Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P<0.0002), no association was noted between the type of germline and somatic NF1 lesion within the same individual.     

Germline and somatic NF1 gene mutations in plexiform neurofibromas

Neurofibromatosis type 1 (NF1), a common autosomal dominant neurogenetic disorder affecting 1 in 4000 individuals worldwide, results from functional inactivation of the 17q11.2-located NF1 gene. Plexiform neurofibroma (PNF) is a congenital benign tumour present in 30-50% of NF1 patients, which in about 10-15% of cases, can develop into a malignant peripheral nerve sheath tumour (MPNST). This study aimed to characterise the NF1 germline and somatic mutations associated with such tumours by DNA analysis in 51 PNFs resected from 44 unrelated NF1 patients. Germline mutations were identified in 35 patients, of which 21 were novel. Somatic NF1 mutations were found in 29 PNF DNAs, which included 9 point mutations, 5 being novel, and 20 tumour DNA samples exhibiting, either loss of heterozygosity (LOH) of the NF1 gene region (16 tumours), or complete or partial NF1 gene deletions analyzed by multiplex ligation-dependent probe amplification (MPLA) analysis. The type of NF1 germline mutations detected in patients with PNF were similar to those detected in most NF1 patients. LOH of the NF1 gene region, as identified by marker analysis and/or MLPA, was detected in only 20/29 (69%) PNFs, compared to the >90% LOH previously found in MPNST. This systematic analysis of the NF1 germline and somatic mutations associated with PNF development suggest that in most such tumours neither the NF1 somatic mutation type, nor its gene location, is influenced by the underlying NF1 germline mutation. Evidence for LOH involving the TP53 gene identified in the PNFs is also reported for the first time.     

Confirmation of mutation landscape of NF1‐associated malignant peripheral nerve sheath tumors

The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss‐of‐function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1‐associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B.     

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci.     

The heterogeneous nature of germline mutations in NF1 patients with malignant peripheral serve sheath tumours (MPNSTs)

Malignant peripheral nerve sheath tumours (MPNSTs) are a major cause of mortality in patients with neurofibromatosis 1 (NF1). We have analysed lymphocyte DNA samples from 30 NF1 patients with MPNSTs to determine their underlying constitutional NF1 gene mutations. Mutations were detected in 27/30 (90%) of these patients. NF1 mutations identified included nonsense, missense, frameshift, splice site mutation and single or multi-exonic deletions and with no obvious clustering of the mutations across the gene. Fourteen of the mutations represent novel gene changes. There did not appear to be any relationship between the mutation type and the level of clinical severity observed. Of the 20 patients with high grade MPNSTs, seven patients had small (<20 bp) and multi-exonic deletions and three had small insertions (<20 bp). Several studies have suggested that NF1 patients with a constitutional 1.5 Mb deletion of the NF1 gene have an increased risk of developing malignant peripheral nerve sheath tumours (MPNSTs). None of our patients had a 1.5 Mb deletion. Larger prospective studies are needed to ascertain whether there is a different spectrum of NF1 mutations in NF1 patients with high grade compared to low grade MPNSTs and of patients with the 1.5Mb deletion, in order to determine the true frequency of MPNST in this sub-group of NF1 patients.     

Biallelic inactivation of TP53 rarely contributes to the development of malignant peripheral nerve sheath tumors

About 10% of the patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNSTs), accounting for half of all MPNST cases. Several nonrandom chromosomal aberrations have been found, but the target genes remain mostly unrecognized. Mutations in the NF1 and TP53 genes have been found in some MPNSTs, and recent data from mouse models support a synergistic effect of these two genes in the development of MPNST. In the present study, we have analyzed 16 MPNSTs, including 11 from patients with NF1 and 5 sporadic cases, for mutations in the coding sequence of the TP53 gene (exons 2-11). We applied denaturing gradient gel electrophoresis and modifications of this technique for analyses of 12 genomic fragments, followed by direct sequencing for identification of the mutated base(s). None of the MPNSTs revealed mutations. The detection of control mutants for each fragment analyzed, the high sensitivity of the technique, the detection of polymorphisms in some samples, and the high content of tumor tissue in the biopsies imply that false negatives are highly unlikely. Although we cannot exclude that deletions including large parts of the gene remain undetected by the mutation analyses, previous comparative genomic hybridization (CGH), cytogenetic banding analysis, and/or loss of heterozygosity studies on 14 of the cases included here had revealed 17p deletions in only three. We thus conclude that TP53 biallelic inactivation is rare in MPNST, and that the potential impact of an altered TP53 pathway on the malignant transformation of a neurofibroma into an MPNST may more frequently occur by changes in other components of that pathway.     

Germline and somatic NF1 mutations in sporadic and NF1‐associated malignant peripheral nerve sheath tumours

Malignant peripheral nerve sheath tumours (MPNSTs) are a malignancy occurring with increased frequency in patients with neurofibromatosis type 1 (NF1). In contrast to the well‐known spectrum of germline NF1 mutations, the information on somatic mutations in MPNSTs is limited. In this study, we screened NF1, KRAS, and BRAF in 47 MPNSTs from patients with (n = 25) and without (n = 22) NF1. In addition, DNA from peripheral blood and cutaneous neurofibroma biopsies from, respectively, 14/25 and 7/25 of the NF1 patients were analysed. Germline NF1 mutations were detected in ten NF1 patients, including three frameshift, three nonsense, one missense, one splicing alteration, and two large deletions. Somatic NF1 mutations were found in 10/25 (40%) NF1‐associated MPNSTs, in 3/7 (43%) neurofibromas, and in 9/22 (41%) sporadic MPNSTs. Large genomic copy number changes accounted for 6/10 and 7/13 somatic mutations in NF1‐associated and sporadic MPNSTs, respectively. Two NF1‐associated and 13 sporadic MPNSTs did not show any NF1 mutation. A major role of the KRAS and BRAF genes was ruled out. The spectrum of germline NF1 mutations in neurofibromatosis patients with MPNST is different from the spectrum of somatic mutations seen in MPNSTs. However, the somatic events share common characteristics with the NF1‐related and the sporadic tumours. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Constitutional NF1 mutations in neurofibromatosis 1 patients with malignant peripheral nerve sheath tumors

Neurofibromatosis type 1 (NF1) patients have 10% of lifetime risk for developing malignant peripheral nerve sheath tumors (MPNST), one of the most aggressive cancers. We examined the spectrum of constitutional NF1 mutations among 24 NF1 patients with MPNST. We found mutations in 18 patients: four megabase deletions involving the NF1 gene, 13 truncating mutations, and only one missense mutation. One deletion included both exonic and intronic sequences. No typical splicing mutation was found. Five of these mutations were novel: c.3686delA, c.197_204+9del17, c.3044T>C (p.Leu1015Pro), c.2497delT, and c.6020_6027dup. The proportion of megabase deletions of the NF1 gene found in patients with MPNST (17%=4/24) was higher than that in a group of unselected NF1 patients (5.4%=27/500).     

Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors.

SUMMARY: Karyotypic complexities associated with frequent loss or rearrangement of a number of chromosome arms, deletions, and mutations affecting the TP53 region, and molecular alterations of the INK4A gene have been reported in sporadic and/or neurofibromatosis type I (NF1)-related malignant peripheral nerve sheath tumors (MPNSTs). However, no investigations addressing possible different pathogenetic pathways in sporadic and NF1-associated MPNSTs have been reported. This lack is unexpected because, despite similar morphologic and immunophenotypic features, NF1-related cases are, by definition, associated with NF1 gene defects. Thus, we investigated the occurrence of TP53 and p16(INK4A) gene deregulation and the presence of microsatellite alterations at markers located at 17p, 17q, 9p21, 22q, 11q, 1p, or 2q loci in MPNSTs and neurofibromas either related (14 cases) or unrelated (14 cases) to NF1. Our results indicate that, in MPNSTs, p16(INK4A) inactivation almost equally affects both groups. However, TP53 mutations and loss of heterozygosity involving the TP53 locus (43% versus 9%), and p53 wild type overexpression, related or not to mdm2 overexpression (71% versus 25%), seem to mainly be restricted to sporadic MPNSTs. In NF1-associated MPNSTs, our microsatellite results are consistent with the occurrence of somatic inactivation by loss of heterozygosity of the second NF1 allele.     

Germline mutations in NF1 patients with malignancies.

We have analyzed 98.5% of the coding region of the NF1 gene at the cDNA level in seven NF1 patients who developed malignant peripheral nerve sheath tumors. Seven germline mutations were detected in six individuals: a 6-bp in-frame deletion in exon 28, a splice acceptor mutation in intron 31 resulting in a premature stop of translation, a missense mutation in exon 38, and three total NF1 gene deletions. In one of the patients with a total NF1 gene deletion, a missense mutation in exon 16 on the other NF1 allele was detected. These data indicate that NF1 patients developing malignant neoplasms can have any type of NF1 germline mutation such as a total gene deletion, a frameshift mutation, an in-frame deletion, or a missense mutation. We conclude that in our series no specific type of NF1 germline mutation was found in NF1 individuals with malignancies, but that large NF1 gene deletions were more frequently found in this group than reported for the general population of NF1 individuals. Genes Chromosomes Cancer 26:376-380, 1999.     

Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation

Patients with neurofibromatosis 1 (NF1) are predisposed to develop multiple neurofibromas (NFs) and are at risk for transformation of NFs to malignant peripheral nerve sheath tumors (MPNSTs). Little is known, however, about the biological events involved in the malignant transformation of NFs. We examined the CDKN2A/p16 gene and p16 protein in NFs and MPNSTs from patients with NF1. On immunohistochemical analysis, all NFs expressed p16 protein. The MPNSTs, however, were essentially immunonegative for p16, with striking transitions in cases that contained both benign and malignant elements. None of the benign tumors had CDKN2A/p16 deletions, whereas three of six MPNSTs appeared to have homozygous CDKN2A/p16 deletions. Methylation analysis and mutation analysis of CDKN2A/p16 in MPNSTs did not reveal any abnormalities. These results show that malignant transformation of NF is associated with loss of p16 expression, which is often secondary to homozygous deletion of the CDKN2A/p16 gene. The findings suggest that CDKN2A/p16 inactivation occurs during the malignant transformation of NFs in NF1 patients and raises the possibility that p16 immunohistochemistry may provide ancillary information in the distinction of NF from MPNST.     

H-ras-1 point mutation in malignant peripheral nerve sheath tumors: polymerase chain reaction restriction fragment length polymorphism analysis and direct sequencing from paraffin-embedded tissues

It has been shown that the NF1 (neurofibromatosis type 1) gene encodes a tumor suppressor which inactivates ras proteins. Among malignant mesenchymal tumors, H-ras-1 mutations have been found in malignant fibrous histiocytoma, leiomyosarcoma and embryonal rhabdomyosarcoma. However, studies on H-ras-1 mutation of many cases of malignant peripheral nerve sheath tumors (MPNST) have not been documented. Therefore, we investigated H-ras-1 mutations of MPNST. In 45 cases of MPNSTs of our files, DNA was extracted from the formalin-fixed paraffin-embedded tissue, and the mutations of the H-ras-1 gene were detected by using PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphisms) method and direct sequencing. We found two cases with H-ras-1 point mutation in MPNST for the first time. Both cases showed the same mutation in codon 13.1 [GGT(Gly) to AGT(Ser) transition]. Interestingly, both cases were associated with NF1. It is possibile that the mutation of the H-ras-1 gene occurred after the mutation of the NF1 gene in the MPNST.     

NF1 Deletions in S-100 Protein-Positive and Negative Cells of Sporadic and Neurofibromatosis 1 (NF1)-Associated Plexiform Neurofibromas and Malignant Peripheral Nerve Sheath Tumors

Although plexiform neurofibroma (PN) is thought to represent a benign neoplasm with the potential for malignant transformation (malignant peripheral nerve sheath tumor; MPNST), its neoplastic nature has been difficult to prove due to cellular heterogeneity, which hampers standard molecular genetic analysis. Its mixed composition typically includes Schwann cells, fibroblasts, perineurial-like cells, and mast cells. Although NF1 loss of heterozygosity has been reported in subsets of PNs, it remains uncertain which cell type(s) harbor these alterations. Using a dual-color fluorescence in situ hybridization and immunohistochemistry technique, we studied NF1 gene status in S-100 protein-positive and -negative cell subpopulations in archival paraffin-embedded specimens from seven PNs, two atypical PNs, one cellular/atypical PN, and eight MPNSTs derived from 13 patients, seven of which had neurofibromatosis type 1 (NF1). NF1 loss was detected in four of seven PNs and one atypical PN, with deletions entirely restricted to S-100 protein-immunoreactive Schwann cells. In contrast, all eight MPNSTs harbored NF1 deletions, regardless of S-100 protein expression or NF1 clinical status. Our results suggest that the Schwann cell is the primary neoplastic component in PNs and that S-100 protein-negative cells in MPNST represent dedifferentiated Schwann cells, which harbor NF1 deletions in both NF1-associated and sporadic tumors.     

A neurogenic tumor containing a low-grade malignant peripheral nerve sheath tumor (MPNST) component with loss of p16 expression and homozygous deletion of CDKN2A/p16: a case report showing progression from a neurofibroma to a high-grade MPNST

Development of malignant peripheral nerve sheath tumors (MPNSTs) is a stepwise process that involves the alteration of many cell cycle regulators and the double inactivation of the NF1 gene. Inactivation of the TP53 gene and deletion of the CDKN2A/p16 gene are known to play an important role in the process. Herein, we present a 19-year-old man with a familial history of neurofibromatosis type 1, in whom the tumor arose from the intercostal nerve and showed 3 components: a neurofibroma, a low-grade MPNST, and a high-grade MPNST. Loss of p16 expression and homozygous deletion of the CDKN2A/p16 gene were observed in both the low-grade and the high-grade MPNST. In contrast to low-grade MPNSTs, high-grade MPNSTs generally tend to lose expression of p16 and harbor homozygous deletion of the CDKN2A/p16 gene. Loss of p16 expression and homozygous deletion of the CDKN2A/p16 gene in low-grade MPNST in our case might be related to its progression to high-grade MPNST. To the best of our knowledge, this is the first study correlating the p16 expression status and CDKN2A/p16 gene alteration in low-grade MPNSTs.     

Microarray-based copy number analysis of neurofibromatosis type-1 (NF1)-associated malignant peripheral nerve sheath tumors reveals a role for Rho-GTPase pathway genes in NF1 tumorigenesis

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumors. Approximately 15% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-Wide Human single-nucleotide polymorphism (SNP) Array 6.0 was used to perform SNP genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH), and copy number neutral-LOH (CNN-LOH) analyses of DNA isolated from 15 MPNSTs, five benign plexiform neurofibromas (PNFs), and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level CNN-LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN-LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA, Ras-related C3 botulinum toxin substrate 1 (RAC1) (7p21-p22), PDGFRL (8p22-p21.3), and matrix metallopeptidase 12 (MMP12) (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho-GTPase pathway genes and several cytoskeletal remodeling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1, ROCK2, PTK2, and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines, whereas wound healing, cell migration, and invasiveness were reduced, consistent with a role for these Rho-GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs.     

A search for evidence of somatic mutations in the NF1 gene

Neurofibromatosis type I (NF1) is an autosomal dominant disorder affecting 1 in 3000 people. The NF1 gene is located on chromosome 17q11.2, spans 350 kb of genomic DNA, and contains 60 exons. A major phenotypic feature of the disease is the widespread occurrence of benign dermal and plexiform neurofibromas. Genetic and biochemical data support the hypothesis that NF1 acts as a tumour suppressor gene. Molecular analysis of a number of NF1 specific tumours has shown the inactivation of both NF1 alleles during tumourigenesis, in accordance with Knudson's "two hit" hypothesis. We have studied 82 tumours from 45 NF1 patients. Two separate strategies were used in this study to search for the somatic changes involved in the formation of NF1 tumours. First, evidence of loss of heterozygosity (LOH) of the NF1 gene region was investigated, and, second, a screen for the presence of sequence alterations was conducted on a large panel of DNA derived from matched blood/tumour pairs. In this study, the largest of its kind to date, we found that 12% of the tumours (10/82) exhibited LOH; previous studies have detected LOH in 3-36% of the neurofibromas examined. In addition, an SSCP/HA mutation screen identified five novel NF1 germline and two somatic mutations. In a plexiform neurofibroma from an NF1 patient, mutations in both NF1 alleles have been characterised.     

Congenital disseminated neurofibromatosis type 1: a clinical and molecular case report

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition with a birth incidence of 1/3,500. Around 50% of cases are due to new mutations. The NF1 gene maps to 17q11.2 and encodes neurofibromin. NF1 is a "classical" tumor suppressor gene. Congenital disseminated NF1 is rare with just two cases previously reported. We present a deceased baby with congenital disseminated NF1 in whom we performed molecular studies. A germline mutation (R461X) in exon 10a of the NF1 gene was found. A 2 bp deletion (3508delCA) in codon 1170 of exon 21 was identified in DNA derived from some tumor tissue. Loss of heterozygosity in NF1 and TP53 was observed in other tumor samples. No microsatellite instability was observed in the tumor samples. This is the first report of molecular analysis of the NF1 locus in a patient with disseminated congenital neurofibromatosis. This case had a de novo germline mutation in NF1 and three documented somatic mutations in the NF1 and TP53 genes in tumor specimens.     

Homozygous inactivation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma

Neurofibromatosis type 1 (NF1) patients are susceptible to tumor development. In the present study we describe a child with NF1 and disseminated neuroblastoma whose death resulted from disease progression. The mother had café-au-lait spots suggesting a familial NF1. Neuroblastoma cells showed MYCN amplification and chromosome 1p36 deletion, common features associated with tumor progression in this malignancy. The NF1 gene displayed a germline T --> C transition of intron 14 in both the proband and mother DNA. This mutation, not yet previously described, occurs in a splicing donor site and produces a new mRNA variant observed together with normal NF1 mRNA. Furthermore, the SSCP analysis of the NF1 gene in tumor cells showed a somatic deletion encompassing the intron 26 and 27b of the paternal NF1 allele. Hence, neuroblastoma cells displayed both somatic and germline mutation of the NF1 gene. Our data suggest that, although rare, neuroblastoma in patients with NF1 may display homozygous gene inactivation.     

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.