Abstract P4-02-16: Prognostic and Predictive Capacity of Tumor Infiltrating Lymphocytes in the MA.20 regional radiotherapy trial
Authors: Riaz Nazia , Chen Bingshu , Bane Anita , Gao Dongxia , Stovgaard Elisabeth , Kos Zuzana , Leung Samuel , Shenasa Elahe , Parulekar Wendy , Chambers Shelley , Nielson Torsten , Whelan Timothy J. 
Published: March 2023

Abstract
Background: The benefit of regional nodal irradiation (RNI) in patients with low burden metastatic axillary disease was established in MA.20 showing that patients randomized to receive adjuvant whole breast irradiation (WBI) plus RNI experienced a significantly better disease free survival (DFS) and distant disease free survival (DDFS) compared to those who received WBI alone and this advantage was maintained in the hormone receptor negative subgroup. Stromal tumor infiltrating lymphocytes (sTILs) have shown prognostic and predictive value in HER2 positive and triple negative breast cancers. To date, clinical importance of immune infiltrates as prognostic and predictive biomarkers in the context of benefit from RNI has not been shown. Methods: 1064 full-face hematoxylin and eosin (H&E) stained sections and formalin fixed paraffin embedded primary tumor blocks assembled into 16 tissue microarrays (TMAs) in quadruplicates linked with clinical data were accessible from the original 1832 patients in the MA.20 trial for this retrospective-prospective translational study conducted according to the REMARK guidelines. sTILs were assessed on scanned images of H&E sections according to the International Immuno-Oncology Working Group method, and on TMAs by CD8 immunohistochemistry (IHC) using a validated assay. Biomarkers were scored by pathologists blinded to the clinical data and analyzed as continuous and categorical variables using prespecified median cutpoints. The median follow-up was 9.5 years. Cox proportional regression modelling was used after adjusting for clinicopathological factors and treatments. Hazard ratios (HR) with 95% confidence intervals (CI) were reported for the primary endpoint of DFS and secondary endpoint of DDFS. Predictive value was assessed by the interaction test between the treatment arm and the biomarkers in the full cohort and an IHC defined non-luminal A subgroup. Results: 1035 cases were evaluable for sTILs on H&E sections. Of these 52.6% (n=544) cases with ≥10% sTILs displayed a significant correlation with age &lt; 50 years, grade III, tumor size &gt;2cm, hormone receptor negative status, and non-luminal A subgroup (p &lt; 0.05). Of the 857 evaluable cases on TMAs, CD8+sTILs (≥16) were observed in 49.8% (n=427) cases and showed a significant association with grade III, ER negativity and non-luminal A status (p &lt; 0.05). For the full cohort, H&E sTILs assessed as a continuous parameter, were not prognostic for DFS (HR 0.993; 95% CI 0.984-1.003; p=0.18) but provided prognostic information for DDFS (HR 0.988; 95% CI 0.977-0.999; p=0.04) in multivariate analyses. H&E sTILs did not show predictive value as a continuous variable. Similarly, using the prespecified cutpoint (≥10%), H&E sTILs were neither prognostic nor predictive. Increasing level of CD8+sTILs was associated with significantly improved DFS (HR 0.99; 95% CI 0.983-0.998; p=0.02) and DDFS (HR 0.98; 95% CI 0.97-0.99; p=0.002) in multivariate analyses. For the full cohort, CD8+sTILs as a continuous variable, showed a significant improvement in DDFS for patients randomized to WBI and RNI (HR 0.979; 95%CI 0.959-0.999; p(interaction) =0.04) compared to WBI alone and a trend (HR 0.977; 95%CI 0.954-1.001; p(interaction) =0.06) for better outcome was observed for the non-luminal A subgroup. CD8+sTILs at the prespecified cutpoint (≥16) were not prognostic or predictive. Conclusions: Pre-treatment tumoral infiltration with stromal lymphocytes provided positive prognostic information for DFS (CD8+sTILs) and DDFS (H&E sTILs and CD8+sTILs) when examined as a continuous variable but failed to do so at prespecified cutpoints. While CD8+sTILs as a continuous variable predicted benefit from RNI, significant prediction results were not seen for prespecified cutpoint or related biomarker H&E sTIL. These results require further validation.
Citation Format: Nazia Riaz, Bingshu Chen, Anita Bane, Dongxia Gao, Elisabeth Stovgaard, Zuzana Kos, Samuel Leung, Elahe Shenasa, Wendy Parulekar, Shelley Chambers, Torsten Nielson, Timothy J. Whelan. Prognostic and Predictive Capacity of Tumor Infiltrating Lymphocytes in the MA.20 regional radiotherapy trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-16.