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Romanian Journal of Morphology and Embryology 2005, 46(3):211–223

Tumoral stroma of salivary pleomorphic – histopathological, histochemical and immunohistochemical study

CL. MĂRGĂRITESCU1), M. RAICA2), CRISTIANA SIMIONESCU1), L. MOGOANTĂ3), M. SURPĂŢEANU4), F. JAUBERT5), FL. BOGDAN6)

1)Department of Pathology, Emergency County Hospital, University of Medicine and Pharmacy of Craiova 2)Department of Cytology and Histology, “Victor Babeş” University of Medicine and Pharmacy, Timişoara 3)Department of Histology, University of Medicine and Pharmacy of Craiova 4)Department of Oral Maxilla Facial Surgery, Emergency County Hospital, University of Medicine and Pharmacy of Craiova 5)Department of Pathology, “René Descartes” University Paris V, Faculty of Medicine, “Necker Enfants Malades” Hospital, France 6)Research Centre for Microscopic Morphology and Immunology, University of Medicine and Pharmacy of Craiova

Abstract The aims of our paper were to establish the main histopathological, histochemical and immunohistochemical aspects of tumoral stroma from salivary pleomorphic . For this purpose we investigated 103 cases by the classical histopathological technique with paraffin embedding and staining with Hematoxylin–Eosin (HE), Hematoxylin–Eosin–Safranin (HES), trichromic Masson, trichromic Goldner Szeckelly, orcein and Periodic Acid Schiff–Blue Alcian (PAS–AA). Immunohistochemically, they were investigated for AE1–AE3, MNF116, CK8, EMA, vimentin, α-actin calponin, S-100, GFAP, collagen IV, and PCNA. The results of our study suggest the key role of neoplastic in the achievement of diverse morphological aspects of stroma in such . Keywords: pleomorphic adenoma, tumoral stroma, histochemistry, immunohistochemistry.

 Introduction comprises 30–50% of the tumor), type II (stroma comprises ~80% of the tumor), type III (stroma The pleomorphic adenoma is a benign comprises ~20–50% of the tumor) and type IV (stroma epithelial , histologically characterized by a attains similar proportion to that of type III, but there is great diversity of morphological aspects. It is the most focal monomorphic differentiation in the epithelial common neoplasm of salivary gland origin; its component) [3]. incidence varies in different statistics from 45% to 74% The stroma of these tumors may have diverse of all the salivary gland tumors [1–4]. appearance: myxoid, chondroid, chondro-myxoid, At the Armed Forces Institute of Pathology (AFIP) hyaline, fibrous, sclero-hyaline and, very rarely, osseous the pleomorphic adenomas represent 60 % of the benign and adipose. These aspects are usually associated tumors from all the salivary gland sites: 61% of with the predominance of one of them in variable the major gland tumors and 54% of the minor gland proportion [5–10]. tumors [5]. The pathognomonic stromal feature of pleomorphic Its structural pleomorphism is given both by the adenoma is the presence of chondro-myxoid stroma. epithelial component, as a result of the cytological Most pleomorphic adenomas, particularly the long- differentiations and the growing patterns, and by the standing lesions present variable amounts of elastic stromal component because of its rich morphological fibers, which are uncommon in other salivary gland and quantitative diversity [5, 6]. tumors [11, 12]. Pleomorphic adenoma is thinly encapsulated. Crystalloids composed of collagenous substance; Prominently myxoid tumors often have incomplete tyrosine and oxalate are more often present in capsules, and tumor tissue is juxtaposed against normal pleomorphic adenoma than in any other salivary gland. This is characteristic of pleomorphic adenoma in gland neoplasm: their incidence varies from 1.5 to the minor glands, where a capsule is rarely well 21% [13–16]. developed. Extracellular stroma is one of the defining  Material and methods components of pleomorphic adenoma, ranging from scanty to abundant. According to the relative proportion Our study comprised 103 cases of pleomorphic of stroma and cellular components Seifert subclassified adenomas selected during 10 years, from 1992 until pleomorphic adenoma into four types: type I (stroma 2001.

212 Cl. Mărgăritescu et al. The surgical specimens were provided by the Oral representing 63.1%), with a female to male ratio of Maxilla Facial Surgery Department of the Emergency 1.71 to 1. County Hospital Craiova, mostly from 15–76 year-old The tumors developed especially in the major females (65 cases), with prevalent parotid localization salivary glands (76 cases in the parotid, 16 cases in the (77 cases). submandibular glands and two cases in the sublingual The surgical specimens were routinely fixed with gland, all representing about 92% of our casuistry). 10% buffered formalin and sent to the Laboratory of The parotid was the most frequently involved gland Cytology and Pathology of the same hospital. (74%). The minor salivary glands were involved They were processed by the classical histopathological in eight cases at the level of the lower (three cases), technique with paraffin embedding and stained with (two cases) and palatine mucosa Hematoxylin–Eosin (HE), Hematoxylin–Eosin–Safranin (three cases). (HES), trichromic Masson, trichromic Goldner Histopathological, histochemical and Szeckelly, orcein and Periodic Acid Schiff–Blue Alcian immunohistochemical aspects of pleomorphic (PAS–AA). adenomas investigations The immunohistochemical process was made in the Histological, Histopathological and Immuno- At the tumoral periphery, the stromal tumoral histochemical Techniques Laboratory from component developed a capsule with variable thickness “Victor Babeş” University of Medicine and Pharmacy which did not separate the tumor completely from the Timişoara, Department of Cytology and Pathology of rest of the salivary gland (Figure 1a). the Hospital “Necker Enfants Malades” from Paris and Thus, from 77 typical pleomorphic adenomas with in the Histological, Histopathological and Immuno- parotid localization 53 presented a capsule that histochemical Techniques Laboratory from University completely surrounded the tumor but with variable of Medicine and Pharmacy of Craiova. thickness in different zones of the tumor. We use DAKO LSAB® 2 System, HRP technique From 16 pleomorphic adenomas with submandibular for AE1–AE3, MNF116, CK8, EMA, vimentin, α-actin localization only 10 were completely encapsulated. and S-100, and ABC/HRP technique for calponin, In all the other cases with parotid, submandibular and GFAP, collagen IV, and PCNA. sublingual localization the tumors were partially The tumors have been diagnosed according to WHO encapsulated, in some regions the tumor coming in classification [17]. direct contact with the surrounding glandular In most cases was made an internal control parenchyma. In three cases, we have noticed some (a positive reaction of the fragments that were placed at tumoral prolongation sent through the capsule into the a suitable distance away from the tumoral tissue). surrounding parenchyma (Figure 1b). As in immunohistochemistry there is no standard or We have not identified the capsula in any of the universal accepted methods to select the histological cases of pleomorphic adenomas with origin in the minor fields in order to interpret the results (with the exception salivary glands of the oral cavity. In a single case of of PCNA) we used the criteria from literature data [18]. pleomorphic adenoma we have noticed small neoplastic The intensity of the immunostaining reaction has cell groups with myoepithelial morphology, but without been described as follows: atypia between the collagen fibers that composed the ▪ (+++), when the immunostaining reaction is tumoral capsula (Figure 1c). intense positive or ”all over” specific distributed, According to the relative proportion between the obvious at small magnification; stromal and the parenchyma tumoral components, the ▪ (++), when the immunostaining reaction is focal or 103 cases of pleomorphic adenomas have been with moderate intensity, evident on average classified in four subtypes (Table 1). magnification; Table 1 – Repartition of the casuistry according to relative ▪ (+), when the immunostaining reaction is weak or proportion between tumoral stroma and parenchyma very focal, visible only at strong magnification; Subtypes of Type Type Type Type No. of all ▪ (±), when the immunostaining reaction is very pleomorphic adenomas I II III IV cases reduced, to limit; No. of cases 57 25 12 9 103 ▪ (−), when the immunostaining reaction is negative. Percentage 55% 24% 12% 9% 100%

 Results In 57 cases of pleomorphic adenomas with stromal predominance the epithelial component was reduced, Epidemiological aspect representing about 20% of the tumoral mass. The neoplastic epithelial proliferations had trabecular, There were selected 103 pleomorphic adenomas tubular and insular patterns; these small cellular groups cases from 283 salivary gland lesions, diagnosed during were often disposed at the periphery of the myoid 1992–2001. stromal component or inside it. In some tumoral areas, In our study the mean age at presentation was the hyaline stromal component created a cylindromatous 48 years, but the age ranges from the second to eight aspect, which required a differential diagnosis with decades. The patients were mostly females (65 cases, adenoid cystic , its tubular variant.

Tumoral stroma of salivary pleomorphic adenoma – histopathological, histochemical and immunohistochemical study 213

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(e) (f) Figure 1 – Typical pleomorphic adenoma: (a) type I – capsula (trichromic Masson staining, ×40); (b) type II – tumoral capsula traversed by a tumoral prolongation (HE staining, ×40); (c) type I – presence of small groups of neoplastic cell into the tumoral capsula (HE staining, ×40); (d) type I – scan image of the histopathological specimen (HES staining); (e) type II – scan image of the histopathological specimen (HES staining); (f) type III – scan image of the histopathological specimen (HES staining) 214 Cl. Mãrgãritescu et al.

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(e) (f) Figure 2 – Typical pleomorphic adenoma of type I: (a) zones with myxoid stromal component and isolated myoepithelial cells with stellate morphology (HE staining, ×200); (b) myxoid aspect of the stromal component with abundant acid mucopolysaccharides and scant neutral mucopolysaccharides (Blue Alcian–PAS–Hematoxylin staining, ×40); (c) myxoid zones, vimentin (+) of the myoepithelial cells with stellate morphology (×400); (d) myxoid zones, GFAP (++) immunostaining of the stellate cells (×400); (e) myxoid zones, AE1/AE3 (+ -) of the isolated stellate cells (×400); (f) myxoid zones, both cytoplasm and nuclear S-100 (+ +) immunostaining of the stellate cells (×200) Tumoral stroma of salivary pleomorphic adenoma – histopathological, histochemical and immunohistochemical study 215 Here and there, inside the myxoid component, there present at the limited areas was micro-, macrocystic, were present myoepithelial cells with stellate shape, cribriform and pseudoangiomatous. The stromal isolated or grouped in small clusters. The stromal component was reduced, representing no more than component realized about 80% of the tumoral mass 20–30% of the tumoral mass. and was mainly represented by the myxoid stroma The most frequent encountered stromal (Figure 1d). differentiation was of myxoid type. In small proportion There were also present other stromal there were associated other stromal differentiations like differentiations, generally in small quantities, but in those of fibrous, fibro-hyaline or fibro-myxoid type. variable proportion from case to case (chondro-myxoid, In 9 cases of typical pleomorphic adenomas the chondroid and fibro-hyaline differentiations). epithelial component predominated, but it presented In 25 cases of pleomorphic adenomas with well- focal monomorphic differentiations of squamous balanced stromal / parenchyma proportion the epithelial (three cases), oncocytic (one case), basaloid (one case), component represented about 50% of the tumoral mass fusiform-myoepithelioma like (two cases) and (Figure 1e). plasmacytoid (one case). The dominant tumoral pattern was tubular and solid- These epithelial differentiations were described at insular. Another pattern was trabecular and fascicular. the cytological differentiations of the epithelial tumoral The stromal component representing about 50% of the component. The stromal component of these tumors was tumoral mass had mainly myxoid, chondro-myxoid and reduced and mainly represented by the myxoid and chondroid differentiations, more often associated. fibro-hyaline differentiation. On the limited areas there have been noticed fibrous, The histopathological study of the 103 typical fibro-hyaline stroma and in one case even an osteoid pleomorphic adenomas revealed the existence of the stromal differentiation. following stromal aspects: myxoid, chondroid, chondro- In 12 cases of typical pleomorphic adenomas the myxoid, hyaline, fibrous, sclerohyalin and osteoid epithelial component predominated, the neoplastic (Table 2). epithelial proliferation realized 70–80% of the tumoral None of these kinds of stromal component was mass (Figure 1f). exclusive in the studied tumors. Most commonly they The most frequent encountered patterns were the were associated in variable proportion, prevailing one of tubular and the insular ones. Another tumoral pattern them. Table 2 – Incidence of the major types of the tumoral stromal component The stromal Myxoid* Chondroid Fibro-hyaline Osteoid No. of all cases component aspects No. of cases 70 45 20 1 103 Percentage 51% 33% 15% 1% 100% *especially the cases in which the myxoid differentiation predominated, because this type of tumoral stroma was present in all cases. The most frequent stromal component had a myxoid In the myxoid matrix, scattered or grouped in small aspect. Thus, in all the 103 cases of typical pleomorphic nests, there were also present modified myoepithelial adenomas we have noticed myxoid stroma but in cells designated as myxoid cells. variable proportion from one case to another. They had a stellate morphology: reduced, basophilic The myxoid stroma prevailed in only 45 cases. cytoplasm, oval, normochromic, central placed nucleus, The myxoid stromal zones were weak basophile, with morphofunctionally modified for mucopolysaccharides variable extension, poor delimited and disposed synthesis (Figure 2a). between the tumoral epithelial structures (Figure 2a). Immunohistochemically all these cells were negative These tumoral areas were rich in ground substance to MNF116, CK8, EMA and calponin, but they which contained a great quantity of acid presented a weak to moderate positive immunoreaction mucopolysaccharides and small quantity of neutral for vimentin and GFAP (Figures 2c and 2d). mucopolysaccharides (Figure 2b), mixed in variable Peculiarly, the modify myoepithelial cells (myxoid proportion (positive for Blue Alcian and variable cells) were weakly positive to AE1–AE3, S-100 and positive for PAS). α-actin (Figures 2e and 2f). The cells from these tumoral zones were rare. They The collagen fibers were usually absent. It has been were mesenchymal cells, reticular cells and modify occasionally noticed a fine network of collagen fibers. myoepithelial cells. The mesenchymal cells were young, Immunoreaction to collagen IV was negative in these undifferentiated connective cells with stellate shape, areas. These zones were often associated with the much prolongation, anastomose between them, chondroid areas, which appeared to differentiate from abundant, light basophile cytoplasm and a big, round the myxoid areas. The chondroid tumoral areas with euchromatic nucleus with 2–4 nucleoli. The reticular variable extension have been noticed in 20 cases but cells were also young connective cells but with a predominated in only eight cases. Histologically, these smaller size than the mesenchymal cells, with a reduced, zones corresponded to some differentiations of basophilic cytoplasm and a round, normochromic, immature or mature, hyaline, cartilaginous type and had central placed nucleus with obvious nucleoli. round to oval shape and poor definite limits.

216 Cl. Mărgăritescu et al. Most frequently the chondroid zones had the aspect The osteoid areas were given by an eosinophil of an immature, hyaline (Figure 3a), where the ground substance with vague lamellar disposition and ground substance had an acidophilic aspect, rare rare osteoblasts-polyhedral cells that have small chondroid lacunae with small, oval chondroblasts cytoplasmic processes, basophilic cytoplasm, (with basophilic cytoplasm, round hypochromatic, PAS positive and round, normochromic, nucleolated central placed nucleus with obvious nucleoli). and central placed nucleus. The stromal zones with mature, hyaline, In only one case we have found crystalloids, cartilaginous differentiation were rare (Figure 3b). presented between neoplastic epithelial proliferations These areas had an abundant extracellular matrix and in the stromal component. They were radially rich in mucopolysaccharides (Figure 3c), with arranged, “petal-shaped”, blunt-ended clusters of accentuate basophile around the chondroid lacunae but eosinophilic tubular structures more probable being acidophilic in the rest, containing a small quantity of tyrosine crystalloids (Figure 4f). mucopolysaccharides but more collagen fibers. Generally, the chondroid lacunae had one or no  Discussions more than two chondrocytes (chondroid lacunae with diad). The chondrocytes were round when isolated and Our study reveals that only 68.31% of pleomorphic adenomas developed in the parotid glands were columnar when disposed in group of two cells; they had clear or weak acidophil cytoplasm, great, hypochromic, completely encapsulated and other 3% of had tumoral round to oval nucleus, nucleolated and placed central or process extended in the normal salivary parenchyma. little eccentric. In the literature quantitative study showed that thickness Immunohistochemically in these areas we noticed a of pleomorphic adenoma capsule ranged from 15–1750 mm [19]. positive reaction for vimentin (with variable intensity from moderate to strong) (Figure 3d), S-100 (an intense When tumors were serially sectioned areas of both cytoplasmic and nuclear reaction) (Figure 3e) and capsular deficiency were seen in all cases [20]. In predominantly mucoid pleomorphic adenomas, the GFAP (a moderate cytoplasm immunoreaction) (Figure 3f), all other reaction were negative. capsule may be virtual-absent and the tumor abuts onto Most of the chondroid differentiations were the adjacent salivary gland. The extension of pleomorphic adenomas into the surrounding tissues surrounded by the myxoid stroma, fact that suggested the possibility of chondroid metaplasia of the myxoid through the capsule can be interpreted as separate stroma. satellite nodules, but they are invariably attached to the main tumor by an isthmus [20, 21]. Another aspect of the stromal component encountered in 12 cases was fibrous, fibro-hyaline or The extension beyond the capsule of these tumors sclerohyalin, represented by fascicles of collagen fibers without nucleo-cytoplasmic atypia and atypical mitotic figures should not be interpreted as a sign of with variable thickness (thicker in sclerohyalin variant), disposed in epithelial proliferations and often suffering malignancy. The tumoral tissue in the capsule may be hyaline dystrophy and even calcium precipitations misunderstood as a sign of malignancy, but in these cases the atypia characteristic to malignant cells was (Figures 4a and 4b). This kind of stroma predominated in only 5 cases, absent. the age of these patients was advanced and these tumors According to the relative proportion between the had a longer evolution. The hyaline aspect was also stromal and the epithelial component of these tumors we present as eosinophil, acellular, homogenous, fine found that 55% of them were of type I, 24% were of type II, 12% were of type III and 9% were of type IV. fibrilar bands scattered through the epithelial structure or surrounding the neoplastic ductal structures, which Seifert G. et al. first submitted such histo- created a cylindromatous aspect for these tumoral pathological classification of the pleomorphic adenomas in 1976. Considering the tumoral stroma / parenchyma regions. This aspect was encountered in two cases of proportion, these authors classified the pleomorphic pleomorphic adenomas with stromal predominance. adenomas in four subtypes: subtype I (30.5% of the cases, in which there were included pleomorphic Few fibroblast cell from these areas were positive only to vimentin, all other immunostaining were negative. adenomas with 30% to 50% of the tumoral stroma), In the old pleomorphic adenomas, we have also subtype II (55% of the cases, with 80% of the stromal component), subtype III (9% of the cases, with 20% to remarked the prevalence of elastic fibers in the tumoral stroma component, highlighted by orcein staining. 30% of the tumoral stroma) and subtype IV (5.5% of the These fibers were scattered through epithelial cases, with poor represented tumoral stroma in a similar proportion to that from subtype III but with focal proliferations like the irregular compact masses or the irregular thin strands (Figures 4c and 4d). monomorphic epithelial differentiations) [22]. In only one case, we have noticed an osteoid This distinction has no therapeutic significance but helps emphasize the broad morphologic spectrum differentiation of the stromal component. In these tumoral areas, we have remarked the presence of possible within this neoplasm. Other observation of our osteoid trabeculae in tight connection with neoplastic study reveals the great diversity of morphological epithelial proliferations (Figure 4e). aspects of stroma in these tumors.

Tumoral stroma of salivary pleomorphic adenoma – histopathological, histochemical and immunohistochemical study 217

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(e) (f) Figure 3 – Typical pleomorphic adenoma of type I: (a) chondroid zones, immature hyaline cartilage (HE staining, ×40); (b) chondroid zones, mature hyaline cartilage (HE staining, ×40); (c) chondro-myxoid zones, immature hyaline cartilage (Blue Alcian–PAS staining, ×40); (d) chondroid zones, vimentin (++) of the lacunar cells (×400); (e) chondroid zones, S-100 (+++) immunostaining (×200); (f) chondroid zones, GFAP (++) immunostaining (×400) 218 Cl. Mãrgãritescu et al.

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(e) (f) Figure 4 – Typical pleomorphic adenoma: (a) type II – fibro-hyaline zones (HE staining, ×100); (b) type II – sclerohyalin zones (HE staining, ×40); (c) old pleomorphic adenomas – elastic fibers in the tumoral stroma (Orcein staining, ×40); (d) old pleomorphic adenomas – irregular thin strands of elastic fibers scattered through epithelial proliferations (Orcein staining, ×100); (e) osteoid differentiation of the stromal component (HE staining, ×100); (f) “petal-shaped” crystalloids between neoplastic epithelial proliferations (HE staining, ×100) Tumoral stroma of salivary pleomorphic adenoma – histopathological, histochemical and immunohistochemical study 219 The most frequent stromal type was the myxoid one, The detection in the ductal cells, by in situ encountered in all cases of pleomorphic adenomas but hybridization, of the genes involved in the aggregan in variable proportion from case to case. This kind of core protein mRNA synthesis and which was positive to stromal component appeared in 51% of the cases. cytokeratins, demonstrated the expression of the Other benign or malign salivary gland tumors may mesenchymal genes into cells with epithelial origin. have on restricted areas tubular epithelial proliferations This fact suggests that the neoplastic ductal cells were associated with myxoid or edematous stroma, as in the involved in the myxoid matrix production from the canalicular adenomas or the polymorphous low-grade stromal areas of the pleomorphic adenomas. adenocarcinomas. In such cases, making seriated This process was at the origin of the structural sections and correlating the histopathological data with pleomorphism of such tumors and represented a the clinical ones it may be avoided a wrong diagnosis. furthermore argument for the single-cell theories, most We also have to bear in mind the tumors of soft probably a myoepithelial origin of all the neoplastic tissue with an important myxoid component such as the cells from these tumors. As well, Ogawa Y. et al., , the myxofibroma, the myxoid and the showed that in the myxoid zones of the pleomorphic myxoid neurofibroma. These tumors with mesenchymal adenomas, the CK14 immunoreaction had a sporadic origin were very rare in the salivary gland character, limited to some tumoral cells with stellate levels, representing about 1.9–5% of all the salivary morphology [30]. tumors [23, 24]. According to Brennan P. A. et al., α- Histochemically, we noticed the prevalence of acid actin in the pleomorphic adenoma is expressed only by mucopolysaccharides in the myxoid areas. When the normal myoepithelial cells and by the so-called referring to the different mucin types present in the modify myoepithelial cells. The weak positive pleomorphic adenomas, the review data have specified immunoreaction of some cells from the myxoid areas the existence of two-mucin types: one of epithelial may be explained by the possible origin of the cells nature with a high content of neutral glycoprotein and from the myofibroblasts [30]. another one of mesenchymal origin rich in sulphated According to Anderson C. et al., the GFAP and non-sulphated glycosaminoglycan [7–9]. immunostaining is present only in the stromal cells from The latter was produced by the modified the myxoid and chondroid tumoral areas [31]. myoepithelial cells depending on the tissue culture and Achtstatter T. et al. consider that the reactivity of the the inoculation studies [25–27]. myoepithelial cells to GFAP is the consequence of a Zhao M et al. in a study concerning the cross-reaction to the intermediate filaments with the glycosaminoglycans (GAGs) localization in same structure and chemical composition as the glial pleomorphic adenoma reveals that both epithelial and fibrillary acidic protein [32]. mesenchymal-like tissues contain GAGs [28]. Ostrzega N. et al. consider that the positive GFAP Myxoid region was consistently stained for both immunoreaction helps in the differential diagnose of the chondroitin 6-sulfate and hyaluronic acid but variably pleomorphic adenomas from the low-grade for chondroitin 4-sulfate, dermatan sulfate and keratan polymorphous adenocarcinoma, where this reaction is sulfate; heparan sulfate was not detected. The chondroid absent [33]. region showed increased staining for chondroitin Study attempt by Aigner Th. and Neureiter D. had 6-sulfate but reduced staining for hyaluronic acid when showed that small quantities of collagen I, III and V compared with the myxoid region. In addition, were present in the myxoid areas and more abundant in chondroitin 4-sulfate, dermatan sulfate and keratan the fibrous zones and tumoral capsule [25, 29]. sulfate were seen both in chondroid cells and the Luna M. G. had proved that type I and II collagens territorial matrix, whereas HS was present only in the were co-distributed in the extracellular matrix of cells. It is suggested that GAGs in PA are mainly myxoid tissue in pleomorphic adenomas [34]. produced by non-luminal cells and influence the In the chondroid tissue, variable immunostaining proliferation, differentiation, secretory activity and patterns of type I and II collagens were also observed. shape of tumor cells, thus contributing to the Some proliferating myoepithelial cells in myxoid and morphological diversity of this tumor [28]. chondroid tissue were immunostained with anti-alpha- The greater hyaluronic acids composition of the smooth muscle actin antibody indicating their pleomorphic adenomas comparative to those from the myoepithelial origin. These results suggested that may be used in the differential proliferating myoepithelial cells may be the origin of the diagnose of these salivary gland tumors [8]. chondroid tissue and the expression of type IX collagen Immunohistochemically we notice the presence of in cartilaginous tissue precedes the expression of type I modify myoepithelial cells (positive to AE1–AE3, and II collagens [34]. S-100 and α-actin) in the myxoid areas, involved in the The second most encountered stroma was the genesis of these areas by epithelial-mesenchymal chondroid (33% of the cases) and chondro-myxoid type, transdifferentiation process. Aigner T. et al. had proved especially the immature, hyaline, cartilaginous one. that the cells from the tubular neoplastic areas had only The tumors rich in chondroid and chondromyxoid epithelial characteristics, while the cells from the stroma imposed a differential diagnosis with tubulo-myxoid zones had a mixed phenotype [29]. cartilaginous type tumors, such as the chondroma or the

220 Cl. Mărgăritescu et al. chondroblastoma, especially in the cases developed on essential roles in chondroid formation in salivary the rest of the heterotopic salivary parenchyma. pleomorphic adenoma [41]. Also, we observed that the chondroid differentiations Thus, CDMP-1 was immunolocalized in the were surrounded by the myxoid stroma, fact that cuboidal neoplastic myoepithelial cells around the suggested the possibility of chondroid metaplasia of the chondroid areas of the pleomorphic adenomas, whereas myxoid stroma. these molecules were not localized in the spindle- According to Aigner Th. et al. the chondroplastic shaped neoplastic myoepithelial cells of the myxoid cells derive from stellate cell of the myxoid areas of the element or the lacuna cells of the chondroid element in tumor, which through mucopolysaccharides synthesis these tumors. developed cartilaginous areas in the pleomorphic CDMP-2 was expressed neither in normal salivary adenomas [29]. glands nor any of the elements of the pleomorphic This fact was sustained by ultrastructural and adenomas. Type-II collagen and aggrecan were immunohistochemical arguments [29, 35–38]. immunolocalized throughout the matrix around the Kusafuka et al. have reported that the genesis of the lacuna cells of the chondroid element, whereas type-X chondroid stromal areas are associated with the collagen was not immunolocalized in any epithelial or overexpression of the bone morphogenetic proteins stromal elements, including the chondroid elements. (BMPs) by the neoplastic myoepithelial cells [39]. Aggrecan was deposited not only on the chondroid This protein belongs to the transforming growing matrix, but also on the myxoid stroma and intercellular factor (TGF)-β superfamily which interferes with the spaces of the tubulo-glandular structures, whereas regulation of the mesenchymal tissue formation. chondromodulin-I was deposited on the chondroid The TGF-β2 is expressed by the neoplastic ductal matrix. The phenotype of the lacuna cells was similar to epithelial cells, while TGF-β3 is expressed by the that of mature to upper hypertrophic chondrocytes of the neoplastic myoepithelial cells from the solid neoplastic authentic cartilage. Also, Kusafuka et al. epithelial proliferating areas, the metaplastic squamous immunohistochemical investigated the expression in cells and the luminal cells from the tubular neoplastic pleomorphic adenomas of two non-collageneous bone proliferating areas. TGF-β is involved in the control of matrix proteins: osteonectin (OSN – glycoprotein the luminal and myoepithelial neoplastic cell involved in the early steps of the mineralization of differentiation [39]. skeletal tissue) and osteopontin (OPN – protein Moreover, immunohistochemical investigation of involved in normal and pathological calcifications) [42]. bone morphogenetic protein-2 (BMP-2) and type II These authors showed a moderate positivity for OSN collagen, two cartilage-associated proteins, made by in the inner layer of tubulo-glandular structures and Zhao M. et al. demonstrated that in pleomorphic modified myoepithelial cells from myxoid areas. adenomas BMP-2 immunoreactivity was detected in the OSN was expressed in all of the lacuna cells in the luminal and non-luminal cells of the tubulo-ductal chondroid areas. OPN was strongly expressed in the structures, plasmacytoid cells, and other scattered tumor stroma of the myxoid and hyaline areas of the cells in solid areas [40]. pleomorphic adenomas, but there was no expression of In addition, tumor cells in chondroid areas in most OPN in the chondroid area. cases, and stellate cells in myxoid areas in many cases, All cases of pleomorphic adenomas expressed type were also intensely labeled for BMP-2. Type II collagen IV collagen. These findings suggested that OSN was was localized in the intercellular matrix of chondroid related to the production of the type IV collagen by areas and in a few chondroid differentiating cells in modified myoepithelial cells, whereas OPN was myxoid areas, confirming its cartilage-specificity. involved in the stromal formation of myxoid or hyaline A proportional relationship was observed between tissues in pleomorphic adenomas [42]. BMP-2 expression and chondroid formation, although Investigation of lumican (a keratan sulphate BMP-2 was also stained in occasional pleomorphic proteoglycan that belongs to the small leucine-rich adenomas without chondroid formation. It is speculated repeat proteoglycans reported to be associated with that BMP-2 might be secreted by tumor cells and play a cartilage formation) in pleomorphic adenomas role in chondroid formation in pleomorphic adenomas demonstrated that this proteoglycan is associated with by inducing some tumor cells to produce type II the formation of “mesenchyme”-like structures in this collagen and other chondroid matricial substances, like type of tumour [43]. glycosaminoglycans. Immunohistochemistry firstly performed with pepsin The expression of BMP-2 is specific to pleomorphic pretreatment, proved that lumican was predominantly adenoma and may possibly be used as a useful marker deposited in the hyaline and fibrous areas and littler in in differentiating pleomorphic adenomas from other the chondroid areas. Without pepsin pretreatment, salivary gland tumors [40]. lumican was identified in myoepithelial cells in myxoid Kusafuka et al. proved by immunohistochemical areas, lacuna cells in chondroid areas, and in the investigation that cartilage-derived morphogenic cytoplasm of inner ductal cells. In situ hybridization proteins (CDMP)-1 and -2 (proteins that belong to the revealed lumican mRNA expression mainly in the bone morphogenetic protein (BMP) family, in the inner cells, the neoplastic myoepithelial cells, and the transforming growth factor (TGF)-β superfamily) play lacuna cells.

Tumoral stroma of salivary pleomorphic adenoma – histopathological, histochemical and immunohistochemical study 221 The fibrous and fibro-hyaline stromal component Skalova A. had showed that extracellular predominated in 15% of the cases. In about 2% of the collagenous crystalloids from pleomorphic adenomas cases, the hyaline was disposed like the homogenous, contain types I and III collagen but not type II, IV, or VI eosinophilic strand around the tubular epithelial collagen [54]. Moreover, cells surrounding collagenous proliferations realizing morphological aspects similar to crystalloids expressed the basement membrane the tubular variant of the adenoid cystic ; molecules laminin and type IV collagen. that is why it was necessary to make a differential As far as the tyrosine crystalloids are concerned, the diagnosis with this entity. review data have specified that they are structures In literature, it has been specified that the existence stained with Millon reagent and not with specific of such modifications even in the limited tumoral areas collagen staining. It has also been specified that some of imposes a multiple section performing and in the these structures would contain much more arginine than absence of atypia, we may conclude that it may be a tyrosine [14, 55] and that they are the result of the [44]. collagen products precipitation secreted by the modified Some long-standing tumors show increasing myoepithelial tumoral cells [56]. hyalinisation and the epithelial component is Tumors that have a lipomatous stromal component progressively effaced. It is important, however, to of 90% or more have been called lipomatous scrutinize the residual epithelial elements of such old, pleomorphic adenomas [57–60]. scarred pleomorphic adenomas as there is a significant In such case we must bear in mind the existence of risk of malignant progression in such tumors [45]. other fat-containing tumors (such as lipoadenoma, Immunohistochemically studies proved that collagen , interstitial lipomatosis, and benign IV and laminin were more intensively localized in mesenchymoma). Occasional, following spontaneous hyaline, fibrous and chondroid types of stroma, whereas infarction or fine needle aspiration can be observed heparan sulfate proteoglycan was more prominent in extensive inflammation and necrosis. In such tumors myxoid areas [46]. may be an increase in mitotic figures and some cellular The results suggest that the stroma contains these atypia [61]. basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that  Conclusions histological variety of the stroma depends on proportion of local contents of each basement Our study demonstrated a great diversity of membrane molecule [46]. morphological aspects of stroma in these tumors; the In such fibrous areas using orcein staining, we have most frequent stromal type being the myxoid one. noticed a great quantity of elastic fibers. Review data The histochemically and immunohistochemically have specified that the presence of these fibers is investigations confirm the key role of myoepithelial relatively characteristic to the pleomorphic adenomas cells in stromal histogenesis. because these fibers may seldom be observed in other Thus, neoplastic myoepithelial cell differentiate types of salivary gland tumors [12, 47, 48]. to stellate cells, which produce myxoid matrix, We have found the osteoid stromal differentiation in generating stromal myxoid areas and farther these one only 1% of the cases. The review data have quoted few differentiate to chondroplastic cells, which through cases of pleomorphic adenomas with osteoid stromal muco-polysaccharides synthesis develop cartilaginous differentiations [5, 10, 49]. areas in the pleomorphic adenomas. In such cases there was observe a tendency of osteoid calcification that realized lacy bony trabeculae References with basophile tint, rare bony lacunae with osteocyte [1] ENEROTH C. M., Salivary gland tumors in the , , and the palate region, Cancer, 1971, (oval cells with reduce pale basophile or even acidophil 27:1415–1418. cytoplasm, oval, normochromic, nucleus, central [2] EVESON J. W., CAWSON R. A., Salivary gland tumours. placed). Still some authors have indicated the presence A review of 2410 cases with particular reference to of morphogenetic bony protein in high percentage in the histological types, site, age and sex distribution, J Pathol, 1985, 146:51–58. pleomorphic adenomas [40–42, 50, 51]. [3] SEIFERT G., MIEHLKE A., HAUBRICH J., CHILLA R., Diseases In about 1% of the cases, we have noticed tyrosine of the salivary glands: diagnosis, pathology, treatment, crystals both in the stromal component and among the surgery, Georg Thieme Verlag, Stuttgart, 1986, epithelial proliferations. According to review data, the 65–130. [4] SPIRO R. H., Salivary neoplasms: overview of a 35-year incidence of such crystalloid structures in pleomorphic experience with 2807 patients, Head Neck Surg, 1986, adenomas is about 1.5–21% [13–16]. 8:177–184. Besides these tyrosine crystalloids, the review data [5] ELLIS G. L., AUCLAIR P. L., (pleomorphic have specified the existence of other two types adenoma). In: Tumors of the salivary glands. Atlas of Tumor Pathology, chapter 4, section 4.1, 3rd series, fascicle 17, of collagen crystalloids (eosinophil, fibrilar structures Armed Forces Institute of Pathology, Washington, 1996, with radial disposal around some annular spaces 39–57. made up of by and sanguine vessels) [6] CHEUK W., CHAN J. K. C., Salivary gland tumors. and crystalloids structures similar to the calcium In: FLETCHER D. M. (ed), Diagnostic histopathology of tumors, chapter 7, volume I, 2nd edition, Churchill oxalate [13, 52, 53]. Livingstone, London, 2000, 231–312.

222 Cl. Mărgăritescu et al.

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Mailing address Claudiu Mărgăritescu, Assistant Professor, M. D., Ph. D., Department of Pathology, Faculty of Dentistry, University of Medicine and Pharmacy of Craiova, Street Petru Rareş no. 2–4, 200 349 Craiova, Romania; Phone +40251–523 654, E-mail: [email protected], [email protected]

Received: 25 November, 2005

Accepted: 10 December, 2005