Association of baseline tumor-infiltrating lymphocytes and cell-cycle regulation markers on prognosis and mortality in patients with advanced breast cancer according to tumor characteristics and treatment type: an observational study

Abstract

Background: Tumor proliferation and immune infiltration are key determinants of breast cancer biology, yet their prognostic value in the advanced setting remains incompletely defined. We evaluated the clinical relevance of tumor‑infiltrating lymphocytes (TILs) and four cell cycle regulation biomarkers-Ki67, MCM2, Cyclin A, and PHH3-across major breast cancer subtypes in a contemporary real‑world cohort.

Methods: We conducted a retrospective analysis of the outcomes of 398 patients with advanced breast cancer treated between 2020 and 2024. Clinicopathological variables were compared across HER2-/HR+, HER2+, and triple‑negative breast cancer (TNBC). Progression‑free survival (PFS) was assessed using Kaplan-Meier analysis and log‑rank tests. Multivariable polytomous logistic regression identified factors associated with PFS < 2 years. Subtype‑specific associations between TILs and PFS > 2 years were evaluated using multivariable Cox models adjusted for age, ECOG status, tumor grade, and prior therapies. Additional Cox regression models assessed predictors of overall survival (OS).

Results: Low TIL density was independently associated with early progression (RRR 2.28, 95% CI 1.19-4.03). Proliferation markers showed consistent associations with PFS: elevated Ki67, MCM2, Cyclin A, and PHH3 each correlated with shorter survival, with MCM2 showing the strongest effect. TIL-PFS associations were subtype‑dependent. In HR+/HER2 - disease, high TILs were linked to shorter PFS (HR 2.27, 95% CI 1.18-4.02), whereas in TNBC, low TILs predicted markedly worse outcomes (HR 2.33, 95% CI 1.88-2.86). TIL levels were not prognostic in HER2 + disease. Across all subtypes, high expression of Ki67, MCM2, Cyclin A, and PHH3 was significantly associated with reduced OS in multivariable models.

Conclusions: Subtype‑specific immune infiltration and elevated proliferation activity are key predictors of disease trajectory in advanced breast cancer. TILs carry divergent prognostic meaning across subtypes, whereas proliferation markers consistently identify high‑risk disease. Integrating immune and proliferative biomarkers may enhance risk stratification and guide treatment tailoring, particularly within TNBC and hormonally driven tumors.

Keywords: Biomarkers; Breast neoplasms; Cell proliferation; Disease-free survival; Ki-67 Antigen; Minichromosome maintenance complex component 2; Molecular subtypes; Tumor; Tumor-infiltrating lymphocytes.