Abstract PS3-04-27: Mutational burden and clonal expansion in lymph nodes correlate with IBC postoperative relapse

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive subtype of breast cancer characterized by early metastasis and poor prognosis. Analyzing mutational patterns between primary tumors and lymph node metastases is critical for understanding postoperative relapse yet remains challenging due to marked intratumoral heterogeneity (ITH). Methods: Whole-exome sequencing (WES) was performed on six paired samples of primary tumors and matched lymph node metastases from patients treated with endocrine therapy and pembrolizumab recruited through a trial (NCT02971748), with two patients remaining disease-free and four experiencing postoperative relapse that ultimately led to death. Somatic variants from WES data were identified using Mutect2, annotated with OpenCRAVAT, and filtered to retain only high-confidence, protein-altering, non-synonymous mutations with sufficient sequencing depth and gene mapping. Clonal architecture and cellular prevalence were analyzed using PyClone. Copy number variations (CNVs) were identified using FACETS, which performs joint segmentation across all samples and classifies CNV segments into structural variant types such as deletions, duplications, and loss of heterozygosity. Results: To explore mutation patterns linked to postoperative relapse, we compared mutated genes between primary breast tumors and matched lymph node metastases (LNM), classifying them as either shared or unique to each tissue. Initial analysis focused on cancer-related genes from the Cancer Gene Census. Genes mutated non-recurrently between the primary tumors and the autologous LNM samples showed low variant allele frequencies, with median VAFs of 7.3% and 11%, respectively, suggesting limited clonal evolution. In contrast, genes mutated in both tissues, such as NF1, PIK3CA, and TP53, tended to show higher VAFs, particularly in the LNMs (median VAF: 22%). Within individual patients, few annotated mutations were shared between the primary tumor and lymph node, with most being site-specific, indicating substantial intra-patient mutational heterogeneity. To extend beyond annotated cancer genes, we analyzed all mutated genes to explore the relationship between mutation patterns and clinical outcomes. In patients with favorable outcomes, the VAF in the LNMs showed a maximum decrease of ∼33% from the primary tumor, while in patients with poor outcomes, VAFs increased by ∼70% in lymph nodes relative to the primary site. This pattern suggests a potential association between higher mutational burden in lymph nodes and postoperative relapse. Clonal structure analysis further revealed outcome-associated differences: in patients with favorable outcomes, one exhibited very low clonal prevalence in the lymph nodes (∼2%), and another showed a ∼40% decrease compared to the primary tumor. In contrast, patients with postoperative relapse displayed mixed patterns, but notably included mutations with clonal prevalence increasing by over 20% in lymph nodes, suggesting potential clonal expansion linked to metastasis. Copy number variation analysis revealed distinct structural variant patterns across patients, with deletion counts increasing from primary tumors to lymph nodes in favorable outcome cases, but decreasing in those with poor outcomes. The increase in deletions was most prominent on chromosome 19 in patients with disease-free, where three overlapping genes, HCN2, CD177P1, and TEX101, were deleted. Conclusion These findings suggest that disease-free HR+ IBC were associated with lower mutation burden and limited clonal expansion in lymph node metastases, while postoperative relapse are linked to increased clonal dominance and structural alterations, particularly in the metastatic site. Future studies can validate such finding as potential biomarker and therapeutic targets. Citation Format: Q. Ye, K. Chen, A. Alexander, R. Upadhyay, S. Krishnamurthy, C. Yam, The MDACC Inflammatory Breast Cancer Team, A. Lucci, W. Woodward, B. Lim. Mutational burden and clonal expansion in lymph nodes correlate with IBC postoperative relapse [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-04-27.