Aberrant methylation of tumor suppressor genes as potential molecular markers for cutaneous squamous cell carcinoma

Aberrant methylation of tumor suppressor genes as potential molecular markers for cutaneous squamous cell carcinoma

P403 Signet ring cell melanoma, Brenner sign, and elevated VEGF Jacqueline Russo, MD, University of Florida College of Medicine, Department of Patholo...

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P403 Signet ring cell melanoma, Brenner sign, and elevated VEGF Jacqueline Russo, MD, University of Florida College of Medicine, Department of Pathology & Laboratory Medicine, Gainesville, FL, United States; Keira Barr, MD, University of California, Davis Health System, Departments of Dermatology and Pathology, Sacramento, CA, United States; Larissa Scanlan, MD, Voluntary Faculty at the University of Miami School of Medicine and Private Practice in Delray, FL, Delray Beach, FL, United States; Vladimir Vincek, MD, PhD, University of Florida College of Medicine, Department of Pathology & Laboratory Medicine, Gainesville, FL, United States Introduction: We present a patient with metastatic signet ring cell melanoma with unknown primary site who presented with Brenner sign and markedly elevated vascular endothelial growth factor (VEGF). We discuss the possible relatedness between Brenner sign and VEGF and their potential utility. Clinical history: A 41-year-old white male presented with a rapidly growing subcutaneous supraclavicular mass with an overlying erythematous macule, consistent with Brenner sign, which extended to the anterior chest wall. A CT scan revealed a 5.0-cm well circumscribed solid mass. Fine needle aspiration and core biopsy showed signet ring cells immunoreactive for HMB-45 and S100 protein; cytokeratin was negative. A subsequent excision and nodal neck dissection revealed a 7.0-cm melanoma with 60 negative lymph nodes. A skin ellipse from the Brenner sign overlying the tumor mass was taken in search of a cutaneous primary site, but revealed only mild spongiotic dermatitis. A CT scan, PET scan, ocular examination, laryngoscopy, bronchoscopy, and esophagoscopy failed to reveal a primary site. The diagnosis of metastatic signet ring cell melanoma was made. At diagnosis, serum VEGF levels were elevated measuring 1117 pg/mL (normal, 31-86 pg/mL) with elevation of white blood cell (WBC) counts to 19.7 3 103 U/L, of which 81% were neutrophils. Clinically, the patient was afebrile with no signs of infection. Once the tumor was removed, WBC and neutrophil counts rapidly returned to normal ranges. Likewise, the Brenner sign disappeared. Unfortunately, VEGF levels postetumor resection were not repeated. Two years later, the patient presented with axillary lymphadenopathy. Axillary lymph node dissection confirmed 2 of 33 lymph nodes positive for metastatic disease. Discussion: Signet ring melanoma is an unusual variant accounting for only 0.5% of melanomas. They present as progressively enlarging pigmented or erythematous plaques. The cells have signet ring histomorphology. Similar morphology can occur in a variety of other cutaneous and noncutaneous neoplasms requiring immunophenotyping for accurate diagnosis. Although rare, patients with melanoma can develop erythematous eruptions at the site of, or at a distance from, their primary melanocytic lesion, termed Brenner sign. It is reported that this sign may be useful for early diagnosis of melanoma. It is hypothesized that the erythema of Brenner sign can be explained by factors responsible for angiogenesis. VEGF is a potent initiator of angiogenesis. In malignancy, angiogenesis is precipitated by an increase in oxygen demand by tumor cells. Newly created vasculature serve to better meet the increased oxygen demand of the tumor while creating an avenue for metastasis. The elevated VEGF in our patient supports previous observations of increased VEGF in patients with melanoma. We present an unusual case of metastatic signet ring cell melanoma with an unknown primary site, in association with Brenner sign and elevated serum VEGF. This case illustrates that elevated growth factors in patients with melanoma may help stratify high risk patients with a worse prognosis, and may provide a useful follow-up marker for disease progression. Further studies into the clinical application of this marker are needed. Commercial support: None identified.

P404 Sentinel lymph node biopsy for cutaneous melanoma: The Washington Cancer Institute experience in 472 patients, 1997-2008 Suzanne Berkman, MD, Washington Cancer Institute/Washington Hospital Center, WA, DC, United States; Gary Peck, MD, Washington Cancer Institute/Washington Hospital Center, WA, DC, United States; Marc Boisvert, MD, Washington Cancer Institute, WA, DC, United States; Rodolfo Chirinos, MD, Washington Cancer Institute/Washington Hospital Center, WA, DC, United States; Saurabh Singh, MD, Washington Hospital Center, WA, DC, United States Background: Lymph node status is the most powerful independent prognostic factor in patients with clinically node-negative melanoma. The staging guidelines of the American Joint Committee on Cancer recommend sentinel lymph node biopsy (SLNB) for melanomas [1.0 mm in Breslow depth (BD) (or \1.0 mm if adverse histopathologic factors are present). Since 1997, we have routinely performed SLNBs for melanomas $ 0.76 mm (or \0.76 mm with adverse histopathologic factors). Objective: To determine the rate of SLNB positivity among 472 patients who underwent SLNB between 1997 and 2008. Secondary goals were to elucidate which factors best predict the risk for SLN metastasis and clarify which patients with thin melanomas (BD \1.0 mm) should undergo SLNB. Methods: We reviewed the Washington Cancer Institute melanoma database and analyzed histopathologic and survival data. Results: Among 472 patients (mean BD, 2.11 mm), 55 (11.7%) patients (mean BD, 3.26 mm) had positive SLNB. Three hundred thirty patients (69.9%) had tumors [1.0 mm (mean BD, 2.68 mm), of which 52 (15.8%) had positive SLNB (mean BD, 3.4 mm) and 278 (84.2%) were negative (mean BD, 2.55 mm; P ¼ .021). One hundred forty-two patients (30.1%) had thin melanomas (mean BD, 0.77 mm), three (2.1%) of which had positive SLNB (mean BD, 0.81mm; no adverse histopathologic

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features reported). The rate of SLNB positivity increased with increasing depth of invasion. BD, Clark level, and nodular subtype were found to be significantly associated with SLNB positivity; ulceration status approached statistical significance. Forty-three of 472 patients (9.1%) developed local or distant recurrence (mean BD, 4.19 mm; mean follow-up, 44.5 months). Of these, 17 (39.5%) survived (mean BD, 3.76 mm; mean follow-up, 57.5 months) and 26 (60.5%) died (mean BD, 4.47 mm; mean follow-up, 35.9 months). Of 417 patients with negative SLNB, 29 (7%), developed recurrence (including 1 thin melanoma) versus 14 (25.4%) of 55 patients with positive SLNB (including 1 thin melanoma). Twelve of 417 (2.9%) SLNB-negative patients and 7 of 55 (12.7%) SLNB-positive patients developed distant metastases (P ¼ .003). Death occurred in 17 (4.1%) SLNB-negative patients and in nine (16.4%) SLNB-positive patients (P ¼.001). Conclusions: SLN positivity is a function of BD, which correlates with disease progression and survival. Given the low rate (2.1%) of positive SLNB in patients with thin melanomas, the decision to perform SLNB in this group should depend on the presence of adverse histopathologic factors. Commercial support: None identified.

P405 Aberrant methylation of tumor suppressor genes as potential molecular markers for cutaneous squamous cell carcinoma Alice Chuang, MD, Johns Hopkins University, Baltimore, MD, United States; David Sidransky, MD, Johns Hopkins University, Baltimore, MD, United States; Joseph Califano, MD, Johns Hopkins University, Baltimore, MD, United States; Nanette Liegeois, MD, PhD, Johns Hopkins University, Baltimore, MD, United States Purpose: Cutaneous squamous cell carcinoma (cSCC) is the second most common type of skin cancer in the United States. Advanced or metastatic cSCC is associated with high morbidity and mortality. Similar to other tumor types, identification of molecular markers has the potential diagnostic and/or prognostic value. Experimental design: We have examined the promoter methylation of 12 candidate genes in 54 primary cSCC tumors and 41 normal skin tissues. Quantitative methylation-specific PCR (QMSP) was applied to determine the methylation status of each gene in all tissue samples. DLC-1 mRNA expression was evaluated by quantitative real-time reverse transcription-PCR. Restoration of DLC-1 expression in vitro was performed by the demethylating agent 5-aza-2’-deoxycytidine (5-Aza-dC) and tumor suppressor properties were tested after ectopic expression in cSCC cell lines. Results: Promoter methylation of the TSGs was more frequently observed among cSCC samples than normal skin samples. The data have demonstrated the value of high sensitivity and specificity in multigene approach in detection of aberrant methylation in cSCC. Additional studies were performed on DLC-1 because QMSP results showed that the DLC1 promoter was methylated in 26 of 54 (48.1%) primary cSCC tumors versus eight of 41 (19.5%) in normal skin tissues. We also found that the expression of DLC-1 was downregulated in cSCC primary tissues. Reactivation of DLC-1 expression was observed after treatment of demethylating agent 5-Aza-dC in cSCC cell lines, confirmed epigenetic inactivation as one major mechanism of DLC1 regulation in cSCC. Moreover, induction of exogenous expression of DLC-1 inhibits cSCC cell proliferation, colony formation, and invasive activities. Conclusion: We have identified a panel of methylated TSGs in primary cSCC tissues that were not reported previously. Early detection of potential aggressive behavior of premalignant skin lesions based on a panel of molecular markers is valuable for minimizing morbidity and mortality of patients. Transcriptional silencing of DLC1 through aberrant methylation may be one of the mechanism in the pathogenesis of cSCC and could have a potential therapeutic application. Commercial support: None identified.

MARCH 2010