Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas

Abstract

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Fig. 1

Molecular characteristics of infant gliomas. a The testing strategy used to identify the molecular driver of the tumor. Samples testing positive in tier 1 did not proceed to tier 2. b Clinical and genomic features of the infant glioma cohort highlighting the molecular alterations and clinical features associated with them including tumor location, grade and outcome. c Alluvial plot (https://rawgraphs.io/) showing the distribution of molecular drivers according to tumor location and histology. Red: high-grade glioma, Purple: mixed high and low-grade glioma, Blue: low-grade glioma. AT/RT: Atypical Teratoid Rhabdoid Tumors, NS: NanoString, CN: copy number, ddPCR: droplet digital PCR, HGG: high-grade glioma, LGG: low-grade glioma, OPHG: optic pathway/hypothalamic glioma

Fig. 2

ALK/ROS1/NTRK/MET fused hemispheric infantile glioma. a Graphical depiction of the newly identified ALK/ROS1/NTRK fusions in infantile glioma. b Circos plot depicting the chromothripsis events with > 10 split-read support from total RNAseq of a PPP1CB-ALK tumor with progressive scaling from full chromosome set to chr 2 p23.1-p23.2 arms. FISH for ALK further showing evidence of ALK translocation and amplification in a tumor positive for PPP1CB-ALK. c, d Examples of PPP1CB-ALK positive tumors—an iLGG diagnosed at 10 months of age (c) and a congenital iHGG (d)—including imaging (MRI axial and coronal T2-weighted images, c; sagittal and coronal head CT, d), hematoxylin and eosin (H&E) staining, and proliferation index (MIB-1). Further examples of large hemispheric congenital iHGG harboring RTK fusions with ETV6-NTRK3 (e) and CLIP2-MET (f). All images are taken at the stated magnification, scale bar = 100 μm for x20 and x40, 200 μm for ×4. TKD: tyrosine kinase domain, LGG: low-grade glioma, HGG: high-grade glioma, H&E: Hematoxylin and eosin stain

Fig. 3

ALK fusions are tumorigenic and susceptible to targeted agents. a Proliferation of iNHA cells overexpressing CCDC88A-ALK compared with iNHA with empty vector (EV). Experiments were conducted in triplicates in four biological replicates (n = 12). Data are represented as mean with SEM, *p < 0.05, paired t test. b Western blot for total and phosphorylated ERK1/2 indicative of MAPK pathway activation in iNHA cells overexpressing CCDC88A-ALK as compared with iNHA EV. c Dose-response curves of iNHA expressing CCDC88A-ALK versus EV upon treatment with ALK inhibitors Ceritinib and Crizotinib. Experiments were conducted in triplicates in two biological replicates (n = 6). Data are represented as mean with SEM, *p < 0.05, paired t test. d In vivo orthotopic xenografts of iNHAs overexpressing CCDC88A-ALK and PPP1CB-ALK resulted in tumor formation with 100% penetrance, p value calculated using the log-rank test. e Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) showing overexpression of FLAG and ALK in the intracranial xenografts. Images are taken at 20x magnification, scale bar = 100 μm. iNHA: immortalized normal human astrocytes, nM: nanomolar, OS: overall survival

Fig. 4

Characteristics of hemispheric glioma in infants. a Histological grade and molecular alterations in Group 1 Hemispheric RTK and Group 2 Hemispheric RAS/MAPK infant gliomas. b Overall survival (OS) of infants according to glioma subgroups, p value calculated using the log-rank test. c Survival of infants with hemispheric gliomas with respect to patient ALK, ROS1, and NTRK status, p value calculated using the log-rank test. d Hematoxylin and eosin (H&E) staining of an infant high grade glioma at diagnosis and second surgery post-chemotherapy, showing a maturing phenotype characterized by lower grade histology. Images are taken at the stated magnification, scale bar = 100 μm for x20, 200 μm for ×4. HGG: high-grade glioma, LGG: low-grade glioma

Fig. 5

Group 3 Midline RAS/MAPK tumors. a Progression-free survival (PFS) of infants with Group 3 tumors as compared with infants with other LGG (non-midline). b The molecular drivers of Group 3 as compared with other infant LGG, highlighting the enrichment for BRAF alterations in Group 3 tumors. c PFS of infant Group 3 according to BRAF status. d Overall survival (OS) of infant OPHG versus non-infant (>1–18 y) OPHG in SickKids cohort. Comparison of OS of BRAF Fused OPHG (e) and BRAFV600E mutated OPHG (f) in infants (Group 3 of infant cohort) vs children/adolescents aged 1–18 y (SickKids cohort). All p-values calculated using the log-rank test. LGG: low-grade glioma

Fig. 6

Graphical summary of the three infant glioma subgroups. HGG: high-grade glioma, LGG: low-grade glioma