Pineoblastoma segregates into molecular sub-groups with distinct clinico-pathologic features: a Rare Brain Tumor Consortium registry study

Abstract

Pineoblastomas (PBs) are rare, aggressive pediatric brain tumors of the pineal gland with modest overall survival despite intensive therapy. We sought to define the clinical and molecular spectra of PB to inform new treatment approaches for this orphan cancer. Tumor, blood, and clinical data from 91 patients with PB or supratentorial primitive neuroectodermal tumor (sPNETs/CNS-PNETs), and 2 pineal parenchymal tumors of intermediate differentiation (PPTIDs) were collected from 29 centres in the Rare Brain Tumor Consortium. We used global DNA methylation profiling to define a core group of PB from 72/93 cases, which were delineated into five molecular sub-groups. Copy number, whole exome and targeted sequencing, and miRNA expression analyses were used to evaluate the clinico-pathologic significance of each sub-group. Tumors designated as group 1 and 2 almost exclusively exhibited deleterious homozygous loss-of-function alterations in miRNA biogenesis genes (DICER1, DROSHA, and DGCR8) in 62 and 100% of group 1 and 2 tumors, respectively. Recurrent alterations of the oncogenic MYC-miR-17/92-RB1 pathway were observed in the RB and MYC sub-group, respectively, characterized by RB1 loss with gain of miR-17/92, and recurrent gain or amplification of MYC. PB sub-groups exhibited distinct clinical features: group 1-3 arose in older children (median ages 5.2-14.0 years) and had intermediate to excellent survival (5-year OS of 68.0-100%), while Group RB and MYC PB patients were much younger (median age 1.3-1.4 years) with dismal survival (5-year OS 37.5% and 28.6%, respectively). We identified age < 3 years at diagnosis, metastatic disease, omission of upfront radiation, and chr 16q loss as significant negative prognostic factors across all PBs. Our findings demonstrate that PB exhibits substantial molecular heterogeneity with sub-group-associated clinical phenotypes and survival. In addition to revealing novel biology and therapeutics, molecular sub-grouping of PB can be exploited to reduce treatment intensity for patients with favorable biology tumors.

Keywords: MYC; PNET; PPTID; Pineoblastoma; RB; miRNA.

Conflict of interest statement

Conflict of Interest

The authors declare that they have no conflict of interest.

Figures

Figure 1. PB comprise five molecular sub-groups

a. Flow diagram of analyses performed: 93 primary tumors with institutional diagnosis of pineoblastoma (PB) or supratentorial PNET (sPNET) were analysed using global methylation profiling and compared against a reference cohort of 1200 pediatric brain tumors to identify and exclude samples that segregated with other brain tumors. A cluster of robust, molecularly confirmed 72 PBs were further characterized using methylation and SNP arrays for copy number alterations, mutational analyses using WES and targeted sequencing, and Nanostring analyses for miRNA expression. Clinical, treatment, and molecular sub-group data available for 46 PB patients treated with curative intent were integrated for clinic-pathologic analyses. b. t-Distributed stochastic neighbour embedding (tSNE) plots of DNA methylation clustering patterns of 93 presumed PB samples relative to 951/1200 representative pediatric brain tumor entities demonstrate PB clusters separately from other tumor entities. Plots using the top 12,500 most varying methylation probes by standard deviation (SD) are shown. Tumors are shown as colored spheres which include atypical teratoid rhabdoid tumor (ATRT), ependymoma posterior-fossa (EP_PF) or supratentorial, RELA-fusion (EP_RELA), embryonal tumor multiple rosettes (ETMR), germ cell tumor (GCT), high-grade glioma (HGG), neuroblastoma (NB), medulloblastoma WNT (MB_WNT), SHH (MB_SHH), group 3 (MB_G3), and group 4 (MB_G4). Black spheres indicate tumors with an institutional diagnosis of PB that segregated with other known brain tumor entities are (n=21). A robust cluster of 72 PBs is boxed; blow-up image of PB cluster on right shows five molecular PB sub-groups designated as 1, 2, 3, RB, and MYC.

Figure 2. PB molecular subgroups have distinct copy number landscapes

a. Pattern of copy number alterations across PB molecular sub-groups as determined using GISTIC analyses of global methylation data. Chromosomal regions with recurrent copy number gains (green) or losses (red) significantly enriched within each PB sub-group are highlighted; asterisk indicates false discovery rate of q<0.05. b, c. Composite circos plots of global methylation profiles showing recurrent copy number gains (green) and losses (red) in 21 group 1 and 11 group 2 PBs. Focal or broad alterations associated with miRNA biogenesis loci DICER1, DROSHA and DGRC8 are highlighted. Higher resolution copy number profiles generated using Conumee, of representative group 1 and group 2 samples with respective focal chr 5p13.3 targeting DROSHA and chr 14q loss associated with DICER1, are shown on the right. d. Composite circos plot of global methylation profiles in 13 group 3 PBs. Higher resolution copy number profile generated using Conumee of a representative group 3 sample is shown on right.

Figure 3. Recurrent copy number alterations in RB and MYC sub-group PBs

a. Composite circos plot of global methylation profiles from 9 RB subgroup PBs depicting recurrent copy number gains (green) and losses (red); recurrent copy number alterations associated with miR-17/92 and RB1 are highlighted. Higher resolution copy number profile of a representative tumor RBTC 1546 with homozygous loss of RB1 at chr13q14.2 and copy number gain encompassing miR-17/92 at chr 13q31.3 is shown on right. b. Copy number driven expression of miR-17/92 in RB sub-group PB. MiRNA expression levels for the miR-17-92, paralogous miR106a-363, miR-106b-25 and unrelated let-7 loci was determined from NanoString(v.3) miRNA expression data from 6 PBs. Plots show relative, normalized probe intensities of miRNAs in PB sub-groups; miRNA expression levels of RBTC746 with focal chr13q13.3 copy number gains targeting miR-17-92 shown in Figure A, is highlighted. c. Composite circos plot of global methylation profiles from 18 MYC subgroup PBs. Recurrent focal chr 8q amplification/gains (green) and chr 16q losses (red) are highlighted. Higher resolution copy number profile of a representative tumor, RBTC 1520, with focal MYC amplification is shown on right.

Figure 4. Recurrent mutations/alterations of miRNA biogenesis genes, RB1 and MYC characterize PB sub-groups.

a. Summary of mutations and copy number alterations associated with miRNA biogenesis gene (DICER1, DROSHA, DGCR8, XPO5, TARBP2), KBTBD2, RB1, miR-17/92, and MYC determined using a combination of targeted sequencing, WES, methylation and SNP array based copy number analyses in individual PBs of different sub-groups with tumor and matched blood DNA available for study. Samples lacking materials for specific assay are indicated by (-); broad copy number alterations determined by methylation or SNP-based copy number analyses are indicated by HT (heterozygous), HM (homozygous), n (normal diploid) or presence (Y) of MYC focal gains or amplification (α) is indicated. Status or specific gene alterations determined by targeted sequencing or WES is indicated as wt (wild-type); * (stop-gain mutation), fs (frameshift insertion or deletion), † (deleterious missense mutation predicted by SIFT and Polyphen2). All predicted truncating gene mutations are highlighted. b. Schema of DICER1 and DROSHA mutations relative to maps of corresponding proteins. Type and location of mutations are shown as colored symbols relative to amino acid sequence numbers and known or predicted functional domains; colors of mutation symbols correspond to tumor sub-group.

Figure 5. Molecular sub-groups of PB have distinct clinicopathologic features

a. Scatterplot of age at diagnosis for PB patients relative to tumor molecular sub-group. Bar indicates median age as determined using Kruskal-Wallis test. b. Frequency of metastatic (M+; M1, −3, −4) and non-metastatic (M0) disease determined as per the Chang staging system is shown relative to PB sub-groups; significance in distribution of M+ versus M0 patients across all PB sub-groups was determined using Fisher exact test. c. Forest plot of Hazard ratio (HR) from univariate Cox proportional hazards regression model of gender (male/M vs. female/F), age, metastatic status (M+ vs M0), radiotherapy (no upfront RT vs. upfront RT), conventional chemotherapy only (chemo) vs. high-dose chemotherapy (HD), and extent of tumor removal (less than gross total resection/GTR vs GTR) on EFS (black) and OS (gray) was performed on data from 46 patients treated with curative intent. Whiskers denote 95% confidence interval. d. Kaplan-Meier survival analyses of event free (EFS) and overall survival (OS) for 46 patients treated with curative intent stratified by PB sub-groups. Plots abbreviated to maximum of 12 years from diagnosis. For patients with group 1-3, RB, and MYC PBs EFS were respectively, 39.5%, 100%, 83.3%, 25.0%, and 14.3%; 5-year OS were 68.0%, 100%, 80%, 37.5%, and 28.6%.

Figure 6

Schematic summary of molecular and clinical features across PB sub-groups