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Wisteria Enterprises Group

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Nolan Williams
Nolan Williams

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All data was publicly available and downloaded from the Gene Expression Omnibus (GEO), NCBI [19] ( ). Three independent experimental cohorts, GSE17705 [20] and GSE6532 [21] (which comprises 2 separate cohorts), were used for discovery and training and are briefly described in Table 1. Patients in all three cohorts were known to have ER+ tumours, were treated with surgical excision of the primary tumour and axillary dissection followed by 5 years of adjuvant tamoxifen. Limited pathological information is available for each sample, but ER and LN status is provided. The development of distant metastases was recorded over 10-years of clinical follow-up and reported as distant metastases free survival (DMFS). DMFS rates for LN- and LN+ patient subgroups were also reported. Patients with HER2 positive tumours were removed from all cohorts, as HER2 is known to be a poor prognostic variable for both LN+ and LN- tumours. Furthermore, in clinical practice patients with HER2+ ER+ tumours of 1 cm or more commonly receive adjuvant chemotherapy and Herceptin. A tumour was considered HER2 positive if either of the two HER2 probes on the Affymetrix chip were overexpressed as calculated using previously published methods [22].

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To extract the data from these cohorts, the raw intensity files (.CEL) comprising each dataset were downloaded and normalized using the Robust Multichip Algorithm (RMA) [23, 24] to generate a single intensity value for each probeset, using GenePattern (Broad Institute, Cambridge, Massachusetts). This preprocessing method has also been shown to yield concordance with qRT-PCR values and has been used in similar studies [24, 25]. Intensity was standardized using a Z score, where probe intensity was averaged among all samples and subtracted from the probe intensity from a single sample, which was then divided by the standard deviation of the probe intensities. Several other peer reviewed articles refer to a similar method to mimic qRT-PCR based assays using microarray gene expression data [25]. 041b061a72


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