RNAscope® ISH to detect lung cancer-related markers

Four-plex detection of biomarker and checkpoint markers in FFPE lung cancer tissues stained with RNAscope<sup>®</sup> Multiplex Fluorescent Assay v2 and RNAscope<sup>®</sup> 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

Four-plex detection of biomarker and checkpoint markers in FFPE lung cancer tissues stained with RNAscope® Multiplex Fluorescent Assay v2 and RNAscope® 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

RNA expression of FOXP3(red), CD274(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of FOXP3(red), CD274(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

RNA expression of CCR5 gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Assay Brown RNA in situ hybridization (ISH)

RNA expression of CCR5 gene in human lung cancer tissue using RNAscope® 2.5 HD Assay Brown

RNA expression of LAG3(red), CD274(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of LAG3(red), CD274(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

Expression of BCL6 in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Assay Brown RNA in situ hybridization (ISH)

Expression of BCL6 in human lung cancer tissue using RNAscope® 2.5 HD Assay Brown

RNA expression of CD8A(red), CD274(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of CD8A(red), CD274(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

RNA expression of IDO1(red), CD274(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of IDO1(red), CD274(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

RNA expression of IDO1(red), CD274(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of IDO1(red), CD274(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

RNA expression of GADD45G gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Assay Brown RNA in situ hybridization (ISH)

RNA expression of GADD45G gene in human lung cancer tissue using RNAscope® 2.5 HD Assay Brown

RNA expression of MET(red), EGFR(green) gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Duplex Assay RNA in situ hybridization (ISH)

RNA expression of MET(red), EGFR(green) gene in human lung cancer tissue using RNAscope® 2.5 HD Duplex Assay

RNA expression of H2AFX gene in human lung cancer tissue using RNAscope<sup>®</sup> 2.5 HD Assay Brown RNA in situ hybridization (ISH)

RNA expression of H2AFX gene in human lung cancer tissue using RNAscope® 2.5 HD Assay Brown

Lung cancer is the leading cause of cancer deaths worldwide. Lung tumors are highly heterogeneous, with great variability between individual patients, cancer types and stages, as well as intra-tumor heterogeneity. This variability poses a considerable challenge in identifying and validating clinically relevant lung cancer biomarkers. Therefore, spatially-mapped gene expression at the cellular level is crucial to understand the cell-to-cell interactions in the tumor and the cellular makeup within the complex tumor microenvironment (TME). 

The RNAscope® assay is a highly specific and sensitive RNA in situ hybridization (ISH) technology that identifies RNA expression at the single cell level with morphological context and with multiplexing capabilities. The RNAscope® ISH assay can be utilized in lung cancer research for:

  • Assessment of predictive markers for lung cancer
  • When antibodies are not available
  • Characterization of secreted proteins
  • Validation of transcriptome data
  • Detection of lncRNAs in lung cancer
  • Detection of immune cell checkpoint and functional lung cancer biomarkers in the TME

Assessment of predictive markers and patient stratification for treatment regimens for lung cancer: FGFR1 amplification is a potential driving oncogene in lung squamous cell cancer (SCC), a subtype of non-small cell lung carcinoma (NSCLC). Lim et al. evaluated the efficacy and tolerability of dovitinib, an FGFR inhibitor, in patients with advanced lung SCC. Treatment with dovitinib demonstrated modest efficacy in patients with advanced SCC with FGFR1 amplification, suggesting that gene amplification may not be the appropriate biomarker for FGFR inhibitor response. RNAscope® ISH showed that, among patient samples with amplified FGFR1, 25% were found to have no FGFR1 mRNA transcripts, demonstrating that expression of FGFR1 mRNA was not associated with the degree of FGFR1 gene amplification. These results suggest that identification of other biomarkers correlated with the efficacy of dovitinib in patients with SCC are necessary. 
Efficacy and Safety of Dovitinib in Pretreated Patients With Advanced Squamous Non-Small Cell Lung Cancer With FGFR1 Amplification: A Single-Arm, Phase 2 Study

When antibodies are not available: The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Brady et al. show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. To confirm that ARNTL2 is expressed within the cancer cells themselves in human lung adenocarcinoma, the authors used RNAscope® ISH to assess ARNTL2 expression in human lung adenocarcinoma sections, as they were unable to identify an anti-ARNTL2 antibody that worked for IHC. ARNTL2 expression was detected specifically in the cancer cells and these findings were consistent with the analysis of gene expression datasets. 
An Arntl2-Driven Secretome Enables Lung Adenocarcinoma Metastatic Self-Sufficiency

Characterization of secreted proteins: STAT3 plays an oncogenic role in several malignancies including lung cancer, making STAT3 a potential target for therapeutic intervention. Grabner et al. demonstrate that STAT3 exhibits an unexpected tumor-suppressive role in KRAS-mutant lung adenocarcinomas. RNAscope® ISH was used to show that NF-kB-induced IL-8 expression is increased when STAT3 is knocked down, thereby promoting tumor growth. They also show that high IL-8 expression is associated with poor prognosis in lung adenocarcinomas with a history of smoking. 
Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

Validation of transcriptome data and detection of lncRNAs in lung cancer: The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung carcinomas (NSCLC) and loss of LKB1 promotes cancer progression. LncRNA microarrays and NanoString assays revealed that the lncRNA LINC00473 expression was significantly different between WT and LKB1-mutant NSCLC samples. The RNAscope® ISH assay validated the expression levels of LINC00473 from NanoString assay results in situ, showing that 54% of mutant LKB1 NSCLC samples were positive for LINC00473. Given the poor specificity of LKB1 antibodies, this paper demonstrates that LINC00473 could be used as a surrogate biomarker for LKB1 in lung cancer samples. 
cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth

Detection of immune cell checkpoint and functional markers in the lung TME: The field of cancer immunotherapy continues to expand rapidly over recent years, with promising clinical results by immune checkpoint inhibitors and other therapeutic approaches. To better stratify patients for immunotherapy treatments, the series of events and biomarkers involved in the cancer‑immunity cycle need to be better understood. In this report the R&D labs at ACD examined 50 selected immune checkpoint and functional markers in multiple tumor types. With the RNAscope® duplex assay we demonstrated localization of PD-L1 and several immune markers in lung cancers. Overall these results demonstrate the utility of the RNAscope® technology in studying tumor immunology and key targets of immunotherapy in the tumor and its complex microenvironment. 
Detection of immune cell checkpoint and functional markers in the tumor microenvironment by the RNA in situ hybridization RNAscope® assay

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