Identifying predictors of treatment response and molecular changes induced by neoadjuvant chemotherapy and endocrine therapy in hormone receptor-positive/HER2-negative breast cancer: the NEOENDO translational study

Abstract

Background: Predictors of response to neoadjuvant chemotherapy (NACT) and endocrine therapy (NET) in hormone receptor-positive (HoR+)/human epidermal growth factor receptor 2 (HER2)-negative breast cancer (BC) are required. Also, pathological and molecular changes induced by both strategies and their impact on patients' outcomes have not been reported so far.

Patients and methods: In a cohort of 186 patients with early-stage HoR+/HER2-negative BC treated with NACT or NET, we assessed the association of baseline main clinicopathological features and PAM50 gene expression (GE), intrinsic subtypes (IS) and risk-of-relapse (ROR-P) score with pathological outcomes according to treatment strategy. Molecular NACT/NET-induced changes were described and compared, along with their associations with event-free survival (EFS). Comparison of the two cohorts after propensity score matching (PSM) was used as sensitivity analysis. Molecular changes were confirmed in cell lines.

Results: NACT was associated with higher rates of residual cancer burden (RCB)-0/I than NET in the overall population (38.2% versus 13.5%, P < 0.001) and after PSM (P = 0.036). PAM50 non-luminal IS were the only independent and positive predictor of RCB-0/I (P = 0.024) in the NACT cohort, while MMP11 messenger RNA levels were the only independent and negative predictor (P = 0.014) in the NET cohort. Both treatments shifted the tumor types toward less aggressive forms (i.e. PAM50 luminal A/normal-like), lowered the risk of recurrence in terms of ROR-P, up-regulated selected immune genes and PAM50 basal-like-related genes/signature and significantly downregulated proliferation-/luminal-/HER2-related genes/signatures, though NACT more than NET. Molecular findings were confirmed after PSM. A net reduction in proliferation-related genes and ROR-P was confirmed in cell lines with chemotherapy and endocrine therapy. Different baseline molecular features associated with diverse kind of responses (ROR-P downstaging, Ki67 reduction or pathological responses) with NACT and NET. Decreasing ROR-P and transitioning the tumor subtype to resemble normal tissue (i.e. PAM50 normal-like) suggested improved EFS.

Conclusions: NACT was more effective in the molecular and dimensional tumor 'downstaging' than NET but baseline molecular features associated with differential responses according to treatment strategy. Examining baseline and post-treatment GE might help tailor more personalized and effective care.

Keywords: PAM50; breast cancer; intrinsic subtypes; molecular downstaging; neoadjuvant chemotherapy; neoadjuvant endocrine therapy.

Figure 1

Baseline GEDs, main surgical and long-term outcomes and treatment-induced PAM50 IS and ROR-P changes according to NET and NACT. (A) Venn diagram reporting differentially expressed and similarly expressed (in the middle) genes, PAM50 IS centroid correlations and signatures between the two treatment cohorts at baseline and companion volcano plot representing baseline GEDs between the two treatment cohorts. (B) Conservative and non-conservative breast surgery rates and pathological response type according to neoadjuvant strategy. (C) Kaplan–Meier curves of EFS and OS according to neoadjuvant therapy. (D) PAM50 IS changes induced by neoadjuvant therapy in the HCB cohort; (E) PAM50 ROR-P changes induced by neoadjuvant therapy in the HCB cohort. In the volcano plot, gray dots represent genes not differentially expressed, while red dots identify significantly differentially expressed genes for an FDR <5%. In Sankey plots, P values are referred to Bhapkar’s tests. Significant if P < 0.05. BCS, breast conservative surgery; EFS, event-free survival; FDR, false discovery rate; GEDs, gene expression differences; HCB, Hospital Clinic of Barcelona; HER2-E, HER2-enriched; IS, intrinsic subtypes; NACT, neoadjuvant chemotherapy; NET, neoadjuvant endocrine therapy; OS, overall survival; pCR, pathological complete response; RCB, residual cancer burden; ROR-P, risk-of-relapse score based on subtypes and proliferation.

Figure 2

Differentially expressed genes, PAM50 signatures/scores and ROR-P after surgery and treatment-induced changes. (A) PAM50 ROR-P score reduction after NET and NACT. Green lines highlight a numerical reduction in mRNA levels after treatment, while burgundy lines represent a numerical increase and blue lines represent stability. Reported P values are referred to paired Student’s t-tests, while the log2 fold changes and FDR are referred to paired pre-/post-SAM analyses. (B) Venn diagrams reporting genes, signatures and PAM50 IS centroid correlations significantly up- and downregulated by NET and NACT and volcano plot of post-surgical gene expression differences between NET versus NACT cohort. Gray dots represent genes not differentially expressed, while red dots identify significantly differentially expressed genes for an FDR <5%. corr, correlation coefficient; FDR, false discovery rate; NACT, neoadjuvant chemotherapy; NET, neoadjuvant endocrine therapy; sig, signature.

Figure 3

Differential gene expression at baseline in different responder subgroups according to neoadjuvant treatment strategy. Supervised clustering of 55 genes, 4 PAM50 signatures, ROR score and the 5 PAM50 intrinsic subtype correlation coefficients across four tumor classes defined according to different response subgroups, according to neoadjuvant treatment. All samples and gene expression data in each category have been combined into a single group. For each gene in a group, we calculated the standardized mean difference between the gene’s expression in that class versus its overall mean expression in the dataset using a four-class Significance Analyses of Microarrays. The red color represents relatively high gene score, blue represents relatively low gene score and white represents median gene score. Basal, basal-like PAM50 intrinsic subtype correlation score; BasalPre, basal-like-related baseline genomic signature; Her2, HER2-enriched PAM50 intrinsic subtype correlation score; HER2Pre, HER2 amplicon-related baseline genomic signature; Int, intermediate; LumA, luminal A PAM50 intrinsic subtype correlation score; LumB, luminal B PAM50 intrinsic subtype correlation score; LuminalPre, luminal-related baseline genomic signature; NACT, neoadjuvant chemotherapy; NET, neoadjuvant endocrine therapy; Normal, normal-like PAM50 intrinsic subtype correlation score; ProliferationPre, proliferation-related baseline genomic signatures; RCB, residual cancer burden; RCB-0, pathological complete response

Figure 4

GEDs in cell lines and baseline and post-surgical PAM50 IS and ROR-P according to survival outcomes. (A) Supervised clustering of 66 genes, 4 PAM50 signatures, ROR-P score and the 5 PAM50 intrinsic subtype correlation coefficients across three treatment groups including two different HoR+/HER2-negative BC cell lines (i.e. T47D and MCF7). All samples and gene expression data in each category have been combined into a single group. For each gene in a group, we calculated the standardized mean difference between the gene’s expression in that class versus its overall mean expression in the dataset using a three-class Significance Analyses of Microarrays. The red color represents relatively high gene score, blue represents relatively low gene score and yellow represents median gene score. (B) Baseline and post-surgical PAM50 intrinsic subtype and ROR categories distribution based on event-free survival events. Basal, basal-like PAM50 intrinsic subtype correlation score; CT, chemotherapy (paclitaxel); ET, endocrine therapy (fulvestrant); GEDs, gene expression differences; Her2, HER2-enriched PAM50 intrinsic subtype correlation score; HER2E, HER2-enriched; HoR+, hormone receptor positive; LumA, luminal A PAM50 intrinsic subtype correlation score; LumB, luminal B PAM50 intrinsic subtype correlation score; Normal, normal-like PAM50 intrinsic subtype correlation score; pCR, pathological complete response; RCB, residual cancer burden; ROR, PAM50 risk of relapse with proliferation and subtype score; sig, gene expression signature.