Molecular profiling of hormone receptor-positive, HER2-negative breast cancers from patients treated with neoadjuvant endocrine therapy in the CARMINA 02 trial (UCBG-0609)

Abstract

Background: Postmenopausal women with large, hormone receptor (HR)-positive/HER2-negative and low-proliferative breast cancer derived a benefit from neoadjuvant endocrine therapy (NET) in the CARMINA02 trial. This study was designed to correlate gene expression and mutation profiles with both response to NET and prognosis.

Methods: Gene expression profiling using RNA sequencing was performed in 86 pre-NET and post-NET tumor samples. Targeted next-generation sequencing of 91 candidate breast cancer-associated genes was performed on DNA samples from 89 patients. Molecular data were correlated with radiological response and relapse-free survival.

Results: The transcriptional profile of tumors to NET in responders involved immune-associated genes enriched in activated Th1 pathway, which remained unchanged in non-responders. Immune response was confirmed by analysis of tumor-infiltrating lymphocytes (TILs). The percentage of TILs was significantly increased post-NET compared to pre-NET samples in responders (p = 0.0071), but not in non-responders (p = 0.0938). Gene expression revealed that lipid metabolism was the main molecular function related to prognosis, while PPARγ is the most important upstream regulator gene. The most frequently mutated genes were PIK3CA (48.3%), CDH1 (20.2%), PTEN (15.7%), TP53 (10.1%), LAMA2 (10.1%), BRCA2 (9.0%), MAP3K1 (7.9%), ALK (6.7%), INPP4B (6.7%), NCOR1 (6.7%), and NF1 (5.6%). Cell cycle and apoptosis pathway and PIK3CA/AKT/mTOR pathway were altered significantly more frequently in non-responders than in responders (p = 0.0017 and p = 0.0094, respectively). The average number of mutations per sample was significantly higher in endocrine-resistant tumors (2.88 vs. 1.64, p = 0.03), but no difference was observed in terms of prognosis. ESR1 hotspot mutations were detected in 3.4% of treatment-naive tumors.

Conclusions: The Th1-related immune system and lipid metabolism appear to play key roles in the response to endocrine therapy and prognosis in HR-positive/HER2-negative breast cancer. Deleterious somatic mutations in the cell cycle and apoptosis pathway and PIK3CA/AKT/mTOR pathway may be relevant for clinical management.

Trial registration: This trial is registered with ClinicalTrials.gov ( NCT00629616 ) on March 6, 2008, retrospectively registered.

Keywords: Breast cancer; Endocrine therapy; Immunity; Lipid metabolism; RNA sequencing; Somatic mutation; TILs; Targeted NGS.

Conflict of interest statement

Ethics approval and consent to participate

The study was authorized by the French National Agency for Medicines and Health Products Safety and was approved by the Ile de France VIII ethics committee. Participating patients completed the informed consent process.

Consent for publication

Not applicable.

Competing interests

I confirm that I have read BioMed Central’s guidance on competing interests and have included a statement indicating that none of the authors have any competing interests concerning this manuscript.

Publisher’s Note

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Figures

Fig. 1

Flow-chart of data processing and analysis. BMI body mass index

Fig. 2

Heatmaps summarizing changes in gene expression over time in two major molecular functional groups in NET responders and non-responders. Samples are ordered from left to right by patient identification for each time point. Red and blue represent relative high and low log2 gene expression values, respectively

Fig. 3

Beanplot summarizing the changes in TILs before and after NET in responders and non-responders. R_pre-NET pre-neoadjuvant endocrine therapy samples in responders, R_post-NET post-neoadjuvant endocrine therapy samples in responders, NR_pre-NET pre-neoadjuvant endocrine therapy samples in non-responders, NR_post-NET post-neoadjuvant endocrine therapy samples in non-responders. NS not significant. **p value < 0.01

Fig. 4

Comparative analysis of lipid metabolism between 7 tumors with relapse and 45 tumors without relapse. a The IPA disease/function analysis confirmed that gene level alterations corresponded to lipid metabolism. The calculated z-score indicates a bio-function with genes exhibiting overall increased mRNA levels (orange bars) or decreased mRNA levels (blue bars). The most significantly altered bio-function bars are highlighted with a green border. b Upstream regulator analysis of PPARγ in lipid metabolism. Upregulated genes are highlighted in red and color depth is correlated with fold change. Orange lines with arrows indicate direct activation. Solid and dashed lines represent direct and indirect interactions, respectively. Yellow and gray lines depict inconsistent effects and no prediction, respectively. c Heatmap summarizing the changes in lipid metabolism-related gene expression in tumors with and without relapse. Red and blue represent relative high and low log2 gene expression values, respectively. NE not evaluated

Fig. 5

Deleterious somatic mutations in 89 tumor samples. Tumors with available mutation data are grouped by radiological response along the x-axis, also showing clinicopathological characteristics for each tumor on the x-axis, the 91 genes of BreastCurie panel are enriched to 10 signaling on the y-axis. The somatic mutations of each tumor are indicated by colored boxes; red boxes indicate pathogenic variants, and blue boxes indicate unknown pathogenic variants

Fig. 6

Somatic mutation frequency and comparison of somatic mutation frequency between responders and non-responders. Data show the percentage of samples with somatic mutations on our 91-gene panel; gray bars indicate non-responders, black bars indicate responders. **p value < 0.01, comparison of PIK3CA mutation frequency between responders and non-responders

Fig. 7

Comparison of somatic mutation frequency grouped by biological pathways in responders and non-responders. Data show the percentage of samples with alteration on ten biological pathways; gray bars indicate non-responders, black bars indicate responders. **p value < 0.01 (**)

Fig. 8

RFS according to PIK3CA/AKT/mTOR pathway status. Kaplan-Meier estimates of RFS according to PIK3CA/AKT/mTOR pathway status in patients; 54 patients with at least 1 mutation in the PIK3CA/AKT/mTOR pathway, 35 patients with no mutation in the PIK3CA/AKT/mTOR pathway