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Liquid Biopsies in HNSCC
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This entry is adapted from the peer-reviewed paper 10.3390/cancers13081874

Head and neck cancers are the seventh most frequent malignancy worldwide, consisting of a heterogeneous group of cancers that develop in the oral cavity, pharynx, and larynx, with head and neck squamous cell carcinoma (HNSCC) being the most common pathology. Due to limitations with screening and physical examination, HNSCC often presents in advanced disease states and is thus associated with poor survival. In this setting, liquid biopsies, or obtaining patient bodily fluid samples for cancer diagnosis and prognosis, may play a dramatic role in optimizing care for HNSCC patients. 

head and neck cancer liquid biopsy circulating tumor cells circulating tumor DNA exosomes head and neck squamous cell carcinoma

1. Introduction

Head and neck cancer is the seventh most common cancer worldwide, with 890,000 new cases and 450,000 deaths in 2018 [1], representing 3% of all cancers and slightly above 1.5% of all cancer deaths in the United States [2]. Head and neck squamous cell carcinomas (HNSCC) are by far the most common form of head and neck cancer, arising from mucosal surfaces of the oral cavity, pharynx, and larynx. Head and neck squamous cell carcinomas have been historically found in older patients with heavy tobacco and alcohol use; recently, there has been a declining global trend in traditional HNSCC cases due to decreased use of tobacco [3][4], counterbalanced with increased cases of HPV-associated oropharyngeal cancer (primarily by HPV type 16) [4][5].

In the era of exome- and genome-wide sequencing, tumor molecular profiling has been important in better understanding the diagnosis, treatment and prognosis of HNSCC in clinical practice. Currently, analysis based on tissue biopsy or cytology samples is still the gold standard in diagnosis of HNSCC. However, tissue biopsy may involve invasive procedures, often in a general anesthetic surgical setting. Thus, there are limitations in tissue access and repeatability of tumor sampling with current biopsy techniques. As there are considerable benefits to repeated biopsies both spatially (to account for tumor heterogeneity) and temporally (to account for dynamic tumor evolution), new methodologies for cancer diagnosis and assessment are needed. Over the last decade, there has been increasing interest in using liquid biopsies to detect cancer-specific biomarkers in patients’ body fluids [6][7]. Liquid biopsy has been reported to play roles in early malignancy detection in diverse tumor types [8][9]. As a rapid and noninvasive approach, liquid biopsies have emerged as an exciting investigational avenue to obtain information on cancer diagnosis, treatment response, and progression [10][11].

2. Circulating Tumor Cells in Head and Neck Cancer

2.1. Characterization and Detection Techniques

Circulating tumor cells (CTCs) represent transient cancer cells originating from a primary tumor or metastatic sites that have the capacity to enter the adjacent vasculature and disseminate to distant sites [12]. Notably, CTCs are extremely rare, with approximately 1–100 CTC/mL among billions of red blood cells [13], making it challenging to detect and capture CTCs from whole blood samples.

Different methods have been established to isolate CTCs, which can be broadly classified as either label-dependent (a positive selection of CTCs making use of specific markers expressed by tumor cells, such as epithelial cell adhesion molecules (EpCAM), cytokeratins (CKs) and integrins) [14] or label-independent (based on physical property differences such as size and density between CTCs and surrounding blood cells) [15]. Integrins, being cell surface receptors highly expressed in many different tumor types, have been an intriguing target for isolating CTCs. Some researchers have applied tumor cell specific ligands, such as LXY30 [14], targeting alpha 3 beta1 integrin, to enrich lung cancer CTCs from patient blood. Similarly, utilizing ligands targeting integrins (also highly expressed in HNSCCs) or other HNSCC cell surface ligands (e.g., Epidermal growth factor receptor which is frequently overexpressed in HNSCC) could increase the isolation and capture of rare CTCs. Biochemical-based schemes have been predominantly adopted for CTC isolation, with the CellSearch system being the only FDA-approved platform so far [16]. Nevertheless, selection bias exists, as CTCs undergoing epithelial-mesenchymal transition (EMT) reduce their expression of EpCAM and CKs, with EpCAM being particularly variable in HNSCC [17]. Conventional methods based on epithelial markers might not capture EMT-transformed CTCs and thus, might underestimate or fail to detect CTCs. To address this issue, negative selection targeting on blood cells for depletion rather than tumor cells for enrichment has been proposed, as CTCs do not express blood cell markers such as CD45 and CD235a [18]. Despite efforts to optimize CTC capture efficiency, satisfying outcomes in cell purity is yet to be achieved [19].

Recently, many new platforms have emerged to overcome the abovementioned challenges. Label-free inertial microfluidics approaches, for example, can avoid underestimation of CTC exhibiting downregulation of surface marker expression, and have been reported to be able to detect a larger CTC pool (3–133 CTC/mL of blood) than what had been previously achieved by the CellSearch system [20]. Researchers have also developed a 2-step CTC isolation and purification method, integrating a negative selection-based CTC isolation scheme and a subsequent 8-day spheroid cell culture for the further purification of CTCs, thereby isolating CTCs with higher purity [21].

2.2. Circulating Tumor Cells as Prognostic Biomarkers

With advances in technologies of CTC isolation and enumeration, there is a growing amount of research focusing on the value and potential use of CTC counts in cancer prognosis and treatment response, many of which have investigated the relationship between CTC quantitative counts and survival outcomes (Table 1). In a prospective clinical follow-up study involving 48 patients with HNSCC, Jatana et al. measured CTC numbers by the negative depletion method and observed an improved disease-free survival with no CTCs present and a worse clinical outcome with >25 CTCs/mL [22]. They reported a significant correlation between CTC presence and decreased disease-free survival (DFS) for the first time in a HNSCC population. However, in a large cohort study of 144 patients with locally advanced HNSCC, results suggested that the presence of CTCs was not predictive for DFS and overall survival (OS) in the cohort. Curiously, CTC detection trended with improved DFS and OS in patients with oropharyngeal carcinomas, while being a prognostic marker of worse DFS and OS in non-oropharyngeal HNSCC patients [23], suggesting CTCs (as currently detected) remain an imperfect prognostic liquid biomarker.

Table 1. Key studies investigating circulating tumor cells (CTCs) as biomarkers in head and neck squamous cell carcinomas (HNSCCs).

Table
Population/Trial Design Detection Methodology Primary
Outcome		 Major Findings Study Strengths Study Limitations References
HNSCC undergoing
definitive surgery		 Negative depletion followed by positive staining (CK) Disease-free survival (DFS)l Patients with no detectable CTCs had
significantly higher DFS		 Patients treated homogenously with surgery Single timepoint study, drawn during surgery [22]
Stage III-IV HNSCC after definitive surgery,
undergoing adjuvant (chemo)radiation (CRT)		 Positive staining (EGFR) 1. DFS
2. Overall survival (OS)		 1. CTCs detectable in 29% of patients
2. Non-OPSCC HNSCC patients with
detectable CTCs have worse DFS and OS		 1. Patients treated homogeneously with surgery, followed by adjuvant therapy
2. Large cohort (n = 144)		 1. Relying on EGFR positivity to define CTC
2. Single timepoint study		 [23]
Treatment-naïve HNSCC ClearCell FX system
Negative depletion (CD45)
Positive staining (CK)		 Progression-free survival (PFS) 1. CTCs detectable in 47.8% of patients
2. Patients with detectable CTCs have worse PFS
3. PD-L1 positive CTCs associated with worse survival		 Analysis of multiple expression markers (PD-L1, ALK, EGFR) 1. Small cohort (n = 23)
2. Single timepoint study
3. Mixed study with various stages, treatments		 [24]
Advanced OSCC after
induction chemotherapy, prior to definitive
treatment		 Positive staining (EpCAM) 1. Recurrence-free survival (RFS)
2.OS		 1. CTCs detectable in 80% of patients
2. Higher baseline CTC and maximal CTC associated with worse RFS and OS		 1. Phase II trial with defined
induction chemotherapy protocol
2.Serial timepoints
3. Homogeneous population of OSCC undergoing treatment		 1. Protocol not reflecting general standard of care
2. Smaller cohort (n = 40)		 [25]
HNSCC patients
undergoing definitive CRT		 Negative depletion 1. PFS
2. OS		 1. CTC reduction associated with response to chemoradiation and improved PFS and OS Serial timepoints Cohort limited to chemoradiation patients [26]
Advanced HNSCC
undergoing definitive CRT		 Negative depletion (CD45)
Positive staining (CK, EpCAM, EGFR)		 Detecting metastases 1. CTCs detected in 42% of patients
2. N2b or higher stage associated with higher CTCs		 1. Robust protocol for CTC
delineation
2. Serial timepoints		 1. Small cohort (n = 26)
2. Cohort limited to
chemoradiation patients
3. No survival analyses		 [27]
Advanced HNSCC
undergoing induction chemotherapy, followed by definitive CRT		 Positive staining (EpCAM) 1. PFS
2. OS		 PD-L1 overexpression on CTC associated with worse PFS and OS 1. Large cohort (n = 113)
2. Investigation of PD-L1 as
biomarker		 Cohort limited to chemoradiation patients [28]

Studies have also begun to illustrate the possible application of CTCs in monitoring disease status by conducting serial measurements of CTC levels. Inhestern et al. assessed and analyzed CTC counts from blood samples of 40 patients with oral cavity SCC (OSCC) before, during, and after treatment [25]. Similarly, Wang et al. investigated CTC counts of 47 HNSCC patients throughout concurrent chemoradiotherapy [26]. Both studies concluded that changes in CTC levels were highly correlative with tumor response to treatment, with persistently high CTC levels corresponding with worse prognosis and treatment response and decreasing CTC levels correlating with improved response and outcomes.

Additionally, CTC count was reported to have a predictive value for regional metastasis in head and neck cancer. In a study that included 42 patients with locally advanced HNSCC, the detection of CTC was discovered to correlate with the nodal stage as well as regional metastasis [27]. Kulasinghe et al. demonstrated in a cohort of 60 head and neck cancer patients that the presence of CTC clusters was significantly associated with distant metastatic disease [24]. Despite these initial encouraging findings, further research is undoubtedly needed to determine the role of CTC as a biomarker for treatment response and tumor state in head and neck cancer.

2.3. Circulating Tumor Cells as Immunotherapeutic Biomarkers

Immunotherapy has become a promising approach for the management and treatment of numerous cancers, including HNSCC, due to the emergence of immune checkpoint inhibitors [29]. There is interest in investigation of the role of CTCs in modulating disease behavior apart from its promise as a prognostic biomarker. Programmed death 1 (PD1) checkpoint inhibitors may block the PD-1/PD-L1 immune checkpoint pathway on CTCs and activate the immune system to eliminate CTCs in circulation, thus potentially reducing the risk of metastasis and disease recurrence.

PD-L1 overexpression in CTCs was detected and used for treatment response monitoring in patients with OSCC [30]. Strati et al. demonstrated (for the first time) that assessment of CTCs overexpressing PD-L1 in liquid biopsies is feasible, with implications for monitoring patients on PD1 inhibitors—and with the potential to provide important prognostic information in a prospective cohort of locally advanced HNSCC patients. By using RT-qPCR, PD-L1 overexpression was found in 24 (25.5%) of 94 patients at baseline, 8 (23.5%) of 34 patients after nonsurgical treatment, including chemoradiation, and 12 (22.2%) of 54 patients at the end of definitive treatment [28]. An application of CTCs in HNSCC was proposed such that patients with high baseline PD-L1 expression be given a monotherapy with a PD-1/PD-L1 inhibitor, and patients with low PD-L1 expression be selected for combination therapy [31]. PD-L1 overexpression at the end of treatment was also revealed as an important independent prognostic factor which correlated with shorter progression-free survival (PFS) and overall survival (OS) compared with PD-L1 negative counterparts [28]. Similarly, a study with an HNSCC cohort treated with nivolumab found that PD-L1-positive CTCs were significantly associated with worse outcomes [24]. Other immune-regulatory molecules in CTCs, including PD-L2 and CD47, were investigated as well. Researchers have suggested that expressions of these three immune-regulatory molecules were positively correlated to one another [32].

3. Circulating Tumor DNA in Head and Neck Cancer

3.1. Characteristics and Detection Techniques

Liquid biopsy techniques have also been focused on cancer-derived non-cellular components that circulate in the bloodstream. Most prominent in these studies have been circulating tumor DNA (ctDNA). Circulating tumor DNA refers to extracellular DNA (cell-free DNA) released into the bloodstream by the sloughing cancer cells, through both pathological and physiological mechanisms, including cellular apoptosis and necrosis from rapidly proliferating cancer cells [33]. cfDNA is present in noncancer states, originating from apoptotic and necrotic cells, which are then phagocytosed by macrophages and other scavenger cells, under normal circumstances. In cancerous states, ctDNA enters circulation in increased amounts when phagocytosis is exhausted or impaired within the tumor [34]. Thus, in patients with cancer, cfDNA is comprised of a combination of noncancer cell DNA from normal cells and ctDNA from cancer cells. Generally, ctDNA represents a small fraction of total cfDNA (usually <1.0%) but may vary from less than 0.1% to over 10% depending on tumor burden, cancer stage, cellular turnover, and response to therapy [35], thus making detection and quantification challenging via traditional sequencing and analysis approaches [36].

The advent of new digital technologies has helped address this challenge, as two major methods (targeted and untargeted ctDNA approaches) are being studied and optimized [6]. Targeted approaches include PCR-based technologies such as BEAMing (beads, emulsion, amplification, and magnetic) and droplet digital PCR (ddPCR), which can optimize samples with low DNA counts or concentration, e.g., ctDNA. Additionally, certain Next-generation sequencing based technologies are being utilized to help isolate and capture ctDNA, including TAm-Seq (tagged amplicon deep sequencing), CAPP-Seq (cancer personalized profiling by deep sequencing), Safe-Seq (safe sequencing system), and AmpliSeq [37][38], each with relative strengths in sensitivity and scalability [39]. Untargeted approaches include other types of NGS-based technologies, including whole genome sequencing (WGS) and whole exome sequencing (WES), which do not require any prior knowledge of molecular alteration but are comparatively less cost-effective [40].

Studies have shown that quantification of copy number aberrations (CNAs), human papillomavirus (HPV) DNA, and somatic mutations in low level ctDNA in plasma from HNSCC patients is technically feasible via TAm-Seq, AmpliSeq, ddPCR and WGS. Combined analyses contribute to a higher sensitivity, which is paramount to utility [41][42][43][44]. Despite current limitations, investigators are having success detecting and isolating minor amounts of ctDNA from body fluids, thus opening the gate to a variety of clinical applications [45]. As technological advances continue, greater sensitivity and specificity may allow for further enhanced clinical application.

3.2. ctDNA as a Biomarker for Disease Staging and Detection

The concept that ctDNA concentration is increased in patients with HNSCC—and correlates with disease severity—is foundational to considering ctDNA as a diagnostic and prognostic tool (Table 2). Mazurek et al. assessed total ctDNA levels in 200 patients with HNSCC, as compared to a noncancer control group. They found that the mean level of total ctDNA was higher in the HNSCC group, especially in oropharyngeal SCCs (OPSCCs), and correlated with nodal status [46]. Similarly, Bettegowda et al. evaluated the detection of ctDNA as a prognostic tool in 359 patients with 15 various cancer types, including HNSCC. By comparing patients with metastatic cancer versus nonmetastatic cancer patients, there was a clear trend with regards to metastatic stage of disease and increased ctDNA quantity [47]. Several feasibility studies have shown that in virally-mediated cancers (e.g., in the head and neck, HPV-related oropharyngeal cancer, and Epstein-Barr virus-mediated nasopharyngeal cancer), circulating HPV and EBV DNA from either plasma or saliva is useful for diagnosing disease at an earlier stage [43][48][49]. This highlights the potential utility of tumor-specific DNA in liquid biopsy for pretreatment phases for head and neck cancer detection and staging, as well as in screening of at-risk populations.

Table 2. Key studies investigating circulating tumor DNA (ctDNA) as biomarkers in HNSCC.

Table
Population/Trial
Design		 Detection Methodology Primary Outcome Major Findings Study Strengths Study Limitations References
Asymptomatic population screening RT-PCR for plasma EBV ctDNA 1. Rate of EBV DNA positivity
2. Rate of NPC in positive EBV DNA patients
3. Sensitivity, specificity of EBV DNA for NPC		 1. 5.5% detectable EBV DNA rate
2. 11% confirmed NPC among EBV DNA positive patients
3. 97.1% sensitivity, 98.6% specificity for NPC		 1. Robust population study (n > 20,000) of endemic population for NPC
2. Demonstration of utility of EBV DNA as a screening test		 1. False positive rates for EBV DNA
2. Challenges to apply to non-endemic populations		 [49]
HNSCC before definitive or salvage therapy PCR (HPV, TP53, PIK3CKA, CDKN2A, FBXW7, HRAS, NRAS mutations) for plasma and/or saliva ctDNA 1. Rate of ctDNA detection in saliva
2. Rate of ctDNA detection in plasma		 1. 96% ctDNA detection rate when both plasma and saliva tested
2. 100% saliva ctDNA identified in OSCC patients		 1. Demonstration of ability to identify salivary ctDNA
2. High detection rates demonstrated
3. Demonstration of detection of non-virally mediated HNSCC		 1. Hetergeneous population of HNSCC patients
2. Not all patients obtained saliva and blood testing
3. No survival analyses		 [43]
OPSCC before and after definitive therapy RT-PCR for plasma and saliva HPV ctDNA 1. Negative predictive value (NPV), positive predictive value (PPV), sensitivity, specificity of HPV ctDNA for tumor detection 1. Sensitivity 76%
2. Specificity 100%
3. NPV 42%
4. PPV 100%		 1. Serial timepoints
2. Demonstration of post-treatment ctDNA positivity correlation with worse survival		 1. Protocol limited to HPV-related HNSCC
2. Poor NPV		 [48]
HNSCC before and after definitive therapy Next-generation sequencing of plasma ctDNA to compare to identified primary tumor DNA mutations Baseline ctDNA mutation rates correlating with primary tumor 75% plasma ctDNA mutation rate Demonstration of utility of mutational profiling with primary tumor 1. Small cohort (n = 8)
2. No evidence of correlation with ctDNA positivity and recurrence		 [50]
p16 positive OPSCC after definitive CRT ddPCR for HPV ctDNA NPV, PPV of HPV ctDNA for cancer surveillance 1. NPV 100%
2. PPV 94%		 1. Large cohort (n = 115)
2. Serial timepoints
3. Demonstration of utility of HPV DNA as a surveillance test		 1. Protocol limited to HPV-related HNSCC [51]

3.3. ctDNA as a Biomarker for Post-Treatment Surveillance

In the posttreatment setting, ctDNA can be used to assess residual disease and stratify patients at variable risk of recurrence following curative-intent therapy. Monitoring disease by ctDNA has increasingly been investigated in a number of malignancies, including breast and colorectal cancers [52][53]. In studies by Wang et al. and Ahn et al., HPV ctDNA from posttreatment HNSCC patients were also analyzed with promising results, providing the possibility of predicting disease recurrence using ctDNA [43][48]. In another study, Hamana et al. compared preoperative and postoperative levels of ctDNA in 64 patients with OPSCC. The number of patients with tumor-specific ctDNA dropped from 40% (28/64) to 20% (13/64) after treatment; moreover, among the 28 patients with measurable ctDNA preoperatively, those with no detectable ctDNA postoperatively had no disease recurrence, whereas 4 patients with postoperative detectable ctDNA subsequently developed distant metastases [54]. Egyud et al. studied the utility of ctDNA as a diagnostic tool for recurrence in 8 HNSCC patients. Their findings showed that two of four patients with recurrence had detectable plasma ctDNA prior to clinical manifestation [50].

Longitudinal monitoring of ctDNA may allow for repeated quick, noninvasive means to assay disease status at multiple timepoints [55]. A recent prospective clinical trial with 115 HPV-positive OPSCC patients measured longitudinal HPV ctDNA levels. Kinetic data measured during curative-intent chemoradiotherapy for a median time of 23 months demonstrated that all patients with recurrence had at least two consecutive abnormal plasma HPV ctDNA levels. Furthermore, of the 87 patients with undetectable HPV ctDNA after completion of chemoradiation, none had recurrences, thus showing high sensitivity and specificity for HPV ctDNA in identifying recurrent HPV-associated oropharyngeal HNSCC [51]. ctDNA has also been investigated as a tool to augment clinical decision-making in determining radiologic presence of disease. In a study by Rutkowski et al., patients with incomplete radiographic response and elevated HPV ctDNA levels were demonstrated by PET-CT to have recurrences, suggesting that more recurrent diseases may be discovered by combining ctDNA with radiographic examination, as opposed to radiographic imaging alone [56]. ctDNA levels were also compared with metabolic response (represented by PET-CT), and their predictive values for assessing treatment response were similar [57]. Thus, the ability to assess treatment response in HNSCC may be enhanced by combining ctDNA data from liquid biopsy with standard imaging results.

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Lingyi Kong
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