Faculty Bibliography

BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16

Despite the increasing use of gene transfer strategies in the study of cellular and molecular biology, melanoma cells have remained difficult to transfect in a safe, efficient, and reproducible manner. In the present study, we report the successful use of nucleofector technology to transfect human melanoma cell lines. This technology uses an empirically derived combination of cell line-specific solutions and nucleofector programmes to electroporate nucleic acid substrates directly into the cell nucleus. Using a colorimetric beta-galactosidase assay, we optimized nucleofection parameters for 13 melanoma cell lines, leading to maximum transfection efficiency and cell survival. The combinations of cell solutions NHEM or T and nucleofector programmes A-24 or U-20 produced the best results. We compared nucleofection with two commercially available lipid-based gene transfer systems, effectene and lipofectamine 2000 using a green fluorescent protein reporter vector. Nucleofection demonstrated a 3- to 40-fold improvement in transfection efficiency when compared with the lipid-based counterparts. Nucleofection was also superior in transfecting small-interfering RNA (siRNA) as determined by Western blot analysis. Lastly, we applied nucleofection to the simultaneous transfection of a p53-dependent luciferase plasmid and p53-siRNA. Experiments using dual transfection showed knockdown of p53 expression and silencing of the reporter plasmid. In conclusion, nucleofection is highly effective for the transfer of nucleic acid substrates, singly or in combination, into human melanoma cell lines

OBJECTIVE: To evaluate the performance of dermoscopists in diagnosing small pigmented skin lesions (diameter </= 6 mm) compared with an automatic multispectral computer-vision system. DESIGN: Blinded comparison study. SETTING: Dermatologic hospital-based clinics and private practice offices. Patients From a computerized skin imaging database of 990 small (</= 6-mm) pigmented skin lesions, all 49 melanomas from 49 patients were included in this study. Fifty randomly selected nonmelanomas from 46 patients served as a control. MAIN OUTCOME MEASURES: Ten dermoscopists independently examined dermoscopic images of 99 pigmented skin lesions and decided whether they identified the lesions as melanoma and whether they would recommend biopsy to rule out melanoma. Diagnostic and biopsy sensitivity and specificity were computed and then compared with the results of the computer-vision system. RESULTS: Dermoscopists were able to correctly identify small melanomas with an average diagnostic sensitivity of 39% and a specificity of 82% and recommended small melanomas for biopsy with a sensitivity of 71% and specificity of 49%, with only fair interobserver agreement (kappa = 0.31 for diagnosis and 0.34 for biopsy). In comparison, in recommending biopsy to rule out melanoma, the computer-vision system achieved 98% sensitivity and 44% specificity. CONCLUSIONS: Differentiation of small melanomas from small benign pigmented lesions challenges even expert physicians. Computer-vision systems can facilitate early detection of small melanomas and may limit the number of biopsies to rule out melanoma performed on benign lesions

OBJECTIVE: To determine the utility of the current diameter criterion of larger than 6 mm of the ABCDE acronym for the early diagnosis of cutaneous melanoma. DESIGN: Cohort study. SETTING: Dermatology hospital-based clinics and community practice offices. Patients A total of 1323 patients undergoing skin biopsies of 1657 pigmented lesions suggestive of melanoma. MAIN OUTCOME MEASURE: The maximum lesion dimension (diameter) of each skin lesion was calculated before biopsy using a novel computerized skin imaging system. RESULTS: Of 1657 biopsied lesions, 853 (51.5%) were 6 mm or smaller in diameter. Invasive melanomas were diagnosed in 13 of 853 lesions (1.5%) that were 6 mm or smaller in diameter and in 41 of 804 lesions (5.1%) that were larger than 6 mm in diameter. In situ melanomas were diagnosed in 22 of 853 lesions (2.6%) that were 6 mm or smaller in diameter and in 62 of 804 lesions (7.7%) that were larger than 6 mm in diameter. Conclusion The diameter guideline of larger than 6 mm provides a useful parameter for physicians and should continue to be used in combination with the A, B, C, and E criteria previously established in the selection of atypical lesions for skin biopsy

BACKGROUND: In patients with T1b-T3b cutaneous melanoma the utility of radiologic imaging at the time of diagnosis is unclear. Whether initial imaging led to a change in stage or treatment plan was investigated. METHODS: The melanoma database was searched for patients with T1b-T3b primary lesions, clinically N0, and asymptomatic for metastatic disease. Radiologic studies conducted before wide local excision +/- sentinel lymph node biopsy as well as all further imaging and investigations were analyzed. Outcome measures included upstaging, change in initial surgical management, true-positive, false-positive, true-negative, and false-negative rates of each imaging modality. RESULTS: In all, 344 preoperative imaging studies (chest x-ray [CXR], computed tomography [CT], positron emission tomography [PET]/CT) were performed on 158 patients, resulting in 49 findings suspicious for metastatic melanoma and 134 findings suggestive of nonmelanoma pathology. Only 1 of 344 (0.3%) studies, a PET/CT, correlated with confirmed metastatic melanoma. The false-positive rates were CXR 5 of 7 (71.4%), chest CT 21 of 24 (87.5%), abdomen/pelvis CT 10 of 11 (90.9%), head CT 2 of 2 (100.0%), PET/CT 3 of 5 (60.0%). No patient was upstaged or had a change in initial surgical management based on preoperative imaging. The cost of all initial imaging and imaging to follow-up abnormal findings was estimated as $555,308 for the 158 patients studied. CONCLUSIONS: Imaging at the time of initial diagnosis of T1b-T3b, clinically N0, M0 melanoma was of low yield with a high false-positive rate, and did not lead to upstaging or change in initial surgical management. These findings suggest that imaging of asymptomatic patients at the time of diagnosis may not be warranted

Cancer/testis (CT) antigens are potential targets for cancer immunotherapy, with NY-ESO-1 being among the most immunogenic. In several clinical trials in malignant melanoma (MM) patients, NY-ESO-1 protein/peptides showed clear evidence of inducing specific immunity. However, little is known about NY-ESO-1 expression in primary and metastatic MM and its relationship to disease progression. We analyzed NY-ESO-1 expression immunohistochemically in a series of primary and metastatic MMs and its relation to prognostic parameters and survival. We studied 61 primary and 63 metastatic MM specimens (from 61 and 56 patients, respectively). The prevalence of NY-ESO-1 expression was significantly higher in metastatic versus primary tumors [18/56 (32%) versus 8/61 (13%), P = 0.015]. There was a significant association between initial stage at presentation and NY-ESO-1 expression [stage I (3.45%), stage II (9.52%) and stage III (45.45%), P = 0.0014]. Primary MMs expressing NY-ESO-1 were significantly thicker than NY-ESO-1 negative cases (median thickness 4.7 mm versus 1.53 mm respectively, P = 0.03). No significant difference was seen in overall survival. In conclusion, NY-ESO-1 is more frequently expressed in metastatic than in primary MM and its expression is associated with thicker primary lesions and a higher frequency of metastatic disease, indicative of a worse prognosis. Our study suggests that patients with metastatic MM who express NY-ESO-1 may benefit from NY-ESO-1-based immunotherapy

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients