Hierarchical clustering defines hypermethylated RSPO2 as early-stage potential biomarker in colorectal cancer

Abstract

Background Early-stage diagnosis, absence of specific reliable biomarkers, and better clinical management of colorectal cancer (CRC) remain major challenges. The Rspondin2 (RSPO2) gene is one of the most potent enablers of Wnt signaling, having contrasting aspects in tumor development and progression.

Aim We aimed to identify the role of RSPO2 as a molecular biomarker by evaluating its methylation and expression profiles in CRC tissue samples to identify biomarker potential.

Methodology Combined bisulfite restriction analysis (CoBRA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access the change in methylation pattern and expression profile of RSPO2 in 52 CRC tissue samples. DNA methylome and transcriptome profiling of CRC samples were hierarchically clustered, and prognostic utility was measured using a Kaplan-Meier (K-M) plot and univariate analysis.

Results RSPO2 was hypermethylated (33/52; 63.46 %) and significantly associated with early-stage (I+II, p = 0.04) CRC tissue and exhibited low RSPO2 expression (42/52; 80.76 %). Hierarchical clustering stratified the samples into two distinct clusters that share some significant clinical and molecular factors specific for cluster 1 such as LN status (p < 0.0001) and lymphovascular invasion (p = 0.0013) while staging (p < 0.0001) and RSPO2 methylation (p = 0.0402) in cluster 2, which are valuable for personalized medicine. K-M plot analysis showed that patients with low RSPO2 expression have poor disease-free survival but could not be a risk factor.

Conclusion RSPO2 may be a potential biomarker for early-stage detection and provide valuable insight into the diagnosis of patients with CRC.

Keywords

Colorectal cancer / RSPO2 / Methylation / Expression / Biomarker / Prognostic

Writing assistance disclosure

No writing assistance was utilized in the production of this manuscript.

Author statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Author contributions

Ankit Srivastava: Data curation, writing - original draft, methodology, literature review, and experiments performed. Gaurav Kant: Literature review and drafting. Manish Pratap Singh: Literature review and drafting. Sameer Srivastava: Supervision, conceptualization, and review & editing.

Ethical conduct of research

Ethical approval for the present study has been granted from the Institute (Ref. no. IEC/2024-25/02) and the hospital ethics committee (Ref. no. Res/BR/TRB-12/2018/14). All procedures performed in the present study were in compliance with institutional guidelines and national/international ethical standards. All the patients who participated in the present study gave their consent according to the institutional guidelines.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors declare that AI-based tools and technologies were not used in this manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors are thankful to the Director, Motilal Nehru National Institute of Technology, Allahabad, India, for providing research facilities to conduct the research and Biorepository, Rajiv Gandhi Cancer Institute & Research Centre (RGCIRC), New Delhi, for providing tissue samples and patient clinicopathological data. The authors are also grateful to the Ministry of Human Resource and Development, Govt. of India, New Delhi, for providing fellowship during this tenure.

Appendix A. Supplementary material

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.gmg.2025.100078.

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