Development and validation of a tumor-derived CXCL1 qPCR assay to support patient selection for anti-CXCL1 therapeutics in bladder cancer

Toru Sakatani; Sunao Tanaka; Kaoru Murakami; Cynthia Jinno; Francisco Aguilar; Catherine Bresee; Owen T M Chan; Daniel J Luthringer; Alberto Contreras-Sanz; Peter Black; Charles J Rosser; Hideki Furuya

doi:10.1016/j.yexmp.2026.105039

Development and validation of a tumor-derived CXCL1 qPCR assay to support patient selection for anti-CXCL1 therapeutics in bladder cancer

Exp Mol Pathol. 2026 Mar 16:146:105039. doi: 10.1016/j.yexmp.2026.105039. Online ahead of print.

Authors

Affiliations

¹ Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
² Biostatistics Shared Resources, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
³ Clinical and Translational Research Program, University of Hawaii Cancer Center, Honolulu, HI, USA.
⁴ Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
⁵ Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada.
⁶ Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Department of Urology, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Nonagen Therapeutics Corp, Los Angeles, CA 90010, USA.
⁷ Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Department of Biomedical Science, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Electronic address: hideki.furuya@cshs.org.

PMID: 41843975
DOI: 10.1016/j.yexmp.2026.105039

Abstract

The CXCR2-CXCL1 axis is critical in tumorigenesis, specifically, CXCL1 is a secreted chemokine implicated in tumor progression, angiogenesis, and immune modulation. Although elevated CXCL1 expression has been associated with poor outcomes in cancers, its evaluation by immunohistochemistry (IHC) is limited by diffusion and stromal expression. We developed a qPCR-based assay to objectively quantify CXCL1 mRNA in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. We analytically validated the CXCL1 qPCR assay using FFPE tissues from muscle-invasive bladder cancer (MIBC) patients. Assay performance was evaluated across preanalytical (tissue preparation, storage conditions), analytical (RNA input, quality, necrosis), and reproducibility parameters. The assay was then applied to a cohort of 47 MIBC patients who underwent radical cystectomy after neoadjuvant chemotherapy, and qPCR expression values were validated against RNA-seq TPM. The assay showed high concordance between fresh frozen and FFPE tissues (P = 0.78), stable RNA profiles under appropriate storage, and robust performance across a 5-100 ng input range when RNA DV200 exceeded 15%. Expression remained reliable in samples with up to 80% necrosis. Inter-operator reproducibility was strong (Pearson r = 0.80). In the clinical cohort, CXCL1 expression by qPCR showed excellent agreement with RNA-seq expression in 47 MIBC patients (Pearson r = 0.88), confirming the assay's robustness and cross-platform validity. We present a robust, analytically validated qPCR assay for quantifying CXCL1 expression in FFPE tumor specimens. The assay demonstrated excellent concordance with RNA-seq expression values, supporting its reliability for measuring CXCL1 mRNA expression in bulk tumor specimens and its potential utility as a companion diagnostic in future CXCR-2-CXCL1-targeted clinical trials.

Keywords: Biomarker validation; Bladder cancer; CXCL1; Chemokine; Companion diagnostic; qPCR assay.