Oral Epithelial Dysplasia: Can Quantifiable Morphological Features

Help in the Grading Dilemma?

Rasha Abu Eid and Gabriel Landini*

Oral Pathology Unit, School of Dentistry, The University of Birmingham
St. Chad's Queensway, Birmingham, England.

ABSTRACT
The epithelial lining of the oral cavity can sometimes experience certain changes that put it at a higher risk of undergoing
malignant transformation. Such changes present clinically as 'premalignant' lesions that at the histological level feature
pathological alterations known as epithelial dysplasia. However, the degree of alteration of tissues is routinely assessed
visually, thus introducing an element of subjectivity to the diagnostic process. The aim of this work was to apply
objective and quantitative image analysis techniques to one problematic area in histopathological diagnosis: the grading
of the severity of epithelial dysplasia. Histopathological diagnosis (which depends to some degree on individual
judgement of histological features by an observer) has been shown to be subject to intra-and inter-observer variations that
affect the accuracy and reproducibility of the diagnostic process. Therefore, more quantitative methods, which do not
depend on human perceptual skills, are preferable. In this paper we present a number of innovative techniques to assess a
number of morphological features of different grades of oral epithelial dysplasia.

Keywords: Epithelial dysplasia , cell morphometry, histopathological diagnosis.

1. INTRODUCTION

The lining of the oral cavity is composed of stratified squamous epithelium made up of several layers of keratinocytes
which might undergo premalignant and malignant changes. A premalignant lesion is an altered tissue, which has a higher
risk of undergoing malignant transformation than its normal counterpart. Histologically, oral premalignant lesions
present various degrees of epithelial dysplasia, that is cellular and tissue atypia with loss of control in the normal
maturation and stratification, short of carcinoma in situ [1].
Traditionally, the severity of the atypic changes (such as cellular and nuclear pleomorphism, abnormal mitosis, and
drop shaped rete-ridges) and height in the epithelium to which these atypic features extend have been used in grading
dysplasia. Mild dysplasia is considered when minimal dysplastic changes are confined to the parabasal layers, while for
moderate dysplasia, more pronounced dysplastic features extend to the middle third and for severe dysplasia, severe
changes are observed through the entire thickness of the epithelium without apparent invasion of the stroma. Although
epithelial dysplasia is generally graded into three classes (mild, moderate and severe), precise grading is a source of
disagreement as the assessment carries a degree of subjectivity [2, 3]. Furthermore, it is still unclear what features (if
any) should be considered to base the grading on.

*
Further author information: (Send correspondence to R.A.)
Rasha Abu Eid: E-mail: r.f.abueid.1@bham.ac.uk, Telephone: (+)44 121 237 2884
Gabriel Landini: E-mail: g.landini@bham.ac.uk, Telephone: , (+)44 121 237 2885
Address: Oral Pathology Unit, School of Dentistry, The University of Birmingham, St. Chad's Queensway, B4 6NN,
Birmingham, England, United Kingdom.
 This research aimed at developing some quantitative objective methods for characterising some morphological
features of oral epithelial dysplasia with the aim of minimising the subjectivity in some aspects of such histopathological
grading.

2. MATERIALS AND METHODS

2.1 Images
Haematoxylin and eosin-stained histological sections from 19 oral epithelial dysplasia biopsy samples were obtained
from the Oral Pathology archives of the School of Dentistry, The University of Birmingham. These included 4 mild, 8
moderate and 7 severe dysplasia cases. Tissue images were captured with an Olympus BX50 Microscope (Olympus
Optical Co. Ltd. Tokyo, Japan) connected to a KY-F55B 3-CCD colour camera (JVC, Tokyo, Japan). The video signal
was digitised by an IT-IC-PCI 24-bit colour frame grabber (Imaging Technology, Bedford, MA, USA). A x20 microscope
objective (n. a. 0.30, resolution 0.67
m) was used to capture images (at this resolution, pixel size was 0.624
m giving
a field width of 479
m).
Twenty eight images (made of collaged images) of the full thickness of the epithelium were analyzed (including 9
mild, 9 moderate and 10 severe dysplasia images).

2.2. Image analysis

Originally the analysis described was developed in a commercial imaging package (Optimas 6.51, Media Cybernetics,
Silver Spring, MD, USA). However the availability of a free, open source package like ImageJ (currently version 1.35p)
allowed us to port such procedures into Java for better multiplatform compatibility and deployment.
The cells in the epithelial compartment were segmented using the method described elsewhere [4]. Because in
haematoxylin and eosin stained sections, the epithelial cell borders cannot be isolated accurately, theoretical cell profile
extents were estimated statistically using a space partition procedure. Cell segmentation was achieved by nuclear
localization based on the optical density of the nuclear stain to define the probable cell extents. The first step consisted of
using a colour deconvolution procedure [5] (written as an ImageJ plugin, available from
http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html) to obtain the optical density of the
haematoxylin stain alone. Colour deconvolution involves the separation of the colour information from images generated
by color subtraction (i.e. light-absorbing dyes such as those used in bright field histology or ink on printed paper) with up
to three non-neutral stains. This is achieved by calculating the contribution of each stain based on the stain-specific RGB
absorption.
The deconvolved image gives the spatial localization of nucleic acids and thus the nuclear location. Following, spatial
partition of the epithelial compartment into areas of influence relative to each nucleus was achieved using the so-called
'watershed transform' [6] (we used D. Sage's watershed segmentation plugin for ImageJ available at
http://bigwww.epfl.ch/sage/soft/watershed/). This procedure divides the nuclear-surrounding space into exclusive
catchment basins belonging to each nucleus. The basic idea behind the transform is to view the digital grey scale image
as a landscape with the pixel grey level as the altitude. If a drop of water hit the surface of this landscape, it would flow
down along the path of steepest descent until it reached a regional minimum. The set of all pixels for which drops of
water end in the same minimum is a “catchment basin”. These were chosen to represent, in theory, the individual
epithelial cell profile extents based on the nuclear locations. The pixels that cannot easily be assigned to a catchment
basin are called watershed lines and represent the theoretical boundaries between cells (Figure 1).
The morphological properties of these algorithmically segmented cells were subsequently analyzed using the
Particles8_Plus plugin for ImageJ (available in the Morphology plugins Collection for ImageJ.
http://www.dentistry.bham.ac.uk/landinig) to extract the following parameters: cell perimeter, area, radius of the
inscribed circle centred at the centre of mass, radius of the enclosing circle centred at the centre of mass, largest axis
length (feret), breadth, convex Hull, area of the convex Hull polygon, radius of the minimal bounding circle, aspect ratio,
roundness, area equivalent diameter, perimeter equivalent diameter, equivalent ellipse area, compactness, solidity,
concavity, convexity, shape, Rfactor, modification ratio, sphericity, feret breadth, rectangularity, and greyscale statistics
(integrated density, minimum, maximum, modal, median, average, average deviation, standard deviation, variance,
skewness, kurtosis and entropy values in the particle). Further details on the calculation and description of these
parameters are available from the same link above.

2.3. Statistical analysis

Statistical analysis of the data was performed using SPSS version 12 (SPSS Inc., Chicago, USA). Two types of
analyses were of interest: the analysis of variance (ANOVA) of the different parameters across the diagnostic groups and
a hierarchical discriminant analysis (with leave one out classification) to disclose the degree of correct classification that
can be achieved using the quantitative parameters in the three classical grades of dysplasia.

A B C D

Figure 1. A) x20 Haematoxylin and Eosin RGB image of dysplastic oral mucosa. B) The Haematoxylin component of A. C) The
segmented epithelial compartment after applying the watershed transform. D) The segmented epithelial compartment after performing
the logical AND operation between A and

3. RESULTS
A total of 33,177 cells were analysed (9,830 cells from mild , 13,805 cells from moderate and 9,542 cells from severe
dysplasia cases).
With the exception of circularity, solidity, concavity, convexity, RFactor, rectangularity, grey value mode, grey
average deviation, grey standard deviation and kurtosis, all the parameters were statistically different between moderate
and severe dysplasia (p<0.05 ANOVA). All the parameters were statistically different between mild and moderate
dysplasia except for convexity (p<0.05 ANOVA).
 Despite the observed differences, cell-wise discriminant analysis using the morphometric parameters showed that
only 52% of the cells could be correctly classified into the original dysplasia grade. Although these results are higher
than the random probability for 3 groups (33.3%), they are not highly reliable. Table 1 shows the detailed classification.
Image-wise, using the averages of the parameters the discriminant analysis was able to correctly classify only 46% of
the images into their diagnosed dysplasia grade. Table 2 shows the detailed classification.

Table 1. The percentage of cell-wise correct classification between the three grades of dysplasia according to the epithelial
morphometric cellular parameters. (Linear discriminant analysis cross validated with leave one out).

Predicted Group Membership

mild moderate severe Total
mild 6616 1675 1539 9830
Cell count moderate 3597 5928 4280 13805
severe 2043 2927 4572 9542
mild 67.3% 17.0% 15.7% 100.0%
Percentage moderate 26.1% 42.9% 31.0% 100.0%
severe 21.4% 30.7% 47.9% 100.0%

Table 2. The percentage of image-wise correct classification between the three grades of dysplasia according to the epithelial
morphometric cellular parameters. (Linear discriminant analysis cross validated with leave one out).

Predicted Group Membership

mild moderate severe Total
mild 7 0 2 9
Image count moderate 2 5 2 9
severe 3 6 1 10

mild 77.8% 0.0% 22.2% 100.0%

Percentage moderate 22.2% 55.6% 22.2% 100.0%
severe 30.0% 60.0% 10.0% 100.0%

4. DISCUSSION
The diagnosis of (oral) epithelial dysplasia implies the possibility of progression to SCC. The presence of dysplasia,
however, does not mean that malignant transformation is inevitable and its absence does not preclude malignancy either
[7]. Nevertheless, the histological presence of dysplasia, even the mild type, suggests an increased risk of transformation
with severe dysplasia having the highest risk.
Unfortunately the diagnosis of dysplasia carries a degree of subjectivity that introduces some inconsistency [2].
Several suggestions have been proposed to overcome this, such as the use of molecular markers [8-10], morphological
descriptors [11, 12] and computer aided analyses [13, 14]. In a review Warnakulasuriya (2001) reported the
inconsistencies in grading oral epithelial dysplasia between different authors and pointed out the contradictory results
concerning the malignant transformation rates reported for each of the dysplasia grades. He also confirmed the existence
of inter-observer variability in reporting dysplasia. While the author proposed the introduction of more objective methods
such as the use of genomic and proliferation markers associated with different histopathological parameters, he
acknowledges that much work is still to be done before such techniques are available and generalized for
histopathological diagnosis [3]. Surprisingly, no mathematical or morphometrical methods were proposed. Bosman
(2001) also emphasized the importance of developing new and better morphological definitions for grading epithelial
dysplasia based on research into the pathogenesis of premalignancy. He suggested that only through correlation between
changes at the molecular level and the clinical outcome, new criteria can be accepted to replace the “gold standard” for
grading epithelial dysplasia [15].
In this work, quantitative reproducible methods were applied to histological images epithelial dysplasia sections with
different grades of severity. The results show that while there are statistical differences on the morphometrical parameters
analysed across the diagnostic classes, these cannot be reliably used for discrimination purposes.
In a previous study, the methods used in this work proved useful in statistically distinguishing other oral epithelial lesions
including normality, epithelial dysplasia (regardless of its grade) and pseudo-epitheliomatous hyperplasia [16]. However
the results shown here seem to indicate that the traditional subclassification of dysplastic changes into 3 grades are not
supported by the morphological evidence we collected. The relatively low success rates in classifying dysplasia grades
emphasizes the problems of using classifiers that have elements of subjectivity. A new quantitative
classification/quantitation system is therefore needed. The use of quantitative methods such as those presented here
might be of use in establishing these new, reproducible and objective grading systems.

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