Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma

Abstract

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%-60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.

Figure 1.

NF1 truncation mutations in three whole-genome-sequenced NF1-PA tumors. The locations of all truncation mutations are designated with a circle mapped to transcript NM_001042492. Truncating mutations were initially identified in WGS data and further validated with targeted approaches. Predicted functional effects are based on transcript NM_001042492. In patient T03, a somatic, copy-neutral LOH of 57.3 Mbp resulted in duplication of the p.W1662* germline variant.

Figure 2.

Mechanism of somatic NF1 inactivation. (A) Immunohistochemistry using rabbit polyclonal neurofibromin antibodies demonstrates positive immunoreactivity in the normal human brain (HBr). (Inset) Single neuron with neurofibromin expression. In each of the three NF1-PA specimens, no neurofibromin expression was detected. Scale bars, 100 μm. Magnification, 400×. Inset, 600×. (B) In T03, a somatic copy-neutral LOH of the q arm of chromosome 17 from position 23.9 Mbp to the telomere results in duplication of the NF1 W1662* germline variant. Shown are the median variant allele frequencies of high-confidence germline heterozygous SNPs identified in the normal DNA in 100,000-bp bins across chromosome 17. The position of the NF1 gene is indicated. (C) Methylation changes in T01 relative to T02 and T03 from Illumina Infinium HumanMethylation450 arrays for 42 probes spanning the NF1 genomic region. T01 exhibits increased methylation (>2 standard deviations) at NCBI 36 chr17:26648522 (probe cg13759778). This location is within 200 bp of the transcription start site of OMG, one of three antisense nested genes within intron 36 of NF1 mRNA NM_001042492.2.

Figure 3.

NF1-PA tumor clonality, cellular heterogeneity, and mutation rate. (A) Somatic mutation clusters were defined based on allele-specific supporting read counts in whole-genome data for mutations identified in nonrepetitive genomic with diploid copy number. Kernel density estimation indicates a monoclonal origin for each tumor and reveals a substantial proportion of stromal cells in the tumor biopsy with estimated tumor purities of 35%, 51%, and 39% for T01, T02, and T03, respectively. (B) CD68 immunostaining confirms the marked cellular heterogeneity of NF1-PA tumors, with 35%–55% of the cells in these tumors representing microglia/macrophages. Representative photomicrographs are included for each NF1-PA tumor. Scale bar, 100 μm. Magnification, 400×. Insets, 600×. (C) BMRs are based on the total sum of somatic mutations identified in all nonrepetitive genomic regions from WGS of three NF1-PA tumors, and compared with pediatric retinoblastoma (N = 4) (Zhang et al. 2012a), pediatric TALL (N = 8) (Zhang et al. 2012b), the M1 and M3 subtypes of adult AML (N = 25) (Welch et al. 2012), and luminal breast cancer (N = 46).