Little is known about the genes and genetic mechanisms underlying ependymoma tumorigenesis, patient relapse and survival. To address these issues we focused our study on chromosome 1q, one of the most commonly gained regions in ependymoma. Using CGH and SAGE profiling we identified CHI3L1 and members of the S100 family as candidate genes in ependymoma. Immunohistochemical analysis on a large cohort of paediatric ependymoma revealed that CHI3L1 protein expression is associated with necrosis and that members of the S100 family are differentially expressed in clinically relevant subgroups. S100A6 is significantly associated with paediatric ependymoma arising in the supratentorial compartment and S100A4 strongly correlates with patients aged less than 3 years at diagnosis.
In this study, different approaches were taken to mine the SAGE data to identify the ependymoma-associated genes on chromosome 1q. Analysis of the effect of de novo 1q gain on gene expression in recurrent ependymoma revealed that CHI3L1 was the most highly expressed gene. Strikingly, four members of the same gene family (S100A10, S100A6, S100A4 and S100A2) were also identified as having distinct gene expression profiles in ependymoma. Comparison of 10 ependymoma SAGE libraries with five normal brain libraries identified S100A10 as the most highly expressed gene on 1q in ependymoma; it was also the second most highly expressed gene in the relapse sample with gain of 1q. S100A6 was also identified as one of the most highly expressed genes in ependymoma when compared with ‘normal' brain. S100A4, was implicated in ependymoma as, again, being one of the most highly expressed genes in ependymoma with very low expression in ‘normal' brain. Analysis of all 1q S100 genes represented in SAGE Genie identified S100A2 as the only S100 gene expressed in ependymoma but not expressed at any level in ‘normal' brain.
S100A10, S100A6, S100A4 and S100A2 are all members of the S100 family of calcium-binding proteins and are located in a cluster on 1q21.3; a region that has been shown to have both high level gains (1q21–q31; Ward et al, 2001
) and an association with tumour recurrence in ependymoma (1q21.1–q23.1; Mendrzyk et al, 2006
). Members of the S100 family show divergent expression patterns in a range of tissues and several have been linked with cancer, including medulloblastoma (Hernan et al, 2003
; Lindsey et al, 2007
) and astrocytoma (Camby et al, 1999
). S100A6 has been shown to clearly distinguish between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumours (Camby et al, 1999
). In ependymoma, we have clearly demonstrated that S100A6 is differentially expressed in tumours arising in different locations of the brain, and is significantly associated with supratentorial tumours (P
Clinically supratentorial ependymoma are associated with better survival rates when compared with posterior fossa tumours (Schiffer and Giordana, 1998
). This survival difference could be because of a number of confounding factors, for example, the resectability of supratentorial when compared with infratentorial tumours (Palma et al, 2000
). There is now evidence that ependymoma arising within different regions of the central nervous system exhibit specific and distinct genetic signatures (Taylor et al, 2005
). For example, genes upregulated specifically in supratentorial tumours include members of the EPHB-EPHRIN and NOTCH cell-signalling systems. Our observation of the differential expression of S100A6 in different regions of the brain adds to this supratentorial gene signature.
In ependymoma we have shown that S100A4 is significantly associated with patients under the age of 3 years at diagnosis in intracranial paediatric ependymoma (P
=0.038). Differences in the genomic profiles between tumours from patients under the age of 3 years and older children have previously been identified. For example, balanced genomes (with no detectable genomic losses and gains) are significantly associated with children younger than 3 years of age at surgery (Dyer et al, 2002
). This finding suggests that ependymoma occurring in patients less than 3 years are biologically distinct from those occurring in older children. It has been hypothesised that tumours occurring in infants may be driven by powerful genetic events that lead to presentation at a young age without the requirement for additional genetic changes (Dyer et al, 2002
). Although S100A4 is one of the best characterised of the S100 genes in terms of its role in cancer (Emberley et al, 2004
; Salama et al, 2007
), no other study has previously reported a link with patient age or explored its role in ependymoma. The significance of S100A4 in ependymomas arising in children under 3 years of age is not clear but its differential expression demonstrates that there is a distinction between genetic events occurring in children of different ages.
S100A4 and S100A6 are clearly differentially expressed in paediatric ependymoma and can be used to distinguish clinically and biologically relevant subgroups. Expanding our study to the gene expression levels across six other brain tumour types showed that for a number of S100 genes the expression levels were notably elevated in a particular tumour type. For example, S100A16 is elevated in grade I astrocytoma and S100A11 in glioblastoma. In contrast, several S100 genes had similar expression levels across multiple tumour types, including S100A14, S100A13 and S100A3. Similar expression profiles were also seen across all tumour types for S100A8 and S100A9 but this could be attributed to their function in which they form a heterodimer complex (Vogl et al, 2006
). These findings highlight the importance of further investigation of specific members of the S100 family to understand their function in ependymoma and other brain tumours.
CHI3L1, located on 1q32.1, was the most overexpressed gene in the relapse pair with 1q gain and was also one of the most upregulated genes in ependymoma when compared with ‘normal' brain. Immunohistochemistry of CHI3L1 revealed a correlation between the immunostaining and necrosis. Notably, the TMAs were constructed by selecting three representative areas from each tumour, in this process we tended to avoid areas of necrosis, however, in five cases necrosis was present. In these five cases staining showed that CHI3L1 was more highly expressed in the cytoplasm of tumour cells adjacent to the necrotic regions (). CHI3L1 encodes for YKL-40, which is a secreted protein that has been reported to be overexpressed in a number of different cancers, including glioma, and has been proposed as a new therapeutic target (Pelloski et al, 2005
; Johansen et al, 2007
). The role of CHI3L1 in cancer is unknown, but it has been suggested that it has a function in a number of pro-survival processes (Johansen et al, 2007
). In glioblastoma (GBM), where necrosis is a characteristic, both CHI3L1 expression and necrosis are associated with poor prognosis (Burger and Green, 1987
; Raza et al, 2002
; Pelloski et al, 2005
; Homma et al, 2006
; Kleihues et al, 2007
). In ependymoma, we did not find a correlation with prognosis, thus, raising the possibility that CHI3L1 is a marker of necrosis rather than of adverse biology per se
. These observations in GBMs and our findings in ependymoma suggest a link between CHI3L1 and necrosis in brain tumours. As the cores represented on the TMA are selected to avoid regions of necrosis, our findings maybe an under-representation and further investigation of CHI3L1 expression on whole tissue sections is necessary.
Previously we identified gain of 1q as one of the most common gains in primary and recurrent ependymoma and demonstrated a tendency that patients with gain of 1q have a poorer outcome (Dyer et al, 2002
). The aim of this study was to investigate 1q in ependymoma to gain insight into the role of genes located in this region. In this study, we have identified members of the S100 family located within the commonly gained amplicon 1q21.3 and provide evidence of their differential expression in clinical subgroups of paediatric ependymoma: S100A4 is associated with patients of a very young age at diagnosis and S100A6 with supratentorial tumour location. We also demonstrated a link between CHI3L1 protein expression and necrosis. However, we are yet to elucidate the underlying mechanism by which 1q gain confers adverse biological behaviour in paediatric ependymoma. We are now extending this study to a larger tumour cohort to further unravel the underlying biology of 1q in this complex tumour.