Involucrin expression in cervical intraepithelial neoplasia: A critical evaluation

GYNECOLOGIC

ONCOLOGY

26, 25-34 (1987)

lnvolucrin Expression in Cervical lntraepithelial Neoplasia: A Critical Evaluation AMIN ELSAYED, M.D. ,* RALPH M. RICHART, CHRISTOPHER P. CRUM, M.D. t fDivision

M.D. ,t AND

of Obstetrical and Gynecological Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, and the *Department of Obstetrics and Gynecology, Zagazig University Medical Center, Zagazig, Egypt

Received August 6, 1985 Involucrin is a keratinocyte envelope protein precursor which is synthesized at an early stage of differentiation in normal squamous epithelium. Recent studies suggest that this protein may be a marker for neoplastic epithelium. To address this issue, we analyzed involucrin expression in 105 biopsies containing 119 areas of normal, condylomatous, and neoplastic epithelium. Overall, 88, 75, and 55% of condylomata, well-differentiated CIN, and poorly differentiated CIN (carcinoma in situ) contained positive staining for involucrin. Excluding lesions with severe inflammation, 100, 88, and 55% of these lesions, respectively, were positive. Staining patterns in neoplastic lesions differed from those in the normal epithelium and condylomata; the staining in CIN tended to be focal, and intensity of staining varied widely from cell to cell in all layers of the epithelium. In high grade CIN, staining correlated with increases in cell size and cytoplasmic differentiation. These studies suggest that involucrin will not differentiate between lesions of low versus high risk for progressing to invasive carcinoma. However, the patterns of involucrin expression confirm the marked differences in patterns of cellular differentiation between classical condylomata and CIN. 0 1987 Academic Press, Inc.

INTRODUCTION

During the late stages of differentiation, human epithelial keratinocytes produce an insoluble protein envelope on the cytoplasmic side of the plasma membrane [l-4]. Involucrin, a soluble protein precursor of the envelope, is synthesized at an earlier stage of differentiation both in the natural epithelium and in cultured keratinocytes [5,6]. The crosslinking of these soluble envelope precursors is catalyzed by a calcium-dependent transglutaminase reaction [7,8]. Involucrin expression is closely dependent upon differentiation which is correlated with increasing cell size as the epithelium matures. Hence, the presence of involucrin has been found to correlate with cell size [9]. Since involucrin is consistently associated with normal squamous differentiation, it has been suggested that involucrin production may be used to distinguish normal squamous epithelium, or its variants, from precancerous squamous lesions [IO]. The latter are often characterized by derangements in maturation and differentiation and, theoretically may exhibit altered involucrin production as well. 25 0090-8258/87$1SO Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form resewed.

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ELSAYED, RICHART, AND CRUM

In a recent study [lo] using immunoperoxidase techniques to localize involucrin in lesions of the uterine cervix, all examples of normal squamous mucosa, all flat and exophytic condylomas, and 97% of squamous metaplasia were positive for involucrin. The majority (81%) of lesions classified as dysplasia were negative. Involucrin production appeared to be influenced by inflammation as well, since in the presence of severe inflammation, only 50-60% of normal or metaplastic epithelia were positive. The purpose of this study was to determine if the histological criteria used to define CIN lesions and distinguish them from condylomata [l l-131 could be correlated with histochemical staining for involucrin. We present evidence that involucrin is frequently produced in lesions which we classify as CIN and that its presence correlates with maturation. However, certain differences exist in the distribution of involucrin expression in neoplastic epithelium which point to differences in patterns of maturation between CIN and nonneoplastic epithelium, including condylomata. MATERIALS AND METHODS Classification of case material. Cervical biopsies were obtained from the files of the Sloane Hospital for women at Columbia University College of Physicians and Surgeons. Each biopsy was reviewed and classified in the following manner: (1) Normal mature squamous epithelium (Fig. I), (2) immature squamous metaplasia

FIG. 1. Normal mature squamous epithelium contains staining for involucrin, seen here as a dark reaction product. The staining is distributed evenly throughout the suprabasal epithelium with little variation in staining intensity from cell to cell. The basal layer is negative (arrows).

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(Fig. 2), (3) flat condyloma (Fig. 3), (4) well-differentiated CIN, with or without koilocytosis (Fig. 4) and (5) poorly differentiated CIN (carcinoma in situ) (Figs. 5-7 [12]. Condylomata contained superficial nuclear atypia (koilocytotic atypia). CIN was distinguished from condylomata by the presence of abnormal mitoses and diffuse nuclear atypia [ 121 but well-differentiated CIN contained conspicuous maturation in at least the upper third of the epithelium, including koilocytotic atypia (Fig. 4). Since expression of involucrin has been correlated with the extent of stromal inflammation, the degree of inflammatory infiltrate was also recorded and graded as 1, 2, or 3 + as described previously [14]. Staining for involucrin. Serial sections from Bouins-fixed biopsies were deparaffinized and stained for involucrin using the avidin-biotin system (Vector Laboratories). Rabbit anti-human involucrin was kindly provided by Dr. Robert Rice of the Harvard School of Public Health and used at a 1:1000 dilution. The purification of this antibody has been described [lo]. The antibody was generated against chromatographically purified involucrin from cultured epidermal cells and its specificity confirmed by. Ouchterlony analysis [lo]. Staining controls included normal skin as a positive control, and substitution of primary antiserum with other antisera to evaluate background and nonspecific staining.

FIG. 2. Immature transformation zone epithelium (squamous metaplasia) with focal maturation in the hematoxylin and eosin stained section (a). Involucrin staining (b) concentrates in this area with sparing of the basal cells.

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ELSAYED, RICHART, AND CRUM

FIG. 3. A flat condyloma contains minimal parabasal cell atypia, orderly maturation, and koilocytosis (a). In a serial section stained for involucrin, staining is intense in the mid portions of the epithelium with focal staining just above the basal lamina. (b). The majority of the basal cell do not stain.

RESULTS Case material. One hundred and five biopsy specimens were evaluated histologically and stained for involucrin. These included 3 with immature squamous metaplasia, 39 with condyloma, 26 with well-differentiated CIN, 34 with poorly differentiated CIN and 3 with invasive squamous cell carcinoma (Table 1). Including biopsies with more than one lesion, a total of 119areas of epithelia were evaluated. Znvolucrin staining. Staining for involucrin as a function of lesion type and degree of associated inflammation is summarized in Table 2. Eighty-eight percent of condylomata were positive, including 100%in which the associated inflammation was O-l + . Fifty-five percent of poorly differentiated UN lesions were positive; an equivalent percentage were positive with O-l + inflammation. Seventy-five percent of well-differentiated CIN lesions were positive; 100% of those containing koilocytosis and O-1 + inflammation were positive. Of 16 lesions in all groups with 2 and 3 + inflammation, only 25% were positive (Table 2). Distribution of involucrin staining. In the normal squamous epithelium both mature and immature, the staining for involucrin was virtually absent in the basal epithelium, in many cases sharply demarcating the suprabasal cells (Figs. 1 and 2) and increasing in intensity as a function of maturation. In all casesof condylomata with minimal inflammation, the staining was prominent, primarily in the mature cells and in the koilocytic cells, concentrating in the periphery of the cells (Fig.

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NEOPLASIA

FIG. 4. This well-differentiated CIN contains diffuse nuclear atypia and conspicuous maturation of cells in all epithelial layers with a few koilocytes (arrow) (a). A serial section stained for involucrin with wide variations in staining intensity in certain areas of the epithelium with intensely staining cells (large arrow). Focal staining is also conspicuous in basal cells (small arrow) (b). The latter appear to contain a moderate amount of cytoplasm, reflecting a greater degree of maturation than usually found in the basal cells of normal epithelium or very high grade UN lesions (compare to Figs. 5-7).

TABLE 1 HISTOLOGICAL

CATEGORIES OF CASES ANALYZED FOR INV~LOCRIN

Lesion Epithelium Metaplasia Condyloma Well differentiated CIN Poorly differentiated CIN Invasive cancer Totals

Major feature

Sole feature

Total

3 39 26

2 10 2

5 49 28

34

0

34

3 105

0 14

3 119

FIG. 5. In high grade CIN with gland involvement (a), there is focal maturation present in the center (arrows). Staining for involucrin (b) is confined to this region.

FIG. 6. This high grade CIN lesion (a) contains subtle maturation in the upper region of the epithelium with greater spacing of nuclei, presumably due to increased cytoplasm in the cells (arrows). The staining for involucrin is confined to this area (b), 30

CERVICAI NEOPLASIA

FIG. 7. This high grade CIN contains virtually no changes in cell density, size, or maturation in the upper cell layers, with the exception of the most superficial cell layer (a). Staining for involucrin is negative throughout the lesion (b).

3). However, in contrast to normal epithelium, occasional staining was present in cells near the basal lamina (Fig. 3). The staining in the well-differentiated and poorly differentiated CIN lesions varied. In CIN lesions with orderly maturation the staining distribution was similar to condylomata and normal epithelium. In most, however, the staining for involucrin tended to be irregular in both distribution and intensity, concentrating primarily in the mid and upper regions of the epithelium, but also in the basal layer (Fig. 4). The pattern of staining suggested considerable variability in cytoplasmic differentiation at each level of the epithelium, as well as premature maturation in areas usually containing undifferentiated cells, such as the basal layer (Fig. 4). TABLE 2 DISTFSBLJTION OF INVOLUCRIN

BY LESION

Lesion (No.)

Positive

With O-l + Inflam.

With 2-3 + Inflam.

Condyloma(49) Well Diff CIN (28) + Koilocytosis - Koilocytosis Poorly Diff CIN (34)

43(88%) 21(75%)

41/41(100%) 22/25(88%) 20/20(100%) 215 16/29(55%)

2/8(25%) O/3(0%) o/ 1(0%) o/20 2/5(40%)

18(55%)

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In poorly differentiated CIN lesions, involucrin staining was less widely distributed than in well-differentiated epithelium. It was usually focal, and correlated with subtle differences in maturation and cell size (Figs. 5-7). Focal staining was frequently observed in pockets of cells in which the nuclear-cytoplasmic ratio increased relative to the undifferentiated portions of the epithelium (Fig. 5), and these changes in cell size were often subtle (Fig. 6). In poorly differentiated CIN without discernible changes in cell density or size, involucrin staining was usually negative (Fig. 7). DISCUSSION

This study demonstrates that, in squamous epithelium, involucrin is a consistent marker for differentiation, but is also present in a high proportion of lesions fulfilling morphological criteria for intraepithelial neoplasia. These findings underscore the importance of applying biologically relevant morphological criteria when grouping lesions for analysis of differences in involucrin expression. Theoretically, the morphological markers for aneuploidy will segregate the majority of squamous lesions of the female genital tract into low and high risk precancer categories. This has been achieved by the use of DNA microspectrophotometry [l 11. With these criteria, conventional classification systems using three (GIN) or four (dysplasia-CIS) categories can be reduced to two; condyloma and “true” CIN. The latter can be divided into well- and poorly differentiated categories so that those lesions resembling condyloma (well-differentiated CIN with koilocytotic atypia) are recognized. In general, flat condyloma, well-differentiated CIN, and poorly differentiated CIN correspond to the conventional designations of CIN I (mild dysplasia), CIN II (moderate dysplasia), and CIN III (severe dysplasiacarcinoma in situ), respectively. However, since traditional classifications employ maturation as the most important criterion for determining lesional grade, many condylomata and well-differentiated CIN lesions would be classified together as CIN I or mild dysplasia (1.5).This is acceptable clinically, since both condylomata and CIN are treated. However, if it were desirable to distinguish condylomata and well-differentiated CIN for other reasons, it would be impossible to do so with involucrin staining. Both condyloma and well-differentiated CIN stained positively, despite the fact that they are biologically different and contain distinctly different types of HPVs in most instances [11,16-181. In fact, in the present study, fifty-five percent of high grade CIN lesions (carcinoma in situ) contained involucrin, a finding at considerable variance with previous reports [lo]. Athough the presence of involucrin by itself does not appear to differentiate between lesions of low versus high malignant potential, the patterns of staining between many benign or condylomatous epithelia and CIN differ and are intriguing. The focal staining in CIN with variable staining from cell to cell may reflect abnormalities in differentiation by which individual cells mature at different rates, giving rise to positive staining in the basal layers of some lesions, and cells staining independent of the adjacent cells in the same layer of the epithelium. In hematoxylin and eosin-stained sections this appears to correspond to maturation in basal or parabasal cells and variations in maturation from cell to cell at a given level of the epithelium. These features are more commonly found in GIN as opposed to nonneoplastic epithelium (Fig. 4).

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Cell culture studies of normal keratinocytes have demonstrated a close correlation between cell size and involucrin expression [6]. This relationship appears to hold true for CIN lesions as well. CIN lesions in which the cells in the mid or superficial areas did not increase in size were usually negative for involucrin (Fig. 7). In contrast many high grade CIN lesions had subtle increases in cell size in the mid and superficial epithelium and these areas often stained strongly for involucrin. These changes in cell size were usually seen as a subtle decrease in nuclear density, presumably reflecting a decreasednucleocytoplasmic ratio of the constituent cells in these areas (Figs. 5 and 6). The relationship between involucrin expression and the presence of HPV antigens has been discussed previously [19]. In our experience and that of others, lesions staining for HPV antigens always stain positive for involucrin as well, particularly in areas in which HPV antigens are present (Amin, unpublished data) [19]. Since involucrin is a marker for differentiation, it will identify cells most likely to be permissive for HPV replication and assembly in benign warts. Whether involucrin staining in neoplastic lesions will correlate with the presence of HPVs (such as HPV 16) in those lesions is unclear. However, it has been demonstrated that well-differentiated CIN lesions containing koilocytotic atypia often contain HPV 16, and in preliminary studies using in siru hybridization there is evidence that HPV16 DNA predominates in areas of maturation and koilocytosis [20]. Hence involucrin expression and HPV replication may be closely related in a neoplastic as well as nonneoplastic HPV-related lesions. REFERENCES 1. Matoltsy, A. G., and Balsamo, C. A. A study of the components of cornified epithelium of human skin, J. Biophys. Biochem. Cytol. 1, 339-360 (1955). 2. Matoltsy, A. G., and Matoltsy, M. N. The membrane protein of horny cells, J. Invest. Dermatol. 46, 127-129 (1966). 3. Green, H. Terminal differentiation of cultured epidermal cells, Cell 11, 405-416 (1977). 4. Rice, R. H., and Green, H. The cornified envelope of terminally differentiated human epidermal keratinocytes consist of cross-linked protein, Ce// 11, 417-422 (1977). 5. Rice, R. H., and Green, H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross linking by calcium ions, Cell 18, 681-694 (1979).

6. Banks-Schlegel, S., and Green, H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia, J. Cell. Bid/. 90, 732-737 (1981). 7. Peterson, L. L., Zetergren, J. G., and Wuepper, K. D. Biochemistry of transglutaminase and cross-linking in skin, J. Invest. Dermatol. 81 (Suppl), 95s-100s (1983). 8. Buxman, M. M., and Wuepper, K. D. Purification and characterization of bovine epidermal transglutaminase, Clin. Res. 22, 157A (1974). 9. Watt, F. M., and Green, H. Involucrin synthesis is correlated with cell size in human epidermal cultures, J. Cell. Eiol. 90, 738-742 (1981). 10. Warhol, M. J., Antonioli, D. A., Pinkus, G. S., Bunkel, L., and Rice, R. H. Immunoperoxidase staining for involucrin: a potential diagnostic aid in cervicovaginal pathology, Hum. Pathol. 13, 1095-1099 (1982). 11. Fu, Y. S., Reagan, J., and Richart, R. M. Definition of precursors, Gynecol.

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13. Fu, Y. S., Braun, L., Shah, K. V., Lawrence, D., and Robboy, S. J. Histologic, nuclear DNA and human papillomavirus studies of cervical condyloma, Cancer 52, 1705-1711 (1983). 14. Crum, C. P., Mitao, M., Winkler, B., Reuman, W., Boone, M., and Richart, R. M. Localizing chlamydial infection in cervical biopsies with the immunoperoxidase technique, Int. J. Gynecol. Parho/. 3, 191-197 (1984). 15. Richart, R. M. Cervical intraepithelial neoplasia: a review, in Pathology annual (S. C. Sommers, Ed.), Appleton-Century-Crofts, New York, p. 301 (1973). 16. Durst, M., Gissman, L., Ikenberg, H., and zur Hausen, H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions, Proc. Nutl. Acad. Sci. USA 80, 3812-3815 (1983). 17. Crum, C. P., Ikenberg, H., Richart, R. M., and Gissman, L. Human papillomavirus type 16 and early cervical neoplasia, N. Engl. J. Med. 310, 880-883 (1984). 18. Gissman, L., Wolnik, L., Ikenberg, H., Koldovsky, U., Schnurch, H. G., and zur Hausen, H. Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers, Proc. Narl. Acad. Sci. USA 80, 560-563 (1983). 19. Warhol, M. J., Pinkos, G. S., Rice, R. H., El-Tavil, G. H., Lancaster, W. D., Jensen, A. B., and Kurman, R. J. Papillomavirus infection of the cervix III: Relationship of the presence of viral structural proteins to the expression of involucrin, lnt. J. Gynecol. Puthol. 3, 71-81 (184). 20. Crum, C. P., Nagai, N., Mitao, M., Levine, R. U., and Silverstein, S. Histological and molecular analysis of early cervical neoplasia, in Pupillomuviruses: molecular und clinical aspects (P. Howley and T. Broker, Eds.), pp. 19-29. Liss, New York (1985).