Genomic analysis of childhood High grade glial (HGG) brain tumors Linda D Cooley Children s Mercy, Kansas City The Children s Mercy Hospital, 2017
Genomic analysis of childhood High grade glial (HGG) brain tumors IRB approved retrospective study Funded by a Children s Mercy Cancer Center grant October 2016-September 2018 Authors declare no conflicts
Pediatric High Grade Glial Tumors High grade gliomas (HGG) comprise ~8 12% of primary pediatric CNS tumors. HGG are aggressive tumors classified by the World Health Organization (WHO) as grade III or IV. Tumor histology - spectrum - with hypercellularity, nuclear atypia, high mitotic activity, plus/minus microvascular proliferation and necrosis. Outcomes remain dismal - 5-year survival ranges from 15 to 35%. Key to improving survival - better understanding of tumor biology. Genetic data show pediatric HGG are distinct from adult HGG.
Specific aims of study 1. Interrogate archival HGG brain tumors for copy number aberrations, loss of heterozygosity, and select somatic mutations using the Affymetrix Oncoscan microarray. 2. Review data from previous clinical Pathology, Cytogenetic, and Molecular reports and review patient Medical Records for clinical characteristics. 3. Correlate genetic alterations with histopathological findings and clinical characteristics.
Archival HGG brain tumors Tumor samples - selected & reviewed by Neuropathologist 42 FFPE samples from CMH archives (1998-2016) 33 unique patients; 4 patients had recurrent tumor resections 2 autopsy and 31 surgical samples
Pathology Pathology 16 supratentorial, 4 infratentorial (cerebellum), 13 midline (brainstem, thalamus, basal ganglia) WHO tumor grade: Grade III (AA) -15 Grade IV (GBM) 18 Gender 15 male : 18 female patients Patient age range 5 months - 17 years Average age 9.7 yrs; median age 10 yr
Methods Affymetrix Oncoscan Molecular Inversion Probe technology DNA isolated using Promega Maxwell RSC FFPE kit Sanger sequencing Microarray analyzed using Affymetrix ChAS software and Biodiscovery Nexus Express software Review previous clinical chromosome, FISH, molecular results Medical records review & data collection by data manager Limited statistical analysis
Chromosomes and microarray 25/33 (76%) tumors had chromosome analysis 7 had no chromosomes ordered, 1 failure 20/25 (80%) had abnormal karyotype 5 normal karyotype 1 had BRAF-KIAA1549 duplication only 33/33 (100%) tumors had abnormal microarray
Number of cases Oncoscan whole chromosome / chromosome arm Copy Number aberrations / LOH 18 16 14 12 10 8 6 4 2 0 1q 2 5p 5q 6p 6q 7 9p 10p 10q 12 13q 17p 18 Gain Loss LOH
Copy Number Aberrations (Percent) Adult Pediatric TCGA/WHO Current Bax 2010 Paugh 2010 1q gain 9 48 (33) 19 (63) 29 (68) chr 7 gain 74 36 19 15 17p LOH/loss ~30 mut 42-13 10q loss 50-70 36 16 38 16q loss 7 6 17.5 22 13q loss 8-12 mut 30-34 CDKN2A del up to 60 42 16 19 PDGFRA amp 13 9 16 12 EGFR amp 35-45 6-3 10
Genomic profiles (as define by Bax, etal. Clin Cancer Res 2010) 3 (9%) Stable genome few (<3), low level, focal changes 7 (21%) Amplifier amplification 8 (24%) Aneuploid large, single copy alterations of whole chromosomes or chromosome arms 15 (46%) Rearranged numerous, low-level, intrachromosomal breaks resulting in multiple gains & losses and a highly rearranged genome
Genomic subtypes of pediatric high-grade glioma have prognostic relevance (Bax). Dorine A. Bax et al. Clin Cancer Res 2010;16:3368-3377
Genome classification vs age, location, grade Stable younger, supratentorial, grade III Aneuploid older, supratentorial, grade III Rearranged older, supratentorial, grade IV Amplifier older, midline, grade IV
Stable genome 3/33 (9%) Stable genome 1 with BRAF-KIAA1549 duplication fusion 1 with htz 9p loss & hmz CDKN2A/B loss 1 with ROS1-GOPC deletion fusion - activation of RTK ROS1 Younger age (5, 6, 15 mo), 2 of 3 supratentorial, 2 of 3 WHO grade III Survival 0, 4, 5 mo Death average survival 3 months
Aneuploid genome favorable 8/33 (24%) Aneuploid LOH/loss 1p Gain 1q, 5p, 7, 12, 19p, 20 1 with ROS1-GOPC deletion fusion Older age, supratentorial location, WHO grade III Death of two pts at 4 mo & 39 mo Survival 6 patients alive @ 13, 9, 6 years, 21, 17, & 7 months average survival 4.7 years
Rearranged genome unfavorable 15/33 (46%) Rearranged LOH 17p Loss 17p,13q, 5q, 9p, 22q, 6q, 8p, 10, 11p, 14q, 18p Gain 1q, 7 Older age, supratentorial location, WHO grade IV Surv 0, 1, 5, 5, 10, 11, 11, 18, 18, 20, 22 months Three patients alive at 7, 24, 42 months, & one patient at 14 years Death of 11 average survival 15 months
Amplifier genome unfavorable 7/33 (21%) Amplifier LOH 17p Loss 10, 11p, 14 Gain 7, 1q, 6p, 8, 12, 21 Amplified genes / regions PDGFRA (4q), CCND1 (11q), CDK4 (12q), 1 with MDM4 (1q), EGFR (7p), MYC (8q), CCND2 (12p) Regions 1p36.13, 3p23p26, 5p13p12, 10q25, 11q13.5q14.1 Older age, midline location, WHO grade IV Death of 5 average survival 7.4 months; two patients alive at 7 months
Amplified genes Case 15 Case 10 PDGFRA CCND1 CDK4 PDGFRA 18
Amplified genes Case 18 Case 32 EGFR EGFR MYC CCND2 MDM4 19
Amplified genes / regions Case 1 Case 31 Case 5 PDGFRA 5p13.1p12 3p 1p - ARHGEF10L 20
Somatic mutations detected by Oncoscan microarray high confidence calls Grade III Anaplastic astrocytoma Grade Age Som mut Survival III 22 mo BRAF Alive 6+ y III 14 yr BRAF Alive ~2 y III 13 yr BRAF Alive 13 y III 15 yr EGFR Alive 7 mo III 5 yr EGFR Alive 7 mo III 12 yr TP53, IDH1 Alive 9 yr Grade IV - Glioblastoma Grade Age Som mut Survival IV 5 yr EGFR 9 mo IV 8 yr EGFR 1 mo IV 8 yr EGFR 11 mo IV 15 mo TP53 4 mo IV 10 yr TP53 1 mo IV 10 yr TP53 5 mo IV 12 yr TP53 Alive 7mo IV 13 yr TP53 11 mo IV 14 yr TP53, IDH1 18 mo IV 14 yr TP53 10 mo
Methylation profiling has identified 6 epigenetically distinct subgroups of GBM Methylation K27 G34 IDH RTK-1 Mesenchymal PXA-like Age Young child Adolescent YA Adult YA All Adolescent YA Young child location Recurrent oncogenic drivers Approx med survival Midline, infratentorial H3 K27, TP53 ATRX PDGFRA ACVR1 FGFR1 mutations Supratentorial Supratentorial Supratentorial Supratentorial supratentorial H3 G34, TP53, ATRX mutations IDH1 or IDH2, TP53, ATRX mutations PDGFRA amp, TP53 mut, CDKN2A/B del, EGFR amp NF1, TP53 mutations CDKN2A/B del EGFR amp PDGFRA amp BRAF V600E CDKN2A del 6 months 1 year > 2 years 1 year 1 year >4 years Expression Proneural Mixed Proneural Proneural Mesenchymal Unknown Modified from Figure 2, Gajjar, J Clin Oncol 2015
Tumor site 4/9 midline tumors H3 K27M mutated Sex Age Grd H3 K27M 17p TP53 Thalamus M 10 y IV Pos LOH mut Other Amplified PDGFRA; triploid Survival 1 mo Thalamus F 12 y IV Pos LOH 6q, 10q, 14 loss, hmz PTEN loss 0 mo Thalamus F 5 y III Pos LOH Amplified 3p22p26, 5p12p13.1; +2,+7,-10 alive 7mo Pons F 5 y IV Pos Loss 1q gain; 10q loss; hypodiploid 5 mo Thalamus M 15 y III Neg -- amplified MDM4, EGFR, MYC, CCND2; 9p, 14 loss; 12p gain alive 7 mo Basal ganglia M 4 y III Neg -- ROS1-GOPC deletion fusion alive 17 m Thalamus F 8 y IV Neg -- 1q, 12p gain; 6q, 9p, 10 loss 1 mo Basal ganglia F 6 m III Neg -- ROS1-GOPC deletion fusion 5 mo Thalamus F 5 y IV Neg LOH Amplified EGFR; 1q gain; 6q, 10, 14 loss 9 mo
Survival - age 24
Survival - tumor location
Survival WHO Grade III vs Grade IV
Ongoing study Further analysis Correlate clinical characteristics and additional pathologic characteristics with the genomic data Additional somatic mutation analysis If possible, whole exome sequencing Collaboration with other pediatric institutions 27
Collaborators Melissa Gener Kevin Ginn Lei Zhang Elena Repnikova John Herriges Midhat Farooqi Vincent Staggs Eugenio Taboada Jessica Nick Tara Bendorf Scott Smith Robin Ryan Stephanie Fiedler Kris Lawrence Children s Mercy Cancer Center 28