January 2013
Volume 54, Issue 1
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Anatomy and Pathology/Oncology  |   January 2013
Expression of Shh and Wnt Signaling Pathway Proteins in Eyelid Sebaceous Gland Carcinoma: Clinicopathologic Study
Author Affiliations & Notes
  • Namju Kim
    From the Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea; the
    Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Korea; the Departments of
  • Ji Eun Kim
    Pathology and
  • Ho-Kyung Choung
    From the Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea; the
    Ophthalmology, Seoul Municipal Government-Seoul National University Boramae Hospital, Seoul, Korea; the
  • Min Joung Lee
    Department of Ophthalmology, Hallym University Sacred Heart Hospital, Pyongchon, Korea; and the
  • Sang In Khwarg
    From the Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea; the
    Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea.
  • Corresponding author: Sang In Khwarg, Department of Ophthalmology, Seoul National University College of Medicine, #28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea; khwarg@snu.ac.kr
Investigative Ophthalmology & Visual Science January 2013, Vol.54, 370-377. doi:10.1167/iovs.12-10671
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      Namju Kim, Ji Eun Kim, Ho-Kyung Choung, Min Joung Lee, Sang In Khwarg; Expression of Shh and Wnt Signaling Pathway Proteins in Eyelid Sebaceous Gland Carcinoma: Clinicopathologic Study. Invest. Ophthalmol. Vis. Sci. 2013;54(1):370-377. doi: 10.1167/iovs.12-10671.

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Abstract

Purpose.: Activation or dysregulation of the Sonic hedgehog (Shh) and Wnt signaling pathways is suggested to lead to the development of many human malignancies. In this study, we investigated Shh and Wnt signaling protein expression in eyelid sebaceous gland carcinoma, and analyzed their correlation with clinical characteristics of the tumor.

Methods.: Patients who underwent surgical resection of eyelid sebaceous gland carcinoma from 1999 to 2011 were recruited for the study. Immunohistochemical stainings of Shh signaling proteins (Shh, Gli-1, Gli-2, Gli-3, and ABCG2) and Wnt signaling proteins (Wnt, glycogen synthase kinase 3β, β-catenin, lipoprotein receptor-related protein [LRP], and c-Myc) were conducted.

Results.: Thirty-seven cases of eyelid sebaceous gland carcinoma were included in this study. Twenty-nine patients showed no metastasis, and eight patients showed lymph node or distant metastasis. Shh, ABCG2, and Wnt proteins were more highly expressed in the group with metastasis than in the group without metastasis (P = 0.031, P = 0.015, and P = 0.001, respectively; χ2 test). Patients showing high ABCG2 expression, Wnt, or LRP expression developed metastasis more commonly than those with low ABCG2, or without Wnt or LRP expression (log rank test, P = 0.019, 0.001, and 0.000, respectively).

Conclusions.: The group with metastasis showed higher expression levels of Shh, ABCG2, and Wnt than did the group without metastasis, and patients with strong ABCG2 expression, Wnt, or LRP expression showed higher cumulative incidence of lymph node or distant metastasis, implying that activation of the Shh and Wnt signaling pathway is associated with aggressive behavior of the tumor.

Introduction
Sebaceous gland carcinoma is one of the most common eyelid malignant neoplasms in Asian. Eyelid sebaceous gland carcinoma is associated with high morbidity and mortality despite complete wide surgical resection because of lymph node metastasis or distant metastasis. Moreover, eyelid sebaceous gland carcinoma tends to be diagnosed in the advanced stage, because this tumor can be easily misdiagnosed as chalazion or chronic conjunctivitis. 1  
For effective management of eyelid sebaceous gland carcinoma, which shows poor prognosis, documenting factors associated with treatment outcome or prognosis would be very helpful for the development of an optimal treatment design. Currently, reported conventional prognostic factors for eyelid sebaceous gland carcinoma are larger lesions, incompletely excised tumors, and histopathologic features, such as poor differentiation, multicentric origin, and pagetoid spread. 2,3 Recently, increasing attention has focused on new molecular markers; stratification of cancer patients according to molecular marker expression permits a more personalized approach that maximizes benefit and minimizes harm. 4,5 Identification of prognostic molecular biomarkers could facilitate effective clinical management of eyelid sebaceous gland carcinoma. 
Sonic hedgehog (Shh) and Wnt signaling, which play an important role in embryonic and organic development, 6,7 regulate the development of the pilosebaceous unit, 8,9 from which sebaceous gland carcinoma arises. Briefly, Shh ligand secreted from the cells binds to the receptor patched, thereby alleviating the suppression of smoothened (Smo). Smo signaling initiates a cascade with the activation of Gli proteins, leading to Gli-1–dependent transcription of proteins such as ABCG2. 10,11 For Wnt signaling, Wnt ligand binds to the receptor frizzled, inactivating glycogen synthase kinase 3β (GSK3β), and thereby blocking the degradation of cytoplastmic β-catenin by protein complex (axin, adenomatous polyposis coli [APC], GSK3β); consequently, β-catenin accumulates in the cytoplasm and nucleus, leading to transcription of Wnt target genes such as cyclin D1 and c-Myc. 11  
Recent studies have demonstrated that reactivation of these pathways later in life can lead to the development of malignancies; for example, studies have shown an association of Shh signaling activation with the development of human cancers such as medulloblastoma, basal cell carcinoma, gastrointestinal tract tumor, ovarian cancer, and breast cancer, 1216 and an association of Wnt signaling activation with the development of human cancers such as colorectal, hepatocellular, breast carcinomas, and nonmelanoma skin tumors. 1720 Moreover, previous studies showed that elevated activity of Shh or Wnt signaling was associated with cancer prognosis, while Shh signaling activity was associated with worse prognosis in prostate, colorectal, and pancreatic cancers, 2123 and β-catenin overexpression was related to the aggressiveness of tumor in hepatoblastoma, pharyngeal squamous cell carcinoma, and breast cancer. 2426  
Although significant expression of Shh/Wnt signaling protein in cancer has been reported, only a few studies have shown Shh/Wnt signaling protein expression in eyelid sebaceous gland carcinoma 27,28 ; moreover, only the expression of the β-catenin was considered for Wnt signaling proteins, and its clinical significance was not clarified. Further, to our knowledge, the expression of Shh pathway proteins has not been investigated in eyelid sebaceous gland carcinoma. 
The purpose of this study was to investigate the expression of Shh and Wnt signaling protein in eyelid sebaceous gland carcinoma, and to correlate their expressions with the clinical features and prognosis of eyelid sebaceous gland carcinoma. 
Methods
Patients
Patients who underwent surgical resection of eyelid sebaceous gland carcinoma in Seoul National University Hospital, Seoul National University Bundang Hospital, and Seoul Municipal Government-Seoul National University Boramae Hospital from 1999 to 2011 were recruited for the study, and their archival paraffin blocks were collected. Thirty-seven paraffin blocks of eyelid sebaceous gland carcinoma were available. 
We reviewed the electronic medical records of 37 patients with eyelid sebaceous gland carcinoma. Wide surgical excision with frozen section control was performed as a primary treatment. Reviewed data included demographic information, histopathologic diagnosis, anatomic tumor location, details of treatment, and outcomes such as local recurrence, distant metastasis, and survival. TMN staging was re-established in all cases according to the 2010 American Joint Committee on Cancer (AJCC) (seventh edition). The hematoxylin and eosin-stained slides were independently reviewed in each case to confirm the original diagnosis. 
This study was approved by the institutional review board of Seoul National University Bundang Hospital. This study was conducted in compliance with the Declaration of Helsinki. 
Tissue Microarray
Tissue microarray (TMA) recipient blocks containing paraffin-embedded eyelid sebaceous gland carcinoma tissues from the archived patient specimens, previously fixed in 10% formaldehyde, were constructed according to established methods. From every archival paraffin block, 1 cylinder of 2.0-mm diameter tissue was taken from representative areas and transferred to the recipient paraffin block than contained 60 tissue cores. The recipient block of eyelid sebaceous gland carcinoma consisted of 37 tumor cores. 
Immunohistochemistry
The TMA block was cut into 4-μm thick serial sections and mounted on glass slides. After deparaffinization and rehydration, heat-induced epitope retrieval was conducted with the Ventana CC1 mild reagent (Ventana Medical Systems, Tucson, AZ), a combination of EDTA and boric acid in Tris buffer for 60 minutes. After the epitope retrieval, the slides were treated with 0.3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase activity. Next, the slides were treated with 10% normal goat serum to block nonspecific antibody binding and incubated with antibody against Shh, Gli-1, Gli-2, Gli-3, ABCG2, Wnt, GSK3β, β-catenin, lipoprotein receptor-related protein (LRP), and c-Myc, followed by incubation with horseradish peroxidase-conjugated multimer antibody reagent (Ventana), using an automated immunostainer (Ventana/BenchMark XT, Tucson, AZ). The list of antibodies used and their conditions are shown in Table 1. The immunoreaction was developed with diaminobenzidine tetrachloride for 5 minutes, and the slides were counterstained with hematoxylin. 
Table 1. 
 
List of Antibodies and Their Conditions used for Immunohistochemistry
Table 1. 
 
List of Antibodies and Their Conditions used for Immunohistochemistry
Antibodies Manufacturer Dilution
Shh Santa Cruz Biotechnology, Santa Cruz, CA 1:150
Gli-1 Santa Cruz 1:50
Gli-2 Abcam, Cambridge, MA 1:50
Gli-3 Santa Cruz 1:200
ABCG2 Abcam 1:200
Wnt Santa Cruz 1:100
GSK3β Santa Cruz 1:100
β-catenin Zymed (Invitrogen), Carlsbad, CA 1:1000
LRP Abcam 1:100
c-Myc Santa Cruz 1:5000
Immunostaining results were interpreted as positive by cytoplasmic staining of Shh, ABCG2, Wnt, GSK3β, and LRP; cytoplasmic or nuclear staining of Gli-1, Gli-2, and Gli-3; nuclear staining of c-Myc; and β-catenin accumulation in the nucleus. The staining intensity was graded as negative, weak positive, and strong positive; the staining-positive tumor cells were counted in relation to the total number of tumor cells in at least three different representative high-power (×400) fields, and staining-positive cells more than 50% was graded as strong positive. For Gli-2 and Gli-3, immunostaining results were recorded as a percentage of positive cells. Immunostaining results for β-catenin were recorded as “not altered” when regular membranous β-catenin was present, “altered by loss” when expression of membranous β-catenin was lost, and “altered by nuclear accumulation” when β-catenin staining was observed in the nucleus. 27 Human tonsils served as positive controls. Primary antibodies were replaced with PBS in negative controls. Twenty normal eyelids obtained during the lateral tarsal strip surgery were also stained for Shh and Wnt signaling molecules to serve as normal controls. The immunostaining and the histology were interpreted by one pathologist (JEK). 
Statistical Analysis
Patients with eyelid sebaceous gland carcinoma were divided into two groups according to the presence of lymph node or distant metastasis. Demographic data, such as sex and age, were compared between the group with metastasis and the group without metastasis. Immunostaining results were compared between the two groups; χ2 test or Fisher's exact test was used to compare nominal variables, and Mann-Whitney U test was used to compare continuous variables without normality or ordinal variables. Correlations between the expressions of the proteins were analyzed with the Spearman correlation test. 
The cumulative incidence of lymph node section or distant metastasis was calculated from the date of surgery until the first day of metastasis detection using the Kaplan-Meier method; patients who had been lost to follow-up without the development of metastasis were considered censored at the date of the last follow-up. Log rank tests were carried out to determine difference in the cumulative incidence of metastasis among comparison groups in univariate analysis. Statistical analyses were performed with SPSS software version 18.0 (SPSS Inc., Chicago, IL). P values less than 0.05 were considered statistically significant. 
Results
Patients' Demographics and Clinical Features
The mean age of the 37 patients with eyelid sebaceous gland carcinoma was 61.7 years (SD, 13.8 years; range, 37–93 years). Among 37 patients, 27 patients showed T2N0 tumors, 8 patients showed T3N0 tumors, 1 patient showed T2N1 tumor, and 1 patient showed T3N1 tumor at initial diagnosis, staged according to the seventh edition of AJCC. Two patients underwent exenterations and 35 patients underwent wide surgical excision. Two patients exhibited lymph node metastasis at initial diagnosis, and six patients showed lymph node metastasis (five patients) and distant metastasis (one patient) at an average of 22.7 months (range, 8–41 months) during a mean follow-up period of 30.4 months (range, 1–97 months). Demographic features, including age and sex, did not differ between the group with metastasis and the group without metastasis (Table 2). 
Table 2. 
 
Clinical Features of Patients with Sebaceous Gland Carcinoma
Table 2. 
 
Clinical Features of Patients with Sebaceous Gland Carcinoma
Sebaceous Gland Carcinoma without Metastasis (n = 29) Sebaceous Gland Carcinoma with Metastasis (n = 8)
Sex, male/female 9:20 1:7 0.404*
Age, y† 63.4 ± 12.5 (37–88) 55.4 ± 17.2 (38–93) 0.079‡
Follow-up, mo† 26.6 ± 22.6 (1–94)  44.0 ± 32.8 (1–97)  0.155‡
Stage (seventh AJCC) (T2N0/T3N0/T2N1/T3N1) 25:4:0:0 2:4:1:1 0.002*
Immunohistochemistry Results: Normal Eyelid
Regarding hedgehog signaling proteins, Shh, Gli-1, and ABCG2 expression was not observed in normal eyelid sebaceous gland. However, Gli-2 and Gli-3 expression was observed in normal sebaceous gland. Regarding Wnt signaling proteins, Wnt, LRP, GSK3β, and c-Myc were not expressed in normal eyelid sebaceous gland. β-catenin was not aberrantly expressed in the nucleus and was expressed only in the cell membrane (Fig. 1). 
Figure 1. 
 
Immunostaining of Shh and Wnt signaling molecules in normal eyelid sebaceous gland. Shh (A, ×200) was not expressed in the cytoplasm of normal sebaceous gland cells. Gli-1 (B, ×400) was not expressed in the nucleus of normal sebaceous gland cells. Gli-2 (C, ×200) and Gli-3 (D, ×200) were expressed in the nucleus of normal sebaceous gland cells. ABCG2 (E, ×200) and Wnt (F, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. β-catenin (G, ×200) was expressed only in the cell membrane and was not expressed in the nucleus of normal sebaceous gland cells. GSK3β (H, ×200) and LRP (I, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. c-Myc (J, ×200) was not expressed in the nucleus of normal sebaceous gland cells.
Figure 1. 
 
Immunostaining of Shh and Wnt signaling molecules in normal eyelid sebaceous gland. Shh (A, ×200) was not expressed in the cytoplasm of normal sebaceous gland cells. Gli-1 (B, ×400) was not expressed in the nucleus of normal sebaceous gland cells. Gli-2 (C, ×200) and Gli-3 (D, ×200) were expressed in the nucleus of normal sebaceous gland cells. ABCG2 (E, ×200) and Wnt (F, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. β-catenin (G, ×200) was expressed only in the cell membrane and was not expressed in the nucleus of normal sebaceous gland cells. GSK3β (H, ×200) and LRP (I, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. c-Myc (J, ×200) was not expressed in the nucleus of normal sebaceous gland cells.
Immunohistochemistry Results: Eyelid Sebaceous Gland Carcinoma
Regarding hedgehog signaling proteins, Shh was positive in 56%, Gli-1 in 41%, and ABCG2 in 39% of the tumors (Table 3). Shh ligand was aberrantly expressed in the cytoplasm of cancer cells, and Gli-1 was expressed in the nucleus of cancer cells showing Shh positivity. Gli-2 and Gli-3 were highly expressed in the nucleus of cancer cells. ABCG2 was also aberrantly expressed in the cytoplasm of cancer cells (Fig. 2). Regarding Wnt signaling proteins, Wnt was positive in 9%, GSK3β in all, LRP in 12%, and c-Myc in 61% of the tumors; β-catenin expression was altered in 50% of the tumors (altered by nuclear expression in 6%, altered by membranous loss in 44%) (Table 3). Wnt was weakly expressed in the cancer cell cytoplasm, and β-catenin was expressed in cancer cell nucleus. GSK3β and LRP were expressed in the cancer cell cytoplasm, and c-Myc was expressed focally or diffusely in the cancer cell nucleus (Fig. 3). 
Figure 2. 
 
Positive immunostaining of Shh signaling molecules in eyelid sebaceous gland carcinoma. (A) Shh ligand was aberrantly expressed in the cytoplasm of cancer cells (×400). (B) Gli-1 was expressed in the nucleus of cancer cells showing Shh positivity (×400). (C, D) Gli-2 (C, ×100) and Gli-3 (D, ×100) were highly expressed in the nucleus of cancer cells. (E) ABCG2 was aberrantly expressed in the cytoplasm of cancer cells (×400).
Figure 2. 
 
Positive immunostaining of Shh signaling molecules in eyelid sebaceous gland carcinoma. (A) Shh ligand was aberrantly expressed in the cytoplasm of cancer cells (×400). (B) Gli-1 was expressed in the nucleus of cancer cells showing Shh positivity (×400). (C, D) Gli-2 (C, ×100) and Gli-3 (D, ×100) were highly expressed in the nucleus of cancer cells. (E) ABCG2 was aberrantly expressed in the cytoplasm of cancer cells (×400).
Figure 3. 
 
Positive immunostaining of Wnt signaling molecules in eyelid sebaceous gland carcinoma. (A) Wnt was weakly expressed in the cytoplasm of cancer cells (×400). (B) β-catenin was expressed normally in the cell membrane and also expressed aberrantly in the nucleus of cancer cells (×400). (C, D) GSK3β (C, ×200) and LRP (D, ×200) were expressed in the cytoplasm of cancer cells. (E) c-Myc was expressed diffusely in the nucleus of cancer cells (c-Myc, ×400).
Figure 3. 
 
Positive immunostaining of Wnt signaling molecules in eyelid sebaceous gland carcinoma. (A) Wnt was weakly expressed in the cytoplasm of cancer cells (×400). (B) β-catenin was expressed normally in the cell membrane and also expressed aberrantly in the nucleus of cancer cells (×400). (C, D) GSK3β (C, ×200) and LRP (D, ×200) were expressed in the cytoplasm of cancer cells. (E) c-Myc was expressed diffusely in the nucleus of cancer cells (c-Myc, ×400).
Table 3. 
 
Expression of Shh/Wnt Signaling Proteins in Eyelid Sebaceous Gland Carcinoma
Table 3. 
 
Expression of Shh/Wnt Signaling Proteins in Eyelid Sebaceous Gland Carcinoma
Signaling Protein Group Protein Expression P Value
Strongly Positive Weakly Positive Negative
Shh signaling pathway
 Shh Total 2 (6%) 17 (50%) 15 (44%) 0.031*
M 2 3 3
NM 0 14 12
 Gli-1 Total 1 (3%) 13 (38%) 20 (59%) 0.662
M 0 4 4
NM 1 9 16
 ABCG2 Total 2 (6%) 11 (33%) 20 (61%) 0.015*
M 2 1 4
NM 0 10 16
Wnt signaling pathway
 Wnt Total 0 (0%) 3 (9%) 31 (91%) 0.001*
M 0 3 5
NM 0 0 26
 GSK3β Total 21 (62%) 13 (38%) 0 (0%) 0.378
M 6 2 0
NM 15 11 0
 LRP Total 1 (3%) 3 (9%) 29 (88%) 0.177
M 1 1 6
NM 0 2 23
 c-Myc Total  7 (22%) 13 (39%) 13 (39%) 0.179
M 2 5 1
NM 5 8 12
Protein Expression
Group AN AL NotA
 β-catenin Total 2 (6%) 14 (44%) 16 (50%) 0.601
M 0 4 3
NM 2 10 13
Shh and ABCG2 were expressed more frequently in the group with metastasis than in the group without metastasis (χ2 test, P = 0.031 and P = 0.015, respectively). Wnt was also expressed more frequently in the group with metastasis than in the group without metastasis (χ2 test, P = 0.001) (Table 3). Spearman correlation test revealed a significant positive correlation between Shh and ABCG2 and between Shh and Wnt expression (P = 0.029 and P = 0.046, respectively) (Table 4). 
Table 4. 
 
Correlation between Expressions of Signaling Proteins of the Shh/Wnt Pathway in Eyelid Sebaceous Gland Carcinoma
Table 4. 
 
Correlation between Expressions of Signaling Proteins of the Shh/Wnt Pathway in Eyelid Sebaceous Gland Carcinoma
Shh Gli-1 Gli-2 Gli-3 ABCG2 Wnt GSK3β β-Catenin LRP
Gli-1 0.321
Gli-2 0.308 0.191
Gli-3 0.407 0.173 0.703
ABCG2 0.387* 0.232 0.174 0.343
Wnt 0.345* 0.350 −0.146 0.000 0.327
GSK3β 0.413 0.179 0.015 0.187 0.468 0.245
β-catenin 0.260 −0.070 −0.219 −0.104 0.344 0.274 0.104
LRP 0.284 0.057 0.156 0.224 0.124 0.195 0.280 −0.034
c-myc −0.148 0.000 −0.074 0.080 −0.026 0.226 0.064 −0.187 −0.138
In univariate analyses using log-rank tests, Shh, Gli-1, Gli-2, Gli-3, ABCG2, Wnt, GSK3 β, β-catenin, LRP, and c-Myc were included as independent parameters. Patients showing high ABCG2 expression developed metastasis more frequently than patients showing low ABCG2 expression (log rank test, P = 0.019; Fig. 4A). Patients showing Wnt expression developed metastasis more commonly than those without Wnt expression (log rank test, P = 0.001; Fig. 4B). Patients showing LRP expression developed metastasis more frequently than those without LRP expression (log rank test, P = 0.000; Fig. 4C). However, other immunohistochemical staining parameters were not associated with the development of metastasis based on univariate analyses. 
Figure 4. 
 
Univariate analyses on risk factors for the development of metastasis. (A) Patients showing high ABCG2 expression developed metastasis more frequently than patients showing low ABCG2 expression (log rank test, P = 0.019). (B) Patients showing Wnt expression developed metastasis more commonly than those without Wnt expression (log rank test, P = 0.001). (C) Patients showing LRP expression developed metastasis more frequently than those without LRP expression (log rank test, P = 0.000).
Figure 4. 
 
Univariate analyses on risk factors for the development of metastasis. (A) Patients showing high ABCG2 expression developed metastasis more frequently than patients showing low ABCG2 expression (log rank test, P = 0.019). (B) Patients showing Wnt expression developed metastasis more commonly than those without Wnt expression (log rank test, P = 0.001). (C) Patients showing LRP expression developed metastasis more frequently than those without LRP expression (log rank test, P = 0.000).
Discussion
Shh and Wnt signaling are the key master regulators of vertebrate development. 7,29 In normal mature tissue, the Shh or Wnt pathway is not activated and their signaling molecules are only found during embryogenesis and in stem cells. It has been postulated that reactivation of signaling pathways that are activated during organogenesis and subsequently inactivated in mature tissue, plays a critical role in tumor development and/or progression. 14,17-20,3033 However, this phenomenon has not been evaluated in eyelid sebaceous gland carcinoma. In this study, we examined the potential involvement of the Shh and Wnt pathways in eyelid sebaceous gland carcinoma by assessing the expression of key downstream effector proteins. 
We found that the Shh and Wnt signaling pathways, which are important in organogenesis of the pilosebaceous unit, were activated in eyelid sebaceous gland carcinoma. A significant correlation was also observed between the expression of Shh and ABCG2, molecules of the Shh signaling pathway, suggesting that once Shh had been activated, the whole canonical pathway of Shh was activated. This finding implies that these signaling pathways could be involved, at least in part, in the development of eyelid sebaceous gland carcinoma. Furthermore, the group with metastasis showed higher expression of Shh, ABCG2, and Wnt than the group without metastasis did, and patients with high ABCG2 expression, Wnt, or LRP expression showed higher cumulative incidence of LN or distant metastasis, implying that activation of the Shh and Wnt signaling pathways is also significantly associated with metastatic potential. This finding suggests that activation of the Shh and Wnt signaling pathways is a possible predictive factor for metastasis in eyelid sebaceous gland carcinoma. 
Given the different treatment approaches for patients with lymph node metastases and those without metastases, identification of patients with a high risk of lymph node metastasis would have significant implications in treatment decisions. Currently, patients with locally advanced eyelid sebaceous gland carcinoma are treated with wide surgical excision and radical neck dissection to decrease the risk of distant metastasis and provide better disease control. 34 However, sentinel lymph node biopsy in eyelid sebaceous gland carcinoma often yields false-negative results, 35 and there is no predictive biomarker for selecting patients with a significant risk of metastasis. Predictive biomarkers of metastasis would provide clinical benefits to patients with eyelid sebaceous gland carcinoma. 
One limitation of our study is that the expression of signaling molecules was not evaluated at the gene or mRNA level. 
In summary, we showed that the Shh and Wnt signaling pathways are frequently activated in eyelid sebaceous gland carcinoma and might be associated with metastasis. Risk stratification for metastasis based on this analysis warrants further validation in prospective cohorts of patients with eyelid sebaceous gland carcinoma. 
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Footnotes
 Supported by Grant number 11-2011-001 from SNUBH Research Fund.
Footnotes
 Disclosure: N. Kim, None; J.E. Kim, None; H.-K. Choung, None; M.J. Lee, None; S.I. Khwarg, None
Figure 1. 
 
Immunostaining of Shh and Wnt signaling molecules in normal eyelid sebaceous gland. Shh (A, ×200) was not expressed in the cytoplasm of normal sebaceous gland cells. Gli-1 (B, ×400) was not expressed in the nucleus of normal sebaceous gland cells. Gli-2 (C, ×200) and Gli-3 (D, ×200) were expressed in the nucleus of normal sebaceous gland cells. ABCG2 (E, ×200) and Wnt (F, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. β-catenin (G, ×200) was expressed only in the cell membrane and was not expressed in the nucleus of normal sebaceous gland cells. GSK3β (H, ×200) and LRP (I, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. c-Myc (J, ×200) was not expressed in the nucleus of normal sebaceous gland cells.
Figure 1. 
 
Immunostaining of Shh and Wnt signaling molecules in normal eyelid sebaceous gland. Shh (A, ×200) was not expressed in the cytoplasm of normal sebaceous gland cells. Gli-1 (B, ×400) was not expressed in the nucleus of normal sebaceous gland cells. Gli-2 (C, ×200) and Gli-3 (D, ×200) were expressed in the nucleus of normal sebaceous gland cells. ABCG2 (E, ×200) and Wnt (F, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. β-catenin (G, ×200) was expressed only in the cell membrane and was not expressed in the nucleus of normal sebaceous gland cells. GSK3β (H, ×200) and LRP (I, ×200) were not expressed in the cytoplasm of normal sebaceous gland cells. c-Myc (J, ×200) was not expressed in the nucleus of normal sebaceous gland cells.
Figure 2. 
 
Positive immunostaining of Shh signaling molecules in eyelid sebaceous gland carcinoma. (A) Shh ligand was aberrantly expressed in the cytoplasm of cancer cells (×400). (B) Gli-1 was expressed in the nucleus of cancer cells showing Shh positivity (×400). (C, D) Gli-2 (C, ×100) and Gli-3 (D, ×100) were highly expressed in the nucleus of cancer cells. (E) ABCG2 was aberrantly expressed in the cytoplasm of cancer cells (×400).
Figure 2. 
 
Positive immunostaining of Shh signaling molecules in eyelid sebaceous gland carcinoma. (A) Shh ligand was aberrantly expressed in the cytoplasm of cancer cells (×400). (B) Gli-1 was expressed in the nucleus of cancer cells showing Shh positivity (×400). (C, D) Gli-2 (C, ×100) and Gli-3 (D, ×100) were highly expressed in the nucleus of cancer cells. (E) ABCG2 was aberrantly expressed in the cytoplasm of cancer cells (×400).
Figure 3. 
 
Positive immunostaining of Wnt signaling molecules in eyelid sebaceous gland carcinoma. (A) Wnt was weakly expressed in the cytoplasm of cancer cells (×400). (B) β-catenin was expressed normally in the cell membrane and also expressed aberrantly in the nucleus of cancer cells (×400). (C, D) GSK3β (C, ×200) and LRP (D, ×200) were expressed in the cytoplasm of cancer cells. (E) c-Myc was expressed diffusely in the nucleus of cancer cells (c-Myc, ×400).
Figure 3. 
 
Positive immunostaining of Wnt signaling molecules in eyelid sebaceous gland carcinoma. (A) Wnt was weakly expressed in the cytoplasm of cancer cells (×400). (B) β-catenin was expressed normally in the cell membrane and also expressed aberrantly in the nucleus of cancer cells (×400). (C, D) GSK3β (C, ×200) and LRP (D, ×200) were expressed in the cytoplasm of cancer cells. (E) c-Myc was expressed diffusely in the nucleus of cancer cells (c-Myc, ×400).
Figure 4. 
 
Univariate analyses on risk factors for the development of metastasis. (A) Patients showing high ABCG2 expression developed metastasis more frequently than patients showing low ABCG2 expression (log rank test, P = 0.019). (B) Patients showing Wnt expression developed metastasis more commonly than those without Wnt expression (log rank test, P = 0.001). (C) Patients showing LRP expression developed metastasis more frequently than those without LRP expression (log rank test, P = 0.000).
Figure 4. 
 
Univariate analyses on risk factors for the development of metastasis. (A) Patients showing high ABCG2 expression developed metastasis more frequently than patients showing low ABCG2 expression (log rank test, P = 0.019). (B) Patients showing Wnt expression developed metastasis more commonly than those without Wnt expression (log rank test, P = 0.001). (C) Patients showing LRP expression developed metastasis more frequently than those without LRP expression (log rank test, P = 0.000).
Table 1. 
 
List of Antibodies and Their Conditions used for Immunohistochemistry
Table 1. 
 
List of Antibodies and Their Conditions used for Immunohistochemistry
Antibodies Manufacturer Dilution
Shh Santa Cruz Biotechnology, Santa Cruz, CA 1:150
Gli-1 Santa Cruz 1:50
Gli-2 Abcam, Cambridge, MA 1:50
Gli-3 Santa Cruz 1:200
ABCG2 Abcam 1:200
Wnt Santa Cruz 1:100
GSK3β Santa Cruz 1:100
β-catenin Zymed (Invitrogen), Carlsbad, CA 1:1000
LRP Abcam 1:100
c-Myc Santa Cruz 1:5000
Table 2. 
 
Clinical Features of Patients with Sebaceous Gland Carcinoma
Table 2. 
 
Clinical Features of Patients with Sebaceous Gland Carcinoma
Sebaceous Gland Carcinoma without Metastasis (n = 29) Sebaceous Gland Carcinoma with Metastasis (n = 8)
Sex, male/female 9:20 1:7 0.404*
Age, y† 63.4 ± 12.5 (37–88) 55.4 ± 17.2 (38–93) 0.079‡
Follow-up, mo† 26.6 ± 22.6 (1–94)  44.0 ± 32.8 (1–97)  0.155‡
Stage (seventh AJCC) (T2N0/T3N0/T2N1/T3N1) 25:4:0:0 2:4:1:1 0.002*
Table 3. 
 
Expression of Shh/Wnt Signaling Proteins in Eyelid Sebaceous Gland Carcinoma
Table 3. 
 
Expression of Shh/Wnt Signaling Proteins in Eyelid Sebaceous Gland Carcinoma
Signaling Protein Group Protein Expression P Value
Strongly Positive Weakly Positive Negative
Shh signaling pathway
 Shh Total 2 (6%) 17 (50%) 15 (44%) 0.031*
M 2 3 3
NM 0 14 12
 Gli-1 Total 1 (3%) 13 (38%) 20 (59%) 0.662
M 0 4 4
NM 1 9 16
 ABCG2 Total 2 (6%) 11 (33%) 20 (61%) 0.015*
M 2 1 4
NM 0 10 16
Wnt signaling pathway
 Wnt Total 0 (0%) 3 (9%) 31 (91%) 0.001*
M 0 3 5
NM 0 0 26
 GSK3β Total 21 (62%) 13 (38%) 0 (0%) 0.378
M 6 2 0
NM 15 11 0
 LRP Total 1 (3%) 3 (9%) 29 (88%) 0.177
M 1 1 6
NM 0 2 23
 c-Myc Total  7 (22%) 13 (39%) 13 (39%) 0.179
M 2 5 1
NM 5 8 12
Protein Expression
Group AN AL NotA
 β-catenin Total 2 (6%) 14 (44%) 16 (50%) 0.601
M 0 4 3
NM 2 10 13
Table 4. 
 
Correlation between Expressions of Signaling Proteins of the Shh/Wnt Pathway in Eyelid Sebaceous Gland Carcinoma
Table 4. 
 
Correlation between Expressions of Signaling Proteins of the Shh/Wnt Pathway in Eyelid Sebaceous Gland Carcinoma
Shh Gli-1 Gli-2 Gli-3 ABCG2 Wnt GSK3β β-Catenin LRP
Gli-1 0.321
Gli-2 0.308 0.191
Gli-3 0.407 0.173 0.703
ABCG2 0.387* 0.232 0.174 0.343
Wnt 0.345* 0.350 −0.146 0.000 0.327
GSK3β 0.413 0.179 0.015 0.187 0.468 0.245
β-catenin 0.260 −0.070 −0.219 −0.104 0.344 0.274 0.104
LRP 0.284 0.057 0.156 0.224 0.124 0.195 0.280 −0.034
c-myc −0.148 0.000 −0.074 0.080 −0.026 0.226 0.064 −0.187 −0.138
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