Reka Toth

Genome-wide genetic and epigenetic analyses of pancreatic acinar cell carcinomas reveal aberrations in genome stability.

Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, to identify potential targets for personalized treatment, we perform integrative genome-wide and epigenome-wide analyses. The results show frequently aberrant DNA methylation, abundant chromosomal amplifications and deletions, and mutational signatures suggesting defective DNA repair. In contrast to pancreatic ductal adenocarcinoma, no recurrent point mutations are detected. The tumor suppressors ID3, ARID1A, APC, and CDKN2A are frequently impaired also on the protein level and thus potentially affect ACC tumorigenesis. Consequently, this work identifies promising therapeutic targets in ACC for drugs recently approved for precision cancer therapy.Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, the authors perform genome- and epigenome-wide analyses from normal and ACC pancreatic tissue that identify aberrations in genome stability and cell cycle control.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome‐wide analysis of DNA methylation in colorectal tumours from 36 never‐smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never‐smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10−5) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never‐smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking‐associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright

RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML.Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors analyse the DNA methylome and mutational profile of JMML to define three subgroups with unique molecular and clinical characteristics.

Genetic Variants in Epigenetic Pathways and Risks of Multiple Cancers in the GAME-ON Consortium

Background: Epigenetic disturbances are crucial in cancer initiation, potentially with pleiotropic effects, and may be influenced by the genetic background. Methods: In a subsets (ASSET) meta-analytic approach, we investigated associations of genetic variants related to epigenetic mechanisms with risks of breast, lung, colorectal, ovarian and prostate carcinomas using 51,724 cases and 52,001 controls. False discovery rate–corrected P values (q values < 0.05) were considered statistically significant. Results: Among 162,887 imputed or genotyped variants in 555 candidate genes, SNPs in eight genes were associated with risk of more than one cancer type. For example, variants in BABAM1 were confirmed as a susceptibility locus for squamous cell lung, overall breast, estrogen receptor (ER)–negative breast, and overall prostate, and overall serous ovarian cancer; the most significant variant was rs4808076 [OR = 1.14; 95% confidence interval (CI) = 1.10–1.19; q = 6.87 × 10−5]. DPF1 rs12611084 was inversely associated with ER-negative breast, endometrioid ovarian, and overall and aggressive prostate cancer risk (OR = 0.93; 95% CI = 0.91–0.96; q = 0.005). Variants in L3MBTL3 were associated with colorectal, overall breast, ER-negative breast, clear cell ovarian, and overall and aggressive prostate cancer risk (e.g., rs9388766: OR = 1.06; 95% CI = 1.03–1.08; q = 0.02). Variants in TET2 were significantly associated with overall breast, overall prostate, overall ovarian, and endometrioid ovarian cancer risk, with rs62331150 showing bidirectional effects. Analyses of subpathways did not reveal gene subsets that contributed disproportionately to susceptibility. Conclusions: Functional and correlative studies are now needed to elucidate the potential links between germline genotype, epigenetic function, and cancer etiology. Impact: This approach provides novel insight into possible pleiotropic effects of genes involved in epigenetic processes. Cancer Epidemiol Biomarkers Prev; 26(6); 816–25. ©2017 AACR.

Robust prediction of gene regulation in colorectal cancer tissues from DNA methylation profiles

ABSTRACT DNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92<AUC<0.97) as well as in individual patient data (average AUC = 0.82), suggesting a robust interplay between methylation and gene regulation. Validation analysis in other cancerous tissues resulted in lower prediction performances (0.69<AUC<0.90); however, it identified genes that share robust dependencies across cancerous tissues. In conclusion, we present a robust classification approach that predicts the gene-specific regulation through DNA methylation in CRC tissues with possible transition to different cancer entities. Furthermore, we present HMGA1 as consistently associated with methylation across cancers, suggesting a potential candidate for DNA methylation targeting cancer therapy.

Abstract 4590: Polymorphisms in cell-cycle related genes modify the effect of NSAIDs on the risk of colorectal cancer

The risk of colorectal cancer (CRC) is determined by the interplay of genetic and environmental factors. The aim of this study was to identify the association between polymorphisms in cell-cycle related genes and risk of colorectal cancer, and to evaluate the interaction with non-steroidal anti-inflammatory drug (NSAID) use. Patients with incident CRC were recruited in the framework of DACHS, a German population based case-control study. Altogether, 1756 cases and 1781 controls were genotyped for 223 candidate or tagging SNPs in 30 cell-cycle related genes using the Illumina GoldenGate Assay. The associations between polymorphisms and the risk of colorectal cancer were assessed with multivariate logistic regression. Effect modification by NSAIDs (use >1/month for >1 year) was tested by using a multiplicative interaction term. Haplotype analysis was performed using the haplo.stats R package. None of the studied SNPs were significantly associated with CRC after multiple test correction. NSAID use lowered the risk of colorectal cancer with an odds ratio (OR) of 0.59 (Confidence interval, CI: 0.51-0.69, p = 9.58*10-11). Several polymorphisms in and near cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) showed interaction with NSAID use. However, only one signal remained significant after FDR correction; the homozygous wild genotype (GG) of rs2069408 neutralizes the protective effect of NSAID use (interaction p A possible mechanism of the interaction between NSAIDs and cell-cycle genes could be mediated via the cell-cycle related effects of MYC, the expression of which is regulated by the Wnt/s-catenin pathway. This pathway plays a central role in colorectal cancer and was previously found to be inhibited by NSAIDs. The analyses of additional SNPs are ongoing to better understand the mechanism of the interaction between NSAID use and cell-cycle related genes. Citation Format: Reka Toth, Yesilda Balavarca, Dominique Scherer, Nina Habermann, Katharina Buck, Akke Botma, Elisabeth J. Kap, Axel Benner, Alexis Ulrich, Michael Hoffmeister, Hermann Brenner, Barbara Burwinkel, Jenny Chang-Claude, Cornelia M. Ulrich. Polymorphisms in cell-cycle related genes modify the effect of NSAIDs on the risk of colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4590. doi:10.1158/1538-7445.AM2015-4590

Abstract 4612: Genetic variants in epigenetic pathways and risk of multiple cancer types in the GAME-ON consortium

Introduction Epigenetic changes are reversible features of the genome that regulate gene transcription and protein expression on several levels including DNA methylation, histone modification or miRNA expression. We investigated the association between inherited variation in genes of key epigenetic processes and risk of multiple cancers within the GAME-ON consortium. Methods We performed a pathway based meta-analysis using genotypes from more than 50,000 cases of breast, lung, prostate, ovarian and colorectal cancer cases and more than 60,000 controls from various genome wide association studies participating in the GAME-ON consortium to estimate associations with cancer risk. Using the 1000GenomeProject database, we selected 505,702 genotyped and imputed single nucleotide polymorphisms in 551 genes (flanking region +/- 250kb) related to DNA methylation, histone modification or chromatin remodeling based on GO and GeneCard databases. In order to allow variants to be associated with only a subset of traits we used subset based meta-analysis. False-discovery rate (FDR) corrected p-values (q-values) lower than 0.05 were considered significant. Results and Discussion 582 SNPs were significantly associated with risk of at least one cancer. We identified nine major regions that showed significant associations with more than one cancer type. Among the most interesting regions was the region around PHC3 (3q36), which showed associations with prostate and colorectal cancer and clear cell ovarian carcinomas. PHC3 is involved in chromatin remodeling and plays a role in epithelial neoplasms. Significant Odds ratios (ORs) ranged from 0.80 to 1.31. The number of risk and protective alleles in this region was similarly distributed (19 and 18, respectively). One of the strongest associations was observed for rs76925190 (intronic in PRKC1), which increased the risk of colorectal and prostate cancer (q-value 4.28*10-10). Variants in this region were previously associated with prostate cancer. Polymorphisms in the region (19q13) around BABAM1 (RISC and BRCA1 A complex member 1), were associated with lung, breast, ovarian and prostate cancer. BABAM1 is associated with the BRCA1-complex. Its function in histone modification and DNA repair emphasizes its importance in carcinogenesis. Significant ORs ranged from 0.88 to 1.14 with similar distribution of risk and protective alleles in this region (19 and 17, respectively). The strongest association was observed for rs4808076 (intronic in ANKLE1), which increased the risk of squamous lung, serous ovarian and ER- -breast cancer (q-value 2.40*10-6). Variants in this region were previously associated with risk of breast and ovarian cancer. Conclusions This study emphasizes the importance of variants in genes of epigenetic processes on cancer risk and further provides insights into novel, pleiotropic epigenetic mechanisms of cancer development. Citation Format: Dominique Scherer, Reka Toth, Linda Kelemen, Angela Risch, Aditi Hazra, Jean Pierre Issa, Victor Moreno, Rosalind A. Eeles, John Quackenbush, Ellen L. Goode, Shuji Ogino, Rayjean Hung, Cornelia M. Ulrich. Genetic variants in epigenetic pathways and risk of multiple cancer types in the GAME-ON consortium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4612. doi:10.1158/1538-7445.AM2015-4612

Abstract 2186: Inflammation- and angiogenesis- related genes: Interaction with NSAID use, and serum inflammatory markers on colorectal cancer risk within the Women's Health Initiative

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Inflammation and angiogenesis are important pathways of colorectal carcinogenesis. We investigated interactions between genes related to both pathways and the use of NSAIDs on CRC risk. Furthermore, we assessed whether specific variants interact with the inflammation biomarkers C-reactive protein (CRP) and serum amyloid A (SAA). From the Womens Health Initiative Observational Study of women aged 50-79 years, we selected 821 Caucasian case/control matched pairs (1993-2008). We genotyped 412 single nucleotide polymorphisms (SNPs) tagging 26 genes related to inflammation and angiogenesis (ALOX5, CRP, IL1B, IL6, PTGIS, PTGS1, PTGS2, TBXAS, TGFB1, TNF, ANGPT1, NGPT2, DKK4, DLL4, VEGFR1, IL10, KDR, MAPK1, NRP1, NRP2, PGF, IK3CA, PIK3CG, TEK, VEGFA) using the Illumina GoldenGate and Sequenom platforms. CRP and SAA were quantified in serum by latex-enhanced nephelometry. CRC risk was estimated by conditional logistic regression based on the co-dominant inheritance model (or dominant if allele count <10). P-values were adjusted for correlated tests (pACT) unless genes were functional candidates. Global P-values were obtained by likelihood ratio tests. Additionally, in interaction analysis we controlled the false discovery rate on the gene level (p-intadj). Interactions between SNPs and serum inflammatory markers were evaluated by testing for different slopes of CRC risk associated with each biomarker across genotype. One SNP in NRP1, rs7895812 was significantly associated with CRC risk (pACT=0.02). Nine of the examined SNPs modified the association between NSAID use and CRC risk, although only two interactions remained significant after multiple comparison adjustment. The protective association of NSAID use was limited to individuals who were wildtype for PTGS2 rs1886233 (rs2383529, intergenic: p-intadj=0.01), whereas for carriers of the variant allele in FLT1 NSAID use was associated with a decreased CRC risk (rs1886233, intronic: p-intadj=0.02). For serum CRP and SAA multiple interactions with genetic variants were observed as well. We showed that genetic variation in inflammation- and angiogenesis-related genes modified the association between NSAID use and CRC risk; the genetic variants also interacted with serum concentrations of CRP and SAA. This information may aid in tailoring prevention strategies to individuals who will obtain the greatest benefit from NSAIDs. Citation Format: Nina Habermann, Elissa Brown, Reka Toth, Dominique Scherer, Katharina Buck, Ting-Yuan David Cheng, Karen W. Makar, Marian L. Neuhouser, Yingye Zheng, David J. Duggan, Shirley A. Beresford, Mark Wener, Heather Ochs-Balcom, Adetunji Toriola, Cornelia M. Ulrich. Inflammation- and angiogenesis- related genes: Interaction with NSAID use, and serum inflammatory markers on colorectal cancer risk within the Womens Health Initiative. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2186. doi:10.1158/1538-7445.AM2014-2186

Abstract 2197: Interaction between genetic variants in one-carbon metabolism and folate biomarkers on colorectal cancer risk: The Women's Health Initiative observational cohort

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Folate-mediated one-carbon metabolism (FOCM) is important in the development of colorectal cancer (CRC), but gene-nutrient interactions in this relationship have not been comprehensively evaluated. We investigated whether folate modified the association of genetic variants of FOCM with CRC risk. Among 821 Caucasian case/control matched pairs in the Womens Health Initiative observational cohort (1993-2008), we genotyped 453 single nucleotide polymorphisms (SNPs) tagging 227 independent loci in 30 genes in FOCM pathways (AARS, ADH1B, ADH1C, BHMT, BHMT2, CBS, DHFR, DNMT1, DNMT3A, DNMT3B, EHMT1, EHMT2, FOLH1, FOLR1, GGH, GNMT, MAT1A, MAT2B, MTHFD1, MTHFD2, MTHFR, MTR, MTRR, PON1, PRDM2, SHMT1, SHMT2, SLC19A1, TCN2, and TYMS). Folate concentrations in plasma and red blood cell (RBC) were measured by radioassays. Multivariable conditional logistic regression was used to estimate odds ratios, and likelihood ratio tests were performed to test the differences in the associations of SNPs with CRC among plasma/RBC folate tertile categories. Nominal P-values were reported for non-synonymous and candidate SNPs. For tagSNPs, we used the Benjamini and Hochberg method to control false discovery rate (FDR) at the gene level. When comparing homozygous variants with wild-types, we observed higher risks of CRC associated with genetic variants in folate absorption and uptake in the presence of low (tertile 1) plasma folate concentrations: GGH (rs16930073, flanking 3′UTR, P-interaction=0.006), FOLH1 (rs10839236, intron, candidate, P=0.018; rs2299650, intron, P=0.016), and FOLR1 (rs651646, intron, P=0.021). In other FOCM pathways (methionine/S-adenosylmethionine reactions, vitamin B12 transport, and DNA methylation), plasma/RBC folate also modified important non-synonymous and candidate SNPs in relation to CRC risk: MTRR (C524T [p.S202L], P=0.045), CBS (rs1801181 [p.A360A], P=0.035), TCN2 (C776G [p.R232P], P=0.040), DNMT1 (A201G [p.I311L], P=0.016), DNMT3A (rs11695471 [intron 22 A>T], candidate, P=0.002), and DNMT3B (283T>C, 5′ near gene, p=0.043). In addition, interactions with plasma/RBC folate were observed for several variants in paraoxonase 1 (PON1), an enzyme generating homocysteine from homocysteine thiolactone: p.L55M (P=0.021), rs8491 (3′UTR, P=0.037), rs854548 (3′ near gene, P=0.033), rs854551 (3′UTR, P=0.045), and rs3917527 (intron, P=0.006). All significant interactions with tagSNPs remained as “noteworthy” at the FDR 25% level. In conclusion, our observations highlight the importance of folate status when investigating FOCM-related genetic influences on CRC risk in postmenopausal women. Citation Format: Ting-Yuan D. Cheng, Karen W. Makar, Marian L. Neuhouser, Joshua W. Miller, Xiaoling Song, Elissa C. Brown, Shirley A.A. Beresford, Yingye Zheng, David J. Duggan, Elizabeth M. Poole, Nina Habermann, Reka Toth, Lynn B. Bailey, Marie A. Caudill, Cornelia M. Ulrich. Interaction between genetic variants in one-carbon metabolism and folate biomarkers on colorectal cancer risk: The Womens Health Initiative observational cohort. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2197. doi:10.1158/1538-7445.AM2014-2197