Molecular Genetics of Paediatric Tumours. Gino Somers MBBS, BMedSci, PhD, FRCPA Pathologist-in-Chief Hospital for Sick Children, Toronto, ON, CANADA

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1 Molecular Genetics of Paediatric Tumours Gino Somers MBBS, BMedSci, PhD, FRCPA Pathologist-in-Chief Hospital for Sick Children, Toronto, ON, CANADA

2 Financial Disclosure NanoString - conference costs for TriCon Molecular Medicine Conference, San Francisco, 2015

3 Molecular Genetics of Paediatric Tumours Introduction and methodologies Paediatric sarcomas* Neuroblastoma* Wilms tumour Concluding remarks

4 Molecular Genetics of Paediatric Tumours Discovery of recurrent genetic abnormalities in paediatric tumours transformational more precise diagnostic accuracy (e.g. EWSR1) discovery of prognostic markers (e.g. NMYC) paradigm shift in tumour classification (e.g. DICER1) better understanding of biology

5 Methodologies Traditional G-banding cytogenetics FISH RT-PCR Newer and Emerging Array technologies NanoString assay Next generation sequencing (NGS)

6 Methodologies Traditional G-banding cytogenetics FISH RT-PCR Newer and Emerging Array technologies NanoString assay Next generation sequencing (NGS)

7 Paediatric Sarcomas

8 Paediatric Sarcomas Two major genetic subtypes: Simple genome with recurrent diagnostic abnormalities (e.g. Ewing sarcoma) Complex genome without recurrent diagnostic abnormalities (e.g. embryonal RMS) Recurrent abnormalities used for diagnosis tumours with fusion transcripts

9 Paediatric Sarcomas Tumors Selected genetic abnormalities Technology Ewing family of tumours Rearrangements of EWSR1; rare non-ewsr1 NanoString/NGS rearrangements (e.g. CIC/DUX4 & FUS/ERG) Synovial sarcoma SYT gene rearrangements NanoString/NGS Rhabdomyosarcoma, alveolar FOXO1 gene rearrangements NanoString/NGS BCOR sarcomas BCOR/CCNB1 gene rearrangement NanoString/NGS Infantile fibrosarcoma ETV6/NTRK3 fusion transcript Osteosarcoma conventional Complex karyotype; ALT; chromothripsis SNP array Osteosarcoma low grade Amplification of MDM2 and CDK4 SNP array Chondrosarcoma extraskeletal myxoid EWSR1/NR4A3 fusion gene NanoString/NGS Chondrosarcoma mesenchymal HEY1-NCOA2 fusion gene NanoString/NGS Chordoma Monosomy 1 and 7; gain of 7q33 SNP array

10 Paediatric Sarcoma fusion transcript detection Input Reverse transcription FISH RT-PCR NanoString Fresh tissue OR FFPE slides 500ng (fresh) to 2ug (FFPE) RNA No Yes No ng RNA, FFPE or fresh Throughput 1 target per assay 1 target per assay 800 targets per assay Cost (CAD) $400 $120 $300 TAT 48 hours 72 hours hours Bioinformatics no no minimal

11 NanoString Assay Reporter Tag Capture Tag Biotin Probe A Probe B Reporter Tag Sequence Unique tag sequence assigned to each target sequence Nucleic acid target (fusion) Universal Capture Tag Sequence Same tag sequence used for all target sequences

12 NanoString Results IMT DSRCT AFH SS EWS Advantages of NanoString Assay: 1. No RT or amplification step 2. Many more probes can be tested 3. Relatively inexpensive Disadvantages of NanoString Assay: 1. Need exact sequence of bpt to hybridize 2. Not able to discover novel transcripts 3. Addition of new probes requires revalidation ARMS Normal

13 Paediatric rhabdomyosarcomas emerging molecular abnormalities Rhabdomyosarcoma: MyoD1 L112R mutations higher frequency in spindle/sclerosing ERMS; poor outcome NCOA2 fusions < 1 year old, spindle cell morphology FOXO1 gene amplification associated with PAX7 partner >> PAX3 partner FGFR4 & pathway gene mutations potential therapeutic targets

14 Paediatric sarcomas emerging molecular abnormalities Ewing sarcoma CDKN2A copy number loss confers a poorer outcome NR0B1 microsatellites role in susceptibility to fusion transcript growth promotion STAG2 mutations seen at increasing clonal frequency with increased stage of disease Synovial sarcoma CCND1 & KRAS mutations in older patients Small percentage with CTNNB1 gene mutations and WNT pathway activation

15 Paediatric Sarcomas - summary Recurrent abnormalities fusion transcript detection Complex genomes copy number changes +++ Emerging molecular abnormalities

16 Neuroblastoma

17 Neuroblastoma Critical to have additional molecular genetic data to classify risk NMYC and 1p the classical abnormalities with prognostic significance Several other changes now associated with prognosis: Segmental copy number changes (especially 11q loss or 17q gain) = unfavorable Whole chromosomal copy number changes = favorable Single gene mutations (ALK, Phox2B, PTPN11, ATRX) Need molecular data for complete report

18 MYCN amplification by FISH MYCN (2p23) Aneuploid tumour MYCN amplification in tumour

19 1p deletion by FISH 1p36 1q control Normal result (2R, 2G) Tumour with 1p deletion (1R, 2G) 1R2G

20 Challenge: FISH vs array technology FISH assay: rapid TAT iterative one probe per assay becomes expensive with > 2 probes Array/SNP assay slower TAT MUCH more data expense inversely proportional to number of loci reported

21 Chromosome 1-1p deletion FISH Signal pattern Patient Control 1q25x2 & 1p36x1 (del1p) 159/200 0/200 1q25x2 & 1p36x2 (normal) 26/ /200

22 Chromosome 2 - MYCN amplification FISH Signal pattern Patient Control MYCN signals/nucleus 16/200 0/200 MYCN CN= MYCN signals/nucleus 90/200 0/200

23 Whole Genome View Example 1 - Numerical chromosome alterations good prognosis cnloh cnloh cnloh cnloh DNA index = 1.35

24 Whole Genome View Example 2 - Segmental chromosome alterations poor prognosis DNA index ~ 1

25 Neuroblastoma Testing Algorithm

26 Neuroblastoma - Single Gene Mutations ALK, ARID1/ARID1A, ATRX poorer outcome Phox2B rare; plays a role in familial neuroblastoma

27 Neuroblastoma Summary Classical abnormalities NMYC, 1p deletion Broader genome-wide abnormalities whole chromosome vs. segmental change Single gene abnormalities

28 Wilms Tumour

29 Wilms tumour genetic abnormalities LOH 1p and 16q: portends a worse prognosis detectable using SNP arrays Mutations in TP53 associated with anaplasia associated with more aggressive disease and poorer outcome Mutations in DROSHA gene Associated with more aggressive blastemal-predominant tumours Inherited syndromes BW syndrome, WT1 mutations

30 Future Directions NGS in the clinic Liquid biopsy 3D assays

31 Approach to Paediatric Tumour Diagnostics Morphology Ancillary Molecular 1 Molecular 2 H&E Immunostains Electron microscopy NanoString FISH SNP NGS RNA seq Liquid biopsy

32 Contact details Address: Division of Pathology Hospital for Sick Children 555 University Avenue Toronto, ON M5G 1X8