- Email: [email protected]
Novel insights into Notum and glypicans regulation in colorectal cancer
S154
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
domain-binding protein 1-like protein, protein Mdm4, Rho guanine nucleotide exchange factor 9, Neuroplastin) previously described as tumor specific proteins were found in DNPC of BCP and no one of them was found in HD. The data obtained demonstrate involvement of number of cellular and extracellular matrix proteins in cfDNA circulation. The meaning of the proteins in cfDNA circulation is unclear but their diagnostic potentialities or use for tumor-specific cfDNA isolation can be potentially explored and employed. No conflict of interest. 690 Transcriptional variations associated with time to breast cancer development among African American women with benign breast disease A.N. Holowatyj1 , J.J. Ruterbusch1 , R. Ali-Fehmi2 , S. Bandyopadhyay2 , G. Dyson1 , D. Radisky3 , M.L. Cote1 . 1 Wayne State University School of Medicine & Barbara Ann Karmanos Cancer Institute, Department of Oncology, Detroit, USA, 2 Wayne State University School of Medicine, Department of Pathology, Detroit, USA, 3 Mayo Clinic Cancer Center, Department of Cancer Biology, Jacksonville, USA Background: Benign breast disease, a known risk factor for subsequent breast cancer, includes a histological spectrum of lesions that can be subdivided based on the overall impression into nonproliferative lesions, proliferative lesions without atypia, and atypical hyperplasia. Yet little is known about benign breast tissue from African American women, who suffer from disproportionately high breast cancer mortality rates. Methods: To better characterize the risk of breast cancer among African American women with benign breast disease, we assessed the benign breast pathology of African American women in our metropolitan Detroit cohort. RNA was extracted from archival formalin-fixed, paraffin-embedded (FFPE) benign breast tissue blocks for 37 of these African American women who subsequently developed breast cancer. Gene expression profiling was performed on these samples using the Affymetrix HTA2.0 exon array chip. Cox proportional hazards modeling was performed to compare time to breast cancer among 67,528 transcripts adjusting for the presence of proliferative disease, atypia, and age at biopsy. Results: Among these 37 patients, median time to breast cancer was 6.4 years, with time to diagnosis ranging from 1 to 15 years. We identified 1,826 transcripts that were associated with a significant hazard for developing cancer after adjusting for patient age at biopsy and overall impression (alpha set at <0.01). Mapping of these 1,826 transcripts resulted in 847 annotated genes, of which the increased expression of 577 genes was associated with a significantly shorter time to diagnosis of a subsequent breast cancer. Using Ingenuity Pathway Analysis (IPA), these 847 genes found in benign tissue were found to be associated with networks of cancer metabolism and cellular development, with cancer among the top diseases and functions. In particular, genes regulated by the estrogen receptor (ER) were found to be higher expressed and significantly associated with shorter time to subsequent breast cancer diagnosis. Conclusions: Our findings highlight the first study to assess the association between transcriptional variations of benign breast disease and subsequent breast cancer development. These results identify increased expression of critical genes and pathways that are significantly associated with shorter time to subsequent breast cancer development. No conflict of interest. 691 Circulating microRNA profiles in plasma: identification of miR-224 as a novel diagnostic biomarker in hepatocellular carcinoma independent of hepatic function W. Okajima1 , S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , T. Ohashi1 , T. Imamura1 , J. Kiuchi1 , H. Taniguchi1 , O. Eigo1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan Background: Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Despite continuous global efforts aimed at eradication and improvements in various treatment techniques, the prognosis of HCC remains poor. However, only a few blood-based molecular biomarkers have been identified in HCC. The present study was designed to identify novel microRNAs (miRNAs) in plasma for detecting and monitoring HCC, independent of hepatic function and background liver diseases with different etiologies. Material and Methods: We performed a systematic review of the NCBI database and selected candidate miRNAs reported as highly expressed in HCC tissue. Results: (1) Four oncogenic miRNAs (miR-151, 155, 191 and 224) with high expression in HCC tissues were selected as candidates. (2) Quantitative RTPCR using plasma samples from 107 HCC patients and 75 healthy volunteers revealed a significantly higher level of plasma miR-224 in HCC patients than in healthy volunteers according to a small-scale analysis (P < 0.0001),
two independent cohort analyses and a large-scale analysis (P < 0.0001, AUC 0.908). (3) miR-224 expression was significantly higher in HCC tissues (P = 0.0011) and HCC cell lines (P = 0.0150) than in normal hepatic tissues and fibroblasts, respectively. (4) Plasma miR-224 reflected tumor dynamics; preoperative plasma levels of miR-224 were significantly reduced in postoperative samples (P = 0.0058), and plasma miR-224 levels were significantly correlated with paired miR-224 levels in HCC tissues (P = 0.0005). (5) Furthermore, plasma miR-224 levels significantly discriminated HCC patients from patients with chronic liver disease (P = 0.0008). A high plasma miR-224 level was significantly correlated with larger tumor size (P = 0.0005), advanced stage (P = 0.0382) and recurrences (P = 0.0027). The plasma miR-224 level could accurately detect small tumors less than 18 mm in size (AUC: 0.814) preoperatively and residual viable HCC tissues following percutaneous ablation therapy or trans-catheter arterial chemoembolization (TACE) therapy (P = 0.0318). Conclusions: Plasma miR-224 may be a sensitive biomarker for screening HCC and monitoring tumor dynamics. No conflict of interest.
Monday 11 July 2016 Poster Session
Prevention and Early Detection II 692 Dose–response relationship between inorganic arsenic exposure and lung cancer among arseniasis area residents with low methylation capacity: A population-based cohort study P.J. Liao1 , K.H. Hsu2 . 1 Oriental Institute of Technology, Department of Health Care Administration, New Taipei City, Taiwan, 2 Chang Gung University, Laboratory for Epidemiology- Department of Health Care Management and Healthy Aging Research Center, Taoyuan City, Taiwan Background: Exposure to inorganic arsenic (InAs)-contaminated water has been documented as a risk factor for lung cancers. This study examined the association between InAs exposure dose, its metabolism pattern, and lung cancer occurrence. Material and Methods: We followed 1300 residents from an arseniasis area in Taiwan, determined urinary InAs metabolites, and identified 39 lung cancer cases. We performed descriptive and univariate statistical analyses and used a Cox proportional hazard model for lung cancer occurrence. Results: The results demonstrated that participants with either the primary methylation index (monomethylarsonic acid [MMA]/InAs) or the secondary methylation index (dimethylarsinic acid [DMA]/MMA) lower than their respective median values were at a higher risk of lung cancer (hazard ratio = 3.41–4.66) than those with high methylation capacity. The incidence density of lung cancer increased from 79.9/100000 (year-1) to 467.4/100000 (year-1) for residents with low methylation capacity when the arsenic exposure dose increased from 2−10 ppb to 200 ppb, respectively. The multiple Cox regression models revealed a dose–response relationship between lung cancer occurrence and increasing arsenic concentrations in drinking water as well as cumulative arsenic exposure (monotonic trend test; P < 0.05 and P < 0.05, respectively) among the residents with low methylation capacity. The relationship between arsenic exposure and lung cancer among the residents with high methylation capacity was nonsignificant. Conclusions: Hypomethylation responses to low concentrations of InAs exposure may dose-dependently increase lung cancer occurrence. The highrisk characteristics observed among those exposed should be considered in future preventive medicine and research on arsenic carcinogenesis. No conflict of interest. 693 Novel insights into Notum and glypicans regulation in colorectal cancer M. De Robertis1,2 , M. Arigoni3 , L. Loiacono4,5 , F. Riccardo3 , R.A. Calogero3 , Y. Feodorova6,7 , D. Tashkova8 , V. Belovejdov8 , V. Sarafian6,7 , F. Cavallo3 , E. Signori1,2 . 1 Laboratory of Genetic and Clinical Pathology, University Campus Bio-Medico of Rome, Roma, Italy, 2 Institute of Translational Pharmacology, National Research Council, Roma, Italy, 3 Molecular Biotechnology Center-University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy, 4 Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, S. Giovanni Rotondo-Foggia, Italy, 5 University of Foggia, Department of Medical and Surgical Sciences, Foggia, Italy, 6 Medical University of Plovdiv, Department of Medical Biology, Plovdiv, Bulgaria, 7 University of Plovdiv, Technological Centre of Emergency Medicine, Plovdiv, Bulgaria, 8 Medical University of Plovdiv, Department of General and Clinical Pathology, Plovdiv, Bulgaria Background: Little is known regarding the fine regulation of the Wnt/b-catenin pathway or its biological functions that might be involved in the pathogenesis of CRC. To identify potential novel CRC biomarkers, the well known
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 azoxymethane (AOM)/ dextran sulfate sodium (DSS) murine model was used to perform a genome-wide expression profiling. The obtained results have been validated in a set of human CRC samples. Material and Methods: 30 Balb/c mice were divided in 4 groups: (1) AOM/DSS treated, (2) AOM treated, (3) DSS treated, (4) untreated. 20 weeks from the cancer induction protocol, 6 mice/group were sacrificed. RNA was isolated from each sample using Trizol® . Genome-wide expression microarray was performed using MouseWG-6v2.0 Expression BeadChip (Illumina, Inc, CA). Microarray data were evaluated by state of the art statistics tools. IPA7 (www.ingenuity.com) was used for Gene Ontology (GO) analysis. 6 AOM/DSS treated mice were investigated at 5th week for early colon lesions. Tissues from 10 patients with non-metastatic CRC staged as T3N0 with a G2 grade of cellular differentiation were analysed. Histopathological analysis and immunohistochemistry (IHC) followed standard procedures. Results: Gene expression profiling showed 2036 differentially expressed genes in AOM/DSS induced tumors, including 1092 upregulated and 944 downregulated genes. In addition to the preeminent activation of the Wnt/b-catenin pathway, GO analyses revealed the enhanced expression of genes encoding Wnt antagonists, which might set up a negative feedback response to activated Wnt/b-catenin signaling in CRC. In particular, we found that the NOTUM gene was significantly up-regulated and two heparan sulfate proteoglycans, Glypican-1 (GPC1) and Glypican-3 (GPC3), which are under Notum control and act as competitive inhibitors of Wnt, were up- and downregulated, respectively. Moreover, the IHC on animal tissues showed a positive correlation of NOTUM overexpression with intracellular bcatenin staining and a correlation with glypicans expression. We revealed an inverse correlation between molecular expression and protein distribution between Notum and GLY3. Conversely, GLY1 showed a very similar expression pattern to Notum in late lesions, although a positive staining was observed in 90% of early lesions. We validated the preclinical results on a set of human colorectal adenocarcinoma. We found a significant alteration of the molecular levels of these Wnt pathway molecular mediators in human CRC samples with respect to normal mucosa. Conclusions: Taken together, our results provide the first demonstration of a perturbed expression in the NOTUM, GPC1 and GPC3 genes in the context of CRC development. Additionally, we show a significant correlation between the expression levels of these molecules and that of b-catenin, suggesting their role as novel biomarkers in CRC. No conflict of interest. 694 Screening and secondary prevention of colorectal cancer among limited contingent as a first step of implementing screening in Belarus I. Rebeko1 , A. Petkevich2 , S. Krasniy3 , I. Abelskaya4 , A. Gerasimovich2 , E. Lobachevskaya2 . 1 N.N. Alexandrov National Cancer Centre of Belarus, Abdominal oncological department, Minsk, Belarus, 2 State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus», Department of Endoscopy, Minsk, Belarus, 3 N.N. Alexandrov National Cancer Centre of Belarus, Research department, Minsk, Belarus, 4 State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus», Administration, Minsk, Belarus Background: Colorectal cancer (CRC) is one of the most significant oncological diseases in context of morbidity and death rate. Screening plays the key role in decreasing the death rate and in decreasing the morbidity of CRC when conducted protractedly and systematically. When implementing screening on the national scale it is vital to consider not only the experience of other countries, but also features of national health services, juridical basis and population. The aim is to estimate how effective is revelation of early phases of CRC and pre-malignant lesions (polyps) on the initial stage of conducting CRC screening and secondary prevention. The CRC incidence rate in the State Institution “Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus” is similar to the CRC incidence in the Republic of Belarus. Accordingly, the results of the study could be used as a model for further CRC screening program in Belarus. Material and Methods: According to study design, from those participants of 45−70 years old without of any intestinal symptoms who are attached to the State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus» two samples of faeces are taken to immunochemical fecal occult blood test (FIT). Regardless of the FIT results, all the participants undergo total colonoscopy (TC) during intravenous anesthesia. All discovered polyps were resected during screening TC. Results: Since 2014, 332 (127 men, 230 women) participants have been included in the screening program. Correlation of participants by age were as follows: 50 cases aged 45−49, 109 aged 50−54, 94 of 55−59 years old, 54 aged 60−64, and 25 subjects aged 65−70.
S155
Polyps were revealed in cases of 172 patients (51.8%, 290 polyps). Adenomatous polyps constituted 47.3%, one patient had the cancer in situ. In the group of patients aged between 45 and 49 twenty-five polyps were revealed in cases of 17 people, from which there were 11 adenomatous (44%). Thirtytwo (9.6%) fecal samples were positive on FIT, among them pathology was revealed in 21 cases (65%). Conclusions: 1. Such research on screening of CRC is conducted for the first time in Belarus. 2. In 51.8% of cases polypous formations were found and resected, 47.3% of which are adenomatous and therefore potentially dangerous with respect to neoplastic transformation. 3. Effectiveness of FIT is limited. For certain assessment more participants are required. 4. In the group of participants aged between 45 and 49 adenomatous polyps were determined in 44% of cases, which can correlate with general population index (48%) and point out the necessity of reconsidering the age interval of screening activities in Belarus. No conflict of interest. 695 Circulating cell free DNA (cfDNA) as a novel tool to monitor Barrett’s esophagus neoplastic progression E. Boldrin1 , E. Rumiato1 , M. Fassan2 , L. Balsamo2 , S. Realdon3 , G. Battaglia3 , M. Rugge2 , A. Amadori4 , D. Saggioro1 . 1 Veneto Institute of Oncology IOV-IRCCS, Immunology and Molecular Oncology, Padova, Italy, 2 University of Padova, Department of Medicine DIMED- Surgical Pathology and Cytopathology, Padova, Italy, 3 Veneto Institute of Oncology IOV-IRCCS, Endoscopy Unit, Padova, Italy, 4 University of Padova, Department of Surgical Sciences- Oncology and Gastroenterology DISCOG, Padova, Italy Background: Barrett’s esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma (EAC). For this reason, BE patients are subjected to endoscopic-based surveillance that, however, is burdensome for the patients and expensive for the national health systems. In this study, we investigated the power of circulating cell free DNA (cfDNA) as a new diagnostic tool in monitoring neoplastic transformation. Material and Methods: We analyzed DNA from longitudinally collected plasma samples and paired formalin fixed paraffin embedded (FFPE) specimens of BE patients who progressed to EAC; we also studied cfDNA from long-term BE patients, as control. Based on previously reported recurrent alterations in BE and EAC studies, samples were investigated for both the presence of loss of heterozygosity (LOH) in areas proximal to TP53, FHIT, CDKN2A and BRCA2 genes, and mutations in exons 4, 5, 7 and 8 of TP53 gene. Results: We found that in the cfDNA of dysplastic BE patients it is possible to detect genetic alterations that not only retrace the time-matched biopsy profile but better represent the total alterations of the dysplastic BE epithelium. No alterations in the cfDNA were found after curative endoscopic mucosectomy, and in the long-term BE patients. BE molecular alterations were mainly localized proximal to, or within, TP53 gene. Conclusions: Although further analyses are required to assess the sensitivity of the LOH analysis in cfDNA extracted from BE patients, these results suggest that the liquid biopsy could become a new, less invasive, diagnostic tool to monitor BE patients by overcoming the limitation of tissue biopsies that only partially depict the complex neoplastic heterogeneity of BE epithelium. No conflict of interest. 696 Prospective validation of a blood-based 9-miRNA profile for early detection of breast cancer in a cohort of women examined by clinical mammography M. Lyng1 , A. Kodahl2 , H. Binder3 , H. Dtizel1 . 1 Molecular medicin, Department of Cancer and Inflammation, Odense, Denmark, 2 Odense University Hospital, Department of Oncology, Odense, Denmark, 3 Institute of Medical Biostatistics, Epidemiology and Biostatistics IMBEI, Mainz, Germany Introduction: Death rates have been declining for breast cancer, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of tumors that might not require treatment, and under-diagnosis of cancers in women with dense breast tissue. Breast cancer screening could be improved by pairing mammography with a blood-based biomarker, of which there are currently none clinically approved. Circulating microRNAs (miRNAs) are increasingly recognized as powerful biomarkers in several pathologies, including breast cancer. We have previously developed a 9-miRNA profile using serum and LNA-based qPCR, which enabled stratification of healthy women vs. breast cancer patients in a retrospective validation cohort (AUC = 0.68, p = 0.012) (Kodahl et al., Mol.Oncol. 2014). Material and Methods: The 9-miRNA profile (miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365, miR-425) was investigated in a prospectively collected cohort of women examined by clinical
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
domain-binding protein 1-like protein, protein Mdm4, Rho guanine nucleotide exchange factor 9, Neuroplastin) previously described as tumor specific proteins were found in DNPC of BCP and no one of them was found in HD. The data obtained demonstrate involvement of number of cellular and extracellular matrix proteins in cfDNA circulation. The meaning of the proteins in cfDNA circulation is unclear but their diagnostic potentialities or use for tumor-specific cfDNA isolation can be potentially explored and employed. No conflict of interest. 690 Transcriptional variations associated with time to breast cancer development among African American women with benign breast disease A.N. Holowatyj1 , J.J. Ruterbusch1 , R. Ali-Fehmi2 , S. Bandyopadhyay2 , G. Dyson1 , D. Radisky3 , M.L. Cote1 . 1 Wayne State University School of Medicine & Barbara Ann Karmanos Cancer Institute, Department of Oncology, Detroit, USA, 2 Wayne State University School of Medicine, Department of Pathology, Detroit, USA, 3 Mayo Clinic Cancer Center, Department of Cancer Biology, Jacksonville, USA Background: Benign breast disease, a known risk factor for subsequent breast cancer, includes a histological spectrum of lesions that can be subdivided based on the overall impression into nonproliferative lesions, proliferative lesions without atypia, and atypical hyperplasia. Yet little is known about benign breast tissue from African American women, who suffer from disproportionately high breast cancer mortality rates. Methods: To better characterize the risk of breast cancer among African American women with benign breast disease, we assessed the benign breast pathology of African American women in our metropolitan Detroit cohort. RNA was extracted from archival formalin-fixed, paraffin-embedded (FFPE) benign breast tissue blocks for 37 of these African American women who subsequently developed breast cancer. Gene expression profiling was performed on these samples using the Affymetrix HTA2.0 exon array chip. Cox proportional hazards modeling was performed to compare time to breast cancer among 67,528 transcripts adjusting for the presence of proliferative disease, atypia, and age at biopsy. Results: Among these 37 patients, median time to breast cancer was 6.4 years, with time to diagnosis ranging from 1 to 15 years. We identified 1,826 transcripts that were associated with a significant hazard for developing cancer after adjusting for patient age at biopsy and overall impression (alpha set at <0.01). Mapping of these 1,826 transcripts resulted in 847 annotated genes, of which the increased expression of 577 genes was associated with a significantly shorter time to diagnosis of a subsequent breast cancer. Using Ingenuity Pathway Analysis (IPA), these 847 genes found in benign tissue were found to be associated with networks of cancer metabolism and cellular development, with cancer among the top diseases and functions. In particular, genes regulated by the estrogen receptor (ER) were found to be higher expressed and significantly associated with shorter time to subsequent breast cancer diagnosis. Conclusions: Our findings highlight the first study to assess the association between transcriptional variations of benign breast disease and subsequent breast cancer development. These results identify increased expression of critical genes and pathways that are significantly associated with shorter time to subsequent breast cancer development. No conflict of interest. 691 Circulating microRNA profiles in plasma: identification of miR-224 as a novel diagnostic biomarker in hepatocellular carcinoma independent of hepatic function W. Okajima1 , S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , T. Ohashi1 , T. Imamura1 , J. Kiuchi1 , H. Taniguchi1 , O. Eigo1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan Background: Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Despite continuous global efforts aimed at eradication and improvements in various treatment techniques, the prognosis of HCC remains poor. However, only a few blood-based molecular biomarkers have been identified in HCC. The present study was designed to identify novel microRNAs (miRNAs) in plasma for detecting and monitoring HCC, independent of hepatic function and background liver diseases with different etiologies. Material and Methods: We performed a systematic review of the NCBI database and selected candidate miRNAs reported as highly expressed in HCC tissue. Results: (1) Four oncogenic miRNAs (miR-151, 155, 191 and 224) with high expression in HCC tissues were selected as candidates. (2) Quantitative RTPCR using plasma samples from 107 HCC patients and 75 healthy volunteers revealed a significantly higher level of plasma miR-224 in HCC patients than in healthy volunteers according to a small-scale analysis (P < 0.0001),
two independent cohort analyses and a large-scale analysis (P < 0.0001, AUC 0.908). (3) miR-224 expression was significantly higher in HCC tissues (P = 0.0011) and HCC cell lines (P = 0.0150) than in normal hepatic tissues and fibroblasts, respectively. (4) Plasma miR-224 reflected tumor dynamics; preoperative plasma levels of miR-224 were significantly reduced in postoperative samples (P = 0.0058), and plasma miR-224 levels were significantly correlated with paired miR-224 levels in HCC tissues (P = 0.0005). (5) Furthermore, plasma miR-224 levels significantly discriminated HCC patients from patients with chronic liver disease (P = 0.0008). A high plasma miR-224 level was significantly correlated with larger tumor size (P = 0.0005), advanced stage (P = 0.0382) and recurrences (P = 0.0027). The plasma miR-224 level could accurately detect small tumors less than 18 mm in size (AUC: 0.814) preoperatively and residual viable HCC tissues following percutaneous ablation therapy or trans-catheter arterial chemoembolization (TACE) therapy (P = 0.0318). Conclusions: Plasma miR-224 may be a sensitive biomarker for screening HCC and monitoring tumor dynamics. No conflict of interest.
Monday 11 July 2016 Poster Session
Prevention and Early Detection II 692 Dose–response relationship between inorganic arsenic exposure and lung cancer among arseniasis area residents with low methylation capacity: A population-based cohort study P.J. Liao1 , K.H. Hsu2 . 1 Oriental Institute of Technology, Department of Health Care Administration, New Taipei City, Taiwan, 2 Chang Gung University, Laboratory for Epidemiology- Department of Health Care Management and Healthy Aging Research Center, Taoyuan City, Taiwan Background: Exposure to inorganic arsenic (InAs)-contaminated water has been documented as a risk factor for lung cancers. This study examined the association between InAs exposure dose, its metabolism pattern, and lung cancer occurrence. Material and Methods: We followed 1300 residents from an arseniasis area in Taiwan, determined urinary InAs metabolites, and identified 39 lung cancer cases. We performed descriptive and univariate statistical analyses and used a Cox proportional hazard model for lung cancer occurrence. Results: The results demonstrated that participants with either the primary methylation index (monomethylarsonic acid [MMA]/InAs) or the secondary methylation index (dimethylarsinic acid [DMA]/MMA) lower than their respective median values were at a higher risk of lung cancer (hazard ratio = 3.41–4.66) than those with high methylation capacity. The incidence density of lung cancer increased from 79.9/100000 (year-1) to 467.4/100000 (year-1) for residents with low methylation capacity when the arsenic exposure dose increased from 2−10 ppb to 200 ppb, respectively. The multiple Cox regression models revealed a dose–response relationship between lung cancer occurrence and increasing arsenic concentrations in drinking water as well as cumulative arsenic exposure (monotonic trend test; P < 0.05 and P < 0.05, respectively) among the residents with low methylation capacity. The relationship between arsenic exposure and lung cancer among the residents with high methylation capacity was nonsignificant. Conclusions: Hypomethylation responses to low concentrations of InAs exposure may dose-dependently increase lung cancer occurrence. The highrisk characteristics observed among those exposed should be considered in future preventive medicine and research on arsenic carcinogenesis. No conflict of interest. 693 Novel insights into Notum and glypicans regulation in colorectal cancer M. De Robertis1,2 , M. Arigoni3 , L. Loiacono4,5 , F. Riccardo3 , R.A. Calogero3 , Y. Feodorova6,7 , D. Tashkova8 , V. Belovejdov8 , V. Sarafian6,7 , F. Cavallo3 , E. Signori1,2 . 1 Laboratory of Genetic and Clinical Pathology, University Campus Bio-Medico of Rome, Roma, Italy, 2 Institute of Translational Pharmacology, National Research Council, Roma, Italy, 3 Molecular Biotechnology Center-University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy, 4 Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, S. Giovanni Rotondo-Foggia, Italy, 5 University of Foggia, Department of Medical and Surgical Sciences, Foggia, Italy, 6 Medical University of Plovdiv, Department of Medical Biology, Plovdiv, Bulgaria, 7 University of Plovdiv, Technological Centre of Emergency Medicine, Plovdiv, Bulgaria, 8 Medical University of Plovdiv, Department of General and Clinical Pathology, Plovdiv, Bulgaria Background: Little is known regarding the fine regulation of the Wnt/b-catenin pathway or its biological functions that might be involved in the pathogenesis of CRC. To identify potential novel CRC biomarkers, the well known
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 azoxymethane (AOM)/ dextran sulfate sodium (DSS) murine model was used to perform a genome-wide expression profiling. The obtained results have been validated in a set of human CRC samples. Material and Methods: 30 Balb/c mice were divided in 4 groups: (1) AOM/DSS treated, (2) AOM treated, (3) DSS treated, (4) untreated. 20 weeks from the cancer induction protocol, 6 mice/group were sacrificed. RNA was isolated from each sample using Trizol® . Genome-wide expression microarray was performed using MouseWG-6v2.0 Expression BeadChip (Illumina, Inc, CA). Microarray data were evaluated by state of the art statistics tools. IPA7 (www.ingenuity.com) was used for Gene Ontology (GO) analysis. 6 AOM/DSS treated mice were investigated at 5th week for early colon lesions. Tissues from 10 patients with non-metastatic CRC staged as T3N0 with a G2 grade of cellular differentiation were analysed. Histopathological analysis and immunohistochemistry (IHC) followed standard procedures. Results: Gene expression profiling showed 2036 differentially expressed genes in AOM/DSS induced tumors, including 1092 upregulated and 944 downregulated genes. In addition to the preeminent activation of the Wnt/b-catenin pathway, GO analyses revealed the enhanced expression of genes encoding Wnt antagonists, which might set up a negative feedback response to activated Wnt/b-catenin signaling in CRC. In particular, we found that the NOTUM gene was significantly up-regulated and two heparan sulfate proteoglycans, Glypican-1 (GPC1) and Glypican-3 (GPC3), which are under Notum control and act as competitive inhibitors of Wnt, were up- and downregulated, respectively. Moreover, the IHC on animal tissues showed a positive correlation of NOTUM overexpression with intracellular bcatenin staining and a correlation with glypicans expression. We revealed an inverse correlation between molecular expression and protein distribution between Notum and GLY3. Conversely, GLY1 showed a very similar expression pattern to Notum in late lesions, although a positive staining was observed in 90% of early lesions. We validated the preclinical results on a set of human colorectal adenocarcinoma. We found a significant alteration of the molecular levels of these Wnt pathway molecular mediators in human CRC samples with respect to normal mucosa. Conclusions: Taken together, our results provide the first demonstration of a perturbed expression in the NOTUM, GPC1 and GPC3 genes in the context of CRC development. Additionally, we show a significant correlation between the expression levels of these molecules and that of b-catenin, suggesting their role as novel biomarkers in CRC. No conflict of interest. 694 Screening and secondary prevention of colorectal cancer among limited contingent as a first step of implementing screening in Belarus I. Rebeko1 , A. Petkevich2 , S. Krasniy3 , I. Abelskaya4 , A. Gerasimovich2 , E. Lobachevskaya2 . 1 N.N. Alexandrov National Cancer Centre of Belarus, Abdominal oncological department, Minsk, Belarus, 2 State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus», Department of Endoscopy, Minsk, Belarus, 3 N.N. Alexandrov National Cancer Centre of Belarus, Research department, Minsk, Belarus, 4 State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus», Administration, Minsk, Belarus Background: Colorectal cancer (CRC) is one of the most significant oncological diseases in context of morbidity and death rate. Screening plays the key role in decreasing the death rate and in decreasing the morbidity of CRC when conducted protractedly and systematically. When implementing screening on the national scale it is vital to consider not only the experience of other countries, but also features of national health services, juridical basis and population. The aim is to estimate how effective is revelation of early phases of CRC and pre-malignant lesions (polyps) on the initial stage of conducting CRC screening and secondary prevention. The CRC incidence rate in the State Institution “Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus” is similar to the CRC incidence in the Republic of Belarus. Accordingly, the results of the study could be used as a model for further CRC screening program in Belarus. Material and Methods: According to study design, from those participants of 45−70 years old without of any intestinal symptoms who are attached to the State Institution «Republican Clinical Medical Centre of the Presidential Administration of the Republic of Belarus» two samples of faeces are taken to immunochemical fecal occult blood test (FIT). Regardless of the FIT results, all the participants undergo total colonoscopy (TC) during intravenous anesthesia. All discovered polyps were resected during screening TC. Results: Since 2014, 332 (127 men, 230 women) participants have been included in the screening program. Correlation of participants by age were as follows: 50 cases aged 45−49, 109 aged 50−54, 94 of 55−59 years old, 54 aged 60−64, and 25 subjects aged 65−70.
S155
Polyps were revealed in cases of 172 patients (51.8%, 290 polyps). Adenomatous polyps constituted 47.3%, one patient had the cancer in situ. In the group of patients aged between 45 and 49 twenty-five polyps were revealed in cases of 17 people, from which there were 11 adenomatous (44%). Thirtytwo (9.6%) fecal samples were positive on FIT, among them pathology was revealed in 21 cases (65%). Conclusions: 1. Such research on screening of CRC is conducted for the first time in Belarus. 2. In 51.8% of cases polypous formations were found and resected, 47.3% of which are adenomatous and therefore potentially dangerous with respect to neoplastic transformation. 3. Effectiveness of FIT is limited. For certain assessment more participants are required. 4. In the group of participants aged between 45 and 49 adenomatous polyps were determined in 44% of cases, which can correlate with general population index (48%) and point out the necessity of reconsidering the age interval of screening activities in Belarus. No conflict of interest. 695 Circulating cell free DNA (cfDNA) as a novel tool to monitor Barrett’s esophagus neoplastic progression E. Boldrin1 , E. Rumiato1 , M. Fassan2 , L. Balsamo2 , S. Realdon3 , G. Battaglia3 , M. Rugge2 , A. Amadori4 , D. Saggioro1 . 1 Veneto Institute of Oncology IOV-IRCCS, Immunology and Molecular Oncology, Padova, Italy, 2 University of Padova, Department of Medicine DIMED- Surgical Pathology and Cytopathology, Padova, Italy, 3 Veneto Institute of Oncology IOV-IRCCS, Endoscopy Unit, Padova, Italy, 4 University of Padova, Department of Surgical Sciences- Oncology and Gastroenterology DISCOG, Padova, Italy Background: Barrett’s esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma (EAC). For this reason, BE patients are subjected to endoscopic-based surveillance that, however, is burdensome for the patients and expensive for the national health systems. In this study, we investigated the power of circulating cell free DNA (cfDNA) as a new diagnostic tool in monitoring neoplastic transformation. Material and Methods: We analyzed DNA from longitudinally collected plasma samples and paired formalin fixed paraffin embedded (FFPE) specimens of BE patients who progressed to EAC; we also studied cfDNA from long-term BE patients, as control. Based on previously reported recurrent alterations in BE and EAC studies, samples were investigated for both the presence of loss of heterozygosity (LOH) in areas proximal to TP53, FHIT, CDKN2A and BRCA2 genes, and mutations in exons 4, 5, 7 and 8 of TP53 gene. Results: We found that in the cfDNA of dysplastic BE patients it is possible to detect genetic alterations that not only retrace the time-matched biopsy profile but better represent the total alterations of the dysplastic BE epithelium. No alterations in the cfDNA were found after curative endoscopic mucosectomy, and in the long-term BE patients. BE molecular alterations were mainly localized proximal to, or within, TP53 gene. Conclusions: Although further analyses are required to assess the sensitivity of the LOH analysis in cfDNA extracted from BE patients, these results suggest that the liquid biopsy could become a new, less invasive, diagnostic tool to monitor BE patients by overcoming the limitation of tissue biopsies that only partially depict the complex neoplastic heterogeneity of BE epithelium. No conflict of interest. 696 Prospective validation of a blood-based 9-miRNA profile for early detection of breast cancer in a cohort of women examined by clinical mammography M. Lyng1 , A. Kodahl2 , H. Binder3 , H. Dtizel1 . 1 Molecular medicin, Department of Cancer and Inflammation, Odense, Denmark, 2 Odense University Hospital, Department of Oncology, Odense, Denmark, 3 Institute of Medical Biostatistics, Epidemiology and Biostatistics IMBEI, Mainz, Germany Introduction: Death rates have been declining for breast cancer, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of tumors that might not require treatment, and under-diagnosis of cancers in women with dense breast tissue. Breast cancer screening could be improved by pairing mammography with a blood-based biomarker, of which there are currently none clinically approved. Circulating microRNAs (miRNAs) are increasingly recognized as powerful biomarkers in several pathologies, including breast cancer. We have previously developed a 9-miRNA profile using serum and LNA-based qPCR, which enabled stratification of healthy women vs. breast cancer patients in a retrospective validation cohort (AUC = 0.68, p = 0.012) (Kodahl et al., Mol.Oncol. 2014). Material and Methods: The 9-miRNA profile (miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365, miR-425) was investigated in a prospectively collected cohort of women examined by clinical