Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors.

We have developed a next-generation sequencing assay to quantify biomarkers of the host immune response in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. This assay aims to provide clinicians with a comprehensive characterization of the immunologic tumor microenvironment as a guide for therapeutic decisions on patients with solid tumors. The assay relies on RNA-sequencing (seq) to semiquantitatively measure the levels of 43 transcripts related to anticancer immune responses and 11 transcripts that reflect the relative abundance of tumor-infiltrating lymphocytes, as well as on DNA-seq to estimate mutational burden. The assay has a clinically relevant 5-day turnaround time and can be conducted on as little as 2.5 ng of RNA and 1.8 ng of genomic DNA extracted from three to five standard FFPE sections. The standardized next-generation sequencing workflow produced sequencing reads adequate for clinical testing of matched RNA and DNA from several samples in a single run. Assay performance for gene-specific sensitivity, linearity, dynamic range, and detection threshold was estimated across a wide range of actual and artificial FFPE samples selected or generated to address preanalytical variability linked to specimen features (eg, tumor-infiltrating lymphocyte abundance, percentage of necrosis), and analytical variability linked to assay features (eg, batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate compared with established orthogonal approaches.