The histological diversity of adenoid cystic carcinoma demonstrated by double immunohistochemistry for p16 and p63 gene products

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1 Orl Med Pthol 16 (2011) 9 The histologicl diversity of denoid cystic crcinom demonstrted y doule immunohistochemistry for p16 nd p63 gene products Mkoto Tnk, Mshiro Wto, Akio Tnk Deprtment of Orl Pthology, Osk Dentl University, Hirkt, Jpn Astrct Bckground: Adenoid cystic crcinom (AdCC) displys morphologicl diversity within its chrcteristic tumor cell nests, which re primrily clssified into tuulr, cririform, nd solid ptterns. Such histologicl diversity should result from the proportionl prolifertion etween neoplstic myoepithelil cells (NMC)/sl cells nd ductl epithelil cells. The im of this study ws to clrify the reltionship etween this morphologicl diversity nd the development of AdCC in the slivry glnd using doule immunostining with monoclonl ntiodies to p16 nd p63 gene products. Methods: We exmined 10 smples of formlin-fixed, prffin-emedded AdCC tissues. Results: In the norml slivry glnd, p16 protein (P16) ws not expressed in ny cells, wheres p63 protein (P63)-positivities were oserved in ductl sl cells nd in cinr myoepithelil cells. In AdCC foci showing the tuulr pttern, P16 expression ws loclized in the inner cells of the ductl structure, while P63 ws loclized in the outer NMC of them within the sme sections. In tumor foci with the cririform pttern, P16 ws expressed in severl cells forming pseudocysts nd in some of the inner cells. P63 ws expressed in mny cells in the cncer nests. In those with the solid pttern, P16 nd P63 were intermixed within the sme foci. Conclusions: Thus, the doule immunohistochemistry for P16 nd P63 ws useful for oserving the morphologicl diversity of AdCC cells within the sme foci on the sme tissue section. These dt suggest tht P16 nd P63 my ply importnt roles in the morphologicl diversity nd development of AdCC tissue rchitectures. [Orl Med Pthol 2011; 16: 9-13 doi: /omp.16.9] Key words: denoid cystic crcinom, immunohistochemistry, p16, p63, slivry glnd Correspondence: Mshiro Wto, Deprtment of Orl Pthology, Osk Dentl University, 8-1, Kuzuhhnzono-cho, Hirkt , Jpn Phone: , Fx: , E-mil: wto@cc.osk-dent.c.jp Introduction Adenoid cystic crcinom (AdCC) is the second most common slivry glnd crcinom nd occurs most often in the hrd plte mong tumors of minor slivry glnd origin. AdCC foci show vriety of histologicl ptterns tuulr, cririform, nd solid. In slivry glnd tumors which re considered to develop from the interclted duct, neoplstic myoepithelil cells (NMC) or sl cells hve een postulted to ply importnt roles in forming tumor-specific tissue rchitectures (1-4). p16 is tumor suppressor gene locted on chromosome 9p21. p16 INK4 is cell cycle inhiitor tht is commonly inctivted in humn tumors nd tumor cell lines. p16 overexpression is found in cervicl cncer of the uterus with immunohistochemicl stining. Aerrnt methyltion of the p16 gene nd loss of expression re consistent with the multistep progression of peripherl-type lung denocrcinom nd slivry glnd crcinom (5-8). In the slivry glnds, the expression of p16 protein (P16) is seen occsionlly in ductl cells, lthough few studies hve exmined P16 expression in slivry glnd tumors (9-11). p63, memer of the p53 fmily, is tumor suppressor gene locted on chromosome 3q The p63 gene plys n essentil role in epithelil development nd in the prolifertion of cells in the lim nd crniofcil structures (12). p63 is expressed in mny norml tissues including squmous epithelium, urothelium, ronchil epithelium, nd myoepithelil lyers of rest, prostte, nd sumucosl glnds, s well s in vrious kinds of tumors including squmous cell crcinom, rest cncer, colorectl cncer, nd slivry glnd tumors (13-16). p63 is expressed in the nuclei of norml slivry glnd myoepithelil nd sl cells. p63 is retined in NMC nd sl cells in oth enign nd mlignnt slivry glnd tumors, suggesting its role in the oncogenesis of slivry glnd tumors (17-18). Doule immunostining for the p63 gene products (P63) nd high moleculr weight cytokertin hs een useful tool for the

2 10 Tnk et l. P16 nd P63 in denoid cystic crcinom Fig. 1. Doule immunostining for p16 gene products (P16) nd p63 gene products (P63) in norml slivry glnds. () Hemtoxylin nd eosin (HE) stin; () douleimmunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In norml slivry glnd, P16 ws not expressed in ny myoepithelil or sl cells of cinr nd ductl structures (), while P63 ws oserved in the nuclei of myoepithelil cells of cini nd sl cells of ducts (). reproducile clssifiction of ppillry rest lesions (19). Doule immunostining hs enled the concurrent expression of P63 nd cytokertin 14 (CK14) in sl cells of the excretory ducts, interclted ducts, nd cini of the slivry glnd (20). Although this method hs een used for differentil dignoses of severl kinds of tumors, until now it hs not een pplied to nlyze the morphologicl diversity of slivry glnd tumors (21-22). To exmine the morphologicl diversity nd development of AdCC cells nd foci, we used doule immunostining method to detect P16 nd P63 coexpression in the sme tissues. Mterils nd methods Tissue specimens We exmined 10 AdCC tissue smples otined from Osk Dentl University Hospitl. They were otined from iopsy specimens nd surgicl resections of six mle nd four femle ptients, whose medin ge ws 62 yers (rnge yers). AdCC ws clssified y the dignostic criteri proposed y WHO clssifiction of tumours for dignosis of slivry glnd tumors. This reserch protocol ws pproved y the Ethics Committee t Osk Dentl University (pprovl numer ). Doule immunohistochemicl stining The specimens were fixed in 10% formlin solution nd emedded in prffin. Four-micrometer-thick sections were deprffinized in L-limonene (Hemo-De, Flm Co., Ltd., Tokyo, Jpn), nd dehydrted through grded ethnol series. Antigen retrievl ws performed y utoclving t 121 t ph 9.0 for 15 min in retrievl uffer (Dko, Glostrup, Denmrk). After heting, the slides were llowed to cool to room temperture nd were then wshed riefly with phosphte-uffered sline. The endogenous peroxidse ctivity ws locked with 3% hydrogen peroxidse, nd nonspecific rections were locked with 2% norml horse serum. The sections were incuted with nti-p63 monoclonl ntiody (7JUL, Novocstr, Newcstle, UK, diluted t 1:25) for overnight t 4. The sections were incuted in polymerized reporter enzyme stining system (ImmPRESS Anti-Mouse Ig (Peroxidse) Polymer Detection Kit, Vector Ls, Burlingme, CA, USA) for 30 min t room temperture nd visulized y incution with 3, 3 -diminoenzidine tetrhydrochloride, which produced rown color. The sections were then immersed in 0.1 mm citrte uffer, ph 6.0, t 98 for 20 min to inctivte the unrected ntiody from the first rection. The sections were incuted with nti-p16 monoclonl ntiody (clone 6H12, Novocstr, 1:20) overnight t 4, nd then incuted using the polymerized reporter enzyme stining system mentioned ove for 30 min t room temperture nd visulized with nickel-dab solution (Vector Ls), which produced chrcol gry color. The negtive controls for immunostining were prepred using norml mouse IgG insted of the primry ntiodies. Results In the doule immunohistochemistry, P16 expression ws oserved s chrcol gry stining in the nuclei of ductl sl cells, while P63 expression ws oserved s rown stining in the nuclei of myoepithelil cells nd NMC. Fig. 2. Doule immunostining for P16 nd P63 in denoid cystic crcinom (AdCC) with tuulr pttern foci. () HE stin; () doule-immunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In tuulr pttern foci of AdCC, P16 ws expressed in the nuclei of inner duct epithelil cells (), while P63 ws expressed in the nuclei of the outer cells of ductl structures ().

3 Orl Med Pthol 16 (2011) 11 Fig. 3. Doule immunostining for P16 nd P63 in AdCC with cririform pttern foci. () HE stin, 100; () doule-immunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In cririform pttern foci of AdCC, P16 ws expressed in some of the inner cells of the solid prt s well s some of the lining cells of pseudocysts (), while P63 ws expressed in most of the peripherl s well s inner cells (). In norml slivry glnds (Fig. 1), P16 ws not expressed in ny myoepithelil cells of cini or in ny sl cells of ducts. P63 expression ws oserved in the nuclei of the myoepithelil cells of cinr nd sl cells of duct (Fig. 1). In AdCC foci with tuulr ptterns (Fig. 2), the doule immunostining showed tht P16 ws expressed in nuclei of the inner tumor cells of ductl structures. P63 ws expressed in nuclei of the outer NMC of the ductl structures (Fig. 2). In AdCC foci with cririform ptterns (Fig. 3), P16 ws expressed in some inner cells of the cncer nests nd in severl cells lining the pseudocysts. P63 ws expressed in mny peripherl nd inner cells of the cncer nests (Fig. 3). P63-positive cells were more predominnt thn P16-positive cells in tumors with cririform pttern. In tumors with solid pttern (Fig. 4), P16- nd P63-positive cells were intermixed in the cncer nests (Fig. 4). P16- nd P63- positive cells hd invded the perineuron (Fig. 5). P16- nd P63-positive cells were metstsized in the lymph nods (Fig. 6). In smples of peripherl nerve invsion nd lymph node metstsis, oth P16- nd P63-positive cells were found in tumors with the tuulr pttern nd cririform pttern. Discussion The present doule immunostining for P16 nd P63 on the sme tissue specimens hs een shown to e useful tool for nlyzing the morphologicl diversity of AdCC tht is relted to the proportionl expression etween P16 nd P63. In norml slivry glnd, P16 hs occsionlly een expressed in duct epithelil cells, ut its expression levels were low in ductl, cinr, nd myoepithelil cells (4, 11). In our study, however, P16-positive cells were not found in ny cell types of norml slivry glnds. The expression levels of P16 hve een known to e lower in norml slivry glnds thn in enign nd mlignnt slivry neoplsms (9). In our present study, AdCCs with the tuulr foci exhiited the P16 overexpression in luminl cells of the ductl structure. In contrst, those with the cririform or solid ptterns contined only few P16-positive inner cells. These results suggest tht P16 is involved in the prolifertion of AdCC cells forming ductl structures. A reltionship etween deletion nd errnt methyltion of p16 nd crcinogenesis hs een reported. Aerrnt methyltion of the p16 gene nd loss of P16 expression re consistent with the multistep progression of peripherl-type lung denocrcinom (5). Sno et l. reported tht overexpression of P16 might e useful dignostic mrker for cervicl neoplstic lesions in routine lortory tests (23). Negri et l. reported tht immunostining for the gene products of p16 INK4 ws useful mrker for identifying neoplstic lesions of the endocervicl glndulr epithelium (24). In squmous cell crcinoms of the cervix uteri, the overexpression of p16 INK4 is considered to e induced y humn ppillom virus nd eventully ssocited with crcinogenesis of the cervicl epithelium (6). Cerilli et l. reported tht inctivtion of p16 is importnt in the development or progression of some slivry crcinoms including slivry duct crcinom (8). Vékony et l. reported tht P16 ws more overexpressed in mlignnt myoepitheliom thn in norml slivry glnd tissues (11). These lines of evidence indictes tht the overexpression of the p16 gene is relted to neoplstic chnges in mny different orgns. Alph-smooth muscle ctin, clponin, P63, nd CK14 Fig. 4. Doule immunostining for P16 nd P63 in AdCC with solid pttern foci. () HE stin, 100; () doule-immunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In solid pttern foci (), P16- nd P63-positive cells were intermixed ().

4 12 Tnk et l. P16 nd P63 in denoid cystic crcinom Fig. 5. Doule immunostining for P16 nd P63 in perineurl invsion of AdCC. () HE stin, 100; () doule-immunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In the site of perineurl invsion of AdCC (), P16/P63-doulepositive cells invded perineuronl spces (). re commonly used s myoepithelil cell mrkers, while epithelil memrne ntigen nd crcinoemryonic ntigen re used s duct epithelil cell mrkers (25-27). P63-positive cells re seen in nuclei of the sl cells in the interclted, strited, nd interloulr ducts: P63 is expressed not only in norml myoepithelil cells ut lso in NMC. Some of the elongted nuclei t the periphery of cini hve een reported to e P63 positive (18). Thus, P63 hs een considered to e relile mrker of myoepithelil cells even in the presence of cytomorphologicl heterogeneity (13). P63 hs recently ecome populr mrker for oth sl nd myoepithelil cells in norml slivry glnds s well s their neoplstic counter prts (16), though we could only immunoloclize P63 in few myoepithelil cells of cini or in sl cells of interclted nd strited ducts in norml slivry glnd. Bil et l. found tht the expressions of P63 nd p73 gene products were correlted with the progression of sloid cells nd NMC of oth enign nd mlignnt slivry glnd tumors (17). The P63 expression retined in NMC nd sl cells of humn slivry glnd tumors suggests its role in the oncogenesis of these tumors with complex tissue rchitectures (18). The sence of P63 in the luminl cells ut its presence in the surrounding non-luminl NMC/sl reserve cells hve een reported in cririform AdCC foci (28). In our study, however, P63 ws expressed in the peripherl cells s well s in the pseudocyst lining cells in the cririform foci, in ddition to in the outer cells of ducts in the tuulr foci. P63-positive cells were more densely distriuted in AdCC thn in norml slivry glnd. Furthermore, P63-positive cells were more enhnced in numer in the cririform nd solid foci thn in the tuulr foci. These findings suggest tht P63 plys role in the prolifertion of NMC in AdCC foci. The doule immunostining method, which we utilized in the present study, hs een used in the differentil dignoses of different kinds of crcinoms. Cmerio et l. reported tht the expressions of P63 nd CK5 were relted to divergent differentition during the development of colorectl cncer (15). Reis-Filho et l. reported the coexpression of the either pir of P63 nd CK5/6 or pir of P63 nd CK14 in the sl myoepithelil cells of slivry glnd tumors (21). Ichihr et l. reported tht P63 nd high moleculr weight cytokertin were useful for reproducily clssifying ppillry rest lesions (19). We hve lso reported tht the comintion of P63 nd CK8 immunostinings is helpful in distinguishing etween NMC nd luminl cells in pleomorphic denom (22). Although these studies used doule immunostining to detect the coexpression of P63 nd cytokertins in different kinds of tumors, there hve een few trils to exmine the morphologicl diversity of AdCC y using doule immunostining for P16 nd P63. In the present study, we were successful in differentil demonstrtion of NMC nd duct epithelil cells y using doule immunostining for P63 nd P16, respectively, in the sme AdCC tissue sections, in which P16 ws shown to e specific to luminl cells nd P63 in NMC. In ddition, oth luminl cells nd NMC/sl cells were shown to e involved in perineurl invsion nd lymph node metstsis. These dt suggest tht the morphologicl diversity nd prolifertion of AdCC cells re relted to the expression levels for P16 nd P63, which ws enled to e demonstrted y this prticulr method of doule immunohistochemistry. Fig. 6. Doule immunostining for P16 nd P63 in lymph node metstsis of AdCC. () HE stin, 100; () doule-immunoperoxidse stin for P16 (chrcol gry) nd P63 (rown), no counterstin, 200. In metsttic AdCC foci in lymph nodes, which were found to e in tuulr nd cririform ptterns (), oth P16- nd P63-positive cells were scttered in mixed fshions ().

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