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ORIGINAL ARTICLE Identification of novel target proteins in sebaceous gland carcinoma Boban M. Erovic, MD,1 Ayman Al Habeeb, MBBS,2 Luke Harris, MD,1 David P. Goldstein, MSc, MD,1 Dae Kim, MBChB, PhD,1 Danny Ghazarian, MD,2 Jonathan C. Irish, MSc, MD1* 1 Department of Otolaryngology–Head and Neck Surgery/Surgical Oncology, Princess Margaret Hospital, University of Toronto, Toronto, Canada, 2Department of Laboratory Medicine and Pathobiology, Princess Margaret Hospital, University of Toronto, Toronto, Canada. Accepted 29 February 2012 Published online 19 June 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23021 ABSTRACT: Background. The aim of this study was to identify new target proteins in sebaceous gland carcinoma. Methods. A tissue microarray containing 115 core biopsies was constructed and stained for proteins involved in carcinogenesis, angiogenesis, inflammation, and cell-to-cell contact. Two investigators independently determined protein expression of all antibodies. Results. Vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor alpha and beta (PDGFR-a/-b), epidermal growth factor receptor (EGFR), cyclooxygenase 1 and 2 (Cox-1/-2), myeloid cell leukemia sequence 1 (Mcl-1), matrix metalloproteinase 1 (MMP-1), CD9, Bmi-1, 14-3-3r, glutathione S- INTRODUCTION Sebaceous gland carcinomas are often aggressive skin malignancies arising most commonly from the meibomian or Zeis glands.1 There is a high tendency for metastatic disease particularly to regional lymph nodes.2 Unfortunately, despite early and aggressive surgical treatment, patients with sebaceous gland carcinomas have a high incidence rate of locoregional recurrence and distant metastatic disease.2 Epidemiological studies have shown that during the past 30 years there has been little change in the stage at which the disease presents, and little improvement in the prognosis for patients with sebaceous gland carcinoma.1 Given the high recurrence rate despite aggressive surgical treatment, an effective systemic adjuvant therapy would represent a major breakthrough in the management of these patients. To the best of our knowledge there are no studies that have looked into novel targeted systemic therapies in the treatment of sebaceous gland carcinoma. In the current study we aimed to identify proteins that are overexpressed in sebaceous gland carcinoma cells, with the hope that they may be amenable to therapeutic targeting. We examined the expression of specific proteins involved in angiogenesis, inflammation, apoptosis, cell proliferation, cell-to-cell contact, and carcinogenesis. *Corresponding author: J. C. Irish, Princess Margaret Hospital, Wharton Head and Neck Centre, 610 University Avenue, 3rd Floor, Toronto, ON M5G 2M9, Canada. E-mail: [email protected] 642 HEAD & NECK—DOI 10.1002/HED MAY 2013 transferase pi (Gstp), and members of the sonic hedgehog (SHH), AKT, and WNT pathways were significantly overexpressed in sebaceous gland carcinomas. Conclusions. We have demonstrated for the first time that proteins related to angiogenesis, inflammation, and cell proliferation are overexpressed in sebaceous gland carcinomas. These proteins may hold promise as novel therapeutic targets for the treatment of sebaceous gland carcinoma. C 2012 Wiley Periodicals, Inc. Head Neck 35: 642–648, 2013 V KEY WORDS: target proteins, sonic hedgehog, angiogenesis, targeted anticancer therapy This was accomplished through the use of a high-throughput tissue microarray containing 35 tumor samples from 20 patients with sebaceous gland carcinoma. Tissue microarrays underwent immunohistochemistry staining and 2 investigators independently determined protein expression. MATERIALS AND METHODS Patients Patients with sebaceous gland carcinoma managed at the Princess Margaret Hospital (PMH) between 1983 and 2009 were identified through the cancer registry. Patients were included if they had a newly diagnosed and previously untreated sebaceous gland carcinoma that was managed at the PMH. Sufficient paraffin-embedded tissue to perform the microarray was an additional criterion for inclusion. Patients with incomplete medical records and those who did not pursue curative treatment were excluded. Approval was obtained from the institutional Research Ethics Board. The extent of disease was classified using the tumor-node-metastasis (TNM) staging system, in accord with the American Joint Committee on Cancer (AJCC) classification. The tumors were classified as periorbital if they occurred within the orbit or on the eyelids and were classified as extraorbital if they arose from another head and neck site. At the PMH, the primary management approach for patients with sebaceous gland carcinoma is primary surgery, with adjuvant radiotherapy offered to those patients with positive margins or nodal metastases. The University TARGET PROTEINS IN SEBACEOUS GLAND CARCINOMAS TABLE 1. Antibodies, sources, and retrieval buffers for each antibody used in this study. Antibody Source Catalog no. Clone Pretreatment Dilution (Incubation) 14-3-3Sigma Bcl-2 alpha Bcl-xl AB-2 Bmi-1 Catenin beta Cdc2p34 CD9 Cytokeratin 7 EGFR Gli-1 Gli-2 Gli-3 Mcl-1 MMP 1 PDGFR alpha PDGFR beta SHH Smo VEGFR2 WISP-1 WISP-2 Gst-p pAKT pmTOR ser2448 FOX-01 Chromogranin p27 p21 p53 Cyclin D1 Rb Cox-1 Cox-2 Thermo Scientific/Neomarkers Thermo Scientific/Neomarkers Thermo Scientific/Neomarkers Millipore Santa Cruz Santa Cruz Novocastra Thermo Scientific/Neomarkers Invitrogen Santa Cruz Aviva Santa Cruz Neomarkers Lab Vision/Neomarkers abcam Thermo Scientific/Neomarkers Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Novocastra Cell Signaling Cell Signaling Novus Biological Neomarkers Transduction Lab Pharmingen Novocastra Lab Vision Pharmingen Santa Cruz (Goat) Santa Cruz (Goat) MS-1185-PO MS-123-PO MS-1334-PO 05-637 Sc-7963 Sc-54 NCL-CD9 MS-1352-PO 28-0005 Sc-20687 ARP31885_T100 Sc-20688 MS-681-PO RB-9225-PO Ab61219 RB-1692-PO Sc-9024 Sc-13943 Sc-6251 Sc-25441 Sc-25442 NCL-GSTpi-438 3787S 4976S NBPI-19825 RB9003-PO 610242 556431 NCL p53-DO7 RM 9104 554136 Sc-1754 Sc-1747 1433S01 100/D5 7D9 F6 TE9 TE9 TE9 TE9 Citrate Citrate TE9 Trypsin Pepsin Citrate TE9 TE9 Trypsin Citrate TE9 TE9 Citrate Trypsin TE9 Citrate Trypsin Citrate Citrate Citrate Citrate Pepsin Citrate TE9 Citrate TE9 Citrate TE9 Citrate 1/400 1/100 1/1000 1/400 overnight 1/50 overnight 1/1000 overnight 1/200 1 hour 1/300 overnight 1/100 1 hour 1/100 overnight 1/100 overnight 1/100 overnight 1/100 overnight 1/800 overnight 1/300 overnight 1/100 overnight 1/400 overnight 1/300 overnight 1/1000 1 hour 1/100 overnight 1/500 overnight 1/300 1 hour 1/50 overnight 1/50 overnight 1/100 overnight 1/500 1 hour 1/1000 1 hour 1/200 overnight 1/300 1 hour 1/200 overnight 1/300 1 hour 1/100 overnight 1/1000 overnight Health Network skin pathology site group (D. Ghazarian and A. Al Habeeb) reviewed all sebaceous gland carcinoma cases. Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS version 15.0; SPSS Inc., Chicago, IL). Demographic and pathologic data were summarized using descriptive statistics. The recurrence and survival rates were calculated from the date of diagnosis to the event of interest. Recurrences were classified as local, regional, or distant. Survival analysis was performed using the Kaplan–Meier method. The log-rank method was used for univariate analysis. All the statistical tests were 2-tailed, and a value of p < .05 was considered significant. Specimens A total of 35 surgical specimens from 20 patients contained sufficient tissue to be sourced for construction of a tissue microarray (TMA). Two skin pathologists reviewed all hematoxylin-eosin–stained tumors. 72F6 OV/TL 12/30 31G7 Polyclon Polyclon Polyclon RC13 Polyclon Polyclon Polyclon Polyclon Polyclon Polyclon LW29 49F9 Tissue microarray construction After examination of all available slides, the most representative blocks were chosen from each of the 35 specimens. TMAs were constructed with 3 cores from each specimen. Each core measured 0.6 mm in diameter. The cores were mounted on linear arrays in a single paraffin TMA block. A character map was constructed delineating the various specimens and their order in the TMA. Tumor samples were categorized as negative, weak, moderate, or high when 0% to 10%, 11% to 30%, 31% to 60%, and 61% to 100% of tumor cells stained positively, respectively. Again, 2 investigators (A.A. and D.G.) independently determined the immunohistochemical expression of all antibodies. Immunohistochemistry staining All details regarding the antibody, source, clone, pretreatment, and dilution are summarized in Table 1. Formalin-fixed paraffin-embedded sections (4 lm) were dewaxed in 5 changes of xylene and brought down to water through graded alcohols. If necessary, antigen retrieval and unmasking procedures were applied (see the following text). Endogenous peroxidase was blocked using 3% hydrogen peroxide. After blocking for 5 minutes with HEAD & NECK—DOI 10.1002/HED MAY 2013 643 EROVIC ET AL. casein, sections were incubated at room temperature with the appropriate primary antibodies in accord with the conditions described in the following text. Incubation was followed by the application of the Biogenex Super Sensitive Kit (cat #HK518-YAK; Biogenex, San Ramon, CA) super enhancer and then the Polymer HRP reagent. After washing in phosphate-buffered saline (PBS), color development was performed with freshly prepared NovaRed solution (cat #SK-4800; Vector Laboratories, Burlingame, CA). Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated in alcohols, cleared in xylene, and mounted in Permount (cat #SP15-500; Fisher Scientific, Fair Lawn, NJ). Cyclooxygenase-1 and -2 staining Staining of cyclooxygenase (Cox)-1 and Cox-2 was performed as follows: 4 lm formalin-fixed paraffin-embedded sections were dewaxed in 5 changes of xylene and brought down to water through graded alcohols. Endogenous peroxidase and biotin activities were blocked respectively using 3% hydrogen peroxide and an avidin/ biotin blocking kit (cat # TA-015-BB; Lab Vision, Fremont, CA). After blocking for 15 minutes with 10% normal serum from the species from which the secondary antibody was obtained, sections were incubated at room temperature with the appropriate primary antibodies using the previously optimized conditions (see Table 1). This was followed with a biotinylated secondary (Vector Laboratories) for 30 minutes and horseradish peroxidase–conjugated ultrastreptavidin labeling reagent (ID Laboratories, London, ON, Canada) for 30 minutes. After washing in PBS, color development was performed as described earlier (Table 1). RESULTS Twenty patients with sebaceous carcinomas of the head and neck were eligible for inclusion in the study. Seventeen patients had a carcinoma of the periorbital region, and 3 patients had a sebaceous carcinoma of other head and neck sites (Table 2). For patients with periorbital sebaceous carcinomas, the upper eyelid was involved in 7 patients (35%) and the lower eyelid was involved in 2 patients (10%). Both eyelids were involved in 2 patients (10%). The remaining periorbital subsites were the medial canthal region (n ¼ 3, 15%) and the entire orbit (n ¼ 3, 15%). The nasal skin (n ¼ 1, 5%) and the scalp (n ¼ 2, 5%) were the subsites for extraorbital sebaceous carcinomas. Of the entire cohort (n ¼ 20), 10 were women and 10 were men. The median age at presentation for patients with periorbital and extraorbital tumors was 72 and 68 years (range, 42–86 years), respectively. TNM classification The distribution of tumors by pathologic T and N classifications is as follows: for the periorbital site, 5% (n ¼ 1) of the carcinomas were classified as T1 and 80% (n ¼ 16) were classified as T3 or T4. At the time of diagnosis, the tumors presenting at extraorbital sites were classified as T1 or T2 in 2 cases and 1 patient presented with T4 disease. Of the entire patient cohort, only 2 patients (scalp HEAD & NECK—DOI 10.1002/HED Characteristics No. (%) Patients Site Periorbital Extraorbital Sex Female Male Age Mean, y Median, y Range, y Margins Positive Negative PNI Postive Negative NA LVI Positive Negative NA ECE Positive NA 20 10 (50) 10 (50) 10 (50) 10 (50) 68 72 42-86 6 (30) 14 (70) 4 (20) 3 (15) 13 (65) 2 (10) 1 (5) 17 (85) 1 (5) 19 (95) Abbreviations: PNI, perineural invasion; LVI, lymphovascular invasion; ECE, extracapsular extension. lesion, 1; both eyelids, 1) had nodal metastases at initial presentation and distant metastases were not noted at presentation in any of the patients. Primary treatment Patient data 644 TABLE 2. Demographic and histopathologic characteristics of 20 patients with sebaceous gland carcinoma. MAY 2013 Of the 20 patients, 14 were treated with primary surgery; 5 of these 14 patients received postoperative radiotherapy. Six of the 20 patients were treated with primary radiotherapy. An elective neck dissection was performed in 1 patient with sebaceous gland carcinoma localized on the scalp. Pathology Positive margins were noted in 6 patients (30%) and negative margins in 14 patients (70%). Four specimens (20%) showed perineural invasion, 1 specimen (10%) showed lymphovascular invasion, and 1 specimen (5%) showed extracapsular lymph node extension. Outcomes The mean and median follow-up times for the entire cohort were 47 and 28 months, respectively, with a range of 4 to 145 months. Overall, 11 patients (55%) developed recurrent disease; of these, 5 patients (45%) developed local and 6 patients (55%) developed regional recurrence. At the last follow-up, 7 patients (35%) were alive without disease, and 3 patients (15%) were alive with disease; 10 patients (50%) died, of which 6 patients died due to sebaceous gland carcinoma. Of the 11 patients with recurrence, 6 patients underwent salvage treatment. Four TARGET PROTEINS IN SEBACEOUS GLAND CARCINOMAS cally, 30 samples (88%) and 2 samples (6%) of 34 showed strong and moderate expression for VEGFR-2, respectively. Platelet-derived growth factor receptor-a and -b The majority of the probes were highly positive for platelet-derived growth factor receptor alpha (PDGFR-a; n ¼ 27, 78%), with only 1 sample (3%) demonstrating weak staining and 3 samples (9%) showing moderate staining. Overexpression of PDGFR-b was found to be high in 27 probes (78%) and weak or moderate in 2 probes (6%). Epithelial growth factor receptor Only 1 sample (3%) was negative for epidermal growth factor receptor (EGFR), with 26, 5, and 2 samples (76%, 15%, and 6%) showing a high, moderate, and weak expression, respectively. Inflammation markers All specimens were positive for cyclooxygenase-1 (Cox-1) and cyclooxgenase-2 (Cox-2). Cytoplasmatic expression of Cox-1 was categorized as moderate in 1 specimen (3%) and high in 31 specimens (97%), respectively. Cox-2 expression was weak, moderate, and high in 2, 4, and 28 of all tumor samples (6%, 12%, and 82%), respectively. Apoptosis markers FIGURE 1. The immunostaining of VEGFR2, PDGFRa and -b, EGFR, Cox-1, Cox-2, Mcl-1, Gli-1, 2-3, Shh, MMP-1, and Gstp in a tissue microarray of sebaceous gland carcinoma are shown. All photomicrographs were taken at original magnification, 200. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] patients with locally recurrent periorbital sebaceous gland carcinomas underwent salvage treatment with surgery and postoperative radiotherapy. Six patients developed regional recurrent disease and were treated either by salvage surgery alone (n ¼ 3) or surgery and radiotherapy (n ¼ 3). One patient refused further therapy. The median (mean) survival after treatment for recurrence was 19 (36) months for the entire cohort. Thirty-three, 15, 17, and 17 samples (100%, 47%, 53%, and 53%) were found to be positive for Mcl-1, Bclx, Bcl-2a, and p53, respectively. The staining was localized to the cell cytoplasm. Looking at each of these proteins independently, Mcl-1 and p53 were highly expressed in 25 samples (76%) and 15 samples (45%), whereas only 4 samples (12%) each were highly positive for Bclx and Bcl-2a. Mcl-1, Bclx, and Bcl-2a were moderately expressed in 4, 2, and 3 samples (12%, 6%, and 9%). WNT pathway Expressions of b-catenin, Wisp-1, and Wisp-2 were localized to the tumor cell cytoplasm, and positive staining was observed in 100% (b-catenin), 30 (91%) (Wisp1), and 11 (34%) (Wisp-2) of the specimens. Immunohistochemistry Shh pathway Original immunohistochemical stains are presented in Figure 1 and the schematic presentation of negative, weak, moderate, and high protein expression in sebaceous gland carcinoma specimens is shown in Figure 2. All sonic hedgehog (Shh) pathway proteins were expressed in the tumor cell cytoplasm. Gli-1, Gli-2, and Gli-3 were detectable in all specimens. Intensity for these 3 proteins was rated as high in 32 (Gli-1), 32 (Gli-2), and 28 (Gli-3) of all investigated samples (97%, 100%, 88%). Moderate intensity was observed in only 3 specimens (1 for Gli-1 and 2 for Gli-3). Shh was highly overexpressed in 28 samples (88%), whereas Smo was not positive in 26 samples (76%). Expression of Shh was weak in 2 specimens (6%) and moderate in 1 specimen (3%). Angiogenesis markers Vascular endothelial growth factor receptor-2 All investigated samples were positive for vascular endothelial growth factor receptor-2 (VEGFR-2). Specifi- HEAD & NECK—DOI 10.1002/HED MAY 2013 645 EROVIC ET AL. FIGURE 2. Schematic representation of negative, weak, moderate, intermediate, and strong staining intensity of 33 different proteins in sebaceous gland carcinoma samples. P/m, primary/metastatic lesion: primary, 1; mets, 2; n, sample number. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Cell adhesion markers Matrix metalloproteinase 1 (MMP-1) was highly overexpressed in all the samples, whereas Keratin-7 was detectable only in 16 samples (50%). For Keratin-7, the intensity was weak or moderate in each of 2 probes (6%), respectively. CD9 was expressed in the cell membrane of 30 specimens (94%) and its intensity was classified as moderate in 4 specimens (12%). No expression for CD9 could be found in 2 specimens (6%). Cell cycle markers Except for the proteins Cdc2p34 and 14-3-3r, which were observed in the cell cytoplasm, all remaining proteins belonging to the cell cycle group were found in the cell nuclei. Cdc2p34 was highly overexpressed in 25 samples (78%) and moderately and weakly expressed in 6 and 1 samples (19% and 3%), respectively. Rb was negative in 18 of all specimens (51%) but was highly overexpressed in 12 samples (34%). Cyclin D1 was positive in only 10% of samples. Cyclin D1 was classified predominantly as negative (61%) and only 2, 1, and 10 samples (6%, 3%, and 30%) showed weak, moderate, and high expression, respectively. The expression of 14-3-3r was significantly overexpressed in 29 samples (85%), moder646 HEAD & NECK—DOI 10.1002/HED MAY 2013 ate in 4 samples (12%), and weak in only 1 sample (3%). The proteins p27 and p21 were predominantly highly positive. In particular, in 27 and 31 of samples (82%, 91%), high overexpression of p27 and p21 was observed with a moderate expression in 5 and 2 samples (15%, 6%), respectively. Bmi-1 was highly overexpressed in all samples. AKT-pathway markers Highly positive staining for pAKT could be observed in 17 samples (52%) and moderate and weak expression was observed in 9 and 3 of the specimens (27%, 9%), respectively. In contrast, FoxO-1 (Figure 1I) was predominantly highly overexpressed in 28 of all samples (82%). Its intensity was weak in 1 sample (3%) and moderate in 2 samples (6%). High and weak expression of mTOR occurred in 23 and 5 specimens (68%, 15%), respectively. Cell-specific markers Glutathione S-transferase pi (Gstp; Figure 1J) was highly positive in 33 of the 34 samples (79%) and only 1 sample was negative for Gstp. TARGET Protein expression in primary and metastatic lesions We compared the protein expression of all 33 antibodies but we were not able to determine a specific expression pattern related to primary or metastatic lesions in this cohort. DISCUSSION Despite significant improvements in our understanding of the molecular biology of cancer and its metastatic behavior, locoregional and distant failures in patients with sebaceous gland carcinoma are still common.2 The cornerstone of treatment is surgical resection of the primary disease with or without adjuvant radiotherapy.2 Unfortunately, to date there is no evidence-based data on the management of patients with distant metastatic disease. In the current English-language literature there are only 3 case reports published.3–5 The choice of chemotherapy in these reports was generally based on a combination of drugs commonly used in the treatment of head and neck carcinomas.3 The aim of this study was to determine the antigen profiles of sebaceous gland carcinomas and to determine if there were any significant characteristics of those tumors that demonstrated metastatic behavior. The most impressive immunostaining in sebaceous gland carcinoma was the expression of VEGFR2, EGFRR2, PDGFR-a, and PDGFR-b. Currently, there are several studies demonstrating that tyrosine kinase receptor inhibitors are effective in patients with squamous cell carcinomas of the head and neck,6 lung,7 and colon.8 Our results may provide the rationale for the use of future targeted therapies, such as sunitinib, cetuximab, bevacizumab, and pazopanib against those proteins. These biologic agents have been shown to be effective in the treatment of head and neck, kidney, lung, and breast malignancies.6,9–11 In addition, we observed a high rate of expression of both Cox-1 and Cox-2, particularly Cox-1, in a significant number of tumor specimens. Multiple studies have been published that demonstrate an antitumor effect of nonsteroidal anti-inflammatories.12–14 Further research is needed to determine whether the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may provide a benefit in patients with metastatic sebaceous gland carcinoma. The hedgehog-signaling cascade is one of the main pathways in human embryogenesis and organogenesis. Mutations that inappropriately reactivate this normally quiescent pathway have been linked to the development of skin, colon, and lung carcinoma.15,16 In our study, we observed high expression of the proteins Gli-1, Gli-2, Gli-3, and Shh. Manipulation of the hedgehog pathway by selective agonists and antagonists, such as cyclophosphamide, may be effective therapeutic modalities in those tumors demonstrating overexpression of hedgehog-signaling pathway proteins.16 Research in prostate cancer and lymphoma has shown that blocking Gli-2 expression with antisense oligonucleotides in vitro leads to a significant delay in tumor progression and an increase in paclitaxel chemosensitivity.17,18 The proteins CD9, MMP-1, and Gstp were also significantly overexpressed in the majority of our sebaceous gland carcinoma samples. CD9 and MMP-1 are proteins PROTEINS IN SEBACEOUS GLAND CARCINOMAS involved in cell motility, invasion, and metastasis.19–21 Selective targeting of CD9 and MMP-1 with specific antibodies has been demonstrated to trigger apoptosis in chemoresistant lung carcinoma cells20 and can suppress the invasion of tumor cells in a 3-dimensional collagen gel.21 Gstp plays a role in the detoxification of platinum-based cytotoxic agents and can promote resistance to platinums. A new target agent named telcyta (TLK)-286 may hold promise by increasing tumor sensitivity to platinums. TLK-286 has demonstrated cytotoxic activity on its own,22 but through its inhibition of Gstp it can enhance the activity of cisplatin in cancer cells.23 A Gstp inhibitor such as TLK-286 may hold promise in patients with sebaceous gland carcinoma through its ability to sensitize tumor cells to cisplatin. Forkhead-box-O (FoxO) transcription factors are involved in multiple signaling pathways and play critical roles in a number of physiological processes as well as tumor pathogenesis.24 FoxO acts as a tumor suppressor, with loss of FoxO function leading to increased cell survival and a predisposition to cancer development.25 Based on current knowledge and studies from the past decade, the development of novel agents, which specifically activate FoxO members, could be useful in the prevention and treatment of epithelial malignancies in particular.25 Our demonstration of significant FoxO-1 overexpression in sebaceous gland cancer cells has suggested that development of FoxO-1 target agents may represent a new effective therapeutic approach. Another very interesting finding of this study was that there is no specific protein expression pattern between primary and metastatic lesions in patients with sebaceous gland carcinoma. Initially we hoped to find a target protein, particularly for metastatic lesions. Conversely, no differential protein expression means that targeting the primary tumor may simultaneously target distant metastases in patients with advanced-stage disease. Although it is recognized that these results are preliminary and that further in vitro and in vivo data are required, we have identified several potentially important protein targets and a target pathway that may be therapeutically exploited for the treatment of sebaceous gland carcinoma. The use of agents that target these proteins and pathways may provide a much-needed systemic treatment for patients with metastatic sebaceous gland carcinoma. REFERENCES 1. Buitrago W, Joseph AK. Sebaceous carcinoma: the great masquerader emerging concepts in diagnosis and treatment. Dermatol Ther 2008;21: 459–466. 2. Shields JA, Demirci H, Marr BP, Eagle RC Jr, Shields CL. Sebaceous carcinoma of the eyelids: personal experience with 60 cases. 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