Dermatopathology Cytologic Abnormalities (ASC-US/LSIL) with High Resolution Anoscopy (HRA) and Biopsy

110A ANNUAL MEETING ABSTRACTS screening guidelines for anal cancer. Here, we describe our anal cytology experience, hysterectomy. The significantly increased detection rate of VAINs in hrHPV-positive with regards to the patient population, the distribution of cytologic results, hrHPV (high group suggests vaginal cytology and HPV co-testing is preferred for follow-up of risk human papillomavirus) detection rates and the histologic outcomes. women with invasive cervical carcinoma after hysterectomy. Design: A database search was conducted from January 2007 to June 2012 for anal cytologic tests (ThinPrep). Clinical information, the results of concurrent hrHPV testing 451 Non-16/18 HR-HPV Genotypes Are Superior to HPV 16/18 for (Hybrid Capture 2) if available, and histologic follow-up within the next 18 months, if Predicting High-Grade Intraepithelial Lesions in LEEP Resections any, were compiled. Also, the development of invasive squamous cell carcinoma during H Zhou, D Mody, M Schwartz, Y Ge. Methodist Hospital, Houston, TX. the study period was recorded. Statistical analysis was performed using chi-square test Background: HPV 16 and 18 are responsible for the majority of cases of high-grade and Fisher’s exact test and a p value of < 0.05 was considered as statistically significant. squamous intraepithelial lesions (HSIL) and cervical cancer. While HPV 16/18 have Results: 443 patients had anal cytologic tests – average age 44.4 years (range 17-80 been extensively studied, more than a dozen non-16/18 high risk HPV (HR-HPV) years), 74% HIV positive (318 men, 4 women), 26% HIV negative (56 men, 65 women). genotypes have been overshadowed by the two leading HPV types and have received The distribution of cytologic diagnoses is depicted below. much less attention. With the current vaccines directed against HPV 16/18, non-16/18 HR-HPV genotypes are expected to play an increasing role in cervical cancer screening and prevention. Therefore, evaluating the predictive value of non-16/18 type HR-HPV is pertinent for early detection and cancer prevention, as well as for future vaccine development. Design: A cohort of 808 SurePath specimens were collected from women who were referred to our institution from 12/2009-4/2011 for abnormal Pap tests. HPV genotypes were determined by DNA microarray against 40 HPV subtypes followed by a confirmatory sequencing assay. Forty-three patients from the cohort had a concurrent or subsequent LEEP procedure. Correlations among HPV genotype, cytologic findings and LEEP findings were analyzed. Results: The HR-HPV infection rate was 81.4% in the 43 patients who underwent a LEEP and 94.4% in patients with HSIL on Pap tests. Cytologic interpretation of HSIL had 88.9% positive predictive value (PPV) for any grade of dysplasia and 83.3% for CIN 2 and above lesions in LEEPs. The PPV of non-16/18 HR-HPV for any grade of dysplasia in LEEPs was 93.3%, which was significantly better than that of HPV 16/18 (65%, p=0.049). For patients with CIN 2 and above lesions in LEEPs, the PPV of non- 16/18 HR-HPV (73.3%) was also better than that of HPV 16/18 alone (58.3%). HPV 31/52/58/39/45 genotypes were commonly detected in women with HSIL in LEEP specimens. Furthermore, all patients with HPV31/52/58 infection documented on Pap Histologic follow-up was available in 173 (39%) patients. High-grade AIN or above was test had HSIL in LEEP (100% positive predictive value). diagnosed in 30.2% ASC-US, 47.6% LSIL, 77.8% HSIL and 72.2% ASC-H/LSIL-H Conclusions: The predictive value of non-16/18 HR-HPV genotypes for HSIL in LEEPs cases. The difference between the detection rates for high-grade AIN or above in the was superior to that of the HPV 16/18 test. The HPV16/18-only test may be not having following groups was not statistically significant – ASC-US vs. LSIL, HSIL vs. ASC-H/ as a high value as perceived, and a negative result may give a false assurance, especially LSIL-H and HIV positive vs. HIV negative patients. 76% ASC-US cases were hrHPV in high risk populations. In addition to HPV 16/18, an expanded test panel of HR-HPV positive, of those that were tested. Invasive squamous cell carcinoma was diagnosed in genotypes to include some of the non-16/18 genotypes with high PPV (such as HPV 6 patients – 5 on immediate follow-up and one 33 months following an initial biopsy 31/52/58) may be warranted. At the present time, testing with a cocktail of HR-HPV with AIN2, 3 were HIV positive, cytologic diagnoses included NILM (1), LSIL (2), types appears to offer a better predictive value for HSIL than using an HPV 16/18 test. LSIL-H (2) and HSIL (1). Conclusions: Our study reveals a high rate of detection of high grade AIN following an abnormal cytology diagnosis and supports the management of patients with lesser Dermatopathology cytologic abnormalities (ASC-US/LSIL) with high resolution anoscopy (HRA) and biopsy. Given the high hrHPV positivity rate in the ASC-US group, hrHPV testing may 452 The Impact on Final Breslow Thickness and Sentinel Lymph not play a significant role in the triage of these patients to HRA. Node Status with Initial Biopsies of Cutaneous Melanomas Transected at the Base 450 Vaginal Cytology and HPV Co-Testing Is Preferred for Follow-Up A Agarwal, CA Torres-Cabala, MT Tetzlaff, DL Stockman, V Chu, VG Prieto, JL Curry. of Women with Invasive Cervical Cancer Treated by Hysterectomy Baylor College of Medicine, Houston, TX; University of Texas MD Anderson Cancer C Zhao, Z Li, S Barron, W Hong, A Karunamurthy. University of Pittsburgh Medical Center, Houston, TX. Center, Pittsburgh, PA; Conemaugh Memorial Medical Center, Johnstown, PA. Background: The histologic evaluation of primary tumor characteristics (Breslow Background: New cervical cancer screening guidelines indicate that women who have thickness (BT), mitotic rate (MR), ulceration) in cutaneous melanoma is critical undergone hysterectomy and no history of cervical intraepithelial neoplasia (CIN) 2+ for staging and classification, prognosis and clinical management decisions. BT is should not be screened for vaginal cancer. Women who have had a hysterectomy for a strong and consistent histologic parameter of sentinel lymph node (SLN) status. invasive cervical cancer/CIN2+ may be at an increased risk of vaginal cancer, but data Melanomas that are superficially sampled with tumor cells present at the deep tissue are very limited. edge may underestimate true BT, pose a risk for positive SLNB, and provide inaccurate Design: A computer-based search of CoPath files was carried out to retrieve cases prognostic information. We report the impact of positive deep margins on initial biopsies with invasive cervical carcinoma treated by hysterectomy with histopathologic and/ of cutaneous melanoma with respect to final BT on wide local excision (WLE) and or cytologic follow-up results during a study period of 10 years. Surgical pathology outcome of SLNB. reports, follow-up hrHPV testing, cytologic, and histopathologic results were recorded. Design: An eighteen month retrospective review was performed in patients diagnosed Results: 147 patients with invasive cervical carcinoma [76 squamous cell carcinoma with primary cutaneous melanoma who undergone WLE and SLNB. Patients were (SqCC), 60 adenocarcinoma (ADC) and 11 adenosquamous carcinoma] treated by categorized into four groups according to status of deep margin on initial biopsy hysterectomy and follow-up results were identified. The average age of these patients and outcome of SLNB. G1=absence of positive deep margin and negative SLNB, was 43 years (range: 29-72) at the time of diagnosis. The average follow-up period G2=absence of positive deep margin and positive SLNB, G3=positive deep margin was 43.3 months (range: 3-139). Two cases (1.4%) of recurrent/residual SqCC were and negative SLNB, G4=positive deep margin and positive SLNB. Comparisons detected in vagina/vulva during follow-up, 1 and 11 months after the hysterectomy, between groups were made with Kruskal Wallis test. Statistical significance was respectively. 20 patients (13.6%) developed vaginal intraepithelial neoplasias (VAINs) considered at p <0.05. during follow-up (table 1). The average interval between hysterectomy and initial Results: 171 patients fulfilled the criteria and were distributed into the four categories diagnosis of VAIN2/3/HSIL (8 cases) was 8.6 months (range: 1-24). 47 women had as follows: G1 (70), G2 (31) G3 (51), G4 (19). Groups with positive SLNB (G2 & G4) hrHPV testing during follow-up and 29.7 % (11/47) had at least one positive hrHPV had greater final BT than patients with negative SLNB (G1 & G3) regardless of the testing. Importantly, VIN was detected in 54.5% (6/11) of patients with hrHPV-positive status of deep tissue edge on initial skin biopsy. The final BT in patients in G4 patients result compared to 16.7% (6/36) of patients with negative hrHPV-negative result. was significantly greater compared to the final BT in the other groups. G4 patients also Vaginal cytologic and histologic follow-up results in 147 patients with invasive cervical had greater tumor deposit size in SLNB compared to G2 patients. carcinoma treated by hysterectomy. Table 1 Case# Carcinoma VIN2/3/HSIL VIN1/LSIL Neg/ASC G1 G2 G3 g Histology 34 2 7 9 16 CL median (range) 4 (2-5) 4 (4-5) 4 (2-5) 4 (3-5) Cytology only 113 0 1 3 109 Initial BT mm* (range) 2.4(0.4-17) 3.12(0.54-15) 2.19(0.20-9.7) 2.86(0.57-5.0) Total 147 2 8 12 125 Final BT mm* (range) 2.55(0.44-17) 3.18(0.54-15) 2.60(0.20-9.7) 3.38(1.5-5.0) Legend: VIN: vaginal intraepithelial neoplasia; H/LSIL: high/low grade intraepithelial lesion; Neg: Tumor deposit size (mm)* in NA 1.63(0.1-5.3) NA 2.77(0.1-15) negative; ASC: atypical squamous cells. SLN* (range) Conclusions: VAIN1+ lesions were identified in 15.0% (22/147) patients with * Average values, p <0.05 invasive cervical cancer treated by hysterectomy, indicating these women were at an Conclusions: Initial biopsies of melanoma with positive deep margins demonstrate risk increased risk of vaginal lesion. All VAIN2+ lesions were identified within 2 years after for greater final BT and increase tumor burden in SLNB. Appropriate initial sampling of melanoma is important in staging since superficially sampled tumors may limit accurate evaluation of histologic parameters which may have significant prognostic implications. ANNUAL MEETING ABSTRACTS 111A 453 Do Cutaneous Leiomyomas Have Similar Morphological and Design: We studied 96 metastatic melanoma patients with AJCC stage IIIc or IV, with Molecular Alterations to Uterine Tumors in the Hereditary Leiomyomatosis a total of 221 samples, including primary melanoma(n=86) and different metastatic and Renal Cell Cancer Syndrome (HLRCC)? localization(n=135).BRAF mutation testing was performed on sections from FFPE LY Ballester, PP Aung, K Lara-Otero, WM Linehan, MJ Merino. NIH/NCI, Bethesda, material using two techniques: HRM analysis followed by Sanger sequencing of variant MD. profiles and anti-BRAFV600E immunostaining. Background: HLRCC is an autosomal dominant syndrome characterized by the Results: As reported by others, IHC was positive in all samples harboring V600E development of cutaneous and uterine leiomyomas (CL) and renal cell carcinoma (RCC). and V600E2 mutations and negative for all other mutations, including V600K(n=3, It has been associated to mutations in the fumarate hydratase gene, in chromosome 1q42- L597S(n=2), K601E(n=3) and pD594N(n=2). The cytoplasmic staining was either 1q43. Loss of heterozigosity (LOH) in this region is evident in the RCC that develop in strongly positive in most tumour cells of V600E and V600E2 mutated cases or these patients. Uterine leiomyomas are frequently found in females with HLRCC, the completely negative. It appeared strong brown with small dots. IHC staining and/or tumors show LOH in 1q42-1q43 and have a histological appearance distinct from the HRM analysis showed a perfect concordance between matched primary melanoma and sporadic leiomyomas (i.e. higher cellularity, eosinophilic nuclei, prominent nucleoli metastasis.The prevalence of each BRAF mutation was V600E(40.6%), V600E2(3.1%), with perinuclear halos and mitosis). CL have been observed in 76% of families with V600K(1.04%) and others(1.04%) respectively. Concordance between the two HLRCC, presenting as firm skin-coloured to light brown-coloured papules and nodules techniques was 99,5%. Sensitivity for IHC taking into account all V600BRAF mutations predominantly in the chest and arms of the patients. was 97% while specificity was 100%. Design: To describe the morphology of CL in HLRCC and assess if they differ from Conclusions: This study confirms the performance of IHC for BRAFV600E and their sporadic counterparts and if they share morphological and molecular characteristics V600E2 mutation detection using the VE1 antibody positivity. It provide evidence that with uterine leiomyomas. We examined the histological features (circumscription, VE1 IHC may be a cost-effective method of BRAFV600 status assessment, although atypia, mitosis, necrosis, size, nuclear morphology, MIB1 labeling, presence of some cases harbouring the V600K mutation were missed in patients who may benefit inflammation) of 100 CL of patients with HLRCC syndrome. Tumors and normal tissues from Vemurafenib. Unlike previous reports, IHC was not found more sensitive for were microdissected and DNA extracted for LOH analysis of the 1q42-1q43 region. BRAFV600E mutation detection than its molecular detection as the two techniques Results: The mean age of the patients was 45.2 years with 66% of the patients being matched perfectly. Moreover, using IHC we report for the first time the concordance female and 34% males. The most common tumor locations were the arm/forearm between BRAF status at primary and metastatic sites that together with the homogeneous (51%), back (30%), chest (11%) and other (4%). The tumors were well-circumscribed staining observed by IHC would suggest tumour homogeneity rather than heterogeneity and localized to the dermis with a Grenz zone. The mean tumor size was 0.34 ± 0.01 at least for this primary genetic alteration. cm. The tumors contained interlacing fascicles of smooth muscle cells with elongated nuclei and abundant eosinophilic cytoplasm. None of the lesions showed the atypical 456 T-Cell Subsets and Plasmacytoid Dendritic Cells in Rosacea features present in uterine leiomyomas. LOH analysis showed loss of heterozigosity and Cutaneous Lupus Erythematosus: A Comparative Immunophenotypic for the 1q42-1q43 region in several of the tumors, similar to the uterine leiomyomas. Analysis Conclusions: Our findings suggest that in HLRCC patients, the CL have the so called T Brown, Jr., K Choi, D Thomas, A Hristov, M Chan. University of Michigan Medical “second hit” present in renal tumors and uterine leiomyomas, as evidenced by LOH for Center, Ann Arbor, MI. 1q42-1q43. However, even when cutaneous leiomyomas share the similar LOH present Background: Distinction of rosacea and cutaneous lupus erythematosus (LE) can be in uterine leiomyomas, they do not show the characteristic atypical morphologic features challenging due to clinical and histologic overlaps. Both conditions may present as of uterine tumors. These findings suggest that other alterations, besides LOH for region facial edema or inflammatory papules, and typically demonstrate perifollicular and 1q may play an important role in the development of leiomyomas in HLRCC patients. perivascular T cell-rich infiltrates histologically. Increased CD8+ cytotoxic T cells, decreased CD4+FoxP3+ regulatory T cells (Tregs), and increased CD123+ plasmacytoid 454 Morphogenesis and Fibrogenesis Pathways for the Distinction dendritic cells (PDCs) have been reported in the skin lesions of LE. Similar data of Slerosing Melanocytic Nevus and Desmoplastic Malignant Melanoma are lacking in rosacea. We hereby explore the utility of immunohistochemistry in A Blanes, L Pozo, SJ Diaz-Cano. University of Malaga School of Medicine, Malaga, differentiating rosacea and LE by comparing their T-cell subsets and the number of Spain; Homerton University Hospital, London, United Kingdom; King’s College PDCs. Hospital, London, United Kingdom. Design: The surgical pathology archive at University of Michigan was searched for Background: Fibroblastic reaction can be observed in benign and malignant melanocytic cases of rosacea and facial LE. CD4, CD8, CD25, and CD123 immunostains were lesions and cause serious diagnostic problems. The mechanism of fibrogenesis and the performed on all cases. The CD4:CD8 ratio in each case was estimated by counting diagnostic utility of fibrogenesis markers have not been investigated in this context. positive cells on digital images taken of at least one representative lymphoid aggregate. Design: We analyzed symmetry, maturation, growth patterns (nested-trabecular, Using the same method, the percentage of CD4+CD25+ regulatory T cells (Tregs) nodular-solid, diffuse), nuclear grade (including chromatin, nucleolus, pleomorphism was determined by dividing the number of CD25+ cells by the number of CD4+ cells and anisokaryosis), stromal reaction, and confluent necrosis in desmoplastic (sclerosing) (CD25/CD4). The percentage of CD123+ PDCs of the total infiltrate was estimated in melanocytic nevi (DMN, 18) and desmoplastic malignant melanoma (DMM, 24), a semi-quantitative manner (0 to 100% in 10% increments). Any clustering of more classified according to the WHO criteria. Cases with history of previous excision, than twenty CD123+ cells was also noted. All data were obtained by multiple authors trauma or histological scarring were excluded. Representative samples were evaluated by blinded to the final diagnoses. quantitative RT-PCR and standard in situ techniques for morphogenesis and fibrogenesis Results: Twenty-seven cases of rosacea and 30 cases of facial LE were selected after pathways (fibrinogen, CD34, CTNNB1, FOXC2, JAG1, RAC1, SMAD2, SNAI1, careful clinicopathologic review. The mean CD4:CD8 ratio is 2.80 in rosacea and SOX10, TGFB1, TGFB2, TGFB3, TWIST1, WNT11, WNT5A, PDGFA, SPARC, 1.74 in LE (p=0.0272). The proportion of CD25+/CD4+ Tregs is 31% in rosacea and PDGFRA, HSPG2). Appropriate controls were run. Fisher’s exact tests and analysis 13% in LE (p<0.0001). The percentage of CD123+ PDCs is 6% in rosacea and 18% of variance (significant if P<0.05) were used for comparison; significant variables were in LE (p=0.0008). Clustering of CD123+ cells is seen in 18% of rosacea, and in 60% then selected for discriminant analysis with cross-validation for diagnostic groups of LE (p=0.0026). (DMN vs. DMM). Conclusions: Cases of facial LE show significantly lower CD4:CD8 ratio, fewer CD25+ Results: Fibrinogen expression was found in both DMN and DMM but with different Tregs, and more CD123+ PDCs than rosacea. These findings support the implication of pattern: perivascular stroma in DMN and in tumour cells and blood vessels with a T cell-mediated cytotoxicity, decreased immune suppression by Tregs, and increased heterogeneous pattern in MM. CD34 was revealed positive in dermal dendritic cells of interferon-producing PDCs in the pathogenesis of LE. We propose that these findings the sclerosed interstitium of DMN, and in the peritumoural stroma of DMM. PDGFRA may serve as useful adjunctive tools in distinguishing rosacea and LE in difficult cases. was expressed in DMN, but not in DMM and HSPG2 (perlecan protein) revealed significantly higher expression in DMM. The remaining markers revealed no statistically 457 Ossifying Fibromyxoid Tumor and Its Non-Ossifying Variant: A significant differences in the expression by DMN and DMM. Diagnostic Challenge for Dermatopathologists Conclusions: The sclerosis seen in DMN is directly correlated with fibrinogen leaking D Buehler, N Atanaskova Mesinkovska, CM McClain, BP Rubin, J Goldblum, SD from blood vessels and it is not a destructive process (preserved intratumour CD34 Billings. Cleveland Clinic, Cleveland, OH; Vanderbilt University, Nashville, TN. positive cells), whereas the fibrogenesis in MM is secondary to tissue destruction (loss Background: Ossifying fibromyxoid tumor (OFMT) is a rare, potentially of intratumour CD34 positive cells and increased turnover of HSPG2) and is enhanced aggressive, tumor that typically arises in the subcutis. Therefore it is encountered by PDGFRA-independent tumour cell expression of fibrinogen. by dermatopathologists and is a source of diagnostic difficulty. In this study, the clinicopathologic features and outcomes of twenty-three subcutaneous OFMT were 455 BRAF V600 Mutation Detection by Immunohistochemistry reviewed. Shows Tumor Homogeneity between Primary and Metastatic Sites Design: Histologic sections of subcutaneous OFMTs were assessed for the morphologic L Boursault, V Haddad, T Jouary, B Vergier, S Verdon, A de Mascarel, JP Merlio. CHU patterns. Prognostic classification as typical, atypical and malignant OFMT was et Université, Bordeaux, France, Metropolitan. assigned. Patient and tumor characteristics and follow up information were obtained Background: Metastatic melanoma is associated with a poor prognosis. First line from institutional records and contributing pathologists. therapy using the BRAFV600E kinase inhibitor increase overal survival of patients Results: Patients (15 F, 8 M) ranged in age from 26 to 88 years (mean 53 years). The with metastatic melanoma and BRAFV600 mutation. Thus, rapid and accurate detection tumors ranged from 0.8 to 8.5 cm (mean 2.7 cm) in size, and all arose in the subcutis of of BRAFV600 mutation is critical in such patients. However, intra or inter-tumor the leg (n=11), trunk (n=7), head/neck (n=3), or arm (n=2). All were well circumscribed heterogeneity may account for different rates of BRAF mutation detection among with a fibrous pseudocapsule. A partial shell of bone was present in 11 of 23 cases while differents sites. We have evaluated the potential use of immunohistochemistry(IHC) for 12 cases were classified as the nonossifying variant. All had a lobular growth pattern detecting BRAFV600 mutations by comparison with molecular detection in a parallel with the tumor cells arranged in combinations of nested, reticular and fascicular patterns. blinded study. Moreover, we also evaluated whether intra and inter tumor heterogeneity Cellularity ranged from low (n=13) to moderate (n=6) to high (n=4). Mild to moderate may account for discrepancies between primary and metastatic material. cytologic atypia was observed in most cases with more cellular cases demonstrating greater nuclear atypia. The average mitotic rate was 6 per 50 HPFs (range 0-46). 13 of 112A ANNUAL MEETING ABSTRACTS 19 cases were positive for S100; focal desmin (4/12) and actin (2/14) immunoreactivity Conclusions: Whole exome sequencing from archival FFPE tissue demonstrated no- was also observed. One case co-expressed focal cytokeratin and CD10, and one case significant difference in the overall number of mutations in MCPyV-positive versus was positive for p63. Two cases met histologic criteria for malignant OFMT, having -negative MCC, however we identified several gene mutations that appeared specific at least two of the following three features: increased cellularity, nuclear atypia and to MCPyV-positive or -negative MCC. In cases with known MCPyV integration sites, mitotic rate >2/50 HPFs, and two other cases were considered atypical. The spectrum we saw no evidence of coding region insertional mutagenesis. Our sequencing results of cytologic features was similar in both ossifying and non-ossifying variants. Clinical identify several mutations in genes not previously associated with MCC that may play follow up was collected for 15/18 patients diagnosed before 2012 (median 48 mo, range a role in disease development. Overall, these findings expand our understanding of 8 to 108 mo). All patients with typical OFMT and two patients with atypical OFMT frequently mutated human genes in MCC. were alive and free of disease. Recurrence occurred in both patients with malignant OFMT, and one patient developed metastases to the lungs. No deaths were recorded. 460 Factor XIIIa Is a Highly Sensitive Nuclear Marker of Sebaceous Conclusions: These results further support criteria for the diagnosis of malignant Differentiation in Normal and Neoplastic Sebocytes OFMT. Dermatopathologists should be aware of this unusual tumor given its superficial LN Clark, HR Elwood, SC Shalin, BR Smoller, JM Gardner. University of Arkansas for presentation and potential for aggressive behavior. Careful attention to the morphologic Medical Sciences, Little Rock, AR. features should allow an accurate diagnosis in most cases. Background: Sebaceous proliferations are commonly encountered cutaneous lesions. Some of these, especially sebaceous carcinoma, can be easily confused with other 458 Why Do Melanomas Ulcerate? A Re-Evaluation after Matching cutaneous neoplasms, particularly poorly differentiated squamous cell carcinoma. for Thickness Few useful or specific immunohistochemical markers for sebaceous differentiation E Castro-Echeverry, K Walker, RT Zreik, A Rao, MP Fernandez. Scott and White are available. We incidentally observed strong nuclear expression in sebaceous glands Memorial Hospital, Temple, TX. by Factor XIIIa, and we hypothesized that this antibody may be a useful marker in Background: Ulceration has long been recognized as an indicator of poor prognosis sebaceous proliferations including sebaceous carcinoma. in melanoma. Multiple morphologic characteristics have been proposed as predictors Design: Our archive files were searched for sebaceous proliferations including of ulceration; however, thickness is a persistent confounder in previous studies. In sebaceous hyperplasia, sebaceous adenoma, sebaceoma and sebaceous carcinoma. the following study, we matched ulcerated melanomas and non-ulcerated melanomas Immunohistochemistry for Factor XIIIa was performed according to the manufacturer’s by Breslow thickness to determine whether any previously described morphologic guidelines. All cases were reviewed by four separate pathologists for accuracy of features provide insight into ulceration. Additionally, we performed extracellular diagnosis, and the presence and intensity of staining. Immunostaining distribution matrix gene expression array analysis in order to describe the extracellular conditions was scored as diffuse, focal or absent nuclear positivity while intensity was scored as that promote ulceration. strongly positive, weakly positive or negative. Normal sebaceous glands and/or dermal Design: Cases of ulcerated melanomas were selected from a prospective cohort of dendritic cells in each case were used as controls. patients diagnosed with melanoma between 1990 and 2000 in central Texas with a Results: Our series included 27 representative cases of sebaceous proliferation as median follow up of 130 months. They were matched by Breslow thickness to non- follows: 7 sebaceous hyperplasias, 8 sebaceous adenomas, 5 sebaceomas, 7 sebaceous ulcerated melanomas from the same cohort. Where possible, they were also matched by carcinomas. Factor XIIIa expression was present in all normal background sebaceous tumor location and age. Slides were reviewed and evaluated for mitotic rate, vascularity, glands and in 100% of sebaceous proliferations with the following scores: sebaceous tumor infiltrating lymphocytes (TIL), regression, solar elastosis, and necrosis. In a subset hyperplasia – 100% strong/diffuse; sebaceous adenoma – 100% strong/diffuse; of recently diagnosed non-ulcerated and ulcerated melanoma, cDNA was extracted from sebaceoma – 60% strong/diffuse, 20% strong/focal, 20% weak/focal; sebaceous micro-dissected tissue and analyzed with the RT^2 Profiler PCR ARRAY (Rotor-Gene carcinoma – 71% strong/diffuse, 29% strong/focal. Additionally, distribution and Format) Human EMT kit. Univariate and multivariate statistical analysis was performed intensity of expression seemed to directly correlate with increased maturation of using SAS 9.1 and SPSS 20. the sebocytes; thus, basaloid cells were typically negative and mature sebocytes Results: Of 401 patients diagnosed with melanoma between 1990 and 2000, 9.7% (39) were strongly/diffusely positive for Factor XIIIa. A small number (n=6) of poorly were ulcerated. In this cohort both ulceration and Breslow thickness were found to be differentiated squamous cell carcinomas were also stained for Factor XIIIa; all showed the strongest independent histologic predictors of disease free survival and overall either negative or weak/focal nuclear expression. survival (p<0.05 by Cox PH). 54 cases were reviewed, 34 with ulcerated and 20 with Conclusions: We report the novel finding of consistent nuclear expression of Factor non-ulcerated melanoma with 14 matched pairs. None of the morphologic features XIIIa in normal, hyperplastic, and neoplastic sebocytes. From our pilot series, Factor mentioned were found to be associated with ulceration, including mitotic rate and XIIIa appears to be a highly sensitive marker of sebaceous differentiation. Our vascularity (p>0.10 by Chi square and McNemar’s test for matched pairs). The PCR preliminary data examining a small number of squamous cell carcinomas suggests gene expression array showed ulceration to be associated with an upregulation in MMP3 Factor XIIIa may have clinical utility as a specific marker to distinguish sebaceous and with a downregulation in CTNNB1. carcinoma from poorly differentiated squamous cell carcinoma. Conclusions: In this matched pair, nested case control study, no morphologic characteristics other than thickness were consistently associated with ulceration. While 461 Spitz Nevi Frequently Show DNA Copy Number Abnormalities ulceration is associated with thickness and, by extension, mitotic rate, this fails to by FISH Using Probes for RREB1 (6p25), MYB (6q23), CEP6 (6 Centromere), explain why some thin melanomas ulcerate and have a poor prognosis while some thick and CCND1 (11q13) melanomas fail to ulcerate. Results from our gene expression array suggest ulceration KJ Craddock, Z Hindi, C Shiau, A Al-Habeeb, D Ghazarian. University Health Network, is the end result of persistent disruption of adherens junction proteins (CTNNB1) and Toronto, ON, Canada. increased breakdown of extracellular matrix proteins (MMP3). Background: Malignant melanoma can be difficult to distinguish from a benign melanocytic lesion by histology. A fluorescence in situ hybridization (FISH) probe set 459 Whole Exome Sequence Analysis of Merkel Cell Carcinoma targeting abnormalities commonly seen in malignant melanoma but not found in benign PJ Cimino, Jr., EJ Duncavage. Washington University School of Medicine, St. Louis, nevi has shown encouraging results in previous studies. However, there is a paucity of MO. data describing the findings of this probe set in Spitz nevi, a benign melanocytic lesion Background: Merkel cell carcinoma (MCC) is a rare neuroendocrine tumor with an that can be difficult to distinguish from melanoma. aggressive clinical course that harbors integrated Merkel Cell Polyomavirus (MCPyV) in Design: Multicolour FISH was performed using a commercially available probeset up to 80% of cases. While mutations in the integrated MCPyV large T antigen sequence (Abbott Laboratories, Abbott Park, IL), on formalin-fixed, paraffin-embedded tissue have been well-documented, little is known about human genomic mutations in MCC, samples from 20 Spitz nevi, diagnosed by histology, all lacking atypical features. beyond Rb1, TP53, and PIK3CA. We sought to determine the human genomic landscape Fluorescent signals for each probe were enumerated by 2 observers in 30 cells each of MCC by exome sequencing. per lesion. An algorithm using signal counts from a combination of 4 probes targeting Design: From a set of 8 previously characterized MCC cases, exome sequencing was chromosome 6p25 (containing RREB1 gene), 6 centromere (CEP6), 6q23 (containing performed using Agilent V4 reagents and HiSeq 2000 paired-end sequencing. DNA MYB gene), and 11q13 (containing CCND1 gene) was used as suggested by the from 6 MCPyV-positive and 2 MCPyV-negative cases was extracted from formalin- manufacturer. The following criteria were used: (1) the average CCND1 or MYB fixed paraffin-embedded (FFPE) blocks. Data was mapped to hg19 reference and all signals per nuclei is greater than or equal to 2.5 or (2) percent loss of MYB relative known SNPs and non-coding variants removed from the files. to CEP6 is greater than or equal to 31% or (3) the percentage of abnormal nuclei for Results: The average number of reads generated per case was 130,477,404 (26GBases). RREB1 is greater than or equal to 63%. If at least one of the three criteria were met, the On average, 94.1% of the exome for each case had ≥ 25x coverage. There were 1,514 specimen was designated FISH positive. If none of the criteria were met, the specimen genes identified with non-SNP mutations found in at least 1/8 cases; 9 genes mutated was designated as FISH negative. (all 8/8 cases); 4 genes (7/8 cases); 9 genes (6/8 cases); 21 genes (5/8 cases); 75 genes Results: FISH results were obtained for all 20 Spitz nevi. Seven (35%) tested positive: (4/8 cases); 232 genes (3/8 cases); 902 genes (2/8 cases); and 262 genes (1/8 cases). 3 showed copy number gain at CCND1, 3 showed gain at MYB (2 showed gain at both Some of the most frequently mutated genes (in at least 7/8 cases) with predicted CCND1 and MYB), and 3 others had loss of MYB. protein damaging mutations include UGT2B15, KLF14, ACVR2A, and CCNE1. Few Conclusions: In contrast to usual benign melanocytic nevi, we found that abnormal cases showed mutations in Rb1 (3/8), TP53 (2/8), and none in PIK3CA. There was no copy number changes at CCND1 and MYB are not uncommonly seen in Spitz nevi; significant difference in the average numbers of mutations between MCPyV-positive and therefore their detection does not aid in the distinction between benign Spitz nevi and -negative cases. There were 15 genes with non-SNP SNVs identified in only the MCPyV malignant melanoma. However, we note a difference in RREB1 findings between Spitz negative cases (2/2) and 575 non-SNP SNVs present in only the MCPyV-positive cases and melanoma. While we did detect copy number gains at RREB1 in a subset of Spitz (at least 2/6). The most frequently mutated gene unique to the MCPyV-positive group cases, none reached the relatively high threshold required for an abnormal result by was PLEC, found in 6/6 MCPyV-positive cases. In 4/6 MCPyV-positive cases we had the manufacturers algorithm; in contrast, we have previously reported RREB1 to be previously determined the MCPyV integration site, and saw no evidence of human significantly gained in 65 % of melanomas. If this FISH probe set is to be used to aid genomic DNA mutation flanking these sites. in the diagnosis of atypical melanocytic lesions, one must be aware that abnormalities may be detected in Spitz nevi, and that the pattern of abnormalities may differ. ANNUAL MEETING ABSTRACTS 113A 462 Clinicopathologic Features of Cutaneous Perineurioma and melanoma triple stain are utilized to exclude metastatic disease. Individual IHC positive Perineurial Cell-Rich Tumors cells are challenging, warranting a clinically significant distinction between isolated MD Cykowski, I Shendrik, CM Magro, JV Pitha, AN Crowson. University of Oklahoma tumor cells and benign nevus cell rests (NCR), as even isolated tumor cells represent Health Sciences Center, Oklahoma City, OK; Regional Medical Laboratories, Tulsa, N1 disease. In this study, the incidence and growth pattern of NCR in lymph nodes from OK; Weill Cornell Medical College, New York City, NY. various anatomic locations are described to form a basis of comparison for isolated IHC Background: Perineuriomas are uncommon, histologically heterogeneous tumors positive cells in sentinel lymph nodes removed for melanoma. with morphologic and immunohistochemical features that recapitulate the normal Design: Triple stain (HMB-45, MART-1, and tyrosinase combination) was performed perineurium. Cutaneous perineuriomas (CPs) involving the dermis and superficial on nodes from axillary (113), inguinal (44), cervical (184), and intraparotid (18) areas subcutis are particularly rare and may be under-recognized. This study aimed to removed for non-melanoma related conditions to simulate regions typically sampled characterize further the clinicopathologic features of CP and associated perineurial during melanoma sentinel node procedures. Additionally, thirteen cases with incidental cell-rich tumors. NCR documented in the diagnosis field were retrieved from the Department of Pathology Design: Eleven cases were retrieved from the pathology archives at three institutions. Anatomic Pathology Information System. Location, size, and histomorphology of Clinical features and routine histology were reviewed. Immunohistochemical stains NCR were recorded. performed on all cases included S-100, EMA, Actin, CD34, Glut-1, CK7, INI-1, and Results: Triple stain positive NCR were identified in 2 of 44 (4.5%) inguinal nodes (1.5 Collagen IV. Additional stains (e.g., claudin-1, factor XIIIa) were available for select mm and 2.0 mm), 1 of 184 (0.5%) cervical nodes (0.5 mm), 1 of 113 (0.9%) axillary cases. nodes (0.2 mm), and 0 of 18 intraparotid nodes. NCR in these cases had an intracapsular Results: Seven CPs (4 soft tissue and 3 sclerosing variants), 2 hybrid schwannoma/ location, a spindle cell morphology mimicking fibroblasts, and were not perceptible on perineuriomas, and 2 perineurial cell-rich neurofibromas were identified. Patients routine stains. NCR documented in the diagnosis field in 13 additional cases had 16 included 7 males and 4 females (median age of 48 years; range of 11 to 65 years). All total NCR with a combined epithelioid and spindle morphology. Thirteen NCR were but one tumor occurred in the extremities. Clinical impressions ranged from digital intracapsular and 3 were subcapsular with a size range of 0.5 – 9.0 mm. fibroma to verruca, epidermal cyst, ganglion cyst and fibrosis. Morphologic features Conclusions: Incidence of NCR is higher in the inguinal location relative to other were heterogeneous ranging from dermal-based, well-demarcated whorled and nested anatomic locations studied. Rests identified only by IHC in our non-melanoma lymph nodules with rare mitoses to larger tumors with a diffuse growth pattern and storiform or node study cases have a spindle cell fibroblastic morphology. This finding in IHC whorled arrangement of spindle cells. Several dermal tumors featured dilated lymphatics stains in melanoma sentinel nodes can create diagnostic confusion with metastatic in the superficial dermis as well as a transition between thin, elongated, EMA-positive melanoma, emphasizing the importance of recognizing this morphologic pattern in perineurial cells and epithelioid cells at the tumor periphery. All CPs were positive NCR. Rests occurred as cell clusters measuring at least 0.2 mm and had a predominant for EMA and Glut-1; 64% were positive for CD34, which highlighted elongated cell intracapsular location, with only three having a subcapsular location. An isolated processes. Claudin-1 and Factor XIIIa were also positive in select dermal-based tumors. cell pattern was not observed in any of our study cases. Our results suggest that an S-100 was negative in pure perineurial tumors and the INI-1 staining pattern was normal. isolated cell pattern identified in melanoma-specific IHC stains should be interpreted Additional stains were largely noncontributory. as metastatic melanoma. Conclusions: CP is a very rare neoplasm with an extensive differential diagnosis that arises in the extremities of predominantly middle-aged adults. The lesion may be 465 Differential Activation of the Mammalian Target of Rapamycin difficult to recognize as being of perineurial origin, both clinically and histologically. (mTOR) Pathway in Poromas and Porocarcinomas We determined that dilated lymphatic channels in the superficial dermis and a peripheral CT Elkins, OH Iwenofu, T Olencki, SB Peters. Ohio State University Medical Center, transition into epithelioid cells from flattened perineurial-type cells may be helpful Columbus, OH. diagnostic clues. For confirmation, Glut-1, CD34, and claudin-1 may be useful for Background: Adnexal carcinomas are rare and treatment options are limited. Recent S-100 negative, EMA-positive dermal tumors with an epithelioid cytology. studies have identified a PIK3CA (c.1633 G>A) mutation in a subset of metastasizing porocarcinomas and have proposed a role for phosphatidiylinositol 3-kinase (PI3K) 463 Marginal Zone Lymphoma of Eyelids: A Clinicopathological pathway inhibitors as treatment for these tumors. The mammalian target of rapamycin Entity. Histopathological and Molecular Analysis of 7 Cases (mTOR), a serine/threonine kinase, represents a key mitogenic output of PI3K A de Mascarel, M Parrens, M Beylot-Barry, O Fitoussi, F Bauduer, S Marouan, S pathyway activation and is an additional potential therapeutic target. Using antibodies Rammeh, B de Barbeyrac, JP Merlio, B Vergier. University Hospital, Bordeaux, France, to PI3K, p-mTOR, p70S6K (an intermediate molecule) and pS6rp (an mTOR surrogate Metropolitan; Hospital of Bayonne, Bayonne, France, Metropolitan; University Hospital marker), we sought to determine the role of the PI3K/mTOR pathway in 6 poromas of Ibn Rochd, Casablanca, Morocco; Charles Nicolle Hospital, Tunis, Tunisia. and 7 porocarcinomas. Background: The MALT lymphomas are the most frequent lymphomas of the ocular Design: An electronic database search for cases of poroma and porocarcinoma yielded 6 adnexa. These lymphomas are localized above all in the conjunctiva but also in the and 7 cases, respectively. Slides were reviewed and sections were stained with previously eyelids. optimized antibodies to PI3K, p-mTOR, p70S6K and pS6rp. Staining was evaluated by Design: 29 ocular MALT lymphomas (OML) are recorded between 2000 and 2011. Out two pathologists. Moderate to intense staining in ≥30% of tumor cells was interpreted of these 29 cases, 7 were located in the eyelids. 30 cutaneous marginal zone lymphomas as positive. Sections with only faint staining were interpreted as background/negative. (CMZL) were studied in parallel. Formalin-fixed biopsies were analyzed by HE stain Results: Differential staining was observed between the two groups. The poroma and immunohistochemistry with CD20, CD5, IgA, IgG, IgM. PCR for the detection group was negative for PI3K, mTOR, and pS6rp staining. Only weak, focal staining of IgH gene rearrangement, MYD88 mutation and Chlamydia Psitacci (Cp) DNA was was observed with PI3K (interpreted as negative). Porocarcinomas were positive for performed on DNA extracted from formalin-fixed fragments. FISH was performed on p-mTOR and pS6rp, and 4/7 were positive for PI3K. Though a threshold of 30% or sections using the MALT1 DNA split signal probes. tumor cells was used for positivity, several of the porocarcinomas showed diffuse Results: Out of these 7 eyelid lymphomas (EL), 3 were bilateral and one was unilateral positive staining. Notably, p70S6 demonstrated uniform strong and diffuse positive but in the upper and lower eyelids. The 7 cases clinically presented only an eyelid edema. nuclear staining in both categories. The 30 CMZL never localized to eyelid, showed papules but no edema. In 4 out of 7 Conclusions: Immunohistochemically, porocarcinomas show increased expression EL, the first biopsy was negative as it was too superficial, the lymphomatous tumor of p-mTOR and pS6rp compared to poromas. Four of seven porocarcinomas showed being in the subcutis. Usually, in the CMZL group, the lymphoma was found located reactivity for PI3K; these results support the previously published finding of PIK3CA in the superficial dermis. The phenotype of 7 EL, 22 non EL OML and 30 CMZL was mutations in half of the porocarcinomas in one study. However, the downstream CD20+, CD5 -. IgM expression was detected in 4/7 EL, 11/22 OML and 0/30 CMZL. In activation of mTOR and its surrogate marker pS6rp in porocarcinomas (including those the 29 OML, 26 displayed IgH rearrangement. In the 3 bilateral EL, the same clone was without increased PI3K expression) is promising and supports further investigation of observed at both sites. The 30 CMZL were all monoclonal. No MYD88 mutation was the use of mTOR inhibitors for these rare adnexal carcinomas. The nuclear expression found in the 7 EL. FISH analysis did not find any MALT1 gene breakpoint (0/18). In all of p70S6K in both poromas and porocarcinomas may suggest an alternate mechanism 29 AOL, Chlamidia psittacci was not detected by PCR, even in the 3 bilateral tumors. of mTOR pathway activation. In the 7 EL, bone marrow was negative but a similar IgH rearrangement was found in the 2 cases where the peripheral blood was studied. By comparaison, the peripheral 466 Microphthalmia-Associated Transcription Factor (MITF) blood of the 30 CMZL exibited a polyclonal profile. The 3 patients with bilateral EL Promiscuity: Staining Patterns in Fibrohistiocytic Lesions received 4 cycles of rituximab and are currently in complete remission respectively 1, N Farahani, D Frishberg, B Balzer, B Catherine, JM Wu. Cedars-Sinai Medical Center, 3 and 6 years from the diagnosis. Los Angeles, CA. Conclusions: The marginal zone lymphoma of eyelids seems to be a different Background: Microphthalmia-associated transcription factor (MITF) is used as a clinicopathological entity from cutaneous marginal zone lymphomas because of: marker of melanocytic differentiation in challenging cases. MITF has also been noted 1) constant edema often bilateral masking the lymphoma, 2) subcutaneous location to focally label histiocytes, which raises the concern of fibrohistiocytic lesions being requiring a deeper biopsy, compared to the other CMZL, 3) IgM expression which confused with malignant melanoma due to MITF staining. Because the specificity of was not observed in CMZL. MITF in fibrohistiocytic lesions has not been rigorously studied, we explored this topic by investigating the presence of MITF labeling in a wide range of fibrohistiocytic lesions 464 Incidence and Description of Nevus Cell Rests and Their in the skin and superficial soft tissue. Distinction from Isolated Tumor Cells in Sentinel Lymph Nodes Removed Design: Ninety four surgical pathology cases of fibrohistiocytic lesions were reviewed for Melanoma Staging and the diagnoses confirmed. The cases included benign fibrous histiocytoma (BFH, LD Duncan, KL DeSouza, JM Lewis. University of Tennessee Medical Center, 29), angiofibroma (11), fibromatosis (14), keloid (10), atypical fibroxanthoma (AFX, Knoxville, TN. 7), dermal scar (9), spindle cell carcinoma (2), and dermatofibrosarcoma protuberans Background: Sentinel lymph node excision is integral to melanoma surgical staging. (DFSP, 12). Immunohistochemical staining was performed on 4-µm tissue sections Immunohistochemical (IHC) stains such as HMB-45, S-100, MART-1, tyrosinase, and using monoclonal MITF antibody (clone 34CA5) from Novocastra (Yerevan, Armenia) at 1:60 dilution. Stain intensity (1-3+), and percentage of positive cells 0, 1 (1-10%), 2 114A ANNUAL MEETING ABSTRACTS (>10-50%), and 3 (>50%) were each evaluated. Focal positive staining was defined as 469 Cutaneous Mantle Cell Lymphoma: A Clinicopathologic Review having either 2+ or 3+ staining intensity and >10% positive cells. Strong diffuse staining AA Gru, LP Dehner, MY Hurley, L Brodell, M Anadkat, JL Frater. Washington University was defined as having 3+ staining intensity and >50% positive cells. in St Louis, St. Louis, MO; Saint Louis University, St. Louis, MO. Results: Benign fibrous histiocytomas and angiofibromas showed strong diffuse staining Background: Mantle cell lymphoma (MCL) is a moderately aggressive lymphoma in the majority of cases (26/29, 90%; 7/11, 64% respectively). A subset of the AFX, believed to arise from the mantle zone or primary lymphoid follicles. Cutaneous MCL keloid, and fibromatosis cases also showed strong diffuse labeling (2/7, 29%; 1/10, is exceedingly rare, and occurs usually as a dissemination of systemic MCL, and rarely 10%; 1/14, 7% respectively). No cases of spindle cell carcinoma, DSFP, or dermal as a primary cutaneous B-cell lymphoma. To date, only ∼16 cases have been described scar showed strong diffuse staining. Focal positive staining was seen in BFH (28/29, in the literature. We report a series of 6 cases of MCL involving the skin, and provide a 97%), keloid (9/10, 90%), angiofibroma (9/11, 82%), AFX (5/7, 71%), fibromatosis clinicopathologic review of the histologic and clinical characteristics of these patients. (10/14, 71%), dermal scar (6/9, 67%), and DSFP (1/12, 8%). No cases of spindle cell Design: Cases of cutaneous MCL were retrieved from our database after performing a carcinoma showed focal positive labeling. search from 1990 till present, after an internal board review approval. Clinicopathologic Conclusions: MITF immunostain labels a wide variety of soft tissue lesions with BFH, characteristics were reviewed by 2 independent hematopathologists, with emphasis and angiofibroma frequently displaying strong diffuse MITF staining. Even DFSP can on the morphology, immunophenotype, and clinical parameters of this population. occasionally have focal MITF positivity. Because of the promiscuity of MITF labeling, Results: 6 cases were identified. Typically, they occurred in older individuals (mean awareness of its pattern in fibrohistiocytic proliferation can avoid potential pitfalls in age=70) and were more frequent in males (n=5, 83%). Half of them presented in the head the diagnosis of spindle cell lesions. and neck region as a mass, and the remainder in the trunk and extremities as cutaneous nodules. All patients had stage IV disease with bone marrow involvement. In 1 of the 6 467 Reducing Block Sampling in Melanoma Re-Excision Specimens cases (17%) the cutaneous lesions preceded the diagnosis of disseminated disease. In C Fives, CCBB Heffron. Cork University Hospital, Wilton, Cork, Ireland. 2/6 cases (33%) involvement of the skin coincided with initial systemic involvement Background: Wide local excisions (WLE) are frequently undertaken in the by MCL, and in 3/6 the skin was a site of recurrence following a diagnosis of systemic management of cutaneous melanoma, however, there is considerable variability between disease(mean interval=57 months). The mean time to recurrence was 45.4 months Histopathology departments in the macroscopic sampling of these common specimens and the overall survival was 66.3 months. Histologically 4/6 cases (67%) had either and definitive College of American Pathologists or Royal College of Pathologist pleomorphic (n=3) or blastoid (n=1) morphology. The mean number of mitosis per 10 guidelines for sampling have not been established. The aim of our study was to high-power fields was 23. All cases showed a dermal-based infiltrate with extension establish evidence based guidelines for the macroscopic handling of these specimens into the subcutis, sparing of the epidermis, and associated perivascular and periadnexal with a subsequent review of the impact on our service following their implementation. accentuation. The immunophenotype included positive cyclin-D1 expression in all Design: The study group comprising of 128 cases which underwent initial biopsy and cases. Limited cytogenetic data showed additional trisomy 14 in one of the cases, in subsequent WLE in our institution in 2010 were identified by a computer generated addition to the t(11;14) translocation. search. From the analysis of this group we derived our guidelines for macroscopic Conclusions: Cutaneous MCL is an exceedingly rare secondary manifestation of sampling. A repeat search was performed over a six month period in 2012 following systemic MCL. The cutaneous lesions can precede or subsequently develop after the the implementation of our recommendations to review the impact these guidelines had diagnosis of systemic MCL. The more aggressive morphologic variants are more on our service. We recorded details of the macroscopic appearance, number of blocks common in the skin, and the proliferation rates are typically high. Ours represents the sampled and margins of the original biopsy. largest single series of cases of MCL with emphasis on the clinicopathologic features Results: In our original study group a wide variation in block numbers sampled (range of this entity. 1-27) was noted with an average 7.5 blocks submitted per case. Residual melanoma was identified in 7 of the cases (5.5%) with clear margins on the original biopsy. Of 470 Low Frequency of Cytogenetic Abnormality, BRAF and NRAS these, four had a pigmented lesion on macroscopic examination and the remaining 3 had Mutations in Borderline Melanocytic Tumors Arising in Deep Penetrating margins of ≤1mm on the original excision. No case required further surgery. Based on Nevi this, our recommendations were, if the margin of the original biopsy was clear by >1mm R Guo, S Li, C Magro, AN Crowson. University of Oklahoma Health Sciences Center, and no macroscopic abnormality was noted grossly a maximum of 3 blocks was to be Oklahoma City, OK; University of Oklahoma and Regional Medical Laboratory, St. John submitted, if a pigmented lesion was present or original margins were involved more Medical Center, Tulsa, OK; Weill Medical College of Cornell University, New York, NY. extensive sampling was advised. On review six months following the implementation Background: A borderline melanocytic tumor arising in deep penetrating nevus of our guidelines we saw an overall block reduction of 36% with an average 2.7 block (B-DPN) has histologic features consistent with a conventional deep penetrating nevus reduction per case. Even with our reduced sampling, our rates of residual melanoma (C-DPN), superimposed upon which are cytologic atypia, deep mitosis and an expansile in the WLE remained comparable with residual melanoma only being detected in nodular growth pattern. A recent study has shown a high incidence of regional lymph those cases with positive original margins, margins <1mm or with a pigmented lesion. node deposits of melanocytes in B-DPN patients. Cytogenetic analysis of B-DPN may Conclusions: We have shown that for WLE specimens with no evidence of a provide valuable information in predicting a risk of aggressive biological behavior. macroscopic lesion and in which margins of the original biopsy were clear by ≥1mm, Design: Oligo-array comparative genomic hybridization (aCGH) assay was applied for little is to be gained from extensive sampling. In these cases, we recommend a maximum cytogenetics analysis. Sanger sequencing was performed to detect the Codon 598-601 of 3 blocks per case. Reduction in sampling based on this evidence would result in saving point mutation of BRAF gene and the Codon 61 point mutation of NRAS gene. A total valuable laboratory resources in already overworked Histopathology departments. 10 cases of C-DPN and 22 cases of B-DPN were studied. Results: The aCGH assay was successfully performed on 7 cases of C-DPN and 13 468 VE1 Is a Sensitive and Specific Marker for BRAF V600E cases of B-DPN. The CGH patterns of the C-DPN cases did not show abnormalities. Mutations in Melanoma In the B-DPN cases, a significant cytogenetic abnormality was only identified in 1 RK Foreman, LM Sholl, JA Barletta, JL Hornick. Brigham and Women’s Hospital, patient, despite the fact that the other 12 B-DPN cases also showed severe atypia, Harvard Medical School, Boston, MA. including 2 of which developed subsequent lymph node metastases. One B-DPN case Background: Around 50% of cutaneous melanomas harbor mutations in the BRAF without obvious cytogenetic abnormality progressed to a lethal metastatic plexiform gene, causing constitutive activation of the MAPK signaling pathway. The majority melanoma a few years later accompanied by an abnormal aCGH pattern at that time. (90%) of BRAF mutations occur at codon 600, with a valine to glutamic acid substitution The BRAF mutation analysis was successfully performed on 29 cases, and showed (V600E). The BRAF V600E inhibitor vemurafenib was recently approved by the Food low frequency in both C-DPN (2/11) and B-DPN (2/18) cases with mostly mosaic and and Drug Administration (FDA) for treatment of BRAF-mutant advanced melanoma. heterozygous patterns. The NRAS mutation analysis was successfully performed on Sequencing is currently performed to identify BRAF mutations prior to initiating 28 cases, and also showed low frequency in both C-DPN (0/10) and B-DPN (1/18) vemurafenib therapy. A monoclonal antibody VE1 directed against V600E-mutant cases with heterozygous patterns. BRAF was recently developed. The aim of this study was to validate the utility of this Brief Summary antibody in detection of the V600E BRAF mutation in melanoma. Frequency Design: Whole tissue sections of 37 melanomas with known BRAF genotypes were Conventional DPN Borderline DPN evaluated (29 metastatic, 8 primary). 17 melanomas had BRAF V600E mutations and 20 CGH Abnormality 0%(0/7) 7.69%(1/13) were BRAF wild-type. 14 BRAF wild-type melanomas also had known NRAS genotype BRAF Mutation 18.18%(2/11) 11.11%(2/18) (3 with codon 61 substitutions and 11 wild-type). One additional BRAF wild-type NRAS Mutation 0%(0/10) 5.56%(1/18) melanoma had a known KIT mutation (at codon 822). Immunohistochemistry (IHC) was Conclusions: The above findings suggest that B-DPN may represent a group of performed following pressure cooker antigen retrieval using the VE1 mouse monoclonal melanocytic lesions with high heterogeneity and low BRAF and NRAS mutation rates. antibody (Spring Bioscience; 1:50 dilution). Cytoplasmic staining was scored as positive The current modality of comparative genomic hybridization does not appear to be a or negative; intensity was graded as weak, moderate, or strong. valid tool in predicting risk of aggressive behavior in these lesions. Results: 15 BRAF V600E-mutant melanomas showed either diffuse strong (13; 76%) or moderate (2; 12%) cytoplasmic staining for VE1 (overall sensitivity 88%). 2 BRAF- 471 Value of Immunocytochemistry for the Detection of the mutant melanomas showed negative staining for VE1. 2 BRAF wild-type melanomas BRAFV600E Mutation in Circulating Tumor Cells from Metastatic Melanoma showed weak, non-specific nuclear staining and were scored negative. All otherBRAF Patients wild-type melanomas were completely negative for VE1 (specificity 100%). V Hofman, M Ilie, E Long-Mira, D Giacchero, C Butori, B Dadone, E Selva, V Gavric- Conclusions: VE1 is a highly sensitive and specific marker for detectingBRAF V600E Tanga, T Passeron, G Poissonnet, J-F Emile, J-P Lacour, P Bahadoran, P Hofman. mutations in melanoma. IHC with VE1 may be useful for rapid identification ofBRAF Pasteur Hospital, Nice, France; Archet Hospital, Nice, France; Antoine Lacassagne mutations and triage of tissue for further genetic analysis. Centre, Nice, France; Ambroise Paré Hospital, Boulogne, France. Background: Therapies targeting the BRAFV600E mutation have transformed the treatment of patients with advanced melanoma. While the BRAFV600E mutation ANNUAL MEETING ABSTRACTS 115A is usually detected in melanoma tissue samples, detection in circulating tumor cells reports, however, MMR status (deficient versus non-deficient) was not associated with (CTCs) would allow non-invasive stratification of patients and examination of the age at presentation (median age, 68 vs. 66), presence of tumor infiltrating lymphocytes effect of targeted therapies. (TILs), or tumor type. Design: We aimed to detect BRAFV600E by immunocytochemistry (ICC) in CTCs Conclusions: Non-head and neck tumors are more likely to be MMR deficient; however, isolated using the isolation by size of epithelial tumor cells (ISET) technology. this criteria alone has a low sensitivity for MMR deficient tumor detection (sensitivity Formalin fixed tumor tissues from 98 metastatic melanoma patients were screened of only 41%). No other clinicopathologic factors (including age, TILs, and tumor type) for BRAFV600E both by pyrosequencing, using the Therascreen BRAF kit, and by were associated with MMR deficiency. We therefore recommend immunohistochemical immunohistochemistry, using the VE1 antibody. Concomitantly and blindly, ICC for testing of all SN for MMR deficiency, regardless of clinicopathologic features. In the BRAF mutation was performed on CTCs isolated by ISET. addition, our findings also confirm the utility of a two antibody approach using MSH6 Results: 53/98 (54%) patients had the BRAFV600E mutation when detected by and PMS2 in screening for MMR deficiency in SN. pyrosequencing in tumor tissue. Among these patients, 51/53 (96%) showed tissue immunostaining with the VE1 antibody. 87/98 (89%) patients demonstrated CTCs, of 474 Duplication and Polysomy: Novel Alternative Pathways for which 54/87 (62%) harbored BRAFV600E detected by ICC. 46/54 (85%) of ICC-CTC BRAF Activation in Primary Melanoma positive patients had BRAFV600E positive tumors and 8/54 (15%) of them showed SR Kandukuri, MJ McFall, J Wu, J Lopategui, E Spiteri. Cedars-Sinai Medical Center, BRAFV600E negative tumors as demonstrated by sequencing. Los Angeles, CA. Conclusions: These results show that CTCs are frequently found in patients with Background: Identification of the BRAF V600E activating mutation on chromosome advanced melanoma when using ISET. Detection of BRAFV600E by ICC was highly 7q in a subset of melanomas (MM) has allowed for the application of targeted therapy. sensitive and reliable. We utilized polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) to identify alternative mechanisms of BRAF activation with potential therapeutic 472 Merkel Cell Polyomavirus Infection Associated with Akt/ implications. Mammalian Target of Rapamycin/4E-BP1 Signaling Pathway Activation Design: A search of our database identified 19 cases of primary MM for which in the Pathogenesis of Merkel Cell Carcinoma hematoxylin and eosin slides were reviewed. Twenty cases of nevi served as controls. T Iwasaki, M Matsushita, D Nonaka, H Nakajima, S Kuwamoto, I Murakami, S Sano, Two 10 µm and 4 µm unstained slides for PCR and FISH were analyzed per case. O Yamamoto, K Hayashi. Tottori University, Yonago, Tottori, Japan; Christie Hospital, PCR analysis was performed with the FDA-approved Roche Cobas 4800 BRAF V600 NHS Foundation Trust, Manchester, United Kingdom; Kochi Universify, Nankoku, mutation assay. For FISH analysis, 40 cells per case were interpreted for signals (gold- Kouchi, Japan. BRAF and green-centromere). A ratio of gold to green signals was calculated per 40 Background: Merkel cell polyomavirus (MCPyV) monoclonally integrates into cells and interpreted as normal (2:2), amplification ≥( 2:1), and polysomy (≥3 green and genomes of approximately 80% human Merkel cell carcinomas (MCCs) and undergoes gold signals with a ratio of less than 2:1 in ≥40% of the cells). Tandem duplication (TD) mutation. It affects clinicopathological features of MCCs, and MCPyV-large T antigen was defined as a ratio of 3:2 in ≥10% of the cells. Five cases of MM and 3 nevi were (MCPyV-LT) plays a pivotal role in pathogenesis by inhibiting cell cycle-regulating excluded due to inadequate specimens for a total of 15 MM and 17 nevi. Statistical function of the retinoblastoma protein. However, the molecular mechanisms involved in analysis was performed with Fisher’s exact test. MCC development after MCPyV infection are unclear. We investigated the association Results: V600E mutation was shown in 8/15 (53%) MM and in 14/17 (82%) nevi of MCPyV infection with the activation of AKT/mammalian target of rapamycin (p=0.128). FISH analysis showed polysomy of BRAF in 4/15 (27%) MM and none in (mTOR)/4E-BP1 signaling pathway in MCC to elucidate the role of signal transduction nevi (p=0.038). TD was detected in 5/15 (33%) MM and none in nevi (p=0.015). Of MCC molecular pathogenesis and to discover molecular targets for MCC treatment. the 7 cases negative for BRAF mutation, polysomy was observed in 1 (14%) case and Design: We analyzed molecular pathological characteristics of 39 MCPyV-positive and TD in 2 (28%) cases. Neither MM nor nevi showed amplification. There were only 26 MCPyV-negative MCCs detected using real-time polymerase chain reaction. PIK3CA 3 cases of MM with V600E and polysomy, one of which also showed TD. One case mutations (exons 9 and 20), mRNA expression of Akt/mTOR/4EBP1 signaling pathway, showed mutation and TD. and clinical data were compared between MCPyV-positive and -negative MCCs, and Conclusions: Polysomy and TD are present in 27% and 33% of MM, respectively. immunohistochemical expression of MCPyV-LT and phosphorylation status of Akt/ Concomitant with their notable absence in nevi, polysomy and TD may represent novel mTOR/4EBP1 signals including Akt and 4E-BP1 were studied. alternative pathways of BRAF activation in MM. The expected frequency of mutation in Results: Heat-map analysis of mRNA expression of the signals in the Akt/mTOR/4E- MM and nevi by PCR was confirmed. Neither MM nor nevi display amplification. TD, BP1 signaling pathway revealed differences in patterns between MCPyV-positive polysomy, and V600E are present in a single case, suggesting that these mechanisms and MCPyV-negative MCCs. MCPyV-negative MCCs showed significantly higher may not always be mutually exclusive. As the sample size of MM cases is small, a expression of some signals including mTOR than MCPyV-positive MCCs (p < 0.05). significant p value was not reached for V600E mutation and polysomy. Additional PIK3CA mutations were found in 6/25 and 0/23 MCPyV-positive and -negative MCCs, research is needed to elucidate the biologic/therapeutic significance of these findings. respectively. Akt/mTOR/4E-BP1 signaling pathway activation was very common in MCCs, as evidenced by the phosphorylation pattern and very low proportion of MCCs 475 Study of Cutaneous Metastases, Experience from Tertiary with negative staining with antibodies (2/61).This pattern was significantly different in Cancer Center MCPyV-positive and -negative MCCs (p = 0.04) but was not associated with differences K Kantekure, D Sariya, S Lessin, H Wu. Fox Chase Cancer Center, Philadelphia, PA. in prognosis. No significant correlation was observed between MCPyV-LT expression Background: It is very important for pathologists to recognize cutaneous metastasis and most signals in Akt/mTOR/4E-BP1 signaling pathway. from systemic malignancies. Due to variable appearances, clinical lesions can mimic Conclusions: These results suggest that MCPyV-positive and -negative MCCs may dermatoses or primary skin tumors. There is also a wide range of histopathologic have different tumorigenic pathways and Akt/mTOR/4E-BP1 signaling pathway signals features. We analyzed clinicopathological characteristics of cutaneous metastases. may be novel targets for targeted therapy in MCC treatment. Design: Retrospective study of 141 patients with biopsy-proven cutaneous metastases from systemic malignancies (excluding that of hematologic or skin cancers) seen over 473 Expanding the Spectrum of DNA Mismatch Repair Protein a 20-year period in a cancer center. Deficiency in Cutaneous Sebaceous Neoplasia Results: 141 cases with biopsy-proven cutaneous metastases (57 men and 84 women). CJ Jessup, M Redston, E Brodell, JDR Reimann. Miraca Life Sciences Research Breast cancer was the most common cause (33/84) in women; lung cancer in men Institute, Newton, MA; Tufts University Medical Center, Boston, MA. (12/57). Most of the skin metastases occurred in patients with known advanced stage of Background: Muir Torre Syndrome (MTS), a variant of Lynch syndrome, is malignancy (60% cases occurred in patients with stage IV disease). However, cutaneous characterized by the presence of cutaneous sebaceous neoplasia plus one or more visceral lesion was the presenting sign prior to a known cancer diagnosis in 11% cases. The malignancies, most commonly colon cancer. The significance of immunohistological most common site of metastasis was abdominal skin (34%), chest (30%) & head & neck detection of DNA mismatch repair (MMR) deficiency in colorectal carcinomas (20%). Due to unusual clinical presentations, 46% of the lesions were not suspected of is well-established, and is recommended as a screening tool for Lynch syndrome being cutaneous metastases at the time of biopsy. A high percentage (48%) of patients identification in newly diagnosed colorectal carcinomas. In comparison, the literature with cutaneous metastases died of underlying malignancy. Mean survival was 17 months on immunohistochemistry (IHC) application to detect MMR protein expression (MLH1, (3 -155 months) after diagnosis of cutaneous metastases. Histologically, the common MSH2, MSH6, PMS2) in sebaceous neoplasia has been less well studied, and has been patterns are nodular tumor or infiltrative growth in a fibrotic dermis. Lymphovascular derived almost exclusively from tertiary care centers. In addition, although recent invasions are not invariably identifiable. data has supported a two-antibody approach using MSH6 and PMS2 to capture MMR Conclusions: Cutaneous metastasis has variable appearance clinically and deficient colon cancers, data are limited for this approach in sebaceous neoplasia. Herein histopathologically. Although most commonly seen in patients with an existing we describe the largest series to date characterizing the clinicopathologic features of diagnosis, it can be the presenting lesion in some patients. Metastatic adenocarcinomas MMR deficient sebaceous neoplasms (SN), as well as the relative frequencies of MLH1, can in general be easily distinguishable from a primary skin tumor. However, there are MSH2, MSH6, and PMS2 deficiency, in a community practice setting. difficult cases that require clinicopathologic correlation and/or immunohistochemical Design: A total of 216 consecutive SN from 216 patients were analyzed (133 sebaceous studies. In the absence of a known primary tumor, it is often difficult to assign a tissue adenomas, 68 sebaceomas, and 15 sebaceous carcinomas). Relevant clinical and of origin of the metastasis based on histology alone. histopathological information was obtained via database searches. MMR protein expression was assessed by IHC. 476 Evaluation of Fumarate Hydratase and Hypoxia-Associated Results: Of the 216 SN, 143 were MMR deficient (66%); 90 MSH2/MSH6 deficient Proteins in HLRCC Syndrome and Sporadic Cutaneous Leiomyomas (63%), 27 MLH1/PMS2 deficient (19%), 22 MSH6 alone deficient (15%), and 4 PMS2 JP Kapil, N Chakravarti, JL Curry, W-L Wang, VG Prieto, MT Tetzlaff, C Torres-Cabala. alone deficient (3%). As in the colon, MMR deficiency was significantly associated MD Anderson Cancer Center, Houston, TX. with anatomic location, with non-head and neck (NHN) tumors more likely to be Background: Hereditary leiomyomatosis and renal cell carcinoma(HLRCC) is a MMR deficient (59/62 NHN vs 84/154 head and neck; P<0.00001). In contrast to prior rare autosomal dominant syndrome with predispositions for cutaneous and uterine 116A ANNUAL MEETING ABSTRACTS leiomyomas and aggressive RCC. It is caused by a germ-line inactivating mutation in tumors and the latter further subclassified as primarily presenting or not with a parotid fumarate hydratase(FH) gene, thought to initiate a pseudohypoxic drive culminating metastasis. Disease-free survival (DFS) and overall survival (OS) were analyzed for in stabilization of hypoxia inducible factor-1a(HIF-1a). Little is known about the all three groups. expression of the proteins involved in the postulated altered molecular pathway in Results: In all, 129 patients were identified as having a metastatic melanoma to the HLRCC-related and sporadic cutaneous leiomyomas. parotid gland or a parotid lymph node. 117 of 129 (90.7%) patients had MKP and 12 Design: 12 cutaneous leiomyomas and 1 smooth muscle tumor of uncertain malignant (9.3%) had MUP. Median follow-up for all patients was 2.1 years. potential(SMTUMP) were examined for IHC expression of FH, HIF-1α, HIF-2α, and Patients with Metastatic Melanoma to the Parotid Region (N=129) α % of Total Alive with Alive Dz Death with Death without HIF-1 -associated downstream proteins lactate dehydrogenase-A(LDH-A) and glucose transporter-1(GLUT-1). Each marker was evaluated for intensity of expression(scale Patients (n) Dz (n) Free( n) Dz (n) Dz (n) MKP 90.7% (117) 20.5% (24) 32.4%(38) 43.5% (51) 3.4% (4) of 0 to 3), percentage of positive cells(0,1:<5%, 2:5-50%, 3:greater than 50%), and MUP, Initial Parotid 6.2% (8) 25% (2) 62.5% (5) 12.5% (1) 0 subcellular localization(nuclear, cytoplasmic, or both). Metastasis (n=8) MUP, Subsequent Parotid Results: The clinical and histologic features of the 13 cases:6 HLRCC pilar leiomyomas, 3.1% (4) 25% (1) 75% (3) 0 0 4 sporadic pilar leiomyomas, 2 angioleiomyomas and 1 SMTUMP were selected from Metastasis (n=4) patients(range 29-89 y/o) with a M:F ratio 4:5. Sites: arm(4), leg(4), shoulder(2), Statistical comparison for p-values by log-rank tests show that the MKP group had trunk(2), and foot(1). HLRCC patients also presented with metastatic renal carcinoma(1) significantly worse OS and DFS from the time of surgery than the MUP group (p=0.02 and uterine leiomyomas(1). and p=0.01, respectively). Comparison of all three groups shows a statistically significant Expression of FH and Hypoxia Markers in HLRCC and Sporadic Cutaneous Leiomyomas longer DFS(p=0.04) and a trend toward longer OS(p=0.06). These data indicate a better Lesions(#Tumors/#Patients) FH* HIF-1α* HIF-2α* LDH-A* GLUT-1* prognosis for those patients with MUP metastatic to the parotid region. HLRCC leiomyomas(6/2) 0,0 3,3 3,3 2.5,3 0,0 Sporadic leiomyomas(4/4) 1,1 2,2.5 3,3 1.5,1.5 0,0.5 Sporadic angioleiomyomas(2/2) 2.5,2.5 2.5,3 3,3 1.5,2 0,0 STUMP(1/1) 2,3 3,3 3,3 2,3 0,0 *All values given as average for intensity and percentage of positive cells(i,%) Conclusions: In comparison to the sporadic counterparts, HLRCC leiomyomas lack expression of FH. This finding may be very useful in clinical practice. Low FH expression in sporadic pilar leiomyomas may indicate a role of FH in these tumors; angioleiomyomas seem to harbor FH-independent mechanisms. Higher expression of LDH-A in HLRCC compared to other tumors supports an intrinsic activation of HIF- 1a. Overexpression of HIF-1a and HIF-2a was detected in all the analyzed lesions, suggesting a common final hypoxic/pseudohypoxic activation in both HLRCC and sporadic smooth muscle lesions. Lack of expression of GLUT-1 in all the leiomyomas— in contrast to that reported in HLRCC renal carcinomas, is intriguing and may suggest a different metabolic final pathway in these lesions.

477 Development and Validation of a Chromogenic RNA In Situ Hybridization Assay for Diagnosis of Atypical Melanocytic Nevi and Malignant Melanoma CL Kauffman, D Stout, X Wu, D Alexandrescu, Y Luo, X-J Ma, P Shitabata. Georgetown Dermpath, Washington, DC; Advanced Cell Diagnostics, Hayward, CA; Dermatopathology Institute, Torrance, CA. Background: Accurately distinguishing benign melanocytic lesions from malignant melanoma remains one of the most difficult challenges in diagnostic pathology. In recent years, a 4-probe fluorescent in situ hybridization (FISH) assay assessing gross genomic aberrations has been developed, but its clinical adoption has been limited due to its focus on Spitz nevi as well as its requirement of specialized equipment. Design: Archival formalin fixed paraffin embedded specimens of melanocytic skin lesions that had been diagnosed by three experienced dermatopathologists were retrieved from two institutions with institutional review board approval. We evaluated 23 genes identified from microarray analysis and the literature in 149 training samples Conclusions: The results of this study suggest that patients who have MUP metastatic (92 melanomas and 57 nevi) to develop a multi-gene RNA ISH assay for classification to the parotid have longer OS and DFS from the time of parotid metastasis as compared of benign nevi and melanomas. The final assay was validated in an independent cohort to those patients who experience a parotid metastasis from MKP. Patients experienced of 171 cases consisting of a broad spectrum of nevi (n=116) and melanomas (n=55). a survival advantage regardless of whether the patient presented with a parotid mass or Results: We identified three melanoma-specific markers (PHACTR1, SPP1 and subsequently following a presenting initial metastasis from another site. PRAME) and three reference genes (MLANA, S100A6 and S100B). Samples with positive staining for at least one of the three melanoma-specific markers were classified as malignant, which had a sensitivity of 97% and a specificity of 93% in the training 479 Wnt and Hedgehog Signaling Pathway Activation in Cutaneous set. Two (22%) of 9 severely atypical nevi in the training set were positive for at least Fibrosing Disorders: A Tissue Microarray Study one of the three melanoma markers. In the validation set, the same assay and scoring K Linskey, J Kay, LM Duncan, RM Nazarian. Massachusetts General Hospital, Boston, algorithm correctly classified 132 (91%) of the 145 unambiguous benign nevi and MA; UMass Memorial Medical Center, Worcester, MA. melanomas (89% sensitivity, 95% CI 78%-96%; 92% specificity, 95% CI 85%-97%). Background: Cutaneous fibrosing disorders represent a dysregulated fibroproliferative Eight (31%) of 26 severely atypical nevi, compared to 2 (4%) of 45 mild and moderate response without a fully elucidated pathogenesis. As Wnt and Hedgehog (Hh) pathways atypia specimens, stained positively for at least one of the 3 melanoma markers (p have been implicated in fibrosis in a number of organ systems, we investigated this < 0.01). A higher number of positive melanoma markers in melanoma cells was pathway in fibrosis in patient skin biopsies. significantly associated with a higher tumor stage (p < 0.01) and Clark level (p=0.05). Design: Using a tissue microarray method and immunohistochemistry, we examined Conclusions: This slide-based assay performs with real-world diagnostic specimens of signaling molecules in the Wnt (Wnt3a, Fz2, WISP1 and WISP2) and Hh (Ihh, Shh, melanocytic lesions, and the results can be viewed under a standard light microscope Ptch, Gli2, Smo) pathways and downstream mediators GSK3β, pGSK3α, Snail and without special equipment. It may serve as a powerful molecular adjunct in the β-catenin in 170 cases of cutaneous fibrosis including keloid (n=58), hypertrophic scar diagnosis of early stage melanoma, especially in the setting of severely atypical nevi. (HScar) (n=57), scleroderma (Scl) (n=22), nephrogenic systemic fibrosis (NSF) (n=9), These melanoma markers may also be prognostic, as evidenced by their correlation scar (n=24), and normal skin (n=8) as controls. with established prognostic factors. Results: Wnt signaling was activated in all fibrosing disorders (Wnt3a, Fz2, and WISP-1 were detected in all cases). WISP-2 was expressed in all Scl cases, with higher expression compared to scar (85%; p=0.0258). HScar demonstrated decreased expression (54%) 478 Metastatic Melanoma to the Parotid Gland: Different Clinical compared to normal skin (100%; p=0.0049) and scar (85%; p=0.0008). Ihh was highly Outcomes Based upon the Presence of Known or Unknown Primary expressed in fibrosis (keloid: 100%, p=0.0001; HScar: 97%, p=0.0045; Scl: 100%, TJ Lawrence, SY Lai, RL Bassett, VG Prieto. University of Texas MD Anderson Cancer p=0.0097), compared to normal (80%). No significant Shh was detected in any cases, Center, Houston, TX. but Gli2 and Smo were highly expressed (100% positive in all case types). GSK3β was Background: Melanoma metastatic to the parotid gland has been reported to have a decreased in Scl (39%) compared to NSF (77%; p=0.0168), normal (91%, p=0.0002) better prognosis in those cases in which there is an unknown primary site. The current and scar (95%, p=0.0001). pGSK3α showed increased expression in keloid (92%, study was designed to study all cases of metastatic melanoma to the parotid gland at p<0.0001), HScar (83%, p<0.0001) and NSF (72%, p=0.0422) compared to normal a single cancer center over a 10-year period to assess possible differences in clinical (40%), which had similar expression to scleroderma (44%). Snail showed expression outcomes. in 100% of keloid, HScar and Scl cases which significantly differed from normal (59%, Design: Surgical pathology reports were reviewed from January 2002 through December p<0.0001 for all). Snail was only moderately expressed in NSF (71%) compared to Scl 2011. Follow-up information was obtained through the institutional electronic medical (p=0.0021). Nuclear localization of β-catenin was increased in keloid (80%, p=0.003), record. Patients were classified as having known (MKP) or unknown primary (MUP) ANNUAL MEETING ABSTRACTS 117A HScar (78%, p=0.027), NSF (100%, p=0.002) compared to normal (46%). β-catenin 482 A Retrospective Histologic and Histochemical Evaluation of staining in Scl (21%) was significantly decreased compared to NSF (p=0.0001), but Cutaneous Calciphylaxis not when compared to normal (p=0.139). M Mochel, R Arakaki, G Wang, D Kroshinsky, M Hoang. Massachusetts General Conclusions: Wnt and Hh (via Ihh and not Shh) signaling was activated in cutaneous Hospital, Boston, MA. fibrosing disorders in human tissue; however unlike keloid, HScar and NSF which Background: Calciphylaxis is a rare and life-threatening disease of vascular display GSK3β and pGSK3α expression as well as β-catenin stabilization, the calcification and cutaneous necrosis. To confirm the diagnosis and exclude other pathogenesis of fibrosis in Scl may occur through an alternate pathway. These results diseases, skin biopsy is performed. However, skin biopsies are often non-diagnostic provide a rationale for targeting of the Wnt and Hh pathways in development of due to inadequate sampling or biopsy site. The objective of this study was to investigate therapeutics. the utility of various histologic features in rendering the diagnosis of calciphylaxis and to compare two histochemical methods, von Kossa (VK) and Alizarin red (AR), for 480 Clinico-Pathological Correlations of BRAF and C-KIT Mutations detecting calcium deposits. in Metastatic Melanoma (MM) Design: The histologic and histochemical features of 56 skin biopsies from 27 MD Lozano, T Labiano, M Montanana, N Gomez, M Aguirre, M Maset, JI Echeveste, consecutive patients with confirmed diagnoses of calciphylaxis were retrospectively MF Sanmamed, A Gurpide, MA Idoate, S Martin-Algarra. University of Navarra, analyzed and compared with those of 19 skin biopsies from 17 patients originally Pamplona, Spain. suspected to have calciphylaxis but determined to have other disease processes. Fisher’s Background: Current advances in the understanding of melanoma gave a solid exact test was used to assess histopathologic features, with two-tailed p-values of <0.05 rationale to introduce different targeted therapies into clinical trials. Patients harbouring deemed significant. BRAF mutations show high response rates after treatment with BRAF inhibitors like Results: Vascular calcification and thrombosis were significantly associated with Vemurafenid or Dabrafenib. Moreover, clinical experience in patients with mucosal or cutaneous calciphylaxis (Table 1). Peri-eccrine calcification, a subtle histologic finding, acral melanomas with gene amplification or activating mutations ofc-KIT treated with was highly specific to calciphylaxis and was the only form of calcium deposition present imatinib, have shown high response rates and prolonged PFS. These results emphasise in four biopsies in the calciphylaxis group. Other epidermal, dermal, and subdermal the importance of molecular information, even in those cases in which the diagnosis was changes were not significantly associated with calciphylaxis. Although VK and AR made on small samples obtained through minimally invasive approaches. Cytology is stains showed comparable detection of calcium in our study, the deposits seen on AR an accurate and cost-effective tool for the diagnosis of metastatic cancer and in many appeared larger and birefringent. times cytological smears are the only samples available from these patients. Table 1: Histologic features of calciphylaxis versus non-calciphylaxis controls Design: We have studied BRAF in 147 samples of MM from 123 patients by direct Histologic features Calciphylaxis (*) Control p-value Sensitivity (*) Specificity Stippled calcification 36/56 (33/43) 0/19 < 0.0001 0.64 (0.77) 1.00 sequencing and cobas®4800 BRAF V600 mutation test. Clinicopathological and Chunky calcification 37/56 (25/43) 5/19 0.0034 0.66 (0.58) 0.74 demographic variables were obtained from our hospital database. DNA extraction was Capillary calcification 28/56 (26/43) 0/19 < 0.0001 0.50 (0.60) 1.00 Any vessel size performed from FFPE tissue sections in 85 cases and directly from cytological smears 43/56 (32/43) 4/19 <0.0001 0.77 (0.74) 0.79 calcification in 62. Comparison between BRAF result in matched paraffin- cytological tumors was Peri-eccrine 6/55 (6/43) 0/19 0.33 0.11 (0.16) 1.00 performed in 9 cases. CKIT mutations were analyzed in 25 patiens by direct sequencing. calcification Results: Sixty-three (51.2 %) patients showed BRAF mutations: 48 V600E (76.2 %), Vascular thrombosis 37/56 7/19 0.033 0.66 0.63 3 V600K (4.76 %), 2 V600R (3.17%) by direct sequencing, and 10 (15.87%) V600 (by *evaluation with AR instead of VK Cobas). Eleven patients were treated with BRAF inhibitors within clinical assay. Two Conclusions: A stippled calcification quality and capillary calcification were highly cases had CKIT mutations, both mucosal melanomas. BRAF and c-KIT mutations are specific to calciphylaxis. Although not statistically significant, peri-eccrine calcification mutally exclusive. BRAF status did not correlated with primary melanoma histology, was highly specific to calciphylaxis and may be useful in the evaluation of biopsies for gender, age at diagnosis, disease free survival, brain metastases rate and overall survival. calciphylaxis when classic features such as small vessel calcification are not present. As Concordance in matched FFPE and cytology was 100%. the VK and AR methods each stained occasional calcium deposits that the other method Conclusions: The frequency of activating mutations in BRAF is 51.2% in this series. missed, performing both stains may increase calcium deposit detection. V600E is more frequent, but V600K appears in 4.76 % of the cases. In our series BRAF mutations do not correlate with any clinico-pathological variable sutudied. Mutational 483 Differential Stathmin 1 Immunoreactivity in Spitz Nevi and analysis of BRAF and c-KIT using cytological samples from patients with metastatic Melanoma melanoma is feasible regardless of the method of PCR used. It can be used to extend J Moore, E Hyjek, T Krausz. University of Chicago Medicine, Chicago, IL. the benefits of targeted therapy to those patients from whom biopsies are not available. Background: The distinction between benign Spitz nevi and malignant melanoma (MM) can at times be difficult or impossible based on morphology and immunohistochemical 481 Clinicopathological Characterization of MCPyV-Infected Merkel analysis. Stathmin 1 plays a role in cell cycle regulation via microtubule dynamics and Cell Carcinomas in Japan and United Kingdom has increased expression in several human malignancies. Both cyclin D1 and stathmin M Matsushita, T Iwasaki, D Nonaka, H Nakajima, S Kuwamoto, M Kato, I Murakami, 1 are thought to be subject to regulation by MiRNA-193b, which is down regulated in S Sano, Y Kitamura, K Hayashi. Tottori University, Yonago, Tottori, Japan; Christie MM and is associated with increased expression of CCND1 in melanomas as compared Hospital, NHS Foundation Trust, Manchester, United Kingdom; Kochi University, to benign nevi. Another recent publication establishes increased stathmin 1 expression in Nankoku, Kochi, Japan. MM as compared to benign nevi and suggests that stathmin 1 is a potential oncogene in Background: Merkel cell polyomavirus (MCPyV) is a novel polyomavirus that is melanoma. Overexpression of cyclin D1 in Spitz nevi compared to melanomas via IHC monoclonally integrated into genomes of up to 80% of Merkel cell carcinomas (MCC). has been reported; however; there are currently no publications investigating stathmin The original association between MCPyV and MCC tumors has been confirmed in 1 expression in Spitz nevi. This study compares immunoreactivity of cyclin D1 and several countries. The aims of this study are to establish the MCPyV phylogenetic stathmin1 antibodies in melanomas, conventional nevi and Spitz nevi. tree and clarify the molecular and clinicopathological differences between Japanese Design: Specimens were selected on the basis of morphological diagnosis from the and Caucasian MCCs. pathology archive. Twenty MM (12 superficial spreading, 5 nodular, 1 spindle cell, 1 Design: We analyzed 45 Japanese [women:31, men:11] and 22 Caucasian (UK) lentigo maligna and 1 metastasis), 10 conventional nevi (5 compound and 5 intradermal), [women:17, men:5] MCC cases. To detect MCPyV-DNA and mRNA expression of and 5 Spitz nevi were stained with antibodies to stathmin 1 (Cell Signaling 3352 Small T (ST) and Large T (LT) antigen, we performed real-time PCR. Analyzed whole 1:50) and cyclin D1 (ThermoFisher SP4 1:30) using established IHC protocols. The sequences of MCPyV-LT and -ST antigen using direct sequence were assembled and melanocytic cells were analyzed for staining pattern and intensity. For stathmin 1, diffuse compared to 50 known MCPyV sequences from NCBI database. Then we establish cytoplasmic staining was considered a positive result. For cyclin D1, reactivity was a phylogenetic tree. Immunohistochemical analyses were performed using CM2B4 quantified as follows: <10% of melanocyte nuclei stained = weak, 10-50% = moderate, (anti-LT antibody) and ISH was also done using DNA and RNA probes targeting ST and >50% = strong expression. or LT mRNA. Results: Diffuse cytoplasmic staining for stathmin 1 was present in 19 of 20 MM cases Results: Rate of MCPyV infection was significantly lower in UK MCCs [32% (7/22)] (95%). One nodular MM specimen showed very weak diffuse cytoplasmic staining. than Japanese MCCs [76% (34/45)] (p=0.001). Combined MCC and SqCC was observed Nine of 10 (90%) conventional nevi and 5/5 (100%) Spitz nevi were negative. One more frequently in UK. Phylogenetic analysis of data base indicated three main clades intradermal nevus showed weak diffuse staining for stathmin 1. Cyclin D1 was strongly (Asian clade and Western clades). All 7 sequences of MCPyV from Japanese MCCs positive in all MM and Spitz nevi, but showed variable reactivity in conventional nevi. clustered in Asian clade. However, there was no significant differences in MCPyV Two nevi showed weak expression, 5 nevi showed moderate reactivity, and 3 showed copy numbers, expressions of LT and ST mRNA and frequency of CM2B4 positivity focally strong expression. between MCPyV-positive cases in Japan and UK. The favorable MCC-specific Conclusions: In this study, cyclin D1 staining was variable in conventional nevi, but survival was longer in MCPyV-positive MCCs than MCPyV-negative MCCs, but strongly positive in both MM and Spitz nevi, limiting its usefulness as a discriminatory the difference was not statistically significant. Expression levels of ST or LT mRNA marker in this setting. Our preliminary findings suggest that stathmin 1 antibody may be were not associated with significant differences in prognosis. The details of ISH will a useful supportive marker in differentiating Spitz nevi from MM in morphologically be shown in the congress. difficult cases; this needs to be validated on a larger number of cases. Conclusions: MCPyV-positive ratio was significantly higher in Japanese than UK MCCs. All 7 MCPyV sequences from Japanese MCCs were belonged to Asian clade. However, MCPyV quantity, LT and ST mRNA expression and LT expression between Japanese and UK–positive cases were neither different and nor associated with prognosis. 118A ANNUAL MEETING ABSTRACTS 484 BAP1 Expression in Melanoma of the Skin Heparanase overexpression creates psoriasis-like phenotype in the mouse skin via R Murali, J Wilmott, V Jakrot, H Al-Ahmadie, T Wiesner, S McCarthy, J Thompson, generation of inflammation-supportive microenvironment, characterized by enhanced R Scolyer. Memorial Sloan-Kettering Cancer Center, New York, NY; Melanoma macrophages accumulation (Figure 2), NF-κB signaling, induction of STAT 3 and Institute Australia, Sydney, NSW, Australia; Royal Prince Alfred Hospital, Sydney, increased vascularization. NSW, Australia. Background: BAP1 is a tumor suppressor gene that was initially thought to function in the BRCA1 growth control pathway, but has more recently been shown to play a role in chromatin modification and transcriptional regulation. Somatic mutation and/ or loss of BAP1 is seen in a variety of tumors, and is associated with poor prognosis in patients with uveal melanoma. In this study, we evaluated immunohistochemical (IHC) expression of BAP1 in cutaneous melanomas, and investigated associations of BAP1 expression with clinico-pathologic parameters and survival. Design: BAP1 IHC was performed in tissue microarrays (TMAs) of selected primary cutaneous melanomas from patients treated at Melanoma Institute Australia between 1992 and 2009. The tumors in the TMAs had been selected to represent a range of thick melanomas of various histologic subtypes. We analyzed associations of BAP1 expression with pathologic and clinical parameters, and with disease-free and melanoma- specific survival. Results: In 158 melanoma patients [median age 72.2 years; 64 females, 94 males], stages at diagnosis were: I (n=15, 9.5%), II (n=90, 57.0%), III (n=43, 27.2%), IV (n=3, 1.9%), and unknown (n=7, 4.4%). Median Breslow thickness was 4.80mm (range 0.80-21.70mm), median mitotic rate was 6/mm2 (range 0-50/mm2), and ulceration was present in 73 (46.8%) tumors. BAP1 expression was absent in 9 (5.6%) tumors. Desmoplastic melanomas were more frequently (21.7%) BAP1-negative than non- desmoplastic melanomas (3%; p<0.0001). There were no statistically significant associations between loss of BAP1 expression and other clinico-pathologic parameters. Higher stage at diagnosis, increasing mitotic rate, presence of LVI and absence of Heparanase-dependent macrophage activation represents a relevant mechanism in BAP1 expression were independently associated with poorer disease-free survival and pathogenesis of psoriasis. melanoma-specific survival. Conclusions: Our results highlight biological significance of heparanase in psoriasis Conclusions: Loss of BAP1 expression was seen in ∼5% of melanomas overall, and reveal possible mechanism of heparanase action. This mechanism involves self- but it was associated with a disproportionately higher percentage of desmoplastic sustained inflammatory circle through which keratinocite-derived heparanase facilitates melanomas than non-desmoplastic melanomas. BAP1 loss was independently associated activation of macrophages, which, in turn, preserve chronic inflammatory conditions with poorer survival. Further, studies are required to explore the spectrum of BAP1 in the skin and in parallel stimulate further production/activation of the enzyme by the expression in desmoplastic melanomas, and to validate the prognostic significance of epithelial compartment. loss of expression of BAP1. 486 Primary Merkel Cell Carcinoma of Lymph Node: A Distinct Entity 485 Heparanase in Psoriasis: Its Role and Mode of Action Has Lower Association with Merkel Cell Polyomavirus (MCPyV) Than Its Cutaneous Counterpart T Neuman, I Lerner, E Zcharia, I Vlodavsky, M Elkin. Hadassah-Hebrew University Medical Center, Jerusalem, Israel; Rappaport Faculty of Medicine, Technion, Haifa, Z Pan, Y Chen, X Wu, V Trisal, S Wilczynski, Q Huang, P Chu, H Wu. University of Israel. Colorado Medical Center, Aurora, CO; City of Hope, Duarte, CA; Memorial Sloan- Background: Heparanase is a predominant mammalian enzyme that cleaves heparan Kettering Cancer Center, New York, NY. sulfate (HS), the key polysaccharide of the ECM and the basement membranes. Background: Rare cases of neuroendocrine tumor have been encountered in lymph Alterations in HS content are characteristic for psoriasis. The role and mode of action node with no skin primary. They show similar morphologic and immunophenotypic of Heparanase in Psoriasis remains to be elucidated. features to those in skin Merkel cell carcinoma (MCC). It is not clear whether the Design: Immunohistochemical staining was used to assess heparanase expression in “nodal MCC” is a primary tumor of lymph node or represents a metastasis from an skin specimens derived from psoriatic patients and healthy individuals. Heparanase- occult tumor elsewhere. Moreover, making an accurate diagnosis of these nodal tumors overexpressing transgenic mice were used to verify the role of heparanase in the can be difficult. pathogenesis of psoriasis utilizing a model of cutaneous inflammation induced by Design: Twenty-two cases of nodal MCC with unknown primary were selected based TPA. Immunofluorescence and quantitative RT-PCR were used to study in depth the on strict criteria. Immunohistochemistry (IHC) stains were performed, including underlying molecular mechanism. chromogranin A, CK AE1/AE3, CK7, CK20, PAX5, synaptophysin, TdT, and TTF1. MCPyV infection was studied by IHC and PCR assay. The clinicopathologic features of Results: Heparanase is preferentially expressed in human psoriatic lesions. our cases were also compared with 526 cases of skin MCC from the literature (Table 1). Results: The nodal MCCs mostly occurred in old patients (mean 65.2 years) with a male predominance (M:F=18:4) from the lymph nodes in head/neck (9 cases), inguina (8) and axilla (5). The tumor cells had nested, organoid or diffuse growth pattern with high N/C ratio, fine chromatin and inconspicuous nucleoli. All our cases were positive for cytokeratin, 20 of which expressed CK20 with characteristic perinuclear “dot-like” staining. The nodal and skin MCCs generally had similar immunohistochemical profiles statistically; however, the nodal cases had much lower MCPyV infection (P = 0.001), which was confirmed by PCR assays. Most of our cases had at least focal expression of PAX5 and TdT, which may mimic hematologic malignancies particularly in cases with a diffuse growth pattern. None of the 19 patients with follow-up developed any skin MCC with an average interval of 23.2 months (2-116 months). Four patients died, 2 of disease and 2 of other causes. Conclusions: Despite sharing similar morphologic and immunophenotypic features, nodal MCCs had significantly lower association with MCPyV than skin MCCs. Therefore, these two entities may arise from overlapping but not identical biological pathways. Table 1 Nodal MCC Cutaneous MCC P-Value Gender (M:F) 18:4 (4.5:1) 326:229 (1.42:1) Mean age (years) 65.2 (N=22) 71.7 (N=526) CK7 3/22 (14%) 10/68 (15%) 1.00 CK20 20/22 (91%) 93/114 (82%) 0.37 Synaptophysin 19/19 (100%) 106/114 (93%) 0.60 Chromogranin A 13/19 (68%) 94/113 (83%) 0.20 TTF1 0/21 (0%) 4/114 (3.5%) 1.00 PAX5 8/13 (62%) 76/91 (84%) 0.12 TdT 10/13 (77%) 55/81 (63%) 0.75 MCPyV 3/13 (23%) 196/261 (75%) 0.001 ANNUAL MEETING ABSTRACTS 119A 487 Biclonality in Cutaneous Marginal Zone Lymphoma examined by at least one of the authors (MCM, RAS) in private consult services or in GG Panse, JM Henneberry. Baystate Medical Center, Springfield, MA. an academic setting. The lesions were subclassified and evaluated for prognostic factors. Background: Biclonality is a rare phenomenon in primary cutaneous marginal zone Morphologic evaluation was performed taking in account architectural (pagetoid spread, B-cell lymphoma (PCMZL). We reviewed the cases of PCMZL from our files and growth pattern, necrosis) as well as cytomorphological (cell type, cytoplasmic, nuclear identified two such cases. characteristics and atypical mitoses) features. Design: The archives of our pathology files were reviewed from January 2000 to Results: Eight patients were female, 4 were male with a mean age at diagnosis of 9 the present for cases of PCMZL, to study their clonal pattern. Thirteen cases were years. Tumor location was extremities in 4 patients, trunk in 3 patients and head and identified, from 8 patients, some with multiple tumors. Five patients had a single tumor neck in 3 patients. Mean time from diagnosis to death was 4 years. Mean Breslow biopsied, one patient had 2 concurrent skin excisional biopsies and 2 patients had 3 thickness was 7.5 mm. and 7 tumors presented as Clark level IV/V lesions. Ulceration biopsies each, over a period of 1 month to 2 years. Immunohistochemistry and in-situ was observed in 4 cases. Mean mitotic rate was 7 per mm2 with 8 cases showing hybridization (ISH) studies for kappa and lambda light chains were performed on all atypical mitoses. Architecturally, 10 cases (84%) showed sheet-like growth pattern with the available skin biopsies. infiltrative borders and the most common (9 cases, 75%) cytomorphological features Results: ISH studies revealed the following: 5 cases demonstrated a clonal population were epithelioid cell type with severe nuclear plemorphism and red macronucleoli. of lymphoid cells with kappa-light chain restriction, a lambda-light chain restriction Conclusions: In summary, in our series of 12 cases we found that the CM features that was identified in one case and two cases demonstrated biclonal populations. Of the are likely to be associated with death are large bulky lesions with sheet-like infiltrative two cases with biclonality, the first case was a 67 year old female, with a history of growth pattern composed of epithelioid cells with severely plemorphic nuclei and red two persistent nodules on the back which were approximately 7 cm apart, ranging macronucleoli, in addition to high mitotic activity with atypical forms. The presence from 1.0 to 1.5 cm in greatest dimension. ISH studies revealed a kappa-restricted of these unequivocal malignant features must prompt the pathologist to confidently clonal population of lymphoid cells in one lesion, and a lambda-restricted population render the diagnosis of malignant melanoma in a pediatric patient. in the second. These results were repeated to confirm the presence of two separate light chain restricted populations. The second patient was a 79 year old gentleman, 490 Prognostic Factors in Mycosis Fungoides with Large Cell with a previously diagnosed PCMZL of the neck and scalp, both tumors showed a Transformation kappa-restricted lymphoid population. Three months later he developed a new lesion MP Pulitzer, PL Myskowski, S Horwitz, S Duzsa, C Querfeld, B Connolly, J Li, R Murali. on the chest which showed a lambda-restricted population of lymphoid cells. All the Memorial Sloan-Kettering Cancer Center, New York, NY. biopsy specimens showed a dense dermal and subcutaneous lymphocytic infiltrate Background: In mycosis fungoides (MF), large cell transformation (LCT-MF) is the with admixed plasma cells and occasional germinal centers. The lymphocytes were histopathologic transformation of atypical lymphocytes to a clonally identical large cell predominantly small to ovoid with hyperchromatic nuclei and focal monocytoid phenotype, which occurs in 20-50% of advanced MF and less commonly in patch/plaque morphology. Immunohistochemical stains were consistent with PCMZL. Primary disease. LCT-MF is thought to be a histologic marker of poor prognosis, associated cutaneous follicle center lymphoma was excluded by either CD10 or BCL6 stains. with a five-year survival of <20%. Some patients with LCT-MF have a more indolent Conclusions: Biclonality has been described in approximately 5% of chronic B-cell course. We sought to identify clinico-pathologic prognostic factors in a large number proliferative disorders. It is proposed that this represents true biclonality (independent of patients with LCT-MF. development of distinct clones) or intraclonal evolution (distinct morphological Design: We identified patients with LCT-MF treated at a referral center for cutaneous variants arising from a common progenitor clone). This study illustrates the unusual lymphoma from January 1990 through March 2012, and who had histopathology nature of biclonality in PCMZL and the importance of working-up each individual slides available for review. Clinical charts were examined, and pathologic features lesion with ISH. analyzed by review of slides and reports on all available cases. Cases in which the diagnosis was not straightforward were reviewed by an interdisciplinary team for 488 Next Generation Sequencing of a Large Cohort of Solid Tumors confirmation. Associations of clinico-pathologic parameters with disease-specific Reveals IDH1 Mutations Present in Melanoma at a Higher Frequency Than survival were analyzed. Expected Results: 51 patients with LCT-MF were identified. Factors significantly associated BP Portier, KP Patel, R Singh, M Routbort, B Handal, BA Barkoh, N Reddy, A Lazar, with shorter survival were: age>60 at diagnosis of MF (p=0.004) and at transformation M Davies, LJ Medeiros, K Aldape, R Luthra. MD Anderson, Houston, TX. (p=0.01); stage III/IV at transformation (p=0.03); concurrent transformation in skin and Background: Systematic genome sequencing has revealed that IDH1 (R132C) nodes (p=0.006); high LDH at transformation (p=0.01); presence of TCR beta (p=0.04) mutations are frequent and provide important prognostic information in glioblastomas. In or gamma (p=0.03) gene rearrangements; high density of infiltrate after transformation addition, IDH1 mutations are observed in approximately 10% of cytogenetically normal (p=0.001); absence of papillary dermal involvement (p=0.04); presence of subcutaneous acute myeloid leukemia cases and are associated with a specific clinicopathologic profile. involvement at transformation (p=0.046); % large cells at transformation (p=0.05); and The prevalence of IDH1 mutation in solid tumors, especially melanomas, is not well presence of follicular mucin at transformation (p=0.02). Factors significantly associated established. In this study, we screened for the presence of IDH1 mutations in a large with a better outcome were: presence of epidermal hyperplasia (p=0.002), and fibrosis cohort of sequentially tested solid tumors utilizing Next Generation Sequencing (NGS). at transformation (p=0.04). Patients presenting with transformation at diagnosis had Design: A total of 910 solid tumor cases were sequentially screened for mutations a better survival compared to those who start with a small cell phenotype (p=0.02). utilizing a 46-gene NGS AmpliSeq™ Cancer Panel (Life Technologies, CA, USA). The Conclusions: In our patients with LCT-MF, values of standard clinical features study group included solid tumors of various types as well as both primary and metastatic (age, stage and LDH) were important prognostically. Further clinical and pathologic tumors. In particular, the study included 208 melanomas and 702 other solid tumors. measures of tumor burden (transformation in viscera or skin/nodes synchronously, Results: Mutations in IDH1 were detected in a total of 14 (1.5%) solid tumors. TCR rearrangements, density, subcutaneous involvement, dermal CD30, and mitotic Specifically,IDH1 mutation R132C was observed in 10 melanomas representing 4.8% activity), follicular mucin, and % LCT were also prognostic. Additional larger studies of all melanomas (1.1% of all tumors tested). IDH1 mutations were also detected in using multivariate models are needed. 4 non-melanocytic solid tumors (lung adenocarcinoma (n=2; R132S and R132L), colonic adenocarcinoma (n=1; R132C), and an undifferentiated carcinoma of unknown 491 Benign Mast Cell Hyperplasia and Atypical Mast Cell Infiltrate primary (n=1; R132C)). In melanomas, IDH1 co-mutations included BRAF, EGFR, (Mastocytosis of Undetermined Significance) in Penile Lichen Planus TP53, CTNNB1, KDR, NRAS, and MLH1 with BRAF co-mutation in 75% of cases. In S Regauer, C Beham-Schmid. Medical School Graz, Graz, Austria. non-melanocytic solid tumors, IDH1 co-mutations included KRAS, NRAS, TP53, and Background: Lichen planus (LP) is a chronic, predominantly lymphocyte-mediated GNAS with KRAS co-mutation in 100% of cases. disease of skin and mucosa. LP occurs in the anogenital region in annular and agminated Conclusions: High throughput NGS is a useful tool for detecting IDH1 mutations and forms, but also as erosive LP on the glans penis. Erosive LP in uncircumcised men often determining their frequency in solid tumors. Using this strategy, we show that IDH1 affect the foreskin and produces a phimosis. In contrast to the lymphocytic infiltrate, mutations are present in approximately 5% of melanomas, a frequency greater than that the presence and type of mast cell infiltrates in LP of the foreskin and glans penis have suggested in earlier studies. Additional mutations are frequently seen in conjunction not received much attention. with IDH1 mutation. The most frequent co-mutations in this cohort were BRAF in Design: 117 foreskins of adult men with LP, removed for phimosis and a histologically melanomas and KRAS in non-melanocytic carcinomas. confirmed diagnosis of LP, were analysed for presence of lymphocytes and mast cells. The infiltrate was characterized immunohistochemically with antibodies to CD 3, 4, 489 Lethal Childhood Melanoma: A Clinicopathological Study of 8, 20, 21, 25, 30, 117c and human mast cell tryptase and analyzed for a point mutation 12 Cases of codon D816V of the c-kit gene and rearrangement of the T-cell receptor gamma CN Prieto-Granada, C Lezcano, A Piris, R Scoyler, M Mihm. Baystate Medical Center, locus (TCR@). correlated with non-erosive and erosive disease forms. Findings were Springfield, MA; Brigham and Women’s Hospital, Boston, MA; Massachusetts General correlated with non-erosive and erosive disease forms. Hospital, Boston, MA; Sydney Medical School, Sydney, NSW, Australia. Results: All foreskins with LP in skin and modified mucosa revealed subepithelial mast Background: Childhood melanoma (CM) is extremely uncommon, representing 3% of cells in lesional skin / mucosa: 33/117 foreskins revealed <15 mast cells/mm2, 22/117 pediatric malignancies and 1-4% of all melanoma cases. The incidence of this type of contained 16-40 mast cells/mm2 and 62/117 foreskins >40 mast cells/mm2 (average 70, melanoma has been recently increasing at a rate of 1-4% yearly. CM has been associated range 40-100). Unaffected skin/mucosa displayed <5 mast cells/mm2. In 27/117 (23%) with better prognosis when compared to adult life melanoma. However, fatal cases mast cells co-expressed CD25 and displayed spindled morphology which we termed have been reported. Melanocytic lesions of childhood can have equivocal morphologic mastocytosis of undetermined significance. Of these 27 patients, 15/27 (56%) had an features, often resulting in “borderline” or “uncertain potential” diagnostic labels. In erosive LP. In 5/27 foreskins a lymphocytic vasculitis of superfical and deep muscular an attempt to identify unambiguous malignant morphologic characteristics of CM, we blood vessels was identified. Neither CD30-expression nor point mutations of the c-kit retrospectively describe the salient features of 12 lesions of CM with fatal outcome. gene at codon D816V were identified in the tissue infiltrates of the foreskin. Only Design: A retrospective clinicopathological review of 12 pediatric ( ≤16 years) one man had a bone marrow biopsy which revealed mast cell granulomas (systemic melanoma patients who died of metastatic disease was performed. The cases were mastocytosis). 92 foreskins with 117 men with LP contained dense lymphocytic infiltrates. 42/88 analysed foreskins revealed T-lymphocytes with a rearranged TCR@. 120A ANNUAL MEETING ABSTRACTS Conclusions: Mast cells are an integral part of the inflammatory infiltrate of penile LP. A 494 Primary Cutaneus Adenoid Cystic Carcinoma: Histological and benign mast cell hyperplasia in lesional skin/mucosa was identified in 2/3 of patients, but Immunohistochemical Findings in a Series of 25 Cases in 23% of men atypical CD25-positive and spindled mast cell infiltrates were identified A Santos-Briz, M Llamas, L Curto, T Mentzel, A Rutten, H Kutzner. Hospital Universitario within lesional foreskins (mastocytosis of undetermined significance)which correlated de Salamanca, Salamanca, Spain; Hospital Universitario de la Princesa, Madrid, Spain; with erosive disease and lymphocytic vasculitis. Mast cells may serve as targets for Hospital del Mar, Barcelona, Spain; Dermatopathologische Gemeinschaftspraxis, innovative therapy options for symptomatic (erosive) penile LP, particularly for those Friedrichshafen, Germany. patients who are not responding to corticosteroid therapy. Background: Primary cutaneous adenoid cystic carcinomas (PCACCs) are rare adnexal tumors, with less than 60 cases reported to date. They are defined as low-grade neoplasms 492 Comparison of Stromal Expression of CD34 and Neoplastic Cell of uncertain histogenesis, characterized by a cribiform pattern, frequent perineural Expression of HMB-45 in Primary Melanomas and Melanocytic Nevi of invasion and repeated local recurrences. Skin Design: A series of 25 cases of PCACCs from the archives of the authors were analyzed S Sabeti, M Khabazian, M Pourabdollah Toutkaboni, F Malekzad. Shahid Beheshti to further characterize the pathologic spectrum and immunohistochemical features of University of Medical Sciences, Loghman Hakim Hospital, Tehran, Islamic Republic this entity. of Iran; Shahid Beheshti University of Medical Sciences, NRITLD, Tehran, Islamic Results: Fifteen patients were female (60%) and 10 were male (40%), with age at Republic of Iran; Shahid Beheshti University of Medical Sciences,Dermatology presentation ranging from 40 to 86 years (median, 60 y). Tumors were located mostly Research Center, Shohadaie Tajrish Hospital, Tehran, Islamic Republic of Iran. in the head (29%), trunk (24%) and upper extremities (24%). All tumors showed poor Background: Melanoma is a clinically and histopathologically challenging circumscription and were composed of a bipopulation of duct-type epithelial cells neoplasm;some of its variants being capable of mimicking banal melanocytic nevi. lining true lumina, and myoepithelial basaloid polygonal cells disposed adjacent to Immunohistochemistry,due to its usefulness, handiness and low cost, has been the most the basement membrane in peripheral, abluminal distribution. Most cases showed useful adjunct to light microscopy for distinguishing this neoplasm from its benign a cribiform pattern, admixed with tubular structures (12 cases, 48%), nodular areas counterparts and other mimickers. Stromal remodeling leading to loss of CD34(+) (3 cases, 12%) or both (6 cases, 24%). One case exhibited a pure tubular pattern. fibrocytes has been extensively studied in many neoplasms and can be a useful adjunct 7 cases (28%) showed perineural infiltration. The depth of the tumors could be in differentiating benign from malignant neoplasms. On the other hand,one of the well- assessed in 17 cases, and ranged from 2.2 to 9.5mm (median, 5.5mm). Most tumors known,highly specific melanoma markers is HMB-45. infiltrated the subcutis. The dimorphic tumor cell population was well appreciated Design: We investigated the rate of immunohistochemical stromal expression of CD34 with immunohistochemistry. In all cases epithelial cells stained with CD117, and in and compared it with the neoplastic cell expression of HMB-45 in malignant melanomas 23 cases (92%) with CK7. Basaloid myoepithelial cells stained in all cases with actin and melanocytic nevi of skin. and in 21 cases (84%) with p63. Results: We examined 22 cases of invasive melanomas and 20 cases of banal Conclusions: Our findings indicate that PCACCs show similar histologic and melanocytic nevi of skin. the sensitivity and specificity obtained for CD34 as a immunohistochemical features to ACCs of other locations. The expression of CD117 discriminant marker were 90.9% and 90.5% and those obtained for HMB-45 were in the epithelial component, and the demonstration of myoepithelial cells could help in 91% and 90%,respectively. the differential diagnosis with other neoplasms, mainly ACC-like basal cell carcinoma. melanoma melanocytic nevus total Positive 20 2 22 495 Comparing BRAF Mutation Status in Corresponding Primary and HMB-45 Negative 2 18 20 Metastatic Cutaneous Melanomas: Implications on Optimized Targeted Total 22 20 42 Therapy Table 1: The rate of IHC expression of HMB-45 in malignant melanomas and melanocytic nevi M Saroufim, R Habib, R Gerges, J Saab, A Loya, S Sheikh, M Satti, C Oberkanins, I Khalifeh. American University of Beirut Medical Center, Beirut, Lebanon; ViennaLab melanoma melanocytic nevus total Diagnostics GmbH, Vienna, Austria; Weill-Cornell Medical Center, New York, NY; positive 2 19 21 Shaukat Khanum Memorial Cancer Center and Research Hospital, Lahore, Pakistan; CD34 negative 20 1 21 total 22 20 42 Dhahran Health Center, SAMSO, Dhahran, Saudi Arabia; King Abdulaziz Medical City, Jeddah, Saudi Arabia. Table 2: The rate of IHC expression of CD34 in malignant melanomas and melanocytic nevi Background: Selective oral BRAF inhibitors show promising results in the treatment Conclusions: These findings suggest CD34 as a useful marker in distinguishing of metastatic melanomas. Consequently, this mandates checking the concordance of malignant melanoma from benign melanocytic nevus. BRAF status in primary (PM) and metastatic (MM) melanomas to optimize individual targeted therapy. 493 Should BRAFV600E Be Tested in Primary or Metastatic Malignant Design: Extended BRAF testing for 9 mutations on 95 lesions from 40 patients Melanoma? (men/women: 27/13; age= 59±13 years) including 40 PM with their corresponding A Santos-Briz, E Godoy, L Arango, P Antunez, E Alcaraz, E Fernandez, MD Ludena. MM (n=42), recurrences (n=9) and second primaries (n=4) was performed. Multiple Hospital Universitario de Salamanca, Salamanca, Spain; Hospital Universitario Morales metastatic sites were present in 9 patients [2 sites (n=6), 3 sites (n=2) and 4 sites (n=1)]. Meseguer, Murcia, Spain. Results: BRAF mutation status was obtained for 85/95 (89.5%) lesions. V600E was Background: The discovery of BRAFV600 mutation as an oncogenic mutation in the only identified mutation type documented in 35.4% of PM vs. 18.9% of MM. cutaneous malignant melanoma (MM) has changed the treatment paradigm for The overall PM-MM BRAF status discordance rate was 32.3% (11/34), and this was melanoma. Selective BRAF inhibitors have demonstrated response rates far higher than significantly more frequent in PM with mutant BRAF (8/12; 67%) versus PM with standard chemotherapeutic options. BRAF mutation analysis is usually performed on wild-type BRAF (3/22; 14%, p=0.005) Patients with metastasis to lymph nodes (3/20; primary tumour tissue; however, no conclusive data are available on the concordance 15%) were less likely to be discordant compared to those with metastasis to other sites of test results between primary tumors and corresponding metastases. (8/14; 57.1%, p=0.023). Females (7/13; 53.8%) were more likely to have discordant Design: Patients with melanoma who underwent surgical resection of the primary PM-MM BRAF status than males (4/21; 19%, p=0.06). Patient age was similar in tumour and sentinel lymph node biopsy or positive lymphadenectomy were patients with concordant and discordant BRAF status (58±13 vs. 62±14 years; p=0.41). retrospectively recruited from our institution. Formalin-fixed, paraffin-embedded PM anatomic location (p=0.23) and time-to-metastasis (p=0.55) were also unrelated to paired samples of primary melanomas and their corresponding lymph node metastases PM-MM BRAF mutation discordance. Discordant BRAF mutation status was predicted were collected from pathologic archives. For each melanoma, hematoxylin and eosin- by multivariate binary logistic regression: 1) the presence of a mutant BRAF in PM stained slides were reviewed. For molecular study, macrodissection of the tumors was [OR (95% C.I.) = 23.4 (2.4-229.7)] and 2) male gender [OR = 0.094 (0.01-0.93)]. performed in all cases. Genomic DNA extraction was conducted from one slide of the MM-MM BRAF concordance was available for 6 possible comparisons and displayed tumor block (5m thickness) using cobas® DNA sample Preparation Kit. Mutation status a 100% concordance rate. was determined by means of a realtime PCR assay (Cobas 4800 BRAF V600 Mutation Conclusions: A high discordant rate implies the need for re-testing of the MM before Test, Roche Molecular Systems). initiation of BRAF targeted therapy. High MM-MM concordance advocates that testing Results: 222 tumor tissues from 152 patients with melanoma were screened (median the most accessible metastatic site is sufficient to obtain accurate BRAF mutation status. Breslow index=2.5mm). Paired samples of primary melanomas and metastases from the same patients (n =60) were included. BRAF mutations were detected in 91 of 152 496 Discrimination of Benign Versus Malignant Melanocytic Skin primary tumors (59.9%). Among the 60 patients who had paired samples of primary Neoplasms by MALDI Imaging Mass Spectrometry (MALDI IMS) and secondary melanomas, 46 (76,6%) showed consistent mutation patterns between A Sepehr, E Seeley, A Harris, S Tahan, R Caprioli. Beth Israel Deaconess Medical primary tumors and metastatic lesions. Among 22 wild-type tumors, only one case Center & Harvard Medical School, Boston, MA; Vanderbilt University, Nashville, TN. (0.45%) developed BRAFV600E metastases. 13 of 38 (34,2%) BRAFV600E melanomas Background: Diagnosis of melanoma is heavily based on histopathologic criteria developed BRAFwild-type metastases. and remains challenging in a large group of cases, as the significance of the criteria Conclusions: Our findings show that there is a high concordance in BRAF status differ among dermatopathologists. Imaging mass spectrometry (IMS) combines the between primary BRAFwild-type tumors and their paired metastases; however discordance measurement capability of mass spectrometers with a surface sampling process that between primary BRAFV600E tumors and metastatic specimens is not a rare finding. It allows probing and mapping of the protein content of a sample. Using MALDI, we have has been suggested that treatment of tumors not harboring a BRAF mutation could applied IMS for the discrimination of benign versus malignant melanocytic skin tumors. result in the hyperactivation of the MAPK pathway and a possible rapid progression. Design: Hematoxylin and eosin (H&E) slides and formalin fixed, paraffin embedded Therefore, although more extensive studies are necessary, the cases presented might (FFPE) tissue blocks of invasive malignant melanomas (MM) and dermal nevi (DN) of suggest that BRAF testing should be performed in metastatic specimens, even in patients patients with clinical follow-up data were retrieved from the archives of the BIDMC. with BRAFV600E primary melanomas. One unstained section per case was prepared. Digital microscope image of the H&E sections were annotated and superimposed to an image of the unstained section. Trypsin ANNUAL MEETING ABSTRACTS 121A and matrix were spotted onto the unstained sections at the annotated locations. Custom 499 MYC Amplification and Overexpression in Primary Cutaneous geometry files were created to allow for the targeting of the specific areas where trypsin Angiosarcoma (AS): A Fluorescence In-Situ Hybridization (FISH) and and matrix have been applied and mass spectral profiles were collected directly from Immunohistochemical (IHC) Study of 39 Cases the tissue sections using MALDI TOF MS. A class prediction model was created using W Shon, SM Jenkins, WR Sukov, AL Folpe. Mayo Clinic, Rochester, MN. a support vector machine algorithm in ClinProTools software. Background: MYC, a proto-oncogene located chromosome 8q24, is involved in control Results: 75 tumors, including 34 MMs and 41 DNs were selected. We generated IMS of cell proliferation and differentiation. Previous studies have documented high-level data and successfully profiled tissues using robotic matrix deposition with a 200 µm MYC gene amplification and MYC expression by IHC in post-irradiation AS, but not resolution and identified candidate peptide peaks (m/z range: 700-4500) which were in primary cutaneous AS or in other radiation-associated vascular proliferations, such preferentially over-expressed in cases of MM or in DN (p < 0.05). When we used a leave as atypical vascular lesions. Prompted by our recent finding of MYC amplification 20% out cross validation genetic algorithm classification with selected peptide peaks, in a primary hepatic AS, we analyzed a large number of well-characterized primary we were able to reach an overall spectral classification of 95% in the training set (n=40). cutaneous AS for MYC gene amplification and protein overexpression. We then applied the candidate peptide peaks to a blindly-selected validation set (n=35) Design: Formalin-fixed, paraffin-embedded blocks from 39 primary cutaneous AS were of MM and DN and reached 80% and 85% spectral classification accuracy, respectively. retrieved from our archives and examined by IHC and FISH, using a commercially Conclusions: We compared the peptide expression profile of MM and DN by MALDI available antibody and probe. Appropriate controls were employed. For FISH, the IMS on FFPE tissues in separate training and validation sets. We developed algorithms number of copies of MYC were compared with the control gene, CEP8 (MYC/CEP8 with overall spectral accuracy of 84% for MM and 89% for DN classes. These spectral ratio). All cases occurred on sun-exposed skin; no patient was known to have a history of rates provide excellent case discrimination capabilities for the diagnosis of melanocytic therapeutic irradiation. Possible correlations with a wide variety of clinicopathological tumors. IMS can be used as an adjunct to routine histopathologic and immunophenotypic variables were evaluated using the logrank test. evaluation of cutaneous melanocytic tumors. Results: Cases occurred in 25 males and 14 females (median age 73 years; range 27-89 years). AS most often involved the head/neck (31), and followed by the leg (4), arm 497 Atypical Spitz Neoplasm: Indolent Clinical Behavior and No Role (3), and chest (1). Clinical follow-up was available on 34 (87.2 %) patients (13 dead for Sentinel Lymph Node Biopsy in Predicting Outcome of unknown cause, 12 dead of disease, 8 alive without disease, 1 dead of other causes; A Sepehr, F Islami. Beth Israel Deaconess Medical Center & Harvard Medical School, mean 4.5 yrs, range 0.13-18.9 yrs). By IHC, MYC overexpression was present in Boston, MA; Mount Sinai School of Medicine, New York, NY. 10/39 (26%) AS-C (2-3+: 8 cases, 21%; rare cells: 2 cases, 5%). By FISH, 2/6 (33%) Background: Atypical Spitz neoplasms (ASN) are rare melanocytic tumors and informative cases with 2-3+ immunostaining showed high-level gene amplification (> ≥ have been the subject of several studies due to difficulties in their histopathologic 8 MYC/CEP8 ratio). One additional case showed lower level amplification ( 2 MYC/ classification, unknown biologic behavior and challenges in the management. Most CEP8 ratio). MYC amplification and MYC overexpression was not correlated with any studies are limited because of small sample size and/or short follow up period. We of the evaluated clinicopathological parameters, including outcome. performed a meta analysis on ASNs and combined results of every study published as Conclusions: We have shown, for the first time, high-level MYC gene amplification of to date to assess any association between clinical and histopathologic parameters and MYC protein overexpression in primary (non-radiation-associated) AS of the with their behavior, and to evaluate the role of sentinel lymph node biopsy (SLNB) in skin. MYC protein overexpression in cases lacking gene amplification likely reflects predicting clinical outcome. other mechanisms of MYC activation. Study of a larger number of primary cutaneous Design: A total of 251 studies were identified. Cases of Spitzoid neoplasm harboring AS showing high level MYC amplification will be necessary to determine whether the unequivocal benign or malignant diagnoses and duplicate cases were excluded. behavior of such cases differs from their more common non-amplified counterparts. Predictors of behavior including age, sex, Breslow depth, mitotic figures, status of the surgical margin, SLNB, completion lymphadenectomy, other treatments and follow 500 CD34-Positive Superficial Myxofibrosarcoma: A Potential up length were documented for each case. Measured outcomes included disease free Diagnostic Pitfall status, recurrence, and stage IV disease/death. SC Smith, A Poznanski, D Fullen, L Ma, RM Patel. University of Michigan, Ann Arbor, Results: From 1989 to 2012, 38 studies were included with a total of 551 patients; MI; Miraca Life Sciences, Glen Burnie, MD. 545 (98.9%) patients were alive at last follow up (mean 59.3 months), and 96.7% were Background: Myxofibrosarcoma (MFS) arises most commonly in the proximal disease-free at last follow up. In 3 patients who died of the disease, Breslow thickness extremities of elderly, where it may extend into the dermis and/or subcutis and be was higher (5.0 mm; SD 3.4) compared to 293 patients who were alive (3.1 mm; SD sampled by superficial punch or shave biopsies. We have encountered superficial 2.1), but this difference was not significant. Mitoses also failed to predict the outcome CD34-positive cases, which may mimic spindle cell/pleomorphic lipoma as well as of ASN. SLNB was positive in 110/281 patients (39.1%) and 2 patients with positive other CD34-positive spindle cell tumors. The prevalence of CD34 immunoreactivity SLNB died of the disease. Among those who were alive/disease-free at the end of in superficial MFS has not been previously assessed. follow up, 40% had a positive SLNB. There was no significant difference in having Design: We performed a systematic review of our institution’s experience with MFS, positive SLNB between those who were alive and those who had died, and between selecting unequivocal MFS cases with superficial (dermal and/or subcutaneous) disease-free patients and those with disease at last follow up. extension, where limited biopsy samples might have proven diagnostically challenging. Conclusions: After a meta analysis on all reported cases of ASN (n=551), predictors of Cases were immunostained for CD34 (Clone QBEnd10, 1:100 dilution) and the tumor behavior such as Breslow depth, mitotic figures, and SLNB failed to predict the immunohistochemical results and clinicopathologic characteristics were tabulated. outcome. Due to low prevalence of ASN, very rare poor outcomes, and relatively short Results: After review of all MFS diagnoses over 5 years (N=56), we identified a follow up, detection of any clinically significant difference is challenging; however, the subset where the pattern of superficial extension might complicate diagnosis (subset data suggest ASN has an indolent course. In the absence of compelling evidence on the N=8). These cases presented clinicopathologic parameters consistent with reported value of therapeutic interventions in predicting the outcome, usefulness of procedures MFS cohorts, including age range (52-88y), 4:3 male:female ratio, and wide anatomic such as SLNB should be revisited. distribution. In 6 of 8 cases, benign diagnoses were favored clinically, while in the two remaining cases, intermediate (dermatofibrosarcoma protuberans) and malignant 498 Immunohistochemical Re-Evaluation of Toker Cells in the (amelanotic melanoma) diagnoses were favored. A sarcoma was not considered Epidermis of the Nipple in the clinical differential for any of these cases. Of eight cases, four (50%; 1 low, M Shimizu, M Nakamura, H Yamaguchi, K Nagata, M Yasuda. Saitama Medical 1 intermediate, and 2 high grade) demonstrated positive staining for CD34, with University International Medical Center, Hidaka, Saitama, Japan. diffuse stain of spindled cells and cellular processes. Four additional cases showed no Background: Toker cells have been attracting attention as a precursor for Paget’s immunoreactivity or equivocal/focal staining for CD34. disease. In 1970 Cyril Toker described clear cells of the nipple epidermis, and these Conclusions: Our study demonstrates that CD34 positivity is prevalent in about half of clear cells are now recognized as Toker cells. Immunohistochemically, Toker cells superficial MFS. Thus, MFS should be considered in the differential diagnosis when a are positive for cytokeratin 7 (CK7), but their reactivity for estrogen receptor (ER), pathologist is confronted with a CD34-positive dermal/subcutaneous spindle cell tumor. progesterone receptor (PR), and HER2 is still controversial. Here we reevaluated CK7- positive cells in the epidermis of the nipple using other immuhohistochemical markers. 501 Histopathologic Features Most Suggestive of Prodromal/ Design: Nipple tissue was obtained from 40 cases of mastectomy specimens for breast Urticarial Bullous Pemphigoid cancer. Serial sections of the formalin-fixed paraffin blocks were made from the nipple. VS Snyder, CL Kinonen, BC Gleason, AB Thomas, TL Cibull. NorthShore University None of the nipple tissues had any breast cancer lesions, and all were histologically HealthSystem, Evanston, IL; Diagnostic Pathology Medical Group, Sacramento, CA. unremarkable. Immunohistochemical staining for CK7, ER, PR, HER2, GCDFP-15, Background: The clinical presentation of bullous pemphigoid (BP) can be highly androgen receptor (AR) were performed on these sections, in addition to hematoxylin- variable, especially in early stages of the disease or in atypical variants such as urticarial eosin (HE) stain. BP. Definitive diagnosis of BP requires correlation between clinical impression, Results: CK7-positive cells in the epidermis were observed in 14 out of the 40 cases histopathology, immunofluorescence (IF), and sometimes ELISA studies. When the (35%), and the number of these cells per section ranged from 3 to 70. These positive presentation is atypical, these patients are occasionally biopsied without concurrent IF cells were sparsely distributed, and small nests consisting of several such cells were also studies. If there are features suggestive of BP, a follow-up biopsy for IF is recommended. found. In addition, these cells were usually located at the basal part of the epidermis. In We compared histopathologic features of cases where the initial H&E findings were these 14 CK7-positive cases, 8 cases were positive for GCDFP-15, 6 cases positive for suggestive of BP and follow-up IF was either positive or negative. ER, and 1 case positive for GCDFP-15. PR was negative in all 14 cases. Design: 20 patients with skin biopsies from NorthShore University HealthSystem Conclusions: Although CK-7-positive cells were found in 35% of mastectomy (2003-2012) had IF performed following an initial H&E biopsy. Patient demographics specimens, the incidence of real Toker cells in the epidermis of the nipple may be and histologic characteristics were reviewed and compared to IF findings. lower. By the use of strict criteria (CK7 positive, and ER, PR, HER2 and GCDFP-15 Results: Patient demographics and results are listed in tables 1 and 2. None of the cases negative) the real Toker cells could be observed in 15% of mastectomy specimens. had a sub-epidermal blister. 122A ANNUAL MEETING ABSTRACTS

Table1: Patient Demographics Conclusions: There are clear patterns of differential gene expression between primary Total Patients Mean Age Female Male cutaneous SCC and metastatic cutaneous SCC. These differentially expressed genes 20 72 (range 41-90) 14 (70%) 6 (30%) and enriched pathways further our understanding of the molecular events involved in the progression of cutaneous SCC to metastasis and may suggest new targets for Table 2: Data Summary prognostication and therapy. Exocytosis of Vacuolization Eosinophils IF Cases Spongiosis Dyskeratosis Vasculitis Eosinophils at DEJ at DEJ IgG, C3 at 504 Keratoacanthoma: A Molecularly Distinct Entity from Squamous Basement 10 10 7 1 7 5 0 Cell Carcinoma Membrane A Su, S Ra, X Li, R Kulkarni, S Binder. UCLA Medical Center, Los Angeles, CA; San Negative 10 9 2 1 6 0 0 Diego Pathologists Medical Group, San Diego, CA. Clinically, BP was suspected in 4/10 cases where IF was positive and 5/10 cases Background: Keratoacanthomas (KA) are crateriform squamous lesions that grow where IF was negative. Most histopathologic features, including the presence/absence rapidly then involute in the sun exposed skin of elderly fair skinned patients. KA is of spongiosis, dyskeratosis, vacuolization at the dermal-epidermal junction (DEJ), a controversial entity with some believing that it represents a variant of squamous and vasculitis, were non-specific. However, eosinophils at the DEJ were found in cell carcinoma (SCC), while others believe it is a distinct benign self-limited 5/10 cases where IF was positive vs 0/10 cases where IF was negative. Exocytosis of squamoproliferative lesion. Our objective was to examine the molecular relationship eosinophils was present in 7/10 cases where IF was positive vs only 2/10 cases where of KA to SCC utilizing DNA microarray technologies. IF was negative. Design: DNA microarray studies were performed on formalin-fixed and paraffin- Conclusions: 1. Spongiosis, vacuolization at the DEJ, and an eosinophilic infiltrate are embedded blocks of skin: Ten cases of SCC arising from actinic damage and ten cases common histopathologic features of BP, but are non-specific. of KA utilizing the AffymetrixU133plus2.0 array. 2. The presence of eosinophils at the DEJ was found only in cases where subsequent Results: Keratoacanthoma demonstrated 1449 differentially expressed genes in IF was positive (5/10). Additionally, exocytosis of eosinophils was more common in comparison with SCC (>5 fold change: p<0.001) with 908 genes upregulated and cases where IF was positive vs negative (7/10 vs 2/10). 541 genes downregulated. The most significantly upregulated genes include CDR1, 3. Even without a sub-epidermal blister, a spongiotic dermatitis with an eosinophil MALAT1, TPM4, CALM1, and TMED2. The most significantly downregulated genes rich infiltrate, particularly in older individuals, should prompt consideration of BP include LOC441461, TYRP1, CEL, INTS6, and WWOX. Functional analysis comparing as a differential diagnosis. If the eosinophils are present at the DEJ and within the KA to normal skin suggests that cellular development, cellular growth and proliferation, epidermis, BP should be more strongly considered and IF should be recommended if cell death, and cell cycle pathways are involved in the pathogenesis of KA. clinically indicated. Conclusions: KA should be classified as a distinct entity separate from SCC based upon its unique molecular profile and clinicopathologic characteristics. 502 Cell Rich Toxic Epidermal Necrolysis (TEN): A Novel Histological Variant with Prognostic Implications 505 PTEN Status in Sporadic and Cowden Syndrome-Associated JJ Speiser, V Mehta, KA Hutchens. Loyola University Medical Center, Maywood, IL. Trichilemmomas: Evaluation of Immunohistochemistry and Fluorescence Background: Toxic epidermal necrolysis (TEN) is a desquamating dermatosis defined In Situ Hybridization by >30% body surface involvement combined with full thickness epidermal necrosis WR Sukov, W Shon, MM Wolz, CN Wieland, LE Gibson, MS Peters. Mayo Clinic, on histology. Mortality for TEN reaches 40% despite therapy. Therapy modalities are Rochester, MN. controversial and range from supportive care only to administration of intravenous Background: PTEN (phosphatase and tensin homolog) acts as a tumor suppressor to immunoglobulin and / or high dose steroids. Classic histology is a cell poor subepidermal negatively control cellular growth and survival via the P13K/AKT signaling pathway. blister with full thickness epidermal necrolysis. The histologic spectrum of TEN and Cowden syndrome (CS) is a rare autosomal dominant disorder linked to germ line its relation to clinical outcome has not been previously examined. Here we describe mutations in PTEN. A few small case series have evaluated the level of PTEN protein a frequently encountered cell rich variant of TEN and compare its outcome with the expression in thyroid nodules, oral papillomas, and cutaneous trichilemmomas, and loss classic pauci-cellular type. of PTEN immunostaining appeared to be both sensitive and specific for CS-associated Design: We analyzed the histologic findings on 23 cases of TEN. All cases were lesions. However, the utility of fluorescence in situ hybridization (FISH) in determining clinically consistent with TEN and were treated with the same standard protocol PTEN gene status has not been studied in cutaneous trichilemmomas. (supportive care only) at the LUMC burn unit. Cases were grouped into either cell Design: Formalin-fixed, paraffin-embedded sections of 14 CS-associated and 19 rich variant, demonstrating full thickness epidermal necrosis with a dense lichenoid sporadic trichilemmomas were immunostained for PTEN (clone 6H2.1, 1:400, Dako) infiltrate, or the classic cell poor variant. Clinical hospital courses within one week using heat-induced epitope retrieval and polymer refine detection system. Appropriate of biopsy were evaluated and separated by complete recovery or morbidity/mortality. controls were employed. Clinical information was obtained from our medical records. Results: 16/23 cases were pauci-cellular and 7/23 were cell rich. 10/16 pauci-cellular PTEN deletion status was analyzed by fluorescence in situ hybridization studies were cases had significant morbidity / mortality (5 morbidity and 5 mortality). 6/7 cell rich performed using a laboratory developed PTEN eneumeration probe and commercially cases had a complete recovery and 1/7 case resulted in mortality. There was a higher available centromere 10 probe (Abbott Molecular). correlation of significant morbidity / mortality with the cell poor variants than the cell Results: Microscopically, all skin biopsies demonstrated typical features of rich variants [p=0.03 (Pearson’s chi-squared test)]. trichilemmoma, characterized by superficial dermal lobules of cuboidal cells with pale Conclusions: The cell rich form of TEN has not been previously well described and eosinophilic or clear cytoplasm. The peripheral nuclear palisading and adjacent thick could be diagnostically challenging for the pathologist. Due to the high mortality and hyaline basement membrane were also present. Complete or heterogeneous loss of morbidity of TEN, it is imperative that pathologists are aware of both the cell poor and PTEN expression was observed in 13/14 (92.9 %) CS-associated trichilemmomas and cell rich variants. Additionally, our study indicates that histological cellularity may be in 2/19 cases (10.5 %) sporadic trichilemmomas. By FISH, 4/7 (57.1%) trichilemmomas a prognostic indicator. Separation TEN by histologic variant is novel and may also be demonstrating PTEN loss, also carried hemizygous PTEN gene deletion (3/6 CS- the missing link needed to align current therapy algorithms. associated and 1/1 sporadic). Of the 18 PTEN-positive tumors, no PTEN abnormality was identified by FISH. 503 Metastatic Cutaneous Squamous Cell Carcinoma: Differential Conclusions: Our study shows that loss of PTEN protein expression is frequently Gene Expression Profiling in Comparison to Primary Cutaneous detected in CS-associated trichilemmomas suggesting that IHC may be a useful Squamous Cell Carcinoma screening test. This preliminary data also indicates that there is no significant correlation A Su, S Ra, X Li, S Binder. UCLA Medical Center, Los Angeles, CA; San Diego between loss of PTEN expression by IHC and PTEN gene deletion by FISH; thus another Pathologists Medical Group, San Diego, CA. mechanism must account for PTEN inactivation. Further studies of larger sample sizes Background: Squamous cell carcinoma (SCC) is the second most common cutaneous are needed to define more clearly the prevalence and significance of PTEN status in malignancy with a substantial minority that metastasize. Regional or distant spread is trichilemmomas. associated with a poor outcome. Lesions that are likely to metastasize require prompt treatment. The molecular mechanisms involved in the progression to metastasis are 506 Skin Punch Biopsy Sectioning: Before or after Tissue incompletely understood. Our goal was to utilize comprehensive DNA microarrays Processing? to investigate the molecular pathways involved in the progression of cutaneous SCC N Sungu, S Orhun, S Marali, S Balci, A Kilicarslan, C Altunkaya, G Guler. Yildirim to metastasis. Beyazit University, Ankara, Turkey. Design: DNA microarray studies with the AffymetrixU133plus2.0 array were performed Background: Punch biopsies (PB) are frequently used in inflammatory dermatoses. on formalin-fixed and paraffin-embedded blocks of skin: 10 cases of primary cutaneous For macroscopic sampling, while the PBs larger than 4mm are recommended to be SCC and 10 cases of metastatic cutaneous SCC to lymph nodes, salivary glands, and cut into two or three equal parts, those smaller than 4mm are either not cut or cut into soft tissue. The data were analyzed using Partek Genomics Suite and Ingenuity Pathway two equal parts. We intended to study whether there is a meaningful difference at Analysis software. microscopic examination between dividing section into two equal parts before tissue Results: Metastatic cutaneous SCC had 106 differentially expressed genes in processing or after. comparison with primary cutaneous SCC (>=2 fold change, FDR p<=0.01) with 92 Design: 400 cases included in the study. Punch biopsies were cut into two pieces genes upregulated and 14 genes downregulated. The most significantly upregulated before tissue processing (first method) in 200 and after tissue processing just before genes included CDR1, TPM4, MALAT1, TMED2, and FN1. The most significantly paraffin embedding (second method) in other 200 cases. We microscopically evaluated downregulated genes included FLG2, LOR, HAL, TYRP1, and FLG. Canonical pathway the appearance of epidermal mesh view (EMV), the presence of cross-cut hair follicle analysis showed that many cancer related pathways that lead to cell migration and (CCHF) and bow shape (BS) due to epidermal angling, presence of two pieces(TP) invasion, including PI3K/AKT signaling, are activated in metastatic cutaneous SCC. on the slide and if there was a distance greater than 2mm(DG) between the parts, the number of new sections(NNS), new slides(NNL), and diameter(D) of biopsies. ANNUAL MEETING ABSTRACTS 123A Results: We determined that the values for CCHFs (p=0.018), EMV(p=0.036), DG number alterations (amp), and select gene fusions/rearrangements. Actionable GA (p=0.008), NNS(p<0.001), and NNL(p<0.001) were considerably higher in first method were defined as those identifying commercially available targeted therapeutics or in than second one but the presence of TP was low(p<0.001) in first method. registered clinical trials (CT). Comparison of 2 methods Results: The 10 cutaneous melanomas included 6 (60%) male and 4 (40%) female 1st method 2nd method p patients with a mean age of 57 years (range 42-83 years). All 10 (100%) of tumors CCHF 12 3 0.018 were Stage IV at the time of NGS. The MM samples tested by NGS were obtained EMV 24 12 0.036 from the liver (25%), lung (8%), other visceral sites (17%), lymph nodes (33%), and BS 17 9 0.105 TP 101 184 0.001 intra-cutaneous metastatic sites (17%). NGS found a total of 24 GA with at least 2 GA DG 8 3 0.008 in all 10 (100%) cases with an average of 2.4 (range 2-4) per tumor impacting multiple Mean NNS 1.53 0.44 0.001 genomic pathways including MAPK, PI3K/mTOR, cell cycle regulation and regulators Mean NNL 0.58 0.15 0.001 of oxidative stress and apoptosis. NGS of 9 (90%) MM had actionable GA and 5 (50%) 2 pieces per 3mm 33/80 98/109 0.001 of MM had GA associated with FDA approved therapies for MM including vemurafenib 2 pieces per 4mm 68/120 86/91 0.001 Total 200 200 targeting BRAF V600E (30% of tumors) V600K (10% of tumors) T599_V600insT (10% of tumors). Four MM (40%) had GA associated with FDA approved therapies Figure 1: The presence of cross-cut hair follicle, bow shape due to epidermal angling, in other tumor types including everolimus targeting NF1 (20% of tumors) and PTEN H&Ex40 (20% of tumors). Conclusions: Deep NGS of MM uncovers an unexpectedly high frequency of actionable GA that have the potential to influence therapy selection and can direct patients to enter clinical trials to study the benefit of anti-MM targeted therapies in a single comprehensive diagnostic test.

508 Immunodetection of Phosphohistone H3 (PHH3) as a Surrogate of Mitotic Figure Count and Clinical Outcome in Cutaneous Melanoma MT Tetzlaff, JL Curry, DL Stockman, D Ivan, W-L Wang, KM Valencia, R Bassett, CA Torres-Cabala, VG Prieto. University of Texas MD Anderson Cancer Center, Houston, TX. Background: In the AJCC-TNM (2009) staging system, the key prognostic factor in cutaneous melanoma is the depth of dermal invasion (Breslow thickness; BT) with further refinement according to the presence of epidermal ulceration and/or dermal mitoses. Immunodetection of phosphohistone H3 (PHH3) has been shown to facilitate the identification of mitotic figures and to function as a surrogate of outcome in various neoplasms. Design: We selected 120 cases of primary cutaneous melanoma from our archives (2002-2005) with completely annotated histopathologic parameters and clinical outcomes and performed double immunohistochemical staining for Mart-1 and PHH3. The association between manual mitotic count and PHH3 mitotic count was assessed by Pearson correlation. The association between the presence of metastasis and mitotic counts (manual and PHH3), Breslow thickness, and ulceration was assessed by logistic Figure 2: Epidermal mesh view,H&Ex100 regression. Results: 113 cases were amenable to anti-PHH3 staining from 66 men and 47 women with mean age of 64 years (9-93). 61 were superficial spreading type, 24 nodular, 6 lentigo maligna, 8 acral lentiginous, and 14 unclassified. The mean BT was 2.53 mm (0.20-25), ulceration was present in 25/113 lesions (22%) and the mean mitotic count was 3.2/mm2 (<1-29). In 27/113 (24%) of the cases, anti-PHH3 failed to highlight mitotic figuresanywhere in the tissue, whereas in 86/113 (76%) anti-PHH3 detected at least 2 mitotic figuressomewhere in the tissue (normal and/or tumor cell). Among the latter, anti-PHH3 did not detect mitoses in tumor cells in 37/86 cases (43%) whereas PHH3 identified at least one dermal melanocytic mitotic figure in 49/86 cases (57%; range:1-66). The relationship between PHH3 and manual mitotic count was statistically significant (Pearson correlation=0.59, p<0.0001) with discrepancies identified in 10 cases: anti-PHH3 did not identify mitoses in 6 cases where they were previously identified, and anti-PHH3 detected mitoses in 4 cases previously described as <1/ mm2. Logistic regression analyses demonstrated an association between the subsequent development of metastatic disease and the following variables: mitotic figures (odds ratio (OR)=5.7; p=0.0001); PHH3-positive mitotic figures (OR)=3.0; p=0.008); Breslow thickness (OR=4.0 per mm; p=0.0002); ulceration (OR)=3.94; p=0.008). Conclusions: The application of PHH3 immunohistochemistry to the description of primary cutaneous melanoma greatly facilitates the identification of mitotic figures and correlates with an increased risk for metastasis.

Conclusions: As macroscopic inspection, proper division, appropriate paraffin 509 The Effects of Eculizumab on the Pathology of Degos Disease embedding and sectioning of biopsies are critical for pinpointing small lesions, we X Wang, CM Magro. Weill Medical College of Cornell University, New York, NY. noted a meaningful difference in quality of microscopic evaluation between first Background: Degos disease is a frequently fatal and largely incurable occlusive and second methods. Better sections were obtained when PBs sectioned after tissue vasculopathy most commonly affecting the skin, the gastrointestinal tract and brain. processing. In addition, decrease in the number of new slides will reduce workload, We have previously published an article in your journal describing that vascular archive work, and cost. C5b-9 deposition and an interferon alpha (INF-α) rich microenvironment are found to be pathogenetically important to the evolution of the vascular changes. We recently 507 Next Generation Sequencing (NGS) Reveals Pathway described the use of Eculizumab as a salvage drug in the treatment of near fatal Degos Activations and New Routes to Targeted Therapies in Metastatic Cutaneous disease. The effects of Eculizumab on the pathology of Degos disease are explored Melanoma (MM) in this manuscript. A Tarasen, JA Carlson, CE Sheehan, G Otto, G Palmer, R Yelensky, D Lipson, A Design: Archival skin and gastrointestinal biopsy material was procured over a period Donahue, K Iwanik, J Buell, S Downing, J Curran, V Miller, P Stephens, JS Ross. of 2.5 years before and after the initiation of Eculizumab therapy in our index case. Albany Medical College, Albany, NY; Foundation Medicine Inc., Cambridge, MA. Routine light microscopy and immunohistochemical assessment for C3d, C4d, C5b- Background: Comprehensive genomic profiling of clinical samples by NGS has the 9, MxA and caspase 3 were examined. Direct immunofluorescent studies were also potential to identify new genomic-derived drug targets of therapy for patients with conducted on select biopsy material. MM in a single diagnostic test. Results: We found that pre-Eculizumab biopsies showed an active thrombotic Design: NGS was performed on hybridization-captured, adaptor ligation based libraries microangiopathy associated with a high INF-α signature and extensive vascular deposits using DNA extracted from 4 formalin-fixed paraffin embedded sections cut at 10 microns of C5b-9 in skin and gastrointestinal biopsy material. Endothelial cell apoptosis as from 10 MM cases. The exons of 182 cancer-related genes were fully sequenced using revealed by Caspase 3 expression was noted. Inflammation comprising lymphocytes and the Illumina HiSeq 2000 at an average sequencing depth of 1098X and evaluated for macrophages along with mesenchymal mucin was observed as well. Post-Eculizumab genomic alterations (GA) including point mutations (mut), insertions, deletions, copy biopsies did not show active luminal thrombosis but only chronic sequelae of prior episodes of vascular injury. There was no discernible caspase 3 expression. After 12 124A ANNUAL MEETING ABSTRACTS months of therapy, C5b-9 was no longer detectable in tissue. The high INF-α signature 512 A Comprehensive Biomarker Study in Spindle Cell- and and inflammation along with mucin deposition was not altered by the drug. In addition, Desmoplastic Melanoma Reveals a Marker-Panel for Diagnostic Distinction there was little effect of the drug on the occlusive fibrointimal ateriopathy. and Identification of Patient Subsets That May Benefit from Molecularly Conclusions: We concluded that complement activation and enhanced endothelial Targeted Therapy cell apoptosis plays an important role in the thrombotic microvascular complications SE Weissinger, P Keil, P Moller, B Horst, JK Lennerz. Ulm University, Ulm, Germany; of Degos disease. However, the larger vessel atherosclerotic-like changes appear to Columbia University, New York, NY. be independent of complement activation and may be on the basis of other upstream Background: The histopathological distinction of spindle cell- (SCM) and desmoplastic mechanisms which in this case was in the context of high levels of INF-α. A more melanoma (DM) is not always straightforward; however, different prognostic and integrated approach addressing upstream and downstream pathways such as those therapeutic implications make this distinction clinically important. While numerous attributable to complement deposition are critical for the successful treatment of studies have assessed individual markers in each subtype, a comparison of diagnostically Degos disease. It does have a role as a salvage drug in acutely ill patients in whom and therapeutically relevant biomarkers has not been performed. Here, we present an a thrombotic diathesis may be pathogenetically significant to a precipitous decline in array-based analysis in two independent cohorts. their clinical state. Design: Archival searches for SCM and DM were performed at both institutions (Ulm/ NYC) and samples were mounted in tissue-microarrays for the assessment of the 510 Nine Cases of Histiocytoid Sweet’s Syndrome following biomarkers: HER,EGFR,cMET,MDM2,p53,ALK,MYC,CCND1,MYB,RREB1 X Wang, CM Magro. Weill Medical College of Cornell University, New York, NY. and corresponding chromosome-enumeration probes by fluorescent in-situ hybridization Background: Histiocytoid Sweet’s syndrome was first described by Requena et al (FISH). In addition, we screened for LAM,S100c,S100n,NSE,VIM,GFAP,CD34,CD56 in 2005 to describe a distinct variant of Sweet’s syndrome with specific morphologic ,CD57,FLI-1,NF,Melan-A/MART-1,MIB-1/Ki-67,COL-IVt,COL-IVn,HMB45,KIT,C features. Its hallmark is an infiltrate of histiocytoid mononuclear cells with an D99,CD68t,CD68n,Desmin,SMA,AE1.3,CAM5.2,HER2,MUM1,EGFR,MET,MDM2 eccentrically disposed C-shaped nucleus. Based on positivity of these cells for ,P53,ALK,MYC,P75, and DOG1 by immunohistochemistry (IHC). Statistical testing myeloperoxidase (MPO), it was hypothesized that the cells were immature neutrophils. included class-correlation as well as Fisher’s/Chi-square tests; significant markers were Although the original series reported underlying myeloproliferative disease in 1 defined as P<0.01 and distinct markers as P<0.05. of 41 cases, subsequent papers described its occurrence in the setting of clonal Results: We assessed a total of 42 cases composed of 16 SCM, 20 DM and 6 SCM/ myeloproliferative disorders. The importance in recognizing this syndrome was to DM in two subsets (Ulm: 9DM/9SM and NYC). Analysis generated 1514 IHC- and expand the histomorphologic spectrum of Sweet’s syndrome and avoid confusion 831 FISH readouts. Intraclass correlation derived a 12 component biomarker signature with leukemia cutis. (CBS:7 significant/5 distinct) composed of 5 biomarkers that were positive in DM Design: Nine cases of histiocytoid Sweet’s syndrome were encountered. Clinical (COL-IV;CD68;p75;Trichrome;CNL2p) and 7 biomarkers that were present in SM features, and light microscopic and phenotypic profile were reviewed. (LAM; Melan-A/MART1;HMB45;KIT;MDM2,p53;CNG8q24). In cross-validation Results: The patients’ ages range from 35 to 79 with 6 males and 3 females. There between Ulm and NYC cases, Melan-A/MART-1, KIT and HMB45 (DM–; SM+) was involvement of arms in 4 and legs in 4 while a truncal distribution was seen in were diagnostically most reliable. Based on the 12-CBS assessment, all but 1 of the 6 1. There was accompanying arthralgias and fevers in 1. Among potential triggers and combined SCM/DM cases were more similar to DM rather than SCM. Marker analysis disease associations were myelodysplastic syndrome in 3, acute myelogenous leukemia of 9 potentially targetable molecular aberrations revealed -with the exception of one in 1, advanced endometrial carcinoma in 1 and marginal zone lymphoma in 1. In the combined MET and KIT-positive case- a total of 16 cases (38%) with mutually exclusive patient with marginal zone lymphoma, the eruption was preceded by herpes zoster. labeling for CKIT/HER2/EGFR/MET, or ALK. One case was related to high doses of Cox 2 inhibitor. The cases showed an interstitial Conclusions: Our comprehensive analysis shows that identification of DM/SCM-patient and perivascular mononuclear infiltrate with a minor component of neutrophils. The subsets that may benefit from molecularly targeted therapy will require more than one mononuclear cells demonstrated a distinctive morphology characterized by eccentrically biomarker. In addition, we provide a diagnostic panel that allows distinction of these disposed C-shaped nuclei with eosinophilic cytoplasm. In 2 cases the process was melanoma subtypes when histological analysis is not straightforward. confined to subcutis. Phenotypically, the cells were not only positive for MPO but also for CD163 and variably positive for MXA, CD16, CD14, CD123, CD4, and CD68. 513 SOX10: A Useful Marker for Identifying Metastatic Melanoma Chloroacetate esterase was negative. in Sentinel Lymph Nodes Conclusions: We found a significant association with underlying myeloproliferative BC Willis, J Wang, C Cohen. Emory University, Atlanta, GA. disease and other malignancies. The extent of histiocytoid infiltration varied however; in Background: SOX10 (Sry-related HMg-Box gene 10) is a key nuclear transcription certain cases the infiltrate was misinterpreted as leukemia cutis. Immunohistochemical factor in the differentiation of neural crest progenitor cells to melanocytes. It has been studies showed that the MPO+ cells were CD163+ and CD14+ confirming their origin shown to be a sensitive and specific marker of malignant melanoma in multiple tissue as a monocyte rather than an immature neutrophil. In this regard the designation types as well as a useful tool in differentiating desmoplastic melanoma from benign of histiocytoid seems appropriate. This exact same cell type has been implicated in dermal scars. Currently, sentinel lymph node biopsy is standard of care in determining Kikuchi’s disease. The MPO+ histiocyte is a specific subset of macrophages that may treatment strategy and prognosis in cases of known malignant melanoma. The aim of be attracted to sites devoid of granulocytes to provide MPO for oxidative processes. our study was to examine the immunostain profile of SOX10 in sentinel lymph nodes potentially involved by metastatic melanoma, and additionally compare it to current 511 Hypopigmented Epitheliotropic T-Cell Dyscrasia as a Form of commonly utilized immunostains (S100 protein, HMB-45, and Melan-A). Prelymphomatous T-Cell Dyscrasia Design: 77 cases of sentinel lymph nodes diagnosed as positive for metastatic X Wang, YC Liu, CM Magro. Weill Medical College of Cornell University, New melanoma and 30 cases negative for metastatic melanoma were examined using S100 York, NY. protein, HMB-45, Melan-A, and SOX10. The four stains were compared for positive Background: Hypopigmented lesions are a common presenting complaint in staining, intensity of staining, and percentage of cells stained. False positives including dermatology clinics and arise in various settings including hypopigmented variants of inflammatory cell staining and subcapsular nevi were screened for in both positive and mycosis fungoides (MF). While the presence of atypical lymphocytes in these infiltrates negative cases. Cases in which metastases were lost on additional levels were excluded raises the diagnostic possibility of hypopigmented MF, there are subsets of patients from the comparison. who fail to meet criteria for fully evolved MF. Results: SOX10 staining showed a statistically significant increase in staining intensity Design: Twenty three cases of hypopigmented T-cell dyscrasia were identified in the when compared to S100 protein, HMB-45, and Melan-A (p=.000, p= .000, and p=.003 data base of Weill Medical College of Cornell University. In each case, routine light respectively). Additionally, there was a statistically significant increase in percentage microscopic analysis and phenotypic and molecular studies were conducted on paraffin of tumor cells stained by SOX10, compared to S100 protein, HMB-45 and Melan-A embedded formal fixed tissue. The comprehensive phenotypic panel included antibodies (p=.015, p=.000, and p=.001 respectively). SOX10 identified metastatic melanoma to beta F1, CD2, CD3, CD4, CD5, CD7, CD8, and CD62L. in all of the cases examined, while the highest number of false negatives was seen in Results: Of the patients studied 16 were females and 7 were male with a mean age of HMB-45 (seven cases, 13%). SOX10 stained background inflammatory cells in two 36 years of age. In children and adolescents, those of African American descendent cases, while HMB-45 and Melan-A stained showed similar patterns in three cases. were seen most commonly. All patients presented with hypopigmented lesions of Conclusions: SOX10 is a useful marker for the identification of metastatic melanoma variable size involving truncal photoprotected areas except in 3 cases where facial in sentinel lymph nodes. It consistently identifies metastases in 100% of cases involvement with hair loss occurred. While the disease was persistent at times over examined, while showing minimal staining of other constituents of normal lymph nodes. years, in only one case was there disease progression to MF; the lesions responded to Subjectively, interpretation was less ambiguous in the SOX10 stained slides due to the topical steroids and light therapy. From a morphologic perspective the cases exhibited nuclear staining pattern and absence of staining of dendritic cells as seen in S100 protein. reproducible light microscopic phenotypic features including the presence of a true We conclude that given the sensitivity and specificity of SOX10 for detecting metastatic vacuous cell poor interface associated with degenerative changes of keratinocytes and melanoma in sentinel lymph nodes, it may play a significant role in supplementing and melanocytes, low grade cerebriform atypia, and paucity of other inflammatory cell potentially replacing traditionally utilized immunostains. elements. Phenotypically the cases showed a significant reduction in the expression of CD7 and CD62L corroborative of their categorization as a form of T cell dyscrasia. 514 Rabbit Polyclonal Anti-SOX10 Is a Reliable IHC Marker for In 50% of the cases the implicated cell type was of the CD8 subset. Polyclonality was Melanoma and Its Mimics seen in almost all cases studied. Clonality was not identified. GG Yang, A Minasyan, J Gordon, M Lacey, K Lundquist. Cell Marque Incorporation, Conclusions: We propose that this entity represents a form of indolent cutaneous Rocklin, CA. lymphoid dyscrasia falling under the designation of the hypopigmented interface variant Background: SOX10 is a transcription factor that participates in melanocytic and of cutaneous lymphoid dyscrasia, a term originally proposed by Guitart and Magro. It Schwann cell differentiation. It has been reported that SOX10 antibody is a sensitive responds well to UVB therapy, like other forms of cutaneous lymphoid dyscrasia and marker for melanomas. However, thus far the primary antibody against SOX10 protein in rarely progresses to overt MF. the research and publications has been antiserum from goat, which hinders the antibody’s ANNUAL MEETING ABSTRACTS 125A acceptance, particularly in laboratories for clinical diagnosis. Here we report the be of less utility and interest than in non-subspecialized departments. The Department assessment of SOX10 expression of melanomas, carcinomas, mesenchymal neoplasms, of Anatomic Pathology at Sunnybrook Health Sciences Centre is subspecialized, but nerve tumors, and mesotheliomas using rabbit polyclonal anti-SOX10 antibody. the area where all pathologists practices overlap is intraoperative consultation (IOC). Design: In this study, we evaluated this rabbit polyclonal anti-SOX10 for benign We therefore decided to abandon our traditional interesting case rounds in favour of a tissue cross-reactivity, neoplastic expression, sensitivity and specificity on whole tissue model which focussed on IOC. sections and tissue microarray by routine immunohistochemistry. Design: IOC rounds commenced in September 2011 after a planning process involving Results: The results are summarized in the table 1. the departmental executive committee and accreditation from the Royal College of Table 1 Physicians and Surgeons of Canada. Rounds occurred weekly, and were limited to Neoplasm Positive cases/Total cases Sensitivity (%) staff pathologists. Cases which had had IOC performed were selected by the Director Melanoma, conventional 37/39 95% of Surgical Pathology and slides were distributed 2-3 days in advance. The pathologist Melanoma, desmoplastic 11/11 100% who performed IOC functioned as the presenter of the case, while the pathologist Melanoma, spindle cell (metastasis to lymph node 3/3 100% Schwannoma 8/8 100% who signed out the final report was the discussant. Presentations focussed on various Malignant peripheral nerve sheath tumor 3/4 75% aspects of IOC, including history, gross findings, microscopic interpretation, and Granular cell tumor 5/5 100% communication with surgeons. Neurofibroma 20/20 100% Results: On average, 11 pathologists attended the rounds weekly. A range of 2 to 6 Carcinomas 0/153 0% cases were presented per session. Evaluation forms were distributed at each rounds; Lymphomas 0/21 0% Mesenchymal tumors 0/16 0% 93% of responses were that the majority of cases were relevant to practice, and Mesothelioma 0/5 0% 100% indicated that the format was conducive to open discussion. Comments were Thirty seven of 39 conventional melanoma cases showed strong and diffuse nuclear positive, and highlighted openness and educational value. In a one year evaluation, all staining with this antibody. All eleven desmoplastic melanoma cases and three spindle respondents indicated that the rounds provided new ideas for practice and enhanced cell melanomas in lymph nodes were all positive for anti-SOX10. Most of malignant their knowledge. 64% identified a practice issue which needed improvement. 82% peripheral nerve sheath tumors (3/4) were strongly reactive for the antibody. All indicated gynecologic pathology as the area where they would most likely ask for schwannoma, neurofibroma, and granular cell tumor were stained with the anti- assistance of a colleague in IOC. SOX10. None of the 153 cases of carcinomas were positive, in which there were 47 Conclusions: IOC rounds provided a non-threatening environment for discussion of breast ductal carcinomas and 43 colorectal carcinomas. No other carcinomas of GI, challenges in IOC. Strong interest was maintained throughout the first year as evidenced respiratory, genitourinary tract primaries were found positive for anti-SOX10. However, by engagement and attendance. These rounds served as a springboard for developing 7 cases of colorectal carcinoma showed weak cytoplasmic staining. All lymphomas, policies improving intraoperative consultation practice and confronting systemic mesenchymal tumors, and mesotheliomas were negative. In normal benign tissue, the difficulties in providing this service in our hospital. We plan to measure effects on antibody stained benign melanocytes, nerve sheath cells, and myoepithelial cells in diagnostic discrepancy in IOC in the future to see the effects our efforts have had on sweat gland, mammary duct and salivary gland. Prostate basal cells are not stained. patient outcome. Conclusions: Rabbit polyclonal anti-SOX10 antibody is a reliable, ready-to-use, sensitive, and specific IHC biomarker for melanoma, especially desmoplastic and 517 Team-Based Learning in Pathology Residency Training spindle cell melanoma, whether it be, in situ, invasive or metastatic. We have found TC Brandler, J Laser, AK Williamson, J Louie, MJ Esposito. Hofstra-North Shore LIJ it also useful in differentiating spindle cell neoplasms/peripheral nerve sheath tumor School of Medicine, Lake Success, NY. from other mesenchymal neoplasm and carcinoma. Background: Team-Based Learning (TBL) has been integrated into the undergraduate and medical education settings in many institutions to date. However, TBL has not 515 Immunohistochemical Expression of Hormone Receptors been widely introduced into graduate medical education. Our study aimed to measure in Melanoma of Pregnant Women, Non-Pregnant Women and Men: A the effect of TBL on promoting learning and teamwork in the setting of pathology Comparison Study residency training. JH Zhou, KB Kim, P Fox, JN Myers, VG Prieto. University of Texas MD Anderson Design: As part of the daily pathology residency training didactic series, four 2-hour TBL Cancer Center, Houston, TX. sessions were held on different days and facilitated by different attending pathologists. Background: In melanoma, the significant survival advantage of female gender and Topics included molecular diagnostics in lung cancer, interstitial lung disease, death reports of both accelerated progression and improved prognosis in pregnancy have certification and delayed hemolytic transfusion reactions. Individual and group readiness lead to studies of the effect of hormones and hormone receptors, but the results have assurance tests were performed during each TBL session and scores were compared been inconclusive. We examined the immunohistochemical expression of Estrogen using wilcoxon matched-pairs signed ranks tests. After three of the four TBL sessions Receptor α, Estrogen Receptor β and Androgen Receptor in melanoma of pregnant 11-16 residents and rotating medical students completed an 18 item validated and women, non-pregnant women and men. reliable team performance survey which measured the quality of team interactions on Design: Archival paraffin embedded melanoma tissue from 18 pregnant women, 18 a scale of 0 (none of the time) to 6 (all of the time). Mean and standard deviation were non-pregnant women and 18 men at MD Anderson Cancer Center (MDACC) from calculated for each item. 1996 to 2011 was used. The non-pregnant women and men patients were stage-matched Results: Scores on the individual versus group readiness assurance tests were found and age controlled to the pregnant patients. All patients were 20–45 years old at the to be significantly different with P values of 0.001, <0.001, 0.003, and <0.001 for the time of diagnosis. Follow-up was from the initial date of diagnosis to death or the last first through fourth TBL sessions, respectively. The team performance survey received follow up at MDACC until the end of July 2012. The immunohistochemical study was mean scores ranging from 5.3 ± 1.1 to 6.0 ± 0.0. performed using the Bond Max automated system of Leica Microsystems (Buffalo Conclusions: The use of TBL promotes teamwork and learning in a pathology residency Grove, IL) according to the manufacture’s protocol. The exact McNemar’s test was setting. Residents scored higher on the readiness assurance tests when working in teams used for the analysis of ERβ expression between the pregnant group and the control showing the power of team learning and achievement. There was a high quality of team groups. Fisher’s exact and Wilcoxon rank sum tests were used to assess the association interaction as shown by the high scores on the team performance survey. Additionally, between ERβ expression and Breslow thickness, primary tumor site, primary tumor or the ACGME competencies of professionalism and interpersonal and communication metastasis, and the stage of disease. skills are further enhanced by incorporating TBL into pathology residency training. Results: There were 22 cases expressing ERβ: 10 (56%) of pregnant women, 7 (39%) of non-pregnant women, and 5 (29%) of men. The percentage of tumor cells positive 518 Knowledge Retention and Learning Benefits from Utilization ranged from 30% to more than 90%. Only 2 cases expressed ERα, one pregnant woman of a Web-Based Learning Module Methodology for Pathology Training and one man. Both patients had acral lentiginous type melanoma in the toe. None of BM Chung, JH James, JV Groth, J Lee, S Sontag, G Chejfec, EL Wiley. University of the cases expressed AR. Statistical analysis showed a trend that ERβ was more likely Illinois Hospital & Health Sciences System, Chicago, IL; Edward Hines, Jr. Veterans to be expressed in pregnant patients than in the male patients (p=0.073). There was no Association Hospital, Hines, IL. significant difference between pregnant and non-pregnant women (p=0.539). There Background: Recent trends in pathology education of medical students and residents has was no association between ERβ expression and Breslow thickness, primary tumor begun to progressively incorporate both passive and active learning digital technologies site, primary tumor or metastasis, and the stage of disease. such as internet-based case studies, learning modules, and virtual microscopy. This Conclusions: Marginal evidence suggests that ERβ is more frequently expressed in trend is likely to continue with improvements in the capabilities of these technologies, pregnant patients than in male patients. The small sample size may have limited the the introduction of telepathology as a viable and more accepted diagnostic practice statistical power of the study. A large-scale study is needed to look into the expression in clinical care, and greater-than-ever exposure with these technologies during post- of ERβ in melanoma and its association with survival. graduate training. Pilot data from a previous study confirmed the utility of employing a web-based learning module format in resident training. However, whether repeated use of the same learning module by trainees confers additional expertise has not been studied. Education Design: 50 representative examples of normal esophageal epithelium, dysplasia (indefinite, low grade, or high grade), and carcinoma were selected by expert pathologists 516 Intraoperative Consultation Rounds: A Pathologist Educational for inclusion in a self-directed, web-based esophageal dysplasia learning module. and Quality Assurance Initiative Residents accessed the training powerpoint and pre- and post-tests, each containing 25 J Babwah, S Raphael, C Rowsell. Sunnybrook Health Sciences Centre, University of snapshot images, on the Blackboard learning environment in 2011 and 2012. Average Toronto, Toronto, ON, Canada. percent correct answers on pre- and post-tests from both years and p-values via student’s Background: Intradepartmental rounds are an important means of pathologist t-test were calculated and analyzed. education, quality assurance and promoting professional interaction. In a subspecialized Results: This study found a significant increase in diagnostic expertise, defined as department, traditional models where pathologists share difficult or interesting cases may average percent correct answers, in first time users (pre-test: 55%, post-test: 75%; p: 5E-4) compared to repeat users (pre-test: 86%, post-test: 82%; p: 0.211) of this module.