[CANCER RESEARCH 59, 5815–5821, November 15, 1999]
Bone Morphogenetic Protein-6 Is a Marker of Serous Acinar Cell Differentiation inNormal and Neoplastic Human Salivary Gland1
A. Kristiina Heikinheimo, 2 Merja A. Laine, Olli V-P. Ritvos, Raimo J. Voutilainen, Brigid L. M. Hogan, andIlmo V. LeivoInstitute of Dentistry, University of Turku, FIN-20520 Turku, Finland [A. K. H., M. A. L.], Haartman Institute, Department of Bacteriology and Immunology, University ofHelsinki, FIN-00014 Helsinki, Finland [O. V-P. R.], Haartman Institute, Department of Pathology, University of Helsinki, FIN-00014 Helsinki, Finland [R. J. V., I. V. L.],Department of Pediatrics, Kuopio University Hospital, FIN-70210 Kuopio, Finland [R. J. V.], and Department of Cell Biology, Vanderbilt University School of Medicine,Nashville, Tennessee [B. L. M. H.]
ABSTRACT
Bone morphogenetic protein (BMP-6, also known as vegetal-pale-gene-related and decaplentaplegic-vegetal-related) is a member of the trans-forming growth factor- b superfamily of multifunctional signaling mole-cules. BMP-6 appears to play various biological roles in developing tissues,including regulation of epithelial differentiation. To study the possibleinvolvement of BMP-6 in normal and neoplastic human salivary glands,we compared its mRNA and protein expression in 4 fetal and 15 adultsalivary glands and in 22 benign and 32 malignant salivary gland tumors.In situ hybridization and Northern blot analysis indicated that BMP-6transcripts are expressed at low levels in acinar cells of adult submandib-ular glands but not in ductal or stromal cells. BMP-6 was immunolocatedspecifically in serous acini of parotid and submandibular glands. Nonewas found in primitive fetal acini or any other types of cell in adultsalivary glands, including mucous acini and epithelial cells of intercalated,striated, and excretory ducts. All 16 cases of acinic cell carcinoma consis-tently exhibited cytoplasmic BMP-6 staining in the acinar tumor cells.Other cell types in these tumors, including intercalated duct-like cells,clear, vacuolated cells, and nonspecific glandular cells, exhibited no cyto-plasmic BMP-6 staining. Other benign and malignant salivary glandtumors lacked BMP-6 immunoreactivity, except in areas of squamousdifferentiation. The results indicate that in salivary glands, BMP-6 ex-pression is uniquely associated with acinar cell differentiation and suggestthat BMP-6 may play a role in salivary gland function. More importantly,our experience of differential diagnostic problems related to salivarygland tumors suggests that the demonstration of consistent and specificBMP-6 immunoreactivity in acinic cell carcinoma is likely to be of clinicalvalue.
INTRODUCTION
Tumors of the salivary glands account for about 5% of all neo-plasms of the head and neck (1). These tumors originate primarily inthe parotid gland, occasionally in the submandibular and intraoralglands, and rarely from the sublingual gland (2). The parotid gland iscomposed of serous acini, and the submandibular gland is composedof mixed serous and mucous acini. The sublingual and intraoral glandsconsist mainly of mucous acini. Salivary gland tumors present ahistomorphological diagnostic challenge to clinical pathologists. Theyare characterized by substantial complexity of morphological featuresand overlapping histological patterns in different tumor entities. Afurther problem is a lack of markers applicable in diagnostic histopa-thology. Little is known about specific transcription and growthfactors involved in human salivary gland tissue morphogenesis andcytodifferentiation (3–7). Identification of such molecules through
basic research is likely to furnish potential new tools for improvingmolecular diagnostics in relation to salivary gland tumors.
The TGF-b3 superfamily of .50 structurally related cytokinesincludes factors important in cell growth and differentiation (8, 9).Apart from TGF-bs, members of this multigene family include mem-bers of the activin/inhibin subunit gene family, members of thedecaplentaplegic-Vg-related gene family, including BMPs, and theMullerian inhibitory substance. TGF-b family members signalthrough heterodimeric complexes of type-I and type-II transmembra-nous serine/threonine kinase receptors (10). Downstream signaling istransduced by unique proteins of theSmadgene family (11). Duringorganogenesis, TGF-b family members regulate cell growth and dif-ferentiation, and in adult tissues, they regulate regeneration and ho-meostasis (9). Some members of the TGF-b family act in a wide rangeof tissues. Others are restricted to specific cell types. For example,BMP-2 and BMP-4 are essential for early embryogenesis (12, 13),BMP-7 is required for eye and kidney development (14, 15), andgrowth and differentiation factor-9 is needed for ovarian function andfemale fertility (16).
BMP-6 (also known as Vgr-1 and DVR-6) is specifically expressedin suprabasal layers of the epidermis, central nervous system, andhypertrophic cartilage (17–19).In vitro, BMP-6 has been shown toinhibit cell division, to promote terminal epithelial differentiation, andto induce endochondral bone formation, osteoblastic differentiation,and neuronal maturation (19–22). Recent BMP-6 gene targeting ex-periments in mice have revealed no overt defects in tissues known toexpress BMP-6, suggesting that other BMPs may compensate for theloss of BMP-6 in such tissues (23). The overexpression of BMP-6function has been implicated in relation to occurrence of psoriasis(24). In prostatic adenocarcinoma, induction of BMP-6 expression hasbeen associated with tumorigenesis (25–28) and the formation ofosteosclerotic deposits in metastatic progression (25, 28). BMP-6mRNA has recently been shown to be expressed in a human salivaryadenocarcinoma cell line (HSG-S8) but not in oral squamous cell,gastric, rectal, bladder, signet-ring cell, or thyroid carcinoma cell lines(29).
Because little was known about the role of TGF-b family membersin human salivary gland differentiation, we initiated a series of studiesaimed at characterizing expression of TGF-b family members indeveloping and adult salivary glands and in salivary gland tumors. Wereport here that BMP-6 is uniquely expressed in normal and neoplasticacinar cells of serous phenotype, thereby providing a new molecularmarker for use in diagnostic histopathology of acinic cell carcinoma.
MATERIALS AND METHODS
Fetal and Adult Salivary Glands. Four fetal and 15 adult salivary glandspecimens were investigated (Table 1). Fetal submandibular gland tissues(15th to 20th gwk) were obtained in connection with legal abortions induced
Received 5/28/99; accepted 9/21/99.The costs of publication of this article were defrayed in part by the payment of page
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1 Supported by the Finnish Cancer Organization, the Finnish Dental Society, and theAcademy of Finland.
2 To whom requests for reprints should be addressed, at Department of Oral andMaxillofacial Surgery, Institute of Dentistry, University of Turku Lemminkaisenkatu 2,FIN-20520 Turku, Finland. Phone: 358-2-333-81; Fax: 358-2-333-8356; E-mail:[email protected].
3 The abbreviations used are: TGF-b, transforming growth factor-b; BMP, bonemorphogenetic protein; gwk, gestational week.
using prostaglandins. The study had been approved by the Ethical Committeeof Helsinki Maternity Hospital, Finland. Normal adult salivary gland tissuesamples were obtained in connection with surgery of the head and neck regionafter permission had been obtained from the Ethical Committee of TurkuUniversity Central Hospital, Finland. The patients were between 19 and 93years of age and had received no long-term medication or radiotherapy beforesurgery.
Salivary Gland Tumors. Twenty-two benign and 32 malignant salivarygland tumors were studied. Tumor samples were obtained during surgicalremoval of tumors in Helsinki University Central Hospital, Finland and clas-sified in accordance with the WHO classification of salivary gland tumors (2).The tumors were mostly primary tumors of the parotid gland (Tables 2 and 3).
Tissue-Sample Preparation.Fresh, normal and neoplastic tissue sampleswere frozen promptly in liquid nitrogen and stored at270°C until subjected toRNA analysis. For routine histology and immunohistochemistry, specimenswere fixed in 10% neutral buffered formalin and embedded in paraffin.
Five-micrometer sections from each tumor were cut on aminoalkylsilane-treated glass slides (Sigma Chemical Co., St. Louis, MO) for immunostainingand histological staining with H&E. Forin situhybridization, cryostat sections(5 mm) were cut from two submandibular gland and two acinic cell carcinomasamples (Table 3, cases 1 and 3) and mounted on glass slides treated with a 2%solution of 3-aminopropyltriethoxysilane (Sigma; Ref. 30).
Probes. A sample of 242-bp human BMP-6 cDNA (31) in a pGEM-3ribovector was kindly donated by Dr. Vicki Rosen (Genetics Institute, Cam-bridge, MA). For in situ hybridization, linearized plasmid templates werelabeled with [35S]a-UTP (1000 Ci/mmol; Amersham, Aylesbury, Bucking-hamshire, United Kingdom) by means ofin vitro transcription using SP6 or T7RNA polymerases (Promega Biotech, Madison, WI; Ref. 32). For Northernblot analysis, a 500-bp cDNA fragment was obtained by reverse transcription-PCR using 59AC(A/G)-GC(A/C)-ACT-AGC-AAT-CTG-TGG-39 and 59(G/A)CA-GCA-(C/T)GG-TTT-GGG-GAC-(G/A)TA-39primers and U2OS-cell-RNA-derived cDNA as a template. The cDNA was subcloned into a pGEM-Tvector and verified by sequencing. A single-stranded antisense BMP-6 cDNAprobe was generated by linear amplification as previously described (33) usingthe purified insert as a template and the above-mentioned antisense oligonu-cleotide as a primer. Theb-actin cDNA was labeled with [32 P]d-CTP (6000Ci/mmol; Amersham) by means of random priming using an Oligolabeling Kit(Pharmacia LKB, Uppsala, Sweden).
RNA Extraction and Northern Blot Analysis. Total cellular RNA wasextracted from eight normal (four submandibular, two parotid, and two sub-lingual) salivary glands, four benign salivary gland adenomas (three pleomor-phic adenomas, one basal cell adenoma), and one acinic cell carcinoma aspreviously described (34). Poly(A)1 RNA was further extracted using thePoly(A) Track Kit (Promega) according to the manufacturer’s instructions. Forcontrol purposes, poly(A)1 RNA was extracted as above from HT1080 and
Table 1 Immunodetection of BMP-6 polypeptides in developing and adult normalhuman salivary glands with antibodiesa DVR-6 (18) to proregion of murine BMP-6
14. 57 M Sl 215. 75 M Sl 216. 49 M Sl 217. 39 M Sl 218. 25 F Sl 219. 35 F Lmg 1b
a Smg, submandibular gland; Par, parotid gland; Sl, sublingual gland; Lmg, labialminor gland;2, all cells negative; M, male; F, female.
b All serous acini positive, all mucous acini negative, occasional ductal cells positive.c All serous acini positive, occasional ductal cells positive.
Table 2 Immunodetection of BMP-6 polypeptides in benign salivary gland tumors
Diagnosis/case no. Age Sex Location BMP-6
Pleomorphic adenoma1. 39 Fa Par 2b
2. 68 F Par 2b
3. 31 M Par 2c
4. 44 F Par 2b
5. 27 F Par 26. 55 F Par 2d
7. 45 F Par 2c
8. 45 F Par 2c
9. 22 M Par 210. 71 M Par 2c,d
11. 65 M Par 212. 19 F Par 2c
Warthin’s tumor13. 48 M Par 2c
14. 49 M Par 215. 50 M Par 2c
16. 50 M Par 217. 69 F Par 2
Basal cell adenoma18. Trabecular/solid 43 F Par 219. Trabecular 43 M Par 220. Trabecular 47 F Par 221. Trabecular/tubular 77 F Par 222. Tubular/solid 49 F Par 2
a F, female; Par, parotid gland;2, all tumor cells negative; M, male.b Weak extracellular staining in some stromal areas.c Areas of squamous metaplasia positive.d Occasional adluminal epithelial ductal tumor cells positive.
Table 3 Immunodetection of BMP-6 polypeptides in malignant salivary gland tumors
Diagnosis/case no. Age Sex Location BMP-6
Acinic cell carcinoma1. Solid 40 Fa Par 112. Papillary-cystic 72 M Par 113. Solid 19 F Par 114. Solid 37 M Par 115. Microcystic 44 M Par 116. Microcystic 45 M Par 117. Solid 80 F Bmg 118. Microcystic 35 M Par 119. Microcystic 36 M Par 11
10. Papillary-cystic 79 M Par 1111. Microcystic 34 M Par 1112. Solid-microcystic 64 M Par 1113. Solid 73 M Par 1114. Microcystic 70 F Par 1115. Microcystic 63 F Par 1116. Papillary-cystic 93 M Par 11
Adenoid cystic carcinoma17. Glandular (cribriform) 44 F Par 2b
18. Tubular 54 F Par 2b
19. Solid/glandular (cribriform) 66 F Par 220. Glandular (cribriform) 21 F Par 2
Mucoepidermoid carcinoma21. 70 F Par 2c
22. 35 M Par 2d
23. 46 F Par 224. 40 M Par 225. 62 F Par 2c
26. 35 M Par 227. 60 F Par 228. 25 F Par 229. 68 F Par 230. 65 F Par 2c
Polymorphous low gradeadenocarcinoma31. 28 F Par 232. 87 F Pmg 2
a F, female; Par, parotid gland;11, all acinar tumor cells positive; M, male; Bmg,buccal minor gland, Pmg, palatal minor gland;2, all tumor cells negative.
U2OS cell lines as well as from fetal kidneys (16–17 gwk) and livers (15–16gwk). For Northern blot analysis, poly(A)1 RNA samples (3mg) were run ina 1.5% agarose gel, transferred onto a nylon filter, and hybridized as previouslydescribed (34).
In Situ Hybridization. In situ hybridization was performed on cryostatsections as previously described (34). Briefly, before hybridization, frozensections were fixed in 4% paraformaldehyde (PFA)/5 mM MgCl2, rinsed inTris-buffered saline, and treated with proteinase-K (0.5mg/ml) for 5 min. Thereaction was stopped by adding glycine (0.1M in PBS), and the sections werepostfixed in 4% PFA/5 mM MgCl2, rinsed in 50% formamide/23SSC at roomtemperature, and acetylated. The prehybridization and hybridization steps wereperformed according to Miettinen and Heikinheimo (34). After posthybridiza-tion washes and RNase A (Sigma) and T1 (Boehringer Mannheim, Mannheim,Germany) treatments, the sections were subjected to autoradiography by dip-ping the slides into film emulsion (NTB-2; Eastman Kodak Co., Rochester,NY) and exposed for up to 5 weeks at 4°C. After developing the slides (D-19developer; Kodak), the sections were counterstained in Harris’ hematoxylinand assessed using dark- and light-field microscopy.
Immunohistochemistry. Affinity-purified polyclonal antibodies,a DVR-6,were used for immunolocation of BMP-6 polypeptide on cryostat sections andformalin-fixed, paraffin-embedded tissue sections. Thea DVR-6 antibodies wereraised against the proregion of a mouse glutathione-S-transferase/BMP-6 fusionprotein (18). Thea DVR-6 antibodies are specific for human, mouse, and chickenDVR-6/BMP-6. Immunostaining was performed using a Vectastain Elite ABCstaining kit (Vector Laboratories, Burlingame, CA), with diaminobenzidine tetra-hydrochloride as the chromogen. Endogenous peroxidase was inactivated bytreating the sections with 10% hydrogen peroxide in methanol for 5 min. Toenhance the availability of the antigenic determinants, sections were pretreatedusing an antigen retrieval system (BioGenex Laboratories, San Ramon, CA). Thesections were treated with 10% normal goat serum in PBS for 30 min at roomtemperature followed by the primary antibody, diluted 1:100 (a DVR-6) in 1%normal goat serum in PBS, and incubated overnight at14°C. Sections werecounterstained in Mayer’s hematoxylin and mounted with Permount (FisherChemicals, Fair Lawn, NJ). The specificity of the immunoreaction was monitoredby replacing the primary antibodies with the corresponding nonimmune sera.
RESULTS
BMP-6 mRNA in Normal and Neoplastic Salivary Glands.Northern blot analysis revealed a single weak band of expected size (4kb) for BMP-6 in two submandibular gland samples and in threetumor samples representing two pleomorphic adenomas and one basalcell adenoma (Fig. 1). Control mRNA from samples includingHT1080 and U2OS cell lines and a human fetal kidney tested positive.Human fetal liver tested negative.In situ hybridization analysis de-tected low levels of BMP-6 mRNA in acinar cells of normal subman-dibular gland epithelium (Fig. 2,A andB). No BMP-6 transcripts were
detected in ductal epithelial cells. The BMP-6 sense probe gave onlya background signal (Fig. 2C). In acinic cell carcinoma, no BMP-6hybridization signal above background level could be detected byinsitu hybridization (data not shown).
BMP-6 Polypeptides in Normal Salivary Glands.Fetal subman-dibular glands, including primitive acini, tested negative (data notshown). In the parotid glands, prominent, uniformly positive cytoplas-mic staining patterns were seen in all serous acini (Fig. 3A). Occa-sional BMP-6-immunoreactive cells were also seen in striated andexcretory ducts of various sizes. Most ductal epithelial cells, however,tested negative. In submandibular (Fig. 3B) and intraoral minorglands, all serous acini or serous portions of mixed acini were con-sistently BMP-6-immunoreactive. All mucous acini and mucous por-tions of acini tested negative. Striated and excretory ducts of the
Fig. 1. Expression of BMP-6 mRNA in adult submandibular glands (Lanes AandB)and salivary gland adenomas (Lanes C–E). Northern blot hybridization with32P-labeledsingle-stranded BMP-6 cDNA probe (exposure time, 2 weeks) shows a weak single bandat 4 kb in the mRNA fraction isolated from the submandibular gland (Aand B),pleomorphic adenoma (C andD), and basal cell adenoma (E). Poly(A) 1 fractions fromHT1080 (F), U2OS (G) cells, and human fetal kidney (H) are positive. Poly(A)1 frac-tions from fetal liver (I) are negative. A cDNA probe forb-actin giving a band of 2 kb wasused as a control for RNA integrity.
Fig. 2. BMP-6 mRNA in an adult submandibular gland, which was detected usinginsitu hybridization.A andB, a weak positive hybridization signal is seen in acinar cells.Cells of intercalated and striated ducts are negative (arrows).C, dark-field illumination ofa section corresponding toA reveals a lack of specific signals when hybridized with asense probe. Exposure time (A–C), 5 weeks.Bars (A–C), 200mm.
submandibular gland exhibited occasional BMP-6-positive cells. NoBMP-6 immunoreactivity was observed in the sublingual gland (Fig.3C). All ducts in these glands also tested negative.
BMP-6 Polypeptides in Benign Salivary Gland Tumors.Ex-pression of BMP-6 was studied in 12 cases of pleomorphic adenoma,most of which showed only little staining (Table 2). In two cases,BMP-6 was occasionally found in adluminal epithelial cells of ductalstructures. In five cases, distinct positivity was noted in areas ofsquamous metaplasia (Fig. 3D). In three cases, extracellular stainingwas seen in some stromal areas, although no cellular source of BMP-6could be detected in these cases.
Monomorphic adenomas studied included Warthin’s tumors andbasal cell adenomas. Five cases of Warthin’s tumor were studied. Nopositivity was seen in the oncocytic epithelium or lymphocyticstroma. In two of these tumors, however, small areas of squamousmetaplasia were noted in the oncocytic epithelium. These displayedpositive staining for BMP-6 (Fig. 3E). We also studied five cases ofbasal cell adenoma and found no staining for BMP-6.
BMP-6 Polypeptides in Malignant Salivary Gland Tumors.Sixteen cases of acinic cell carcinoma were stained for BMP-6 (Table3). They included five solid tumors, seven tumors with microcysticpatterns of growth, three with papillary-cystic patterns, and one witha solid-microcystic pattern. All exhibited consistent strong cytoplas-mic staining in acinar tumor cells (Fig. 4,A–D; Fig. 5,A andB). Noneof the other cell types in these tumors, including intercalated duct-likecells, clear, vacuolated cells, and nonspecific glandular cells werepositive. In the papillary-cystic variant, only low amounts of posi-tively stained acinar cells were detected among the nonspecific glan-dular cells and intercalated duct-like cells (Fig. 5,A andB). In any ofour acinic cell carcinomas, there was no apparent correlation foundbetween the intensity of lymphocytic inflammatory infiltrate typicalof these tumors and amounts of BMP-6.
Four cases of adenoid cystic carcinoma were studied. They in-cluded two tumors with glandular (cribriform) patterns, one with a
solid/glandular pattern, and one with a tubular pattern. No staining forBMP-6 was seen in the tumor cells of the two glandular (cribriform)cases (Fig. 4E). One case of tubular and one glandular (cribriform)type of adenoid cystic carcinoma exhibited weak positive staining inoccasional adluminal epithelial cells; the surrounding myoepithelialcells were negative (Fig. 4F).
Ten cases of mucoepidermoid carcinoma were stained for BMP-6.Six exhibited no positivity for the protein. In two cases, stratifiedsquamous carcinoma tumor cells were positive for BMP-6. In oneother case, the squamous carcinoma tumor component was positive,although no stratification was found. In one case, individual well-differentiated squamous carcinoma cells occasionally tested positive(Fig. 4G). In all mucoepidermoid carcinomas, the mucin and mucouscell components and the intermediate cells consistently tested nega-tive for BMP-6.
Two cases of intraoral polymorphous low-grade adenocarcinomawere studied. No positivity for BMP-6 was seen in either case. Aconsistently negative staining pattern was observed in all specimensstudied when the primary antibodies were replaced by nonimmunecontrol sera (Fig. 4D).
DISCUSSION
BMP-6 was immunolocated in this study, specifically in serousacini of human adult parotid and submandibular glands. Fetal aciniand all other cell types in adult salivary glands, including mucousacini and epithelial cells of intercalated, striated, and excretory ducts,tested negative. The level of BMP-6 transcription in the acinar cellswas, however, low, as measured byin situhybridization and Northernblot analysis. Alternatively, the half-life of the transcript was short.Studies have shown that BMP-6 regulates growth and differentiationof epithelial tissue (18, 20, 24). In newborn mouse skin, BMP-6 waslocated in suprabasal but not in basal cell layers, suggesting that itsexpression is associated with terminal differentiation of keratinocytes
Fig. 3. Immunolocation of BMP-6 polypeptide insalivary glands (A–C) and salivary gland adenomas(D–E).A, in the parotid gland, all serous acini exhibitstrong positive immunoreaction with BMP-6 anti-bodies. Myoepithelial and stromal cells are negative.B, in the submandibular gland, staining for BMP-6 isseen in serous acini and serous portions of mixedacini. All mucous acini, mucous portions of mixedacini, and ducts are negative.C, the sublingual glandremains negative.D andE, positive immunoreactionfor BMP-6 is evident in areas of squamous metapla-sia of pleomorphic adenoma (D) and Warthin’s tu-mor (E). Hematoxylin counterstain (A–D).Bars, 100mm (A–C) and 200mm (D andE).
(18). In vitro, BMP-6 inhibited cell growth and induced keratinocytedifferentiation, suggesting that BMP-6 function is a primary step inkeratinocyte differentiation (20). It is concluded that in adult humansalivary glands, BMP-6 expression is characteristic of serous acinarcells, which are highly specialized epithelial cells with a secretoryfunction. BMP-6 originating in the saliva may be needed for theregeneration of oral mucous membranes, as demonstrated duringkeratinocyte differentiation (20).
The results of the work described here show that the 22 benignsalivary gland tumors included in the study mostly lacked BMP-6immunoreactivity. Occasional areas of squamous metaplasia in 5/12of the pleomorphic adenomas and 2/5 of the Warthin’s tumors exhib-ited positive BMP-6 staining. This finding is in agreement withfindings from previous work indicating a coexpression of BMP-6 withcytokeratin pair 1/10 of the suprabasal cytokeratins typical of corni-fying stratified epithelia,e.g.,in skin (20). Two cases of pleomorphic
Fig. 4. Immunolocation of BMP-6 in malignant salivary gland tumors.A–C, in acinic cell carcinoma, a positive immunoreaction with BMP-6 antibodies is seen in the acinar tumorcells. Other cells, including intercalated duct-like cells, nonspecific glandular cells, and lymphocytic inflammatory cells, are negative.D, control section stained with normal rabbitserum is negative.E, adenoid cystic carcinoma of the glandular (cribriform) type is negative, but serous acinar cells of the normal parotid gland exhibit positive immunostaining withBMP-6 antibodies.F, weak positive immunoreaction is seen in occasional adluminal epithelial cells of a case of a tubular type of adenoid cystic carcinoma.G, individualwell-differentiated squamous cells in a case of mucoepidermoid carcinoma are positive for BMP-6. Hematoxylin counterstain (A–G).Bars, 100mm (A–D) and 200mm (E–G).
adenoma and one case of basal cell adenoma expressed low levels ofBMP-6 transcripts as detected by Northern blot analysis. The positivesignal most likely originated from serous acini of the adjoining normalgland. Results of previous studies have suggested that BMP-2 inparticular is responsible for the formation of the chondroid matrix inpleomorphic adenoma (29, 35–37). In the study described here, noBMP-6 immunoreactivity was detected in the chondroid areas of thepleomorphic adenomas, suggesting that BMP-6 expression in suchtumors is associated more with epithelial than with chondroid differ-entiation.
In the various malignant salivary gland tumors we studied, all 16cases of acinic cell carcinoma consistently exhibited distinct cytoplas-mic BMP-6 staining in the acinar tumor cells. Other cell types,including intercalated duct-like cells, clear, vacuolated cells, andnonspecific glandular cells, tested negative. The level of BMP-6transcription in the neoplastic acinar cells was, however, lower than intheir normal cellular counterparts as measured byin situhybridizationand Northern blot analysis. Alternatively, BMP-6 mRNA degradationcould be faster in the neoplastic acinar cells. The remaining malignantsalivary gland tumors mostly lacked BMP-6 immunoreactivity, withthe exception of 2/4 of the adenoid cystic carcinomas and 4/10 of themucoepidermoid carcinomas in which occasional areas of BMP-6immunoreactivity were detected in adluminal epithelial ductal tumorcells and in squamous tumor epithelium, respectively. Acinic cellcarcinoma is the second most frequently encountered malignant epi-thelial tumor of the parotid gland after mucoepidermoid carcinoma.Histologically, acinic cell carcinoma displays cytological differentia-tion toward acinar cells. The parotid gland is the predominant site ofoccurrence (81% of cases). The results we report show that BMP-6immunoreactivity, which is found in normal serous acinar cells,persists in the malignant counterparts of these cells, making BMP-6 auseful new marker of acinic cell carcinoma in diagnostic histopathol-ogy. In our experience of differential diagnostic problems relating tosalivary gland tumors, this marker has already proved to be of clinicalvalue.
Recent studies have shown that BMP-6 is expressed in humanmalignant prostatic tissues and in benign prostatic hyperplasia, espe-cially in basal cells and areas of basal-cell hyperplasia, suggesting thatBMP-6 may be involved in the differentiation of prostatic epithelialcells and in the genesis of prostatic hyperplasia and adenocarcinoma(25, 27, 28). It has been also suggested that BMP-6 function inmalignant prostatic tissue may in part be responsible for the formationof calcified deposits in metastatic tumor sites in bone (25, 28).However, the findings in our study on acinic cell carcinoma show thatBMP-6 expression need not have any association with hard-tissue
formation or metastasis. It would be of interest to find out whetherBMP-6 activity is related to the formation of the lymphatic infiltratetypical of acinic cell carcinoma in a manner analogous to the chemo-tactic attraction of monocytes by BMP-3 and BMP-4 (38).
Overall, we have shown that BMP-6 is uniquely associated with theserous acinar cell phenotype in normal and neoplastic human salivaryglands. This shows that expression of certain members of the TGF-bsuperfamily may be characteristic of distinct cell types in both normaland neoplastic tissues. We conclude that BMP-6 is a useful newmarker of acinic cell carcinoma in diagnostic histopathology.
ACKNOWLEDGMENTS
We are grateful to Dr. Vicki Rosen (Genetics Institute Inc., Cambridge,MA) for her donation of human BMP-6 cDNA. The technical assistance ofRitva Koutu-Koskinen, Ritva Javanainen, Merja Haukka, and Jarmo Koskinenis acknowledged.
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Fig. 5. Close-up of a case of acinic cell carcinoma with papillary-cystic growth pattern.A, part of papillary structure with mostly nonspecific glandular cells and some microcysticlumina. Some acinar cells are pointed out (arrows). H&E stain.B, immunolocation of BMP-6 in the adjacent section. Positive immunoreaction is seen in a number of acinar cells(arrows). Hematoxylin counterstain.Bars, 200mm (A andB).
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1999;59:5815-5821. Cancer Res A. Kristiina Heikinheimo, Merja A. Laine, Olli V-P. Ritvos, et al. GlandCell Differentiation in Normal and Neoplastic Human Salivary Bone Morphogenetic Protein-6 Is a Marker of Serous Acinar
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