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5-Fluorouracil (5-FU) is an essential component of systemic chemotherapy for colorectal cancer (CRC) in the palliative and adjuvant settings. Over the past four decades, several modulation strategies including the implementation of 5-FU-based combination regimens and 5-FU pro-drugs have been developed and tested to increase the anti-tumor activity of 5-FU and to overcome the clinical resistance. Despite the encouraging progress in CRC therapy to date, the patients' response rates to therapy continue to remain low and the patients' benefit from 5-FU-based therapy is frequently compromised by the development of chemoresistance. Inter-individual differences in the treatment response in CRC patients may originate in the unique genetic and epigenetic make-up of each individual. The critical element in the current trend of personalized medicine is the proper comprehension of causes and mechanisms contributing to the low or lack of sensitivity of tumor tissue to 5-FU-based therapy. The identification and validation of predictive biomarkers for existing 5-FU-based and new targeted therapies for CRC treatment will likely improve patients' outcomes in the future. Herein we present a comprehensive review summarizing options of CRC treatment and the mechanisms of 5-FU action at the molecular level, including both anabolic and catabolic ways. The main part of this review comprises the currently known molecular mechanisms underlying the chemoresistance in CRC patients. We also focus on various 5-FU pro-drugs developed to increase the amount of circulating 5-FU and to limit toxicity. Finally, we propose future directions of personalized CRC therapy according to the latest published evidence.

Made available in DSpace on 2019-10-06T17:16:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 There is increasing evidence indicating a role for Fusobacterium nucleatum (F. nucleatum) in colorectal cancer (CRC) development and prognosis. This study evaluated F. nucleatum as a prognostic biomarker, by assessing its association with post-diagnosis survival from CRC. From September 2008 to April 2012 CRC patients (n = 190) were recruited from three hospitals within the Czech Republic. F. nucleatum DNA copies were measured in adjacent non-malignant and colorectal tumor tissues using quantitative real-time PCR. Cox Proportional Hazards (HR) models were applied to evaluate the association between F. nucleatum DNA and overall survival, adjusting for key confounders. Risk prediction modeling was conducted to evaluate the ability to predict survival based on F. nucleatum status. High, compared with low, levels of F. nucleatum in colorectal tumor tissues were associated with poorer overall survival (adjusted HR 1.68, 95% CI 1.02–2.77), which was slightly attenuated after additional adjustment for microsatellite instability status. However, inclusion of F. nucleatum in risk prediction models did not improve the ability to identify patients who died beyond known prognostic factors such as disease pathology staging. Although the increased presence of F. nucleatum was associated with poorer prognosis in CRC patients, this may have limited clinical relevance as a prognostic biomarker. Centre for Public Health Queen’s University Belfast Department of Biology São Paulo State University UNESP Institute of Biology and Medical Genetics First Faculty of Medicine Charles University Department of Molecular Biology of Cancer Institute of Experimental Medicine of the Czech Academy of Sciences Department of Surgery General University Hospital in Prague Department of Surgery First Faculty of Medicine Charles University and Thomayer Hospital Biomedical Centre Faculty of Medicine in Pilsen Charles University Department of Oncology First Faculty of Medicine Charles University and Thomayer Hospital Cancer Biology and Therapeutics Group School of Biomolecular and Biomedical Science UCD Conway Institute University College Dublin Department of Biology São Paulo State University UNESP

Anaesthesiologists as essential members of medical surgical operation teams are occupationally exposed to substantial concentrations of volatile anaesthetics. Additionally, the other members of the surgical operation teams (such as surgeons and nurses) and the patient may also be exposed. The mutagenic and carcinogenic effects of such exposures have been suggested in literature and they pose a potential genotoxic burden and health problems for individuals in various branches of medicine as well as for patients. The lymphocyte cytokinesis–block micronucleus (CBMN) assay represents a marker of biological effects and has been applied in in vivo biomonitoring studies of humans exposed either environmentally or occupationally to genotoxic chemicals. The main aim of the present report is to systematically review the published studies investigating the use of the lymphocyte CBMN assay to determine DNA damage in subjects exposed to anaesthetic gases. We also compared the performance of the CBMN assay with other DNA damage assays employed. The results on the genotoxicity of anaesthetic gases in humans were also compared with those obtained in in vitro and animal experiments. Despite the evident genotoxic effects of anaesthetic gases, the understanding of the mechanisms by which these events lead to chromosomal instability and eventually malignant transformation is incomplete and requires more research.

Non-specific structural chromosomal aberrations (CAs) observed in peripheral blood lymphocytes of healthy individuals can be either chromosome-type aberrations (CSAs) or chromatid-type aberrations (CTAs) depending on the stage of cell division they are induced in and mechanism of formation. It is important to study the genetic basis of chromosomal instability as it is a marker of genotoxic exposure and a predictor of cancer risk. For that purpose, we conducted two genome-wide association studies (GWASs) on healthy individuals in the presence and absence of apparent genotoxic exposure from the Czech Republic and Slovakia. The pre-GWAS cytogenetic analysis reported the frequencies of CSA, CTA and total CA (CAtot). We performed both linear and binary logistic regression analysis with an arbitrary cut-off point of 2% for CAtot and 1% for CSA and CTA. Using the statistical threshold of 1.0 × 10−5, we identified five loci with in silico predicted functionality in the reference group and four loci in the exposed group, with no overlap between the associated regions. A meta-analysis on the two GWASs identified further four loci with moderate associations in each of the studies. From the reference group mainly loci within genes related to DNA damage response/repair were identified. Other loci identified from both the reference and exposed groups were found to be involved in the segregation of chromosomes and chromatin modification. Some of the discovered regions in each group were implicated in tumourigenesis and autism.

BACKGROUND Telomeres, repetitive DNA capping ends of eukaryotic chromosomes, are important in the maintenance of genomic integrity. Perturbed telomeres are common features of many human malignancies, including colorectal cancer. METHODS Telomere length (TL), measured by a Monochrome Multiplex Real-Time qPCR, was investigated in tumour tissues, adjacent mucosa, and blood from patients with colorectal cancer with different clinicopathological features and its impact on patient survival. TL was also measured in a limited number of liver metastases, non-cancerous liver tissues or corresponding tissues from the same patients. RESULTS TL in tumour tissues was shorter than in the adjacent mucosa (P

The lymphocyte cytokinesis-block micronucleus (CBMN) assay has been applied in hundreds of in vivo biomonitoring studies of humans exposed either environmentally or occupationally to genotoxic chemicals. However, there is an emerging need to re-evaluate the use of MN and other biomarkers within the lymphocyte CBMN cytome assay as quantitative indicators of exposure to main classes of chemical genotoxins. The main aim of the present report is to systematically review published studies investigating the use of the lymphocyte CBMN assay to determine DNA damage in subjects exposed to anaesthetic gases. We also compared performance of the CBMN assay with other DNA damage assays employed and identified strengths and weaknesses of the published studies. We have retrieved 11 studies, published between 1996 and 2013, reporting MN associated with occupational exposures (operating room personnel). The individual job categories were often described (anaesthesiologists, technicians, radiologists) among cases, as well as duration of exposure. All studies reported the compounds present at the workplace and, in some instances, the exposure levels were measured. Controls were usually recruited among personnel at the hospital not exposed to anaesthetics or they were healthy unexposed subjects from general population. The number of investigated subjects, due to the character of the occupation, was relatively smaller than those investigated in other occupational monitoring settings. Overall, the majority of the studies were age- and gender- matched (or investigated only males or females) while less attention was given to lifestyle confounders. Appropriate measurement of exposure, available in approximately half of the studies only, was compromised by the lack of the personal dosimetry-based determinations. In all studies, higher MN frequencies were observed in exposed individuals. The meta-analysis of mean MN frequency of combined studies confirmed this tendency (log mean ratio = 0.56 [0.34-0.77]; P = 3.51 × 10−7). Similar differences between the exposed and controls were also observed for other biomarkers.

Genomic instability is a characteristic of a majority of human malignancies. Chromosomal instability is a common form of genomic instability that can be caused by defects in mitotic checkpoint genes. Chromosomal aberrations in peripheral blood are also indicative of genotoxic exposure and potential cancer risk. We evaluated associations between inherited genetic variants in 33 mitotic checkpoint genes and the frequency of chromosomal aberrations (CAs) in the presence and absence of environmental genotoxic exposure. Associations with both chromosome and chromatid type of aberrations were evaluated in two cohorts of healthy individuals, namely an exposed and a reference group consisting of 607 and 866 individuals, respectively. Binary logistic and linear regression analyses were performed for the association studies. Bonferroni-corrected significant p-value was 5 × 10-4 for 99 tests based on the number of analyzed genes and phenotypes. In the reference group the most prominent associations were found with variants in CCNB1, a master regulator of mitosis, and in genes involved in kinetochore function, including CENPH and TEX14, whereas in the exposed group the main association was found with variants in TTK, also an important gene in kinetochore function. How the identified variants may affect the fidelity of mitotic checkpoint remains to be investigated, however, the present study suggests that genetic variation may partly explain interindividual variation in the formation of CAs.

Colorectal cancer (CRC) ranks as the second most common non-sex-specific cancer worldwide and the second cause of cancer deaths. Screening programs for CRC based on the fecal immunochemical test (FIT) are effective in average-risk population for the tumor detection but has low sensitivity for the recognition of advanced adenomas. We identified microRNA (miRNA) signatures by next-generation sequencing in fecal samples that could detect patients with adenomas or CRC. The same signatures were also evaluated in plasma extracellular vesicles (EVs) from the same patients. A genome-wide miRNA expression profiling was initially performed in 221 subjects (80 healthy subjects; 43 adenoma patients; 41 individuals with inflammatory bowel disease [IBD]; and 57 CRC) recruited in a cross-sectional study in Italy where stool and plasma samples have been consecutively collected at colonoscopy. Several differentially expressed miRNAs, mostly in stool samples, have been identified both in common or peculiar of specific comparison among the 3 categories of patients vs healthy subjects. Subsequently, a similar profiling has been performed for a set of 24 paired colorectal tumor and adjacent non-tumor samples from the group of CRC patients already samples for stool and plasma. Several miRNAs (n=137) resulted altered in cancer tissues compared to non-tumor; on the other hand, 91 miRNAs resulted altered in stool and only 6 in plasma EVs. Analyzing stool samples from the whole Italian cohort we observed a good overlap of the altered miRNAs observed in tissues (miR-148a-3p, miR-182-5p, miR-143-3p, miR-12136-3p; miR-378a-3p, miR-1290-5p, miR-21-5p, miR-7704-3p). The identified differentially expressed miRNAs in stool and plasma EVs were validated in an independent cohort from the Czech Republic (22 healthy, 19 Inflammation, 21 Adenoma, 34 CRC) with a similar approach. Preliminary results in this independent cohort showed that 19, 12 and 7 miRNAs were differentially expressed when comparing CRC with healthy, CRC and adenoma and inflammation individuals, respectively. In the comparison of CRC versus healthy subjects we observed a good overlap of miRNAs differentially expressed between the Czech and Italian cohorts: 10 miRNAs (miR-148a-3p, miR-1181-3p, miR-1290-5p, miR-4488-3p, miR-1246-3p, let-7i-5p, let-7b-5p, miR-4497-3p, miR-21-5p, miR-149-3p) were significantly altered in the same directions in the 2 cohorts. Moreover, hsa-miR-1290-5p and hsa-miR-4488-3p resulted significantly upregulated when going from healthy, inflammation, adenoma and CRC both in the Italian and in the Czech cohorts. Finally, with the application of a multi-class machine learning approach, we identified a signature of 13 miRNAs whose expression was able to accurately classify (AUC = 0.90) the four classes of subjects in combination with the information of patient age and sex. These results showed that there could be a potential application of the analyses of nucleic acids in stool as adjuvant in non-invasive screening. Citation Format: Sonia Tarallo, Antonio Francavilla, Giulio Ferrero, Francesca Cordero, Gaetano Gallo, Andrew Maltez Thomas, Paolo Manghi, Veronika Vymetalkova, Ludmila Vodickova, Nicola Segata, Pavel Vodicka, Barbara Pardini, Alessio Naccarati. Whole miRNome profiling in fecal and plasma exosome samples for the diagnosis of colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 257.

Colorectal cancer (CRC) is one of the most frequent cancers worldwide. The available screening tests have reduced the incidence and mortality for this cancer; however, the research of new potential molecular biomarkers to use concomitantly could improve advanced adenomas/CRC early detection. Small non-coding RNAs (sncRNAs) are stable, easy detectable molecules that have been found dysregulated in several diseases including CRC. Besides the most studied microRNAs (miRNAs), sncRNAs include also P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transfer RNAs (tRNAs) and other miscellaneous RNAs, all with key roles in post-transcriptional regulation. The aim of the present study was to identify by next-generation sequencing (NGS) signatures of sncRNAs other than miRNAs, in fecal and plasma extracellular vesicles (EVs) samples potentially able to detect patients with adenomas or CRC. We initially performed a small RNA-sequencing in samples from 221 subjects collected at colonoscopy. The group included: i) 80 healthy subjects with negative colonoscopy results; (ii) 43 adenoma patients; (iii) 41 individuals with inflammatory bowel disease and (iv) 57 newly diagnosed CRC patients. A computational pipeline for the identification of sncRNAs after miRNA mapping reads was previously implemented by our group. In stool samples, comparing the categories of interest mentioned above, we identified 395 differentially expressed sncRNAs (DEsncRNAs), mainly piRNAs (46.3%) and tRNAs (43.6%) (likelihood ratio test adjusting for sex and age, FDR adjusted p ≤ 0.05). Similarly, a group of 32 DEsncRNAs was detected in plasma EVs with piRNAs (41%) and tRNAs (44%) resulting among the most altered. Small RNA-sequencing has also been performed in a subset of 24 paired colorectal tumor and adjacent non-malignant tissues from the CRC patients for whom stool and plasma samples were analysed. Fifty-seven DEsncRNAs were found in tumor vs. non-malignant adjacent tissue with a similar distribution of dysregulated sncRNA biotypes. Some DEsncRNAs were shared between stool and tissues (n=35), plasma EVs and tissues (n=11) and, interestingly, among the profiles of the three different biospecimens (n=10). To validate our findings, we also sequenced stool samples from an independent cohort from the Czech Republic (at present 22 healthy subjects, 19 with inflammations, 21 with adenomas and 34 CRC patients). A total of 100 DEsncRNAs were detected in this validation cohort, among which 43 overlapped with those detected in the Italian cohort. In conclusions, we show that besides the most widely performed miRNA profiling, also the detection of sncRNAs in surrogate tissues by NGS may help to identify reliable and comparable results with those of primary tissues. To further investigate these molecules may provide new accurate markers for CRC diagnostic purposes. Citation Format: Antonio Francavilla, Sonia Tarallo, Giulio Ferrero, Francesca Cordero, Gaetano Gallo, Veronika Vymetalkova, Ludmila Vodickova, Pavel Vodicka, Barbara Pardini, Alessio Naccarati. Small non-coding RNA profiling in stool and plasma samples to explore potential biomarkers for colorectal cancer diagnosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 269.

Colorectal cancer (CRC) is a worldwide health burden, with nearly 1.2 million new cases expected each year. Significant efforts are currently being employed to find patients who will benefit from chemotherapy. In fact, drug resistance is a limiting factor for the efficacy of chemotherapy in CRC treatment. In the present work, we aimed at identifying molecular markers in plasma cell-free DNA that could be associated with good/poor response and chemoresistance, comparing good responders (i.e., benefitting from chemotherapy, not having side effects like toxicity, no relapses, and having a complete response with no residual cancer) from non-/poor responders (patients with chemoresistance and lacking any response, or developing toxicity). Paired plasma samples from 10 colon cancer patients collected before and after adjuvant 5-fluorouracil (5-FU)-based therapy were profiled by a whole-exome sequencing (WES) approach in order to identify mutations associated with therapy response. In concomitance, DNA from tumor tissue from the same patients was also profiled by WES for mutation burden concordance. Analyses were performed on colon cancer patients treated with the same 5-FU-based therapy. The first sampling was performed at the time of the diagnosis (i.e., active disease), and the second after 6-9 months depending on patients’ conditions (i.e., covering the tumor resection, administration of adjuvant chemotherapy, etc.). After follow-up, 3 patients were clinically classified as “good responders” and 7 as “non-/poor responders.” All prepared libraries passed the quality control checkpoints and were sequenced. The number of total reads exceeds 2,57 × 109. Mean read length is about 100 nt. Alignment to human reference genome (hg38) is > 99.8%. The bioinformatics analyses are currently ongoing to profile the two different groups over time. Detailed results of the study will be presented during the meeting. The significance of the project lies in the improvement of the diagnosis and therapy efficacy in colon cancer patients. Supported by grant AZV MZ 17-30920A. Citation Format: Veronika Vymetalkova, Barbara Pardini, Marketa Urbanova, Klara Cervena, Pavel Vodicka, Alessio Naccarati. Cell-free tumor DNA profiling of colon cancer patients: Searching for mechanisms of chemoresistance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A35.

Colorectal cancer is the third most common cancer and the second leading cause of cancer death in Europe. Therefore, the search for new biomarkers to enable facilitate early diagnosis, prognosis, and individualized treatment is particularly warranted. From a clinical point of view, malignancies in the colon and the rectum (RC) present two distinct entities that require different treatment strategies, so CRC might not be considered as a single enteropathogenic entity. MicroRNA (miRNAs) are small noncoding RNA molecules involved in the post-transcriptional and translational regulation of gene expression. The discovery that miRNAs may act either as oncogenes or tumor suppressors has initiated extensive research. It was recently discovered that extracellular miRNAs circulate in the bloodstream and that such circulating miRNAs are remarkably stable. This has raised the possibility that miRNAs might serve as novel markers for disease diagnosis and prognosis. In the present study, we aimed to identify circulating miRNAs enabling early diagnosis and prognosis of RC. MiRNA expression levels (from plasma and plasma exosomes) were characterized by next-generation sequencing (NGS) in 24 patients with RC collected at 2-time intervals. The first sampling was collected at the time of diagnosis and the second sampling was conducted around 1 year after the diagnosis. The expression levels of miR-122 and miR-142 were identified to be associated with overall survival in RC patients. Their functional characteristics were tested in vitro on rectal cancer cell lines HRA16 and SW1463. Increased levels of miRNAs were associated with decreased long-term survival of cells. Identified miRNAs are currently being verified on a larger population of RC patients. This has raised the question whether miRNA can serve as biomarkers for rectal cancer, but prior to its clinical applications, clinical trials are warranted. Acknowledgments: This work was supported by AZV 17-30920A, GA17-16857S, and GA UK 302119. Citation Format: Klara Cervena, Alena Opattova, Barbara Pardini, Alessio Naccarati, Pavel Vodicka, Veronika Vymetalkova. Identification of circulating miRNA signatures in rectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A56.

Interindividual differences in DNA repair systems may play a role in modulating the individual risk of developing colorectal cancer. To better ascertain the role of DNA repair gene polymorphisms on colon and rectal cancer risk individually, we evaluated 15,419 single nucleotide polymorphisms (SNPs) within 185 DNA repair genes using GWAS data from the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), which included 8,178 colon cancer, 2,936 rectum cancer cases and 14,659 controls. Rs1800734 (in MLH1 gene) was associated with colon cancer risk (p-value = 3.5 × 10-6 ) and rs2189517 (in RAD51B) with rectal cancer risk (p-value = 5.7 × 10-6 ). The results had statistical significance close to the Bonferroni corrected p-value of 5.8 × 10-6 . Ninety-four SNPs were significantly associated with colorectal cancer risk after Binomial Sequential Goodness of Fit (BSGoF) procedure and confirmed the relevance of DNA mismatch repair (MMR) and homologous recombination pathways for colon and rectum cancer, respectively. Defects in MMR genes are known to be crucial for familial form of colorectal cancer but our findings suggest that specific genetic variations in MLH1 are important also in the individual predisposition to sporadic colon cancer. Other SNPs associated with the risk of colon cancer (e.g., rs16906252 in MGMT) were found to affect mRNA expression levels in colon transverse and therefore working as possible cis-eQTL suggesting possible mechanisms of carcinogenesis.

The development of minimally invasive and low-cost assay enabling early diagnosis, treatment response and prognosis in cancer patients may provide a promising alternative to tumor biopsy. Circulating cell-free DNA (cfDNA) is probably the most promising tool among all components of liquid biopsy. This review includes studies exploring cfDNA as the diagnostic, prognostic or predictive biomarker for all types of cancer. In this article, we systematically reviewed the relevant literature from PubMed about cfDNA. All articles presented higher cfDNA concentration in cancer patients when compared with patients with benign disease or healthy individuals. Most of the articles showed a connection between cfDNA and prognosis. The presence of high cfDNA level in serum or plasma was associated with worse overall patient’s survival. This review supports the idea that the cfDNA analysis represents a promising research area and hopefully in the future, could be applied as a new biomarker for cancer detection, prognosis determination and prediction of the response to therapy.

New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.

Mucins and their glycosylation have been suggested to play an important role in colorectal carcinogenesis. We examined potentially functional genetic variants in the mucin genes or genes involved in their glycosylation with respect to colorectal cancer (CRC) risk and clinical outcome. We genotyped 23 single nucleotide polymorphisms (SNPs) covering 123 SNPs through pairwise linkage disequilibrium (r2>0.80) in the MUC1, MUC2, MUC4, MUC5AC, MUC6, and B3GNT6 genes in a hospital-based case-control study of 1532 CRC cases and 1108 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall survival and event-free survival in a subgroup of 672 patients. Among patients without distant metastasis at the time of diagnosis, two MUC4 SNPs, rs3107764 and rs842225, showed association with overall survival (HR 1.40, 95%CI 1.08–1.82, additive model, log-rank p = 0.004 and HR 0.64, 95%CI 0.42–0.99, recessive model, log-rank p = 0.01, respectively) and event-free survival (HR 1.31, 95%CI 1.03–1.68, log-rank p = 0.004 and HR 0.64, 95%CI 0.42–0.96, log-rank p = 0.006, respectively) after adjustment for age, sex and TNM stage. Our data suggest that genetic variation especially in the transmembrane mucin gene MUC4 may play a role in the survival of CRC and further studies are warranted.

DNA repair processes are involved in both the onset and treatment efficacy of colorectal cancer (CRC). A change of a single nucleotide causing an amino acid substitution in the corresponding protein may alter the efficiency of DNA repair, thus modifying the CRC susceptibility and clinical outcome. We performed a candidate gene approach in order to analyze the association of non-synonymous single nucleotide polymorphisms (nsSNPs) in the genes covering the main DNA repair pathways with CRC risk and clinical outcome modifications. Our candidate polymorphisms were selected according to the foremost genomic and functional prediction databases. Sixteen nsSNPs in 12 DNA repair genes were evaluated in cohorts from the Czech Republic and Austria. Apart from the tumor-node-metastasis (TNM) stage, which occurred as the main prognostic factor in all of the performed analyses, we observed several significant associations of different nsSNPs with survival and clinical outcomes in both cohorts. However, only some of the genes (REV3L, POLQ, and NEIL3) were prominently defined as prediction factors in the classification and regression tree analysis, therefore, the study suggests their association for patient survival. In summary, we provide observational and bioinformatics evidence that even subtle alterations in specific proteins of the DNA repair pathways may contribute to CRC susceptibility and clinical outcome.

The DNA-damaging agent 5-fluorouracil represents the most commonly used chemotherapeutic drug for colorectal cancer patients. DNA lesions associated with 5-fluorouracil therapy are primarily repaired by base excision repair (BER) and mismatch repair (MMR) pathways. Published evidence suggests that the individual DNA repair capacity (DRC) may affect a patient’s prognosis and response to chemotherapy. With this in mind, we designed a prospective study of which the main aim was to investigate BER-DRC in relation to 5-fluorouracil response as potential predictive and/or prognostic biomarker. BER-DRC was supplemented by a microsatellite instability (MSI) analysis which represents an indirect marker of MMR activity in the tumor. All parameters were measured in paired samples of tumor tissue and non-malignant adjacent mucosa of 123 incident colon cancer patients. Our results indicate that BER-DRC in non-malignant adjacent mucosa was positively associated with overall survival (P = 0.007) and relapse-free survival (P = 0.04). Additionally, in multivariate analysis, good therapy responders in TNM stage II and III with an elevated BER-DRC in mucosa exhibited better overall survival. Moreover, the overall survival of these patients was even better in the presence of a decreased BER-DRC in tumor tissue. The ratio of BER-DRC in tumor tissue over BER-DRC in mucosa positively correlated with advanced tumor stage (P = 0.003). With respect to MSI, we observed that MSI-high tumors were mostly localized in proximal colon; however, in our cohort, the MSI status affected neither patients’ prognosis nor survival. In summary, the results of the present study suggest that the level of BER-DRC is associated with patients’ survival. BER-DRC represents a potential prognostic biomarker, applicable for prediction of therapy response and useful for individual approach to patients.

Constitutive activation of interferon signaling pathways has been reported in colorectal cancer (CRC), leading to a strong CD8+ T cell response through stimulation of NLRC5 expression. Primed CD8+ T cell expansion, however, may be negatively regulated by PD-L1 expression. Additionally, aberrant PD-L1 expression enables cancer cells to escape the immune attack. Our study aimed to select potential regulatory variants in the NLRC5 and PD-L1 genes by using several online in silico tools, such as UCSC browser, HaploReg, Regulome DB, Gtex Portal, microRNA and transcription factor binding site prediction tools and to investigate their influence on CRC risk in a Czech cohort of 1424 CRC patients and 1114 healthy controls. Logistic regression analysis adjusted for age and gender reported a moderate association between rectal cancer risk and two NLRC5 SNPs, rs1684575 T>G (OR: 1.60, 95% CI: 1.13-2.27, recessive model) and rs3751710 (OR: 0.70, 95% CI: 0.51-0.96, dominant model). Given that a combination of genetic variants, rather than a single polymorphism, may explain better the genetic etiology of CRC, we studied the interplay between the variants within NLRC5, PD-L1 and the previously genotyped IFNGR1 and IFNGR2 variants, to evaluate their involvement in the risk of CRC development. Overall we obtained 18 pair-wise interactions within and between the NLRC5 ad PD-L1 genes and 6 more when IFNGR variants were added. Thirteen out of the 24 interactions were below the threshold for the FDR calculated and controlled at an arbitrary level q*T-NLRC5 rs289747 G>A (P

Human malignancies are often hallmarked with genomic instability, which itself is also considered a causative event in malignant transformation. Genomic instability may manifest itself as genetic changes in the nucleotide sequence of DNA, or as structural or numerical changes of chromosomes. Unrepaired or insufficiently repaired DNA double-strand breaks, as well as telomere shortening, are important contributors in the formation of structural chromosomal aberrations (CAs). In the present review, we discuss potential mechanisms behind the formation of CAs and their relation to cancer. Based on our own studies, we also illustrate how inherited genetic variation may modify the frequency and types of CAs occurring in humans. Recently, we published a series of studies on variations in genes relevant to maintaining genomic integrity, such as those encoding xenobiotic-metabolising enzymes, DNA repair, the tumour suppressor TP53, the spindle assembly checkpoint, and cyclin D1 (CCND1). While individually genetic variation in these genes exerted small modulating effects, in interactions they were associated with CA frequencies in peripheral blood lymphocytes of healthy volunteers. Moreover, we observed opposite associations between the CCND1 splice site polymorphism rs9344 G870A and the frequency of CAs compared to their association with translocation t(11,14). We discuss the functional consequences of the CCND1 gene in interplay with DNA damage response and DNA repair during malignant transformation. Our review summarizes existing evidence that gene variations in relevant cellular pathways modulate the frequency of CAs, predominantly in a complex interaction. More functional/mechanistic studies elucidating these observations are required. Several questions emerge, such as the role of CAs in malignancies with respect to a particular phenotype and heterogeneity, the formation of CAs during the process of malignant transformation, and the formation of CAs in individual types of lymphocytes in relation to the immune response.

Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphocytes of healthy volunteers. © 2015 Wiley Periodicals, Inc.

BACKGROUND:\ud Soluble mesothelin-related peptide (SMRP) is a promising diagnostic biomarker for malignant pleural mesothelioma (MPM), but various confounders hinder its usefulness in surveillance programmes. We previously showed that a single nucleotide polymorphism (SNP) within the 3'untranslated region (3'UTR) of the mesothelin (MSLN) gene could affect the levels of SMRP.\ud OBJECTIVES:\ud To focus on SNPs located within MSLN promoter as possible critical genetic variables in determining SMRP levels.\ud METHODS:\ud The association between SMRP and SNPs was tested in 689 non-MPM subjects and 70 patients with MPM. Reporter plasmids carrying the four most common haplotypes were compared in a dual luciferase assay, and in silico analyses were performed to investigate the putative biological role of the SNPs.\ud RESULTS:\ud We found a strong association between serum SMRP and variant alleles of rs3764247, rs3764246 (in strong linkage disequilibrium with rs2235504) and rs2235503 in non-MPM subjects. Inclusion of the genotype information led to an increase in SMRP specificity from 79.9% to 85.5%. Although not statistically significant, the group with MPM showed the same trend of association. According to the in vitro luciferase study, rs3764247 itself had a functional role. In silico approaches showed that the binding sites for transcription factors such as Staf and ZNF143 could be affected by this SNP. The other SNPs were shown to interact with each other in a more complex way.\ud CONCLUSIONS:\ud These data support the suggestion that SMRP performance is affected by individual (ie, genetic) variables and that MSLN expression is influenced by SNPs located within the promoter regulatory region.

Background Genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1‑4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut. Methods We performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries. Results We did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (Ptrend of = 0.0071). Conclusions Our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts. Electronic supplementary material The online version of this article (10.1186/s12876-017-0659-9) contains supplementary material, which is available to authorized users.

DNA repair in blood cells was observed to be suboptimal in cancer patients at diagnosis, including colorectal cancer (CRC). To explore the causality of this phenomenon, we studied the dynamics of DNA repair from diagnosis to 1 yr followup, and with respect to CRCtreatment. Systemic CRC therapy is targeted to DNA damageinduction and DNA repair is thus of interest. CRC patients were blood-sampled three times in 6-mo intervals, starting at the diagnosis, and compared to healthy controls. DNA repair was characterized by mRNA levels of 40 repair genes, by capacity of nucleotide excision repair (NER), and by levels of DNA strand breaks (SBs). NER and base excision repair genes were significantly under-expressed (P

According to the Vogelstein's model of colorectal carcinogenesis, genetic variations in highly penetrant genes may be involved in the colorectal cancer (CRC) pathogenesis. Similarly, aberrant function and/or altered expression of microRNAs (miRNAs) often occur in CRC. In this context, polymorphisms in miRNA-binding sites (miRSNPs) may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and increased susceptibility to common diseases. To explore this phenomenon, we have mined the 3' untranslated regions (3'UTRs) of genes known to be frequently mutated in CRC to search for miRSNPs and tested their association with CRC risk and clinical outcome. Eight miRSNPs (rs1804191, rs397768, rs41116 in APC; rs1137918, s227091, rs4585 in ATM; rs712, rs1137282, rs61764370 in KRAS; rs8674 in PARP1 and rs16950113 in SMAD7) were tested for their association with CRC risk in a case-control study (1111 cases and 1469 healthy controls). The role of these miRSNPs was also investigated in relation to clinical outcome on a subset of patients with complete follow-up. rs8679 within PARP1 was associated with CRC risk and patients' survival. In the dominant model, carriers of at least one C allele were at a decreased risk of cancer (P = 0.05). The CC genotype in rs8679 was also associated with an increased risk of recurrence/progression in patients that received 5-FU-based chemotherapy (log-rank test P = 0.03). Carriers of the homozygous variant genotype TT for rs712 in KRAS gene were associated with a decreased risk of rectal cancer (odds ratio (OR) = 0.65, 95% confidence intervals (CI) 0.43-1.00, P = 0.05) while individuals with colon cancer carrying the heterozygous GT genotype showed a longer overall survival (OS) (P = 0.04). We provide the first evidence that variations in potential miRNA-binding target sites in the 3' UTR of PARP1 gene may modulate CRC risk and prognosis after therapy. Further studies are needed to replicate our finding and assess miRSNPs as predictive biomarkers in independent populations.

Colorectal cancer (CRC) is a complex disease that develops as a consequence of both genetic and environmental risk factors in interplay with epigenetic mechanisms, such as microRNAs (miRNAs). CRC cases are predominantly sporadic in which the disease develops with no apparent hereditary syndrome. The last decade has seen the progress of genome-wide association studies (GWAS) that allowed the discovery of several genetic regions and variants associated with weak effects on sporadic CRC. Collectively these variants may enable a more accurate prediction of an individual's risk to the disease and its prognosis. However, the number of variants contributing to CRC is still not fully explored.SNPs in genes encoding the miRNA sequence or in 3'UTR regions of the corresponding binding sites may affect miRNA transcription, miRNA processing, and/or the fidelity of the miRNA-mRNA interaction. These variants could plausibly impact miRNA expression and target mRNA translation into proteins critical for cellular integrity, differentiation, and proliferation.In the present chapter, we describe the different aspects of variations related to miRNAs and other non-coding RNAs (ncRNAs) and evidence from studies investigating these candidate genetic alterations in support to their role in CRC development and progression.

Aim: The aim of the present study is to address a genome-wide search for novel methylation biomarkers in the rectal cancer (RC), as only scarce information on methylation profile is available. Materials & methods: We analyzed methylation status in 25 pairs of RC and adjacent healthy mucosa using the Illumina Human Methylation 450 BeadChip. Results: We found significantly aberrant methylation in 33 genes. After validation of our results by pyrosequencing, we found a good agreement with our findings. The BPIL3 and HBBP1 genes resulted hypomethylated in RC, whereas TIFPI2, ADHFE1, FLI1 and TLX1 were hypermethylated. An external validation by TCGA datasets confirmed the results. Conclusion: Our study, with external validation, has demonstrated the feasibility of using specific methylated DNA signatures for developing biomarkers in RC.

Non-specific chromosomal aberrations (CAs) are microscopically detected in about 1% of lymphocytes drawn from healthy persons. Causes of CAs in general population are not known but they may be related to risk of cancer. In view of the importance of the mitotic checkpoint machinery on maintaining chromosomal integrity we selected 9 variants in main checkpoint related genes (BUB1B, BUB3, MAD2L1, CENPF, ESPL1/separase, NEK2, PTTG1/securin, ZWILCH and ZWINT) for a genotyping study on samples from healthy individuals (N = 330 to 729) whose lymphocytes had an increased number of CAs compared to persons with a low number of CAs. Genetic variation in individual genes played a minor importance, consistent with the high conservation and selection pressure of the checkpoint system. However, gene pairs were significantly associated with CAs: PTTG1-ZWILCH and PTTG1-ZWINT. MAD2L1 and PTTG1 were the most common partners in any of the two-way interactions. The results suggest that interactions at the level of cohesin (PTTG1) and kinetochore function (ZWINT, ZWILCH and MAD2L1) contribute to the frequency of CAs, suggesting that gene variants at different checkpoint functions appeared to be required for the formation of CAs.

Colorectal cancer (CRC) represents a serious health problem with high incidence and mortality worldwide. The main reason of high mortality is the late diagnosis. For the Duke stage I, the average 5-years survival rate is 90%. In comparison to patients diagnosed at IV stage, the 5-years survival rate decreases to 3%. Mucinous CRC is generally defined as having greater than 50 % of the tumor area with a mucinous differentiation by histological examination. The increased incidence occurs in proximal colon and among younger patients, as compared to nonmucinous adenocarcinoma. Mucinous CRCs have been found to have a higher Duke stage at diagnosis, and, consequently a poorer prognosis. Mucines are surface or secreted glycoproteins with high molecular weight. Dysregulation of their expression may lead to malignant transformation. MUC13 overexpression influences colon cancer tumorigenesis and metastasis via multiple oncogenic proteins. Recently, we have observed that rs1532602 polymorphism in microRNA (miRNA) binding sites in MUC13 gene was associated with significantly decreased risk of CRC. The aim of the present study is to investigate regulation in vitro by miRNAs as a potential mechanism for increased MUC13 expression in CRC. Two different miRNAs were selected in this investigation. MiRNA-4647 is predicted to bind to the rs1532602 in MUC13. MiRNA-137 functions as a tumour suppressor in colon. Additionally, we investigated the impact of miRNAs mentioned above on regulation of MUC13 in a cohort of 113 patients with sporadic CRC. The present work provides important insights into role of miRNAs which bind to MUC13. Definition of functions, mechanisms of MUC13 regulation and its impact on CRC may contribute to better prediction and individual treatment of CRC. Acknowledgement: Grant Agency of the Ministry of Health of the Czech Republic (AZV MZ 15-26535A) and Grant Agency of the Czech Republic GACR 17-16857S. Citation Format: Linda Bartu, Alena Opattova, Pavel Vodicka, Veronika Vymetalkova. Regulation of MUC13 by microRNA in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 478.

Colorectal cancer (CRC) is a worldwide health burden with nearly 1.2 million new cases expected each year globally. When CRC is identified early, there is a five-year survival rate of about 90% but it drops to almost 12% once there are distant metastases. Drug resistance is a limiting factor of the efficacy of chemotherapy in CRC treatment. Significant efforts are currently being employed to discriminate patients who will benefit or not from chemotherapy. In fact, there is no current 'gold standard' to differentiate responders from non-responders. In the present work, we focused on good responders (those who benefit from the chemotherapy, not having side effects like toxicity, no relapses and having a complete response with no residual cancer) and non-/bad responders (patients with acquired chemoresistance and lack any response, or developing toxicity). The main aim was to identify markers/mutations in plasma cell-free DNA that could be associated with good/bad responders and chemoresistance. Paired plasma samples from colon cancer patients collected before and after therapy and the relative cell-free tumor DNA (ctDNA) was profiled by a whole exome sequencing approach in order to identify novel acquired mutations. Plasma ctDNA was isolated by QIAamp Circulating Nucleic Acid Kit. The SureSelectXT HS Reagent Kit was used for library preparation and libraries were sequenced by the HiSeq2500 system. Analysis was performed on nine patients (stage II and III) treated with the same 5-FU based therapy. The first sampling was collected at the time of diagnosis (i.e., active disease), and the second after 6-9 months depending on patient` s conditions (i.e., covering the tumor resection, administration of adjuvant chemotherapy). After follow up, three patients were classified as good responders and six as non/poor responders. The bioinformatics analysis is currently running. The number of total reads exceeded 990x106. Total length of all exons was 221,924,089bp. After trimming, the average read length was 113bp. Alignment to human reference genome (hg38) is > 99.8%. Coverage of exons ranges from 17 to 34. Results of the study will be presented during the meeting. Supported by grant AZV MZ 17-30920A, 15-26535A and GACR 17-16857S. Citation Format: Veronika Vymetalkova, Barbara Pardini, Katerina Jiraskova, Marjketa Urbanova, Miroslav Levy, Veronika Veskrnova, Pavel Vodicka, Alessio Naccarati. Repeated whole-exome sequencing for cell-free tumor DNA profiling of colon cancer patients: Searching for mechanisms of acquired chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1235.

e15607Background: Secreted mucins form the physical barrier which play an important role dor maintaining gut homeostasis, while transmembrane mucins contribute to the protective mucous gel. Dysregu...

Polymorphisms in microRNA (miRNA) binding sites may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and susceptibility to common diseases. Mucins have been identified as markers of adverse prognosis. We hypothesized that genetic variations in miRNA binding sites located in mucin genes may modulate signaling response and the maintenance of genomic stability ultimately affecting cancer susceptibility, efficacy of chemotherapy and survival. In this study, we analyzed the association of single nucleotide polymorphisms in predicted miRNA target sites (miRSNPs) of mucin genes with colorectal cancer (CRC) risk and clinical outcome. Thirteen miRSNPs in 9 genes were assessed in 1111 cases and 1469 controls. No strongly significant associations were observed in the case-control study. Patients carrying the CC genotype of rs886403 in MUC21 displayed a shorter survival and higher recurrence risk when compared with TT carriers [overall survival (OS): hazard ratios (HR) 1.69; 95% confidence intervals (CI) 1.13-2.46; P = 0.01 and event-free survival (EFS): HR 1.99; 95% CI 1.38-2.84; P = 0.0002, respectively]. The observed associations were more striking after stratification for tumor site (in patients with colon cancer, OS: HR 2.63; 95% CI 1.69-4.10; P < 0.0001 and EFS: HR 2.65; 95% CI 1.72-4.07; P < 0.0001). In contrast, rectal cancer cases carrying the CC genotype of rs4729655 in MUC17 displayed a longer survival (OS: HR 0.27; 95% CI 0.14-0.54; P = 0.0002) than those with the most common genotype. To our knowledge, this is the first study investigating miRSNPs potentially affecting miRNA binding to mucin genes and revealing their impact on CRC susceptibility or patient's survival.

// Alessio Naccarati 1, 2 , Fabio Rosa 3 , Veronika Vymetalkova 2, 4 , Elisa Barone 5 , Katerina Jiraskova 2, 4 , Cornelia Di Gaetano 3, 6 , Jan Novotny 7 , Miroslav Levy 8 , Ludmila Vodickova 2, 4 , Federica Gemignani 5 , Tomas Buchler 9 , Stefano Landi 5 , Pavel Vodicka 2, 4, * , Barbara Pardini 3, 6, * 1 Molecular and Genetic Epidemiology Research Unit, Human Genetics Foundation, Turin, Italy 2 Department of Molecular Biology of Cancer, Institute of Experimental Medicine, Prague, Czech Republic 3 Genomic Variation in Human Populations and Complex Diseases Research Unit, Human Genetics Foundation, Turin, Italy 4 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic 5 Department of Biology, University of Pisa, Pisa, Italy 6 Department of Medical Sciences, University of Turin, Turin, Italy 7 Department of Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic 8 Department of Surgery, First Faculty of Medicine, Charles University and Thomayer University Hospital, Prague, Czech Republic 9 Department of Oncology, Thomayer Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic * These authors contributed equally to this work Correspondence to: Alessio Naccarati, e-mail: alessio.naccarati@hugef-torino.org Keywords: double-strand break repair (DSBR) genes, colorectal cancer risk and clinical outcomes, miRNA binding sites, 3′UTR polymorphisms, MRE11A Received: July 07, 2015 Accepted: December 09, 2015 Published: December 31, 2015 ABSTRACT Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome.

Colorectal cancer (CRC) is a complex disease that develops as a consequence of both genetic and environmental risk factors in interplay with epigenetic mechanisms, such as microRNAs (miRNAs). CRC cases are predominantly sporadic in which the disease develops with no apparent hereditary syndrome. The last decade has seen the progress of genome-wide association studies (GWAS) that allowed the discovery of several genetic regions and variants associated with weak effects on sporadic CRC. Collectively these variants may enable a more accurate prediction of an individual’s risk to the disease and its prognosis. However, the number of variants contributing to CRC is still not fully explored.

Human cancers are often associated with numerical and structural chromosomal instability. Structural chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL) arise as consequences of direct DNA damage or due to replication on a damaged DNA template. In both cases, DNA repair is critical and inter-individual differences in its capacity are probably due to corresponding genetic variations. We investigated functional variants in DNA repair genes (base and nucleotide excision repair, double-strand break repair) in relation to CAs, chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) in healthy individuals. Chromosomal damage was determined by conventional cytogenetic analysis. The genotyping was performed by both restriction fragment length polymorphism and TaqMan allelic discrimination assays. Multivariate logistic regression was applied for testing individual factors on CAs, CTAs and CSAs. Pair-wise genotype interactions of 11 genes were constructed for all possible pairs of single-nucleotide polymorphisms. Analysed individually, we observed significantly lower CTA frequencies in association with XPD Lys751Gln homozygous variant genotype [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.48-0.85, P = 0.004; n = 1777]. A significant association of heterozygous variant genotype in RAD54L with increased CSA frequency (OR 1.96, 95% CI 1.01-4.02, P = 0.03) was determined in 282 subjects with available genotype. By addressing gene-gene interactions, we discovered 14 interactions significantly modulating CAs, 9 CTAs and 12 CSAs frequencies. Highly significant interactions included always pairs from two different pathways. Although individual variants in genes encoding DNA repair proteins modulate CAs only modestly, several gene-gene interactions in DNA repair genes evinced either enhanced or decreased CA frequencies suggesting that CAs accumulation requires complex interplay between different DNA repair pathways.

Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. It is routinely cured by a 5-fluorouracil (5-FU)-based chemotherapy which improves outcomes in patients. We investigated the effect of single nucleotide polymorphisms (SNPs) in two microRNA (miRNA)-encoding genes that have been previously reported as important in prognosis in patients with stage III CRC and treated with 5-FU-based chemotherapy. Two SNPs (rs4919510 in miR-608 and rs213210 in miR-219-1) were genotyped in 1083 CRC patients recruited in the Czech Republic to evaluate their effect on clinical outcomes. Carriers of the variant T allele in rs213210 and receiving 5-FU chemotherapy were associated with a significantly worse survival [hazard ratio (HR) = 2.18; 95% confidence interval (CI): 1.20-3.98; adjusted P = 0.01] and an increased risk of relapse (HR = 1.94; 95% CI: 1.16-3.25; adjusted P = 0.01). After further stratification for tumor grading, stage III patients carrying the G allele of rs4919510 and undergoing adjuvant chemotherapy were at decreased risk of relapse (HR = 0.44; 95% CI: 0.20-0.94; adjusted P = 0.03). The present study confirms that variations in miRNA-encoding genes may be an important factor for modulating CRC prognosis and predicting therapy response.

Variations in the TP53 gene have been suggested to play a role in many cancers, including breast. We previously observed an association between TP53 haplotypes based on four polymorphisms (rs17878362, rs1042522, rs12947788, and rs17884306) and the risk of colorectal and pancreatic cancer. Based on these results, in the present study, we have investigated the same polymorphisms and their haplotypes in 705 breast cancer cases and 611 healthy controls in relation to the disease risk, histopathological features of the tumor and clinical outcomes. In comparison to the most common haplotype A1-G-C-G, all the other identified haplotypes were globally associated with a significantly decreased breast cancer risk (P = 0.006). In particular, the A2-G-C-G haplotype was associated with a marked decreased risk of breast cancer when compared with the common haplotype (P = 0.0001). Moreover, rs1042522 in patients carrying the GC genotype and receiving only the anthracycline-based chemotherapy was associated with both overall and disease-free survival (recessive model for overall survival HR = 0.30 95% CI 0.11–0.80, P = 0.02 and for disease-free survival HR = 0.42 95% CI 0.21–0.84, P = 0.01). Present results suggest common genetic features in the susceptibility to breast and gastrointestinal cancers in respect to TP53 variations. In fact, similar haplotype distributions were observed for breast, colorectal, and pancreatic patients in associations with cancer risk. Rs1042522 polymorphism (even after applying the Dunn-Bonferroni correction for multiple testing) appears to be an independent prognostic marker in breast cancer patients.

Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was2% and for CSA and CTA the limit was1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphocytes of healthy volunteers.

Mutations in the mutL homolog 1 (MLH1) gene are frequent in patients with hereditary non-polyposis colorectal cancer (CRC). The MLH1 gene was screened for mutations in patients with sporadic CRC. The nucleotide sequences for all 19 exons of MLH1 were analyzed by high resolution melting and sequenced in a group of 104 sporadic CRC patients, and the results were verified in a replication group of 1,095 patients and 1,469 controls. Different melting profiles for exon 2 of the MLH1 gene were observed in the germline DNA of one patient. Sequencing of the patient’s DNA resulted in the identification of a heterozygous C>G variant at c.204, which resulted in an Ile68Met change in the amino acid. A detailed search of the National Center for Biotechnology Information and the 1000 Genomes databases indicated that the detected variant was unique. According to the SIFT and PolyPhen-2 algorithms, the substitution of Ile to Met was predicted to decrease the activity of the MLH1 protein. The newly identified, functional germline variant was not present in any other CRC patient or control. Thus, a novel germline variant in the MLH1 gene was identified, representing a rare event in sporadic CRC. The occurrence and relevance of this mutation in other types of cancer requires additional investigation.

DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC). CRC is routinely cured by 5-fluorouracil (5-FU)-based chemotherapy, with a prognostic effect and resistance to such therapy conferred by MMR status. In this study, we aimed to analyse the effect of genetic variants in classical coding regions or in less-explored predicted microRNA (miRNA)-binding sites in the 3' untranslated region (3'UTR) of MMR genes on the risk of CRC, prognosis and the efficacy of 5-FU therapy. Four single nucleotide polymorphisms (SNPs) in MMR genes were initially tested for susceptibility to CRC in a case-control study (1095 cases and 1469 healthy controls). Subsequently, the same SNPs were analysed for their role in survival on a subset of patients with complete follow-up. Two SNPs in MLH3 and MSH6 were associated with clinical outcome. Among cases with colon and sigmoideum cancer, carriers of the CC genotype of rs108621 in the 3'UTR of MLH3 showed a significantly increased survival compared to those with the CT + TT genotype (log-rank test, P = 0.05). Moreover, this polymorphism was also associated with an increased risk of relapse or metastasis in patients with heterozygous genotype (log-rank test, P = 0.03). Patients carrying the CC genotype for MSH6 rs1800935 (D180D) and not undergoing 5-FU-based chemotherapy showed a decreased number of recurrences (log-rank test, P = 0.03). No association with CRC risk was observed. We provide the first evidence that variations in potential miRNA target-binding sites in the 3'UTR of MMR genes may contribute to modulate CRC prognosis and predictivity of therapy.

Colorectal cancer (CRC) has one of the highest mortality due to the late diagnosis and a lack of proper predictive and prognostic markers. According to the current knowledge, DNA repair processes are involved both in the onset of CRC and in the treatment efficacy. In the present study, we analyzed the link between functional genetic polymorphisms (SNPs) in DNA repair genes covering the main DNA repair pathways (relevant for therapy response) in relation with the risk of CRC and clinical outcomes. Our set of candidate polymorphisms was selected according to different functional and genomic databases (FSNP, GAD, Linkage Disequilibrium, Gerp++, SiPhy). FSNP database provides integrated information about the functional effects of SNPs which are predicted and indicated at several levels (protein coding, splicing regulation, transcriptional regulation, post translation). We have focused on those affecting protein coding. We hypothesize that these modified proteins modulate the function/efficiency of DNA repair, and thus may have an effect on CRC. Sixteen polymorphisms in twelve DNA repair genes (C19orf40, EME1, FANCI, MUS81, NEIL3, POLE, POLN, POLQ, RAD51D, REV1, REV3L, RPA1) were analyzed in DNA samples of 1080 cases and 1442 controls from the Czech Republic. Clinical data at diagnosis and complete information on follow up were provided for all patients. Genetic variations in several DNA repair genes were associated with clinical outcome. In particular, CRC patients carrying the AG heterozygous genotype for rs5030755 in RPA1 gene displayed a longer survival and decreased recurrence risk (Overall Survival (OS): HR 0.74; 95% CI 0.56-0.98; p = 0.04 and Event-Free Survival (EFS): HR 0.72; 95% CI 0.54-0.95; p = 0.02). This association with better OS was more pronounced in individuals with colon and sigmoideum cancer (HR 0.58; 95% CI 0.4-0.83; p = 0.0035). Understanding the SNPs effect on the treatment response, resulting ultimately into low-cost and low-invasive markers, is regarded as very important for the possibility to tailor patient specific treatment strategy. Individualized therapy will eventually help to improve therapeutic efficacy and to minimize toxicities. The present results identified plausible candidate DNA repair gene variants potentially affecting clinical outcome in relation to CRC patient's survival. Supported by grant GA UK 112515, GA CR 15-14789S Citation Format: Katerina Jiraskova, Jana Slyskova, Fabio Rosa, Cornelia Di Gaetano, Barbara Pardini, Veronika Vymetalkova, Pavel Vodicka. Nonsynonymous functional variants in DNA repair genes in sporadic colorectal cancer: searching for predictive and prognostic markers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3409.

Human cancers arise from cells unable to maintain genomic and chromosomal stability, mainly as a sequential consequence of altered DNA repair mechanisms (base and nucleotide excision, mismatch and double-strand breaks). Chromosomal aberrations (CAs) are a marker of cancer risk and many specific CAs represent causative events in malignant transformation. Non-specific CAs arise as a result of direct DNA damage by ionizing radiation (chromosome-breaks; CSA) or replication on a damaged DNA template (CSA and chromatide-breaks; CTA). Frequencies of CAs in blood lymphocytes (PBL) are predictive for cancer risk in prospective epidemiological studies and patients with many types of cancer show elevated CAs at diagnosis. We have recently disclosed associations of CAs with variants in genes encoding DNA repair and xenobiotic metabolizing enzymes on 1800 healthy subjects exposed and unexposed to potentially carcinogenic compounds. Notably, lower frequencies of CAs and CTA are associated with high activity EPHX1 and XPD Lys751Gln homozygous variant genotypes and several pair-wise interactions significantly modulated CA, CTA and CSA frequencies. On the contrary, increased CAs and CSA frequencies were observed in subjects bearing splicing A variant in CCND1 G870A. Current investigations aim at understanding the genetics underlying CAs as intermediary cancer biomarkers, such as variants in genes encoding kinetochores, mitotic apparatus regulating enzymes, variants in genes encoding polymerases in DNA repair synthesis and the CAs dynamics in subjects repeatedly analysed over time. We have also explored miRNA binding sites in 3′UTR of DNA repair genes (BER, NER and DSB) both in colorectal cancer patients and in healthy subjects with CAs. Since shortening of telomeres in each cell division may lead to telomere crisis and complex CAs, relative telomere length (RTL) was determined in 187 individuals and compared to their CA count in PBL. Further analyses investigated RTL in incident cancer patients (breast, colorectum and lungs) and in patients (breast and colorectum) with the estimated double-strand breaks repair capacity. CAs detected in patients with above solid cancers were associated with clinicopathological characteristics and analysed as a potential prognostic factors. Our studies suggest that CAs in PBL may represent perspective transient marker in carcinogenesis and we hereby provide a biological basis for the link between CAs and cancer risk along with the genetic control of the overall CA frequency. Grant support: GA CR 15-14789S, AZV 15-27580A and COST LD14050. Citation Format: Pavel E. Vodicka, Ludmila Vodickova, Zdena Polivkova, Ludovit Musak, Maria Dusinska, Sona Vodenkova, Veronika Vymetalkova, Michal Kroupa, Alessio Naccarati, Rajiv Kumar, Kari J. Hemminki. Chromosomal damage as markers of genotoxicity and carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 801.

Mucins, high molecular weight glycoproteins predominantly expressed at the epithelial surface of tissues, provide protection for colon surface under normal physiological conditions. Mucinous colorectal carcinoma is generally defined as having greater than 50% of the tumor area with a mucinous differentiation by histologic examination. MiRNAs have recently emerged as important regulators for altered mucin expression during malignant development. MiRNAs are short non-coding RNAs that regulate gene expression by binding to the 3’untranslated regions (3’UTR) of target mRNA thereby hampering protein translation or inducing mRNA destabilization. Aberrant miRNA expression and/or function are frequently observed in colorectal cancer (CRC). Polymorphisms in miRNA binding sites may affect miRNA binding to target genes, resulting in differential mRNA and protein expression and susceptibility to common diseases. We hypothesize that variations in mucin genes may modulate signaling response affecting cancer susceptibility, cancer survival and efficacy of chemotherapy. Thirteen polymorphisms in nine mucin genes (MUC6, MUC7, MUC13, MUC14, MUC15, MUC17, MUC20, MUC21 and MUC24) were analyzed in DNA samples of 1111 cases and 1469 controls from the Czech Republic. Investigated variants were also studied in association with clinical outcome in all patients provided with detailed information on follow up. Genetic variations in mucin genes were associated with clinical outcome. In particular, CRC patients carrying the CC genotype for rs886403 in MUC21 gene displayed a shorter survival and higher recurrence risk (HR 1.69; 95% CI 1.13-2.46; p = 0.01 and EFS: HR 1.99; 95% CI 1.38-2.84; p = 0.0002, resp.). The observed association was strikingly pronounced in colon cancer patients while individuals with rectal cancer and carrying variant CC genotype of rs4729655 in MUC17 displayed better overall survival (HR 0.27; 95% CI 0.14-0.54; p = 0.0002). Expanding our knowledge on mucin involvement in CRC may help us better understand the etiopathogenesis of this disease and thereby contribute to the development of new treatment strategies. The present results identified plausible candidate SNPs potentially affecting miRNAs that target mucin genes in relation to CRC patients’ survival. Work supported by IGA MZ: NT 15-26535A Citation Format: Petra Bendova, Veronika Vymetalkova, Barbara Pardini, Fabio Rosa, Cornelia di Gaetano, Ludmila Vodickova, Tomas Buchler, Alessio Naccarati, Pavel Vodicka. Role of mucin gene variations in microRNA binding sites in modulating colorectal cancer susceptibility and clinical outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3411.