No association of TGFB1 L10P genotypes and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a multi-center cohort study
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- Rebbeck, T.R., Antoniou, A.C., Llopis, T.C. et al. Breast Cancer Res Treat (2009) 115: 185. doi:10.1007/s10549-008-0064-8
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Background The transforming growth factor β-1 gene (TGFB1) is a plausible candidate for breast cancer susceptibility. The L10P variant of TGFB1 is associated with higher circulating levels and secretion of TGF-β, and recent large-scale studies suggest strongly that this variant is associated with breast cancer risk in the general population. Methods To evaluate whether TGFB1 L10P also modifies the risk of breast cancer in BRCA1 or BRCA2 mutation carriers, we undertook a multi-center study of 3,442 BRCA1 and 2,095 BRCA2 mutation carriers. Results We found no evidence of association between TGFB1 L10P and breast cancer risk in either BRCA1 or BRCA2 mutation carriers. The per-allele HR for the L10P variant was 1.01 (95%CI: 0.92–1.11) in BRCA1 carriers and 0.92 (95%CI: 0.81–1.04) in BRCA2 mutation carriers. Conclusions These results do not support the hypothesis that TGFB1 L10P genotypes modify the risk of breast cancer in BRCA1 or BRCA2 mutation carriers.
KeywordsBRCA1BRCA2Risk modifiersHereditary cancer
Consortium of Investigators of Modifiers of BRCA1/2
Breast Cancer Association Consortium
Breast Cancer Family Registries
Epidemiological Study of Familial Breast Cancer
German Consortium of Hereditary Breast and Ovarian Cancer
Helsinki Breast Cancer Study
Hospital Clinico San Carlos
Interdisciplinary Health Research International Team Breast Cancer Susceptibility
The Kathleen Cunningham Consortium for Research into Familial Breast Cancer
Modifier study of quantitative effects on disease
National Cancer Institute Study
Ontario Cancer Genetics Network Study
Genetic Modifiers of cancer risk in BRCA1/2 mutation carriers study
Transforming growth factor-β
Transforming growth factor-β (TGF-β) is encoded by the TGFB1 gene (OMIM 190180; Chromosome 19q13.1), and regulates normal mammary gland development and function by activating the TGF-β signaling pathway . TGF-β appears to both inhibit the development of early benign breast tumors as well as promote tumor invasion and metastasis once somatic mutations have abrogated the normal TGF-β tumor suppressor function [1–3].
A number of studies have suggested that TGFB1 L10P (rs1982073) is involved in the etiology and severity of sporadic breast cancer. L10P is associated with higher circulating levels of acid-activatable TGF-β  and increased TGF-β secretion in vitro , which led to the hypothesis that this variant plays a functional role in breast carcinogenesis. A number of epidemiological association studies have been published, but the results of these studies have not been consistent [3, 5–17]. Data from large case–control series, including a recent multi-center study of 12,946 cases and 15,109 controls by the Breast Cancer Association Consortium , identified small but statistically significant associations with breast cancer risk, with a per-allele increase in risk of 8% (95%CI 1.04–1.31). This study also reported some evidence of a stronger association among cases diagnosed below age 40 years. Given the biological and epidemiological evidence that TGFB1 L10P may be associated with early onset breast cancer, we undertook a large multi-center cohort study of BRCA1 and BRCA2 mutation carriers to determine if the L10P variant modulates breast cancer risk on the background of these mutations.
Materials and methods
Eligibility was restricted to female carriers of pathogenic mutations in BRCA1 or BRCA2 who were 18 years old or older. Information collected included the year of birth, exact mutation description, family membership, country of residence, age at last follow-up, ages at breast and ovarian cancer diagnosis and information on bilateral prophylactic mastectomy. Details of the CIMBA initiative and information about the participating centers can be found elsewhere . Women were included in the analysis if they carried mutations that were pathogenic according to generally recognized criteria (see also: http://research.nhgri.nih.gov/projects/bic/). All carriers participated in clinical and research studies at the host institutions under IRB-approved protocols.
Association of TGFB1 L10P with breast cancer by BRCA1/2 mutation status from retrospective likelihood analysis: per-allele effects
12 df Wald χ2 test of heterogeneity among centers (P-value)
15.512 (P = 0.22)
28.372 (P = 0.005)
After considering genotyping quality control criteria (described below), a total of 3,442 BRCA1 and 2,095 BRCA2 mutation carriers yielded a TGFB1 genotype for analysis. Six individuals carried a mutation in both BRCA1 and BRCA2. For the purposes of this analysis, these six women were included in the BRCA1 mutation group only, since the breast cancer risk in BRCA1 mutation carriers has been noted to be higher than BRCA2 mutation carriers at young ages . Individuals were classified according to their age at diagnosis of breast cancer or their age at last follow-up. For this purpose, individuals were censored at the age of the first breast cancer diagnosis (n = 3,113), ovarian cancer diagnosis (n = 486), bilateral prophylactic mastectomy (n = 112) or the age at last observation (n = 1,826). Only individuals censored at breast cancer diagnosis were assumed to be affected.
Laboratory analysis to determine L10P (i.e., T29C) genotypes was undertaken in each center using a PCR-based Taqman assay. Allele specific oligonucleotide probes were added to the PCR reaction and are cleaved by the 5′ exonuclease activity of Taq polymerase only if genomic DNA includes the corresponding variant of the polymorphism. The probes used were either an MGB probe or were labeled at the 5′ end with the reporter dye (FAM or VIC) and at the 3′ end with the quencher dye (TAMRA). The PCR primers were 5′-CCCACCACACCAGCCCTGTTC-3′ in the forward direction and 5′-TTCCGCTTCACCAGCTCCATGT-3′ in the reverse direction. The probe for the T allele was 5′-TAGCAGCAGCAGCAGCAGCCGC-3′ and for the C allele was 5′-TAGCAGCAGCGGCAGCAGCCG-3′. The annealing temperature was 60°C.
Because the assays were performed in multiple laboratories, a series of quality control measures were implemented to maximize genotype integrity. All centers duplicated genotyping on at least 2% of their samples, included no template (water) controls in every plate, and all plates were set up to have a random mixture of affected and unaffected carriers. A study was included in the analysis only if the genotype call rate was over 95%. The concordance between duplicates had to be at least 98%. We also evaluated deviation from Hardy–Weinberg equilibrium (HWE) among a subset of unrelated subjects for each study separately. The genotype frequencies among unrelated individuals for all studies were consistent with HWE at the 5% significance level. Fifteen centers initially provided data to this study. However, data from two studies were excluded because they did not meet the criteria for inclusion because their data exceeded the required discordance rate among duplicates of 2% and a failure rate >5%. Therefore, data on 13 centers were included in this study.
The data were analyzed within a survival analysis framework by modeling the retrospective likelihood of the observed genotypes, conditional on the disease phenotypes . This method corrects for the likely over-sampling of affected individuals, since carriers are recruited through genetic clinics . The effect of each SNP was modeled as a per-allele hazard ratio (HR). In addition, we estimated the effect of heterozygote and homozygote classes for BRCA1 and BRCA2 mutation carriers separately. HRs were assumed to be independent of age (i.e., by assuming a Cox proportional hazards model). Baseline age-specific incidence rates in the Cox proportional hazards model were chosen such that the overall breast cancer incidence rates, averaged over all genotypic categories, agreed with external estimates of BRCA1 and BRCA2 incidence rates . Between-study heterogeneity was examined by comparing the models which allowed for study specific log-hazard ratios against models in which the same log-hazard ratio was assumed to apply to all studies. All analyses were stratified by study group and country of residence (where numbers were sufficiently large), and used calendar-year and cohort-specific breast cancer incidence rates for BRCA1 and BRCA2 . Compound BRCA1 and BRCA2 mutation carriers were assumed to develop the disease according to the BRCA1 incidence rates, since the penetrance of BRCA1-associated breast cancer is known to be earlier in age than that of BRCA2-associated breast cancer . Therefore, on average cancers diagnosed in BRCA1 mutation carriers would precede those diagnosed in BRCA2 mutation carriers. A robust variance estimation approach was used to allow for the non-independence among related carriers [22, 23]. Analyses were carried out using the pedigree analysis software MENDEL .
Results and discussion
In our data, the frequency of the 10P variant allele was 41.6% in BRCA1 mutation carrier controls with no cancer diagnosis, LL, LP, and PP genotype frequencies of 34.4, 48.1, and 17.5%, respectively. In BRCA2 mutation carrier controls with no prior cancer diagnosis, the 10P allele frequency was 40.2%, with LL, LP, and PP genotype frequencies of 36.5%, 47.3%, and 16.1%, respectively. The frequency of L10P variants did not differ between controls with no history of cancer between BRCA1 and BRCA2 mutation carriers (χ2 = 1.91, df = 2, P = 0.385). Overall and in each center, the frequency of L10P alleles did not deviate significantly from Hardy–Weinberg proportions.
There was no significant evidence of association between TGFB1 L10P and breast cancer risk in either BRCA1 or BRCA2 mutation carriers (P-trend: 0.821 and 0.24, respectively; Table 1). The per-allele HR was estimated to be 1.01 (95%CI: 0.92–1.11) among BRCA1 carriers and 0.92 (95%CI: 0.81–1.04) among BRCA2 mutation carriers. In BRCA1 mutation carriers, the HR associated with the heterozygote LP genotype was 1.04 (95%CI: 0.91–1.20), and the HR associated with the homozygote PP genotype was 0.98 (95%CI: 0.81–1.17). In BRCA2 mutation carriers the heterozygote and homozygote HRs were 0.89 (95%CI: 0.74–1.07) and 0.88 (95%CI: 0.68–1.13), respectively.
There was no evidence for heterogeneity of effects by center in BRCA1 mutation carriers (P-value = 0.22), but there was some evidence for heterogeneity of effects across centers in BRCA2 mutation carriers (P = 0.005). However, this appears to be in part explained by the statistically significant increased risk in the DKFZ study, which involved a sample size of only 50 BRCA2 mutation carriers.
Our results do not support the hypothesis that TGFB1 L10P influences breast cancer risk in BRCA1 or BRCA2 mutation carriers. We have reported a lack of statistical significance for such an association, and also have observed that the direction of the L10P genotype effects in most of our contributing centers, the HR estimate for the P allele is below unity, i.e. in the opposite direction to the estimated OR in the population studies. The upper bound of the 95%CI for BRCA1 does not exclude previous estimates of TGFB1 L10P effect including the results published by the Breast Cancer Association Consortium (BCAC) , which reported a per-allele OR = 1.08 (95%CI 1.04–1.11) in a sample of 15,109 cases and 12,946 controls, and a previous meta-analysis of published studies involving 4,021 cases and 8,253 controls , which report a per-allele OR = 1.04. However, the upper bound of the 95%CI estimate for BRCA2 is on the lower 95% confidence limit from the BCAC study.
The differences between the risk estimates from the current study and those from the large case–control studies of women not selected for BRCA1/2 mutations [16, 17] can be interpreted in a number of ways. First, it is possible that TGFB1 L10P does influence breast cancer risk in the general population, but that this variant does not affect the risk of breast cancer in BRCA1 or BRCA2 carriers (or does so to a weaker extent). The L10P proline allele, associated with increased TGF-β secretion, may be associated with a reduced risk of in situ tumors but an increased risk of invasive cancer [1, 2, 16]. Breast tumor characteristics in BRCA1 mutation carriers, including histopathological features related to disease aggressiveness, differ substantially from those in the general population . Similarly, there is some evidence that the phenotypic characteristics of BRCA2 tumors may also differ from sporadic cases . Therefore, the biological effect of TGFB1 in BRCA1- and possibly BRCA2-associated tumors may be different than in tumors arising from the general population. It is also worth noting that the same relative risk would only be expected if BRCA1/2 and TGFB1 combine multiplicatively. It is possible that TGFB1 L10P does increase risk in carriers, but does not “interact” multiplicatively, so that its effect is not detectable in carriers who are already at very high risk.
Second, it is possible that TGFB1 L10P is not causally associated with breast cancer risk in the general population, and the previous reports may represent false positive associations. Although the large BCAC study found a highly statistically significant effect of L10P, with no evidence for heterogeneity of effects across studies, the level of significance (P = .00003) is not beyond reasonable doubt in the context of genetic association studies . The literature contains reports of positive and inverse associations as well as null results for TGFB1 L10P [3, 5–15, 28], so there is not complete consistency across studies or populations with respect to the role of this variant in breast cancer etiology. Finally, it is possible that there is a true association, but that it is weaker than previously reported. A per allele odds ratio of 1.02–1.03 would then be statistically compatible with the HR estimates seen in carriers. Further large association studies would be required to evaluate this possibility. These results emphasize that very large studies are essential to evaluate the effects of genetic polymorphisms on disease risk.
The evidence that TGFB1 L10P is associated with breast cancer risk in the general population has been mixed and no strong conclusions have been reached about the role of this variant in breast cancer etiology. The present study provides no evidence that this variant is a modifier of breast cancer risk in BRCA1 or BRCA2 carriers.
ACA, HAP and the CIMBA data management are funded by Cancer Research––UK. Douglas F. Easton is a Principal Research Fellow of Cancer Research––UK. Timothy R. Rebbeck is supported by R01-CA102776. Boris Pasche is supported by R01-CA112520.
Timothy R. Rebbeck, Douglas E. Easton, Antonis C. Antoniou, Amanda B. Spurdle, Georgia Chenevix-Trench, Boris Pasche, Virginia Kaklamani, Fergus J. Couch made substantial contributions to conception and design. Antonis C. Antoniou, Susan Peock, Margaret Cook, Douglas E. Easton, Jean-Philippe Peyrat, Joëlle Fournier, Philippe Vennin, Claude Adenis, Danièle Muller, Jean-Pierre Fricker, Michel Longy, Olga M. Sinilnikova, Dominique Stoppa-Lyonnet, Ute Hamann, Amanda B. Spurdle, Georgia Chenevix-Trench, Patricia A. Harrington, Alan Donaldson, Allison M. Male, Carol Anne Gardiner, Heli Nevanlinna, Helen Gregory, Lucy E. Side, Anne C. Robinson, Louise Emmerson, Ian Ellis, Boris Pasche, Virginia Kaklamani, Rita K. Schmutzler, Beatrix Versmold, Christoph Engel, Alfons Meindl, Karin Kast, Dieter Schaefer, Ursula G. Froster, Trinidad Caldes Llopis, Kristiina Aittomäki, Fergus J. Couch, Jacques Simard, Lutecia H. Mateus Pereira, Mark H. Greene, Irene L. Andrulis made substantial contributions to acquisition of data. Timothy R. Rebbeck, Antonis C. Antoniou, Douglas E. Easton, made substantial contributions to analysis and interpretation of data. Timothy R. Rebbeck, Antonis C. Antoniou, Douglas E. Easton, Georgia Chenevix-Trench, Boris Pasche, Jacques Simard, Irene L. Andrulis were involved in drafting the manuscript or revising it critically for important intellectual content. All authors have given final approval of the version to be published.
Participating CIMBA centres
Breast Cancer Family Registry (BCFR)
BCFR was supported by the National Cancer Institute, National Institutes of Health under RFA # CA-95-011 and through cooperative agreements with members of the BCFR, including Cancer Care Ontario (U01 CA69467, PI: Irene A. Andrulis), Columbia University (U01 CA69398, PI: Mary Beth Terry), Fox Chase Cancer Center (U01 CA69631, PI: Mary Daly), Huntsman Cancer Institute (U01 CA69446, PI: Saundra Buys), Northern California Cancer Center (U01 CA69417, PI: Esther M. John), University of Melbourne (U01 CA69638, PI: John L. Hopper), Research Triangle Institute Informatics Support Center (RFP No. N02PC45022-46). The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the CFR, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or the CFR.
German Consortium of Hereditary Breast and Ovarian Cancer (GC-HBOC)
GC-HBOC is supported by a grant of the German Cancer Aid (grant107054) and the Center for Molecular Medicine Cologne (grant TV93) to Rita K. Schmutzler. We thank Juliane Köhler for her excellent technical assistance and the 12 centers of the GC-HBOC for providing samples and clinical data.
Helsinki Breast Cancer Study (HEBCS)
HEBCS was supported by the Academy of Finland (110663), Helsinki University Central Hospital Research Fund, the Sigrid Juselius Fund and the Finnish Cancer Society. We thank Tuomas Heikkinen and Kati Kämpjärvi for their contribution in the molecular analyses and Drs. Kirsimari Aaltonen and Carl Blomqvist, for their help in patient sample and data collection.
Hospital Clinico San Carlos (HCSC)
Trinidad Caldes is funded by FMMA/06 and RTICC06/002/0021 HCSC-Spain. We thank Miguel de la Hoya, Pedro Perez-Segura and Elena Oliveira for their contribution.
Interdisciplinary Health Research International Team Breast Cancer susceptibility (INHERIT)
Jacques Simard, Francine Durocher, Rachel Laframboise, Marie Plante, Centre Hospitalier Universitaire de Québec & Laval University, Québec, Canada; Peter Bridge, Jilian Parboosingh, Molecular Diagnostic Laboratory, Alberta Children’s Hospital, Calgary, Canada; Jocelyne Chiquette, Hôpital du Saint-Sacrement, Québec, Canada; Bernard Lesperance, Roxanne Pichette, Hôpital du Sacré-Cœur de Montréal, Montréal, Canada. This work was supported by the Canadian Institutes of Health Research for the INHERIT BRCAs program, the CURE Foundation and the Fonds de la recherche en Santé du Quebec/Reseau de Medecine Genetique Appliquee.
The Kathleen Cunningham Consortium for Research into Familial Breast Cancer (KConFab)
We wish to thank Heather Thorne, Eveline Niedermayr, Helene Holland, Xiaoqing Chen and Jonathan Beesley, all the KConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (funded by NHMRC grants 145684 and 288704) for their contributions to this resource and its management, and the many families who contribute to KConFab. KConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. Amanda B. Spurdle and Georgia Chenevix-Trench are NHMRC Career Development Awardee and Senior Principal Research Fellow, respectively.
Mayo Clinic Study (MAYO)
The Mayo Clinic study was supported by the U.S. Army Medical Research and Materiel Command (W81XWH-04-1-0588), the Mayo Clinic Breast Cancer SPORE (P50-CA116201) and NIH grant CA122340 to Fergus J Couch. We wish to thank Noralane Lindor and Linda Wadum for their contributions.
Modifier Study of Quantitative Effects on Disease (Mod-Squad)
Csilla I. Szabo is partially supported by a Susan G. Komen Foundation Basic. Clinical and Translational Research Grant (BCTR0402923). Research Project of the Ministry of Education, Youth and Sports of the Czech Republic No MSM0021620808 to Michal Zikan, Zdenek Kleibl and Petr Pohlreich. We acknowledge the contributions of Michal Zikan, Petr Pohlreich and Zdenek Kleibl (Department of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University Prague, Czech Republic) and Lenka Foretova, Machakova Eva Lukesova Miroslava (Department of Cancer).
National Cancer Institute study (NCI)
We acknowledge the contributions of Dr. Jeffery Struewing and Marbin A. Pineda from the Laboratory of Population Genetics. Drs. Greene and Struewing were supported by funding from the Intramural Research Program of the US National Cancer Institute. Their data collection efforts were supported by Contracts NO2-CP-11019-50 and N02-CP-65504 with Westat, Rockville, MD.
Ontario Cancer Genetics Network study (OCGN)
We thank the staff and all of those who participate in the OCGN. We acknowledge the contributions of the provincial familial cancer clinics and molecular diagnostic laboratories and Mona Gill for excellent technical assistance. The OCGN study was supported by funding from Cancer Care Ontario and the National Cancer Institute of Canada with funds from the Terry Fox Run.
Deutsches Krebsforschungszentrum study (DKFZ)
The DKFZ study was supported by the DKFZ. We thank Diana Torres and Muhammad U. Rashid for providing DNA samples and supplying data. We thank Antje Seidel-Renkert and Michael Gilbert for expert technical assistance.
Epidemiological study of BRCA1 & BRCA2 mutation carriers (EMBRACE)
Margaret Cook, Susan Peock and EMBRACE are funded by Cancer Research–UK. DFE is the PI of the study. EMBRACE Collaborating Centers are: Coordinating Centre, Cambridge: Susan Peock, Margaret Cook, Alexandra Bignell. North of Scotland Regional Genetics Service, Aberdeen: Neva Haites, Helen Gregory. Northern Ireland Regional Genetics Service, Belfast: Patrick Morrison. West Midlands Regional Clinical Genetics Service, Birmingham: Trevor Cole, Carole McKeown. South West Regional Genetics Service, Bristol: Alan Donaldson. East Anglian Regional Genetics Service, Cambridge: Joan Paterson. Medical Genetics Services for Wales, Cardiff: Alexandra Murray, Mark Rogers. St James’s Hospital, Dublin & National Centre for Medical Genetics, Dublin: Peter Daly, David Barton. South East of Scotland Regional Genetics Service, Edinburgh: Mary Porteous, Michael Steel. Peninsula Clinical Genetics Service. Exeter: Carole Brewer, Julia Rankin. West of Scotland Regional Genetics Service, Glasgow: Rosemarie Davidson, Victoria Murday. South East Thames Regional Genetics Service, Guys Hospital London: Louise Izatt, Gabriella Pichert. North West Thames Regional Genetics Service. Harrow: Huw Dorkins. Leicestershire Clinical Genetics Service, Leicester: Richard Trembath. Yorkshire Regional Genetics Service, Leeds: Tim Bishop, Carol Chu. Merseyside & Cheshire Clinical Genetics Service. Liverpool: Ian Ellis. Manchester Regional Genetics Service, Manchester: D Gareth Evans, Fiona Lalloo, Andrew Shenton. North East Thames Regional Genetics Service, NE Thames: Alison Male, James Mackay, Anne Robinson. Nottingham Clinical Genetics service, Nottingham University Hospitals, Nottingham: Carol Gardiner. Northern Clinical Genetics Service, Newcastle: Fiona Douglas, John Burn. Oxford Regional Genetics Service, Oxford: Lucy Side, Lucy Walker, Sarah Durell. Institute of Cancer Research and Royal Marsden NHS Foundation Trust: Rosalind Eeles. North Trent Clinical Genetics Service, Sheffield: Jackie Cook, Oliver Quarrell. South West Thames Regional Genetics Service, London: Shirley Hodgson. Wessex Clinical Genetics Service. Southampton: Diana Eccles, Anneke Lucassen.
Genetic Modifiers of cancer risk in BRCA1/2 mutation carriers study (GEMO)
We wish to thank Laure Barjhoux for management the GEMO samples and carrying out genotyping, and all the GEMO centers for their contributions to this resource. The GEMO study was supported by the Programme Hospitalier de Recherche Clinique AOR01082, by Programme Incitatif et Coopératif Génétique et Biologie de Cancer du Sein, Institut Curie, and by the Association “Le cancer du sein, parlons-en!” Award.