Reactivity of monoclonai antibodies 17,13 and 63,12 with 141 orai mucosai iesions
C, A, Migliorati', R, RankenS M, J, Kaplan^ and S, Silverman Jr/ Departments of 'Oral Diagnostic Sciences, Division of Oral Medicine, tjniversity of Fiorida Coiiege of Dentistry, Gainesville, 'Intek Diagnostics Inc., tvlillbrae, CA, 'Otolaryngology, School of t\/ledicine. University of California - San Francisco, 'Stomatology, Division of Oral Medicine, School of Dentistry, University of California San Francisco, USA
Migliorati CA, Ranken R, Kaplan MJ, Silverman S Jr: Reactivity of monoclonal antibodies 17,13 and 63,12 with 141 oral mucosai lesions, J Oral Pathol Med 1992; 21:412^17, We studied the reactivity of monoclonal antibodies (MAbs) 17,13 and 63,12 with normal and diseased human oral mucosa by means of the immunoperoxidase technique. The specimens included: 22 normal oral tissues, 20 benign tumors, 17 lichen planus, 23 focal keratosis and epithelial hypcrplasias, 18 proliferative verrucous leukoplakias, 20 dysplasias, and 21 squamous cell carcinomas. In most cases of normal mucosa and benign lesions, MAb 17,13 stained basal epithelial cells only, whereas MAb 63,12 stained all cell layers above the basal cells. In the premalignant and malignant lesions MAb 17,13 stained above the basal cells and MAb 63,12 either stained areas not stained by MAb 17,13 or the staining was absent. Based on the different staining patterns observed, there appears to be a potential value of these new reagents in diagnostic histopathology regarding specimens with equivocal cellular morphology.
In previous reports (1 3) we demonstrated the reactivity of monoclonal antibody 17.13 (MAb 17.13) with human squamous cell carcinoma and its potential application to head and neck tumor diagnosis. MAb 17.13 was derived from a fusion of splenocytes of a BALB/c mouse immunized with a surgically resected poorly differentiated human recurrent laryngeal squamous cell carcinoma with SP2/0 mouse myeloma cells. It is an IgM antibody that consistently reacts with an unidentified cytoplasmic antigen present in oral squamous tumor cells. In normal tissues and benign lesions, the antibody reacts with another unidentified antigen present in the cytoplasm of basal epithelial cells only. MAb 63.12, another newly developed reagent, was generated from the fusion of SP2/0 mouse myeloma cells with splenocytes of a mouse that had been immunized with tumor cells derived from a squamous carcinoma of the floor of the mouth. This IgM antibody reacts with an antigen present in the cytoplasm of non-basal epithelial cells. Its reaction pattern is a mirror image of the pattern displayed by MAb 17,13, as demonstrated by our previous studies, whereas MAb 63,12 stained all suprabasal epithelial layers in nomial oral mucosai tissues and benign oral lesions.
Based on these findings, we studied by means of the immunoperoxida,se technique the reactivity of MAbs 17,13 and 63,12 in a larger and diverse group of oral mucosai lesions to further confirm the reliability of the staining pattern of these antibodies. In turn, this would help assess their capability of marking tissues that possess the potential of possibly becoming malignant or already expressing malignant transformation at a stage when histologic morphology appears normal or equivocal.
Key words: cancer diagnosis: immunohlstopathology: monoclonal antibodies: mouth, diseases: oral histopathology: oral premalignancy; oral cancer. Cesar A. Migliorati, Department of Oral Diagnostic Sciences. University of Florida College of Dentistry, Box 100414 JHMHC; Gainesville, FL 32610, USA. Accepted for publication May 8. 1992.
in liquid nitrogen for the immunop)eroxidase reaction. Monocionai antibodies and Immunoperoxidase reaction
Three commercially unavailable murine MAbs obtained with the specificity of hybridoma technology (4) were utilized in this study: MAbs 17,13, 63,12, and 88,25, the latter, also an IgM type, serving as a negative control. For a complete review of the antibodies used in this study, see MIGLIORAII et al, (1) and RANKEN et al. (2, 3),
Material and methods Tissue sampies
Biopsy specimens from 141 oral mucosai lesions were obtained from patients seen at the Oral Medicine Clinic, University of California, San Francisco, The specimens included 22 normal oral mucosae, 20 benign tumors and benign pigmented lesions, 17 lichen planus, 23 focal keratoses and epithelial hyperplasias, 18 proliferative verrucous leukoplakia, 20 dysplasias, and 21 squamous cell carcinomas. The specimens were divided into two equal parts. One half was fixed in IO'MJ formalin and processed for routine histopathologic diagnosis and the other half was frozen immediately
For each specimen, 5 nm serial frozen sections were cut on the cryostat (Slee, London) and mounted on formaldehyde-gelatin-coated glass slides. Tissue sections were immediately fixed in cold acetone and air dried. Sections were reacted with the monoclonal antibodies by tneans of the immunoperoxidase technique (5), The procedure has been described previously (1, 4), In brief it entails the following steps: Glass slides were coated with gelatin. Frozen tissue sections were mounted and fixed with cold acetone. The tissues were blocked with normal horse serum and hydrogen peroxide and incubated with the primary monoclonal antibodies. After reacting the tissues with biotinylated
Monoclonal antibodies 17.13 and 63.12 413 17.13 reaction Basal cells only
n Normal oral mucosa Benign tumor, benign pigmented lesions Lichen planus Focal keratosis, epithelial hyperplasia Proliferative verrucous leukoplakia Dysplasia Squamous cell carcinoma
horse anti-rnouse IgG heavy and light chain specific, slides were incubated with avidin-biotin-peroxidase complex. A mixture of diaminobenzidine and hydrogen peroxide was used lor providing color to the reaction and hematoxylin Was used as a counterstain. All specimens were reacted with the 3 MAbs. Any brown staining that was more intense than the negative control background ob.served with MAb 88.2,S Was considered a positive reaction. Staining patterns were recorded and the results were compared with the independently read routine histopathologic slides. Resuits
Table 1 presents a summary of the results obtained among the 141 specimens, grouped within 7 categories. Figs. 1 3 illustrate representative staining re-
A
22 20
20 14 1 13 2
17
23 18 20 21
Above basal cells
63.12 (nonnal) reaction Above basal cells HomoFocal geneous patchy NEG
88.25 (control) reaction
POS
NEG
22 20 17 23 18 20 21
2
8
14
0
0
6 16 10 16
16 3 12 2
4 14 11
0 0 0 16 20 21
0 0 0 0 0
20 21
actions of MAbs 17.13 and 63,12. The staining results were as follows: Group I: nortnal oral muco.sa
The
normal oral mucosa group included 18 samples from the gingiva, 2 from the buccal mucosa, and 2 from the tongue. The staining pattern of MAb 17,13 in normal oral mucosa was characterized by a positive reaction of the basal cells in the majority of cases (91%), In two cases of gingival tissue (9%), the parabasal and spinous cells reacted positively. All samples were positive with MAb 63,12, This staining pattern was always present above the basal cell layer, and was either homogeneous (36%) or focal/patchy (64%) in distribution. Group 2: benign tumors and henis;n
reactive lesions - This group included 10 fibromas, 4 squamous papillomas, 2 pyogenic granulomas, 1 focal melanosis, 1 peripheral ossifying fibroma, 1 mucocele, and 1 gingival fibromatosis.
0
The staining pattern of MAb 17,13 was observed solely in the epithelial basal cell layer in 14 (70%i) cases. In six cases (2 papillomas, 2 fibromas, 2 pyogenic granulomas) MAb 17,13 stained cells above the basal cell layer, MAb 63,12 stained all specimens. The staining was always above the basal cell layer and was homogeneous in 16 (80'V,,) cases and focal/patchy in 4 (20%), Group 3: liehen planus - In all but one of the 17 cases, the MAb 17,13 stained suprabasal epithelial cells. All specimens also exhibited a positive staining reaction with MAb 63,12, This reaction was focal/patchy in 14 cases (82"o) and homogeneous in 3 (18%), Of interest, when a subepithelial lymphocytic infiltrate was present, both MAbs stained suprabasally. Occasionally, areas of lichen planus not exhibiting the sub)epithelial lymphocytic infiltrate presented a staining pattern comparable with that
B
I'^ig L Normal oral mucosa. A. M&F. staining (F., epithelium. CT, connective tissue), x 400. B, staining pattern with M.Ab 17.13. Note that only basal epithelial cells react with antibody (arrows), x 400. C, staining pattern with MAb 63.12. Nole that basal epithelial cells are the only cells that do not react with antibody (arrows), x 400.
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Fig. 2. Focal keratosis. A, H&E staining (E, epithelium. CT, connective tissue), x 200. B, Staining pattern with MAb 17.13, Observe similar pattern as lor normal oral mucosa. Basal epithelial cells are the only cells reacting with antibody (arrows), x 200. C. Staining pattern with MAb 63.12. Observe that basal epithelial cells are the only cells not reacting with antibody (arrows), x 200,
observed for normal oral mucosa (basal cells only with the MAb 17,13 and supra-basal cells with the MAb 63,12), Group 4: foeal keratosis, epithelial hyperplasia - This group comprised 21 cases of focal keratosis and 2 cases of epithelial hyf)erplasia. In 13 cases (57%i) the reaction with MAb 17,13 was positive in the basal cell layer. The other 10 cases (4.3'V;>) stained both the basal cell layer and suprabasal epithelial cells. All specimens exhibited positive suprabasal staining with the MAb 63.12. Twelve stained homogeneously and 11 stained focal / patchy. Groups 5-7: proliferative verrucous leukoplakia (PVL), dysplasia, and squamous eell eareinoma (SCC) These three lesions are presented together due to the similar results observed. This group comprised a total of 59 specimens. The reaction with MAb 17.13 was positive in suprabasal epithelial cells in 57 (97"/i) of the cases. However, in two case of PVL, the basal cells stained positively with MAb 17,13, The reaction with MAb 63,12 was a "mirror image" of that observed with MAb 17,13, In 57 cases, no reaction was observed in suprabasal epithelial cells. In the 2 cases
of PVL where MAb 17,13 stained the basal cells only, the staining pattern for MAb 63,12 was suprabasal and homogeneously positive. In other words, the two cases diagnosed as early PVL stained in the same manner as the normal oral mucosa or benign lesions. In this group of 59 lesions, the staining pattern of MAbs 17,13 and 63,12 was irrespective of the presence or absence of an inflammatory infiltrate in the underlying connective tissue.
Discussion The potential role of cell markers in oral histopathology has been discussed in the recent literature (6-9), Traditionally pathologists have relied on morphologic diagnostic techniques involving varying descriptive criteria that lead to subjective judgments. In some instances (e,g, premalignant lesions, superficial invasion, margins), this fosters uncertain diagnoses and increases the risk for inadequate treatment (10, II), New immunoeytochemical techniques and monoclonal antibodies (MAbs) facilitate the recognition of molecules present on cell
membranes and within the cytoplasm. This provides additional insight for cell and tissue typing. Hopefully, these techniques will significantly add to the accuracy of recognizing cells at increased risk for malignant transformation or those cells that may be expressing biochemical malignant behavior prior to recognizable morphologic changes (II 13), In the current study, immunoperoxidase staining patterns (mirror image) observed in frozen sections of oral .squamous epithelium by MAbs 17,13 and 63,12 appear to be useful in distinguishing benign epithelium from dysplasia or malignancy. Our results confirm that the antigen recognized by MAb 17,13 in normal oral mucosa and benign lesions is confined to the basal epithelial cells only. In contrast, in lesions representing epithelial dysplasia or squamous cell carcinoma, MAb 17.13 will stain all dysplastic and squamous tumor cells, whereas the reaction with MAb 63.12 is negative. Therefore, in areas of dysplasia and squamous cell carcinoma, it appears that the 17.13 antigen(s) not only persists but is also expressed in all the epithelial areas of dysplasia. On the
Monoclonal antibodies 17.13 and 63.12 415
Eig. 3. Epithelial dysplasia. .\. H&E, staining (E, epithelium. CT, connective tissue), x 2(K). B. staining pattern with M.Ab 17.13. Observe that, as opposed to the normal oral mucosa and local keratosis, several epithelial cell layers are reacting with anlibody (arrows), x 200. C. Staining pattern with MAb 63.12. Observe that most of reaction is negative. There are only few cells showing positive staining (arrows), x 200,
other hand, the 63,12 antigen(s) decreases or is completely absent in the same areas. Of importance is the fact that antigens can be masked during tissue fixation. However, because we Used frozen tissue, masking of the antigens probably did not occur. Furthermore, the staining pattern observed with dysplastic and malignant lesions Was not observed with any of the other lesions studied. In the benign oral lesions, the predominant staining pattern observed with the MAb 17,13 was the ba,sal cell layer only. In the same lesions, the MAb 63.12 stained suprabasul epithelial cells. However, in some benign lesions and 'n apparently normal oral mucosa, a suprabasal staining pattern with MAb 17.13 was observed. There is as yet no explanation for this finding. However, because of the apparent specificity, the slight variations in binding indicate the presence of antigen(s) that in follow-up fnay have significance. The fact that the suprabasal epithelial eells of lichen planus (LP) stained positively with the MAb 17.13 is of interest. The suprabasal staining observed with I^Ab 17,13 seemed to correlate with the presence of a mononuclear cell infiltrate
in the underlying connective tissue. Furthermore, epithelial areas staining with MAb 17,13 also stained with MAb 63,12, suggesting a simultaneous and mixed expression of both antigens. Extensive changes in cytokeratin expression patterns in affected human gingiva have been demonstrated, including cytokeratin 19 (15), It has been suggested that these changes in the cytokeratins expression in human epithelia may be induced by inflammatory processes. Expression of cytokeratin 19 has also been reported to be induced in oral dysplastic lesions, and to have a similar staining pattern as MAb 17,13 (16), However, the staining pattern observed in lichen planus is different from that observed with the dysplastic and malignant lesions. Nonetheless, it must be remembered that LP is associated with a risk for malignant transformation (17, 18), and by some is even considered a "precancerous" lesion. It might be that LP malignant transformation is a slow-field canccrization process and that the detection of this so far unidentified antigen(s) by MAb 17,13 has a significance in abnormal cell regulation. Another interesting observation was noted with PVL lesions, PVL exhibits
epithelial changes varying from benign hyperkeratosis, stages of dysplasia, to carcinoma (19), The risk for eventual malignant transformation is high. Therefore, the strong and homogeneous staining reaction observed in PVL lesions with MAb 17,13 and the lack of staining with MAb 63,12 would support this possibility, and would add to the concept that MAb 17,13 is capable of detecting early epithelial changes that may be associated w ith malignant transformation. Two PVL lesions stained with MAb 17,13 failed to demonstrate positive staining in the suprabasal epithelial cells. Possible explanations might include an incorrect clinical/microscopic diagnosis, or the areas biopsied were non-representative of early PVL changes. Early changes may mimic focal hyperkeratosis and will stain similarly (basal cells only). This finding adds to the importance of accurate and careful follow-up of all white lesions of the oral mucosa as well as the potential value of reliable cell markers. It has been demonstrated that cell markers are useful in diagnostic histopathology (6), A more precise identification of the nature of pathologic processes has become possible with the use
416
MIGLIORATI et al.
of reagents capable of recognizing antiReferences gens on cell membranes and within the 1. MiciuORATi CA, RANKKN R , KAPLAN M , cytoplasm (7 9). Several studies using Sii.vKRMAN SJR. The use of monoclonal monoclonal antibodies directed against antibody 17.13 in the diagnosis of human a variety of epithelial antigens have been oral squamous cell carcinoma. J Oral performed. These studies have aided our Pathot Med 1990; 19: 414 8, understanding of the normal develop2. RANKEN R , WHITI; CF, GOTTFRIKD TG, ment of oral epithelium and the changes et at. Reactivity of monoclonal antibody in antigenic expression that may occur 17,13 with human squamous cell carciduring the development of disease pronoma and its application to tumor diagcesses (20-27), Difference in cytokeratin nosis. Cancer Res 1987; 47: 5684 90, expression have been observed between 3. RANKKN R , KAPLAN M , SILVI;RMAN SJR, gingival epithelium and adjacent alveoet at. A monoclonal antibody to squamous cell carcinoma. Laryngoscope lar mucosa (20), and keratinized and 1987; 97: 657 62. non-keratinized mucosa (15, 21-22), 4. KoHLER G, MiLSTEiN C. Continuous culOthers have shown differences in keratures of fused cells secreting antibody of tin expression between benign and mapredefined specificity. Nature 1975; 256: lignant oral lesions (23-25, 27), 495 7. We believe that the MAb 17,13 and 5. Hsu S, RAINH L, FANGFR H . U.se of avi63,12 are directed to cytoplasmic antidin-biotin-peroxidase complex (ABC) in gens present in oral squamous epithelial immunoperoxidase techniques. J Histocells and that their immunoperoxidase ehem Cytochem 1981; 29; 577-80. staining patterns are indicative of dif- 6. SEIFERT G , The importance of tumor markers in oral pathology II. Cell memferent disease processes. With these rebrane and cytoplasmic antigens as tumor sults, it is tempting to postulate that in markers. Pathot Res Pract 1985; 179: the absence of connective tissue inva625-8. sion by epithelial cells, the full thickness 7. CoiNi>RE JM, TANCUV F, MERLK) JP, (•/ staining of oral epithelial lesions with at. The value of immunohistological MAb 17.13 and the absence of staining techniques in undifferentiated cancers. with MAb 63.12 would predict a greater Tumori 1986; 72: 539 44. potential for the development of malig8. WILLIAMS DM, DANIELS TE. The present nancy. If true, the value could be seen and potential role of cell markers in oral in the evaluation by the lesions in group diagnosis. J Orat Pathot 1987; 16: 186-8. 4 (focal keratosis and epithelial hyper9. MORGAN PR, SHIRLAW PJ, JOHNSON NW, et at. Potential applications of antiplasia). Some lesions in this group keratin antibodies in oral diagnosis. J stained positively with the MAb 17,13 Orat Pathot 1987; 16: 212 22. in the suprabasal epithelial cells. By 10. PlNDBORCi JJ, Ri:iBIil J, HOI.MSTRtIP P. means of currently available histopathSubjectivity in evaluating oral epithelial ologic techniques, one cannot observe dysplasia, carcinoma in situ and initial histopathologic differences between carcinoma. J Orat Pathot 1985; 14: lesions that stained the basal cell only 698 708. or those that stained suprabasal cells. 11. MArTHEWS JB. Immunoeytochemical Such findings might well designate the methods: a technical overview. J Orat leukoplakias that in time are most likely Pathot 1987; 16: 189-95. to transform into malignancy, thereby 12. S( HOEPKE HG. The prospective of the aiding clinical management. In order to biotechnology industry on changing technology in pathology. Arch Pathot investigate this possibility, long-term Lab Med 1987; 111: 596 600. prospective follow-up studies are under way. On the other hand, retrospective 13. MiisillKA M, MiWA T, StiztiOKi Y, et at. Detection of proliferative cells in dysplastudies using formalin-fixed tissue are sia, carcinoma in .situ, and invasive carcialso being conducted. noma of the uterine cervix by monocloOther MAbs have demonstrated the nal antibody against DNA polymerase. capability of detecting premalignant Cancer 1988; 61: 1182 6, and malignant changes in lesions of epi- 14. SILVERMAN SJR, GORSKY M , LOZADANuR F, GiANNOTi K, A prospective study thelial origin (16, 28 33). Although the of findings and management in 214 paantigens recognized by MAbs 17.13 and tients with oral lichen planus. Orat Surg 63.12 are not yet known, the staining Orat Med Orat Pathot 1991; 72: 665 70. pattern differences observed among 15. BOSCH F X , OUHAVUN JP, BADER BL, et these reagents appear to indicate that at. Extensive changes in cytokeratin exMAbs 17,13 and 63,12 are detecting pression patterns in pathologically afunique cytoplasmic epitopes. The nafected human gingiva, Virchows Archiv ture of these antigens is of particular B Ceii Pathot 1989; 58: 59 77, interest and studies for their identifica- 16. LiNDBERG EK, RHEINWALD JG, Supration are ongoing. basal 40kd keratin (KI9) expression as
an immunohistological marker of premalignancy in oral epithelium. Am J Pathot 1989; 134: 89 98, 17. SILVERMAN S JR, GORSKY M , LOZADA-
NiiR F. A prospective follow-up study of 570 patients with oral lichen planus; persistence, remission, and malignant association, Orat Surg Orat Med Orat Pathot 1985; 60: 30 4, 18. MuRri PR, DAI rARY RB, BHONSLE PC, ('/ at. Malignant potential of oral lichen planus: observations in 722 patients from India. J Orat Pattwt 1986; 15: 71 7. 19. HANSEN LS, OLSON JA, SILVKRMAN SJR.
Proliferative verrucous leukoplakia. Orat Surg Orat Med Orat Pathot 1985; 60: 285-98. 20. OUHAYOUN JP, GOSSELIN F, FoREST N , et
at. Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and adjaeent alveolar mucosa. Differentiation 1985; 30; 123-9. 21. CLAUSEN H , VEHTOFTE P, MOE D , et at.
Differentiation-dependent expression of keratins in human oral epithelia.,/ Invest Dermatot 1986; 86: 249 54. 22. CLAUSEN H , MOPE D , BUSC HARD K , DA-
BELSTEEN E. Keratin proteins in human oral mucosa, J Orat Pathot 1986; 15: 36^2, 23. REIBEL J, CLAUSEN H , DAHELSIEEN E.
Staining patterns of human premalignant oral epithelium and squamous cell carcinomas by monoclonal anti-keratin antibodies. Aeta Pathot Mierobioi Immunot Seand Sect A 1985; 93: 323 30. 24.
MORI M , NAKAI M , HYUN KH, et al
Distribution of keratin proteins in neoplastic and tumorlikc lesions of .squamous epithelium. Orat Surg Oral Med Orat Pathot 1985; 59: 63 9. 25. NAKAI M , MORI M . Immunohistochemi-
cal distribution of monoclonal antibdoies against keratin in papillomas and carcinomas from oral and nasopharyngeal regions. Orat Surg Orat Med Orat Pathot 19X6; 62: 292 302. 26. TATEMOTO Y, SAKA
M , TANIMURA T,
MORI M , Immunoeytochemical observations on binding of monoclonal antibody to epithelial membrane antigen in epithelial tumors of the oral cavity, Orat Surg Orat Med Orat Pattwt 1987; 64: 721 6. 27.
HEYBFN A , HUITFEI.DI HS, KOPPANO HS, THRANE PS, BRYNE M , BRANirrzAEG
P, Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions, J Orat Pathot Med 1992; 2 1 : 7 11, 28.
BOHEIM K, SCHWARZEURTNER H , BOHEIM
C, et at. Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQMI, Acta Otolaryngol 1986; 102: 333 40, 29. W
C, A, Migliorati', R, RankenS M, J, Kaplan^ and S, Silverman Jr/ Departments of 'Oral Diagnostic Sciences, Division of Oral Medicine, tjniversity of Fiorida Coiiege of Dentistry, Gainesville, 'Intek Diagnostics Inc., tvlillbrae, CA, 'Otolaryngology, School of t\/ledicine. University of California - San Francisco, 'Stomatology, Division of Oral Medicine, School of Dentistry, University of California San Francisco, USA
Migliorati CA, Ranken R, Kaplan MJ, Silverman S Jr: Reactivity of monoclonal antibodies 17,13 and 63,12 with 141 oral mucosai lesions, J Oral Pathol Med 1992; 21:412^17, We studied the reactivity of monoclonal antibodies (MAbs) 17,13 and 63,12 with normal and diseased human oral mucosa by means of the immunoperoxidase technique. The specimens included: 22 normal oral tissues, 20 benign tumors, 17 lichen planus, 23 focal keratosis and epithelial hypcrplasias, 18 proliferative verrucous leukoplakias, 20 dysplasias, and 21 squamous cell carcinomas. In most cases of normal mucosa and benign lesions, MAb 17,13 stained basal epithelial cells only, whereas MAb 63,12 stained all cell layers above the basal cells. In the premalignant and malignant lesions MAb 17,13 stained above the basal cells and MAb 63,12 either stained areas not stained by MAb 17,13 or the staining was absent. Based on the different staining patterns observed, there appears to be a potential value of these new reagents in diagnostic histopathology regarding specimens with equivocal cellular morphology.
In previous reports (1 3) we demonstrated the reactivity of monoclonal antibody 17.13 (MAb 17.13) with human squamous cell carcinoma and its potential application to head and neck tumor diagnosis. MAb 17.13 was derived from a fusion of splenocytes of a BALB/c mouse immunized with a surgically resected poorly differentiated human recurrent laryngeal squamous cell carcinoma with SP2/0 mouse myeloma cells. It is an IgM antibody that consistently reacts with an unidentified cytoplasmic antigen present in oral squamous tumor cells. In normal tissues and benign lesions, the antibody reacts with another unidentified antigen present in the cytoplasm of basal epithelial cells only. MAb 63.12, another newly developed reagent, was generated from the fusion of SP2/0 mouse myeloma cells with splenocytes of a mouse that had been immunized with tumor cells derived from a squamous carcinoma of the floor of the mouth. This IgM antibody reacts with an antigen present in the cytoplasm of non-basal epithelial cells. Its reaction pattern is a mirror image of the pattern displayed by MAb 17,13, as demonstrated by our previous studies, whereas MAb 63,12 stained all suprabasal epithelial layers in nomial oral mucosai tissues and benign oral lesions.
Based on these findings, we studied by means of the immunoperoxida,se technique the reactivity of MAbs 17,13 and 63,12 in a larger and diverse group of oral mucosai lesions to further confirm the reliability of the staining pattern of these antibodies. In turn, this would help assess their capability of marking tissues that possess the potential of possibly becoming malignant or already expressing malignant transformation at a stage when histologic morphology appears normal or equivocal.
Key words: cancer diagnosis: immunohlstopathology: monoclonal antibodies: mouth, diseases: oral histopathology: oral premalignancy; oral cancer. Cesar A. Migliorati, Department of Oral Diagnostic Sciences. University of Florida College of Dentistry, Box 100414 JHMHC; Gainesville, FL 32610, USA. Accepted for publication May 8. 1992.
in liquid nitrogen for the immunop)eroxidase reaction. Monocionai antibodies and Immunoperoxidase reaction
Three commercially unavailable murine MAbs obtained with the specificity of hybridoma technology (4) were utilized in this study: MAbs 17,13, 63,12, and 88,25, the latter, also an IgM type, serving as a negative control. For a complete review of the antibodies used in this study, see MIGLIORAII et al, (1) and RANKEN et al. (2, 3),
Material and methods Tissue sampies
Biopsy specimens from 141 oral mucosai lesions were obtained from patients seen at the Oral Medicine Clinic, University of California, San Francisco, The specimens included 22 normal oral mucosae, 20 benign tumors and benign pigmented lesions, 17 lichen planus, 23 focal keratoses and epithelial hyperplasias, 18 proliferative verrucous leukoplakia, 20 dysplasias, and 21 squamous cell carcinomas. The specimens were divided into two equal parts. One half was fixed in IO'MJ formalin and processed for routine histopathologic diagnosis and the other half was frozen immediately
For each specimen, 5 nm serial frozen sections were cut on the cryostat (Slee, London) and mounted on formaldehyde-gelatin-coated glass slides. Tissue sections were immediately fixed in cold acetone and air dried. Sections were reacted with the monoclonal antibodies by tneans of the immunoperoxidase technique (5), The procedure has been described previously (1, 4), In brief it entails the following steps: Glass slides were coated with gelatin. Frozen tissue sections were mounted and fixed with cold acetone. The tissues were blocked with normal horse serum and hydrogen peroxide and incubated with the primary monoclonal antibodies. After reacting the tissues with biotinylated
Monoclonal antibodies 17.13 and 63.12 413 17.13 reaction Basal cells only
n Normal oral mucosa Benign tumor, benign pigmented lesions Lichen planus Focal keratosis, epithelial hyperplasia Proliferative verrucous leukoplakia Dysplasia Squamous cell carcinoma
horse anti-rnouse IgG heavy and light chain specific, slides were incubated with avidin-biotin-peroxidase complex. A mixture of diaminobenzidine and hydrogen peroxide was used lor providing color to the reaction and hematoxylin Was used as a counterstain. All specimens were reacted with the 3 MAbs. Any brown staining that was more intense than the negative control background ob.served with MAb 88.2,S Was considered a positive reaction. Staining patterns were recorded and the results were compared with the independently read routine histopathologic slides. Resuits
Table 1 presents a summary of the results obtained among the 141 specimens, grouped within 7 categories. Figs. 1 3 illustrate representative staining re-
A
22 20
20 14 1 13 2
17
23 18 20 21
Above basal cells
63.12 (nonnal) reaction Above basal cells HomoFocal geneous patchy NEG
88.25 (control) reaction
POS
NEG
22 20 17 23 18 20 21
2
8
14
0
0
6 16 10 16
16 3 12 2
4 14 11
0 0 0 16 20 21
0 0 0 0 0
20 21
actions of MAbs 17.13 and 63,12. The staining results were as follows: Group I: nortnal oral muco.sa
The
normal oral mucosa group included 18 samples from the gingiva, 2 from the buccal mucosa, and 2 from the tongue. The staining pattern of MAb 17,13 in normal oral mucosa was characterized by a positive reaction of the basal cells in the majority of cases (91%), In two cases of gingival tissue (9%), the parabasal and spinous cells reacted positively. All samples were positive with MAb 63,12, This staining pattern was always present above the basal cell layer, and was either homogeneous (36%) or focal/patchy (64%) in distribution. Group 2: benign tumors and henis;n
reactive lesions - This group included 10 fibromas, 4 squamous papillomas, 2 pyogenic granulomas, 1 focal melanosis, 1 peripheral ossifying fibroma, 1 mucocele, and 1 gingival fibromatosis.
0
The staining pattern of MAb 17,13 was observed solely in the epithelial basal cell layer in 14 (70%i) cases. In six cases (2 papillomas, 2 fibromas, 2 pyogenic granulomas) MAb 17,13 stained cells above the basal cell layer, MAb 63,12 stained all specimens. The staining was always above the basal cell layer and was homogeneous in 16 (80'V,,) cases and focal/patchy in 4 (20%), Group 3: liehen planus - In all but one of the 17 cases, the MAb 17,13 stained suprabasal epithelial cells. All specimens also exhibited a positive staining reaction with MAb 63,12, This reaction was focal/patchy in 14 cases (82"o) and homogeneous in 3 (18%), Of interest, when a subepithelial lymphocytic infiltrate was present, both MAbs stained suprabasally. Occasionally, areas of lichen planus not exhibiting the sub)epithelial lymphocytic infiltrate presented a staining pattern comparable with that
B
I'^ig L Normal oral mucosa. A. M&F. staining (F., epithelium. CT, connective tissue), x 400. B, staining pattern with M.Ab 17.13. Note that only basal epithelial cells react with antibody (arrows), x 400. C, staining pattern with MAb 63.12. Nole that basal epithelial cells are the only cells that do not react with antibody (arrows), x 400.
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Fig. 2. Focal keratosis. A, H&E staining (E, epithelium. CT, connective tissue), x 200. B, Staining pattern with MAb 17.13, Observe similar pattern as lor normal oral mucosa. Basal epithelial cells are the only cells reacting with antibody (arrows), x 200. C. Staining pattern with MAb 63.12. Observe that basal epithelial cells are the only cells not reacting with antibody (arrows), x 200,
observed for normal oral mucosa (basal cells only with the MAb 17,13 and supra-basal cells with the MAb 63,12), Group 4: foeal keratosis, epithelial hyperplasia - This group comprised 21 cases of focal keratosis and 2 cases of epithelial hyf)erplasia. In 13 cases (57%i) the reaction with MAb 17,13 was positive in the basal cell layer. The other 10 cases (4.3'V;>) stained both the basal cell layer and suprabasal epithelial cells. All specimens exhibited positive suprabasal staining with the MAb 63.12. Twelve stained homogeneously and 11 stained focal / patchy. Groups 5-7: proliferative verrucous leukoplakia (PVL), dysplasia, and squamous eell eareinoma (SCC) These three lesions are presented together due to the similar results observed. This group comprised a total of 59 specimens. The reaction with MAb 17.13 was positive in suprabasal epithelial cells in 57 (97"/i) of the cases. However, in two case of PVL, the basal cells stained positively with MAb 17,13, The reaction with MAb 63,12 was a "mirror image" of that observed with MAb 17,13, In 57 cases, no reaction was observed in suprabasal epithelial cells. In the 2 cases
of PVL where MAb 17,13 stained the basal cells only, the staining pattern for MAb 63,12 was suprabasal and homogeneously positive. In other words, the two cases diagnosed as early PVL stained in the same manner as the normal oral mucosa or benign lesions. In this group of 59 lesions, the staining pattern of MAbs 17,13 and 63,12 was irrespective of the presence or absence of an inflammatory infiltrate in the underlying connective tissue.
Discussion The potential role of cell markers in oral histopathology has been discussed in the recent literature (6-9), Traditionally pathologists have relied on morphologic diagnostic techniques involving varying descriptive criteria that lead to subjective judgments. In some instances (e,g, premalignant lesions, superficial invasion, margins), this fosters uncertain diagnoses and increases the risk for inadequate treatment (10, II), New immunoeytochemical techniques and monoclonal antibodies (MAbs) facilitate the recognition of molecules present on cell
membranes and within the cytoplasm. This provides additional insight for cell and tissue typing. Hopefully, these techniques will significantly add to the accuracy of recognizing cells at increased risk for malignant transformation or those cells that may be expressing biochemical malignant behavior prior to recognizable morphologic changes (II 13), In the current study, immunoperoxidase staining patterns (mirror image) observed in frozen sections of oral .squamous epithelium by MAbs 17,13 and 63,12 appear to be useful in distinguishing benign epithelium from dysplasia or malignancy. Our results confirm that the antigen recognized by MAb 17,13 in normal oral mucosa and benign lesions is confined to the basal epithelial cells only. In contrast, in lesions representing epithelial dysplasia or squamous cell carcinoma, MAb 17.13 will stain all dysplastic and squamous tumor cells, whereas the reaction with MAb 63.12 is negative. Therefore, in areas of dysplasia and squamous cell carcinoma, it appears that the 17.13 antigen(s) not only persists but is also expressed in all the epithelial areas of dysplasia. On the
Monoclonal antibodies 17.13 and 63.12 415
Eig. 3. Epithelial dysplasia. .\. H&E, staining (E, epithelium. CT, connective tissue), x 2(K). B. staining pattern with M.Ab 17.13. Observe that, as opposed to the normal oral mucosa and local keratosis, several epithelial cell layers are reacting with anlibody (arrows), x 200. C. Staining pattern with MAb 63.12. Observe that most of reaction is negative. There are only few cells showing positive staining (arrows), x 200,
other hand, the 63,12 antigen(s) decreases or is completely absent in the same areas. Of importance is the fact that antigens can be masked during tissue fixation. However, because we Used frozen tissue, masking of the antigens probably did not occur. Furthermore, the staining pattern observed with dysplastic and malignant lesions Was not observed with any of the other lesions studied. In the benign oral lesions, the predominant staining pattern observed with the MAb 17,13 was the ba,sal cell layer only. In the same lesions, the MAb 63.12 stained suprabasul epithelial cells. However, in some benign lesions and 'n apparently normal oral mucosa, a suprabasal staining pattern with MAb 17.13 was observed. There is as yet no explanation for this finding. However, because of the apparent specificity, the slight variations in binding indicate the presence of antigen(s) that in follow-up fnay have significance. The fact that the suprabasal epithelial eells of lichen planus (LP) stained positively with the MAb 17.13 is of interest. The suprabasal staining observed with I^Ab 17,13 seemed to correlate with the presence of a mononuclear cell infiltrate
in the underlying connective tissue. Furthermore, epithelial areas staining with MAb 17,13 also stained with MAb 63,12, suggesting a simultaneous and mixed expression of both antigens. Extensive changes in cytokeratin expression patterns in affected human gingiva have been demonstrated, including cytokeratin 19 (15), It has been suggested that these changes in the cytokeratins expression in human epithelia may be induced by inflammatory processes. Expression of cytokeratin 19 has also been reported to be induced in oral dysplastic lesions, and to have a similar staining pattern as MAb 17,13 (16), However, the staining pattern observed in lichen planus is different from that observed with the dysplastic and malignant lesions. Nonetheless, it must be remembered that LP is associated with a risk for malignant transformation (17, 18), and by some is even considered a "precancerous" lesion. It might be that LP malignant transformation is a slow-field canccrization process and that the detection of this so far unidentified antigen(s) by MAb 17,13 has a significance in abnormal cell regulation. Another interesting observation was noted with PVL lesions, PVL exhibits
epithelial changes varying from benign hyperkeratosis, stages of dysplasia, to carcinoma (19), The risk for eventual malignant transformation is high. Therefore, the strong and homogeneous staining reaction observed in PVL lesions with MAb 17,13 and the lack of staining with MAb 63,12 would support this possibility, and would add to the concept that MAb 17,13 is capable of detecting early epithelial changes that may be associated w ith malignant transformation. Two PVL lesions stained with MAb 17,13 failed to demonstrate positive staining in the suprabasal epithelial cells. Possible explanations might include an incorrect clinical/microscopic diagnosis, or the areas biopsied were non-representative of early PVL changes. Early changes may mimic focal hyperkeratosis and will stain similarly (basal cells only). This finding adds to the importance of accurate and careful follow-up of all white lesions of the oral mucosa as well as the potential value of reliable cell markers. It has been demonstrated that cell markers are useful in diagnostic histopathology (6), A more precise identification of the nature of pathologic processes has become possible with the use
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of reagents capable of recognizing antiReferences gens on cell membranes and within the 1. MiciuORATi CA, RANKKN R , KAPLAN M , cytoplasm (7 9). Several studies using Sii.vKRMAN SJR. The use of monoclonal monoclonal antibodies directed against antibody 17.13 in the diagnosis of human a variety of epithelial antigens have been oral squamous cell carcinoma. J Oral performed. These studies have aided our Pathot Med 1990; 19: 414 8, understanding of the normal develop2. RANKEN R , WHITI; CF, GOTTFRIKD TG, ment of oral epithelium and the changes et at. Reactivity of monoclonal antibody in antigenic expression that may occur 17,13 with human squamous cell carciduring the development of disease pronoma and its application to tumor diagcesses (20-27), Difference in cytokeratin nosis. Cancer Res 1987; 47: 5684 90, expression have been observed between 3. RANKKN R , KAPLAN M , SILVI;RMAN SJR, gingival epithelium and adjacent alveoet at. A monoclonal antibody to squamous cell carcinoma. Laryngoscope lar mucosa (20), and keratinized and 1987; 97: 657 62. non-keratinized mucosa (15, 21-22), 4. KoHLER G, MiLSTEiN C. Continuous culOthers have shown differences in keratures of fused cells secreting antibody of tin expression between benign and mapredefined specificity. Nature 1975; 256: lignant oral lesions (23-25, 27), 495 7. We believe that the MAb 17,13 and 5. Hsu S, RAINH L, FANGFR H . U.se of avi63,12 are directed to cytoplasmic antidin-biotin-peroxidase complex (ABC) in gens present in oral squamous epithelial immunoperoxidase techniques. J Histocells and that their immunoperoxidase ehem Cytochem 1981; 29; 577-80. staining patterns are indicative of dif- 6. SEIFERT G , The importance of tumor markers in oral pathology II. Cell memferent disease processes. With these rebrane and cytoplasmic antigens as tumor sults, it is tempting to postulate that in markers. Pathot Res Pract 1985; 179: the absence of connective tissue inva625-8. sion by epithelial cells, the full thickness 7. CoiNi>RE JM, TANCUV F, MERLK) JP, (•/ staining of oral epithelial lesions with at. The value of immunohistological MAb 17.13 and the absence of staining techniques in undifferentiated cancers. with MAb 63.12 would predict a greater Tumori 1986; 72: 539 44. potential for the development of malig8. WILLIAMS DM, DANIELS TE. The present nancy. If true, the value could be seen and potential role of cell markers in oral in the evaluation by the lesions in group diagnosis. J Orat Pathot 1987; 16: 186-8. 4 (focal keratosis and epithelial hyper9. MORGAN PR, SHIRLAW PJ, JOHNSON NW, et at. Potential applications of antiplasia). Some lesions in this group keratin antibodies in oral diagnosis. J stained positively with the MAb 17,13 Orat Pathot 1987; 16: 212 22. in the suprabasal epithelial cells. By 10. PlNDBORCi JJ, Ri:iBIil J, HOI.MSTRtIP P. means of currently available histopathSubjectivity in evaluating oral epithelial ologic techniques, one cannot observe dysplasia, carcinoma in situ and initial histopathologic differences between carcinoma. J Orat Pathot 1985; 14: lesions that stained the basal cell only 698 708. or those that stained suprabasal cells. 11. MArTHEWS JB. Immunoeytochemical Such findings might well designate the methods: a technical overview. J Orat leukoplakias that in time are most likely Pathot 1987; 16: 189-95. to transform into malignancy, thereby 12. S( HOEPKE HG. The prospective of the aiding clinical management. In order to biotechnology industry on changing technology in pathology. Arch Pathot investigate this possibility, long-term Lab Med 1987; 111: 596 600. prospective follow-up studies are under way. On the other hand, retrospective 13. MiisillKA M, MiWA T, StiztiOKi Y, et at. Detection of proliferative cells in dysplastudies using formalin-fixed tissue are sia, carcinoma in .situ, and invasive carcialso being conducted. noma of the uterine cervix by monocloOther MAbs have demonstrated the nal antibody against DNA polymerase. capability of detecting premalignant Cancer 1988; 61: 1182 6, and malignant changes in lesions of epi- 14. SILVERMAN SJR, GORSKY M , LOZADANuR F, GiANNOTi K, A prospective study thelial origin (16, 28 33). Although the of findings and management in 214 paantigens recognized by MAbs 17.13 and tients with oral lichen planus. Orat Surg 63.12 are not yet known, the staining Orat Med Orat Pathot 1991; 72: 665 70. pattern differences observed among 15. BOSCH F X , OUHAVUN JP, BADER BL, et these reagents appear to indicate that at. Extensive changes in cytokeratin exMAbs 17,13 and 63,12 are detecting pression patterns in pathologically afunique cytoplasmic epitopes. The nafected human gingiva, Virchows Archiv ture of these antigens is of particular B Ceii Pathot 1989; 58: 59 77, interest and studies for their identifica- 16. LiNDBERG EK, RHEINWALD JG, Supration are ongoing. basal 40kd keratin (KI9) expression as
an immunohistological marker of premalignancy in oral epithelium. Am J Pathot 1989; 134: 89 98, 17. SILVERMAN S JR, GORSKY M , LOZADA-
NiiR F. A prospective follow-up study of 570 patients with oral lichen planus; persistence, remission, and malignant association, Orat Surg Orat Med Orat Pathot 1985; 60: 30 4, 18. MuRri PR, DAI rARY RB, BHONSLE PC, ('/ at. Malignant potential of oral lichen planus: observations in 722 patients from India. J Orat Pattwt 1986; 15: 71 7. 19. HANSEN LS, OLSON JA, SILVKRMAN SJR.
Proliferative verrucous leukoplakia. Orat Surg Orat Med Orat Pathot 1985; 60: 285-98. 20. OUHAYOUN JP, GOSSELIN F, FoREST N , et
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