141. Inflammation-mediated Vascular Changes in Breast Cancer-related Lymphedema

PURPOSE: Breast cancer-related lymphedema (BCRL) is a chronic disease characterized by Th2 biased immune responses. While studies have explored the lymphatic vasculature changes during BCRL progression, blood vasculature changes remain unexamined. We hypothesized that inflammatory markers in blood endothelial cell (BEC) markers would be upregulated in the lymphedematous skin of BCRL patients. METHODS: Skin punch biopsies were collected from surgical breast cancer patients at Memorial Sloan Kettering Cancer Center from both lymphedematous and contralateral arms. Samples underwent protein detection via immunohistochemistry (IHC) and transcriptomic analysis through single-cell RNA sequencing (scRNA-seq). IHC sections were stained using DAPI and antibodies to CD31 (PECAM1), E-selectin, VCAM-1, ICAM-1, and Endothelin, with LYVE1 and CD31 used to distinguish BECs from lymphatic endothelial cells (LECs) respectively. Protein expression was quantitatively analyzed in ImageJ software, and statistical significance was determined with the Wilcoxon signed rank test. scRNA-seq data of paired skin samples were analyzed with ICARUS, version 3.0. Cell populations in Seurat-generated clusters were identified and labelled using CellMarker 2.0. RESULTS: We obtained upper arm biopsy samples of 14 BCRL patients, with a mean age of 58.9, mean BMI of 27.4, mean volume differential between limbs of 33%, and mean time since BCRL diagnosis of 60 months. In comparison to the contralateral arm, the lymphedematous skin biopsies demonstrated significantly higher expression of VCAM1 (p=0.0031), ICAM1 (p=0.0166), and endothelin (p=0.0107); there was no difference in CD31 (p=0.058) or E-selectin expression. The BCRL patient in preliminary scRNA-seq analyses had an L-Dex of 18.9 and volume differential of 20%. We identified multiple cell clusters for ongoing analyses, including BECs as a distinct population from LECs. GO:Biological Process gene set enrichment analysis demonstrated significant differences between lymphedematous and contralateral BEC populations in extracellular matrix remodeling. Clear trends in differential regulation of inflammation and fibrosis-related markers were observed. CONCLUSION: Our study shows specific inflammation-related changes in lymphedema skin BECs at the protein and RNA levels. These findings complement the current knowledge that BCRL is an inflammation-mediated disease. Importantly, our study suggests that BCRL-related inflammation affects blood vasculature in addition to the lymphatics, providing a plausible mechanistic link to why patients with BCRL can suffer from vascular-related comorbidities, recurrent infections, and chronic pain. We aim to add additional patient transcriptomic data to our analyses and ultimately deepen our understanding of BCRL pathophysiology to inform better treatments.