molecular testing in tumor samples - AIOM€¦ · Form a presentation by Dr Anne-Claire HARDY-BESSARD. Rapport DPN, IMG, DPI et formes héréditaires de cancers ... 1 642 2 445 3

Implications of

BRCA1/2

molecular testing

in tumor samples

Dr Etienne ROULEAU

SITUATION

27/05/2019TITRE DU DIAPORAMA Général

BRCA1/2 up-to-2016

Genes BRCA1 et BRCA2

Breast and ovarian cancer predisposition

Few therapeutic applications…

27/05/2019 TITRE DU DIAPORAMA Général

R Cancer

At 70

year-old

BRCA1 BRCA2

Breast Ovarian Breast

Controlat.

Breast Ovarian Breast

Controlat.

Mavaddat

& al. 2013

60%

(44-75)

59%

(43-76)

83%

(69-94)

55%

(41-70)

16.5%

(7.5-34)

62%

(44-79.5)

Form a presentation by Dr Anne-Claire HARDY-BESSARD

Rapport DPN, IMG, DPI et formes héréditaires de cancers

K breast, 31

year-old

K

breast,

44 year-

old

K prostate,

59 year-old

46 year-old 45 year-old 58 year-old

50 year-old89 year-old39 year-old89 year-old

63 year-old55 year-old

30 year-old 34 year-old 24 year-old

2 year-old

4 year-old

K ovary, 43

year-old

23

Leukemia

K

breast,

40

year-

old

BRCA1/2 testing in France

Germline testing in France – 16 labs

27/05/2019

2014 2015 2016

Total number of consultations dedicated to breast-ovarian syndrome

39 133 45 430 51 971

Among them, many consultations dedicated to isolated ovarian cancers

1 642 2 445 3 374

Regional average of the number of consultations per 100,000 inhabitants 55 63 72

Index case that has been genetically tested for breast-ovarian syndrome 11 688 14 700 17 821

Index cases identified as carriers of a mutation related to breast-ovarian syndrome 1 207 1 610 1 762

Proportion of carriers 10% 11% 10%

Screening timeline in weeks 33 34 24

Rapport d’activité de l’oncogénétique en France 2017, INCa.


O’Connor, Molecular Cell, 2015

Tumour cells harbouring deleterious BRCA1/2mutations are highly sensitive to PARP inhibition1

Efficacy of PARP inhibitors is clinically proven in ovarian cancer2-4, breast5 and prostate cancer6…

Inactivation process1

> Germline/somatic mutation

> Loss of wild-type allele

> (methylation of BRCA1)

Contrary to germline testing, tumour testing has additional considerations

Somatic testing in France > 25 labs 1. Ashworth A. J Clin Oncol 2008;26:3785–3790

2. Tutt A et al. Lancet 2010;376:235–244

3. Kaufman B et al. J Clin Oncol 2015;33: 244–250

4. Ledermann J et al. Lancet Oncol 2014;15: 852–861

5. Tutt, A et al J Clin Oncol 2009

6.Mateo, J., et al NEJM 2015

6.

To a therapeutic impact

Acceleration in BRCA testing

December 2014 – january 2018 Olaparib AstraZenecaTreatment / maintenance of mutated BRCA ovarian cancer

Treatment of mutated BRCA breast cancer

December 2016 Rucaparib Clovis Treatment mutated BRCA ovarian cancer

March 2017 Niraparib Tesaro Maintenance in ovarian mutated BRCA

October 2018 Talazoparib Pfizer Treatment of mutated BRCA breast cancer

Committee for Medicinal Products for Human Use (CHMP)

Summary of opinion post authorisationMEA

FDA

FDA approval - Ashworth 2019 AACR

New indications

Extension of indications in 2018 for French labs

At least 2 - Pancreas / Breast / Prostate 11/22

Breast 5/22

No extension / on demand 5/22

Post mortem 1/22


TEST CHALLENGING

Comparaison between blood and tumor

Blood – 99,2% concordant (Local / Central)

Tumor– 82,9% concordant (Local / Central)


392 Case included

2 WT 1 UV 388 MT

392 cases includes

23 no sample 27 failure

(7%)

341

324 MT

12WT

5 UV

Supplement to: Moore K, Colombo N, Scambia G, et al. Maintenance

olaparib in patients with newly diagnosed advanced ovarian cancer.

N Engl J Med.


cbioportal / 2016

20kb

Challenges to tumour testing

Heterogeneity of the material

> Limit of detection1,2

> Tumour samples are a mix of tumoral and normal cells3–5

> Tumour heterogeneity3–5

DNA degradation with fixation (FFPE – process from

operating theatre)6

> Adaptation of methods for detection

Low quantity of DNA extracted from sample6

> Choose methods with low DNA quantity

> Development of NGS

1. Ellison G et al. J Clin Pathol 2013;66:79–89

2. Lee HJ et al. Korean J Pathol 2013;47:52–60

3. de Bruin EC et al. Science 2014;346:2516

4. Gerlinger M et al. N Engl J Med 2012;366:883–892

5. Piotrowska Z et al. Cancer Discov 2015;5:713–722

6. Sah S et al. Genome Med 2013;5:77

DNA quality and quantity

Figure : Tapestation profiles of 3 DNA with the average size of

DNA A) usually profile 2kb, B) high quality C) Low quality

A)

B) C)

Quality impact

Size of the operative piece

Age of the block

Chemotherapy treatment

Various factors on the pre-analytical block

Co

nce

ntr

ation

ng

/µL

Cases n= 145

10% 4

20% 5

30% 4

2mm² 1

5mm² 1

Neoplastic cells

Surface

Genetiss 2018 – minimum requirement

survey of BRCA1/2 practices

Gustave Roussy – experience in DNA concentration

No requirement (9/22)

Germline testing (3/22)

Minimum cells (13/22)

Minimum surface

Minimum age

Performing the testing

BRCA1/2 = NGS screening

QUALITY STANDARD> Full coding sequence coverage

• 11/24 300X to more

• 12/24 give the result with the coverage if below to 300X

> Stability of the enrichment

> Sensitivity• 12/24 up to 5% - 11/24 up to 10%

> Ability to detect rearrangement or allelic drop-out

BIOINFORMATIC SOLUTION> Limit of detection: 10%

> Alterations• Insertion/deletion (>20 pb)

• Large rearrangements

Detection of large rearrangement

No 16

Bioinformatic analysis 6

Germline testing 1

MLPA 1

Genetiss 2018 – time to report

Less than 15 days 3

Less than 21 days 5

Less than 1 month 5

1 to 2 months 10

Genetiss 2018 – survey of BRCA1/2 practices

Performing the testing

Mainly amplicon and dedicated to BRCA1/2


2017 2018

Ion Proton (Life Technologies) 0 1

Ion S5 system (Life Technologies) 4 4

MiniSeq (Illumina) 1 1

MiSeq (Illumina) 14 14

NextSeq (Illumina) 1 2

PGM IonTorrent (Life Technologies) 2 2

2017 2018

Multiplicom 10 13

Agilent Sureselect 2 2

Qiagen 2 1

Thermofisher 5 5

Fluidigm 0 1

Sophia Genetics 1 2


LARGE REARRANGEMENTS

Large rearrangements

Lack of Enrichment - Large Rearrangements

> Coverage failure

> Enrichment - medium size rearrangements

Elimination during bioinformatic alignment

Imperfect compensation:

> Bioinformatic algorithm

> MLPA27/05/2019 TITRE DU DIAPORAMA Général

BRCA1/2

BRCA1/2

BRCA1/2

RGT

SNP

WT

Detection of large rearrangements

A) Deletion of exon 2 BRCA1 with MLPA, B) Deletion of

exon 2 BRCA1 from NGS normalization, C)

Duplication of exons13-14-15 BRCA1 from NGS

normalization

Technique XT Sureselect Agilent® - panel de 12 gènes

A)

B)

C)

QUANTITATIVE VARIATIONS

27/05/2019TITRE DU DIAPORAMA Général

Loss of heterozygosity

Main mechanism of inactivation : LOH + mutation

100% BRCA1 and 76% BRCA21

81% BRCA1 and 72% BRCA22

Secondary mutation <1%; 1/1363

Quantitative variation1

> Heterogeneity of alterations

> LOH of the mutated allele will enrich in mutated allele

> Disease progression can increase the allelic frequency of tumour

mutations, favouring detection of the mutated allele

LOH, loss of heterozygosity

1. Kanchi KL et al. Nat Commun 2014;5:3156;

2. Cancer Genome Atlas Research Network. Nature 2011;474:609–615; 3. TGCA ovarian cancer -

bioportal

B

A

50% A

50% B100% B

n=21

Note any obvious LOH on the allelic frequency of the mutation. 5

It is not necessary to report the allele frequency on the reports 9

Others 7


Resistance mechanisms

Risk of reversion to ‘wild-type’ BRCA1,2

> Potentially following platinum-based chemotherapy or PARP inhibitor treatment

> Loss of the mutated allele

> New mutations and/or loss of mutated allele

> Following resistance to treatment original germline mutation may no longer be detectable in tumour samples

23 - -

1. Ashworth A. J Clin Oncol 2008;26:3785–3790

2. Sakai W et al. Nature 2008;451:1116–1120

Resistance by

synthetic lethality

TAA

No protein

STOP codon

TAA

Mutation inframeGermline mutation with

premature STOP codon Truncated protein

Functionally active

Resistances tracking

Quigley D, et al.

Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of BRCA2 Reversion Mutations

Associated with Resistance to PARP Inhibitors. Cancer Discov. 2017 Sep;7(9):999-1005.

BRCA1/2 EQA

Gen&tiss experience

Some data presented here are preliminary results – thanks not to diffuse them


Numéro

d'échantillonCellularité % BRCA1 BRCA2 EEQ Divers (hors EEQ)

18-O-16 90%

c.2601del

p.(Gln867fs) VAF: 75%

Délétion des exons 14 à 24*

WT GENOTYPETP53: c.358A>G p.(Lys120Glu)

VAF:75%

18-O-17 80% WT/NC***

c.8487+1G>A

p.(?)

VAF: 90%

/ NC***

GENOTYPETP53.c.1024del p.(Arg342Glyfs*3)

VAF:75%

18-O-18 90% WT WT GENOTYPE -

18-O-19 90% WT WT GENOTYPE

TP53: c.713G>C; p.(Cys238Ser)

RAD51C : c.577C>T; p.(Arg193*)

VAF:95%

18-O-20 90% WT WT GENOTYPE

TP53 c.713G>A p.(Cys238Tyr)

RAD51D:c.803G>A (p.(Trp268*)

VAF:95%

Echantillon

éducatif "OVAIRE

2018"

NA Délétion des exons 1 à 14 WT EDUCATIFTP53: c.818G>A;p.(Arg273His)

VAF:75%

Genotyping

2017 : 3 false negative – 2,7%

> 110 analysis

> 22 participants

2018 : 3 false negative / 4 false positive – 5,6%

> 125 analysis

> 25 participants

Other genes 2018

> 2018 – TP53 – 5 participants – no error

> RAD51C – 3 participants detected

> RAD51D – 1 participant detected


Educative sample

2017

> BRCA1: 11/20 (55.0%) participants miss a class III variant

c.1303G>T; p.(Asp435Tyr) VAF 7%

> BRCA2: 10/20 (50.0%) participants miss a class V variant

c.8021dup; p.(Ile2675Aspfs*6) VAF 10%

2018

> Large rearrangement in BRCA1 : 5 / 21 (educational)

> Large rearrangement in BRCA1 : 2 / 25 (case 1)


VALIDATION AND REPORTING

THE RESULTS

Variants identified in BRCA1/2

Inactivating mutations – more than 75% are STOP

mutations

UMD BRCA1/2 database 2013 – French population – germline mutations

N=683 variants in BRCA1; N=624 deleterious variants in BRCA2

7% splicing

2% RGT

89% STOP

3% missense

8% splicing

11% RGT

77% STOP

Variants interpretation

Many databases (clinvar to national)

Currently, the assessment of variants

is done only for high-risk families and

only for this level of risk

Process of variant assessment

> High-risk family and co-segregation

> Histological data

> Functional data (RNA, protein)

> Co-occurrence (no bi-allelic BRCA1, rare bi-

allelic BRCA2)

Classe 1

= NEUTRE

= POLYMORPHISM

Classe 5

= DELETERE

Questions on the report

Tumor BRCA1/2 mutated / no germline reported

- 10/24 proposal to a new germline test to check

- 14/24 proposal to oncogenetic consultancy

Tumor BRCA1/2 non mutated / no germline testing

- 17/24 proposal to an oncogenetic consultancy

- 12/24 proposal to a germline testing for large

rearrangement

Variants

Any variant (UV-3) must be reported on the report - 20/23

A variant can be rendered with a therapeutic application, but without

application for the family (class 4) - 13/23

Oncogenetic consultation systematically for any variant (3, 4, 5) - 17/23

Variant interpretation

Estimation of the impact of a variant: 3 dimensions

Absence of

cancer predisposition

Presence of

cancer predisposition

Absence of a

therapeutic effectPresence of a

therapeutic effect

SENSITIVITY

RESISTANCE

Interpretation of variants

Gen&tiss 2017 – Interpretation 10 variants in the BRCA1/2 genes assessment from 19 labs.


BRCA1 BRCA1 BRCA1 BRCA1 BRCA1 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2

c.5408G>C c. 5545G>T c.4485-1G>T c. 68_69delExon 20

duplicationc.10137T>A c.8725A>T c.9609C>G c.8167G>C Exon 3 deletion

p.(Gly1803Ala) p.(Glu1849*) splicing p.(Glu23Valfs*17) LRG p.(Tyr3379*) p.(Lys2909*) p.(Tyr3203*) p.(Asp2723His) LRG

Class PARP-Inhibitor Familial counselling V V V V V I V V V V

V-Causal Yes Yes 20 11 17 24 9 1 15 21 25 22

IV -Likely causal Yes No 1 2 0 0 3 0 2 1 0 1

IV -Likely causal Yes Yes 2 5 8 1 5 0 6 2 0 2

IV -Likely causal No No 0 2 0 0 0 0 0 0 0 0

III -Unclassified variant No No 2 3 0 0 8 10 2 1 0 0

I- Polymorphism/Neutral No No 0 2 0 0 0 14 0 0 0 0

Score 1,72 1,16 1,68 1,96 1,04 1,56 1,52 1,8 2 1,88

BRCA1 BRCA1 BRCA1 BRCA1 BRCA1 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2

c.5408G>C c.562G>T c.4485-13

_4485-1delc.5221_5238del c.5117G>A c.10137T>A c.6842_6937del c.9609C>G c.8167G>C Exon 3 deletion

p.Gly1803Ala p.Glu188* splicing p.Val1741_His1746del p.Gly1706Glu p.Tyr3379* p.Glu2282_Gly2313del p.Tyr3203* p.Asp2723His LRG

Class PARP-Inhibitor Familial counselling V V IV III V I III V V V

V-Causal YES YES 16 15 3 1 4 3 3 18 18 14

IV -Likely causal YES YES 0 1 8 3 9 0 0 1 1 2

IV -Likely causal YES NO 1 3 4 2 6 0 1 0 0 1

IV -Likely causal NO NO 0 0 3 3 0 1 1 0 0 0

III -Unclassified variant NO NO 1 0 1 10 0 7 14 0 0 2

I- Polymorphism/Neutral NO NO 1 0 0 0 0 8 0 0 0 0

Score 1,74 1,79 1,79 1,21 1,21 1,21 1,47 1,95 1,95 1,63

Gen&tiss 2018 – Interpretation 10 variants in the BRCA1/2 genes assessment from 25 labs.

Interpretation of variants


Gen&tiss – comparison between 2017 and 2018

BRCA1 BRCA2 BRCA2 BRCA2 BRCA2

c.5408G>C c.10137T>A c.9609C>G c.8167G>C Exon 3 deletion

p.(Gly1803Ala) p.(Tyr3379*) p.(Tyr3203*) p.(Asp2723His) LRG

2017 1,74 1,21 1,95 1,95 1,63

2018 1,72 1,56 1,80 2,00 1,88

N %

Better 9 50%

Worse 5 28%

Same 3 17%

Twice full score 1 6%

Only 1 participation 8

EXTENSION TO OTHER GENES

What is the best HRD panel !


Présentation de Myriad Genetics

Beyond BRCA1/2

Panel HRD

Roy R, Chun J, Powell SN. BRCA1 and BRCA2: different roles in a common pathway of

genome protection. Nat Rev Cancer. 2011 Dec 23;12(1):68-78.

Che R, Zhang J, Nepal M, Han B, Fei P. Multifaceted Fanconi Anemia Signaling.Trends

Genet. 2018 Mar;34(3):171-183.

PARP

BRCA1BRCA2

Different panels

Thermofisher

Oncomine Comprehensive Assay v3

16 gènes impliqués dyear-old l’HRD

>30 gènes

TSO500, Illumina

Mutations and tissue

Cancer Genome Atlas Research Network Nature

474:609–615, 201

Heeke AL, Lynce F, Baker T, et al J Clin Oncol

35:1502, 2017 (suppl 15)

Prithard 2016

Norquist CCR2018

Girard, E et al. International Journal of Cancer

144, no. 8 (April 15, 2019): 1962–74.

Ovary

Prostate

Breast

Gènes Ovaire Sein Prostate

ATM

ATR

BARD1

BRCA1

BRCA2

BRIP1

BLM

CDK12

CHEK1

CHEK2

FAM175A

FANCA

FANCD2

FANCI

FANCL

FANCM

FANCC

MRE11A

NBN

PALB2

RAD50

RAD51

RAD51B

RAD51C

RAD51D

RAD52

ATRX

WHAT IS THE LEVEL OF

EVIDENCE ?

Limited clinical trials

Swisher EM et al. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1):

an international, multicentre, open-label, phase 2 trial. Lancet Oncol. 2017 Jan;18(1):75-87.

Limited functional data

Swisher. Lancet Oncology

Volume: 18 Issue 1 (2017)

Rucaparib in relapsed,

platinum-sensitive high-grade

ovarian carcinoma (ARIEL2

Part 1): an international,

multicentre, open-label,

phase 2 trial.

Limited functional data

No concordant data

Abstract 2476: Preclinical

assessment of the PARP inhibitor

rucaparib in homologous

recombination deficient prostate

cancer models

Minh Nguyen, Andrew D. Simmons

and Thomas C. Harding

BARD1, CDK12, FANCA, PALB2,

RAD51, RAD51C, RAD51D, and

RAD54L increased rucaparib

efficacy ≥ 2-fold in at least 1 cell line

examined

RAD51C

RAD51D

Effect on confirmed ovarian cancer risk

Seconds in frequency after BRCA1/2 in ovarian cancer RAD51C: 0.4 to 2% - constitutional mutations 3% - somatic mutations 3% RAD51D: 0.3 to 1.1% - constitutional mutations 5% - somatic mutations 0%

Kondrashova O et al Secondary Somatic Mutations Restoring <i>RAD51C</i> and <i>RAD51D</i> Associated

with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma. Cancer Discov.

2017 Sep;7(9):984-998

GREAT

12/21 labs want to move a large panel in 2019

Genomic BRCA and Extensive ovArian cancer Testing

> To date 66 declared centers / 23 associated platforms

> Ovarian cancer – prospective cohort

> Supporting the capture approach

> Basic panel : BRCA1/2 + RAD51C/D

> Largest panel possible

> No intervention – follow-up of variants for clinical outcomes

> Support by ARCAGY-GINECO


CONCLUSION

Conclusion

Improve again the quality of tumour testing

> Large rearrangement / Limit of detection

> Reversion detection

Extend molecular testing to other biomarkers

> Beyond BRCA1/2 : difficulty of scarcity - clinical evidence =>

Collecting data

Improve the variant interpretation

Improve the report harmonization

diane-latrille.com

http://www.diane-latrille.com/portfolio-item/pandore/

Thanks

Comité de pilotage

Jean-Christophe SABOURIN

Aude LAMY

Céline GARREC

Frederique PENAULT-LLORCA

Cécile AUBE

Anne CAYRE

Cédric LEMARECHAL

Laurent DOUCET

Clotilde DESCARPENTRIES

Isabelle QUINTIN-ROUE

Hélène BLONS

Jean-François EMILE

Jean-François COTE

Antoinette LEMOINE

Valérie DURANTON-TANNEUR

Yves DENOUX

Karen LEROY

Isabelle SOUBEYRAN

Véronique HADDAD

Paul HOFMAN

Florence PEDEUTOUR

Alexandre HARLE

Ludovic LACROIX

Alexander VALENT

Marc-Antoine BELAUD-ROTUREAU

Pierre-Jean LAMY

AFAQAP

Caroline Egele

Jean-Pierre Bellocq

Dominique Fetique

Gustave Roussy

Jean-Yves Scoazec

Isabelle Miran

Catherine Richon

Ludovic Lacroix

Sophie Cotteret

Birama Ndiaye

Biomedical Quality Assurance

Research unit of the University

of Leuven

Cleo Keppens

Kelly Dufraing

Els Dequeker

Lien Tembuyser

Veronique Tack

Departement of Pathology

of the Radboud University

Nijmegen Medical Centre

Marjolijn Ligtenberg

Han van Krieken

Institut National du Cancer

Frédérique Nowak

Etienne Lonchamp

Support financier :

UFR de Médecine Marseille

Genetics and Bioinformatics team

Christophe BEROUD

David SALGADO

Jean-Pierre DERIVES

UFR de Médecine - Nantes

Laboratoire de Biochimie

Marc DENIS

http://en.wikipedia.org/wiki/File:KUL.gif

Projects

Variant interpretation

Report harmonization

Standardization - artificial sample

> Large rearrangement

> Low allelic frequency on different mutations

> Reversion cases


Documents

molecular testing in tumor samples - AIOM€¦ · Form a presentation by Dr Anne-Claire HARDY-BESSARD. Rapport DPN, IMG, DPI et formes héréditaires de cancers ... 1 642 2 445 3