Low mutation burden and frequent loss of CDKN2A/B and SMARCA2, but not PRC2, define premalignant neurofibromatosis type 1-associated atypical neurofibromas

Abstract

Background: Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft-tissue sarcoma, often arising from preexisting benign plexiform neurofibromas (PNs) and atypical neurofibromas (ANFs). ANFs are distinct from both PN and MPNST, representing an intermediate step in malignant transformation.

Methods: In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of 3 MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of 5 ANFs and 5 MPNSTs.

Results: We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED and SUZ12 were frequently mutated, deleted, or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3, and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only.

Conclusions: The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction and cell-cycle and pluripotency self-renewal genes.

Keywords: atypical neurofibromas; benign-to-malignant transformation; malignant peripheral nerve sheath tumor; neurofibromatosis type 1; plexiform neurofibromas.

Fig. 1

MRI and fluorodeoxyglucose (FDG) PET evaluation of 2 distinct nodular lesions within a single large PN. (A, B) A 9-year-old boy with NF1 and newly diagnosed PN involving the right retroperitoneum, pelvis, and thigh. MRI evaluation at the NCI revealed 2 distinct nodular lesions, one in the right inguinal area (A; sample ANF14-2), and one in the right paraspinal area (B; sample ANF14-1). (C‒E) Volumetric MRI analysis demonstrated faster growth rates for the nodular lesions (black contour) compared with the PN (green contour). (F, G) FDG-PET demonstrated FDG avidity of the nodular lesions with minimal uptake in the surrounding PN (F and G, inguinal and paraspinal lesions, respectively). Core biopsy of the inguinal lesion showed ANF; core biopsy of the paraspinal lesion showed neurofibroma. The inguinal lesion was resected, and pathology confirmed atypical neurofibroma. Follow-up with MRI continues to demonstrate more rapid growth of the remaining paraspinal lesion compared with the PN (C, D).

Fig. 2

CNV in ANF and MPNST. (A) GISTIC analysis of 26 ANFs (upper panel) and 28 MPNSTs (lower panel). Gains and losses are shown in blue and red, respectively. Statistically significant CNVs (false discovery rate <0.25) are denoted with gray vertical bars. Chromosome numbers are shown on top of each panel. (B) CNV in chromosome 9p in 26 ANFs (left panel) and 28 MPNSTs (right panel). Gains and losses are shown in blue and red, respectively. Homozygous losses are shown in dark red and thick bars. Vertical rectangles in the center denote minimally overlapped regions in ANF and MPNST that both include the CDKN2A and CDKN2B genes. Left vertical rectangle in the ANF panel denotes SMARCA2. Arrows show samples (A11_ANF7, A12, ANF11-7) in which deletion of SMARCA2 is clearly independent from CDKN2A/B.

Fig. 3

Expression of genes frequently affected by CNV/mutations, genes identified by GSEA leading edge analysis, and select biologically relevant genes in normal tissues (n = 39), PN (n = 23), ANF (n = 5), and MPNST (n = 5) as determined by RNAseq analysis. Expression values are shown in fragments per kilobase per million (FPKM) reads, log2 transformed, log2(FPKM) units. Blue, red, gray, and orange bars correspond to normal tissues, PN, ANF, and MPNST, respectively. Asterisks indicate statistically significant difference (by 2-tailed t-tests) between normal and PN, ANF, or MPNST expression values at P < 0.0001.