Intra-tumor heterogeneity-resistant gene signature predicts prognosis and immune infiltration in breast cancer

Haixing Shen¹Qing Zheng²Jie Pan³Yukai Jin³Xiaohong Zheng³Yueqing Yuan⁴Da Tang⁴Zhou Qiang⁴Jingzhi Wang^5,6*Tianmiao Sun^1*

• ¹Affiliated Cixi Hospital, Wenzhou Medical University, Ningbo, Zhejiang 315300, China
• ²Department of Gastrointestinal Surgery, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, China
• ³Cixi Biomedical Research Institute, Wenzhou Medical University, Ningbo, Zhejiang, 315300, China
• ⁴Department of Neurosurgery, Shanghai Tenth People's Hospital of Tongji University, Shanghai 200072, China
• ⁵Department of Radiotherapy Oncology, The Affiliated Yancheng First Hospital of Nanjing University Medical School, Yancheng, Jiangsu 224000, China
• ⁶The First People's Hospital of Yancheng, Yancheng, Jiangsu 224000, China

Background: Breast cancer (BC) remains a significant threat to human health, with substantial variations in prognosis and treatment responses. Intra-tumor heterogeneity (ITH) presents a critical challenge in developing reliable prognostic models. Methods: This study integrated multi-region RNA sequencing data from BC patients with the TCGA BC dataset. Genes resistant to sampling bias were identified by evaluating inter-patient heterogeneity (IPH) and ITH. A machine learning framework incorporating ten algorithms was used to construct a prognostic signature. The expression of prognostic genes was validated through RT-qPCR. Results: The signature, comprising CFL2 and SPNS2, demonstrated stable predictive performance in training and validation cohorts (C-index > 0.6). High-risk patients exhibited enriched immune infiltration, particularly CD8+ T cells, and higher expression of immune checkpoint molecules, suggesting sensitivity to immunotherapy. A nomogram integrating risk score with clinical variables further improved prognostic accuracy. Dysregulation of signature genes was confirmed in BC cell lines. Conclusion: By minimizing ITH interference, this study developed a robust prognostic signature for BC, offering insights into the tumor immune microenvironment and potential therapeutic strategies.