal cancer risk of 63% (HR 0.37, 0.15–0.98) compared to men in the low-activity group. PA in adulthood was not associated with cancer risk among women. These results emphasize the role of weight maintenance and high-intensity PA throughout the lifespan in cancer prevention, particularly in men with LS

Epidemiological, clinical and molecular characterization of Lynch-like syndrome : A population-based study

Colorectal carcinomas that are mismatch repair (MMR)-deficient in the absence of MLH1 promoter methylation or germline mutations represent Lynch-like syndrome (LLS). Double somatic events inactivating MMR genes are involved in the etiology of LLS tumors. Our purpose was to define the clinical and broader molecular hallmarks of LLS tumors and the population incidence of LLS, which remain poorly characterized. We investigated 762 consecutive colorectal carcinomas operated in Central Finland in 2000-2010. LLS cases were identified by a stepwise protocol based on MMR protein expression, MLH1 methylation and MMR gene mutation status. LLS tumors were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations in 578 cancer-relevant genes. Among 107 MMR-deficient tumors, 81 (76%) were attributable to MLH1 promoter methylation and 9 (8%) to germline mutations (Lynch syndrome, LS), leaving 14 LLS cases (13%) (3 remained unclassified). LLS carcinomas were diagnosed at a mean age of 65 years (vs. 44 years in LS, p <0.001), had a proximal to distal ratio of 1:1, and all were BRAF V600E-negative. Two somatic events in MMR genes were identifiable in 11 tumors (79%). As novel findings, the tumors contained an average of 31 nonsynonymous somatic mutations/Mb and 13/14 were CIMP-positive. In conclusion, we establish the epidemiological, clinical and molecular characteristics of LLS in a population-based study design. Significantly more frequent CIMP-positivity and lower rates of somatic mutations make a distinction to LS. The absence of BRAF V600E mutation separates LLS colorectal carcinomas from MLH1-methylated colorectal carcinomas with CIMP-positive phenotype.Peer reviewe

Body Weight, Physical Activity, and Risk of Cancer in Lynch Syndrome

Lynch syndrome (LS) increases cancer risk. There is considerable individual variation in LS cancer occurrence, which may be moderated by lifestyle factors, such as body weight and physical activity (PA). The potential associations of lifestyle and cancer risk in LS are understudied. We conducted a retrospective study with cancer register data to investigate associations between body weight, PA, and cancer risk among Finnish LS carriers. The participants (n = 465, 54% women) self-reported their adulthood body weight and PA at 10-year intervals. Overall cancer risk and colorectal cancer (CRC) risk was analyzed separately for men and women with respect to longitudinal and near-term changes in body weight and PA using extended Cox regression models. The longitudinal weight change was associated with an increased risk of all cancers (HR 1.02, 95% CI 1.00–1.04) and CRC (HR 1.03, 1.01–1.05) in men. The near-term weight change was associated with a lower CRC risk in women (HR 0.96, 0.92–0.99). Furthermore, 77.6% of the participants retained their PA category over time. Men in the high-activity group had a reduced longitudinal cancer risk of 63% (HR 0.37, 0.15–0.98) compared to men in the low-activity group. PA in adulthood was not associated with cancer risk among women. These results emphasize the role of weight maintenance and high-intensity PA throughout the lifespan in cancer prevention, particularly in men with LS

Immunoprofiles and DNA Methylation of Inflammatory Marker Genes in Ulcerative Colitis-Associated Colorectal Tumorigenesis

Immunological and epigenetic changes are interconnected and contribute to tumorigenesis. We determined the immunoprofiles and promoter methylation of inflammation-related genes for colitis-associated colorectal carcinomas (CA-CRC). The results were compared with Lynch syndrome (LS)-associated colorectal tumors, which are characterized by an active immune environment through inherited mismatch repair defects. CA-CRCs (n = 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274, NTSR1, PPARG, PTGS2, PYCARD, SOCS1, and SOCS2), the repair gene MGMT, and eight standard marker genes for the CpG Island Methylator Phenotype (CIMP) were investigated for promoter methylation in CA-CRCs, LS tumors (n = 29), and paired normal mucosae by multiplex ligation-dependent probe amplification. All but one CA-CRCs were microsatellite-stable and all LS tumors were microsatellite-unstable. Most CA-CRCs had a high ICS (55%) and a positive CD274 expression in immune cells (52%). NTSR1 revealed frequent tumor-specific hypermethylation in CA-CRC and LS. When compared to LS mucosae, normal mucosae from patients with CA-CRC showed significantly higher methylation of NTSR1 and most CIMP markers. In conclusion, CA-CRCs share a frequent ICShigh/CD274pos expression pattern with LS tumors. Elevated methylation in normal mucosa may indicate field cancerization as a feature of CA-CRC-associated tumorigenesis

Immunoprofiles and DNA Methylation of Inflammatory Marker Genes in Ulcerative Colitis-Associated Colorectal Tumorigenesis

Immunological and epigenetic changes are interconnected and contribute to tumorigenesis. We determined the immunoprofiles and promoter methylation of inflammation-related genes for colitis-associated colorectal carcinomas (CA-CRC). The results were compared with Lynch syndrome (LS)-associated colorectal tumors, which are characterized by an active immune environment through inherited mismatch repair defects. CA-CRCs (n = 31) were immunohistochemically evaluated for immune cell scores (ICSs) and PDCD1 and CD274 expression. Seven inflammation-associated genes (CD274, NTSR1, PPARG, PTGS2, PYCARD, SOCS1, and SOCS2), the repair gene MGMT, and eight standard marker genes for the CpG Island Methylator Phenotype (CIMP) were investigated for promoter methylation in CA-CRCs, LS tumors (n = 29), and paired normal mucosae by multiplex ligation-dependent probe amplification. All but one CA-CRCs were microsatellite-stable and all LS tumors were microsatellite-unstable. Most CA-CRCs had a high ICS (55%) and a positive CD274 expression in immune cells (52%). NTSR1 revealed frequent tumor-specific hypermethylation in CA-CRC and LS. When compared to LS mucosae, normal mucosae from patients with CA-CRC showed significantly higher methylation of NTSR1 and most CIMP markers. In conclusion, CA-CRCs share a frequent ICShigh/CD274pos expression pattern with LS tumors. Elevated methylation in normal mucosa may indicate field cancerization as a feature of CA-CRC-associated tumorigenesis

No Difference in Penetrance between Truncating and Missense/Aberrant Splicing Pathogenic Variants in MLH1 and MSH2: A Prospective Lynch Syndrome Database Study

Background. Lynch syndrome is the most common genetic predisposition for hereditary cancer. Carriers of pathogenic changes in mismatch repair (MMR) genes have an increased risk of developing colorectal (CRC), endometrial, ovarian, urinary tract, prostate, and other cancers, depending on which gene is malfunctioning. In Lynch syndrome, differences in cancer incidence (penetrance) according to the gene involved have led to the stratification of cancer surveillance. By contrast, any differences in penetrance determined by the type of pathogenic variant remain unknown. Objective. To determine cumulative incidences of cancer in carriers of truncating and missense or aberrant splicing pathogenic variants of the MLH1 and MSH2 genes. Methods. Carriers of pathogenic variants of MLH1 (path_MLH1) and MSH2 (path_MSH2) genes filed in the Prospective Lynch Syndrome Database (PLSD) were categorized as truncating or missense/aberrant splicing according to the InSiGHT criteria for pathogenicity. Results. Among 5199 carriers, 1045 had missense or aberrant splicing variants, and 3930 had truncating variants. Prospective observation years for the two groups were 8205 and 34,141 years, respectively, after which there were no significant differences in incidences for cancer overall or for colorectal cancer or endometrial cancers separately. Conclusion. Truncating and missense or aberrant splicing pathogenic variants were associated with similar average cumulative incidences of cancer in carriers of path MLH1 and path_MSH2.Peer reviewe

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Instituto Multidisciplinario de Biología Celula