No detectable truncating mutations in large T antigen (LT-Ag) sequence of Merkel cell polyomavirus (MCPyV) DNA obtained from porocarcinomas.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC). In tumor cells the MCPyV large T antigen (LT-Ag) is frequently found truncated and this is considered a major tumor-specific signature. The role of MCPyV in other, non-MCC tumours, is little known. Viral DNA and/or tumour-specific mutations have been sometimes detected in different tumours, but such data are not unequivocal and the involvement of the virus in the tumorigenesis is not clear. In a previous study, we demonstrated a significantly higher prevalence of MCPyV DNA in formalin fixed paraffin embedded (FFPE) porocarcinoma tissues compared to the normal skin. In the present study, we investigated the presence of truncating mutations in MCPyV LT-Ag coding region in porocarcinoma specimens. Using several overlapped PCR primer pairs, the complete LT-Ag sequence from two biopsies were obtained. No truncating mutations were detected. The lack of truncating mutations in LT-Ag sequence does not seem to support the role of MCPyV in porocarcinoma oncogenesis. However, an oncogenetic mechanism, different from that proposed for MCC and not associated with the LT-Ag mutations/deletions, cannot be excluded. Further studies of more sequences coding for LT-Ag would be needed to verify this hypothesis.

Clear-Cell Dermal Duct Tumor: Case Report and Literature Review.

ABSTRACT: Clear-cell dermal duct tumor is a benign adnexal neoplasm composed of dermal multiple solid islands of clear cells, displaying ductal differentiation. Histopathologically, lesions can be subdivided into 2 distinct subgroups: (1) "pure" clear-cell dermal duct tumors, entirely composed of clear cells, and (2) "mixed" clear-cell dermal duct tumors, showing an associated conventional poroid component. Such a subclassification may be significant for the differential diagnosis: the less frequent "mixed" variant may be more easily recognized because of the presence of poroid and cuticular cells and the more frequent "pure" variant is to be distinguished from many other benign and malignant dermal clear-cell epithelial tumors.

Spitz Tumors and Melanoma in the Genomic Age: A Retrospective Look at Ackerman's Conundrum.

After 25 years, "Ackerman's conundrum", namely, the distinction of benign from malignant Spitz neoplasms, remains challenging. Genomic studies have shown that most Spitz tumors harbor tyrosine and serine/threonine kinase fusions, including ALK, ROS1, NTRK1, NTRK2, NTRK3, BRAF and MAP3K8, or some mutations, such as HRAS and MAP3K8. These chromosomal abnormalities act as drivers, initiating the oncogenetic process and conferring basic bio-morphological features. Most Spitz tumors show no additional genomic alterations or few ones; others harbor a variable number of mutations, capable of conferring characteristics related to clinical behavior, including CDKN2A deletion and TERT-p mutation. Since the accumulation of mutations is gradual and progressive, tumors appear to form a bio-morphologic spectrum, in which they show a progressive increase of clinical risk and histological atypia. In this context, a binary classification Spitz nevus-melanoma appears as no longer adequate, not corresponding to the real genomic substrate of lesions. A ternary classification Spitz nevus-Spitz melanocytoma-Spitz melanoma is more adherent to the real neoplastic pathway, but some cases with intermediate ambiguous features remain difficult to diagnose. A prognostic stratification of Spitz tumors, based on the morphologic and genomic characteristics, as a complement to the diagnosis, may contribute to better treatment plans for patients.

Folliculosebaceous Cystic Hamartoma with Spindle Cell Lipomatous and Neural Components.

Folliculosebaceous cystic hamartoma is a cutaneous malformation composed of a cystic folliculosebaceous structure associated with mesenchymal elements, generally consisting of fibrous stroma, adipocytes and small vascular channels. We report the case of a 55-year-old female patient with a cutaneous nodule of the right nasal wing. Microscopically, the lesion showed a dilated hair follicle with multiple sebaceous glands, surrounded by a mesenchymal component composed of fibromyxoid stroma, spindle cells, mature-appearing adipocytes and collagen bundles, resembling spindle cell lipoma, associated with an additional neural component, consisting of small nerve bundles. In folliculosebaceous cystic hamartoma, the association of spindle cell lipomatous and neural components has not previously reported.

Conceptual Evolution and Current Approach to Spitz Tumors.

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.

Eruptive melanocytic nevi in a patient with amelanotic melanoma: a paraneoplastic phenomenon?

Eruptive melanocytic nevi (EMN) describes the sudden onset of cutaneous nevi over weeks or months. Such a clinical event is generally seen in young adult patients and may be related to several possible causes. We report here a case of EMN in an old male patient followed up for a thick amelanotic cutaneous melanoma. A few months after the eruption, multiple hepatic masses, diagnosed as melanoma metastasis, were detected. The presented case may suggest that EMN may be a paraneoplastic phenomenon of alert in patients being followed for melanoma or other malignancies.

NGS-Based Analysis of Atypical Deep Penetrating Nevi.

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 ß-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 ß-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.

Melanocytic Skin Neoplasms: What Lesson From Genomic Aberrations?

Studies on the genomic aberrations in melanocytic neoplasms have shown a complex genomic landscape. In nevi and melanomas, a MAP-kinase pathway activation was generally found, produced by different chromosomal aberrations, including BRAF, NRAS, HRAS, GNAQ, GNA11, BAP1, CTNNB1, MAP2K1, PRKAR1A, and NF1 mutations, and ALK, ROS1, NTRK1, RET, MET, BRAF, NTRK3, and PRKCA fusions. Melanomas also showed a variable number of additional mutations ablating tumor-suppression mechanisms and activating other oncogenic pathways, including CDKN2A loss, PTEN loss, as well as TP53 and TERT-promoter mutations. Moreover, borderline melanocytic tumors displayed the same chromosomal aberrations, but more mutations than nevi and fewer than melanomas. In this context, the notion that melanocytic neoplasms can be classified as benign/malignant is hardly supportable, because all neoplasms harbor a certain number of mutations and the progression risk, that is, the malignant potential, is related and proportional to the burden of pathogenic mutations. Moreover, from the genomic analysis, in parallel to the current diagnostic categories of "nevi," "melanomas," and "melanocytomas," some aggregations or classes of tumors based on the characteristic types of driver mutations/fusions emerge as possible and more rationale, including Spitzoid neoplasms, blue neoplasms, BAP1-inactivated melanocytic neoplasms, deep penetrating melanocytic neoplasms, pigment-synthesizing melanocytic neoplasms, and "common" melanocytic neoplasms. Each of these classes, showing the same driver mutations/fusions, demonstrates to have the same pathogenesis and may be genetically considered as a single tumor, although with a variable amount of progression risk. Histologic features, being an expression of the mutational state, could be used to obtain an approximate risk assessment in each single tumor.

Enhancing the prognostic role of melanoma sentinel lymph nodes through microscopic tumour burden characterization: clinical usefulness in patients who do not undergo complete lymph node dissection.

This study aimed to investigate the sentinel lymph node (SLN) tumour burden to predict the non-SLN positivity rate and the survival of melanoma patients to evaluate whether SLN microstaging could predict the prognosis, similar to what is currently performed by examining the lymph nodes excised by complete lymph node dissection. Of 1130 consecutive melanoma patients who underwent SLN biopsy, 226 were tumour-positive and 204 were included in this study. SLN metastases were classified on the basis of dimensional (Rotterdam) and topographic (Dewar) criteria either separately or combined. SLN metastases more than 1 mm in diameter had the highest non-SLN positivity rate (31%) compared with metastases 0.1-1 mm (10%) and less than 0.1 mm (4%). The non-SLN positivity rate was 45% for extensive metastases, 5% for subcapsular metastases and 23-29% for parenchymal, combined and multifocal classes, therefore suggesting a simplification of the parenchymal SLN metastases into only two classes: extensive and 'not extensive'. The dimension of the metastasis was correlated with a different non-SLN positivity rate only when the metastasis was in the parenchyma (20-36%) and not when it was in the subcapsular location (4-7%). Interestingly, the 5-year melanoma-specific survival (MSS) was 89% for patients with subcapsular less than 0.1 mm metastases and 45% for patients with nonsubcapsular more than 1 mm metastases (P=0.017). In the parenchyma, larger metastases (>1 mm) were related to a lower 5-year MSS (46%) than smaller (<1 mm) metastases (MSS 77%). SLN tumour burden characterization can be simplified and it can provide prognostic information on non-SLN positivity and survival, which is especially useful in patients who do not undergo complete lymph node dissection.

ERK5 is activated by oncogenic BRAF and promotes melanoma growth.

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.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. OBJECTIVE: To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. METHODS: Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. RESULTS: The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥105 copies per reaction, while the upper limit of ddPCR was 104 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). CONCLUSIONS: The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome.

Time to reconsider Spitzoid neoplasms?

BACKGROUND: Spitzoid neoplasms may pose significant diagnostic problems because in a fraction of them it is quite difficult or impossible to establish if they are benign or malignant lesions. An extraordinarily large number of studies have been made in attempts to solve this problem; regrettably, the histological criteria proposed and the various special sophisticated techniques employed have proven to be ineffective in making this distinction with confidence. OBJECTIVES: To explore the possible causes for this diagnostic failure and an attempt to identify the source of this problem. METHOD: A historical and technical analysis of the specialized literature is performed, critically evaluating the main points of this controversial topic. RESULTS: The reasons for the diagnostic failure in Spitzoid neoplasms are not clear but could be the result of inappropriate conceptual representation. The analysis of available data and a rational review of old and new assumptions and concepts may suggest a different representation for Spitzoid neoplasms: Spitz nevus, atypical Spitz tumor and Spitzoid melanoma, rather than being three different tumors that are difficult or impossible to distinguish with assurance, could be viewed as one unique entity, Spitz tumor (ST). This tumor is a low-grade malignant neoplasm, in which the amount of intrinsic risk is variable, ranging from very low to high (ST1, ST2, ST3), and malignant potential could be estimated. CONCLUSIONS: The proposed alternative representation of Spitzoid neoplasms as a unique tumor may help in overcoming the difficulty in diagnosis of these tumors.