Preliminary work in the liquid biopsy area has shown promise in translating this science into clinically useful measures and possible screening tools. Despite these advances, significant challenges remain. ctDNA and other liquid biomarker techniques will benefit from improved sensitivity, specificity, and overall accuracy required for widespread, meaningful clinical use. While solid tissue biopsies presently remain the gold standard and reference for most ctDNA analyses, as demonstrated by the above studies, even this presents controversy. ctDNA may be derived from areas of tumors that were not biopsied from the index lesion and may contain a divergent genomic composition limiting targeted approaches [133]. Questions remain how representative liquid biopsies can be of solid tumors with respect to intra-tumor heterogeneity. This may not present an issue in virally mediated HNC as HPV and EBV ctDNA are specific biomarkers that can reliably be detected. However, this does pose a challenge in non-viral HNC, as driver mutations may be heterogeneously distributed throughout a primary tumor. Liebs et al. illustrated the concordance of the mutational profiles between tumor tissue and cfDNA in HNC patients to only be 11%, highlighting the significant heterogeneity of HNC [134]. While CTCs did not perform better, they were interestingly able to detect specific mutations not detected in the tissue samples, suggesting a potential advantage of liquid biopsies. While they may not fully capture the intra-tumor heterogeneity and tumor microenvironments of primary tumors, liquid biopsies may have the potential to detect genomic data from micrometastases and other subclones that contribute to treatment resistance [135]. For example, in patients with metastatic breast cancer, Chu et al. utilized ctDNA sequencing to detect ESR1 mutations, which would suggest tumor resistance to endocrine therapy, which was not identified in the corresponding solid biopsies of metastatic lesions [136]. This highlights a similar limitation of spot core biopsies and fine-needle aspirations of solid tumors, as they also do not capture tumor heterogeneity or specific niches of tumor microenvironment. Nonetheless, the pursuit of capturing tumor heterogeneity is an important field of research, and tumor heterogeneity likely contributes to why reliable assessable mutations in non-virally associated HNC have not yet been identified. Thus, further studies are needed to clarify these associations and identify mutations, cell surface markers, or other metabolites which may increase the ability to more accurately isolate liquid biopsy targets among various HNC tumors and subsites. Potential opportunities include ctDNA mutational panels, or expression signatures.

Currently, numerous assays to detect ctDNA exist; however, no single ideal technique or assay has been universally adopted. Concerns for inter-laboratory variability in accuracy and interpretation have hobbled mainstream clinical adoption. Further studies evaluating agreement of different assays in ctDNA detection are needed, similar to how O’Leary et al. demonstrated good agreement between BEAMing and ddPCR [20]. This will clarify whether there is sufficient reproducibility for clinical use and if analyses among different studies can appropriately be made. Additionally, by improving the associated sensitivity and specificity of these techniques and further understanding ctDNA biology, more accurate detection of HNC, disease burden, surveillance, and prognostication may be possible [137].

Through combinatorial approaches and future technological refinements, integration of ctDNA data with various other circulating biomarkers may ultimately broaden the scope of ctDNA applications in HNC and improve its utility as a clinical tool. Several studies have investigated the combined role of ctDNA and CTC detection in solid tumors, but to date, none have been conducted in a solely HNC sample [132,138]. Furthermore, advancements in platforms similar to Bu et al. that can simultaneously capture ctDNA, exosomes, and CTCs and provide combinatorial analysis of their expression profiles through machine learning will ease this path of investigation [139].

ctDNA has been increasingly studied as a biomarker for HNSCC post-treatment surveillance and used to assess residual disease, monitor for recurrence, and stratify patients at increased risk for relapse of disease [26,140,141,142]. Despite the early integration of ctDNA and similar biomarkers in treatment algorithms, further large-scale prospective and randomized clinical trials in HNC cohorts are needed to fully integrate ctDNA data into treatment stratification paradigms. Indeed, several clinical trials are underway including a phase II trial using ctDNA testing to determine the optimal time to begin routine treatment in patients with HPV-positive HNC [143,144,145]. While clinical validation of ctDNA use in non-viral HNC is continuing [146], clinical validation in virally mediated HNC cohorts have demonstrated excellent detection of viral ctDNA as biomarkers for screening, treatment response and surveillance (as noted above by Chera et al. in HPV-related HNC and Lo et al. in EBV-related HNC, among others; Table 1 and Table 2) [26,53]. Additionally, although not yet standard of care, HPV ctDNA is already being used in patient care as a biomarker [147]. ctDNA assays have also already been clinically validated in several cancer types, including non-small-cell lung cancer, colorectal cancer, and pancreatic cancer, representing the robustness of the technology and ctDNA as a biomarker [148,149,150]. The studies presented in this review do reveal areas in need of improvement in the field of ctDNA usage for HNC, including more cost-effective detection options, enhanced accuracy, expanded gene targets, and broadened combinatorial approaches with other liquid biopsy targets. Yet, with the break-neck speed of research in this field, these areas are to be strengthened quickly. While the American Society of Clinical Oncology (ASCO) and College of American Pathologists published a joint review in 2018, concluding at the time that ctDNA assays did not possess the evidence to suggest clinical utility outside of clinical trials [151], a re-evaluation of new literature is warranted and may soon suggest otherwise.