secretory andneoplastic mucosa - journal of clinical...

J Clin Pathol 1990;43:901-904

Immunohistochemical demonstration ofpancreatic secretory trypsin inhibitor in normaland neoplastic colonic mucosa

H Bohe, M Bohe, C Lindstrom, K Ohlsson

AbstractSpecimens of normal and neoplasticcolonic mucosa from 52 patients wereanalysed by immunohistochemistryusing a monospecific polyclonal anti-serum against human pancreaticsecretory trypsin inhibitor (PSTI). Innormal colonic mucosa PSTI was foundin the goblet cells in the basal parts ofthe crypts. In adenomas of tubular,villous, and tubulo-villous types PSTIwas also found in the upper parts of thepolyps, usually occurring in the re-generation zone. There was a moreintense staining reaction in polyps withincreased atypia. Carcinomas of dif-ferent types and of various grades ofdifferentiation and of in situ type did notcontain PSTI.These findings indicate that PSTI

could be a marker for adenomatousrather than carcinomatous epithelium inthe colon. Furthermore, the absence ofthe inhibitor in malignant cells mightfacilitate tissue invasion by malignantcells because of deficient protease inhibi-tion.

Department ofSurgery, University ofLund, Malmo GeneralHospital,S-21401 Malmo,SwedenH BoheM BoheDepartment ofSurgicalPathophysiologyK OhlssonDepartment ofPathologyC LindstromCorrespondence to:Dr H H BoheAccepted for publication2 July 1990

Pancreatic secretory trypsin inhibitor (PSTI)was first described by Kazal as a product ofthe acinar cells of the pancreas.' The mainfunction of PSTI is believed to be that ofpreventing the autodigestion of the pancreaticgland by inhibiting preactivated trypsin. Anincreasing body of evidence has accumulatedwhich indicates that PSTI may be producedbeyond the pancreas.23 The presence ofimmunoreactive PSTI (irPSTI) has beenshown in and isolated from the normalgastrointestinal mucosa."5 In the stomachirPSTI is found in the foveolar cells of thegastric pits; in gastric metaplastic mucosa withintestinal metaplasia irPSTI is also found inPaneth cells6; in the small intestine andduodenum irPSTI is found in Paneth cells7and goblet cells7 8; in the colon irPSTI islocated in goblet cells.7 IrPSTI has also beenshown in colonic adenomas.7 The aim of thisstudy was to analyse normal and neoplasticcolorectal mucosa for the presence and local-isation of irPSTI.

MethodsSpecimens of surgically resected colonic

mucosa were obtained from 52 patientsundergoing surgery because of colonic car-cinoma or adenoma. The specimens were fixedin buffered 10% formalin and embedded inparaffin wax. They were then analysed usingthe peroxidase-antiperoxidase method (PAP)described by Sternberger et al,9 with somemodifications.'o The antiserum used was amonospecific polyclonal rabbit antiserumagainst human PSTI." The antiserum wasused in serial dilution: 1 in 2000 gave the beststaining reaction. Control staining was perfor-med by using the antiserum previously ab-sorbed by PSTI and by using non-immunerabbit serum instead of the specific antiserum.The following types of mucosa were analysed:colonic mucosa of the normal type (n = 10),colonic adenomas of the tubular type (n = 8),of the villous type (n= 6), of the tubulo-villous type (n= 6), colonic carcinomas of thein situ type or of intramucosal type (n = 10),well differentiated colonic carcinomas (n = 6),and poorly differentiated colonic carcinomas(n=6).The terms carcinoma in situ (basement

membrane unbroken) and intramucosalcarcinoma (intramucosally infiltrating car-cinoma) have been used to delineate thosetumours in early carcinomatous change with-out signs of infiltration through the muscularismucosae, but with pronounced cellular atypiaand cribriform structures in the tumour.

Swine anti-rabbit IgG, peroxidase-anti-peroxidase (PAP), and normal swine serumwere obtained from Dako Immunoglobulins,Copenhagen, Denmark. Human pancreaticsecretory trypsin inhibitor was purified fromhuman pancreatic juice."The staining reaction was completely

absent when the antiserum was absorbed byPSTI at higher concentrations than 1 ,ug/ml(of diluted antiserum 1 in 2000).

ResultsNormal colonic mucosa contained irPSTI inthe goblet cells in the basal parts of the crypts(fig 1). In adenomas irPSTI was also shown inthe upper part of the polyps corresponding tothe reversed mode of epithelial regenerationin adenomas. The staining reaction was oftenrelated to the grade of atypia, with a moreintense staining reaction in cases with morepronounced atypia (fig 2).

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Bohe, Bohe, Lindstrom, Ohlsson

Figure 1 Normal colonicmucosafrom the stalk of acolonic polyp. PSTI islocated in the epithelium ofthe basal parts of thetubular crypts.(Immunohistochemiealstaining using anantiserum against humanPSTI.)

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Colonic carcinomas of the in situ orintramucosal types did not contain irPSTI (figs3 and 4).

Poorly differentiated, infiltrating coloniccarcinomata did not contain irPSTI (fig 5). Intwo of the six specimens of well differentiatedcolonic carcinomas, there were small areas witha few cells with positive staining for irPSTI.

DiscussionThe findings of this study show that irPSTI is

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found not only in the goblet cells of normalcolonic mucosa but also in colonic adenomas.Negative controls using non-immune rabbitserum or antiserum previously absorbed byPSTI showed the specificity of the stainingreaction.

In normal colonic mucosa the cells contain-ing irPSTI were found predominantly in thebasal parts of the crypts. In the adenomas theywere also found in the upper parts of theadenomas. Thus the goblet cells containingirPSTI seemed to occur at the zone of re-

Figure 2 Part of atubular adenoma. Outerpart of the stalk in theright hand side of thepicture. There is a shift inthe PSTI contentfrom thebasal parts of the crypts inthe stalk with normalmucosa to the surface partsof the adenomatous glands.(Immunohistochemicalstaining using anantiserum against humanPSTI.)

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Immunohistochemical demonstration ofpancreatic secretory trypsin

Figure 3 Part of anadenomatous polyp with afocus of intramucosalcarcinoma. Theadenomatous part (lower)has PSTI staining whilethe carcinomatous part(upper) does not expressPSTI.

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generation. In normal colonic mucosa theepithelium regenerates from the basal parts ofthe tubular crypts while in adenomas there is areversed mode of epithelial regeneration local-ised in the luminal areas. The staining reactionalso seemed to be associated with the degree ofepithelial atypia, with an increased stainingreaction in polyps with more pronouncedatypia. On the other hand, cells containingirPSTI were not found in in situ carcinoma,intramucosal carcinoma, or in invasive car-cinomas, except for in a few cells in small areas

Figure 4 Detail offigure3. The borderline zone ofthe adenomatous polypwith afocus of earlyintramucosalcarcinomatous changewithout penetration of themuscularis mucosae. Thereis a distinct borderline(arrow) between benignadenomatous epitheliumwith a high content ofPSTI and thecarcinomatous epithelium(upper) in which there isno PSTI expression.(Immunohistochemicalstaining with an antiserumagainst human PSTI.)

in well differentiated carcinomas. This shiftfrom an intense staining reaction in adenomaswith a more pronounced atypia to an almostentire lack of irPSTI in carcinomas is difficultto explain. The apparent absence of theinhibitor could, however, facilitate tissue infil-tration by cancer cells-that is, the proteasesproduced by the cancer cells'2 can more easilydegrade surrounding tissue and thus facilitateinvasion. More studies, however, will berequired to establish the exact function ofPSTI.'k''+r^ 4.;0-"'St-* f

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Bohe, Bohe, Lindstrom, Ohlsson

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The nomenclature of early carcinomatouschanges in colorectal adenomas correspondingto the stages of carcinoma in situ andintramucosally infiltrating carcinoma varies.Some judge the intraepithelial (in situ) and theintramucosal stages to be carcinomatouschanges,""'5 but others classify them as

severely dysplastic.16 Our present findingswould rather support the opinion that theseearly stages are true carcinomatous changes.The pronounced shift from adenomatous

epithelium with a high PSTI content to car-cinomatous epithelium without PSTI, alreadyprogressed to carcinoma in situ, is of greatinterest both from a theoretical and a practicalpoint of view. In the future PSTI might be a

marker for the adenoma-carcinoma sequence inthe epithelium of the large bowel.

This study was supported by the Swedish Cancer Society (No1300-B89-04XA), the Medical Faculty, University of Lund,Albert Palsson's foundation, Agnes Nils Nilssons foundation,and the Foundation of Malmo General Hospital for Cancer.

1 Kazal LA, Spicer DS, Brahinsky RA. Isolation of a crys-talline trypsin inhibitor-anticoagulant protein from pan-creas. J Am Chem Soc 1948;70:3034-40.

2 Bohe M. Immunoreactive pancreatic secretory trypsininhibitor in sera from patients with acute ulcerative colitis.Res Exp Med 1987;187:55-8.

3 Matsuda K, Ogawa M, Murata A, Kitahara L, Kosaki G.Elevation of serum immunoreactive pancreatic secretorytrypsin inhibitor contents in various malignant diseases.Res Commun Chem Pathol Pharmacol 1983;40:301-5.

4 Bohe M, Lindstrom C, Ohlsson K. Immunoreactive pan-creatic secretory trypsin inhibitor in gastrointestinalmucosa. Adv Exp Med Biol 1989;240:101-5.

5 Shibata T, Ogawa M, Matsuda K, Miyauchi K, YamamotoT, Mori T. Purification and characterization of pancreaticsecretory trypsin inhibitor in human gastric mucosa. ClinChim Acta 1986;159:27-36.

6 Bohe M, Lindstrom CG, Ohlsson K. Varying occurrence ofgastroduodenal immunoreactive pancreatic secretorytrypsin inhibitor. J Clin Pathol 1987;40:1345-8.

7 Bohe M, Borgstrom A, Lindstrom C, Ohlsson K. Pancreaticendoproteases and pancreatic secretory trypsin inhibitorimmunoreactivity in human Paneth cells. J Clin Pathol1986;39:786-93.

8 Fukayama M, Hayashi Y, Koike M, Ogawa M, Kosaki G.Immunohistochemical localization ofpancreatic secretorytrypsin inhibitor in fetal and adult pancreatic and extra-pancreatic tissues. J Histochem Cytochem 1986;34:227-35.

9 Sternberger LA, Hardy Jr PH, Cuculis JJ, Meyer HG. Theunlabelled antibody enzyme method of immuno-chemistry. Preparations and properties of soluble antigen-antibody complex (horse-radish peroxidase-antihorse-radish peroxidase) and its use in identification of spiro-chetes. J Histochem Cytochem 1970;18:315-33.

10 Fryksmark U, Ohlsson K, Polling A, Tegner H. Distribu-tion of antileukoprotease and characterization of humanantileukoprotease in serum. Ann Otol Rhinol Laryngol1982;91:268-7 1.

11 Eddeland A, Ohlsson K. Purification and immunochemicalquantitation of human pancreatic secretory trypsininhibitor. Scand J Clin Lab Invest 1978;38:261-7.

12 Mullins DE, Rohrlich ST. The role ofproteinases in cellularinvasiveness. Biochim Biophys Acta 1983;695:177-214.

13 Cotran RS, Kumar V, Robbins SL, eds. In: Robbins'Pathologic Basis of Disease. 4th ed. Philadelphia: W BSaunders Co, 1989:894.

14 Lindstrom CG. Experimental colo-rectal tumours in the rat.A histopathologic study with special reference toimmunologic and radiographic aspects. (Munksgaard,Copenhagen.) Acta Pathol Microbiol Scand 1978 (suppl268) Section A:20-21.

15 Fenoglio-Preiser CM, Lantz PE, Listrom MB, et al. In:Gastrointestinal pathology. An atlas and text. New York:Raven Press, 1989:737-8.

16 Jass JR. In: Morson BC, ed. Systemic pathology. 3rd ed. Vol3: Alimentary tract. Edinburgh: Churchill Livingstone,1987:359.

Figure 5 Coloniccarcinoma infiltrating thesubserosalfat tissue. Theinfiltrating malignantglands with cribriformstructures, lacking PSTI,are clearly shown. Thedark areas around themalignant structuresconsist of condensed stromaand some surroundinginflammatory cells.

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