Amplification of Mutant NRAS in Melanocytic Tumors With Features of Spitz Tumors.

NRAS activating mutations are prevalent in melanocytic neoplasia, occurring in a subset of common acquired melanocytic nevi and ∼30% of cutaneous melanomas. In this study, we described a cohort of 7 distinctive melanocytic tumors characterized by activating point mutations in codon 61 of NRAS with amplification of the mutant NRAS allele and shared clinicopathologic features. These tumors occurred predominantly in younger patients, with a median age of 20 years (range, 6-56 years). They presented as papules on the helix of the ear (4 cases) or extremities (3 cases). Microscopically, the tumors were cellular, relatively well-circumscribed, compound, or intradermal proliferations. The tumor cells often extended into the deep reticular dermis and involved the superficial subcutaneous fat in some cases. The melanocytes were epithelioid to spindled with moderate amounts of cytoplasm and conspicuous nucleoli. They were arranged in short plexiform fascicles, nests, and cords. Some cases had occasional pleomorphic and multinucleated melanocytes. Rare dermal mitotic figures were present in all cases. The dermis contained thick collagen bundles and minimal solar elastosis. Follow-up data were available for 5 patients, with a median period of 4.2 years (range, 1-9 years), during which no recurrences or metastases were reported. Our series highlights a clinicopathologically and molecularly distinctive subset of NRAS-mutated tumors with amplification of the mutant NRAS allele.

Examination of Bovine Red Blood Cell Death in Vitro in Response to Pathophysiologic Proapoptotic Stimuli.

BACKGROUND: Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. METHODS: Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. RESULTS: Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. CONCLUSIONS: Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

Primary cutaneous perivascular epithelioid cell tumor: a clinicopathological and molecular reappraisal.

BACKGROUND: Perivascular epithelioid cell tumor (PEComa) is a rare neoplasm of uncertain histogenesis with a mixed myomelanocytic immunophenotype, rarely arising in the skin (primary cutaneous PEComa [pcPEComa]). OBJECTIVE: We analyzed the clinicopathological features of 8 pcPEComas, assayed for DNA copy number changes and for initiating mutations common in melanocytic neoplasms. METHODS: pcPEComas were evaluated using immunohistochemistry, comparative genomic hybridization, and DNA sequencing. RESULTS: pcPEComas were erythematous nodules, mostly in the lower extremities of women (5/8), composed of large pale-staining epithelioid cells. The patient's age range was 26 to 67 (mean 46) years. The percentages of tumors staining positively were as follows: micro-ophthalmia-associated transcription factor, NKI/C3, bcl-1, E-cadherin, and cathepsin K (100%); HMB-45, 4E-binding protein 1, and CD68 (88%); smooth muscle actin and muscle-specific actin (40%); S100 (38%); calponin (20%); desmin (13%); and melan-A, SOX10, and keratin (0%). No chromosomal copy number changes or initiating mutations were identified. LIMITATIONS: Small sample size is a limitation. CONCLUSIONS: pcPEComas have a different molecular signature than extracutaneous tumors and are unrelated to tuberous sclerosis. However, the common expression of 4E-binding protein 1 points to a role of the mTOR pathway in their pathogenesis. Because pcPEComas are diagnostically challenging, we propose that micro-ophthalmia-associated transcription factor, NKIC3, smooth muscle actin, desmin, bcl-1, cathepsin K, and 4E-binding protein 1 can be used when evaluating a possible pcPEComa.

Ambiguous melanocytic tumors with loss of 3p21.

Germline loss-of-function mutations in BAP1 are associated with the development of cutaneous melanocytic tumors with some histopathologic characteristics seen in Spitz nevi. Similar melanocytic tumors occurring in a sporadic setting have been demonstrated to have biallelic loss of BAP1. In some of these sporadic tumors, loss of BAP1 occurs through mutation of 1 allele and genomic loss of the other. We screened our database of comparative genomic hybridization profiles of ambiguous melanocytic tumors to identify cases with a single genomic event involving loss of the BAP1 locus. The prevalence of tumors with a single genomic event involving loss of BAP1 was 6.7% in our study population. We further characterized the BAP1 status in 17 of these tumors with available additional material, confirming loss of BAP1 in all cases. We describe BAP1 loss in a blue nevus-like melanoma and further expand the histopathologic spectrum of spitzoid melanocytic neoplasms with BAP1 loss.

DNA copy number changes in tumors within the spectrum of cellular, atypical, and metastasizing fibrous histiocytoma.

BACKGROUND: Cutaneous fibrous histiocytoma (FH) is a common mesenchymal neoplasm. Metastasis is rare, disproportionately occurring among the aneurysmal, cellular, atypical, and deep variants. OBJECTIVE: We determined whether DNA copy number changes occurred in atypical FH (AFH), and whether they were similar to those in metastasizing FH (MetFH) and benign cellular FH (CFH). METHODS: Five primary tumors of MetFH were evaluated by array-based comparative genomic hybridization analysis, with tissue from local recurrences and lung metastases in 2 and 2 patients, respectively. Seven indolent AFH and 5 CFH were identified for comparison. RESULTS: Substantial differences between the groups were found both in the frequency of chromosomal aberrations (higher among MetFH and absent or solitary in CFH) and array-based comparative genomic hybridization profiles (frequent gains of 7 and 8q and losses of Xq in MetFH; recurrent losses of chromosomes 9 and 22 in AFH; isolated loss of 5q and gain in chromosome 20 in 2 CFH). Fatal MetFH cases (2 of 5 cases) exhibited the highest rate of chromosomal aberrations. LIMITATIONS: This study included a small sample size with a short-term follow-up. CONCLUSIONS: Benign CFH, indolent AFH, and MetFH represent distinct biological entities within the spectrum of FH; array-based comparative genomic hybridization may be a tool in recognizing FH cases with metastatic potential and increasingly aggressive behavior.

Neurofibroma-like spindle cell melanoma: CD34 fingerprint and CGH for diagnosis.

We present a case that highlights the use of two maneuvers useful in the diagnosis of spindle cell melanoma. A shave biopsy from the cheek of a 58-year-old man demonstrated a thin invasive melanoma of 0.3 mm thickness with a less than 2-mm-wide intra-epidermal component. Below this melanocytic lesion, but not contiguous with it, there was a transected S-100-positive and Melan-A-negative spindle cell proliferation. Upon re-excision, no residuum of conventional melanoma was identified, but a residual spindle cell neoplasm that was 4 mm in diameter, nodular, well-circumscribed, cytologically bland, and S-100 positive was noted. At our consensus conference, our group favored neurofibroma but agreed that spindle cell melanoma could not be excluded based on histopathologic features alone. To further address the differential diagnosis, we performed CD34 staining that demonstrated lack of a CD34 fingerprint. We also completed array-based comparative genomic hybridization, which demonstrated gain of chromosome 6p, loss of 6p and gain of 7. These two methods of analysis support a diagnosis of spindle cell melanoma.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm.

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.