Molecular Genetics of Paediatric TumoursGino Somers MBBS, BMedSci, PhD, FRCPAPathologist-in-ChiefHospital for Sick Children, Toronto, ON, CANADA

Financial Disclosure

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

Molecular Genetics of Paediatric Tumours

Introduction and methodologies

Paediatric sarcomas*

Neuroblastoma*

Wilms tumour

Concluding remarks

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

Methodologies

Traditional

G-banding cytogenetics

FISH

RT-PCR

Newer and Emerging

Array technologies

NanoString assay

Next generation sequencing (NGS)

Methodologies

Traditional

G-banding cytogenetics

FISH

RT-PCR

Newer and Emerging

Array technologies

NanoString assay

Next generation sequencing (NGS)

Paediatric Sarcomas

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

Paediatric Sarcomas

Tumors Selected genetic abnormalities Technology

Ewing family of tumours Rearrangements of EWSR1; rare non-EWSR1

rearrangements (e.g. CIC/DUX4 & FUS/ERG)

NanoString/NGS

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

FISH RT-PCR NanoString

Input Fresh tissue OR FFPE slides

500ng (fresh) to 2ug (FFPE) RNA

50-200ng RNA, FFPE or fresh

Reversetranscription

No Yes No

Throughput 1 target per assay 1 target per assay 800 targets per assay

Cost (CAD) $400 $120 $300

TAT 48 hours 72 hours 72-120 hours

Bioinformatics no no minimal

Paediatric Sarcoma – fusion transcript detection

Reporter Tag SequenceUnique tag sequence assigned to each target sequence

Universal Capture TagSequenceSame tag sequence used for all target sequences

Biotin

Nucleic acid target (fusion)

Capture Tag

Probe A Probe B

Reporter Tag

NanoString Assay

Normal

DSRCT

AFH

IMT

SS

EWS

ARMS

NanoString Results

Advantages of NanoString Assay:1. No RT or amplification step2. Many more probes can be tested3. Relatively inexpensive

Disadvantages of NanoString Assay:1. Need exact sequence of bpt to hybridize2. Not able to discover novel transcripts3. Addition of new probes requires revalidation

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

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

Paediatric Sarcomas - summary

Recurrent abnormalities – fusion transcript detection

Complex genomes – copy number changes +++

Emerging molecular abnormalities

Neuroblastoma

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

Aneuploid tumour MYCN amplification in tumour

MYCN

(2p23)

MYCN amplification by FISH

1p deletion by FISH

1R2G

Tumour with 1p deletion (1R, 2G)

1p36

1q control

Normal result (2R, 2G)

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

Chromosome 1 - 1p deletionFISH Signal pattern Patient Control

1q25x2 & 1p36x1 (del1p) 159/200 0/200

1q25x2 & 1p36x2 (normal) 26/200 199/200

Chromosome 2 - MYCN amplification

MYCN CN=37

FISH Signal pattern Patient Control

10-20 MYCN signals/nucleus 16/200 0/200

20-50 MYCN signals/nucleus 90/200 0/200

Whole Genome View

DNA index = 1.35

cnLOH cnLOH cnLOH cnLOH

Example 1 - Numerical chromosome alterations – good prognosis

Example 2 - Segmental chromosome alterations – poor prognosis

DNA index ~ 1

Whole Genome View

Neuroblastoma Testing Algorithm

Neuroblastoma - Single Gene Mutations

ALK, ARID1/ARID1A, ATRX – poorer outcome

Phox2B – rare; plays a role in familial neuroblastoma

Neuroblastoma Summary

Classical abnormalities – NMYC, 1p deletion

Broader genome-wide abnormalities – whole chromosome vs. segmental change

Single gene abnormalities

Wilms Tumour

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

Future Directions

NGS in the clinic

Liquid biopsy

‘3D’ assays

Approach to Paediatric Tumour Diagnostics

Morphology

• H&E

Ancillary

• Immunostains

• Electron microscopy

Molecular 1

• NanoString

• FISH

• SNP

Molecular 2

• NGS

• RNA seq

• Liquid biopsy

Contact details

Email: gino.somers@sickkids.ca

Address: Division of Pathology

Hospital for Sick Children

555 University Avenue

Toronto, ON M5G 1X8