A 6-gene signature identifies four molecular subgroups of neuroblastoma

Neuroblastoma (NB) is a childhood tumour of the sympathetic nervous system, and is the most common cancer diagnosed during infancy. The prognosis of NB patients depend upon clinical factors as stage [1], age at diagnosis [2], tumour histopathology [3], and several genetic factors as MYCN amplification (MNA) status [4] and DNA index [5]. Generally, children diagnosed before the age of 18 months with a localized tumour have a favourable outcome, whereas older children with metastasised tumours show poor prognosis and low survival rate. However, MNA is proven to be strongly associated with rapid progression and poor prognosis in all patients despite age and stage of disease [4, 6] and is therefore central to the risk stratification system in all clinical trial groups [7]. It is still important to emphasize that the majority of metastatic tumours do not show amplification of the MYCN oncogene, and other chromosomal aberrations are being evaluated for the International Neuroblastoma Risk Group (INRG) classification system [8].

Studies show that sporadic NB tumours can be assigned to three major subtypes based on their genomic profile, and these molecular signatures also categorize risk groups of NB patients [9]. Type 1 comprise low-risk tumours with triploid DNA content, numeric alterations, and high expression of the nerve growth factor TrkA [10, 11], Type 2A involve intermediate-risk tumours with high occurrence of 11q-deletion (del11q) and 17q gain (gain17q) but no MNA, and Type 2B comprise high-risk MYCN amplified tumours with high occurrence of gain17q and 1p-deletion (del1p) [12, 13]. The outcome prediction of intermediate-risk tumours still remains uncertain and some tumours cannot be definitively assigned to any of the three major groups, indicating this division to be only broadly outlined.

Genome-wide transcriptome microarray analysis enables the possibility to investigate the expression of all genes in a tumour simultaneously. De Preter and colleagues established a 132-gene classifier that discriminates the three major genomic NB subtypes reflecting inherent differences in gene expression between these subtypes [14]. Several studies have established predictive gene signatures of NB tumours [14‐20], and others have focused on the differential expression of genes between tumour subsets [21‐29]. The three genes MYCN[30], ALK[31], and PHOX2B[32] have been directly linked to NB pathogenesis; MYCN is amplified in a subgroup of aggressive metastasizing tumours, activating mutations of ALK or amplification is seen in approximately 7% of sporadic cases [33, 31, 34‐37], and PHOX2B is mutated in a subset of familial cases and in a small percentage of sporadic cases [32, 38].

In the present study, we explored subtype discoveries by unsupervised expression profiling using Principal Components Analysis (PCA). The analyses identified four distinct PCA clusters in two independent data sets, which were verified in a third larger data set by PCA and unsupervised hierarchical clustering. This study presents a new alternative way of subtype discrimination which will hopefully facilitate the search for subtype-specific therapeutic targets and the development of personalized medicine for children with neuroblastoma.

A large number of publications prove that cancer can be classified through gene expression profiling. Principal components analysis (PCA) is a useful tool to reduce the dimensions of data to be able to identify and visualize hidden patterns. PCA has been widely used in genome expression studies to discriminate tumour subtypes. For example, Yeoh and colleagues identified prognostically important subtypes and a novel subgroup of pediatric acute lymphoblastic leukaemia (ALL) by PCA of gene expression data [43]. In order to develop more effective and less toxic cancer treatment it is necessary to identify and correctly classify the molecular subtypes, as well as to unravel the underlying oncogenic driving pathways for each type.

In the current study, subtypes of neuroblastoma were explored by expression profiles from four microarray studies [22, 25, 28, 39]. In the first step, PCA was performed on two independent data sets and four distinct clusters were identified in both sets. Prognostic factors such as high INSS stage, MNA, and del1p, differed significantly between clusters. Three of the four clusters (i.e. p1-p3) corresponded well to the previously established genomic subtypes 1, 2A, and 2B. Remarkably, a fourth novel cluster (p4) with a considerable different expression profile appeared independently in both data sets, and has not been described elsewhere. This new cluster was found to encompass mainly high stage tumours with poor outcome and high frequency of del11q and del1p, but low frequency of MNA. In data set 1 (De Preter) all 3 cases assigned to cluster p4 were found to be of clinical INSS stage 4, and in data set 2 (McArdle/Wilzén) 3 out of 5 patients died from disease. However, samples assigned to the p4 cluster showed a significantly lower expression of MYCN and ALK compared to the MNA-specific cluster p3 (table 2), which indicate an alternative progression pathway within tumours assigned to the fourth cluster.

In the second step, the existence of four groups could be verified by an unsupervised hierarchical clustering and PCA of a third data set (Wang data set [28]) using a discriminative gene set of 74 genes from the first step. All tumour samples assigned to the h3 cluster were MNA tumours of clinical stage 4, and tumours assigned to the h2 and h4 clusters comprised high stage tumours with no MNA but with high content of del11q tumours. The lowest survival probability and highest relapse rates were seen in cluster h3, followed by clusters h2 and h4 in descending order (Figure 3C). A comparison between the survival probabilities of the hierarchical and PCA sub-divisions indicated that the h4 cluster constituted less unfavourable tumours compared to those assigned to the p4 cluster. The heterogeneous results seen between data sets may be explained by the small number of cases in data sets 1 and 2 as well as different tumour cohorts. As for example, the Wang data set does not include any clinical stage 2 tumours which might indicate that the cases in this data set were not randomly selected. This would of course affect the subgroup characteristics and might explain the divergent outcome seen in patient cohorts from the different datasets. Also, since the fourth subgroup (p4, h4) is a very small group it is difficult to say if the frequencies of different genetic alterations are stably distributed between the subgroups. Moreover, six patients of the h4 cluster in the Wang data set were lost for follow up, which makes it difficult to draw any major conclusions.

The hierarchical clustering also identified five gene clusters. Nervous system developmental genes, including NTRK1 and DBH were found to be highly expressed in the favourable tumour cluster h1. Cell-cycle related genes including BIRC5, CCNB1 and MCM-genes were found to be highly expressed in clusters h2 and h3. Not surprisingly, the MYC gene cluster (g3) was specifically found to be over-expressed in the MNA-specific group h3. Westermann and colleagues recently defined a core set of MYCN/c-MYC downstream target genes which were associated with malignant progression in NB [44]. In line with their results, we found MYCN and c-MY C to be significantly negatively correlated in all three data sets. We also wondered whether the c-MYC over-expression could be specifically connected to any of the four groups. However a heat map showed the c-MYC over-expression to be evenly distributed among all groups except for the MNA-specific group p3/h3 (Additional file 8). The transcription factor LMO3 (LIM domain only 3) found in gene cluster g5 has been significantly associated with a poor prognosis in NB [45]. This is concordance with the present study in which the highest expression of LMO3 was found in the most unfavourable tumour group h3. Interestingly, LMO3 has been shown to interact and act as a co-repressor of p53 [46].

The fourth novel tumour group (h4) was found to be characterized by high expression of several brain-specific and nervous system developmental genes. The Erbb receptors (e.g. Erbb3) and the SoxE family (Sox8, Sox9 and Sox10) are essential for development of the sympathetic nervous system and the development of neural crest cells. Interestingly, Leon and colleagues reported that Sox10 and Phox2b act together with the NK2 homeobox Nkx2-1 to modify RET signalling and suggest this interaction to contribute to HSCR (Hirschprungs disease) susceptibility [47]. The growth arrest-specific 7, Gas7, is regulated by Sox9 and the ERK1/2 MAP kinase and is involved in chondrogenesis, and has been reported to form a MLL/GAS7 fusion protein in a pediatric case of B-cell acute lymphoblastic leukaemia [48]. In addition, the g4 cluster comprised the suggested 3p tumour suppressor gene SEMA3B[49]. The subgroup discrimination properties of SEMA3B found in the present study, as well as the significantly lower expression observed in tumour groups p2/h2 and p3/h3, could support its tumour suppressor function.

The validation test of the four PCA clusters using unsupervised and unfiltered global transcripts clearly shows that the four subgroups exist in all three data sets (Additional file 7). Moreover, the PCA of the 6-gene signature in all three data sets convincingly show that this expression profile is sufficient for NB subtype discrimination (Figure 4A). Overall, our results indicate that the most unfavourable group displaying MNA (corresponding to Type 2B) and the most favourable group with high NTRK1 expression (corresponding to Type 1) can be easily discriminated by their expression profiles. Moreover, our data indicates that the del11q tumours (corresponding to Type 2A) are divided into at least two expression subgroups (see table 4). Interestingly, the existence of two del11q expression subgroups has recently been reported by two other research groups [23, 50]. Fischer and colleagues studied the gene expression patterns of del11q tumours divided into two clinical groups of favourable and unfavourable biology using their previously described prognostic 144-gene expression classifier. They found that the clinical groups clustered using unsupervised PCA and hierarchical clustering [23]. Also, Buckley and colleagues identified a 15-miRNA signature that discriminates two distinct biological subtypes of del11q tumours [50]. Our current study differs from these two studies in one important aspect- it does not rely on any prior subtype division (e.g. genomic subtypes, clinical groups etc.), which means that it is entirely unbiased. As stated by Fischer and colleagues the 11q-deletion is most likely a secondary event, and it is possible that the decision between favourable and unfavourable neuroblastoma is made by a yet undefined transformation event, for example ALK. This hypothesis is completely consistent with our finding, where ALK expression is significantly elevated in subgroup p2/h2 (see Figure 1 and 4B). In order to clarify and relate our subgroup discoveries to these recent findings, we performed a PCA of the Wang data showing tumours marked by their del11q status and coloured by their h-group belongings (Figure 5). Deletion of 11q was found to be distributed through all tumour groups except for the MNA-specific h3-group in which only two cases with 11q-deletion could be found (Figure 5A). In line with Fischer et al. [23] we filtered out all cases with MNA and/or del1p, and ended up with a PCA on 74 cases (Figure 5B). This left us with three expression groups of del11q tumours, one favourable group (h1), and two unfavourable groups (h2 and h4, see Figure 3C). In the last step we removed the h4 expression subgroup, leaving us with two groups of del11q tumours, one favourable (h1) and one unfavourable (h2). These results suggest that our subgroup discoveries are not contradictory to the findings by Fischer et al. [23] and Buckley et al. [50], but rather indicate that there are three del11q expression subgroups instead of two; one favourable with high NTRK1 expression (h1), one unfavourable with high ALK, BIRC5 and CCND1 expression (h2), and one smaller group (h4) characterized by high expression of nervous system developmental genes (e.g. ERBB3, SOX10).

The discriminative power of the six NB genes strengthen the fact that these genes are indeed important in neuroblastoma development. ALK was recently recognized as the NB predisposition gene and has thereafter also been found to be affected in sporadic tumours, either though mutations of the tyrosine kinase domain or by genomic amplification [31, 34‐37]. Interestingly, Passoni and colleagues investigated the ALK expression and protein phosphorylation status and found that over-expression of either mutated or wild-type ALK defines poor prognosis patients [51]. In this study, we found elevated expression of ALK in the p2/h2 group, and the highest expression level was found in the MNA-specific group p3/h3. Moreover, various cell-cycle related genes, including BIRC5 and CCND1, were found to be highly expressed in sample groups p2/h2 and p3/h3, but low in group p4/h4. The CCND1 gene region on 11q has been shown to be amplified in a subset of primary neuroblastic tumours [13, 40], and several cases have been found to show an extensive over-expression of cyclin D1 which correlates with histological subgroups [52]. Moreover, CCND1 is used as a marker for minimal residual disease of NB [53]. The anti-apoptotic gene BIRC5 (also known as survivin) is located in the often gained region on 17q (gain17q), and has previously been found to be associated with poor prognostic factors and low survival probability in NB [41, 54]. Recently, Eckerle and colleagues found that BIRC5 is a direct transcriptional target of activating E2Fs, and that BIRC5 is indirectly induced by N-myc [55]. These findings are supported by the significantly higher expression of both BIRC5 and MYCN found in tumour group p3/h3 in the current study. Moreover, an elevated expression of BIRC5 was found in sample group p2/h2, and a significant down-regulation of both MYCN and BIRC5 was found in group p4/h4 (p < 0.05, Welch t-test). The MNA-specific p3/h3 group was also characterized by a very low expression of NTRK1, whereas the favorable tumour group p1/h1 showed the highest expression of NTRK1. TrkA (or NTRK1) is a well-known marker of favorable NB tumours and its expression has been linked to several cancer forms [56].

In conclusion, by expression profiling of 148 NB tumours from four different Affymetrix-based microarray studies, our data suggest the existence of at least four molecular subgroups of neuroblastoma tumours. Three of the expression-based tumour groups corresponded well to the previously postulated genomic subtypes and a fourth novel group was identified which has not been described elsewhere. The novel tumour group comprised high-stage 11q-deleted tumours with low expression of ALK and MYCN, but high expression of various CNS and nervous system developmental genes. Our findings suggest an alternative classification system based on expression profiling of a 6-gene signature. Further studies of the novel subgroup's specific characteristics are warranted, and will hopefully lead to discoveries on new specific therapeutic targets for children with neuroblastoma.