Abstract PO5-14-03: Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer
Authors: Cronister Catie , Schweitzer Brock , Gilmore Hannah , Nielsen Tyler 
Published: May 2024

Abstract
Discussion: Breast cancer is the most common malignancy diagnosed amongst women, with more the two million new cases identified globally each year. Thus, considerable resources have been devoted to a better understanding of the disease and how to optimize outcomes for patients. Genetics and genomics have become part of routine care with the latter used to inform treatment planning for patients with ER+ breast cancer (ER+ BC). A multitude of assays may be used to determine the need for adjuvant chemotherapy but none of the current tests on the market are guideline-recommended for making decisions related to neoadjuvant chemotherapy, surgery, or radiation. The 24-gene StemPrintER assay was developed to measure the “stemness” of tumors, or how much a tumor behaves like stem cells, which could have important and novel implications for breast cancer treatment planning. The assay has been validated for predicting prognosis using formalin-fixed paraffin-embedded (FFPE) tissue blocks from several clinical cohorts, including TransATAC. The goal of current and future research will be to determine whether the assay can determine the ideal surgical intervention for ER+BC patients. Early in silico work demonstrated a trend toward this predictive capacity for the assay and suggests the assay may be able to identify the benefit of mastectomy versus quadrantectomy. Here, we report on the ability to set up and run the assay in our lab using synthetic oligonucleotides and archival tissue blocks.
Methods: Synthetic oligonucleotides and archival FFPE breast cancer tissue were used to set up and analytically validate the assay’s performance in two phases. The initial phase was intended to confirm the individual assay design performance by assessing linearity and precision and the ability to amplify RNA from FFPE, which confirmed compatibility with standard extraction methods. The second phase was intended to determine whether these methods could be implemented to reliably extract and amplify RNA from FFPE &gt;10 years old (range: 10-14 years) in order to generate a StemPrintER score. The assay was run according to standard operating procedures for StemPrintER. Analyses were conducted Python 3.9.12.
Results: Two assay runs with duplicate samples for each run using a 7-point dilution series demonstrated linearity (coefficient of correlation, r2) results ranging from 0.978 to 0.999 for each of the 24 genes in the assay. Amplification efficiency was between 100-101% for each gene. Using 14 FFPE samples from patients with triple-negative breast cancer, we demonstrated a consistent extraction yield of RNA from tumor blocks which produced amplification of each of the targets. Analysis of intra- and inter-run precision resulted in coefficients of variation (CV) ranging from 0.2-1.07% and inter-run CV’s ranging from 0.66%-5.56%. Utilizing FFPE &gt;10 years old from ER+BC patients demonstrated that the lab could extract RNA from 100% of samples and amplify the material to obtain binary StemPrintER results.
Conclusion: These data demonstrate that StemPrintER can be run reliably on FFPE tissue up to 14 years old. In addition, our laboratory has demonstrated that assay results are highly linear and reproducible. Additional studies are planned to build on preliminary surgical prediction data and determine whether the assay can meet the unmet need of identifying the ideal surgical intervention in patients with early-stage ER+/HER2- breast cancer.
Citation Format: Catie Cronister, Brock Schweitzer, Hannah Gilmore, Tyler Nielsen. Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-03.