Mechanisms of Disease: genetic predictors of response to treatment in brain tumors

DIFFUSE ASTROCYTOMAS

Diffuse astrocytic tumors include astrocytomas (WHO malignancy grade II), anaplastic astrocytomas (WHO malignancy grade III) and glioblastomas (WHO malignancy grade IV). The age at which the incidence of grade II astrocytomas peaks is 25–50 years, while in highly malignant glioblastomas the peak age of incidence is 45–70 years. The age at which the incidence of anaplastic astrocytomas peak overlaps with the peak age of incidence for grade II astrocytomas and the peak age of incidence of glioblastomas. All tumor types are more common in males than females, with a 1.25:1 male:female ratio. Glioblastomas are the most common form of astrocytic tumor and are categorized into those that develop from a previously diagnosed astrocytoma (i.e. secondary glioblastomas, which are relatively rare) and those that seem to develop de novo.

Primary and secondary glio blastomas show some differences in their patterns of genetic abnormality, although the genes involved in glioblastoma development encode components of the same cellular signaling pathways. The median survival of patients with an astrocytoma is around 7 years, while patients with anaplastic astrocytomas have a median survival of 3–4 years. Patients with glioblastoma have a very poor prognosis, with a median survival of 14.6 months reported in a trial that used concomitant radiotherapy and temozolomide followed by six monthly cycles of adjuvant temozolomide. In the case of the astrocytic tumors, the only major prognostic factor after histopathological diagnosis is age; in general, younger patients survive longer.

Diffuse astrocytomas (WHO grade II)

In adults, diffuse astrocytomas WHO grade II are relatively monomorphic tumors with moderately increased cellularity compared with normal white matter, and consist of tumor cells with the characteristics of astrocytes. The loss of one TP53 allele and mutation of the DNA binding region in the retained allele is the only common genetic abnormality of diffuse astrocytomas, and is found in over 60% of these tumors. The retained mutated allele is often duplicated. In a small percentage of tumors, there may be two individually mutated alleles or one retained wild-type allele together with a mutated TP53 allele. All of these scenarios will lead to a functionally abnormal p53 pathway. Astrocytic tumors are one of the tumor types seen in Li–Fraumeni syndrome, where approximately 70% of individuals have inherited one mutated allele of TP53. Cytogenetics, metaphase comparative genomic hybridization (CGH), and molecular genetic techniques, have identified many other chromosomal copy number aberrations in astrocytic tumors, but have not yet revealed other commonly involved genes. The technique of array CGH should provide the resolution required to identify specific genes and thus the signaling pathways that are involved. Few array CGH and expression studies have encompassed the diffuse astrocytomas (WHO grade II); however, expression of both the ligands and receptors of the plateletderived growth factor (PDGF) and epidermal growth factor systems have been reported in these tumors. There are no well-established genetic predictors of response to therapy for WHO grade II diffuse astrocytomas.

Anaplastic astrocytomas (WHO grade III)

Anaplastic astrocytomas (WHO grade III) are more cellular than diffuse astrocytomas (WHO grade II) and show nuclear atypia with mitoses . They do not have microvascular proliferation or spontaneous tumor necrosis, characteristics that are indicative of glioblastomas. Anaplastic astrocytomas show similar TP53 mutation rates to diffuse astrocytomas (WHO grade II) but have greater allelic losses and chromosome/chromosomal region copy number abnormalities. Abnormalities in 19q have been reported to be common in this grade, but gene amplification is uncommon. Approximately 13% of diffuse astrocytomas show abnormalities in the genes coding for protein components of the RB1 pathway that are common in glioblastomas (WHO grade IV). In anaplastic astrocytomas, loss of both wildtype copies of any of the three tumor-suppressor genes CDKN2A, CDKN2B and RB1 or amplification and overexpression of CDK4, which disrupts the RB1 pathway , have been linked to shorter overall survival (P = 0.009) and this association was confirmed in age-adjusted multivariate analysis (P = 0.013). These findings suggest a poor prognosis for patients who have anaplastic astrocytomas with RB1 pathway aberrations and receive conventional therapy.

Glioblastomas (WHO grade IV)

Glioblastomas are pleomorphic cellular tumors with high mitotic rates and spontaneous necrosis that usually show a marked microvascular proliferation . Most glioblastomas are primary, and in contrast to the greater than 60% TP53 mutation rate seen in astrocytomas and anaplastic astrocytomas, primary glioblastomas have TP53 mutations in only approximately 30% of tumors. Secondary glioblastomas have TP53 mutations as frequently as the tumors from which they are derived i.e. the astrocytomas and anaplastic astrocytomas. Despite the low rates of TP53 mutation relative to astrocytomas and anaplastic astrocytomas, more than 70% of primary glioblastomas have disruption of the p53 pathway. This disruption can involve either TP53 or genes coding for proteins upstream of p53. Such genes include p14ARF (30–40% homozygous deletions), MDM2 (10% amplification and overexpression) and sometimes MDM4 (<1% amplification and over expression). These genes code for proteins that control the molar quantities of p53 in the cell and the abnormalities seen in these genes would result in decreased cellular levels of wild-type p53. In almost all glioblastomas, only one of the genes coding for components of the p53 pathway is abnormal.

The RB1 pathway in glioblastomas

The RB1 pathway in glioblastomas is disrupted by genetic and epigenetic events affecting one or several of the genes coding for its component pathway proteins. Homozygous deletions of CDKN2A (coding for p16) and CDKN2B (coding for p15), occur in 30–40% of glioblastomas.

Both genes are on chromosome 9p and have been implicated in the melanoma-astrocytoma syndrome. Other tumors have amplification and overexpression of CDK4 (located at 12q14), and sometimes of CDK6, CCND1 or CCND3. Further abnormalities include homozygous deletion of RB1 (located at chromosome 13q14) or hemizygous deletion with mutations occurring in the retained allele resulting in a nonfunctional RB1 protein.

Hypermethylation of the RB1 promoter, resulting in transcriptional silencing of the gene has also been documented. The loss of expression of CDKN2A and CDKN2B proteins would result in all CDK4/ CDK6 kinases being free to form heterodimers with CCND1 (cyclin D1) or CCND3 (cyclin D3), resulting in the potential of phosphorylation of the RB1 protein. Mutation or lack of expression of RB1 or inappropriate phosphorylation of the RB1 protein all result in loss of control of the transcription factors central to the regulation of the restriction point in G1 of the cell cycle, causing progression into the S phase of the cell cycle. In a comprehensive study of four RB1-pathway genes and three p53- pathway genes in 136 glioblastomas, 76% of tumors were found to have mutations resulting in a disrupted p53 pathway and 67% a disrupted RB1 pathway. In astrocytomas WHO grade II, 67% were found to have mutations resulting in a disrupted p53 pathway while none was found to have mutations causing disruption to the RB1 pathway. In anaplastic astrocytomas, 72% showed mutations disrupting the p53 pathway and 13% showed mutations disrupting the RB1 pathway. The adjacent location of genes coding for components the p53 and RB1 pathways (i.e. CDKN2A, p14ARF and CDKN2B [9p21] or MDM2 and CDK4 [12q14]) may explain the differences in the genetic aberrations found in primary/de novo and secondary glioblastomas. In de novo glioblastomas, coamplification with overexpression of MDM2 and CDK4 genes at 12q14 would require only a single genetic event to disrupt both pathways. Two genetic events would be required for homozygous deletion of both copies of the region on chromosome 9p21 encompassing CDKN2A, CDKN2B and p14ARF, which are the most-common mutation patterns found in de novo glioblastomas. Targeting individual tumor-suppressor genes in each of the pathways (e.g. TP53 and RB1) would require four mutation events, a finding common in secondary glioblastomas. As yet there are no treatments targeting these pathways.

Growth factor receptors and ligands involved in glioblastoma development

Innovative rational therapy design has focused on abnormalities of growth factor receptors and signal transduction pathways in tumors. The earliest genetic abnormality detected in glioblastomas was amplification of the EGFR at 7p11-12, which encodes a tyrosine kinase growth factor receptor. Amplification occurs in ≤35% of mostly de novo glioblastomas and in a few percent of anaplastic astrocytomas, but not in astrocytomas. The amplified EGFR is always overexpressed but overexpression is also found in a further 15% of glioblastomas in the absence of gene amplification. The amplicon is located on double minutes, i.e. extrachromosomal fragments, and as some tumors amplify sequences adjacent to the EGFR gene there may be additional gene targets in the 7p11-12 region. About half of tumors with amplification of EGFR have rearrangements of the amplified gene. The most common rearrangement results in an aberrantly spliced in-frame transcript, which codes for a transmembrane tyrosine kinase that has lost 267 amino acids from the extracellular domain and does not bind its ligand, its cytoplasmic tyrosine kinase domain, however, is constitutively activated

Other rearrangements of the amplified EGFR, which occur less frequently, result in amino acid abnormalities in the region of the cytoplasmic domain, involved in down regulation of the receptor. Rearrangements affecting the sequences coding for the extracellular and cytoplasmic domains of EGFR can occur in the same tumor. Transcripts that code for ligands of EGFR have been reported in tumor tissue, providing a basis for autocrine/juxtacrine or paracrine stimulation of tumor-cell proliferation. Retrospective analysis of individual cases treated with conventional therapy have generally not found rearrangements or amplification of the wild-type EGFR to be significant prognostic indicators for patients with glio blastoma. High-level expression of PDGF ligands and receptors have been observed in all grades of astrocytic gliomas, suggesting the presence of autocrine and/or paracrine signaling. In a small subset (~8%) of glioblastomas, amplification of PDGFRA has been noted. Hence, constant growth stimulation can be produced in glioblastomas by normal and mutated (constitutively activated) growth factor receptors at the cell surface, as well as by ligands that activate wild-type receptors .

There are a number of tyrosine kinase inhibitors in preclinical or clinical development that target PDGFR and EGFR . The clinical value of these agents will generally depend on them having a reasonable therapeutic index and manageable side effects. The usefulness of such agents will be determined by the genetic/ epigenetic abnormalities present in an individual’s tumor cells.