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Over the past several decades, the study of Spitz neoplasms has flourished, with expanded conceptualization and refined terminology, providing a framework for the assessment and classification of Spitz nevi, atypical Spitz Tumors, and Spitz melanoma. Cancer genomics have generated concepts such as driver and passenger genes and clonal evolution, which can be applied to Spitz tumors. Herein, we provide a historical perspective, followed by a summary of current knowledge and clinical approaches for these challenging tumors.

Simple Summary The recent WHO classification of melanocytic tumors requires the implementation of combined phenotypic–genotypic diagnostics. For rare tumors, such as atypical deep penetrating nevi, there is insufficient information regarding genetic status, and it is not yet clear whether the observed unusual morphological cyto-architectures reflect a distinct genomic profile or are associated with an increased metastatic potential and aggressive clinical behavior. We report herein a comprehensive next-generation sequencing (NGS) analysis of a series of atypical DPNs, showing their mutational profile with some specific signatures for these rare and diagnostically challenging tumors. Abstract Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that β-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the β-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Background: Recent studies have shown an increasing incidence of cutaneous adnexal carcinomas (CACs). Objectives: The aim of our study was to evaluate incidence and survival for cases of CACs and investigate their association with other skin neoplasms. Methods: We conducted a population-based study. Data on incident cases of CACs were obtained from the Tuscany Cancer Registry between 1985 and 2010. In order to determine whether the occurrence of squamous cell carcinoma (SCC) among patients with CAC is higher or lower than expected in the general population, the standardized incidence ratio (SIR) was calculated. Results: A total of 242 patients with CAC were observed; the age-standardized incidence rate was 3·8 cases per million person-years. From 1997 to 2010 crude incidence rates increased by 159%. Age-specific incidence was higher in men over 80 years old than in women of the same age and younger individuals. Carcinomas of sweat gland origin prevailed; the most common histotype was porocarcinoma and the most frequently affected site was the head/neck. Overall, 88% of CACs were diagnosed at a localized stage. The 5-year overall survival and disease-specific survival rates were 59% [95% confidence interval (CI) 53–65] and 94% (95% CI 91–98), respectively. In the observation cohort, the number of SCCs was significantly higher than expected as the SIR was calculated to be 33·7 (P < 0·001). Conclusions: Increasing incidence warrants awareness and early diagnosis of CACs. Increased SCC incidence among patients with these tumours highlights the relevance of careful skin examination and follow-up.

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

Cutaneous melanoma is one of the most aggressive type of skin tumor. Early stage melanoma can be often cured by surgery; therefore current management guidelines dictate a different approach for thin (4mm) melanomas. We have carried out whole-exome sequencing in 5 thin and 5 thick fresh-frozen primary cutaneous melanomas. Unsupervised hierarchical clustering analysis of somatic copy number alterations (SCNAs) identified two groups corresponding to thin and thick melanomas. The most striking difference between them was the much greater abundance of SCNAs in thick melanomas, whereas mutation frequency did not significantly change between the two groups. We found novel mutations and focal SCNAs in genes that are embryonic regulators of axon guidance, predominantly in thick melanomas. Analysis of publicly available microarray datasets provided further support for a potential role of Ephrin receptors in melanoma progression. In addition, we have identified a set of SCNAs, including amplification of BRAF and ofthe epigenetic modifier EZH2, that are specific for the group of thick melanomas that developed metastasis during the follow-up. Our data suggest that mutations occur early during melanoma development, whereas SCNAs might be involved in melanoma progression.

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

Histopathological evaluation of melanocytic tumours usually allows reliable distinction of benign melanocytic naevi from melanoma. More difficult is the histopathological classification of Spitz tumours, a heterogeneous group of tumours composed of large epithelioid or spindle-shaped melanocytes. Spitz tumours are biologically distinct from conventional melanocytic naevi and melanoma, as exemplified by their distinct patterns of genetic aberrations. Whereas conventional naevi and melanoma often harbour BRAF mutations, NRAS mutations, or inactivation of NF1, Spitz tumours show HRAS mutations, inactivation of BAP1 (often combined with BRAF mutations), or genomic rearrangements involving the kinases ALK, ROS1, NTRK1, BRAF, RET, and MET. In Spitz naevi, which lack significant histological atypia, all of these mitogenic driver aberrations trigger rapid cell proliferation, but after an initial growth phase, various tumour suppressive mechanisms stably block further growth. In some tumours, additional genomic aberrations may abrogate various tumour suppressive mechanisms, such as cell-cycle arrest, telomere shortening, or DNA damage response. The melanocytes then start to grow in a less organised fashion, may spread to regional lymph nodes, and are termed atypical Spitz tumours. Upon acquisition of even more aberrations, which often activate additional oncogenic pathways or reduce and alter cell differentiation, the neoplastic cells become entirely malignant and may colonise and take over distant organs (spitzoid melanoma). The sequential acquisition of genomic aberrations suggests that Spitz tumours represent a continuous biological spectrum, rather than a dichotomy of benign versus malignant, and that tumours with ambiguous histological features (atypical Spitz tumours) might be best classified as low-grade melanocytic tumours. The number of genetic aberrations usually correlates with the degree of histological atypia and explains why existing ancillary genetic techniques, such as array comparative genomic hybridisation (CGH) or fluorescence in situ hybridisation (FISH), are capable of accurately classifying histologically benign and malignant Spitz tumours, but are not very helpful in the diagnosis of ambiguous melanocytic lesions. Nevertheless, we expect that progress in our understanding of tumour genomics and progression will refine the classification of melanocytic tumours in the near future. By integrating clinical, pathological, and genetic criteria, distinct tumour subsets will be defined within the heterogeneous group of Spitz tumours, which will eventually lead to improvements in diagnosis, prognosis and therapy.

Background Mucosal melanomas (MM) represent a heterogeneous tumour population that exhibits site-specific molecular profiles. Aims In a multicentre retrospective study, we investigated KIT aberrations in primary anorectal (AR) melanomas compared with melanoma metastatic to the gastrointestinal (GI) tract. Methods Primary AR MM (n=31) and GI metastatic melanoma (n=27) were studied for KIT mutations on exons 11, 13, 17 and 18 by high-resolution melting analysis, direct sequencing and c-KIT expression by immunohistochemistry. Selected cases were also investigated for increased KIT gene copy number by fluorescent in situ hybridisation. Results Functional KIT mutations were demonstrated in 11/31 (35.5%) of AR melanomas and in 1/26 (3.8%) of GI melanoma metastases (p=0.004). A significant difference emerged between primary and metastatic MM with regards to KIT-positive immunostaining (p=0.002). Immunohistochemical c- KIT protein overexpression did not correlate with KIT mutational status. Increased KIT copy number was demonstrated in 5/20 AR primary cases. Conclusions The rate of functional mutations in KIT is significantly higher in AR MM than in GI metastatic melanoma. KIT protein overexpression does not correlate with KIT mutations and cannot be used for screening purposes. Recognising the molecular heterogeneity of MM helps to identify patients who require a different therapeutic approach.

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.

Context.—Several aspects of sweat gland carcinomas (incidence, classification, diagnosis, and behavior) have not been definitively clarified and need to be studied further. Objective.—The clinicopathologic findings of a large series of sweat gland carcinomas, collected during a period of 15 years, are presented. Methods.—Sixty sweat gland carcinomas (41 porocarcinomas, 3 syringomatous carcinomas, 8 ductal carcinomas, 5 adenoid cystic carcinomas, and 3 mucinous carcinomas) were analyzed histologically and immunohistochemically. Results.—Porocarcinomas were composed of eosinophilic and clear atypical cells arranged in solid-cystic lobular masses. These tumors were divided into 2 subgroups: horizontal porocarcinomas, showing a prominent intraepidermal component, and nodular porocarcinomas, which demonstrated predominant nodular growth. Syringomatous carcinomas presented keratinizing and nonkeratinizing cysts, dilated tubules (sometimes with a “tadpole” appearance), small neoplastic ducts, solid islands, and cellular cords. Ductal carcinomas were characterized by a prominent formation of tubules, solid islands, and cellular cords. Adenoid cystic carcinomas presented a characteristic pattern, showing basaloid monomorphous cells with moderately atypical nuclei, arranged in cribriform or solid islands and in tubular structures. Mucinous carcinomas were composed of moderately atypical cells with eosinophilic vacuolated cytoplasm, forming solid and cystic islands floating in large mucin pools. Immunohistochemically, cytokeratin was found in neoplastic cells in all cases, carcinoembryonic antigen was detected in 73% of cases, and actin-positive (myoepithelial) cells were not found. Conclusions.—Although numerous studies have been published in recent years, the histologic features, histogenesis, and classification of sweat gland carcinomas still remain controversial and need to be clarified by further studies.

Electrochemotherapy (ECT) is a novel treatment for recurrent or in-transit unresectable melanoma metastases based on the administration of anti-neoplastic drugs followed by cancer cell electroporation. Whether ECT can also induce anti-tumour immunity is unclear. We addressed this issue investigating the presence of dendritic cells (DCs) in the inflammatory infiltrate of ECT-treated lesions. Biopsies from melanoma patients (n = 9) were taken before ECT (T0), at d7 and d14 after treatment and studied by immunofluorescence with DCs-related antibodies. Epidermal Langerin+ Langerhans cells (LCs) were the most represented subset before treatment. ECT induced a significant reduction in epidermal LCs number at d7 (p < 0.001), while they were completely replaced at d14. Similarly, the few LCs observed intermingled with metastatic melanoma cells at T0 decreased after treatment (p < 0.001), suggesting an ECT-induced activation of LCs. Consistently, at d1 after ECT (n = 3 patients), LCs were found to express CCR7, which mediates LCs migration to regional lymph nodes, and CD83, the typical DCs maturation marker. In contrast, plasmacytoid DCs (pDCs) were not present at T0, but significantly increased after ECT both in melanoma metastasis (p < 0.001) and perilesionally (p < 0.05). Similarly, CD1c+ dermal DCs (dDCs), observed in low number before ECT, strongly increased at d7 and even more at d14 (p < 0.05 and p < 0.001, respectively). Notably, some dDCs expressed CD83. These data suggest that ECT promotes LCs migration from the tumour to draining lymph nodes and pDCs and dDCs recruitment at the site of the lesion. These findings may help to design new strategies of in situ DCs vaccination in cancer patients.

The special types of breast cancer seem to have not only distinct morphological features but also distinct biological features. Women diagnosed with a first primary invasive breast cancer in the 2004–2005 period were identified through Tuscan Cancer Registry. Information on age, tumor size, lymph node status, histological type and grade, hormonal receptors, HER2 immunohistochemical expression were collected. Five subtypes were defined: luminal A, luminal B HER2+, luminal B HER2−, triple negative, and HER2 positive. The association between the histological type and molecular subgroups was assessed by a Fisher’s exact test, and a multinomial logistic regression model was used. Out of 1,487 patients, 34 % were luminal A subtype, 25 % luminal B HER2−, 11 % luminal B HER2+, 19 % triple negative, and 10.2 % HER2+; 58.5 % of cancers were ductal NOS types. With luminal A as reference, histological types distribution was significantly different between the subgroups. Mucinous, tubular, and cribriform histotypes were found among luminal A cancers more than in other subgroups; all medullary carcinomas were triple negative cancers. Pathological stage at diagnosis was more advanced, and histological grade was lower among subgroups other than luminal A. Significant association between breast cancer histotypes and molecular subgroups was found.

In a population-based screening program, a percentage of tumors remain undetected; these tumors comprise a heterogeneous group, and they are more likely to have adverse prognostic features. The aim of this study was to identify differences in biological characteristics of screen-detected versus interval breast cancers in a population-based screening program according to molecular subtypes.We analyzed the population-based data from a long-running screening program in the area of Florence. Data on screening history and on age, T and N status, grade, histotype, hormonal status and Ki-67 and HER2 expression were retrieved. Subtypes of breast cancer were defined on the expression of ER, PR, Ki-67 and HER2: luminal A if ER/PR+, HER2- and Ki6714 %, luminal B (HER2 negative) if ER/PR+, HER2- and Ki67 ≥14 %, luminal B (HER2 positive) if ER/PR+ and HER2+, triple negative if ER/PR-and HER2-, HER2 positive if ER/PR- and HER2+. Association between molecular subtypes and mode of detection will be evaluated by a logistic regression model adjusted for the potential confounding variables.Information about biomarkers was known for 277 cases, 211 screening-detected and 66 interval cancers. Among interval cases, the triple-negative cancers were more represented than luminal A (OR = 3.52; CI, 1.112-11.13; p = 0.0319), while the proportion of HER2+ was quite similar (OR = 1.57; p = 0.4709).Although made on a small number of cases, our results suggest a difference in distribution of molecular subtypes according to mode detection, confirming the results of earlier studies.

To the editor: The specific function of Langerhans cells (LCs) in the skin immune system is controversial and a matter of intense debate.[1][1] As epidermal LCs are potential targets for cancer immunotherapy, the topic is of primary importance.[2][2] Recently, van de Ven et al showed that LCs are

The aim of this study is to evaluate the prognostic values of some biological parameters in a population based series of female breast cancer patients. Through the Tuscan Cancer Registry all the invasive breast cancer cases diagnosed during the period 2004–2005 in the provinces of Florence and Prato, central Italy, were retrieved. Molecular subtypes were analyzed defined by immunohistochemical markers, by age, tumor size, lymph node status, histotype, grade of differentiation and proliferative marker. Out of 1487 patients 70.3% were luminal A subtype (ER/PR + HER2-), 15.6% luminal B (ER/PR + HER2+), 8.1% triple negative (ER/PR-HER2-), 6.0% HER2+ (ER/PR-HER2+); the 3 year survival rates were 93.3%, 89.5%, 86.3%, 82.7% respectively (p < 0.001). Analysis of survival by the Cox proportional hazards model showed an independent prognostic value of molecular classification. Our study revealed significant differences in clinicopathological characteristics among breast cancer molecular subtypes and confirmed their prognostic independent role.

Background: We tested the relevance of clinical information in the histopathologic evaluation of melanocytic skin neoplasm (MSN). Methods: Histopathologic specimens from 99 clinically atypical MSN were circulated among ten histopathologists; each case had clinical information available in a database with a five-step procedure (no information; age/sex/location; clinical diagnosis; clinical image; dermoscopic image); each step had a histopathologic diagnosis (D1 through D5); each diagnostic step had a level of diagnostic confidence (LDC) ranging from 1 (no diagnostic certainty) to 5 (absolute diagnostic certainty). The comparison of the LDC was employed with an analysis of variance (ANOVA) for repeated measures. Findings: In D1 (no information), 36/99 cases (36.3%) had unanimous diagnosis; in D5 (full information available), 51/99 cases (51.5%) had unanimous diagnosis (p for difference between proportions

AIMS: The histological features used in the diagnosis of melanoma may be present in benign naevi, but quantitative data are not available. The aim of this study was to establish the real prevalence of such features in naevi. METHODS: Ten dermatopathologists, from nine Italian institutions, studied a series of naevi. Eleven histological parameters currently used in melanoma diagnosis were analysed: asymmetry, poor circumscription, predominance of single melanocytes, irregular confluent nests, suprabasal melanocytes, hair follicle involvement, absence of maturation, cytological atypia, dermal lymphocytic infiltrate, mitoses, and necrosis. RESULTS: Ninety one naevi were examined: 22 junctional, 59 compound, and 10 intradermal. None of the studied parameters was seen in 22 of the benign naevi studied. One or more investigated features were found in 69 naevi. Poor circumscription was found in 49 cases, single melanocytic predominating in 42, asymmetry in 41, irregular confluent nests in 16, cytological atypia in 14, suprabasal melanocytes in seven, and hair follicle involvement in seven; absence of maturation, mitoses and necrosis were not found. CONCLUSIONS: The histological features used for the histological diagnosis of melanoma are often present in benign melanocytic naevi. This suggests a critical, non-mechanical use of them in melanoma diagnosis.

Two growth phases, radial and vertical, are commonly identified in cutaneous melanoma. In the radial growth phase, melanoma appears as an irregular plaque; cells may invade the dermis, but do not form a nodule. In the vertical growth phase, the lesion develops vertically, forming a true tumour.1 Growth phases, generally reputed to be sequential stages of tumour development, are considered to be characteristic of skin melanoma and are not generally recognised in other tumours. However, analysing other skin neoplasms, some analogies can be noted. Squamous cell carcinoma originates in the epidermis as squamous cell carcinoma in situ, which shows a radial growth …

Trabecular carcinoma has been renamed as Merkel cell carcinoma because, ultrastructurally, tumour cells resemble Merkel cells of the skin. Recently, evidence of exocrine (sweat gland) differentiation in some trabecular carcinomas has been reported. These findings raise two possibilities: (1) trabecular carcinomas are a heterogeneous group of lesions; or (2) that trabecular carcinomas are glandular tumours, in which exocrine features are rarely detected.

A case of sclerosing epithelial hamartoma in a 19-year-old female is reported. The lesion, presenting the distinctive clinical and histologic features, was associated with a melanocytic compound nevus. This unusual and interesting event is discussed.