Christian Hafner

Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi

Epidermal nevi are common congenital skin lesions with an incidence of 1 in 1,000 people; however, their genetic basis remains elusive. Germline mutations of the FGF receptor 3 (FGFR3) cause autosomal dominant skeletal disorders such as achondroplasia and thanatophoric dysplasia, which can be associated with acanthosis nigricans of the skin. Acanthosis nigricans and common epidermal nevi of the nonorganoid, nonepidermolytic type share some clinical and histological features. We used a SNaPshot multiplex assay to screen 39 epidermal nevi of this type of 33 patients for 11 activating FGFR3 point mutations. In addition, exon 19 of FGFR3 was directly sequenced. We identified activating FGFR3 mutations, almost exclusively at codon 248 (R248C), in 11 of 33 (33%) patients with nonorganoid, nonepidermolytic epidermal nevi. In 4 of these cases, samples from adjacent histologically normal skin could be analyzed, and FGFR3 mutations were found to be absent. Our results suggest that a large proportion of epidermal nevi are caused by a mosaicism of activating FGFR3 mutations in the human epidermis, secondary to a postzygotic mutation in early embryonic development. The R248C mutation appears to be a hot spot for FGFR3 mutations in epidermal nevi.

Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern

Activating mutations of the p110 α subunit of PI3K (PIK3CA) oncogene have been identified in a broad spectrum of malignant tumors. However, their role in benign or preneoplastic conditions is unknown. Activating FGF receptor 3 (FGFR3) mutations are common in benign skin lesions, either as embryonic mutations in epidermal nevi (EN) or as somatic mutations in seborrheic keratoses (SK). FGFR3 mutations are also common in low-grade malignant bladder tumors, where they often occur in association with PIK3CA mutations. Therefore, we examined exons 9 and 20 of PIK3CA and FGFR3 hotspot mutations in EN (n = 33) and SK (n = 62), two proliferative skin lesions lacking malignant potential. Nine of 33 (27%) EN harbored PIK3CA mutations; all cases showed the E545G substitution, which is uncommon in cancers. In EN, R248C was the only FGFR3 mutation identified. By contrast, 10 of 62 (16%) SK revealed the typical cancer-associated PIK3CA mutations E542K, E545K, and H1047R. The same lesions displayed a wide range of FGFR3 mutations. Corresponding unaffected tissue was available for four EN and two mutant SK: all control samples displayed a WT sequence, confirming the somatic nature of the mutations found in lesional tissue. Forty of 95 (42%) lesions showed at least one mutation in either gene. PIK3CA and FGFR3 mutations displayed an independent distribution; 5/95 lesions harbored mutations in both genes. Our findings suggest that, in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of skin tumors lacking malignant potential. The remarkable genotype–phenotype correlation as observed in this study points to a distinct etiopathogenesis of the mutations in keratinocytes occuring either during fetal development or in adult life.

Clonality of multifocal urothelial carcinomas: 10 years of molecular genetic studies

Multifocal occurrence and frequent recurrence are characteristic features of urothelial carcinomas of both the urinary bladder and the upper urinary tract. To describe the clonal nature of these tumors, 2 theories have been proposed. The monoclonality hypothesis describes the multiple tumors as descendants of a single genetically transformed cell spreading throughout the urothelium. In contrast, field cancerization caused by carcinogen exposure of the urothelium may lead to independent development of synchronous or metachronous nonrelated tumors at different sites of the urothelial tract. In the last 10 years, a multitude of molecular genetic studies have investigated the clonality of multifocal urothelial carcinomas. The majority of studies revealed a monoclonal origin of the multiple tumors. However, most of these studies investigated advanced invasive carcinomas. A small but significant proportion of multifocal urothelial carcinomas appear to arise from different clones, supporting the field‐cancerization hypothesis. Oligoclonal tumors might be more common in precursor lesions and early tumor stages. The frequent monoclonality found in patients with advanced tumors could be due to outgrowth of 1 tumor cell clone with specific genetic alterations. Two important mechanisms appear to be important for the spread of malignant cells: intraluminal seeding and intraepithelial migration. Investigation of the entire urothelial lining in patients with urothelial tumors should provide further insight into the development of multifocal urothelial carcinomas.

Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome

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.

Evidence for oligoclonality and tumor spread by intraluminal seeding in multifocal urothelial carcinomas of the upper and lower urinary tract

Multifocality and recurrence of urothelial carcinoma may result from either the field effect of carcinogens leading to oligoclonal tumors or monoclonal tumor spread. Previous molecular studies, favoring the monoclonality hypothesis, are mostly limited to the urinary bladder. We investigated genetic alterations in a total of 94 synchronous or metachronous multifocal tumors from 19 patients with at least one tumor both in the upper and lower urinary tract. Loss of heterozygosity (LOH) was determined using eight markers on chromosome 9 and one marker on 17p13 (p53). Microsatellite instability was investigated at six loci and protein expression of MSH2 and MLH1 was evaluated by immunohistochemistry. In addition, exons 5–9 of the p53 gene were sequenced. Deletions at chromosome 9 were found in 73% of tumors and at 17p13 in 18% of tumors. There was no significant difference in the frequency of LOH in the upper and lower urinary tract. Deletions at 9p21 were significantly correlated with invasive tumor growth. The pattern of deletion revealed monoclonality of all tumors in nine patients. In five patients there were at least two tumor clones with different genetic alterations. In four of these patients the different clones occured in the bladder and subsequently in the ureter and renal pelvis. All four patients with p53 mutations revealed identical mutations in all tumors. Thus, multifocal urothelial carcinomas are frequently monoclonal, whereas others show oligoclonality, providing molecular evidence for field cancerization. Intraluminal tumor cell seeding appears to be an important mechanism of multifocal occurence and recurrence of urothelial carcinomas.

A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene.

BackgroundActivating mutations in the PIK3CA gene have been identified in a variety of human malignancies and are commonly detected in hotspot codons located in the helical and kinase domains in exons 9 and 20. Existing methodologies for the detection of PIK3CA mutations are time-consuming and/or expensive. In the present study we describe the first application of a PIK3CA SNaPshot assay to the screening of frequent mutations in these exons.FindingsA SNaPshot assay for the simultaneous detection of four frequent PIK3CA hotspot mutations (E542K, E545G, E545K and H1047R) has been developed and evaluated. The assay combines multiplex PCR amplification with a multiplex primer extension assay to allow targeted detection of all four mutations in one reaction. The method was tested using samples that had previously been analysed for mutations by high-resolution melting analysis and sequencing. All mutations detected were concordant and no false positive results were obtained. Sensitivity tests showed that the SNaPshot assay could detect mutant DNA when it represents 5–10% of the total DNA present. The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.ConclusionThe SNaPshot assay described here offers a fast, sensitive, inexpensive and specific approach to the analysis of frequent PIK3CA mutations in both fresh and archival patient samples.

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

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.

Expression profile of Eph receptors and ephrin ligands in human skin and downregulation of EphA1 in nonmelanoma skin cancer

Eph receptors and ephrin ligands represent the largest family of receptor tyrosine kinases. Beyond their well-defined meaning in developmental processes, these molecules also have important functions in adult human tissues and cancer. However, the Eph/ephrin expression profile in human skin is only marginally studied. We therefore investigated the mRNA expression of 21 Eph receptors and ephrin ligands in adult human skin in comparison to 13 other adult human tissues using quantitative real-time RT-PCR. In addition, immunohistochemistry was established for some members (EphA1, EphA2 and EphA7) to confirm the results of the RT-PCR and to identify the expressing cells in the skin. We found all investigated family members expressed in human skin, but at highly varying levels. EphA1, EphB3 and ephrin-A3 turned out to be most prominently expressed in skin compared to other adult human tissues. EphA1 was exclusively expressed in the epidermis. We therefore investigated the expression of EphA1 in nonmelanoma skin cancers derived from the epidermis (56 basal cell carcinomas and 32 squamous cell carcinomas). As demonstrated by immunohistochemistry, both skin cancers displayed a significant downregulation of EphA1 compared to the normal epidermis. In squamous cell carcinoma, the EphA1 downregulation was associated with increased tumor thickness, although this was not significant. Our results indicate that Eph receptors and ephrin ligands are widely expressed in the adult human skin, particularly in the epidermis, and may play an important role in skin homeostasis. EphA1 seems to be a marker of the differentiated normal epidermis and its downregulation in nonmelanoma skin cancer may contribute to carcinogenesis of these very frequent human tumors. EphA1 represents a new potential prognostic marker and therapeutic target in nonmelanoma skin cancer.

Phacomatosis Pigmentokeratotica Is Caused by a Postzygotic HRAS Mutation in a Multipotent Progenitor Cell

Phacomatosis pigmentokeratotica (PPK) is a rare epidermal nevus syndrome characterized by the co-occurrence of a sebaceous nevus and a speckled lentiginous nevus. The coexistence of an epidermal and a melanocytic nevus has been explained by two homozygous recessive mutations, according to the twin spot hypothesis, of which PPK has become a putative paradigm in humans. However, the underlying gene mutations remained unknown. Multiple tissues of six patients with PPK were analyzed for the presence of RAS, FGFR3, PIK3CA, and BRAF mutations using SNaPshot assays and Sanger sequencing. We identified a heterozygous HRAS c.37G>C (p.Gly13Arg) mutation in four patients and a heterozygous HRAS c.182A>G (p.Gln61Arg) mutation in two patients. In each case, the mutations were present in both the sebaceous and the melanocytic nevus. In the latter lesion, melanocytes were identified to carry the HRAS mutation. Analysis of various nonlesional tissues showed a wild-type sequence of HRAS, consistent with mosaicism. Our data provide no genetic evidence for the previously proposed twin spot hypothesis. In contrast, PPK is best explained by a postzygotic-activating HRAS mutation in a multipotent progenitor cell that gives rise to both a sebaceous and a melanocytic nevus. Therefore, PPK is a mosaic RASopathy.

Keratinocytic epidermal nevi are associated with mosaic RAS mutations

Background Activating RAS mutations in the germline cause rare developmental disorders such as Costello syndrome. Somatic RAS mutations are found in approximately 30% of human cancers. Keratinocytic epidermal nevi (KEN) represent benign congenital skin lesions arranged along Blaschkos lines. A subgroup of KEN is caused by hotspot oncogenic FGFR3 and PIK3CA mutations in mosaicism, but the majority lack these mutations. Methods This study screened 72 KEN for activating mutations in RAS genes and other oncogenes. Results Activating RAS mutations were identified in 28/72 (39%) of KEN. HRAS was the most commonly affected oncogene (86%), with the HRAS p.G13R substitution representing a new hotspot mutation. Conclusion These results indicate that activating RAS somatic mutations leading to mosaicism result in benign KEN of the skin. Given the prevalence of KEN, mosaic HRAS mutations appear to be more common in patients than germline ones. These findings identify KEN as a mosaic RASopathy and lend further support to the notion that genetic mosaicism is an important contributor to disease.