Implications of BRCA testing in cancer

(1)

Implications of BRCA1/2

molecular testing in tumor samples

Dr Etienne ROULEAU

(2)

SITUATION

27/05/2019TITRE DU DIAPORAMA Général

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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.

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

(4)

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-old 89 year-old

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

2 year-old

4 year-old

K ovary, 43 year-old

3 2

Leukemia

K

breast, 40 year- old

(5)

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.

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O’Connor, Molecular Cell, 2015

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⚫ Tumour cells harbouring deleterious BRCA1/2

mutations are highly sensitive to PARP inhibition¹

⚫ Efficacy of PARP inhibitors is clinically proven in ovarian cancer^2-4, 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

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Acceleration in BRCA testing

December 2014 – january 2018 Olaparib AstraZeneca Treatment / 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

(9)

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

(10)

TEST CHALLENGING

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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.

(12)

cbioportal / 2016

20kb

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Challenges to tumour testing

⚫ Heterogeneity of the material

> Limit of detection^1,2

> Tumour samples are a mix of tumoral and normal cells^3–5

> Tumour heterogeneity^3–5

⚫ 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

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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)

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Quality impact

⚫ Size of the operative piece

⚫ Age of the block

⚫ Chemotherapy treatment

⚫ Various factors on the pre-analytical block

Concentration 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

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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

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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

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LARGE REARRANGEMENTS

(19)

Large rearrangements

⚫ Lack of Enrichment - Large Rearrangements

> Coverage failure

> Enrichment - medium size rearrangements

⚫ Elimination during bioinformatic alignment

⚫ Imperfect compensation:

> Bioinformatic algorithm

> MLPA

BRCA1/2 BRCA1/2 BRCA1/2 RGT

SNP WT

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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)

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QUANTITATIVE VARIATIONS

27/05/2019TITRE DU DIAPORAMA Général

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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% B

100% 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

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Resistance mechanisms

Risk of reversion to ‘wild-type’ BRCA^1,2

> 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 inframe Germline mutation with

premature STOP codon Truncated protein

Functionally active

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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.

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BRCA1/2 EQA

Gen&tiss experience

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

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Numéro

d'échantillon Cellularité % BRCA1 BRCA2 EEQ Divers (hors EEQ)

18-O-16 90%

c.2601del p.(Gln867fs) VAF: 75%

Délétion des exons 14 à 24*

WT GENOTYPE TP53: c.358A>G p.(Lys120Glu) VAF:75%

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

c.8487+1G>A p.(?) VAF: 90%

/ NC***

GENOTYPE TP53.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 EDUCATIF TP53: c.818G>A;p.(Arg273His)

VAF:75%

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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

(28)

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)

(29)

VALIDATION AND REPORTING

THE RESULTS

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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

(31)

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

(32)

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

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Variant interpretation

Estimation of the impact of a variant: 3 dimensions

Absence of cancer predisposition

Presence of cancer predisposition Absence of a

therapeutic effect Presence of a

therapeutic effect

SENSITIVITY RESISTANCE

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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_69del Exon 20

duplication c.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

_4485-1del c.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.

(35)

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

(36)

EXTENSION TO OTHER GENES

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What is the best HRD panel !

Présentation de Myriad Genetics

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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

BRCA1

BRCA2

(39)

Different panels

Thermofisher

⚫ Oncomine Comprehensive Assay v3

⚫ 16 gènes impliqués dyear-old l’HRD

(40)

>30 gènes

TSO500, Illumina

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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

(42)

WHAT IS THE LEVEL OF

EVIDENCE ?

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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.

(44)

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.

(45)

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

(46)

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

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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

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CONCLUSION

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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

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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

(51)

Projects

⚫

Variant interpretation

⚫

Report harmonization

⚫

Standardization - artificial sample

> Large rearrangement

> Low allelic frequency on different mutations

> Reversion cases