The analysis of biomarkers in biological fuids, also known as liquid biopsies, is seen with great potential to diagnose complex diseases such as cancer with a high sensitivity and minimal invasiveness. Although it can target any biomolecule, most liquid biopsy studies have focused on circulating nucleic acids. Historically, studies have aimed at the detection of specifc mutations on cell-free DNA (cfDNA), but recently, the study of cell-free RNA (cfRNA) has gained traction. Since 2020, a handful of cfDNA tests have been approved for therapy selection by the FDA, however,
no cfRNA tests are approved to date. One of the main drawbacks in the feld of RNA-based liquid biopsies is the low reproducibility of the results, often caused by technical and biological variability, a lack of standardized protocols and insufcient cohorts. In this review, we will identify the main challenges and biases introduced during the diferent stages of biomarker discovery in liquid biopsies with cfRNA and propose solutions to minimize them.
Cite this article
Author information
- Institut de Biologia Evolutiva, Universitat Pompeu Fabra, Barcelona, Spain
- Lluc Cabús & Esther Lizano
- Flomics Biotech, Barcelona, Spain
- Lluc Cabús, Julien Lagarde, Joao Curado & Jennifer Pérez-Boza
Contributions
LC was the main contributor to the manuscript. JPB designed this manuscript. LC and JPB contributed to the final version of the manuscript. JPB, JL, JC and EL revised the manuscript. The authors read and approved the final manuscript.
Corresponding author
Correspondence to Jennifer Pérez-Boza.
Funding
Abbreviations
cfRNA: Cell-free RNA
miRNAs: MicroRNAs
mRNAs: Messenger RNAs
lncRNAs: Long non-coding RNAs
EVs: Extracellular vesicles
RNA-seq: RNA sequencing
UMIs: Unique Molecular Identifiers
ML: Machine learning
GBSI: Global Biological Standards Institute
Ethics declarations
Not applicable.
Consent for publication
Not applicable.
Competing interests
LC, JL and JPB are employees, JC and EL are co-founders of Flomics, a biotechnology company providing human plasma cell-free RNA sequencing research and services.
Rights and permissions
Reprints and permissions
According to a recent market research study, the liquid biopsy industry is expected to exceed 5.8 billion dollars by 2026 [81], although few cfDNA tests are already in use in the clinical practice [82]. In 2020, the FDA already approved three cfDNA-based tests and, of these, Guardant Health’s Guardant360 CDx is the frst one to use next generation sequencing for diagnosis [83]. Since the feld of cfRNA is still young, there are no diagnostic tests based on RNA approved for the use in the clinical practice yet. Tere are many challenges to be addressed to translate an RNA-based liquid biopsy biomarker into the clinic, although several studies are currently undergoing clinical trials [84–88]. A robust and standardized methodology needs to be established, assessing all the possible biases that can alter the results. Tis will lead to more reproducible results and more robust statistical models.
One of the main challenges in the feld of liquid biopsies is caused by a limitation on the number of donors in the training cohorts. Most of the studies comparing cases with controls use small retrospective cohorts to detect a disease after it is clinically reported [52, 89–91], which
is suboptimal for biomarkers for early diagnosis. Only a handful of prospective studies have attempted population screening to fnd undiagnosed patients [92]. Tis approach, although optimal for diagnostic biomarker discovery and validation, requires the screening of a signifcant part of the population (depending on disease prevalence) and a great budgetary efort. Te technical
and economical requirements for such an attempt are beyond the grasp of most research centers and the biomarkers discovered in the frst cohorts are not strong enough to pass an initial step of validation and attract big-pharma companies. Although multiple collaborative consortia are created to compile biological data for specifc pathologies (such as the NCI Cohort Consortium for
cancer), the lack of standardized methods often leads to results that highlight technical diferences over biologically relevant biomarkers.
Another important aspect for the incorporation of liquid biopsies into the clinical practice is the ability of the physicians to be able to interpret the results of the biomarker model. Tis can be an arduous task for the medical staf that is not profcient in bioinformatics and statistical analysis. For this purpose, several research centers and companies have created cloud computing
pipelines that take directly the sequencing data and generate comprehensive reports
Te feld of liquid biopsies, and more specifcally cell-free long RNA liquid biopsies is promising, but still young. With a relatively reduced number of studies published, there are candidate biomarkers undergoing clinical trials, but none have been approved by the regulatory agencies at the moment. In the last few years, there has been an increasing interest in liquid biopsy-based biomarkers using RNAs. However, it has been only in the last 5 years that the focus has started to switch from miRNAs to long RNAs, leading to the discovery of new disease-associated RNAs. Although there is still much work left to do to translate long cfRNA into clinical practice, a number of recent promising results suggest that long cfRNA-based liquid biopsies could be one of the next big revolutions in the feld of screening and diagnosis.
References
2. Rasmussen M, Reddy M, Nolan R, Camunas-Soler J, Khodursky A, Scheller NM, et al. RNA profiles reveal signatures of future health and disease in pregnancy. Nature. 2022;601(7893):422–7.
3. Moufarrej MN, Vorperian SK, Wong RJ, Campos AA, Quaintance CC, Sit RV, et al. Early prediction of preeclampsia in pregnancy with cell-free RNA. Nature. 2022;602(7898):689–94.
4. Mathios D, Johansen JS, Cristiano S, Medina JE, Phallen J, Larsen KR, et al. Detection and characterization of lung cancer using cell-free DNA fragmentomes. Nat Commun. 2021;12(1):5060.
5. Vandekerkhove G, Lavoie JM, Annala M, Murtha AJ, Sundahl N, Walz S, et al. Plasma ctDNA is a tumor tissue surrogate and enables clinical-genomic stratification of metastatic bladder cancer. Nat Commun. 2021;12(1):184.
6. Ladabaum U, Alvarez-Osorio L, Rösch T, Brueggenjuergen B. Cost-effectiveness of colorectal cancer screening in Germany: current endoscopic and fecal testing strategies versus plasma methylated Septin 9 DNA. Endosc Int Open. 2014;2(2):E96–104.
7. Lee B, Mahmud I, Marchica J, Dereziński P, Qi F, Wang F, et al. Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery. Sci Rep. 2020;10(1):3716.
8. Dudley JC, Schroers-Martin J, Lazzareschi DV, Shi WY, Chen SB, Esfahani MS, et al. Detection and surveillance of bladder cancer using urine tumor DNA. Cancer Discov. 2019;9(4):500–9.
9. Hossein-nezhad A, Fatemi RP, Ahmad R, Peskind ER, Zabetian CP, Hu SC, et al. Transcriptomic Profiling of Extracellular RNAs Present in Cerebrospinal Fluid Identifies Differentially Expressed Transcripts in Parkinson’s Disease. J Parkinsons Dis. 2016;6(1):109–17.
10. Kopkova A, Sana J, Machackova T, Vecera M, Radova L, Trachtova K, et al. Cerebrospinal fluid MicroRNA signatures as diagnostic biomarkers in brain tumors. Cancers. 2019;11(10):1546.
11. Pessoa LS, Heringer M, Ferrer VP. ctDNA as a cancer biomarker: a broad overview. Crit Rev Oncol Hematol. 2020;155:103109.
12. Chattopadhyay I. Application of Circulating Cell-free DNA for Personalized Cancer Therapy. In: Precision Medicine in Oncology. Wiley; 2020. p. 83–97. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119432487.ch3 [cited 7 Mar 2022].
13. Cowling T, Loshak H. An Overview of Liquid Biopsy for Screening and Early Detection of Cancer. In: CADTH Issues in Emerging Health Technologies. Ottawa: Canadian Agency for Drugs and Technologies in Health; 2016. Available from: http://www.ncbi.nlm.nih.gov/books/NBK555478/ [cited 7 Mar 2022].
14. Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and death of circulating cell-free DNA. Cancer Biol Ther. 2019;20(8):1057–67.
15. Zaporozhchenko IA, Ponomaryova AA, Rykova EY, Laktionov PP. The potential of circulating cell-free RNA as a cancer biomarker: challenges and opportunities. Expert Rev Mol Diagn. 2018;18(2):133–45.
16. Yi Z, Ma F, Rong G, Liu B, Guan Y, Li J, et al. The molecular tumor burden index as a response evaluation criterion in breast cancer. Signal Transduct Target Ther. 2021;6(1):1–8.
17. Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, et al. Exosome RNA Unshielding couples stromal activation to pattern recognition receptor signaling in Cancer. Cell. 2017;170(2):352–66 e13.
18. Byron SA, Van Keuren-Jensen KR, Engelthaler DM, Carpten JD, Craig DW. Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Nat Rev Genet. 2016;17(5):257–71.
19. El Marabti E, Younis I. The Cancer Spliceome: reprograming of alternative splicing in Cancer. Front Mol Biosci. 2018;5:80.
20. Ben-Aroya S, Levanon EY. A-to-I RNA editing: an overlooked source of Cancer mutations. Cancer Cell. 2018;33(5):789–90.
21. Glinge C, Clauss S, Boddum K, Jabbari R, Jabbari J, Risgaard B, et al. Stability of circulating Blood-based MicroRNAs – pre-analytic methodological considerations. PLoS One. 2017;12(2):e0167969.
22. Gu M, Zheng A, Tu W, Zhao J, Li L, Li M, et al. Circulating LncRNAs as novel, non-invasive biomarkers for prenatal detection of fetal congenital heart defects. Cell Physiol Biochem. 2016;38(4):1459–71.
23. Feng L, Liao YT, He JC, Xie CL, Chen SY, Fan HH, et al. Plasma long non-coding RNA BACE1 as a novel biomarker for diagnosis of Alzheimer disease. BMC Neurol. 2018;18:4.
24. Lin Y, Leng Q, Zhan M, Jiang F. A plasma long noncoding RNA signature for early detection of lung Cancer. Transl Oncol. 2018;11(5):1225–31.
25. Frankish A, Diekhans M, Jungreis I, Lagarde J, Loveland JE, Mudge JM, et al. GENCODE 2021. Nucleic Acids Res. 2021;49(D1):D916–23.
26. Kozomara A, Birgaoanu M, Griffiths-Jones S. miRBase: from microRNA sequences to function. Nucleic Acids Res. 2019;47(D1):D155–62.
