Virchows Arch [Cell Pathol] (1986) 52:11%128 I" Arc/dv B �9 Springer-Verlag 1986

Histochemistry of small intestinal dysplasia in familial polyposis coli

Paolo RomagnolP, Francesco Nardi 2, Francesco Tonelli 3, and Paolo Bechi 4 1 Istituto di Istologia ed Embriologia Generale, Universitfi di Firenze 2 Istituto di Anatornia e Istologia Patologica I, Universit~t di Roma, La Sapienza 3 Cattedra di Anatomia Chirurgica e Corso di Operazioni, Universit~i di Firenze 4 Istituto di Clinica Chirurgica I, Universit~ di Firenze, Italia

Summary. Biopsies of duodenal and ileal mucosa from patients with familial polyposis coli were studied. Areas of atypia were identified in the duodenum of six patients and in the ileum of three patients. Grade I atypia was characterized by crowding and elongation of cells and nu- clei, a slight reduction in the number of goblet cells and the presence of a brush border; grade II atypia was further characterized by pseudo- or pluristratification of cells, a marked reduction in the number of goblet cells and the absence of a brush border. In areas of atypia, columnar cells often contained PAS-positive apical granules, which were diastase- resistant and unstained by alcian blue at any pH; the brush border, even where recognizable in haematoxylin-eosin and PAS-stained sec- tions, was unreactive histochemically for alkaline phosphatase. Goblet cells were few in areas of atypia, but those present were regularly stained by PAS and alcian blue pH 2.6. Apical granules, similar in their histo- chemical characteristics to those observed in columnar cells in areas of atypia, were also found in otherwise normal mucosal areas, even in some patients with no overt areas of atypia in the biopsies studied. These granules have been interpreted as an abnormality, possibly preced- ing the onset of atypia. Hyperplasia of goblet cells, secreting mucins with the same staining pattern as in normal intestine, was found in some patients, either adjacent to areas of atypia or independent of them. Intervening columnar cells had a normal morphology, alkaline phospha- tase-reactive brush borders and no sign of mucus secretion. This goblet cell hyperplasia has been interpreted as a reactive, nonspecific alteration of the mucosa.

Key words: Duodenal polyps - Ileal polyps - Intestinal dysplasia - Intes- tinal mucosa - Polyposis coli

Offprint requests to: P. Romagnoli, Istituto di Istologia ed Embriologia Generale, Viale Pierac- cini, 18, 1-50139 Firenze, Italia

118 P. Romagnoli et al.

Introduction

The histological and histochemical features of adenomatous polyps of the large intestine are well known and different degrees of atypia have been recognized (Filipe et al. 1980; Gabbert and H6hn 1980; BalS.zs 1980; Oo- hara et al. 1980, 1982). Grade I atypia is characterized by the presence of tall columnar cells, often mucus secreting, and goblet cells; columnar ceils have an incompletely differentiated brush border; the nuclei are rodlike and pseudostratified. Grade II atypia is characterized by a lack of goblet cells and a great reduction of mucus production by columnar cells. These cells are tall and the nuclei rodlike and pseudostratified and the brush border practically absent; glycogen deposits may be found in some columnar cells. Grade III atypia is characterized by irregular multistratification of the epi- thelium, large polymorphic nuclei and the absence of mucus secretion and brush borders (Gabbert and H6hn 1980). Grade III atypia is therefore characterized by cells which resemble those of adenocarcinoma and is con- sidered to be an advanced stage in carcinogenesis (Lane et al. 1971). A relative increase in sialomucin compared with sulphomucin secretion has been reported to occur in the apparently unaffected colonic mucosa of pa- tients with adenomatous polyps and to represent a condition prone to evolve into atypia as defined above (Filipe et al. 1980). However, this indication has been denied by other authors, who believe that these qualitative modifi- cations in mucus secretion are reactive and do not evolve to atypia or carcinoma (Gad 1979; Listinsky and Riddell 1981).

On the other hand, there has been little work on the histology and histochemistry of small intestinal polyps occurring in patients with familial polyposis coli (Hamilton et al. 1979; Goldman and Antonioli 1982; Dirsch- mid and Schobel 1983). The purpose of this research was to analyse these features in close detail to understand better the histogenesis of adenomatous polyps of the small intestine and to evaluate their possible premalignant significance, which has been suggested but not yet confirmed on clinical grounds (Heffernon et al. 1962; Ross and Mara 1974; Roth and Logio 1982).

Materials and methods

Twentytwo patients with familial polyposis coli, aged 17 to 67 and belonging to 21 families were studied. Duodenal biopsies were obtained at endoscopy from 13 patients. Biopsies of the distal ileum (up to 15 cm from the ileal anastomosis) were obtained at endoscopy from all 22 patients, all of whom had undergone colectomy. Ileal biopsies (within 15 cm from the ileocaecal valve) were also obtained in eight patients at colectomy. Details of the number and site of the biopsies are given in Table 1.

The biopsies were fixed in phosphate-buffered 10% formalin, dehydrated in graded ace- tones, diaphanized in xylene and embedded in paraplast; infiltration of the tissue with paraplast and embedding were carried out at a temperature below 60 ~ C. Histological sections (5 lam thick) were stained with the following methods: haematoxylin-eosin, gallocyanin-chromalum, periodic acid Schiff (P.A.S.), diastase-P.A.S., alcian blue pH 2.6 and pH 1.0 and Feulgen's reaction. Some sections were methylated before staining with alcian blue in order to abolish the staining of the carboxylic and sulphate groups of mucins (Gabe 1976). Alkaline phosphatase activity was demonstrated in sections from each specimen with the following method derived

Polyposis coli with small intestinal involvement 119

Fig. 1. Adenomatous polyp of the ileal mucosa, which was taken "in toto" at endoscopy. A certain degree of villous atrophy occurred in this specimen, as is frequently seen in the ileal mucosa after colectomy (Bechi et al. 1981). The mucosal surface is largely devoid of epithelium, an artefact often occurring during biopsy of larger samples. Haematoxylin and eosin x 40

Fig. 2. Biopsy of ileal mucosa appearing normal at endoscopy, where histological signs of atypia were found. The figure represents the transition between normal (below right) and atypical (adenomatous: above left) mucosa. This figure and Fig. 3 are from a specimen fixed in Bouin's fluid, unlike all the other specimens. Haematoxylin and eosin x 140

120 P. Romagnoli et al.

from that of Reale and Luciano (1967). Sections were deparaffined and hydrated. They were incubated for 30 min at room temperature in a medium containing: (1) 20 ml, 0.2 M CaC12 ; (2) 20ml, 0.1 M Na barbiturate; (3) 5 ml, 0.1 M Na - fl-glycerophosphate; (4) 5 ml, 0.05 M MgC12. After rinsing in chilled distilled water, the sections were treated for 5 min with chilled 0.05 M PbNO3, rinsed again in distilled water and treated for 2 min at room temperature with a dilute aqueous solution of (NH4)2S (3 drops of (NH4)2S in 40 ml distilled water). Stained sections were dehydrated, diaphanized and mounted in Permount. Control sections were incubated without the substrate.

Results

Areas with histological features of atypia were identified in the duodenal biopsies of six patients and in the ileal biopsies of one of these and two further patients (Figs. 1 and 2). Five of these biopsies (two duodenal and three ileal) had been taken from endoscopically normal mucosa, two of them (duodenal) from velvet-like mucosa and the others from mucosal po- lyps. Two grades of atypia were recognized in sections stained with haema- toxylin and eosin. Grade I atypia was characterized by the presence of tall and crowded columnar cells, with large, elongated nuclei and basophilic cytoplasm. A brush border was recognized in haematoxylin and eosin- stained sections. Goblet cells were slightly less frequent than normal (Fig. 3). Grade II atypia was characterized by pseudo- or pluristratification of colum- nar cells, which were also tall, crowded, and having large elongated nuclei and basophilic cytoplasm. A brush border could not be recognized in haema- toxylin and eosin-stained sections and goblet cells were much less frequent than normal (Fig. 4). Atypia grade I and grade II coexisted in each affected biopsy and areas with features intermediate between grade I and grade II atypia were also seen (Fig. 5). Areas with large polymorphic nuclei and

Fig. 3. Same biopsy as Fig. 2 (opposite side of atypical mucosa). The epithelium between the arrows shows the features of grade I atypia (crowding and elongation of cells and nuclei, slight reduction in the number of goblet cells, presence of a brush border). Haematoxylin and eosin x 350

Fig. 4. Glands with features of grade II atypia in a polyp of the ileal mucosa (same biopsy as Fig. 1). The glands are enlarged and the nuclei of epithelial cells crowded and sometimes stratified. A brush border is not recognizable and goblet cells are scanty. Haematoxylin and eosin x 270

Fig. 5. Duodenal biopsy. Gland cells near the mucosal surface with intermediate features be- tween grade I and grade II atypia (crowding, elongation and pseudostratification of cells and nuclei, reduction in the density of goblet cells, presence of a brush border). Columnar cells contain many PAS-positive, diastase-resistant granules in the supranuclear cytoplasm, mainly at the cell apex. Diastase - PAS - haematoxylin • 700

Fig. 6, Duodenal biopsy; gland cells (near the mucosal surface) with features of grade II atypia (crowding, elongation, marked pseudostratification of cells and nuclei, reduction in the density of goblet cells, lack of a brush border). Columnar cells contain scanty, PAS-positive, diastase-resistant granules in the supranuclear cytoplasm. Diastase - PAS - haematoxylin x 700

Polyposis coli with small intestinal involvement 121

122 P. Romagnoli et al

Polyposis coli with small intestinal involvement 123

Table 1. Sites of biopsies, endoscopical aspect of the mucosa and respective relevant histological and histochemical findings

Endoscopical aspect at the site of biopsy

Site of biopsy

Duodenum (54 biopsies) Ileum (50 biopsies)

Polyps Velvet-like Normal Polyps Normal mucosa mucosa mucosa

Atypia ~ 8 2 2 5 3

Apical PAS + granules 4 2 10 4 4 in columnar cells of normal areas a

Unusually numerous 1 0 0 3 3 goblet cells

No abnormality 0 0 27 4 31

The mucosa was defined normal or atypic on the basis of haematoxylin and eosin stained sections. In areas of atypia, columnar cells were tall and crowded, with elongated nuclei; sometimes they were stratified and lacking a brush border. Goblet cells were less frequent than normal

complete absence of goblet cells were not recognized in any of the sections examined.

Histological evidence of atypia was not found in the ileal biopsies of four patients with polyp-like mucosa at endoscopy. In these biopsies the polyp-like appearance was caused by heavy infiltration of the lamina propria by lymphoid cells.

The histochemical features of the areas of atypia were as follows. Most columnar cells contained small, scanty apical granules reactive with P.A.S. and diastase-resistant, unstained by alcian blue at any pH. These granules seemed to be more abundant in the gland epithelium than surface epithelium and in the areas of grade I atypia than in those of grade II atypia (Figs. 5 and 6). The epithelial cells of the areas of atypia were unreactive for alkaline

Fig. 7. Glands from an area of duodenal mucosa, which appeared normal in sections stained with haematoxylin and eosin. Columnar cells contain PAS-positive granules in the supranuclear cytoplasm; in some cells (arrows) these granules appear localized to the Golgi region. PAS and haematoxylin x 700

Fig. 8. Same biopsy as Fig. 7. The apical granules of columnar cells are diastase resistant. Diastase - PAS - haematoxylin, • 700

Fig. 9. Area of ileal mucosa with goblet cell hyperplasia. Alcian blue pH 2.6, no counterstain x 270

Fig. 10. Same area of Fig. 9, from a serial section of the specimen. The brush border has an intense alkaline phosphatase activity. Alkaline phosphatase histochemistry, no counterstain x 270

124 P. Romagnoli et al.

Table 2. Number of biopsies and areas of atypia in and outside polyps respectively

a Four of these biopsies were on velvet- like mucosa

b Two of these biopsies were on velvet- like mucosa

Duodenum Ileum

Number of biopsies 11 9 on polyps Number of biopsies 43 a 41 outside polyps Areas of atypia 8 5 in polyps Areas of atypia 4 b 3 outside polyps

Table 3. Relevant histochemical findings in areas of atypia and in areas normal when stained with haematoxylin and eosin

Brush border of columnar and Apical granules Secretory goblet cells of columnar granules of

cells goblet cells Areas of Normal atypia" areas Areas of atypia Areas of atypia

and normal and normal areas areas

P.A.S. + + + + Diastase-P.A.S. + + + + Alcian blue, pH 2.6 + / - + / - - + Alcian blue, pH i.0 . . . . b Alkaline phosphatase - + - -

a The brush border was absent from areas with grade II atypia u The secretory granules of ileal goblet cells were lightly stained by alcian blue at pH 1.0.

Occasionally, this was true also for duodenal goblet cells

phosphatase, even when a brush border was identified in sections stained with haematoxylin and eosin and P.A.S. Goblet cells were always stained by P.A.S. and alcian blue at pH 2.6. They were also very lightly stained at pH 1.0 in the ileum and, occasionally, in the duodenum.

Apical granules which were P.A.S.-reactive, diastase-resistant and un- stained by alcian blue at any pH (Figs. 7 and 8), were found in the columnar cells of the glands of some areas, which appeared normal in sections stained with haematoxylin and eosin; these granules were found in the duodenum of 11 patients (all those with areas of atypia in the duodenum and in five other ones) and in the ileum of seven of the same patients (two with areas of atypia in the ileum and five others).

Areas of the surface epithelium and outer portion of glands with an unusually high density of goblet cells were found in the duodenal biopsy of one patient and in the ileal biopsies of five patients. In the duodenal and two of the ileal biopsies, these areas were adjacent to areas of atypia. All the goblet cells in these areas were stained by alcian blue at pH 2.6 (Fig. 9) and a few of them also at pH 1.0. Alkaline phosphatase activity

Polyposis coli with small intestinal involvement 125

was demonstrated in the brush border between the goblet cell stomata (Fig. 10). Paneth cells were regularly found in the bottom of the glands of these areas.

No alterations of the surface or gland epithelium were detected in the duodenal mucosa of two patients and in the ileal mucosa of 12 patients.

Details of the histological and histochemical findings in the biopsies under study are given in Tables 1, 2 and 3.

Controls of the histochemical reactions

The sections methylated before staining with alcian blue were completely unstained by this dye at any pH.

No reaction product was seen in the brush border, or elsewhere in the tissue, in the sections incubated for alkaline phosphatase without the sub- strate.

Discussion

This report provides a detailed description of the histochemistry of duodenal and ileal areas of atypia in patients with familial polyposis coli. Moreover, a peculiar histochemical alteration was found in areas of the duodenal and ileal mucosa, appearing normal when stained by haematoxylin and eosin. This was characterized by the presence of P.A.S.-positive apical granules in the columnar cells of the glands. No sign of atypia and an absence of apical P.A.S.-positive granules from columnar cells were found in the ileal mucosa of about half of the patients under study, whereas in the duode- nal mucosa this was true only for a minority of the patients who had under- gone biopsy of the duodenum.

Two grades of atypia were found in this study. Grade I atypia was characterized by the presence of crowded columnar cells with elongated nuclei, containing P.A.S.-positive, diastase-resistant, alcian blue-unstained apical granules (presumably neutral mucins) and lacking alkaline phospha- tase activity in the brush border; in these areas, goblet cells were slightly less frequent than normal. These areas have features resembling those of grade I atypia of the colon. Grade II atypia was characterized by the pres- ence of pseudo- or pluristratified columnar cells, sometimes with scanty apical granules and devoid of a brush border, and goblet cells were much less frequent than normal. Except for the presence of some goblet cells, the latter areas resemble those indicated as grade II atypia in the colon, which are considered to be prone to evolution to grade III atypia (character- ized by large, polymorphic nuclei and lack of mucus secretion) and later to malignancy (Lane et al. 1971; Gabbert and H6hn 1980). Intermediate features between grade I and grade II atypia were also found.

A grading of atypia among the different biopsies was impossible, since different grades were found close to each other in all the specimens contain- ing areas of atypia. This suggests the possibility of a progression from grade I to grade II atypia, particularly since gradations between grades

126 P. Romagnoli et al.

I and II were also present. Transition to grade III atypia might be suspected to be the further step, but areas with grade III atypia were not found in this study. Considering the premalignant significance given in the litera- ture to grade III atypia (Lane et al. 1971), the findings in this research could be considered consistent with the lower incidence of small intestinal compared with large intestinal cancer in the patients with familial polyposis coli.

Secretion of mucins by the columnar cells of non-familial adenomatous polyps has been described in the colon, although these mucins have not been characterized thoroughly by histochemical methods (Filipe et al. 1980). In the stomach, adenomatous polyps contain columnar cells resembling those described here and secreting small amounts of mucins; the types of these mucins vary from case to case, often comprising sialo- and sulphomu- cins as well as neutral mucins (Jass and Filipe 1979; Romagnoli 1985). In the biopsies of the small intestine reported here, columnar cells in areas of atypia contained only granules with the features of neutral mucins, while the scanty goblet cells in the same areas, and those of apparently normal mucosa, also contained sialo- and (in the distal ileum) sulphomucins. There- fore the types of mucins secreted by the columnar cells of adenomatous polyps or the so-called incomplete type of intestinal metaplasia of gastric mucosa (Jass and Filipe 1979; Romagnoli 1985; Filipe etal. 1985) and those secreted by the columnar cells in areas of atypia of the small intestine found in this research differ from each other, but are always abnormal when compared with the mucins demonstrated in the normal areas of the same organs. Several attempts have been made to correlate the pattern of mucin secretion with early stages of evolution towards malignancy (Filipe et al. 1980; Matsukura et al. 1980; Jass and Filipe 1981; Arslan Pagnini et al. 1983), but have not yet been widely validated (Gad 1979; Dawson 1981 ; Grigioni et al. 1983). The significance of the pattern of mucin secretion might be better understood if considered together with all the other histolog- ical and histochemical features of the mucosa. In the cases reported here, as well as in those with abnormal mucus secretion by gastric (Jass and Filipe 1979; Filipe et al. 1985; Romagnoli 1985) or large intestinal mucosa (Filipe et al. 1980), altered patterns of mucin secretion were associated with other nuclear and cytoplasmic features (crowding of the cells, stratification and elongation of nuclei, secretion of mucins by columnar cells, lack of detectable alkaline phosphatase in the brush border), indicating that muco- sal cells have features not corresponding to any normal cell type and are therefore "neoplastic" as defined by Riddell et al. (1983).

The modifications of the type of mucins secreted by goblet cells, on the contrary, may well be a reactive phenomenon to several conditions; this has been studied especially in the large intestine, where areas of so-called transitional mucosa (Filipe and Branfoot 1974) are a common feature not only in patients with neoplasia, but also with conditions neither neoplastic nor prone to malignancy (Listinsky and Riddell 1981). In these instances, however, mucins are secreted only by goblet cells and not by intervening columnar cells.

A peculiar alteration was detected by histochemical methods in apparent-

Polyposis coli with small intestinal involvement 127

ly normal (when stained with haematoxylin and eosin) areas of all but one patient with areas of atypia, and in three other patients. This alteration was characterized by the presence of small granules in the apical cytoplasm of columnar cells of some glands. These granules had the same histochemical characters as those found in the columnar cells of areas of atypia and, since they were unstained by alcian blue, were different from those of adja- cent goblet cells. These granules might therefore be the first sign of the onset of grade I atypia.

In some specimens there was an unusually high density of goblet cells in the mucosal surface and outer portion of glands. These goblet cells had a typical morphology and secreted mucins with the histochemical character- istics of normal small intestinal mucosa. This feature does not seem to represent a condition preceding and leading to atypia, in view of the reduc- tion in number of goblet cells in areas of atypia and of the normal appear- ance of the nuclei, alkaline phosphatase activity and absence of secretion in intervening columnar cells in areas of goblet cell crowding. A reactive origin of goblet cell hyperplasia has already been described in the human ileum (Bechi et al. 1981). These areas cannot be considered as the expression of "colonic" metaplasia, since villi remained detectable on the surface of the mucosa, Paneth cells were found in the depth of the glands and the histochemical features of goblet cell secretion were small rather than large intestinal in type.

Areas of atypia were also found in the absence of polyps at endoscopy and gross examination; two such areas were found in velvet-like duodenal mucosa, which should, therefore, be considered a possible step in the devel- opment of polyps. This finding suggests that endoscopical examination should be performed repeatedly and multiple biopsies taken at random from the small intestine of these patients.

Acknowledgements. The authors thank Prof. E. Reale (Hanover Medical School, Hanover, Germany) for discussion of the manuscript. The authors also acknowledge the skillful assistance of the Technicians of the Institute of Histology and General Embryology of the University of Florence (Italy). P. Romagnoli completed this research while he was a guest researcher of the Foundation Alexander von Humboldt in the Laboratory of Cell Biology and Electron Microscopy of Hanover Medical School (FRG). Research funds were received by Regione Toscana.

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Received February 3 / Accepted July, 1986