cases. Details for the TMAs set may be found at http:// cdp.nci.nih.gov/breast/prognostic_cs.html. A summary of patients’ clinical and pathological characteristics for the TMAs is provided in Analysis of SRM Dataset SRM raw data were pre-processed using MultiQuant 1.0 software to identify and derive peak areas for SRM transitions. A 2-point Gaussian smoothing procedure was applied to all peaks. Mean peak 
areas for each transition was compared between LN negative and LN positive groups using a test assuming unequal variance at a two-tailed significance threshold of 0.10. Analyses were conducted using R software. In-House Human Breast Cancer Tissue Microarrays and Immunohistochemistry Tissue microarrays were constructed at UHN from samples obtained from primary breast cancer patients admitted to Princess Margaret Hospital between January and December of 2006. For all TMAs, 4 mm formalin-fixed, paraffin embedded sections were dewaxed in five changes of xylene and brought down to water through graded alcohols. Tissue sections were microwaved in Tris-EDTA Buffer for antigen retrieval. After blocking for 15 minutes, sections were incubated at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22182733 room temperature overnight with the appropriate primary antibodies using previously optimized dilutions. Primary antibody incubation was followed by incubation with a biotinylated secondary antibody for 30 minutes and 3 Breast Cancer Odanacatib Decorin, HSP90B1 Metastases Survival Name Endoplasmin precursor 40S ribosomal protein S25 Hemoglobin subunit alpha Actin, alpha cardiac muscle 1 GTP-binding nuclear protein RAN 14-3-3 protein zeta/delta Accession P14625 P62851 P69905 P68032 P62826 P63104 Peptide used for SID SILFVPTSAPR LITPAVVSER LRVDPVNFK SYELPDGQVITIGNER AAQGEPQVQFK NLLSVAYK Confirmed Yes Yes Yes Yes No Yes List of peptides for which SID SRM-MS was used to confirm their identification. UniProtKB accession number and amino acid sequence are shown. SID: Stable isotope dilution. SRM-MS: selected reaction monitoring mass spectrometry. doi:10.1371/journal.pone.0030992.t001 using Aperio image analysis. TMAs were analyzed blindly and independently by two pathologists using a four point semi-quantitative scale for intensity: 3+, 2+, 1+, and 0 . In case of disagreement cores were reviewed together and consensus was reached. TMAs were scanned at 206 and the ImageScope Positive Pixel Count algorithm version 9.1 used for software-based analysis on the entire core. An average intensity of positive pixels is calculated by the software generating a continuous variable of intensity scores in which higher scores mean lower staining intensity. Given the heterogeneity and relatively low DCN epithelial staining, any strong even if focal, cancer cell staining was considered high expression, while due to the more diffuse moderate to strong HSP90B1 positivity in the majority of cores, we required both diffuse and strong HSP90B1 positivity to categorize it as high expression. For analytical purposes, 3+ and 2+ staining were considered high expression for both DCN and HSP90B1; and 1+ and 0 staining were considered low expression. Evaluation of ER and HER2 staining was performed using current recommendations,. HER2 cases that were considered equivocal were omitted from the analysis since fluorescence in situ hybridization staining for HER2 was not available. Breast cancer molecular subtypes were defined by IHC expression of ER, and Ki-67 as suggested by Cheang et al and Hugh et al: Luminal A, luminal B, HER2 and basal-like . a va
