This document summarizes current diagnostic methods for oral cancer. It discusses visual oral examination and biopsy as current standards but notes limitations. Several novel diagnostic technologies are emerging, including vital staining with toluidine blue or Lugol's iodine, oral cytology using exfoliated cells, and optical imaging technologies using autofluorescence, chemiluminescence or multispectral analysis. New diagnostic methods under development include DNA methylation biomarkers like ZNF582 and PAX1 and mRNA biomarkers like OAZ1, SAT and DUSP1 which show promise as adjunct tests to improve early detection of oral cancer.

1. Current Insights into Oral Cancer
Diagnostics
Su, Y.-F.; Chen, Y.-J.; Tsai, F.-T.; Li, W.-C.; Hsu, M.-L.; Wang, D.-H.; Yang, C.-C. Current Insights
into Oral Cancer Diagnostics. Diagnostics 2021, 11, 1287. https:// doi.org/10.3390/diagnostics11071287

2. 1. INTRODUCTION
• Oral cancer is one of the most common head and neck
malignancies and has an overall 5-year survival rate.
• Oral cancer is generally preceded by OPMD but determining the
risk of OPMD progressing to cancer remains a difficult task.
• Several diagnostic technologies have been developed to
facilitate the detection of OPMD and oral cancer, and
• Some of these have been translated into regulatory-approved in
vitro diagnostic systems or medical devices.

3. • The 5-year survival rate of oral cancer is 50% because most
oral cancers have progressed to an advanced stage, at the time
of diagnosis.
• By way of contrast, the survival rate of oral cancer can exceed
80% if it is diagnosed and treated early.
• Oral squamous cell carcinoma arises from the epithelial layer,
accounting for more than 90% of oral malignant diseases.

4. • The main sites of oral cancer are the lip,
tongue, gum, floor of the mouth, palate, and a
range of other parts of the oral cavity.
• Furthermore, the rapid development of novel
biomarkers, electronic systems, and artificial
intelligence helped to develop a new era
where OPMD and oral cancer are detected at
an early stage

5. AIMS AND OBJECTIVE
• The purpose of this article is to review the currently available
diagnostic methods for oral cancer as well as possible future
applications of novel promising technologies to oral cancer
diagnosis.
• This will potentially increase diagnostic options and improve
our ability to effectively diagnose and treat oral cancerous-
related lesions.

6. • At present, a visual oral examination (VOE) is the routine
screening method used to identify oral mucosal lesions.
• It depends on the physician’s experience, because several
OPMDs, such as white or red lesions and persistent ulcers, are
often indistinguishable in their clinical presentation.
• A summary of the global oral cancer forum in 2016 depicted
the shortcomings of VOE, that it depends on the examiner's
quality, requires training and calibration of screeners, and is
indistinguishable from benign lesions, cancer, OPMDs, etc.

7. • Currently, a biopsy of the tissues followed by histopathology
remains the gold standard for differentiating and diagnosing
different types of OPMDs and oral cancers.
• Among all OPMDs, oral epithelial dysplasia is a more advanced
lesion type that is characterized by its histological features.
• Despite the well-established and systemic approaches of
histopathologic examination, some clinical conditions may face
challenges in determining an accurate diagnosis.

8. • The mucosa affected by the effects of field cancerization or
several lesions under various cancer-developing statuses may
require multiple biopsies.
• Nevertheless, due to its invasiveness, many patients fear
repeated biopsies
• The clinical observer’s subjectivity and variability during the
VOE and histopathological examination pose limitations on
OPMD detection and oral cancer diagnosis

10. 2. Current Available Techniques
• There are several diagnostic methods that are used routinely in
clinical practice, these include vital staining, oral cytology, and
optical imaging technology
• 2.1.1. Vital Staining - Toluidine blue dye
- Lugol’s iodine

11. Toluidine blue
• A hospital-based diagnostic accuracy study was carried out to
evaluate the efficacy of Toluidine blue staining, which served as an
adjunct tool to standard clinical examination to facilitate early
detection of the oral cavity and oropharynx malignant lesions.
• Fifty-five subjects with OPMDs or malignant lesions were subjected
to detailed clinical examination and toluidine blue staining.
Gupta M, Shrivastava K, Raghuvanshi V, Ojha S, Gupta
A, Sasidhar S. Application of in vivo stain of methylene
blue as a diagnostic aid in the early detection and
screening of oral cancerous and precancerous lesions. J
Oral Maxillofac Pathol 2019;23:304

12. • By comparing the staining results with the histopathologic
examination, the toluidine blue test detected malignancy with a
sensitivity of 92.6% and a specificity of 67.9%.
• The overall diagnostic accuracy was 80%. The result indicates
that the toluidine blue staining method can be a valuable
adjunctive diagnostic process for oral and oropharyngeal
cancers.
• In practice, it is a useful way of identifying lesions with
possible malignant change

13. Lugol’s iodine
• In a comparative study done to test
the efficacy of sensitivity and specificity of TBS (1%), LIS (50%
dilution) and AA(3%) for the detection of oral lesions of Leukoplakia
and erosive lichen planus in the South-Indian population.
• TBS and LIS showed high sensitivity in diagnosing PMD like OL
and erosive OLP compared to AA.
• None of the vital stains have both the parameters of an acceptable
level, it is indicated that a combination of staining may yield better
results.
Efficacy of
Toluidine Blue,
Lugol’s Iodine
and Acetic Acid
for Detecting
Oral Lesions of
Leukoplakia
and Erosive
Lichen Planus –
A Cross-
Sectional Study

14. 2.1.2. Oral Cytology
• Oral cytology was first utilized to evaluate
human oral mucosal lesions in 1963 as a
screening method for oral precancer and
oral cancer.
1. Exfoliative oral cytology is a conventional
method that collects oral mucosal cells by
scrapping, brushing, or rinsing the exfoliative
cells using a tongue blade or brush.
Srilatha T, Manthapuri S, Shylaja
S, Ramanand OV, Reddy ES,
Vamshi VR. Cytomorphometric
analysis of exfoliated buccal
mucosal cells in smokers and
patients with hypertension: A
quantitative analysis. J Int Oral
Health 2021;13:53-9

15. • The collected oral mucosal cell
specimens are then fixed and
stained, and their morphology is
examined and interpreted by an
experienced pathologist under a
microscope.
• This method is derived from a
cervical pap smear, and is simple,
non-aggressive, and relatively
painless
Srilatha T, Manthapuri S, Shylaja S, Ramanand OV,
Reddy ES, Vamshi VR. Cytomorphometric analysis
of exfoliated buccal mucosal cells in smokers and
patients with hypertension: A quantitative analysis.
J Int Oral Health 2021;13:53-9

16. • Over the years, oral cytology has
undergone substantial
improvements
2. Brush cytology is also a method
that is applied to individuals who
have difficulties opening their mouth
for scalpel biopsies, which confirm a
lesion site

17. Advances
3. OralCDx® Brush Test (CDx Diagnostics,
New York, NY, USA)
• OralCDx® is an advanced complementary
form of exfoliative oral cytology that is
coupled with an artificial intelligence
computer-assisted tissue analysis.
• Has an adjunctive role as a diagnostic or
screening tool for the identification of
OPMD at an early stage

18. • Several studies done by Sciubba, J.J.; Mehrotra, R.; Svirsky, J.A.;
reported that OralCDx® has high sensitivity and specificity in the
detection of OSCC and precancerous lesions compared to a
surgical biopsy.
• There are also studies done by Svirsky, J; Rick, G.M; Greenberg,
M.S. that questioned the previously reported high performance of
the OralCDx® system

19. 2.1.3. Optical Imaging
• Light-based technologies for detecting
OPMDs and oral cancer have been used
in clinical practice for several years.
• Chemiluminescence or fluorescent light
is used as an intraoral detector to
pinpoint oral mucosal lesions.
• The color of the light reflected from the
oral epithelium is used to assess the
condition of the oral epithelium
Credit: Journal of Biomedical
Optics (2023). DOI:
10.1117/1.JBO.28.1.016002

20. Autofluorescence-Based
• Autofluorescence refers to when
the light of a particular
wavelength interacts with cells
and causes excitation and re-
emission of light of different
wavelengths.
• Autofluorescence emitted from
tissues is produced by
fluorophores that naturally occur
in human tissues,
Tatehara, Seiko & Satomura, Kazuhito. (2020). Non-
Invasive Diagnostic System Based on Light for Detecting
Early-Stage Oral Cancer and High-Risk Precancerous
Lesions—Potential for Dentistry. Cancers.

21. • Collagen, tryptophan, elastin, keratin, hemoglobin, and NADH
are naturally occurring fluorophores.
• The concentration of these fluorophores alters in OPMDs and
cancerous lesions, which causes alterations of natural light
scattering and absorption properties of the tissues
• VELScope®Vx (LED Dental, British
Columbia, BC, Canada) is a
approved medical device
that noninvasively screens for
alterations in oral mucosal autofluorescence

22. • It is a handheld camera device that emits
blue light (400 nm and 460 nm
wavelengths) that is combined with
proprietary optical filtering in order to
visualize oral abnormalities.
• The normal oral mucosa produces green
autofluorescent light, while anomalous
oral mucosal lesions absorb the
autofluorescent light and appear as dark
areas that contrast with the adjacent tissue

23. • One clinical study has shown that the use of the
VELScope®Vx is beneficial for confirming the presence of
OPMDs, including erythroplakia, leukoplakia, and other oral
tissue disorders; however, it was unable to discriminate
between low-risk and high-risk lesions
• Another study found that the VELScope®Vx alone performed
relatively poorly when detecting epithelial dysplasia;

24. Chemiluminescence-Based
• Refers to the emission of the visible range of light radiation after the
electrons, excited by a chemical exergonic reaction, return from an
excited to ground state; light photons are released upon the transition
of electronic potential energy within the molecules.
• This technique is based on the reflectance phenomenon that
indicates the proportion of incident light that a given surface can
reflect.
• This technique has been used for many years as a diagnostic aid in
the examination of oral mucosa for the detection of OPMDs or
malignant lesions

25. • The ViziLite® Blue oral examination kit (Zila Pharmaceuticals, Arizona,
AZ, USA) is an FDA 510(k)-
• It is a handheld disposable chemiluminescence-based device that utilizes
light illumination at wavelengths of 430 nm, 540 nm, and 580 nm inside
the oral cavity.
• A 1% acetic acid wash is used before starting the light emission to
remove surface debris and improve the visibility of epithelial cell nuclei.
• Abnormal oral mucosa is distinctively white (acetowhite) in appearance,
whereas normal oral mucosa is lightly bluish.
• A number of studies have shown varying results with the ViziLite®. This
device is useful when detecting lesions that have not been identified by
standard VOE.

26. Multispectral Fluorescence- and Reflectance-Based
• Identafi® (DentalEZ, Pennsylvania, PA, USA) is an FDA
approved, probe-like medical device designed for multispectral
screening of OPMDs.
• Uses three light sources: white light, violet light (405 nm), and
green-amber light (545 nm); these can be used sequentially during
an oral mucosal examination.
• The white light provides an optimal view for a VOE of the oral
mucosa, while the violet light excites endogen fluorophores,
enhancing the fluorescence of normal tissues; this results in a dark
appearance when a suspicious oral lesion is present.

27. • Meanwhile, the green-amber light excites hemoglobin
molecules in the blood, which allows the visualization of
diffuse or dilated vasculature through reflectance spectroscopy.
• The third wavelength is used mainly to distinguish between
benign and malignant oral lesions, being likely to have
abnormal vascular structures

28. • In another study, Identafi® white light produced equivalent
lesion visibility and border distinctness to that obtained
using VOE.
• Its violet light demonstrated a specificity of 85.4%, but a
low sensitivity of 12.5% when detecting oral epithelial
dysplasia (OED).
• Finally, the green-amber light was able to identify 40.9%
of lesions with vasculature visibility.
• Thus Identafi® has the potential as an aid to VOE when
detecting and visualizing oral mucosal lesions via its
combination of multispectral light sources.

29. 2.2. New Methods under Development for Clinical
Application
• At present, there are only a few clinical-validated and FDA-,
CE,CLIA-approved biomarker tests are available for clinical
applications of oral cancer detection.
• These include -DNA methylation biomarkers,
-mRNA- and protein-based expression
biomarkers.

30. 2.2.1. DNA Methylation Biomarker -ZNF582 and
PAX1
• The physician collects oral exfoliate cells from a suspicious
lesion, and the cell specimens are then subjected to genomic
DNA extraction and DNA bisulfite conversion
• At which unmethylated cytosine is then converted into uracil,
whereas the methylated cytosine remains unchanged.
• After the completion of DNA bisulfite conversion,
methylation-specific PCR is then performed to detect the
methylation level (methylated cytosines) of both genes.

31. ZNF582 belongs to a large family of transcriptional regulators that
encode Krüppel associated box zinc finger proteins (KRAB-ZFPs) .
• A recent report has revealed that ZNF582 acts as a tumor
suppressor gene in nasopharyngeal carcinoma (NPC) by regulating
the transcription and expression of adhesion molecules Nectin-3
and NRXN3.
• Hypermethylation of ZNF582 promotes NPC metastasis via the
regulation of these adhesion molecules.

32. • The tumor suppressor role of ZNF582 was also observed in
anal and esophageal carcinoma, and its methylation seems to
have potential as a cancer detection biomarker
• PAX1 is known for its paired-box domain and plays a crucial
developmental role during embryogenesis as well as acting as
a tumor suppressor gene.
• A high level of PAX1 methylation induces tumorigenesis,
including oral cancer and cervical cancer

33. • Both DNA methylation biomarkers were developed as in vitro
diagnostics under the name EpiGene ZNF582 DNA Detection Kit
and the EpiGene PAX1 DNA Detection Kit (iStat Biomedical,
New Taipei City, Taiwan), and both are CE-approved in vitro
diagnostics.
• This diagnostic test detects the risk level of oral precancerous
lesions and oral cancers by measuring the methylation level of
ZNF582 and PAX1.
• It is an adjunctive test that assists physicians with VOE, and which
helps to determine a follow-up decision as to whether to pursue a
histopathological examination.

34. 2.2.2. mRNA Biomarker - Multipanel mRNA OAZ1, SAT and
DUSP1
• A case-control study of seven mRNAs (OAZ1, SAT, H3F3A, IL-1 β,
IL-8, SP100P, and DUSP1) and three proteins (IL-8, M2BP, and IL-1
β) was conducted to validate whether the biomarkers are capable of
discriminating patients with OSCC from healthy subjects in five
independent cohorts investigated by the National Cancer Institute-
Early Detection Research Network-Biomarker Reference Laboratory.
• A total of 395 subjects were enrolled from five independent cohorts.
• All seven mRNA and three protein expression levels were increased
in OSCC versus controls in all five cohorts.

35. OAZ1, Ornithine decarboxylase antizyme, is induced by a
polyamine-dependent mechanism and has been found to be
important in DNA repair and linked to the metastatic potential
of human OSCC cell lines.
DUSP1 is a subtype of type I cysteine-based protein tyrosine
phosphatase and is involved in various signaling pathways.
• Salivary DUSP1 mRNA is significantly increased in OSCC
patients compared with normal controls

36. SAT is the rate-limiting acetyltransferase in the polyamine
metabolism catabolic pathway.
• The enzyme is involved in regulating the intracellular
concentration of polyamines and their transport out of cells.
• The expression of SAT has been shown to be significantly
higher in prostate and oral cancer

37. 2.2.3. Protein Biomarker
• CD44 is a cell surface glycoprotein that is involved in many
cell regulation pathways, including cell proliferation and
migration .
• It is also a tumor-initiating and stem cell-associated biomarker
and has been shown to cause tumor initiation in different
tumors, including overexpression in oral cancer.
• In normal epithelium, CD44 expression occurs primarily in the
basal and suprabasal region.
• When dysplasia progresses, CD44 expression migrates into the
superficial layers, implicating its role in the early stages of
carcinogenesis.

38. • A point-of-care (POC) IVD was developed to detect the presence
of CD44 in a saliva sample to evaluate the risk of oral cancer.
• The BeVigilantTM Rapid Test (Vigilant Biosciences, Florida, FL,
USA) is a CE-approved POC IVD, which qualitatively identifies
the presence of soluble CD44 (sCD44) and total protein levels in
patients at risk of oral cancer.
• This POC is a dual-marker lateral flow test that contains two strips
anchored with monoclonal antibodies for CD44 and total protein.
• One monoclonal captures sCD44, and the other captures a
different binding domain of human CD44.

39. S100A7 is a calcium-binding protein and a member of the
multigenic calcium-modulated S100 family
• It is expressed in the upper well-differentiated spinous layer of
normal epithelium .
• Overexpression of S100A7 is found in high-risk dysplastic oral
lesions linked to cancer development.
• S100A7 is a laboratory-developed test StraticyteTM (Proteocyte
AI, TO, Canada).
• This test quantitatively measures the S100A7 biomarker in
biopsy tissues of patients at risk of oral cancer in conjunction
with the proprietary algorithms, advanced imaging, and digital
pathology.

40. 3. Developing Future Technologies
3.1. Artificial Intelligence (AI)-Based System
• Artificial intelligence (AI) technology has grown in recent years
and has been used to improve diagnoses, which can be useful for
the early diagnosis of oral cancer
ML DL

41. • The smartphone-based probe imaging system, combined with
OSCC risk factors, provides triage guidance for the oral
mucosa screener.
• The algorithm classified intraoral-lesion images and pairs into
‘suspicious’ and ‘not suspicious’ with sensitivity, specificity,
positive predictive value, and negative predictive values
ranging from 81% to 95%

42. • In a study by Wang et al., a partial least squares and artificial
neural network (PLS-ANN) classification algorithm was
utilized to discriminate the autofluorescence spectra of
premalignant and malignant tissues (dysplasia and SCC) from
benign tissues (healthy volunteer, OSF, and hyperkeratosis).
• Results showed that PLS-ANN could differentiate
premalignant and malignant tissues from benign tissues with a
sensitivity of 81%, specificity of 96%, and a positive
predictive value of 88%

43. 3.2. Lab-on-Chip
• Is a unique microelectromechanical
system that can be used to detect protein
and ribonucleic acid biomarkers.
• It applies microfluidics engineering
technologies that integrate all aspects of
a range of lab procedures such as
specimen preparation and separation of
saliva, serum, blood, or urine, signal
amplification, and signal detection on a
single chip in an automated manner
https://www.researchgate.net
/publication/281080765_Lab-
on-a-
Chip_Devices_and_Micro-
Total_Analysis_Systems_A_Pra
ctical_Guide

44. • No specialized laboratory personnel are required for analysis and this
approach can be easily used in remote regions or large-scale screening.
• A wide range of molecular diagnostics, biochemical techniques, and
immunoassays have been applied using LOC platforms, including
nucleic acid assays, protein assays, cell sorting, etc.
• Advantages of LOC largely reduce reagent consumption, decrease the
use of hazardous materials and exposure to infectious agents, and
minimize the risk of specimen contamination.
• LOC also increases reproducibility and consistency, as well as
generating a relatively low cost

45. Conclusion
• Considering the size of the research and development in the
field of oral cancer diagnostics, the diagnostic methods
described in this article are mostly those already in clinical
use or commercially available and are showing great promise
in clinical application.
• Greater efforts to intensify and optimize clinical performance
during oral cancer diagnosis are highly encouraged to help
with the effective identification of OPMDs and oral cancer

46. R
ef
er
e
n
ce
• https://www.researchgate.net/publication/281080765_Lab-on-a-
Chip_Devices_and_Micro-
Total_Analysis_Systems_A_Practical_Guide
• Tatehara, Seiko & Satomura, Kazuhito. (2020). Non-Invasive
Diagnostic System Based on Light for Detecting Early-Stage Oral
Cancer and High-Risk Precancerous Lesions—Potential for Dentistry.
Cancers. 12. 3185. 10.3390/cancers12113185.
• Journal of Biomedical Optics (2023). DOI: 10.1117/1.JBO.28.1.016002
• Srilatha T, Manthapuri S, Shylaja S, Ramanand OV, Reddy ES, Vamshi
VR. Cytomorphometric analysis of exfoliated buccal mucosal cells in
smokers and patients with hypertension: A quantitative analysis. J Int
Oral Health 2021;13:53-9
• Mathew P et al. Efficacy of toluidine blue, lugol's iodine and acetic acid
for detecting oral lesions of Leukoplakia and erosive lichen planus–A
cross- sectional study. Journal of Indian Academy of Oral Medicine and
Radiology. 2020 Jul 1;32(3):253-258

47. Thank you