UMEÅ UNIVERSITY MEDICAL DISSERTATIONSNew Series No 179- ISSN 0346-6612

From the Departments o f Otorhinolaryngology - Head and Neck Surgery, Histology and Cell Biology, and Anatomy,

University o f Umeå, Umeå, Sweden

SALIVARY GLAND NEOPLASMSStudies on the cytoskeleton, the secretory apparatus and

the nuclear DNA content

by

Hans Gustafsson

Umeå 1986

SALIVARY GLAND NEOPLASMS Studies on the cytoskeleton, the secretory apparatus and the

nuclear DNA content

Akademisk avhandling som med vederbörligt t i l l s t å n d av Rektorsäm­bete t vid Umeå uni vers it é t för avläggande av medicine doktorsexamen kommer a t t offentligen försvaras i Rosa salen, Tandläkarhögskolan, 9 t r , fredagen den 12 december 1986, kl 09.00.

av

HANS GUSTAFSSON

UMEÅ 1986

UMEÅ UNIVERSITY MEDICAL DISSERTATIONSNew S e r ie s No 179 - ISSN 0346-6612

Salivary gland neoplasms. Studies on the cytoskeleton, the secretory apparatus and the nuclear DNA content.

Hans Gustafsson, Departments of Otorhinolaryngology - Head and Neck Surgery, Histology and Cell Biology, and Anatomy, University of Umea, S- 901 85 Umeå, Sweden

ABSTRACT

The heterogeneity of salivary gland neoplasms have made c la s s i f ic a t io n and prognostication of these tumours sometimes d i f f i c u l t , and the in ­troduction of techniques, such as enzyme and carbohydrate histochemis­try and electron microscopy have only to a certa in extent increased our knowledge in these respects . In the present study immunohistochemical methods have been used to identify intermediate filament proteins (IFP) in normal fe ta l and adult parotid glands, as well as in salivary neo­plasms. The intermediate filaments (IF) make up the cytoskeleton in eucaryotic c e l l s . Epithelia l t i s su e contains IF composed of d if fe ren t cytokeratins (CK 1-19) whilst mesenchymal t is su e generally contains IF composed of vimentin, and the IFP pattern is very stab le even during cell transformation. I t would thus be possible to fu r ther c la r i fy the histogenesis of sa livary neoplasms by identify ing IFP, in addition the IFP pattern would probably be useful in tumour typing. Furthermore, ul t ra s t ru c tu ra l cytochemical s tud ies , mi ero spec tor photometry on nuclear DNA as well as enzyme secretory studies of certa in tumour types were carried out, in order to fu r ther charac terize the biology of salivary neoplasms.The immunohistochemical investigations showed tha t in normal parotid t is su e , the d if fe ren t cell types d iffe red in IFP expression: acinar c e l l s express mainly CK 18 and myoepithelial c e l l s mainly CK 17 and 19, whilst duct c e l l s contained a broad range of CK. Vimentin could in ad­dition to CK be detected in myoepithelial c e l l s and basal c e l l s of ex­cretory ducts. Fetal parotid c e l l s showed a sim ilar CK pattern as mature duct c e l l s . In addition, vimentin could be found in some basal c e l l s of the terminal tubules of the fe ta l glands. Salivary neoplasms could be divided into three types with regard to th e i r IFP pattern:1. Acinic cell carcinomas showed a CK-pattern sim ilar to normal acinar c e l l s but a co-expression of CK and vimentin was present in some c e l l s .2. Adenoid cys tic carcinomas, mixed tumours and basal cell adenomas showed a CK-pattern of normal duct or myoepithelial c e l l s . The p e r i ­pheral c e l l s were also vimentin pos it ive . 3. Mucoepidermoid carcinomas and adenocarcinomas had a s im ilar CK-pattern as duct c e l l s , and no tu ­mour c e l l s contained vimentin. This indicates th a t typing of IFP may be useful for subgrouping of sa livary neoplasms.By stereological measurements, the c e l l s of acinic cell carcinomas were found to be very s im ilar to normal parotid acinar c e l l s . Furthermore, they contained amylase and a f te r stimulation by norepiphrine a secre­tory response was induced, with a r ise in in t ra c e l lu la r cAMP as well as a release of amylase. By single cell measurements of nuclear DNA con­te n t , no difference was found between acinic cell carcinomas with de­f in i t e metastasis and those without recurrence, both in paraffin sec­tions and cytological smears.

Key words: Salivary gland neoplasms, intermediate filaments, immuno- histochemistry, u l t ra s t ru c tu re , DNA, development, h is togenesis , progno­sis

UMEÂ UNIVERSITY MEDICAL DISSERTATIONSNew S e r ie s No 179 - ISSN 0346-6612

From the Departments of Otorhinolaryngology - Head and Neck Surgery, Histology and Cell Biology, and Anatomy,

University of Umeå, Umeå, Sweden

SALIVARY GLAND NEOPLASMS Studies on the cytoskeleton, the secretory apparatus and the

nuclear DNA content

by

HANS GUSTAFSSON

UMEÂ 1986

2

CONTENTS

ABBREVIATIONS ................................................................................................ 4

ABSTRACT ................................................................................................................... 5

ORIGINAL PAPERS .................................................................................................... 6

INTRODUCTION ............................................................. 7

C lass if ica t ion of sa livary neoplasms ................................................... 8S tructure of the salivary glands ........................................................... 10Tumour histogenesis ...................................................................................... 14New approaches in cell b io lo g y ................................................. 15

OBJECTIVES OF THE STUDY .................................................................................... 18

MATERIAL AND METHODS .......................................................................................... 19

Morphology (I-VI) .......................................................................................... 19Imraunohistochemistry (I-V) ........................................................................ 19Immunoblots (IV) ......................................................... 20Immunostaining (I-V) .................................................................................... 20Stereological methods (VI) ........................................................................ 20Studies on secretion and cyclic AMP accumulation (VI) ................. 21Amylase and cyclic AMP assay (VI) .................................. 21DNA measurement (VII) .................................................................................. 21

RESULTS 23

Cytoskeletal proteins in normal sa livary glands ( I-IV) ....................... 23Cytoskeleton of developing parotid glands (IV) ....................................... 23Cytoskeleton of benign sa livary neoplasms ( I , I I I , V) ......................... 23Cytoskeleton of malignant salivary neoplasms ( I I , I I I , V) ................. 24Morphometry and secretory behaviour of ACC (VI) ..................................... 27DNA levels in acinic cell carcinomas (VII) ........................ 27

3

DISCUSSION ............................................................................................................... 28

Intermediate filament proteins in normal salivary glands .......... 28Intermediate filaments in fe ta l parotid glands ................ 31Intermediate filaments in salivary gland neoplasms ................. 32Aspects of histogenesis as a basis for tumour t y p i n g .................. 36The stimulus-secretory coupling in acinic cell cancer ................. 37Measurement of DNA content and determination of long-term prognosis .......................................................................................................... 38

GENERAL SUMMARY AND CONCLUSIONS .................................................................... 40

ACKNOWLEDGEMENTS .............................................................................. 43

REFERENCES .............................................................................................................. 44

PAPERS I-VII

4

ABBREVIATIONS

PA - pleomorphic adenoma (mixed tumour)MA - monomorphic adenomaACC - acinic cell carcinomaMEC - mu coepidermoid carcinomaCPA - carcinoma in pleomorphic adenoma (carcinoma in mixed tumours)AdCC - adenoid cys tic carcinomaSDC - sa livary duct carcinomaAb(s) - antibody (antibodies)mAb(s) - monoclonal antibody (antibodies)pAb(s) - polyclonal antibody (antibodies)IF(s) - intermediate filament (filaments)IFP(s) - intermediate filament protein (proteins)CK(s) - cy tokera t in (s) , p rekera tin(s)kD - kiloDalton

5

ABSTRACT

The heterogeneity of sa livary gland neoplasms have made c la s s i f ic a t io n and prognostication of these tumours sometimes d i f f i c u l t , and the in ­troduction of techniques, such as enzyme and carbohydrate histochemis­try and electron microscopy have only to a ce rta in extent increased our knowledge in these respects . In the present study immunohistochemical methods have been used to iden tify intermediate filament proteins (IFP) in normal fe ta l and adu lt parotid glands, as well as in sa livary neo­plasms. The intermediate filaments (IF) make up the cytoskeleton in eucaryotic c e l l s . Epithelial t i s su e contains IF composed of d if fe ren t cytokeratins (CK 1-19) w hilst mesenchymal t is su e generally contains IF composed of vimentin, and the IFP pattern is very stab le even during cell transformation. I t would thus be possible to fu r the r c la r i fy the histogenesis of sa livary neoplasms by identify ing IFP; in addition the IFP pattern would probably be useful in tumour typing. Furthermore, u l t ra s t ru c tu ra l cytochemical s tud ies , microspectorphotometry on nuclear DNA as well as enzyme secretory studies of ce r ta in tumour types were ca rr ied out, in order to fu r th e r charac terize the biology of sa livary neoplasms.The immunohistochemical investigations showed th a t in normal parotid t i s su e , the d if fe ren t cell types d iffe red in IFP expression: acinar c e l l s express mainly CK 18 and myoepithelial c e l l s mainly CK 17 and 19, w hilst duct c e l l s contained a broad range of CK. Vimentin could in ad­dition to CK be detected in myoepithelial c e l l s and basal c e l l s of ex­cretory ducts. Fetal parotid c e l l s showed a sim ilar CK pattern as mature duct c e l l s . In addition , vimentin could be found in some basal c e l l s of the terminal tubules of the fe ta l gland. Salivary neoplasms could be divided into three types with regard to th e i r IFP pattern:1. Acinic cell carcinomas showed a CK-pattern sim ilar to normal acinar c e l l s but a co-expression of CK and vimentin was present in some c e l l s .2. Adenoid cystic carcinomas, mixed tumours and basal ce ll adenomas showed a CK-pattern of normal duct or myoepithelial c e l l s . The p e r i ­pheral c e l l s were also vimentin pos it ive . 3. Mucoepidermoid carcinomas and adenocarcinomas had a s im ilar CK-pattern as duct c e l l s , and no tu ­mour c e l l s contained vimentin. This indicates th a t typing of IFP may be useful for subgrouping of salivary neoplasms.By stereological measurements, the c e l l s of acinic cell carcinomas were found to be very s im ilar to normal parotid acinar c e l l s . Furthermore, they contained amylase and a f te r stimulation by norepiphrine a secre­tory response was induced, with a r i s e in in t r a c e l lu la r cAMP as well as a release of amylase. By single cell measurements of nuclear DNA con­te n t , no difference was found between acin ic cell carcinomas with de­f in i t e metastasis and those without recurrence, both in paraffin sec­tions and cytological smears.

Key words: Salivary gland neoplasms, Intermediate filam ents, immuno- histochemistry, u l t ra s t ru c tu re , DNA, development, h is togenesis , progno­s is

6

ORIGINAL PAPERS

This thes is is based upon the following publications, reference to which will be made by c i ta t io n of the appropriate Roman numerals.

I Gustafsson H, Kjörell U, Carlsöö B.Cytoskeletal proteins in oncocytic tumours of the parotid gland. Arch Otolaryngol 1985;111:99-105.

II Gustafsson H, Carlsöö B, Kjörell U, Thornell L-E. Immunohistochemical and u l t ra s t ru c tu ra l observations on adenoid cys t ic carcinoma of salivary glands with special reference to in­termediate filaments and proteoglycan p a r t ic le s .Acta Otolaryngol 1986;102:152-60.

III Gustafsson H, Carlsöö B, Kjörell U, Thornell L-E.U ltras truc tu ra l and immunohistochemical aspects of carcinoma in mixed tumours.Am J Otolaryngol 1986;7:218-30.

IV Gustafsson H, Kjörell U, Eriksson A, Virtanen I , Thornell L-E. Distribution of intermediate filament proteins in fe ta l and adult parotid glands in man.Submitted

Y Gustafsson H, Virtanen I , Thornell L-E.The intermediate filament protein pattern of salivary gland ca rc i­nomas as revealed with monoclonal antibodies.Submitted

VI Gustafsson H, Carlsöö B, Henriksson R.U ltras truc tu ra l morphology and secretory behaviour of acin ic cell carcinoma.Cancer 1985;55:1706-10.

YII Gustafsson H, Lindholm C, Carlsöö B.DNA cytophotometry of ac in ic cell carcinoma and i t s re la tion to prognosis.Acta Otolaryngol (in press) .

7

INTRODUCTION

Salivary gland neoplasms, a r is in g in major or minor salivary and mucous glands, co n s t i tu te a very heterogeneous group of tumours. For a long time, d ifferences in opinion with regard to the histogenesis of these lesions as well as the d i f f i c u l t i e s encountered in d i f fe re n t ia t in g malignant tumours from benign ones have prevented a universally applic­able c la s s i f ic a t io n .

Other d i f f i c u l t i e s in es tab lish ing such a c la s s i f ic a t io n are:1. These tumours are ra re ; the annual incidence for malignant neoplasms being only 1.0/100,000 inhabitants (Cancer Incidence in Sweden, 1958- 1981).

2. They show a wide his to logica l v a r ia b i l i ty , not only between d i f f e r ­ent tumours but also within one and the same tumour. A single neoplasm can simultaneously show d if fe ren t ia t io n of not only squamous and gland­ular c e l l s , but also of a varie ty of mesenchymal t is su e types.

3. The morphology and c l in ica l behaviour do not always co r re la te . Neo­plasms, such as acinic cell tumours - with c e l l s very akin to normal acinar c e l l s - or well d i f fe ren t ia ted mucoepidermoid tumours, may occa­sionally metastasize. Moreover, even in cases of "benign" mixed tu ­mours, secondary metastasis has been reported.

Foote & Frazell (1953) were the f i r s t to c la ss i fy a large ser ies of tu ­mours on the basis of th e i r biological behaviour, and th e i r work s t i l l forms the basis for c la s s i f ic a t io n of sa livary tumours. The works of for example, Eneroth (1964), Evans & Cruickshank (1970) and S e ife r t & Donath (1976), have fu r ther increased our knowledge. In 1972 the cur­ren tly most used c la s s i f ic a t io n was established (WHO, Thackray A Sobin) (Table I ) .

8

Table I . C lass if ica t ion of sa livary neoplasms (WHO 1972)

I , Epithelial tumours

A. Adenomas1. Pleoaorptilc adenowa (mixed tumour). A circumscribed tumour char­ac terized microscopically by i t s pleomorphic or "mixed" appearance; c lea r ly recognizable ep i th e l ia l t i s su e being intermingled with t is su e of mucoid, rrçyxoid, or chondroid appearance. (74%)*

2. Nonoaorphlc adénouas: The monomorphic adenomas are benign growths in which the epithelium forms a very regular, usually glandu­l a r , pattern and in which there is no evidence of the mesenchyme-like t i s su e th a t is so ch a ra c te r is t ic a component of the pleomorphic adeno­ma.

(<a) Adenolymphoma: A tumour composed of glandular and often cystic s t ru c tu re s , sometimes with pap il la ry cystic arrangement, l ined by ch a ra c te r is t ic eosinophilic epithelium. The stroma contains a v a r i ­able amount of lymphoid t i s su e with f o l l i c l e s . (4.1%)*

(b) Oxyphilic adenoma: A benign tumour consisting of large c e l l s with granular eosinophilic cytoplasm (oncocytes). There is very l i t t l e stroma, and although occasionally lymphocytes may be present, lymphoid f o l l i c l e s are ra re ly seen, in con trast to adenolymphoma. (0.9%)*

(£) Other types of monomorphic adenomas. There are other types of ade­noma composed, in varying proportions, of ep i th e l ia l c e l l s of duct- l in ing type and of c e l l s resembling those of a basal cell skin tu ­mour (myoepithelial c e l l s ) , arranged in a tubular, alveolar or t rabecular pa t te rn , with or without cysts or papillary formations. In one type the c e l l s and th e i r arrangement so resemble th a t of a basa l-cell carcinoma of the skin th a t i t has been named a basal- ce ll adenoma. There is a lso a very rare type of tumour containing duct- l ike s truc tu res with an ep i th e l ia l l in ing surrounded by c lea r c e l l s in the position normally occupied by the myoepithelial c e l l s .

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B. Mucoepidermoid tumourA tumour characterized by the presence of squamous c e l l s , mucus-secret­ing c e l l s , and c e l l s of intermediate type. (4.9%)*

C. Acinic cell tumourA tumour consisting of c e l l s sim ilar to the serous c e l l s of sa livary glands. (2.7%)*

D. Carcinoma1. Adenoid cy stic carcinoma: An i n f i l t r a t i v e malignant tumour hav­ing a very ch a ra c te r is t ic cribriform appearance. The tumour c e l l s are of two types, duct-l in ing c e l l s and c e l l s of myoepithelial type, and are arranged as small duc t-l ike s tructu res or la rger masses of myoepi­th e l ia l c e l l s disposed around cys t ic spaces to give a cribriform or lace - l ik e pa tte rn . (4.7%)*

2. Adenocarcinoma: A malignant ep i th e l ia l tumour showing some tubule or pap illary glandular formation. No remnants of pleomorphic adenoma are present. (2.3%)*

3. Epidermoid carcinoma: A malignant ep i th e l ia l tumour with c e l l s forming keratin or having in te rc e l lu la r bridges; mucous secretion is not present. (0 . 8%)*

4. Undifferentiated carcinoma: A malignant tumour of ep i th e l ia l s tructu re th a t is too poorly d if fe ren t ia ted to be placed in any of the other groups of carcinoma. (3.9%)*

5. Carcinoma in pleomorphic adenoma (malignant mixed tumour): Tu­mours showing d ef in i te evidence of malignancy, such as invasive growth and cytological changes appropriate to carcinoma, and in which areas c h a rac te r is t ic of pleomorphic adenoma can s t i l l be found. (1.5%)*

I I . Non ep i th e l ia l tumoursI I I . Unclassified tumoursIV. Allied conditions

*Frequency of all salivary neoplasms (from Biörklund & Eneroth, 1980).

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A drawback with the WHO c la s s i f ic a t io n is the lack of discrimination between benign and malignant les ions , due to the use of an intermediate term, tumour. The two types of neoplasms c la s s i f ie d under th is heading usually behave benignly. However, high grade mucoepidermoid tumours have a very bad prognosis and for ac inic cell tumours the 20-year de­terminate survival ra te is only 56% (Eneroth e t al 1966). For both these tumour types, most authors nowadays use the designation "cancer" (Blanch 1974, Batsakis 1979, Biörklund & Eneroth 1980).

New e n t i t i e s , such as sa livary duct carcinoma (Kleinsasser e t al 1968), c lea r cell carcinoma (e .g . Echewarria 1967), ep ithelial-m yoepithelial carcinoma of in terca la ted ducts (Donath e t al 1972) and terminal duct carcinoma (Batsakis e t al 1983a) have been introduced, making modern c la s s i f ic a t io n even more voluminous (Table I I ) .

Structure of the sa livary glandsAll the major and minor sa livary glands have the same principal s t ru c ­tu re . A large excretory duct is repeatedly dichotomously divided into smaller ducts terminating into ac in i . The stroma divides the parenchyme into lobes and lobules. Vessels and nerves mainly follow the ducts.

Current tex t books (Geneser 1985, Fawcett 1986) divide salivary glands into serous, mucous or seromucous. Serous glands are the parotid , and the posterior lingual glands of von Ebner. Mixed mucous and serous are the submandibular, sublingual, la b ia l , buccal, an te r io r lingual and the posterior lingual (except von Ebner's ) . The gl ossopalatine and palatine and the glands of the root of the tongue are pure mucous glands. Some "serous" c e l l s , however, ac tua lly contain material which has histochem- ical ch a ra c te r is t ic s of mucous.

The salivary ducts are divided in to in te rca la ted ducts close to the ac in i , s t r i a te d ducts which are purely in tra lobu la r and excretory ducts located ex tra lobu la rly .

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Table I I . C lass if ica t ion of ep i th e l ia l salivary gland tumours (from Batsakis 1979)

Type of lesion Variations

Benign Mixed tumour (pleomorphic adenoma)Papilla ry cystadenoma lymphomatosum (Warthin's tumour)Oncocytoma (oncocytosis)Monomorphic tumours

Basal ce ll adenoma Glycogen rich adenoma (?)Clear ce ll adenoma Membranous adenoma Myoepithelioma

Sebaceous tumours Adenoma Lymphadenoma

Papilla ry ductal adenoma (papilloma)Benign lymphoepithelial lesion Unclassified

Malignant Carcinoma ex pleomorphic adenoma (carcinoma a r is in gin a mixed tumour)Malignant mixed tumour (biphasic malignancy)Mucoepidermoid carcinoma

Low-grade Intermedi ate-grade High-grade

Adenoid cys t ic carcinoma Acinous cell (acinic) carcinoma Adenocarcinoma

Mucus-producing adenopapillary and non-papillary carcinomaSalivary duct carcinoma (ductal carcinoma)Other adenocarcinomas

Oncocytic carcinoma (malignant oncocytoma)Clear cell carcinoma (non-mucinous and glycogen-con­ta in ing or non-glycogen-containing)Primary squamous cell carcinomaHybrid basal cell adenoma/adenoid cys t ic carcinomaU ndifferentiated carcinomaEpithelial-m yoepithelial carcinoma of in te rca la ted ductsMiscellaneous (includes sebaceous, Stensen1s duct, melanoma and carcinoma ex lymphoepithelial lesion) Metastatic Unclassified

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Acini. The epithelium of the acini consists of mucous and serous c e l l s (Fig. la ) ; sometimes intermingled within the same ac in i . Peripherally located myoepithelial c e l l s also co n s t i tu te an important part of the sa livary acinus. The serous c e l l s are pyramidal in shape and contain extensive rough endoplasmic reticulum a t the base and an apex loaded with zymogen granules (PAS posit ive but mucicarmine negative). Mucous c e l l s appear generally more "swollen" but have principa lly the same a rch itec tu re . A confluent network of mucinous substances pushes the nucleus basally . In routine preparations the c e l ls appear pale, but the granules are stained by both PAS and mucicarmine.

Myoepithelial c e l l s are found between the acinar or duct c e l l s and the basal lamina (Figs la , b). They are spindle-shaped, with numerous long thin pro jec tions, and contain large numbers of cytoplasmic filaments. The myoepithelial c e l l s are fa r more numerous in the submandibular gland than in the parotid . They are found spread from the acini to the d is ta l part of the s t r i a t e d duct (Cutler e t al 1977).

Ducts. In terca la ted ducts are composed of small cuboidal c e l ls sur­rounding a duct lumen. They are poor in organelles and are often en­closed by myoepithelial cell projections (Fig. lb ) . The s t r ia te d ducts are composed of cy lindrical c e l l s with a large nucleus. In the basal par t of these c e l l s , para l le l rows of numerous mitochondria (Fig. l c ) , separated by invaginations of the cell membrane, are c lea rly apparent. Small zymogen granules can be seen apically (Cutler e t al 1977). The excretory ducts are located en tire ly within the connective t is su e septa, and are lined by pseudostra tif ied epithelium tha t is gradually transformed into s t r a t i f i e d squamous epithelium.

Embryology. As numerous theories on the origin of salivary neoplasms have concerned und iffe ren tia ted c e l l s , a recapitu la tion of the develop­ment is necessary. The parotid anlage a r ises from the epithelium of the stomodeal ectoderm. The anlagen of the other major glands a r ise near the zone where the buccopharyngeal membrane once marked the junction of ectoderm and entoderm (Batsakis 1979). The development of the acinar- duct unit has been most thoroughly studied in the submandibular gland of r a t and hamster. The pattern of d i f fe re n t ia t io n , however, appears

Fig. 1 a) Serous (S) and mucous (M) ce l ls of a sublingual acinus. (ME = myoepithelial c e l l , b) Submandibular in te rca la ted duct, c) Typical s t r i a te d duct ce l l s with numerous mitochondria.

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sim ilar for most salivary glands, i r re spec tive of species, although differences in timing may occur. In b r ie f , the hamster submandibular gland a t gestational day 13 is composed of p lu r ipo ten tia l (undifferen t­iated) stem c e l l s . At day 14, cytomorphodifferentiation into "terminal tubules", composed of secretory c e l l s , myoepithelial cell precursors and und iffe ren tia ted stem c e l l s take place. From the undifferen tia ted c e l l s of the terminal tubules the "terminal buds" a r ise which are fo re­runners of the ac in i . Both the terminal tubules and the buds are sup­ported by short s ta lks which la t e r are transformed into in te rca la ted ducts. The acini are fu l ly developed 2-3 weeks post partum (Chaudhry e t al 1985).

Myoepithelial ce ll precursors in the terminal tubules are rich in ribo­somes, polyribosomes, and mitochondria. After b ir th a s ig n if ican t in ­crease in myoepithelial ce ll precursors, d is tr ibu ted between the basal lamina and other c e l l s of the terminal tubules occurs. Deposits of de­l ic a te cytofilaments can be seen in t ra c e l lu la r ly shortly a f te r b i r th . After two weeks the cytoplasms are v i r tu a l ly f i l l e d with myofilaments. The development of myoepithelial c e l l s thus occurs in several d iscre te steps prior to the synthesis and deposition of myofilaments and once th is re la t iv e ly advanced stage of maturation is reached the c e l l s lose th e i r a b i l i ty to divide (Chaudhry e t al 1983a).In man a large number of c i l i a t e d c e l l s occur early in the primitive ducts but by week 20 the only remnants are within the ac in i . Triangular myoepithelial c e l l s are found as early as week 12, but before week 25 they contain only minute amounts of myofilaments (Donath e t al 1978).

Tumour histogenesisThe polymorphism of sa livary neoplasms has given r i s e to a number of theories concerning th e i r o rig in . Tumour c la s s i f ic a t io n in general is based on the presumed origin of the neoplasia, e .g . malignant tumours from mesenchymal t is su e are ca lled sarcomas, whereas ep i th e l ia l malig­nancies are ca lled carcinomas or cancers.

The f i r s t h is to logica l descrip tions of salivary neoplasms were publish­ed by B illro th (1859) and Virchow (1863). The neoplasms were presumed to a r ise from connective t i s su e elements and were named enchondromas.

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The theory of a mesenchymal origin pers is ted for decades. The term mixed tumour was coined by Minssen (1882) to indicate a dual ep i th e l ia l and mesenchymal o rig in . Later on an ep i th e l ia l origin theory became more and more prominent and the name was changed to pleomorphic adenoma (Willis 1953). Of the ep ith e l ia l c e l l s , the rnyoepithelial cell has par­t ic u la r ly a t t ra c ted in te re s t as a progenitor (for review, see e .g . Mylius 1960, Hamperl 1970, Hübner e t al 1971, Batsakis e t al 1983b).

The issue of tumour origin is closely associated with sa livary gland cell regeneration and renewal. In man, the regenerative and p r o l i f e r ­a t ive capacity is proposed to reside within the in te rca la ted duct (Batsakis 19,79). After pa r tia l acinar cell degeneration, induced by ethionine, the recovery phase manifests both acinar ce ll p ro l i fe ra t io n and the appearance of acinar c e l l s as a re s u l t of the d if fe ren t ia t io n of the most terminal portion of the sa livary duct system. However, a f te r complete acinar destruction following ionizing radia tion there is no evidence of acinar regeneration even a f te r 75 weeks (Leeb 1975).

Present theories mostly favour und iffe ren tia ted duct reserve c e l l s as progenitors (Eversole 1971, Batsakis 1980, Chaudhry e t al 1983a, b, 1984, 1985, 1986). On the other hand, Dardic s tressed the renewing po­ten t ia l of acinar as well as duct c e l l s and proposed th a t the origin for mixed tumours was from a mature duct or acinar ce ll or a complete acinar-duct un it ra ther than an und iffe ren tia ted duct cell (Dardic e t al 1982, 1983a, b).

New approaches in cell biologyToday's pathology is based primarily on morphological studies of c e l l s and t i s su e s . The introduction of molecular markers may change the na­ture of diagnostic pathology from a subjective appraisa l , whose accura­cy is dependent upon the sk i l l and experience of the individual patho­lo g is t , to a multi méthodologie and analy tic science based on objective­ly demonstrable data.

Immunohistochemistry. Immunological techniques have opened up great p o s s ib i l i t i e s for the id en t i f ic a t io n of d if fe ren t proteins and other tumour products in both serum and tumour t is su e . Two major types of

16

probe have been used to iden tify such cell type-specific components: antibodies and, to a le sse r ex ten t, nucleic acid sequences. Up to now most studies have been concerned with the presence of e .g . onco-fetal antigens, m etalloproteins, hormones, enzymes and a number of other nor­mal membrane and in t r a c e l lu la r constituents (for studies concerning sa livary gland tumours, see e .g . S e i fe r t & Caselitz 1983, Murase e t al 1985, Warner e t al 1985).

The se le c t iv i ty of c e l l s in which the antigens appear is a p re requ is ite for a wide ap p l icab il i ty of whatever immunological marker is employed. Furthermore, the expression of the antigen investigated must be r e la ­t iv e ly constant during cell transformation, otherwise fa lse-negative re su lts may be too frequent. Moreover, the antigen expression must d i­vide c e l l s in a meaningful manner, e .g . discriminate between d if fe ren t types of t i s su e or d i f fe re n t ia te benign lesions from malignant ones. Studies on intermediate filament proteins (IFP) have in these respects been promising.

Different types of proteins co n s t i tu te these filaments and by immuno­logical methods f ive classes have been distinguished. U1 t ra s t ru c tu ra l ly the filaments cannot be separated as they all have a diameter of 7-11 nm. The t is su e s p ec if ic i ty of IF is remarkable. Cytokeratins (CKs) are found in e p i th e l ia l c e l l s of ectodermal or endoderma! orig in . Most mesenchymal c e l l s contain vimentin but, in s t r ia te d and some vascular smooth muscle c e l l s , desmin is also found. Neurofilaments and g l ia l f i b r i l l a r y ac id ic protein (GFA) are found in neurons and g lia l t i s su e , respectively . Vimentin, desmin and GFA are single pro te ins . On the other hand, three d if fe ren t neurofilament proteins ex is t and as fa r as CKs are concerned, a large group of a t le a s t 19 d if fe ren t proteins are presently known in man. During the present inves tiga tions, the patterns of CKs in d if fe ren t ep i th e l ia were characterized (Moll e t al 1982, 1983) and were found to be constant. The pattern of IFPs, both as a group and the pattern of individual CKs, have been found to be highly stab le during neoplastic transformation (Moll e t al 1982, Quinlan e t al 1985). These properties would make IFPs valuable tools for studies of histogenesis and tumour typing (Gabbiani e t al 1981, Ramaekers e t al

17

1982, 1983, 1985b, Osborn & Weber 1982, 1983, Kahn e t al 1983, M ie tt i ­nen e t al 1984b, Cooper e t al 1985, Virtanen e t al 1985).

With regard to salivary gland and salivary neoplasm IFP pa tte rns , a number of studies have been performed using d if fe ren t immunohistochemi- cal techniques. The re su lts on the normal parotid have, however, been divergent, making evaluation of the tumour sta in ing d i f f i c u l t .

Measurements of nuclear DNA conten t. The introduction of quan tita t ive microspectrophotometric methods by Caspersson (1936), made determina­tion of DNA content of individual c e l l s possible . At present a number of techniques for DNA determination ex is t : in cell suspensions, flow cytometry can be used and in sections or smear, single cell measure­ments are used.

For a number of neoplasms a corre la tion between c e l lu la r DNA content and prognosis has been documented. For salivary neoplasms, Eneroth & Zetterberg (1973, 1974a, b, 1975, 1976, Eneroth e t al 1974) found a corre la tion between malignancy and DNA content. However, only a few studies have been performed on the prognostic value of DNA levels in sa livary gland tumours.

Receptor charac terization and stimulus-secretion coupling. A vast num­ber of secretory products have been described in tumour t is su e . Simi­la r ly , receptors in a few neoplasms have been characterized and in mam­mary neoplasms, for instance, estrogen receptor content has been cor­re la ted to prognosis and d if fe ren t ia t io n (Hartveit e t al 1980, Mohammed e t al 1986). Only a few descrip tions of adrenoceptors, primarily in p i tu i ta ry adenomas, have been published (Heisler e t al 1983, Reisine e t al 1983, Ramsdell e t al 1985). Likewise, in t r a c e l lu la r events during the secretory process, such as changes in cyclic AMP lev e ls , have been studied (de Carni I l i e t al 1980, Reisine e t al 1983, Heisler e t al 1983). Comparisons of such reactions with th a t of the t is su e origin may be a way of determining the degree and direction of d i f fe ren t ia t io n .

Furthermore, the above discussed studies on tumour t is su e may be a way of evaluating the influence of in te rac t ion between cell types in normal t is su e when the t is su e of origin is complex with regard to cell type and innervation.

18

OBJECTIVES OF THE PRESENT STUDY

The main objectives of the present study were to determine:

- the intermediate filament pattern of developing and mature normal sa livary gland

- whether the IFP pattern of sa livary neoplasms is re la ted to any par­t i c u la r cell (adult or f e ta l ) with regard to histogenesis

- whether the IFP pattern can be used for typing of sa livary gland tumours

- whether ce r ta in tumour c e l l s which closely resemble normal acinar c e l l s respond to secretory stimuli

- whether the nuclear DNA content of neoplastic c e l l s in acinic cell carcinoma has any p red ic tive value in se lec ting pa tien ts with good pro­gnosis as re f lec ted by long-term survival.

19

MATERIAL AND METHODS

Morphology (I-VI)Minute specimens were taken from salivary t is sue and tumour t is su e a t the time of the operation and fu r ther processed. Fetal parotid t is su e was obtained from aborted fe tuses (IY). For immunological s tud ies , specimens were immediately frozen by immersion in isopentane precooled with liqu id nitrogen. For electron microscopy, specimens were immedi­a tely fixed in 2.5% or 4% glutaraldehyde in phosphate ( I - I I I , VI) or cacodylate ( I I ) buffer, rinsed in buffer, post-fixed in 1% O S O 4 , de­hydrated in graded ethanol so lu tions , propylene oxide and embedded in Yestopal or Epon 812 res in .

For id en t i f ica t io n of mucous substances, t i s su e from adenoid cystic carcinomas was fixed in 2.5% glutaraldehyde in cacodylate buffer with 2 000 p.p.m. ruthenium red added overnight, rinsed in buffer containing 1 000 p.p.m. ruthenium red, post-fixed in 1% OSO4 with 500 p.p.m. ruthenium red, dehydrated and embedded in Epon ( I I ) . Specimens were also processed to demonstrate periodate reactive substances by the periodic acid - chromic acid - s i lv e r methenamine method and the phos- photungstic acid method ( I I ) . U ltrath in sections were cut using an LKB Ultrotome and were mounted on copper or gold grids and examined e i th e r unstained or a f te r s ta in ing with uranyl acetate and lead c i t r a t e in a Philips EM 300 or a Jeol CX 100 electron microscope.

Immunohistochemistry (I-V)Antibodies;. Three mouse monoclonal antibodies d irected against cyto- keratins were used (PKK1, PKK2 and PKK3). These antibodies have been characterized elsewhere (Holthöfer e t al 1983, 1984).

Two mouse monoclonal antibodies to vimentin were used: one was ra ised against bovine lens vimentin (vim 9) and was purchased from Sanbio (Nistelrhode, the Netherlands) and the other monoclonal antibody was against vimentin from human f ib ro b las ts from clone FW24 BA6 (vim 24) (Labsystems Oy, Helsinki, Finland).

20

Guinea pig and rabbit polyclonal antibodies against human sole keratins were prepared according to Kjörell & Östberg (1984) and DNase 1 a n t i ­bodies were prepared according to Wang & Goldberg (1978), Preimmuniza­tion serum or sera from non-immunized animals were also processed as control samples ( I - I I I ) .

Immunoblots (IV)Pieces of normal parotid t is su e were f i r s t extracted in 0.5% Triton X-100 (50 mM Tris-HCl, pH 7 .4 , supplemented with proteolysis in h ib i­to r s ) . Thereafter, the samples were dissolved in electrophoresis sample buffer (Laemmli 1970) and were subjected to polyacrylamide gel e le c t ro ­phoresis in the presence of sodium dodecyl sulphate. Then, the polypep­t ides were transferred onto n i tro ce llu lo se sheets according to Towbin e t al (1979). Amidoblack was used for protein sta in ing . Culture super­natant of the monoclonal antibodies was used for immunoreaction. The binding of the antibodies was detected by peroxidase-coupled rabbit and mouse antiserum (1:100, Dakopatts, Glostrup, Denmark). The re su lts with parotid t is su e were in terp re ted by using cytoskeletal preparation of standard cell l ines as a control: UCF7 and A431 c e l l s with a known pat­tern of cytokeratin expression (Quinlan e t al 1985).

Immunostaining (I-V)Serial frozen sections were used for immunostaining a f te r acetone f ix a ­tion a t -20°C for 5 min. The primary antibodies were used in a concen­t ra t io n of 20-50 mg/1 a t room temperature. The DNase 1 anti-DNase 1 in ­cubation has been described by Wang & Goldberg (1978). After thorough washing, the sections were exposed to fluorescein isothiocyanate (FITC coupled) or tetramethylrhodamine isothiocyanate (TRITC conjugated) labe lled antibodies or processed by the peroxidase-anti peroxidase (PAP)-method (Sternberger e t al 1970). The sections were mounted in Moviol 4-88 (Hoechst, Frankfurt am Main, West Germany), pH 8.5 and viewed in a Leitz Orthoplan microscope equipped with epifluorescent optics or an Olympus VAN0X microscope.

Stereological methods (VI)The stereological analyses were carried out on randomly taken electron micrographs of parotid acinar c e l l s and tumour c e l l s using Wei b e l ' s

21

(1979) point counting method employing a multipurpose grid . The d ia­meters of the secretory granules in the electron micrographs were mea­sured in a Zeiss TGZ-3 p a r t ic le s ize analyser. The stereological calcu­la t ions are described in deta il in VI. I t should be pointed out th a t values obtained by stereological methods are subject to various inher­ent e r ro rs , due mainly to t is su e preparative procedures including sec­tioning. In comparative s tud ies , however, these errors diminish in im­portance.

Studies on secretion and cyclic AMP accumulation (VI)Gland specimens were t ransfe rred to a Krebs-Henseleit bicarbonate buf­fe r (pH 7 .4 ) . The buffer was supplemented with pyruvate, glutamate and fumarate as well as albumin (1 mg/ml) and glucose (0.6 mg/ml). Before incubation a piece of t i s su e was taken for analyses of to ta l anylase and cyclic AMP contents. The secretory studies in v i t ro were performed with a batch-incubati on technique described in deta il elsewhere (VI; Carlsöö e t al 1981).

Amylase and cyclic AMP assay (VI)Amylase was assayed by a micromodification of the 3 ,5 -d in i t ro sa l ic y la te method (Danielsson 1974). One un it of amylase is defined as the ac t iv ­i ty l ib e ra t in g reducing groups which correspond to 1 umol of maltose monohydrate per minute a t 25°C. Amylase re lease was expressed as the percentage of amylase in medium in re la t ion to the to ta l anjylase con­ten t in medium plus homogenate. Cyclic AMP was extracted from the t i s ­sue and assayed radio-immunologically, th is being expressed as pmol/mg t is su e dry weight. For d e ta i l s , see VI.

DNA measurement (VII)Representative areas of the orig inal paraffin-embedded tumour specimens were selected and two consecutive sections (4 urn) were cut. One section was stained with haematoxylin-eosin, the other was Feulgen-stained by the method of Duijndam & van Duijn (1973) a f te r acid hydrolysis in 5 N HC1 for 1 hour. Computerized stage scanning cytophotometry was perform­ed using a Leitz MPV 3 cytophotometer and the HISTOSCAN program (Bjel- kenkrantz e t al 1981). Within each specimen a representa tive area of the tumour was selected and within th is area nuclei, not overlapping

22

each other, were systematically measured. About 100 tumour c e l l s and about 50 control c e l l s were measured. The control c e l l s , i . e . lympho­cytes and/or acinar c e l l s , were used to define the normal, diploid (2C) DNA value.

For f ive acinic cell tumours, DNA analysis was performed on asp ira tion biopsy smears. After destai n ing in methanol and hydrolysis in 5 N HC1, the smears were stained in an 0.02% acri f i avine-S02 solution (Tanke & van Ingen 1980). Measurements were made using a Leitz MPY 3 cytophoto- meter and the FLU0RA program (Bjelkenkrantz e t al 1983). In these spec­imens, granulocytes were used as control c e l l s .

To distinguish d iploid/near diploid from aneuploid cell populations, the percentage of tumour cell nuclei with DNA values exceeding 2.5C was determined for each specimen. Furthermore, mean and modal values were calculated from the histograms.

23

RESULTS

Cytoskeletal proteins in normal sa livary glands (I-IV)In sections of normal sa livary glands labelled with CK-pAbs, intense sta in ing was found in a ll types of duct and myoepithelial c e l l s , w hils t acinar c e l l s were stained mainly a t the apex and la te ra l side ( I - I I I ) . A sim ilar reaction was obtained for CK-mAb PKK1. CK-mAb PKK2 reacted strongly with myoepithelial and duct c e l l s , w hilst PKK3 reacted moder­a tely with duct and acinar c e l l s (IV).Vimentin pAbs did not reac t with any ep i th e l ia l or myoepithelial c e l l , nor did mAb vim 9 (I I-IY ). On the contrary, vim 24 c lea r ly stained nrçyo- ep i th e l ia l and basal c e l l s of excretory ducts (IY). No difference was detected between parotid , submandibular, or palata l glands, or between mucous or serous acinar c e l l s . Actin was detected 1n myoepithelial c e l l s and a t luminar surfaces of s t r i a t e d duct and acinar c e l l s by the DNase 1/antiDNase 1 method. Along the in te rc e l lu la r c a n a l icu l i , s ta in ­ing was also evident between acinar c e l ls ( I ) . At those locations where IFP or ac tin could be found immunologically, fine filaments could also be id en t i f ied by electron microscopy.

Cytoskeleton of developing paro tid glands (IY)CK and vimentin were determined by mAbs in developing paro tid gland.In general, a moderate to strong s ta in ing was found in a ll fe ta l gland c e l l s for PKK1 and PKK2, a t gestational week 18. For PKK3, the s ta in ing in ten s i ty was somewhat weaker. The s ta in ing reactions were most Intense in the la rger ducts for PKK1 and PKK2. At gestational week 22, the d i f ­ferences in s t a in a b i l i ty between large and small ducts was more pro­nounced, although all ep i th e l ia l c e l l s s t i l l stained by a l l CK-mAbs. In terminal tubules and buds, sca tte red tr ian g u la r basal c e l l s were found sta inable with both vim 9 and vim 24. In la rge r ducts no vimentin con­ta in ing c e l l s were found.

Cytoskeleton of benign salivary neoplasms ( I , I I I , Y)In mixed tumours CK-pAbs reacted with v ir tu a l ly a ll tumour c e l l s ( I I I ) , and mAbs PKK1 and PKK2 reacted strongly with v i r tu a l ly all tumour c e l l s , while PKK3 gave a fa in te r reaction and the reaction was most pronounced a t the duct lumina.

24

Y i menti n was detected by pAbs ( I I I ) and mAbs (vim 9, vim 24) in c e l l s of both mesenchymal and ep i th e l ia l character. In the ep ith e l ia l areas, i t was mainly the peripheral c e l l s of cords th a t were reactive .

Basal cell adenomas gave with mAbs almost the same reaction as did the ep i th e l ia l areas of mixed tumours (V, unpublished data) .The CK antibodies, PKK1 and PKK2, were strongly reac tive , while a fa in t to moderate PKK3 reaction was found a t duct lumina. Vimentin (vim 9, vim 24) was detected in peripheral c e l l s .

In oncocytomas two cell types were distinguished: one more numerous and large cell type and one small elongated type. Both types reacted strongly with PKK1 and weakly with PKK3, while the l a t t e r cell type was much more reactive to both CK-pAbs and PKK2 than was the former. Vimen­t in was not detected in tumour c e l l s (pAbs, vim 9, vim 24), and actin (DNase 1/ antiDNase 1) was only revealed a t luminal surfaces ( I , unpub­lished data) .

In the ep i th e l ia l parts of adenolymphomas the small basal cell and the large apical c e l l s reacted d if fe ren t ly with CK-pAbs: the basal c e l l s were intensely stained and the apical c e l l s moderately so ( I ) . With CK-mAbs th is d ifference was less marked, with PKK1 and PKK2 the d i f f e r ­ence being barely v is ib le while with PKK3 the basal c e l l s were less reac tive (unpublished da ta) . Vimentin was not detected in tumour c e l l s (pAbs, vim 9, vim 24). Actin (DNase 1/antiDNase 1) was found only a t the apical surface of the columnar c e l l s ( I ) .

Cytoskeleton of malignant sa livary neoplasms ( I I , I I I , V)By using pAbs, CK was detected in adenoidcystic carcinomas and carcino­mas in mixed tumours ( I I , I I I ) .

CK and vimentin were determined by mcAbs in 16 consecutive salivary tumours which were i n i t i a l l y judged as malignant. The neoplasms were re c la s s i f ie d by one pathologis t and the r e l i a b i l i t y of the diagnoses evaluated. Ten diagnoses were judged as d ef in i te (2 AdCC, 3 MEC, 2 ACC, 2 SDC and 1 poorly d if fe ren t ia ted c lea r-ce l l adenocarcinoma) while for

25

two other tumours i n i t i a l l y judged as AdCC, the d if fe ren t ia l diagnosis of basal cell adenoma and mixed tumour, respectively , could not be com­p le te ly ruled out. One tumour could have been MEC or a mucous-producing adenocarcinoma. For one probable ACC, the d if fe ren t ia l diagnosis was a m etastasis . One neoplasm was probably a MEC and f in a l ly one AdCC was re c la s s i f ied as a carcinoma in mixed tumour, where the malignant par t was tha t of an adenoid cystic carcinoma. The immunoreactivity of mAbs against CKs and vimentin is given in Table I I I .

With respect to PKK2, PKK3 and vim 9 re a c t iv i ty , the tumours could be divided into three groups.

Group A consisted of neoplasms with stronger s t a in a b i l i ty for PKK3 than for PKK2. They a lso contained vim 9 posit ive tumour c e l l s .This group consisted of two ACC and one probable ACC.

In Group B neoplasms, a markedly stronger reaction for PKK2 was noted than for PKK3. For vim 9 the cell population was non-homogeneous. In the periphery, towards the basal membrane, c e l l s were strongly vim 9 pos it ive but in the central portions and towards secretory lumina, the ce l l s were vim 9 negative. This group of neoplasm contained two AdCC, three probable AdCC, one AdCC ex mixed tumour and also one probable MEC.

In Group C, PKK2 stained more intensely than PKK3. Vim 9 was negative. The tumours in th is group consisted of two salivary duct carcinomas, one c lea r-ce l l adenocarcinoma and three MEC and one MEC or adenocarci­noma.

Polygonal p a r t ic le s of proteoglycans in pseudocysts of AdCC could be id en t i f ied by electron microscopy a f te r conventional glutaraldehyde fixation and osmium p os t-f ixa t ion . Addition of ruthenium red to the f ix a t iv e resu lted in a more intense sta in ing of the p a r t ic le s and they could be id en t i f ied without lead and uranium s ta in ing . In specimens fixed in OSO4 without p r io r aldehyde f ix a t io n , the polygonal p a r t i ­cles could not be demonstrated. In periodic acid - chromic acid - s i lv e r methenamine stained, Vestopal-embedded sec tions, the deposits

26

were found mainly along in t rac y s t ic collagen bundles and basal lamina. A fa in t diffuse sta in ing was reg is tered over the amorphous or f inely f i b r i l l a r ground substance of the cys ts , but was d i f f i c u l t to evaluate because of the s ize of the s i lv e r p a r t ic le s . Phosphotungstic acid stained the most la te ra l par t of the basal lamina network and the amorphous material of the cysts .

Of the four carcinomas in mixed tumours investigated u l t ra s t ru c tu ra l ly and compared with IFP pa tte rn , squamous d if fe ren t ia t io n with to n o fi la - ments and glandular d if fe ren t ia t io n with secretory granules were simul­taneously found in two carcinomas.In one neoplasm only glandular c e l l s were found, while in the fourth the c e l l s were devoid of ep i th e l ia l s t ru c tu re s , such as basal lamina and desmosomes. In the l a t t e r neoplasm, c e l l s were often elongated and resembled f ib ro b las ts ( I I I ) .

The three carcinomas te s ted immunohistochemically displayed d if fe ren t IFP pa t te rns , two with a d i s t in c t coexpression of vimentin and CK, though both exhibited d if fe r in g histo logica l s truc tu res : one showed a predominant mesenchymal d i f fe ren t ia t io n and the other was of a purely ep i th e l ia l nature. In the th ird tumour, the c e l l s expressed CKs only

( I I I ) .

Table I I I . Immunoreactivity of monoclonal antibodies against CKs and vimentin.

PKK1 PKK2 PKK3 Yim 24 Yim 9

Mixed tumour 2-3 2-3 0-2 0-2* 0-2*Adenolymphoma 2-3 2-3 1 0 0Oncocytoma 2 1-3** 1 0 0Basal cell adenoma 3 3 0-1 0-2* 0-2*Acinic cell carcinoma 3 2 3 0-2 0-2Mucoepidermoid carcinoma 1-3 1-3 0-2 0 0Adenoid cys t ic carcinoma 3 3 0-1 0-2* 0-2*Adenocarcinoma 3 3 2 0-1 0

♦Peripheral c e l l s are vimentin-positive, central negative**Two cell types, one strongly s ta inab le , the other weakly0=no reaction , l=weak reaction , 2=moderate reaction , 3=strong reaction

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Morphometry and secretory behaviour of ACC (VI)For the tumour c e l l s from three ACC, compared s te reo log ica lly with nor­mal parotid gland acinar c e l l s , the morphometric study revealed th a t the granule density was increased. The c e l l s contained more granules but the diameter was considerably less compared to normal contro ls . No difference in cell volume or nuclear volume density could be found.

In the batch incubation system, amylase and cAMP were detected in t i s ­sue from the three ACC, as well as in normal parotid gland t is su e . Even though a high basal (non-stimulatory) amylase output was noted from tu ­mour specimens, noradrenalin induced a marked increase in the amylase re lease . Noradrenalin stimulation also caused an enhanced cAMP accumu­la tion in the tumour t is su e . Similar quan ti ta t ive a l te ra t io n s were ob­served in the normal gland t i s su e .

DNA levels in acinic cell carcinomas (VII)Cytology smears and paraffin blocks were processed for DNA measurements from 13 patien ts : 9 patien ts without signs of recurrence in the 6-year follow-up period, 2 with local recurrences, and 2 patien ts with méta­stasés and/or had died of disease. Measurements on sections (8, 2, 2 patien ts in the groups, respectively) as well as on smears (3, 0, 2) revealed th a t all tumours were d iploid or near-diploid within the range (2C +_ 10%). Two recurrent and two metastasizing tumours had, however, modal values between 2.2C and 2.3C. The percentages of tumour c e l l s ex­ceeding the 2.5C leve l , as a measure of non-diploidity , were 6.4% for non-recurrent, 2.5% for recurrent and for neoplasms with métastasés 16%. Measurements carried out on smears gave higher corresponding values than those on sec tions, 25% for non-recurrent tumours and 24% for metastasizing neoplasms.

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DISCUSSION

Intermediate filament proteins in normal sa livary glands In order to evaluate tumour behaviour, tumour histogenesis and tumour d if fe re n t ia t io n , a thorough knowledge of the d is t r ib u tio n of immuno­logical markers present in the various normal c e l l s , and how these s tructures develop and mature is required.

Cytokeratins. The composition of cytokeratins varies between d if fe ren t kinds of ep i th e l ia l c e l l s . By peptide mapping and two-dimensional e lec ­trophoresis , catalogues of the CK-content of d i f fe ren t ep i th e l ia have been established (Moll e t al 1982). Cytokeratins can, according to th e i r charges, immunoreacti vi t i e s , mRNA hybridization, peptide mapping pa t te rn , aminoacid sequences and re la tionship to wool kera tins be d iv i­ded in to an ac id ic type I and a (n eu tra l- to -) basic type II (Cooper e t al 1985, Quinlan e t al 1985). Tissue d is tr ibu tion data have established th a t a t le a s t one member from each is present in all ep i th e l ia , and i t has been proposed th a t one ac id ic and one basic cytokeratin is a prere­q u is i te for cytokeratin filament formation (Cooper e t al 1985). There are indications th a t spec if ic keratin pairs e x is t , where the basic mem­ber is always la rger than the acidic one. Each pa ir is spec if ic for a ce rta in ep i th e l ia l t i s su e type (Cooper e t al 1985).

Our immunoblots have shown, th a t vimentin as well as CKs are present in sa livary gland t is su e . The immunohistochemical s tudies fu r ther revealed th a t the CK composition of various sa livary c e l l s d i f fe r s . Duct c e l l s contain a complex of CKs strongly s tainable with all CK-Abs (PKK1, PKK2, PKK3 and a number of pAbs) w hils t the acinar c e l l s are most reac­t iv e for antibodies against CK18 (mAbs PKK1 and PKK3). The myoepithe­l i a l c e l l s also contain CKs, but these do not reac t with the antibody spec if ic against CK18 (mAb PKK3). Q ua lita t ive ly , the IFP composition is basica lly s im ilar in the parotid , the submandibular and the palata l glands.

Squamous ep i th e l ia mainly expresses la rger cytokeratins as 50/58 kD CKs supplemented by another pa ir according to d if fe ren t ia t io n ; ke ra t in ­ized epidermis contain also 56.5/65-67 kD CKs, cornea contains addi-

29

t io n a l ly 55/64 kD CKs and esophageal or tongue type of ep î th e l la makes 51/59 kD CKs. Simple epithelium expresses 2 to 4 of the smallest CKs (45/52 kD and 40 and 54 kD CKs). Complex ep i th e l ia l s truc tu res such as glands usually express a mixture of CK patterns of simple and s t r a t i ­fied ep i th e l ia (Cooper e t al 1985). The exact CK composition in n\yoepi- th e l ia l c e l l s has not ye t been determined.

With regard to the polymorphism of cytokeratins , i t is understandable tha t minor differences in antigen preparation and immunization may give d if fe ren t re su lts regarding CK lo ca l iza tio n . The immunological findings of d i f fe ren t studies on normal parotid gland are summarized in Table IV. Polyclonal antibodies have been used by C aselitz e t al (1982), Krepier e t al (1982), Nathrath e t al (1982), Saku e t al (1984), Kahn e t al (1985), Takai e t al (1985) and Hosaka e t al (1985) as well as in parts of the present study ( I , II and I I I ) . The only studies in which monoclonal antibodies were used are the present study (IV), Erlandsson e t al (1984), Palmer e t al (1985) and Caselitz e t al (1986) (Table I I I ) . Erlandsson e t al (1984), however, using formalin-fixed pepsin- digested paraffin sec tions, were unable to demonstrate CKs in acinar and myoepithelial c e l l s . Palmer e t al (1985) used a mAb (16a) spec if ic for 45-46 kD CK and found re a c t iv i ty only in basal excretory duct c e l l s . The sta in ing pattern of a mAb spec if ic for CK 18 (mAb CK 5) was most recently reported on by C aselitz e t al (1986a), and the s ta in ing pattern in sa livary glands was v ir tu a l ly identical to our findings with PKK3 (IV). In the present study (IV), several mAbs were employed and CKs could be demonstrated in a ll ep i th e l ia l and myoepithelial c e l l s .

Vimentin, in con trast to cy tokeratins , i s a single gene product and one would consequently expect a more uniform sta in ing pa t te rn . Most s tudies fa i l to detect vimentin in ep i th e l ia l and myoepithelial c e l l s . However, Caselitz e t al (1982) found a posit ive s ta in ing in basal t r ian g u la r c e l l s of ducts, and Erlandsson e t al (1984) and Kahn e t al (1985) found re ac t iv i ty within a few myoepithelial c e l l s . Hosaka e t al (1986) claim the existence of vimentin in a ll ductal c e l l s of sa livary glands. How­ever, they used non-digested formaldehyde-fixed paraffin blocks and pAbs. They had s im ilar locations for a ll other antigens tes ted and judging by published p ic tu re s , th e i r re su lts may well be a r t i f a c t s due

30

to overstaining. In all these s tud ies , pAbs were employed. Using a mAb (vim 24) we were able to demonstrate vimentin in both these cell types. The other mAb (vim 9) stained neither ep i th e l ia l nor myoepithelial c e l l s (IV). The most probable explanation for th is d ispari ty in vimen- t in re a c t iv i ty between the two monoclonal antibodies may be some kind of antigen masking, for example, by phosphorylation. By immunoblotting i t was further shown (V) th a t with vim 24, no cross-reaction occurred with any other polypeptide. The findings in all studies on vimentin in sa livary gland c e l l s are summarized in Table V.

Table IV. Cell types in which CKs have been detected

Type of Ab

AC MEC ICDC SDC EDC EDBC

Krepier e t al 1982 pAbs + + + + ? ?

Caselitz e t al1981, 1982 pAbs - + + + + +1986a, b Ln 5 + + + + + +

Kl 1 + + + + + +CK 5 + - + + + +

Nathrath e t al AK1 _ + _ . -

1982 AK + + + + + ?

Kahn e t al 1985 pAbs + + + + + +

Saku e t al 1984 pAbs + - + + + +

Erlandsson e t al AE1 . _ + + + +1984 AE2 - - - - - -

AE3 - - + + + +

Palmer e t al pAbs - - + + + +1985 16a - - - - - +

Takai e t al 1985 pAbs - - + + ? ?

Hosaka e t al 1986 pAbs - - + + + +

Gustafsson e t al pAbs + + + + + +1985, 1986 PKK1 + + + + + +

PKK2 - + + + + +PKK3 + — + + + +

AC * acinar c e l l s , MEC = rnyoepithel ia l c e l l s , ICDC = in terca la ted duct c e l l s , SDC = sa livary duct c e l l s , EDC = excretory duct c e l l s , EDBC = excretory duct basal c e l l s .

31

Table V. Cell types in which vimentin has been detected

AC MEC IDC SDC EDC EDBC

Krepier e t al 1982 pAbs - - - - ? ?Caselitz e t al 1982 pAbs - - - - - +Erlandsson e t al 1984 pAbs - (+) - - - ?Kahn e t al 1985 pAbs - (+) - - - ?Hosaka e t al 1985 pAbs - - + + + +Gustafsson e t al pAbs - - - - - -1985, 1986 vim 9 - - - - - -

vim 24 - + - - - +

In adult t i s su e , coexpression of vimentin and cytokeratin is very ra re . The only reported t i s su e , except sa livary glands, with a dual filament expression is the t e s t i s . The Serto l i c e l l s are CK-negative and vimen- t in -p o s i t iv e , w hilst the opposite re ac t iv i ty is found in the c e l l s of the re te t e s t i s (Paranko e t al 1986).

In summary, the present study showed th a t the sa livary gland has a com­plex s truc tu re of ep i th e l ia l c e l l s , displaying d if fe ren t patterns of IF expression. The acinar c e l l s have a pattern s im ilar to simple ep ithe­lium with a main expression of CK 18. Myoepithelial c e l l s display most­ly CK 17 and 19. Duct c e l l s have a broad range of CKs as they are heavily stained by most CK antibodies, both monoclonal and polyclonal.

Furthermore, two cell types, the myoepithelial ce ll and the basal t r i ­angular cell of excretory ducts, display a coexpression of cytokeratins and vimentin, a phenomenon v i r tu a l ly unique among ep i th e l ia l glandular c e l l s . Both these cell types have been widely discussed with regard to tumour h istogenesis .

Intermediate filaments in fe ta l paro tid glandsAt gestational week 18 only CKs are expressed 1n the fe ta l paro tid , whereas four weeks l a t e r some basal t r ian g u la r c e l l s of the terminal tubules displayed a d i s t in c t reaction for both vim 9 and vim 24. Larger

32

ducts were, however, always vimentin mAbs unreactive. At th i s stage of development, the shape and form of these vimentin co-expressing c e l l s correspond to th a t of myoepithelial c e l l s (Donath e t al 1978). As the vim 9 reactive epitop has not been found in the adult gland, the ex­pression thus appears t ran s ien t during development. A temporary CK- vimentin co-expression has been described in other cell types during development such as S erto li c e l l s and ce rta in renal c e l l s (Holthöfer e t al 1983, Ramaekers e t al 1985a, Waldherr & Schwechheimer 1985).

Intermediate filaments in sa livary gland neoplasmsCytokeratins are undoubtably expressed in all ep i th e l ia l sa livary gland neoplasms. In the present study ( I - I I I , Y) CKs could be demonstrated in mixed tumours, oncocytomas, adenolymphomas, basal cell adenomas, muco- ep idermoid carcinomas, ac in ic cell carcinomas, adenoid cystic carcino­mas, adenocarcinomas, and in carcinomas in mixed tumours. The presence of CK in mixed tumours (Caselitz e t al 1981, 1982, 1986b, Krepier e t al 1982, Nathrath e t al 1982, Erlandsson e t al 1984, Saku e t al 1984, Kahn e t al 1985, Palmer e t al 1985), adenolymphomas (Caselitz e t al 1982, 1986, Saku e t al 1984), monomorphic adenomas (Nathrath e t al 1982, Saku e t al 1984), mueoepi dermoid carcinomas (Caselitz e t al 1982), acinic cell carcinoma (Warner e t al 1985), and adenoid cystic carcinomas (Caselitz e t al 1984, 1986b) has previously been demonstrated by im- munohistochemical techniques.

In general, pAbs can be useful for demonstration of cytokeratins as a group, as these antibodies are not dependent on a single antigenic ep i­tope, which may be hidden or a l te red under ce rta in circumstances. How­ever, for the id en t i f ic a t io n of individual cytokeratin proteins the c ro ss - rea c t iv i ty between Cks makes i t almost impossible to identify individual CKs with pAbs. The ideal antibody would, of course, be one against a single cytokeratin . Such antibodies e x is t only for a few cytokeratins (Ramaekers e t al 1985b, Palmer e t al 1985, Cooper e t al 1985) and some are not y e t commercially ava ilab le . Applications of d i f ­fe ren t anti-CK pAbs to reveal individual c e l l s in pleomorphic adenomas have been performed and show d if fe ren t IFP p a t te rns , depending on cell type (Krepier e t al 1982, Nathrath e t al 1982). Erlandsson e t al (1984)

33

used two mAbs against CK but could only show a difference in in ten s i ty , not in sp e c if ic i ty .

At le a s t two tumour cell types could be id en t i f ied immunohistochemical- ly in oncocytomas and adenolymphomas ( I ) . The two cell types displayed d if fe r in g a f f in i t i e s for CK pAbs, viz. one was weakly and the other strongly s ta inab le . The morphological resemblance of the two tumours is thus fu r ther substan tia ted on the immunohistochemical leve l . However, i t does not imply a d if fe ren t origin for the two ce ll types, as poly­clonal antibodies only identify quan ti ta t ive d ifferences.

Three mAbs, all with a well defined sp ec if ic i ty against CKs, typical for simple ep i th e l ia were used in V. In the normal parotid acinar c e l l s , a predominance of CK 18 was generally found, and the same pre­dominance was also observed in ACC. Adenocarcinomas and mu coepidermoid carcinomas showed a preponderance of CKs 17 or 19 and th is tendency was even more pronounced in adenoid cystic carcinomas. This s ta in ing pat­tern is the same as in normal gland ducts or myoepithelial c e l l s . At present, very few investigations have been published concerning mono­clonal antibodies and sa livary neoplasms. Palmer e t al (1985) used a mAb 16a against 45-46 kD CK which stained only basal c e l l s of excretory ducts in normal glands and the outer cell layer of ducts in pleomorphic adenomas. CK 18 has been detected in luminal c e l l s of AdCC tubularce l l s in mixed tumours and oncocytic cell of adenolymphomas by C aselitz e t al (1986b). They a lso used an antibody (CK Bl), which in normal exo­crine glands se lec t ive ly reacts with myoepithelial c e l l s and basalc e l l s of excretory ducts. CK Bl reacts with spindle-shaped and poly­gonal c e l l s in mixed tumours, "myoepithelial" c e l l s of AdCC and cer ta in basal c e l l s in adenolymphomas. The sp ec if ic i ty of the l a t t e r antibody has, however, not yet been defined by immunoblotting.The d if fe ren t s ta in ing re a c t iv i ty could be of value for tumour typing(vide in f ra ) .

Vimentin . For demonstration of vimentin in tumour t i s su e , immunohisto­chemical methods are superior to biochemical techniques. Application of mAbs revealed the presence of v imentin in a number of salivary neo­plasms, viz. mixed tumours, basal cell adenomas, adenoid cystic c a rc i ­

34

nomas, acinic cell carcinomas and ce r ta in carcinomas in mixed tumours; ( I I , I I I , V). Previously, vimentin has only been detected in mixed tu ­mours (Caselitz e t al 1981, 1982, Krepier e t al 1982, Kahn e t al 1985) and adenoid cys tic carcinomas (Caselitz e t al 1984). The antivimentin mAb vim 9 did not reac t with tumour c e l l s of adenocarcinomas. In addi­t io n , most mueoepi dermoid carcinomas and one carcinoma in pleomorphic adenoma were unreactive in th is mAb ( I I I , V). In the l a t t e r neoplasm the malignant par t was tha t of a poorly d i f fe ren t ia ted adenocarcinoma.

A dual filament expression is rare in tumours, benign or malignant, but a coexpression of vimentin and cytokeratins has been described for renal adenocarcinomas, some seminomas, mesotheliomas, Wilm's tumours and ce rta in thyroid carcinomas (Herman e t al 1983, Holthöfer e t al 1983, Waldherr & Schweichheimer 1985, McNutt e t al 1985, Ramaekers e t al 1985a, Miettinen e t al 1984a, b, 1986, Biobei e t al 1985). Para­thyroid neoplasms and a few neuroendocrine tumours have also been found to express CK and neurofilaments simultaneously (Miettinen e t al 1985a, 1985b, Lehto e t al 1985).

C lin ica l ly , the vimentin-negative salivary neoplasms mucoepidermoid carcinoma and adenocarcinoma have an increased tendency to lymphatic node metastasis compared with the vimentin-positive tumours, although both types can give r i s e to d is tan t metastasis . Batsakis (1979) has pointed out th a t adenoid cys t ic carcinomas and acinic cell carcinomas (both vimentin posit ive neoplasms) involve regional lymph by continuous spread fa r more often than by the lymphatic route.

Grawitz tumours of kidney are generally regarded as adenocarcinomas, but as mentioned above about half of these tumours exhib it a CK-vimen- t in coexpression. The tumour exhibits a marked res is tance to chemo­therapy and a tendency for blood vessel invasion and lung métastasés, all features typical of sarcomas (Ramaekers e t al 1985b). Therefore, these facts as well as the IFP pattern prompted Ramaekers e t al (Herman e t al 1983, Ramaekers e t al 1985b) to propose th a t renal cell tumours should be considered as carcinosarcomas. Furthermore, the re la t iv e quan tit ie s of cytokeratin and vimentin in renal carcinoma may be in-

35

dicati ve of whether haematogenous or lymphatic spread is to be expected in a certa in case (Ramaekers e t al 1985b).

Certain vimentin-positive sa livary neoplasms display mesenchymal fea­tu res , and the diagnosis of both mixed tumours and adenoid cystic ca r­cinomas re l ie s on such features: areas of myxoid or chondroid s truc tu re and pseudocysts, respectively . In both these tumours, s tructures have been found to contain glycosaminoglycans of mesodermal type ( I I ) (Azzopardi & Smith 1959, Bloom e t al 1977, 1982, Toida e t al 1985, Lam 1985). However, the corre la tion between l ig h t and u l t ra s t ru c tu ra l mor­phology and filament pattern is not absolute. For carcinomas in mixed tumours ( I I I ) i t was shown th a t a coexpression of both cytokeratins and vi menti n ex is ts in sp i te of a pure ep ith e l ia l or a pure mesenchymal tumour cell d i f fe ren t ia t io n .

Tumour heterogeneity . I t appears more and more evident th a t in many respects the biological behaviour of tumours is controlled by minor tumour cell subpopulations (Yarbro 1985). Not only have differences in morphology and c l in ica l behaviour been described, but also divergent DNA contents have been measured between d if fe ren t tumour cell subpopu­la t io n s .

Studies in v itro have shown th a t d if fe ren t clones are not phenotypical- ly s tab le but tend to d i f fe re n t ia te fu r the r when iso la ted , e .g . as in a recently developed m etastasis . Furthermore, these d if fe ren t cell popu­la t ions in te ra c t , and in general the phenotypic d iv e rs i f ic a t io n of clones declines as the number of subpopulations increases (Fidler & Poste 1985).

In many tumours, prognosis may be re la ted to subtle c h a rac te r is t ic s of subpopulations th a t are not always apparent in h is to logica l sec tions. These tumour cell populations may have a d if fe ren t growth pattern and may require d if fe ren t therapy (Yarbro 1985). The behaviour of subgroups might be re la ted to submicroscopi cal features or expression of, for example, IFP, as has been suggested by Ramaekers e t al (1985b). In the present study, a wide heterogeneity with regard to IFP content within one and the same tumour could be found for several types of neoplasms,

36

sometimes c lea r ly re la ted to other h isto logica l findings. In oncocyto­mas, small elongated c e l l s seem to contain more CKs than others and a higher content of CK was found in c e l l s of the basal layer of Warthin's tumour ( I ) . In AdCC and basal cell adenoma, the peripheral c e l l s ad­jacen t to basement membranes show a dual filament expression ( I I , V), w hils t CK 18 (PKK3) was localized mainly a t the ductal lumina in a num­ber of neoplasms (V). In other neoplasms, however, differences in vimentin content or CK expression could not be re la ted to l ig h t micro­scopical findings. No fundamental differences were found between p r i ­mary or recurrent tumours, nor between primary tumours and métastasés (Y). For the detection of subpopulations, IFP determination alone can­not a t present be of any c l in ica l use, and in the future combined ap­proaches are more l ik e ly . At present, the ap p l icab il i ty of th is im- munohistochemical method is mainly for tumour typing.

In the present stud(y the malignant tumours could be d is tr ibu ted into the following three groups:Group A PKK3 > PKK2 = ACCGroup B PKK2 » PKK3 and vimentin 9 posit ive c e l l s peripherally = AdCC Group C PKK2 > PKK3 and vimentin negative = MEC and adenocarcinomas.

However, future studies concentrated on subpopulations in , for in ­stance, métastasés, may reveal tumour cell l ines responsible for the malignant behaviour and may offer new prospects of determining tumour prognosis.

Aspects of histogenesis as a basis for tumour typing The ap p l icab il i ty of a marker in applied histogenetic studies depends on i t s s t a b i l i t y during neoplastic change. As fa r as the IFP expression is concerned, i t has been generally accepted th a t th is is s tab le during neoplastic changes (Osborn & Weber 1982, 1983, Miettinen e t al 1984b). However, in the l ig h t of recent findings of a dual filament expression in a number of t i s su e s , th is opinion has been questioned. Certain IFP patterns may be expressions of d i f fe ren t ia t io n ra ther than of cell lineage (Cooper e t al 1985).

37

The IFP pattern in sa livary neoplasms is so characterized th a t ce r ta in well defined tumour types express a vim 9 reactive epitope, whereas others do not. In the developing gland a t r ian g u la r vim 9 reactive basal cell can be seen. This observation is in correspondence with the histogenical theories of Eversole (1971) and Batsakis (1980): A vim 9 reactive embryonic p lu ripo ten tia l in te rca la ted duct reserve cell might give r i s e to the vimentin-containing neoplasms, whereas a corresponding vim 9 nonreactive excretory duct cell might be the orig in of the vimen­t i n -negative neoplasms.

However, as no vim 9 pos it ive c e l l s were observed in the normal adu lt gland, the presence of vim 9 pos i t ive c e l l s in ce rta in neoplasms may well be explained by a reexpression of an IFP pattern from an e a r l i e r stage of gland development.

Demonstration of immunohistochemical s im ila r i t ie s between tumour types, such as vimentin expression, th eo re t ic a l ly provides a new po ten tia l ly meaningful basis for tumour c la s s i f ic a t io n (Ramaekers e t al 1985b, Gould 1986). The vim entin-posit ive sa livary tumours seldom give r i s e to regional node métastasés, but have a tendency towards hematogenous spread sim ilar to other vimentin and CK coexpressing tumours (renal adenocarcinomas). The vim entin-posit ive tumours are, furthermore, slow- growing neoplasms. The 5-year prognosis appears good but there i s a tendency for la te recurrences for both tumour types (ACC and AdCC). C lass if ica t ion of sa livary gland tumours relying on objectively demon­s trab le d if fe ren t ia t io n ra the r than presumed cell lineage may give a b e t te r co rre la tion between diagnosis and prognosis (cf Gould 1986). Accepting the biological p roperties of a tumour as a basis for c l a s s i ­f ic a t io n , instead of the presumed h istogenesis , must, however, involve more determinants than ju s t IFP (Ramaekers e t al 1985b).

The stimulus-secretory coupling in acin ic cell cancer The concept of secretory c e l l s includes not only the a b i l i ty to syn­thes ize secretory substances but also the a b i l i ty to discharge them. In ce r ta in salivary neoplasms, various secretory proteins have been found. The main secretory macromolecule of serous sa livary gland is the enzyme amylase. Salivary neoplasms lack most s truc tu res th a t are of im-

38

portance for secretion . Although both glandular and ductal s tructures may be found, no excretory system e x is ts . Even the neoplasms most akin to normal glands, the ACCs, are devoid of s tructu res such as myoepithe­l i a l c e l l s and nerve terminals.

In the present study (VI) tumour t i s su e was found not only to contain amylase, but an amylase discharge could also be induced from tumour t is su e by a common salivary secretagogue - noradrenalin. A concomitant r i s e in cyclic AMP levels as in normal sa livary exocytotic processes could furthermore be documented.

The r e la t iv e q u an ti ta t ive amounts of amylase are lower in tumour t is su e than in the normal parotid . The tes ted ACC were all well d i f fe ren t ia ted tumours with large amounts of granules, as shown by the stereological measurements. In the biochemically tes ted tumours, no myoepithelial c e l l s or neural elements could be detected whatsoever and from a phys­iological point of view i t is in te re s t in g th a t development of a secre­tory apparatus and a secretory response can be e l i c i t e d without the p a r tic ipa tion of innervation or of myoepithelial c e l l s .

Whether th is kind of measurement of biological responsiveness in neo­p la s t ic t is su e will be an aid in tumour typing or in se lec ting t r e a t ­ment remains to be seen.

Measurements of DNA content and determination of long time prognosis The current c la s s i f ic a t io n (WHO 1972) in most cases provides a good aid to prognostication. Some e n t i t i e s , such as anaplastic carcinomas and carcinomas in pleomorphic adenomas, run a rapid downhill course, w hilst adenoid cystic carcinomas exhib it a more protracted course, but end up in a 20-year determinate survival ra te of only 13% (Blanck e t al 1967). Grading of mu eoe pi dermoid carcinomas can give a good idea of the pro­gnosis: high-grade tumours have a cure ra te of 27$, while the corre­sponding figure for low-grade tumours is 92% (Spiro e t al 1978).

Most acinic cell carcinomas behave benignly but a number of neoplasms metastasize and the 20-year survival ra te is no b e t te r than 56% (Ene-

39

roth e t al 1966). A malignant and a benign variant of th is tumour type has been suggested by Kleinsasser e t al (1967) but neither electron microscopy, cytological grading nor c l in ica l staging has given any sup­port for such a d if fe re n t ia t io n . Eneroth & Zetterberg (1976) have de­scribed two i n f i l t r a t i v e ACC with aneuploid c e l lu la r DNA content, sug­gesting th a t DNA measurement could be a possible aid in prognosis of th is p a r t ic u la r tumour. However, in the present study no difference in nuclear DNA content could be found between neoplasms without recur­rences and those with métastasés, e i th e r with modal DNA values, or when the percentage of nuclei with increased DNA content (> 2.5 C) was e s t i ­mated.

In the present study, measurements were made by a s ing le-ce ll technique on smears and sec tions. Since th is method is so time consuming, i t is possible to assess only a limited number of nuclei. Flow cytometry, on the other hand, allows measurement of large numbers of c e l l s .

This has a t le a s t two great advantages:1. Determination of ce ll f rac t ions in individual phases of the ce ll cycle can be performed. The S-phase in p a r t ic u la r has been corre la ted to m itotic index and prognosis, see e .g . Tribukait (1984).

2. There is an increased prospect of detecting a minor cell population with d if fe ren t leve ls of ploidly. I t has been shown (Vindeltfv e t al 1980, Raber e t al 1982, Sjindergaard e t al 1983, Ljungberg e t al 1985) for tumours from a number of t is su e s th a t a s ig n if ican t number of neo­plasms show heterogeneity with respect to nuclear DNA content, and th a t subpopulations may be responsible for th e i r biological behaviour (Fidler & Poste 1985).

Flow cytometry is normally performed on fresh m ateria l, which makes i t an unsuitable technique for re trospec tive long-term s tud ies . However, techniques in th is f i e ld are improving, hopefully opening up new pos­s i b i l i t i e s , as indicated above.

40

GENERAL SUMMARY AND CONCLUSIONS

1. A number of antibodies against various intermediate filament pro­te in s were used to stud|y the morphology and histogenesis of salivary glands and neoplasms.

U ) For normal sa livary glands, all ep i th e l ia l and myoepithelial c e l l s contained CKs. By using mAbs against CK, d if fe ren t patterns were observed in acinar c e l l s , myoepithelial c e l l s and duct c e l l s . All ep i th e l ia l and myoepithelial c e l l s were negative for all the an t i - v imentin antibodies, except for one, mAb vim 24; myoepithelial c e l l s and duct c e l l s being reactive for th is antibody. Fetal c e l l s display the CK pattern of duct c e l l s . Vimentin could in fe ta l gland be detected in basal c e l l s of terminal tubules and buds by all mAbs tes ted .Application of antibodies against IFPs should provide new informa­tion on cytoskeletal s t ru c tu re s . As these s truc tu res have been found to be very s tab le during tumour transformation, th is knowl­edge concerning the cytoskeletal s truc tu re during normal develop­ment and under normal adult conditions may provide a framework for studies on d if fe ren t ia t io n or histogenesis of neoplasms and hope­fu l ly also be of great importance in the investigation of non-neo­p la s t ic d iseases.

(j>) In tumour t i s su e , CKs are found in a ll sa livary ep i th e l ia l t i s su e , te s ted by both pAbs and mAbs. The mAbs-CK stain ing pattern of acinic cell carcinoma is sim ilar to th a t of normal acinar c e l l s , w hilst adenoid cystic carcinoma, mucoepidermoid carcinoma and adenocarcinoma showed e sse n tia l ly the same pattern as duct or myo­ep i th e l ia l c e l l s .Vimentin was demonstrated by a mAb (vim 9) in acinic cell carcinoma (ACC), adenoid cys t ic carcinoma (AdCC), monomorphic adenomas, pleo­morphic adenomas and carcinoma ex mixed tumours - e ssen tia l ly those tumours claimed to a r is e from in te rca la ted duct reserve c e l l s . Vimentin 9 reac tive c e l l s were found in the terminal parts of the developing gland duct system. This finding is consis ten t with two p o s s ib i l i t i e s with regard to tumour development.

41

1. An origin of vim 9 posit ive neoplasms from a pi uri potential re ­serve cell of the peripheral duct system.2. The vim 9 re ac t iv i ty of these neoplasms is due to a dedifferen­t ia t io n and reexpression of an IFP pattern found during an e a r l i e r period of gland development.However, the former theory requires an additional documentation of vim 9 reactive c e l l s p e rs is ten t into adulthood.Furthermore, vimentin expression can be lo s t for mixed tumours during mal igni f i ca tion . The u l t ra s t ru c tu ra l morphology and IF pat­tern in carcinomas in mixed tumours do not always co r re la te .By combining mAbs against CKs and vimentin (PKK2, PKK3 and vim 9) malignant salivary neoplasms could be divided into three groups with d if fe ren t s ta in ing c h a rac te r is t ic s :- ac inic cell carcinomas- adenoid cys tic carcinomas- adenocarcinomas and mucoepidermoid carcinomas.

(£) The sa livary neoplasms often showed heterogeneous cell populations with respect to IFP content. This phenomenon could partly be r e la ­ted to morphology. In oncocytomas the strongest CK-Abs reaction was found in small elongated tumour c e l l s , in Warthin's tumours the basal c e l l s displayed a more intense CK sta in ing than the columnar c e l l s . Furthermore, in AdCC, tumour c e l l s apposing basement mem­branes exhibited a coexpression of CKs and vimentin. In other neo­plasms, heterogeneity was not re la ted to any difference in l ig h t microscopic morphology. The significance of such differences has h i the r to not been studied, but they may increase our knowledge in the f ie ld of tumour heterogeneity and clonal in te rac tion and might also be a factor of importance when determining the degree of ma­lignancy.

2 .U) In well d i f fe ren t ia ted acinic cell carcinomas, tumour c e l l s marked­

ly resembling normal acinar c e l l s were frequently encountered. In certa in cases only minor d ifferences could be detected even by u l t ra s t ru c tu ra l stereological measurements. Furthermore, an ade­quate secretory stimulus response was obtained a f te r stimulation

42

with noradrenalin, i . e . an increase in in t ra c e l lu la r cAMP and re ­lease of arçylase. Thus, the development and function of the secre­tory process in sa livary acinar c e l l s does not seem to be dependent on myoepithelial c e l l s or innervation.

(b) Microspectrophotometric analysis of DNA in acinic cell carcinomas was compared with the prognosis. All tumours investigated were d i­ploid and there were few hyperploid or aneuploid c e l l s in all tu ­mours. At present, DNA measurements do not seem to have any predic­t iv e value for prognosis concerning th is tumour type.

43

ACKNOWLEDGEMENTS

I would l ik e to express my sincere gra ti tude to all those, mainly a t . the Departments of Otorhinolaryngology - Head and Neck Surgery, Histo­logy and Cell Biology, and Anatomy, who guided, ass is ted and supported me during my thes is work. In p a r t ic u la r , I wish to express my gra ti tude to:

Professor Matti Anni ko, Head of the Department of Otorhinolaryngology - Head and Neck Surgery, for providing excellent working conditions and for his personal support.

Associate Professor Bengt Carlsöö, my tu to r , who in i t i a t e d the work with th is th e s is . His in te re s t in and knowledge of u l t ra s t ru c tu re and salivary glands inspired to a number of s tudies.

Professor Lars-Eric Thornell, my tu to r , who introduced me to the f ie ld of immunohistochemistry, for his never f a i l in g in te re s t and support.

Associate Professor Anders Eriksson, Associate Professor Roger Henriks­son, Doctor Uno K jöre ll , Doctor Christer Lindholm, and Professor Ismo Virtanen, for pleasant co llaboration .

Professor Gunnar D. Bloom, former Head of the Department of Histology, who introduced me in to the world of science.

Ann-Louise Grehn, Siv Domeij, Monica Andersson, Lena Söderberg, Eva Wikman, Örjan Stenman and Kent Persson for sk i lfu l technical a s s i s t ­ance.

Andrea Lotz, Ingrid Nilsson and Loi ornai Örnehult for excellent secre­ta r i a l ass is tance .

Harmeth Sjögren and Max Brandt for thoroughly l in g u is t ic revision of the manuscripts.

This work was supported by grants from the Lions Fund, The Mangberg Fund and the Medical Faculty, University of Umeå.

44

REFERENCES

Azzopardi JG, Smith OD. Salivary gland tumours and th e i r mucins. J Path Bact 1959;77:131-40.

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