Head and neck cancers are a group of aggressive and genetically complex cancers arising from diverse anatomical sites of the head and neck (1). According to the GLOBOCAN 2020 database, out of an estimated 19.3 million new cancer cases, there were a cumulative number of 878348 (4.5%) new cases of cancer of the lip and oral cavity, larynx, nasopharynx, orpharynx, and hypopharynx reported in 2020, worldwide (2). The incidence rate of lip and oral cavity cancer (Age Standardized Rate 10.2 per 100,000) follows that of prostate and lung cancer as the leading cancers among males in the lower Human Development Index (HDI) countries, and is the leading cause of cancer-related death among males in India. The high incidence of cancers of the lip and oral cavity cancers in South Central Asia and Melanesia (Papua New Guinea) is largely attributed to the popularity of betel nut chewing in these regions. On the other hand, the high incidence rates in Eastern and Western Europe and in Australia/New Zealand have been attributed to alcohol consumption, tobacco smoking and HPV infection for cancers of the oropharyngeal region, and to ultraviolet radiation from sunlight exposure for lip cancer (2). While most of these cancers, collectively known as head and neck squamous cell carcinoma (HNSCC) are derived from the mucosal epithelium of the oral cavity, pharynx and larynx (1, 3), their heterogeneity at the molecular level has proved to be a major impediment in the identification of drug targets and targeted therapeutics (1). Thus, the 5-year overall survival (OS) rate of HNSCCs has remained approximately at 40–50% without much improvement over the past decade, largely due to poor availability of effective therapeutic options for HNSCC patients with recurrent disease (4). Additionally, locoregional recurrences occur in 15%-50% of patients with HNSCC and is a major factor contributing towards deaths because of the complexities associated with treatment of recurrent HNSCC, which include the effects of prior treatment on tumor cells and the infiltrative and the multifocal nature of HNSCC itself (5).

Majority of HNSCC patients present with advanced stage cancer (3) which is difficult to treat even using a combinatorial approach (1), thereby underscoring the significance of early detection. Careful physical examination and early recognition of oral pre-malignant lesions (OPLs) which present as leukoplakia (white patches) or erythroplakia (red patches), and may progress to invasive cancer, remain the cornerstone of the early detection of HNSCC (3). Indeed, identification of early, asymptomatic oral squamous cell carcinoma (OSCC) during routine oral examinations by community-based dentists and physicians followed by oral brush biopsy could increase the 5 year OS rate to a remarkable 94% (6). Prognosis of the cancer and determination of the likelihood of disease recurrence or progression is equally important. Thus, the recent years have seen the development of significant interest in the formulation of strategies based on specific markers and molecular signatures for early detection and/or clinical prognosis of HNSCC (4, 7), with non-invasive diagnostic testing holding particular appeal (7). Key amongst these are “liquid biopsies,” which involve analysis of body fluid samples such as blood or other accessible fluids like ascites, pleural effusions, saliva, or urine. Most liquid biopsy-based diagnostic tests for solid malignancies are based on blood, serum or plasma specimens, which are less invasive, and have the potential to detect HNSCC, tumor recurrences or metastases at initial stages (7, 8). The primary markers being investigated from liquid biopsies are circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), tumor antigens, cell free coding and noncoding RNAs, metabolites as well as extracellular vesicles and exosomes (7, 8).

High throughput transcriptome analysis techniques have recently revealed circular RNAs (circRNAs) as potentially useful markers for the prognosis of cancer from body fluids. CircRNAs, also known as competitive endogenous RNAs (ceRNAs), are a large family of covalently closed, single-stranded, stable RNA molecules which regulate gene expression by regulating micro RNAs (miRNAs), binding with RNA-binding proteins (RBPs) or by getting directly translated into proteins. They exist stably and at high levels in body fluids, including plasma, serum, exosomes, and urine, and exhibit distinct expression patterns between patients with cancer and healthy controls (9, 10). Different circRNAs have been identified as functional prognostic biomarkers in lung, breast, colorectal, prostate, gastric, cervical, thyroid, bladder and ovarian cancers, hepatocellular carcinoma and esophageal squamous cell carcinoma (ESCC) (9, 11, 12). The circRNAs were found to be either significantly upregulated or downregulated in the body fluids of cancer patients in comparison to healthy controls with high discriminatory accuracy, and the expression level of most circRNAs were associated with tumour size, tumour differentiation, distant metastasis, and TNM stage (9).

The circular RNA, circPVT1, was proposed to play a pivotal and oncogenic role in the pathogenesis of HNSCC. CircPVT1 was found to be over-expressed in tumors compared to matched non-tumoral tissues, with particular enrichment in patients with TP53 mutations, and circPVT1 upregulation and downregulation were respectively associated with an increase or decrease in the malignant phenotype of HNSCC cell lines (13), indicating its relevance as a prognostic marker. Dysregulated circRNA expression has been reported in different types of HNSCC like ESCC, OSCC, laryngeal squamous cell carcinoma (LSCC), nasopharyngeal cancer and hypopharyngeal cancer (14–18).

In addition to the high heterogeneity of the molecular factors associated with HNSCC, inconsistencies are likely to arise among the findings of different studies on the potential of using circRNA as a biomarker of HNSCC due to variation in population ethnicity, sample size, specimens and control sources, which can become a hurdle to the application of circRNA in clinical practice (19). Therefore, we have performed a meta-analysis in order to investigate the relationship between the dysregulated expression of circRNAs and the prognosis and clinicopathological characteristics of HNSCC with the aim of determining whether circRNAs may be candidate biomarkers for HNSCC prognosis.

The therapies for HNSCC currently available and administered alone or in combination are radiotherapy, chemotherapy, targeted drugs and immunotherapy. While radiotherapy and chemotherapy remain highly toxic, the efficacy of EGFR inhibitors which are the only approved targeted drugs, is limited by inherent and acquired resistance (1). While newer strategies have contributed towards a significant improvement of the quality of life in the past two decades (43), there is no improvement in OS rates of patients in recent decades. Thus, an improved understanding of the molecular mechanisms underlying HNSCC is necessary to enhance therapeutic efficacy. Besides this, the requirement to identify patients who are more susceptible to metastases and recurrences has led to a pressing need for more compelling biomarkers for the identification of individuals who are at higher risk. This indicates the critical requirement of novel biomarkers to more precisely predict outcome of HNSCCs and help in the decision of therapeutic strategies. Studies indicate that circRNA play a vital role in the regulation of various molecular events which determine the occurrence, progression, and metastasis of various malignant tumors (44). Studies have also demonstrated that circRNAs are relatively stable because of their covalently closed circular structures with neither 5’ caps nor 3’ tails; consequently, most circRNAs are resistant to RNA exonucleases, and are more stable when compared with linear transcripts (45), making them good candidates for biomarkers.

A number of meta-analysis studies have established circRNA as a prognostic biomarker for cancer (19), such as hepatocellular carcinoma (46), colorectal cancer (47) and breast cancer (48). However, to the best of our knowledge, no meta-analysis has been performed to determine the prognostic significance of circRNAs in HNSCCs. In performing this meta-analysis, we included 9 studies with a total of 982 HNSCC cases in order to uncover the connection between the expression of different types of circular RNA and their clinicopathological and prognostic significance in HNSCC. Our results indicate that in HNSCC patients, the high expressions of seven circRNAs (circPVT1, circCOROC1, circPARD3, circ_0000199, circMYC, circ_0102272 and circCUX1) and low expressions of two circRNAs (circ0092125 and circ00072387) in tumors, in comparison to the levels in adjacent normal tissue were associated with poor clinicopathological features such as poor TNM staging, histological grading, lymph node metastasis, and recurrence in HNSCC and showed inferior OS [increased circRNA expression (univariate analysis: HR = 3.40, 95% CI: 2.66-4.36, p < 0.0001, I² = 0%; multivariate analysis: HR = 3.33, 95% CI: 2.54-4.38, p < 0.0001, I² = 0%), decreased circRNA expression (univariate analysis: HR = 2.83, 95% CI: 1.73-4.65, p < 0.0001, I² = 57.8%; multivariate analysis: HR = 2.35, 95% CI: 1.42-3.89, p = 0.0009, I² = 0%)].

The miRNAs that were targeted by these circRNAs were also identified in six of the nine included studies, revealing the mechanisms through which the circRNAs were involved in tumorigenesis and cancer progression. CircPARD3 acts as a sponge of the tumor-suppressor miR-145-5p in LSCC (22), while circCORO1C sponges let-7c-7p whose normal function is to inhibit the proliferation, migration and invasion of LSCC cells and to promote their apoptosis (21). Circ0000199 and circ0072387 interact simultaneously with the tumor suppressor miR-145-5p & miR-29b-3p and hsa-miR-129-3p, hsa-miR-141-3p, & hsa-miR-29-3p respectively in OSCC cells thereby regulating cell proliferation, growth and differentiation (24, 34). In nasopharyngeal carcinoma, circMYC is reported to interact with miR-20b-5p and let-7e-5p simultaneously, which in turn target argonaute RISC component 1 (AGO1) and cryptochrome circadian regulator 2 (CRY2) (42). Both AGO1 and CRY2 tightly regulate cell development, proliferation and migration (49, 50). Finally, circPVT1 binds to miR-497-5p through a specific binding site and affects its expression, leading to downregulation of miR-497-5p in HNSCC tumors with mutant p53 (13). The downregulation of both circ_00072387 and circ_0092125 was reported to significantly affect tumor size, TNM stage, and lymph node metastasis in OSCC patients by interacting with respective miRNAs (33, 34). Circ_00072387 binds with multiple miRNAs such as miR-129-3p, miR-141-3p, and miR-29-3p which regulate several tumor related pathways viz; MAPK signaling pathway, Ras signaling pathway, and Hippo signaling pathway (34). Similarly, circ_0092125 interacts with miR_1184, miR_1205, and miR_1322 which are reported to be involved in the occurrence and progression of OSCC (33). In nasopharyngeal carcinoma, interaction of circMYC with miR-20b-5p and let-7e-3p regulates tumor development as both these miRNAs exhibit tumor suppressor functions in a variety of cancers (42). Luo et al., 2020 also reported that miR-20b-5p and let-7e-3p jointly target AGO1 and CRY2. Various reports indicate the crucial role played by AGO1 in solid tumors and the involvement of CRY2 in epithelial-mesenchymal transition, Akt signaling and p53 signaling (42).

In addition, some of the included studies also revealed other molecular mechanisms through which the identified circRNAs contributed towards the cancer phenotype. The upregulation of circCUX1 was associated with radiotherapy resistance of hypopharyngeal tumors attributed to the binding of circCUX1 to the 3’UTR-of caspase-1, thereby inhibiting its expression and resulting in a decrease in inflammatory factors, thus leading to tolerance to radiotherapy (18). Similarly, circPARD3 promoted the proliferation, migration, invasion and chemoresistance of LSCC by inhibiting autophagy via the PRKC1-Akt-mTOR pathway (22). CircCORO1C promoted the proliferation, migration and invasion of LSCC cells by specifically upregulating the expression of the target PBX3 gene and affecting the epithelial mesenchymal transition (EMT) process. Indeed, dysregulation of PBX3 has also been reported in prostate, gastric, cervical and liver cancer (21). CircPVT1 was significantly upregulated in tumors with mutant p53 compared to those with wild type p53, and only in HNSCC patients with p53 mutations. In fact, the expression of circPVT1 was found to be regulated at the transcriptional level by the mut-p53/YAP/TEAD complex which resides on the promoter of circPVT1, such that increased expression of mutant p53, YAP and TEAD1 can increase the nascent circPVT1 expression. Simultaneously, circPVT1 might act within a positive self-regulating loop, controlling and enhancing its own nuclear expression, and thus regulating tumorigenesis (13).

Limitations: Our current study has some limitations which should be considered: (a) Based on our inclusion criteria, eight out of nine studies were only from Asian population (b) The sample size and number of the enrolled studies in this analysis were relatively small (c) Studies with good quality diagnostic data could not be retrieved because of which it was not possible to determine the potential of circRNAs as diagnostic markers of HNSCC (d) Begg’s funnel plot and Egger’s test value could not be generated for studies reporting downregulation of circRNAs in HNSCC due to the small number of studies. Thus, more high-quality studies on larger sample sizes and diverse population groups are required in order to firmly establish circRNAs as a good prognostic marker of HNSCC.

Taken together, our meta-analysis indicates that the nine circRNAs (circPVT1, circCOROC1, circPARD3, circ_0000199, circMYC, circ_0102272, circCUX1, circ_0092125 and circ_00072387) are potential prognostic biomarkers of HNSCC.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

MN and DR contributed to the design of study, performed the statistical analyses and drafted the manuscript. YC designed the study, provided intellectual input, approved the protocols to be followed in the study and drafted the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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