OncoKids SM Pediatric Cancer Panel

Transcription

1 Detection of mutations and gene fusions in pediatric hematologic malignancies and solid tumors with the OncoKids SM next generation sequencing based assay Jaclyn Biegel, Timothy Triche, D. Gigi Ostrow, Jonathan Buckley, Tracy Busse, Xiaowu Gai, Jianling Ji, Alexander Judkins, Dennis Maglinte, Gordana Raca, and Matthew Hiemenz

2 OncoKids SM Pediatric Cancer Panel Diagnostic mutations or gene fusions that will help determine prognosis and guide therapy Monitor disease remission and recurrence Distinguish relapse or metastasis from second tumor Identify novel therapeutics based on molecular features

3 OncoKids SM Pediatric Cancer Panel Primer-based target enrichment (Ampliseq) Interrogate DNA and RNA isolated from fresh, frozen, and FFPE tissue Small input (20 ng DNA and RNA) Sequencing on the Ion Torrent S5XL (2 hours) Analysis with Ion Reporter and custom tool for indels 3 day turn-around time

4 OncoKids SM was Designed Specifically for Use in Pediatric Cancers

5 OncoKids SM Validation 237 unique tumor samples (fresh bone marrow, frozen tumor, FFPE, isolated cdna, DNA) DNA component of the assay 102 single or multiple nucleotide variants (MNVs) 49 indels 9 gene amplification RNA component of the assay 51 fusions in 50 leukemia RNA samples (gene rearrangements detected by karyotype, FISH, array, RT-PCR, or NGS) 41 solid tumor RNA samples with fusions

6 OncoKids SM is Sensitive and Specific Single Nucleotide Variant: C to T InDel: 24 base deletion SMARCB1 c.118c>t, p.arg40* SMARCB1 c.20_43delgcaagaccttcgggcagaagcccginst (p.ser7ilefs*56)

7 Detection of DNA Amplifications is Highly Specific MYCN

8 Accuracy (DNA) 101 of 102 SNVs and MNVs were detected (99%) Low level germline mosaic (<5%) in RB1 was not detected 44 of 49 indels were detected (90%) ASXL1 c.1934dupg in an 8 G homopolymer track in 4 cases was not detected ASXL2 c.2090dupg in a 5 G homopolymer track in one case was not detected 9 of 9 amplification events detected as high copy number gains

9 A diverse range of fusions in leukemia samples were detected ATF7IP-JAK2 ETV6-NTRK3 P2RY8-CRLF2 RCSD1-ABL2 BCR-ABL1 ETV6-RUNX1 PAG1-ABL2 SSBP2-JAK2 BCR-JAK2 FIP1L1-PDGFRA PAK5-JAK2 STIL-TAL1 CRLF2-P2RY8 FOXP1-ABL1 PML-RARA TERF2-JAK2 EBF1-PDGFRB MLL Rearrangement RANBP2-ABL1 ZC3HAV1-ABL2 ETV6-ABL1 NUP214-ABL1 RBM15-MKL1 ZEB2-PDGFRB ETV6-JAK2 NUP98-NSD1 RCSD1-ABL1 ZMIZ1-ABL1

10 Representative solid tumor fusions CCDC6-RET Cllorf95-RELA EML4-ALK ETV6-NTRK3 EWSR1 Rearrangement FUS-CREB3L2 GOPC-ROS1 KIAA1549-BRAF NPM1-ALK PAX3-FOXO1 SS18-SSX1 Lung adenocarcinoma Ependymoma Lung adenocarcinoma Congenital mesoblastic nephroma Ewing sarcoma Fibromyxoid sarcoma High grade glioma Pilocytic astrocytoma Anaplastic large cell lymphoma Alveolar rhabdomyosarcoma Synovial sarcoma

11 Case example: pediatric patient with high grade glioma 4 y/o girl presented with 6 months of increasing left-sided weakness resulting in falls and ataxia MRI demonstrated a large right, intraventricular mass Pathology showed glioblastoma, small cell variant Most recent yrs showed no evidence of recurrent tumor

12 Single interstitial deletion with breakpoints in ROS1 and GOPC(FIG) ROS1 GOPC (FIG)

13 OncoKids SM Showed a GOPC-ROS1 Fusion in 283,317 Reads GOPC ROS1

14 GOPC-ROS1 Fusion Positive Glioblastoma May Respond to Crizotinib..GAT AAG GAA CTG GCA GGA AGT ACT CTT CCA ACC CAA GAG....Asp Lys Glu Leu Ala Gly Ser Thr Leu Pro Thr Gln Glu. Cancer Growth Metastasis. 2015; 8:51-60.

15 Accuracy (RNA) 47 of 51 fusions in the leukemia samples were detected ETV6 exon 5-RUNX1 exon 2 missed in 4 cases (primers not in the original design) 47 of 47 detected (100%) 33 of 41 fusions were detected in the solid tumor samples 4 brain tumors with KIAA1549-BRAF fusions, 2 tumors with CIC-DUX4 fusions primers not included in the assay so not detected 2 poor quality samples with predicted EWSR1 fusions were false negatives 33 of 35 (94%)

16 Performance and reporting criteria DNA Average read depth 5,000 X Average read length 120 bp 98% coverage at 250X Report SNVs at >5% allele frequency Report indels (<20 bp) at >10% allele frequency Report amplification at >8 copies RNA Evaluate expression of targeted and non-targeted fusions compared with 4 internal control genes Optimum read length 115 bp Read count for fusions varies from ,000 All novel fusions are confirmed by RT-PCR

17 Clinical test launched June 20, samples analyzed to date 11 cases with Tier 1 variants of strong clinical significance Metastatic solid pseudopapillary carcinoma with B-catenin mutation Angiomatoid fibrous histiocytoma with EWSR1-ATF1 fusion Diffuse midline glioma with H3F3A, ATRX, KRAS, PIK3CA and PPM1D mutations Sertoli leydig cell ovarian tumor with DICER1 mutation

18 Acknowledgements Jon Sherlock, Susan Ewald, John Bishop, Scott Myrand, Nick Khazanov, Jeff Smith, Janice Au Young, Chaitali Parikh, Jingwei Ni, Seth Sadis, Vinay Mittal and Dinesh Cyanam from Thermo Fisher Scientific. Cindy Fong, Jennifer Han and Sherry Yen for technical assistance. Moiz Bootwalla and Alex Ryutov for bioinformatics support. Clinical collaborators from University of Chicago, COG Biopathology Center, UCLA, University of Pennsylvania, Denver Children s Hospital, Children s Healthcare of Atlanta, Lucille Packard Hospital, OmniSeq, and ARUP who provided samples for analysis.