Technical considerations in PCR-based assay design for diagnostic DNA methylation cancer biomarkers

Maartje Massen, Kim Lommen, Kim A.D. Wouters, Johan Vandersmissen, Wim van Criekinge, James G. Herman, Veerle Melotte, Leo J. Schouten, Manon van Engeland, Kim M. Smits*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Background: DNA methylation biomarkers for early detection, risk stratification and treatment response in cancer have been of great interest over the past decades. Nevertheless, clinical implementation of these biomarkers is limited, as only < 1% of the identified biomarkers is translated into a clinical or commercial setting. Technical factors such as a suboptimal genomic location of the assay and inefficient primer or probe design have been emphasized as important pitfalls in biomarker research. Here, we use eleven diagnostic DNA methylation biomarkers for colorectal cancer (ALX4, APC, CDKN2A, MGMT, MLH1, NDRG4, SDC2, SFRP1, SFRP2, TFPI1 and VIM), previously described in a systematic literature search, to evaluate these pitfalls. Results: To assess the genomic assay location, the optimal genomic locations according to TCGA data were extracted and compared to the genomic locations used in the published assays for all eleven biomarkers. In addition, all primers and probes were technically evaluated according to several criteria, based on literature and expert opinion. Both assay location and assay design quality varied widely among studies. Conclusions: Large variation in both assay location and design hinders the development of future DNA methylation biomarkers as well as inter-study comparability.

Original languageEnglish
Article number56
JournalClinical Epigenetics
Issue number1
Publication statusPublished - 27 Apr 2022

Bibliographical note

Funding Information:
This work was supported by KWF Kankerbestrijding [UM-2-13-6075] and by SU2C-DCS International Translational Cancer Research Dream Team Grant [Stand Up To Cancer (SU2C)-AACR- DT1415, MEDOCC].

Publisher Copyright: © 2022, The Author(s).


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