Neither Tumor-Infiltrating Lymphocytes nor Cytotoxic T Cells Predict Enhanced Benefit from Chemotherapy in the DBCG77B Phase III Clinical Trial

Abstract

Recent studies have shown that immune infiltrates in the tumor microenvironment play a role in response to therapy, with some suggesting that patients with immunogenic tumors may receive increased benefit from chemotherapies. We evaluated this hypothesis in early breast cancer by testing the interaction between immune biomarkers and chemotherapy using materials from DBCG77B, a phase III clinical trial where high-risk premenopausal women were randomized to receive chemotherapy or no chemotherapy. Tissue microarrays were evaluated for tumor-infiltrating lymphocytes (TILs) assessed morphologically on hematoxylin and eosin-stained slides, and by immunohistochemistry for CD8, FOXP3, LAG-3, PD-1 and PD-L1. Following REMARK reporting guidelines, data analyses were performed according to a prespecified statistical plan, using 10-year invasive disease-free survival as the endpoint. Differences in survival probabilities between biomarker groups were evaluated by Kaplan-Meier and Cox proportional hazard ratio analyses and prediction for treatment benefit by an interaction test. Our results showed that stromal TILs were associated with an improved prognosis (HR = 0.93; p-value = 0.03), consistent with previous studies. However, none of the immune biomarkers predicted benefit from chemotherapy in the full study set nor within major breast cancer subtypes. Our study indicates that primary tumors with higher immune infiltration do not derive extra benefit from cyclophosphamide-based cytotoxic chemotherapy.

Keywords: adjuvant chemotherapy; breast cancer; cyclophosphamide; immuno-oncology; immunohistochemistry; lymphocyte biomarkers; tumor immune microenvironment.

Figure 1

CONSORT diagram of DBCG77B phase III clinical trial immune biomarker study.

Figure 2

Violin plot showing the distribution of immune biomarkers, with the table summarizing the median values and predefined cut points, obtained from previous studies. White dots represent the median value for each marker. H&E sTIL was included in the primary objective as a continuous variable and in the exploratory analysis as a categorical variable. Cutoff point for CD8 iTIL was adjusted for the core size differences between this study (2.0 mm) and previous papers (0.6 mm). Median value was used as a cutoff point for CD8 sTILs. N: number of evaluable cases, H&E: hematoxylin and eosin, sTIL: stromal tumor-infiltrating lymphocyte, iTIL: intraepithelial tumor-infiltrating lymphocyte.

Figure 3

Prognostic significance of H&E sTIL in the full study set. Invasive disease-free survival plot and unadjusted hazard ratio for a 10-point difference in continuous H&E sTIL score (expressed as percentage) for the full study set (n = 681), using 10-year IDFS as the end point. HR: hazard ratio, CI: confidence interval, H&E: hematoxylin and eosin, IDFS: invasive disease-free survival, sTIL: stromal tumor-infiltrating lymphocyte.

Figure 4

Forest plot showing adjusted hazard ratios and corresponding 95% confidence intervals for a 10-point difference in continuous H&E sTIL according to the treatment regimens. These analyses were performed for the full study set (primary objective), in the non-luminal A subset (secondary objective), and in the basal and non-luminal subsets (exploratory). Due to the limited number of the events, unadjusted estimates are presented for the basal subset. The box sizes represent the sample size (number of the cases in each subgroup analysis). CI: confidence interval, HR: hazard ratio, H&E: hematoxylin and eosin, sTIL: stromal tumor-infiltrating lymphocyte.