OmniSeq Inc scientists 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 immunological tumor microenvironment as a guide for therapeutic decisions on patients with solid tumors. The assay relies on RNA-seq to semi-quantitatively measure the levels of 43 transcripts related to anticancer immune responses and 11 transcripts reflecting the relative abundance of tumor-infiltrating lymphocytes, as well as on DNA-seq to estimate mutational burden. The assay has a clinically relevant five-day turnaround time and can be conducted on as little as 2.5 ng RNA and 1.8 ng 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 with respect to gene-specific sensitivity, linearity, dynamic range, as well as detection threshold was estimated across a wide range of actual and artificial FFPE samples selected or generated to address i) pre-analytical variability linked to specimen features (eg, tumor-infiltrating lymphocyte abundance, percent of necrosis), and ii) analytical variability linked to assay features (eg, batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate as compared to established orthogonal approaches.
Overview of the assay
A: The assay relies on a next-generation sequencing (NGS) platform to simultaneously measure gene expression (GEX) of 54 transcripts related to tumor-infiltrating lymphocytes (TILs) and other immunological functions in the tumor microenvironment, as well as neoplastic mutational burden (MuB). B: The workflow consists of a first evaluation of slides by a pathologist, optional macrodissection of tumor tissue, single-step RNA and DNA extraction, followed by target amplification and NGS of both RNA and DNA libraries in a unique sequencing run. NGS data are processed through the bioinformatics pipeline with run quality control, reporting and pathologist sign-out in a custom laboratory information system. C: Performance is evaluated for several pre-analytical and analytical parameters by a series of reproducibility studies, as well as by experiments demonstrating sensitivity, accuracy, and specificity across a broad range of tumor types.