L’utilizzo delle nuove tecnologie genomiche nel carcinoma ovarico in pazienti BRCA mutati: quali informazioni?

L’utilizzo delle nuove tecnologie genomiche nel carcinoma ovarico in pazienti BRCA mutati: quali

informazioni?

Ettore Capoluongo*

*Full Prof. of Clinical Biochemistry and Clinical Molecular Biology

Head of Laboratory of Genomics and Molecular Diagnostics – Catholic University and Policlinico Gemelli Foundation and Research Hospital - Rome Italy, ID: Z4-10878 , Date of preparation Jun 2018; Date of expiry Jun 2019

1. Ovarian cancer and homologous recombination system

2. BRCA1/2 germline and somatic pipeline: issues and pitfalls 3. Extended gene panel in the era of PARP-1 inhibitor treatment 4. Our Experience on about 4000 OvCa patients

Agenda

Ovarian cancer and homologous

recombination system

+

• On 10,000 BRCA carriers: The cumulative risks of

developing OvCa prior to age 80 yrs were estimated to be:

– 44% for BRCA1 – 17% for BRCA2

• Highlighting the importance of these genes in the development of OvCa

Evidence on the potential importance of

 Family history

 Mutation location in risk assessment

BRCA1/2-related cancer risk

 ~ 25-30% of sporadic OvCa pts carry a BRCA mut

 ~ 8% carries a SOMATIC mutation in absence of a germline

 More than 50% of germline showed LOH at somatic level

 Contribution of other LOW-PENETRANT genes (?):under investigation

BRCA 1 - BRCA 2 and OvCa

Ovarian cancer Patients

The most common types of MUTATIONS ARE:

 SMALL INSERTION/DELETION FRAMESHIFT

 NONSYNONYMOUS TRUNCATION

 DISRUPTION OF SPLICE SITE

LEADING TO ENTIRE NONFUNCTIONAL BRCA PROTEINS

Large genomic rearrangements (LGRs) comprise about 10% of all mutations occurring in BRCA1 (due to its pseudogene)

BRCA 1 and BRCA 2

Genetic alteration

It is predicted that around 1 in 423 women carries a PV in either one of these genes

Approximately 50% of all HGSCs contain defects in HR at the time of diagnosis

GENE GERMLINE

PATHOGENIC VARIANTS

SOMATIC MUTATIONS (AVERAGE)

GENE FREQUENCY OF

PATHOGENIC VARIANTS

BRCA1 8-12% 5% (3-9%) BRCA1 0,05%

BRCA2 5-7% 3% (3-4%) BRCA2 0,068%

Our experience in more than 3,500 OvCa pts = 27% gBRCA positive + 6% somatically mutated

The commonest causes:

HGSC – High-Grade Serous Carcinoma PV – Pathogenic Variants Capoluongo E. Eur J Hum Genet 2016 Sep;24 Suppl 1:S1-2.

Evans DG et al. J Med Genet 2017.

Total BRCA1/2 mutation frequency by TUMOUR TYPE and in OvCa

Huang KL et al. Cell 2018. Gao J et al. Sci Signal 2013. Cerami E et al. Cancer Discov 2012.

23%

11%

10%

9%

8%

8%

7%

6%

6%

6%

6%

mBRCA1/2 frequency (%)

Ovarian Endometrial Gastroesophag ealBreast

CRC NSCLC Prostate Melanoma Cervix

Somatic BRCA in ovarian cancer: ~ 7%

Hollis RL and Gourley C. Cancer Biol Med 2016;13:236-47.

Patch AM et al. Nature 2015;521:489-94.

What about Large Genomic Rearrangements?

• Genomic rearrangements are composed of Structural Variations (SV)

 Deletions



 Inversions



 Umbalanced Translocations (transpositions)

 ……and others

• SV manifests as a join (or ‘breakpoint junction’) linking two segments of the genome not normally juxtaposed, typically associated with an increase or decrease in the copy number of adjacent genomic regions

Breast and Ovarian cancers can have

hundreds of tandem duplications

However,

LOF – Loss Of Function NGS – Next Generation Sequencing

• ARIEL2/3 study results suggest that biallelic LOF may bring about genomic instability

• Approximately 80–90% of gBRCA mt OvCa carriers show LOH of the wild-type allele

• If NGS-based assays are used on tumour tissue to play as predictive biomarkers, it may be appropriate to consider screening for biallelic germline loss-of-function in:

•

RAD51C and RAD51D

• BRCA1 and BRCA2

• NGS can predict such imbalances between BRCA1 and BRCA2

LOH and large BRCA1/2 rearrangements (2% in our experience)

BRCA1/2 germline and somatic pipeline: issues and pitfalls

Germline and somatic BRCA mutations are clinically relevant in OvCa: Great debate

1. National Cancer Institute. http://www.cancer.gov/dictionary?cdrid=46384 [accessed January 2018].

2. National Cancer Institute. http://www.cancer.gov/dictionary?CdrID=46586. [accessed January 2018].

3. Vergote I et al. Euro J Cancer 2016; 69:127-1.

Germline BRCA mutations

Tumour BRCA mutations

- Tumour sample

- Acquired mutations (somatic) found only in tumour cells² - 5-8% of ovarian cancer patients harbour BRCA

somatic mutation

BRCA mutations can be either germline or somatic

- Blood sample

- Inherited mutations found in all body cells¹

Germline BRCA mutations can be detected in a blood

sample³

Somatic BRCA mutations can be detected only in the

tumour sample³

A patient with ovarian cancer who is BRCA wild-type on germline testing would need subsequent tumour testing to

establish whether or not she has a somatic BRCA mutation, to determine management decisions

A patient with OvCa who has a BRCA mutation identified from tumour testing would

need subsequent germline testing to determine whether

there are implications for her relatives

Germline test

Tumour test

Tumour test

Germline test Negative Positive

Adapted from: Current perspectives on recommendations for BRCA genetic testing in ovarian cancer patients.

Vergote I et al. Eur J Cancer 2016;69:127-34.

Beginning with a tumour test, rather than a germline test, could be more cost-effective

2

Issue

Type of BRCA1/2 assay: Blood or tumour first?

tBRCA1/2 CNV is challenging

• Methods used to detect larger GERMLINE DELETIONS or DUPLICATIONS may not directly translate to analysis of FFPE-derived DNA due to:

– smaller DNA fragment size

– chemical modification of the DNA

– chromosomal copy number changes (aneuploidies) – instability, often present in tumour samples

FFPE – Formalin Fixed Paraffin Embedded NGS – Next Generation Sequencing CNV – Copy Number Variation

• Frequency of somatically acquired deletions and rearrangements in tumour tissue is UNKNOWN

• Bioinformatic approaches using NGS data analysis of CNV from capture enrichment strategies is recommended, if previously validated!

A pipeline established and validated on gBRCA1/2 testing

 MAY NOT be SUITABLE for tBRCA1/2 testing

 MAY REQUIRE MODIFICATION, depending on molecular pipeline

ISO 27001 is an internationally

recognised best practice framework for an

information security management system

Sanger method cannot be used to screen for LRGs

PIPELINE:

• The assay was unable to differentiate between g/sBRCA mutation: Need to check blood for germline mutations

CLINICAL SIGNIFICANCE:

• Neither the Myriad myChoice® HRD test nor Foundation Medicine T5 NGS assay could fully predict which patients were likely to benefit from

maintenance options

• The above topics raise important questions about the utility of genetic assays as

ARIEL3 STUDY

Coleman RL et al. Lancet 2017;390:1949-61.

Type of BRCA1/2 assay: BRCA or HRD-related genes?

• Germline mutations in BRCA1/2: The first biomarkers of HRD phenotype

• Literature data showed that NGS methods generally are highly accurate

• Sanger cannot be used in high throughput settings

• Main challenge: Characterization of the clinical significance of BRCA1/2 variants is under continuous monitoring and revision

• Germline LGR could be also detected by combining:

– MLPA – CGH

– Long PCR-based methods – SNP array

– Microsatellite analysis

– Long-reads NGS methods

NGS – Next Generation Sequencing HRD – Homologous Recombination Deficiency

NGS germline testing: Rationale

TUMOUR BRCA TESTING BLOOD BRCA TESTING

ADVANTAGES

• Can detect both somatic and germline mutations

• Identifies a greater number of patients who may benefit from maintainance options

• Validated methods are available and professionals are experienced in testing and interpreting variants

• Potentially requires less extensive genetic counselling at the outset, and less involvement for the wider family

• Reverted BRCA1/2 mutations can identify patients resistant to specific options

• Patient protocols, pathways and procedures are well established

• Analysis feasible in 100% of cases

• Useful for TMB • Sample is easily obtained and contains high-quality DNA

DISADVANTAGES

• Validated methods not yet widely available • Genetic profile of the tumour may change with disease progression

• Types of mutations not well defined: Increased number of VUS

• Sample with sufficiently high percentage of tumour cells/quality of DNA may be hard to obtain, leading to the need for repeat testing and/or biopsy

• Analysis not always possible for technical reasons

• Investment in new NGS technology may be required as most traditional methods are unsuitable due to limited DNA

• Requires additional expertise in pathology to determine sample adequacy

tBRCA1/2

tBRCA1/2

Free adaptation from Capoluongo E et al. Semin Oncol 2017;44:187-97.

BRCA1/2 tumour testing: Approach

How to choose the best technology?

CNV – Copy Number Variation LOH – Loss of Heterozygosity

AMPLICON HYBRID-CAPTURE

Workflow Moderate (between 1-2 days) Moderate (between 1-2 days) Cost per sample More expensive Less expensive

Pros Commercial solutions Scaleability

Failure rate around 1%

Cons Failure rate

Design +++

Off target

Few commercial solutions

DNA input 20 ng (Q-PCR) 10-50 ng (Qubit)

Applications Mutations Mutations

CNV

Large Genomic Rearrangements (depending on the captured regions) LOH?

• BRCA1/2 FFPE tumour testing should be performed on PRIMARY

• Fresh-frozen specimens (FFS) provide better

quality DNA = 70% of our processed samples

• 30%: FFPE

• Unfortunately, FFS are

FFS – Fresh Frozen Sections FFPE – Formalin Fixed Paraffin Embedded

What is the ideal sample to perform tBRCA1/2 assay?

Can BRCA1/2 FFPE analysis be implemented in routine practice?

High-quality diagnostic availability of NGS-based BRCA1/2-

Authors provide recommendations for BRCA1/2 PREDICTIVE TESTING of tumour tissue to clinical

decision making in OvCa pts

FFPE – Formalin Fixed Paraffin Embedded NGS – Next Generation Sequencing Endris V et al. Virchows Arch 2016;468:697-705.

TUMOR TESTING

HIGH QUALITY TUMOR

TESTING IS FUNDAMENTAL

3. Extended gene panel in the era of PARP-1

inhibitor treatment

What about multipanel genes ?

+

7.4%

Literature sometimes discordant

Frey and Pothuri, Gynecologic Oncology Research and Practice (2017) 4:4

34

Low S, Cancer Science. 2018;109:497–506

Risk assessment: Different panels available

The most frequently mutated genes in hereditary BC (combining all studies both somatic and germline):

ATM, RAD50, CHEK2, TP53, PALB2, MRE11A, CDKN2B-AS1, AP3K1, CASP8,

RAD51B, FGF10, NOTCH2, CCDC170, CYP1B1, LEPR, MYHFR and NQO1

Genetic alterations & phenotypic effect

Clinical importance of BRCA1/2

mutations has had the greatest impact in the management of Ovarian Cancer

Nesic et al., J Pathol. 2018;244(5):586-597

g/sBRCA1/2 testing still remains a MUST!

 Harmonisation of tBRCA testing: To be strongly encouraged

 Need to focus on the following critical questions:

• Type of starting samples: Fresh or FFPE tissue? And which tumour cell percentage?

• Fixation and tissues processing: What TAT and how many tissue slices?

• Type of pipelines: NGS alone or NGS coupled to MLPA?

• Chemistry: Target enrichment or PCR-based methods?

• Minimum acceptable coverage: Unique or method dependent?

• Gene panel: BRCA1/2 testing alone or BRCA coupled to HRD in case of BRCA1/2 negative results?

• Type of variants: How to undoubtedly discriminate the germline from somatic variants?

• Internal and external quality controls: Commercial, homemade or referred to international frameworks?

To partially respond to the first question (tumour versus germline), it would be interesting to underline

NGS – Next Generation Sequencing MLPA – Multiplex Ligation-dependent Probe Amplification VUS – Variants of Uncertain Significance TAT – Turn Around Time

Need of consensus paper

Our experience

NGS Lab Working Group – My «Golden Team»

Dr. Paola CONCOLINO Dr. Elisa DE PAOLIS Dr. Maria DE BONIS

Dr. Giorgia MAZZUCCATO Dr. Angelo MINUCCI

Dr. Roberta RIZZA

Dr. Concetta SANTONOCITO Dr. Giovanni Luca SCAGLIONE

Through the AZFastnet platform

About 7,000 gBRCA1/2 in the last 48 months

• 1500 fresh OvCa tissues: Under analysis (only tumour testing)

• 600 FFPE OvCa tissues corresponding to gBRCA1/2 mut+/-

• 90 ascitic liquids

3500 OvCa pts (G+S)

Peculiar case report 1

 OvCa HGSOC pts: 40 yr

 gBRCA1 ex 20-21 del assessed by MLPA+MAQ

 FFPE analysis (40% NC): only a slight bioinformatic prediction of CNV change at ex 20-21

 Additional BRCA1 somatic variant: p.Lys223Argfs (ex 10; c.668delA); Allele cov: 4500x;

28% AF During the study

setting

In this case, without germline story:

 The germline deletion would not probably have been identified (family and personal risk assessment missing)

 PARP-1i treatment would have been ensured by FFPE BRCA assay

Reasons:

1. Tumor relapse (not Primary tumor) 2. Clonal switch?

3. Germline story is very important!

FFPE Relapse Primary Fresh tumour

Devyser BRCA kit, an NGS-based mutation detection system for BRCA1/2 testing

Large tissue rearrangement (LTR) – Case report 2

B1 loss or B2 gain?

Fluctuation ranging 0.6≤1.0≤1,6:

BRCA1/2 CNV not imbalanced 99% senstivity

and secificity

PREDICTION reached on FFPE tBRCA1 Exons 18 e 19

Large germline rearrangement (LGR) – case 3

Confirmed on GERMLINE 0.2

corresponding

to LOH

Reversal mutation: how to check for?

 New strategies are needed to overcome tumor resistance mechanisms, such as secondary mutations that revert BRCA genes to wildtype, to both platinum agents and PARP-inhibitors

Gynecol Oncol Rep. 2017 Aug; 21: 57–60

STOP CODON IN BRCA2

GERMLINE BRCA SAMPLE (BLOOD)

One of our reverting patients – Case 4

GERMLINE SOURCE TISSUE SOURCE

Somatic BRCA testing on III recurrence HGSOC: NGS data

BLOOD: BRCA GERMLINE (c.2494G>T in heterozygous status) I RELAPSE: FFPE

(c.2494G>T in heterozygous status)

II RELAPSE: FFPE (c.2494G>T in heterozygous status)

III RELAPSE: FFPE

SHIFT IN MELTING PROFILE DUE TO COEXISTENCE OF c.2494G>T (p.GLU832TER) AND c.2494G>C (p.GLU832GLN)

NGS data confirmed by targeted High Resolution Melting Analysis (HRMA):

Comparison between blood and relapses HGSOC (I-II-III)

All confirmed by Sanger

Peculiar Case 5: SOMATIC BRCA SAMPLE

(FRESH FROZEN TISSUE)

INTERPRETATION STILL REMAINS THE BIGGEST CHALLENGE

SOMATIC?

Germline with SOMATIC LOH?

SOMATIC with LOH?

Driver or Passenger?

Same criteria for the evaluation of G o S variants??

Continous management of VUS and new variants

56

VUS: Class 3

Which findings?

57

tissue

blood

Tumor-specific mutations

(a) The 20 most common mutations in OvCa in the COSMIC database

(b) The most commonly mutated genes in recurrent ovarian cancer in this study, the ranking of mutant gene with higher frequency rate in drug-resistant recurrent ovarian cancer and sensitive recurrent OvCa.

Main issues: TAT

Type of issues Level of importance

Hospital organisation: Surgeon + pathologist inside High Number of patients managed/year High

Dedicated personnel High

Presence of certified and centralised molecular lab Very high Presence of oncology department Moderate

Presence of clinical genetic unit High

TUMOR BOARD HIGH and MANDATORY

TAT: SHORTER IF FRESH TISSUES ARE USED

Type of Hospital organisation does matter

Stakeholder management can be challenging as new stakeholders are

involved compared to gBRCA testing (e.g. surgeons, pathologists)

“It is important that cancer centres, pathology

departments and molecular diagnostic laboratories develop effective communication strategies and

standard operating procedures (SOPS) for the biomarker testing and reporting of results”

PREMISE

Stefania Gori, Ettore Capoluongo, Antonio Russo, et al;

«Recommendations for the implementation of BRCA testing in ovarian cancer patients and their relatives »

Critical Reviews in Oncology-Hematology, 2019 in press