Type of issue	Bottleneck	Potential workaround/solution
Logistical issues	Many hospitals face reimbursement issues when it comes to liquid biopsy testing and costs may be difficult to justify and thus are not covered for every ordered test.	This will hopefully change when significant progress is made in the analytical and clinical validity as well as clinical utility of plasma-based assays, especially as evidence from appropriately designed clinical trials accumulates. To date, there are no cost-effectiveness data on cfDNA assays and models for evaluating the economic impact on health must be considered in order for healthcare payers to cover such liquid biopsy solutions [176]
	Access to testing	Those who face reimbursement issues from large, commercial providers may find a solution in partnering with academic labs who perform validated assays routinely
Pre-analytical/analytical factors	Despite advances in liquid biopsy technology, pre-analytical and analytical standards are still lacking and may vary between testing labs. The 2018 joint review by ASCO and the College of American Pathologists determined that data on pre-analytic variables, analytical validity, interpretation and reporting are still insufficient to justify widespread adoption for the majority of ctDNA solid tumor assays [96].	Previous works [60, 62, 128, 177–182] have detailed the influence of pre-analytical factors such as blood collection tubes, plasma versus serum, transit time, centrifugation protocols, storage conditions, cfDNA purification, quantification, characterization and analysis platforms. Several collaborative efforts are currently underway to evaluate these findings, devise the criteria for standardizing liquid biopsy workflows, put these into context for providers and end users of cfDNA assays, as well as to bridge the gap between academia and industry to enable a transition of research findings into clinical implementation: The European Liquid Biopsy Society (ELBS); European Liquid Biopsy Academy (ELBA); International Society of Liquid Biopsy (ISLB); BloodPac
Negative results	According to the ASCO Post, the 2021 National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for molecular and biomarker analysis of NSCLC advises that cfDNA/ctDNA testing should not replace standard histologic diagnosis via tissue testing, unless the patient is unable to undergo invasive tissue sampling [183].	A recent consensus statement released by the International Association for the Study of Lung Cancer (IASLC) concluded that ctDNA analysis is an acceptable initial approach detection of biomarkers at diagnosis as well as for monitoring targeted therapies [184].
	Both technical and biological factors can influence the concordance between tumor tissue results and plasma DNA analysis.	The risk of false-positive and false-negative results from liquid biopsy testing have not completely eradicated the necessity of reflex tissue testing, such that the FDA recommends follow-up testing after receiving negative results, including those from commercial providers such as Guardant360 CDx and FoundationOne Liquid CDx
Diversity of NGS panels	Generally, there are no harmonized minimal requirements for NGS panels, although most harbor biomarkers within guidelines of national consortia, e.g. NCCN.	Implementation of broader NGS panels, e.g. those that evaluate mutations outside of the canonical hotspots, as these may reflect emerging biomarkers or inclusion criteria for recruiting/ongoing clinical trials
Clinical validity	Determination of clinical validity for broad NGS panels is challenging, as they are applied in multiple tumor types. For both of the use cases of therapy monitoring and early-stage cancer, evidence of clinical validity is still emerging, whereas there is still no evidence available for cancer screening purposes.	As summarized by Ignatiadis et al., further clinical validation of ctDNA assays will be conducted in the form of large-cohort phase III trials, which should include mandatory blood sampling and emphasize the need to report the results of liquid biopsy assays as official trial results [52].
Clinical utility	Currently, liquid biopsy assays lack consistency and precision. In fact, clinical validity and clinical utility have not yet been shown for the vast majority of assays.	Sophisticated multicenter clinical validation studies and regulatory guidelines are lacking but must be established to ensure responsible future application of liquid biopsies in precision oncology.
	It is not yet known whether interventional treatment based on detection of ctDNA relapse via liquid biopsy will actually improve patient outcome, i.e. cure, or whether systemic treatment will simply delay onset of metastatic disease [52].	Design and conducting of suitable clinical trials with the goal of improving outcomes of patients with detectable ctDNA
Bioinformatics	Increasing complexity of bioinformatics algorithms	Need for increased usability, e.g. through user-friendly graphical user interfaces with clear and concise instruction for use and interpretation of results
Education	Lack of structured, formal training available for clinicians regarding the implementation of genomic medicine: which tests to order for which patients; understanding the benefits and limitations of NGS assays; when to order liquid biopsy; how to interpret the clinical reports, prioritize variants and evidence and convert potential actionable insight into clinical care.	Such questions may be answered within the context of an appropriately set up molecular tumor board (MTB), where a panel of diverse experts, e.g. oncologists, pathologists, clinical geneticists and bioinformaticians meet on an ongoing basis to deliver the most suitable precision oncology approaches. Additionally, innovative education platforms, particularly geared towards young oncologists, may provide the fundamental awareness of such testing modalities early on in a clinician’s training. Molecular profiling and liquid biopsy education should be integrated into university training programs.
Interpretation bottlenecks	Lack of MTB infrastructure within hospital	With the advent of virtual molecular tumor boards, it may be possible to partner with experienced MTBs to discuss difficult patient cases
	Clinical decision support tools are still in their infancy and harmonization efforts regarding the interpretation of genomic variants have only just begun [124–126, 185] .	Various strategies of decision support software may lead to discrepancies in pathogenicity, actionability and treatment matching when interpreting genomic variants. For this reason, data must be evaluated and prioritized at a multidisciplinary MTB to derive the most suitable treatment decision.
	The interpretation and prioritization of variants at an MTB may vary from clinic to clinic and some MTBs may still be unfamiliar with data derived from ctDNA assays.	Clinics should strictly employ standardized criteria to define actionability and must rely on validated evidence-based scales, such as the FDA-approved content of OncoKB and European ESCAT guidelines [186–188]