Immunohistochemical characterization of the differentiation state of basal cell carcinomas with special interest for infiltrating relapsing tumors

ARTICLE

Basal cell carcinomas (BCC) are locally invasive tumors of slow progression and are seldom metastatic [1]. Because of these characteristics, doubts have been raised regarding their malignancy. Occasional cases of highly invasive [2], sometimes metastatic [3] forms are nevertheless observed. Some histological subtypes, e.g. infiltrative aggressive-type, seem to have a higher potential for recurrence. It could be assumed that the proliferative potential of some tumors is inversely related to their degree of cellular differentiation.

As a basal keratinocyte differentiates along its journey to the skin surface, transcription of cytokeratins K5 and K14 is down-regulated whereas new sets of differentiation-specific genes are up-regulated, including cytokeratins K1 and K10, profilaggrin, cornified cell envelope components and new desmosomal proteins.

To study the differentiation stage of tumoral epithelial cells, many studies demonstrating the usefulness of anti-cytokeratin [4-18] and anti-desmosomal protein [19] antibodies have been reported. However, no antibody has been found to specifically label BCC. Two-dimensional gel analysis of tumor cell proteins [6] confirmed the absence of any specific tumoral cytokeratin in these carcinomas. Moreover, numerous antibodies that label all BCC were also found to stain squamous cell carcinomas [9-19].

The aim of this study was to try to differentiate groups of BCC by their labeling with new monoclonal antibodies (MoAbs) directed against epidermal differentiation markers. Among the large number of markers described in the last few years, we chose cytokeratins K1 and K10, and corneodesmosin. The study was performed on a large collection of 66 BCC biopsies obtained from patients whose subsequent evolution was recorded. All samples were reexamined in order to determine their histological type. The histological and immunohistochemical results are discussed in relation to the clinical evolution of the tumors.

Materials and methods

Tumors

Sixty-six BCC biopsies obtained from well characterized patients (41 males, 25 females, mean age 69 years, range: 43-91 years, at the time of diagnosis) before any therapeutic regime was instigated, and stored in the collection of the Cancer Center of Nancy (France) were used in this study. Twelve were from clinically aggressive BCC that had relapsed once or more in spite of extensive surgery. Fifty-four were from BCC that had been successfully treated by surgery, radiotherapy, electrocoagulation or local chemotherapy, without any relapse for at least 5 years. Samples of skin from a fetus of 3 months were used as controls.

Histology

All samples were immersed in Bouin's fixative upon removal, and embedded in paraffin. Fresh 4-µm sections were prepared and stained with hematoxillin-eosin. The classification criteria proposed by Kirkham [20] were used to describe tumoral architecture.

Immunohistochemistry

Antibodies. The three MoAbs used in this study and described below are part of a series of antibodies directed against epidermal differentiation antigens, produced and characterized in our laboratory, after immunization of mice with an homogenate of human plantar stratum corneum [21-27]. Analyzed by indirect immunofluorescence (IIF), F12-19 (IgG1-kappa) labels all the human epidermal cell layers as well as sebaceous and sudoral gland cells and all the cells of the hair follicle outer root sheath. All human cytokeratins are stained by this MoAb on Western blotting, indicating that it is specific for an epitope shared by all cytokeratins [21]. EE21-06 (IgG1-kappa) is specific for human squamous cornified epithelia: epidermis, gingiva and hard palate. It labels all the suprabasal epidermal cell layers in inter- and intra-follicular epidermis but not cutaneous appendages. The epitope recognized is shared by cytokeratins K1, K2, K9, and K10-11, as identified by Western blotting [22]. G36-19 (IgG1-kappa) specifically labels the stratum granulosum and the lower stratum corneum of cornified squamous epithelium and the hair follicle inner root sheath. This antibody stains corneodesmosin, a 52-56 kDa basic glycoprotein synthesized in the stratum granulosum, and transported via lamellar bodies to the extracellular space where it is detected in the desmosome core. Then the protein is located on the dense plug of corneodesmosomes, the modified desmosomes specifically observed in the stratum corneum of cornified epithelia [23-25]. Hyperkeratotic lesions, which are characterized by an increased number of corneodesmosomes, also show an increased expression of corneodesmosin [26]. Moreover, proteolysis of the protein is thought to be a key event in desquamation [24, 27].

Indirect immunofluorescence. Fresh, 4-µm-thick sections were prepared for each sample and deparaffinized by immersion for 10 min in toluene at 37° C. They were then sequentially hydrated in graded ethanol baths, washed in phosphate-buffered saline (PBS), and incubated with the appropriate MoAb diluted in PBS, for 30 min at 37° C. Upon incubation, the slides were washed, first in PBS supplemented with 0.5% Tween 20, then in PBS. The secondary antibody (fluorescein isothiocyanate-labeled rabbit antibodies to mouse IgG (H+L), Zymed, San Francisco, CA) was then added, and the slides were further incubated for 15 min at 37° C. The same series of washes was performed, and the sections were then examined under UV light using a Leitz microscope equipped with Ploem epi-illumination. Controls included incubations with the fluorescent antibody alone, and staining of 4 normal skin samples.

Results

Histology

Out of the 54 non-relapsing tumors, the classification proposed by Kirkham [20] allowed us to distinguish 22 BCC of the circumscribed type (nodular BCC), 11 adenoid BCC, 4 keratotic BCC, 9 superficial BCC, 4 Pinkus fibroepithelial tumors, and 4 solid infiltrative aggressive-type BCC which appeared as thin, non-anastomotic trabeculae, deprived of peripheral palissadic cells, and invading the dermis. The latter aspect, characteristic of "an aggressive growth pattern" of BCC, as described by Jacobs et al. [28], and also reported by Sexton et al. [29], was also observed in all the 12 relapsing tumor samples of our series. Among the nodular BCC, large nodules were always observed. In only 3 cases, were they associated with micro-nodules.

Immunohistochemistry

To evaluate the differentiation stage of the epithelial cells in the various histological types of BCC, IIF analysis was then performed with MoAbs directed against differentiation-related antigens. Results are summarized in Table I.

F12-19, an MoAb directed against an epitope shared by all the cytokeratins, homogeneously labeled the center of nodular BCC, whereas there was no labeling of the palissadic ring (Fig. 1). F12-19 also labeled all the tumor masses of keratotic BCC, with a reinforcement of the fluorescence intensity on the concentric keratinized areas, and brightly stained all the trabeculae of the adenoid BCC. Cell-to-cell heterogeneity was noted in the labeling intensity of infiltrative aggressive-type BCC (data not shown). In contrast, superficial BCC, and basocellular buds sprouting from the trabeculae of Pinkus fibroepithelial tumors were not labeled. Lastly, F12-19 did not react on embryonic primary epithelial germs (data not shown).

EE21-06, an MoAb specific for the terminal differentiation-related cytokeratins K1, K2, K9 and K10-11, labeled most nodular, adenoid (Fig. 2) and keratotic BCC, with patterns similar to that reported above for F12-19. Infiltrative aggressive-type BCC were inconsistently labeled, with bright heterogeneous reactive patches within a given tumor (Fig. 3). Neither superficial BCC, basocellular buds of Pinkus fibroepithelial tumors, nor primary epithelial germs, were labeled by this MoAb.

G36-19, an MoAb specific for the cornification-related protein corneodesmosin, labeled the concentric keratinized areas and/or hair follicle-like structures of keratotic BCC and some individual cells in the center of nodular BCC (Fig. 4). Superficial BCC, basocellular trabeculae of adenoid BCC and of infiltrative aggressive-type BCC, and primary epithelial germs, were not labeled by G36-19.

Discussion

In this paper, the histological and immunohistological characteristics of BCC were investigated in a series of 66 samples from patients whose response to BCC-specific therapy had been recorded for at least 5 years. A retrospective study of their histological features showed that all clinically aggressive tumors relapsing after surgical treatment, were of the infiltrative aggressive-type, as previously shown in other series [5, 28-32]. All these BCC displayed similar characteristics: thin, non-anastomotic trabeculae without palissadic cells, without peripheral palleal dermis and directly infiltrating the reticularis dermis. This homogeneity could be due to the selection criteria we used, requiring both examination of the initial biopsy and relapse after extensive surgery.

One of the major concerns with immunohistochemical analysis performed with MoAbs is whether a negative result reflects masking of the epitope or a real absence of the antigen. In particular, masking of cytokeratin epitopes has already been described. This may explain why F12-19, an MoAb directed to all cytokeratins including K5 and K14, did not label either superficial BCC, some parts of the tumor masses or embryonic epithelial germs. This could be related to an immature state of the tumor cells, characterized by a specific organization of intermediate filaments masking the F12-19 epitope. Indeed, superficial BCC as well as embryonic epithelial germs were labeled by N40-13, an MoAb likely directed against a conformational epitope of cytokeratins K5/K14 (unpublished observation of the authors).

Our immunohistological analysis, as well as data already published [28], supports the conclusion of previous morphological studies in discriminating three major groups of BCC: (1) superficial, (2) nodular, including keratotic and adenoid variants, and (3) infiltrative aggressive-type BCC. Previous studies of the differentiation stage of BCC showed that epithelial BCC cells are labeled by antibodies directed to epidermal basal cell cytokeratins [11-13, 15, 17, 18] and suggested that most BCC are undifferentiated, except keratotic BCC [9, 15, 16]. In our study, the use of EE21-06 and G36-19 (MoAbs specific for the differentiation-related cytokeratins K1, K2, K9 and K10-11, and for the cornification-related antigen corneodesmosin, respectively) allowed various degrees of BCC cell differentiation to be more precisely determined. Superficial, undifferentiated BCC appear somewhat similar in their antigenic expression to embryonic primary epithelial germs, as previously reported by Van Cauwenberge et al. [33]. EE21-06 strongly labeled the keratinized areas and/or the hair follicle-like structures of keratotic BCC, the center of the tumor masses in nodular BCC and trabeculae in Pinkus fibroepithelial tumors. In a recent study however [34], expression of cytokeratin K10 has not been observed in nodular (primary or recurrent) BCC. Therefore, these BCC could rather only express cytokeratin K1 or K9. More likely, this could be due to differences in specificity or sensitivity of the MoAbs used or to tumor heterogeneity. The keratinized areas and/or the hair follicle-like structures of keratotic BCC, a few patches of cells within nodular BCC, and cells in the center of trabeculae in Pinkus fibroepithelial tumors were labeled by G36-19, displaying characteristics of granular keratinocytes. The maturation of nodular and keratotic BCC thus appears centripetic, from peripheral undifferentiated cells to central granular cells. Use of other MoAbs would be necessary to test whether these cells cornify or not. Conversely, adenoid BCC cells appeared to be homogeneously differentiated, since all trabeculae were brightly labeled by EE21-06 but were not stained by G36-19 and did not show signs of granular maturation. Infiltrative aggressive-type BCC are characterized by the heterogeneous cell-to-cell labeling produced by F12-19 anf EE 21-06. A wide range of monospecific antibodies should be used to precisely determine the pattern of cytokeratin expression in these BCC. Such IIF labeling heterogeneity has previously been reported [18, 34, 35], and is also displayed by squamous cell carcinomas [15, 17]. This similarity with squamous cell carcinomas could bear some relationship to the greater aggressiveness of these BCC.

CONCLUSION

This retrospective histological and immunohistological study performed with a new series of MoAbs, confirms and completes previous reports on BCC classification [28] and suggests the discrimination of three major types of BCC: superficial, nodular (including adenoid and keratotic) and infiltrative aggressive-type. In particular, some nodular BCC cells are differentiated and even show characteristics of granular keratinocytes. Infiltrative aggressive-type BCC present cell-to-cell heterogeneity, similar to that observed in squamous cell carcinomas. This feature, that is not observed in other types of BCC, correlates with greater malignancy and relapse risk. We suggest that an immunohistochemical analysis with antibodies specific for different stages of keratinocyte differentiation may help to indicate surgical treatment of infiltrative aggressive-type BCC.

Acknowledgements

The research was supported by grants from the "Association pour la Recherche contre le Cancer" and from the "Fédération Nationale des Centres de Lutte contre le Cancer".

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