- Email: [email protected]
IKZF1 gene deletions are strongly associated with Ikaros dominant-negative isoforms expression in acute lymphoblastic leukemia
S150
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
(OR = 1.566), however the association did not reach statistical significance (p = 0.1197). We also evaluated a possible genotype-smoking association and found that C allele carrying individuals with smoking within the last 5 years had a significant risk of CRC (OR = 2.284; p = 0.0427). Conclusion: It has been revealed that CYP1A1 allele frequencies show large differences across different populations. Evaluation of an association between CYP1A1*2A and cancer susceptibility is more difficult and needs large number of participants in Caucasians. The data from this new study should be useful for understanding CRC aetiology and in identifying individual risk of developing CRC. Reference(s) [1] Inoue H, et al (2000). Cancer research, 60(14), 3749–3752. [2] Sivaraman L, et al (1994). Cancer research, 54(14), 3692–3695. [3] Welfare MR, et al (1997). Carcinogenesis, 18(7), 1351–1354. No conflict of interest. 671 Carcinogenic and non-carcinogenic risk assessment for children exposed to DDTs residues in pasteurized cow milk from Iran market N. Rastkari1 , M. Zare Jeddi2 , R. Ahmadkhaniha3 , M. Yunesian4 . 1 Tehran University of Medical Sciences, Center for Air Pollution Research CAPRInstitute for Environmental Research IER, Tehran, Iran, 2 Tehran University of Medical Sciences, Institute for Environmental Research, Tehran, Iran, 3 Tehran University of Medical Sciences, Department of Human EcologySchool of Public Health, Tehran, Iran, 4 Tehran University of Medical Sciences, Department of Environmental Health − School of Public Health, Tehran, Iran Background: Milk-producing animals accumulate pesticides from contaminated feed and by inhaling contaminated air. Organochlorine pesticides due to their lipophilic properties are initially stored in fat-rich tissues and subsequently translocated and excreted in milk. Therefore, the consumption of dairy products together with other contaminated food may expose consumers to unexpected levels of organochlorine pesticides. Organochlorine pesticides being endocrine disrupting chemicals are believed to produce a wide variety of adverse health outcomes such as cancers. Dichlodiphenyl-trichloroethane (DDT), a previously widely used OCP, and some of its major metabolites, is officially classified according to Integrated Risk Information System (IRIS) of United States Environmental Protection Agency (USEPA) as probable human carcinogens (Group B2) and responsible for causing damage to organs through repeated and prolonged exposure. Material and Methods: The objectives of the present study were to evaluate children’s exposure to DDTs (DDT and its metabolites) via dietary milk consumption in Iran, and to assess the respective potential risks to children’s health in terms of carcinogenic and non-carcinogenic effects. Residue levels were determined by GCMS analysis in 60 cow milk samples of Full fat (3%) pasteurized commercial types. The estimated daily intake (EDI) was calculated for different ages (1, 3, 5 and 7 years old children) based on the respective milk consumptions and finally Hazard quotient (HQ) for non-carcinogenic effects and hazard rate for carcinogenic effect were estimated. Results and Discussion: In all of the samples DDT metabolites were detected in the range of 0.0015 to 0. 2796 mg/L with the mean of 0.047±0.03, 0.15±0.08, 0.085±0.05, 0.05±0.02 mg/L for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD, respectively. The calculated EDIs (0.002–0.03 mg/kg-day) for all age categories and all compounds were lower than the RfDs. Consequently, the hazard quotients calculated in these groups for DDTs were far less than 1. For estimation of the cancer risk values, the cancer benchmark concentrations (CBC) were derived using oral slope factors and their hazard ratios (HR) in all age categories. Hazard ratio obtained for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD in Full fat (3%) pasteurized milk could not pose potential carcinogenic risk to young children (1, 3, 5 and 7 years old) since all of the HRs were far less than 1. Conclusion: This study has shown that cow milk is not a major source of human exposure to DDTs and from this perspective is safe for consumption. No conflict of interest. 672 Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: Gene−environment interaction H. Masaoka1,2 , H. Ito3,4 , N. Soga5 , A. Yokomizo2 , M. Eto2 , K. Matsuo1,4 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Kyushu University Graduate School of Medical Sciences, Department of Urology, Fukuoka, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan, 4 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 5 Aichi Cancer Center Hospital, Department of Urology, Nagoya, Japan Background: Although a range of chemical exposures (cigarette smoking and occupational exposure) are recognized risk factors for the development of
bladder cancer (BCa), many epidemiological studies have demonstrated that alcohol drinking is not associated with BCa risk. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms impact the accumulation of acetaldehyde, resulting in an increased risk of various cancers. To date, however, no studies evaluating the association between BCa risk and alcohol drinking have considered these polymorphisms. Here, we conducted a matched case–control study to investigate whether ALDH2 and ADH1B polymorphisms influence BCa risk associated with alcohol drinking. Material and Methods: Cases were 74 BCa patients and controls were 740 first-visit outpatients without cancer at Aichi Cancer Center Hospital between January 2001 and December 2005. Odds ratio (OR), 95% confidence interval (CI) and gene–environment interaction were assessed by conditional logistic regression analysis with adjustment for potential confounders. Results: ALDH2 Glu/Lys was associated with a significantly increased risk of BCa compared with Glu/Glu (OR 2.03, 95% CI 1.14–3.62). In contrast, ALDH2 Glu/Lys showed no increase in risk among the stratum of never drinkers compared with Glu/Glu, indicating a gene–environment interaction. ADH1B His/Arg had an OR of 1.98 (1.20–3.24) compared with His/His. ADH1B Arg+ showed a similar OR and 95% CI. Individuals with ALDH2 Glu/Lys and ADH1B Arg+ had the highest risk of BCa compared with ALDH2 Glu/Glu and ADH1B His/His (OR 4.00, 95% CI 1.81–8.87)]. Conclusions: Those with the ALDH2 Glu/Lys and ADH1B Arg+ genotype are at increased risk of BCa. Gene–environment interaction between ALDH2 Glu/Lys and alcohol drinking might suggest acetaldehyde contributes to the carcinogenesis of BCa. No conflict of interest. 673 Association between night shift work and methylation status of circadian rhythm genes among Polish nurses and midwives − preliminary results A. Bukowska1 , E. Wieczorek2 , B. Peplonska1 , M. Przybek2 , E. Jablonska2 , E. Reszka2 . 1 Nofer Institute of Occupational Medicine, Department of Environmental Epidemiology, Lodz, Poland, 2 Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Lodz, Poland Background: It has been suggested that rotating night shift work may contribute to epigenetic changes in the circadian rhythm genes. The results of previous studies in this topic are sparse and inconsistent. The aim of the study was to assess the potential relationship between current rotating night shift work and changes in levels of 5-methylcytosine in the promoter regions of selected circadian rhythm genes. Material and Method: The cross-sectional study included as many as 710 nurses and midwives (348 working on rotating night shifts and 362 working only during the day) aged 40−60 years. During in person interviews, information about occupational history and potential confounders was collected. DNA was isolated from peripheral blood samples and promoter methylation status of core circadian rhythm genes (PER1, PER2, PER3, BMAL1, CLOCK, CRY1, CRY2, NPAS2) was determined using quantitative methylation-specific real-time PCR assay (qMSP) method. Univariate and multivariate regression models were used to assess the association between current work at night and epigenetic changes, with adjustment for age and folate intake. Results: Current rotating night work was significantly inversely associated with hypermethylation of promoter region PER2, with OR = 0.7 (95% CI: 0.5−1.0). Moreover, increased odds ratio for high promoter methylation level of gene CRY2 (OR = 1.4 (95% CI: 1.0−2.1) was observed. There were no statistical significant association between night shift work and epigenetic changes in other circadian rhythm genes. Conclusions: Our study suggests that rotating night shift work may modify methylation level of PER2 and CRY2 genes. Further analyses are warranted to explore this under investigated topic. No conflict of interest.
Monday 11 July 2016 Poster Session
Molecular and Genetic Epidemiology II 674 IKZF1 gene deletions are strongly associated with Ikaros dominantnegative isoforms expression in acute lymphoblastic leukemia V. Vshyukova1 , A. Meleshko1 . 1 Belarussian Center for Paediatric Oncology and Haematology, Laboratory of genetic biotechnologies, Minsk, Belarus Background: In most cases IKZF1 deletions lead to the expression of dominant-negative Ikaros splice variants (Ik-DN isoforms), and significantly correlate with an increased relapse rate and poor outcome in acute lymphoblastic leukemia (ALL) patients. Since detection of IKZF1 deletions by PCR and sequencing associated with a number of technical difficulties due to oligoclonality of tumor cells, RQ-PCR quantification of Ik-DN isoforms is on potential benefits for IKZF1 status assessment.
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Material and Methods: Totally 220 bone marrow (BM) samples from primary ALL patients were included in the study. Control group included of 32 BM samples from healthy donors. RQ-PCR quantification of Ikaros isoforms expression was performed in 9 separate reactions for each sample. We worked up RQ-PCR estimation of functional (Ik1, 2, 3) and Ik-DN (Ik4, 5, 6, 8, 9 and 10) isoforms expression relative to control gene − Abelson’s tyrosine-kinase. Ik-Ratio between a functional isoforms (Ik1+Ik2+Ik3) and the sum of all isoforms expression levels was calculated. IKZF1 deletions of exons 1−6 (Ex1−6) and exons 3−6 (Ex3−6) were assessed by PCR amplification followed by sequencing. Results: The thresholds for Ik-DN overexpression were set empirically on the level 0.2 for Ik6, 0.1 for Ik9 and Ik10, and 0.8 for Ik-Ratio. Totally, IkDN overexpression has been identified in 96 ALL samples. Determined by the threshold, 29.2% of ALL were characterized by overexpression of Ik6 only, and 8.3% overexpressed Ik9. Combined overexpression of Ik6 and 9 was estimated in 50.0% of ALL samples, Ik6 + 9 + 10 − in 8.3%, and Ik6 + 10 − in 4.2%. The presence of clonal IKZF1 deletion was followed by Ik6−10 overexpression in 100% of cases, whereas subclonal deletions came to Ik-DN in 40% of cases only. To be noticed is that 10.9% of ALL with IKZF1 wild type also registered overexpression Ik6 or 9. Compared to Ik-DN isoforms quantification, the IkRatio have demonstrated major specificity for the presence of IKZF1 deletions, including subclonal deletions: only 4.2% (n = 4) of cases with IKZF1 wild type were determined as false positive results. Conclusions: Our results demonstrate feasibility of RQ-PCR quantification of Ik-DN, in particular the calculation of Ik-Ratio, as an additional method for IKZF1 gene testing in ALL patients. No conflict of interest. 675 Development of a prediction model and estimation of cumulative risk for upper aerodigestive tract cancer based on aldehyde dehydrogenase 2 (ALDH2) genotype and alcohol consumption in a Japanese population K. Matsuo1 , Y. Koyanagi2 , H. Ito3 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan Background: Alcohol consumption and aldehyde dehydrogenase 2 (ALDH2) polymorphism are associated with Upper aerodigest tract (UAT) cancer risk, and a significant gene–environment interaction between the two has been confirmed in a Japanese population. To aid the development of a personalized prevention strategy, we developed a risk prediction model and estimated absolute risks stratified by a combination of ALDH2 genotype and alcohol consumption. Materials and Methods: We conducted two age- and sex-matched case– control studies at Aichi Cancer Center, one (630 cases and 1,260 controls) for model derivation and the second (654 cases and 654 controls) for external validation. Based on data from the derivation study, a prediction model was developed by fitting a conditional logistic regression model using the following predictors: age, sex, smoking, drinking, and ALDH2 genotype. A fitting of models were done for UAT cancer, esophageal cancer and head and neck cancer separately. Then, fitted models were applied in the validation study. Discriminative ability was assessed by the value of the area under the receiver operating characteristic (ROC) curve (AUC), which is also known as the concordance (c) statistic. Cumulative risks were estimateded by combining odds ratios estimated from the risk model with the age-specific incidence rate and population size as described by Peto et al (Peto et al. BMJ 2000;321:323– 329). Results: The risk model, including a combination of ALDH2 genotype and alcohol consumption, provided high discriminatory accuracy and good calibration in both the derivation and validation studies for UAT cancer: C statistics were 0.82 (95% confidence intervals 0.80–0.84) and 0.83 (0.81– 0.85), respectively, and the calibration plots of both studies stayed close to the ideal calibration line. Discrimination accuracy was better in esophageal cancer (0.94 for derivation and 0.91 for validation) and slightly worse in head and neck cancer (0.72 for derivation and 0.73 for validation). For heavy drinkers with a heterozygous genotype, cumulative risk at age 80 for UAT cancer was above 20%. In contrast, risk in the other groups was less than 5%. Conclusion: We developed simple risk prediction models of UAT cancer among Japanese population and they were acceptable accuracy. Estimation of cumulative risk revealed that a specific population, heavy drinkers with ALDH2 heterozygous, would be highly benefitted from personalized modification of alcohol consumption. Although application of genetic information is still on the way in cancer prevention, our results would be one of the nice applications. No conflict of interest.
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676 Genome-wide AFB1-induced mutational signature in cells, mice and human tumors − implications for molecular epidemiology W. Yu1 , M. Huang1 , W.W. Teoh2 , M. Ardin3 , S. Villar3 , A. Jusakul4 , R. Othman2 , K. Sabapathy2 , J. Zavadil3 , S. Rozen1 . 1 Duke-NUS Medical School, Centre for Computational Biology and Programme in Cancer and Stem Cell Biology, Singapore, Singapore, 2 National Cancer Centre Singapore, Division of Cellular & Molecular Research- Humphrey Oei Institute of Cancer, Singapore, Singapore, 3 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 4 Duke-NUS Medical School, Programme in Cancer and Stem Cell Biology, Singapore, Singapore Introduction: Aflatoxin B1 (AFB1), found in foodstuffs throughout the world, is an IARC Group 1 carcinogen that causes hepatocellular carcinoma (HCC). The mutagenic effects of AFB1 on TP53 and reporter genes have been studied experimentally and in HCCs. Here we present first-of-its-kind data on the extended, genome-wide AFB1 mutagenesis using a set of complementary human and mouse in vivo and in vitro experimental systems. Materials and Methods: We determined genome-wide mutation patterns induced by AFB1 in two clonally expanded human liver cell-lines HepaRG and HepG2, in a mouse model of HCC driven by AFB1 treatment, and in own and public data from primary HCCs associated with aflatoxin exposure. Results and Discussion: The cell-line mutational patterns were remarkably stable across replicates, but differed somewhat between cell lines. Mutational patterns in the mouse tumours were more variable across replicates, possibly reflecting variability in the physiological clearance of the toxin in mice or random events during tumourigenesis. However, the overall pattern was consistently dominated by G>T mutations with a preference for TGC>TTC and substantial mutation enrichment on the non-transcribed strand. We next integrated these results with publicly available human HCC data and newly generated genomic HCC data from a known geographical region of aflatoxin exposure. Like the experimental systems, the human HCCs showed high rates of G>T mutations and strong transcriptional strand bias, providing evidence that the HCCs were direct consequences of AFB1 exposure. However, they differed from the experimental systems in that the most prominent mutations were GGC>GTC. This difference may be due to exposure to other aflatoxins, to other mutagens, or to differences in biochemical processing of AFB1. Human HCCs may harbour mutational signature of tobacco smoking that is similarly dominated by G>T mutations although it is typically associated with an enrichment in GG>TT dinucleotide substitutions. Indeed, we observed low GG>TT to G>T ratios (<0.005) in the AFB1-treated cell lines and tumours from AFB1-treated mice, whereas in several of the HCCs patients a marked increase of the GG>TT to G>T ratio (>0.01) suggested additional smokingassociated DNA damage on the target guanines. Conclusions: The experimental cell-based clonal expansion systems and mouse models used in our study of genome-wide mutagenic impact of AFB1 present innovative tools allowing to determine mutational signatures of candidate mutagenic carcinogens. We propose that the described experimental, multi-system approach can be used more broadly in support of molecular epidemiology studies aimed at cancer prevention. Acknowledgment: Funding obtained from IARC; ITMO CANCER − INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1; Singapore A*STAR and MOH via Duke-NUS and NMRC/CIRG/1422/2015. No conflict of interest. 677 Implementation of liquid biopsies for detection of activating EGFR mutations in Bulgarian patients with NSCLC S. Bichev1 , A. Savov1 . 1 National Genetics Laboratory, University Hospital Of Obstetrics And Gynecology, Sofia, Bulgaria Background: Activating somatic mutations in tyrosine kinase domain of EGF receptor are well established predictive biomarker for patient’s response to targeted therapies. For this reason NSCLC patients have to be tested for these mutations. The preferred material for EGFR testing is FFPET samples or cytology samples. However in many cases the initial material is insufficient and often re-biopsy is not possible due to patient’s status or patient’s will. In such cases an alternative method for EGFR genotyping should be used. In recent years it becomes clear that tumor cells release cell free tumor DNA (CtDNA) in bloodstream. Molecular characterization of ctDNA may provide a strategy for EGFR genotyping. Material and Method: For a period of 3 months 15 patients have been screened for EGFR activating mutations. From all patients, ctDNA was isolated from plasma samples, immediately after collecting the blood. Cell free tumor DNA was extracted with QiaAmp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. To achieve better results from DNA extraction we used QiaVac 24 plus system (Qiagen, Hilden, Germany). EGFR genotyping was performed with therascreen EGFR Plasma RGQ PCR Kit. Results and Discussion: DNA extraction was successful in all 15 cases. The quality and concentration of extracted DNA fulfilled the requirements of
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
(OR = 1.566), however the association did not reach statistical significance (p = 0.1197). We also evaluated a possible genotype-smoking association and found that C allele carrying individuals with smoking within the last 5 years had a significant risk of CRC (OR = 2.284; p = 0.0427). Conclusion: It has been revealed that CYP1A1 allele frequencies show large differences across different populations. Evaluation of an association between CYP1A1*2A and cancer susceptibility is more difficult and needs large number of participants in Caucasians. The data from this new study should be useful for understanding CRC aetiology and in identifying individual risk of developing CRC. Reference(s) [1] Inoue H, et al (2000). Cancer research, 60(14), 3749–3752. [2] Sivaraman L, et al (1994). Cancer research, 54(14), 3692–3695. [3] Welfare MR, et al (1997). Carcinogenesis, 18(7), 1351–1354. No conflict of interest. 671 Carcinogenic and non-carcinogenic risk assessment for children exposed to DDTs residues in pasteurized cow milk from Iran market N. Rastkari1 , M. Zare Jeddi2 , R. Ahmadkhaniha3 , M. Yunesian4 . 1 Tehran University of Medical Sciences, Center for Air Pollution Research CAPRInstitute for Environmental Research IER, Tehran, Iran, 2 Tehran University of Medical Sciences, Institute for Environmental Research, Tehran, Iran, 3 Tehran University of Medical Sciences, Department of Human EcologySchool of Public Health, Tehran, Iran, 4 Tehran University of Medical Sciences, Department of Environmental Health − School of Public Health, Tehran, Iran Background: Milk-producing animals accumulate pesticides from contaminated feed and by inhaling contaminated air. Organochlorine pesticides due to their lipophilic properties are initially stored in fat-rich tissues and subsequently translocated and excreted in milk. Therefore, the consumption of dairy products together with other contaminated food may expose consumers to unexpected levels of organochlorine pesticides. Organochlorine pesticides being endocrine disrupting chemicals are believed to produce a wide variety of adverse health outcomes such as cancers. Dichlodiphenyl-trichloroethane (DDT), a previously widely used OCP, and some of its major metabolites, is officially classified according to Integrated Risk Information System (IRIS) of United States Environmental Protection Agency (USEPA) as probable human carcinogens (Group B2) and responsible for causing damage to organs through repeated and prolonged exposure. Material and Methods: The objectives of the present study were to evaluate children’s exposure to DDTs (DDT and its metabolites) via dietary milk consumption in Iran, and to assess the respective potential risks to children’s health in terms of carcinogenic and non-carcinogenic effects. Residue levels were determined by GCMS analysis in 60 cow milk samples of Full fat (3%) pasteurized commercial types. The estimated daily intake (EDI) was calculated for different ages (1, 3, 5 and 7 years old children) based on the respective milk consumptions and finally Hazard quotient (HQ) for non-carcinogenic effects and hazard rate for carcinogenic effect were estimated. Results and Discussion: In all of the samples DDT metabolites were detected in the range of 0.0015 to 0. 2796 mg/L with the mean of 0.047±0.03, 0.15±0.08, 0.085±0.05, 0.05±0.02 mg/L for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD, respectively. The calculated EDIs (0.002–0.03 mg/kg-day) for all age categories and all compounds were lower than the RfDs. Consequently, the hazard quotients calculated in these groups for DDTs were far less than 1. For estimation of the cancer risk values, the cancer benchmark concentrations (CBC) were derived using oral slope factors and their hazard ratios (HR) in all age categories. Hazard ratio obtained for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD in Full fat (3%) pasteurized milk could not pose potential carcinogenic risk to young children (1, 3, 5 and 7 years old) since all of the HRs were far less than 1. Conclusion: This study has shown that cow milk is not a major source of human exposure to DDTs and from this perspective is safe for consumption. No conflict of interest. 672 Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: Gene−environment interaction H. Masaoka1,2 , H. Ito3,4 , N. Soga5 , A. Yokomizo2 , M. Eto2 , K. Matsuo1,4 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Kyushu University Graduate School of Medical Sciences, Department of Urology, Fukuoka, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan, 4 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 5 Aichi Cancer Center Hospital, Department of Urology, Nagoya, Japan Background: Although a range of chemical exposures (cigarette smoking and occupational exposure) are recognized risk factors for the development of
bladder cancer (BCa), many epidemiological studies have demonstrated that alcohol drinking is not associated with BCa risk. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms impact the accumulation of acetaldehyde, resulting in an increased risk of various cancers. To date, however, no studies evaluating the association between BCa risk and alcohol drinking have considered these polymorphisms. Here, we conducted a matched case–control study to investigate whether ALDH2 and ADH1B polymorphisms influence BCa risk associated with alcohol drinking. Material and Methods: Cases were 74 BCa patients and controls were 740 first-visit outpatients without cancer at Aichi Cancer Center Hospital between January 2001 and December 2005. Odds ratio (OR), 95% confidence interval (CI) and gene–environment interaction were assessed by conditional logistic regression analysis with adjustment for potential confounders. Results: ALDH2 Glu/Lys was associated with a significantly increased risk of BCa compared with Glu/Glu (OR 2.03, 95% CI 1.14–3.62). In contrast, ALDH2 Glu/Lys showed no increase in risk among the stratum of never drinkers compared with Glu/Glu, indicating a gene–environment interaction. ADH1B His/Arg had an OR of 1.98 (1.20–3.24) compared with His/His. ADH1B Arg+ showed a similar OR and 95% CI. Individuals with ALDH2 Glu/Lys and ADH1B Arg+ had the highest risk of BCa compared with ALDH2 Glu/Glu and ADH1B His/His (OR 4.00, 95% CI 1.81–8.87)]. Conclusions: Those with the ALDH2 Glu/Lys and ADH1B Arg+ genotype are at increased risk of BCa. Gene–environment interaction between ALDH2 Glu/Lys and alcohol drinking might suggest acetaldehyde contributes to the carcinogenesis of BCa. No conflict of interest. 673 Association between night shift work and methylation status of circadian rhythm genes among Polish nurses and midwives − preliminary results A. Bukowska1 , E. Wieczorek2 , B. Peplonska1 , M. Przybek2 , E. Jablonska2 , E. Reszka2 . 1 Nofer Institute of Occupational Medicine, Department of Environmental Epidemiology, Lodz, Poland, 2 Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Lodz, Poland Background: It has been suggested that rotating night shift work may contribute to epigenetic changes in the circadian rhythm genes. The results of previous studies in this topic are sparse and inconsistent. The aim of the study was to assess the potential relationship between current rotating night shift work and changes in levels of 5-methylcytosine in the promoter regions of selected circadian rhythm genes. Material and Method: The cross-sectional study included as many as 710 nurses and midwives (348 working on rotating night shifts and 362 working only during the day) aged 40−60 years. During in person interviews, information about occupational history and potential confounders was collected. DNA was isolated from peripheral blood samples and promoter methylation status of core circadian rhythm genes (PER1, PER2, PER3, BMAL1, CLOCK, CRY1, CRY2, NPAS2) was determined using quantitative methylation-specific real-time PCR assay (qMSP) method. Univariate and multivariate regression models were used to assess the association between current work at night and epigenetic changes, with adjustment for age and folate intake. Results: Current rotating night work was significantly inversely associated with hypermethylation of promoter region PER2, with OR = 0.7 (95% CI: 0.5−1.0). Moreover, increased odds ratio for high promoter methylation level of gene CRY2 (OR = 1.4 (95% CI: 1.0−2.1) was observed. There were no statistical significant association between night shift work and epigenetic changes in other circadian rhythm genes. Conclusions: Our study suggests that rotating night shift work may modify methylation level of PER2 and CRY2 genes. Further analyses are warranted to explore this under investigated topic. No conflict of interest.
Monday 11 July 2016 Poster Session
Molecular and Genetic Epidemiology II 674 IKZF1 gene deletions are strongly associated with Ikaros dominantnegative isoforms expression in acute lymphoblastic leukemia V. Vshyukova1 , A. Meleshko1 . 1 Belarussian Center for Paediatric Oncology and Haematology, Laboratory of genetic biotechnologies, Minsk, Belarus Background: In most cases IKZF1 deletions lead to the expression of dominant-negative Ikaros splice variants (Ik-DN isoforms), and significantly correlate with an increased relapse rate and poor outcome in acute lymphoblastic leukemia (ALL) patients. Since detection of IKZF1 deletions by PCR and sequencing associated with a number of technical difficulties due to oligoclonality of tumor cells, RQ-PCR quantification of Ik-DN isoforms is on potential benefits for IKZF1 status assessment.
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Material and Methods: Totally 220 bone marrow (BM) samples from primary ALL patients were included in the study. Control group included of 32 BM samples from healthy donors. RQ-PCR quantification of Ikaros isoforms expression was performed in 9 separate reactions for each sample. We worked up RQ-PCR estimation of functional (Ik1, 2, 3) and Ik-DN (Ik4, 5, 6, 8, 9 and 10) isoforms expression relative to control gene − Abelson’s tyrosine-kinase. Ik-Ratio between a functional isoforms (Ik1+Ik2+Ik3) and the sum of all isoforms expression levels was calculated. IKZF1 deletions of exons 1−6 (Ex1−6) and exons 3−6 (Ex3−6) were assessed by PCR amplification followed by sequencing. Results: The thresholds for Ik-DN overexpression were set empirically on the level 0.2 for Ik6, 0.1 for Ik9 and Ik10, and 0.8 for Ik-Ratio. Totally, IkDN overexpression has been identified in 96 ALL samples. Determined by the threshold, 29.2% of ALL were characterized by overexpression of Ik6 only, and 8.3% overexpressed Ik9. Combined overexpression of Ik6 and 9 was estimated in 50.0% of ALL samples, Ik6 + 9 + 10 − in 8.3%, and Ik6 + 10 − in 4.2%. The presence of clonal IKZF1 deletion was followed by Ik6−10 overexpression in 100% of cases, whereas subclonal deletions came to Ik-DN in 40% of cases only. To be noticed is that 10.9% of ALL with IKZF1 wild type also registered overexpression Ik6 or 9. Compared to Ik-DN isoforms quantification, the IkRatio have demonstrated major specificity for the presence of IKZF1 deletions, including subclonal deletions: only 4.2% (n = 4) of cases with IKZF1 wild type were determined as false positive results. Conclusions: Our results demonstrate feasibility of RQ-PCR quantification of Ik-DN, in particular the calculation of Ik-Ratio, as an additional method for IKZF1 gene testing in ALL patients. No conflict of interest. 675 Development of a prediction model and estimation of cumulative risk for upper aerodigestive tract cancer based on aldehyde dehydrogenase 2 (ALDH2) genotype and alcohol consumption in a Japanese population K. Matsuo1 , Y. Koyanagi2 , H. Ito3 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan Background: Alcohol consumption and aldehyde dehydrogenase 2 (ALDH2) polymorphism are associated with Upper aerodigest tract (UAT) cancer risk, and a significant gene–environment interaction between the two has been confirmed in a Japanese population. To aid the development of a personalized prevention strategy, we developed a risk prediction model and estimated absolute risks stratified by a combination of ALDH2 genotype and alcohol consumption. Materials and Methods: We conducted two age- and sex-matched case– control studies at Aichi Cancer Center, one (630 cases and 1,260 controls) for model derivation and the second (654 cases and 654 controls) for external validation. Based on data from the derivation study, a prediction model was developed by fitting a conditional logistic regression model using the following predictors: age, sex, smoking, drinking, and ALDH2 genotype. A fitting of models were done for UAT cancer, esophageal cancer and head and neck cancer separately. Then, fitted models were applied in the validation study. Discriminative ability was assessed by the value of the area under the receiver operating characteristic (ROC) curve (AUC), which is also known as the concordance (c) statistic. Cumulative risks were estimateded by combining odds ratios estimated from the risk model with the age-specific incidence rate and population size as described by Peto et al (Peto et al. BMJ 2000;321:323– 329). Results: The risk model, including a combination of ALDH2 genotype and alcohol consumption, provided high discriminatory accuracy and good calibration in both the derivation and validation studies for UAT cancer: C statistics were 0.82 (95% confidence intervals 0.80–0.84) and 0.83 (0.81– 0.85), respectively, and the calibration plots of both studies stayed close to the ideal calibration line. Discrimination accuracy was better in esophageal cancer (0.94 for derivation and 0.91 for validation) and slightly worse in head and neck cancer (0.72 for derivation and 0.73 for validation). For heavy drinkers with a heterozygous genotype, cumulative risk at age 80 for UAT cancer was above 20%. In contrast, risk in the other groups was less than 5%. Conclusion: We developed simple risk prediction models of UAT cancer among Japanese population and they were acceptable accuracy. Estimation of cumulative risk revealed that a specific population, heavy drinkers with ALDH2 heterozygous, would be highly benefitted from personalized modification of alcohol consumption. Although application of genetic information is still on the way in cancer prevention, our results would be one of the nice applications. No conflict of interest.
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676 Genome-wide AFB1-induced mutational signature in cells, mice and human tumors − implications for molecular epidemiology W. Yu1 , M. Huang1 , W.W. Teoh2 , M. Ardin3 , S. Villar3 , A. Jusakul4 , R. Othman2 , K. Sabapathy2 , J. Zavadil3 , S. Rozen1 . 1 Duke-NUS Medical School, Centre for Computational Biology and Programme in Cancer and Stem Cell Biology, Singapore, Singapore, 2 National Cancer Centre Singapore, Division of Cellular & Molecular Research- Humphrey Oei Institute of Cancer, Singapore, Singapore, 3 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 4 Duke-NUS Medical School, Programme in Cancer and Stem Cell Biology, Singapore, Singapore Introduction: Aflatoxin B1 (AFB1), found in foodstuffs throughout the world, is an IARC Group 1 carcinogen that causes hepatocellular carcinoma (HCC). The mutagenic effects of AFB1 on TP53 and reporter genes have been studied experimentally and in HCCs. Here we present first-of-its-kind data on the extended, genome-wide AFB1 mutagenesis using a set of complementary human and mouse in vivo and in vitro experimental systems. Materials and Methods: We determined genome-wide mutation patterns induced by AFB1 in two clonally expanded human liver cell-lines HepaRG and HepG2, in a mouse model of HCC driven by AFB1 treatment, and in own and public data from primary HCCs associated with aflatoxin exposure. Results and Discussion: The cell-line mutational patterns were remarkably stable across replicates, but differed somewhat between cell lines. Mutational patterns in the mouse tumours were more variable across replicates, possibly reflecting variability in the physiological clearance of the toxin in mice or random events during tumourigenesis. However, the overall pattern was consistently dominated by G>T mutations with a preference for TGC>TTC and substantial mutation enrichment on the non-transcribed strand. We next integrated these results with publicly available human HCC data and newly generated genomic HCC data from a known geographical region of aflatoxin exposure. Like the experimental systems, the human HCCs showed high rates of G>T mutations and strong transcriptional strand bias, providing evidence that the HCCs were direct consequences of AFB1 exposure. However, they differed from the experimental systems in that the most prominent mutations were GGC>GTC. This difference may be due to exposure to other aflatoxins, to other mutagens, or to differences in biochemical processing of AFB1. Human HCCs may harbour mutational signature of tobacco smoking that is similarly dominated by G>T mutations although it is typically associated with an enrichment in GG>TT dinucleotide substitutions. Indeed, we observed low GG>TT to G>T ratios (<0.005) in the AFB1-treated cell lines and tumours from AFB1-treated mice, whereas in several of the HCCs patients a marked increase of the GG>TT to G>T ratio (>0.01) suggested additional smokingassociated DNA damage on the target guanines. Conclusions: The experimental cell-based clonal expansion systems and mouse models used in our study of genome-wide mutagenic impact of AFB1 present innovative tools allowing to determine mutational signatures of candidate mutagenic carcinogens. We propose that the described experimental, multi-system approach can be used more broadly in support of molecular epidemiology studies aimed at cancer prevention. Acknowledgment: Funding obtained from IARC; ITMO CANCER − INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1; Singapore A*STAR and MOH via Duke-NUS and NMRC/CIRG/1422/2015. No conflict of interest. 677 Implementation of liquid biopsies for detection of activating EGFR mutations in Bulgarian patients with NSCLC S. Bichev1 , A. Savov1 . 1 National Genetics Laboratory, University Hospital Of Obstetrics And Gynecology, Sofia, Bulgaria Background: Activating somatic mutations in tyrosine kinase domain of EGF receptor are well established predictive biomarker for patient’s response to targeted therapies. For this reason NSCLC patients have to be tested for these mutations. The preferred material for EGFR testing is FFPET samples or cytology samples. However in many cases the initial material is insufficient and often re-biopsy is not possible due to patient’s status or patient’s will. In such cases an alternative method for EGFR genotyping should be used. In recent years it becomes clear that tumor cells release cell free tumor DNA (CtDNA) in bloodstream. Molecular characterization of ctDNA may provide a strategy for EGFR genotyping. Material and Method: For a period of 3 months 15 patients have been screened for EGFR activating mutations. From all patients, ctDNA was isolated from plasma samples, immediately after collecting the blood. Cell free tumor DNA was extracted with QiaAmp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. To achieve better results from DNA extraction we used QiaVac 24 plus system (Qiagen, Hilden, Germany). EGFR genotyping was performed with therascreen EGFR Plasma RGQ PCR Kit. Results and Discussion: DNA extraction was successful in all 15 cases. The quality and concentration of extracted DNA fulfilled the requirements of