Carina Pereira

Functional polymorphisms of Toll-like receptors 2 and 4 alter the risk for colorectal carcinoma in Europeans

BACKGROUND Colon carcinogenesis is associated with increased expression levels of Toll-like receptor 2 and Toll-like receptor 4. AIM To determine in a Caucasian population the role of Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in colorectal cancer development. METHODS Hospital based multicentre case control study involving 193 colorectal cancer patients and 278 healthy individuals. DNA samples were extracted from blood cells and genotyping of TLR2+597T>C, TLR2-4760T>C, TLR4-3745A>G, TLR2Arg753Gln, TLR4Asp299Gly was performed. Functionality of risk polymorphisms was evaluated through production of TNF-α in cell culture and Toll-like receptors levels quantified by real-time RT-PCR. RESULTS TLR2+597CC homozygous had 5-fold decreased risk (odds ratio (OR)=0.21, 95% CI: 0.09-0.50, p<0.001) and TLR4 299Gly homozygous 3-fold increased risk of colorectal cancer (OR=3.30, 95% CI: 1.18-9.28, p=0.015). In stratified analysis, TLR2+597CC genotype protective effect was even higher in overweight individuals (OR=0.17, 95% CI: 0.06-0.53, p<0.001) and in never smokers (OR=0.11, 95% CI: 0.02-0.51, p=0.001). Also, the increased risk effect for TLR4 299Gly homozygous genotype was higher in overweight individuals (OR=8.67, 95% CI: 1.11-87.85, p=0.011). TLR2+597T>C polymorphism conferred 41% less (p=0.03) and TLR4Asp299Gly 65% more TNF-α production (p=0.02) with no differences in Toll-like receptors levels. CONCLUSION Functional Toll-like receptor 2 and Toll-like receptor 4 polymorphisms significantly alter the risk to have colorectal cancer. Obesity and smoking may influence the risk for colorectal cancer in individuals presenting these genetic profiles.

Cyclooxygenase polymorphisms in gastric and colorectal carcinogenesis: are conclusive results available?

Objective Cyclooxygenases (COX) are important enzymes not only in the maintenance of mucosal integrity but also in pathological processes, namely in inflammation and tumor development in the gastrointestinal tract. Our goal was to understand whether there is a clear role for COX polymorphisms in gastric and colorectal carcinogenesis. Methods A systematic review was conducted on observational studies assessing the involvement of COX polymorphisms at the onset of gastric or colorectal lesions, retrieved through a MEDLINE database search by May 2008. The dominant genetic model was assumed for each polymorphism and a random-effect model was used for pooling results. Results Twenty-two studies were retrieved reporting a total of 26 COX polymorphisms (nine in COX1 and 17 in COX2 genes). Carriers of −1329A, −899C alleles, and *429TT genotype revealed increased risk for gastric cancer [odds ratio (OR)=1.83; 95% confidence interval (CI): 1.07–3.10, OR=2.02; 95% CI: 1.00–4.10 and OR=1.34; 95% CI: 1.06–1.71, respectively). For colorectal lesions, the −899G>C and −1329G>A polymorphisms also showed an increased risk for cancer (OR=1.35; 95% CI: 1.01–1.81 and OR=1.36; 95% CI: 1.11–1.66, respectively). Furthermore, C allele carriers of V102V single nucleotide polymorphisms presented a decreased risk for colorectal adenoma onset (OR=0.77; 95% CI: 0.58–1.03). Conclusion Although further studies, namely cohorts and/or adequately matched case–control studies, are required to unravel the impact of most COX polymorphisms, clearly there are evidences that support the involvement of −899G>C and −1329G>A COX2 polymorphisms in either gastric or colorectal carcinogenesis. These markers could be used to optimize management strategies (follow-up and/or chemoprevention).

Genetic Variability in Key Genes in Prostaglandin E2 Pathway (COX-2, HPGD, ABCC4 and SLCO2A1) and Their Involvement in Colorectal Cancer Development

The pro-carcinogenic effects of prostaglandin E2 (PGE2) in colonic mucosa are not only regulated by the rates between Cyclooxygenase-2 (COX-2) biosynthesis and 15-Hydroxyprostaglandin Dehydrogenase (15-PGDH)-dependent degradation but also the steady-state levels of PGE2 in extracellular microenvironment, maintained by key specific prostaglandin transporters, the Multidrug Resistance Protein (MRP4) (efflux carrier) and Prostaglandin Transporter (PGT) (influx carrier). To understand the contribution of genetic variability in genes coding for COX-2/15-PGDH/MRP4/PGT proteins in CRC development, we conducted a hospital-based case-control study involving 246 CRC patients and 480 cancer-free controls. A total of 51 tagSNPs were characterized using the Sequenom platform through multiplexed amplification followed by mass-spectrometric product separation or allelic discrimination using real-time PCR. Seven tagSNPs were implicated in CRC development: the rs689466 in COX-2 gene, the rs1346271 and rs1426945 in 15-PGDH, the rs6439448 and rs7616492 in PGT and rs1751051 and rs1751031 in MRP4 coding genes. Upon a stratified analysis a measurable gene-environment interaction was noticed between rs689466 and smoking habits, with individuals ever-smokers carriers of rs689466 GG homozygous genotype having a nearly 6-fold increased susceptibility for CRC onset (95%CI: 1.49–22.42, P = 0.011). Furthermore, the multifactor dimensionality reduction (MDR) analysis identified an overall four-factor best gene-gene interactive model, including the rs1426945, rs6439448, rs1751051 and rs1751031 polymorphisms. This model had the highest cross-validation consistency (10/10, P<0.0001) and an accuracy of 0.6957 and was further associated with a 5-fold increased risk for CRC development (95%CI: 3.89–7.02, P<0.0001). In conclusion, specific low penetrance genes in the pro-carcinogenic PGE2 pathway appear to modulate the genetic susceptibility for CRC development. A clearer understanding on CRC etiology through the identification of biomarkers of colorectal carcinogenesis might allow a better definition of risk models that are more likely to benefit from targeted preventive strategies to reduce CRC burden.

Family History and Breast Cancer Hormone Receptor Status in a Spanish Cohort

Background Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women. Materials and Methods A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles. Results Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER−&PR−. Women with a family history of breast cancer were more likely to have ER−&PR− tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91–2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34–5.81). Conclusions An increased proportion of ER−&PR− breast cancer was observed among younger Spanish women with a family history of the disease.

The −1195G allele increases the transcriptional activity of cyclooxygenase‐2 gene (COX‐2) in colon cancer cell lines

Up‐regulation of cyclooxygenase‐2 (COX‐2) is an early and key event in human colorectal carcinogenesis (CRC). Nevertheless, the molecular mechanisms leading to this over‐expression are largely unknown. We previously reported an association between the −1195G allele and higher predisposition for CRC in a Caucasian population. The biological explanation for the involvement of this polymorphism in CRC remains elusive. We aimed to functionally characterize the influence of the −1195A>G promoter region polymorphism on COX‐2 transcription activity in colon cancer cell lines. Luciferase reporter assays were performed to assess whether the −1195A/G alleles influenced COX‐2 transcription. The COX‐2 promoters region containing either the −1195A or −1195G alleles was cloned into pGL3‐basic reporter vector. The reporter vectors were transiently co‐transfected with the pGL4.73 control plasmid to HCT‐116 and HCA‐7 colon cancer cell lines. The levels of reporter gene expression driven by the −1195G allele‐containing COX‐2 promoter were significantly higher in both colon cancer cell lines. A 2.2‐fold increase in promoter activity was observed in the HCT‐116 cell line (P < 0.001), and this over‐expression was even more noticeable in the HCA‐7 COX‐2 expressing cell line with a threefold higher transcriptional activity (P = 0.001). The −1195G allele appeared to enhance COX‐2 transcription, providing a molecular basis underlying the increased susceptibility for CRC and potentially a new mechanism for COX‐2 overexpression.

Predictive clinical model of tumor response after chemoradiation in rectal cancer

Survival improvement in rectal cancer treated with neoadjuvant chemoradiotherapy (nCRT) is achieved only if pathological response occurs. Mandard tumor regression grade (TRG) proved to be a valid system to measure nCRT response. The ability to predict tumor response before treatment may significantly have impact the selection of patients for nCRT in rectal cancer. The aim is to identify potential predictive pretreatment factors for Mandard response and build a clinical predictive model design. 167 patients with locally advanced rectal cancer were treated with nCRT and curative surgery. Blood cell counts in peripheral blood were analyzed. Pretreatment biopsies expression of cyclin D1, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and protein 21 were assessed. A total of 61 single nucleotide polymorphisms were characterized using the Sequenom platform through multiplex amplification followed by mass-spectometric product separation. Surgical specimens were classified according to Mandard TRG. The patients were divided as: “good responders” (Mandard TRG1-2) and “poor responders” (Mandard TGR3-5). We examined predictive factors for Mandard response and performed statistical analysis. In univariate analysis, distance from anal verge, neutrophil lymphocyte ratio (NLR), cyclin D1, VEGF, EGFR, protein 21 and rs1810871 interleukin 10 (IL10) gene polymorphism are the pretreatment variables with predictive value for Mandard response. In multivariable analysis, NLR, cyclin D1, protein 21 and rs1800871 in IL10 gene maintain predictive value, allowing a clinical model design. Conclusion: It seems possible to use pretreatment expression of blood and tissue biomarkers, and build a model of tumor response prediction to neoadjuvant chemoradiation in rectal cancer.

Genetic variants as ovarian cancer first-line treatment hallmarks: A systematic review and meta-analysis

BACKGROUND The potential predictive value of genetic polymorphisms in ovarian cancer first-line treatment is inconsistently reported. We aimed to review ovarian cancer pharmacogenetic studies to update and summarize the available data and to provide directions for further research. METHODS A systematic review followed by a meta-analysis was conducted on cohort studies assessing the involvement of genetic polymorphisms in ovarian cancer first-line treatment response retrieved through a MEDLINE database search by November 2016. Studies were pooled and summary estimates and 95% confidence intervals (CI) were calculated using random or fixed-effects models as appropriate. RESULTS One hundred and forty-two studies gathering 106871 patients were included. Combined data suggested that GSTM1-null genotype patients have a lower risk of death compared to GSTM1-wt carriers, specifically in advanced stages (hazard ratio (HR), 0.68; 95% CI, 0.48-0.97) and when submitted to platinum-based chemotherapy (aHR, 0.61; 95% CI, 0.39-0.94). ERCC1 rs11615 and rs3212886 might have also a significant impact in treatment outcome (aHR, 0.67; 95% CI, 0.51-0.89; aHR, 1.28; 95% CI, 1.01-1.63, respectively). Moreover, ERCC2 rs13181 and rs1799793 showed a distinct ethnic behavior (Asians: aHR, 1.41; 95% CI, 0.80-2.49; aHR, 1.07; 95% CI, 0.62-1.86; Caucasians: aHR, 0.10; 95% CI, 0.01-0.96; aHR, 0.18; 95% CI, 0.05-0.68, respectively). CONCLUSION(S) The definition of integrative predictive models should encompass genetic information, especially regarding GSTM1 homozygous deletion. Justifying additional pharmacogenetic investigation are variants in ERCC1 and ERCC2, which highlight the DNA Repair ability to ovarian cancer prognosis. Further knowledge could aid to understand platinum-treatment failure and to tailor chemotherapy strategies.

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2–6 sialic acids

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2–6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.

Pharmacogenomics in epithelial ovarian cancer first-line treatment outcome: validation of GWAS-associated NRG3 rs1649942 and BRE rs7572644 variants in an independent cohort

The identification of predictive biomarkers for the first-line treatment of epithelial ovarian cancer (EOC) remains a challenge. Although genome-wide association studies (GWAS) have identified several genetic polymorphisms as predictors of EOC clinical outcome, the subsequent validation has not yet been performed. This study aims to validate the influence of Neuregulin 3 (NRG3) rs1649942 and Brain and reproductive organ-expressed (TNFRSF1A modulator) (BRE) rs7572644 GWAS-identified variants in an independent cohort of EOC patients from the North region of Portugal (n = 339) submitted to first-line treatment. Polymorphism genotypes were determined by real-time PCR using validated assays. Patients carrying the NRG3 rs1649942 A allele presented a significantly longer overall survival (OS) when compared to GG-genotype patients (log-rank test, P = 0.011) in the FIGO IV stage subgroup. No impact was observed for early-stage patients or considering disease-free survival (DFS) as an outcome. For FIGO I/II stage patients, BRE rs7572644 C allele carriers exhibit a decreased OS (P = 0.014) and DFS (P = 0.032) when compared to TT-homozygous patients. Furthermore, a Multivariate Cox regression analysis revealed a three-fold increase in the risk of death (HR, 3.09; P = 0.015) and recurrence (HR, 3.33; P = 0.009) for FIGO I/II C allele carriers. No significant impact was observed for late-stage patients. The BRE rs7572644 and NRG3 rs1649942 genetic variants were validated in an independent cohort of EOC Portuguese patients, particularly in specific subgroups considering FIGO staging. Further functional post-GWAS analyses are indispensable to understand the biological mechanisms underlying the observed results.

Influence of Genetic Polymorphisms in Prostaglandin E2 Pathway (COX-2/HPGD/SLCO2A1/ABCC4) on the Risk for Colorectal Adenoma Development and Recurrence after Polypectomy

Objectives:Deregulation of prostaglandin E2 (PGE2) levels reported in colorectal carcinogenesis contributes to key steps of cancer development. Our aim was to evaluate the influence of the genetic variability in COX-2/HPGD/SLCO2A1/ABCC4 PGE2 pathway genes on the development and recurrence of colorectal adenomas.Methods:A case-control study was conducted gathering 480 unscreened individuals and 195 patients with personal history of adenomas. A total of 43 tagSNPs were characterized using the Sequenom platform or real-time PCR.Results:Ten tagSNPs were identified as susceptibility biomarkers for the development of adenomas. The top three most meaningful tagSNPs include the rs689466 in COX-2 (odds ratio (OR)=3.23; 95% confidence interval (CI): 1.52–6.86), rs6439448 in SLCO2A1 (OR=0.38; 95% CI: 0.22–0.65) and rs1751051 in ABCC4 genes (OR=2.75; 95% CI: 1.58–4.80). The best four-locus gene–gene interaction model included the rs1346271, rs1863642 and rs12500316 single nucleotide polymorphisms in HPGD and rs1678405 in ABCC4 genes and was associated with a 13-fold increased susceptibility (95% CI: 3.84–46.3, P<0.0001, cross-validation (CV) accuracy: 0.78 and CV consistency: 8/10). Interesting, in low-risk patients the ABCC4 rs9524821AA genotype was associated not only with a higher hazard ratio (HR=2.93; 95% CI: 1.07–8.03), but half of these patients had adenoma recurrence at 60 months, considerably higher than the 21% noticed in low-risk patients.Conclusions:Genetic polymorphisms in COX-2/PGE2 pathway appear to contribute to the development of colorectal adenomas and influence the interval time to adenomas recurrence. The definition of risk models through the inclusion of genetic biomarkers might improve the adherence and optimization of current screening and surveillance guidelines for colorectal cancer prevention.