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Amplified Loci on Chromosomes 8 and 17 Predict Early Relapse in ER-Positive Breast Cancers

2012-06-02 18:38:45

PLoS One; 2012 June; 7(6):e38575


Erhan Bilal, Kristen Vassallo, Deborah Toppmeyer, Nicola Barnard, Inga H. Rye, Vanessa Almendro, Hege Russnes, Anne-Lise Børresen-Dale, Arnold J. Levine, Gyan Bhanot, Shridar Ganesan



Introduction



Hormone therapy is widely used for treatment of estrogen receptor positive (ER+) breast cancer and has been shown to result in significantly improved survival and lower rates of recurrence. However, a significant subset of ER+ breast cancer patients treated with adjuvant hormone therapy suffer early disease recurrence. These poor prognosis ER+ tumors tend to have higher grade and show higher proliferative indices and may not be “addicted” to ER –dependent signalling, making them resistant to hormone therapy and prone to early relapse. A better understanding of the mechanisms underlying the early relapse of some ER+ breast cancers may lead to better prognostic assays, and to new targeted therapeutic strategies for these poor prognosis cancers.



Several assays have been developed to distinguish ER+ patients likely to do well with hormonal therapy from those likely to have early disease progression. The best validated of these is the Oncotype Dx® assay from Genomic Health, Inc., based on RT-PCR measurement of mRNA levels of 21 genes. ER+ breast cancer patients whose tumors have low ODx Recurrence Scores (RS) do well with adjuvant hormonal therapy alone, while tumors with high ODx RS are more likely to benefit from the addition of chemotherapy to hormonal therapy. Other panels of genes, such as the Genomic Grade Index panel, and clinical markers such as histological grade, are also used to classify patients into good or poor prognosis classes. In addition, molecular signatures from clustering methods applied to gene-expression data are also able to separate ER+ breast cancers into good prognosis (Luminal A) and poor prognosis (Luminal B) classes. However, several studies have shown that, when the prognostic assays are compared to the gene expression based sub-classification of breast cancers, these assays are essentially identifying Luminal A tumors (low grade, highly ER+ breast cancers, HER2-) as being good prognosis, and Luminal B, ER+ breast cancers (which are ER+, mostly intermediate-to-high grade, some with HER2 amplification) as poor prognosis.



Although gene expression based assays such as Oncotype Dx have prognostic and predictive utility, they do not identify the biologic pathways driving resistance in the poor prognosis tumors. Moreover the optimal strategy for “Intermediate Risk” ODx RS, found in up to 30% of ER+ cancers, is not clear at present. In contrast, the presence of the HER2 amplicon, in ER+ breast cancers, has both clear prognostic value and identifies a clear and effective therapeutic target. ER+ breast cancers with HER2-amplification tend to have early recurrence if treated with hormonal therapy alone, likely because the activation of the HER2 pathway leads to independence from ER- mediated signalling. Moreover, therapy that specifically targets HER2 has been shown to dramatically improve outcome in HER2+ patients. Thus all breast cancers are now routinely tested for the presence of HER2 amplification.



As HER2 amplicon genes are part of the 21 gene panel used in determining the Oncotype Dx recurrence score (RS), breast cancers with HER2 amplification generally have high RS, high histological grade, and a high genomic grade and are easily and correctly identified as poor prognosis by the assay. However, the majority of poor prognosis ER+ cancers with high ODx RS do not have HER2 amplification. Indeed only patients with ER+ tumors and no evidence of HER2 amplification have Oncotype DX assays performed in most clinical settings. At present there is little insight into the mechanism driving estrogen independence and growth in poor prognosis ER+/HER2- breast cancers.



In order to gain insight into the biology of these poor prognosis ER+/HER2- breast cancers, we analyzed a public gene expression data set of early stage ER+ breast cancers treated with tamoxifen using a novel method. Sets of outlier genes whose expression correlated with clinical outcome were analyzed to identify either molecular pathways or enrichment of chromosomal regions. Four separate regions of the genome were identified whose amplification was highly predictive of poor prognosis in early stage ER+ breast cancers treated with tamoxifen. As expected, one of these was the HER2 amplicon on 17q12; validating our methods as being able to identify relevant amplicons. The other three amplification regions were in 8q24.3, 8p11.2 and 17q21.33-q25.1. Although these loci have previously been identified as regions of amplification in subsets of breast cancer, their association with tamoxifen resistance in ER+/HER2- breast cancers is novel. The presence of these amplicons in ER+/HER2- breast cancer and their association with poor prognosis was validated in several independent data sets. Taken together, these findings demonstrate that these amplicons are strong predictors of early relapse in ER+ breast cancers.



Results



Outlier Genes and Patterns Associated with Tamoxifen Treatment Response



A gene expression dataset (published by Loi et al) containing 268 patients with early stage ER+ breast cancers treated with local therapy and adjuvant tamoxifen with 9+ years of available clinical follow-up data, was analyzed. Clinical characteristics of this set have been previously described (Table S1).



Genes whose expression values were outliers in at least 10 samples in this dataset were identified and analyzed for their correlation with distant metastasis free survival. Outlier genes for which there was a significant difference in distant metastasis free survival between samples having outlier expression when compared to samples with normal expression, were identified and retained (see Methods for details). Table S2 has the set of outlier genes, hazard ratios, log-rank P values and outlier scores.



Principle component analysis (PCA) demonstrated that the outlier genes separated into 3 clusters (Figure 1A). Survival analysis of these clusters showed that one cluster contained genes whose over-expression associated with poor prognosis, and the other two contained genes over-expressed in good prognosis samples. The set of outlier genes in each cluster was analysed using Gene Ontology (GO) to identify pathways and potential chromosomal amplifications associated with outcome (Table S3). Pathways enriched in over-expressed outliers associated with good prognosis included development, cell adhesion, and immune response genes. Of note, no clusters of outliers associated with good prognosis suggestive of an underlying amplicon were detected. Outlier genes whose over-expression was associated with poor prognosis had a significant enrichment of genes in cell cycle pathways. Analysis of outliers for clustering by chromosomal location identified putative amplification of four chromosomal regions associated with poor prognosis: 17q12, 17q21.33-q25.1, 8p11.2 and 8q24.3. The presence of genomic amplification in any of these regions leads to outlier expression of their genes, and is a marker of poor prognosis in ER+ breast cancer.



Cell cycle pathway outliers contained genes associated with proliferation and included many of the genes used to define the Genomic Grade Index. This confirms prior observations that proliferation-associated genes are strong markers of poor prognosis in ER+ breast cancer. The known amplicon on 17q12 associated with poor prognosis was also identified. This amplicon contains the HER2 gene and is known to be associated with relative resistance to hormonal therapy and poor prognosis in ER+ breast cancer. The other putative amplicons in 17q21.33-q25.1, 8p11.2, and 8q24.3 have been previously reported as amplified in subsets of breast cancers but their association with tamoxifen resistance in ER+ breast cancer is a novel finding. The full list of outlier genes identified in these amplified chromosomal regions is listed in Table 1, with potential oncogenes highlighted in red. Some of these genes have been previously identified as playing a role in tumorigenesis or cancer progression including, WHSC1L1, CLTC, HSF1, and LSM1. Of note the FGFR1 which has been implicated in hormonal resistance in ER+ breast cancer, is present at the edges of the 8p11.2 amplicon, but is not present in our minimal amplicon defined by our analysis (see Table S2). Similarly MYC, another oncogene reported to induce hormone resistance in breast cancer, is upstream of the 8p24.3 amplicon defined by our analysis, and is not associated with poor outcome in this dataset (see Table S2).



For under-expressed outliers, a similar analysis showed that relative under-expression of the cell cycle pathway was associated with good prognosis, while under-expression of the immune response and cell adhesion pathway was associated with poor prognosis (Figure 1B). This mirrors the results for over-expressed outlier genes and confirms the strong association of the cell cycle, immune response and cell adhesion pathways with prognosis in ER+ breast cancers.



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