Involucrin in Squamous and Basal Cell Carcinomas of the Skin: an Immunohistochemical Study

Involucrin in Squamous and Basal Cell Carcinomas of the Skin: An Immunohistochemical Study

JONATHAN W. SAID, M .D., AARON F. SASSOON, B.S., I. PETER SHINTAKU, PH.D., AND SusAN BANKS-SCHLEGEL, PH.D.

Division of Anatomic Pathology, Cedars-Sinai Medical Center (JWS. IPS), Los Angeles, Califom.ia: Laboratory of Human Ca rcinogene~is, National Cancer Institute, National Institutes of Health (SB-SJ, Bethesda. Maryland; and Department of Biology. University of Southern California (AFS), Los Angeles, California, U.S. A.

Involucrin is a precursor of the cross-linked envelope MATERIALS AND METHODS protein of human stratum corneum, and its appearance Cases of skin tumors (24 squamous cell and 21 basal cell carcinomas) in the upper layers of the epidermis is a function of the were retrieved from the files of the surgica l pathology division of normal differentiation of the keratinocyte. Cases of Cedars-Sinai Medical Center. In selected cases, sections were reviewed basal cell and squamous cell carcinoma were evaluated independently by a dermatopathologist not otherwise involved with the for the presence of involucrin using immunoperoxidase study (Or. Theodore H. Kwan, Beth Israel Hospital, Boston, Massa techniques on paraffin sections. Basal cell carcinomas chusetts). Cases of basal cell carcinoma were subclassified according to were negative for involucrin with staining restricted to conve ntwnal histologic cri teria [10] into solid type (7 cases), superficial squanwus horn cysts, while squamous cell carcinomas multifocal (3 cases), adenoid (3 cases), sclerosing (4 cases), keratotic (:J stained strongly, particularly in large keratinized cells. cases), and cystic ( l case). Cases of squamous cell carcinoma compri sed Cases of squamous cell carcinoma in situ (Bowen's dis 14 cases of squamous cell carcinoma in situ (Bowen's disease) and 10 ease) r e vealed increased staining for involucrin with cases of inftlt.rat.ing squamous cell carcinoma. Thirteen cases revealed evidence of sun damage in t he surrou nding epidermis with solar elas staining of dyskeratotic cells at all levels in the epithe tosis and/or solar keratosis. lium. Abnormal patterns of staining were also noted in Details of preparation and characterization of the a nt i-in volucrin non- neoplastic epidermis adjacent to carcinomas. Im antiserum (k indly supplied by Dr. Howard Green) have been previously munohistochemical staining for involucrin identifying reported [5]. Briefl y, a soluble envelope precursor protein was purified abnormal or premature keratinization is a sensitive from cul tures of human epidermal keratinocytes and antiserum was marker for dyskeratosis in squamous epithelia and may prepared in rabbits by injection of thi s purified protein dissolved in have applications in the histopathologic evaluation of complete Freund's adjuvant. Formalin-fixed paraffin-embedded blocks ~v e r e recut. and 4-l'm sections were mounted on glue-coated slides fo r skin specimens. 1mmunoperoxidase staining as previously described [11]. Parall el sec tions were evaluated with and without. trypsin digestion (0.12 mg/ml trypsi n at. 37"C for 30 min) with si milar resul ts. Foll owing hydration During the late stages of differentiation, human epidermal of paraffin sections, they we re incubated for 15 min in a solution of keratinocytes develop an insoluble protein envelope that con methanolic peroxide ro consume endogenous peroxidase activity. Sec tributes to the protection of the skin against the environment t ions were rinsed wi t h phosphate-buffered saline (PBS) at pH 7.4 and mcubated with swine serum to reduce background staining. T he sec [1-4). Involucrin, one of the soluble precursor proteins of this twns were then incubated sequentially with antibodies to involucrin envelope isolated from cultured human epidermal keratinocytes produced in rabbits a nd diluted 1:1000, swine anti rabbit serum protein, (5], is found in the cytoplasm of t he outer squamous cell layers and horseradish peroxidase-rabbit-antihorseradish peroxidase soluble of t he epidermis and in the envelope of the stratum corneum complexes (Dako Corporation, Santa Barbara, California). Antibody (5,6). A 36K soluble precursor named keratolinin ~ as re_cent ly localization was effected by incubation with a solution of 6 m" 3 3' been isolated from the bovine snout and human epidermis, and di amino benzidine in 10 ml PBS to which 0.1 ml of hydrogen pe~o~ id e does not cross-react with involucrin (7]. The cross-linking of was added just before use. Sections were counterstained with methyl these soluble envelope protein precursors to form insoluble green and mounted in Permount. Immunoflu orescence microscopy was polymers is catalyzed by transglutaminase in a calcium-de performed on unfixed cryostat sections incubated sequentially with normal swine serum, primary ant iserum, and fluorescein-conjugated pendent reaction [7,8). Tissue culture studies have shown that swine anti rabbit immunoglobulins. Sections were examined with a Zeiss squamous cell carcinoma Jin es produce involucrin but reveal microscope equipped with an epifluorescent condenser, primary (440- aberrations in terminal differentiation and formation of cor 490 nm) and secondary (520 nm) filters, and a dichroic reflector (510 nified envelopes [9) . Presence of involucrin in skin neoplasms nm). may therefore be expected to serve as a marker for squamous Similar to previous reports [5, 12] when frozen sections of normal differentiation, and alterations in involucrin synthesis might epidermi s were stained with ant iserum to involucrin, the envelope be anticipated in dysplastic or preneoplastic squamous epithe precursor was seen throughout the cell cytoplasm, but was also concen lium . In this study immunoperoxidase techniques are used to trated at the cell periphery of t he outer spinous and granul ar cell s, and localize involucrin in histologic sections of basal cell and squa appeared diffuse in t he cell s of the stratum corneum (data not shown). In fo rmaldehyde-fixed sections, the outer spinous and cr ranular ce lls mous cell carcinomas in order to assess patterns of distribution still stained well but staining of the stratum corneum wabs suppressed, in these neoplasms and the surrounding epidermis. presumably because t he formaldehyde fix ation prevented penetration of the antiserum (Fig l ) [5,6,13]. While formaldehyde fixation also reduced the staining of t he peripheral uncross-linked involucrin. it appeared to have li ttle effect on the cytoplasmic staining (Fig 1), as Manuscript received July 25, 1983; accepted for publication October previously noted [5,6]. Negative cont rols consisted of parallel sections 20. 1983. with elimination of the primary antiserum and substitution with preim Reprint requests to: Jonathan W. Said, M.D., Division of Anatomic mune serum or anti-involucrin serum which had been adsorbed with Pathology, Cedars-Sinai Medi cal Center, 8700 Beverly Boulevard, Los purified envelopes. These controls were included for each case and Angeles, Cali forn ia 90048. revealed no staining.

449 450 SAID ET AL Vol. 82, No.5 RESULTS creased in areas of extensive ulceration and necrosis. One case showed a variation in staining for involucrin in that the major Squamous Cell Carcinomas ity of the tumor was negative, with highly keratinized foci Twenty-four cases of squamous cell carcinoma were evalu staining strongly. ated (14 cases of invasive squamous cell carcinoma and 10 cases Ten cases of squamous cell carcinoma in situ of the epidermis of carcinoma in situ). In 11 cases, normal skin was present on (Bowen's disease) were evaluated, and all showed an abnormal the sections away from the tumors and showed the normal pattern of staining for involucrin. In 8 cases a diffuse pattern pattern of localization of involucrin to the upper layers of the of staining for involucrin was observed in all cell layers of the epidermis similar to results illustrated in Fig 1. Thirteen cases neoplastic epidermis down to the basal layer. In 3 tumors, of infiltrating carcinoma showed diffuse cytoplasmic staining staining was focal and was restricted to nests of dyskeratotic both in the intraepidermal and infiltrating component of the cells at all levels within the neoplastic epidermis (Fig 4). tumors regardless of extent of infiltration (Fig 2a). In unfixed The epidermis immediately adjacent to but not involved by frozen sections, staining was predominantly located at the cell carcinoma was also evaluated in all 24 cases. In 2 cases we periphery (Fig 2b ). Staining was of greatest intensity in the found a normal pattern of localization to the upper cell layers. center of squamous nests, particularly in larger keratinized However, in the remaining cases staining was abnormal, ex cells (Fig 3). Staining was unaffected by minor degrees of tending to the basal layer in 18 cases and two-thirds of the way inflammation, although in 2 cases staining was focally de- to the base of the epidermis in 4 cases (Fig 5). These abnormal patterns of increased staining for involucrin were present in dyskeratotic and hyperplastic epidermis, but also in cases where the epidermis adjacent to the tumors appeared histologically normal. Basal Cell Carcinomas Twenty-one cases of basal cell carcinoma were evaluated. The tumors were negative for involucrin in all cases with the exception of squamous horn cysts which stained strongly for involucrin in 7 cases (Fig 6) . In 4 cases individual keratinized cells also stained strongly. Unlike cases of squamous cell car cinoma, the skin adjacent to the basal cell carcinomas showed a normal pattern of staining. The skin immediately overlying the basal cell carcinomas, however, showed an abnormal pat tern of staining in 17 cases with stain extending to the base in . . ·- ... _ -· . 15 cases and two-thirds of the thickness of the epidermis in 2 FIG 1. Formalin-ftxed section of normal epidermis s howing locali cases (Fig 7). These changes were particularly evident at sites zation of involucrin (blach) to the superficial layers of the epidermis by of attachment between the basal cell carcinomas and overlying immunoperoxidase technique (methyl green counterstain, x 80). epidermis.

FIG 2. a, Infiltrat ing squamous cell carcinoma shows nests of tumor infiltrating into the deep dermis and staining with variable intensity for involucrin (blach) (methyl green counterstain, X 200). b, Unfixed frozen section of infiltrating squamous ce ll carcinoma shows localization of staining for involucrin predominantly at the cell periphery {X 200). May 1984 INVOLUCRIN IN SKIN TUMORS 451 DISCUSSION carcinoma in situ, with loss of polarity and increased staining of cells at all levels of the epidermis, suggest that the orderly is one of the soluble protein precursors of the Involucrin o~ te~minal differentiation is defective in these epi envelope found in normal stratum corneum (4-6] . seq~ence cross-linked m~ense staining for involucrin and high-molec of soluble precursor proteins including involucrin thelia. ~1m1lar Cross-linking keratms occurs in cases of squamous cell carcinoma insoluble polymers is catalyzed by ular-Weight and keratolinin [7,8] into 's disease, corresponding to the presence of in a calcium-dependent reaction. in situ and Bowen epidermal transglutaminase numerous dyskeratotic cells. The appearance of mRNA for involucrin and synthesis of the The presence of increased staining for involucrin in squa is an orderly function of normal terminal maturation, protein mous cell carcmoma of the skin differs from that described in which correlates with the size of keratinocytes and their level cervical intraepithelial neoplasms, which demonstrate lack of the epidermis [5,6,14,15]. Alterations in involucrin syn within staining for involucrin in comparison with normal epithelium have been demonstrated in squamous carcinoma cell thesis .l17]. These differences may be explained by biologic differences lines [9] and with changes in the cell microenvironment m these squamous epithelia. Squamous cell carcinoma of the (16,17]. cervix is predominantly characterized by undifferentiated ma In this study strong staining for involucrin was noted in lignant cells of basal type without surface maturation or kera cell carcinomas of the skin. Staining for involucrin squamous tinization [20], unlike the numerous dyskeratotic cells with most intense in the larger neoplastic cells, paralleling was prominent tonotilament bundles present in squamous cell car in cultured human epidermal keratinocytes which have findings cinomas of the skin (10]. In our laboratory, although small and demonstrated that accumulation of mRNA for involucrin and for ~arge eel.! anaplastic carcinomas of the cervix are negative of the protein correlates with cell size [15] . Rheinwald synthesis mvolucnn, large cell keratinizing squamous cell carcinomas in (9] demonstrated reduced cornified envelope for and Beckett situ and infiltrating squamous cell carcinomas with keratini in squamous cell carcinoma cell lines in culture, and mation zation stain strongly for involucrin (unpublished observations). that this alteration might induce malignant transfor suggested Basal cell carcinomas were strikingly different from squa by evading mechanisms for limiting cell growth. Our mation m?us cell carcinomas in their patterns of staining for involu indicate that in human epidermal neoplasms inappro results crm. These tumors were uniformly negative except for individ or premature terminal differentiation may occur, and priate ual keratinized cells and squamous horn cysts. This result is sence of involucrin in these lesions correlates with that pre expected since normal basal cells lack involucrin. of neoplastic keratinocytes and presence of large differentiation o.f intere~t ~ere the abnormal patterns of increased staining keratins (18] . m the lower spinous cell layers of the epidermis of involucrin in squamous cell carcinomas of fo~ mvolucnn The presence to squamous cell carcinomas and overlying basal cell accordance with the presence of numerous cells adJa~ent the skin is in Th.ese changes apparently represent staining of which are strongly labeled with antisera to carcu;tomas. in these neoplasms With faulty or premature keratinization either as keratins [18,19] . Altered patterns of keratmocytes high-molecular-weight a reaction ~o the tumors or as a feature of the dys~lastic or staining for involucrin in Bowen's disease and squamous cell preneoplastlc state. Deep epidermal staining for involucrin

i· FIG 5. Non-neoplastic epidermis adj acenr. to squamous cell carc involucrin, with staining FIG 3. Infiltrating squamous cell carcinoma showing strong staining noma shows abnormal pattern of staining for ck) (methyl green counter of large keratinizin g tumor cells for involucrin (black) (m ethyl green of all layers a bove the stratum basale (bla , X 250). counterstain, X 400). stam

FIG 4. Squamous cell carcinoma in s itu with nes ts of cells at varying levels in the neoplastic epidermis staining s trongly for involucrin (black) (methyl green counterstain, X 200).

4. 452 SAID .ET AL VoL 82, No.5 involving all differentiated cell layers of the epidermis is not specitic for neoplasia, and is also encountered in various benign epidermal hyperplasias including verruca and solar k eratosis (unpublished observations). In summary, this study demonstrates staining for involucrin in in-situ and infiltrating squamous cell carcinomas but not in basal cell carcinomas of the skin. Increased staining was also noted in dyskeratotic squamous epithelium and in epidermis ...... ~'!. ."? , ,., ,= # .::.- -. adjacent to carcinomas. Immunohistochemical staining for in • : f" ... ·- - -- rf* ~ .. ~ _,. volucrin represents a sensitive marker for abnormal or prema ...... ~ ::... ,. ture s quamous maturation and an adjunct to the histopatho -- ~ - logic evaluation of skin lesions.

The authors a re g rateful for the word processing skill s o f Mavis '/ ...... Thompson and Meyer Bekhore, and the photographic assistance of Kai :' .. Chi en and Robert Heuser. We thank Dr. Theodore H. Kwan, Beth Israel Hospital, Boston, Massachusetts for review of the histology 111 selected cases. REFERENCES -. f L Matoltsy AG, Balsamo CA: A study of t he components of cornified epithelium of human skin. J Biophys Biochem Cytol 1:339-360, 1955 2. Matoltsy AG, Matoltsy MN: The membrane protein of horny cells. J Invest Dermatol 46: 127- 129, 1966 3. Green H: Terminal differentiation of cultured huma n epidermal cells. Cell 11:405- 416, 1977 ·. 4. R ice RH, Green H: The cornified envelope of terminally differen tiated human epidermal keratinocytes consists of a cross- linked . -- ..... protein. Ce ll 11:417- 422, 1977 ··~ .... . - 5. Rice RH, Green H: Presence in human epidermal cells of a soluble ' . .. protein precursor of t he cross-lin ked envelope: activation of the ! ·:., : cross linking by calcium ions. Cell 18:681- 694, 1979 .!' · " y -, 6. Banks-Schlegel S, Green H: lnvo lucrin synthesis and tissue assem 6' bly by keratinocytes in natural and cultured human epitheli a. J Cell Bioi 90:732- 737, 1981 F1r: 6. Basal ce ll carcinoma showing absence of staini ng for invol 7. Peterson LL, Zetergre n JG, Wuepper KD: Biochemistry of trans uc rin except for squamous horn cysts (a rrowheads). The overlying glu taminase and cross-lin king in the s kin . J Invest Dermatol epidermis shows a normal pattern of staining for involu crin (methyl 8l(suppl):95s- 100s, 1983 green co unterstain, x 150) . 8. Buxman MM, Wuepper KD: P urification and characteri zation of bovine epidermal transglu t.a minase. Clin Res 22:157 A, 1974 9. Rheinwald JG, Beckett MA: Defective terminal differentiation in cul ture as a consistent and selectable c haracter of malignant human keratinocytes. Cell 22:629- 632, 1980 10. Lever WF, Schaumburg-Lever G: Histopathology of the Skin, 5th ed. Phil adelphia. JB Lippincott, 1975 11. Said JW, Nash G, Tepper G, Banks-Schl egel S: Keratin proteins and carcinoembryonic a ntigen in lung carcinoma: an immune peroxidase study of fifty-four cases, with ul t rastructural corre lations. Hum Pathol 14:70- 76, 1983 12. Kubil us J , Baden HP: Isolation of two immunologically related transglu taminase 14 substrates from cu ltured human keratino cytes. ln Vitro 82:44 7- 455, 1982 13. Banks-Schlegel SP, Green H : Formation of epidermis by serially cultivated human epidermal cell s transplanted as an epithelium to athymic mice. Transplant 29:308- 313, 1980 14. Banks-Schlegel SP, Green H : Studjes on the development of t he deiinitive ce ll type of embryonic epidermis usin g the cross linked envelope as differentiation marker. Dev Bioi 74:274-285, 1980 15. Watt FM, Green H: lnvo lucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Bioi 90:738- 742, 1981 16. Green 1-1, Watt FM: Regulation by vitamin A of envelope cross linking in cultured keratinocytes derived from different human epi t helia. Mol Ce ll Bioi 2: 1115-1117, 1982 17. Warhol MJ, Antonioli DA, P inkus GS, BurkeL, Rice RH: Immu noperoxidase staining for in vo lucrin : a potential diagnostic aid in cervi covaginal pathology. Hum Pathol 13:1095- 1099, 1982 18. Thomas P, Said JW, Nash G, Banks-Schelegel S: Profiles of keratin proteins in basal a nd squamous cell carcinomas of t he s kin: an immunohistochemi cal study. Lab Invest, 50:36- 41, 1984 19. Loning T, Staquet M-J, Thivo let ,1, Seifert G: Keratin polypeptide F IG 7. Epidermis overlying basal cell carcin oma shows abnormal distribution in normal and di seased human epidermis a nd oral staining for involucrin (bloch) which extends to the junction wit h the mucosa. Virch Arch [Pathol Anat] 388:273- 288, 1980 underlying tumor. T he basal ce ll carcinoma is unstained except for 20. Blaustein A: Pathology of the Female Genital Tract, 2d ed., New squamous horn cyst (arrowhead) (methyl green counterstain, X 225). York, Springe r-Verlag, 1982