Heat-map of significantly differentially expressed miRNAs associated with outcome after adjuvant Tamoxifen treatment.

<p>Data is based on the discovery set (p<6.6e-4, FDR 2.5% and variance >0.1). The green symbols above the heat-map indicate samples from patients with no recurrence, whereas the red symbols indicate samples from patients with recurrence. The heat-map is a standardized intensity plot with the intensities ranging from −2 (green) to +2 (red).</p

Characteristics of included patients and their breast cancer tumor (N = 152).

a<p>the average and median were calculated only for the tumors defined as positive, i.e. staining was observed in ≥ 10% of tumor cells by immunohistochemistry.</p>b<p>If the actual percentage was not provided, patients were deemed positive if ER staining was observed in ≥ 10% of tumor cells by immunohistochemistry and/or target protein (ER or PgR) was >10 fmol/mg total protein as determined by biochemistry.</p>c<p>the 5 ER- tumors had a PgR status of 90%, 50%, 90%, 80% and IHC+ (i.e. >10%) respectively.</p><p>Abbreviations: R: patients with recurrence. N: patients without recurrence. Disc.: Discovery set. Test#1: Test set#1. Test#2: Test set#2. Avg: average IDC: invasive ductal carcinoma. ILC: invasive lobular carcinoma. TTR: time to recurrence.</p

Association of miR-7 with tumor grade.

<p>Grade 1 vs. 3 and Grade 2 vs. 3: p = 0.01, and Grade 1 vs. 2: p = 0.02). N = 52 patients (Discovery set).</p

Kaplan-Meier plots of the 10 miRNAs identified in the discovery set.

<p>The bold line represents the patients with a good prognosis, whereas the dotted line represents the poor-prognosis patients. A) Probability of recurrence. B) Probability of overall survival.</p

Joint distribution of the ΔΔCt values of <i>BCL2</i> and <i>CDKN1A</i>.

<p>The diagonal line corresponds to the rule determined by conditional logistic regression. Pairs to the right of the line are correctly classified with respect to their outcome (recurrence/non-recurrence) (accuracy of 75%), whereas pairs left of the line are classified incorrectly.</p

Characteristics of patients and their tumor included in the study.

a<p>Cut-off: ≥1% staining of tumor cells was denoted positive.</p>b<p>HER2 amplification was investigated by both IHC and FISH.</p

Survival analysis of the 2-gene signature.

<p>Kaplan-Meier curves of recurrence-free survival according to model-based prediction of outcome using the 2-gene signature (<i>BCL2-CDKN1A</i>) for the independent gene expression dataset GSE2990. Grey line (top) indicates the good outcome signature, whereas the black line (bottom) indicates the poor outcome signature. Only data from post-menopausal (>50 years) and ER+ breast cancer patients were included in the analysis. Data was adjusted for clinical variables. A) Tamoxifen-treated patient samples (N = 58). B) Untreated patient samples (N = 62).</p

The most significant genes exhibiting altered expression in the recurrent vs. non-recurrent patient samples identified using single gene analysis, p<0.1.

<p>The genes are ranked by the p-value of the Wilcoxon signed rank test (p_wil).</p

Genes identified and their expression pattern.

<p>A) The 2-, 8- and 9-gene signatures identified by various statistical analyses. B) ΔΔCt of the genes present in the 2-, 8- and 9-gene signatures. <i>BCL2</i> overlap in all three, whereas <i>CDKN1A</i> is in the 2- and 9-gene signatures, and <i>PRKCE</i> and <i>EGFR</i> are in both the 8- and 9-gene signatures. A positive ΔΔCt_median value denote that the expression of the gene is highest in the tumor sample from patients without recurrence, whereas a negative value means the expression is higher in the tumor samples from patients with recurrence.</p

Performance of the identified genes.

<p>The capabilities of the identified 2-, 8- and 9-gene signatures to predict recurrence was evaluated in 6 independent gene expression datasets. A) Summarized results of accuracy (%), along with sensitivity/specificity in parenthesis (both given as %), of the identified signatures to predict recurrence. B–G) Dot-plots of the identified 2-gene signature (<i>BCL2-CDKN1A)</i> illustrating the probability of recurrence. The vertical line separates the cases, i.e. patients with recurrence (left of the line) from controls (right of the line). The horizontal line refers to the cut-point used, hence the upper left and lower right corners includes the correctly classified patients. X-axis denotes the patient index in the study (same random order as original study). The Y-axis is the SVM probability of recurrence. B) GSE1378 C) GSE1379 D) GSE9893 E) GSE12093 F) GSE6532-GPL96 and G) GSE6532-GPL570.</p