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Inherited BRCA1 and BRCA2 (BRCA1/2) mutations confer elevated breast cancer risk. Knowledge of factors that can improve breast cancer risk assessment in BRCA1/2 mutation carriers may improve personalized cancer prevention strategies.
A cohort of 5,546 BRCA1 and 2,865 BRCA2 mutation carriers was used to evaluate risk of breast cancer associated with BARD1 Cys557Ser. In a second non-independent cohort of 1,537 of BRCA1 and 839 BRCA2 mutation carriers, BARD1 haplotypes were also evaluated.
The BARD1 Cys557Ser variant was not significantly associated with risk of breast cancer from single SNP analysis, with a pooled effect estimate of 0.90 (95%CI: 0.71-1.15) in BRCA1 carriers and 0.87 (95%CI: 0.59-1.29) in BRCA2 carriers. Further analysis of haplotypes at BARD1 also revealed no evidence that additional common genetic variation not captured by Cys557Ser was associated with breast cancer risk.
Evidence to date does not support a role for BARD1 variation, including the Cy557Ser variant, as a modifier of risk in BRCA1/2 mutation carriers.
Interactors of BRCA1/2 have been implicated as modifiers of BRCA1/2-associated cancer risk. Our finding that BARD1 does not contribute to this risk modification may focus research on other genes that do modify BRCA1/2-associated cancer risk.
There is substantial inter-individual variability in age at cancer diagnosis in BRCA1 and BRCA2 mutation carriers, which persists even among relatives that carry the same BRCA1 and BRCA2 mutation (1). Variation in genes that interact with BRCA1 and BRCA2 in the recognition and repair of DNA damage are strong candidates for study as genetic modifiers of BRCA1 and BRCA2 cancer risk. The BRCA1-BARD1 heterodimer is known to be important for BRCA1 function, with interaction mediated through the ring finger domains of the two proteins (2). In addition, although there is no evidence for a direct interaction between BARD1 and BRCA2, they do operate in the same DNA repair processes, exemplified by the fact that the BRCA2 partner RAD51, BARD1 and BRCA1 all relocate to proliferating cell nuclear antigen structures after irradiation (3).
The BARD1 Cys557Ser SNP (rs28997576) was first reported as a germline alteration in a sporadic breast/uterine tumour (4). This variant lies between the ankyrin repeats and BRCT domains of BARD1, and the ectopically expressed Cys557 protein has growth suppression and pro-apoptotic effects relative to 557Ser (5). This SNP (minor allele frequency in Europeans: 0.025) has been reported to be associated with both breast cancer in the general population and familial breast cancer, but results have not shown consistent across all studies (6-12). Stacey et al (6) initially reported that the Cys557Ser variant was associated with increased breast cancer risk in 756 Icelandic mutation carriers who carry BRCA2 999del5 founder mutation (odds ratio (OR)=3.1; 95% CI 1.2-8.4). However, subsequent studies reported no elevated risk in 228 Nordic BRCA1 and BRCA2 carriers (OR=0.8, 95%CI: 0.3-2.0) (8), or in 1,207 Polish BRCA1 mutation carriers (OR=0.9, 95%CI: 0.4-2.2) (10). There have been no previous haplotype-based studies assessing the role of BARD1 variation in breast cancer risk in BRCA1 and BRCA2 carriers specifically.
In order to resolve whether BARD1 is a modifier of BRCA1 and BRCA2-associated breast cancer risk, we undertook a large study to comprehensively assess the association of BARD1 Cys557Ser as well as haplotypic variation with cancer risk in BRCA1 and BRCA2 carriers.
The design for this study has been described in detail previously (13). Briefly, eligible participants included adult women with documented disease-associated inherited mutations in BRCA1 or BRCA2. Mutations were included in the analysis if they were pathogenic according to generally recognized criteria (14, 15). Two overlapping cohorts of women with disease-associated BRCA1 and BRCA2 mutations were identitied (Table 1). First, a cohort of 5,546 BRCA1 and 2,865 BRCA2 mutation carriers from the multicenter CIMBA consortium (13) was used to evaluate risk of breast cancer associated with BARD1 Cys557Ser. Second, a cohort of 1,537 of BRCA1 and 839 BRCA2 mutation carriers participating in the MAGIC consortium was used to further explore the relationship between BARD1 haplotypes and breast cancer risk. Recruitment and genetic studies were approved by relevant ethics committees at all sites, and informed consent was obtained from each participant.
For analysis of the BARD1 Cys557Ser SNP, existing genotype data from BRCA1 and BRCA2 mutation carriers was requested from members of the CIMBA consortium. The primary methods used for genotyping were Sequenom iPlex (EMBRACE, -HEBON, kConFab, SWE-BRCA, PISA, Penn, Austria, Mayo, FCCC, GEMO, Georgetown, HEBCS) and by Taqman assays (OUH, Baylor, Beth Israel, City of Hope, Creighton, Dana Farber, NorthShore, IHCC, UCLA, University of Chicago, University of Texas Health Science Center, University of Utah, and Women’s College Hospital; (16)) Genotypes for the INHERIT samples were typed by direct sequencing using an ABI Prism 3730xl DNA Analyser automated sequencer, with version 3.1 of the Big Dye fluorescent method according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using the Staden preGap4 and Gap4 programs. Samples from IHCC were typed by PCR-RFLP(10). SNP quality control measures included >95% success rate, Hardy-Weinberg Equilibrium P>0.005, In addition, concordance of more than 98% for duplicate samples was required for studies that had included 2% duplicated samples for quality control purposes (all studies undergoing Sequenom iplex for BARD1 Cys557Ser, and all samples included in the haplotype sub-study).
For studies of BARD1 haplotypic variation, 11 haplotype tag SNPs were identified and assayed at the University of Pennsylvania as previously described(16). The rs IDs were as follows: rs6712055, rs16852689, rs280621, rs13021937, rs13423596, rs10190829, rs6751923, rs4234006, rs28997576, rs3768708, rs1374230.
To assess the relationship between BARD1 SNPs and breast cancer risk, proportional hazards models were used as previously described(16, 17). Briefly, participants were followed from the time of genetic testing or study ascertainment until the first diagnosis of breast cancer (the primary event in this analysis) or were censored at ovarian cancer. Participants who developed breast cancer were censored at bilateral prophylactic mastectomy if it occurred more than a year prior to the cancer diagnosis. This is to avoid censoring at bilateral mastectomies at which occult tumors were detected, but ages are rounded. The remaining participants were censored at the age at last observation. To address the problem of non-random sampling of mutation carriers with respect to the disease phenotype, analyses used the weighted Cox regression approach (17), where affected and unaffected individuals were differentially weighted such that observed breast cancer incidence rates in the study sample are consistent with established breast cancer risk estimates for BRCA1 and BRCA2 mutation carriers (18). Analyses assessing the association of the BARD1 Cys557Ser SNP combined heterozygote and homozygote variant carriers under a dominant model due to the rare frequency of this variant. Analyses were assessed separately for BRCA1 and BRCA2 mutation carriers, adjusted for Study group, ethnicity (non-Jewish Caucasian, Jewish or other), and year of birth cohort (decade of birth, categorized as <1940, 1940-1949, 1950-1959, 1960-1969, 1970-1989). There were 3047 breast cancer events out of 5546 total for BRCA1 (55%) and 1578 breast cancer events out of 2865 total for BRCA2 (55%) for the Cys557Ser censored analysis datasets. The remainder were censored for analysis. Secondary analyses adjusted for prophylactic oophorectomy, or assessed risk for the subset of carriers with mutations determined to result in unstable transcripts/proteins (class 1 loss of function mutations). R version 2.7.0 was used for single SNP statistical analyses.
To investigate haplotype effects, the Estimation-maximization algorithm (19, 20) was used to estimate haplotype frequencies as implemented in R version 2.1.1 subroutine haplo.em(21) as previously described (16). In this analysis, we included 607 breast cancer cases and 863 censored observations for BRCA1, and 813 breast cancer cases and 423 controls for BRCA2.
The frequency of the Cys557Ser SNP in the combined dataset (Table 1) was similar to published reports, with 4.4% of individuals carrying at least one rare allele (4.5% in BRCA1 carriers, 4.2% in BRCA2 carriers). There were no significant associations of Cys557Ser and breast cancer risk for carriers of BRCA1 mutations (hazard ratio (HR)=0.90, 95%CI: 0.71-1.15) or BRCA2 mutations (HR=0.87, 95%CI: 0.59-1.29). There was no evidence for heterogeneity by center for either BRCA1 or BRCA2 analyses (P>0.5). There was also no evidence for association with additional adjustment for prophylactic oophorectomy, or when analyses were restricted to Class 1 mutations. For example, the HR for the subset of 3882 individuals with BRCA1 Class 1 mutations was 0.84 (0.62-1.15), and for the 2668 individuals with BRCA2 class 1 mutations was 0.96 (0.64-1.45).
For the haplotype analysis (Table 2), we also observed no overall effect of variation at BARD1 in either BRCA1 false discovery rate (FDR)-corrected p-value=0.152) or BRCA2 (FDR-corrected p-value=0.134). No single BARD1 haplotype was significantly associated with breast cancer risk. Cys557Ser is represented by SNP 16 in Table 2 (BRCA1 haplotype 8 and BRCA2 haplotype 10). Since this variant was relatively rare (approximately 2% in both BRCA1 and BRCA2 carriers), estimates of its effect were not made in our primary analysis. When we fit a model that allowed the estimation of effects for haplotypes with at least 1% frequency in controls, no single haplotype was significantly associated with risk. The haplotype that contained the 557Ser allele was also not significantly associated with risk in either BRCA1 (HR=0.91, 95%CI: 0.45-1.85) or BRCA2 (HR=0.69, 95%CI: 0.28-1.72). Indeed, neither of these estimates was associated with increased risk of breast cancer as previously reported.
The data presented here do not provide evidence that neither the BARD1 Cys557Ser SNP nor additional haplotypic variability not captured by Cys557Ser is associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers. Our sample size had>99% power to detect the effect size reported by Stacey et al. (6) of OR=3.1. The study had >80% power to detect risk ratios of 0.89 (or 1.13) for BRCA1 carriers and 0.86 (or 1.17) for BRCA2 carriers. The upper 95% confidence limits on the rate ratio in our analysis exclude any substantial risk.
Our study found no evidence to support substantial associations of BARD1 variation with increased breast cancer risk in BRCA1 and BRCA2 carriers.
The CIMBA data management is supported by Cancer Research- UK. ACA is a Cancer Research – UK, Senior Cancer Research Fellow. TRR was supported by R01-CA102776 and R01-CA083855.
MAGIC: The MAGIC Consortium includes the following centers and individuals: Baylor-Charles A. Sammons Cancer Center (Joanne L. Blum, M.D. PhD, Estelle Brothers, RN, Gaby Ethington), Baylor College of Medicine (Claire Noll, Sharon Plon, M.D., Ph.D.), Beth Israel Deaconess Medical Center (Nadine Tung, M.D.), City of Hope National Medical Center (Veronica Lagos, Jeffery Weitzel, M.D.), Creighton University (Carrie Snyder, B.A., Henry T. Lynch, M.D., Patrice Watson, Ph.D.), Dana Farber Cancer Institute (Kathryn Stoeckert, Judy E. Garber, M.D., M.P.H.,), Duke University (Sydnee Crankshaw, Joellen Schildkraut, Ph.D.), NorthShore University HealthSystem Center for Medical Genetics (Wendy S. Rubinstein, M.D., Ph.D., Christina G. Selkirk, MS CGC),Fox Chase Cancer Center (Mary B. Daly, M.D., Ph.D., Andrew Godwin, PhD), Queensland Institute of Medical Research (Georgia Chenevix-Trench), Georgetown University (Claudine Isaacs, M.D.), Jonsson Comprehensive Cancer Center at the University of California, Los Angeles (Joyce Seldon, MS CGC, Patricia A. Ganz , M.D.), Mayo Clinic College of Medicine (Linda Wadum, Fergus Couch, Ph.D.), University of Chicago (Shelly Cummings, Olufunmilayo Olopade, M.D.), University of California, Irvine (Susan L. Neuhausen, Ph.D., Linda Steele), University of Pennsylvania Health System: (Susan Domchek, MD, Katherine Nathanson M.D., Tara Friebel, M.P.H., Timothy Rebbeck, Ph.D.), University of Texas, Southwestern (Gail Tomlinson, M.D.), University of Vienna (Christian Singer, M.D.), Women’s College Hospital (Steven A. Narod, MD). This publication was supported in part by revenue from Nebraska cigarette taxes awarded to Creighton University by the Nebraska Department of Health and Human Services. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Nebraska or the Nebraska Department of Health and Human Services. Support was also received from NIH grants 5UO1 CA86389 (to HTL) and R01-CA083855, R01-CA74415 (to SLN) and R01-CA102776 and R01-CA083855 (to TRR).
Austria MUV: We wish to thank Daniela Muhr, Christine Fuerhauser-Rappaport, all the Hereditary Breast and Ovarian Cancer counselling clinics in Austria, and the many families who contributed to this study. This study was supported by the MUV Comprensive Cancer Center (Cluster Genetics and Epigenetics) and by the Austrian Society for Endocrinological Oncology.
EMBRACE: Epidemiological study of BRCA1 and BRCA2 mutation carriers (EMBRACE): Douglas F. Easton is the PI of the study. EMBRACE Collaborating Centers are: Coordinating Center, Cambridge: Susan Peock, Margaret Cook, Clare T. Oliver, Debra Frost. North of Scotland Regional Genetics Service, Aberdeen: Helen Gregory, Zosia Miedzybrodzka. Northern Ireland Regional Genetics Service, Belfast: Patrick Morrison, Lisa Jeffers. West Midlands Regional Clinical Genetics Service, Birmingham: Trevor Cole, Carole McKeown, Kai-Ren Ong, Laura Boyes. 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 T. Rogers, Emma McCann. St James’s Hospital, Dublin & National Centre for Medical Genetics, Dublin: M. John Kennedy, David Barton. South East of Scotland Regional Genetics Service, Edinburgh: Mary Porteous. Peninsula Clinical Genetics Service, Exeter: Carole Brewer, Emma Kivuva, Anne Searle, Selina Goodman. West of Scotland Regional Genetics Service, Glasgow: Rosemarie Davidson, Victoria Murday, Nicola Bradshaw, Lesley Snadden, Mark Longmuir, Catherine Watt, Sarah Gibson. South East Thames Regional Genetics Service, Guys Hospital London: Louise Izatt, Gabriella Pichert, Chris Jacobs, Caroline Langman. North West Thames Regional Genetics Service, Kennedy-Galton Centre, Harrow: Huw Dorkins. Leicestershire Clinical Genetics Service, Leicester: Julian Barwell. Yorkshire Regional Genetics Service, Leeds: Carol Chu, Tim Bishop, Julie Miller. Merseyside & Cheshire Clinical Genetics Service, Liverpool: Ian Ellis, Catherine Houghton. Manchester Regional Genetics Service, Manchester: D Gareth Evans, Fiona Lalloo, Felicity Holt. North East Thames Regional Genetics Service, NE Thames: Alison Male, Lucy Side, Cheryl Berlin. Nottingham Centre for Medical Genetics, Nottingham: Jacqueline Eason, Rebecca Collier. Northern Clinical Genetics Service, Newcastle: Fiona Douglas, Oonagh Claber. Oxford Regional Genetics Service, Oxford: Lisa Walker, Diane McLeod, Dorothy Halliday, Sarah Durrell, Barbara Stayner. The Institute of Cancer Research and Royal Marsden NHS Foundation Trust: Ros Eeles, Susan Shanley, Nazneen Rahman, Richard Houlston, Elizabeth Bancroft, Lucia D’Mello, Elizabeth Page, Audrey Ardern-Jones, Kelly Kohut, Jennifer Wiggins. Elena Castro, Anita Mitra, Lisa Robertson. North Trent Clinical Genetics Service, Sheffield: Jackie Cook, Oliver Quarrell, Cathryn Bardsley. South Essex Cancer Research Network, Southend: Anne Robinson. South West Thames Regional Genetics Service, London: Shirley Hodgson, Sheila Goff, Glen Brice, Lizzie Winchester. Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton: Diana Eccles, Anneke Lucassen, Gillian Crawford, Emma Tyler, Donna McBride. EMBRACE is funded by Cancer Research-UK Grants C1287/A10118 and C1287/A8874. Don Conroy is supported by Cancer Research UK Grant C8197/A10123. D.Gareth Evans and Fiona Lalloo are supported by an NIHR grant to the Biomedical Research Centre, Manchester. The Investigators at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust are supported by an NIHR grant to the Biomedical Research Centre at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust. Ros Eeles, Elizabeth Bancroft and Lucia D’Mello are also supported by Cancer Research UK Grant C5047/A8385.
GEMO: The GEMO study (Cancer Genetics Network “Groupe Génétique et Cancer”, Fédération Nationale des Centres de Lutte Contre le Cancer, France) is supported by the Ligue National Contre le Cancer ; Association for International Cancer Research Grant (AICR-07-0454) ; and the Association “Le cancer du sein, parlons-en!” Award.
We wish to thank all the GEMO collaborating groups for their contribution to this study. GEMO Collaborating Centers are: Coordinating Centres, Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Hospitalier Universitaire de Lyon / Centre Léon Bérard, & UMR5201 CNRS, Université de Lyon, Lyon: Olga Sinilnikova, Laure Barjhoux, Sophie Giraud, Mélanie Léone, Sylvie Mazoyer; and INSERM U509, Service de Génétique Oncologique, Institut Curie, Paris: Dominique Stoppa-Lyonnet, Marion Gauthier-Villars, Bruno Buecher, Claude Houdayer, Virginie Moncoutier, Muriel Belotti, Antoine de Pauw. Institut Gustave Roussy, Villejuif: Brigitte Bressac-de-Paillerets, Audrey Remenieras, Véronique Byrde, Olivier Caron, Gilbert Lenoir. Centre Jean Perrin, Clermont–Ferrand: Yves-Jean Bignon, Nancy Uhrhammer. Centre Léon Bérard, Lyon: Christine Lasset, Valérie Bonadona. Centre François Baclesse, Caen: Agnès Hardouin, Pascaline Berthet. Institut Paoli Calmettes, Marseille: Hagay Sobol, Violaine Bourdon, Tetsuro Noguchi, François Eisinger. Groupe Hospitalier Pitié-Salpétrière, Paris: Florence Coulet, Chrystelle Colas, Florent Soubrier. CHU de Arnaud-de-Villeneuve, Montpellier: Isabelle Coupier, Pascal Pujol. Centre Oscar Lambret, Lille: Jean-Philippe Peyrat, Joëlle Fournier, Françoise Révillion, Philippe Vennin, Claude Adenis. Centre René Huguenin, St Cloud: Etienne Rouleau, Rosette Lidereau, Liliane Demange, Catherine Nogues. Centre Paul Strauss, Strasbourg: Danièle Muller, Jean-Pierre Fricker. Institut Bergonié, Bordeaux: Michel Longy, Nicolas Sevenet. Institut Claudius Regaud, Toulouse: Christine Toulas, Rosine Guimbaud, Laurence Gladieff, Viviane Feillel. CHU de Grenoble: Dominique Leroux, Hélène Dreyfus, Christine Rebischung. CHU de Dijon: Cécile Cassini, Laurence Faivre. CHU de St-Etienne: Fabienne Prieur. Hôtel Dieu Centre Hospitalier, Chambéry: Sandra Fert Ferrer. Centre Antoine Lacassagne, Nice: Marc Frénay. CHU de Limoges: Laurence Vénat-Bouvet. Creighton University, Omaha, USA: Henry T. Lynch.
HEBON: The Hereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON) Collaborating Centers: Coordinating center: Netherlands Cancer Institute, Amsterdam: Frans B. L. Hogervorst, Senno Verhoef, Martijn Verheus, Laura J. van ‘t Veer, Flora E. van Leeuwen, Matti A. Rookus; Erasmus Medical Center, Rotterdam: Margriet Collée, Ans M.W. van den Ouweland, Agnes Jager, Maartje J. Hooning, Madeleine M.A. Tilanus-Linthorst, Caroline Seynaeve; Leiden University Medical Center, Leiden: Christi J. van Asperen, Juul T. Wijnen, Maaike P. Vreeswijk, Rob A. Tollenaar, Peter Devilee; Radboud University Nijmegen Medical Center, Nijmegen: Marjolijn J. Ligtenberg, Nicoline Hoogerbrugge; University Medical Center Utrecht, Utrecht: Margreet G. Ausems, Rob B. van der Luijt; Amsterdam Medical Center: Cora M. Aalfs, Theo A. van Os; VU University Medical Center, Amsterdam: Johan J.P. Gille, Quinten Waisfisz, Hanne E.J. Meijers-Heijboer; University Hospital Maastricht, Maastricht: Encarna B. Gomez-Garcia, Cees E. van Roozendaal, Marinus J. Blok; University Medical Center Groningen University: Jan C. Oosterwijk, Annemarie H van der Hout, Marian J. Mourits; The Netherlands Foundation for the detection of hereditary tumours, Leiden, the Netherlands: Hans F. Vasen. The HEBON study is supported by the Dutch Cancer Society grants NKI 1998-1854, NKI 2004-3088 and NKI 2007-3756.
HEBCS: HEBCS study was supported by Helsinki University Central Hospital Research Fund, Academy of Finland (132473), the Finnish Cancer Society and the Sigrid Juselius Foundation. We would like to thank Drs. Kristiina Aittomäki, Carl Blomqvist and Kirsimari Aaltonen for their help with patient data and samples.
IHCC: Support was provided by Grant PBZ_KBN_122/P05/2004
INHERIT: We would like to thank Dr Martine Dumont for sample management, Martine Tranchant for skillful technical assistance and Dr Frédéric Guénard for genotyping. We would also like to thank Dr Jacques Simard, Director of the INHERIT BRCAs research program, which is supported by the Canadian Institutes of Health Research (CIHR). This work was also supported by the Fonds de la Recherche en Santé du Québec (FRSQ)/Réseau de Médecine Génétique Appliquée (RMGA), the CURE Foundation and the Canadian Breast Cancer Research Alliance (CBCRA). F.D. is a recipient of a chercheur-boursier from the Fonds de la Recherche en Santé du Québec (FRSQ) and a Research Career Award in the Health Sciences from CIHR/Rx&D Health Research Foundation.
kConFab: We wish to thank Heather Thorne, Eveline Niedermayr, 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, 288704 and 454508) for their contributions to this resource, 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. ABS and GCT are supported by a NHMRC Senior Research and Principal Research Fellowships, respectively.
Mayo: This research was supported by NIH grant CA128978, and NCI breast cancer specialized program of research excellence (SPORE) CA116201, and grants from the breast Cancer Research Foundation and the Komen Foundation for the cure. We wish to thank Zachary Fredericksen, Robert Tarrell and Vernon S. Pankratz for their assistance.
OUH: Thomas Sydenham, Anne-Marie Gerdes and Torben A Kruse are thanked for their contribution to this project.
Penn: This work was supported by HHSN21620074400C (to SMD), the Breast Cancer Research Foundation (to KLN), and R01-CA102776 and R01-CA083855 (to TRR).
PISA: We wish to thank the “Fondazione Cassa di Risparmio di Pisa” for a grant to MAC. All the surgeons (Dr M. Roncella; Dr. E. Rossetti) and clinicians (Dr. A. Cilotto, C. Marini) who allowed us the identification of patients
SWE-BRCA collaborators: Per Karlsson, Margareta Nordling, Annika Bergman and Zakaria Einbeigi, Gothenburg, Sahlgrenska University Hospital; Marie Stenmark-Askmalm and Sigrun Liedgren, Linköping University Hospital; Åke Borg, Niklas Loman, Håkan Olsson, Ulf Kristoffersson, Helena Jernström, Katja Harbst and Karin Henriksson, Lund University Hospital; Annika Lindblom, Brita Arver, Anna von Wachenfeldt, Annelie Liljegren, Gisela Barbany-Bustinza and Johanna Rantala, Stockholm, Karolinska University Hospital; Beatrice Melin, Henrik Grönberg, Eva-Lena Stattin and Monica Emanuelsson, Umeå University Hospital; Hans Ehrencrona, Richard Rosenquist Brandell and Niklas Dahl, Uppsala University Hospital.