2932 - Spatial Protein Profiling of Irradiated Breast Cancer Involved Lymph Nodes Suggests Nodal Irradiation Modulates Immune Function
Presenter(s)
S. O. Dudzinski1, W. Ma2, E. N. Barrientos3, B. D. Smith4, S. F. Shaitelman5, S. D. Hernandez3, A. Serrano3, K. B. Khan3, L. V. Kostousov3, W. Lu3, R. Zacharia1, N. I. Comeaux6, L. M. Solis Soto3, A. A. Sahin7, G. M. Raso3, J. Wang8, K. E. Hoffman5, and W. A. Woodward5; 1Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 2Department of Bioinformatics & Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 3Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, 4Department of Breast Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 5The University of Texas MD Anderson Cancer Center, Houston, TX, 6Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 7Department of Anatomical Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, 8Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX
Purpose/Objective(s): We hypothesized the systemic benefit of regional nodal irradiation (RNI) for breast cancer derives from immune stimulation in involved nodes. A pilot study was performed to compare immune signals in pathologically involved nodes from patients treated on a prospective trial of pre-operative and post-operative conventional versus hypofractionated RNI regimens.
Materials/Methods: Nodal tissues from 24 patients (17 evaluable) with ER+, pN+ BC were analyzed using a multiplex analysis platform's Digital Spatial Profiler (DSP): 4 pre-operative RNI short-course (Pre-short); 5 pre-op standard RNI (Pre-standard); 6 post-op (Post); 3 post-op after neoadjuvant chemotherapy (Post-NACT). Slides were stained for PanCK, CD45, CD3e, and SYTO 13, and processed with five immune-specific protein DSP panels (n=58 total immune-related proteins). Up to 5 Regions of interest (ROIs), each measuring 550x550 µm, were selected and categorized as “Tumor” (PanCK+, T-ROI) or “Immune” (CD45+, I-ROI) compartments. Oligonucleotides were collected and quantified after UV exposure. Data was normalized and background corrected. Pearson’s correlations compared protein expression within ROIs, both between patients and within an individual patient. T-Test and KS tests were performed on expression of proteins in all ROIs by compartment type and groups, FDR < 0.1, P value < 0.05. In addition, signals with a Log2 difference of -1 to 1 were examined using adjusted linear mixed model (LMM) analyses to examine patient level differences. Patient level principal component analyses (PCA) of ROIs by compartment type were created.
Results: Intrapatient ROIs and ROIs within groups are highly correlated (all P < 0.001). Considering all Pre vs Post ROIs, 27 differentially expressed proteins (DEPs) were significantly upregulated in Post T-ROIs. None were significantly downregulated. After correction by LMM, 11 markers were up in Post cases and none downregulated. Both methods identified lower expression of SMA and immune cell markers including CD4 (T cells), CD45RO (Memory T cells), CD14 (monocytes), CD11c (dendritic/myeloid cells), TIM-3 and B7-H3 (immune checkpoint inhibitors) in the Pre nodes. No significant DEPs remain in I-ROIs after LMM correction. PCA was applied to identify dominant patterns of variation. The first two principal components accounted for 45.8% of the total variance, with PC1 largely separating samples based on Pre RNI. Exploratory analysis of Pre-short vs Pre-standard ROIs showed 9 increased and 0 decreased proteins in Pre-short including immune markers CD27, Sting, CD40, CD11c, CD25.
Conclusion: Significantly different immune signals in pathologically involved irradiated nodes suggest a less immunosuppressive phenotype in irradiated nodes and potential immune activation with nodes receiving Pre-short RNI. However, the small sample size limits definitive conclusions. Further functional immune studies are warranted to determine optimal radiation regimens. (SAPHIRE NCT02912312).