Simplified diagram of interactions between the proteins of the Rb1 pathway (above) and the p53 pathway (below). The genes coding for p16 and p15 proteins are CDKN2A and CDKN2B, respectively. The genes for all of the proteins underlined have been shown to be abnormal in the astrocytic and some other gliomas as well as many other tumour cell types in other organs.
In the vast majority of cases where a pathway is disrupted in a tumour it is due to only one of the genes coding for a protein in that pathway being abnormal (loss of both wild type copies in the case of most tumour suppressor genes or amplification and overexpression in the case of proto-oncogenes). Thus it appears that pathways are targeted in oncogenesis and progression, and can be disrupted in many ways by losing, mutating, or amplifying the genes coding for the protein components of the pathway.
The numbers of cases of anaplastic astrocytomas (malignancy grade III) studied are also limited. Mutations of the TP53 gene also occur at approximately the same frequency as is found in the astrocytomas malignancy grade II. Thus in the anaplastic astrocytomas the p53 pathway is also non-functional, and in the majority of cases (more than 60%) this is due to mutations of the TP53 gene. Cytogenetics, CGH, and molecular genetic techniques all show that the losses of alleles on 6q, 13q, 17p and 22q, as seen in the astrocytoma malignancy grade II, occur at similar or higher frequencies in the anaplastic astrocytomas.
With the sole exception of losses of alleles on 19q (targeted gene unknown) there are no conclusively demonstrated abnormalities specific to this malignancy grade. Around 20% of anaplastic astrocytomas show similar genetic abnormalities to those found in glioblastomas involving other components of the p53 pathway (that is, MDM2 and p14ARF) and lead to disruption of the Rb1 pathway (fig 5), and these are discussed in the glioblastoma section below
De novo glioblastomas are common and this has ensured their study in considerable numbers. Secondary glioblastomas are less frequent and very less commonly studied. Such patients will frequently have been treated by irradiation and/or with cytotoxic drugs. Glioblastomas show the greatest numbers of genetic abnormalities among the astrocytic tumours and clear patterns of genetic aberrations are emerging.
The p53 pathway in glioblastomas is targeted through mutation of the TP53 gene (approximately 37%), as is seen in astrocytomas and anaplastic astrocytomas, but also by targeting other genes coding for proteins that control cellular p53 levels. The two genes whose products are involved in controlling p53 levels are p14ARF and MDM2. p14ARF controls the activity of MDM2, which in its turn controls the breakdown of p53.
Loss of both copies of the p14ARF gene or amplification and over-expression of MDM2 will lead to the rapid breakdown of wild type p53 protein resulting in a cell with little or no wild type p53. The vast majority of glioblastomas (> 70%) have either no wild type p53 or no p14ARF or over express MDM2 as mutually exclusive genetic abnormalities. Methylation of the p14ARF promoter with decreased or non-expression are further mechanisms that have been shown to be involved in some tumours. In glioblastomas additionally the retinoblastoma pathway and the PI3 kinase–Act pathway are also targeted.
In a similar manner one or other of the genes coding for proteins involved in the control of entry into the S phase of the cell cycle (the retinoblastoma pathway) are mutated in glioblastomas (fig 1). Entry into S phase is normally initiated by the release of transcription factors from newly phosphorylated Rb1 at the restriction point in G1. At the end of the cell cycle Rb1 is unphosphorylated. Unphosphorylated Rb1 normally sequesters the E2F transcription factors.
Loss of wild type RB1 gene resulting in no functional RB1 or inappropriately phosphorylated Rb1 will result in any expressed E2F being free to initiate transcription of the genes necessary for entry into S phase. Inappropriate phosphorylation may be achieved in glioblastomas with wild type Rb1 by either loss of wild type p16 expression or over-expression of CDK4 caused by amplification of its gene. These would make inappropriate phosphorylation of a wild type Rb1 more likely with the release of the E2Fs. p16 normally binds CDK4 and thus inhibits the formation of the CDK4/cyclin D1 heterodimer.
In the absence of p16 all expressed CDK4 is available for heterodimer formation. When CDK4 is overexpressed in the presence of normal levels of p16 there will be excess CDK4 available for heterodimer formation. One or the other of these abnormalities are present in over 90% of glioblastomas and are, with very few exceptions, mutually exclusive.
While disruption of the p53 and Rb1 pathways seem essential for glioblastomas, the ways in which the pathways are rendered dysfunctional may confer slightly different biological characteristics on the individual glioblastoma. In addition to the genetic abnormalities resulting in the disruption of the p53 and Rb1 pathways, over 90% of glioblastomas lose alleles from 10q.
The regions consistently lost include the variously named PTEN/MMAC1/TEP1 tumour suppressor gene at 10q23–24. PTEN has been shown to be mutated in up to 45% of glioblastomas. The gene is a dual specificity phosphatase (necessary for its ability to function as a tumour suppressor) and has homology to the cytoskeletal protein tensin.
One of its major substrates is phosphatidylinositol-3, 4, 5-triphosphate (PIP3) and lack of control of PIP3 is likely to have a major effect on the activation of the Akt pathway, affecting among other things apoptosis and HIF-1 activity.
Other genes coding for proteins involved in the PI3K/AKT pathway have recently been shown to be mutated, albeit infrequently. This is supported by reports on the affect of Akt activation in an animal model of astrocytoma.
Amplification of the epidermal growth factor receptor (EGFR) gene (7p11–12) is found in about 35% of glioblastomas. When amplified this gene is always over-expressed but may also be over-expressed in glioblastomas without amplification. Rearrangements of the amplified gene occur in almost half of the tumours with amplification.
The most common rearrangement results in a transcript that is aberrantly spliced, remains in frame, and codes for a mutated EGFR that has lost 267 amino acids of its extracellular domain and does not bind ligand. This mutated EGFR is constitutively activated and attempts are ongoing to target treatment to this aberrant cell surface molecule.
Other rearrangements of the amplified EGFR gene occur less frequently and may result in abnormalities of the cytoplasmic domain Glioblastomas can develop from an astrocytoma or as a de novo glioblastoma. It is tempting to try to sort all these findings into a series of events explaining the development of the two forms of glioblastoma.
Both have disrupted the normal p53 and Rb1 pathways, but in different ways. The de novo tumours do this by a single genetic event when amplification of the 12q14 region encompassing the CDK4 and MDM2 genes results in their over-expression and the disruption of both pathways. Two genetic events are required to disrupt the two pathways when homozygous deletion of the region on 9p encompassing the genes coding for p16 (CDKN2A), p15 (CDKN2B), and p14ARF (p14AR)) occurs (requires loss of both autosomes).
Occasionally de novo tumours may also show more complex patterns of mutations with loss of one allele of each of TP53 and RB1, with mutation of the retained alleles, requiring four genetic mutational events. However, in de novo glioblastomas these are in the minority.
Secondary glioblastomas generally have no wild type p53 due to loss of one allele and mutation of the retained allele, and lose a functional Rb1 pathway in a similar manner. Other correlations are that EGFR amplification is unusual in cases with no wild type p53, although this does occur occasionally. In addition to the abnormalities of the genes listed there are likely to be many other genetic changes affecting other regions of the genome that have been found to be manifestly abnormal in these tumours by deletion or amplicon mapping. The genes targeted by these changes have yet to be identified.
Oligodendrogliomas
Oligodendrogliomas occur mainly in the cerebral hemispheres of adults. They are believed to derive from oligodendrocytes. They consist of moderately cellular, monomorphic tumours with round nuclei, often artefactually swollen cytoplasm on paraffin section (fig 6), few or no mitoses, no florid microvascular proliferation or necrosis, and are classified as malignancy grade II according to the WHO.
Classically they show a “chicken wire” pattern of capillaries. They do not express any antigen characteristic of normal oligodendrocytes and may express GFAP. Grade II oligodendrogliomas are relatively indolent, although they usually recur at the primary site and may display a tendency for subependymal spread with a 5% incidence of cerebrospinal fluid (CSF) seeding. Oligoastrocytomas consist of tumour cells with either astroctytic or oligodendroglial morphological characteristics.
Tumour cells with these two phenotypes can be either diffusely mixed or combined as discrete areas in an individual tumour. The morphological borderlines between astrocytomas, oligoastrocytomas, and oligodendrogliomas are difficult and controversial issues.
