Primary endocrine carcinoma the parotid salivary ...With the Grimelius method the majority of the cells appeared impregnated, to varyingdegrees, with the exception of the duct-lining

J Clin Pathol 1982;35:611-616

Primary endocrine carcinoma of the parotid salivarygland associated with a lung carcinoid: a possible newassociationV EUSEBI,* S PILERI,* L USELLINI,t A GRASSIGLI,t C CAPELLAt

From the *Istituto di Anatomia e Istologia Patologica, Universitai di Bologna, the tCentro di DiagnosticaIstopatologica, Istochimica ed Ultrastrutturale, Universitai di Pavia a Varese, and the tClinica Chirurgica e

Terapia Chirurgica Generale, Universita di Bologna, Italy

SUMMARY An endocrine carcinoma of the left parotid salivary gland in a 58-year-old woman isreported. The tumour displayed a large argyrophilic cell-component and at ultrastructural levelendocrine-like granules (ELG) were evident.As endocrine-paracrine cells are not normally present in the parotid, it is suggested that the

endocrine elements may have been derived from an anomalous differentiation of the ductal epithelialstem cells.A bronchial carcinoid, removed seven years previously, proved structurally, cytologically and

histochemically different from the tumour of the parotid salivary gland. It is proposed that theoccurrence of the two tumours might be an as yet undescribed association which is more thanfortuitous.

Endocrine tumours of the parotid salivary gland arevery rare. Nicod' in 1958 described a typical argen-taffin carcinoid of the left parotid gland, in a 51-year-old woman. In 1963 an "undifferentiated"carcinoma of the parotid gland associated withCushing's syndrome was reported;2 Koss et at3described 14 cases of small cell (oat-cell) carcinomaof minor salivary gland origin; and lastly one caseof neuroblastoma of the parotid gland, barelydistinguishable from an undifferentiated carcinoma,has been described by Mackay et al in 1976.4Here we report a case of an endocrine carcinoma

of the parotid salivary gland, also studied ultra-structurally.

Case report

A 58-year-old female was admitted in November1979 with a six-month history of a painful lump inthe left parotid region. On physical examination afirm mass, fixed to the deep tissues, was found in theupper pole of the parotid gland. After excision thenodule was oval in shape and measured 1-5 cm.across. It was firm in consistency, appeared greyishin colour with numerous small haemorrhagic foci on

Accepted for publication 11 November 1981

the cut surface and was surrounded by salivary glandtissue.The past medical history of the patient revealed

that seven years previously she underwent, inanother hospital, a lower right lobectomy for apulmonary "carcinoma" of a large bronchus. Thepatient had remained in good health until January1979 when a subcutaneous nodule 1 cm across wasexcised from the right axilla. It was not in a lymphnode and was regarded as a metastatic carcinomaconsistent with mammary origin. Subsequently aright modified radical mastectomy was performedbut no malignant changes could be found within thebreast. At the time of writing the patient is alive andwell 14 months after excision of the parotid noduleand no further signs of tumour recurrence haveappeared. The radioimmunoassay for calcitoninperformed a month after the parotid operationproved negative.

Material and methods

Sections from the tumour of the parotid glandtogether with the subcutaneous nodule and thepulmonary lesion were stained with haematoxylinand eosin (HE), alcian blue (AB) method at pH 2 5,periodic acid-Schiff (PAS) before and after diastase

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Eusebi, Pileri, Usellini, Grassigli, Capella

digestion, the Gomori method for reticulin, theGrimelius method for argyrophilic cells, the Masson-Fontana method for argentaffin cells, and the Congored stain for amyloid.An immunoperoxidase method5 using anti-actin

antiserum (kindly supplied by Prof G Bussolati),antihuman ACTH antiserum (Wellcome), anti-calcitonin antiserum (kindly supplied by Prof ESolcia and JM Polak), anti-5-hydroxytryptamineantiserum (provided by Dr R Buffa), antigastrinantiserum (provided by Prof E Solcia and Dr JWalsh), antiglucagon antiserum 4842 (provided byProf E Solcia and Dr VLW Go), antibovine pan-creatic peptide (BPP) antiserum (Lilly) and anti-met-enkephalin antiserum (provided by ProfE Solcia andDr JM Polak) were applied to the tumour of theparotid gland.For electron microscopy, small blocks from the

parotid nodule fixed in Carson's solution6 werepostfixed in 1% osmium tetroxide, dehydrated andembedded in an Epon-Araldite mixture. Thin sectionswere stained with uranyl acetate and lead citrate andobserved in a Zeiss EM 10 electron microscope.

MORPHOLOGY

Tumour of the parotid glandThe tumour was surrounded by a thick fibrouscapsule which separated it from normal parotidtissue. By studying serial sections, it was possible toestablish that the tumour was not in an intraparotidor paraparotid lymph node. The neoplastic cells wereclumped in nests, or in large sheets frequentlyseparated by an amorphous hyaline material whichstained faintly with Congo red. However, no greenbirefringence was observed by polarising micro-scopy. The neoplastic elements were round or spindlein shape displaying a finely eosinophilic granularcytoplasm. The overall superficial appearance of theneoplasm somewhat resembled that of a medullarycarcinoma of the thyroid (Fig. 1). The nuclei variedfrom round to ovoid and exhibited prominenteosinophilic nucleoli. Mitoses were scanty.

Frequently, ductal structures were scatteredthrough the tumour either isolated singly in thestroma or within the solid areas (Fig. 2). The cellslining ducts were cuboidal but occasionally ap-peared flattened (Fig. 3). Their round nuclei werecentrally placed and displayed small nucleoli. Theductal lumina frequently contained eosinophilicPAS-positive material.With the Grimelius method the majority of the

cells appeared impregnated, to varying degrees, withthe exception of the duct-lining cells (Fig. 4). Theargyrophilic cells sometimes appeared singly placedat the base or wedged between the non-argyrophilic

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Fig. 3 A glandular space lined by flattened epithelium isevident in the centre of this neoplastic area.Haematoxylin and eosin x 175.

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Fig. 4 The Grimelius-positive elements contrast with thecuboidal cells of the ducts which appear unstained. x 175.

Fig. 6 Electron micrograph of a spindle cell areashowing elongated endocrine-like cells containingmoderate numbers of cytoplasmic dense-core granules.x 5600.

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Fig. 5 Argyrophilic cells appear wedged between thenon-argyrophilic elements of this duct. Grimelius x 200.

elements of the ducts (Fig. 5). No Grimelius-positivecells could be found in the normal salivary tissue,nor did the tumour cells stain with the Masson-Fontana reaction.With the immunoperoxidase methods tumour

cells were negative with all the antisera used. Onlyscattered actin-positive elements were seen borderingthe ducts of the normal parotid tissue.On electron microscopic examination the neo-

plastic cells were arranged in nests and sheets oraround ductal structures which contained secretorymaterial within their lumina. The nests and sheetswere composed of electron-translucent medium-sized polygonal or fusiform cells with ovoid or roundnuclei (Fig. 6). The cell membranes were distinct anddesmosome-like attachments were occasionally ob-served between adjacent cells. In about 70% of these

cells endocrine-like granules (ELG) were seen. Theirnumber varied from scanty in some cells to numerousin others. The granules were small (averaging 145 nmin diameter) and appeared uniform in size (Fig. 7).They displayed a dense core and a thin electron-lucid halo below the limiting membrane. The ELGwere mostly concentrated at the stromal pole of thecells. The granular endoplasmic reticulum com-ponent was variable in amount but usually not veryprominent.The cells forming the ductal structures varied from

columnar to cuboidal and displayed numerousmicrovilli on their luminal surface. In the cytoplasmnumerous large, irregularly outlined dense secretorygranules were located immediately below the luminalsurface. These granules closely resembled thosefound in the cells lining the normal human salivarygland ducts.7 Clumps of endocrine-like cells wereinterposed between the exocrine elements and anevident basal lamina (Fig. 8).

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Fig. 7 Densely granulated endocrine-like cell containingnumerous round osmiophilic granules with a thin haloseparating the membrane from the core. x 28 000.

In addition, a population of rather small round orovoid cells with scarce cytoplasm containingabundant ribosomes, few mitochondria and oc-casional filaments, was observed. These cells, sparsein number and mostly located at the periphery ofthe ducts, were interpreted as immature ductal cells(stem cells). The stroma was abundant and containedfibroblasts, bundles of collagen and a dense networkof irregularly orientated fibrils of varying thickness,which appeared very similar to the fibrils found inthe adenoid cystic carcinomas of salivary gland8 andin a gastric carcinoid recently described by Wilanderet al.9

Skin noduleThe skin nodule appeared structurally and cyto-logically identical to the tumour of the parotidgland, but no ductal structures were evident (Fig. 9).The Grimelius method was positive in most of thecells (Fig. 10). In the stroma amorphous eosinophilicmaterial was also present which stained weakly withCongo red, but no dichroism could be seen.

Tumour of the lungThe pulmonary tumour slides were reviewed. Theneoplastic proliferation consisted of solid alveolar

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Fig. 8 Duct lined exocrine cells showing microvilli andlarge irregularly outlined, apical granules. These cells aredirectly surrounded by endocrine-like cells lailing on aductal basal lamina. x 5600.

structures composed of uniform polygonal cellsseparated by thin fibrovascular septa. No ductal orglandular structures were observed. The neoplasticcells were small and displayed a clear or faintlyacidophilic cytoplasm. The nuclei were round with-out nucleoli (Fig. 11). No cells appeared impregnatedby the Grimelius method nor could AB or PAS-positive material be seen. The stroma was negativewith Congo red staining.

In view of these histological features the tumourwas regarded as a pulmonary carcinoid with para-ganglioid features.10The two hilar lymph nodes available appeared

reactive,

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Endocrine carcinoma of the parotid salivary gland

Fig. 11 The "paraganglioid" structure of the tumour ofthe lung is well evident. Haematoxylin and eosin x 75.

Fig. 9 The neoplastic elements of the axillarymetastatic nodule appear spindle in shape as in theprimary tumour. Haematoxylin and eosin x 100.

Fig. 10 Grimelius positive cells in the axillarymetastatic tumour. x 175.

Discussion

The tumour of the parotid gland was composed ofround to spindle-shaped argyrophilic elements con-taining small dense-core granules at ultrastructurallevel.Although the neoplastic cells were not positive

with any of the antihormone sera used, the histo-

chemical and ultrastructural data are consistent withthe tumour cells having an endocrine nature; theirgranules being similar to those of P cells normallypresent in the human bronchial and gastric mucosa.11

Nests and sheets of argyrophil cells were in directcontact with ductal structures lined by exocrine cellswhich were ultrastructurally similar to those de-scribed in normal salivary gland ducts.7 Furthermorethe endocrine-paracrine elements and the ductal cellswere so often contained within the same basementmembrane that a primary ductal origin seems verylikely.The pulmonary carcinoid exhibited a para-

ganglioid structure, with small non-argyrophil cellsand scanty Congo red-negative stroma. It wouldappear therefore that the two tumours are structur-ally, cytologically and histochemically sufficientlydifferent to suggest that they represent two indepen-dent endocrine tumours developing in the samepatient.

Multiple endocrine adenomatosis is a well knownsyndrome with ever increasing associations, beingdescribed.'2 13 It seems possible that the occurrenceof a pulmonary carcinoid together with an endocrine-paracrine tumour of the parotid gland mightrepresent yet another unrecognised example ofmultiple endocrine tumours.The skin nodule was practically synchronous with

the tumour of the parotid gland, appearing bothstructurally and cytologically very similar exceptthat it lacked residual glandular structures. The cellswere also argyrophilic and hyaline Congo red-positive material was seen in the stroma. Thereforeit seems justifiable to regard it as a probable met-astasis from the tumour of the parotid gland ratherthan as secondary from the pulmonary carcinoidremoved seven years previously.

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Moreover, in view of the aggressive behaviour ofthe parotid tumour as well as its structure andcytochemistry, we think that the designation ofendocrine carcinoma is preferable to that ofcarcinoid.The origin of the endocrine-paracrine tumours of

the parotid gland is open to question. Carcinoids andendocrine carcinomas are generally believed to arisefrom cells of the diffuse endocrine system (DES) ofFeyrter'4 or APUD system of Pearse'5 which are

diffusely distributed in the digestive, respiratory andurinary tract. Argentaffin cells have been observed indog submandibular glands'6 and somatostatin-immunoreactive cells have more recently beendetected in monkey submandibular glands.'7 Todate, no cells belonging to the DES have beenidentified in normal human salivary gland, andtherefore the theory of origin from DES cells cannotyet be applied to parotid endocrine tumours.An alternative hypothesis, elaborated elsewhere,'8

is that the endocrine cells may represent an

anomalous second line of differentiation derivedfrom ductal stem cells. This view is supported by thecoexistence, in the parotid tumour studied, ofargyrophil cells, exocrine ductal cells as well as ofundifferentiated elements which probably representstem cells.We think that the existence, albeit rare, of an

endocrine carcinoma of the parotid salivary glandhas to be taken into consideration and such a tumourdistinguished from the spindle-cell (myoepithelial)carcinoma19 by the Grimelius method, electron-microscopy and immunohistochemistry employinganti-actin antisera.

The authors wish to thank Prof JG Azzopardi forconfirming the diagnosis. Acknowledgement is alsodue to Prof R Lattes for critically reading themanuscript.

References

Nicod JL. Carcinoide de la parotide. Bull Assoc FrancL'Etude Cancer 1958;45:214-22.

2 Clinicopathological Conference. Cushing's syndromeassociated with a parotid gland tumour. Am J Med 1963;34:394-406.


Koss LG, Spiro RH, Hajdu S. Small cell (oat cell)carcinoma of minor salivary gland origin. Cancer 1972;30:737-41.

'Mackay B, Luna MA, Butler JJ. Adult neuroblastoma.Electron microscopic observation in nine cases. Cancer1976;37:1334-51.

Sternberger LA. Immunocytochemistry. 2nd ed. New York:John Wiley, 1979.

6 Carson FL, Martin JH, Lynn JA. Formalin fixation forelectron microscopy: a re-evaluation. Am J Clin Pathol1973 ;49:365-73.

7 Tandler B. Ultrastructure of chronically inflamed humansubmandibular glands. Arch Pathol Lab Med 1977 ;101:425-9.

Tandler B. Ultrastructure of adenoid cystic carcinoma ofsalivary gland origin. Lab Invest 1971 ;24:504-12.

9 Wilander E, Westermark P, Grimelius L. Intracellular andextracellular fibrillar structures in gastroduodenalendocrine tumours. Ultrastruct Pathol 1980;1 :49-54.

10 Capella C, Gabrielli M, Polak JM, Buffa R, Solcia E,Bordi C. Ultrastructural and histological study of 11bronchial carcinoids. Virchows Archiv [Pathol Anat]1979 ;381 :313-29.

Solcia E, Capella C, Buffa R, Usellini L, Frigerio B,Fontana P. Endocrine cells of the gastrointestinal tractand related tumours. In: Ioachim H. Pathobiology annual.Vol 9. New York: Raven Press, 1979:163-204.

12 Williams ED, Celestin IR. The association of bronchialcarcinoid and pluriglandular adenomatosis. Thorax1967;17:120-7.

13 Newsome HH. Multiple endocrine adenomatosis. SurgClin North Am 1974;54:387-93.

4 Feyrter F. Uber die peripheren endokrinen (parakrinen)Drusen des Menschen. Wien: Mandrich, 1953.

5, Pearse AGE. The cytochemistry and ultrastructure ofpolypeptide hormone producing cells of the APUDseries and the embryologic, physiologic, and pathologicimplications of the concept. J Histochem Cytochem 1969;17:303-13.

16 Godlowski ZZ, Calandra YC. Argentaffin cells in thesubmaxillary glands of dogs. Anat Record 1961 ;100:45-7.

17 Girod C, Dubois MP, Durand N. Immunocytochemicalevidence for the presence of somatostatin-like immuno-reactivity in scattered cells of the duct system of thesubmandibular glands in the monkey macaca irus.Histochemistry 1980;69 :137-43.

18 Eusebi V, Azzopardi JG. Lobular endocrine neoplasia infibroadenoma of the breast. Histopathology 1980 ;4:413-28.

19 Hamperl H. The myothelial (myoepithelial cells). Normalstate; regressive changes; hyperplasia; tumours. CurrTop Pathol 1970;53:161-220.

Requests for reprints to: Dr V Eusebi, Istituto diAnatomia e Istologica Patologica, Policlinico S Orsola,Via Massarenti 9, 40100 Bologna, Italy.



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Primary endocrine carcinoma the parotid salivary ...With the Grimelius method the majority of the cells appeared impregnated, to varyingdegrees, with the exception of the duct-lining