Patients with cell cycle pathway activation or outliers patterns consistent with amplification of 17q12, 17q21.33-q25.1, 8p11.2 and 8q24.3 show poor outcome under tamoxifen treatment.

<p>A) Kaplan-Meier curves of the samples in the primary dataset (GSE6532) enriched for over-expressed cell cycle genes versus the rest of samples that don’t show this feature. Patients with cell cycle activated genes show a significant decrease in distant metastasis free survival rate (HR  = 9.71, 95% CI  = 3.3–28.6; P<0.0001). B) Kaplan-Meier curves of the ER+ samples in the primary dataset (GSE6532) stratified by presence of putative amplicons in 17q12, 17q21.33-q25.1, 8p11.2 and 8q24.3. Patients that show any one of the chromosomal amplifications have significantly higher relapse rates when compared to samples without any amplifications: 17q12 (HR  = 4.09, 95% CI  = 3.84–21.99; P  = 6.3e−07), 17q21.33– q25.1 (HR  = 3.14, 95% CI  = 2.17–13.62; P  = 3.0e−04), 8p11.2 (HR  = 3.75, 95% CI  = 3.18–18.31; P  = 5.7e−06), and 8q24.3 (HR  = 4.29, 95% CI  = 4.32–34.08; P  = 2.2e−06). C) Analysis of combined gene expression data of 624 ER+ breast cancers from multiple published data sets. Outlier analysis was performed to identify cases with evidence of amplification at 17q12, 17q22, 8p11.2, and 8q24.3 and those without evidence of any amplification. Kaplan-Meier curves of relapse free survival for ER+ samples with each of the four amplicons, and samples containing no amplicon are plotted: 17q12 (HR  = 2.30, 95% CI  = 1.45–3.64; P  = 4.0e−04), 17q22 (HR  = 3.07, 95% CI  = 1.99–4.73; P<1.0e−04), 8p11.2 (HR  = 1.96, 95% CI  = 1.23–3.13; P  = 4.9e−3), 8q24.3 (HR  = 2.38, 95% CI  = 1.60–3.55; P<1.0e−04) D) Kaplan-Meier curves of overall survival for the ER+ samples in the test CGH dataset (GSE22133) with each of the 4 amplicons, as well as samples that don’t have any of the chromosomal amplifications. Analysis of the CGH data identified amplification peaks at each of the four regions that overlap with the previously identified loci. Patients that show any one of the chromosomal amplifications have significantly higher event rates than those without any of the amplifications: 17q12 (HR  = 2.61, 95% CI  = 1.51–5.51; P  = 6.8e−04), 17q22 (HR  = 3.02, 95% CI  = 1.76–5.18; P  = 7.3e−05), 8p11.2 (HR  = 2.65, 95% CI  = 1.48–4.74; P  = 1.3e−03), and 8q24.3 (HR  = 2.12, 95% CI  = 1.24–3.65; P  = 6.7e−03). Log-rank tests were used to calculate all the P values.</p

Over-expressed genes in chromosomal regions 17q12, 17q21.33-q25.1, 8p11.2 and 8q24.3 associated with early relapse in ER+ breast cancers treated with tamoxifen.

<p>List of genes associated with early relapse on chromosomes 8 and 17. Highlighted in bold are cancer related genes of interest.</p

Multivariate Cox analysis of Age, Tumor size, Tumor grade, Lymph node status, Progesteron status, Oncotype Dx recurrence score, Her2 amplicon (17q12) and Any amplicon (17q12, 17q21.33-q25.1, 8p11.2 or 8q24.3).

<p>219 samples from the primary data set (GSE6532) had clinical information for all analyzed covariates. Cox proportional-hazard regression was performed on the reduced data set (with and without ‘Any amplicon’ covariate) resulting in a significant overall model fit (P  = 0.0005 and P  = 0.0001).</p

Analysis of amplicon status using multiplexed FISH in a cohort of tamoxifen treated ER+/HER2- breast cancers.

<p>ER+/HER2- samples with systemic relapse events were probed for the amplification of 8p11.2, 17q22 and 8q24.3 by multiplexed FISH assay as described in Methods. A) Out of 36 samples, 15 had at least one region amplified. Kaplan-Meier curves for relapse free survival is shown for cancers having at least one amplicon vs cancer having no amplicons (HR  = 2.31, 95% CI  = 0.66–8.06; P  = 0.1041 (Gehan-Willcoxon) or P  = 0.1886 (Mantel-Cox)). B) A typical image of multicolor FISH in a breast cancer specimen. This cell has evidence of amplification of both 17q23.1 and 8p11 loci, but normal 8q24 loci.</p

Immunohistochemical staining patterns of WRAP53 in breast cancer.

<p><b>A.</b> and <b>B.</b> Positive nucleus/positive cytoplasma, <b>C.</b> and <b>D.</b> positive nucleus/negative cytoplasma (with normal mammary epithelial cells), <b>E.</b> positive nucleus/negative cytoplasm (invasive lobular carcinoma), and <b>F.</b> negative nucleus/negative cytoplasm.</p

Analysis of intermediate grade tumors by presence of amplicons.

<p>Kaplan-Meier curves comparing distant relapse rates for intermediate grade cancers with any of the 4 amplicons versus cancers with none of the amplicons (A) in the training set GSE6532 (HR  = 3.22, 95% CI  = 1.6–6.5; P  = 0.0012). Also shown Kaplan-Meier curves comparing overall survival for intermediate grade cancers with any of the 4 amplicons versus cancers with none of the amplicons (B) in the test set GSE22133 (HR  = 3.01, 95% CI  = 1.2–7.6; P  = 0.0200).</p

FISH scores for ER+/HER2- breast cancer tissue samples.

<p>Fluorescence in situ hybridization (FISH) results for 14 paraffin embedded ER+/HER2- breast cancer samples. Scores were calculated as the average number of spots over 20 cancer cells for each chromosomal location and separated into amplified, not amplified and borderline classes as follows: (>4 amplified; 2–4 borderline; <2 not amplified). The last column lists the associated Oncotype Dx score for each sample, 8 have high scores (>31) while 6 have low scores (<18).</p

Multivariate Cox regression analysis (BCSS).

<p>¹ Tumor grade is included as ordinal variablePrognostic impact of <i>WRAP53</i> in patients stratified for ER- and <i>TP53-</i>status</p><p>Multivariate Cox regression analysis (BCSS).</p

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer - Fig 3

<p>Kaplan-Meier curves showing Breast Cancer Specific Survival (BCSS) of WRAP53 IHC staining of nucleus and cytoplasm in combination, <b>A</b>. in the test set and <b>B.</b> in the validation set. Numbers at risk are listed below each chart.</p