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Importance Approximately 10% to 25% of patients treated with BRAF inhibitors develop cutaneous squamous cell carcinoma (SCC), but the mechanism responsible has not yet been determined. We report what we believe to be the first case in which Merkel cell polyomavirus (MCPyV) and human papillomavirus subtype 17 (HPV-17) were associated with cutaneous SCC that developed during treatment with the BRAF inhibitor dabrafenib. Observations A 62-year-old woman with V600E BRAF -mutant metastatic melanoma enrolled in a phase 1 trial of dabrafenib, a selective inhibitor of V600-mutant BRAF kinase. During the first 6 weeks of treatment, the patient developed multiple skin lesions, including a 6-mm crusted papule on the left eyebrow, which was resected and, on pathology examination, revealed SCC. The DNA extracted from paraffin-embedded tissue was amplified by polymerase chain reaction for detection of MCPyV and epidermodysplasia verruciformis HPV (EV-HPV) types. Analysis of the cloned and sequenced polymerase chain reaction products revealed the presence of MCPyV and HPV-17 DNA. Other EV-HPV subtypes were not detected. Conclusions and Relevance To our knowledge, this is the first report demonstrating the coexistence of MCPyV and HPV-17 in cutaneous SCC. Because both viruses have oncogenic potential, their role in the development of BRAF inhibitor–related SCC merits further investigation.

The CD44 antigen (ECMRIII, Hermes antigen) is a highly glycosylated cell-surface polypeptide involved in diverse cellular functions, including cell adhesion and lymphocyte-homing receptor activity. CD44 is also expressed in vivo by several tumors, including astrocytomas, meningiomas, and colonic adenocarcinomas. In addition, it has been shown that expression of CD44 appears to confer metastatic potential to cell lines derived from certain adenocarcinomas. In the skin, CD44 is normally expressed in epidermal keratinocytes and hair follicular, sebaceous, and eccrine epithelial cells. However, there have been few data with regard to the expression in vivo of CD44 in primary cutaneous neoplasms. Furthermore, there have not been studies in vivo of the possible differential expression of CD44 in primary versus metastatic tumors in the skin. We have examined by immunohistochemistry the expression of CD44 in cutaneous invasive and metastatic squamous cell carcinomas, metastatic adenocarcinomas, and basal cell carcinomas. All invasive and metastatic squamous cell carcinomas, as well as metastatic adenocarcinomas, strongly expressed CD44 ; however, basal cell carcinomas were nonreactive or showed only focal, minimal reactivity. Adjacent normal skin demonstrated CD44 immunoreactivity throughout the epidermis, including the basal layer. In addition, hair follicles and sebaceous and eccrine glands expressed CD44. The results suggest that expression of CD44 in these cutaneous epithelial tumors is not related to malignant transformation, but instead may be related to tumor progression and the ability to metastasize.

It has been hypothesized that the ability for neoplastic growth of epithelial-derived neoplasms depends upon the stroma. There are currently some studies which show that the stroma surrounding basal cell carcinomas (BCC) is derived from the tumor. In contrast, other studies provide evidence that the stroma is a host-derived response to the tumor. In order to further examine the nature of stroma enveloping cutaneous epithelial neoplasms, we examined a series of tumors which contain abundant stroma, including morpheic type BCC (MBCC), desmoplastic trichoepitheliomas (DTE), and microcystic adnexal carcinomas (MAC). The spindle-shaped cells surrounding the epithelial islands of the two malignant tumors, MBCCs and MACs, were negative in 70% and 100% of cases, respectively, for CD34. In contrast, the spindle-shaped cells surrounding the islands of the benign DTEs were positive for CD34 in 80% of cases. The results suggest that whereas stromal cells surrounding DTEs resemble the CD34-positive perifollicular cells, the spindle-shaped stromal cells surrounding MBCC and MAC are CD34 negative, and may be derived from sources other than the normal mesenchymal tissue surrounding cutaneous appendages.

Cutaneous melanoma (CM) is the most serious form of skin cancer. DNA repair has been implicated in the etiology of CM in patients of xeroderma pigmentosum (XP), an autosomal recessive genetic disorder of DNA repair. We have reported that reduced cellular DNA repair capacity (DRC, as assessed by an in vitro lymphocyte-based assay) for removing UV-induced DNA damage is an independent risk factor for sporadic CM and may contribute to susceptibility to sunlight-induced CM in the general population. Recently, we completed a genome-wide association study (GWAS) of melanoma and we also have data on DRC phenotype measured as luciferase activity in the in vitro host-cell reactivation assay using study subjects' lymphocytes for 1042 CM patients. The life-style questionnaire data are also available for these patients included in the GWAS. This is a unique study population and opportunity for us to identify genetic predictors of DRC in CM patients using a genome-wide approach. We found that the DRC varies 6 folds among 1042 CM patients and that the history of sunburn modified the DRC levels. However, we did not find the significant difference within categories of other demographic, exposure and clinical factors including age, sex, smoking and drinking status, the presence of moles and dysplastic nevi, family history of any cancer in first-degree relatives, eye color, hair color, skin color, freckling in the sun as a child, tanning ability, tumor stage, thickness, clark level, ulceration and anatomic site. We applied a generalized linear model with SNPs as predictors and DRC (a continuous variable) as the outcome. The covariates of age, sex, DRC assay-related variables were adjusted in the model. Several suggestive loci contributing to the DRC phenotype have been identified at the significance level of 10−5, including rs1799787 (intron) and rs1799793 (codon 312) in ERCC2/XPD in all CM patients, and rs12342322 near RASEF in 931 CM patients with stage I – IV. The results indicate that DRC phenotype is genetically determined by the genes involved in the nucleotide excision repair pathway and that newly identified SNPs may help understand the underlying mechanism in DRC's role in CM initiation and progression [This work was supported by National Institute of Health grants R01CA100264 (Q. Wei), P50CA093459 (E. Grimm), and P30 CA016672 (The University of Texas M. D. Anderson Cancer Center)]. Citation Format: Li-E Wang, Hongliang Liu, Jeffrey E. Gershenwald, Victor G. Prieto, Elizabeth A. Grimm, Jeffery E. Lee, Christopher I. Amos, Qingyi Wei. Genetic predictors of DNA repair capacity in melanoma patients: A genome-wide approach. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 66.

8537 Background: The advent of targeted therapy has revolutionized the treatment of cancer. The mutant BRAFV600E protein is found in over 50% of melanomas and thyroid carcinomas, resulting in elevated kinase activity, increased mitogen-activated protein kinase (MAPK) pathway signaling, and cell proliferation. Vemurafenib and PLX4720 were designed to selectively inhibit the BRAF kinase, and clinical trials of vemurafenib in metastatic melanoma have demonstrated a response rate of over 50% and an overall survival advantage over standard dacarbazine therapy. Approximately 20-30% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) highlighting the importance of understanding toxicities associated with this drug. Paradoxical ERK activation in BRAF wild-type, RAS-mutant cells is thought to be the major mechanism by which this occurs, as evidenced by the presence of RAS mutations in 60% of such lesions. Methods: Using a combination of BRAF-wild-type cSCC cell lines, primary human keratinocytes, as well as a UV mouse model of cSCC and human cSCC samples, we identified novel effects of BRAFi on apoptosis. Results: Here we show an unexpected and novel effect of vemurafenib and PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases. JNK signaling and apoptosis are suppressed in cSCC lesions arising in vemurafenib-treated patients as well as in irradiated mouse skin. This occurs independently of paradoxical ERK signaling and in the presence of MEK inhibitor. Treatment with PLX4720 greatly accelerates the development of UV-induced cSCC in mice without Ras mutations. Kinome screening identified ZAK and MKK4 (MEK4 / MAP2K4) kinases as inhibited by vemurafenib, leading to suppression of MKK4 and MKK7 (MAP2K7) phosphorylation. Knockdown of inhibited off-target kinases recapitulates these anti-apoptotic effects of vemurafenib. Conclusions: Our results implicate suppression of JNK signaling, independent of ERK activation, as an additional, complementary mechanism of adverse effects of vemurafenib. This has broad implications for combination therapies with other modalities that induce apoptosis and for the long-term use of vemurafenib in the adjuvant setting.

Melanoma is an aggressive malignancy with a relatively high metastatic rate, responsible for almost 60% of lethal skin tumors. Despite numerous efforts, there is still a need to better understand the molecular pathways involved in malignant melanoma metastasis. In recent years, there have been several studies highlighting the role of deregulated microRNAs (miRNAs) expression in tumor progression and metastasis. DICER is a central regulator of miRNA maturation and recently, has been shown to be upregulated in cutaneous melanoma at both protein and mRNA level. Arur et al. [1], reported DICER to be one of the ERK substrate, and this activation results in its nuclear translocalization (personal communication). However, it is unknown whether differential cellular expression of DICER contributes to the pathogenesis of melanoma. In order to test the hypothesis that aberrant nucleo-cytoplasmic expression of DICER plays a significant role in melanoma progression, we have performed semiquantitative immunohistochemical analysis of DICER in benign nevi (n=17), primary melanomas without metastasis (n=38) and with metastasis (n=27), and melanoma metastases (n=34 cases). A significant positive correlation was observed between the percentage of positive cells (p=0.003) and staining intensity (p=0.01) for nuclear DICER with progression from benign nevi to primary melanoma. Interestingly, primaries without metastasis had significantly higher levels of DICER immunopositivity in tumor cells than primaries with metastasis (p=0.02). Furthermore, metastatic melanomas showed significantly larger number of cells having intense immunoreactivity for DICER protein in nucleus than the primary melanomas (p=0.02). Significantly larger percentage of tumor cells showing DICER expression in the nucleus were observed in those lesions having ulceration (p=0.007) and vertical growth phase, which are well-established markers of poor clinical outcomes in patients with cutaneous melanoma. On the other hand, lesions showing no vascular invasion expressed higher levels of DICER in the cytoplasm (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1104. doi:1538-7445.AM2012-1104

Derangement of the p53 tumor suppressor gene has been implicated in the aetiology of a wide range of human neoplasias. We have previously determined that overexpression and mutation of the p53 gene in cultured metastatic melanomas is low (11%). However, two recent immunohistochemical studies have reported that > 85% of malignant melanoma specimens overexpress mutated p53 protein. In an effort to resolve this contradiction in the published literature, we have re-evaluated a range of cultured and non-cultured melanocytic lesions for the occurrence of point mutations in the p53 gene using DNA- and RNA-dependent single strand conformation polymorphism (RNA-SSCP) and direct DNA sequencing of polymerase chain reaction (PCR) amplified DNA, and overexpression of the p53 protein using immunohistochemistry. We found point mutations in 25% (9 of 36) of cultured melanomas and 0% in 34 fresh melanoma biopsies; however, increased p53 expression was found in 42% of paraffin-embedded melanoma specimens and 7% of benign lesions. The low frequency of p53 point mutations and high frequency of p53 expression suggests that derangement of the p53 gene by point mutations is not a common perturbation in the majority of melanoma cells, and that overexpression of p53 in this tumour type is due to a mechanism other than point mutation.

Melanoma is an aggressive malignancy with a relatively high metastatic rate, responsible for almost 60% of lethal skin tumors. Despite numerous efforts, there is still a need to better understand the molecular pathways involved in malignant melanoma metastasis. FPR and its variant FPRL1, which belong to the GPCR family, have been shown to be involved in metastasis of glioma and ovarian tumor cells via activation of various signaling cascades. It is unknown whether expression of these receptors contributes to the pathogenesis of melanoma. In order to test the hypothesis that aberrant expression of these GPCRs plays a significant role in melanoma progression, we have performed semiquantitative immunohistochemical analysis of FPR and FPRL1 in benign nevi (n=49), primary melanomas with and without metastasis (n=130), and melanoma metastases (n=47 cases). A significant positive correlation was observed between the percentage of positive cells and staining intensity for FPR (p=0.001) and FPRL1 (p=0.009) with progression from benign nevi to primary melanoma. Significantly higher levels of FPR were observed in those lesions having higher Breslow thickness (p=0.044) and ulceration (p=0.019), which are well-established markers of poor clinical outcomes in patients with cutaneous melanoma. On the other hand, lesions showing a radial growth pattern expressed low levels of FPR (p=0.027). A gain in FPRL1 staining was seen in lesions displaying microscopic satellitosis (p=0.051). Disease-specific survival was significantly shorter in patients with increased cytoplasmic expression of FPR (log rank test, p=0.022). Thus, overexpression of FPR and FPRL1 correlates with disease progression and high-risk clinical and histologic features in melanoma. In addition, FPR expression is associated with shorter disease-free survival. Therefore, these markers may be an important therapeutic target in patients with melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2011-5220

Lichen planus-like keratosis (LPLK) (benign lichenoid keratosis) is a common skin lesion that shows some morphologic features of lichen planus (LP) and lichenoid actinic keratosis (LAK). To try to detect differences among these three entities, immunohistochemical staining for S100 protein and HLA-DR (with the antibody LN3) was performed in 31 cases of LPLK, 26 of LAK, and 25 of LP. Langerhans cells (LC) were counted per linear mm of epidermis in the S100 and LN3 slides. With anti-S100 staining, LP cases showed higher numbers of LC (mean = 25.3, SE = 2.84, median = 21.2) than did LPLK (mean = 17.3, SE = 2.26, median = 14.5) and LAK (mean = 9.7, SE = 1.5, median = 5.4). With LN3 stains, LP cases also showed higher numbers of LC than did LPLK and LAK. These results suggest that the involvement of LC in the production of lesions may be different in these three entities. However, due to the overlap in distribution of values observed, the use of these stains does not allow a definite diagnosis to be made exclusively based on the number of LC.

Lichen planus-like keratosis (LPLK) is a common skin lesion that has some morphologic features of lichen planus (LP) and lichenoid actinic keratosis (LAK). Although most authors consider LPLK to be a distinct lesion, surgical pathologists are often unfamiliar with it. We examined in detail the clinical and histologic features of LPLK in 100 consecutive cases. In our series, LPLK lesions occurred mostly in late-middle-aged individuals, on the trunk (64%) and extremities (31%) and predominantly in women (73%). Among the 14 histologic parameters that were studied, most LPLK had hyperkeratosis (81%) with hypergranulosis (77%), focal acanthosis (75%), and focal parakeratosis (59%). Solar elastosis was observed in 48% of the cases. Only a few cases had eosinophils (15%) or plasma cells (7%) in the inflammatory infiltrate. The adjacent skin was available for study in 71% of the specimens, and only 26.8% of the cases had an adjacent lesion, most frequently seborrheic keratosis (8.4%), solar lentigo (7%), and actinic keratosis (5.6%). According to our results, the most consistent features observed in LPLK are a lichenoid lymphocytic infiltrate with hyperkeratosis, hypergranulosis, focal acanthosis, and focal parakeratosis, without prominent atypia of keratinocytes.

e21034 Background: Although it is well-established that melanomas can elicit immune responses, they more often produce factors that adversely impact or block antitumor immunity. Suppressive immune cells such as Foxp3+ regulatory T cells (Treg) and tumor-infiltrating macrophages have been implicated as poor prognostic indicators in other cancer etiologies, but their prognostic value in melanoma has remained unclear. Methods: We performed an immunohistochemical (IHC) analysis of 62 melanoma tumors comprising a spectra of disease stages to assess tumor infiltration by T cells (CD3+, CD4+, CD8+ and Foxp3+ subsets), B cells (CD19+), and macrophages (CD68+). To assess the potential negative impact of tumors on regional immunity, we also analyzed a set of uninvolved regional lymph nodes (LN) harvested from sentinel node positive basins of 28 stage III melanoma patients. All immune markers were correlated with overall survival by univariate Cox regression analyses. Results: Overall, relatively few Foxp3+ T cells ...

8556 Background: Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous tumor that has a propensity for locoregional and distant metastasis. Despite this overall aggressive biology, little is ...

Cutaneous melanoma is an increasingly important public health problem, as the incidence of this disease has increased 15-fold during the last four decades, more than any other malignancy. There is a great need for biomarkers that can be used both for both diagnosis and prognosis in melanoma. Identification of such biomarkers may provide insight into the pathogenesis of this disease, and also suggest rational therapeutic approaches. Protein translation has been shown to play a significant role in carcinogenesis in various organ systems. Several pathways upstream of protein translation are deregulated during melanoma development, including the RAS-RAF-MAPK and the PI3K-AKT kinase cascades. It is unknown whether dysregulated translational machinery contributes to the pathogenesis of melanoma. In order to test the hypothesis that dysregulation of protein translation plays a significant role in melanoma, we have performed immunohistochemical analysis to evaluate expression of phospho-4E-BP1 (p4E-BP1), phospho-eIF4E (peIF4E) and total eIF4E in benign nevi (n=49), primary melanomas (n=130), and melanoma metastases (n=47 cases) specimens obtained from the Department of Pathology of The University of Texas M. D. Anderson Cancer Center. A significant positive correlation was observed between the percentage of positively staining cells and staining intensity for p4E-BP1 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1486.

9022 Background: Activating mutations of BRAF are highly prevalent in melanoma. However, multiple lines of evidence suggest that other pathways must also contribute to this disease. Activation of the PI3K-AKT pathway has been implicated in melanoma by mutations of NRAS and PTEN. Little direct information is known about the activation of the PI3K-AKT signaling pathway in melanoma, particularly in metastases. Methods: Proteins isolated from 99 frozen melanoma tumors were measured by reverse phase protein arrays (RRPA). A total of 53 proteins were assessed using validated antibodies. Activating mutations were assessed by mass spectroscopy-based genotyping. Activation of AKT (phospho-AKT) was compared to mutations and anatomic sites. Results: Samples from 75 regional metastases (LN or in-transit) and 24 distant metastases were analyzed. Technical replicates (same lysate) and biological replicates (same tumor, different lysates) demonstrated average Pearson correlation coefficients (r) of > 0.90, supporting the high technical quality of the analysis. A positive correlation was observed between levels of p-AKT and known AKT substrates p-GSK3 and p-TSC2 (p< 0.001 for both), and a negative correlation with PTEN (p < 0.01), supporting the maintenance of phosphorylation events during sample processing. Relative differences in p-AKT by RPPA were also confirmed by immunohistochemistry of representative tumors. Tumors with BRAF mutations had higher levels of p-AKT than tumors with NRAS mutations (p = 0.03). Indeed, tumors with NRAS mutations had p-AKT levels similar to tumors wild-type for BRAF and NRAS (p =0.73). Detailed analysis demonstrated that all tumors with elevated p-AKT had low PTEN expression. Similar results were seen in human melanoma cell lines. Analysis of distant metastases demonstrated that brain metastases had higher levels of p-AKT, p-GSK3, and p-TSC2, and lower levels of PTEN, compared to metastases to the lung or liver. Conclusions: AKT activation in melanoma correlates with PTEN expression, and does not correlate with NRAS mutation. Activation of the PI3K-AKT pathway may contribute to the aggressiveness of brain metastases. These findings have clinical implications for targeting this pathway, and demonstrate the feasibility and potential of RPPA analysis of signaling pathways in melanoma. No significant financial relationships to disclose.

3021 Background: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a promising therapy for the treatment of metastatic melanoma. Trials at the NCI have shown a 50% response rate. However, outside of the NCI, ACT has been quite limited and little is known about how patient characteristics and prior treatment affect large-scale TIL expansion needed for treatment. Methods: As part of a phase II melanoma ACT clinical trial at M. D. Anderson Cancer Center, we evaluated how clinical characteristics such as age, gender, biopsy site, melanoma type, primary tumor location, and prior treatment of patients (N=134) affected TIL expansion rates. 159 isolated metastatic melanomas (19 patients having >1 tumor harvest) were analyzed for the ability to expand a minimum of 40 million TIL, considered a minimum number for the subsequent rapid expansion protocol (REP) needed for TIL infusion. Effects of these parameters on TIL anti-tumor reactivity and TIL/tumor fragment were also analyzed. Statistical analysis was done by either Fisher's Exact test, Wilcoxon Rank Sum test, or Kruskal-Wallis Test. Results: 54.7% of the samples evaluated generated ≥ 40 million TIL, and 70% of 70 samples tested had anti-tumor reactive TIL. There was a significant relationship between age categories (p=0.0216) and gender (p=0.0164) on TIL expansion success. Younger patients did the best and females did better than males (odds 2.19, p=0.044). The site of the primary melanoma affected success of TIL expansion (p=0.0059), while no effect of tumor biopsy site and histological type was found. Prior chemotherapy (or biochemotherapy) greatly reduced TIL growth (odds 0.32, p=0.010) when compared to no prior therapy. Multivariate linear regression analyses showed that prior chemotherapy decreased TIL/tumor fragment yield by 6.19 million (p=0.0004) while immunotherapy increased it by 3.51 million (p=0.16) compared to no prior therapy. Total TIL yield or TIL/tumor fragment did not correlate with patient overall survival. Conclusions: Parameters such as age, sex, primary melanoma site, and prior therapy significantly affect TIL expansion and should be considered in selecting patients for melanoma ACT. No significant financial relationships to disclose.

Sezary syndrome (SS) is a T-cell lymphoproliferative disorder involving the blood, skin and lymph nodes associated with erythroderma. Other cutaneous manifestations of SS include palmoplantar keratoderma, ectropion, and alopecia. Two clinical patterns of SS can be defined: “secondary” SS arising from systemic spread of long-standing mycosis fungoides (MF) and “primary” SS with a much shorter prodromal phase characterized by pruritus, xerosis and scaling culminating in erythroderma. Most previous studies examining the histological features of SS have not distinguished between these two different clinical patterns, and have emphasized that the histological features of SS mostly resemble those of typical MF, including epidermotropism and/or band-like superficial dermal lymphoid infiltrates. In this study, we have concentrated on the histological findings of primary SS and correlated them with the clinical and hematologic parameters at the time of skin biopsy. We note that dermal perivascular lymphoid infiltrates predominate in a majority of primary SS cases, raising the differential diagnosis of benign dermatoses, rather than resembling the classical pattern of MF. This impression is further complicated by commonly observed epidermal changes such as spongiosis, acanthosis and parakeratosis that are likely secondary to tumor-associated pruritus and secondary lichenification.

p63, a recently identified homologue of p53 gene, has been reported to be essential in the development of epithelia and is mainly expressed by basal and myoepithelial cells. The purpose of this study was to investigate the pattern of p63 expression in cutaneous adnexal neoplasms and to assess its possible value in the differential diagnosis of primary cutaneous neoplasms versus adenocarcinomas metastatic to the skin. Immunohistochemical analysis using p63 monoclonal antibody (clone 4A4) was performed on formalin-fixed, paraffin-embedded archival tissue from 20 benign adnexal tumors, 10 malignant adnexal tumors, and 14 adenocarcinomas metastatic to the skin. The expression of p63 was evaluated in epidermal cells, skin appendages and metastatic tumor cells. p63 was consistently expressed in the basal/suprabasal cells of epidermis and cutaneous appendages, including the basal/myoepithelial cells of sweat glands. Thirteen out of 20 (65%) benign adnexal tumors showed strong p63 expression; the remaining 7(35%) cases had over 50% p63-positive nuclei. All primary cutaneous carcinomas, including adenocarcinomas, expressed p63. In contrast, none of the metastatic adenocarcinomas to the skin was positive for p63 (p

Background: MAPK, AKT, and c-Kit are major signaling molecules. This study evaluates the expression of the activated forms of these molecules in melanocytic lesions. Design: Tissue microarrays were constructed using formalin-fixed, paraffin-embedded archival tissue from 14 benign nevi (BN), 16 dysplastic nevi (DN), 18 malignant melanomas (MM), and 25 metastatic malignant melanomas (MMM). Two control cases (nevus and melanoma) were included in all three microarray blocks. Immunohistochemical analysis of p-MAPK, p-AKT and p-c-kit was performed using standard technique. Both the intensity and the number of labeled cells were recorded in a semiquantitative fashion. Results were analyzed using Chi-square test, with p Results: c-kit expression was highest in BN compared to DN, MM and MMM that showed similar expression. Among MMM, the visceral metastases had the strongest c-kit expression. p-AKT showed decreased expression with progression of the melanocytic lesions. The differences for p-c-kit and p-AKT were highly significant (0.04 and 0.0001 respectively). There was no statistically significant difference in expression of p-MAPK. Conclusion: Since the observed changes apply to the activated forms of AKT and c-kit these molecules are likely involved in melanoma progression. These findings may help determine the applicability of targeted therapies such as gleevec.

Protein tyrosine kinases (PTK) are proteins that may play importants roles in survival, proliferation and progression to metastasis in several tumors, including melanoma. There is little information on the expression pattern of the phosphorylated (i.e. activated) forms of such kinases in melanoma and their impact on overall survival (OS). We sought to determine the expression, by immunohistochemistry, of phosphorylated MAP kinase (pMAPK), Akt (pAkt) and C-kit (pCkit) in primary and metastatic melanoma, and their correlation with OS. Fifteen patients with Stage III and Stage IV (metastatic melanoma) disease were randomly selected from the UT MD Anderson database. Expression was rated by two investigators on intensity (negative, weak, moderate, and strong) and proportion of reactive tumor cells (0 = 25–75%, 3 = >75%). Eleven of the 15 tumors (primary and/or metatasis) exhibited pMAPK expression (73%). Corresponding percentages were 100% for pAKT and

20024 Background: With current therapies, the estimated 10-year survival rate for stage IIIC melanoma is < 30%. Our previous neoadjuvant studies of combination chemotherapy either alone or in combi...

Purpose Caspase 8 is an apoptosis-related cysteine peptidase that is mainly involved in the death receptor (FAS/FASLG) pathway. Studies have shown that the CASP8 D302H minor variant genotypes protect against breast cancer risk in different ethnic groups and that the minor functional -652 6N del allele protected against several types of cancer in a Chinese population. This study was to identify such an association in Caucasians. Methods We conducted a hospital-based case-control study of 805 cutaneous melanoma and 1023 squamous cell carcinoma of the head and neck (SCCHN) and 1584 cancer-free controls to assess the association between two selected genetic polymorphisms (-652 6N ins/del and D302H) of the CASP8 gene and risk of melanoma and SCCHN. We genotyped these two polymorphisms by the PCR-based assays and estimated related haplotypes for all subjects and detected camptothecin-induced apoptotic cells in cultured lymphocytes from 170 cancer-free controls. We used multivariate unconditional logistic regression analysis to calculate the odds ratios and their 95% confidence intervals and assessed gene-environment interactions. Results We found that the CASP8 -652 6N del variant genotypes or haplotypes carrying the -652 6N del allele protected against risk of both melanoma (adjusted OR = 0.74, 95% CI = 0.58 to 0.95) and SCCHN (adjusted OR = 0.73, 95% CI = 0.57 to 0.95), whereas CASP8 302H variant genotypes protected against risk of melanoma (adjusted OR = 0.70, 95% CI = 0.57 to 0.85) but not SCCHN (adjusted OR = 1.06, 95% CI = 0.87 to 1.30), compared to the common homozygous genotypes. We also found that the number of the CASP8 -652 6N del (but not 302H) variant tended to be correlated with increased levels of camptothecin-induced apoptosis. Conclusion The CASP8 -652 6N del variant is risk factor for the studies cancers in Caucasians and may have an effect on apoptosis in response to DNA damage through the mitochondrial pathway, in addition to the FAS/FASLG-mediated pathway. Further validation by population-based case-control studies and mechanistic studies are warranted.

8548 Background: EMD 121974 is a selective antagonist of avβ3 integrin, which promotes the proliferation of tumor-associated endothelial cells and potentially the survival of melanoma cells expressing avβ3 integrin. We conducted a randomized phase II trial of cilengitide in pts with MM to evaluate the clinical efficacy at 2 different doses. Methods: Pts with stage IV or unresectable stage III non-choroidal melanoma who had no more than 1 prior systemic therapy were enrolled. Pts at least 18 years of age and with ECOG performance status of 0 to 2 were eligible. All pts underwent baseline tumor biopsy and were randomly assigned to either 500 mg or 2,000 mg intravenous (IV) EMD 121974 twice weekly, using the following stratification factors: 1) prior systemic treatment; 2) visceral metastases; 3) serum lactate dehydrogenase level; 4) tumor avβ3 overexpression, where overexpression is defined as > 25% of melanoma cells staining positive. The primary objective of this study was to determine the progression-free survival rate at 8 weeks. Results: Twenty-nine pts were enrolled, and 26 pts (14 at 500 mg; 12 at 2,000 mg dose) were treated. Patient characteristics for 500 mg and 2,000 mg arm, respectively, are as follows: median age, 57 and 61; percentage (% age) of male, 50% and 50%; % age of ECOG performance status of 0, 79% and 58%; % age of stage IV, 79% and 75%; % age of tumor avβ3 overexpression, 21% and 25%. Three of 26 pts were progression-free at 8 weeks (2 at 500 mg; 1 at 2,000 mg dose). One pt at 2,000 mg had a prolonged partial response after initial 28% enlargement of target lesions. There were no grade 3 or 4 adverse events (AEs) except one pt with grade 3 lymphopenia at 2,000 mg. Although both doses of EMD 121974 were well tolerated, the 2,000 mg was associated with higher incidences of grade 2 fatigue, arthralgia, lymphopenia, peripheral neuropathy, and GI AEs. Optional tumor biopsies were performed on day 8, and the correlative studies to examine the molecular changes in the tumor are currently in progress. Conclusions: IV EMD 121974, 500 or 2,000 mg twice weekly, was well tolerated but had minimal clinical efficacy as a single-agent for MM. Supported by NCI grant N01 CM-17003 and CA16672 No significant financial relationships to disclose.

8050 Background: Given the reported presence of frequent mutation in NRAS and BRAF in many melanocytic lesions, the association of mutations activating the RAS/RAF/MAPK pathway with tumor progression in melanoma remains unclear. We compared the NRAS and BRAF mutation status with the levels of activated MAPK in paired primary and metastatic melanomas. Methods: 15 patients with material from both primary and metastatic lesions were assessed. Four patients had metastasis at initial diagnosis, the remaining patients developed metastasis from 8.5 to 66 months (mos) following initial diagnosis (median 24.1 mos). Mutations in BRAF (exons 11 and 15) and KRAS and NRAS (exons 1 and 2) were assessed by quantitative pyrosequencing. MAPK levels were assessed by immunohistochemistry on paraffin sections using phosphor-44/42 (Thr202/Tyr204) polyclonal antisera. Results: Activating NRAS point mutations were found in 3 of 15 patients, with 2 detected in the metastatic lesions; both of these had upregulation of activated MAPK compared to the primary lesions. BRAF V599 point mutations were found in 3 of 15 patients and present in both the primary and cognate metastatic lesions. All cases with BRAF or NRAS mutations had moderate to high levels of activated MAPK, as compared to only 2 of 9 patients without such mutations. The correlation of mutational status and clinical course revealed that NRAS/BRAF mutations and/or MAPK activation did not negatively impact overall survival. Conclusions: Molecular alterations in the RAS signaling pathway are associated with activated MAPK in both primary and metastatic melanoma. NRAS mutations and MAPK activation may be preferentially associated with disease progression, as opposed to BRAF mutations. However, neither NRAS/BRAF mutation nor activation of MAPK signaling pathway in primary and/or metastatic melanoma was significantly associated with poor disease outcome. Further molecular dissection using the archived material from two large melanoma neoadjuvant trials conducted in 1988 and 1994 is ongoing to determine the prognostic and predictive power of these biomarkers. No significant financial relationships to disclose.

8004 Background: Therapeutic lymph node dissection (TLND) has long been the standard of care for melanoma metastatic to the regional LNs and is commonly used for patients (pts) with microscopic disease identified by SLN biopsy. We and others have demonstrated that additional histologically positive (pos) nodes are identified in 8%-33% of completion LN dissection (CLND) specimens. The purpose of this study was to determine predictors of additional LN involvement in pts. with pos SLNs. Methods: Pts with clinically node-negative melanoma who underwent SLN biopsy at our institution between 1991 and 2003 and had pos SLNs were identified. Patient and primary tumor factors, number of pos SLNs, and extent of microscopic disease within the SLN were evaluated as potential predictors of pos NSLNs. Results: Overall, 359 (16%) of 2,203 pts had a pos SLN. Pos NSLNs were identified in 48 (14%) of the 343 pts with pos SLNs who underwent CLND. On univariate analysis, several markers of SLN microscopic tumor burden, tumor thickness >2mm, age >50 years, and Clark level >III were predictive of additional pos nodes in the TLND specimen. Primary tumor ulceration was not predictive. On multivariable logistic regression analysis, markers of microscopic tumor burden (large SLN met focus, large SLN tumor square area, presence of SLN extracapsular extension, and synchronous subcapsular and intramedullary SLN deposits) were the most powerful independent predictors of pos NSLNs; tumor thickness >2mm also predicted additional disease. A model was established using tumor thickness and SLN tumor burden to identify pts with low, intermediate, or high risk for NSLN involvement (Table). Conclusions: In melanoma pts with ≥1 pos SLN, SLN tumor burden and primary tumor thickness may be useful in identifying a group at low risk for pos NSLNs. This information is valuable in designing clinical trials in which pts may be randomly assigned to TLND versus observation alone after removal of a pos SLN. [Table: see text] No significant financial relationships to disclose.

9530 Background: Gastrointestinal stromal tumors (GISTs) originate from interstitial cells of Cajal and represent the most common mesenchymal tumor of GI tract. The activating mutations of KIT and platelet-derived growth factor receptor α (PDGFRA) have been demonstrated in 88% and 6% of GISTs respectively. Multiple genetic events are involved in tumor initiation and progression. Activation of an oncogene can result from a single hit of gain-of-function mutation, but two hits leading to loss of heterozygosity (LOH) are necessary for inactivation of a tumor suppressor gene for the initiation of neoplasia. Little is known about the significance and mechanism of LOH of oncogenes in tumor progression. The LOH of activating-oncogenes is not infrequent, i.e. in more than 8% of GIST. These GIST clones with LOH prevail and dominate, strongly suggestive of proliferative and metastatic advantages. However, the significance and mechanisms of LOH acquiring homozygous activating-oncogene mutation remain unknown. Methods: Genomic DNA and cDNA sequencing analysis of KIT, single-nucleotide polymorphisms (SNPs), immunohistochemistry of GISTs clones, and 3D structural analysis of the mutated KIT. Results: Real-time genetic studies in GIST show initial development of overexpression of KIT without mutation, to coexistence of various heterozygous-activating mutations; eventually, one clone dominates. Comparisons within the same patient show that clonal evolution from heterozygous to LOH acquiring homozygous (diploid) KIT-activating mutation results in dominance of the latter, with augmented KIT signaling and doubling of mitotic figures. Using SNPs for allelotyping, we found that mitotic nondisjunction, rather than mitotic recombination, represents an important mechanism of the second hit. Conclusion: GIST clones with LOH acquiring homozygous KIT-activating mutation gain a selective advantage over the heterozygous counterpart. Mitotic nondisjunction, in addition to the commonly recognized mitotic recombination, is an important and perhaps the primary mechanism of LOH acquiring homozygous KIT-activating mutation during GIST progression. No significant financial relationships to disclose.

7528 Background: Imatinib mesylate (Gleevec™), an oral inhibitor of phosphorylation of certain tyrosine kinases (TK), is active in gastrointestinal stromal tumors which are chemotherapy resistant. Perhaps MM may respond to Gleevec, especially if the tumor cells or microenvironment express one of the known TK targets: KIT, PDGF receptors α or β, c-abl, or ARG. Methods: Tumor biopsies from MM patients (pts) were screened by immunohistochemistry (IHC) for TK expression in more than 25% of the cells, as a condition for enrollment in a Phase II trial testing Gleevec @ 400 mg bid. Results: All 31 pts screened met this criterion and 21 of them were treated with Gleevec (median age 58, male [13]; stage IV [19]; PS 0–1 [20]; prior Rx: none [8], Intron-A [8], one chemotherapy [5]). The one patient with strongest expression of KIT in over 75% of tumor cells tolerated treatment for one year and achieved near complete response in in-transit, inguinal/iliac, and lung metastases with marked PET scan improvement as early...

7257 Background: SCLC cell lines commonly express c-kit and its ligand, suggesting an autocrine loop promoting cell growth. IM inhibits c-kit kinase activity. SCLC cells treated with IM in vitro undergo cell cycle arrest. In this study we investigated the feasibility of combining IM with cytotoxic chemotherapy for extensive disease SCLC. Methods: Six patients received P (30 mg/m2) and I (65 mg/m2) on days 1 and 8 every 21 days for four cycles. IM (300 mg) was given daily during chemotherapy and then as maintenance until progression of disease. Immunohistochemistry (IHC) for potential IM targets was done on patients' pre-treatment biopsies. Results: The first 3 patients experienced significant neutropenia that required GCSF support. One patient died during treatment from neutropenic fever. GCSF was added prophylactically to the subsequent 3 patients who did not experience significant neutropenia. Five patients had grade 2 diarrhea and one had grade 3 diarrhea. One patient had a deep vein thrombosis. This p...

REPORT OF A CASE A 71-year-old white man with a history of multiple actinic keratoses, basal cell carcinomas, and squamous cell carcinomas of the head, trunk, and extremities presented with a slightly raised, erythematous, poorly circumscribed, scaly plaque (4×2 cm in diameter) on the right side of his chest ( Figure 1 ), of uncertain duration. A biopsy specimen was obtained from the center of the lesion. Histologic findings are depicted in hematoxylin-eosin-stained sections ( Figure 2 ). What is your diagnosis? DIAGNOSIS: Malignant melanoma in situ, amelanotic type. HISTOPATHOLOGIC FINDINGS Routine histologic examination showed a markedly atrophic epidermis with hyperkeratosis and focal parakeratosis. There was a diffuse, broad proliferation of melanocytes arranged as single cells or in irregular nests along the dermoepidermal junction and at higher levels of the epidermis. The cells had abundant, clear cytoplasm without evident melanin pigment and hyperchromatic nuclei with prominent eosinophilic nucleoli. The dermis contained abundant

Background and Design: Trichoepithelioma is a benign skin tumor with follicular differentiation, which sometimes is difficult to distinguish clinically and histologically from basal cell carcinoma. One of the most helpful differences is the histologic appearance of the stroma. CD34 is an antigen known to stain the spindle-shaped cells located around the middle portion of normal hair follicles. We have stained formalin-fixed, paraffin-embedded sections of 16 trichoepitheliomas and 19 basal cell carcinomas for CD34 (anti-HPCA-1, Becton Dickinson, San Jose, Calif) to detect differences in the staining pattern and to facilitate discrimination of these two types of tumors. Results: The spindle-shaped cells surrounding the islands of trichoepithelioma cells were focally strongly positive for CD34. In all basal cell carcinomas, the spindle-shaped cells surrounding the nests of tumor cells were negative; in these areas only the blood vessels were positive with this antibody. Conclusions: CD34 staining pattern differentiates between trichoepithelioma and basal cell carcinoma. CD34 stain may be helpful in distinguishing between these two tumors on small punch biopsies or in difficult diagnostic cases. (Arch Dermatol. 1994;130:589-592)

The 2005 National Institute of Health (NIH) Consensus Conference outlined histopathological diagnostic criteria for the major organ systems affected by both acute and chronic graft-versus-host disease (GVHD). The 2014 Consensus Conference led to this updated document with new information from histopathological studies of GVHD in the gut, liver, skin, and oral mucosa and an expanded discussion of GVHD in the lungs and kidneys. The recommendations for final histological diagnostic categories have been simplified from 4 categories to 3: no GVHD, possible GVHD, and likely GVHD, based on better reproducibility achieved by combining the previous categories of "consistent with GVHD" and "definite GVHD" into the single category of "likely GVHD." Issues remain in the histopathological characterization of GVHD, particularly with respect to the threshold of histological changes required for diagnostic certainty. Guidance is provided for the incorporation of biopsy information into prospective clinical studies of GVHD, particularly with respect to biomarker validation.