Alternative lengthening of telomeres in molecular subgroups of paediatric high-grade glioma

Abstract

Purpose: The maintenance of telomere length prevents cancer cell senescence and occurs via two mutually exclusive mechanisms: (a) reactivation of telomerase expression and (b) activation of alternative lengthening of telomeres (ALT). ALT is frequently related to alterations on ATRX, a chromatin-remodelling protein. Recent data have identified different molecular subgroups of paediatric high-grade glioma (pHGG) with mutations of H3F3A, TERTp and ATRX; however, differences in telomere length among these molecular subgroups were not thoroughly examined.

Methods: We investigated which genetic alterations trigger the ALT mechanism in 52 IDH-wildtype, 1p/19q-wildtype pHGG. Samples were analysed for telomere length using Tel-FISH. ATRX nuclear loss of expression was assessed by IHC, H3F3A and TERTp mutations by DNA sequencing, and TERTp methylation by MS-PCR.

Results: Mutant H3.3 was found in 21 cases (40.3%): 19.2% with K27M mutation and 21.1% with G34R mutation. All H3.3G34R-mutated cases showed the ALT phenotype (100%); on the opposite, only 40% of the H3.3K27M-mutated showed ALT activation. ATRX nuclear loss was seen in 16 cases (30.7%), associated sometimes with the G34R mutation, and never with the K27M mutation. ATRX nuclear loss was always related to telomere elongation. TERTp C250T mutations were rare (5.4%) and were not associated with high intensity Tel-FISH signals, as TERTp hyper-methylation detected in 21% of the cases. H3.3/ATRX/TERTp-wildtype pHGG revealed all basal levels of telomere length.

Conclusion: Our results show a strong association between H3.3 mutations and ALT, and highlight the different telomeric profiles in histone-defined subgroups: H3.3-G34R mutants always trigger ALT to maintain telomere length, irrespective of ATRX status, whereas only some H3.3-K27M tumours activate ALT. These findings suggest that acquiring the gly34 mutation on H3.3 might suffice to trigger the ALT mechanism.

Keywords: Alternative lengthening of telomeres; H3.3; Paediatric high-grade gliomas; Telomeres; pHGG.

Fig. 1

Relevant clinico-pathological, genetic and molecular features of pHGG in our cohort, showing the increased telomere fluorescence intensity and ALT activation in different molecular subgroups of cases with H3G34R mutations, H3K27M mutations, ATRX nuclear loss with H3.3-wt, TERTp mutations or no alterations on these genes

Fig. 2

Panel of representative cases analysed for telomere elongation using Tel-FISH (magnification × 100). Green signals represent telomeres. a pHGG with large, very bright intranuclear foci of telomere signals in single cells. b Cases with telomere elongation featuring very bright signals in large neoplastic regions. c Cases with small and homogeneous telomere foci, indicative of basal-level telomere length and no ALT

Fig. 3

Telomere length assessed in 52 pHGG. a Telomere fluorescence intensity quantified in different subgroups with H3.3-G34R mutations (red), H3.3-K27M mutations (orange), ATRX nuclear loss (yellow), TERT promoter mutations (light blue) or no alterations on these genes; positive and negative controls (grey boxes) were included to establish a threshold (dotted black line). b Number of cases with H3.3-G34-mut, H3.3-K27-mut, H3.3-wt with ATRX loss and H3.3/ATRX-wt, stratified according to ALT activation