Bridging and analytical validation of the Prosigna ® Breast Risk of Recurrence Test as a whole-transcriptome NGS lab developed test

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Abstract Background The Prosigna Breast Risk of Recurrence test is based on the PAM50 classifier and was originally validated as an in vitro diagnostic (IVD) test on the Dx enabled nCounter ® Analysis System. The Prosigna test is intended for early-stage, hormone receptor+ (HR+) breast cancer and provides the risk of recurrence (ROR) score (0-100), intrinsic subtype (Luminal A, Luminal B, HER2-enriched, and Basal-like), and the 10-year probability of distant recurrence. We describe the performance of the Prosigna test as a whole transcriptome RNA sequencing laboratory developed test (LDT) for measuring the Prosigna ROR score and intrinsic subtypes on tissue from surgical resection and core needle biopsy as compared to the Prosigna test on the nCounter system. Methods We evaluated three separate breast cancer cohorts to 1) bridge the IVD test on the nCounter system and NGS LDT test (n = 245), 2) validate the bridged algorithm on an independent biobank sample set (n = 187), and 3) retrospectively test performance on long-term archival samples from a previous study (n = 109). Results Bridging analysis showed minimal score variability and robust correlation of Prosigna ROR scoring in surgical resections (SR) (2.459, SD; 0.981, R 2 ) and core needle biopsy (CNB) (2.338, SD; 0.970, R 2 ) samples. In the validation set, the Prosigna NGS LDT ROR scores maintained high correlation to the scores of the nCounter system (SR = 0.968, CNB = 0.966, R 2 ), exhibited minimal score variability (SR = 2.488, CNB = 2.558, SD), and demonstrated high concordance in subtype classifications (SR = 92.3% CNB = 92.8%). Further testing demonstrated comparable performance across tumor fractions, a lower limit of detection (LLOD) of 5 ng, and robustness to exogenous ethanol or genomic DNA contamination. When testing previously extracted RNA from the clinical cohort, we observed high correlation (0.974, R 2 ) and low variance (3.078, SD) of ROR scores with original values on the nCounter system, along with strong risk group (95.4%) and subtype (94.5%) concordance. Conclusions This study describes the analytical validation of the Prosigna NGS-based LDT measuring the Prosigna ROR score and intrinsic subtypes with robust analytical performance on SR and CNB specimens, providing confidence for clinicians utilizing the NGS-based version of this well-established test.

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