Abstract PS2-03: Comparison of pathologist reads of sp142 and sp263 with quantitative measurement of protein and mRNA in triple negative breast cancer
Authors: Gupta Swati , Yaghoobi Vesal , Fernandez Aileen , McCann Leena , Weidler Jodi , Bates Michael , Reisenbichler Emily , Rimm David 
Published: February 2021

Abstract
Background: PD-L1 SP142 immunohistochemistry (IHC) assay has been approved as a companion test by the US Food and Drug Administration (FDA) to identify eligibility for atezolizumab therapy in patients with advanced triple negative breast cancer (TNBC) but a number of studies suggest the assay suffers from poor reproducibility. Using readings of 70 TNBC chromogenic FDA approved assays from 19 pathologists in a previous study as a baseline, we compared pathologist reads to quantitatively measured mRNA and protein expression Methods: Formalin-fixed paraffin-embedded (FFPE) slides representing primary invasive triple negative breast cancer (stage I-III) from 100 patients between 2012-16 were selected from the Yale Pathology archives. Slides were macrodissected to tumor enrichment for quantitative assessment of CD274 (PD-L1 mRNA) measured using a closed-system, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) research use only (RUO)* prototype assay on the GeneXpert® instrument. We also measured protein expression levels using the AQUA method of quantitative immunofluorescence (QIF) in both the tumor and non-tumor compartments on full sections by QIF stained using SP142 in a lab derived test (LDT). The IHC stained slides were prepared using SP142 and SP263 assays prepared exactly according the FDA approved label followed by reading by 19 pathologists. This study was approved by Yale Human Investigation IRB protocol ID 9505008219.Results: Previous work from our group showed overall percent agreement for both the SP142 and SP263 IHC assays read by pathologists was in the 40-50% range. We used the median CD274 score to compare positive (IC≥1) vs negative (IC&lt;1) and found that the levels of mRNA were not statistically significantly different between the two categorical scores. However, quantitative measurement of protein expression (including both tumor and non-tumor regions) showed statistically significant differences between pathologist read PD-L1 positive/negative scoring for SP142 (p=0.0004) and SP263 (p=0.0185). Concordance of quantitative PD-L1 measurement between protein QIF scores and transcript RT-qPCR levels was modest, with a Spearman coefficient r = 0.18. Conclusions: By chromogenic IHC, using the FDA approved assays, pathologist read scoring shows no difference to mRNA for CD274. Quantitative continuous scoring of protein expression show that, on average, when pathologists score IC≥1, there is more protein present than when they score IC&lt;1. Further studies are needed to determine if RNA and protein for PD-L1 are concordant and to determine which assay(s) and cutoff values are best correlated with clinical outcomes on atezolizumab therapy.
*For Research Use Only - Not for use in diagnostic procedures. Not approved or reviewed by any regulatory body
Citation Format: Swati Gupta, Vesal Yaghoobi, Aileen Fernandez, Leena McCann, Jodi Weidler, Michael Bates, Emily Reisenbichler, David Rimm. Comparison of pathologist reads of sp142 and sp263 with quantitative measurement of protein and mRNA in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-03.