Project Summary We have developed the sensitivity to endocrine therapy (SETER/PR) index of gene expression to predict the degree of sensitivity to endocrine therapy for patients with metastatic breast cancer, to guide the selection of treatment after disease progression. The assay measures the expression of estrogen receptor (ER) gene (ESR1), the frequency of ESR1 transcripts with activating mutation in a hotspot region of the ligand-binding domain, and a signature representing the expression of genes most strongly correlated with expression of both estrogen (ER) and progesterone receptor (PR) genes. Therefore, the SETER/PR index summarizes 18 selected transcripts relative to 10 reference transcripts that represent the range of gene expression. Using RNA from high-quality fresh samples of breast cancer, the SETER/PR index was highly reproducible under diverse analytical and pre-analytical conditions, and was prognostic for PFS and overall survival (OS) when tested in biopsies of metastatic breast cancer from patients who then receive endocrine therapy. We have recently translated the assay for use with sections from routine formalin-fixed paraffin-embedded (FFPE) tissue biopsies. We know that it will be important to define the pre-analytical conditions under which an RNA-based assay can be accurately applied to usual clinical samples of metastatic breast cancer. Although it is usual to immediately preserve small biopsies of metastatic cancer in clinical practice, there are major pre-analytical conditions to consider: the use of fine needle aspiration cytology (several sample preservation protocols are possible), dilutive and contaminating effects from host tissue RNA within a cytologic or tissue biopsy of metastasis, and the ability to perform the assay using malignant effusion samples (larger volumes are not immediately fixed). We have designed our studies to provide insights into whole transcriptome assays and, separately, our customized, targeted RNA sequencing assay for the SETER/PR index. Our first objective is to identify the transcripts with measurement that is robust to the effects of routine fixation and processing on small biospecimens, compared to the high-quality RNA from matched samples in RNA preservative. We will use whole transcriptome RNA sequencing (RNAseq) to measure every transcript under every clinical procedure for preserving a tumor tissue or cytologic biopsy, compared to high-quality RNA as the reference. We will similarly, compare these pre-analytical conditions using our customized RNAseq assay for the SETER/PR index (droplet-based reverse transcription of targets, followed by sequencing). This will define the best conditions for RNA-based testing of cytology and tissue samples, and will provide data for all transcripts in addition to the specifically targeted transcripts in the SETER/PR index. Our second objective is to define a panel of host organ transcripts that will be used to estimate the extent of the host organ RNA within a clinical biopsy sample obtained from any metastatic site. This involves the identification of organ-specific transcripts from RNAseq data and existing microarray data sets, so that a measurement of the SETER/PR index from a metastatic biopsy is accompanied by a measurement to estimate the extent of host organ RNA that was included within the sample, and hence the purity of cancer. This will serve as a useful quality control measure, and potentially enable corrected reporting for the tumor population. Our third objective is to analytically validate and test the feasibility and clinical validity of the SETER/PR index using the diagnostic assay with biopsy samples of metastatic breast cancer collected from a clinical trial. We will finalize the SETER/PR index assay, including analytes to estimate the extent of host organ contamination, an complete the analytical validation studies to establish this as a CLIA-compliant clinical test. Then, we will test the clinical performance of the SETER/PR index test using samples of metastatic breast cancer from the Metastatic Breast Cancer (MBC) trial, specifically in the treatment arms that compared endocrine- based treatments for progression-free and overall survival. The MBC trial is a basket trial of randomized treatment comparisons that is being developed by the Food and Drug Administration, academic oncology community, and the National Cancer Institute. It aims to coordinate predictive diagnostic development with a network of clinical trials of new treatments. We will complete the development of an assay that accurately measures the expression, functional sequence integrity, and transcriptional product of ER within a biopsy of metastatic breast cancer – to predict the sensitivity to endocrine therapy at any point in the treatment course of metastatic breast cancer.

Project Narrative This proposal is to integrate biospecimen studies in order to minimize any pre-analytical effects on the predictive accuracy of a novel gene expression assay that we are developing to predict tumoral sensitivity to endocrine therapy (SETER/PR index) from clinical biopsies of a patient's metastatic breast cancer. The results will also be informative for other applications to develop RNA-based assays to test metastatic biopsies. Our overall goal is to maximize the analytical and clinical validity of this assay when using standard clinical biopsy samples, in preparation for it use as a predictive test within large clinical trials, and as a future clinical test.