
==== Front
Discov Oncol
Discov Oncol
Discover Oncology
2730-6011
Springer US New York

39259454
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10.1007/s12672-024-01308-2
Review
Does circulating tumor DNA apply as a reliable biomarker for the diagnosis and prognosis of head and neck squamous cell carcinoma?
Ghiyasimoghaddam Negin 1
Shayan Navidreza 2
Mirkatuli Hanieh Alsadat 3
Baghbani Mohammadhasan 2
Ameli Nima 4
Ashari Zeynab 5
http://orcid.org/0000-0002-1262-4668
Mohtasham Nooshin mohtashamn@mums.ac.ir

6
1 https://ror.org/00fafvp33 grid.411924.b 0000 0004 0611 9205 Department of Emergency Medicine, Bohlool Hospital, Gonabad University of Medical Sciences, Gonabad, Iran
2 grid.411768.d 0000 0004 1756 1744 Department of Medical Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran
3 grid.469938.9 Department of Medical Sciences, Shahrood Branch, Islamic Azad University, Shahrood, Iran
4 https://ror.org/04sfka033 grid.411583.a 0000 0001 2198 6209 Sinus and Surgical Endoscopic Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
5 https://ror.org/02558wk32 grid.411465.3 0000 0004 0367 0851 Department of Cellular and Molecular (Genetic), Faculty of Biology, Qom Branch, Islamic Azad University, Qom, Iran
6 https://ror.org/04sfka033 grid.411583.a 0000 0001 2198 6209 Oral and Maxillofacial Diseases Research Center, Mashhad University of Medical Sciences, P.O. Box: 9177948959, Mashhad, Iran
11 9 2024
11 9 2024
12 2024
15 4277 11 2023
3 9 2024
© The Author(s) 2024
2024
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Oral cavity cancer is the most common type of head and neck cancer. There is no definitive standard diagnosis, prognosis, or treatment response biomarker panel based on simple, specific, non-invasive, and reliable methods for head and neck squamous cell carcinoma (HNSCC) patients. On the other hand, the frequent post-treatment biopsies make it challenging to discriminate residual disease or recurrent tumors following postoperative reparative and post-radiation changes. Saliva, blood plasma, and serum samples were commonly used to monitor HNSCC through liquid biopsies. Based on the evidence, the most prominent molecular-based fluid biomarker, such as circulating tumor DNA (ctDNA), has potential applications for early cancer diagnosis, screening, patient management, and surveillance. ctDNA showed genomic and epigenomic changes and the status of human papillomavirus (HPV) with the real-time monitoring of tumor status through cancer therapy. Due to the intra and inter-tumor heterogeneity of tumor cells like cancer stem cells (CSCs) and tumor microenvironment (TME) in HNSCC, the tiny tissue biopsy cannot reflect all genomic and transcriptomic abnormality. Most liquid biopsies are applied to detect circulating molecular biomarkers consisting of cell-free DNA (cfDNA), ctDNA, microRNA, mRNA, and exosome for monitoring tumor progression. Based on the results of previous studies, liquid biopsy can be applied for comprehensive multi-omic discovery by assessing the predictive value of ctDNA in both early and advanced cancers. Liquid biopsy can be used to evaluate molecular signature profiles in HNSCC patients, with great potential to help in early diagnosis, prognosis, surveillance, and treatment monitoring of tumors. These happen by designing longitudinal extensive cohort studies and the utility of organoid technology that promotes the context of personalized and precision cancer medicine.

Keywords

Circulating tumor DNA
Biomarkers
Squamous cell carcinoma of head and neck
Cell-free nucleic acids
issue-copyright-statement© Springer Science+Business Media, LLC 2024
==== Body
pmcIntroduction

Head and neck cancer (HNC) is the sixth most common cancer type all around the world, with more than 870,000 new cases and 440,000 deaths in 2020 [1]. More than 90% of HNCs comprise head and neck squamous cell carcinoma (HNSCC), which originates from the mucosal epithelium of the oral cavity, pharynx, and larynx [2]. There is a diversity of therapeutic advances in HNSCC besides conventional approaches (surgery and chemo/radiotherapy), including cetuximab (an epidermal growth factor receptor (EGFR) antibody) applied in the end stage, nivolumab and pembrolizumab which are anti-programmed death-1 (PD-1) inhibitors use in relapse or metastatic cases, and disease progression on or after platinum-containing chemotherapy, and pembrolizumab recommended as both monotherapy in PD-L1 expressing tumors, or in combination with chemotherapy [3]. Immunotherapy provides a new avenue for long-term remissions in metastatic cases and locoregionally advanced settings. In addition, various ranges of immune checkpoint inhibitors (ICIs), excluding previous limitations by targeting co-stimulatory and co-suppressive checkpoints in immune cells, have been evaluated as the new target [4, 5]. Inter- and intratumor heterogeneity of HNSCC, besides the self-renewal and re-growth of the cancer stem cell (CSC) population, promotes therapeutic resistance. In addition, late-stage diagnosis and high recurrence rate remain the main challenges in the clinical management of patients, so 5-year overall survival (OS) and disease-free survival (DFS) rates did not significantly change in the past years [6].

Although it is well-known that the high-risk factors of carcinogenesis are exposure to smoking, alcohol consumption, and human papillomavirus (HPV) infection, the genetic background should not be underestimated [2]. Recent studies based on multi-omics analysis of genomics, transcriptomics, proteomics, metabolomics, and epigenomics proposed that body fluid encompasses valuable molecular biology markers that showed potential for the early monitoring, diagnosis, prognosis, and precision medicine therapy of HNSCC patients [7, 8]. Due to the heterogenicity of tumor cells like CSCs and tumor microenvironment (TME) in HNSCC, the tiny tissue biopsy cannot reflect all genomic and transcriptomic abnormality. However, body fluids, including saliva, plasma, and serum, provided easy access and showed the accurate signature profile of biomarkers ranging from extracellular vesicles to circulating tumor cells (CTCs) in cancer studies. Most liquid biopsies are applied for the detection of circulating molecular biomarkers consisting of cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), microRNA, mRNA, and exosome for monitoring tumor progression (Fig. 1) [9]. In previous studies, the saliva and blood (plasma and serum) samples were prominently used for HNSCC monitoring in different liquid biopsies. Saliva sampling provides benefits including simple and non-invasive collection, appropriate safety, less time consuming, and economical with great early diagnostic and prognostic potential for oral diseases. Evaluation of saliva as a source of biomarkers named “Salivaomics” provides five different connections between genome and epigenome, transcriptomics, metabolomics, lipidomics, proteomics, and microbiota that give scientists the opportunity for precision medicine [10]. Extensive clinical studies showed serum and plasma could be valuable sources based on biomolecules, such as nucleic acids and protein or exosomes and other types of extracellular vesicles for risk assessment, monitoring and early diagnosis of cancer, accurate diagnosis, prognosis, evaluation of response to therapy, and cancer surveillance and monitoring response [11, 12].

Fig. 1 Schematic overview of liquid biopsy for HNSCC monitoring in patients

In contrast to some other cancers, there is no definitive standard diagnosis, prognosis, or treatment response biomarker panel based on simple, specific, non-invasive, and reliable methods for HNSCC patients. On the other hand, the frequent post-treatment biopsies make it challenging to discriminate residual disease or recurrent tumors following postoperative reparative and post-radiation changes [13]. For these reasons, we aimed this study to review the most critical molecular-based fluid biomarker, such as ctDNA, and its changes that represent a promising strategy for diagnosis, prognosis, and HNSCC monitoring.

Application of ctDNA in HNSCC

CtDNAs are originated from tumor cells released into the circulatory body fluid through a passive process by apoptosis and necrosis or an active process due to the micro-vesicles that contain DNA fragments. They are fragments of tumor DNA cells released through physiologic and pathologic features, demonstrating the genetic profile of somatic mutations in the tumor genome, and are comprised of < 1.0% total cfDNA [14].

The main approaches are applied for the evaluation of driver mutation of ctDNAs based on polymerase chain reaction (PCR) techniques: first, droplet digital PCR (ddPCR) with high sensitivity in samples with low DNA quantity and the ability to discover DNA at concentrations as little as 37 copies per 20 µL [15]. Second, the BEAMing (beads, emulsion, amplification, and magnetic) high-throughput analysis realizes infrequent genetic events, such as mutant and wild-type sequences, at ratios less than 1:10,000 [16]. The main sequencing methods include tagged-amplicon deep sequencing (TAm-Seq), cancer personalized profiling by deep sequencing (CAPP-Seq), whole genome bisulfite sequencing (WGBS-Seq), whole exome sequencing (WES), and whole genome sequencing (WGS) [17]. The WGS and WES techniques can discover a wide range of genetic variants at once and need more DNA amount in the 200–1000 ng range compared to the one ng for BEAMing and ddPCR. In addition, they present results as ratios, while BEAMing and ddPCR demonstrate absolute quantification [18].

Based on the evidence, ctDNA has potential applications for early cancer diagnosis, screening, patient management, and surveillance. Evaluation of two different ctDNA detection approaches in plasma and oral rinses of HNSCC patients at early and late stages identified five genes in 42% of plasma cases, and 67% were early-stage cases. It was approved potentially of ctDNA targeted mutation discovery in plasma of early stages HNSCC for early diagnosis. However, only 28% (a low-rate mutation) of the overall mutations were assessed because of cfDNA degradation through prolonged storage [19]. The tumor locoregional plays a pivotal role in the optimization of cfDNA discovery. Wang et al. [20] discovered 100% and 80% ctDNA in saliva and plasma samples of oral cavity cancers, respectively. They studied rare somatic mutation with a Safe-Sequencing System and HPV DNA fragments with dPCR in patients. They proposed that saliva encompasses considerable ctDNA for assessing oral cavity cancers, while plasma is significantly enriched for ctDNA from the other sites. They suggested plasma samples were better in oropharyngeal squamous cell cancer (OPSCC) patients by detecting ctDNA in 91% versus 47% of saliva. Likewise, plasma samples performed better than saliva samples in laryngeal (86% compared to 70%) and hypopharyngeal (100% compared to 67%) SCCs [20]. The urine DNA (uDNA) can be helpful in the evaluation of patients at all stages of urologic malignancy by next-generation sequencing (NGS), but among liquid biopsies, the saliva, serum, and plasma sampling were more prominent in HNSCC in comparison to the other biofluids [21].

It was suggested that combining multiple biofluid ctDNA analyses increased sensitivity and specificity. A study evaluated 72 colorectal cancer cases, 14 patients with benign colorectal tumors, and 14 healthy controls for triple tumor liquid biopsy biomarkers, including CTCs, exosomes, and cfDNA by multimodal liquid biopsy (MMLB) system [22]. MMLB analysis demonstrated notably promoted accuracy in diagnosing malignancies, determining the pathological status of cases, forecasting survival rates, and identifying gene mutations. Similar approaches improve liquid biopsy assays to achieve personalized medicine [22].

In a study, ctDNA was analyzed for assessment of therapeutic response and recurrence in HNSCC patients by SCC panel and dPCR method. Fourteen of 24 patients (58.2%) showed TP53 as the more common mutated gene. Eleven patients with negative ctDNA through follow-up after beginning curative treatment demonstrated a significantly better prognosis than 7 cases reverted to ctDNA positivity. The result of this study illustrated that individualized ctDNA screening by the mentioned strategy and method could be a novel and promising biomarker for HNSCC monitoring [23]. A comparison of 22 OPSCC patients’ plasma ctDNA with tDNA showed that non-responders’ mutational signatures of OPSCC patients using NGS could be detected by evaluating cfDNA [24]. In a study, the ctDNA of 170 HNSCC patients was compared to the tDNA and analyzed for 18 DNA damage repair (DDR) gene mutations. It was concluded that the addition of ctDNA to the standard tDNA analysis increased mutations in one or more genes in each of the study subsets of DDR genes in HNSCC with older than 60 years, laryngeal primaries, advanced stage, and chemo/radiotherapy pre-treatment.

Furthermore, HNSCC cases with DDR gene mutations demonstrated fewer primary tumors in the oropharynx or HPV-positive status. HNSCC patients with ctDNA mutations in all subsets of DDR genes showed notably worse OS [25]. Altogether, recent studies are focused on selecting the highest accuracy method among advanced technologies that detect ctDNA genomic and epigenomic changes in both early and advanced stages of HNSCC, besides having predictive potential during disease monitoring.

Detection of HPV and EBV status by ctDNA in HNSCC patients

The ctDNA can be applied to diagnosing HPV status and prognosis of HPV-positive HNSCC patients. For the first time, it was reported that ctHPV16 DNA is detectable in pre-diagnostic plasma at least several years before diagnosing HPV16-positive HNSCC for a subset of individuals later diagnosed with HPV-positive HNSCC. The plasma samples were gathered six months before the HNSCC diagnosis [26]. Results of a meta-analysis study that comprised 457 HPV-positive HNSCC patients showed repeated liquid biopsy of HPV-DNA due to the follow-up can apply in the future as a diagnostic biomarker for the monitoring post-treatment surveillance of these patients [27]. However, larger sample sizes and the similarity in standard protocols and methodology should be applied to establish better application in clinical practice.

It was reported that assessing response to therapy using real-time biofluid markers could promote outcomes by finding an early treatment change in metastatic HPV-positive OPSCC patients. In addition, ctDNA therapeutic responses illustrated earlier than conventional imaging allowed early detection of treatment failure and can shift the patients to clinical trials or alternative therapies [28].

Different detection techniques may have varying sensitivity and specificity in various body fluids. Approximately 80% of OPSCCs manifested following HPV infection, and cfDNA assessment promotes earlier detection than their typical presentation. In this regard, a comparison between plasma samples and oral rinses of 66 HPV-positive OPSCC patients was performed by NGS, ddPCR, and quantitative PCR (qPCR) to identify the best method for HPV ctDNA detection [29]. HPV cfDNA detection by NGS and ddPCR in plasma cases demonstrated 70% sensitivity compared to the qPCR (20.6%). In oral rinse, NGS showed 75% sensitivity compared to the ddPCR (8.3%) and qPCR (2.1%). In addition, follow-up identified cfDNA HPV in plasma by NGS but not ddPCR or qPCR, reflecting disease remission or progression [29]. A fundamental restriction in the study was that the extracted DNA was previously stored in banked plasma samples and oral rinses, and the cfDNA may be degraded over time. In Table 1, we present more studies related to the ctDNA that was considered as a biomarker for HPV-positive HNSCC.

Table 1 A summary of human papillomavirus circulating tumor DNA analysis in HPV-positive HNC patients

Author (Year)	Sample	ctDNA
Diagnostic	Target	Patients	Sensitivity
(%)	Specificity
(%)	Refs.	
Retting et al. (2022)	Plasma	ddPCR	HPV16, 18, 31, 33, 35	12	35.7	99.1	[26]	
Siravegna et al. (2022)	Plasma	ddPCR	E7 of HPV16, 18, 33, 35, 45	70	98.4	98.6	[67]	
Akashi et al. (2022)	Plasma	ddPCR	E6/E7 of HPV16	25	54	NA	[68]	
Berger et al. (2022)	Plasma	ddPCR	HPV16, 18, 31, 33, 35	1076	56.7	99.7	[69]	
Haring et al. (2021)	Plasma	ddPCR	E6 of HPV16	16	88.9	88.9	[28]	
Sastre-Garau et al. (2021)	Plasma	NGS	HPV16, 18, 31, 33, 35, 45, 51, 11, 35, 39, 52, 53, 56, 59, 68, 69	80	95.0	98.1	[70]	
Tanaka et al. (2021)	Plasma	ddPCR	E6/E7 of HPV16	48	93.0	97.0	[71]	
Veyer et al. (2020)	Plasma	ddPCR	E6 of HPV16	66	71.0	NA	[72]	
Rutkowski et al. (2020)	Plasma	qPCR	HPV16	66	100.0	98.0	[73]	
Reder et al. (2020)	Plasma	qPCR	HPV16 E6, E7	23	100.0	83.0	[74]	
Hanna et al. (2019)	Saliva	ddPCR	HPV16,18,31,33,45 E7	21	87.0	67.0	[75]	
Fakhry et al. (2019)	Saliva	qPCR	E6/E7 of 37 HPV subtypes	202	79.7	NA	[76]	
Chera et al. (2019)	Plasma	ddPCR	E7 of HPV16	103	89.0	97.0	[77]	
Damerla et al. (2019)	Plasma	ddPCR	E6/E7 of HPV16 and 33	97	95.6	100.0	[78]	
Lee et al. (2017)	Plasma	NGS	E7 of HPV16	27	100.0	92.7	[79]	
Ahn et al. (2014)	Oral rinse	qPCR	E6/E7 of HPV16	72	25.0	98.3	[80]	
Plasma	52	62.5	97.7	
Oral rinse and Plasma	46	66.7	95.0	
Cao et al. (2012)	Plasma	qPCR	E6/E7 of HPV16 and 18	40	65.0	100.0	[81]	
Chuang et al. (2008)	Oral rinse	qPCR	E6/E7 of HPV16	20	50.0	100.0	[82]	

In addition to the strong correlation between HPV and OPSCC, another virally mediated HNC-Epstein Barr virus (EBV) showed a relation with nasopharyngeal cancer (NPC). The ctEBV DNA is known as an independent dynamic biomarker for prognosis of a subset of malignancies and is recommended by the National Comprehensive.

Cancer Network (NCCN) for assessing the response of EBV tumor status through or before therapeutic approaches [30, 31]. For example, manifestation of ctEBV DNA after therapy is correlated to a worse prognosis and is being assessed as a marker for consolidation chemotherapy [32]. ctEBV DNA affects clinical decision-making in both therapeutic approaches and surveillance for EBV-related NPC that provide a template for HPV-related OPSCC. In large-size screening studies, discovering ctEBV DNA revealed earlier detection of NPC [33] such as earlier diagnosis of recurrence in the posttreatment setting [31]. By screening more than 20,000 participants’ plasma, EBV DNA was discovered in 1112 participants (5.5%), and 309 (1.5% of all participants and 27.8% of those who initially tested positive) with sensitivity and specificity 97.1% and 98.6% for nasopharyngeal carcinoma, respectively. Results of their study showed detection of plasma EBV DNA can be applied for early diagnosis and better outcomes in participants who were identified by screening compared to those in a historical cohort [33].

Genomic alternations in HNSCC

Heterogeneity in HNSCC nature is a significant concern in patients, and various genomic changes that activate oncogenes or repress tumor suppressor genes drive tumors in different treatment responses with different prognoses. Studying 528 HNSCC cases based on The Cancer Genome Atlas (TCGA) revealed that 3491 genes were deregulated. The somatic copy number variations were more frequent in 5 genes, including cyclin-dependent kinase inhibitor 2 A (CDKN2A) and CDKN2B deletions in 32.03% and 28.34% of patients, respectively. Amplification of PTPRF interacting protein alpha 1 (PPFIA1), Fas-associated protein with death domain (FADD), and Anoctamin-1 (ANO1) were in 26.02%, 25.63%, and 25.44% of patients, separately. Furthermore, TP53 mutation (72%) was more frequent than titin (TTN) (39%), FAT atypical cadherin 1 (FAT1) (23%), and mucin-16 (MUC16) (19%) of the cases. Moreover, a mutual co-alternation pattern was observed between the TP53 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations, and also CDKN2A co-changes with the TP53 and FAT1 [34].

The genome-wide profiling of 279 HNSCC patients by generating a comprehensive multi-platform for identifying somatic mutation has been well-investigated by TCGA [35]. Among premature mutation termination of the protein TP53 (86%) and CDKN2A (correlated to cell cycle and survival), FAT1 and AJUBA (affect Wnt/β-catenin pathway) were more frequent in HPV−HNSCC patients. While mutations of the oncogene PIK3CA, new changes related to the loss of TNF receptor-associated factor 3 (TRAF3) and amplification of the cell cycle gene E2F transcription factor 1 (E2F1) were more frequent in HPV+ cases. In addition, TP53 mutation, CDKN2A loss of function, chromosome 3q amplification, and change of genes involved in oxidative stress pathway, including kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid-derived 2 like 2 (NFE2L2), or cullin-3 (CUL3), were correlated to the heavy smoking signature and anatomic tumor sites in laryngeal tumors. Interestingly, it was shown that the mutation rates were unaffected by HPV status. However, transversions at CpG sites were more predominant in HPV− and TpC mutations in HPV+ patients [35]. It was estimated that up to 70% of mutation in HNSCC patients was related to the TP53, then after were PI3K-PTEN-AKT pathway, PIK3CA (6–21%), FAT1 (12–23%), and CDKN2A (9–22%) [36].

Application of ctDNA as a biomarker for genomic changes in HNSCC

For the first time, in a cohort of 75 HNSSC patients, tissue DNA, and ctDNA sequencing were compared for prognosis and precision oncology treatments. It was estimated that 65.3% of patients burden actionable ctDNA alterations, and the more commonly changed genes were TP53, CDKN2A, telomerase reverse transcriptase (TERT), breast cancer 2 (BRCA2), and neurogenic locus notch homolog protein 1 (NOTCH1). More detected ctDNA changes significantly correlated with reduced O.S. In contrast to ctDNA, there was no correlation between DNA repair gene alterations in tDNA and patient prognosis. Interestingly, no ctDNA of patients showed NOTCH1 alterations, identified in 20% of tDNA. Unfortunately, there was a wide variety of time gaps between the comparison of ctDNA and tDNA [37]. Analysis of 60 ctDNA HNC cases with recurrent and metastatic (R/M) by NGS showed TP53 (68%), PIK3CA (34%), NOTCH1 (20%), and ARID1A (15%) were the most frequent mutations that consistent with tumor sequencing data that TP53 (48%) and PIK3CA (24%) were the most common [38].

A study of 40 HNSCC cases and 20 control plasma by low-coverage whole genome sequencing (lcWGS) detected 52% copy number aberrations (CNAs), 67% more common somatic mutations of 12 driver genes in patients, and 100% human papillomavirus (HPV) DNA in HPV-positive tumors. However, higher sensitivity results revealed a 78% detection rate when combined with analysis of ctDNA in plasma and strongly associated with the tumor stage. In addition, HPV status and location of the primary tumor did not affect the detection of CNAs or somatic mutations in plasma. This outcome is valuable because lcWGS can reduce costs [39]. A comparison of 121 oral squamous cell carcinoma (OSCC) and 50 healthy controls showed a significant correlation between increased plasma cfDNA levels and larger tumor size, cervical lymph node metastasis, and late-stage OSCC with a poor prognosis of OSCC. In addition, 75% of the OSCC cases demonstrated a significant reduction in cfDNA after tumor resection, providing diagnostic and prognostic value [40].

Application of ctDNA as biomarkers for other cancers

Based on the results of a meta-analysis study, liquid biopsy can be applied for comprehensive multi-omic discovery by assessment of the predictive value of ctDNA in early breast cancer cases. Identifying ctDNA as a baseline after completing neoadjuvant therapy (NAT) significantly correlated to worse relapse-free survival (RFS) and O.S. However, the discovery of ctDNA did not relate to the probability of achieving a pathologic complete response (pCR). In addition, ctDNA assessment during NAT for early breast cancer merits further evaluation as a stratification risk factor in prospective trials to improve individualized patient treatment [41]. The phase II clinical trial study reported that ctDNA levels were significantly correlated with OS and outperformed radiologic evaluations in predicting survival of non-small-cell lung cancer (NSCLC) in stage IIIA. In addition, low levels of ctDNA and undetectable ctDNA levels were significantly associated with promoted progression-free survival and OS in pre-treatment patients and after neoadjuvant treatment, respectively [42]. In a cohort study, somatic mutations in tDNA and plasma ctDNA samples of NSCLC were identified with NGS and applied for ctDNA-based molecular residual disease (MRD) analysis. It was shown that perioperative ctDNA was strongly correlated to lower recurrence-free survival. In addition, ctDNA-based MRD demonstrated a higher correlation to RFS prediction than to all clinicopathologic parameters like the TNM stage. Moreover, MRD-positive patients who received adjuvant therapies showed promoted RFS compared to those who did not. Altogether, ctDNA can be used as a biomarker for early diagnosis of MRD and prediction of postoperative relapse in resected NSCLC [43].

Previous studies showed increased specificity and sensitivity of ctDNA due to improved technology. It was proposed that ctDNA has the potential to become a ‘game-changer’ in the treatment of early-stage colon cancers and can apply as a strong predictor for early detection after recurrence and survival after surgery and adjuvant therapy [44]. In a prospective longitudinal cohort study, 299 cases with stage I to III colorectal cancer were assessed for 6 DNA methylation markers in ctDNA plasma samples. The results showed that longitudinal evaluation of ctDNA methylation might enable early Detection of MRD and Risk Stratification of recurrence, potentially, and postoperative treatment of colorectal cancer (CRC) patients [45]. Serial liquid biopsy and longitudinal monitoring of ctDNA provide this opportunity for clinical management of ovarian cancer patients that late clinical manifestation remains the challenge resulting in poor survival. ctDNA screening can be applied for early diagnosis, predicting prognosis, screening for recurrence, and response to therapeutic approaches [46].

Detection of epigenetic changes by ctDNA in HNSCC

Hypermethylation of promoter inhibits the expression of tumor suppressor genes and promotes the tumorigenesis process. Conversely, hypomethylation can activate some elements, like non-coding RNAs, that provide genetic instability and improve oncogene activity. A comparison of 317 primary HNSCC, 225 matched normal mucosa tissues, and plasma ctDNA demonstrated high hypomethylation of long interspersed nucleotide element-1 (LINE-1) correlated to the poor prognosis and early oral cavity cancer relapse. Hypomethylation of LINE-1 increased retrotransposon activity and induced genome instability, suggesting it can be applied as a predictive, reliable biomarker [47]. It was reported that methylation in TP53, CDKN2A, death-associated protein kinase 1 (DAPK1), ras association domain family member 1 (RASSF1), and p15 genes was more frequent in HNSCC patients than in the control. In addition, increasing the number of ctDNA genetic methylations increases diagnostic sensitivity accuracy [48]. In a study, methylation-based markers were evaluated in promoters of cyclin A1 (CCNA1), DAPK, cadherin 8 (CDH8), and tissue inhibitors of metalloproteinase 3 (TIMP3) genes that play a role in clinical features by ddPCR. The methylated plasma ctDNA was found in 73.3% of OPSCC patients and detected in 71% of tissues that proposed the potential application of ctDNA in patient management [49].

In a study, a comparison of 100 HNSCC patients and 50 healthy control serum samples showed endothelin receptor type B (EDNRB) hypermethylation was a particular but not sensitive serum biomarker by quantitative methylation-specific polymerase chain reaction (qMSPCR) method [50]. Detection of methylation and mutation-based ctDNA by CAPP-seq and cell-free methylated DNA immuno-precipitation and high-throughput sequencing (cfMeDIP-seq) methods demonstrated a strong correlation between ctDNA abundance and worse OS independent of clinical stage and recurrence in HNSCC patients. The cfMeDIP-seq showed 941 ctDNA-derived hypermethylated regions enriched for CpG islands that can apply as HNSCC-specific methylation patterns [51]. There was concordance in the result of different algorithms between relative quantification, absolute quantification, and quasi-digital PCR in clinical performance based on the analysis of short-stature homeobox 2 (SHOX2) and septin-9 (SEPT9) methylation in cfDNA biomarkers [52]. A study illustrated that cfDNA can be applied as specific prognostic and diagnostic biomarkers, comparing methylome profiles before and after surgery of OSCC plasma samples. Among 200 HNSCC-specific differential methylated regions (DMRs) based on the TCGA data, five areas were located in the promoter of PENK, neurexophilin 1 (NXPH1), zinc finger protein interacting with k protein 1 (ZIK1), T-Box transcription factor T (TBXT), and cysteine dioxygenase 1 (CDO1) genes, respectively. The genome-wide cfDNA DMR analysis further revealed candidate biomarkers in secreted frizzled-related protein 4 (SFRP4), SRY-box transcription factor 1 (SOX1), interferon regulatory factor 4 (IRF4), and protocadherin 17 (PCDH17) genes [53]. Generally, evaluation of ctDNA methylation status can be promised by the determined specific panel for target genes that play a pivotal role in the pathogenesis of HNSCC following the deactivation of tumor suppressor genes or activation of oncogenes.

Application of exosomes-mediated biomarkers in HNSCC

Exosomes are extracellular vesicles with 40 to 160 nm dimension containing proteins, miRNA, mRNA, and DNA that accumulate within the tumor microenvironment and are released in saliva, blood, urine, and cerebrospinal fluid with notable differences between HPV-positive and HPV-negative cell lines. They can play a role in tumorigenesis, tumor growth, metastasis, and drug resistance by transferring cargo, including nucleic acids and proteins [54]. The serum, tumor tissues, and transforming growth factor-beta (TGFβ) content of circulating exosomes increased in parallel to the tumorigenesis process in HNSCC patients. However, this TGFβ overexpression did not associate with clinicopathological indices or survival. Interestingly, exosome-associated TGFβ reflected tumor progression and correlated with tumor size [55].

Exosomal miRNAs derived from cancer cells are critical in regulating the cellular environment. It identified high quantities of oncogenic miRNAs, including miR-21 and miR-27, in the extracellular vesicles of OSCC patients’ s plasma that target tumor suppressive genes involved in proliferation, apoptosis, and invasion. miR-21 stimulates inflammation cascade and actives NF-κB and increases interleukin 6 (IL-6), C-C motif chemokine ligand 2 (CCL2), prostaglandin E2 (PEG2), and matrix metalloproteinase-9 (MMP9) levels. These results illustrated the role of extracellular vesicle-associated miR-21 in regulating the immune response in monocytes [56]. It was demonstrated that expression of miR-221 in OSCC cell line-derived exosome increased, as well as tumor tissue and cell line. In addition, OSCC-derived exosomal miR-221 could target and downregulate phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) expression and promote vascular endothelial cell migration and angiogenesis [57]. The study of 47 OSCC patients showed that the cancer‑associated fibroblast (CAF) density in tumor tissues was correlated to the upregulation of miR‑382‑5p, OSCC lymph node metastasis, invasion, and TNM stage. In addition, it was shown that CAF‑derived exosomes transported miR‑382‑5p to OSCC cells. The results approved a new mechanism of CAF‑facilitated OSCC progression that can be applicable for detecting new cancer therapeutic targets [58]. Finally, the exosomes demonstrated promising strategies by having therapeutic potential as cancer vaccines, drug carriers, or tools to reverse the drug resistance of HNSCC.

Discussion

Although the evaluation of liquid biomarker methods showed high sensitivity, specificity, and overall accuracy required for widespread clinical application, tissue biopsies remain the gold standard for most ctDNA assessments, and this issue is a questionable controversy. The ctDNA originated from areas of tumors that may not encompass divergent genomics through biopsy, but it is unclear how it can reflect intra-tumor heterogeneity [59]. This issue does not apply to HPV and EBV, which contain specific DNA biomarkers. However, it remains a challenge in non-viral HNC when driver mutations play a pivotal role in the heterogeneously of a primary tumor. In addition, although the half-life of ctDNA is fewer than 2 h, which is shorter than protein biomarkers, it can provide real-time monitoring of tumor status through cancer therapy. Another challenge is the contamination of genomic DNA during ctDNA isolation with bio-fluid proteins [60].

Furthermore, the level of ctDNA or cfDNA in bio-fluid can be affected by infections, inflammation, drug utility, or percutaneous endoscopic gastrostomy placement during radiochemotherapy, and it is suggested to be combined with other biomarker proteins, leucocytes, and clinical visitations to mirror the nature of tumor burden when scientists evaluate ctDNA levels [40]. Analysis of bio-fluid can be affected by patients’ metabolic activity and speed of ctDNA decomposition. The safety of ctDNA should be clarified before clinical application by developing more clinical research centralizing the whole metabolic chain regulating the level of circulating DNA in the blood [13]. Moreover, liquid biopsy provides massive molecular data for a procession. Thus, the application of machine learning to discover and detect disease signatures in the biofluid biopsy can promote the accuracy and efficiency of cancer diagnosis and screening, such as the web-based automated machine [61].

In contrast to other studies, some research results showed that liquid biopsy cannot be considered a reliable strategy for HNSCC patient management. Galot et al. [62] reported that ctDNA was discovered only in 51% of HNSCC cases, and it was more frequent in metastatic (70%) than locoregional recurrent patients (30%). They suggested liquid biopsies cannot cover the complete mutation signature of the HNSCC patients. However, they can consider actionable mutations or variants not found in matched tumor tissue when it is impossible to perform a tissue biopsy. In addition, tissue tumor variants in 81% of total HNSCC cases and 58% of metastatic patients were not identified in plasma [62]. The concordance of the mutational signatures between tumor tissue and cfDNA in HNSCC cases was reported at 11%, while it was 63% and 55% in advanced CRC and melanoma, respectively. This result illustrated notable heterogeneity of HNCSCC [63]. In addition, it was proposed that the utility of ctDNA as a biomarker for early detection of possible HNSCC relapse because the post-treatment follow-up can be more appropriate than diagnostic markers in early cancers [13].

Nevertheless, more research investigations are required for HNSCC with a larger cohort of patients with a clearly defined area of interest, standardized protocol methods, and optimized bio-fluid collection, processing, and storage conditions. Moreover, the application of ctDNA as a biomarker for evaluating HNSCC relapse due to the post-treatment follow-up can be more suitable than its use as a diagnostic marker of primary cancer.

CTC-derived organoids provide a pre-clinical model that maintains matched mutation of their original tissues and burden biomarkers that widely apply in 3D organoids for drug screening, disease monitoring, metastatic progression, building biobanks, and capturing of different stages. Thus, developing this technology to evaluate HNSCC patients can serve beneficial clinical practice in the future [64]. Evaluation of ctDNA demonstrates several benefits in comparison to the traditional tumor biopsy. Liquid biopsy shows real-time monitoring of response to therapeutic approaches, this provides an opportunity for monitoring those patients with tumors in anatomically challenging locations. In addition, the assessment of biofluid markers gives us the risk of relapse at diagnosis, disease surveillance, and predicting recurrence [65]. For future perspective, NGS of tumor tissue DNA is revealed as a promising avenue to comprehensively determine tumor mutation burden and their correlation with improved clinical outcomes for HNSCC patients. Since the tumor heterogeneity, liquid biopsy-based applications are revolutionizing the management of HNSCC patients. It can be approved whether ctDNA recapitulates de novo tumor tissue genomic landscape by comparing tumor tissue DNA from a HNSCC patient to their ctDNA derived from blood. For instance, when a drug targets a specific mutation, analyzing whether ctDNA burdens the same mutation, would allow more precise delivery of treatment, providing a precision medicine approach. In the future, ctDNA analysis will become part of the routine clinical management of HNSCC patients, thereby enhancing outcomes through targeted therapies [66].We propose longer-term studies in large-scale individuals and apply more reliable liquid biomarkers for HNSCC patients by organoid technologies for further evaluation of clinical applications of the CTCs, ctDNA, exosomes, and other biomarkers in HNSCC. In addition, we suggest detecting new biomarkers due to the high risk of relapse in locoregional disease after initial treatment and the restricted therapeutic options in the metastatic status. These biomarkers should discover minimal residual disease, evaluate treatment response, screen disease status and genomic profile signature, and identify resistance mechanisms. These suggestions can be achieved by analyzing ctDNA with the blood and saliva samples to provide high sensitivity and specificity in HNSCC patients. Based on the results of ctDNA studies, more investigations can reveal whether a change in therapeutic approaches will promote patient management as the primary endpoint compared to conventional methods. Only when these data are available can the assessment of molecular fluid biomarkers through liquid biopsy provide a valuable, cost-effective, and non-invasive strategy for cancer diagnosis, prognosis, and monitoring.

Conclusion

Liquid biopsy can be applied to evaluate molecular signature profiles in HNSCC patients, with great potential to help in early diagnosis, prognosis, surveillance, and treatment monitoring of tumors. Detection of more accurate predictive biomarkers for HNSCC patient management can happen by designing longitudinal extensive cohort studies and the utility of organoid technology that promotes the context of personalized and precision cancer medicine.

Acknowledgements

None.

Author contributions

All authors contributed to the study’s conception and design. Nooshin Mohtasham performed supervision. Hanieh Alsadat Mirkatuli, Mohammadhasan Baghbani, and Nima Ameli performed investigations. The first draft of the manuscript was written by Negin Ghiyasimoghaddam, Nooshin Mohtasham, Navidreza Shayan and all authors commented on previous versions. Nooshin Mohtasham performed editing and revising. Zeynab Ashari and Hanieh Alsadat Mirkatuli designed the figure. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

Data sharing does not apply to this article as no datasets were generated or analyzed during the current study.

Code availability

Not applicable.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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