Transcriptomic validation of <i>ALK</i> fusions by RNA STEP with associated NaPi2b enrichment in lung adenocarcinoma.

Abstract

e20763 Background: The RNA Salah Targeted Expression Panel (RNA STEP) is an exploratory clinical transcriptomic assay capable of directly quantifying transcripts of 204 oncogenic, pre-selected genes in clinical samples. This study analyzes RNA STEP gene expression of ALK fusion positive ( +) samples to investigate RNA STEP as an orthogonal and complementary assay to next-generation sequencing (NGS). SLC34A2 is a gene that codes for the sodium-phosphate cotransporter 2B (NaPi-IIb), crucial for regulating phosphate levels (homeostasis) by transporting sodium and phosphate ions across cell membranes, mainly in lung alveolar cells for surfactant production. SLC34A2 mutations are linked to the rare lung disease pulmonary alveolar microlithiasis (PAM) with calcium phosphate buildup. SLC34A2 is frequently overexpressed in various cancers, including lung cancer, with promotion of cell growth and metastasis. Methods: Primary lung cancer samples (N = 524 total, 24 ALK +) were tested with both clinical NGS (Illumina TSO500 platform) and RNA STEP (nanoString platform). Gene expression by RNA STEP (log2 ratio) versus ALK fusion unique supporting reads by NGS were compared with Pearson correlation. Four RNA STEP cutoffs were compared for positive percentage agreement and accuracy with NGS. Differences in gene expression were compared for the ALK + versus ALK- samples with box and whisker plots and Mann-Whitney U test for significance. Results: Strong correlation was observed for ALK gene expression by RNA STEP versus supporting reads by NGS (R = 0.777 for 24 ALK+ samples). An ALK RNA STEP cutoff of log2 ratio ≥2 yielded a positive percentage agreement of 87.5% and accuracy of 97.9%. RNA STEP and NGS results correlated and were concordant with each other (optimized cut-off: log2 ratio ≥2), supporting the accuracy and value of RNA STEP as an orthogonal and complementary assay to NGS. Median expressions of the ALK , ROS1 , and SLC34A2 genes were significantly higher in the ALK + samples versus ALK- samples (p &lt; 0.001). Some ALK inhibitors also inhibit ROS1 and that when both ALK and ROS1 gene expression are high in an ALK fusion positive lung cancer, perhaps an inhibitor that targets both might be considered. Conclusions: Wild-type ALK gene expression correlated with ALK fusion supporting reads and positive gene expression demonstrated strong concordance with ALK fusion presence. RNA STEP provides clinically useful orthogonal transcriptomic validation of ALK fusions, particularly in cases with borderline results or atypical fusion partners or breakpoints beyond classic EML4::ALK . ROS1 and SLC34A2 (NaPi2b) expression were high in the ALK+ cohort in agreement with the literature, highlighting these markers as possible therapeutic targets for patients with ALK + lung adenocarcinoma.