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. 2024 Aug 28;5(11):100723.
doi: 10.1016/j.jtocrr.2024.100723. eCollection 2024 Nov.

Unveiling the Molecular Features of SCLC With a Clinical RNA Expression Panel

Hilal Ozakinci  1 , Aileen Y Alontaga  1 , Pedro Cano  2 , John M Koomen  2   3 , Bradford A Perez  4   5 , Amer A Beg  6 , Alberto A Chiappori  1 , Eric B Haura  1 , Theresa A Boyle  1   2
Affiliations

Unveiling the Molecular Features of SCLC With a Clinical RNA Expression Panel

Hilal Ozakinci et al. JTO Clin Res Rep. .

Abstract

Introduction: The translation of gene expression profiles of SCLC to clinical testing remains relatively unexplored. In this study, gene expression variations in SCLC were evaluated to identify potential biomarkers.

Methods: RNA expression profiling was performed on 44 tumor samples from 35 patients diagnosed with SCLC using the clinically validated RNA Salah Targeted Expression Panel (RNA STEP). RNA sequencing (RNA-Seq) and immunohistochemistry were performed on two different SCLC cohorts, and correlation analyses were performed for the ASCL1, NEUROD1, POU2F3, and YAP1 genes and their corresponding proteins. RNA STEP and RNA-Seq results were evaluated for gene expression profiles and heterogeneity between SCLC primary and metastatic sites. RNA STEP gene expression profiles of independent SCLC samples (n = 35) were compared with lung adenocarcinoma (n = 160) and squamous cell carcinoma results (n = 25).

Results: The RNA STEP results were highly correlated with RNA-Seq and immunohistochemistry results. The dominant transcription regulator by RNA STEP was ASCL1 in 74.2% of the samples, NEUROD1 in 20%, and POU2F3 in 2.9%. The ASCL1, NEUROD1, and POU2F3 gene expression profiles were heterogeneous between primary and metastatic sites. SCLCs displayed markedly high expression for targetable genes DLL3, EZH2, TERT, and RET. SCLCs were found to have relatively colder immune profiles than lung adenocarcinomas and squamous cell carcinomas, characterized by lower expression of HLA genes, immune cell, and immune checkpoint genes, except the LAG3 gene.

Conclusions: Clinical-grade SCLC RNA expression profiling has value for SCLC subtyping, design of clinical trials, and identification of patients for trials and potential targeted therapy.

Keywords: Biomarker; Clinical testing; RNA expression; Small cell lung cancer; Transcriptomics.

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Conflict of interest statement

Dr. Chiappori received funding from 10.13039/100002491Bristol-Myers Squibb for the clinical trial (MCC19163). Dr. Boyle and Dr. Koomen declare grants/contracts with Bristol-Myers Squibb unrelated to this research. Dr. Perez declares receiving grants from Bristol-Myers Squibb; providing consulting services for AstraZeneca, Bristol-Myers Squibb, G1 Therapeutics, and Novocure; and having board membership in Out of Zion. Dr. Haura declares providing consulting services for Kanaph Therapeutics and ORI Capital II; receiving research funding from 10.13039/100019364Revolution Medicines; and providing advisory services for RevMed and Janssen, all unrelated to this research. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) RNA STEP. Panel targets include 204 genes. (B) Overview of the samples and methods included in the study. RNA STEP, RNA Salah Targeted Expression Panel.
Figure 2
Figure 2
Comparison of RNA STEP, RNA-Seq, and IHC results. (A) RNA STEP and RNA-Seq revealed a statistically significant correlation for four transcriptional factors of SCLC (n = 17, p-values all <0.05). Numbers in the x and y axis are illustrated as log2 values of RNA-Seq TPM values and log2 ratio of RNA STEP, respectively. (B) Statistically significant correlations were observed in the comparison of RNA STEP (log2 ratio) and IHC (H score) results for the NEUROD1 and POU2F3 genes (n = 12, p < 0.05). (C) In the comparative analyses of RNA STEP and IHC results, the PPA or sensitivity reached 100% for the ASCL1 and NEUROD1 genes, whereas the NPA or specificity achieved 100% for the POU2F3 and NEUROD1 genes. The YAP1 gene was not assessable for PPA or PPV due to the absence of any positive results. IHC, immunohistochemistry; NPA, negative percent agreement; PPA, positive percent agreement; RNA-Seq, RNA sequencing; RNA STEP, RNA Salah Targeted Expression Panel.
Figure 3
Figure 3
ASCL1, NEUROD1, POU2F3, and YAP1 gene expression profile of SCLC. (A) RNA expression levels of ASCL1, NEUROD1, POU2F3, and YAP1 genes in the tested 35 tumor samples by RNA STEP. RNA STEP results of samples 1 to 31 and 32 to 33 were ordered by highest to lowest ASCL1 and NEUROD1 results, respectively. (B, C) Comparison of ASCL1, NEUROD1, POU2F3, and YAP1 gene expression levels in primary and metastatic tumors of same patient (#32, 54 y old, male) by RNA STEP. The distinct expression profiles observed in primary and metastatic tissues highlight substantial intrapatient heterogeneity. (B) Visual representation illustrating the gene expression profile of each tumor site. Created using BioRender.com. (C) Gene expression levels assessed by RNA STEP. RNA STEP, RNA Salah Targeted Expression Panel.
Figure 4
Figure 4
Gene expression analysis in SCLC and LUAD. RNA STEP revealed high expression of 26 genes in SCLC (mean log2 ratio ≥ 1 by RNA STEP). Among these, 22 genes exhibited significantly higher expression in SCLC compared with LUAD (p < 0.0001). The black bars represent the mean values. ∗No statistically significant difference was detected for the MAGEA1, MAGEA3, CDK4, and PTK74 genes between SCLC and LUAD. LUAD, lung adenocarcinoma; RNA STEP, RNA Salah Targeted Expression Panel.
Figure 5
Figure 5
Gene expression profile of SCLC (n = 35), adenocarcinoma (n = 160), and SqCC (n = 25). ∗Genes exhibiting elevated expression level in SCLC (mean log2 ratio ≥) and having a statistically significant higher in expression in SCLC compared with LUAD (p < 0.0001). LUAD, lung adenocarcinoma; SqCC, squamous cell carcinoma.
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