Comment[ArrayExpressAccession] E-GEOD-49481 MAGE-TAB Version 1.1 Public Release Date 2015-08-01 Investigation Title RNA Profiles Reveals Familial Aggregation of Molecular Subtypes in non-BRCA1/2 Breast Cancer Families Comment[Submitted Name] RNA Profiles Reveals Familial Aggregation of Molecular Subtypes in non-BRCA1/2 Breast Cancer Families Experiment Description In more than 70% of families with a strong history of breast and ovarian cancers, pathogenic mutation in BRCA1 or BRCA2 cannot be identified, even though hereditary factors are expected to be involved. It has been proposed that tumors with similar molecular phenotypes also share similar pathophysiological mechanisms. Grouping into molecularly homogeneous subsets may therefore be of potential value for further genetic analysis in order to identify new high penetrance breast cancer genes. In the current study, the aim was to investigate if global RNA profiling can be used to identify functional subgroups within breast tumors from families tested negative for BRCA1/2 germline mutations and how these subgroupings relate to different breast cancer patients within the same family. By analyzing a collection of 70 breast tumor biopsies from 58 families, we show that distinct functional subgroupings, similar to the intrinsic molecular breast cancer subtypes, exist. The distribution of subtypes was markedly different from the distribution found among BRCA1/2 mutation carriers. From 11 breast cancer families, breast tumor biopsies from more than one affected family member were included in the study. Notably, in 8 of these families we found that patients from the same family shared the same tumor subtype, showing a tendency of familial aggregation of tumor subtypes (p-value = 1.7e-3). Our finding indicates involvement of hereditary factors in these families in which family members may carry genetic susceptibility not just to breast cancer but to a particular subtype of breast cancer. Using our previously developed BRCA1/2-signatures, we identified 7 non-BRCA1/2 tumors with a BRCA1-like molecular phenotype and provide evidence for epigenetic inactivation of BRCA1 in three of the tumors. In addition, 7 BRCA2-like tumors were found. This is the first study to provide a biological link between breast cancers from family members of high risk non-BRCA1/2 families in a systematic manner, suggesting that future genetic analysis may benefit from subgrouping families into molecularly homogeneous subtypes in order to identify new high penetrance susceptibility genes. Gene expression profiling of 253 breast tumor samples. Breast tumor tissue from 125 patients with germline mutations in BRCA1 (n = 33) or BRCA2 (n = 22) or with no detectable germline mutation in BRCA1 or BRCA2 (n = 70) were included in the study. Serving as a representative control group, primary breast tumor samples (n = 128) were randomly selected among available samples originating from the same department and time period as for the hereditary samples. The study was conducted using Agilent-029949 Custom SurePrint G3 Human GE 8x60K Microarray platform. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Larsen Larsen Thomassen Tan Lænkholm Bak Sørensen Andersen Kruse Gerdes Person First Name Martin Martin Mads Qihua Anne-Vibeke Martin Kristina Mette Torben Anne-Marie Person Mid Initials Jakob J P K A Person Email martin.larsen@rsyd.dk Person Affiliation Odense University Hospital Person Address Dept. of Clinical Genetics, Odense University Hospital, Sdr. Boulevard 29, Odense C, Denmark Person Roles submitter Protocol Name P-GSE49481-1 P-GSE49481-3 P-GSE49481-4 P-GSE49481-2 P-GSE49481-5 Protocol Description Scanned images were quantified using Agilent Feature Extraction Software (version 10.7.3.1). Bad quality features flagged during feature extraction were removed and the remaining data were pre-processed. Data were background corrected (normexp, offset=50), then within-array normalized by loess normalization method and between-array normalizatized by the quantile method. The normalized values were used to calculate log2 transformed Cy5/Cy3 ratios. Replicate probes were collapsed by calculating the median. Probes without gene symbol annotation were filtered out. In cases of multiple probes per gene symbol only the probe with the maximum mean (Cy5) intensity was kept. Missing expression values were imputed by k-nearest neighbors averaging (k = 10). ID_REF = VALUE = Log2 transformed ratios(Cy5/Cy3) representing test/reference RNA was amplified and labeled using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion) according to the manufacturer’s protocol. Hybridization and washing were performed according to manufacture’s recommendations in a low ozone environment. Total RNA was extracted from freshly frozen tumor tissue using Trizol Reagent (Invitrogen) and RNeasy Micro Kit (Qiagen). Scanned using a Agilent G2565CA Microarray scanner Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name pam50agilent menopause_status mutation_hgvs lumb_brca2_pred_agilent lumb_brca2-like_pred pr_dbcg basal_brca1_pred_agilent general_brca2_pred_agilent grade age group who her2_dbcg general_brca1_pred_agilent er_dbcg er her2 tumor_size meth_brca1 basal_brca1-like_pred family_id pr Experimental Factor Type pam50agilent menopause_status mutation_hgvs lumb_brca2_pred_agilent lumb_brca2-like_pred pr_dbcg basal_brca1_pred_agilent general_brca2_pred_agilent grade age group who her2_dbcg general_brca1_pred_agilent er_dbcg er her2 tumor_size meth_brca1 basal_brca1-like_pred family_id pr Comment[SecondaryAccession] GSE49481 Comment[GEOReleaseDate] 2015-08-01 Comment[ArrayExpressSubmissionDate] 2013-08-01 Comment[GEOLastUpdateDate] 2015-08-01 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-49481.sdrf.txt