Molecular genetic alterations in radiation-induced astrocytomas

Abstract

Astrocytic tumors occasionally arise in the central nervous system following radiotherapy. It is not clear if these gliomas represent a unique molecular genetic subset. We identified nine cases in which an astrocytoma arose within ports of previous radiation therapy, with total doses ranging from 2400 to 5500 cGy. Irradiated primary lesions included craniopharyngioma, pituitary adenoma, Hodgkin's lymphoma, ependymoma, pineal neoplasm, rhabdomyosarcoma, and three cases of lymphoblastic malignancies. Patients ranged from 9 to 60 years of age and developed secondary tumors 5 to 23 years after radiotherapy. The 9 postradiation neoplasms presented as either anaplastic astrocytoma (3 cases) or glioblastoma multiforme (6 cases). Two of the latter contained malignant mesenchymal components. We performed DNA sequence analysis, differential polymerase chain reaction (PCR), and quantitative PCR on DNA from formalin-fixed, paraffin-embedded tumors to evaluate possible alterations of p53, PTEN, K-ras, EGFR, MTAP, and p16 (MTS1/CDKN2) genes. By quantitative PCR, we found EGFR gene amplification in 2 of 8 tumors. One of these demonstrated strong immunoreactivity for EGFR. Quantitative PCR showed chromosome 9p deletions including p16 tumor suppressor gene (2 of 7 tumors) and MTAP gene (3 of 7). Five of 9 tumors demonstrated diffuse nuclear immunoreactivity for p53 protein. Sequencing of the p53 gene in these 9 cases revealed a mutation in only one of these cases, a G-to-A substitution in codon 285 (exon 8). Somewhat unexpectedly, no mutations were identified in PTEN, a commonly altered tumor suppressor gene in de novo glioblastoma multiformes. Unlike some radiation-induced tumors, no activating point mutations of the K-ras proto-oncogene or base pair deletions of tumor suppressor genes were noted. These radiation-induced tumors are distinctive in their high histological grade at clinical presentation. The spectrum of molecular genetic alterations appears to be similar to that described in spontaneous high grade astrocytomas, especially those of the de novo type.

Figure 1.

Gadolinium-enhanced magnetic resonance images of a patient who was treated with 4500 cGy of radiation therapy for a residual pituitary adenoma (A, arrow) following incomplete surgical resection. Isodose curves of radiotherapy (shown in red, with percentages of maximal dose in white) demonstrate the isocenter of radiation therapy at the residual adenoma. Fifteen years after surgery and radiotherapy, the patient developed a glioblastoma multiforme involving the left and right hemispheres, crossing at the corpus callosum (B). The new lesion was radiographically distinct from the residual adenoma (B, arrow) and occurred within the port of previous radiation therapy.

Figure 2.

Multiplex PCR reaction products of EGFR gene and reference genes (top, β-globin; bottom, cystic fibrosis) resolved by polyacrylamide gel electrophoresis and stained with ethidium bromide. Preferential PCR amplification of the EGFR gene fragment compared to reference gene was seen in tumors T309 and T312. Cell lines SK-MG3, MDA-468, and A-431 are known to contain EGFR amplification and served as positive controls.

Figure 3.

Strong cell membrane and cytoplasmic immunoreactivity for EGFR protein was seen in one of the two tumors that contained amplified EGFR gene by differential PCR (T309).