Abstract P4-06-03: Assessment of intratumoral heterogeneity in early stage estrogen receptor (ER) positive breast cancer
Authors: Sofia Mastoraki, Juliana Navarro-Yepes, Tuan Tran, Aysegul Sahin, Kelly Hunt, Nicholas Navin, Khandan Keyomarsi
Journal: Cancer Research (2020)

Abstract Introduction: Tumor heterogeneity is a hallmark of cancer and its underlying clinical relevance has been well established across different tumor types. In the context of ER-positive breast cancer, variation in ER expression among different tumors or distinct cell populations within a single tumor are predicted to account for differences in clinical behavior, treatment response, and disease recurrence. However, a clear understanding of the molecular and cellular mechanisms of tumor heterogeneity that are relevant to the prognosis and therapy of early stage ER-positive breast cancer has not been established. Previous results from bulk RNA-sequencing (RNA-seq) of ER-positive biopsies represent an average of gene expression patterns; this might obscure biologically relevant differences between cells. Single-cell RNA-sequencing (scRNA-seq) is an approach to overcome this problem, allowing assessment of intratumoral cell populations and biological systems at unprecedented resolution. In this study, our aim is to compare gene expression profiles of bulk RNA-seq and scRNA-seq from tumor biopsies of early stage ER-positive patients. Methods: Tumor and normal biopsies obtained from ER-positive patients were divided into 3 parts; two being dissociated by enzymatic disaggregation and one by direct total RNA isolation. Tumor tissues were subjected to both scRNA-seq and bulk RNA-seq analyses while single-cell suspension of the normal matched tissue was used for bulk RNA-seq alone. Furthermore, patient-derived organoids were generated from both normal and tumor samples. Single-cell isolation and barcoding were assessed using the 10x Genomics technology followed by RNA sequencing with the Illumina NovaSeq6000 system (50PE), whereas bulk RNA-seq was performed using an Illumina PE150 strategy. Results: Our preliminary data represents an assessment of tumor and normal adjacent tissues collected after mastectomy from an 80-year old ER-positive, PR-positive, HER2-negative patient diagnosed with early stage infiltrating ductal carcinoma in situ (stage IB). scRNA-seq of tumor tissue from this patient identified 10 distinct clusters of cells, consisting of both immune and non-immune stromal populations (epithelial, endothelial, fibroblasts, and immune cells). 95% of single cells were luminal. However, there was a minor group (2%) of cells showing a basal-like signature, which can lead to disease recurrence. Interestingly, although the patient was clinically characterized as HER2-negative by IHC and FISH, HER2 overexpression is observed in the vast majority (95%) of single cells isolated. Furthermore, organoid cultures recapitulated the features of patients' tumors and presented similar transcriptomic profiles. We have adapted similar sequencing and downstream analyses for an additional number of patient biopsies. Our ongoing study is geared towards comparing bulk and single cell transcriptome profiles from these ER-positive cases and identifying overlapping populations that can predict recurrence or novel therapeutic vulnerabilities. Conclusions: Bulk RNA-seq approaches lack the resolution to visualize the true extent of stromal heterogeneity and may mask rare populations or cellular phenotypes that could be critical for tumor survival. ScRNA-seq highlights the dynamic and adaptive nature of all cellular populations within an evolving tumor microenvironment and reveal potential crosstalk between these two compartments. Lastly, establishment of organoid cultures presents the opportunity of high-throughput drug screening studies and the identification of new, patient-tailored therapeutic strategies. Citation Format: Sofia Mastoraki, Juliana Navarro-Yepes, Tuan Tran, Aysegul Sahin, Kelly Hunt, Nicholas Navin, Khandan Keyomarsi. Assessment of intratumoral heterogeneity in early stage estrogen receptor (ER) positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-03.