In this study, molecular profiling of AT/RTs was performed to determine whether distinct subgroups with therapeutic or prognostic relevance exist in this diagnostic category. With use of gene expression microarray data as input to an unbiased agglomerative hierarchical clustering algorithm, 4 clusters of AT/RTs were identified that showed differing gene expression profiles. This molecular clustering also corresponds with several observable histopathological features. Tumors composed predominantly of small blue cells with few or no observed rhabdoid cells grouped together in cluster 2 and showed significant upregulation of genes associated with neurogenesis. Cluster 4 tumors showed downregulation of genes associated with neurogenesis, brain development, and axonogenesis and higher proliferation rates (as measured by MIB1). Cluster 4 also showed specific upregulation of genes associated with choroid plexus epithelium, including transthyretin (TTR
); potassium inwardly rectifying channel, subfamily J, member 13 (KCNJ13
); 5-hydroxytryptamine (serotonin) receptor 2C (HTR2C
); and coagulation factor V (F5
The delineation between AT/RTs and choroid plexus tumors, particularly choroid plexus carcinomas, has long been a subject of debate.30–33
Our results suggest that, at least for some tumors, there is overlap at the gene expression level and at the histopathological level. This is also supported by a recent report showing membraneous expression of KCNJ13, previously thought to be specific to tissues with choroid plexus lineage and found by us to be upregulated in cluster 4, in 2 of 8 AT/RTs.34
To our knowledge, our study is the first to find an association with this subset of AT/RTs and shorter survival.
In addition to different gene expression profiles, the clusters were associated with different overall survival times. Patients in clusters 3 and 4 experience significantly shorter overall survival than did those in clusters 1 and 2. Analysis of the differentially expressed genes indicates that upregulation of BMP pathway-related genes is strongly associated with shorter survival, with BMP4 showing the most significant association. Although this study did not directly measure activation of BMP signaling, the upregulation of multiple direct gene targets is suggestive that BMP signaling is active in cluster 3 and 4 tumors.
BMP4 belongs to the TGF-β super-family of secreted growth factors. This super-family consists of 2 branches, the BMP/GDFs and the TGF-β/activin/nodal genes. BMP signaling elicits a wide diversity of transcriptional programs and physiological responses that are highly variable both across different cell types and across signaling intensities. It plays a crucial role in normal development and maintenance of many tissues. In the brain, appropriate BMP signaling levels are critical to neural tube induction,35
formation of neural crest cell populations,36,37
development of the dorsal-most forebrain including the choroid plexus,38,39
dorsal-ventral patterning of the spinal cord,40,41
cell-fate decisions in neural precursors,42,43
and generation and differentiation of cerebellar granule cells,44,45
among other processes.
The pleiotropic effects of BMP signaling are accomplished through 3 effectors: SMAD1, SMAD5, and SMAD8. SMADs are thought to exert their effects primarily through chromatin remodeling.46,47
SWI/SNF has been shown to be essential for transcription of several TGF-β and BMP downstream signaling targets47–49
This raises the question of whether loss of the SWI/SNF component SMARCB1, as occurs in rhabdoid tumors, could affect BMP signaling.
In cancer, addition of BMP4 has been shown to down-regulate proliferation and promote cell differentiation in both a glioma cell line50
and in Sonic hedgehog-driven models of medulloblastoma.51
On the other hand, BMP4 was overexpressed in malignant melanoma, where it promoted cell invasion and migration,52
thus reinforcing that effects of BMP signaling are highly dependent on cell context and other factors. For AT/RTs, it remains to be determined whether the upregulated BMP pathway gene expression seen in this study contributes to malignancy or is merely a marker of tumors with a different biological profile. Both differentiated choroid plexus cells and precursor cell populations, from which choroid plexus arises, are known to produce BMPs.39,53,54
Consistent with this, one of our molecularly defined clusters (cluster 4) showed both upregulated BMP expression and upregulation of genes normally associated with choroid plexus lineage.
Molecular profiling of CNS tumors has led to insights into signaling pathways that drive different subsets of these tumors and to identification of possible precursor cells for these tumors.55–57
In addition, multiple studies have identified gene signatures prognostic for survival in brain tumors.11,14–16,18
Most of these studies have analyzed 50–100 or more samples, and published data from other institutions have often been available for corroboration of results. In contrast, our research examines a comparatively rare pediatric brain tumor and is based on a sample size of only 18 patients. There are currently no other published AT/RT gene expression data available for corroboration. Therefore, the findings presented here must be considered to be preliminary and will need to be evaluated further using additional patient cohorts. It would also be of interest to evaluate these findings in older patients, who are known to show better response to treatment,4,7,8
because our cohort included only 2 patients >3 years of age. In addition, this study is specific to AT/RTs (RTs of the brain) and did not assess RT tumors from other locations, particularly the kidney and soft tissues, which may show other molecular characteristics.
In conclusion, this study identifies molecular factors that define subsets of AT/RTs with distinct gene expression signatures. Our data indicate that, although loss of SMARCB1 is common to all of the AT/RTs studied, other differences in underlying biology distinguish subsets of these tumors, and these differences may affect overall survival under current therapeutic regimens. As with other pediatric brain tumors, molecular profiling of RTs is a promising avenue of research that may aid in the search for improved treatment strategies for this devastating disease.