Supplementary Methods
RNA extraction, Transcriptome Amplification and Labeling
RNA was extracted from freshly frozen biopsies as previously described(1).
Isolated total RNA and Stratagene Universal Reference RNA (Stratagene, La Jolla,
CA) was examined for integrity and purity using a Bioanalyzer 2100 (Agilent
Technologies, Inc., Santa Clara, CA) and a NanoDrop ND-1000 (Kiesker-Biotech,
Steinfurt, Germany).
Amplification of mRNA for microarray experiments was performed according
to the TAcKLE protocol(2) and consistency of cDNA size distribution was monitored
on a Bioanalyzer. Tumor and reference cDNA were both labeled separately with Cy3
and Cy5 for color switch replicate experiments. Comparable label efficiency for each
match was verified on a NanoDrop.
Microarrays, Quality Control and Data Preprocessing
Global gene expression profiling was conducted on Agilent Whole Human Genome
4x44 K Oligo Microarrays. Tumor and reference samples were combined for two
color hybridizations with blocking agent and 2x GE hybridization buffer from Agilent.
Hybridization conditions and washing procedures were performed following
manufacturers recommendations. Microarray read out was accomplished in a twocolor Agilent Scanner G25505B with automatically adjusting PMT voltages. Raw
intensity tables of recorded images were generated with Feature Extraction 9.1
software.
Microarray signal intensities were assessed for uniform signal and background
behavior and congruence of color switch replicate hybridizations were verified by
joined channel scatter plot illustrations using the in-house ChipYard analysis
framework (http://www.dkfz.de/genetics/ChipYard/). A Bland Altman visualization
method provided statistical quality control.(3, 4) Data were pre-processed by spot
filtering, normalization, and imputation, which resulted in 32,911 data points
representing gene specific oligomeres for statistical analysis (Supplementary
Methods).
For further quality assessment of the gene expression data, we applied the 50-gene
subtype predictor (5) to obtain the gene expression–based “intrinsic”
subtypes luminal A, luminal B, HER2-enriched, normal-like and basal-like. Intrinsic
subtyping prediction is based on a nearest shrunken centroid system using 50 genes,
called the Pam50 classifier. Achieving pCR rates in the subtypes for luminal A <10%,
luminal B around 10-20%, basal-like and erbB2+ around 30-50% (without
Trastuzumab) would conclude that the gene expression data is of sufficient quality (57). To incorporate measurement bias a median gene centering as calibration method
was performed before the Pam50 classifier was applied.
CD24 is represented by oligo A_23_P114457, which perfectly matches the CD24
sequence on chromosome 6q21.
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