RAF1 Gene Fusions as a Possible Driver Mechanism in Rare BAP1-Inactivated Melanocytic Tumors: A Report of 2 Cases.

BRCA1-associated protein (BAP1)-inactivated melanocytic tumor (BIMT) is a group of epithelioid melanocytic neoplasms characterized by the loss of function of BAP1, a tumor suppressor gene located on chromosome 3p21. They occur sporadically or in the setting of an autosomal-dominant cancer susceptibility syndrome that predisposes to the development of different internal malignancies. Most of these cutaneous lesions are associated with a BRAF-mutated melanocytic nevus and therefore are included in the group of combined nevi in the last WHO classification of skin tumors. Apart from a BRAF mutation, an NRAS mutation has been reported in rare cases, whereas in some lesions no driver mutation has been detected. Here, we report 2 cases of BIMTs with a BAP1 mutation and a RAF1 fusion. Both lesions proved to be BRAF and NRAS wild type and were associated with a conventional melanocytic nevus with dysplastic junctional features. We suggest that RAF1 fusions can represent an underlying driver genetic event in these cases. Our study extends the morphological and molecular spectrum in BIMTs.

Combination of Congenital and Deep Penetrating Nevus by Acquisition of ß-Catenin Activation.

Deep penetrating nevus (DPN) is an intradermal, sometimes compound benign melanocytic lesion, which involves the reticular dermis, occasionally reaching the subcutis, which can raise concern for melanoma both clinically and histologically. Recently, it has been genetically defined by the combination of MAPK activating and ß-catenin activating mutations. We sought to investigate genetic alterations in 2 cases of combined nevi of congenital melanocytic and DPN. Case 1 was a 16-year-old woman with a pigmented lesion on the trunk since birth, which was completely excised. Histopathological examination revealed a combined congenital nevus with a DPN. Comparative genomic hybridization showed no major genetic alterations, except for gain of 6q11.1 and point mutation of B-RAF V600E. Case 2 was a 62-year-old woman with a congenital pigmented lesion on the back. The lesion was diagnosed as a combined nevus of congenital and DPN. Comparative genomic hybridization showed no genetic alterations, and the NRAS Q61K was detected in both components. DPN is in most cases part of a combined nevus. Our cases showed strong and uniform nuclear expression of ß-catenin and cyclin D1 in the DPN component suggesting the evolution of the congenital nevus to the DPN clone by acquiring ß-catenin activating mutation.

Tyrosinase and nestin immunohistochemical expression in melanocytic nevi as a histopathologic pattern to trace melanocyte differentiation and nevogenesis.

While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.

Differential Clinicopathological Features in Spontaneous Regression of Melanomas and Melanocytic Naevi.

The aim of this study was to determine the clinical, histological and/or immunohistochemical features that enable differential diagnosis of regression of melanocytic naevi from regression of melanomas. All melanocytic neoplasms with histologically-confirmed regression diagnosed in our hospital between 2002 and 2009 were reviewed retrospectively. Lamellar and delicate fibrosis were associated with melanocytic naevi (p <0.0001 and p = 0.021, respectively). Compact fibrosis, high vessel density and higher number of granzyme B+ lymphocytes were associated with malignant melanoma (p = 0.011, p = 0.005 and p = 0.013, respectively). Density of inflammatory infiltrate (p = 0.016), vascular proliferation (p = 0.005), epidermal atrophy (p = 0.009), rate of apoptosis (p = 0.046) and granzyme B immunoreactivity (p = 0.013) was more common in severe-dysplastic naevi and melanomas than in the remaining melanocytic naevi. Logistic regression demonstrates that 5 variables (age, lamellar fibrosis, melanophages, vessel density, and granzyme B immunostaining) would serve to classify appropriately 87% of melanomas among melanocytic lesions with complete regression.

Use of New Techniques in Addition to IHC Applied to the Diagnosis of Melanocytic Lesions, With Emphasis on CGH, FISH, and Mass Spectrometry.

Melanoma remains one of the most aggressive forms of cutaneous malignancies. While its diagnosis based on histologic parameters is usually straight forward in most cases, distinguishing a melanoma from a melanocytic nevus can be challenging in some instances, especially when there are overlapping clinical and histopathologic features. Occasionally, melanomas can histologically mimic other tumors and even demonstration of melanocytic origin can be challenging. Thus, several ancillary tests may be employed to arrive at the correct diagnosis. The objective of this review is to summarize these tests, including the well-established and commonly used ones such as immunohistochemistry, with specific emphasis on emerging techniques such as comparative genomic hybridization, fluorescence in situ hybridization and imaging mass spectrometry.

A Symmetric Eczematous Eruption Harboring Thousands of Melanocytic Lesions.

IMPORTANCE: The abrupt appearance of melanocytic lesions is a unique phenomenon that can occur in the setting of eruptive nevi or epidermotropic melanoma metastases. OBJECTIVE: To examine the immunohistochemical and genetic mutative features of a novel case of an eczematous reaction followed by the abrupt appearance of melanocytic lesions. DESIGN, SETTING, AND PARTICIPANT: Case report of a 48-year-old woman with no significant medical history who first presented with an eczematous dermatitis on her torso, extremities, and buttocks and who subsequently developed thousands of pinpoint, histologically atypical melanocytic tumors and invasive melanoma within the areas of inflammation. MAIN OUTCOMES AND MEASURES: Immunohistochemical and mutational analyses of the patient's melanocytic tumors were conducted. RESULTS: Mutational analysis of the pigmented lesions did not identify any activating mutations in BRAF, PTEN, NRAS, KRAS, and HRAS. Immunohistochemical analyses of 9 biopsied pigmented lesions all showed normal expression of the tumor suppressors p16 and PTEN and no expression of mutated BRAF V600E protein. CONCLUSIONS AND RELEVANCE: To our knowledge, this is a previously unreported eruption comprising 2 distinct components: an eczematous reaction and a wave of melanocytic proliferations within the inflammatory regions. Possible explanations for this patient's condition, include immune stimulation leading to nevogenesis, benign "nevic" metastases, eruptive nevi, and epidermotropic metastatic melanoma.

Proliferation indices correlate with diagnosis and metastasis in diagnostically challenging melanocytic tumors.

The diagnosis of melanocytic lesions remains a formidable challenge in dermatopathology. For diagnostically challenging lesions, ancillary tests are available to inform the diagnosis, including immunohistochemistry and molecular testing (particularly fluorescence in situ hybridization [FISH]). However, the test result that most robustly informs the diagnosis remains controversial. Thirty-seven diagnostically challenging melanocytic lesions from our consultation service were reviewed. Histopathologic, immunohistochemical, and second-generation FISH results (NeoGenomics; probes 6p25, 8q24, 11q13, 9p21, and centromere 9) were correlated with the final consensus diagnosis and clinical follow-up using logistic regression and Fisher exact test. Based on histopathologic and immunohistochemical features, cases were designated as "favor benign" (n=19) or "favor malignant" (n=18) by a consensus group of up to 7 dermatopathologists. The sensitivity of FISH for the diagnosis of melanoma was 39%, and the specificity was 84%. Univariate logistic regression models for a final diagnosis of melanoma showed that only increased Ki-67-positive dermal tumor cells (≥5%; P=.01) significantly correlated with the diagnosis of melanoma. FISH result did not correlate with the final diagnosis (melanoma or nevus; P=.26). Follow-up (range, 8-29months) was available for 35 cases (19 diagnosed as nevus and 16 as melanoma), and metastases (restricted to sentinel lymph nodes) were detected from 5 melanomas (3 FISH negative and 2 FISH positive). Only increased dermal mitotic figures (>1/mm(2)) correlated with metastases to sentinel lymph nodes (P=.04). Thus, in the classification of diagnostically challenging melanocytic lesions, indices of proliferation emerge as the most informative diagnostic adjuncts-correlating with diagnosis and clinical behavior, respectively.

Quantitative Analysis of Formaldehyde-induced Fluorescence in Paraffin-embedded Specimens of Malignant Melanomas and Other Melanocytic Lesions.

Inter-observer agreement is problematic in the histopathological diagnosis of melanoma and melanocytic naevi, even among expert pathologists. Formaldehyde-induced fluorescence (FIF) has been used for histochemical demonstration of catecholamines, 5-hydroxytryptamine and their immediate precursors. FIF can detect melanogenic activity and may be useful in differentiating malignant melanoma from other melanocytic lesions. The fluorescence of various types of melanocytic lesions has been previously studied quantitatively in formalin-fixed and paraffin-embedded sections. This study compared 2 sets of excitation and emission bands: 450-490 nm excitation/510-560 nm absorption filters (filter unit A) and 480 nm excitation/510< nm absorption filters (filter unit B). Higher FIF was observed with filter unit A than with filter unit B. FIF intensity of central regions was found to be higher than that of the peripheral regions. Mean FIF was significantly higher in malignant melanomas than in naevi. Fluorescence imaging with filter unit A gave better diagnostic performance. In conclusion, quantitative measurement of FIF is a useful marker of malignant potential.

A deep penetrating facial congenital melanocytic tumor with bone involvement and ipsilateral eye blindness.

Bone involvement has been described in tumors with melanocytic differentiation such as melanotic neuroectodermal tumor of infancy, and very rarely in cellular blue nevi and neurocristic cutaneous hamartoma. We present an unusual case of facial congenital melanocytic tumor that involved the underlying bones and maxillary sinus and led to unilateral blindness. A newborn with a large red bluish patch with peripheral brown and black macules overlying marked swelling on the left side of his face was presented. The tumor was shown by magnetic resonance imaging, scintigraphy, and histopathology to invade the underlying bones and maxillary sinus and to compress the left eyeball resulting in blindness. Histopathology, immunohistochemistry, morphometric computerized microscopy, molecular genetic mutation analysis, and fluorescent in situ hybridization studies were more congruent with a melanocytic nevus. An 8.5-year follow-up was uneventful, with spontaneous partial shrinkage of the tumor.

Development of a novel noninvasive adhesive patch test for the evaluation of pigmented lesions of the skin.

BACKGROUND: The accurate clinical assessment of melanocytic neoplasms is a challenge for clinicians. Currently, obtaining a biopsy specimen and conducting a histologic examination is the standard of care. The incidence of melanoma in white populations is high, resulting in a large number of biopsy specimens. OBJECTIVE: The objective of this study is to develop a noninvasive genomic method using mRNA to classify pigmented skin lesions as either benign or malignant. METHODS: An adhesive patch method was used to obtain cells from the surface of melanocytic lesions. mRNA was extracted and a genomic signature was formulated in a training set of benign and malignant melanocytic neoplasms and subsequently tested in a validation set. RESULTS: A 2-gene signature assessing the expression levels of CMIP and LINC00518 was able to differentiate melanomas from nevi in an independent validation set of 42 melanomas and 22 nevi with a sensitivity of 97.6% and specificity of 72.7%. LIMITATIONS: Larger and more diverse sets of melanomas and nevi are needed for additional validation of the molecular expression profiling in various subsets of melanocytic neoplasms. CONCLUSION: Our data suggest that mRNA molecular signatures can serve as a highly useful noninvasive method of differentiating melanoma from nevi and decrease the number of unnecessary biopsies.

Proliferative nodules of undifferentiated spindle cells arising in a large congenital melanocytic naevus.

Proliferative nodules (PN) are benign lesions that arise in large congenital melanocytic naevi (LCMN). Clinically and histologically they can be difficult to differentiate from malignancies, which are also associated with LCMN. The PN in this case consisted of undifferentiated spindle cells and exhibited unusual histological features including negative stains for melanocytic markers (S100, HMB45 and MelA), negative stain for c-Kit, high mitotic index and unusual morphology of the lesional cells. As a result, a firm histological classification could not be made, which posed a challenge for the clinical management.

Histopathologic clues for the diagnosis of Wiesner nevus.

The dermatologic hallmark of a recently described BAP1-associated cancer susceptibility syndrome is a dome-shaped nevus with distinct clinicopathological features, first delineated by Wiesner and colleagues. Here we describe the leading histopathological criteria of Wiesner nevus. Wiesner nevus is composed of various nevomelanocytic populations all showing different degrees of atypia ranging from hyperchromatic nevus cell-like to large atypical epithelioid cells. Immunohistochemically, Wiesner nevus is BAP1 negative and VE1 positive.

Management of small and intermediate congenital nevi: a nationwide survey in Italy.

OBJECTIVE: Small- and intermediate-sized congenital nevi (SCN and ICN) undergo periodic clinical monitoring or surgical excision. We analyzed the management of SCN and ICN in the Italian hospital network. METHODS: A nationwide survey of a representative sample of centers was conducted. Data were analyzed grouping centers by melanoma incidence into high-volume (>25 diagnoses per year) and low-volume (≤ 25 diagnoses per year). RESULTS: In the pediatric population, 11% of SCN and 22% of ICN are excised, the remainder undergoing clinical monitoring at intervals of 6 months to 2 years (SCN) and of 6 months to 1 year (ICN). In adults, 24% of SCN and 41% of ICN are excised. Clinical monitoring of SCN varies from 6-monthly (most common among low-volume hospitals) to every 2 years; preferred strategies for ICN are follow-up at 1 year (51%) or follow-up at 6 months (42%). For prophylactic surgery, complete excision is preferred. CONCLUSIONS: The Italian hospital network values management and treatment of SCN and ICN. In most cases natural evolution prompts prophylactic excision. Clinical examination is an important monitoring tool, though follow-up frequency depends on the clinician's experience and practice.

Melanocytic differentiation is present in a significant proportion of nonpigmented diffuse neurofibromas: a potential diagnostic pitfall.

Whereas the pigmented (melanotic) variant of diffuse neurofibroma (DNF) with positivity for melanocytic markers is well recognized, expression of melanocytic markers in nonpigmented DNF has not been systematically studied. We analyzed 28 unselected consecutive DNFs for expression of melanocytic markers, including melan A, microphthalmia transcription factor (MITF), and HMB-45 antigen. For comparison, we also analyzed 40 localized skin neurofibromas and 7 intraneural neurofibromas. One case of nonpigmented DNF was analyzed by electron microscopy. Of the 28 DNFs studied by immunohistochemistry, 3 were pigmented and 25 nonpigmented. The 3 pigmented DNFs and 9 of 25 (36%) nonpigmented DNFs expressed melan A, MITF, and HMB-45 antigen. These markers were expressed either focally or more diffusely, typically in a minority of the lesional cells, and usually both in the dermal and subcutaneous portion of the DNF. Melan A was expressed in the largest number of the lesional cells (up to 50%), whereas only a small fraction of the melan A-positive cells (from 5% to 10% in most cases) also expressed HMB-45 antigen. None of the 47 non-DNFs expressed these markers. Ultrastructurally, melanosomes were present in some cells in nonpigmented DNF that expressed the melanocytic markers. Twenty-three of 28 (82%) DNFs, including 10 of 12 (83%) DNFs with melanocytic differentiation, were associated with neurofibromatosis type 1. Expression of melanocytic markers, including melan A, HMB-45 antigen, and MITF in DNF is a potential pitfall in differential diagnosis with melanocytic lesions that may clinically or histopathologically resemble DNF, in particular congenital melanocytic nevus with neurotization and neurofibroma-like melanoma.

Reticulin and NM23 staining in the interpretation of lymph nodal nevus rests.

Melanocytic nevus rests in lymph nodes are a known diagnostic challenge, especially in patients with a history of melanoma. Reticulin and NM23 have been studied in this context. The pattern of reticulin staining in melanomas surrounds groups/nests of melanocytes but individual cells in benign nevi. NM23, a metastasis-suppressor gene, has an association with metastatic potential in melanomas and some carcinomas. Twenty-eight cases (14 cases of metastatic melanoma to lymph nodes and 14 cases of lymph node nevus rests, all confirmed with Melan-A staining) were stained with reticulin and NM23. The pattern of reticulin staining was reported as surrounding groups if staining was noted in approximately 5-10 melanocytes in greater than 50% of the lesion but was otherwise reported as surrounding individual melanocytes. Cytoplasmic staining was considered to represent reactivity for NM23. Reticulin staining around groups of melanocytes was identified in all 14 cases of metastatic melanoma. Regarding nodal nevus rest cases, 12 of 14 cases (86%) demonstrated staining around individual melanocytes, whereas in 2 cases, reticulin surrounded melanocytic groups. NM23 staining was equivocal in all cases. Reticulin staining reliably invests groups of melanocytes in cases of metastatic melanoma, whereas in nodal nevus rests, it predominantly surrounds individual melanocytes. NM23 demonstrated no discriminatory value in this analysis. In cases in which a collection of melanocytes is present within a lymph node, reticulin deposition around individual melanocytes supports a diagnosis of lymph nodal nevus rest.