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Home , Choroid plexus carcinoma, Ganglioglioma, Gliomatosis cerebri, Medulloblastoma, Pilocytic astrocytoma

Impact of RNA-sequencing analysis in refining diagnosis in pediatric neuro-oncology Kathleen M. Schieffer1, Katherine E. Miller1, Stephanie LaHaye1, James Fitch1, Kyle Voytovich1, Daniel C. Koboldt1, Benjamin Kelly1, Patrick Brennan1, Anthony Miller1, Elizabeth Varga1, Kristen Leraas1, Vibhuti Agarwal2, Diana Osorio2, Mohamed S. AbdelBaki2, Jonathan L. Finlay2, Jeffrey R. Leonard3, Alex Feldman4, Daniel R. Boue4, Julie M. Gastier-Foster1, Vincent Magrini1, Peter White1, Catherine E. Cottrell1, Elaine R. Mardis1, Richard K. Wilson1 1. Institute for Genomic Medicine, Nationwide Children’s Hospital, Columbus, OH; 2. Department of Pediatrics, Nationwide Children’s Hospital, Columbus, OH; 3. Department of , Nationwide Children’s Hospital, Columbus, OH; 4. Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, OH

Background and demographics Methods Case 2: Refine diagnosis

The Nationwide Children’s Hospital Institute for Genomic Medicine (IGM) translational research protocol Transcriptomic analysis was performed from 70 tissue sections collected from 56 patients: snap frozen (n=48), A 10-month-old was diagnosed with a sella/suprasellar tumor, most consistent with a CNS embryonal tumor. No enrolls pediatric patients with high-risk, relapsed, refractory, or difficult to classify tumors. The goal of formalin-fixed paraffin-embedded (FFPE) tissue (n=21), or disassociated cells (n=1). When possible, total RNA- constitutional or somatic variations or copy number aberrations were noted. The tissue was also sent to an this protocol is to refine the patient diagnosis, identify targeted therapeutics relevant to the individual sequencing was performed (n=60); however, when the sample was poor quality or low input, cDNA capture was outside institution for the Infinium MethylationEPIC 850K array, reporting a classification of pineoblastoma group tumor biology and eliminate unsuitable treatments, and determine eligibility for clinical trials. Findings utilized to enrich for exonic regions (n=8). RNA was unavailable for 2 patients. We aimed for a total of ≥80 million A/intracranial (score=0.99). are reported and new cases identified for enrollment at weekly multidisciplinary tumor boards, mapped reads per sample, with 4 samples falling below that threshold (average mapped reads: 197,334,031 represented by oncology, surgery, pathology, radiology, radiation oncology, and IGM researchers. 148,456,775 reads). The analysis pipeline is described in Figure 3. A B Exon 1 2 3 4 5 6 7 8 9 - 11 12 13 14-17 18 19 20 21 22-23 24 25-27 Medically actionable findings are CLIA validated to allow for return of results to the medical record. To date, 56 unique pediatric patients with (CNS) tumors have undergone comprehensive DNA-sequencing RNA-sequencing and gene-level expression quantification Exon 1 2 3 4 5 6 7 8 9 - 11 12 13 14-17 18 19 20 21 22-23 24 25-27 genomic and transcriptomic analysis (Figure 1A). A majority of sequenced patients are male (60%) and between Constitutional and somatic Salmon tximport package variation RNA-sequencing Transcript-level the ages of 1-5 and 11-17 years-old (Figure 1B). Multiple tissue sections or time points were sequenced from Gene-level expression Assess for nonsense-mediated Tumor tissue(s) expression some patients, thus a total of 70 unique tissues were evaluated, with 66% of tissues from the primary tumor quantification decay and aberrant splicing quantification (Figure 1C).

Constitutional and somatic Outlier analysis (protein-coding genes only) variation Identify reference cohort D A Yolk sac tumor (1) Single nucleotide variation, Differential C UCSC Treehouse cohorts Outlier metric Outlier criteria indels, copy number expression Germinoma (1) 1. Nervous system (n=434) Mahalanobis |Log2 fold change| (3) variation/loss of heterozygosity, Quantile normalize Atypical teratoid rhabdoid tumor (1) 2. Non-nervous system distance and chi- >3.5 structural variation and calculate log2 Diffuse (1) (n=194) squared P-value P <0.005 CNS neuroblastoma (1) fold change Ectopic retinoblastoma (1) Diffuse midline glioma (1) 3. Hematologic (n=479) ETMR (1) 3% multiforme (4) (7) 20% (1) Assess biological relevance 20% (1) Clinical validation of medically actionable targets Therapeutic Pathway analysis Functional Clustering patterns Tumor cellularity targets Ingenuity Pathway Annotation Principal component Pineoblastoma (3) ESTIMATE 5% n=56 DGIdb Analysis DAVID analysis Atypical mixed glioneuronal tumor (1) 18% (8) Literature review to evaluate potential therapeutic targets CNS glioneuronal tumor (1) 16% Pleomorphic xanthoastrocytoma (2) Figure 3: IGM translational cancer protocol comprehensive DNA/RNA sequencing analysis pipeline. RNA-sequencing is performed on tumors to aid in DNET (2) 7% (3) 11% refining the patient diagnosis, identifying therapeutic targets, and confirming the transcriptional effects of constitutional and somatic DNA variation. High-grade neuroepithelial tumor (1) Rosette-forming glioneuronal tumor (1) (4) carcinoma (5) Case 1: Refine diagnosis (1) A 12-year-old was diagnosed with medulloblastoma, suggestive of Group 3 or Group 4, due to presence of B C isochromosome 17q. The patient recurred 3 years later. Six years after the initial diagnosis, the patient presented 25 3% 3% 3% Primary with a cerebellar lesion “most consistent with recurrent medulloblastoma”, with comment recommending genomic studies to confirm the morphologic impression. Figure 5: Refinement of a retinoblastoma diagnosis in the absence of constitutional RB1 variation. (A) RB1-SIAH3 fusion with fusion breakpoints at 20 exon 17 of RB1 and exon 2 of SIAH3. (B) Sashimi plot demonstrating a drastic reduction in spliced read depth following the fusion breakpoint at exon 17 of RB1 in the Recurrent described retinoblastoma case and consistent spliced read depth in a pineoblastoma patient with wild-type RB1. (C) Unsupervised three-dimensional principal component A +6 years Molecular refinement of analysis of the top 500 most variable protein-coding genes. (D) Unsupervised hierarchical clustering of the St. Jude-Pediatric Cancer Genome Project retinoblastoma cases 15 diagnosis (n=19) and our described case using 79 retina-associated genes. Metastatic +3 years Secondary diagnosis Female 25% Primary diagnosis Recurrence “most consistent with recurrent Secondary glioblastoma count 10 Male Medulloblastoma Medulloblastoma medulloblastoma” +6 years, 9 months 66% Residual Patient deceased Case 3: Identify targeted therapy 5 Second Gross total resection Resection Resection A 1-year-old with was treated with gross total resection and chemoradiation. Genomic 0 malignancy Radiation + adjuvant Proton radiation + salvage chemotherapy Clinical trial + everolimus <1 y 1-5 y 6-10 y 11-17 y ≥18 y IGM sequencing of primary and analysis identified a somatic TP53 mutation and 17p loss of heterozygosity. Comprehensive transcriptomic secondary tumors analysis revealed overexpression of multiple pathways (mTOR, FGF, and PDGF signaling) consistent with other

Figure 1: Demographics of the IGM translational research protocol CNS cohort. (A) Primary diagnoses from 56 unique pediatric patients with CNS B Primary Secondary IGM choroid plexus carcinoma cases and a 2017 case report (Cornelius et al. 2017 Front. Pharmacol.). tumors who underwent comprehensive genomic and transcriptomic sequencing on our IGM translational cancer protocol. Patient diagnosis is segregated by WHO Treatment on sunitinib (PDGF inhibitor), everolimus (mTOR inhibitor), and thalidomide (FGF inhibitor) was classification of CNS tumors. ETMR: embryonal tumor with multilayered rosettes; DNET: dysembryoplastic neuroepithelial tumor. (B) Age and sex at primary tumor C diagnosis. (C) Distribution of tumor occurrence for the sequenced tissue sections. initiated. Hematoxylin A B Cohort findings and eosin

A B C Li-Fraumeni syndrome 8.9% 5 D 7.1% 4 3.6% 3 Klippel-Feil syndrome with 3.6% 3.6%

1.8% count nemaline myopathy 2 1.8% 1.8% 1.8% 1.8% Olig2 1 Nemaline myopathy 1.8% 0 F 1.8% Noonan syndrome D E QKI-RAF1 ST7-MET RB1-SIAH3 0 1 2 3 4 5 6 EP300-BCOR E 0.45 FGFR1-TACC1 F count C11orf95-RELA KIAA1549-BRAF 0.40

0.35 C 5% D 5% 0.30 5% FGF 0.25 ExampleExample EGF Refinement of diagnosis (5.4%) Overlap 26% 0.20 CaseCase 11 PDGF ”most consistent with medulloblastoma” à secondary 0.15

11% Secondarytumor VAF MET glioblastoma 0.10 NTRK CNS embryonal tumor à retinoblastoma 11% DNMT3B Low-grade neuroepithelial tumor à rosette-forming 0.05 n=17 (30.3%) 21% PI3K glioneuronal tumor 0.00 16% KIT 0.00 0.20 0.40 0.60 0.80 Primary tumor VAF Figure 6: Transcriptomic analysis identifies therapeutically-relevant targets in a pediatric choroid plexus carcinoma patient. (A) Manhattan plot visualizing genome-wide differential expression relative to the UCSC Treehouse nervous system cohort (n=434). Outliers are shown as a blue dot. Outliers that are targetable, as determined by DGIdb, are shown as a red dot. (B) Distribution of DESeq2 normalized counts for FGFR2 within the IGM translational research protocol Figure 4: Comprehensive sequencing of primary and secondary tumors refined diagnosis from medulloblastoma to secondary glioblastoma. cohort. (C) Same as (B) but for MTOR. (D) Boxplots showing the distribution of log2(TPM+1) values for FGFR2 among varying cohorts. The described case is shown Figure 2: Summary cohort findings. (A) Constitutional disorders identified through genomic sequencing. (B) Medically actionable and biologically-relevant novel (A) Patient disease course from primary diagnosis. Changes made due to enrollment on the IGM translational research protocol are shown in red. (B) Morphologic and as a red line. (E) Same as (D) but for MTOR. (F) Overexpression of the PDGF signaling pathway, shown by Ingenuity Pathway Analysis. fusions identified through RNA-sequencing. (C) Potentially targetable gene overexpression. (D) Cases where genomic and transcriptomic sequencing helped to refine the immunohistochemical similarities (H&E and synaptophysin) and minor differences (Olig2) between the primary and secondary tumors are shown. (C) Somatic copy number patient diagnosis. variation (CNV) and loss of heterozygosity (LOH) from the primary tumor. (D) Somatic CNV/LOH from the secondary tumor. (E) Rare coding somatic variation in the primary tumor and secondary tumor showing vastly different genomic profiles. (F) Unsupervised three-dimensional principal component analysis of the top 500 most variable protein coding genes. VAF: variant allele frequency; MB: medulloblastoma; GBM: glioblastoma. We are grateful to the patients and their families for their participation.