Histol Histopathol (1 997) 12: 69-77 Histology and Histopathology

An evaluation of the role of nuclear cytoplasmic ratios and nuclear volume densities as diagnostic indicators in metaplastic, dysplastic and neoplastic lesions of the human cheek F.H. White', Y. Jin and L. Yang2 'Department of Anatomy, Faculty of Medicine, University of Hong Kong, Hong Kong,2Department of Oral Pathology, Qin Du Stomatological College, Fourth Military Medical University, Xian, Shaannxi Province, People's Republic of China

Summary. The increase in nuclear cytoplasmic (NIC) ratio is one of the features of cellular atypia which is used in the histopathological assessment of premalignant lesions of the oral mucosa. Since this feature is readily quantifiable using morphometry, we have analysed both N/C and nuclear volume densities in basal and spinous cells from human cheek lesions with and without malignant potential in order to ascertain the validity of this parameter as a predictor. Using a strictly standardised sampling procedure, measurements of cellular and nuclear areas of basal and spinous cells from normal and pathological human cheek mucosa were made on haematoxylin and eosin-stained sections using a VIDAS image analyser. Cases examined comprised fibrous hyperplasia (FH), traumatic inflammation (IF), benign hyperkeratosis (HK), lichen planus (LI), leukoplakia with dysplasia (DYS), squamous cell papilloma (PP), dysplastic epithelium from the edges adjacent to invasive carcinoma (CE) and islands from invasive squamous cell carcinoma (CI). In basal cells, NIC ratios and nuclear volume densities were lower than values obtained for the normal controls. In spinous cells, these parameters were elevated in the potentially premalignant lesions (DYS, CE) as well as in CI but values were similarly elevated in FH, IF, HK and PP, lesions which appear to have no malignant potential.

The NIC ratio is of no value as a predictor of malignant potential in basal or spinous cells from cheek lesions. The putative increase in NIC which has been previously described qualitatively is probably due to increased nuclear hyperchromatism, which may provide an illusory increase in relative nuclear size at the expense of the cytoplasm.

Offprint requests to: Dr. F.H. White, Deparíment of Anatomy, FacuHy of Medicine, University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong

Key words: Cellular atypia, Dysplasia, Epithelium, Morphometry, Nuclear cytoplasmic ratio, Nuclear volume density, Oral cancer, Oral mucosa, Squamous cell carcinoma, Squamous cell papilloma

lntroduction

Squamous cell carcinoma of the oral mucosa is the most life threatening of the oral diseases. While the incidence of this neoplasm is low when compared with other malignancies, the disease is important because of its morbidity and high mortality (Binnie and Rankin, 1984). Despite strenuous research efforts and an enormous literature. there are no clear indications that cancer therapy is ihproving patient survival (Kolata, 1985).

hile prevention is better than cure, it is dificult to prevent the disease when aetiological factors have proved thus far to be elusive (Binnie et al., 1983). There has been a considerable research effort directed at one aspect of prevention, that is, the identification and elimination of precursor conditions, the so-called precancerous or premalignant lesions. While elimination of these lesions would be relatively straightforward, there are no reliable features to assist the histo- pathologist in their diagnosis.

For the histopathologist, the diagnosis of oral squamous cell carcinoma generally present few problems. Some difficulties may occur in establishing the histogenesis of poorly-differentiated or anaplastic lesions, in identifying recurrent or residual tumour cells at excision margins or at sites of previous surgery or irradiation, in recognising the presence of variants, in determining the presence or absence of pseudo- epitheliomatous change and in dealing with the problem of the inadequate biopsy (Smith et al., 1990). However, there are many more difficulties in classifying and

N/C ratio in human cheek lesions

diagnosing precancerous lesions. In the oral cavity, squamous cell carcinomas are

often associated with or may arise from leukoplakias, white patches or plaques that cannot be characterised clinically or pathologically as any other disease. The WHO (1978) has stressed that the term leukoplakia should be used only as a clinical description and should carry no histological connotations. There are a number of cytological features, referred to individually as cellular atypia, which may be present in epithelium and which are suggestive of premalignant change. These produce general disturbances of the tissue architecture which are known collectively as epithelial dysplasia. The features of epithelial atypia have been documented by the WHO (1978), inter alia.

In the assessment of premalignancy, the histo- pathologist determines the presence of these features and their degree of change within a given lesion using qualitative observation, but the precise relationship of these atypical features to subsequent malignant development has yet to be resolved. Difficulties arise since we still do not appreciate whether particular atypical features have greater or lesser significance; whether the grade (mild, moderate or severe) of dysplasia plays a role as a diagnostic or prognostic indicator; precisely what criteria are used for the grading system of dysplasia; and finally, the fact that the interpretation of such alterations may be subject to marked observer variation (Pindborg et al., 1985). There is obviously a requirement for improving objectivity in the assessment of established criteria for dysplastic lesions, as well as for determining which criteria are important in predicting their future development.

The nuclear cytoplasmic ratio is allegedly increased in premalignant lesions (see for example WHO, 1978) and is one of the features of cellular atypia which lends itself to an objective approach. In this preliminary morphometric report, we have estimated the nuclear cytoplasmic ratio and the nuclear volume densities for basa1 and spinous in cells from a variety of dysplastic and neoplastic epithelial lesions of the human cheek mucosa with a view to assessing whether this commonly used qualitative parameter has any potential value as a diagnostic indicator for this site. In effect, we are testing the commonly held hypothesis that nuclear cytoplasmic ratio is increased in premalignant and malignant lesions when compared with normal tissues.

Materials and methods

The material for this retrospective study of buccal mucosa1 lesions was obtained from the files of the Department of Oral Pathology, Qin Du Stomatological College, Fourth Military Medical University, Xian People's Republic of China. Al1 tissues had been fixed for 18-24 hours in 10% formalin, dehydrated, cleared in chloroform and embedded in paraffin wax.

Blocks were sectioned and sections were stained with haematoxylin and eosin. Four representative

sections from each of the blocks were examined. The diagnostic groups were assigned to the following histological categories after independent assessment by the three authors: normal buccal mucosa (N; n=ll) ; keratosis with varying degrees of inflammation (INF; n=ll) ; keratosis with minimal signs of inflammation (HK; n=ll) ; fibrous hyperplasia (FH; n=l l ) ; lichen planus (LI; n=12); varying leukoplakia (DYS; n=6); squamous cell papilloma (PP; n=5); dysplastic epithelium immediately adjacent to the margins of a squamous cell carcinoma (CE; n=5); and islands of squamous cell carcinoma (CI; n=ll). Al1 lesions were assigned to diagnostic groups after unanimous agreement had been reached. Some further comments on the diagnostic criteria for the groups under investigation are outlined below. These are based on the WHO (1978) guidelines.

Normal epithelium (N) was obtained from the margins of lesions which were diagnosed clinically as frictional keratosis or histopathologically as hyper- keratosis with inflammation. Al1 specimens were non- keratinised and free from inflammatory infiltrate.

The inflammatory group (INF) comprised lesions which clinically were diagnosed as frictional keratosis since they were related to some obvious traumatic factor (in most cases, to fractured teeth or restorations, or to the clasp of a partial denture). The degree of intra and subepithelial inflammation was highly variable, but al1 lesions showed metaplastic change as evidenced by ortho or parakeratosis.

The hyperkeratotic group (HK) was also derived from frictional keratoses. Histopathologically, lesions showed marked keratosis but were relatively free from inflammatory infiltrate or from any signs of cellular aiypia.

The fibrous hyperplasia group (FH) possessed a marked increase in the fibrocellular content of the lamina propria. Epithelial changes observed were atrophy, metaplastic keratosis and occasional mild inflammatory infiltration.

The dysplasia group (DYS) consisted clinically of localised whitish plaques (leukoplakias) and was similar to the HK group macroscopically. However, histo- pathological examination showed varying degrees of dysplasia and marked ortho or parakeratosis. A chronic inflammatory infiltrate was present which was variable in concentration and extent. Neither microabscesses nor Candidal invasion of the surface cells were detected.

Lichen planus lesions (LI) presented clinically as small whithis papules coalescing to form plaques or reticular patterns. Several exhibited Wickham's striae radiating from the main body of the lesions. Histologically, many of the lesions had saw tooth rete ridges and a prominent well-defined band of chronic inflammatory cells lying close to the epithelium, which showed evidence of hydropic degeneration. In some cases atrophic epithelium was present. A layer of ortho- or parakeratin was always present.

Squamous cell papillomas (PP) were usually small

N/C ratio in human cheek lesions

Table 1. Sarnpling procedures adopted for the rnorphometric analysis.

No. groups No. blocksi No. sectionsi No. fieldsl Total No. fieldsi Pt block secüon Pt

(<5 mm) nodular exophytic lesions. Histologically, many possessed an epithelial covering of normal morphology, usually with a keratinised surface and a stromal core of lamina propria of essentially normal appearance but containing foci of chronic inflarnrnatory cells.

The epithelium adjacent to squamous cell carcinoma (CE) was usually mildly or moderately dysplastic. A chronic inflammatory cell infiltrate was usually present and some keratinisation was observed.

Islands of squamous cell carcinoma (CI) ve re located at various levels in the lamina propria of the cheek and were often present deep within the subjacent muscle. The majority were well differentiated, having a tendency to form central whorls of keratin, but others were only moderately differentiated with little or no keratin formation. In a few cases, poorly differentiated lesions were present, with epithelial islands consisting of highly atypical cells exhibiting marked nuclear and cellular pleomorphism and hyperchromatism, many abnormal mitotic figures and little structural difference between peripheral and central cells. Al1 carcinomas were pooled without regard to the degree of differentiation of the lesions.

Morphometric procedures

The sectioned material was subjected to morpho- metric analysis using a VIDAS (Carl Zeiss) semi- automated image analysis system. A video camera (JVC TK5310) was attached to a Zeiss light rnicroscope and microscopical images were put directly into the system and viewed on a monitor. A light cursor and digitising tablet were used to outline basal and spinous cells and their nuclei. The system was set up to provide files for handling nuclear, cellular and cytoplasrnic areas and to determine the nuclear-cytoplasmic ratio and nuclear volume density for each patient in each of the histopathological groups.

Preliminary measurements to determine minimal sample size were made on four sections each from patients with normal epithelium and on four sections each from patients with dysplasia using the cumulative mean plot. At a magnification of x500, measurements from eight random fields from one representative section yielded a final mean value which fe11 consistently within 15%. Thus, for the remainder of the analysis, eight fields from one representative section of each lesion were analysed. Sarnpling procedures are summarised in Table 1.

Areas of basal and spinous cells (Acell) and their nuclei (Anuc) were measured for each representative section from each of the histopathological groups. The

VIDAS system provided for the incorporation of the individual primary parameters and, following completion of the measurements for an individual patient, calculated the nuclear cytoplasmic rations (N/C) and nuclear volume densities (VvN) from the following equations:

N/C = Anuc Anuc VvN=- Acell - Anuc Acel 1

During measuring procedures, non keratinocytes such as intraepithelial clear cells and infiltrating lymphocytes were excluded, as were keratinocytes undergoing mitosis or in an apparent state of degeneration. No attempt was made to take into account the intercellular space, which was incorporated into the measurements of the area of the cells.

For statistical purposes, final group means and standard deviations were calculated from the mean values obtained from individual patients within each group. Data were subjected to a Duncan multiple range test and a Student's t test. In order to clarify the extent of a particular change in numerical value for a given parameter in any given lesion, pathological alteration ratios were also calculated (White and Gohari, 1984~; White et al., 1985).

Results

The results of the quantitative analysis (Tables 2-4) indicate that in normal controls and in al1 of the pathological conditions investigated, highly significant reductions in both the N/C ratio and in VvN occurred during differentiation between basal and spinous layers, with p values obtained by a paired t-test always being less than 0 .O 1.

Comparisons between normal cheek and metaplastic, dysplastic, benign and malignant lesions revealed marked differences between values obtained for basal and spinous cell layers. In basal cells, al1 pathological specimens had values which were lower, and in some cases significantly lower (Table 2), than those obtained for normal basal cells. In particular, values for N/C and VvN were both substantially and significantly reduced (Table 4) in LI and in DYS. VvN was also significantly reduced in these lesions as well as in HK lesions with minimal inflammation when compared with normal tissues. The N/C ratio was also significantly reduced in DYS lesions when compared with IF lesions.

The pattern of alterations was less consistent when comparing pathological groups with normal controls in spinous cells (Table 3). In general, with the exception of LI, in which values were slightly reduced, al1 pathological groups had higher values when compared with the normal controls. Many of these values were significantly evaluated, with p values of less than 0.05 (Table 4). For both N/C and VvN, the highest values were obtained for CI, with PP and FH having the next highest values, while LI lesions consistently had the lowest values.

N/C ratio in human cheek lesions

Table 2. Nuclear volume densities and nuclear cytoplasmic ratioc for basa1 cells from human cheek lesions.

N FH IF HK PP LI DYS CE CI

N: normal; FH: fibrous hyperplasia; IF: traumatic inflammation; HK: benign hyperkeratosis; PP: squamous cell papilloma; LI: lichen planus; DYS: leukoplakia with dysplasia; CE: epithelium adjacent to squamous cell carcinoma; CI: islands of squamous cell carcinoma.

Table 3. Nuclear volume densities and nuclear cytoplasmic ratios of spinous cells from human cheek lesions.

N FH 1F HK PP LI DYS CE CI

N: normal; FH: fibrous hyperplasia; IF: traumatic inflammation; HK: benign hyperkeratosis; PP: squamous cell papilloma; LI: lichen planus; DYS: leukoplakia with dysplasia; CE: epithelium adjacent to squamous cell carcinoma; CI: islands of squamous cell carcinoma.

Tabie 4. Results of statistical analysis.

NIC N vs LI pc0.01 N vs DYS pc0.05 IF vs DYS pc0.05

VvN N vs HK pc0.05 N vs LI pcO.01 N vs DYS pc0.05

Sphlous CeIIs NIC N vs lF pc0.02

N vs FH pc0.005 N vs HK pc0.01 N vs DYS pc0.05 N vs PP p<0.05 N vs CI pc0.001 IFvsLI pc0.05 IF vs LI pc0.01 FH vs LI pc0.005

VvN N vs lF p0.05 N vs FH pc0.002 N vs HK pc0.01 N vs PP pc0.05 N vs DYS pc0.05 N vs CI pc0.001 IF vs LI pcO.01 lF vs Cl pc0.01 FH vs Ll pc0.002

FH vs Cl pc0.05 HK vs Ll pc0.01 HK vs CI pc0.001 Ll vs PP pc0.05 Ll vs CE pc0.05 LI vs CI pc0.001 DYS vs CI pc0.001 PP vs Cl pc0.05 CEvsCI pc0.05

FH vs Cl pc0.01 HK vs Ll pc0.005 HK vs CI pc0.005 LI vs DYS pc0.05 Ll vs PP pc0.05 L1 vs CI pco.001 DYS vs CI pc0.002 CE vs CI pc0.05

have no tendency to undergo malignant change (FH, IF, PP) were similar to those obtained for the CI group. Spinous cell PAR values for NIC ratio and VvN (Table 6) for FH, IF and PP lesions had values which were lower than for the CI group but PAR values for DYS lesions or others which have some documented malignant potential (LI and CE) were actually lower than those for the benign group.

In comparison between basal and spinous cells, al1 values were significantly reduced in spinous cells, with p values of at least 0.01. N: normal; FH: fibrous hyperplasia; IF: traumatic inflammation; HK: benign hyperkeratosis; PP: squamous cell papilloma; LI: lichen planus; DYS: leukoplakia with dysplasia; CE: epithelium adjacent to squamous cell carcinoma; CI: islands of squamous cell carcinoma.

The pathological alteration ratio (PAR) provides an indication of the degree of pathological change for a particular morphometric parameter which is independent of methodological variables (White and Gohari, 1984c; White et al., 1985) and enables comparisons to be made with other studies despite differences in preparative procedures. In Table 5, basal cell PAR values derived for NIC ratios and VvN for purely benign lesions which

Discussion

The application of morphometric methods to pathological epithelial tissues can greatly enhance descriptions of tissue architecture (White, 1983, 1986) and can confirm or refute previously suspected alterations or indeed in many instances can be used to detect previously unsuspected alterations. For more than two decades, morphometry and stereology have been used to describe alterations in normal and experimentally treated or pathological tissues and cells and a number of publications have reported quantitative structural alterations in normal and pathological oral epithelia in an attempt to increase objectivity. A number of reports have described various aspects of the structure of oral epithelium of humans (Schroeder and Munzel- Pedrazzoli, 1970; Meyer and Schroeder, 1975; Bernimoulin and Schroeder, 1977; Landay and Schroeder, 1977; Muller-Glauser, 1981) and in experimental animal models (Andersen and Schroeder, 1978; Bale and White, 1982; Gohari and White, 1984; Thompson et al., 1984; White and Gohari, 1983a,b, 1984d, 1985).

There is some information available on the morphometry of human oral cancer and precancer (Klein-Szanto et al., 1976; Longmore and Cowpe, 1982; Keszler and Cabrini, 1983; Abdel-Salam et al., 1987, 1990; Shabana et al., 1987,1989; Ogden et al., 1990; Jin et al., 1993, 1995a,b; Tipoe et al., 1996) as well as a number of reports on experimental carcinogenesis of the oral cavity (Eveson and MacDonald, 1978, 1981; Franklin et al., 1980; Franklin and Smith, 1980; White

N/C ratio in human cheek lesions

Table 5. Pathological alteration ratios: Basal cells. Table 6. Pathological alteration ratios: Spinous cells.

FH IF HK PP LI DYS CE CI FH IF HK PP L1 DYS CE CI

FH: fibrous hyperplasia; IF: traumatic inflammation; HK: benign FH: fibrous hyperplasia; IF: traumatic inflammation; HK: benign hyperkeratosis; PP: squarnous cell papilloma; LI: lichen planus; DYS: hyperkeratosis; PP: squamous cell papilloma; LI: lichen planus; DYS: leukoplakia with dysplasia; CE: epithelium adjacent to squamous cell leukoplakia with dysplasia; CE: epithelium adjacent to squamous cell carcinoma; CI: islands of squarnous cell carcinoma. carcinoma; CI: islands of squamous cell carcinoma.

Table 7. Results of previously published morphometric estimates of the N/C ratio in normal and pathological epithelum.

AUTHOR SlTE SPECIES LESION STRATUM

B S G PB LS MS SF T

White and Gohari, 1981a

Jin et al.. 1995a

Klein Szanto et al.. 1976

Cheek pouch

Cheek pouch

FOM

Cheek

Landay and Schroeder, 1977 Cheek

Shabana et al., 1987 Cheek

Franklin and Craig, 1980

Franklin and Srnith, 1980

Boysen and Reith, 1983

Hamster

Hurnan

Human

Human

Human

Nasal mucosa Human

Hamster N 0.397 0.242 0.088 HYP 0.414 0.275 0.081 DYS 0.403 0.282 0.106 C A 0.440 0.392 0.169

N 0.618 0.234 FH 0.648 0.244 INF 0.600 0.196 HK 0.648 0.200 PP 0.552 0.220 CE 0.558 0.188 C1 0.650 0.302

N 0.57 LP 0.65 LP 0.62

N 0.40

N 0.784 TK 0.808 LI 0.692 LP 0.809 CD LP 0.845 RG 0.753

Cheek pouch Hamster N 0.21 Turpentine 0.16 DMBA 0.16

Cheek pouch Hamster N 0.21 BEN HYP 0.19 DMBA HYP 0.25 DMBA HYPIDYS 0.32 DMBA ATRIDYS 0.31 DMBA CA 0.39

PSE 0.31 SCE 0.30 SCISSE 0.33 SSNKE 0.29 SSKE 0.34 DYS 0.27

B: basa1 layer; S: spinous layer; G: granular layer; PB: parabasal layer; LS: lower spinous layer; MS: middle spinous layer; SF: surface layer; N: normal; HK: hyperkeratosis; FH: fibrous hyperplasia; INF: inflammation; PP: squarnous cell papilloma; CE: dysplastic epithelium from edge of carcinoma; TK: traumatic keratosis; LI: lichen planus; LP: leukoplakia; CD-LP: Candidal leukoplakia; RG: risk group; T: total epithelium; DMBA: dimethylbenz(a)anthracene; BEN HYP: benign hyperplasia; HYP: hyperplasia; HYPIDYS: hyperplastic dysplasia; ATFUDYS: atrophic dysplasia; CI: carcinoma islands; PSE: pseudostratified epithelium; SCE: simple cuboidal epithelium; SCISSE: simple cuboidal stratifiod squamous epithelium; SSNKE: simple stratified squamous non-keratinized epithelium; SSKE: stratified squamous keratinised epithelium; DYS: dysplasia; CA: carcinoma.

and Gohari, 1981a-c, 1984a-c, 1986; White and Al- which include both light and electron microscopy, a Azzawi, 1982, 1983; Tarpey and White, 1984; White et variety of experimental animal models as weil as human al., 1985; Rich and Reade, 1988; Rich et al., 1991). tissues and a veritable plethora of morphometric and These reports have used a variety of stmctural methods, stereological parameters. The overall impression of

N/c ratio in hurnan cheek lesions

Table 8. Pathological alteration ratios calculated for NIC ratios of potentially premalignant and malignant cells from published reports.

Klein-Szanto et al., 1976

Shabana et al., 1987

FOM Cheek

Chwk

Human Human

Human

AUTHOR SlTE SPECIES LESION STRATUM

B S G PB LS MS SF T

White and Gohari, 1981 a Cheek pouch Hamster HYP 1.04 1.14 0.92 DYS 1.02 1.17 1.20 C A 1.11 1.62 1.92

Jin et al., 1995a Cheek pouch Hamster FH 1 .O5 1.04 INF 0.97 0.84 HK 1.05 0.85 PP 0.89 0.94 CE 0.90 0.80 CI 1.05 1.29

LP 1.14 LP 1.55

TK 1 .O3 LI 0.88 LP 1.15 CD LP 1 .O8 RG 0.96

Turpentine DMBA

BEN HYP DMBA HYP DMBA HYPIDYS DMBA ATWDYS DMBA CA

SCE SCISSE SSNKE SSKE DYS

Franklin and Craig, 1980

Franklin and Smith, 1980

Boysen and Reith, 1983

Cheek pouch

Cheek pouch

Nasal mucosa

Hamster

Hamster

Human

B: basal layer; S: spinous layer; G: granular layer; PB: parabasal layer; LS: lower spinous layer; MS: middle spinous layer; SF: surface layer; N: normal; HK: hyperkeratosis; FH: fibrous hyperplasia; INF: inflammation; PP: squamous cell papilloma; CE: dysplastic epithelium from edge of carcinoma; TK: traumatic keratosis; LI: lichen pianus; LP: leukoplakia; CD-LP: Candidal leukoplakia; RG: risk group; T: total epithelium; DMBA: dimethylbenz(a)anthracene; BEN HYP: benign hyperplasia; HYP: hyperplasia; hYP1DYS: hyperplastic dysplasia; ATWDYS: atrophic dysplasia; CI: carcinoma islands; PSE: pseudostratified epithelium; SCE: simple cuboidal epithelium; SCISSE: simple cuboidal stratified squamous epithelium; SSNKE: simple stratified squamous non-keratinized; SSKE: stratified squamous keratinised epithelium; DYS: dysplasia; CA: carcinoma.

evaluating data from these reports is that generally, electron microscopical morphometry can provide important biological data such as on normal and abnormal epithelial differentiation but is too time- consuming and has far less applicability to the diagnostic aspects of precancer and to the possibility of providing objective indications of the behaviour of malignant lesions. Hence, the application of simple, reliable histological descriptors, if they exist, is far more likely to be useful.

In the present preliminary study, we have selected lesions which could be considered under five separate groupings. The normal controls comprised the first group. Non neoplastic lesions with no known malignant potential comprised the second group. These were chronic inflammation, FH and HK. The third group included leukoplakia with dysplasia , dysplastic epithelium from the edges of carcinomas and LI lesions. The dysplastic epithelium adjacent to squamous cell carcinoma has been considered as an «at risk» group (Wright and Shear, 1985) on the assumption that an

altered field extends peripherally from the carcinoma. LI, a lesion considered innocuous for so long, now appears to have malignant potential (Silverman et al., 1985; Murti et al., 1986; Holmstrup et al., 1988). There are numerous reports which confirm that oral leukoplakia is a precancerous condition (Pindborg et al., 1968; Silverman and Rosen, 1968; Silverman et al., 1976, 1984; Banoczy, 1977). It is this third group that we would consider as being potentially malignant. The fourth group consisted of squamous cell papillomas, which are benign neoplasms, and to our knowledge, there are no reports of malignant transformation in these lesions. The fifth group contained neoplasms which were frankly malignant and which showed extensive invasion of the underlying voluntary muscle.

Within these groupings, we intend to analyse systematically a number of quantitative parameters which can be generated reliably with facility by even the most inexperienced operators in order to determine the relationship of these features to subsequent malignant development. In particular, we need to assess whether

N/C ratio in human cheek lesions

particular quantifiable epithelial atypical features which have already been defined have greater or lesser diagnostic significance. Subsequently, we will attempt to evaluate whether other quantifiable features may have a beneficia1 role to play in cancer diagnosis and prognosis; whether the grade (mild, moderate or severe) of epithelial dysplasia can be quantified with simple structural markers and whether this information can be used as a diagnostic or prognostic indicator. If we can establish reliable quantitative indicators of premalignant change, we may be able to define the grading system of dysplasia and cancer more precisely, to reduce the marked observer variation that currently exists in the interpretation of the histopathological diagnosis of these lesions (Pindborg et al., 1985) and ultimately to improve the prognosis for the patient.

An increase in the nuclear cytoplasmic ratio is cited regularly as a feature of cellular atypia and, as such, is considered as one of the features which is suggestive of premalignant change in a given suspicious lesions. On the basis of the groupings outlined above, we would have expected that normal controls would have had the lowest nuclear cytoplasmic ratio and the malignant group would have had the highest. Non-neoplastic lesions without malignant potential and benign neoplasms would hypothetically have ratios similar to those of the normal control group, whereas we would have expected that the potentially premalignant group would have values substantially higher than the controls, and perhaps somewhat lower than or similar to the overtly malignant group.

For basal cells, the normal controls had a higher ratio than any of the lesions analysed, a situation totally contrary to expectations. In spinous cells, trends were somewhat different. Normal controls had the lowest ratios and squamous cell carcinomas had the highest. However, the non neoplastic and benign neoplastic groups had values which were higher, and often significantly so, than those obtained for the lesions with malignant potential. The studies of Franklin and Smith (1980) and White and Gohari (198 la), performed using the hamster cheek pouch model treated with the carcinogen DMBA, both showed consistent elevations in DMBA-induced hyperplasia, dysplasia and carcinoma, although in the former study, consistent statistical differences were detected, whereas in the latter they were not.

From the data presented in Tables 7 and 8, it is apparent that the alterations in NIC ratio detected quantitatively are not reliable either in their magnitude or in their direction (i.e. whether there is an increase or decrease). More importantly, the specificity of this alteration is poor. In the only work which is directly comparable to the present report, Jin et al. (1995a) were unable to detect significant differences in NIC ratios of basal cells between normal palatal epithelium and any of the pathological groups investigated. Shabana et al. (1987) described alterations in basal cells from human cheek lesions. While increases in NIC ratio were

detected in Candidal leukoplakia, this parameter was actually decreased in what they cailed the «risk gmup», defined as patients who either had white lesions that had undergone malignant change or who had lesions coexisting with carcinomas on first examination.

Wright and Shear (1985) found that only 46.2% of dysplastic lesions adjacent to squamous cell carcinomas had an increased NIC ratio detected qualitatively. In their list of features of epithelial atypia, the presence of disturbed epithelial polarity, nuclear hyperchromatism, basal cell hyperplasia, disturbed epithelial maturation, loss of cell adherence and inflammation were noted in more than 50% of their cases of epithelial dysplasia. Preliminary quantitative analyses might best be commenced on some of the features which appear to have a higher prevalence and which readily lend them- selves to morphometric analysis, for example basal cell hyperplasia. A further problem with the application of the features of cellular atypia to diagnosis is that the vast majority are only useful when severa1 of these features are present together. Individual features have little if any value when considered in isolation, inflarnmation being an obvious example. In some lesions, notably carcinoma in situ, al1 of the features may be present. It is thus likely that if morphometric methods are to have diagnostic usefulness, analyses will have to address the evaluation of objective descriptors in their multiplicity rather than individually.

The results of our analysis would suggest that there is no justification for including increase in nuclear cytoplasmic ratio or in nuclear volume density, a closely related parameter, as one of the features of cellular atypia when assessing the malignant potential of cheek lesions, since not only it is not always present but also, and more importantly, it seems to be a feature of non- neoplastic and benign lesions. The inclusion of this parameter in the WHO (1978) paper might result from the illusion that NIC ratio is increased because of increased nuclear hyperchromatism, which of course tends to increase the prominence of nuclei on tissue sections.

Acknowledgements. This work was carried out while Dr. Jin was the recipient of a Sino-British Trust Visitorship held in the Departrnent of Anatomy, University of Hong Kong. We appreciate the support and encouragement given to us by Professor Brian Wheatherhead and are grateful to Dr. G.L. Tipoe for his assistance with the operation of the VIDAS systern. Part of this work was supported by the Cornrnittee on Research and Conference Grants, The Universily of Hong Kong.

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Accepted July 16, 1996