Jennifer Hauenstein Oncology Cytogenetics Emory University Hospital Atlanta, GA

Transcription

1 Comparison of Genomic Coverage using Affymetrix OncoScan Array and Illumina TruSight Tumor 170 NGS Panel for Detection of Copy Number Abnormalities in Clinical GBM Specimens Jennifer Hauenstein Oncology Cytogenetics Emory University Hospital Atlanta, GA

2 Objectives Comparison of Illumina TruSight Tumor 170 and ThermoFisher/Affymetrix Oncoscan assays for Glioblastoma - Workflows and Genomic content - Data comparison of Emory Glioblastoma (GBM) dataset with TCGA - Evaluation of the reportable results for GBM tumors

3 Workflow Comparison NA QC TST170 Tissue QC DNA and RNA isolation Nucleic Acid (NA)QC DNA QC OncoScan Tissue QC DNA isolation DNA QC Library Prep (40 ng) DNA Library Prep (Hybrid Capture) 151 SNVs and indels 59 Copy Number (Amplifications) RNA Library Prep (Hybrid Capture) 55 fusions and splice variants Library Prep (80 ng) Molecular Inversion Probe (MIP) 217,454 MIP probes with 74 mutation probes Concentration on ~900 cancer genes Sequencing NextSeq NGS sequencing (Illumina NextSeq) Array ThermoFisher/Affymetrix Scanner Data Analysis BaseSpace (SNV, CN, Fusions) Custom Workflows (OpenSource) 3 rd Party Software (e.g. DNANexus) Data Analysis ChAS (Copy Number) 3 rd Party Software (e.g. Nexus)

4 Whole Genome View of TST170 and Oncoscan Coverage TST170 List chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrx PIK3CD ALK VHL FGFR3 TERT CCND3 CARD11 PPP2R2A JAK2 KIF5B HRAS CCND2 FGF9 RAD51B RAD51 TSC2 TP53 SMAD4 STK11 SRC ERG SMARCB1 AR MTOR MYCN PPARG PDGFRA RICTOR ROS1 PMS2 NRG1 MLLT3 RET CCND1 FGF23 FLT3 AKT1 FGF7 SLX4 NF1 BCL2 GNA11 GNAS TMPRSS2 CHEK2 BTK PAX7 GEN1 RAF1 KIT FGF10 ESR1 ETV1 FGFR1 CDKN2A PTEN FGF19 FGF6 FLT1 MAP2K1 CREBBP RAD51D MAP2K2 EWSR1 ARID1A EML4 MYD88 KDR PIK3R1 EGFR NBN GNAQ FGF8 FGF4 KRAS BRCA2 NTRK3 PALB2 CDK12 JAK3 EP300 MYCL1 DNMT3A MLH1 FGF5 MSH3 PIK3CG MYC NTRK2 FGFR2 FGF3 ERBB3 RB1 FANCI CDH1 ERBB2 NOTCH3 MPL MSH2 CTNNB1 FAM175A APC CDK6 PTCH1 MRE11A CDK4 FGF14 IDH2 BRCA1 CCNE1 MUTYH MSH6 BAP1 TET2 FGF1 MET ABL1 ATM MDM2 LAMP1 ETV4 CEBPA RAD54L FANCL PIK3CB FGF2 CSF1R SMO TSC1 KMT2A PTPN11 RAD51C AKT2 NRAS IDH1 FOXL2 INPP4B PDGFRB BRAF NOTCH1 CHEK1 HNF1A RPS6KB1 AXL NOTCH2 ERBB4 ATR FBXW7 NPM1 EZH2 ETS1 BRIP1 CD79A MCL1 BARD1 PIK3CA FGFR4 XRCC2 FLI1 CD79B ERCC2 NTRK1 PAX3 ETV5 ERCC1 DDR2 BCL6 MDM4 TFRC AKT3 Total Genes Black: SNVs & indels Green: Amplifications Red: Fusions Bold: Multiple

5 Emory GBM OncoScan Cohort (n=143) MDM4 AKT3 PDGFRA EGFR MYC WT1 CDK4 MDM2 CDKN2C ARID1A CDKN2A/B PTEN RB1 NPAS3 TCGA GBM Cohort (n=580) MDM4 AKT3 MYCN PDGFRA FGFR3 SOX2 EGFR MYC CCND2 CDK4 MDM2 CDKN2C CDKN2A/B PTEN RB1 NPAS3

6 Evaluation of TST170 and Oncoscan Array (4) Emory GBM Tumors

7 Oncoscan Whole Genome Views From ChAS GBM1 GBM2 Gain of 7 EGFR Amp FGFR3 Amp Gain of 7 CDK4 Gain CDKN2A Loss Loss of 10 CDKN2A Loss Loss of 10 GBM3 GBM4 MYCN Gain 17p CN-LOH MDM4 Amp PDGFRA Amp CDKN2A Loss CDKN2A Loss PTEN loss

8 Comparison of GBM 1 WGV Regions Chr 7 EGFR CDKN2A PTEN Concordant Oncoscan Copy Number TST170 Copy Number TST170 Mutation and Fusions 7p( )x41 EGFR Amp 7pq x4 (CDK6, MET, BRAF gain) 10pq x1-2 (RET, FGF8, FGFR2, PTEN loss) 15q( )x1 (FGF7 loss ) 22pq x1 (CHEK2 loss) 1p( )x1-2 17p( )x2 1p( )x1 17q( ) 4p( )x1 22pq x1 7pq x4 9p( )x1-2 10pq x1-2 15q( )x1 16q( )x1 EGFR amp (chr 7) CDK6, MET, BRAF gain (chr 7) RET, FGF8, FGFR2, PTEN loss (chr 10) FGF7 loss (chr 15) CHEK2 loss (chr 22) MCL1 c.c205t [p.l69f] EGFR viii: reported as slice variant EGFR: chr7:

9 Comparison of GBM2 WGV Regions FGFR3 Chr 7 Chr 10 CDK4 Concordant Oncoscan Copy Number TST170 Copy Number TST170 Mutation and Fusions 3q( )x3 TFRC Gain 4p( )x6 - FGFR3 Amp 7pqx4 - EGFR, CDK6, MET, BRAF Gain 10pqx1 - RET, PTEN, FGF8, FGFR2 Loss 12q( )x3 CDK4 Gain 22pqx1 - CHEK2 Loss 3q( )x3 4p( )x6 6q( )x3 6q( )x3 7pqx4 9p( ) x1 9p( )x0 9q( )x3 10pqx1 11p( )x3 12q( )x3 13q( )x3 14q( )x3 22pqx1 TFRC Gain (3q) FGFR3 Amp (4p) EGFR, CDK6, MET, BRAF Gain (chr7) RET, PTEN, FGF8, FGFR2 Loss (chr10) CDK4 Gain (12q) CHEK2 Loss (chr22) Fusion: FGFR3-TACC3 (chr4)

10 Comparison of GBM3 WGV Regions IDH1 ROS1 TP53 NF1 IDH1:p.R132H:c.395G>A IDH1:p.R132H:c.395G>A Concordant Oncoscan Copy Number TST170 Copy Number TST170 Mutation and Fusions 2p( )x3 - MYCN, ALK Gain 5q( )x1 - FGF1, PDGFRB, FGFR4 Loss 6q( )x1 - ESR1 loss 9p( )x1 - JAK2 Loss 11q( )x3 - FGF19, FGF4, FGF3, ATM, CHEK1, CCND1 Gain 15q( )x1-2 - FGF7 loss 19pq( )x1 -AKT2, ERCC2, ERCC1, CCNE1 loss 1p( )x1-2 1p( )x1 1p( )x1 1p( )hmz 1q( )x1 2p( )x3 2p( )hmz 2q( )hmz 2q( )x1 3p( )hmz 4p( )x1-2 4q( )hmz 4q( )x1 5q( )x2-3 5q( )x1 6p( )hmz 6p( )hmz 6q( )x3 6q( )x1-2 6q( )x3 6q( )x1 6q( )x3 6q( )x1 8q( )hmz 9p( )x1 9p( )x0-1 9p( )x1 9q( )x1-2 10p( ) 11p( )x2-3 11p( )x3 11p( )x1 11q( )x3 12q( )hmz 12q( )x3 14q( )x1 15q( )x1-2 15q( )hmz 15q( )x1 15q( )hmz 15q( )x3 16p( )hmz 17p( )hmz 18p( )x1 18q( )x1 19p( )x3 19p( )hmz 19pq( )x1 22q( )hmz IDH1p.R132H:c.395G>A MYCN, ALK Gain (chr2) FGF1, PDGFRB, FGFR4 Loss (chr5) ESR1 loss (chr6) JAK2 Loss (chr9) FGF19, FGF4, FGF3, ATM, CHEK1, CCND1 Gain (chr11) FGF7 loss (chr15) AKT2, ERCC2, ERCC1, CCNE1 loss (chr19) IDH1:p.R132H:c.395G>A ROS1:c.5435_5446del:p.1812_1816del TP53:c.C700T:p.R234C NF1:c.A1985G:p.K662R

11 Comparison of GBM4 WGV Regions BCL6 PDGFRA CDKN2A TP53 Concordant Oncoscan Copy Number TST170 Copy Number TST170 Mutation and Fusions 1p( )x3 MYCL1 Gain 1q( )x5-6 - MDM4 Amp 3q( )x3 - PIK3CB Gain 3q( )x3 - PIK3CA, & TFRC Gain 4q( )x12 PDGFRA & KIT Amp 4q( )x3 - FGF5 Gain 6q( )x2 - ESR1 2 copies 7q( )x3 - CDK6, MET, BRAF Gain 9p( )x2 - JAK2 2 copies 19q( )x1-2 - CCNE1 Loss 1p( )x2 1p( )x3 1p( )x3 1q( )x3 1q( )x5-6 1q( )x3 1q( )x1-2 2q( )x3 2q( )x1-2 3p( )x3 3q( )x3 3q( )x3 3q( )x2 3q( )x3 4p( )x3 4p( )x3 4p( )x3 4pq( )x3 4q( )x12 4q( )x3 4q( )x3 4q( )x3 6q( )x2 7p( )x3 7q( )x3 9p( )x2 9pq( )x3 10q( )x3 10q( )x2 12pq( )x3 12q( )x5 14q( )x3 14q( )x2 17p( )hmz 18q( )x3 19p( )x5 19p( )x2 19p( )x5 19p( )x2 19p( )x5 19p( )x2 19p( )x2-3 19p( )x2 19q( )x1-2 19q( )x5 19q( )x2 19q( )x2 19q( )x2 19q( )x5 19q( )x5 19q( )x2 20q( )x3 21q( )x3 22q( )x3 22q( )x2 Xpq( )x2 Xq( )x3 Xq( )x2 MDM4 Amp (chr 1q) MYCL1 Gain (chr1) PIK3CB, PIK3CA, & TFRC Gain (chr3) PDGFRA, KIT Amp (4q) FGF5 Gain (chr4) ESR1 2 copies (chr6) CDK6, MET, BRAF Gain (chr7) JAK2 2 copies (chr9) CCNE1 Loss (chr 19) BCL6:c.G1724A:p.R575H PDGFRA:c.663_674del:p.221_225del CDKN2A:c.83_100del:p.28_34del TP53:c.G594A:p.M198I

12 Summary This is a small study, only 4 GBMs specimens. More detailed comparisons with a larger number of specimens are necessary to make any firm conclusions about using OncoScan vs TST170 for GBMs and gliomas There were some limitations in calling loss, particularly for CDKN2A, with TST170, however, we observed concordance for many copy number abnormalities TST170 had some clear advantages over OncoScan, particularly the ability to detect mutations in regions of CN- LOH (e.g. TP53 p.m198i) and identifying gene fusions (e.g. FGFR3-TACC3)

13 Emory Oncology Cytogenetics Lab Mike Rossi Debra Saxe Jean Williams Carol Phillips Kim Thompson Beth Matthews Cindy O Hare Adrianna Liebenberg Emory Molecular Pathology Lab Mike Rossi Charles E. Hill Omid Rouhi Geoff Smith Heather Jones Thank you!!! Emory Neuropathology Stewart Neill Jose E. Velazquez Vega Matthew Sneiderjan Daniel Brat Emory Neurosurgery Jeffrey Olson Illumina Ali Kuraishy Jenn Lococo Danny Chou Beena Murthy Cody Chivers James Mellott James Hudson