Abstract LB394: Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer
Authors: Kristensen Vessela N. , Alnaes Grethe I. , Vernhoff Patrik , Schmiester Leonard , Ghannoum Salim , Fongaard Marie , Bjornstad Paal M. , Selsaas Knut , Geisler Stephanie , Seyedzadeh Manouchehr , Buvarp Unn-Cathrin , Luders Torben , Lambrechts Diether , Lyngra Marianne , Frigessi Arnoldo , Tekpli Xavier , Geilser Jurgen 
Published: April 2024

Abstract
BackgroundAromatase inhibitors (AIs) are commonly used in the management of estrogen receptor-positive (ER+) breast cancer as they effectively reduce estrogen levels which inhibits tumor growth and recurrence. Aromatase inhibitors are increasingly used in selected patients as neoadjuvant therapy.
Clinical trialThe NEOLETEXE is a neoadjuvant, randomized, open-label, intra-patient, cross-over, single-center phase II clinical trial, aimed to treat postmenopausal patients with locally advanced breast cancer. Most patients presented large T3/T4 tumors and/or N2/N3 involvement. Patients were randomly assigned to neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane (25 mg daily) for 3 months followed by a cross-over to the alternative AI for another 3 months followed by surgery. A total of 102 patients were enrolled in the NEOLETEXE trial.
Aim of the studyWe aimed to compare clones derived from bulk exome sequencing with single-cell analyses at DNA and RNA level from the same tumors at different time points of treatment in order to characterize evolutionary trajectories of cancer and immune cells in 23 patients at scRNA level and 16 patients at scDNA level from the NEOLETEXE trial.
MethodsTumor biopsies taken before treatment (baseline) at crossover (after 3 months) and end of neoadjuvant therapy (after 6 months), were analyzed by single-cell DNA (MissionBio) and scRNA (10xGenomics), T cell receptor (TCR), and B cell receptor (BCR) sequencing.
Results We employed the The Mission Bio Tapestri Platform, which enables targeted scDNAseq of nuclei isolated from fresh frozen tumor tissue using microfluid droplet technology. A custom panel of 497 amplicons covering 528 sequence variants was designed to study treatment response selected by the following criteria: 1) Up to 10 mutations from each sublonal clusters defined by analysing 32 samples (3 time points) from 11 NeoLetExe patients with whole exome sequencing (WES) data prioritizing mutations in genes part of the Cancer Gene Census (COSMIC), 2) known mutations covering all chromosomes to enable CNV analysis and 3) “Hand-picked” mutations with significance for breast cancer (e.g. hotspot ESR1 mutations). We performed SNV and CNV analysis and reconstruction of mutational lineages with user developed tools. On average, 81% of the clusters defined by WES was validated by scDNAseq and copy number profiles matching between the two data sets. Fishplot with variants defining cell clusters in WES using data from scDNAseq show clonal development during treatment.The scRNA analyses revealed cell types were identified through clustering of the scRNA data. We focused on the epithelial cells to first distinguish between the normal and malignant epithelial cells using the InferCNV algorithm. We next examined CD4, CD8, NK cells, and macrophages, using RNA velocity and diffusion pseudotime using CellRank and scVelo to delineate the differentiation trajectories of immune cell types.
Conclusions We created a detailed map that provides a high-resolution view of the diverse clones and cell types present in tumor biopsies from the NEOLETEXE trial. We characterize the impact of therapies on the evolutionary dynamics of both cancer and immune cells. Our analyzes sought insights into the underlying factors that contribute to the sensitivity and resistance to aromatase inhibitors.
Citation Format: Vessela N. Kristensen, Grethe I. Alnaes, Patrik Vernhoff, Leonard Schmiester, Salim Ghannoum, Marie Fongaard, Paal M. Bjornstad, Knut Selsaas, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Torben Luders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Xavier Tekpli, Jurgen Geilser. Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB394.