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,TFPI1andVIM), 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.

Although population-wide screening programs for several cancer types have been implemented in multiple countries, screening procedures are invasive, time-consuming and often perceived as a burden for patients. Molecular biomarkers measurable in non-invasively collected samples (liquid biopsies) could facilitate screening, as they could have incremental value on early diagnosis of cancer, but could also predict prognosis or monitor treatment response. Although the shift towards biomarkers from liquid biopsies for early cancer detection was initiated some time ago, there are many challenges that hamper the development of such biomarkers. One of these challenges is large-scale validation that requires large prospectively collected biobanks with liquid biopsies. Establishing those biobanks involves several considerations, such as standardization of sample collection, processing and storage within and between biobanks. In this perspective, we will elaborate on several issues that need to be contemplated in biobanking, both in general and for certain specimen types specifically, to be able to facilitate biomarker validation for early detection of cancer.

The 5-yr survival of early-stage renal cell carcinoma (RCC) is approximately 93%, but once metastasised, the 5-yr survival plummets to 12%, indicating that early RCC detection is crucial to improvement in survival. DNA methylation biomarkers have been suggested to be of potential diagnostic value; however, their current state of clinical translation is unclear and a comprehensive overview is lacking. To systematically review and summarise all literature regarding diagnostic DNA methylation biomarkers for RCC. We performed a systematic literature review of PubMed, EMBASE, Medline, and Google Scholar up to January 2019, according to the Preferred Reporting Items for Systematic Review and Meta-Analysis of Diagnostic Test Accuracy Studies (PRISMA-DTA) guidelines. Included studies were scored according to the Standards for Reporting of Diagnostic Accuracy Studies (STARD) criteria. Forest plots were generated to summarise diagnostic performance of all biomarkers. Level of evidence (LoE) and potential risk of bias were determined for all included studies. After selection, 19 articles reporting on 44 diagnostic DNA methylation biomarkers and 11 multimarker panels were included; however, only 15 biomarkers were independently validated. STARD scores varied from 4 to 13 out of 23 points, with a median of 10 points. Large variation in subgroups, methods, and primer locations was observed. None of the reported biomarkers exceeded LoE III, and the majority of studies reported inadequately. None of the reported biomarkers exceeded LoE III, indicating their limited clinical utility. Moreover, study reproducibility and further development of these RCC biomarkers are greatly hampered by inadequate reporting. In this report, we reviewed whether specific biomarkers could be used to diagnose the most common form of kidney cancer. We conclude that due to limited evidence and reporting inconsistencies, none of these biomarkers can be used in clinical practice, and further development towards clinical use is hindered.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

Background: Very few (<0.1%) of DNA methylation biomarkers are eventually translated into clinical practice, even though over 5,000 have been published over the last decades. In an attempt to create an overview of the current evidence on these markers, we performed two systematic reviews on diagnostic DNA methylation biomarkers in liquid biopsies, for colorectal cancer (CRC) and renal cell carcinoma (RCC) (1). Here, we present the evidence of these systematic reviews and provide novel recommendations to improve the current clinical translation of DNA methylation biomarkers. Methods: For CRC, we identified 109 bodily fluid biomarker studies published before January 2019 in PubMed, Embase, Cochrane Library, or Google Scholar. For RCC, we identified 6 liquid biopsy studies up to January 2019 in these databases. Data extraction (study design, patient characteristics, disease stage, tumor location, technical assays, diagnostic measures) was performed on published reports. STARD criteria and Level of Evidence (LoE) were registered to assess reporting quality and strength for clinical translation, and forest plots were generated to summarize diagnostic performance of the biomarkers. Findings: Our systematic literature search revealed multiple issues that hamper the development of DNA methylation biomarkers for RCC and CRC diagnosis, including methodologic and technical heterogeneity and lack of validation or clinical translation. Among the most important issues were a lack of translation from tissue into liquid biopsy; for CRC 88/389 (23%) CRC markers were studied in liquid biopsies, and for RCC these numbers were 15/44 (34%). In addition, results showed a lack of independent validation, with 37/88 (42%) CRC markers and 9/15 (60%) RCC markers in liquid biopsies studied in more than one study or study population. Also, inappropriate marker identification and primer design, lack of true clinical need definition, and low reporting quality were issues that were recognized in our systematic literature searches. These issues all hamper the development of the field, keep the LoE low, and hinder the translation of DNA methylation biomarkers into clinical tests. Interpretation: Our systematic literature searches revealed that major requirements to develop clinically relevant diagnostic DNA methylation markers are often lacking. To avoid the resulting research waste, clinical needs, intended biomarker use, and independent validation should be better considered prior to study design. In addition, improved reporting quality would facilitate meta-analysis, thereby increasing LoE and enabling clinical translation. Reference: 1. Lommen et al. Eur Urol Oncol 2019; https://doi.org/10.1016/j.euo.2019.07.011. Citation Format: Kim Lommen, Zheng Feng, Cary J.G. Oberije, Alouisa J. P. van de Wetering, Selena Odeh, Alexander Koch, Maureen J. B. Aarts, Joep G. van Roermund, Leo J. Schouten, Egbert Oosterwijk, Nathalie Vaes, Ad A. M. Masclee, Beatriz Carvalho, Gerrit A. Meijer, Maurice P. Zeegers, James G. Herman, Vivianne C. Tjan-Heijnen, Veerle Melotte, Manon van Engeland, Kim Smits. Clinical translation of liquid biopsy DNA methylation biomarkers: Lessons from two systematic reviews [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A62.