Molecular diagnosis of

developmental tumors:

Opportunities and challenges of NGS

Dra. Carmen de Torres

Department of Oncology. Developmental Tumor Biology Laboratory

Hospital Sant Joan de Déu, Barcelona

2

Adult cancer versus

developmental tumors

Marshall GM, et al. Nat Rev Cancer 2014

3

NGS: Somatic alterations in

developmental tumors

Lawrence MS, et al. Nature 2013

4

Pediatric and adult

high-grade gliomas

Jones and Baker, Nature Rev Cancer 2014

5

Diagnosis-Research

Diagnosis

• Somatic alterations

• Diagnosis

• Prognosis

• Minimal residual disease

• Therapeutic targets

• Germline predisposition to cancer

Research

- Neuroblastoma

- Sarcomas

- CNS tumors

- Retinoblastoma

- Leukemia

6

Somatic alterations: Diagnosis

SMALL ROUND BLUE

CELL TUMORS

➢ EWING’S SARCOMA/PNET

➢ RHABDOMYOSARCOMA, other

sarcomas

➢ NEUROBLASTOMA

➢ WILMS

➢ LEUKEMIA, LYMPHOMA

7

Somatic alterations: Diagnosis

• Translocation-associated sarcomas

- Chimeric transcription factors

- Chimeric tyrosine kinases

• Karyotypically complex sarcomas

Lineage

Prognosis

Driver alterations

Taylor BS, et al. Nat Rev Cancer 2011

8

Somatic alterations: Diagnosis

KIAA1549-BRAF

7q34

Forshew T, et al. J Pathol 2009

Dougherty MJ, et al. Neuro Oncol 2010

9

Somatic alterations: Diagnosis

Butrynski JE, et al. N Engl J Med 2010

10

Translocations: Methods

11

Translocations: Methods

Interphase fluorescent in situ hybridization (i-FISH)

?

12

Translocations: Methods

Pierron G, et al. Nat Genet 2012

13

Translocations: Methods

1: A673 (EWSR1-FLI1 type 1)

2: SK-ES-1 (EWSR1-FLI1 type 2)

3: A4573 (EWSR1-FLI1 type 3)

4: Biopsy

5: NTC

Messahel B, et al. Lancet Oncol 2005

RT-PCR and sequencing

TBP

EWSR1-FLI1

14

Translocations: Methods

RNA-seq

15

Somatic alterations: Prognosis

Huang M, et al. Cold Spring Harb

Perspect Med 2013

Brodeur GM. Nat Rev Cancer 2003

16

Somatic alterations: Prognosis

Northcott PA, et al. Nature 2012

Robinson G, et al. Nature 2012

17

Somatic alterations: Prognosis

18

- Precise staging

- Local-regional tumors with MRD

- Involvement of CNS

- Response to treatment

- Early detection of relapses

Minimal residual disease: clinical relevance

19

Minimal residual disease: methods

20

MRD: Neuroblastoma and retinoblastoma

y = -1.1773x + 5.9808R² = 0.9989

0

1

2

3

4

5

6

7

0 1 2 3 4 5

dC

T

Log Y79 dilution

CRX mRNA

y = 3.8747x - 3.0715R² = 0.9994

-4

-2

0

2

4

6

8

10

12

14

0 1 2 3 4 5

dC

T

Log SK-N-JD dilution

PHOX2B mRNA

21

Liquid biopsy

• BRAF V600E cfDNA

• CRX mRNA CSF

0

100

200

300

400

500

600

0 2 4 6 8N

um

be

r o

f co

pie

sWeeks on treatment

MRD: Langerhans cell

histiocytosis

22

MRD: ddPCR vs RT-qPCR

• Hayashi M, et al. Highly personalized detection of minimal Ewing sarcoma

disease burden from plasma tumor DNA. Cancer. 2016;122:3015-23 – tumor-

specific EWS-ETS breakpoints; animal models

• Cavalli M, et al. Comparative analysis between RQ-PCR and digital droplet

PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone

marrow of early stage follicular lymphoma. Br J Haematol 2017;177(4):588-

596

23

MRD: NGS vs RT-qPCR

Ladetto M, et al. Next-generation sequencing and real-time

quantitative PCR for minimal residual disease detection in B-cell

disorders. Leukemia 2014;28:1299-307

- compared immunoglobulin heavy-chain-gene-based minimal

residual disease (MRD) detection by real-time quantitative PCR

(RQ-PCR) and next-generation sequencing (NGS)

- NGS showed at least the same level of sensitivity as

allele-specific oligonucleotides-PCR, without the need for patient-

specific reagents

24

Precision medicine

25

Somatic alterations: Therapeutic targets

4 weeks on dabrafenib

Courtesy O. Cruz

Pleomorphic xanthoastrocytoma

BRAF V600E

26

NGS: Somatic alterationsin developmental tumors

Nat Genet. 2014;46:457-61

Proc Natl Acad Sci U S

A. 2014;111:E5564-73

Cancer Discov. 2014;4:1326-41

27

Diffuse intrinsic pontinegliomas (DIPG)

Nat Genet. 2014;46(5):457-61

- Not curable

- First human

disease driven by a

histone mutation

- ACVR1

28

DIPG

Nat Genet. 2014;46(5):457-61

Two molecular and

clinical subgroups

• H3.3 (K27M or

G34V/R)

• H3.1 (K27M) and

ACVR1

H3.3 and H3.1 - Nat Genet. 2012;44(3):251-3

29

Complementary genomic approaches

highlight the PI3K/Mtor pathway as

common vulnerability in osteosarcoma

Proc Natl Acad Sci U S A. 2014 ;111:E5564-73.

30

NGS: Germline alterations in

developmental tumors

Bardai A, et al. N Engl J Med 2015

31

NGS: Diagnosis, prognosis and

therapeutic targets

Worst BC, et al. Eur J Cancer 2016

Next-generation personalised medicine for high-risk paediatric cancer

patients - The INFORM pilot study

32

33

First molecular tumor

board: selection of

case-specific best

options

Second molecular

tumor board: final

decision

34

Robinson et al. J Clin Oncol 2015

35

FGFR1

p.N577K - GOF

Missense

Pkinase_Tyr

Int J Mol Med. 2016 Jul;38(1):3-15

dcc.icgc.org

36

Nat Commun. 2015; 6: 7557

Germline and somatic / variants and CNVs

• Diagnosis, prognosis

• Therapeutic targets

• Germline predisposition

Pediatric solid tumor

panels

37

Variants and CNAs

Somatic and germline

Diagnosis, prognosis,

therapyInterpretation

Precisionmedicine for

pediatric solidtumors

38

Data

Interpretation

39

• Patient

• Sample

• Date of biopsy – clonal evolution

• Quality

• Liquid biopsy - metastasis - primary tumor

• Sequencing

• Panels / exome / genome

• Variants / Copy number alterations

• Quality

• Variant calling

NGS for therapy selection:

Challenges

40

NGS for therapy selection:

Challenges

• Interpretation of variants: LOF / GOF

• Treatment selection:

• Level of evidence

• Context – tumor dependent

• Avatar

• Multidisciplinary team

• Report: Context!

• TAT

41

Gen Variante Tumor Fármaco Evidencia Ensayos clínicos

Evidencia en el

mismo tipo de

tumor

-

Evidencia en

otros tumoresCCND2 p.W149* Pulmón Palbociclib Ensayo clínico I/II NCT02154490

PIK3R1 p.Y580C Endometrio, otros Inhibidores PI3K Preclínica, caso

42

Acknowledgements

HSJD lab team

HSJD clinical team

IMEGEN

N López-Bigas

CNAG

J Biegel

C Jones

Broad Institute

MSKCC

B Royer-Pokora

43

Muchas gracias

© 2015 IMEGEN – Información confidencial. Todos los derechos reservados.

Instituto de Medicina Genómica SL

Agustín Escardino 9,

Parc Científic de la Universitat de València

46980 Paterna (Valencia, España)

+34 963 212 340

Imegen.es

© 2015 IMEGEN – Información confidencial. Todos los derechos reservados.