Personalized Medicine in Oral Oncology: Imaging Methods and Biological Markers to Support Diagnosis of Oral Squamous Cell Carcinoma (OSCC): A Narrative Literature Review

For decades, oral squamous cell carcinoma (OSCC) has been one of the most prevalent and mortal cancers worldwide. The gold standard for OSCC diagnosis is still histopathology but this narrative multidisciplinary review has the aim to explore the literature about conventional OSCC prognostic indicators related to the pTNM stage at the diagnosis such as the depth of invasion and the lymphovascular invasion associated with distant metastasis as indicators of poor life expectancy. Despite its multifactorial nature and recognizable precursors, its diagnosis at the early stages is still challenging. We wanted to highlight the importance of the screening as a primary weapon that a stomatologist should consider, intercepting all at-risk conditions and lesions associated with OSCC and its early stages. This narrative review also overviews the most promising imaging techniques, such as CT, MRI, and US-echography, and their application related to clinical and surgical practice, but also the most-investigated prognostic and diagnostic tissue and salivary biomarkers helpful in OSCC diagnosis and prognostic assessment. Our work highlighted remarkable potential biomarkers that could have a leading role in the future. However, we are still far from defining an appropriate and concrete protocol to apply in clinical practice. The hope is that the present and future research will overcome these limitations to benefit patients, clinicians, and welfare.


Introduction
The oral cavity may be affected by various malignant neoplasias, mainly of epithelial origin, such as oral squamous cell carcinoma (OSCC) and lymphoepithelial carcinoma [1], but also those arising from the derailment of oral melanocytes (oral melanomas) [2] and other soft tissues, such as sarcomas and angiosarcomas, adenocarcinoma, and adenocarcinoma not otherwise specified (NOS), as well as other rare tumors [3][4][5][6][7][8][9][10], and, on clinical bases, it is often difficult to distinguish them from benignant neoplasias and conditions.
OSCC can arise from previously clinically healthy oral mucosa or on precursor lesions and conditions, previously classified as potentially malignant lesions and conditions, respectively, and these were recently reclassified as oral potentially malignant mucosal disorders or OPMDs.By definition, the precancerous lesion is a circumscribed oral mucosal lesion with a higher probability of malignant transformation than the mucosa surrounding the lesion; the precancerous condition is a generalized condition in which all the oral mucosa has more chance of malignant transformation than a subject without that condition.Last, OPMDs include conditions that affect the oral mucosa with an increased risk of malignancy, thus assuming, de facto, both precancerous lesions and conditions under the same group.Leukoplakia, erythroplakia, lichen planus, and submucous fibrosis are the most common OPMDs [12][13][14].
Some authors consider that various viruses have been associated with oral cancerogenesis [26][27][28][29][30][31].Among them, HPV is historically and universally responsible for head and neck cancers of the oropharynx (OPC) and, as a matter of fact, from a clinical standpoint the evaluation of HPV-related markers is a valid parameter in the potential adjuvant or neoadjuvant treatment of oropharyngeal cancer regarding radiotherapy.In detail, HPV-16 is responsible for almost 90% of HPV-positive oropharyngeal cancers [32][33][34], mainly in men.Regarding the HPV-related cancers of the oral cavity proper, and thus excluding the OPCs, more than 97% of OSCCs are HPV-negative [27], and those that are positive could represent a subgroup of OSCCs of the base tongue [31].Hence, HPV's pathogenic role in OSCC is still debated.However, the literature reports a better prognosis in HPV-positive OSCC than HPV-unrelated OSCC, despite the finding that HPV in the oral mucosae is rarer than in the pharynx [26,33,35,36].
The main issue related to OSCC is its late diagnosis, responsible for poor 5-year survival and substantial disabilities and bad health-related quality of life of OSCC survivors, who frequently may encounter recurrences or second primary tumors due to field cancerization [67][68][69][70][71][72] To date, the conventional OSCC prognostic indicators are related to the pTNM stage at the diagnosis and mainly the depth of invasion and the lymphovascular invasion [1,[73][74][75][76], both responsible for distant metastasis and poor life expectancy [77,78].
The recognition of OSCC at its early stage is possible through different measures related to the various degrees of prevention: -Primary prevention (counselling at-risk subjects (stop smoking, stop drinking)); -Secondary prevention (screening and early diagnosis of OPMDs); -Tertiary prevention (strict follow-up of OSCC survivors to intercept recurrences, metastasis, and/or second primary tumors).

Imaging Techniques
Clinically similar conditions may underlie lesions utterly different from each other in nature and prognosis [81].The possibility to benefit from imaging devices capable of noninvasively detecting signs of early cancer allows the strengthening of diagnosis parallel to less invasive treatments and better survival and quality of life.
Among the most-investigated imaging tools in oral oncology, confocal microscopy The selected articles were grouped by topic and kind of marker studied.In this way, for convenience, the results have been presented in several sections, dealing, respectively, with imaging techniques-focusing on those innovative noninvasive and real-time ones-tissue markers, and salivary biomarkers-mainly adhesion molecules, enzymes, proteins, genes, various types of RNA, and metabolites involved in cancerogenesis or associated with cancer aggressiveness.Further research summarized the role of oral dysbiosis in the occurrence or maintenance of the OSCC microenvironment and reported the mostinvestigated microorganisms associated with OSCC onset and prognosis.
Mendeley Reference Manager software was used to export and manage the references.

Imaging Techniques
Clinically similar conditions may underlie lesions utterly different from each other in nature and prognosis [81].The possibility to benefit from imaging devices capable of noninvasively detecting signs of early cancer allows the strengthening of diagnosis parallel to less invasive treatments and better survival and quality of life.
Among the most-investigated imaging tools in oral oncology, confocal microscopy (CM), optical coherence tomography (OCT), ultrasound echography (US-ECO), and tissue fluorescence (TF) [82] may be advantageously used to complement conventional RMN and CT scans [83] or to anticipate the diagnosis of small masses or early tumor changes unrecognized by these standard techniques.
Briefly, confocal microscopy (CM) and optical coherence tomography (OCT) are based on an incident harmless laser source penetrating the epithelial layers.Then, an associated detector collects the backscattered co-planar or coherent light, respectively, thus allowing the digital rebuilding of virtual horizontal (in CM) or transversal (in OCT) sections of the tissue at a microscopic resolution [82,[84][85][86].
Both CM and OCT applications in the imaging of the oral cavity are well documented in the literature [87][88][89][90][91].
Images similar to those gained by OCT imaging can be obtained by high-frequency US-echography, which is based on the transduction of sound waves backreflected from the tissue to a transducer capable of giving a visual image of the echoes, thus offering digital pictures of the body structures.In the oral field, this technology has been used to describe benign oral neoformations [121], as well as to characterize both healthy oral mucosa [121,122] and oral squamous cell carcinomas [123][124][125][126][127]. Furthermore, the high-frequency USs have been proven more accurate in estimating the thickness of thin carcinomas of the tongue when compared to MRI [128].The potential application of high-frequency US-echography for the evaluation of the depth of invasion of OSCC is also an interesting point of reflection that is now emerging from a few studies.As a matter of fact, as reported in the eighth edition of the American Joint Committee on Cancer (AJCC) staging of oral cancer, the depth of invasion or DOI (≤5 mm, >5 mm but ≤10 mm, and >10 mm) in the new criteria has a fundamental role when categorizing a lesion from T1 to T4a and is strongly associated with neck node metastasis.US-echography, together with MRI and CT, could be very helpful in order to determine the preoperative DOI criteria and represent an important support for an adequate surgical preoperatory planning [129][130][131][132][133].
Each of the imaging techniques mentioned above has its advantages and limitations.Romano et al. [82] attempted to define the main functional characteristics of each one based on their capability to allow:

Tissue Markers
The most conventional approach to studying cancerogenesis considers genetic and epigenetic changes occurring in tumoral tissues.For this purpose, histological, immunohistochemical, and immunofluorescent studies focus on specific molecules, proteins, genes, or pathways differing from healthy normal cells.
Being a stratified epithelium, the healthy oral mucosa histologically features the expression of cytokeratin types, peculiar to each layer and anatomical site, and each keratinocyte is well anchored and linked to the others by variously organized junctional proteins.
Hence, a way through which an epithelial cancer cell may acquire motility and capability to metastasize is given when it loses the anchorage with other cells.Examples of altered expression of anchorage and cytoskeleton proteins involved in oral cancerogenesis are offered by alterations in cadherin family proteins and the interrelated proteins, such as integrins and vimentins [144][145][146][147]. E-cadherin alterations have been proven to provide cancer cells high propensity to invade and metastasize.This event is associated with its downregulation and delocalization from the membrane to the cytoplasm in cancer cells and is correlated to aggressive, poorly differentiated, high-grade OSCC and lower patient survival [148].Furthermore, in a vicious circle, the delocalization of E-cadherin to cytoplasm seems due not only to the hypermethylation of its gene promoter, thus enabling E-cadherin synthesis, but also to the increase in epidermal growth factor receptor (EGFR) expression in OSCC that seems to favor E-cadherin internalization [148].EGFR is, in fact, overexpressed in OSCC, which is associated with poor prognosis [149][150][151].The evidence is such that several studies proposed anti-EGFR target therapies [152,153].
However, numerous other pathway dysregulations have been considered involved in OSCC, such as WNT/β-catenin pathway dysregulation.To better understand, it must be considered that, under physiological conditions, Wnt/β-catenin signaling regulates cell differentiation, proliferation, and apoptosis [154,155].Hence, abnormal activation of this signaling promotes OSCC progression and metastasis [156].The pivotal event leading to this dysregulation is caused by the aberrant expression of β-catenin which is not regulated by transmembrane WNT and can uncontrollably migrate to the nucleus, thus triggering a series of antiapoptotic WNT target genes, such as c-Myc, cyclin D1, and Bcl-2, thus upregulating proliferation and migration [157][158][159][160][161][162].Furthermore, in these events, EGFR is also involved since it forms a complex with β-catenin and further promotes the cancer cell's ability to invade and metastasize [163].
OSCC has also been proven to express higher than normal levels of cyclooxygenase type 2, conventionally associated with chronic inflammation [164][165][166].
Table 2 summarizes the evidence from the literature about the association between the most-investigated tissue markers and OSCC.

Circulating Markers
Since one of the goals of early diagnosis is the noninvasiveness of the procedure, another approach is offered by identifying biological markers from biological fluids, such as blood and saliva.
In detail, the use of saliva is convenient and noninvasive.Many studies reported hundreds of potential saliva biomarkers for OSCC and various targets identified by various methods.
All the technologies used to identify and quantify these biomarkers are based on the so-called omics sciences: a term that has recently become widespread to comprehensively define each branch of molecular techniques focusing on specific targets [167].By the definition given by Vailati-Riboni et al., the aim of omics sciences is "to identify, characterize, and quantify all biological molecules that are involved in the structure, function, and dynamics of a cell, tissue, or organism" [167].
In any case, a liquid biomarker can be investigated for diagnostic or prognostic purposes since some markers have been recognized as indicative of cancer (diagnostic value), while others are associated with the best or worst prognosis (prognostic values).In both cases, the presence/absence may be indicative, but its quantification (higher or lower expression) and the onset period (during the primary tumor stage, its recurrences or metastasis) should also be considered.

Salivary Biomarkers
Concerning OSCC, its salivary biomarkers can be proteins, genes, various types of RNA, and metabolites.In each case, it must be considered that each molecule could be variously considered a diagnostic or prognostic marker.In the first case, the target-marker's presence/absence or quantity indicates cancer; in the second case, its presence/absence or level of expression is associated with a better or worse life expectancy.
In 2022, Shaw et al. [168] meta-analyzed the scientific literature for the quantitative analysis of diagnostic accuracy and applicability of four classes of salivary biomarkers and the relative capability of polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) to detect them in the saliva.The authors reported results of detecting salivary biomarkers better by PCR than by ELISA; furthermore, the more sensitive and specific biomarkers associated with OSCC were mRNAs, miRNA, and IL-8 (sensitivity and specificity over 89%).Data were analyzed from over 1000 patients in thirteen eligible studies.
In the field of omics sciences, the role of salivary proteins associated with OSCC has been widely investigated.Various systematic reviews and meta-analyses have recently summarized this research topic's state of the art [168][169][170][171][172].
In detail, Arroyo et al. [172] analyzed the diagnostic capacity of salivary proteins as biomarkers for the differential diagnosis of OPMDs and OSCC compared to healthy controls.Their research was published in 2021 and considered, at the end of the selection, eight papers eligible for their purpose, thus reporting a series of potential markers, of which those significantly higher in OSCC/OPMDs than healthy controls, and therefore with diagnostic significance, were carcinoembryonic antigen (CEA) [173,174] and the soluble fragment of cytokeratin 19 (CYFRA21) [170,171].In all these cases, ELISA was used as a helpful technique to discover and quantify proteins in a sample [175][176][177][178][179].
AlAli et al. [180] also found the prognostic significance of higher levels of IL-8 in cancer patients, whose overexpression, together with matrix metalloproteinase 9, MMP-9, was confirmed by other authors as associated with the worst prognosis.Later, Elmahgoub found the diagnostic significance of IL-1β, IL-6, and IL-8 for identifying early OSCC and OPMDs, as their levels were significantly higher than in healthy controls [181].A similar approach to identifying the most significant diagnostic and prognostic salivary markers associated with OSCC and their different levels of expression varying during different stages of the disease was also reported by Riccardi et al. [171], who focused their systematic review and meta-analysis on a series of salivary proteins potentially helpful in screening oral neoplasia and predicting the prognosis of OSCCs.The authors grouped the main salivary OSCC biomarkers into five classes, such as cytokines (IL-1a, IL-1b, IL-6, IL-8, IL-10, TNF-α), other acute-phase response proteins associated with an inflammatory tumoral microenvironment (AATa, HPX, C3, TTR, serotransferrin, and RETN), growth factors (vascular endothelial growth factor-a, VEGF-a), matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9), and proline-rich proteins (PRPs) [171].In detail, the prognostic role of cytokines like IL-1α, IL-6, IL-8, VEGF-a, and TNF-α was confirmed and remarkably accurate for tongue OSCC, with a poorer survival expectancy when overexpressed [182].Furthermore, Riccardi et al. [171] summarized that early OSCC and OPMDs moving toward cancer exhibit overexpression of IL-6 and IL-1β.At the same time, TNF-α and IL-10 seemed to be more indicative of advanced OSCC, where they act by sustaining the inflammatory microenvironment where OSCC grows and acquires malignant and more aggressive behavior.
Similarly, salivary levels of MMP-1 and MMP-3 have been proven to be up to 10-fold overexpressed in OSCC compared with healthy controls, and the increase seemed to be proportional to the OSCC stage, thus adding crucial prognostic information [183].At the same time, MMP-2 and MMP-9 should be more indicative as screening biomarkers, peculiarly present in OSCC and those OPMDs moving toward malignant transformation [184,185].Furthermore, among the proteins expressed in acute inflammation and cancer, there is broad evidence on how the inflammatory microenvironment is linked to cancer development and progression, mainly through the pathway involving COX-2 [164,165,186].
Moreover, among other kinds of salivary proteins investigated, resistin (RETN), a derived peptide hormone, seemed highly correlated with advanced OSCC and metastases, particularly in smokers and betel-chewers [187] and, similarly to other well-known hormones, such as the sexual ones, responsible for a gender-related difference in the onset and prognosis of specific subsets of OSCC [15,19,20].
The discovery of salivary biomarkers is accelerating from the advent and diffusion of other automated high-throughput techniques capable of massively sequencing genomes from tissues or fluids, known as next-generation sequencing (NGS).Used for sequencing both entire genomes and specific genetic sequences, NGS allows the identification of targeted sequences of cancer-related genes and RNAs.In the case of OSCC salivary biomarkers identified by NGS, the most-considered ones are genomic markers (genomic sequences indicative of genes involved in cancerogenesis) and proteomic markers, as described previously [188].
In the case of gene analysis, NGS aims to discover mutations at the gene level or methylation regarding promoter for oncosuppressor genes, known to silence oncosuppressor transcription, thus reducing their protein expression [148].When used to find protein markers, NGS analyzes the transcriptomes (mRNA and small miRNA) to establish any relevant changes occurring in cancer.

Blood Markers and Circulating Cancer Cells
The various groups of "circulating markers" comprise a series of molecules-mainly tumoral cells, proteins, and nucleic acid compounds-whose presence in the blood has been proven indicative of OSCC at various stages.
Recently, Zhu et al. [189] monitored, over a 5-year follow-up, the blood fibrinogen and albumin in OSCC patients to determine their prognostic value.In a cohort of over 150 patients suffering from OSCC, the increase in the fibrinogen-to-albumin ratio was reported to be an independent prognostic factor, negatively associated with the cancerspecific survival of the patients considered.These results are similar, and the evidence is reinforced, by the work published, in the same year, on bladder cancer by Barone et al. [190].
Other than inflammatory and nutritional circulating markers, other authors investigated the number and content of circulating extracellular vesicles carrying nucleic acids and proteins associated with OSCC.As reported by Brocco et al., extracellular vesicles (EVs) are "particles naturally delivered into the extracellular microenvironment, containing a rich cargo of DNA, RNA, miRNAs, proteins, lipids, and metabolites," and they allow the transfer of their molecular cargoes, mediating short-and long-distance signaling, during physiological or pathological processes [191].EVs are grouped by size and origin into exosomes, originating from the endosomal system and with a diameter of 30-150 nm, microvesicles, 100-1000 nm in diameter, released by plasma membrane budding, and apoptotic bodies, produced by apoptotic cells, with a variable diameter of 200-5000 nm [191].Tumor cells have been proven to produce and release more EVs than healthy cells, and in cancer, they participate in various biological processes involved in tumoral neoangiogenesis, progression, and metastases [191].Brocco et al. identified the EV concentrations and specific content from the peripheral blood samples of 106 oncologic patients and 25 healthy controls, thus proving the utility of EVs as predictors and diagnostic markers [191].
Furthermore, while the work by Brocco et al. focused on metastatic and locally advanced nonhematological cancer, Sun et al. [192] studied the EVs' types by a proteomic approach, in salivary samples from 30 OSCC patients and 30 matched healthy controls, thus reporting the occurrence of 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC.This work has been very recently published, and further developments are expected.
Other than circulating proteins, both free or conveyed by vesicles, some researchers focused on cancer cells and circulating tumor nucleic acids, through a methodology known as "liquid biopsy".This method focuses on the finding in body fluids, mainly plasma but also saliva, of potential biological markers associated with OSCC, like oncogenic microRNAs (miRNAs), small single-chain noncoding RNA molecules involved in mRNA inhibition for oncosuppressor proteins [193][194][195], circulating cell-free DNA (cfDNA), and mitochondrial DNA (mtDNA), both proportionally higher with OSCC aggressiveness and metastasis [196], or proper tumor cells.
In this last case, the literature agrees with the finding that plasma-circulating cancer cells and cancer stem cells can be considered a valuable diagnostic and prognostic OSCC marker since their identification and amount are strictly and significatively associated with the TNM staging [197], with sensitivity, specificity, and accuracy globally over 95% [198].

Oral Microbiota Changes
A recent branch of oral oncology studies the oral microbiome and microbiota associated with OSCC.By definition, the microbiota is the microbic population specifically living in specific anatomical environments, such as the mouth and its niches, while the term "microbiome" refers to all genomic material distinct from the human genome [44,199].
Arising evidence has proven that specific changes in oral microbiota composition or oral microbiome expression occur associated with OSCC, as well proven for other extra-oral diseases and cancer [55,57,[200][201][202].
The most-investigated oral dysbiosis occurring in OSCC is associated with the overexpression of periodontopathogen bacteria such as Fusobacterium nucleatum and Porphyromonas gingivalis, responsible for chronic inflammation, bone resorption, hyperglycemia, and local and systemic toxicity, and an increase in oxidative stress resulting in overproduction of free radicals with oncogenic derailment [20,45,55,207,208].These events can be directly and indirectly linked to bacterial proliferation and bacterial toxins, which may damage keratinocyte DNA beyond their repair capacities, thus allowing the accumulation of irreversible damage which leads to cancerogenesis.
In general, oral dysbiosis is responsible for a microecological imbalance in the oral cavity and chronic inflammation and immune stimulations, which may trigger a stream of events that promote cancerogenesis [209].
In detail, F. nuclatum and P. gingivalis have been proven to enhance local inflammation at periodontal sites, thus exacerbating the production of those inflammatory cytokines previously described as associated with OSCC developments, mainly IL-6, TNF-α, and MMP-9, responsible for tissue dissolution and cancer progression, and proven to be overexpressed in OSCC [171,184,210].
Although the literature has reported the co-occurrence of oral dysbiosis in subjects suffering from OSCC, it is still unknown whether the bacteria anticipate and are directly responsible for OSCC or if peculiar dysbiosis occurs later, thus favored by the tumoral microenvironment.
Some recent research has tried to respond to this question.
Regarding the dysbiosis mechanisms involved in oral tumorigenesis, Pignatelli et al. [211] hypothesized three different mechanisms through which oral dysbiosis empowers oral tumorigenesis: the imbalance of keratinocyte proliferation and death; -immune dysregulations; -alterations of metabolisms of food compounds, drugs, and host metabolite.
Substantial efforts have been made to identify specific microbiota associated with OSCC.For this purpose, as an example, Nie et al. [212] characterized the microbiota composition in different oral niches of OSCC in a sample of 65 OSCC patients.In their work, the microbiota identification of each niche in each subject was performed by 16S rDNA amplification and sequencing by PCR.Their results showed a noticeable difference in the microbiota composition of the tumoral niche compared with the neighboring and contralateral healthy areas.In detail, tumor tissue was peculiarly overcolonized by a greater abundance of Fusobacteria, Prevotella spp., Porphyromonas, Campylobacteria, Aggregatibacteria, Treponema, and Peptostreptococcus.Conversely, Neisseria and Veillonella were predominantly present in healthy tissues [212].Interestingly, the species overexpressed at the tumoral sites were all anaerobic/facultative anaerobes.Furthermore, the authors associated the microbiota with clinical parameters, such as tumor size, histological tumor grading, TNM staging, and presence/absence of metastases, thus concluding on significant species predictive for metastases (but not for all the other indicators) by the significantly different identification and overexpression of Parvimonas micra, Prevotella pallens, Luteimonas marina, Peptostreptococcus stomatis, and Pyramidobacter piscolens [212].
These findings were shared by Pignatelli et al., who reported significantly higher levels of periodontitis-related taxa in OSCC subjects from the literature.Among the species most represented, the authors considered P. gingivalis, F. nucleatum, C. rectus, and P. stomatis, parallel to a significant decrease in Veillonellae [211].
Furthermore, most species were proven capable of metabolizing or fermenting host proteins into sulfides and nitrosamines, known as potent cell mutagens [213].Among the most-reported species involved in this event, F. nucleatum, P. gingivalis, A actinomycetemconcomitans, and Prevotella intermedia produce the genotoxic and mutagenic agent hydrogen sulfide, responsible for chronic inflammation and cell migration and invasion, as well as cell proliferation and tumor angiogenesis [211].
Pignatelli et al. gave a further in-depth explanation of periodontopathogen bacteria with cancerogenic properties as due to the capability to produce volatile sulfur products responsible for the increase in ROS production and promoting collagen degradation of the basement membrane, thus favoring not only the cancer initiation but also invasion [211].
Various species have been considered able to interact with each other to amplify the oncogenic risk.This is the case for F. nucletum, which is capable of co-aggregating and interacting with various other bacteria, or P. gingivalis, which upregulates a series of genes responsible for loss of bacterial adhesion, thus reducing biofilm formation, as well as an increase in cell motility in tumor sites [214], so its salivary overabundance was proven to be correlated with advanced OSCC stages [215].
Otherwise, the acid production by Lactobacilli spp.seems to favor an acidic environment favorable to tumor growth, and their levels increase parallel to advancing TNM cancer stages [216].Similarly, some Streptococcus spp.are associated with an increased risk of metastasis since they produce hydrogen peroxide and nitrogen dioxide, which are responsible for lowering pH and the development of hypoxia in the tumoral environment [217].
Lastly, the interaction between bacteria and alcohol or tobacco consumption must also be considered an additional factor in oral cancerogenesis.For example, Neisseria spp.are high producers of acetaldehyde.Hence, in an alcohol drinker, the alcohol consumed can be efficiently metabolized by these bacteria into acetaldehyde, of proven cancerogenic properties [218].

Discussion
As in past decades, OSCC is still one of the most prevalent and mortal cancers worldwide [1].Despite the well-known multifactorial origin and the presence of recognizable and indicative precursors, its diagnosis at the early stages is still a challenge due to the unclear features at the initial stages and confounding factors or co-morbidities that can be underestimated by the subjects suffering this neoplasia.
In order to better identify the early signs of OSCC and prognosticate its behavior once diagnosed, OSCC can be approached by different technologies and methods.
This work has overviewed the current knowledge in the primary fields related to this issue, thus reporting the experiences from the scientific literature regarding noninvasive imaging techniques and diagnostic or prognostic markers of different natures and origins.
Table 3 and Figure 2 summarize the potential clinical support offered and the analyses obtained by each methodology, respectively.
Noninvasive imaging techniques showed the capability to support early and real-time appropriate diagnosis.Moreover, they could also guide toward the most appropriate therapeutical approach.Identifying a series of targeted molecules can be advantageous to indicate OSCC diagnosis and prognosis based on their presence or absence, increase or decrease, and structural or functional alterations associated with OSCC onset, recurrence, stages, or prognosis.Among the imaging techniques, high-frequency ultrasound imaging, confocal microscopy, optical coherence tomography, and vascular imaging offered promising examples in the literature of advantageous helpfulness to defining, before surgery and before treatment, the nature of lesions suspected to be oral cancer.
Moreover, histological evaluation after surgery is still the gold standard for diagnosing OSCC, grading its histological disarray, staging its TNM features, and preliminarily hypothesizing a prognosis according to the classical parameters.In this context, scientists are constantly researching some tissue markers specific for OSCC, which could add further indications regarding the potential for malignant transformation in the case of OP-MDs or that could be associated with aggressive forms of advanced OSCC.
However, the identification of tissue markers has a biopsy as a prerequisite and can be adopted only in the case of suspected lesions that require diagnostic insights through a conventional diagnostic pathway.Hence, the study of tissue markers is not applicable for screening programs or follow-up purposes.
In these latter cases, identifying other markers from fluids is more convenient; for this reason, scientists can draw on easily accessible bodily fluids such as blood and saliva.Leaving aside the blood, whose sampling is, in any case, invasive, the saliva remains, which is the ideal fluid, as it is easily accessible, collectible without invasive procedures, and in direct contact with any oral tumors or OPMDs.For these reasons, any changes in its composition can be easily detected, with simplicity and good patient compliance, giving reliable estimates of any metabolic or other changes in the course of oral carcinogenesis.
A salivary marker is each protein, RNA, or metabolite whose presence or absence or hypo-or hyperexpression is indicative of tumoral changes in the oral cavity.However, in any case, a universal marker may not be found due to heterogeneity across different ethnicities and populations [219].Among the imaging techniques, high-frequency ultrasound imaging, confocal microscopy, optical coherence tomography, and vascular imaging offered promising examples in the literature of advantageous helpfulness to defining, before surgery and before treatment, the nature of lesions suspected to be oral cancer.
Moreover, histological evaluation after surgery is still the gold standard for diagnosing OSCC, grading its histological disarray, staging its TNM features, and preliminarily hypothesizing a prognosis according to the classical parameters.In this context, scientists are constantly researching some tissue markers specific for OSCC, which could add further indications regarding the potential for malignant transformation in the case of OPMDs or that could be associated with aggressive forms of advanced OSCC.
However, the identification of tissue markers has a biopsy as a prerequisite and can be adopted only in the case of suspected lesions that require diagnostic insights through a conventional diagnostic pathway.Hence, the study of tissue markers is not applicable for screening programs or follow-up purposes.
In these latter cases, identifying other markers from fluids is more convenient; for this reason, scientists can draw on easily accessible bodily fluids such as blood and saliva.Leaving aside the blood, whose sampling is, in any case, invasive, the saliva remains, which is the ideal fluid, as it is easily accessible, collectible without invasive procedures, and in direct contact with any oral tumors or OPMDs.For these reasons, any changes in its composition can be easily detected, with simplicity and good patient compliance, giving reliable estimates of any metabolic or other changes in the course of oral carcinogenesis.
A salivary marker is each protein, RNA, or metabolite whose presence or absence or hypo-or hyperexpression is indicative of tumoral changes in the oral cavity.However, in any case, a universal marker may not be found due to heterogeneity across different ethnicities and populations [219].
For this purpose, a series of omics sciences have been developed and used to study the primary salivary markers with diagnostic or prognostic significance in oral oncology.
Among them, the study of the microbiome is the most recent omics science and studies the alteration in microbial, mainly bacterial, content, expression, and interaction, occurring during or before oral cancer development.
The investigations of all these kinds of molecules and bacteria shed light on the silent killer of OSCC and they could be considered, once further studies have concluded on the identification of more specific host molecules or bacteria, as indicative and noninvasive diagnostic and prognostic markers to accelerate diagnosis of early tumoral changes and predict the best therapeutical and follow-up approaches.
Despite the arising knowledge, many mechanisms of action and interaction between bacteria and host and some crucial cancerogenic markers are yet to be identified.Indeed, only a few studies have addressed the combinatorial value of these markers or performed statistical analysis to include interaction terms among oral dysbiosis, tissue changes, and salivary markers.These correlations may be considered through metabolomic studies, which were recently considered concerning OSCC.In this regard, the term "salivaomics" has been coined, and the interest in this branch has been exponentially increasing during the last few years [236,237].
Last, but not least, oral brush biopsy is another technique, less invasive than histology, to obtain crucial information through the cytology of the exfoliated cells from the oral mucosa.This modern and innovative technique is highly suitable for surgeons and well-tolerated by patients, as the collection of samples is usually painless and uneventful.The procedure includes the use of one or usually more brushes that are used by making a rotational movement for 10-15 s around the suspicious oral lesions or frank OSCC; it could be useful to also take a sample from healthy tissue or dysplastic tissue in order to make a comparison between normal and pathological tissue markers.Their collection from suspicious oral lesions or frank OSCC allows for analysis with various downstream methods depending on the marker to focus on and is widely versatile to analyze any cytological change in genetic content (DNA aneuploidies, altered mRNA expressions, methylation patterns, genes expression, and anomalies) as well in protein levels of expression or anomalies of location.For example, through a recent systematic review, Datta et al. investigated the effectiveness of DNA image cytometry (DNA-ICM) from brushings in differentiating OPMDs from benign/inflammatory lesions during screening and in predicting malignant transformation [238].The authors concluded that there was still limited evidence in the literature about the success of DNA-ICM as an oral cancer screening tool and recommended further longitudinal and extensive community screening studies.

Conclusions
In conclusion, although each of the reported studies highlighted some potential prognostic and diagnostic biomarkers, we are still far from their advantageous use in clinical practice and, at present, none of the adjunctive tests reported and none of the biomarkers investigated can be recommended as a replacement for the current gold standard for diagnosis, which is still a surgical biopsy with the classical histological assessment.
We encourage further multicentric and comprehensive studies and hope that they will allow us to reach this critical goal by excluding a holistic approach and considering a multivariate analysis of the co-occurring biological, histological, metabolic, and microbiological changes occurring in cancerogenesis or proposing digital-supported analyses of big data or algorithms, through procedures or sample collections with low invasiveness.This is the case offered by the promising results from the study by The et al. [239], who recently assessed the quantitative Malignancy Index Diagnostic System (qMIDS) based on a multigene RT-qPCR method for the contemporary detection of a series of genes involved in cancerogenesis and immune dysregulations occurring during cancer, from tiny 1 mm 3 minimally invasive biopsies.

Figure 1 .
Figure 1.Sample scheme of the keyword search strategy.

Figure 1 .
Figure 1.Sample scheme of the keyword search strategy.

Figure 2 .
Figure 2. The potential analyses obtained by each methodology.

Figure 2 .
Figure 2. The potential analyses obtained by each methodology.

Table 1 .
The potential support in each step of OSCC management by each imaging tool.

Table 2 .
The tissue markers mainly associated with OSCC.

Table 3 .
The potential support in each step of OSCC management by each adjunct.Identification, characterization, and quantification of microbiota composition peculiar to the tumor microenvironment before OSCC diagnosis, associated with prognostic indicators and for follow-up purposes