Markers of survival and metastatic potential in childhood CNS primitive neuro-ectodermal brain tumours: an integrative genomic analysis

Abstract

Background: Childhood CNS primitive neuro-ectodermal brain tumours (PNETs) are very aggressive brain tumours for which the molecular features and best treatment approaches are unknown. We assessed a large cohort of these rare tumours to identify molecular markers to enhance clinical management of this disease.

Methods: We obtained 142 primary hemispheric CNS PNET samples from 20 institutions in nine countries and examined transcriptional profiles for a subset of 51 samples and copy number profiles for a subset of 77 samples. We used clustering, gene, and pathway enrichment analyses to identify tumour subgroups and group-specific molecular markers, and applied immunohistochemical and gene-expression analyses to validate and assess the clinical significance of the subgroup markers.

Findings: We identified three molecular subgroups of CNS PNETs that were distinguished by primitive neural (group 1), oligoneural (group 2), and mesenchymal lineage (group 3) gene-expression signatures with differential expression of cell-lineage markers LIN28 and OLIG2. Patients with group 1 tumours were most often female (male:female ratio 0·61 for group 1 vs 1·25 for group 2 and 1·63 for group 3; p=0·043 [group 1 vs groups 2 and 3]), youngest (median age at diagnosis 2·9 years [95% CI 2·4-5·2] for group 1 vs 7·9 years [6·0-9·7] for group 2 and 5·9 years [4·9-7·8] for group 3; p=0·005), and had poorest survival (median survival 0·8 years [95% CI 0·5-1·2] in group 1, 1·8 years [1·4-2·3] in group 2 and 4·3 years [0·8-7·8] in group 3; p=0·019). Patients with group 3 tumours had the highest incidence of metastases at diagnosis (no distant metastasis:metastasis ratio 0·90 for group 3 vs 2·80 for group 1 and 5·67 for group 2; p=0·037).

Interpretation: LIN28 and OLIG2 are promising diagnostic and prognostic molecular markers for CNS PNET that warrant further assessment in prospective clinical trials.

Funding: Canadian Institute of Health Research, Brainchild/SickKids Foundation, and the Samantha Dickson Brain Tumour Trust.

Figure 1. Molecular sub-groups of CNS-PNET exhibit distinct cell lineage and signalling signatures

A. Unsupervised cluster analyses were performed on human HT-12v4 expression array (Illumina) data from 51 primary CNS-PNET samples to identify the most stable tumour grouping with a minimal gene set (Supplemental Figure 1). Heat map shows the most highly expressed cell lineage genes in each sub-group identified using a supervised t-test adjusted for multiple testing (FDR≤0.05), relative to a hierarchical cluster map of all tumours. Magnitude and significance of cell lineage genes most significantly up-regulated in each tumour sub-groups are denoted respectively by fold change and p-values. B. Signalling pathways enriched in each tumour sub-group were determined by Ingenuity Pathways Analyses of group-specific gene sets derived from supervised analyses. Most significantly altered canonical pathways determined from analyses of 343, 276 and 325 genes respectively in groups 1, 2 and 3 are represented in relation to tumour sub-group. Proportion of up or down regulated genes within each category are respectively indicated in red and green.

Figure 2. Cell lineage markers, LIN28 and OLIG2, identify molecular sub-groups of CNS-PNET

A. Enrichment of specific lineage genes in CNS-PNET sub-groups was confirmed by qRT-PCR analyses of 51 primary CNS-PNET profiled by gene expression arrays (Supplemental Figure 2). Meanexpression levels of LIN28, OLIG2 and IGF2 (n=3 replicas), which were most highly enriched respectively in group 1, 2 and 3 CNS-PNETs, are represented with SEM (horizontal bars). Transcript levels within CNS-PNET sub-groups 1, 2 and 3 are indicated by green, blue and purple bars or spheres respectively. B. Distinct expression patterns of LIN28 and OLIG2 in molecular sub-groups of CNS-PNET was validated and further characterized by immuno-histochemical analyses in a larger cohort of CNS-PNET (n=72) (Supplemental Figure 2). Characteristic LIN28 and OLIG2 immuno-stains (10× magnification) in CNS-PNET sub-groups are shown in relation to a hematoxylin and eosin (H&E) stain. Corresponding tissue microarray core is shown at low magnification in inset

Figure 3. Age and gender distribution in molecular sub-groups of CNS-PNET

Demographic information available on 108 primaries CNS-PNET (Table 1, 2 and supplemental Table 1) was examined to determine tumour sub-group specific correlation with: A- Gender and, B- Age at diagnosis. Significance was determined using ANOVA (gender) and Chi-square analyses (age). Number of patients in each category is indicated within the bar graphs.

Figure 4. Molecular sub-groups of CNS-PNET exhibit distinct clinical phenotypes

Clinical information available on 108 primaries CNS-PNET (Table 1, 2 and supplemental Table 1) was analysed to determine tumour sub-group specific correlation with A - Metastatic status at diagnosis, B- Age and metastatic status at diagnosis, C- Overall survival and D- Overall survival and age. Significance was determined using 2-sided Fisher's exact test (metastatic status at diagnosis) and log-rank (survival) analyses. Number of patients in each category is indicated within the bar graphs.