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Desmoplastic melanoma (DM) is an uncommon subtype of melanoma with distinct clinicopathological features. It is classified into pure desmoplastic melanoma (PDM) when the proportion of desmoplastic melanoma is ≥90% of the dermally-invasive component, and mixed desmoplastic melanoma (MDM) when the proportion of desmoplastic melanoma is <90%. Studies have reported a lower sentinel lymph node biopsy (SLNB)-positivity rate in PDM compared to MDM and non-DM. As a result, some have recommended not performing SLNB in PDM patients. When PDM is identified in a partial biopsy of a melanoma, there is a risk that sampling bias may under-recognise MDM, but to the best of our knowledge this has not been previously assessed or quantified. The aim of this study was to assess the concordance of the proportion of desmoplastic melanoma in an initial partial biopsy of PDM with the proportion in the entire tumour following complete excision, in patients with cutaneous melanoma. A secondary aim was to determine how frequently this potentially resulted in a patient not receiving a SLNB. Seventy-eight cases of cutaneous melanoma were identified from the Melanoma Institute Australia (MIA) database and 23 cases from the Memorial Sloan Kettering Cancer Centre (MSKCC), where an initial biopsy contained PDM and a subsequent wide excision had residual invasive melanoma. Clinicopathological features were analysed in all patients, including whether a SLNB was performed, the results of SLNB, and any subsequent recurrence. Ninety percent (91/101) of cases were still classified as PDM in the complete wide excision specimen while 10% (10/101) of cases were reclassified as MDM, which was a significant change in classification of final desmoplastic melanoma subtype (p<0.001). The proportion of desmoplastic melanoma was also significantly different between the initial and excisional biopsies (p=0.004). Forty-eight (48/101) patients had a SLNB, of which two (4.5%) were positive for metastatic melanoma; both cases were PDM in the excision specimen. Of the 10 cases demonstrating MDM in the excision specimen, the initial biopsy was a punch biopsy in six cases, shave biopsy in two cases and subcutaneous tissue was sampled in two patients (one punch biopsy, one incisional biopsy). Four of these 10 patients underwent SLNB which was negative in all cases. Twenty-two patients developed recurrence in the follow-up period (median 30 months, range 1-192 months), three with MDM in their excision specimen. One patient did not have a SLNB and developed regional lymph node recurrence. In this study there was a 10% risk that the percentage of desmoplastic melanoma in an initial biopsy of PDM was not representative of the entire lesion, resulting in reclassification as MDM in the excision specimen. If a SLNB is not performed in such cases, a positive SLNB may be missed (one patient in our study) which could impact treatment options for the patient. We recommend caution in not offering a SLNB in the setting of an initial biopsy of PDM if the biopsy is small compared with the overall lesion. If a SLNB is not procured at the time of wide excision in such cases, the SLNs should still be mapped by lymphoscintigraphy to facilitate careful follow up and to enable earlier detection and treatment of nodal disease., (Copyright © 2023 Royal College of Pathologists of Australasia. All rights reserved.)

Desmoplastic melanomas (DMs) are often challenging to diagnose and ancillary tests, such as immunohistochemistry, have limitations. One challenge is distinguishing DM from benign desmoplastic melanocytic neoplasms. In this study, we explored the utility of next-generation sequencing data in the diagnosis of DMs versus desmoplastic Spitz nevi (DSN) and desmoplastic nevi (DN). We sequenced 47 cases and retrieved 12 additional previously sequenced clinical cases from our dermatopathology database. The 59 total cases were comprised of 21 DMs, 25 DSN, and 13 DN. The DMs had the highest tumor mutation burden at 22 mutations/megabase (m/Mb) versus the DSN (6 m/Mb) and DN (8 m/Mb). Truncating mutations in NF1 resulting in a loss-of-function were exclusive to the DM cohort, identified in 8/21 (38%) cases. Importantly, missense mutations in NF1 were nonspecific and seen with similar frequency in the different cohorts. Other mutations exclusive to the DMs included truncating mutations in TP53 , CDKN2A , and ARID2 . Among the DSN, 17/25 (68%) had an HRAS mutation or receptor tyrosine kinase fusion consistent with other Spitz tumors. Two cases in the DN cohort had missense mutations in BRAF without additional progression mutations and 2 other cases had mutations in GNAQ , supporting a diagnosis of a sclerosing blue nevus. The remainder of the DN had nonspecific mutations in various signaling pathways with few progression mutations. Overall, our study provides preliminary data that next-generation sequencing data may have the potential to serve as an ancillary diagnostic tool to help differentiate malignant and benign desmoplastic melanocytic neoplasms., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the IDP Foundation and the Greg and Anna Brown Family Foundation. P.G. has served as a consultant for Myriad Genomics, DermTech Int., Merck, and Castle Biosciences and has received honoraria for this. For the remaining authors none were declared., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Genomic classification of melanoma has thus far focused on the mutational status of BRAF , NRAS , and NF1 . The clinical utility of this classification remains limited, and the landscape of alterations in other oncogenic signaling pathways is underexplored., Methods: Using primary samples from the InterMEL study, a retrospective cohort of cases with specimens collected from an international consortium with participating institutions throughout the United States and Australia, with oversampling of cases who ultimately died of melanoma, we examined mutual exclusivity and co-occurrence of genomic alterations in 495 stage II/III primary melanomas across 11 cancer pathways. Somatic mutation and copy number alterations were analyzed from next-generation sequencing using a clinical sequencing panel., Results: Mutations in the RTK-RAS pathway were observed in 81% of cases. Other frequently occurring pathways were TP53 (31%), Cell Cycle (30%), and PI3K (18%). These frequencies are generally lower than was observed in The Cancer Genome Atlas, where the specimens analyzed were predominantly obtained from metastases. Overall, 81% of the cases had at least one targetable mutation. The RTK-RAS pathway was the only pathway that demonstrated strong and statistically significant mutual exclusivity. However, this strong mutual exclusivity signal was evident only for the three common genes in the pathway ( BRAF , NRAS , and NF1 ). Analysis of co-occurrence of different pathways exhibited no positive significant trends. However, interestingly, a high frequency of cases with none of these pathways represented was observed, 8.4% of cases versus 4.0% expected ( P < .001). A higher frequency of RTK-RAS singletons (with no other pathway alteration) was observed compared with The Cancer Genome Atlas. Clonality analyses suggest strongly that both the cell cycle and RTK-RAS pathways represent early events in melanogenesis., Conclusion: Our results confirm the dominance of mutations in the RTK-RAS pathway. The presence of many mutations in several well-known, actionable pathways suggests potential avenues for targeted therapy in these early-stage cases.

Relatlimab and nivolumab combination immunotherapy improves progression-free survival over nivolumab monotherapy in patients with unresectable advanced melanoma 1 . We investigated this regimen in patients with resectable clinical stage III or oligometastatic stage IV melanoma (NCT02519322). Patients received two neoadjuvant doses (nivolumab 480 mg and relatlimab 160 mg intravenously every 4 weeks) followed by surgery, and then ten doses of adjuvant combination therapy. The primary end point was pathologic complete response (pCR) rate 2 . The combination resulted in 57% pCR rate and 70% overall pathologic response rate among 30 patients treated. The radiographic response rate using Response Evaluation Criteria in Solid Tumors 1.1 was 57%. No grade 3-4 immune-related adverse events were observed in the neoadjuvant setting. The 1- and 2-year recurrence-free survival rate was 100% and 92% for patients with any pathologic response, compared to 88% and 55% for patients who did not have a pathologic response (P = 0.005). Increased immune cell infiltration at baseline, and decrease in M2 macrophages during treatment, were associated with pathologic response. Our results indicate that neoadjuvant relatlimab and nivolumab induces a high pCR rate. Safety during neoadjuvant therapy is favourable compared to other combination immunotherapy regimens. These data, in combination with the results of the RELATIVITY-047 trial 1 , provide further confirmation of the efficacy and safety of this new immunotherapy regimen., (© 2022. The Author(s).)

Competing Interests: Conflicts of interest Drs Yamada and Prow have a conflict of interest with Trajan Scientific & Medical Pty Ltd as they are currently commercializing microbiopsy. Drs Jain, Busam, Marchetti, and Halpern and authors Autuori, Everett, Harris, and Orlow do not have any conflicts of interest to disclose.

The current classification of Spitz neoplasms in the World Health Organization (WHO), Fourth Edition defines Spitz neoplasms as melanocytic proliferations with characteristic Spitz morphology and a Spitz-associated genomic fusion or HRAS mutation. In contrast, melanocytic neoplasms with BRAF mutations are considered typical of common acquired nevi, dysplastic nevi, and melanomas from intermittent sun-damaged skin. However, increased utilization of ancillary testing methods such as BRAFV600E immunohistochemistry and sequencing studies have made apparent a subgroup of benign-grade and intermediate-grade melanocytic neoplasms with Spitzoid morphology that harbor BRAFV600E mutations. We refer to these cases as BRAF mutated and morphologically Spitzoid (BAMS) nevi and tumors. Two experienced dermatopathologists reviewed a series of 36 BAMS nevi/tumors. Cases in which a diagnosis of melanoma was favored were excluded. The histomorphologic, clinical, and molecular findings were assessed by immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing using validated gene panels. Characteristics of BAMS nevi/tumors were compared with a control set of Spitz tumors with previously reported fusion proteins. BAMS nevi/tumors had a decreased proportion of Kamino bodies (P=0.03) and a higher proportion of cytoplasmic pigmentation (P<0.00001). There were no differences in other morphologic features such as the silhouette, epidermal hyperplasia, pagetosis, and cytologic atypia compared with fusion-induced Spitz tumors. In 6/17 cases where next-generation sequencing studies were available, recurrent mutations in the KMT gene family were seen. This was higher than the proportion of such mutations seen in fusion Spitz tumors and lower than the frequency in cutaneous melanoma., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the IDP Foundation Inc. P.G. has served as a consultant for Myriad Genomics, DermTech Int., Merck, and Castle Biosciences and has received honoraria for this. For the remaining authors none were declared., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Nevus-associated melanomas (NAM) account for 30% of all melanomas and are associated with younger age and with thinner Breslow thickness. Previous studies of NAM dermoscopy found conflicting results., Objective: To compare the clinical and dermoscopic features of NAM and de novo melanomas (DNM), stratified by melanoma thickness, in a relatively large cohort of patients., Methods: A cross-sectional study of all melanomas biopsied between 2004 and 2019 at a large cancer centre. Lesions were categorized as in situ and invasive NAM or DNM. Dermoscopic images were reviewed and annotated. Associations between melanoma subtype and dermoscopic features were analysed via logistic regression modelling. Bivariate analyses were conducted using non-parametric bootstrap and chi-squared methods., Results: The study included 160 NAM (86 in situ and 74 invasive) and 218 DNM (109 in situ and 109 invasive). NAM were associated with younger age, greater likelihood of being present on the torso, and thinner Breslow thickness. NAM were 2.5 times more likely to show a negative pigment network than DNM. In situ NAM were 2.1 and two times more likely to display dermoscopic area without definable structures and tan structureless areas than DNM, respectively. In situ melanomas were more likely to present a pigment network, and invasive melanomas more commonly presented scar-like depigmentation and shiny white structures. Streaks, blotches and shiny white structures were associated with deeper Breslow depth., Conclusions: Even though the nevus component of NAM could not be identified dermoscopically in the current series, negative pigment network, tan structureless areas and areas without definable structures are dermoscopic clues for NAM., (© 2021 European Academy of Dermatology and Venereology.)

Desmoplastic melanoma can be difficult to diagnose and on average have a significantly higher T stage at the time of diagnosis compared with conventional melanomas. Histologically, these tumors typically consist of spindle cells in a fibrous matrix. The spindle cells may display fibroblast and/or Schwann cell-like features. In this study, we describe the features of 12 cases of desmoplastic melanoma closely simulating neurofibroma. Although the spindle cells in these tumors may be indistinguishable from those of neurofibroma, features such as prominent fibroplasia (12/12), poor lateral circumscription (8/9), diffuse infiltration of subcutaneous tissue (7/9), and lymphoid aggregates (10/12) may be helpful clues to the diagnosis. No immunohistochemical markers were reliable in distinguishing neurofibroma-like desmoplastic melanomas from neurofibroma. Clinical follow-up was available in 8 cases, of which 4 were initially misdiagnosed as benign neoplasms and given no further re-excision. All 4 of these cases recurred; 2 of which showed transformation to a more aggressive phenotype.

Background: Lentigo maligna/lentigo maligna melanoma (LM/LMM) poses a treatment and surgical challenge given unpredictable subclinical extension resulting in incomplete excision., Objectives: To describe the demographic, clinical and pathologic characteristics of incompletely excised LM/LMM. To evaluate the potential role of reflectance confocal microscopy (RCM)., Patients and Methods: A retrospective review of a melanoma database at a tertiary cancer centre for patients referred with 'incompletely excised LM/LMM' or 'incompletely excised melanoma' between October 2006 and July 2017. We recorded clinical and pathological data and surgical margins needed to clear the residual LM/LMM. The second part consisted of a prospective cohort of patients in which RCM was performed when presenting with incompletely excised LM/LMM., Results: We included a total of 67 patients (retrospective + prospective cohort); mean age was 64.9 (standard deviation: 11.3) years and 52.2% were males. For the retrospective cohort (n = 53), the mean scar size was 3.4 cm. The average initial margins excised prior to presentation were 4.8 mm (range 3-7 mm). The average additional margin needed to clear the residual, incompletely excised LM/LMM was 7.8 mm. For the prospective cohort (n = 14), there were no differences in age, gender or size when compared to the retrospective cohort. RCM had a diagnostic accuracy of 78.6%, a sensitivity of 90.9%, a specificity of 33.3% and a positive predictive value of 83.3% for the detection of incompletely excised LM/LMM., Conclusions: Incompletely excised LM/LMM is a poorly characterized clinical-pathological scenario that may require considerable extra margins for microscopic clearance. RCM may emerge as a valuable tool for the evaluation of patients with incompletely excised LM/LMM., (© 2020 European Academy of Dermatology and Venereology.)

Background: Multiple BRCA1-associated protein 1 (BAP1)-inactivated melanocytic tumors (BIMTs) have been associated with a familial cancer syndrome involving germline mutations in BAP1., Objectives: We sought to describe the clinical and dermoscopic features of BIMTs., Methods: This was a retrospective, multicenter, case-control study. Participating centers contributed clinical data, dermoscopic images, and histopathologic data of biopsy-proven BIMTs. We compared the dermoscopic features between BIMTs and control patients., Results: The dataset consisted of 48 BIMTs from 31 patients (22 women; median age 37 years) and 80 control patients. Eleven patients had a BAP1 germline mutation. Clinically, most BIMTs presented as pink, dome-shaped papules (n = 24). Dermoscopically, we identified 5 patterns: structureless pink-to-tan with irregular eccentric dots/globules (n = 14, 29.8%); structureless pink-to-tan with peripheral vessels (n = 10, 21.3%); structureless pink-to-tan (n = 7, 14.9%); a network with raised, structureless, pink-to-tan areas (n = 7, 14.9%); and globular pattern (n = 4, 8.5%). The structureless with eccentric dots/globules pattern and network with raised structureless areas pattern were only identified in BIMT and were more common in patients with BAP1 germline mutations (P < .0001 and P = .001, respectively)., Limitations: Limitations included our small sample size, retrospective design, the absence of germline genetic testing in all patients, and inclusion bias toward more atypical-looking BIMTs., Conclusions: Dome-shaped papules with pink-to-tan structureless areas and peripheral irregular dots/globules or network should raise the clinical suspicion for BIMT., (Copyright © 2018 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Evidence on the relationship between the vitamin D pathway and outcomes in melanoma is growing, although it is not always clear. We investigated the impact of measured levels of sun exposure at diagnosis on associations of vitamin D receptor gene (VDR) polymorphisms and melanoma death in 3336 incident primary melanoma cases. Interactions between six SNPs and a common 3'-end haplotype were significant (p < .05). These SNPs, and a haplotype, had a statistically significant association with survival among subjects exposed to high UVB in multivariable regression models and exerted their effect in the opposite direction among those with low UVB. SNPs rs1544410/BsmI and rs731236/TaqI remained significant after adjustment for multiple testing. These results suggest that the association between VDR and melanoma-specific survival is modified by sun exposure around diagnosis, and require validation in an independent study. Whether the observed effects are dependent or independent of vitamin D activation remains to be determined., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: One of the main problems in the diagnostics of pediatric melanomas is the differentiation from benign dermal lesions typical for this age group, such as Spitz nevus. The biological behavior of pediatric melanomas differs considerably from that of melanomas in adults., Material and Methods: Cancer testis (CT) antigens are named after their typical expression pattern since they are present in various types of malignant tumors but in normal adult tissues are solely expressed in testicular germ cells. Because of this tumor-associated expression pattern, CT antigens are regarded as potential targets for vaccine-based immunotherapy of cancer and might be used as diagnostic tools in surgical pathology. In adults, melanoma is among the tumors showing a high incidence of CT antigen expression; however, while there is ample knowledge about adult melanomas, little is known about the presence of CT antigens in pediatric melanomas. Consequently, the expression of CT antigens MAGE-A1, MAGE-A4, CT7/MAGE-C1, NY-ESO-1, and GAGE was analyzed in a series of pediatric melanomas. The study was restricted to cases of metastatic disease and/or fatal outcome. A total of 12 cases were available and immunohistochemically analyzed with monoclonal antibodies (mAb)., Results: The expression of CT antigens was generally low and present in only 4 of 12 cases. This is in stark contrast to the expression of these antigens in adult melanomas. Moreover, the extent of expression was very limited with most cases showing only a focal CT antigen expression and only marked in very small tumor areas (<5%)., Conclusion: Despite the low case numbers this study indicates that CT antigens are most likely not useful as diagnostic markers in pediatric melanomas or as targets for vaccine-based immunotherapy. It supports the notion that pediatric melanomas show a different biological behavior than their adult counterparts.

Background: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions., Methods: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis., Results: The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%., Conclusions: These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617-628. © 2016 American Cancer Society., (© 2016 Myriad Genetics, Inc. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.)

Background: Clinical and histopathologic assessment of pigmented skin lesions remains challenging even for experts. Differentiated and accurate noninvasive diagnostic modalities are highly desirable., Objective: We sought to provide clinicians with such a tool., Methods: A 2-gene classification method based on LINC00518 and preferentially expressed antigen in melanoma (PRAME) gene expression was evaluated and validated in 555 pigmented lesions (157 training and 398 validation samples) obtained noninvasively via adhesive patch biopsy. Results were compared with standard histopathologic assessment in lesions with a consensus diagnosis among 3 experienced dermatopathologists., Results: In 398 validation samples (87 melanomas and 311 nonmelanomas), LINC00518 and/or PRAME detection appropriately differentiated melanoma from nonmelanoma samples with a sensitivity of 91% and a specificity of 69%. We established LINC00518 and PRAME in both adhesive patch melanoma samples and underlying formalin fixed paraffin embedded (FFPE) samples of surgically excised primary melanomas and in melanoma lymph node metastases., Limitations: This technology cannot be used on mucous membranes, palms of hands, and soles of feet., Conclusions: This noninvasive 2-gene pigmented lesion assay classifies pigmented lesions into melanoma and nonmelanoma groups and may serve as a tool to help with diagnostic challenges that may be inherently linked to the visual image and pattern recognition approach., (Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Factors known to affect melanoma survival include age at presentation, sex and tumor characteristics. Polymorphisms also appear to modulate survival following diagnosis. Result from other studies suggest that vitamin D receptor (VDR) polymorphisms (SNPs) impact survival in patients with glioma, renal cell carcinoma, lung, breast, prostate and other cancers; however, a comprehensive study of VDR polymorphisms and melanoma-specific survival is lacking. We aimed to investigate whether VDR genetic variation influences survival in patients with cutaneous melanoma. The analysis involved 3566 incident single and multiple primary melanoma cases enrolled in the international population-based Genes, Environment, and Melanoma Study. Melanoma-specific survival outcomes were calculated for each of 38 VDR SNPs using a competing risk analysis after adjustment for covariates. There were 254 (7.1%) deaths due to melanoma during the median 7.6 years follow-up period. VDR SNPs rs7299460, rs3782905, rs2239182, rs12370156, rs2238140, rs7305032, rs1544410 (BsmI) and rs731236 (TaqI) each had a statistically significant (trend P values < 0.05) association with melanoma-specific survival in multivariate analysis. One functional SNP (rs2239182) remained significant after adjustment for multiple testing using the Monte Carlo method. None of the SNPs associated with survival were significantly associated with Breslow thickness, ulceration or mitosis. These results suggest that the VDR gene may influence survival from melanoma, although the mechanism by which VDR exerts its effect does not seem driven by tumor aggressiveness. Further investigations are needed to confirm our results and to understand the relationship between VDR and survival in the combined context of tumor and host characteristics., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: We previously reported a significant association between higher UV radiation exposure before diagnosis and greater survival with melanoma in a population-based study in Connecticut. We sought to evaluate the hypothesis that sun exposure before diagnosis was associated with greater survival in a larger, international population-based study with more detailed exposure information., Methods: We conducted a multicenter, international population-based study in four countries-Australia, Italy, Canada, and the United States-with 3,578 cases of melanoma with an average of 7.4 years of follow-up. Measures of sun exposure included sunburn, intermittent exposure, hours of holiday sun exposure, hours of water-related outdoor activities, ambient ultraviolet B (280-320 nm) dose, histologic solar elastosis, and season of diagnosis., Results: Results were not strongly supportive of the earlier hypothesis. Having had any sunburn in 1 year within 10 years of diagnosis was inversely associated with survival; solar elastosis-a measure of lifetime cumulative exposure-was not. In addition, none of the intermittent exposure measures-water-related activities and sunny holidays-were associated with melanoma-specific survival. Estimated ambient UVB dose was not associated with survival., Conclusion: Although there was an apparent protective effect of sunburns within 10 years of diagnosis, there was only weak evidence in this large, international, population-based study of melanoma that sun exposure before diagnosis is associated with greater melanoma-specific survival., Impact: This study adds to the evidence that sun exposure before melanoma diagnosis has little effect on survival with melanoma., (©2014 American Association for Cancer Research.)

Predicting clinical behavior of atypical Spitz tumors remains problematic. In this study, we assessed interobserver agreement of diagnosis by 13 expert dermatopathologists for atypical Spitz tumors (n=75). We determined which histomorphologic features were most heavily weighted for their diagnostic significance by the experts and also which histomorphologic features had a statistically significant correlation with clinical outcome. There was a low interobserver agreement among the experts in categorizing lesions as malignant versus nonmalignant (κ=0.30). The histomorphologic features that were given the most diagnostic significance by the experts were: consumption of the epidermis, atypical mitoses, high-grade cytologic atypia, and mitotic rate. Conversely, the histomorphologic features that most correlated with disease progression were: frequent mitoses, deep mitoses, asymmetry, high-grade cytologic atypia, and ulceration. The presence and/or pattern of pagetoid spread, consumption of the epidermis, and lymphoid aggregates demonstrated no association with clinical behavior. The results support the assertion that there is a lack of consensus in the assessment of atypical Spitz tumors by expert dermatopathologists. Importantly, many features used to distinguish conventional melanoma from nevi were not useful in predicting the behavior of atypical Spitz tumors. This study may provide some guidance regarding histologic assessment of these enigmatic tumors.

A rare germline variant in the microphthalmia-associated transcription factor (MITF) gene, E318K, has been reported as associated with melanoma. We confirmed its independent association with melanoma [odds ratio (OR) 1.7, 95% confidence interval (CI) = 1.1, 2.7, P = 0.03]; adjusted for age, sex, center, age × sex interaction, pigmentation characteristics, family history of melanoma, and nevus density). In stratified analyses, carriage of MITF E318K was associated with melanoma more strongly in people with dark hair than fair hair (P for interaction, 0.03) and in those with no moles than some or many moles (P for interaction, <0.01). There was no evidence of interaction between MC1R 'red hair variants' and MITF E318K. Moreover, risk of melanoma among carriers with 'low risk' phenotypes was as great or greater than among those with 'at risk' phenotypes with few exceptions., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The complex lymphatic drainage in the head and neck makes sentinel lymph node biopsy (SLNB) for melanomas in this region challenging. This study describes the incidence, and location of additional positive nonsentinel lymph nodes (NSLN) in patients with cutaneous head and neck melanoma following a positive SLNB., Methods: A retrospective review was performed using a single institution prospective database. Patients with a primary melanoma in the head or neck with a positive cervical SLNB were identified. The lymphadenectomy specimen was divided intraoperatively into lymph node levels I-V, and NSLN status determined for each level., Results: Of 387 patients with melanoma of the head and neck who underwent cervical SLNB, 54 had a positive SLN identified (14%). Thirty six patients (67%) underwent immediate completion lymph node dissection (CLND) of whom eight patients (22%) had a positive NSLN. The remaining 18 patients (33%) did not undergo CLND and were observed. Half of positive NSLNs (50%) were in the same lymph node level as the SLN and 33% were in an immediately adjacent level; only two patients were found to have NSLNs in non-adjacent levels. The only factor predictive of NSLN involvement was the size of the tumor deposit in the SLN>0.2 mm (p = 0.05). Superficial parotidectomy at CLND revealed metastatic melanoma only in patients with a positive parotid SLN., Conclusions: A positive NLSN was identified in 22% of patients undergoing CLND after a positive SLNB. The majority of positive NSLNs are found within or immediately adjacent to the nodal level containing the SLN., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Benign melanocytic nevi and cutaneous melanomas can be difficult to differentiate by means of routine microscopic analysis. Recent evidence has suggested that cytogenomic analysis may be a useful diagnostic method for evaluation of melanocytic proliferations. We investigated the array-based comparative genomic hybridization (aCGH) platform for DNA copy number analysis of formalin-fixed, paraffin-embedded (FFPE) tissues in melanocytic tumors and compared aCGH analysis with fluorescence in situ hybridization (FISH) assays in diagnosis of melanoma. aCGH findings and FISH results were interpreted independently in a blinded fashion. Positive findings were not noted in any benign nevi at aCGH analysis, whereas substantial unbalanced genomic aberrations were revealed in 92% of melanomas. Positive results were obtained in 72% of melanomas via the four-probe FISH assay (RREB1/MYB/CEP6/CCND1). A few additional FISH studies were performed to verify some aCGH findings of focal amplification of oncogenes and homozygous deletion of tumor suppressor genes. The overall concordance in aberrations detected using the two methods was 90%. Most discrepancies were due to a minor abnormal clone identified via FISH that was below analytical sensitivity of the FFPE aCGH test. Our study demonstrated that copy number analysis of FFPE tumor samples via aCGH is a robust and reliable method in diagnosis of melanoma and that aCGH and FISH tests should be used as complementary methods to improve the accuracy of genetic evaluation of melanocytic tumors., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Death due to melanoma in childhood (up to 20 y of age) is a rare event, with an average of 18 cases reported annually in the United States. In this study we evaluated 2 subgroups of high-risk melanocytic neoplasms in childhood, specifically atypical Spitz tumors (ASTs) with chromosomal copy number changes and conventional melanomas. We analyzed the clinical, histologic, and molecular features of all cases and performed the Fisher exact test, logistic regression, and multivariate analysis to evaluate features associated with aggressive clinical behavior in these cases. Among the ASTs, all of which had 1 or more chromosomal copy number aberrations, the presence of homozygous 9p21 deletions and a positive sentinel lymph node were each found to be correlated with tumor extension beyond the sentinel lymph node, with P-values of 0.046 and 0.01, respectively. Two patients with ASTs that had homozygous 9p21 deletions developed brain metastasis, one of whom died of disease. Among the 21 conventional melanomas, 3 patients developed distant metastasis and died of disease. Chromosomal copy number aberrations evaluated by fluorescence in situ hybridization were present in the majority of the cases (16/18). Among conventional melanomas, we did not identify any clinical, histologic, or molecular features associated with aggressive behavior. The presence of 8q24 gains was seen almost exclusively in 6 amelanotic small cell melanomas in children of whom 1 died of disease. Characteristic chromosomal copy number aberrations may occur in specific subtypes of melanocytic neoplasms in children and may help with the classification and prognostication of these rare tumors.

Background: The tumor microenvironment after treatment with ipilimumab is not well described. Furthermore, the safety of surgery for patients being treated with ipilimumab for metastatic melanoma has not been well reported. This study analyzed the safety of surgery and the immune phenotype of tumors resected while on ipilimumab., Methods: From our prospective melanoma database, we identified patients undergoing surgery for any indication within 30 days of receiving a dose of induction ipilimumab or while on maintenance ipilimumab therapy. Surgical toxicity was graded 1-5 by the Clavien classification. Tumor-infiltrating lymphocytes were classified by flow cytometry and compared with peripheral blood., Results: 23 patients were identified who underwent 34 operations a median of 27 weeks after initiation of ipilimumab (1-123 weeks). Subcutaneous resections were the most frequent, followed by intra-abdominal and nodal procedures. Grade 1 or 2 wound complications were seen in 22% of patients. No Grade 3-5 complications were seen. Analysis of the T cell infiltrate and matched peripheral blood from ten patients showed an elevated % of CD4+FOXP3+ T-regulatory cells and a 2.8-fold lower ratio of CD8+/CD4+FOXP3+ in the tumor compared with blood (p=0.02). In addition, all CD8+ T cells had a higher expression of PD-1 in the tumor, compared with peripheral blood., Conclusions: Surgery for patients on ipilimumab is safe. This study highlights the immunosuppressive phenotype in tumors not responding to immunotherapy. The high percentage of T-regulatory cells and low T-effector cells in progressive tumors suggests a possible mechanism of immune escape.

Importance: Little is known about survival after a diagnosis of a second or higher-order (multiple) primary melanoma, and no study has explored survival in a population-based sample that included patients with single primary melanomas (SPMs) and multiple primary melanomas (MPMs) of any stage. Because people with a first primary melanoma are known to have an increased risk of being diagnosed with another, evidence for prognosis is needed., Objective: To determine whether survival after diagnosis was better in patients with MPMs than with SPMs, as suggested in a recent study. DESIGN Survival analysis with median follow-up of 7.6 (range, 0.4-10.6) years., Setting: The Genes, Environment, and Melanoma Study enrolled incident cases of melanoma from population-based cancer registries in Australia, Canada, Italy, and the United States. Multiple primary melanomas were ascertained during a longer period than SPM., Participants: Two thousand three hundred seventy-two patients with SPM and 1206 with MPM., Exposure: Diagnosis with melanoma., Main Outcomes and Measures: Melanoma-specific fatality hazard ratios (HR) and 95% confidence intervals associated with clinical and pathological characteristics of SPM, MPM, and both in Cox proportional hazards regression models., Results: Melanoma thickness was the main determinant of fatality (HR for >4 mm, 7.68 [95% CI, 4.46-13.23]); other independent predictors were ulceration, mitoses, and scalp location. After adjustment for these other predictors, we found little difference in fatality between MPM and SPM (HR for MPM relative to SPM, 1.24 [95% CI, 0.91-1.69; P = .18]). Thicker SPM, however, had higher fatality (HR for >4 mm, 13.56 [95% CI, 6.47-28.40]) than thicker MPM (2.93 [1.17-7.30])., Conclusions and Relevance: Although overall fatalities due to SPM and MPM were similar, relative fatality for thicker SPM was greater than that for thicker MPM. This finding may offer support for a difference in outcome between patients with SPM and MPM related to factors other than closer surveillance and earlier diagnosis. The better outcomes are worth further exploration.

Objective: The authors evaluated the cost-effectiveness of a FISH assay in melanoma diagnosis in the USA., Method: A model was developed simulating the addition of FISH to the diagnosis of suspected melanoma. A decision analytic module simulated diagnosis using microscopic assessment alone versus addition of FISH (sensitivity: 92%; specificity: 94%). The authors simulated a clinical setting in which an initial excisional biopsy microscopic assessment (sensitivity: 73%; specificity: 78%) was followed by dermatopathologist assessment (sensitivity: 89%; specificity: 79%) for inconclusive results. Diagnostic strategies 1 and 2 added FISH to the initial and dermatopathologist assessments, respectively. A Markov outcomes module simulated patients' remaining lifetime, including treatment., Results: In diagnostic strategies 1 and 2, the cost per quality-adjusted life year gained was US$14,930 and 43,925, respectively, versus no FISH. Cost per misdiagnosis avoided was US$3292 and 3759, respectively. Sensitivity and specificity without FISH were both ≥88%; however, addition of FISH exceeded US$100,000/quality-adjusted life year., Conclusion: In specific clinical settings, FISH could be cost effective for melanoma diagnosis.

Proliferative (cellular) nodules (PN) which mimic malignant melanoma clinically and histologically are described in congenital melanocytic nevi (CMN) and may pose significant diagnostic challenges. We report the case of a 10-day-old male with a giant congenital nevus involving the neck, upper chest, back, and left shoulder containing several nodular lesions, some crusted. Biopsy of a nodule revealed densely packed nevus cells with hyperchromatic round to oval and occasionally irregularly shaped nuclei. There was no necrosis or pushing border, and the nodule blended with the adjacent nevus; however, the lesion demonstrated a significant number of mitoses (27 per mm2) and a 60% labeling index with Ki-67. Further analysis by fluorescence in situ hybridization (FISH) with a 4-color probe set targeting 6p25, 6q23, 11q13, and centromere 6 revealed increased chromosomal copy numbers of all 4 probes, which was interpreted as evidence of polyploidy. In addition, analysis of DNA copy number changes using a single nucleotide polymorphism microarray (Affymetrix, Santa Clara, CA) showed no chromosomal aberrations. The diagnosis of PN in a giant congenital nevus was eventually rendered. At 13-month follow-up, the nodules showed no evidence of growth. Our case illustrates that PNs in the neonatal period might demonstrate extreme mitotic activity. This feature is worrisome when encountered in melanocytic lesions; however, it should not trigger by itself a diagnosis of melanoma in the absence of other histologic criteria of malignancy. In addition, we document polyploidy by FISH in PN, which can potentially be misinterpreted as a FISH-positive result.

Clear cell sarcoma (CCS) of tendons and aponeuroses, also known as melanoma of soft parts, represents an aggressive rare malignancy that is characterized by a nested or fascicular pattern of spindled cells and a pathognomonic reciprocal translocation, t(12;22)(q13;q12), that results in the fusion of EWSR1 and ATF1 genes. Numerous recent studies have recognized the importance of a cutaneous CCS variant that can mimic a broad spectrum of entities, including spindle cell melanoma, spindle cell squamous carcinoma, cutaneous leiomyosarcoma and atypical fibroxanthoma. We report a case of a 13-year-old boy with cutaneous CCS who presented with a few months history of an asymptomatic papule on the lower lip that was suggestive of a mucocele. Biopsy of the lesion showed a wedge shaped neoplasm arranged in nests and fascicles of epithelioid- to oval-shaped cells with pale cytoplasm, open chromatin and prominent nucleolus. The superficial component was closely opposed to the basal epithelium resembling the junctional nests of a melanocytic neoplasm. The process extended into and involved the striated muscle of the lip. The cells expressed S-100, CD99 and synaptophysin by immunohistochemistry, and there was focal HMB-45 and microphthalmia transcription factor (MiTF) positivity as well. Fluorescence in situ hybridization confirmed the presence of the t(12;22) (ESWR1-ATF1) translocation., (© 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Background: Desmoplastic melanoma (DM), a variant of spindle cell melanoma, has a higher propensity for local recurrence and a lower incidence of nodal metastasis. In this retrospective review, we evaluated the risk for regional nodal metastases and the need for sentinel lymph node biopsy (SLNB) in patients with head and neck DM., Methods: We identified 103 patients with DM from an institutional database of patients with head and neck melanomas treated between 1985 and 2009. Forty-seven patients had their primary treatment at Memorial Sloan-Kettering Cancer Center, and 56 patients were treated for recurrent or metastatic disease., Results: Of the 47 study patients, 27 were men and 20 were women with a median age of 71 years. All patients underwent wide excision, and 21 (44 %) underwent SLNB. None of the patients who underwent SLNB had positive nodes. The mean Breslow thickness for the 45 reported patients was 6.1 mm, with 84 % of tumors >2 mm in thickness and 55 % >4 mm. All known Clark thickness levels (n = 40) were IV or V. The overall survival was 73 %, with disease-specific survival of 84 %, local recurrence-free survival of 75 %, and neck recurrence-free survival of 97 % at 5 years., Conclusions: Although DM is diagnosed at higher Breslow thickness and Clark level, neck metastases are rare and prognosis is favorable compared to conventional melanoma. The low incidence of lymphovascular invasion, high frequency of histopathologically negative sentinel lymph nodes, and low neck recurrence rates indicate that staging of neck disease by SLNB is not necessary in patients with pure DM of the head and neck.

Through in vivo selection of human cancer cell populations, we uncover a convergent and cooperative miRNA network that drives melanoma metastasis. We identify miR-1908, miR-199a-5p, and miR-199a-3p as endogenous promoters of metastatic invasion, angiogenesis, and colonization in melanoma. These miRNAs convergently target apolipoprotein E (ApoE) and the heat shock factor DNAJA4. Cancer-secreted ApoE suppresses invasion and metastatic endothelial recruitment (MER) by engaging melanoma cell LRP1 and endothelial cell LRP8 receptors, respectively, while DNAJA4 promotes ApoE expression. Expression levels of these miRNAs and ApoE correlate with human metastatic progression outcomes. Treatment of cells with locked nucleic acids (LNAs) targeting these miRNAs inhibits metastasis to multiple organs, and therapeutic delivery of these LNAs strongly suppresses melanoma metastasis. We thus identify miRNAs with dual cell-intrinsic/cell-extrinsic roles in cancer, reveal convergent cooperativity in a metastatic miRNA network, identify ApoE as an anti-angiogenic and metastasis-suppressive factor, and uncover multiple prognostic miRNAs with synergistic combinatorial therapeutic potential in melanoma., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Selective inhibition of mutant BRAF by using class I RAF inhibitors in patients with metastatic melanoma has resulted in impressive clinical activity. However, there is also evidence that RAF inhibitors might induce carcinogenesis or promote tumor progression via stimulation of MAPK signaling in RAF wild-type cells. We analyzed melanocytic lesions arising under class I RAF inhibitor treatment for dignity, specific genetic mutations, or expression of signal transduction molecules., Patients and Methods: In all, 22 cutaneous melanocytic lesions that had either developed or considerably changed in morphology in 19 patients undergoing treatment with selective BRAF inhibitors for BRAF-mutant metastatic melanoma at seven international melanoma centers within clinical trials in 2010 and 2011 were analyzed for mutations in BRAF and NRAS genes and immunohistologically assessed for expression of various signal transduction molecules in comparison with 22 common nevi of 21 patients with no history of BRAF inhibitor treatment., Results: Twelve newly detected primary melanomas were confirmed in 11 patients within 27 weeks of selective BRAF blockade. In addition, 10 nevi developed of which nine were dysplastic. All melanocytic lesions were BRAF wild type. Explorations revealed that expression of cyclin D1 and pAKT was increased in newly developed primary melanomas compared with nevi (P = .01 and P = .03, respectively). There was no NRAS mutation in common nevi, but BRAF mutations were frequent., Conclusion: Malignant melanocytic tumors might develop with increased frequency in patients treated with selective BRAF inhibitors supporting a mechanism of BRAF therapy-induced growth and tumorigenesis. Careful surveillance of melanocytic lesions in patients receiving class I RAF inhibitors seems warranted.

Background: Early diagnosis of melanoma remains of paramount importance, because it has been widely demonstrated that survival is strongly related to Breslow thickness. Several studies have shown that dermatoscopy improves accuracy in the diagnosis of melanoma. Although histopathology is considered the gold standard to differentiate melanoma from nevi, there are some cases of melanoma in which the histopathologic features are less than definitive. It has also been demonstrated that fluorescence in situ hybridization can be used to differentiate melanomas from nevi based on chromosomal copy number aberrations., Objective: In this study we present a case series to demonstrate the value of combining fluorescence in situ hybridization and dermatoscopy/clinical history to enhance diagnostic capability for selected cases of early melanoma., Methods: Cases were identified that had dermatoscopic findings or clinical history highly suggestive of melanoma and fluorescence in situ hybridization evaluation positive for melanoma, but histopathologic features that were less than definitive. Two dermatopathologists performed independent histologic analysis of specimens and two dermatologists experienced in dermatoscopy reviewed dermatoscopic and clinical data., Results: Nine cases meeting inclusion criteria were identified. In 6 cases the histologic differential diagnosis was dysplastic nevus versus early melanoma whereas in 3 cases the differential diagnosis included Spitz nevus versus early melanoma., Limitations: Limitations of this study include restrictive inclusion criteria and study design restricted to a case series., Conclusion: This exploratory study demonstrates that in a subset of early melanoma cases, combining multiple diagnostic modalities such as dermatoscopy and molecular techniques with histology enhances detection of early melanoma., (Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Melanoma accounts for the majority of deaths from skin cancer. Women tend to be diagnosed at a younger age and have better survival than men. A tumor-host interaction might be responsible for these gender-specific differences. Recently, a functional single-nucleotide polymorphism in the promoter of the human homolog of mouse double minute 2 (MDM2) gene was characterized: single-nucleotide polymorphism (SNP)309 increases the MDM2 transcription. In melanoma, the effects for SNP309 and the related tumor protein p53 (TP53) Arg72Pro are inconsistent among published reports. This study investigated the association between SNP309 (RefSNP accession ID (rs)2279744) and TP53 codon 72 (rs1042522) polymorphisms, with outcome in a hospital-based cohort of 990 patients with melanoma. We assessed whether these polymorphisms were associated with clinicopathological and phenotypic characteristics and whether these SNPs affect the age of onset of the disease, recurrence, and survival. No significant associations were found between the SNPs and survival. However, women carrying the SNP309 GG genotype were less likely to be diagnosed at a younger age: odds ratio(adjusted<50) 0.52 (0.29-0.92). Our results suggest that women carrying the SNP309 GG genotype might be at lower risk of developing melanoma at a younger age compared with those carrying TG or TT. Further studies are needed to determine whether a nearby functional polymorphism is responsible for this effect in premenopausal women.

The vitamin D receptor (VDR) gene has been associated with cancer risk, but only a few polymorphisms have been studied in relation to melanoma risk and the results have been inconsistent. We examined 38 VDR gene single nucleotide polymorphisms (SNPs) in a large international multicenter population-based case-control study of melanoma. Buccal DNAs were obtained from 1,207 people with incident multiple primary melanoma and 2,469 with incident single primary melanoma. SNPs with known or suspected impact on VDR activity, haplotype tagging SNPs with ≥ 10% minor allele frequency in Caucasians, and SNPs reported as significant in other association studies were examined. Logistic regression was used to calculate the relative risks conferred by the individual SNP. Eight of 38 SNPs in the promoter, coding, and 3' gene regions were individually significantly associated with multiple primary melanoma after adjusting for covariates. The estimated increase in risk for individuals who were homozygous for the minor allele ranged from 25 to 33% for six polymorphisms: rs10875712 (odds ratios [OR] 1.28; 95% confidence interval (CI), 1.01-1.62), rs4760674 (OR 1.33; 95% CI, 1.06-1.67), rs7139166 (OR 1.26; 95%CI, 1.02-1.56), rs4516035 (OR 1.25; 95%CI, 1.01-1.55), rs11168287 (OR 1.27; 95%CI, 1.03-1.57) and rs1544410 (OR 1.30; 95%CI, 1.04-1.63); for two polymorphisms, homozygous carriers had a decreased risk: rs7305032 (OR 0.81; 95%CI 0.65-1.02) and rs7965281 (OR, 0.78; 95%CI, 0.62-0.99). We recognize the potential false positive findings because of multiple comparisons; however, the eight significant SNPs in our study outnumbered the two significant tests expected to occur by chance. The VDR may play a role in melanomagenesis., (Copyright © 2011 UICC.)

Objectives: To describe the computed tomography (CT) features in patients with biopsy-proven hepatic metastases of uveal melanoma and correlate these findings with survival., Methods: The medical records of patients with uveal melanoma evaluated at Memorial Sloan-Kettering Cancer Center from January 1998 to September 2009 were reviewed. Inclusion criteria were biopsy-proven liver metastasis and CT scan images available within 2 months of biopsy. Exclusion criteria were prior systemic or liver-directed therapy for uveal melanoma. Statistical analyses were carried out using the t test, χ(2) test, and Kaplan-Meier log-rank analyses., Results: Of 505 medical records reviewed, 76 were selected for study. Characteristic CT findings included multiple (68 patients [90%]), hypodense (100%), heterogeneous (100%), and enhancing (100%) hepatic lesions with a mean dominant lesion size of 46.8 cm(2). Eighteen patients (24%) exhibited hepatomegaly. Predominant lesion size larger than 100 cm(2), hepatomegaly, and ascites correlated with a lower survival rate (P = .008, P < .001, and P < .001, respectively). Radiographic evidence of extrahepatic metastases was present in 40 patients (53%). However, the presence of extrahepatic metastases did not affect survival. The results of at least 1 liver function test were abnormal in 46 of 67 patients (69%), and elevation of at least 1 serum transaminase and elevation of alkaline phosphatase were associated with larger lesions (P = .009 and P = .004, respectively) and hepatomegaly (P < .001 for both)., Conclusions: Radiographic evidence of predominant lesion size larger than 100 cm(2), hepatomegaly, and ascites-but not radiographic evidence of extrahepatic metastases-correlate with a lower survival rate in patients with biopsy-proven hepatic metastases of uveal melanoma.

Background: Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response., Methods: To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape., Results: Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules., Conclusion: Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially "boosting" the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

Background: Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas., Methods: We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi., Results: We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway., Conclusions: Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).

Background: Microscopic satellitosis in melanoma is uncommon. The role of regional basin staging/therapy in patients with this high-risk feature has not been well defined., Methods: Patients presenting from 1996 to 2005 with clinically localized melanoma containing microscopic satellitosis were identified from a prospective, single-institution database. Multiple factors were analyzed to determine their predictive value for recurrence. The management of the draining nodal basin was evaluated to determine its impact on recurrence and survival., Results: Thirty-eight patients presented to our institution during this time period with clinically localized melanoma containing microscopic satellitosis. The 5-year overall and disease-free survivals in these patients were 34% and 18%, respectively. Sixty-eight percent had pathologically involved regional nodal metastases. With median follow-up of 21 months, 68% recurred, with a median time to recurrence of 9 months. Lymphovascular invasion (LVI) (p = 0.01), tumor regression (p = 0.04), and positive regional lymph nodes (p = 0.02) were associated with an increased risk of recurrence. Of the 31 patients who underwent sentinel lymph node (SLN) biopsy, 22 had metastasis in the SLN (71%). Fifteen of these patients underwent completion lymphadenectomy (CLND) and seven were observed. There was no difference in disease-free survival (DFS), disease-specific survival (DSS), or overall survival (OS) between these groups (p = 0.42)., Conclusions: Pathological lymph node metastases were more prevalent (68%) than in any group previously defined. Regional nodal status predicted recurrence but not nodal recurrence. In SLN-positive patients, CLND did not improve DFS, DSS, or OS, although the number of patients was small. Further studies are needed to determine the utility of regional nodal staging/therapy in these high-risk patients.

While sentinel lymph node biopsy (SLN) is a highly accurate and well-tolerated procedure for patients with cutaneous melanoma, the role of the completion lymph node dissection (CLND) for patients with positive SLN biopsy remains unknown. This study aimed to look at the prognostic value of a positive nonsentinel lymph node (NSLN). A prospectively maintained database identified 222 patients with cutaneous melanoma and a positive SLN biopsy, without evidence of distant disease. All of these patients underwent CLND, and 37 patients (17%) had positive NSLN. With median follow-up of 33 months, patients with negative NSLN had median survival of 104 months, while patients with positive NSLN had median survival of 36 months (p < 0.001). There were no survivors in the patients with positive NSLN beyond 6 years. When patients with an equal number of positive nodes were analyzed, the presence of a positive NSLN was still associated with worse melanoma-specific survival (66 months for NSLN- versus 34 months for NSLN+, p = 0.04). While increasing age, tumor thickness, and male sex were associated with an increased risk of death on multivariate analysis, a positive NSLN was the most important predictor of survival (hazard ratio 2.5). We conclude that positive NSLN is an independent predictor of disease-specific survival in patients with cutaneous melanoma.

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.

Purpose: We conducted a phase II trial of extended-dose temozolomide (TMZ) in patients with melanoma to test the hypothesis that the approximately 30% response rate observed in patients treated with extended-dose TMZ with antiangiogenic agents was caused by TMZ alone. We hypothesized that expression of methylguanine methyltransferase (MGMT) in the tumor would correlate with drug resistance to TMZ., Patients and Methods: Patients with stage IV or unresectable stage III melanoma were treated with TMZ 75 mg/m(2)/d for 6 weeks followed by a 2-week rest period. Cycles were repeated until progression. Patients were stratified by M1c disease or not. The primary end point was objective response proportion. MGMT expression was assessed by methylation-specific pyrosequencing of the promoter and by immunohistochemistry., Results: Forty-nine patients (25 with M1c disease) were assessable. Three patients (12.5%) in each cohort experienced partial responses; there were no complete responses. Ten patients (21%) had stable disease lasting more than 24 weeks. Median time to progression was 3.3 months. Median survival was 10.1 months; survival was similar in the two cohorts. The estimated 18-month survival was 27%. There was no correlation between response and either immunohistochemistry staining for MGMT or for MGMT promoter methylation. Seventy-five percent of patients developed CD4(+) lymphopenia after three cycles., Conclusion: Extended-dose TMZ therapy did not result in a 30% responses rate, which has been observed using extended-dose TMZ with antiangiogenic agents. Response did not correlate with MGMT expression or promoter methylation as a continuous variable, suggesting that other resistance mechanisms are important.

Consumption of the epidermis (COE), defined as thinning of the epidermis with attenuation of basal and suprabasal layers and loss of rete ridges adjacent to collections of melanocytes, is a recently coined term encompassing changes of the epidermal architecture associated with melanoma. To evaluate this feature as an additional diagnostic criterion for melanoma, we examined COE in 453 melanocytic lesions, including 213 invasive melanomas from a population-based series and 240 suspicious pigmented lesions from a clinic-based series, excluding halo and Spitz nevi. In the population-based series, COE was identified in 92/213 (43%) invasive melanomas and became progressively more frequent with increasing Breslow depth (P < 0.0001) and Clark level (P = 0.0002). COE was more frequent when mitotic figures (P < 0.0001), ulceration (P = 0.005), or vertical growth phase (P = 0.009) were present, but it was not significantly associated with age, gender, site, regression, or tumor-infiltrating lymphocytes. In the clinic-based series of pigmented lesions, COE was present in 2/25 (8%) in situ melanomas, 1/29 (3%) lesions classified as melanoma in situ/high-grade dysplastic nevi, and 1/40 (2.5%) high-grade dysplastic nevi. COE was not identified in 146 low-grade dysplastic, congenital, or common nevi. In the combined datasets, 94/96 (98%) lesions exhibiting COE were classified as melanoma. This study demonstrates that COE is frequently present in invasive melanomas, is associated with more aggressive histopathologic features (including increased Breslow depth and ulceration) and may be a useful supplementary diagnostic criterion for melanoma. Furthermore, the process leading to COE may be the first step in a progression to ulceration.

Background: The matrix metalloproteinases (MMPs) are enzymes that cleave various components of the extracellular matrix (ECM) and basement membranes. MMPs are expressed in melanocytes and their overexpression has been linked to tumor development, progression and metastasis. At the genetic level, the following functional promoter polymorphisms are known to modify the gene transcription: -1306 C/T and -735 C/T in the MMP2 gene, and -1171 5A/6A in the MMP3 gene. Functional polymorphisms in MMP genes' promoter regions may modulate the risk for melanoma progression., Methods: We evaluated MMP2 and MMP3 germline polymorphisms in a group of 1002 melanoma patients using PCR-based methods, including fragment size analysis and melting temperature profiles. Two-sided Chi-Square, Cochran-Armitage tests for trend, Fisher's exact tests, and Kendall's Tau tests were performed to evaluate the associations between genotype and various clinical and epidemiologic factors. Multivariate analyses were conducted using logistic regression, adjusting for known melanoma confounders such as age, sex, phenotypic index, moles, freckles, and race. Survival estimates were computed using the Kaplan-Meier method and differences in survival were assessed using the log rank test., Results: All genotypes were in Hardy-Weinberg equilibrium. After adjustment for age, sex and phenotypic characteristics of melanoma risk, no significant associations were identified with the clinical, pathological, and epidemiological variables studied. The melting profile for MMP2 -735 C/T identified a new change in one sample. A new PCR-amplification followed by direct sequencing confirmed a heterozygote G to A substitution at position -729., Conclusion: This study does not provide strong evidence for further investigation into the role of the MMP2 and MMP3 variants in melanoma progression.

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.

Cyclin-dependent kinase inhibitor type 2A (CDKN2A) has been identified as a major melanoma susceptibility gene based on the presence of germline mutations in high-risk melanoma families. In this study, we sought to identify and characterize the spectrum of CDKN2A mutations affecting p16 inhibitor of cyclin-dependent kinase type 4 (INK4a) in individuals with melanoma using a population-based study design. DNA samples from 1189 individuals with incident multiple primary melanoma (MPM) and 2424 with incident single primary melanoma unselected for family history of melanoma were available for screening of CDKN2A (p16INK4a) mutations. Variants were classified for functional impact based on intragenic position, existing functional data, sequence, and structural analysis. The impact of individual mutations and functional groupings was assessed by comparing frequencies in cases of MPM versus cases with a single first primary melanoma, and by comparing the reported incidence rates in first-degree relatives. Our results show that mutations occur infrequently in these high-risk groups, and that they occur mainly in exons 1alpha and 2. Rare coding variants with putative functional impact are observed to increase substantially the risk of melanoma. With the exception of the variant in position -34 of CDKN2A of known functional consequence, the remaining rare variants in the non-coding region have no apparent impact on risk.