Background: Cancer-testis antigens (CTAs) have restricted expression in normal adult tissues but have been found to be overexpressed in multiple tumors.  This and their ability to elicit spontaneous cellular and humoral immune responses have rendered CTAs as good candidate targets for cancer immunotherapy. CTA overexpression is variable and influenced by factors such as tumor stage, grade, treatment and detection methods.  Here we report the use of a targeted RNA sequencing panel that accurately detects CTAs in solid tumors as a  screening tool for cancer vaccine and cell-based immunotherapies.

Methods: Studies were designed to characterize the analytical performance of an RNA sequencing assay targeting expression of six CTAs which have shown potential as prognostic biomarkers and immunotherapeutic targets: CTAG1B (NY-ESO-1), CTAG2 (LAGE-1A), MAGEA1, MAGEA3, MAGEA4, and SSX2. Performance variables with respect to gene-specific amplicon specificity, linearity and limits of detection were estimated with various mixing studies and input RNA levels.  The effects of the tumor micro-environment (adjacent benign tissue, necrosis) on CTA expression was evaluated by including these potential interferents in the assay.  Analytical precision including intra-assay, inter-assay, and inter-operator reproducibility was measured by testing replicate RNA isolates. Accuracy was determined by comparing the CTA transcripts with those from established IHC, RT-PCR and whole transcriptome assays.  CTA transcript stability in FFPE specimens was evaluated in serial sections from blocks with routine storage and compared to originating matched fresh frozen specimens.

Results: RNA stability was demonstrated by high degree of CTA expression correlation between matched frozen and FFPE samples. Analytic accuracy, sensitivity and specificity was demonstrated by high correlation between CTA RNA-Seq and RT-PCR results, and IHC where available. Reproducibility results show little variation between runs and operators.  The studies established a baseline threshold of ≥20 reads per million (RPM) for the lower limit of gene expression detection and is the threshold used to interpret the CTA expression as “Positive” or “Negative”.

Conclusion: The analytical performance of the RNA sequencing assay for reporting CTAs has been validated for clinical use using FFPE  specimens from multiple tumors .  With ability to process many samples within a single run and a 10ng RNA input each, , the assay is a robust method for identifying tumors  that overexpress  tumor-specific CTAs as potential targets for  immunotherapies including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors.