Analytical performance of OncoPrism-HNSCC, an RNA-based assay to inform immune checkpoint inhibitor treatment decisions for recurrent/metastatic head and neck squamous cell carcinoma

While immune checkpoint inhibitor (ICI) therapies can significantly improve outcomes for patients with recurrent/metastatic head and neck squamous cell carcinoma (RM-HNSCC), only about 15–20% benefit from such treatments. Clinical tests that guide the use of ICIs are therefore critically needed. OncoPrism-HNSCC was developed to address this need. The assay combines next generation RNA sequencing-based immunomodulatory gene expression signatures with machine learning algorithms to generate an OncoPrism Score that classifies patients as having low, medium, or high likelihood of disease control in response to ICI treatment. Also, OncoPrism-HNSCC leverages the same FFPE patient tumor RNA used for ICI response prediction to identify rare cases where oncogenic rearrangements in NTRK1/2/3 or ALK genes, which may indicate the use of potentially highly effective targeted therapies. The clinical performance of OncoPrism-HNSCC has been validated. Here, we report its analytical performance in the presence of potentially confounding sources of variation. Intermediate precision. Technical replicates of RNA-seq libraries were made varying the operator, the reagant lot, the day the library was made, and the sequencer the library was sequenced on. %DV200 study: FFPE RNA with decreasing %DV200 values was generated using the NEBNext® Magnesium RNA Fragmentation Module (New England Biolabs, Ipswich, MA). For each fragmentation time point, 500 ng RNA in a volume of 18 µl water was combined with 2 µl 10X RNA Fragmentation Buffer in thin-walled PCR tubes. Tubes were transferred to a preheated (94˚C) thermocycler and incubated for 0, 1, 2, 3, 4, or 5 minutes. Fragmentation was terminated by transferring tubes to an ice-cold aluminum block and immediately adding and mixing 2 μl 10X RNA Fragmentation Stop Solution. Fragmented RNA was purified using the Zymo RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA) according to manufacturer’s instructions. %DV200 was calculated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). gDNA was spiked-in at increasing percentages on a per mass basis (0, 5, 10, 20, 30%) RNA input study: libraries were made using different RNA input amounts 10-80 ng. Technical replicates annotated as being from the "Reproducibility" study were sequenced on one sequencer using 40ng input.