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

Abstract

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.

Figure 1.

A and B: Neurofibroma from a patient with NF1, showing bland-looking spindle cells separated by a myxoid and collagenous background (A). Immunohistochemical staining illustrates diffuse nuclear and faint cytoplasmic staining for p16 (DAB chromogen with hematoxylin counterstain) (B). C and D: “Atypical” neurofibroma showing increased cellularity and nuclear atypia (C). No mitotic figures are present. Immunohistochemical staining for p16 demonstrates nuclear and faint cytoplasmic staining of the neoplastic cells, including “atypical” cells (D). E and F: MPNST (E) showing marked increased cellularity, fasicular and herringbone growth pattern, nuclear atypia, and marked mitotic activity. Immunohistochemical staining shows no immunoreactivity for p16 (F). G and H: MPNST arising in association with a neurofibroma (G). An abrupt transition from the neurofibroma (right) to MPNST (left) is seen. Immunohistochemical staining of the same tumor (H) shows that the benign neurofibromatous component (right) stains for p16 protein, whereas the malignant component (left) is immunonegative for p16 protein.

Figure 2.

Comparative multiplex analysis of the CDKN2A/p16 gene. Homozygous deletion (HD) is present in three tumors (lanes 3, 5, and 10), as evidenced by preferential amplification of the control amplicon (top band) with minimal amplification of the CDKN2A/p16 amplicon (bottom band). M, pUC18/HaeIII digest size marker. Lane 0: Negative control (“no DNA”). Lane 1: Normal control DNA. Lanes 2 and 3: DNA from the same tumor, showing the benign (NF) component (lane 2) and the malignant component (MPNST; lane 3), the latter showing HD for the CDKN2A/p16 gene. Lanes 4 and 5 show findings similar to those of lanes 2 and 3, with DNA from the different areas in another tumor (lane 4, NF; lane 5, MPNST). Lane 5 shows HD for the CDKN2A/p16 gene. Lane 6: Neurofibroma from a patient with NF1. Lanes 7–10: MPNSTs from four different NF1 patients. Lanes 7–9 do not show HD, whereas the MPNST in lane 10 shows HD for the CDKN2A/p16 gene.