Comparison of flow cytometry findings with treatment response in breast cancer patients receiving neoadjuvant chemotherapy

Introduction and Aim: Breast cancer is one of the most frequently diagnosed malignancies among women worldwide and represents a major public health concern. Approximately 2.25 million new cases are diagnosed globally each year. Reliable biomarkers are needed to improve breast cancer screening and prognosis. Previous studies have shown that neoadjuvant chemotherapy (NACT) can affect the percentages and absolute counts of peripheral blood lymphocyte subsets in cancer patients. The aim of this study was to investigate the dynamic effects of NACT on peripheral blood lymphocyte subpopulations in patients with breast cancer and to evaluate the possible correlations between these immune markers and pathological complete response (pCR). Materials and Methods: Between October 2024 and October 2025, a total of 23 patients who were histopathologically diagnosed with breast cancer and initiated on neoadjuvant chemotherapy at the Department of Medical Oncology of our hospital were prospectively evaluated. Peripheral venous blood samples were collected from each patient before NACT (baseline, month 0), and at the 3rd and 6th months of treatment. Peripheral blood lymphocyte subsets were analyzed using the flow cytometry method. Results: During neoadjuvant chemotherapy, significant time-dependent changes were observed in T and NK cell subpopulations. According to the Friedman test results, significant linear increases were detected in total lymphocyte, CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁺CD8⁺CD28⁺, CD4⁺, CD8⁺ T lymphocyte, CD133+ lymphocyte cell ratios (p≤0.002), whereas CD3⁺CD56⁺ NK-like and CD56dimCD16bright NK cell subsets exhibited significant quadratic trends (p≤0.003). Linear increases were also observed in CD56dimCD16brightCD133⁺ and CD56dimCD16brightCD28⁺CD133⁺ populations (p<0.05). Significant differences were found between treatment groups in CD3⁻CD56⁺ and CD56brightCD16⁻ NK cell ratios (p<0.05). Univariate logistic regression analysis revealed no statistically significant immunophenotypic or clinical predictors (p>0.05), although NK and CD8⁺CD28⁻ T cells showed a tendency to reduce event probability. Similarly, Cox regression analysis identified no statistically significant factors; however, higher CD8⁺ T cell levels and increased CD56brightCD16⁻ NK cells demonstrated protective trends for survival, which were considered biologically meaningful. Conclusion: In this study, dynamic changes observed in lymphocyte subsets throughout the "pre-treatment, 3rd month, and 6th month" timeline demonstrated the immunomodulatory effects of neoadjuvant chemotherapy on the immune system. Higher baseline levels of NK cells and CD8⁺ T lymphocytes showed a protective trend for progression-free survival. These findings support the role of innate and adaptive antitumoral immune components in limiting tumor progression and suggest that these immune cell subsets may serve as potential prognostic biomarkers in breast cancer. Keywords: Breast cancer, Neoadjuvant chemotherapy, Lymphocyte subsets, natural killer (NK) cells, CD8⁺ T lymphocytes, Flow cytometry.