Lan Kluwe

Treatment of ADHD in neurofibromatosis type 1

Forty-six of 93 children with neurofibromatosis type 1 (NF1) were found to satisfy the diagnostic criteria for attention-deficit-hyperactivity disorder (ADHD). Detailed comparisons were made among 20 children with NF1 and ADHD (12 males, 8 females; mean age 10.7 years, SD 2.2), 26 control children with NF1 (15 males, 11 females; mean age 11.3 years, SD 2.3), 14 control children with ADHD (7 males; mean age 9.9 years, SD 1.9), and 14 normally developing control children (7 males; mean age 11.2 years, SD 2.8). Children with NF1 and ADHD had the lowest IQ scores among the four groups. Test of Variables of Attention (TOVA) scores were poorer in the NF1-ADHD and ADHD control groups than in the two non-ADHD groups. Those with NF1 and ADHD were rated significantly poorer on the Child Behavior Checklist (CBCL) than were the NF1 control group. By administrating low doses (5 to 15 mg) of methylphenidate to the NF1-ADHD group, significantly improved TOVA scores were obtained. One-year follow-up yielded significantly improved CBCL scores. Our results show a high incidence of ADHD in NF1 and support an association between ADHD and learning and social problems in children with NF1. It was demonstrated that stimulant medication can lead to improvement in cognitive, academic, and social problems of children with NF1 and ADHD.

Large-Scale Molecular Comparison of Human Schwann Cells to Malignant Peripheral Nerve Sheath Tumor Cell Lines and Tissues

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.

Migration of Human Glioma Cells on Myelin

Histoanatomically invading astrocytoma cells appear to migrate along distinct structures within the brain. Astrocytoma invasion may occur along extracellular matrix (ECM) protein-containing structures, such as blood vessels, but most frequently occurs along tracts of myelinated fibers. This behavior most likely is a consequence of the use of constitutive extracellular ligands expressed along the pathways of preferred dissemination. Enzymatic modification of the extracellular space or deposition of ECM by the tumor cells may also create a more permissive environment. Established human glioma cell lines and two preparations of primary cells isolated from glioblastoma biopsies were studied with the use of cell adhesion and monolayer migration assays to investigate whether crude human central nervous system myelin extracts present specific cell adhesion ligands that promote glioma attachment and cell migration. Two cell lines showed high levels of adhesion and migration on central nervous system myelin similar to levels of migration on the ECM protein merosin, which has previously been shown to be a highly permissive substrate for cultured astrocytoma cells. Two other cell lines showed lower but specific migratory response; one cell line did not attach or specifically migrate on crude myelin extracts. For both glioblastoma primary cell preparations, myelin and merosin were the most permissive substrates for attachment and migration. Other ECM proteins (collagen type IV, fibronectin, and vitronectin) were moderate or nonpermissive substrates. Our findings indicated that astrocytoma cells may be able to use oligodendrocyte membrane-associated ligands as well as ECM proteins of the basement membranes for invasion of normal brain.

Allelic Losses at 1p, 9q, 10q, 14q, and 22q in the Progression of Aggressive Meningiomas and Undifferentiated Meningeal Sarcomas

Meningiomas are usually benign tumors; however, they can recur after surgical resection and occasionally show histological progression to a higher malignancy grade. Five such rare cases of aggressively recurring meningiomas were present in our departmental cohort of 923 primary meningeal neoplasms operated over a 17-year period. Four other aggressively recurring meningeal tumors with a very similar clinical and histomorphological appearance (three undifferentiated meningeal sarcomas, one hemangiopericytoma) was also included in this study. We investigated whether disease progression can be traced by genetic alterations and whether a pattern of genetic alterations is specific for meningiomas. A total of 40 specimens from primary tumors and multiple recurrences of the nine patients were analyzed with 26 polymorphic allelic markers for deletions on 1p, 1q, 9q, 10q, 14q, and 22q. Loss of heterozygosity (LOH) at 22q was observed in all meningiomas cases at the earliest time point analyzed. Allelic loss at 1p was seen in the original tumor in two cases and upon meningioma recurrence in two others. Deletion on 10q occurred during tumor progression in two cases, and on 9q and 14q in one case. While allelic loss at 22q appears to be an early event in aggressive meningioma disease, there is a clear correlation of further deletions on chromosome arms 1p, 9q, 10q, and 14q with histopathological and clinical progression, as shown in these intraindividual trackings. None of these genetic findings were present in the non-meningiomatous meningeal tumors, indicating that meningothelial cells have their own lineage-specific genetic pathways towards clinical malignancy.

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 × 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.

Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis

Schwannomatosis is a third major form of neurofibromatosis that has recently been linked to mutations in the SMARCB1 (hSnf5/INI1) tumor suppressor gene. We analyzed the coding region of SMARCB1 by direct sequencing and multiplex ligation‐dependent probe amplification (MLPA) in genomic DNA from 19 schwannomatosis kindreds. Microsatellite markers in the SMARCB1 region were developed to determine loss of heterozygosity (LOH) in associated tumors. We detected four alterations in conserved splice acceptor or donor sequences of exons 3, 4 and 6. Two alterations that likely affect splicing were seen in introns 4 and 5. An additional four alterations of unclear pathogenicity were found to segregate on the affected allele in eight families including two non‐conservative missense alterations in three families. No constitutional deletions or duplications were detected by MLPA. Nine of 13 tumors examined showed partial LOH of the SMARCB1 region consistent with ‘second hits.’ Alterations were detected in tumors both with and without somatic NF2 gene changes. These findings support the hypothesis that SMARCB1 is a tumor suppressor for schwannomas in the context of familial disease. Further work is needed to determine its role in other multiple and single tumor syndromes.

Loss of NF1 allele in schwann cells but not in fibroblasts derived from an NF1‐associated neurofibroma

Neurofibromas, the hallmark of neurofibromatosis 1, are composed mainly of Schwann cells and fibroblasts. Inactivation of both NF1 alleles is the cause of these benign tumors, but it is unknown which cell type is the progenitor. In this study, we selectively cultured Schwann cells from an NF1‐associated neurofibroma. Fibroblasts were also obtained by culturing the tumor cells under standard conditions. Using four intragenic markers, we genotyped the NF1 locus in the original tumor and in the derived Schwann cells and fibroblasts. Loss of heterozygosity for two informative markers, which indicates loss of one NF1 allele, was found in Schwann cells but not in fibroblasts. This result suggests that genetic alterations of the NF1 gene in Schwann cells are responsible for the development of neurofibromas. Genes Chromosomes Cancer 24:283–285, 1999.

Clinical characterisation of 29 neurofibromatosis type-1 patients with molecularly ascertained 1.4 Mb type-1 NF1 deletions

Background Large deletions of the NF1 gene region occur in ∼5% of patients with neurofibromatosis type-1 (NF1) and are associated with particularly severe manifestations of the disease. However, until now, the genotype–phenotype relationship has not been comprehensively studied in patients harbouring large NF1 gene deletions of comparable extent (giving rise to haploinsufficiency of the same genes). Method We have performed the most comprehensive clinical/neuropsychological characterisation so far undertaken in NF1 deletion patients, involving 29 patients with precisely determined type-1 NF1 (1.4 Mb) deletions. Results Novel clinical features found to be associated with type-1 NF1 deletions included pes cavus (17% of patients), bone cysts (50%), attention deficit (73%), muscular hypotonia (45%) and speech difficulties (48%). Type-1 NF1 deletions were found to be disproportionately associated with facial dysmorphic features (90% of patients), tall stature (46%), large hands and feet (46%), scoliosis (43%), joint hyperflexibility (72%), delayed cognitive development and/or learning disabilities (93%) and mental retardation (IQ<70; 38%), as compared with the general NF1 patient population. Significantly increased frequencies (relative to the general NF1 population) of plexiform neurofibromas (76%), subcutaneous neurofibromas (76%), spinal neurofibromas (64%) and MPNSTs (21%) were also noted in the type-1 deletion patients. Further, 50% of the adult patients exhibited a very high burden of cutaneous neurofibromas (N≥1000). Conclusion These findings emphasise the importance of deletion analysis in NF1 since frequent monitoring of tumour presence and growth could potentiate early surgical intervention thereby improving patient survival.

Assessment of benign tumor burden by whole-body MRI in patients with neurofibromatosis 1

People with neurofibromatosis 1 (NF1) have multiple benign neurofibromas and a 10% lifetime risk of developing malignant peripheral nerve sheath tumors (MPNSTs). Most MPNSTs develop from benign plexiform neurofibromas, so the burden of benign tumors may be a risk factor for developing MPNST. We studied 13 NF1 patients with MPNSTs and 26 age- and sex-matched controls (NF1 patients who did not have MPNSTs) with detailed clinical examinations and whole-body MRI to characterize their body burden of internal benign neurofibromas. Internal plexiform neurofibromas were identified in 22 (56%) of the 39 NF1 patients studied. All six of the NF1 patients with MPNSTs under 30 years of age had neurofibromas visualized on whole-body MRI, compared to only 3 of 11 matched NF1 controls under age 30 (p < 0.05). Both the median number of plexiform neurofibromas (p < 0.05) and the median neurofibroma volume (p < 0.01) on whole-body MRI were significantly greater among MPNST patients younger than 30 years of age than among controls. No significant differences in whole-body MRI findings were observed between NF1 patients with MPNSTs and controls who were 30 years of age or older. Whole-body MRI of NF1 patients allows assessment of the burden of internal neurofibromas, most of which are not apparent on physical examination. Whole-body imaging of young NF1 patients may allow those at highest risk for developing MPNST to be identified early in life. Close surveillance of these high-risk patients may permit earlier diagnosis and more effective treatment of MPNSTs that develop.

High-Resolution DNA Copy Number Profiling of Malignant Peripheral Nerve Sheath Tumors Using Targeted Microarray-Based Comparative Genomic Hybridization

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition that predisposes to benign and malignant tumors. The lifetime risk of a malignant peripheral nerve sheath tumor (MPNST) in NF1 is ∼10%. These tumors have a poor survival rate and their molecular basis remains unclear. We report the first comprehensive investigation of DNA copy number across multitude of genes in NF1 tumors using high-resolution array comparative genomic hybridization (CGH), with the aim to identify molecular signatures that delineate malignant from benign NF1 tumors. Experimental Design: We constructed an exon-level resolution microarray encompassing 57 selected genes and profiled DNA from 35 MPNSTs, 16 plexiform, and 8 dermal neurofibromas. Bioinformatic analysis was done on array CGH data to identify concurrent aberrations in malignant tumors. Results: The array CGH profiles of MPNSTs and neurofibromas were markedly different. A number of MPNST-specific alterations were identified, including amplifications of ITGB4, PDGFRA, MET, TP73, and HGF plus deletions in NF1, HMMR/RHAMM, MMP13, L1CAM2, p16INK4A/CDKN2A, and TP53. Copy number changes of HMMR/RHAMM, MMP13, p16INK4A/CDKN2A, and ITGB4 were observed in 46%, 43%, 39%, and 32%, respectively of the malignant tumors, implicating these genes in MPNST pathogenesis. Concomitant amplifications of HGF, MET, and PDGFRA genes were also revealed in MPNSTs, suggesting the putative role of p70S6K pathway in NF1 tumor progression. Conclusions: This study highlights the potential of array CGH in identifying novel diagnostic markers for MPNSTs.