(A) and (B) represent the S100A1 and S100B expression on conjunctival naevi, respectively. (C) and (D) show the expression of S100A1 and S100B on conjunctival melanoma, respectively. (E–H) represent the S100A1, S100B, CEA, and (more ...)
S100A1
S100A1 positively stained more than 10% of the lesional cells of 16 of the 18 conjunctival naevi (89%), of 10 of 14 PAM (71%), of all conjunctival melanomas (n=14, 100%), and all uveal melanoma (n=20, 100%) (fig 2). Significantly more cells were positive in conjunctival melanoma compared to conjunctival naevi (71.4%, and 30.6%, respectively) (p=0.001 Mann‐Whitney) (fig 1), while the PAM reacted intermediately (45.0% positive cells). | Figure 1Percentage positive cells after staining for antibodies against S100A1 (A), S100B (B), and MelanA (C) in conjunctival naevus, PAM, conjunctival melanoma, and uveal melanoma specimens. Each dot represents a single case, the line represents (more ...) |
S100A6
In all uveal melanomas more than 10% of the cells stained positively; the average percentage of positive cells was very high (72.1%). All conjunctival tissues showed severe non‐specific staining, and could therefore not be scored: most epithelial cells stained positive, which has been described previously.21,22S100B
More than 10% of the cells scored positively with S100B of 17 of the 18 conjunctival naevi (94%), of eight of 14 PAM (57%), of all conjunctival melanoma (n=14, 100%), and of 15 of 20 uveal melanoma (75%). Comparing the average scores for S100B, naevus and conjunctival melanoma scored significantly higher than PAM (p<0.001) (fig 1). The average percentage positive cells in conjunctival melanoma was significantly higher than in the uveal melanoma (62.9%, 18.5%, respectively) (p<0.0001, Mann‐Whitney) MelanAMelanA stained more than 10% of the cells in 16 of the 18 naevi (89%), nine of 13 PAM (69%), nine of 16 conjunctival melanoma (56%), and 19 of 20 uveal melanoma (95%). Most naevi and uveal melanoma had a good expression of MelanA, while in PAM and conjunctival melanoma expression was very variable (fig 1).
CEA
CEA was completely negative in most lesions, but in one naevus , two conjunctival melanomas, and 15 uveal melanomas, groups of non‐melanocytic cells were positive (fig 2). The total number of CEA positive cells was very low in all categories.The atypia stage of the PAM lesions did not correlate with the expression levels of the tested antibodies. No significant difference in expression was seen between uveal or conjunctival melanomas in patients with or without metastasis, although for S100A1 and S100A6 there was a trend towards higher expression in uveal melanoma patients with metastatic disease.
Discussion In this study, we set out to determine whether expression patterns of various markers may differentiate between benign and malignant conjunctival pigmented lesions, as previous studies did not yield a positive result.14,15,16,17 In our study, S100A1 staining differed between a naevus and a conjunctival melanoma. The naevi had low S100A1 expression (mean 30.6%), while in most conjunctival melanomas expression was strong (mean 71.4%) (figs 1 and 2). It must be noted that because of the rarity of histological sections of conjunctival naevi and conjunctival melanomas, the sample size is small. Since no other studies have been published about S100A1 staining of melanocytic conjunctival lesions, confirmation by others will be awaited. S100B staining did not differ between conjunctival naevi and conjunctival melanoma, however, a lower staining was seen in the PAM lesions (fig 1). Others also found no difference in S100 expression between conjunctival naevi and conjunctival melanoma.16,17,26 Steuhl et al did also find slightly lower expression of S100 in epithelial melanosis than in conjunctival naevi or conjunctival melanoma.16 However, Hitzer et al, and Sharara et al did not find any difference between PAM and conjunctival naevi , and conjunctival melanoma.17,26 The former studies used the “general” S100 antibody which probably represents the S100B used in our study. To our surprise MelanA was not present in seven of the 16 conjunctival melanomas, while the expression in PAM and conjunctival naevi was higher. Others found higher expression of MelanA in conjunctival melanoma,12,13 although Heegaard et al found weak staining of the MelanA in most of the conjunctival melanomas.12 Our findings indicate that MelanA is not a good marker for conjunctival melanoma. Whether conjunctival melanoma is biologically more closely related to the skin melanoma or the uveal melanoma is still a topic of discussion. We included uveal melanoma samples as a control and to compare expression of the conjunctival lesions with this tumour. The S100B was as abundantly expressed in conjunctival melanomas as the S100A1, while in uveal melanomas the expression of S100A1 was also high, but S100B was low. Others also found higher expression of S100B in conjunctival melanoma than in uveal melanoma.12,13 Iwamoto claims that conjunctival melanoma is most similar to the epithelioid phenotype of the cutaneous melanoma.13 Table 1 compares the S100A1, S100A6, and S100B data from this study with data on cutaneous melanoma in the literature. All lesions have similarities for one or two antibodies, but one cannot conclude that conjunctival melanoma resembles cutaneous melanoma more closely than uveal melanoma. Perhaps it is better to see them as three separate identities, although clinically the conjunctival melanoma has more similarities with cutaneous melanoma, since both have the tendency to metastasise to the regional lymph node first. | Table 1Expression of S100A1, S100A6, and S100B of conjunctival melanoma, uveal melanoma, and cutaneous melanoma |
The tested antibodies may not only be used for differentiation between lesions but may also be potential serum metastases markers. Especially molecules with a high expression in the malignant lesion may be good candidates. Serum levels of S100B have proved to be reliable serum markers to follow metastasis in cutaneous melanoma.23 For both uveal and conjunctival melanoma good serum markers may also be of help in detecting and following primary and metastastic disease. For uveal melanoma, S100A1 and S100A6 could be candidate serum markers, since they are abundantly expressed on uveal melanoma, for conjunctival melanoma S100A1 and S100B could be good candidates to test in the serum. Van Ginkel already implied that S100A6 could be of influence on malignant transformation of the uveal melanoma.27 Serum S100B was not able to detect metastatic disease in patients with uveal melanoma,28 probably since S100B is not well expressed in uveal melanoma, but serum S100A1b was able to detect metastatic disease in uveal melanoma patients (GS Missotten et al, submitted). As far as we know, no serum markers have yet been analysed in conjunctival melanoma. In 1976 Michelson et al reported a slightly elevated blood CEA levels in 45% of the uveal melanoma patients, although the increase of CEA levels was only marginal.29 We also investigated the CEA expression of conjunctival and uveal lesions, but could not find any tumour labelling, although in some tumours we did find staining of a few non‐melanocytic cells (fig 2), which were microscopically identified as melanophages. As CEA is not expressed, using it as a serum marker seems inefficient. In conclusion, we think that of those we studied S100A1 is the best marker to differentiate between a naevus and conjunctival melanoma, although it does not provide an absolute cut‐off value. Because of the small sample size, further studies will be needed to confirm these findings. |
Acknowledgements The authors thank Professor WJ Mooi and his laboratory for the S100A1 and S100B staining. We also appreciate the help of ER Barthen and CJM Krose with the S100A6, MelanA, and CEA stainings. |
Abbreviations DAB - 3,3 diaminobenzidine PAM - primary acquired melanosis PBS - phosphate buffered saline |
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