RNA-seq Profiling of Draining and Metastatic Lymph Nodes in HER2-Positive Breast Cancer Identifies POSTN as a Prognostic Marker

Summary

We reveal an extracellular-matrix and PI3K-AKT-centred program underlying lymph-node dissemination in HER2-positive breast cancer and identify POSTN as a poor-prognosis marker, supporting risk stratification and pathway-directed therapeutic strategies.

Abstract

HER2-positive breast cancer is clinically aggressive and frequently disseminates to regional lymph nodes, yet the transcriptomic programs associated with nodal spread remain incompletely defined. Here, we profiled matched tumor-draining lymph nodes (TDLN) and metastatic tumor-involved lymph nodes (TMLN) from six patients with HER2-positive breast cancer using RNA sequencing (RNA-seq). Differential expression analysis was performed with DESeq2, applying Benjamini-Hochberg correction (FDR < 0.05) and an absolute log2 fold-change threshold (|log2FC| ≥ 1). We identified 237 differentially expressed genes (182 upregulated and 55 downregulated in TMLN vs. TDLN). Functional enrichment analyses highlighted extracellular matrix (ECM)-receptor interaction, PI3K-AKT signaling, focal adhesion, and cholesterol metabolism as prominent pathways associated with metastatic lymph nodes, suggesting coordinated ECM remodeling and signaling activation during nodal dissemination. Among the most upregulated genes, POSTN emerged as a key candidate and was further linked to adverse clinicopathologic characteristics. In an independent validation cohort (n = 120), higher POSTN expression was associated with inferior disease-free survival. Collectively, these results nominate POSTN as a potential prognostic biomarker and support ECM-centered and PI3K-AKT-related mechanisms as actionable biological features of lymph-node metastasis in HER2-positive breast cancer.

Introduction

Breast cancer (BC) is the most frequently diagnosed cancer in women and a leading cause of cancer mortality worldwide, with a substantial and rising burden in China¹. Approximately 15%-20% of BCs are HER2-positive (HER2+), characterized by ERBB2 amplification/overexpression and an aggressive phenotype with higher risks of recurrence and death^2,3. Although anti-HER2 systemic therapy has markedly improved outcomes, a considerable proportion of patients still relapse or develop metastases, underscoring the need for biologically informed risk stratification and new therape....

Protocol

This study was approved by the institutional ethics committee and conducted in accordance with the Declaration of Helsinki and local regulations governing the use of human biospecimens. This study was approved by the ethics committee of Boai Hospital of Zhongshan (KY-2020-012-124). Written informed consent was obtained from all participants before any study-specific procedure.

Study cohorts and samples
The discovery cohort comprised paired tumor-draining lymph node (TDLN) and metastatic lymph node (TMLN) tissues collected intraoperatively from 6 patients with HER2-positive breast cancer. For each patient, the matched ....

Results

Cohorts and clinicopathologic characteristics
The discovery cohort consisted of 6 paired TDLN/TMLN samples, and the validation cohort comprised 120 archival lymph-node specimens with longitudinal follow-up. Baseline clinicopathologic characteristics for both cohorts, including age, tumor size, grade, receptor status, and nodal status, are summarized in Table 1.

Sequencing quality and global data structure
All 12 discovery libraries.......

Discussion

HER2-positive breast cancer is characterized by enhanced invasiveness, frequent lymph-node metastasis (LNM), and inferior outcomes despite modern systemic therapy¹⁵. Conventional clinicopathologic factors only partially explain prognosis in this subtype, leaving a gap for biologically grounded markers that capture the risk of nodal dissemination¹⁶. By comparatively profiling matched tumor-draining lymph nodes (TDLN) and tumor-metastatic lymph nodes (TMLN) with RNA-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bioanalyzer	2100-BA	Agilent, USA	Equipment for assessing RNA integrity and quality control.
DESeq2	N/A	R Bioconductor	Differential expression analysis software for RNA-seq data.
FastQC Software	N/A	Babraham Bioinformatics	Quality control software for RNA-seq data to assess sequence quality.
HEK293 Cell Line	ATCC-CRL-1573	ATCC, USA	Human embryonic kidney cell line used for transfection experiments.
qRT-PCR Kit	4385616	ThermoFisher, USA	Quantitative PCR kit for validating gene expression.
RNA Extraction Kit	74204	Qiagen, Germany	RNA extraction kit used to isolate high-quality RNA from tissue samples.
RNA Sequencing Kit	RS-122-2001	Illumina, USA	RNA library preparation kit for sequencing RNA samples.
Sequencing Platform	NovaSeq 6000	Illumina, USA	High-throughput RNA sequencing platform for sequencing RNA libraries.
Tissue Freezing Equipment	TFE-300	Thermo Fisher, USA	Equipment for freezing tissue samples in liquid nitrogen.
Tympanic Thermometer	55678-TT	ThermoScience, Japan	Accurate thermometer used for body temperature measurement.