The molecular landscape of premenopausal breast cancer

Abstract

Introduction: Breast cancer in premenopausal women (preM) is frequently associated with worse prognosis compared to that in postmenopausal women (postM), and there is evidence that preM estrogen receptor-positive (ER+) tumors may respond poorly to endocrine therapy. There is, however, a paucity of studies characterizing molecular alterations in premenopausal tumors, a potential avenue for personalizing therapy for this group of women.

Methods: Using TCGA and METABRIC databases, we analyzed gene expression, copy number, methylation, somatic mutation, and reverse-phase protein array data in breast cancers from >2,500 preM and postM women.

Results: PreM tumors showed unique gene expression compared to postM tumors, however, this difference was limited to ER+ tumors. ER+ preM tumors showed unique DNA methylation, copy number and somatic mutations. Integrative pathway analysis revealed that preM tumors had elevated integrin/laminin and EGFR signaling, with enrichment for upstream TGFβ-regulation. Finally, preM tumors showed three different gene expression clusters with significantly different outcomes.

Conclusion: Together these data suggest that ER+ preM tumors have distinct molecular characteristics compared to ER+ postM tumors, particularly with respect to integrin/laminin and EGFR signaling, which may represent therapeutic targets in this subgroup of breast cancers.

Fig. 1

Differences in outcome and gene expression comparing premenopausal (preM) and postmenopausal (postM) tumors. a Principal component analysis (PCA) using RNA seq data from The Cancer Genome Atlas (TCGA). Tumor: purple triangle, estrogen receptor-negative (ER–)/preM; red diamond, ER–/postM; orange star, estrogen receptor-positive (ER+)/preM; pink square, ER+/postM. Normal: light blue triangle, ER–/preM; black diamond, ER–/postM; green star, ER+/preM; dark blue square, ER+/postM. b Heatmap showing genes differentially expressed (n = 1,609) between ER+/preM and. ER+/postM, using RNA-Seq data. Each gene is normalized to standard normal distribution with green indicating lower expression, and red indicating higher expression. PC principal component

Fig. 2

Differences in methylation and copy number variation (CNV) between premenopausal (preM) and postmenopausal (postM) estrogen receptor-positive (ER+) tumors: a Heatmap representing significant differences in methylation of 1,738 probes between preM (n = 75) and postM (n = 233) tumors. Red and green indicate higher and lower levels of methylation, respectively. b Heatmap representing significant changes in CNV at gene level between preM and postM ER+ tumors (772 genes). White indicates diploid normal copy, gray indicates single copy deletion (LOH), blue indicates homozygous deletion, yellow indicates low-level copy number amplification, and red indicates high-level copy number amplification. c Peaks of significant amplification (top panels) and deletion (bottom panels) in preM and postM ER+ tumors. Peaks were identified by GISTIC 2.0. The x-axis represents the G score (top) and Q value (bottom). The vertical green line represents the significance cutoff q value = 0.25. Stars indicate significantly different regions of amplifications or deletions comparing preM and postM ER+ tumors. d Mutation distribution in the top five differentially mutated genes (CDH1, GATA3, MLL3/KMT2C, GPS2, PIK3CA). Red, black, and gray indicate truncating, in-frame and other mutations, respectively. Figures were generated with cBio MutationMapper [49, 50]

Fig. 3

Pathways activated in premenopausal (preM) breast tumors. Top 50 entities in superPathway analysis using Agilent array, copy number variant (CNV) and mutation analysis of The Cancer Genome Atlas (TCGA) data (preM, n = 65 and postM, n = 239). Green indicates low activity score and red indicates high activity score. Red stars refer to pathways related to integrin/laminin signaling, and blue stars label EGFR and AREG signaling

Fig. 4

Sub-clusters within premenopausal (preM) estrogen receptor-positive (ER+) tumors. a Clustering result of The Cancer Genome Atlas (TCGA) gene expression data by sparse k-means yielded three clusters (n₁ = 36, n₂ = 29, n₃ = 4). b Sparse k-means clustering in Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) preM ER+ tumors using genes selected by TCGA (n₁ = 56, n₂ = 19, n₃ = 43). c Kaplan-Meier survival curve using clustering result from sparse k-means analysis from METABRIC data. d Semi-supervised clustering of METABRIC gene expression data from preM ER+ tumor. e Kaplan-Meier survival curve of three groups from semi-supervised clustering of gene expression from d. f Kaplan-Meier survival curves of groups resulting from four different clustering algorithms, with the p value from the log-rank test. Lum luminal, Her2 human epidermal growth factor receptor 2