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Preoperative (preop) partial breast radiotherapy (RT) has been shown to upregulate the expression of immune and inflammatory genes in breast cancer (BC). Although there is no evidence of such effect beyond this clinico-pathological low-risk BC, a handful of clinical trials in the high-risk early-stage setting are currently based on the hypothesis of a synergistic effect between preop RT and immunotherapy. The purpose of this study is to investigate the changes in gene expression induced by preoperative RT in early-stage breast cancer, across all molecular subtypes.

The pilot clinical study YOUNGSTER recruited 20 early-stage BC patients. The inclusion criteria were (i) eligibility for breast-conserving surgery followed by whole-breast irradiation and RT boost to the tumor bed and (ii) no prior treatment. The intervention consisted in delivering the RT boost (5 x 2,67 Gy/fx) preoperatively followed by a new core-needle biopsy of the tumor 3-5 days after the RT boost. After intervention, patients followed the standard of care.

To date, 17 paired (pre/post RT boost) core-needle biopsy samples were analyzed. The RNA was profiled using the NanoString nCounter Elements XT panel, a customized 72-gene panel that includes the 50 genes within the PAM50 signature and other immune genes such as CD3G, CD4, CD8A, CD18, CD68, CD84, CD86, CD274 and PDCD1. Differential gene expression (pre- vs post-RT) was identified using a two-class paired SAM analysis with a false discovery rate (FDR) < 5%. Tumor proliferation (Ki67) and the percentage of tumor-infiltrating lymphocytes (TILs) were also assessed by immunohistochemistry and HE staining, respectively.

The baseline PAM50 intrinsic subtype of the 17 paired-samples was: Luminal A (n=7), Luminal B (n=3), HER2-Enriched (n=4) and Basal-like (n=3). We found that the preop RT boost significantly downregulated the mRNA levels of proliferative genes, including MKI67 (FDR=0), the PAM50 proliferation signature score (p=0.003) and % of Ki67 IHC staining (p=0.010) in Luminal A, Luminal B and HER2-Enriched tumors, but not in Basal-like tumors. On the other hand, the preop RT boost significantly upregulated the expression of MDM2 (FDR=0), CD68 (FDR=0) and CD86 (FDR<3%), for all molecular subtypes. No differential gene expression was observed for other immune markers, in particular the T-cell marker CD274/PD-L1. HE staining did not show an increase of TILs either.

The results of this work seem to suggest that radiotherapy slows down tumor proliferation in early-stage Luminal A/B and HER2-Enriched BC, while it selectively upregulates the expression of certain immune genes such as CD68 and CD86, for all molecular subtypes. Furthermore, no evidence was found of a radiotherapy-induced upregulation of PD-L1 in early-stage BC. This latter result calls into question the therapeutic strategy of combining neoadjuvant PD-L1 inhibitors with a RT boost in high-risk early-stage BC, which is currently under investigation in several clinical trials.