Amplified loci on chromosomes 8 and 17 predict early relapse in ER-positive breast cancers

Abstract

Adjuvant hormonal therapy is administered to all early stage ER+ breast cancers, and has led to significantly improved survival. Unfortunately, a subset of ER+ breast cancers suffer early relapse despite hormonal therapy. To identify molecular markers associated with early relapse in ER+ breast cancer, an outlier analysis method was applied to a published gene expression dataset of 268 ER+ early-stage breast cancers treated with tamoxifen alone. Increased expression of sets of genes that clustered in chromosomal locations consistent with the presence of amplicons at 8q24.3, 8p11.2, 17q12 (HER2 locus) and 17q21.33-q25.1 were each found to be independent markers for early disease recurrence. Distant metastasis free survival (DMFS) after 10 years for cases with any amplicon (DMFS = 56.1%, 95% CI = 48.3-63.9%) was significantly lower (P = 0.0016) than cases without any of the amplicons (DMFS = 87%, 95% CI = 76.3% -97.7%). The association between presence of chromosomal amplifications in these regions and poor outcome in ER+ breast cancers was independent of histologic grade and was confirmed in independent clinical datasets. A separate validation using a FISH-based assay to detect the amplicons at 8q24.3, 8p11.2, and 17q21.33-q25.1 in a set of 36 early stage ER+/HER2- breast cancers treated with tamoxifen suggests that the presence of these amplicons are indeed predictive of early recurrence. We conclude that these amplicons may serve as prognostic markers of early relapse in ER+ breast cancer, and may identify novel therapeutic targets for poor prognosis ER+ breast cancers.

Figure 1. PCA plots of high and low outliers.

Principal component analysis of high outlier genes (A) and low outlier genes (B) associated with differential distant metastasis free survival are shown. The figure represents the projection of each gene’s outlier profile on the first two principal components of the corresponding matrix. Gene clusters associated with good prognosis are circled in blue while gene clusters associated with bad prognosis are circled in red. Over-expressed genes associated with poor prognosis, which map to the chromosomal regions 8q24.3, 8p11.2, 17q21.33-q25.1 and 17q12, and are associated with specific GO pathways are labeled with different colors.

Figure 2. 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.

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.

Figure 3. Analysis of intermediate grade tumors by presence of amplicons.

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).

Figure 4. Analysis of amplicon status using multiplexed FISH in a cohort of tamoxifen treated ER+/HER2- breast cancers.

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.