Path. Res. Pract, 178,555-561 (1984)

Combined Production of Mucus, Amines and Peptides byGoblet-Cell Carcinoids of the Appendix and Ileum

H. HoflerInstitute of Pathology, Univ. of Graz (Austria)

G. KloppelInstitute of Pathology, Univ. of Hamburg (FRG)

Ph. U. HeitzDepartment of Pathology, Univ. of Basel (Switzerland)

SUMMARY

Globlet-cell caranoids areparticular mucus-producing tumorscombining features of typi­cal carcinoids and adenocarcinomas. The immunoreactivity of five goblet-cell carcinoidsof the appendixand one tumor of the ileum for 5-hydroxytryptamine (S-RT, serotonin),glucagon, somatostatin, substance P (SP), neuron-specific enolase (NSE), lysozyme, secre­tory component (SC) and carcino-embryonic antigen (CEA) was compared with that ofthe mucosa of the appendix (n = 24) and ileum (n = 12), and of typical carcinoids(appendix: n = 10; ileum: n =3). The goblet-cell carcinoids wereconsistently lysozyme-,SC- and CEA-reactive and contained weakly NSE reactive endocrine cells, while typicalcarcinoids were lysozyme-, SC- and CEA-negative, but strongly NSE- reactive. Twogoblet-cell carcinoids wereglucagon-reactive, one displayed SP-reactivity, one malignanttumor was reactive to the a-chain of glycoprotein hormones; six of ten typical appendixcarcinoids wereSPreactive, aswerethe three typtcal ileumcarcinoids. Using the immune­gold technique combined with the alaan-blue reaction, the presence of 5-hydroxytrypta­mine (S-RT) and mucus was demonstrated within the same cell. These findings suggesthistogenetic differences between goblet-cell carcinoids and typical carcinoids; the formerare possibly derived from undifferentiated stem cells, whereas the latter probably arisefrom endocrine cells in the mucosal stroma.

Introduction

Mucus-producing solid tumors of the appendix andileum displaying some features of typical carcinoids havebeen designated synonymously mucinous carcinoids'r ",goblet-cell carcinoids'" and amphicrine carcinoids". Theyare characterized by the combined occurrence of endoc-

© 1984 by GustavFischerVerlag,Stuttgart

rine cells taking silver stains, and mucus-producing cells.In addition, using light- and electron-microscopy, the pres­ence of mixed endo-exocrine or amphicrine cells was re­ported': 12, 20, 21, 26.

It is important to distinguish goblet-cell carcinoids fromcarcinomas because of their less aggressive and malignantbiologic behavior".

0344-0338/84/0178-0555$3.50/0

556 . H. Hofler, G. Kleppe] and Ph. U. Heitz

The purpose of this study was to analyze goblet-cellcarcinoids by immunocytochemical methods; 1) to studythe peptide an d 5-hydroxytryptamine content of theendocrine component of goblet-cell carcinoids of theappendix and ileum, 2 ) to compare the peptide patternand the reactivity for the secretory component (sq ,lysozyme and carcinoembryonic antigen (CEA) in goblet­cell and typica l carcinoids, and the normal mucosa of theappendix and the ileum, 3) to determine whether mucusand secretory granules are present in the same tumor cell,and 4) to study the usefulness of the techniques for thedifferentiation of goblet-cell carcinoids from carcinomas.

M aterial and Methods

Five goblet-cell carcinoids of the appendix, ten typical car­cinoids of the appendix and three of the ileum, twenty-fourmucosal samples of normal appendices and twelve of the normalileum were examined after fixation in 10% liquid, buffered for­maldehyde or Bouin's solution. One ileal goblet-cell carcinoidwas quenched in melting isopentane at -160 °C, freeze-driedovernight at -40 °C in a thermoelectr ic freeze-dryer, then vapor­fixed with paraformaldehyde, p-benzoquinone and diethylpy­rocarbonate for 3 hours at +60 °C and embedded in paraffin . Allspecimens were stained as follows: HE, PAS, silver stainingaccording to Masson, Grimelius and Sevier-Munger combinedwith alcian-blue.

The unlabeled antibody-enzyme method (Sternberger, 1979)was used. All reagents were diluted with phosphate-bufferedsaline (PBS 0.5 M; Grube, 1980). The histochemical reaction forthe peroxidase was performed using 3',3-diaminobenzidine-tet­rahydrochloride (0.05% w/v) and hydrogen peroxide (0.01% v/v)in Tris buffer (0.05 M). The sections were then postfixed in aque­ous osmium tetroxide (1%). In some sections, the contrast of theproduct of the peroxidase reaction was enhanced by addition ofcobalt chloride (1%) and nickel ammonium sulfate (1%) to theincubation solution. (Adams, 1981). For the localization of SC,lysozyme, CEA and somatostatin, the sections were treated withprotease type VII (0.1%, Serva) for 30 minutes at 37 °C (Denk etal., 1977) prior to the first antibody.

The following primary antisera were used:

Antibody to working Origindilution

5-hydroxytryptamine 1:5000 Immunonuclear(5-HT; serotonin ) (USA)pancreatic glucagon, 1 : 10000 Novo (Denmark)porcinesubstance P, synthetic 1 : 2000 Seralab (FRG)(monoclonal), cloneNCI/34 HLcarcinoembryonic antigen 1 : 2000 Dako (Denmark)(CEA), humansecretory component (sq , 1 : 2000 Dako (Denmark)humanlysozyme (muramidase), 1: 2500 Dako (Denmark)humanvasoactive intestinal 1: 1250 Milab (Sweden)polypeptide, porcinesomatostatin, synthetic 1: 7500 Immunonuclear

(USA)pancreatic polypeptide 1 : 80000 Dr. R. E. Chance(BPP), bovine (Indianapolis, USA)human chorionic 1: 7500 National Institute ofgonadotropin, alpha child health andsubunit (alpha-hCG) human

development (USA)neuron-specificenolase 1 : 4000 Dr. P. Marangos,(rat), E.e. 4.2.1.11) National Institute

of Health (USA)

Small samples of three-goblet-cell carcinoids of the appendixwere dewaxed and reembedded in Epon 812. They were thenalternately cut into semithin (1 um) and thin sections (60 nm).The Epon was removed from the semithin sections with sodiumethoxide (Major et al., 1961; Lane and Europa, 1965) and incu­bated with anti-5-HT and anti-glucagon sera. 5-HT-Immuno­reactivity was localized on thin sections using the immunogoldtechnique (Gold-part icle size 20 nm, De Mey, 1983); the sectionswere then contrasted with uranyl acetate and lead citrate.

Table 1. Findings in normal mucosa and in typical carcinoids

silver FIF 5-HT SP GLU SOM PP VIP a-chain NSE SC LYS CEAstain

Normal appendix + + + + + + (+ ) + +mucosa (n = 24)Normal ileum + + + + + + (+) + + +mucosa (n = 12)Typical carcinoid 10/10 10/10 10/10 6/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 1/10of the appendix(n = 10)Typical carcinoid 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 3/3 0/3 0/3 3/3of the ileum(n = 3)

(+) weak reaktionAbbreviations: FIF: Formaldehyde-induced fluorescence; 5-hydroxytryptamin; SP: Substance P; GLU: Glucagon; SOM: Somatosta-tin; PP: Pancreatic polypeptide; VIP: Vasoactive intestinal polypeptide; a-chain: a-chain of glycoprotein hormones; NSE: Neuron-specific enolase; SC: Secretory component; LYS: Lysozyme; CEA: Carcino-embryonic antigen

Goblet-Cell Carcinoid . 557

Table 2. Goblet-cell carcinoids mvestigated

Age/Sex Localization Macroscopic Findings Therapy

appendix thickened wall, obliterated tipileum multicentric tumor, max. diameter 6 em, exulcerationappendix and ileum thickened wall of the appendix; tumor in terminal ileum, diameter

4 cm; metastases in mesenteric lymph nodes

8/812/8251/82

52/8255/8261/82

55 m58 m80 m

57 m66 f67 m

appendixappendixappendix

tumor at the tip, diameter 1 emthickened wall, induration of the mesenteriolumtumor at the np, diameter 2 em

appendectomyhemicolectomyresection of theterminal ileum,cecum and appendixappendectomyappendectomyappendectomy

ControlsThe following controls were carried out; 1) normal serum

instead of the first antibody, 2) preabsorption of the anti-5-HTserum with serotonin-creatinine sulfate (004 mM, Fluka), of anti­glucagon serum with synthetic glucagon (1 mM, Serva), of sub­stance P antibody with synthetic substance P (5 pM; Bachem), ofanti-CEA serum with CEA (100 nM; Hoffman-La Roche), ofanti-lysozyme serum with human lysozyme (1 0M; Worthington,USA), and 3) omission of hydrogen peroxide from the incubationmedium for the peroxidase reaction . Normal mucosa adjacent tothe tumors was used as positive control.

Results

The distribution pattern of endocrine cells containingpeptides and S-HT was found to be identical in the normalmucosa of the appendix and ileum, as reported previ­ously". The reactivity of SC and CEA was also similar,whereas lysozyme-reactivity was confined to Paneth cells,which are absent from the epithelium of the normalappendix. The findings in the mucosa (used as positivecontrols) and in the typical carcinoids of the appendix andileum are summarized in Table 1.

Goblet-cell carcinoids (Tables 2 and 3): All tumorsextended through the entire appendix or ileum wall. Themucosa was intact over all but one tumor, in which it wasulcerated. Typical tumor tissue consisting of individualcells and large cell groups was found in the mucosa, dis­placing the crypts from the muscularis mucosae. The bulkof tumor tissue was localized in the submucosa. In alltumors exocrine and endocrine cells were present , but

Table 3. Findings in goblet-cell carcinoids

mucus-producing, goblet-cell-like tumor cell predomi­nated. True glandular differentiation was exceptional.Cells resembling signet-ring cells were observed, especiallyin the mucosa (Fig. 1 to 3). All mucus-producing cellswerefound to be PAS- and alcian-blue-reacrive, Two tumorsformed large mucus pools in the submucosa and musclelayer, closely resembling mucinous adenocarcinomas.

The distribution of the endocrine cells within the tumorswas highly variable. In the mucosa, single endocrine cellswere often observed incorporated in the exocrine mucus­producing tumor tissue. They were located at theperiphery of tumor cell clusters, showed often long cellprocesses and looked as if they were "deformed" by themucus-producing tumor cells. Virtually pure endocrineareas resembling typical carcinoids were found in only onecase. Generally, a clear distinction could be made betweenendocrine and exocrine cells at light microscopy, but thepresence of transitional or mixed endo-/exocrine cellscould be proved only in semithin sections stained with acombination of the immunocytochemical reaction for 5­HT and alcian-blue. Round alcian-blue-positive inclusionscould be recognized in serotonin-reactive cells (Fig. 1).

All tumors showed silver-staining' and S-HT-reactivetumor cells (Fig. 1,). Two tumors contained glucagon­reactive cells (Fig. 2), one, SP-reactive cells, and a furthertumor, a -chain immunorectivity (Table 3). All goblet-cellcarcinoids displayed extensive SC-reactivity (Fig. 3),which was not limited to mucus-producing cells, but wasalso found in solid, non-silver-staining, alcian-blue andPAS-negative areas. Lysozyme-reactivity was found to behighly variable from one tumor to another and within thesame tumor (Fig. 4). CEA-reactivity was uniformly dis-

silver FIFstain

5-HT SP GLU SOM PP VIP a-chain NSE SC LYS CEA

8/81 + n.d. + (+) + +2/82 + + + + + (+) + + +

51/82 + + + + (+) + + +52/82 + + + (+) + + +55/82 + + + + (+) + (+) +61/82 + n.d. + (+) + + +

(+): weak reaction; n.d.: not done; abbreviations as in Table 1

558 H. Hofler, G. Kloppel and Ph. U. Heitz

Fig. 1. Goblet-cell carcinoidof the ap­pendix, 55/82. Immuno-cytochemicallocalization of 5-HT-reactivity intumor cells and epithelial endocrinecells (large arrow). Mucous globules(small arrows) may be seen in an (al­cian-blue positive) endocrine tumorcell. Semithin section, unlabeled anti­body enzyme method for5-HT, counterstainedwith alcian-blue.Differential interference contrast op­tics. X 360.

Fig. 2. Sametumor as Fig. 1 - Gluca­gon-Immunoreactivity in tumor cellslocalized in the muscle layer. X 360.

tributed in all tumors. SC-, lysozyme- and CEA-reactivitywas strictly limited to the exocrine tumor areas and wasnot found in endocrine cells of the normal mucosa.

Electron-microscopic FindingsPure endocrine or exocrine cells could clearly be found

in two tumors. In addition, several cells displaying both,endo- and exocrine features were observed. In these"mixed" cells, cytoplasmic secretory granules and mucusoccurred in variable amounts. Cells containing round tooval endocrine granules with a diameter of 200 to 400 nmwere found to be 5-HT-reactive (Fig. 6). In one tumor,cells with round granules (diameter up to 500 nm; Fig. 6,

inset) were found to be glucagon-immunoreactive at light­microscopy, but attempts to demonstrate glucagon at theultrastructural level were unsuccessful.

The control reactions 1-3 (see above) were consistentlynegative (Fig. 5).

Discussion

All goblet-cell carcinoids examined in this senesdisplayed the characteristics described previ­ously': 3, 15,21,24,25. In general, the tumors can be easilydifferentiated from typical carcinoids by their conspicuous

Fig. 3. Same case as in Figs. 1 and 2­SC-immunoreactivity in a largenumber of tumor cells in the mucosa.X 180.

Fig.4. Goblet-cell carcinoid of the ap­pendix, 44/82 Lysozyme-im­munoreactivity in tumor cells presentin the muscle layer.X 360.

Fig. 5. Same case as Figs. 1, 2 and 3 ­Control section: absence of im­munoreact ion product after preab­sorption of the diluted anti-serum to 5­HT with 5-HT-creatinine sulfate(0,4 mM). Differential interferencecontrast optics. X 320.

Goblet-Cell Carcinoid . 559

production of mucus, despite the occasional occurrence ofareas typical of carcinoids. It is considerably more difficultto distinguish goblet-cell carcinoids from adenocar­cinomas or goblet-cell carcinomas, which are rare in theileum and appendix. The conspicuous features of goblet­cell are 1) the isomorphous tumor structure, 2) the pres­ence of adenoid tumor cell goups in the intestinal wall, 3)the intact mucosa overlying the tumor, and 4) the presenceof endocrine cells.

The most important feature is the intricacy of exocrineand endocrine cells demonstrated by specific imrnuno­staining. Endocrine cells could be shown unequivocally inthis series by the visualization of a biogenic amine, of

peptides, and of neuron-specific enolase". In both goblet­cell and typical carcinoids, only peptides present in thenormal mucosa of the appendix and ileum could be found.The reaction for neuron-specific enolase was somewhatweaker in goblet-cell carcinoids than in typical carcinoids.

We were also able to show the presence of 5-HT andmucus within the same tumor cell. In addition, 5-HTimmunoreactivity could be localized to secretory granulesof a mucus-producing tumor cell at the ultrastructurallevel. This finding strongly suggests the occurrence ofmixed exo-endocrine cells (amphicrine cells) in goblet-cellcarcinoids. To date other workers were unable to demon­strate mixed cells in these tumors': 25. If our findings are

560 . H. Hofler, G. Kloppel and Ph. U. Heitz

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...~f'

.~,

I'

i ""

.. ':~;-: "..

I'<~"·....:Ji• 0 0

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Fig. 6. Same case as Figs. 1, 2, 3, and5. - Immunocytochemical localizationof serotonin in a mucus-producing(amphicrine) tumor cell. M = mucus.X 23250 . Inset - Endocrine cell withlarger secretory granules (im­munoreactive of glucagon in semi -thinsection), N = nucleus. Immunogoldtechnique for 5-HT, contrasted withlead citrate and uranyl acetate. x18 000.

confirmed by others, the presence of amphicrine cells mustbe considered as one important feature of goblet-cell car­cinoids.

On the basis of the heterogeneity of tumor cells, i.e. thepresence of exocrine cells containing, SC and lysozyme, ofendocrine and of mixed exo- endocrine cells, we suggestthat goblet-cell carcinoids are clearly different from typicalcarcinoids from the morphologic, and possibly from thehistogenetic point of view. We suggest goblet-cell car-

cinoids to be derived from undifferentiated stem cells("crypt-cell carcinomas",15). If so, endocrine, amphicrineand exocrine tumor cells represent various lines of differ­entiation. The differentiation of a stem cell was also shownto occur in non-tumorous tissue" ll, and in rat adenocar­cinemas".

Goblet-cell carcinoids are different from typical car­cinoids, which are probably derived from endocrine cellsin the mucosal stroma13, 22. This view appears to be sup-

ported by the differences of biologic behaviour betweenq'pical carcinoids, goblet-cell carcinoids and adenocar­cinomas.

Acknowledgements. We thank Prof. H. Mitschke, Saarbriicken(FRG) for providing us with two cases. We are also grateful toMrs. G. Hel1eis for technical assistance, Mrs. H. Schleich andC. Stockli for typing the manuscript and Mr. 1. Georgiev forphotomicrographs.

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Key words: Goblet-Cell Carcinoid - Mucocarcinoid - Amphicrine Cells - Neuropeptides - Immunocytochemistry

Doz. Dr. Heinz Hofler, Institute of Pathology, Auenbruggerplatz 25, A-8036 Graz, Austria