A major emphasis has historically been placed on stratifying LGGs diagnosis or therapy on the basis of pathological and molecular genetic criteria. However, the increasing application of molecular approaches is transforming the way to categorize these tumours, since it seems that histologically comparable lesions may exhibit diverse patterns of gene expression and genomic alterations [5–7, 9, 19, 20]. This investigation has focused on the identification of a specific gene signature based on high-throughput techniques that provide a genome-wide snapshot of LGGs with respect to both distinct lesion site in the brain and histotype.
Although an abundance of data is available on gene expression profiles of LGGs, they are often conflicting. Indeed, statistical methods for evaluation and interpretation of microarray data are still evolving. We successfully adopted an analysis workflow (Figure 1) able to overcome a major criticality in high-throughput studies, that is to find robust, reproducible and biologically sound results [16, 46]. Details of the workflow description are reported in Additional file 1.
Brain region-specific gene signature among LGGs
Question (1) was used to assess the procedure and represent the first example of biologically validated l1l2 framework with an independent methodology. Indeed, this query is the one with more samples available as well as the one already investigated in previous works [9, 19]. The provided outcome from l1l2 was a list of 331 probe sets (Additional file 3), corresponding to 206 loci, above 70% of frequency. l1l2 produces a multi-gene model and only a multidimensional representation can correctly visualize its strong discriminative power (Figure 2b). The figure shows that the infratentorial tumours group is spatially separated from the supratentorial counterpart.
Our analysis identified various interesting genes which encode cell adhesion molecules, ECM, extracellular matrix, lipid metabolism, CNS development, cell differentiation, transcription regulation, and invasion-related proteins. Unlike Potter et al. reported , our results are in line with previous findings that clearly defined the existence of PA subgroups. Indeed, 14 out of 206 differentially expressed genes (reported in bold in Table 2) were reported by previous studies [9, 19, 20]. Wong and colleagues identified two subgroups of PA reporting a list of significant differentially expressed genes involved in cell adhesion, regulation of cell growth, cell motility, and angiogenesis . Sharma and colleagues reported differential expression of genes playing a role in forebrain development as LHX2 and nuclear receptor subfamily 2, group E, member 1 (NR2E1), and hindbrain development as paired box gene 3 (PAX3) and iroquois homeobox protein 2 (IRX2), able to stratify infratentorial from supratentorial PAs . The comparison with the Sharma’s data, the only comparable, inasmuch as homologous for case selection, sample processing and Affymetrix platform, allowed us, even using our own statistical approach, to identify five genes (LHX2, NR2E1, PAX3, IRX2, and zinc finger homeobox 4, (ZFHX4) common to both analyses.
To investigate paediatric LGG development related to site of lesion (infratentorial vs. supratentorial) , we next proceeded by selecting those candidate genes that were most represented among all the high-ranked pathways for the validation process by using our in-house designed qPCR systems on 52 samples (34 samples belonged to dataset 1, while 18 samples were from dataset 2). Finally, the list of candidates comprised 19 probe-sets corresponding to 15 loci in total (Table 3). We validated the generalization ability of the 15 gene signature by applying a multivariate statistical model on the qPCR data of dataset 1 (34 samples). Such multivariate model, obtained with a RLS analysis, was used to assign the samples to a group and the classification results were compared to the l1l2 microarray-based model (Figure 3b). The two independent methods have good performances, being able to associate 33 out of 34 samples to the right class. Moreover, 5 out of 15 genes emerged from the univariate Mann–Whitney test on the qPCR data, confirming and enhancing the LGG differences in infratentorial as compared with supratentorial regions, see Table 4 and Figure 3a. As shown in Figure 3a, a group of 4 genes (ARX, GPR17, LHX2 and CXCL14) well stratified LGGs between infratentorial and supratentorial tumours. ARX is a homeobox-containing gene expressed during development. This gene is involved in CNS development and in cell proliferation in forebrain [GO:0021846]. Mutations in this gene cause X-linked mental retardation and epilepsy. To the best of our knowledge, ARX was never associated with LGGs. GPR17 is a G-protein involved in signal transduction [GO:0007165]. LHX2 is downregulated in infratentorial tumours as already reported . CXCL14 is a chemokine associated with tumour development [GO:0006995], and PTDG2S whose functions are associated to lipid metabolism [GO:0006633], might be involved in controlling the proliferation rate of LGGs.
Additionally, the predominant terms related to pathways consisted of MAPK signaling pathway, containing at least 12 genes, followed by chemokine signaling pathway with 8 genes enriched. These findings reinforce the observations of several consecutive articles about aberrant activation of the mitogen-activated protein kinase (MAPK) pathway in LGGs . The identification of a brain region-specific gene signature suggests that LGGs at different sites may be distinct in terms of biological properties and tumorigenesis despite the same histology. KIAA1549:BRAF fusions were analyzed in the LGG cohort and we found the gene fusion slightly more frequent in infratentorial (38.5%) versus supratentorial (25%) tumours, while we didn’t note any difference for BRAF V600E mutation. Moreover, we did not identify significantly improved progression-free survival in tumours with gene-fusions or BRAF V600E mutation.
Identification of a subgroup of 19 genes specifically related with PA histotype
Next, to molecularly characterize PA able to distinguish infratentorial versus supratentorial, l1l2 analysis were conducted only on 27 PAs out of 37 LGGs, whose 17 arising in infratentorial and 10 in supratentorial regions, see Table 1. A gene signature of 82 genes (see Additional file 5) well distinguishes PA arising supratentorial versus infratentorial regions (Figures 4a,b). Significant biological processes represented include GO terms of nervous system development, cell morphogenesis, cell differentiation and cell adhesion, MAPKKK cascade, chemotaxis, and regulation of neurogenesis. We found that, together with ARX, forkhead box G1 (FOXG1) was strongly represented in PA. FOXG1 is an oncogenic transformer which could play an important role in controlling both cell proliferation and forebrain cell differentiation in PA [21, 49–51].
Through the comparison of gene lists between LGG and PA, we found 19 genes specifically related with PA histotype as a group (genes in bold in Additional file 5). The functional analysis showed that several genes create a network within the (TGF-β)-signaling pathway. This pathway possess a dual role in oncogenesis. In some tumour types, i.e., in high-grade gliomas, TGF-beta becomes an oncogenic factor, while it is also considered a tumour suppressor factor in normal epithelial cells and astrocytes. Moreover, noncanonical TGF-beta signaling pathways interact, through RSmads molecules, with MAPK signaling pathway . Thanks to this interaction, it is likely to assume an active involvement of TGF-beta signaling pathway in the PA development.
Our analysis shows a strong difference between supratentorial and infratentorial PAs. In fact, cerebellar PAs, corresponding to the classical description of the biphasic tumour with compact areas with piloid cells and Rosenthal fibers and microcistic areas with granular eosinophilic bodies , seem to be defined by a specific gene signature versus supratentorial PAs. Therefore, this molecular fingerprint is able to better sub-classify such a morphologically heterogeneous tumours.
Neurogenesis, cell motility and cell growth genes dichotomize mixed glial-neuronal tumours versus PAs
Finally, the analysis on 22 supratentorial LGGs identified a list of 70 genes (see Additional file 6) able to dichotomize mixed glial-neuronal tumours versus PAs (Figure 4c,d). The signature consists of genes encoding adhesion, ECM-receptor interaction, matrix extracellular organization, neurogenesis, immune response, and metabolic proteins. Several genes are components of collagen gene family whose functions are associated with extracellular matrix (ECM) reorganization. Intriguingly, changes in expression of genes controlling neurogenesis (DLX1, DLX2), cell growth such as insulin-like growth factor 2 (IGF2), insulin-like growth factor binding protein 6 (IGFBP6) and latent transforming growth factor beta binding protein (LTBP2), cell motility such as l1 cell adhesion molecule (L1CAM), COL3A1 and integrin, alpha 8 (ITGA8), and interactions with the surrounding environment such as lumican (LUM), COL1A1, COL6A3 and periostin, osteoblast specific factor (POSTN) appear to be linked to the presence of neuronal cell component.
Because of their rare occurrence, little is yet known about the molecular pathology of mixed glial-neuronal neoplasms and the cytogenetic and molecular genetic studies reported are very few [53–55]. Our findings show the complexity and vitality of these tumours, shedding some light on features such their richness in connective tissue and, they point to some interesting candidate genes (i.e., DLX1, DLX2) worth further investigations that could help the pathologists in the differential diagnosis.
From a biological point of view, it is remarkable that the mixed glial-neuronal tumours are strikingly separated from PAs, allowing us to look differently at mixed glial-neuronal tumours in which, generally, the glial component catches the attention of the pathologists and contributes to grading. Our findings, indeed, shed some light on the biological complexity of the mixed glial-neuronal tumours, still poorly known. It remains to be established if mixed glial-neuronal tumours differ from PAs because of their ganglion-like component or because of their glial one or both. What seems indubitable is that the ganglion cell component is not a bystander. Future functional studies are needed to evaluate these targets in paediatric mixed glial-neuronal tumours versus PAs but evidence supports a role for these gene candidates in tumorigenesis.