Abstract P2-08-06: Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study
Authors: Budhathoki Nibash , Fa'ak Faisal , D'Abreo Nina , Dhage Shubhada , Soe Phyu Phyu , Holmes Dennis , Haan Josien , Wang Shiyu , Dauer Patricia , Menicucci Andrea , Audeh William , Marks Douglas K 
Published: March 2022

Abstract
Introduction: Early lymph node (LN) metastasis often precedes systemic metastasis and corresponds with a 35% decrease in 10-year survival compared to patients without LN metastasis. Understanding biologic pathways involved in early LN metastasis could identify promising drug targets for early-stage breast cancer (EBC) treatment. While tumor size modestly correlates with the probability of clinical or occult axillary metastases, small T1 tumors are often found to have LN metastases, while many large T3 tumors are often LN negative. We compared large tumors without evidence of LN metastasis (pT2-3N0) and small tumors with LN metastasis (pT1N1-3) by whole transcriptome analysis to elucidate molecular biological differences associated with the presence or absence of early LN metastasis at diagnosis. Methods: The FLEX study enrolls patients with EBC who undergo standard of care MammaPrint (MP) and BluePrint (BP) testing, and consent to clinically annotated full transcriptome data collection. MP classifies tumors as having High Risk (HR) or Low Risk (LR) of distant recurrence. MP combined with BP subtype tumors as either Luminal A-type (MP LR; 56.8%), Luminal B-type (MP HR; 36.4%), HER2-type (1.5%), or Basal-type (5.3%). In our primary comparison, 355 patients had small, LN-positive tumors (pT1N1-3), and 235 had large, LN-negative tumors (pT2-3N0). Control groups consisted of small, LN-negative (pT1N0; n=1384) and large, LN-positive tumors (pT2-3N1-3; n=375). R package ‘limma’ was used for quantile normalization and differential gene expression (DGE) analyses. Differentially expressed genes (DEGs) with a false discovery rate &lt; 0.05 and fold change &gt; 1.4 were considered significant for this exploratory analysis. Gene set enrichment analysis (GSEA) was performed using the Hallmark gene sets (MSigDB). Results: Principal component analysis of each sample’s transcriptional profile revealed more gene profile differences between MP risk and BP subtype than by pathological stage. Overall, DGE analysis comparing pT2-3N0 to pT1N1-3 identified 30 DEGs; of which MUCL1 had a &gt; 2-fold decrease. Within MP LR and BP Luminal A-type tumors, no DEGs were identified comparing pT2-3N0 to pT1N1-3. HR tumors had 73 DEGs (33 upregulated and 40 downregulated); two of 40 downregulated genes, including MUCL1, had &gt; 2-fold change. Within Luminal B tumors, 34 DEGs were identified when comparing pT2-3N0 with pT1N1-3, although none had &gt; 2-fold change. Basal and HER2 tumors were not analyzed due to small numbers. GSEA showed upregulated proliferation and oxidative phosphorylation pathways, and downregulated epithelial to mesenchymal transition (EMT) and immune signatures in pT2-3N0 relative to pT1N1-3 tumors overall, within HR, and Luminal B tumors. Conclusion: This study provides a foundation for understanding the mechanisms that promote LN metastasis. Studies have previously correlated LN metastasis with EMT, immune function, and gene overexpression (BCL2L1, AURKA, CDKN2A, MCL1, MYC). While we did not find significant differences in these genes, our results indicated similar differentially regulated pathways. Overall, we found most biological differences within MP HR and Luminal B tumors. Proliferation-related pathways were upregulated and EMT and immune-related pathways were downregulated in pT2-3N0 tumors compared with pT1N1-3 tumors, suggesting these pathways distinguish small, node-positive tumors from large, node-negative tumors and are involved in early LN metastasis. Interestingly, MUCL1 which has been reported to promote migration and invasion in breast cancer, was consistently downregulated in pT2-3N0 relative to pT1N1-3 tumors. Future studies will investigate antineoplastic therapies for EBC that modulate these dysregulated pathways to reduce early LN metastasis and subsequent systemic metastasis.
Citation Format: Nibash Budhathoki, Faisal Fa'ak, Nina D'Abreo, Shubhada Dhage, Phyu Phyu Soe, Dennis Holmes, Josien Haan, Shiyu Wang, Patricia Dauer, Andrea Menicucci, William Audeh, Douglas K Marks. Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-06.