Abnormalities of p16 gene and its expression in human brain tumors.

我们分析了来自日本的75例脑肿瘤（3例室管膜瘤、9例少突胶质细胞瘤、12例星形细胞瘤、11例间变性星形瘤、1例髓母细胞瘤和39例多形性胶质母细胞瘤）中候选肿瘤抑制因子p16基因的表达状况。利用p16基因外显子2引物和对照染色体9qSTS引物进行定量PCR分析，发现7例p16基因纯合缺失；11个间变性星形细胞瘤中有1个（hho III级），39个多形性胶质母细胞瘤中有6个（iv级），但没有1或II级肿瘤。应用单链构象多态性技术，对8例（星形细胞瘤1例、间变性星形细胞瘤1例、多形性胶质母细胞瘤6例）的PCR产物进行了迁移性转移观察。对异常迁移产物的DNA测序显示，3例多形性胶质母细胞瘤发生突变，导致密码子66、143和145的氨基酸替换。多克隆抗p16抗体免疫组化分析显示p16表达频繁缺失。45例（室管膜瘤1例，少突胶质细胞瘤4例，星形细胞瘤5例，间变性星形细胞瘤6例，多形性胶质母细胞瘤29例），星形细胞瘤1例，间变性星形细胞瘤2例，多形性胶质母细胞瘤6例，70%以上的肿瘤细胞不表达p16。结论：p16基因的纯合缺失、突变和异常表达在高级别恶性肿瘤中较为常见。此外，本研究显示的低频率纯合缺失与以往报道的p16基因在白种人恶性胶质瘤中频繁缺失有很大不同，提示胶质瘤的发生机制可能存在种族差异。

We have analyzed the status of candidate tumor suppressor p16 gene in 75 brain tumors (3 ependymomas, 9 oligodendrogliomas, 12 astrocytomas, 11 anaplastic astrosytomas, 1 medulloblastoma and 39 glioblastoma multiforms) from Japanese patients. With quantitative multiplex polymerase chain reaction (PCR) assay using the exon 2 primers of the p16 gene and control chromosome 9qSTS primers, we found homozygous deletion of the p16 gene in 7 cases; in 1 out of 11 anaplastic astrocytomas (HWO grade III), 6 out of 39 glioblastoma multiforms (gradeIV), but in none of the tumors of grade I or II. Using the single strand conformation polymorphism technique, mobility shift of PCR products were observed in 8 cases (1 astrocytoma, 1 anaplastic astrocytoma, 6 glioblastoma multiforms). DNA sequencing of the aberrantly migrated products revealed that 3 cases of glioblastoma multiform had mutations which caused amino acid substitutions in codon 66, 143 and 145.Immunohistochemical analysis using polyclonal anti-p16 antibody revealed frequent loss of p16 expression. Of 45 cases analyzed (1 ependymoma, 4 oligodendrogliomas, 5 astrocytomas, 6 anaplastic astrocytomas, 29 glioblastoma multiforms), 1 astrocytoma, 2 anaplastic astrocytomas, 6 glioblastoma multiforms did not express p16 in over 70% of tumor cells. In conclusion, homozygous deletion, mutation and abnormal expression of p16 gene was frequent in high grade malignancy. In addition, the low frequency of homozygous deletions shown in this study is quite different from previous reports that demonstrated frequently deleted p16 gene in malignant gliomas from Caucasian patients and suggesting a possible racial difference in the mechanism of the tumorigenesis of gliomas.