Abstract 1568: Effects of melanin on NanoString gene and miRNA expression assays for utilization in biomarker analysis for melanoma
Authors: Kurmis Alexis , Kelly Eileen , Valencia Nancy , Wahl Justin , Laing Christian , Riccitelli Nathan , Pollner Reinhold 
Published: September 2020

Abstract
Purpose
Formalin-fixed, paraffin-embedded (FFPE) tumor tissue, while standard for biological preservation, can significantly impact nucleic-acid quality via degradation and cross-linking. The low quality of FFPE-treated samples, coupled with the presence of native small molecules inhibitors in various tissue types, yields significant challenges for PCR-based technologies. Alternatively, NanoString RNA assays combine straightforward workflows with multiplexed detection of up to 800 targets via hybridization-based chemistry that is not reliant on enzymatic processes, and thus may offer advantages in a melanin-rich environment. For utilization in a melanoma disease setting, we evaluated the impact of different melanin-levels in FFPE samples on the performance of NanoString mRNA and miRNA assays
Methods
One FFPE cell line block was sectioned and spiked with zero or a range of biologically-relevant levels of melanin, both before and after total nucleic acid isolation with the Qiagen AllPrep FFPE procedure. Total RNA content was measured by NanoDrop and fluorescent-based methods. Abundance of individual RNA molecules was assessed by the NanoString IO360 gene expression assay and the NanoString Human v3 miRNA panel.
Results
Melanin concentration was observed to directly impact quantification results, with higher concentrations correlating to elevated estimates of total RNA yield by NanoDrop, but reduced RNA by fluorescent-based measurement. Different melanin concentrations did not impact RNA expression measurements in the enzyme-free IO360 gene expression assay. For the v3 miRNA assay, however, increased melanin concentration resulted in significant reduction in miRNA signal, but as observed with the IO360 assay, did not affect endogenous mRNA transcript detection. The reduction in miRNA levels was significantly greater for the samples spiked with melanin post-extraction compared with those aliquots spiked prior to isolation. Tumor inflammation signature (TIS) scoring was not impacted by elevated melanin levels.
Conclusions
NanoString hybridization reactions exhibited minimal sensitivity to melanin content. Conversely, NanoString assays requiring enzymatic processing yielded substantially reduced counts at elevated melanin concentrations. Isolation procedures were effective in removing lower levels of melanin, but could not fully mitigate melanin impact at all tested concentrations. Significantly, classification of tumor immune-environment by NanoString was not affected by melanin, potentially making TIS scoring an attractive tool for biomarker analysis of checkpoint inhibitor therapies in melanoma.
Citation Format: Alexis Kurmis, Eileen Kelly, Nancy Valencia, Justin Wahl, Christian Laing, Nathan Riccitelli, Reinhold Pollner. Effects of melanin on NanoString gene and miRNA expression assays for utilization in biomarker analysis for melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1568.