Background and Purpose: The field of immuno-oncology (IO) covers a broad set of research disciplines and presents a highly diverse set of experimental requirements. The challenges include sample types of varying quality and quantity of material (including both small fixed samples and blood products) as well as an expanding multiplicity of targets to assay for immunological response. The HTG EdgeSeq system combines HTG's proprietary quantitative nuclease protection assay chemistry with a Next Generation Sequencing (NGS) platform to enable semiquantitative analysis of hundreds of targeted genes in a single assay. The novel HTG EdgeSeq Immuno-Oncology Assay measures 549 IO-related genes and can be used with most clinically relevant samples.

Methods: An internal verification study was performed to evaluate a five-point, two-fold titration series (6 concentrations) in a variety of sample types with as few as a 250 cells, 32 μl of PAXgene, or 1.56 mm2 of a 5 μm FFPE section. FFPE sample types included: melanoma; DLBCL; and tissue from lung, breast, colon and renal carcinomas. Additionally, examples of the biological relevance of this data are provided by examination of expression from several pairs of samples, each profiled using a single 5 μm FFPE section.

Results: Equivalent expression across the dynamic range was obtained within each of the sample types (Pearson correlations ranging between 0.84 to 0.98). For DLBCL, while most canonical markers of immune cells showed similar patterns of expression, different patterns of expression of T cell and NK cell genes were obtained, likely indicating a different composition of the immune cell infiltrates within these tumors. Specific to the two different melanoma tumors the lymphocyte infiltrates appear to be similar in these tumors. Interestingly, a markedly different immune response appears to be occurring in the melanoma tumors. One tumor appears to be mounting a significant type I interferon response, which is not as apparent in the second tumor. Differential expression of other well-known metastasis-associated genes are also observed between the two tumors. These observations suggest that patterns of differential expression could be used to assist in directing patients to targeted therapies.

Conclusion: The HTG EdgeSeq Immuno-Oncology Assay provides a valuable tool for researchers exploring the immune response to tumors across a wide variety of tissues. Combining highly reproducible results with very small sample input amounts allows the assay to be utilized for the very small and precious samples available to researchers in this field.

Citation Format: Monica M. Reinholz, Debrah Thompson, James Cooley, Xiao-Bo Chen, Iris Howlett, John Luecke, Patrick Roche, Bonnie LaFleur. Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2237.