This covers the hard palate and gingivae and in places is bound directly to bone forming a mucoperiosteum. A fatty submucosa is present in the centre of the hard palate. The microanatomy of masticatory mucosa is related to its function of resisting compressive forces. Large connective tissue papillae are characteristic and the oral epithelium is cornified, most often showing parakeratosis. The keratin layer imparts a pale pink colour and if the keratin layer increases in thickness due to chronic irritation or trauma, the mucosa appears white and slightly spongy.

This covers the ventral tongue, floor of mouth, soft palate and the buccal, labial and vestibular surfaces of the oral cavity. Again, the microanatomy of lining mucosa is related to its functions, shaping food for chewing and swallowing, and allowing movement for speech and other oral functions. Lining oral epithelium is non-keratinising in health and is almost transparent. Consequently, lining mucosa appears clinically red in colour due to blood in the vessels of the lamina propria. Lining oral epithelium is quite variable in thickness. Floor of mouth mucosa has poorly developed connective tissue papillae and the epithelium is thin. This contrasts with buccal mucosa where the connective tissue papillae are long, slender and curved, extending through a much thicker squamous epithelium. Most lining mucosa is located over a submucosa that contains minor mucus salivary glands.

This is found on the dorsum of the tongue. The mucosa is covered by filiform papillae and there are also larger fungiform, foliate and circumvallate papillae at specific locations. The dorsal lingual epithelium is keratinising and bacterial biofilm can be abundant on the surface of the filiform papillae.

Collection devices suitable to obtain cells from the superficial and intermediate layers may be conventional brushes, such as the CytoBrush, Orca-brush or others. Signs of dysplasia will be detected in the upper layers due to the principle of disturbed cell maturation that occurs in dysplasia in which a degree of nuclear abnormality (dyskaryosis) in the surface layers reflects the degree of disturbance of maturation of the whole thickness of the epithelium, i.e. dysplasia. It is the task of a cytopathologist to identify nuclear abnormalities in the cells collected in this way to predict the histological grade of dysplasia. The diagnostic criteria used are similar to cervical exfoliative cytology and are well known (Figs 6.1–6.3) (see Ch. 21).

Fig. 6.1 The Orcellex® device is placed firmly onto the oral mucosa and rotated 10 times to collect an adequate sample.

Fig. 6.2 LBC preparation of normal floor of mouth mucosa showing superficial and intermediate squamous cells.

Fig. 6.3 LBC preparation of normal floor of mouth mucosa using the Orcellex® brush showing intermediate and basal cells.

Screening for oral cancer and precursor lesions can be performed by dentists and other healthcare professionals. Tolonium chloride rinsing has been advocated in the past to distinguish between normal and abnormal oral mucosa and more recently tissue fluorescence has been employed effectively.⁴ Exfoliative cytology is advocated for evaluation of suspicious lesions that are detected clinically by screening. This may be followed by a mucosal biopsy and transepithelial sampling. Exfoliative cytology of oral lesions may replace tissue biopsy in lesions that are clinically not obviously suspicious for malignancy but nevertheless need surveillance. As tissue biopsy is associated with low compliance (9%) and brush-biopsy not (100%),⁵ this approach may lead to a higher rate of oral cancers identified in early stages.

An arbitrary distinction is often made between oral premalignant conditions and oral premalignant lesions. The former include a group of generalised mucosal disorders linked by epithelial atrophy and where there is an increased predisposition to oral cancer when mucosa is exposed to the known major risk factors such as sunlight exposure, smoking and alcohol consumption, areca or betel nut and also syphilis and sideropenic dysphagia.⁷ Recently, strong evidence for an aetiological relationship between human papilloma virus and a subset of head and neck cancers has been noted.⁸ Oral cancer risk is also associated with low socioeconomic status and related to lifestyle risk factors.⁹ Where risk factors cannot be eliminated and lesions persist, then regular clinical follow-up should be considered. The need for follow-up and interval between visits will vary in individual cases. In contrast to the oral premalignant conditions, oral premalignant lesions are morphologically abnormal solitary or multiple areas of mucosa that are typically white, discoloured white, red, speckled or verrucous in appearance (Fig. 6.4). The WHO classification¹⁰ combines leukoplakia and erythroplakia into ‘precursor lesions’, with the 6.8% estimated rate of transformation of oral leukoplakias to cancer.¹¹ It identifies proliferative verrucous leukoplakia (PVL) as a separate high-risk lesion with minimal cytological atypia.

Fig. 6.4 Leukoplakia in the floor of the mouth.

There are several schemes for grading dysplasia in biopsies of oral precursor lesions. The WHO classification provides a five point system: hyperplasia, mild, moderate and severe dysplasia followed by carcinoma in situ.¹⁰ The system can be mapped onto both the Ljubljana classification and the widely used three point squamous intraepithelial neoplasia (SIN) system (Table 6.1). For the first time the WHO set out criteria for diagnosis by defining both the architectural and cytological features of dysplasia.^10,11 However, there is good evidence in the literature favouring a binary system (‘high-grade’ and ‘low-grade’ categories) over multipoint scales for prediction of malignant transformation (Figs 6.5, 6.6).^12,13

Table 6.1 Current histological grading systems for oral dysplasia/squamous intraepithelial neoplasia (SIN)

Fig. 6.5 Low-grade epithelial dysplasia in the oral mucosa. Changes are restricted to the lower one-third of the oral epithelium. In an intra-observer study, there was variation between mild and moderate grades, but consensus that this lesion was low grade.

Fig. 6.6 High-grade epithelial dysplasia in the oral mucosa. In an intra-observer study, there was variation between moderate, severe and severe amounting to CIS, but consensus that this lesion was high grade.

Squamous cell carcinoma will develop from antecedent dysplastic oral mucosal lesions if an early diagnosis has not been made and treatment given. Early diagnosis within stages I and II correspond to a vastly improved 5-year survival rate when compared with more advanced stage III and IV lesions.^14,15

An accumulating body of evidence exists to show that oral cytology is a valuable technique for the assessment of oral premalignant lesions.^1,16,17 Exfoliative cytology has been shown to detect dysplasia in suspicious oral lesions with high sensitivity and specificity by several groups.^18–20 The use of auxiliary methods such as DNA image cytometry, AgNOR analysis, cytomorphometry²¹ and cell cycle immunohistochemistry and semiautomated multimodal cell analysis using brush biopsies can increase accuracy even further.^{17–19,22–24} Molecular genetics of each of the precursor lesions is being investigated for genetic alterations, which have been demonstrated in oral squamous cell carcinoma, to define their location on the continuum of changes, which lead to malignant transformation.²⁵

Although the degree of dysplasia can be predicted on cytological samples, tissue biopsy is usually performed when dysplasia is detected, to confirm its grade and exclude the presence of invasion. The latter cannot be reliably assessed by exfoliative cytology alone. However, observer variability in the histological assessment of oral premalignant lesions is well described.²⁶

Up to 5–14% of oral brush biopsies may yield equivocal cytological diagnoses.^18,28 Underlying diagnoses are mild, moderate or marked dysplasia, abnormal regenerating squamous epithelium or just scarcity of abnormal cells. In these cases, ancillary methods are desirable, that allow more definite, correct cytological diagnoses. Two such methods are: DNA image cytometry (DNA-ICM) and AgNOR analysis.

Diagnostic DNA-ICM is based on DNA measurements of several hundred atypical cells in routine cytological specimens. It aims to distinguish true prospectively malignant lesions from microscopically atypical or otherwise doubtful lesions. The biological basis of this ancillary method is chromosomal aneuploidy which is an accepted marker of malignant transformation of cells. Its cytometric equivalent, DNA aneuploidy, is assumed if gains or losses of chromosomes or their parts result in a plus or minus of more than 10% of nuclear DNA mass in a growing cell population (stemline-aneuploidy) or if extremely high nuclear DNA values (single cell aneuploidy) occur. Measurements may be performed on previously stained slides after de-staining and Feulgen re-staining. Morphologically suspicious cells are interactively selected on a monitor and internal calibration is performed with normal (e.g. intermediate squamous) cells. The method has been internationally standardised and is applicable to many different epithelial dysplasias.²⁹ Papanicolaou, Feulgen and silver nitrate staining and respective measurements may sequentially be applied also on the same cells (multimodal cell analysis¹⁹) (Fig. 6.7).

Fig. 6.7 Triplets of multimodal cell analysis on few identical atypical oral squamous cells, stained (A(i), B(i)) according to Papanicolaou for subjective inspection, (A(ii), B(ii)) according to Feulgen for DNA-ICM and (A(iii), B(iii)) with silver nitrate for AgNOR-analysis. Respective euploid (diploid) DNA-histogram in (A(iv)) and aneuploid with abnormal stemlines at 3.5c and 7.0c in (B(iv)). Mean AgNOR-counts per nucleus were 3.7 in (A) and 5.4 in (B). Final histological diagnoses were ulcer in (A) and in situ squamous cell carcinoma in (B). The displacements of images in (B) are due to computerised relocations of cells after repeated staining.

Remmerbach et al.²⁸ reported a frequency of 13.9% of doubtful or suspicious oral cytological diagnoses due to different grades of squamous dysplasia or abnormal regenerating epithelium. Applying DNA aneuploidy as a marker for prospective malignancy on identical slides, they could improve diagnostic sensitivity for the detection of oral cancer from 91.3% to 97.8% and specificity from 95.1% to 100%. Thus 29.4% of oral cancers that clinically appeared as leukoplakias or erythroplakias were detected in stages Tis or T1. In a similar study, Maraki et al.¹⁷ described a sensitivity of 100% and specificity of 97.4% for the combined cytological and DNA cytometric evaluation of oral leukoplakias and erythroplakias. Some 8.1% of her cytological diagnoses had been equivocal. DNA-ICM was only applied, if one of the above mentioned diagnoses had occurred. Seven cases in which combined cytological/DNA cytometric diagnosis of early oral cancer was achieved up to 2.5 years before definitive biopsy diagnosis have been published.^17,23 Thus DNA-ICM may help to predict the prospective behaviour of cytological suspicious lesions, as the positive predictive values of DNA aneuploid findings was reported to be 100% and the negative value 98.1%.^18,23 DNA-ICM may be used for identification of tumour-free resection margins instead of histology.^30,31

Another auxiliary method that allows assessment of potential malignancy of dysplastic or regenerating cells is AgNOR analysis. Remmerbach et al.^19,24 showed that counting the number of silver nitrate-stained nucleolar organiser regions (AgNORs) in about 100 atypical squamous cells allows 100% sensitivity and specificity of oral cancer detection on brush biopsies using a cut-off value to identify the precancerous from already cancerous cells among all dysplastic cells.³⁰ The AgNOR method can also be useful for grading dysplasia, for example a cut-off value of 2.3 separates mild and moderate dysplasia.³²

Both methods, DNA-ICM and AgNOR analysis, may even be performed sequentially on identical cells. This type of multimodal cell analysis is especially useful, if only few atypical cells are available.¹⁹ Thus, AgNOR analysis can be combined with DNA-ICM if the latter does not yield an unequivocal diagnosis (Fig. 6.7).

Immunohistochemical detection of cell cycle markers has also been proposed as a potential method of detecting cytological abnormality but no data on specificity and sensitivity in the routine clinical setting are available.^33,34 Molecular markers and techniques such as loss of heterozygosity in exfoliated cells can be used to detect clinically non-visible lesions in early dysplastic oral lesions.³⁵