Introduction
Brain tumours are the largest group of solid tumours and the leading cause of tumour-related death in children [
4]. In particular, tumours arising in midline structures of the brain including the brainstem and thalamus present significant challenges for physicians in regards to their therapeutic approach. At present, tumours arising in the brainstem, known as diffuse intrinsic pontine glioma (DIPG), have been extensively investigated and stratified into disease subtypes [
6,
20]. Whereas DIPG is heavily researched, thalamic tumours, accounting for approximately 5 % of all paediatric brain neoplasms, are largely unexplored [
8‐
10,
14,
27,
28]. Currently, the outcome of patients with thalamic tumours is predicted based on the histological grade and the potential for surgical resection of the tumour. High grade tumours classified as World Health Organization (WHO) grade III or IV are associated with worse clinical outcome as compared to grade I or II low grade tumours [
23,
24]. Due to their precarious midline location and the vital functions of the thalamus and nearby structures, these tumours, particularly the diffuse gliomas, often cannot be completely resected, which is associated with a worse clinical outcome [
2,
10,
22,
27]. Further, limited resections also potentially lead to sampling related errors in histological grading due to small biopsy specimens [
12,
26]. As such, there is a need to identify genetic biomarkers to supplement histological grading and extent of surgical resection to aid in the diagnosis and management of thalamic tumour cases.
Recent studies have identified several biomarkers that aid in disease diagnosis and are important in predicting patient outcome in childhood gliomas. Recurrent mutations in histone H3 in which lysine 27 is substituted for methionine (H3K27M) were first described in patients with paediatric high grade glioma, primarily DIPG [
20,
31,
33]. When correlated to clinical outcome, the presence of H3K27M in DIPG was associated with worse overall survival as compared to those with wild type H3 (H3WT) status, regardless of the histology of the tumour [
5,
20,
21]. Subsequent studies have identified H3K27M mutations in other high grade midline tumours, including those in the thalamus, but have not looked directly at its impact on patient survival [
5,
31,
33]. In addition to H3K27M, genetic aberrations affecting the RAS-MAPK pathway including KIAA1549-BRAF and other BRAF, RAF and FGFR fusion events, as well as BRAF and FGFR1 point mutations have been described primarily in hemispheric low grade gliomas in adults and children [
7,
18,
19,
30,
34]. KIAA1549-BRAF fusions have previously been shown to be associated with better patient survival [
3,
15] while BRAFV600E has been linked to increased likelihood of tumour progression and transformation in low grade glioma [
17,
25]. FGFR1 aberrations including N546K and fusion events with TACC were reported in low grade paediatric astrocytomas [
18,
34] and the former shown to be a negative prognostic marker in a small cohort of grade I pilocytic astrocytomas [
3]. However, thalamic gliomas are often under-represented in glioma cohorts because their midline location often means only a biopsy is performed and there is little tissue available to study. Thus a comprehensive study of genetic markers and their role relative to histologic and clinical risk factors has not been performed for thalamic glioma. To address this limitation, we assembled a cohort of concisely defined paediatric thalamic glioma. We investigated the diagnostic and prognostic roles of defined genetic, clinical and histologic markers.
Discussion
Thalamic tumours present a significant challenge for clinicians in terms of accurate diagnosis and an appropriate therapeutic approach. This study investigated the prognostic potential of molecular alterations and clinical factors in a concisely defined thalamic glioma cohort.
As expected, our results show the presence of H3K27M mutations in 50 % of high grade paediatric thalamic tumours [
5,
20,
31,
33]. Interestingly, 12 % of patients diagnosed with low grade thalamic tumours also tested positive for H3K27M despite previous reports suggesting it to be exclusive to high grade cases when not located in the brainstem [
31,
33]. Of note, two of five low grade cases positive for H3K27M later transformed to high grade malignancies at the time of second surgery, a rare occurrence in paediatric low grade glioma and consistent with the diagnosis of secondary HGG [
25]. While it is possible that these H3K27M positive thalamic gliomas were under-graded histologically based on sampling bias, the significant survival difference observed between low grade and high grade H3K27M tumours supports the idea that these tumours were indeed distinct from their high grade counterparts. Further, there is a lack of any distinguishing MRI characteristics to suggest under-grading in these cases. Importantly, under the new World Health Organization classifications published recently, these tumours would be classified as diffuse midline glioma, H3-K27M mutant, further supporting their unique identity as compared to non-H3K27M low grade tumours [
24].
Patients harbouring H3K27M showed significantly worse overall survival when compared to H3WT cases. Once separated based on histological grade, both low and high grade tumours maintained a significantly worse survival in the presence of H3K27M. Of note, high grade thalamic tumours, regardless of H3K27M status, yielded dismal survival with those harbouring the mutation succumbing to their disease in a slightly faster timeframe. This result coincides with our previous work in DIPG [
5,
20], where we found H3K27M to be a negative prognostic marker in DIPG, albeit independent of tumour histology. Previous research investigating the impact of H3K27M on high grade adult midline tumours found a correlation between H3K27M and poor survival in the brainstem, but not the thalamus [
1,
13]. Similar to this study, in our cohort high grade histology was associated with a poor outcome in both H3K27M and H3WT patients. However, in our cohort, several longer term survivors with H3WT high grade gliomas were present making the overall survival slightly better for H3WT patients. Work investigating paediatric glioblastoma (Grade IV) identified H3K27M positive cases as showing poor survival in midline cases including those in the brainstem and thalamic regions, consistent with our findings [
21].
The presence and effect of H3K27M mutations in low grade malignancies on patient outcome has not previously been shown in malignancies outside the brainstem. In this study, patients with low grade thalamic gliomas had good overall survival with 79 % of patients alive upon the completion of this study (mean follow-up 14.03 years), consistent with previous studies [
3,
11,
15,
29,
30]. However, all 5 patients whose low grade gliomas were positive for H3K27M succumbed to their disease, with increased latency compared to high grade H3K27M cases. These findings substantiate the knowledge that H3K27M mutations do exist in low grade tumours and that H3K27M status can supplement histological grading in determining the clinical progression of the tumour. Importantly, the poor survival of patients with H3K27M low grade glioma suggests that they should be treated as high grade glioma; receiving more aggressive adjuvant therapies than would be typically given to low grade glioma patients.
MAPK pathway activation was detected in 44 % of patients. BRAFV600E was detected in 10 (16 %) of the thalamic tumour samples, a slightly lower percentage than previously reported [
29,
30]. However, in these reports, the thalamus was grouped with all diencephalon-based structures, potentially altering the true prevalence. Furthermore, this study identified the presence of BRAFV600E mutations in 3 grade I malignancies for the first time, in contrast with previous reports [
11]. In the absence of H3K27M, BRAFV600E cases were associated with a good clinical outcome. Previously we reported that low grade tumours having BRAFV600E were at a higher risk for developing secondary high grade glioma [
25]. However, only 3 patients within this initial study were identified as thalamic, of which one co-occurred with H3K27M and another with CDKN2A deletion, potentially predisposing to tumour transformation. Furthermore, although the results here suggest robust survival in our BRAFV600E cohort, our median follow-up was 10.27 years and it is possible that with longer latency these patients may develop high grade glioma. Therefore, patients harboring low grade BRAFV600E tumours should be monitored closely post-treatment for disease progression. In the presence of H3K27M, BRAFV600E patients had an outcome similar to BRAFWT, H3K27M-positive cases, suggesting that H3K27M is the dominant prognostic indicator. KIAA1549-BRAF fusion events were exclusive to low grade thalamic glioma and were found in 39 % of tested tumours. This percentage is similar to previous reports in midline paediatric low grade glioma [
11,
15]. However, it should be noted that these studies included non-thalamic locations, including the cerebellum, hypothalamus, optic pathway and brainstem. Patients with a KIAA1549-BRAF fusion showed robust survival, with all 14 patients alive at the time this study was completed (median follow-up: 11.55 years). FGFR1N546K was detected in 4 patients (6 %) and was seen in both low and high grade cases. FGFR1N546K mutations have been previously linked to decreased patient survival [
3]. The small number of patients harbouring this mutation in this cohort means conclusions related to its prognostic significance cannot be drawn. However, of the 4 FGFR1 mutated patients, the 2 also harbouring H3K27M showed poor survival in accordance with the H3K27M phenotype. Alternately, FGFR1 mutated patients without H3K27M behaved in accordance with their histological grade, suggesting that histology and H3K27M status are the main phenotypic contributors in these cases. We did not identify FGFR1-TACC1 or FGFR3-TACC3 fusions in our cohort as described in [
34].
The presence of MAPK pathway activation was found in 28 patients within our cohort, 89 % of which were low grade glioma. Importantly, of the 3 high grade cases with MAPK activation, 2 were also H3K27M and behaved in accordance with the H3K27M phenotype. The remaining high grade, MAPK activated sample shows a prolonged survival in comparison to other high grade tumours (3.75 years) and was alive upon the completion of this study. In low grade tumours, the presence of MAPK activation, regardless of fusion or hotspot mutation event was related to prolonged survival. Taken together, H3K27M and MAPK pathway activation effectively stratifies thalamic tumours into survival groups. MAPK pathway activation in the absence of H3K27M confers long-term survival across the entire cohort irrespective of tumour histology. Tumours wild type for the genetic targets tested here behave in close accordance with their histological grade. Further testing is required to identify additional genetic marks capable of further stratifying this group. Importantly, it must be recognized that a limitation of this study in respect to MAPK activation is the inclusion of pilocytic astrocytoma and ganglioglioma within the low grade histology category. This saturation of circumscribed and non-invasive lesions positive for MAPK activation may partially explain the robust survival seen. However, in the case of thalamic tumours, the finding appear consistent across histological grades and as such, remain an important clinical predictor of patient outcome. Lastly, tumours harbouring H3K27M, regardless of histology or MAPK activation show dismal survival. In this respect, H3K27M is one of the most critical factors in predicting patient outcome in thalamic glioma cases and must be considered equally important as tumour histology in primary prognostic categorization.
Acknowledgements
This research was supported by an operating grant from the Canadian Cancer Society Research Institute [MOP 702296]. Scott Ryall is a recipient of the Canadian Institutes of Health Research Frederick Banting and Charles Best Canada Graduate Scholarships Award. Rahul Krishnatry is a recipient of the Society of Neurooncology International Research Development Award. We would like to thank the patients and their families for donating biological material to allow this study to be conducted.