JOURNALOF PATHOLOGY, VOL.
161: 201-208 (1990)
MORPHOMETRIC DEFINITION AND GRADING OF GASTRIC INTESTINAL METAPLASIA P. TOSI*, M. I. FILIPE~,J. P. A. B A A K ~ ,P. LUZI*, R. SANTOPIETRO*, T. hfEGHA*
c. MIRACCO*, v. SFORZA*
AND
*Institute of Pathological Anatomy and Histopathology, University of Siena, Italy; fDepartment ofHktopathology, Guy's Hospital Medical School, London, U.K.; $Pathological institute, Free University Hospital, Amsterdam. The Netherlands Received 22 January 1990 Accepted 19 March I990
SUMMARY Type I and type 111intestinal metaplasia in gastricmucosa have been examined using morphometricmethods. Tissue (volume per cent gland, lumen, epithelium,goblet cell vacuoles) and nuclear parameters (area, with related standard deviation,and form factors) were used as indicators of gland crowding, nuclear-cytoplasmicratio, nuclear atypia, and pleomorphism. In type 111 intestinal metaplasia, there is significantly (i) greater nuclear pleomorphism, (ii) a higher nuclear-cytoplasmicratio, and (iii) smaller and less numerous goblet cell vacuoles in both the upper and the lower parts of the crypts. These two parameters have significantly higher values in the lower than in the upper part of individual crypts. No cell population with large pleomorphic nuclei characterized type 111 metaplasia, though there was more variation in nuclear size. KEY WORDS-Stomach,
intestinal metaplasia, morphometry,grading.
INTRODUCTION The definition, recognition, and grading of precancerous lesions are fundamental to successful screening for gastric cancer. There is strong evidence and general agreement on the possible progression of changes from chronic atrophic gastritis to intestinal metaplasia (IM), dysplasia, and finally intestinal type carcinoma (for a review, see refs 1-3) even if a number of intestinal metaplasia-mild dysplasia cases remain stable for years or regre~s.4.~ The problem facing the pathologist is the interpretation of a biopsy showing IM or mild forms of dysplasia. How does one distinguish a reactive from a preneoplastic phenotype? How does one define the stage of a lesion in terms of cancer risk? Answers to these questions would determine the clinical management of the patient. Since the routine qualitative assessment and grading of dysplastic lesions are highly subjective and their reproducibility is low, the introduction Addressee for correspondence:Professor Piero Tosi, Istituto Anatomia ed Istologia Patologica, Universita degli Studi di Siena, Via delfe Scotte, 53100 Siena, Italy.
0022-341 7/90/070201-08 $05.00 0
1990 by John Wiiey & Sons. Ltd.
of quantitative (morphometrical) cyto-histological parameters could provide a useful adjunct to pathological diagnosis in this field. It has been postulated'.6 that IM characterized by incomplete cell differentiation and by sulphomucin secretion (type I11 IM) is closely related to intestinal type carcinoma, whereas other non-sulphomucin-secreting types (types I and 11) are predominant in situations where the risk of cancer is low. It has also been hypothesized that IM types I and I11 are part of a dynamic process with possible transition from type I to the more immature type 111, antedating dysplasia and associated with a greater risk of malignan~y.~.' In the present study we have attempted to define and grade variants of IM more objectively by quantitatively (morphometrically) evaluating their cyto-histological features. METHODS Details of patients and biopsies A total of 54 gastric mucosal areas at low magnification ( x 460), including type I IM (22 areas) and type I11 IM (32 areas), and 108gastricmucosal areas
202
P. TOSI ET AL.
Fig. I-Absence of architectural derangement and the presence of mature absorptive and numerous goblet cells characterize type I intestinal metaplasia(a). Absorptive cells are prevalentlynon-secreting.HID/AB (b)
at high magnification ( x 2800) (44 type I, 64 type 111) were analysed in 25 patients (14 males and 1 1 females; age range 43-85, mean 62.5). Material was obtained from gastrectomy specimens and/or biopsies taken from antrum, corpus, or fundus. The diagnoses confirmed histologically were peptic ulcer (3 cases), early gastric carcinoma (1 1 cases), advanced gastric carcinoma (6 cases), adenoma (1 case), and IM associated with severe dysplasia (4 cases). All tissues were fixed in 10 per cent buffered formalin (pH 7.2) and routinely processed. Paraffin sections were cut at 5 pm, stained with haematoxylin and eosin (H&E), and used for histological and morphometrical analysis. Mucin staining (alcian blue/PAS; high iron diamine (HID)/alcian blue (AB)) was performed in parallel sections to identify IM type I (complete, non-sulphated) and 111 (incomplete, sulphated). Areas showing type I or I11 IM were selected on sections stained for mucins, marked on the parallel H&E-stained sections, and photographed. Photographs were taken of both the upper and the lower thirds of each intestinalized crypt. The neck region, corresponding to the middle third level, was excluded to avoid the proliferative area of stem cells.’ Paneth cells were also excluded.
Photography
This was done on a Leitz Dialux microscope with the following specifications: objective: x 63 Leitz NLP Fluotar; eyepiece: x 12.5 Leitz Paniplan GF. The areas and IM crypts within the areas to be analysed by morphometry were marked on photographs at low magnification ( x 175). Dejhition of types I and 111 intestinal metaplasia
Type I (also termed ‘mature’, ‘complete’, or ‘small intestinal’ type) is characterized by regular architecture and straight crypts lined by mature absorptive and goblet cells. Goblet cells secrete sialomucinsand occasionallyneutral mucins and/or sulphomucins. Absorptive cells are non-secretory and have a brush border. Paneth cells are often seen (Figs l a and lb). Type 111 (immature, ‘incomplete’ or ‘colonic’ type) shows a variable degree of architectural distortion and tortuosity of crypts, with marked cellular dedifferentiation. Columnar cells predominantly secrete sulphomucins, and goblet cells contain sialoand/or sulphomucin; Paneth cells are inconspicuous (Figs 2a and 2b).9
MORPHOMETRY OF INTESTINAL METAPLASIA
203
Fig. 2-Type 111 intestinal metaplasia. Some architectural derangement is evident. Few absorptive and goblet cells but predominant columnar mucus-secreting cells constitute the gland epithelium (a). Sulphomucins (black) are present in columnar mucous cells, while sialo- (grey) and/or sulphomucins (dark grey/black)are secreted by goblet cells. HID/AB (b)
Morphometry
The architectural parameters measured were: volume per cent glands ( W G L A ) and volume per cent stroma (VVSTR) on the entire mucosa; (ii) volume per cent epithelium (VVEPI) and lumen (VVLU) on all the glands; (iii) volume per cent nuclei/epithelium ( W N U / EPI); and (iv) volume per cent goblet cell vacuoles (WGCV) on epithelium cytoplasm. The nuclear features studied were nuclear profile area, with related standard deviation (SD), and nuclear shape factors. Gland crowding and the quantity of epithelium, lumen, stroma, and goblet cell vacuoles were evaluated by means of the parameter volume percentage (VV%). Nuclear area and SD as well as nuclear ellipsoidity, regularity, and roundness (measured by the shape factors form Ell. form Ar, and form Pe, respectively-for details see below) are expressions of nuclear size, shape, and pleomorphism. WNU/EPI is here considered as the nuclear-cytoplasmic ratio. (i)
In order to obtain reproducible measurements, glands, epithelium, lumen, and stroma were delineated previously with a waterproof marker on the photographs at low magnification. Lumina were defined as the area totally circumscribed by epithelium when glands were transversely sectioned, or as the area under a line connecting tops of foveolae between neighbouring glands in the case of longitudinally sectioned glands. For the parameters W N U / E P I and VVGCV, basal membrane and vacuoles were also delineated by means of a waterproof marker on high magnification photographs. Since the basal membrane is often vague, or absent or difficult to detect because of vacuolization, in this study it was defined as a line connecting basal ends of nuclei which are basally located. In order to distinguish goblet cell vacuoles from other small cytoplasmic vacuoles, goblet cell vacuoles were defined as large, oval to round vacuoles, with clear lateral demarcation, in contact with basally placed nuclei and the lumen, or with basal nuclei and not in contact with the lumen, but in the latter case they still had to constitute more than 75 per cent of cell cytoplasm.
204
P. TOSl ET AL.
Point counting was used for measuring volume percentage. A multipurpose test (Weibel's M 168 grid)" was superimposed on photographs printed at x 460 (for the parameters VVGLA, W L U , VVSTR and WEPI) and at x2800 (for VVNU/EPI and WGCV). Six fields (1008 points) were sufficient to cover the whole photograph. A graphic tablet cursor system was used for evaluating nuclear area (with related SD), nuclear ellipsoidity (form Ell), nuclear regularity (form Ar), and nuclear roundness (form Pe) on the photographs printed at x 2800. Nuclear ellipsoidity (form Ell) was calculated by the ratio minimum/maximum diameter, where these diameters correspond to those of an elliptical type structure having the same moment of inertia as the structure to be measured. Its value is 1 for a circle and less than 1 for elongated structures. Form Ar is a size-independent indicator of the regularity of a profile, calculated by the following formula: area/n/4 x major diameter x minor diameter, where the major and minor diameters are calculated by the moment of inertia of an elliptical type structure. Its value is 1 for a circle and an ellipse, and less than 1 for irregular structures. Form Pe is calculated by the following formula: (4n x area)/perimete?. Its value is 1 for a circle and less than 1 for irregular structures. All well-defined epithelial nuclei were measured on photographs at high magnification. In each case, the number of measured nuclei was higher than 100. For the assessment of architectural parameters a limit of 840 points was sufficient to obtain an intraand inter-observer coefficient of variation within 10 per cent. For the assessment of nucleometric parameters 50 nuclei were sufficient to have an inter-observer variation within 5 per cent.
by repeating the measurements seven times. The Student's r-test was applied to determine any consistent intra- and inter-observer differences, but no such differences were detected for all the quantitative parameters used. RESULTS
Type I and type I11 IM show significantly different nucleometric and tissue architectural features. Nuclear area is not discriminant between the two groups, but its SD is much higher in type 111. It may therefore be deduced that nuclei are more anisokaryotic and more pleomorphic in type 111, as also shown by the lower values of shape factors (form Ell, form Ar and form Pe): in type 111, nuclei are more elongated (elliptical), have more irregular profiles, and are less round. The per cent of nuclear area as related to total epithelial area is much higher in type 111, while the per cent of area occupied by vacuoles of goblet cells as related to total epithelial cytoplasmic area is significantly higher in type I. Significant differences were shown between the lower and upper parts of the individual crypts both in type I and in type 111. In type I, the discriminant parameters are SD of nuclear area (nuclei in the lower part are more pleomorphic), shape factors (nuclei in the lower part are more elongated, less round, and their profile is less regular), and per cent of nuclear area on epithelial area (higher in the lower part of the crypts). In type 111, the only discriminant parameters are per cent of nuclear area on total epithelium area (higher in the lower part of the crypts) and area occupied by goblet cells as a per cent of total epithelial cytoplasmic area (goblet cells are less numerous in the lower part). In type 111 IM, at both crypt levels, nuclei are Statistics more elliptical and irregular and the nuclei/epiThe Student's t-test was applied to assess the thelial ratio is greater than that in type I, while significant differences between mean values of the vacuoles are more numerous and larger in type I parameters. P
161: 201-208 (1990)
MORPHOMETRIC DEFINITION AND GRADING OF GASTRIC INTESTINAL METAPLASIA P. TOSI*, M. I. FILIPE~,J. P. A. B A A K ~ ,P. LUZI*, R. SANTOPIETRO*, T. hfEGHA*
c. MIRACCO*, v. SFORZA*
AND
*Institute of Pathological Anatomy and Histopathology, University of Siena, Italy; fDepartment ofHktopathology, Guy's Hospital Medical School, London, U.K.; $Pathological institute, Free University Hospital, Amsterdam. The Netherlands Received 22 January 1990 Accepted 19 March I990
SUMMARY Type I and type 111intestinal metaplasia in gastricmucosa have been examined using morphometricmethods. Tissue (volume per cent gland, lumen, epithelium,goblet cell vacuoles) and nuclear parameters (area, with related standard deviation,and form factors) were used as indicators of gland crowding, nuclear-cytoplasmicratio, nuclear atypia, and pleomorphism. In type 111 intestinal metaplasia, there is significantly (i) greater nuclear pleomorphism, (ii) a higher nuclear-cytoplasmicratio, and (iii) smaller and less numerous goblet cell vacuoles in both the upper and the lower parts of the crypts. These two parameters have significantly higher values in the lower than in the upper part of individual crypts. No cell population with large pleomorphic nuclei characterized type 111 metaplasia, though there was more variation in nuclear size. KEY WORDS-Stomach,
intestinal metaplasia, morphometry,grading.
INTRODUCTION The definition, recognition, and grading of precancerous lesions are fundamental to successful screening for gastric cancer. There is strong evidence and general agreement on the possible progression of changes from chronic atrophic gastritis to intestinal metaplasia (IM), dysplasia, and finally intestinal type carcinoma (for a review, see refs 1-3) even if a number of intestinal metaplasia-mild dysplasia cases remain stable for years or regre~s.4.~ The problem facing the pathologist is the interpretation of a biopsy showing IM or mild forms of dysplasia. How does one distinguish a reactive from a preneoplastic phenotype? How does one define the stage of a lesion in terms of cancer risk? Answers to these questions would determine the clinical management of the patient. Since the routine qualitative assessment and grading of dysplastic lesions are highly subjective and their reproducibility is low, the introduction Addressee for correspondence:Professor Piero Tosi, Istituto Anatomia ed Istologia Patologica, Universita degli Studi di Siena, Via delfe Scotte, 53100 Siena, Italy.
0022-341 7/90/070201-08 $05.00 0
1990 by John Wiiey & Sons. Ltd.
of quantitative (morphometrical) cyto-histological parameters could provide a useful adjunct to pathological diagnosis in this field. It has been postulated'.6 that IM characterized by incomplete cell differentiation and by sulphomucin secretion (type I11 IM) is closely related to intestinal type carcinoma, whereas other non-sulphomucin-secreting types (types I and 11) are predominant in situations where the risk of cancer is low. It has also been hypothesized that IM types I and I11 are part of a dynamic process with possible transition from type I to the more immature type 111, antedating dysplasia and associated with a greater risk of malignan~y.~.' In the present study we have attempted to define and grade variants of IM more objectively by quantitatively (morphometrically) evaluating their cyto-histological features. METHODS Details of patients and biopsies A total of 54 gastric mucosal areas at low magnification ( x 460), including type I IM (22 areas) and type I11 IM (32 areas), and 108gastricmucosal areas
202
P. TOSI ET AL.
Fig. I-Absence of architectural derangement and the presence of mature absorptive and numerous goblet cells characterize type I intestinal metaplasia(a). Absorptive cells are prevalentlynon-secreting.HID/AB (b)
at high magnification ( x 2800) (44 type I, 64 type 111) were analysed in 25 patients (14 males and 1 1 females; age range 43-85, mean 62.5). Material was obtained from gastrectomy specimens and/or biopsies taken from antrum, corpus, or fundus. The diagnoses confirmed histologically were peptic ulcer (3 cases), early gastric carcinoma (1 1 cases), advanced gastric carcinoma (6 cases), adenoma (1 case), and IM associated with severe dysplasia (4 cases). All tissues were fixed in 10 per cent buffered formalin (pH 7.2) and routinely processed. Paraffin sections were cut at 5 pm, stained with haematoxylin and eosin (H&E), and used for histological and morphometrical analysis. Mucin staining (alcian blue/PAS; high iron diamine (HID)/alcian blue (AB)) was performed in parallel sections to identify IM type I (complete, non-sulphated) and 111 (incomplete, sulphated). Areas showing type I or I11 IM were selected on sections stained for mucins, marked on the parallel H&E-stained sections, and photographed. Photographs were taken of both the upper and the lower thirds of each intestinalized crypt. The neck region, corresponding to the middle third level, was excluded to avoid the proliferative area of stem cells.’ Paneth cells were also excluded.
Photography
This was done on a Leitz Dialux microscope with the following specifications: objective: x 63 Leitz NLP Fluotar; eyepiece: x 12.5 Leitz Paniplan GF. The areas and IM crypts within the areas to be analysed by morphometry were marked on photographs at low magnification ( x 175). Dejhition of types I and 111 intestinal metaplasia
Type I (also termed ‘mature’, ‘complete’, or ‘small intestinal’ type) is characterized by regular architecture and straight crypts lined by mature absorptive and goblet cells. Goblet cells secrete sialomucinsand occasionallyneutral mucins and/or sulphomucins. Absorptive cells are non-secretory and have a brush border. Paneth cells are often seen (Figs l a and lb). Type 111 (immature, ‘incomplete’ or ‘colonic’ type) shows a variable degree of architectural distortion and tortuosity of crypts, with marked cellular dedifferentiation. Columnar cells predominantly secrete sulphomucins, and goblet cells contain sialoand/or sulphomucin; Paneth cells are inconspicuous (Figs 2a and 2b).9
MORPHOMETRY OF INTESTINAL METAPLASIA
203
Fig. 2-Type 111 intestinal metaplasia. Some architectural derangement is evident. Few absorptive and goblet cells but predominant columnar mucus-secreting cells constitute the gland epithelium (a). Sulphomucins (black) are present in columnar mucous cells, while sialo- (grey) and/or sulphomucins (dark grey/black)are secreted by goblet cells. HID/AB (b)
Morphometry
The architectural parameters measured were: volume per cent glands ( W G L A ) and volume per cent stroma (VVSTR) on the entire mucosa; (ii) volume per cent epithelium (VVEPI) and lumen (VVLU) on all the glands; (iii) volume per cent nuclei/epithelium ( W N U / EPI); and (iv) volume per cent goblet cell vacuoles (WGCV) on epithelium cytoplasm. The nuclear features studied were nuclear profile area, with related standard deviation (SD), and nuclear shape factors. Gland crowding and the quantity of epithelium, lumen, stroma, and goblet cell vacuoles were evaluated by means of the parameter volume percentage (VV%). Nuclear area and SD as well as nuclear ellipsoidity, regularity, and roundness (measured by the shape factors form Ell. form Ar, and form Pe, respectively-for details see below) are expressions of nuclear size, shape, and pleomorphism. WNU/EPI is here considered as the nuclear-cytoplasmic ratio. (i)
In order to obtain reproducible measurements, glands, epithelium, lumen, and stroma were delineated previously with a waterproof marker on the photographs at low magnification. Lumina were defined as the area totally circumscribed by epithelium when glands were transversely sectioned, or as the area under a line connecting tops of foveolae between neighbouring glands in the case of longitudinally sectioned glands. For the parameters W N U / E P I and VVGCV, basal membrane and vacuoles were also delineated by means of a waterproof marker on high magnification photographs. Since the basal membrane is often vague, or absent or difficult to detect because of vacuolization, in this study it was defined as a line connecting basal ends of nuclei which are basally located. In order to distinguish goblet cell vacuoles from other small cytoplasmic vacuoles, goblet cell vacuoles were defined as large, oval to round vacuoles, with clear lateral demarcation, in contact with basally placed nuclei and the lumen, or with basal nuclei and not in contact with the lumen, but in the latter case they still had to constitute more than 75 per cent of cell cytoplasm.
204
P. TOSl ET AL.
Point counting was used for measuring volume percentage. A multipurpose test (Weibel's M 168 grid)" was superimposed on photographs printed at x 460 (for the parameters VVGLA, W L U , VVSTR and WEPI) and at x2800 (for VVNU/EPI and WGCV). Six fields (1008 points) were sufficient to cover the whole photograph. A graphic tablet cursor system was used for evaluating nuclear area (with related SD), nuclear ellipsoidity (form Ell), nuclear regularity (form Ar), and nuclear roundness (form Pe) on the photographs printed at x 2800. Nuclear ellipsoidity (form Ell) was calculated by the ratio minimum/maximum diameter, where these diameters correspond to those of an elliptical type structure having the same moment of inertia as the structure to be measured. Its value is 1 for a circle and less than 1 for elongated structures. Form Ar is a size-independent indicator of the regularity of a profile, calculated by the following formula: area/n/4 x major diameter x minor diameter, where the major and minor diameters are calculated by the moment of inertia of an elliptical type structure. Its value is 1 for a circle and an ellipse, and less than 1 for irregular structures. Form Pe is calculated by the following formula: (4n x area)/perimete?. Its value is 1 for a circle and less than 1 for irregular structures. All well-defined epithelial nuclei were measured on photographs at high magnification. In each case, the number of measured nuclei was higher than 100. For the assessment of architectural parameters a limit of 840 points was sufficient to obtain an intraand inter-observer coefficient of variation within 10 per cent. For the assessment of nucleometric parameters 50 nuclei were sufficient to have an inter-observer variation within 5 per cent.
by repeating the measurements seven times. The Student's r-test was applied to determine any consistent intra- and inter-observer differences, but no such differences were detected for all the quantitative parameters used. RESULTS
Type I and type I11 IM show significantly different nucleometric and tissue architectural features. Nuclear area is not discriminant between the two groups, but its SD is much higher in type 111. It may therefore be deduced that nuclei are more anisokaryotic and more pleomorphic in type 111, as also shown by the lower values of shape factors (form Ell, form Ar and form Pe): in type 111, nuclei are more elongated (elliptical), have more irregular profiles, and are less round. The per cent of nuclear area as related to total epithelial area is much higher in type 111, while the per cent of area occupied by vacuoles of goblet cells as related to total epithelial cytoplasmic area is significantly higher in type I. Significant differences were shown between the lower and upper parts of the individual crypts both in type I and in type 111. In type I, the discriminant parameters are SD of nuclear area (nuclei in the lower part are more pleomorphic), shape factors (nuclei in the lower part are more elongated, less round, and their profile is less regular), and per cent of nuclear area on epithelial area (higher in the lower part of the crypts). In type 111, the only discriminant parameters are per cent of nuclear area on total epithelium area (higher in the lower part of the crypts) and area occupied by goblet cells as a per cent of total epithelial cytoplasmic area (goblet cells are less numerous in the lower part). In type 111 IM, at both crypt levels, nuclei are Statistics more elliptical and irregular and the nuclei/epiThe Student's t-test was applied to assess the thelial ratio is greater than that in type I, while significant differences between mean values of the vacuoles are more numerous and larger in type I parameters. P
