Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

Abstract

Introduction: The status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8(+) TILs and FOXP3(+) TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).

Methods: One hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8(+) TIL and FOXP3(+) TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8(+) TIL and FOXP3(+) TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.

Results: TNBC patients with high CD8(+) TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8(+) TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537-6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029-4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8(+) TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499-9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8(+) TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).

Conclusions: This is the first study to demonstrate that high CD8(+) TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Fig. 1

Immunohistochemical double staining of tumor-infiltrating CD8⁺ (blue) and FOXP3⁺ (brown) lymphocytes. The representative tumor tissue of high (a) and low (b) infiltration of lymphocytes on surgical specimens after neoadjuvant chemotherapy (black arrow points to CD8⁺ lymphocytes, and red arrow points to FOXP3⁺ lymphocytes)

Fig. 2

Recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) in patients with different status of CD8⁺ tumor-infiltrating lymphocyte (TIL), FOXP3⁺ TIL, and CD8/FOXP3 ratio. Estimated Kaplan-Meier curves of RFS (a) and BCSS (b) in patients with high or low CD8⁺ TIL, those of RFS (c) and BCSS (d) in patients with high or low FOXP3⁺ TIL, and those of RFS (e) and BCSS (f) in patients with high or low CD8/FOXP3 ratio. pCR pathological complete response

Fig. 3

The cases with high change rates of CD8⁺ TIL and CD8/FOXP3 ratio before and after neoadjuvant chemotherapy (NAC). The cutoffs of a high or low rate of changes were defined as near the median ratio as follows: CD8⁺ TIL, 1.4 and CD8/FOXP3 ratio, 1.8. Immunohistochemical images of tumor-infiltrating CD8⁺ (blue) and FOXP3⁺ (brown) lymphocytes in pretreatment biopsy specimens (a) and residual tumors after NAC (b) of the same patient (black arrow points to CD8⁺ lymphocytes, and red arrow points to FOXP3⁺ lymphocytes). c Changing levels of CD8⁺ TIL in each case; 9 of 38 cases experienced cancer recurrence, and 7 patients died. d Changing levels of CD8/FOXP3 ratio in each case; 8 of 37 cases experienced cancer recurrence, and 5 patients died

Fig. 4

The cases with low change rates of CD8⁺ TIL and CD8/FOXP3 ratio before and after neoadjuvant chemotherapy (NAC). The cutoffs of a high or low rate of changes were defined as near the median ratio as follows: CD8⁺ TIL, 1.4 and CD8/FOXP3 ratio, 1.8. Immunohistochemical images of tumor-infiltrating CD8⁺ (blue) and FOXP3⁺ (brown) lymphocytes in pretreatment biopsy specimens (a) and residual tumors after NAC (b) of the same patient (black arrow points to CD8⁺ lymphocytes, and red arrow points to FOXP3⁺ lymphocytes). c Changing levels of CD8⁺ TIL in each case; 20 of 40 cases experienced cancer recurrence and 14 patients died. d Changing levels of CD8/FOXP3 ratio in each case; 21 of 41 cases experienced cancer recurrence, and 16 patients died

Fig. 5

Recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) in patients with different change rates of CD8⁺ tumor-infiltrating lymphocyte (TIL), FOXP3⁺ TIL, and CD8/FOXP3 ratio. Estimated Kaplan-Meier curves of RFS (a) and BCSS (b) in patients with high or low change rates of CD8⁺ TIL before and after neoadjuvant chemotherapy, those of RFS (c) and BCSS (d) in patients with high or low change rates of FOXP3⁺ TIL, and those of RFS (e) and BCSS (f) in patients with high or low change rates of CD8/FOXP3 ratio