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The p16INK4a gene product acts as a negative regulator of the cell cycle by binding to cyclin-dependent kinases (CDKs) 4 and 6, thereby inhibiting the formation of an active CDK/cyclin D complex. Deletion of the p16 locus has been observed in tumor cell lines and, less frequently, in primary human neoplasms. We analyzed 31 glioblastomas and identified 6 cases with hemizygous and 6 with homozygous deletions of the p16 locus. Eight of these cases showed a concurrent amplification of the EGFR gene (epidermal growth factor receptor) while the overall frequency was 35%. This close correlation suggests that deletion of the p16 chromosomal region constitutes another genetic hallmark of the primary glioblastoma, which rapidly develops de novo, without a less malignant precursor lesion and for which EGFR amplification is a characteristic genetic change. The p16 protein was not detectable in 15 of 22 glioblastomas but only 4 of these showed homozygous deletion of the gene. The alternative transcript p16 beta, for which a growth-suppressing function has been suggested, was co-expressed with p16 alpha mRNA in most cases. Hypermethylation of CpG islands in the 5' region of the p16 gene was identified in only 1 case, suggesting that this alternative mechanism of gene silencing is rarely responsible for loss of p16 expression in glioblastomas. Likewise, only 1 glioblastoma carried a p16 mutation and in addition, unexpectedly, a homozygous deletion of p16 in approximately 80% of tumor cells. This mutation, Arg24Pro, has previously been identified in a melanoma kindred.

Glioma models have provided important insights into human brain cancers. Among the investigative tools, MRI has allowed their characterization and diagnosis. In this study, we investigated whether diffusion MRI might be a useful technique for early detection and characterization of slow-growing and diffuse infiltrative gliomas, such as the proposed new models, LN-2669GS and LN-2540GS glioma sphere xenografts. Tumours grown in these models are not visible in conventional T2 -weighted or contrast-enhanced T1 -weighted MRI at 14.1 T. Diffusion-weighted imaging and diffusion tensor imaging protocols were optimized for contrast by exploring long diffusion times sensitive for probing the microstructural alterations induced in the normal brain by the slow infiltration of glioma sphere cells. Compared with T2 -weighted images, tumours were properly identified in their early stage of growth using diffusion MRI, and confirmed by localized proton MR spectroscopy as well as immunohistochemistry. The first evidence of tumour presence was revealed for both glioma sphere xenograft models three months after tumour implantation, while no necrosis, oedema or haemorrhage were detected either by MRI or by histology. Moreover, different values of diffusion indices, such as mean diffusivity and fractional anisotropy, were obtained in tumours grown from LN-2669GS and LN-2540GS glioma sphere lines. These observations highlighted diverse tumour microstructures for both xenograft models, which were reflected in histology. This study demonstrates the ability of diffusion MRI techniques to identify and investigate early stages of slow-growing, invasive tumours in the mouse brain, thus providing a potential imaging biomarker for early detection of tumours in humans.