Perry A, Nobori T, Ru N, Anderl K, Borell T J, Mohapatra G, Feuerstein B G, Jenkins R B, Carson D A
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Neuropathol Exp Neurol. 1997 Sep;56(9):999-1008. doi: 10.1097/00005072-199709000-00005.
The p16 protein plays a key role in cell cycle control by preventing CDK4 from inactivating the retinoblastoma protein (pRb). The corresponding tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicated in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 methods of detecting p16 deletion. Thirty diffuse gliomas were analyzed for p16 gene dosage. Dual color fluorescence in situ hybridization (FISH) was performed on cytologic preparations using paired centromeric (CEN) and locus-specific probes for CEN9/p16, CEN8/RB, and CEN12/CDK4. Quantitative PCR was performed using primers for p16, MTAP, and reference genes. Eleven cases were also studied using comparative genomic hybridization (CGH). Abnormalities of the p16-CDK4-RB pathway were identified in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis further revealed tetraploidy/aneuploidy in 14 (47%), RB deletion in 11 (37%) and CDK4 amplification in 1 (3.3%). There were 94% and 100% concordance rates between CGH and FISH or PCR, respectively. Quantitative PCR was noninformative in 4 cases. Although FISH and quantitative PCR are both reliable techniques, each has limitations. PCR is likely to miss p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysis may miss small deletions. Replacement of the latter with a cosmid probe may improve the sensitivity of FISH in future experiments.
p16蛋白通过阻止细胞周期蛋白依赖性激酶4（CDK4）使视网膜母细胞瘤蛋白（pRb）失活，在细胞周期调控中发挥关键作用。相应的肿瘤抑制基因（p16/MTS1/CDKN2）最近被认为与星形细胞瘤的恶性进展有关，并且有可能作为患者预后和指导特定治疗策略的重要标志物。我们进行了一项研究以评估检测p16缺失的两种方法。对30例弥漫性胶质瘤进行了p16基因剂量分析。使用针对CEN9/p16、CEN8/RB和CEN12/CDK4的配对着丝粒（CEN）和基因座特异性探针，对细胞涂片进行双色荧光原位杂交（FISH）。使用针对p16、MTAP和参考基因的引物进行定量PCR。还使用比较基因组杂交（CGH）对11例病例进行了研究。通过FISH和/或PCR在21例（70%）病例中鉴定出p16-CDK4-RB途径异常。其中包括15例（50%）p16缺失，其中9例通过两种技术均检测到，3例仅通过FISH检测到，3例仅通过PCR检测到（一致率=81%）。FISH分析进一步显示14例（47%）为四倍体/非整倍体，11例（37%）为RB缺失，1例（3.3%）为CDK4扩增。CGH与FISH或PCR之间的一致率分别为94%和100%。定量PCR在4例病例中无信息价值。虽然FISH和定量PCR都是可靠的技术，但每种技术都有局限性。当存在明显的正常细胞污染或克隆异质性时PCR可能会遗漏p16缺失，而用于FISH分析的p16酵母人工染色体（YAC）探针可能会遗漏小的缺失。在未来的实验中用黏粒探针替代后者可能会提高FISH的敏感性。
