Expression of stromelysin 3 in basal cell carcinomas

ARTICLE

Stromelysin-3 (ST3) belongs to the metalloproteinase family [1]. Matrix metalloproteinases are mediators of tissue remodelling which play a role in various processes such as development, tissue repair and tumour progression [2, 3]. These molecules are capable of degrading most macromolecular components of the extracellular matrix. Nevertheless, no structural substrate in the extracellular matrix has been identified for ST3 [4]. It has been suggested that ST3 could have an anti-apoptotic activity, therefore enhancing the survival of tumoral cells [5].

ST3-mRNA is expressed in the majority of human carcinomas [6], but the expression is restricted to the fibroblastic cells surrounding the epithelial part of these cancers. By contrast, ST3 has seldom been identified in benign tumours [6]. Although the biological functions of ST3 are still unclear, this protein could play an active role in the invasion of healthy tissue by the malignant cells. It has been demonstrated that the level of expression of ST3 is linked to the prognosis of breast carcinoma, as an increased level of expression correlates with a poorer prognosis [7]. Such a link has also been shown in other types of carcinomas, such as head and neck squamous cell carcinoma [8].

ST3 is expressed in the skin during wound healing and in cutaneous carcinomas [9], but not in normal fibroblasts. ST3-mRNA was demonstrated in the stroma of basal cell carcinomas, especially in the most aggressive cases [9, 10]. In situ hybridisation methods showed an increased expression in the areas where the cells had a tendency to loose their peripheral palisading [9]. We have shown by immunohistochemistry that squamous cell carcinomas express ST3 more frequently and more intensely than keratoacanthomas [11]. Moreover, we also observed a correlation between the expression of this metalloproteinase and the prognosis of squamous cell carcinomas [11]. The expression of ST3 has already been demonstrated in the stroma of basal cell carcinomas (BCC), either by in situ hybridisation, or by immunohistochemistry [12, 13]. Nevertheless, the histological subtype of BCC was not specified in some of these works [6, 14] and the staining was not quantified in others [12]. Moreover, these studies did not take into account the possibility of false positive results that could be due to a previous biopsy. As ST3 is expressed in inflammatory scars and wound healing, the biopsy could induce a transitory expression of ST3 which would not be related to the carcinoma. Biopsies are very often carried out in the most aggressive cases such as morpheiform BCCs, that were very often described as ST3-positive [10].

The purpose of the present work was a systematic immunohistochemical study of ST3 expression in the four major types of basal cell carcinomas, including cases with deep invasion, in order to evaluate a possible link between ST3 and the prognosis of those carcinomas.

Patients and methods

To avoid a possible bias due to changes induced by a previous partial excision or biopsy we included in this study cases of primary BCCs that were fully excised, without any previous biopsy. All cases included were typical both clinically and histologically. We selected for the immunohistochemistry study slides showing both the tumour and the healthy margins on both sides. We excluded cases sent to us for advice, incompletely removed tumours and those with regressive changes. All slides were obtained from the collection of the Laboratoire d'Histopathologie Cutanée, between the period 1977-1999.

Therefore, 40 cases of Pinkus' fibroepithelial tumour, 40 cases of superficial BCCs, 40 cases of nodular BCCs and 38 cases of morpheiform BCCs were included. In addition, we also selected 10 cases of BCC showing a very deep invasion in the fat tissue and/or in the underlying muscle. The patient characteristics are detailed in table I. The deeply invasive cases were of the nodular (5 cases) and the morpheiform types (5 cases). Nine of the 10 cases were excised on the face, in areas that are usually associated with a poorer prognosis (nasal, perioral and periocular areas) and one case originated from the back. Invasion of the underlying muscle was found in the 5 nodular BCCs and subcutis penetration was noticed in the 5 morpheiform cases, one showing in addition a perineural invasion.

The histological diagnosis of all tumours was established independently by two of us (BC, EG), after the slides were selected according to the diagnosis of the laboratory files and put in random order by GN.

Immunohistochemistry

We performed a conventional streptavidin-biotin technique, using the commercial kit LSAB2 (Dako). The anti-ST3 monoclonal antibody (5ST-4A9 clone, IgG1, lambda) was kindly provided by Marie-Christine Rio, IGBMC, Strasbourg France, as it is not commercially available. It was obtained by immunisation of BALB/c mice with recombinant human ST3 that was extracted from inclusion bodies of Escherichia coli cells (BL 21) strain, expressing cDNA inserted in a pET-3 vector [15]. The reactivity pattern of the antibody is similar on frozen and formalin fixed biopsies, when microwave antigen retrieval is performed. Since the antibody was characterized [15] numerous studies have been carried out using the same method [11-13].

Five micron sections of each tumour were submitted to immunohistochemical technique using Superfrost plus slides (Menzel, Gläser, Germany). After microwave antigen retrieval (17 min for formaldehyde-fixed samples and 25 min for those fixed in Bouin's solution), the sections were incubated with the antibody (1/4,000) for 24 hrs at room temperature. Positive control consisted of ST3-positive breast carcinoma sections. Negative controls consisted of sections that were similarly processed except that no primary antibodies were used. Using this method, we never observed unwanted background stain in the stroma of epithelial tumours, neither in the present study nor in our previous work [11].

We quantified ST3 staining using a simple semiquantitative scale: 0 (absence of positive cells), 1 (few isolated positive cells), 2 (intermediate between 1 and 3) and 3 (diffuse positivity or staining of multiple groups of cells). The slides were processed in a random order and analysed by two of us (BC and GN) to quantify the staining.

Results

Positively stained cells were present only in the stroma, but the tumoral cells were ST3-negative in all cases. A reinforcement in positively stained cells was observed around the most aggressive lobules (Fig. 1) or groups of neoplastic cells, mainly those located in the deep dermis or in the subcutis. ST3 expression was present in the cytoplasm of the stromal cells, which were most often located close to the malignant epithelial cells (Fig. 2).

The proportion of positive cases was 27.5% of fibroepithelial tumours (n = 11), 65% of superficial BCCs (n = 26), 72.5% of nodular BCCs (n = 31), 86.9% of morpheiform BCCs (n = 35) and 100% of deeply invasive BCCs (n = 10). The semiquantitative results are detailed in table I.

We observed an increase in the proportion of tumours exhibiting a strong staining (class 2 and 3) in the most aggressive groups of BCCs. A strong staining was noticed in 7.5% of fibroepithelial tumours, 20% of superficial BCCs, 45% of nodular BCCs, 63.2% of morpheiform BCCs and 100% of deeply invasive BCCs.

Discussion

This systematic study of the four major types of BCCs shows that ST3 expression is not only more frequent but also stronger in the most aggressive cases.

We present for the first time a study of a homogeneous group of fully excised tumours, with clear-cut characterisation of the histological subtypes prior to immunohistochemical study. To the best of our knowledge, Pinkus' fibroepithelial tumour has not been investigated in the previous studies which included cases of BCCs. This is particularly interesting, as Pinkus' tumour was considered as a precursor of BCCs for years and is now classified among the most indolent forms of BCCs [16]. ST3 staining was weak and inconstant in this group of well circumscribed BCCs. We observed an increasing proportion of positively stained tumours in our five groups, the most aggressive cases being constantly positive, especially the invasive morpheiform type. A reinforcement of the staining was observed in the stroma surrounding the invasive strands of tumoral cells in this type, which usually lack peripheral palisading. This was already noticed in a small series of cases studied by in situ hybridisation [9, 10].

The analysis of the literature shows that the expression of ST3 in BCCs was analysed in 7 different studies [6, 9, 10, 12-14, 17], using both in situ hybridisation and immunohistochemistry. The global rate of "positive" tumours was approximately 60% in these studies. BCCs were often mixed together with other cancers or benign lesions, and the staining was not quantified in the immunohistochemical studies. As it was demonstrated that ST3 can be expressed in inflammatory scars [9], the study of morpheiform BCCs should take into account the scar induced by a biopsy prior to complete excision. This factor could be responsible for false positive results in those carcinomas biopsied because of misleading clinical appearance. To rule out this confusing factor, we included only BCCs that were fully excised and that were not submitted to a previous biopsy. We can therefore affirm that the higher expression of ST3 in morpheiform BCCs is not artefactual.

Our study shows an increasing proportion of strongly positive cases in the five groups of tumours. Moreover, the majority of 31 tumours with the strongest staining (class 3) were found in the morpheiform and deeply invasive groups (21 cases, 68%). These results suggest that a strong ST3 expression (i.e. numerous positive cells in the stroma) is associated with a poorer prognosis of BCCs. In their study of various skin tumours, Thewes et al. [12] also found a higher rate of positively stained cases in their nodular BCCs compared to their superficial tumours (47% vs 30%). The differences between the study of Thewes et al. [12] and ours, i.e. a lower rate of positively stained tumours, are probably not relevant and could be due only to the technique of immunohistochemistry, as Thewes et al. did not use microwave antigen retrieval.

Our impression is that the level of expression of ST3 evaluated by the number of positively stained cells in the stroma, is of greater importance than the global rate of positivity, as the differences among the various groups of BCCs were more clear-cut when analysing the semi-quantitative results. We had already noticed that those squamous cell carcinomas which led to metastasis exhibited the strongest ST3 expression [11], suggesting once more a correlation between ST3 expression and prognosis. The present study shows a link between the histological subtype of BCC and ST3 expression.

CONCLUSION

As such a link has been shown in so many types of human carcinomas, it can be hypothesised that ST3 is not only a marker of poor prognosis, but could play a direct role in tumour progression. The inhibition of apoptosis by ST3 has already been demonstrated [5] and could be responsible for a longer survival of malignant cells. Moreover, the transfection of both sense and anti-sense ST3 in nude mice has shown that ST3 expression promotes tumour take in nude mice [18]. Other experiments have demonstrated that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells [19]. It was recently shown in an animal model that ST3 favours the cancer cells survival, by decreasing cell death through both apoptosis and necrosis [20]. A direct role of ST3 in human cutaneous carcinomas remains to be established by further experiments.

Article accepted on 28/6/01

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