Eyelid sebaceous carcinoma: a novel mutation in lymphoid enhancer‐binding factor‐1

DEAR EDITOR, Sebaceous gland carcinoma (SGC) is a tumour of the pilosebaceous unit associated with hair follicles, and occurs predominantly in the eyelid due to the abundance of sebaceous glands (meibomian and Zeiss glands). A higher incidence of SGC has been reported in China and India. Wnt/b-catenin signalling plays an important role in the formation of sebaceous glands. Developmentally high levels of b-catenin lead to the formation of hair follicles, whereas lower levels favour differentiation of sebocytes in mammalian epidermis. Activating b-catenin mutations are seen in hair follicle tumours such as pilomatrixomas. Conversely, a transgenic mouse expressing a defective b-catenin-binding site in the Lef-1 protein (with resultant inability to activate transcription efficiently) spontaneously developed sebaceous skin tumours. First discovered in lymphocytes, lymphoid enhancer-binding factor (LEF)-1 is a member of the LEF/T-cell factor family of high-mobility group (HMG) transcription factors. The human LEF1 gene spans at least 140 kb, and contains 12 exons and 11 introns. Two major protein isoforms of LEF-1 have been reported. The full-length isoform (55 kDa) contains three functional domains, namely the b-catenin-binding domain at the N-terminus, the context-dependent activation domain and the HMG DNA-binding domain near the Cterminus (Fig. 1a). The truncated or short isoform (33 kDa) is transcribed from a promoter within the intron between exons 2 and 3, which lacks the b-catenin-binding domain. The short isoform exhibits dominant negative activity by suppressing, rather than enhancing, the Wnt pathway by preventing b-catenin recruitment to the Wnt response element. LEF-1 is expressed in developing thymus and is involved in thymocyte differentiation and development of hair follicles, teeth and the mammary gland. It has been found to be involved in the differentiation of multipotent skin stem cells. Benign sebaceomas and sebaceous adenomas of the skin harbour dual mutations in the LEF1 gene, encoding a key transcription factor and binding partner of b-catenin within the nucleus. Expression of the N-terminal-truncated LEF1 gene (DN Lef1) in transgenic mice leads to spontaneous tumours with sebaceous differentiation, thereby suggesting a causal role of LEF1 in sebaceous neoplasms. The status of LEF-1 in human eyelid sebaceous carcinoma has not been reported so far. In vitro functional expression studies by Takeda et al. revealed that the mutant LEF1 stimulated the expression of sebocyte markers, suggesting that mutant LEF-1 may determine the differentiated characteristics of sebaceous tumours. The purpose of this study was to analyse the LEF1 gene for mutations in 36 histologically proven cases of SGC. In addition, the immunoexpression and mRNA expression patterns were correlated with the high-risk features of eyelid SGC. Sequencing of exon 1 of LEF1 in our study revealed two novel mutations in the N-terminal region (b-catenin-binding domain) of the LEF1 gene, in seven of 36 cases (19%) (Fig. 1c). The first in exon 1 of LEF1 (K27X) resulted in a premature stop codon in six cases (Fig. 1d). The second mutation resulted in the substitution of isoleucine for valine at amino acid position 64 in a single case. The average age of patients with SGC with mutant LEF1 in our study was > 60 years. Increased frequency of LEF1 mutations has been reported previously in patients of advanced age with benign sebaceous tumours. There was no association between LEF1 mutations and reduced disease-free survival in the present study. The functional significance of mutations in the LEF1 gene affecting the amino-terminal (b-catenin-binding domain) segment of the LEF-1 protein was first described in an SV40-immortalized human sebocyte line, SZ95, by Takeda et al. Complete loss of LEF-1 immunostaining was observed in 69% (25 of 36) of cases. Of the 11 with LEF-1 expression, cytoplasmic localization was detected in 91% (10 of 11) (Fig. 2b), while both nuclear and cytoplasmic reactivity was observed in only one case (9%) (Fig. 2c). LEF-1 loss was significantly associated with reduced disease-free survival (P = 0 035, log-rank analysis) and with tumour recurrence in seven of nine cases (78%) (Fig. 2e). These results indicate dysregulation of the Wnt/b-catenin signalling pathway. In the present study mutations in LEF1 resulted in loss of LEF-1 expression in six of 36 cases (17%) observed. However, absence of LEF-1 expression was also seen in 20 of 29 cases (69%) with wildtype LEF1. In murine epidermal stem cells, expression of the dominant negative isoform (DN Lef1) suppresses hair cell differentiation, but stimulates sebocyte differentiation, epidermal cysts and skin tumours. Thus, DN Lef1 acts as an inhibitor of proliferative signals emanating from the canonical Wnt pathway in some cells, but promotes the differentiation of other cells. The potential mechanism for the loss of full-length LEF1 in periocular sebaceous carcinoma could be due to repression of the endogenous (full-length) LEF1 gene by a silencing mechanism, as has been shown in B-lymphocyte cell lines. It could