Pyogenic granuloma (PG) is a common benign vascular skin lesion presenting as a rapidly growing angiomatous papule. The pathogenesis of most sporadic PGs and PGs associated with port wine stains ...(PWSs) remains elusive. We report that of 10 PGs secondarily arisen on a PWS, 8 showed a BRAF c.1799T>A (p.(Val600Glu)) and 1 a NRAS c.182A>G (p.(Gln61Arg)) mutation. The GNAQ c.548G>A mutation was identified in the PG and in the respective underlying PWS, indicating that PGs originate from cells of the PWS. In contrast to PG, 12 papulonodular lesions, which had developed in the PWSs of seven patients, showed a RAS and BRAF wild-type status. In sporadic PG we identified the BRAF c.1799T>A mutation in 3 of 25, a BRAF c.1391G>A mutation in 1 of 25, and a KRAS c.37G>C mutation in 1 of 25. Mutation-specific immunohistochemical detection of BRAF p.(Val600Glu) confirmed endothelial cells as carriers of the mutation in secondary and sporadic PG.
Our study identifies the BRAF c.1799T>A mutation as a major driver mutation in the pathogenesis of, particularly, secondary PG. These data shed light on the hitherto undetermined genetic basis of PG and classify PG as a benign neoplasm.
Nevus sebaceous is a common congenital cutaneous malformation. Affected individuals may develop benign and malignant secondary tumors in the nevi during life. Schimmelpenning syndrome is ...characterized by the association of nevus sebaceous with extracutaneous abnormalities. We report that of 65 sebaceous nevi studied, 62 (95%) had mutations in the HRAS gene and 3 (5%) had mutations in the KRAS gene. The HRAS c.37G>C mutation, which results in a p.Gly13Arg substitution, was present in 91% of lesions. Nonlesional tissues from 18 individuals had a wild-type sequence, confirming genetic mosaicism. The HRAS c.37G>C mutation was also found in 8 of 8 associated secondary tumors. Mosaicism for HRAS c.37G>C and KRAS c.35G>A mutations was found in two individuals with Schimmelpenning syndrome. Functional analysis of HRAS c.37G>C mutant cells showed constitutive activation of the MAPK and PI3K-Akt signaling pathways. Our results indicate that nevus sebaceous and Schimmelpenning syndrome are caused by postzygotic HRAS and KRAS mutations. These mutations may predispose individuals to the development of secondary tumors in nevus sebaceous.
Summary Trichoblastoma (TB) and syringocystadenoma papilliferum (SCAP) are both rare adnexal skin lesions occurring either sporadically or as secondary neoplasms in sebaceous nevi. TB and SCAP ...associated with sebaceous nevi have been shown to carry the same HRAS mutation as the underlying nevus. However, the genetic background of sporadic TB and SCAP has remained unknown. Therefore, we screened 18 sporadic TBs and 23 sporadic syringocystadenoma papillifera from 41 patients for the presence of activating mutations in RAS genes and other oncogenes. Using a RAS SNaPshot assay, HRAS mutations were detected in 2 (11%) of 18 sporadic TB and 6 (26%) of 23 sporadic syringocystadenoma papillifera. A KRAS mutation was identified in 1 sporadic SCAP. High-throughput oncogene mutation profiling furthermore identified BRAF V600E mutations in sporadic syringocystadenoma papillifera, which could be validated in 12 (52%) of 23 lesions using a BRAF SNaPshot assay. BRAF and RAS mutations were mutually exclusive in sporadic syringocystadenoma papillifera. No BRAF mutation could be detected in 3 syringocystadenoma papillifera secondarily arisen from a sebaceous nevus as well as in sporadic TB. In 14 lesions carrying an oncogenic mutation, nonlesional control tissue from the epidermal margin revealed a wild-type sequence, thus proving the somatic character of the mutation. Our results indicate that activation of the RAS-mitogen-activated protein kinase pathway by BRAF and RAS mutations contributes significantly to the tumorigenesis of sporadic SCAP and, less frequently, of sporadic TB.
A frequent adverse effect of mutation-specific BRAF inhibitor therapy is the induction of epithelial proliferations including cutaneous squamous cell carcinomas. To date, the only factor identified ...contributing to their development is the activation of the mitogen-activated signal transduction cascade by mutations in the RAS genes. However, these mutations explain only 60% of the tumors; hence, it is important to identify what is causing the remaining tumors.
To test for the presence of human papillomaviruses (HPVs) and the recently identified human polyomaviruses (HPyVs), Merkel cell polyomavirus (MCPyV), and trichodysplasia spinulosa-associated polyomavirus (TSPyV), as well as HPyV-6, HPyV-7, HPyV-9, and HPyV-10, in epithelial proliferations occurring after BRAF inhibitor therapy to determine whether these oncogenic viruses may contribute to BRAF inhibitor-induced skin tumors.
Retrospective study at a university hospital in Austria of epithelial proliferations that developed in patients with melanoma after initiation of treatment with the BRAF inhibitor vemurafenib. Samples were analyzed for (1) presence of the most frequently observed RAS mutations by SNaPshot technology, (2) detection of the viruses by real-time polymerase chain reaction, and (3) presence of capsid proteins of the most abundantly detected virus by immunohistochemical analysis.
RAS mutational status, as well as HPV and HPyV presence, in BRAF inhibitor-induced epithelial proliferations.
Eighteen biopsy samples from 6 patients were retrieved from our hospital's archive. We identified RAS mutations in 10 (62%) of the 16 samples with clear results. DNA of HPyV-9, HPyV-10, and TSPyV were virtually absent in the samples. MCPyV DNA was present in 13 of 18 samples, and HPV, HPyV-6, and HPyV-7 DNA were present in all samples. In general, the amount of DNA encoding the latter viruses was rather low, with the exception of HPyV-6 in several samples of 1 individual patient. Notably, the relevance of the presence of HPyV-6 in the epithelial proliferation was underlined by immunohistochemical detection of the core protein VP1 of HPyV-6.
The presence of both high HPyV-6 DNA load and VP1 protein suggests that polyomaviruses may contribute to the epithelial proliferations observed in patients receiving BRAF inhibitor therapy, albeit the relative impact as compared with that of RAS mutations appears circumstantial.
The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of ...MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.
"RASopathies" are a group of developmental syndromes with partly overlapping clinical symptoms that are caused by germline mutations of genes within the Ras/MAPK signaling pathway. Mutations ...affecting this pathway can also occur in a mosaic state, resulting in congenital syndromes often distinct from those generated by the corresponding germline mutations. For syndromes caused by mosaic mutations of the Ras/MAPK signaling pathway, the term "mosaic RASopathies" has been proposed. In the following article, genetic and phenotypic aspects of mosaic RASopathies will be discussed.
Seborrheic keratosis (SK) represents a frequent epidermal skin tumor. Although lacking a malignant potential, these tumors reveal multiple oncogenic mutations. A previous study identified activating ...mutations in 89% of SK, particularly in FGFR3 and PIK3CA genes. The aim of this study was to identify further oncogenic mutations in human SK. Therefore, we screened for mutations in EGFR, FGFR2, PIK3R1, HRAS, KRAS, and NRAS genes using both Sanger sequencing of selected exons and a multiplex SNaPshot assay in 58 SK of 14 patients. We identified a somatic EGFR p.L858R mutation in 1 SK. Furthermore, the HRAS mutations p.G13R (2/58 SK) and p.Q61L (2/58 SK) were found. These mutations have not been described in human SK yet. In addition, 1 SK revealed the KRAS p.G12V mutation, which has already been reported in SK. No mutations were detected in FGFR2, PIK3R1, and NRAS genes. The results of this study suggest that activating mutations of EGFR, HRAS, and KRAS contribute to the pathogenesis of human SK, although at a lower frequency than FGFR3 and PIK3CA mutations. FGFR2, PIK3R1, and NRAS mutations obviously do not have a significant role in the development of SK.
Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be ...extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11R183C and GNA11Q209L in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11R183C under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.
Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in ...KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.
PDF
