The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas

Abstract

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Figure 1.

Distribution of germline (upper panel) and somatic (lower panel) NF1 mutations among 23 plexiform neurofibromas. Black and green dots represent truncating (nonsense, frame-shifting indels and splice-site substitutions) and missense mutations, respectively. Green and red rectangles represent the RAS-GAP and CRAL/TRIO (SEC14-like) domains, respectively. Note that germline substitutions K583R and K1423K result in splicing defects and therefore depicted with black dots. The germline nonsense mutation R816X was found in two unrelated individuals, and the germline frame-shifting mutation L2112fs was found in monozygotic twins and was counted for both individuals. Large deletions are not shown on the diagram (for details see Supplementary Tables S2A and B).

Figure 2.

NF1 expression in PN and normal Schwann cells. (a) Table shows sample or cell identification, type of the germline and somatic mutations in NF1 and expression values compared with that in normal Schwann cells (%). The PN sample with the median expression value (53%) is highlighted with red font. CN-LOH: Copy Neutral Loss Of Heterozygosity; PGD: Partial Gene Deletion; TGD: Total Gene Deletion. (b) Bar graph of the values shown in (a). Sample with the median expression value (53%) is shown in red. NA, not applicable.

Figure 3.

Gene Set Enrichment Analysis of genome-wide expression in PN. Enrichment plots of five significant and seven trending sets of genes involved in DNA replication initiation and strand elongation, telomere maintenance and extension, cell proliferation and differentiation, as well as gene sets comprising targets of MYC and E2F, and down-regulated by activated KRAS. FDR, false discovery rate; NES, normalized enrichment score.

Figure 4.

Expression of 60 Gene Set Enrichment Analysis leading-edge genes in PN and normal Schwann cells. Expression values of genes that were found in at least two significant sets are shown. Blue bars represent normal Schwann cells, and red bars represent PN. Fifty-two genes (POLA2 through XRCC6) are up-regulated and eight genes (DTX2 through TMEM8B) are down-regulated in PN compared with normal Schwann cells (SC). Note that raw median expression value of KRT16 in PN is 0.51, which results in negative value of −0.97, when log2 transformed.