Quantitative multiplex immunofluorescence analysis identifies infiltrating PD1+ CD8+ and CD8+ T cells as predictive of response to neoadjuvant chemotherapy in breast cancer

Abstract

Background: This study aimed to explore the potentially predictive role and dynamic changes of immune checkpoints on T cell subsets in patients with breast cancer receiving neoadjuvant chemotherapies.

Methods: Fluorescent multiplex immunohistochemistry (mIHC) was used to stain CD4, CD8, PD1, TIM3, and cytokeratins simultaneously in paired breast cancer samples before and after neoadjuvant therapies (NAT) in a prospective cohort (n = 50). Singleplex IHC was conducted to stain for CD3 in 100 cases with inclusion of extra retrospective 50 cases. Cell levels were correlated with clinicopathological parameters and pathological complete response (pCR).

Results: In pretreatment tumors, the percentages of infiltrating CD8⁺ , PD1⁺ , PD1⁺ CD8⁺ , and the ratio of PD1⁺ CD8⁺ /CD8⁺ cells, were higher in pCR than non-pCR patients in either the stromal or intratumoral area, but PD1⁺ CD4⁺ , TIM3⁺ CD4⁺ , TIM3⁺ CD8⁺ cells and CD4⁺ /CD8⁺ ratio was not. Multivariate analyses showed that the percentage of intratumoral CD8⁺ cells (OR, 1.712; 95% CI: 1.052-2.786; P = 0.030) and stromal PD1⁺ CD8⁺ /CD8⁺ ratio (OR, 1.109; 95% CI: 1.009-1.218; P = 0.032) were significantly associated with pCR. Dynamically, reduction in the percentages of PD1⁺ , CD8⁺ and PD1⁺ CD8⁺ cells after therapy strongly correlated with pCR. Notably, incremental percentages of PD1⁺ CD8⁺ cells, rather than TIM3⁺ CD8⁺ , were shown in tumors from non-pCR patients after NAT. CD3 staining confirmed the percentage of T cells were associated with pCR.

Conclusions: PD1⁺ CD8⁺ rather than TIM3⁺ CD8⁺ cells are main predictive components within tumor-infiltrating T cells in NAT breast cancer patients. Dynamically incremental levels of PD1⁺ CD8⁺ cells occurred in non-pCR cases after NAT, suggesting the combination of chemotherapy with PD1 inhibition might benefit these patients.

Key points: SIGNIFICANT FINDINGS OF THE STUDY: PD1⁺ CD8⁺ , rather than TIM3⁺ CD8⁺ , T cells are the main component to predict the response of neoadjuvant therapies in breast cancer.

What this study adds: Incremental levels of PD1⁺ CD8⁺ T cells in non-pCR post-NAT tumors suggest PD1 inhibition might benefit in the neoadjuvant setting.

Keywords: Breast cancer; PD1; T cell; neoadjuvant chemotherapy; tumor-infiltrating lymphocytes.

Figure 1

T cell subsets in tumor microenvironment (TME) by mIHC in pre‐NAT tumors. (a) Representative mIHC images of preneoadjuvant (left) and post‐neoadjuvant treatment (right) in one pCR patient (top) and one non‐pCR patient (bottom). CD4 (red), CD8 (magenta), PD1 (green), TIM3 (yellow), and CKs (cyan) were stained by fluorescent multiplex IHC, and the percentages of different immune cell subtypes were determined by AI‐assisted methods. (b) The percentages of infiltrating CD8⁺, PD⁺ and PD1⁺CD8⁺ cells, and the ratio of PD1⁺CD8⁺/CD8⁺ in the whole tumor were higher in preneoadjuvant specimens from pCR patients compared to non‐pCR patients (P = 0.096, P = 0.069, P = 0.013, and P = 0.012, respectively). The percentages of infiltrating CD8⁺, PD⁺ and PD1⁺CD8⁺ cells, and the ratio of PD1⁺CD8⁺/CD8⁺ also increased in the stroma (P = 0.203, P = 0.037, P = 0.011, and P = 0.003) and the intratumoral region (P = 0.005, P = 0.052, P = 0.426, and P = 0.582). (c) The ratio of CD4⁺/CD8⁺ T cells were not significantly different in either the stromal or intratumoral areas between the pCR patients and non‐pCR patients. (d) The percentages of TIM3⁺CD4⁺, TIM3⁺CD8⁺ T cells, and the ratio of TIM3⁺CD8⁺/CD8⁺ T cells, were not significantly different in either the stromal or intratumoral areas between the pCR patients and non‐pCR patients. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (b) () PD⁺, () CD8⁺, () PD1⁺CD8⁺, () PD1⁺CD8⁺/CD8⁺; (d) () TIM3⁺CD4⁺, () TIM3⁺CD8⁺, () TIM3⁺CD8⁺/CD8⁺

Figure 2

ROC curve analysis of T cell subsets in the tumor microenvironment (TME) in relation to response to NAT. Subsets of PD1⁺ (a), CD8⁺ (b), PD1⁺CD8⁺ (c) T cells, and PD1⁺CD8⁺/CD8⁺ ratio (d) were analyzed by receiver operating characteristics (ROC) curve test. The area under the curve for intratumoral CD8⁺ cells, stromal and whole tumor PD1⁺CD8⁺ cells, stromal and whole tumor PD1⁺CD8⁺/CD8⁺ ratio were 0.822 (P = 0.007), 0.779 (P = 0.013), 0.775 (P = 0.014), 0.839 (P = 0.005), and 0.793 (P = 0.014), respectively. (a) () AUC = 0.706, P = 0.052, () AUC = 0.750, P = 0.037, () AUC = 0.725, P = 0.067; () Intratumoral, () Stromal, () Whole tumor. (b) () AUC = 0.822, P = 0.007, () AUC = 0.629, P = 0.194, () AUC = 0.675, P = 0.096; () Intratumoral, () Stromal, () Whole tumor. (c) () AUC = 0.636, P = 0.410, () AUC = 0.779, P = 0.013, () AUC = 0.775, P = 0.014; () Intratumoral, () Stromal, () Whole tumor. (d) () AUC = 0.575, P = 0.561, () AUC = 0.839, P = 0.005, () AUC = 0.793, P = 0.014; () Intratumoral, () Stromal, () Whole tumor.

Figure 3

Dynamic changes of percentages of CD8⁺, PD1⁺, PD1⁺CD8⁺ T cell subsets, and the PD1⁺CD8⁺/CD8⁺ ratio in post‐NAT tumors compared with self‐paired pre‐NAT tumors. (a) Comparison between paired pre‐NAT and post‐NAT samples of all patients. The percentages of PD1⁺ cells in the whole tumor, stromal, and intratumoral areas significantly decreased post‐NAT (P = 0.004, P = 0.037, and P < 0.001), yet the percentages of CD8⁺ T cells increased (P = 0.038, P = 0.019, and P = 0.832). (b) Comparison between paired pre‐NAT and post‐NAT samples of pCR patients. The percentages of PD1⁺, CD8⁺, and PD1⁺CD8⁺ T cells, as well as the PD1⁺CD8⁺/CD8⁺ ratio, decreased in the whole tumor (P = 0.028, P = 0.173, P = 0.075, and P = 0.028) and in stromal area (P = 0.028, P = 0.249, P = 0.028, and P = 0.028). (c) Comparison between paired pre‐NAT and post‐NAT samples of non‐pCR patients. CD8⁺ T cells in all parts of the tumor (P = 0.005, P = 0.004, P = 0.081) and PD1⁺CD8⁺ T cells in the whole tumor area (P = 0.047) increased. The percentages of PD1⁺CD8⁺ T cells in the stroma and intratumoral area (P = 0.105, and P = 0.554, respectively) and the ratio of PD1⁺CD8⁺/CD8⁺ in all parts (P = 0.925, P = 0.838, and P = 0.626, respectively) increased without statistical significance; however, the level of whole area and intratumoral area PD1⁺ cells significantly decreased post‐NAT (P = 0.041, P = 0.009). (d) The percentages of TIM3⁺CD4⁺, TIM3⁺CD8⁺ T cells, and the ratio of TIM3⁺CD8⁺/CD8⁺ T cells, were not significantly increased in either the stromal or intratumoral areas after NAT in non‐pCR patients. Pre, pre‐NAT; Post, post‐NAT. (a) () PD1⁺ () CD8⁺ () PD1⁺CD8⁺ () PD1⁺CD8⁺/CD8⁺. (b) () PD1⁺ () CD8⁺ () PD1⁺CD8⁺ () PD1⁺CD8⁺/CD8⁺. (c) () PD1⁺ () CD8⁺ () PD1⁺CD8⁺ () PD1⁺CD8⁺/CD8⁺. (d) () TIM3⁺CD4⁺ () TIM3⁺CD8⁺ () TIM3⁺CD8⁺/CD8⁺.

Figure 4

AI‐assisted analyses of CD3⁺ T cells in TME of pre‐NAT and post‐NAT samples. (a) Representative CD3⁺ T cells images preneoadjuvant (left) and post‐neoadjuvant treatment (right) in one pCR patient (top) and one non‐pCR patient (bottom). (b) The percentage of CD3⁺ T cells was higher in pCR compared to non‐pCR patients in preneoadjuvant therapy specimens. This was the case in the whole tumor (P = 0.001), tumor stroma (P = 0.004), and intratumoral region (P = 0.002). (c) Receiver operating characteristics (ROC) curve analyses showed that the area under the curve values for the percentages of CD3⁺ T cells in the whole tumor, stroma, and intratumoral region were 0.762 (P = 0.001), 0.739 (P = 0.004), and 0.747 (P = 0.003), respectively. (d) The total levels of CD3⁺ T cells in the whole tumor, stroma, and intratumoral region were not significantly increased after NAT (P = 0.795, P = 0.169, P = 0.401, respectively). In pCR patients, the levels of CD3⁺ T cells decreased significantly (P = 0.004, P = 0.002, and P < 0.001, respectively). In non‐pCR patients, they increased only significantly in the stromal area (P = 0.186, P = 0.012, P = 0.388, respectively). Statistical significance was determined by Wilcoxon rank‐sum test. All_CD3, CD3⁺ T cells in whole tumor; Pre_All, pre‐NAT whole tumor area; Post_All, post‐NAT whole tumor area; Pre_Str, pre‐NAT stromal area; Post_Str, post‐NAT stromal area; Pre_Tum, pre‐NAT intratumoral area; Post_Tum, post‐NAT intratumoral area.