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Clinical significance of novel DNA methylation biomarkers for renal clear cell carcinoma

  • Original Article – Cancer Research
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Objective

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor characterized by the highest mortality rate of the genitourinary cancers, and, therefore, new diagnostic and/or prognostic biomarkers are urgently needed.

Methods

Based on genome-wide DNA methylation profiling in 11 pairs of ccRCC and non-cancerous renal tissues (NRT), the methylation at regulatory regions of ZNF677, FBN2, PCDH8, TFAP2B, TAC1, and FLRT2 was analyzed in 168 renal tissues and 307 urine samples using qualitative and quantitative methylation-specific PCR (MSP).

Results

Significantly higher methylation frequencies for all genes were found in ccRCC tissues compared to NRT (33–60% vs. 0–11%). The best diagnostic performance demonstrated a panel of ZNF677, FBN2, PCDH8, TFAP2B & TAC1 with 82% sensitivity and 96% specificity. Hypermethylation of ZNF677 and PCDH8 in the tissue samples was significantly related to numerous adverse clinicopathologic parameters. For the urine-based ccRCC detection, the highest diagnostic power (AUC = 0.78) was observed for a panel of ZNF677 & PCDH8 (with or without FBN2 or FLRT2) with 69–78% sensitivity and 69–80% specificity, albeit with lower values in the validation cohort. Besides, methylation of PCDH8 was significantly related to higher tumor stage and fat invasion in the study and validation cohorts. Moreover, PCDH8 was strongly predictive for OS (HR, 5.7; 95% CI 1.16–28.12), and its prognostic power considerably increased in combination with ZNF677 (HR, 12.5; 95% CI 1.47–105.58).

Conclusion

In summary, our study revealed novel, potentially promising DNA methylation biomarkers of ccRCC with the possibility to be applied for non-invasive urine-based ccRCC detection and follow-up.

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Availability of data and materials

All data supporting the results reported in the article is available from the corresponding author upon a reasonable request.

Abbreviations

AUC:

Area under the curve

ASC:

Asymptomatic control

ccRCC:

Clear cell renal cell carcinoma

CI:

Confidence interval

MC:

In vitro methylated control

MSP:

Methylation-specific polymerase chain reaction

qMSP:

Quantitative methylation-specific polymerase chain reaction

NRT:

Non-cancerous renal tissue

NTC:

No-template control

HR:

Hazard ratio

UC:

Unmethylated control

M:

Methylated

U:

Unmethylated

ROC:

Receiver operating characteristic

AUC:

Area under the curve

DSe:

Diagnostic sensitivity

DSp:

Diagnostic specificity

WHO/ISUP:

World health organization/International Society of Urological Pathology

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Acknowledgements

The authors would like to thank Aušra Šumskaitė, Rūta Matuliavičiūtė, and Eugenijus Ganža for their assistance in DNA samples preparation and MSP analysis.

Funding

This work was funded by the 2014–2020 European Union Structural Funds according to the activity "Intelligence. Joint science-business projects" grant No. J05-LVPA-K-04–0029. SJ and KZ were supported by the European Social Fund according to the activity "Development of students' ability to carry out R&D activities" under Measure No.09.03.3-LMT-K-712 "Development of Scientific Competences of Scientists, other Researchers and Students through Practical Research Activities" (grant No. 09.03.3-LMT-K-712–15-0214 to SJ). The study was also partially supported by the Research Council of Lithuania (RCL) grant No. S-MIP-17/54.

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Authors and Affiliations

  1. Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania

    Raimonda Kubiliūtė, Kristina Žukauskaitė, Algirdas Žalimas & Sonata Jarmalaitė

  2. National Cancer Institute, Santariskiu 1, LT‑08406, Vilnius, Lithuania

    Raimonda Kubiliūtė, Kristina Žukauskaitė, Algirdas Žalimas, Albertas Ulys, Rasa Sabaliauskaitė, Arnas Bakavičius & Sonata Jarmalaitė

  3. Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

    Arnas Bakavičius, Arūnas Želvys & Feliksas Jankevičius

  4. Faculty of Medicine, Institute of Clinical Medicine, Vilnius University, Vilnius, Lithuania

    Arūnas Želvys & Feliksas Jankevičius

Authors
  1. Raimonda Kubiliūtė
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  2. Kristina Žukauskaitė
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  3. Algirdas Žalimas
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  9. Sonata Jarmalaitė
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Contributions

RK: performed the DNA methylation and gene expression analysis in the tissue samples and urine samples of the validation cohort, analyzed the experimental and clinical data and drafted the manuscript; KZ: performed the DNA methylation analysis in urine samples of the study cohort, contributed to gene expression analysis; AZ, AU, RS and AB: collected the clinical data and revised the analysis; RS and AZ: critically revised the manuscript; AU and FJ: coordinated the patient selection, supervised the clinical data analysis and was involved in the conception of the study; SJ: designed the research, supervised the analysis of the clinical and experimental data, and revised the manuscript critically for important intellectual content. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved.

Corresponding author

Correspondence to Sonata Jarmalaitė.

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Competing interests

RK and SJ are inventors of the patent application No. PCT/IB2021/052532. The remaining authors declare that they have no conflict of interest.

Ethics approval and consent to participate

The study was approved by the Lithuanian Bioethics Committee (Nr. 158200˗18/12˗1077˗585 for Study cohort and Nr. 158200-18/12-1077-585 for Validation cohort), and written informed consent was obtained from all patients.

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Kubiliūtė, R., Žukauskaitė, K., Žalimas, A. et al. Clinical significance of novel DNA methylation biomarkers for renal clear cell carcinoma. J Cancer Res Clin Oncol 148, 361–375 (2022). https://doi.org/10.1007/s00432-021-03837-7

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