Common genetic variation and mammographic density: risk factors for breast cancer By Stine Margarethe Biong Cancer Genome Variation Group Department of Genetics Institute for Cancer Research Oslo University Hospital Radiumhospitalet © Stine Margarethe Biong, 2012 Series of dissertations submitted to the Faculty of Medicine, University of Oslo No. 1361 ISBN 978-82-8264-354-2 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Inger Sandved Anfinsen. Printed in Norway: AIT Oslo AS. Produced in co-operation with Unipub. The thesis is produced by Unipub merely in connection with the thesis defence. Kindly direct all inquiries regarding the thesis to the copyright holder or the unit which grants the doctorate. Table of contents Acknowledgements Aims List of papers 1 Introduction ............................................................................................................................. 1 1.1 The breast .......................................................................................................................... 1 1.1.1 Breast anatomy ........................................................................................................... 1 1.1.2 Breast histology .......................................................................................................... 3 1.1.3 Normal breast development and physiology .............................................................. 6 1.1.4 Steroid hormones ........................................................................................................ 9 1.2 Single nucleotide polymorphisms ................................................................................... 13 1.2.1 SNPs in the estrogen pathway .................................................................................. 14 1.2 Breast cancer .................................................................................................................... 16 1.2.1 Incidence and mortality ............................................................................................ 17 1.2.2 Mammographic screening program ......................................................................... 18 1.2.3 Breast cancer risk ..................................................................................................... 18 1.3 Mammographic density ................................................................................................... 27 1.3.1 Mammographic density classification ...................................................................... 27 1.3.2 Causes of MD variation ............................................................................................ 29 2 Materials and methods ......................................................................................................... 32 2.1 Sample population ........................................................................................................... 32 2.1.1 TMBC ....................................................................................................................... 32 2.1.2 MDG ......................................................................................................................... 32 2.2 Blood samples and plasma analyses ................................................................................ 33 2.2.1 TMBC ....................................................................................................................... 33 2.2.2 MDG ......................................................................................................................... 34 2.3 Biopsy collection ............................................................................................................. 34 2.3.1 MDG ......................................................................................................................... 34 2.4 Mammograms .................................................................................................................. 34 2.4.1 TMBC ....................................................................................................................... 34 2.4.2 MDG ......................................................................................................................... 35 2.4.3 Mammogram assessment ......................................................................................... 35 2.5 In silico analyses: candidate gene and SNP selection ..................................................... 36 2.5.1 Gene selection .......................................................................................................... 36 2.5.2 SNP selection ........................................................................................................... 36 2.6 SNP genotyping ............................................................................................................... 37 2.6.1 Taqman real-time PCR genotyping .......................................................................... 37 2.6.2 Sequenom technology and genotyping .................................................................... 38 2.7 Microarray technology .................................................................................................... 40 2.7.1. Gene expression ....................................................................................................... 40 2.7.2 GWAS ...................................................................................................................... 42 2.8 Statistics ........................................................................................................................... 43 2.8.1 ANOVA ................................................................................................................... 43 2.8.2 Correlation ................................................................................................................ 43 2.8.3 Chi-square and Fishers exact test ............................................................................. 43 2.8.4 Gene expression analysis: SAM ............................................................................... 44 2.8.5 SNP analysis: haplotype estimation ......................................................................... 44 2.8.6 Regression analysis .................................................................................................. 45 2.8.7 FDR multiple testing correction ............................................................................... 47 2.9 Gene ontology databases ................................................................................................. 47 2.9.1 DAVID ..................................................................................................................... 47 3 Results in brief ....................................................................................................................... 48 Paper I ......................................................................................................................................... 48 Paper II ....................................................................................................................................... 49 Paper III ...................................................................................................................................... 51 4 Discussion ............................................................................................................................... 53 4.1 Discussion of main findings ............................................................................................ 53 4.1.1 Involvement of hormone and growth factor pathways in MD ................................. 53 4.1.2 Integrated analyses and complex diseases ............................................................... 57 4.2 Methodological considerations ........................................................................................ 58 4.2.1 Heterogeneity within and between studies. .............................................................. 58 4.2.2 Study selection bias .................................................................................................. 59 4.2.3 Assessment of menopausal status ............................................................................ 59 4.2.4 MD measurement ..................................................................................................... 60 4.2.5 Blood vs Tissue ........................................................................................................ 60 4.2.6 SNP quality control .................................................................................................. 61 4.2.7 SNP analysis ............................................................................................................. 61 4.2.8 Integrated analysis ................................................................................................... 64 5 Main conclusion and future perspectives ........................................................................... 65 5.1 Integrated analyses .......................................................................................................... 65 5.2 Translation into the clinic ................................................................................................ 66 5.3 Over diagnosis ................................................................................................................