Twenty-one gene panel and calculation of recurrence score. (a) The final gene list (16 cancer-related and five reference genes) and summary score (Recurrence Score) algorithm for this assay were developed by analyzing the results of three independent preliminary breast cancer studies (for instance, training sets) with a total of 447 patients . The Recurrence Score, on a scale from 0 to 100, is derived from the reference-normalized expression measurements in four steps. In the first step the expression for each gene is normalized relative to the expression of the five reference genes (b-actin, GAPDH, GUS, RPLPO, and TFRC). Reference-normalized expression measurements range from 0 to 15, where a 1-unit increase reflects approximately a twofold increase in RNA. (b) In the second step the HER2 Group Score, the ER Group Score, the Proliferation Group Score, and the Invasion Group Score are calculated from individual gene expression measurements. (c) In the third step the Recurrence Score unscaled (RSU) is calculated using coefficients that were pre-defined based on regression analysis of gene expression and recurrence in the three training studies (Providence, Rush, and NSABP B-20 ). A plus sign indicates increased expression is associated with increased recurrence risk. A minus sign indicates that increased expression is associated with decreased recurrence risk.