Abstract 2529: Comparison of small cell lung cancer transcriptional factor expression by immunohistochemistry, multiplex immunofluorescence, and a targeted RNA expression panel

Abstract Recent studies have identified distinct molecular subtypes of small cell lung cancer (SCLC) (SCLC-A, SCLC-N, SCLC-P, and SCLC-Y) based on ASCL1, NEUROD1, POU2F3, and YAP1 expression. In this study, we investigated the correlation between immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and RNA Salah Targeted Expression Panel (RNA STEP) for ASCL1, NEUROD1, POU2F3, and YAP1 in formalin-fixed-paraffin-embedded (FFPE) samples from 14 SCLC samples collected under an IRB-approved protocol (MCC19163). For the mIF evaluation, FFPE samples were immunostained using the AKOYA Biosciences OPAL TM 7-Color Automation IHC kit on the BOND RX autostainer. For RNA STEP, nanoString nCounter TagSet chemistry was used to measure gene expression levels of test FFPE samples relative to a reference pooled human normal mRNA control. All three methods demonstrated significant correlation for ASCL1 levels (Table 1). IHC and RNA STEP NEUROD1 results exhibited significant correlation. However, mIF NEUROD1 results did not correlate with the results of other methods likely due to non-specific positivity of the NEUROD1 mIF antibody. No strong correlation was observed between any of the methods for POU2F3 and YAP1. Only 2 of the 14 samples displayed positive POU2F3 protein expression by IHC; one of these also had elevated POU2F3 expression by RNA STEP. Importantly, the sample with the highest POU2F3 protein expression by IHC also had the highest values by mIF and RNA STEP. All samples were negative for YAP1 by IHC and RNA STEP. Notably, ASCL1 staining was primarily observed in CD56+ POU2F3- cells by mIF. These findings highlight the reliability and utility of IHC and RNA STEP in assessing transcriptional factors in SCLC. Also, mIF presents an innovative method for the simultaneous analysis of multiple markers using a single tissue section, enabling the concurrent detection of multiple markers within individual cells. Table 1. Statistical Significance (p-values) and Correlation Analysis (Spearman's Coefficients) IHC vs RNA STEP IHC vs mIF RNA STEP vs mIF p r P r p r ASCL1 0. 021 0. 62 0. 0001 0. 91 0. 010 0. 67 NEUROD1 0. 002 0. 78 0. 517 -0. 14 0. 671 0. 12 POU2F3 0. 201 0. 36 0. 422 0. 18 0. 045 0. 55 YAP1 0. 299 0. 30 0. 364 0. 20 0. 295 0. 30 Citation Format: Hilal Ozakinci, Aileen Alontaga, Jonathan V Nguyen, Carlos M Moran Segura, Daryoush Saeed-Vafa, Jhanelle Gray, Eric Haura, Alberto A Chiappori, Theresa Boyle. Comparison of small cell lung cancer transcriptional factor expression by immunohistochemistry, multiplex immunofluorescence, and a targeted RNA expression panel abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2529.