molecular testing the why,the when and which tests? · molecular testing the why,the when and ......

Molecular Testing

the Why,the When and

Which tests?

Samuel Murray PhDBioMarker Solutions Ltd

London, UK

Molecular Pathology Board – EMQN, UK

Disclosures

• Consultancy/Advisory/Contractual

• Amgen, AstraZeneca, Bayer Healthcare, BioCartis, Biodesix, Biomarker Solutions, BioPath, BMS, Boehringer Ingelhiem, Celgene, GSK, Horizon Dx, Life Technologies, Merck, Nanostring, Phillips, Siemens, Thermo Fisher

Molecular Testing

Why?

Biomarker criteria A biomarker is a characteristic that is objectively measured and

evaluated as an indicator of normal biologic processes,

pathogenic processes, or pharmacologic responses to a

therapeutic intervention

Based on sound scientific evidence

– Reproducible in different tumour types

– Understood mechanistically

Can be measured reproducibly with high sensitivity and

specificity using the patient material

Can be repeated over time, on subsequent biopsies/samples

Has a clinically relevant impact on treatment

Biomarkers Definitions Working Group, Clin Pharmacol Ther 2001;69(3):89-95

Level I P phaseIII/M-ALevel II P phaseIIILevel III RetrospectiveLevel IVLevel V

Why do we need biomarkers?

A: Reduce otherwise unnecessary exposure to

treatment

B: reduce toxicity burden

C: cost benefit obvious to health provider

D: improve TTP and OS

Standard vs individualized treatment

Prognostic vs Predictive:an important distinction

Some markers can have both predictive and

prognostic value

Prognostic

Provides information

on overall patient

outcome, regardless

of which treatment is

used

Predictive

Provides information

on efficacy of a

specific therapy

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

YEARS

SU

RV

IVA

L

318 205 112 67 48 25260 229 143 77 37 24360 254 123 61 44 26

driverNoTxdriverTx

noDriver

(A)(B)(C)

Courtesy of Scagliotti AIOM

Lung Cancer ConsortiumSurvival by group

Squamous cell cancer

Non squamous cancer

Platinum + 3rd generation agent

EGFR WT, ALK neg,

ROS 1 neg

EGFR mut +

ALK/ROS1rearranged

EGFR-TKI

Crizotinib

clinically selected

clinically unselected

Platinum + Pemetrexed

Platinum-based doublet

+ Bevacizumab

Oncogene addicted

KRAS mut+

Advanced NSCLC in 2013

Squamous cell cancer

Platinum + 3rd generation agent

Non-oncogene addicted

EGFR mut +

ALK/ROS1rearranged

EGFR-TKI

ALK-TKI

clinically selected

Platinum + Pemetrexed

Oncogene addicted

RET fusion Cabozantinib

HER2 mut +

clinically unselected

BRAF mut +

Trastuzumab/Afatinib/Dacomitinib

Vemurafenib/Dabrafenib ±Selumetinib/Trametinib

Platinum-based doublet + Bevacizumab

Non-oncogene addicted

Non squamous cancer

Oncogene addicted

FGFR-1 amplification

DDR2 mut +

FGFR-1 inhibitors

Dasatinib

PI3KCA mut + or amplified BKM120

PI3KCA mut + or amplified

BKM120

KRAS mut+

Immune regulatory PDL/PDL1 + MoAbs + smi’s

What we thought of 2016 in 2013

"Here's my sequence”

Cancer Treatment TODAY!!

Basket Diagnostics

to match

Basket Drugs

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

YEARS

SU

RV

IVA

L

318 205 112 67 48 25260 229 143 77 37 24360 254 123 61 44 26

driverNoTxdriverTx

noDriver

(A)(B)(C)

IPASS: Comparison of PFS by mutation status within treatment arms

EGFR M+

HR=0.48, 95% CI 0.36, 0.64

p<0.0001

EGFR M-

HR=2.85, 95% CI 2.05, 3.98

p<0.0001

0 4 8 12 16 20 24

Time from randomisation (months)

0.0

0.2

0.4

0.6

0.8

1.0

PF

SGefitinib EGFR M+ (n=132)Gefitinib EGFR M- (n=91)Carboplatin / paclitaxel EGFR M+ (n=129)Carboplatin / paclitaxel EGFR M- (n=85)

Treatment by subgroup

interaction test, p<0.0001

HR 2.85 HR 0.48

Curtesy of Dr Tony Mok

HR = 0.48

Maemondo M, et al. NEJM 2009

Mitsudomi T, et al. Lancet Oncol 2009

RAS

Sartori-Bianchi A, et al. PLoS One 2009

KRAS is not alone

Sartori-Bianchi A, et al. PLoS One 2009

Reflex testing in Colorectal Cancer

KRAS

NRAS

BRAF

PIK3CA

PTEN

Molecular Testing

Which?

Molecular Pathology EQA Programmes - Oncology

inc:tRNAtDNAtCNV

Liquid BiopsiesSignaturesGenetics

IHCGCN

.European Thoracic Oncology Platform (ETOP).

European Society of Medical Oncology (ESMO).

United Kingdom National External Quality Assessment for Molecular Genetics (UKNEQAS).

European Society of Pathology (ESP).

Italian Association of Medical Oncology (AIOM).

European Molecular Genetics Quality Network (EMQN).

Global..

FDA – EMA

• For Molecular Pathology

- typically look for a 510K submission leading to CE-IVD

• This requires in general:

a) Precision Analysis = reproducibility

b) Clinical Validation = equivalence to an existing Dx

(this is OK for EGFR for example BUT for a Novel marker?)

Sensitivity and Specificity are included within these categories

Circumvention in USA = Lab Developed Test (LDT) under

CLIA conditions

ISO15189 and EQA

• Any diagnostic TEST (MDx) even if CE-IVD, requires to

fall under ISO15189 regulations

• Part of this process is to EXAMINE the laboratory

performing the specific test

• In most countries this requires, or shall soon mandatorily

require participation in an External Quality Assessment

Scheme (EQA)

What do patients/oncologists want?

1. Genotyping from clinically relevant samples

2. A quick turn around

3. A true answer

4. A clear report

At present Molecular

Pathology EQA schemes SCORE 21 different report

specifications

WHEN?

WHICH?

How Often?

WHERE?

issues around what to test

How much tumour is needed? What % of the sample

should be tumour?

Discordance in mutations

between primary tumour

and metastatic disease

Mutational

Heterogeneity within the

same lesion

Does the test sample

ADEQUATELY represent

the patient’s disease?

How many tumour cells?

BRAF Melanoma heterogeneity

WT n=423 MUT n=246 Primary

WT n=358

84.6%

36.7%

MUT n=173

70.3%

3.1%Alter Genotype

Metastasis

30.1% ALTER GENOTYPE UPON METASTASIS

3.0% (20)Mixed Genotype

67

15.5%Mut - WT

38

9.0%WT - Mut

WT = 132 (57.4%) Mutant = 97 (42.2%)

Mutant-Mutant77 (79.4%)

WT-Mutant

18 (13.6%)

Mut-WT15

(15.5%)

Primary

WT-WT107 (81.1%)

Metastasis

Mutant = 95 (41.3%)

WT = 123 (53.5%)

Total “Switch” = 20.0%

Mixed= 12 (5.2%)

1 7 5

Mixed-WT: 1WT-Mixed: 7 (5.3%)Mutant-Mixed: 5 (5.2%)

1 (0.4%)

EGFR NSCLC Heterogeneity

Heterogeneity

VEGFR-2(FLK-1/KDR)

pY1175pY1214

VEGFR-1(FLT-1) VEGFR-3

(FLT-4)sVEGFR-1 NRP-1 NRP-2

Raf

MEK-1/2

ERK-1/2

Ras

PI-3K

PLCg

PKC

Akt

S6K

Src

eNOS

Rac

CDC42

p38 MAPKFAK

Paxillin

Migration Vascular Permeability SurvivalProliferation

VEGF-CVEGF145VEGF165PlGF-2

VEGF-AVEGF-CVEGF-D

VEGF-CVEGF-D

VEGF165VEGF-BPlGF-2

VEGF-AVEGF-B

PlGF

p42/ p44 MAPK

DNA RNA ProteinTranscription Translation

ReverseTranscription

Degradation

Regulation

(Genetic Code)

Pallis A, Murray S, et al. Curr Med Chem. 2011;18: 1613-28.

Lack of information on, & understanding

of interaction!!!

Little integration of Genetic and Epigenetic

data via Decisional Algorithms

EGFR Gene Gain35%

NSCLCSomatickRAS

Mutations20%

SomaticEGFR

Mutations15-32%

MET gene Gain2-5%

IGF-1R?

VEGF/R?

EML4-ALK 3-5%

BRAF3-5%

HER23-5%

PTEN?

MET3-5%

PI3CA2%

FGFR42%

P53?

Pallis A, et al. CMC2011

Predictive Biomarkers in NSCLC

…more than 50% of NSCLCs harbor mutations in known oncogenes for which targeted therapies have been

developed…

Somatic CRAF1%

Predictive Biomarkers in Melanoma

SomaticNRAS 10%

SomaticERBB4

Mutations20%

Somatic ARAF1%

Somatic FLT3%

Somatic cKIT7%

SomaticBRAF

Mutations43%

Somatic PIK3A10%

Somatic PTEN10%

PTENLoss10%

?

Somatic GNA11

6% SomaticGNAQ3%

?

SomaticMEK1

1%

SomaticMEK2

1%

70-75-80% Coverage

NSCLCSomatickRAS

Mutations20%

SomaticEGFR

Mutations15-32%

EGFR Gene Gain35%

MET gene Gain2-5%

IGF-1R?

VEGF/R?

EML4-ALK 3-5%

BRAF3-5%

HER23-5%

PTEN?

MET3-5%

PI3CA2%

FGFR42%

P53?

Predictive Biomarkers

www.somaticmutations-EGFR.net

Exon Total

18(41 aa’s)

19(32 aa’s)

20(61 aa’s)

21(52 aa’s)

(186 aa’s)

Amino acids affected 27

(65.8%)

27

(84.4%)

39

(63.9%)

46

(88.5%)

139

(74.7%)

Insertions &/or Duplications 0 5 35 0 40

Deletions &/or Deletional-Insertions 3 94 18 2 117

Point Mutations 50 34 51 67 202

Total 53 133 104 69 359

GXGXXG K DFG L L Y

G719 L858K745-I759 T790

687 728

729

762

761

823

824 875

18 19 20 21

L861

2 5 7 13

18-24 25-28EGFR Exons

EGF

Binding

EGF

Binding TMTyrosine kinase domain Autophosphorylation

domain

17

Exon 18

Exon 19

Exon 20Exon 21

Unknown

www.somaticmutations-EGFR.net; Murray S, et al. J Thorac Oncol 2008; 3:832-839

Linardou H, et al. Nat Rev Clin Oncol 2009;6:352-366.]

Spectrum and Complexity of Biomarkers

CK2/PAK/Src

439KTLGRRDSSDDWEIPDGDQITVGQRIGSGSFGTVYKGKWHGDVAVKL485

* * * * ** * * *

PKCa

578KSNNIFLHEDLTVKIGDFGLATVKSRWSGSHQFEQLSGSILWM620

********* * * ********* ********* ******

S140

G-loop

ATP bindingdomain

CR3

Activationsegment

C764N Kinase Domain

CR1 CR2RBD Z-CRD

V600

ERK1/2S397T404S409

K483

S729

N-region

AKT/PKAS365

B-Raf

S750S126

80% BRAF V600

However: there are over 60 different mutations involving 40 codons

Clinical utility?

Unknown

Bypass tracksEGFR MTKRAS MT

No ALKamp or mut

ALKamp

ALKmut

ALK+

Mechanisms of resistance

Anti-EGFR Anti-ALK




3. A true answer

4. A clear report



specifications

day 0 day 1 day 2

Weekend

day 5 day 6 day 7 day 8

Guidelines sample-to-result workflow

day 3 day 4

Path2Dx

1

1. Request

2. Sample On-site/Off-site?

3. Pathology retrieval and annotation

4. MDx LIMS

5. Dx

6. Reporting

1 2 3 4 5 6

TAT = 5-6 days




3. A true answer

4. A clear report



specifications

25% of the

Worlds BEST

Molecular

Pathology labs

FAIL at EGFRTesting

?? What will happen with

a SIGNATURE

Technique Sensitivity Mutations

(% of mutant DNA)

Direct sequencing 20 Known and new

TaqMan PCR 10 Known only

Loop-hybrid mobility shift assay 10 Known only

Pyrosequencing 5 Known and new

PCR-SSCP 5 Known and new

dHPLC 5 Known and new

Cycleave PCR 5 Known only

PCR-RFLP and length analysis 5 Known only

MALDI-TOF MS-based genotyping 5 Known only

Scorpions ARMS - Thera screen 1 Known only

PNA-LNA PCR clamp 1 Known only

Single-molecule sequencing 0.1 Known and new

Mutant-enriched sequencing 0.1 Known only

SMAP 0.1 Known only

Medium sensitivity

High sensitivity

Low sensitivity

assessing EGFR mutations

Exon 18

Exon 19

T790M

Resistance Mutation

Exon 21

Coverage 82-87%

Are ALL CE-IVD

EGFR kits the same?

(TheraScreenEGFR29 Mutation Kit; DxS/Roche)

9/25 exon 19 microdeletions missed!.....(35 %)

Penzel R, et al. Virchows Arch 2011:458:95-98

Sequencing Vs

ARMS

In a multicentre analysis of 1,047 cases

163 patients harbored mutations

32% of the mutations

were not detected by ARMS!!!!




3. A true answer

4. A clear report



specifications

Oncotype Patient Report

ER+ve, HER2-ve, Node –ve/ +ve, Pre- and Post-menopausal

Page 1: Risk of Recurrence

Page 2: Chemotherapy Benefit

Page 3: Quantitative ER, PR and HER2

Chemotherapeutic

agent(s) CMF

Does this relate to

‘Todays’ standard

of care??

No Clinical

Indication for

mRNA

quantification of

ER, PR or HER2

Summary

1. MDx must be clinically relevant – prognostic

- predictive

- differential Dx

2. Short TAT

3. Available to many

4. CE-IVD (or LDT under ISO15189)

5. Include relevant biomarkers (exclude irrelevant biomarkers)

6. Legible to Clinician and Patient

7. Reproducible (specimens and inter-intra-laboratory)

8. TRUE

Molecular Testing

Which & When?

NSCLC Testing Algorithm

Test for

EGFRTest for

ALK/ROS1/RET

NEXT

BRAFHER2

-ve

Test for

KRAS

+ve

No anti-EGFR

0% RR

15% W

10% A

-ve

-ve

+ve20% W

40% A

EGFR inhibitor+ve

12% W

12% A

ALK inhibitor

35-60%

T790M

35-60%

ALK mutationsPD

Phase III

Stage III/IV

at Diagnosis

NGS panel

± FISH

LOD >1%

Liquid biopsies?

Also REQUIRE

1. Re-Testing at relapse

2. Liquid biopsy retesting as 30% fail rate

all Biomarkers and Resistance markers

Predictive Biomarkers for chemotherapy in NSCLC

Mostly RETROSPECTIVE analyses evidence for PREDICTIVE markers in advanced

NSCLC studies

in general, low levels = sensitivity • ERCC1 - platinum • Thymidylate Synthase (TS) - pemetrexed • RRM1 - gemcitabine • BRCA 1 - platinum

Chemotherapy

CRC Screening Algorithm

RAS mutation WT

WTBRAF mutation

Anti-EGFR MoAb

HL MSI+

BRAFMut

Sporadic

HNPCC

Screening HNPCC

MSH2 MLH1 MSH6 PMS2

1/3 incidence in

MSI-H/BRAFWTOncotype

‘Stage II’

L H

Surveilance

RAS typically

Codons

12,13,59,61,117,146

Test RAS

at

diagnosis

Mini NGS panel

(5 genes + MSI)

or

conventional

ASPCR

LOD not below 1%

EPCAM

50% BRAF No KIT20% NRAS

10% BRAF 2% KIT10% NRAS



25% GNAQ50% GNA11

Melanoma Testing Algorithm

Metastatic Melanoma

Up front NGS panel

testing(5 genes)

Liquid Biopsies will be an option very shortly for :

Diagnosis+ Monitoring

We need POC devices

High level of intra-inter

tumor heterogeneity

questions repeat testing

if WT

Note: Only BRAF

Validated

Biomarker When Test

cerbB2/HER2 @Diagnosis ISH

Signature @Diagnosis (see below)

BRCA1/2 (hereditary) + Counselling

(see below)

TNBC BRCA1/2 following 2nd ER/PR/HER2 check

BrCa Testing Algorithm

Germline mutational analysis

APC

HNPCC

BRAC1/2

CDKN2A

RET

etc

Hereditary Genetics

ASCO Policy Statement on Genetic Testing for Cancer Susceptibility

Genetic testing should be offered when the following conditions apply:

an individual has a personal or family history suggestive of a genetic cancer susceptibility syndrome

the results of the test can be interpreted

testing will influence medical management

Genetic testing

Affects all family

psychological…

ethical…

social…

legal…

genetic

discrimination…

privacy…

testing children?…

Therefore:MANDATORY Informed Consent and Counselling

BEFORE and AFTER genetic testing

BRCA1 PenetranceLIFETIME RISK

early onset Breast Cancer 65%

(55-85% by age 70,

up to half by age 50)

Ovarian Cancer40%

(95% CI 18-54%)

Second Primary 40-60%

Breast Cancer

( 5% per yr … vs…

1% per yr for sporadic Br Ca)

Also, greater risk (2-3 fold) for :

Ca cervix, uterus, fallopian tube,

pancreas, stomach, colon, prostate

BRCA2 PenetranceLIFETIME RISK

Breast Cancer

45%(40-85% by age 70)

Ovarian Cancer 25%

Male

Breast Cancer

6-8%

BRCA2 families (2-5 fold increased risk):

Ca pancreas, melanoma and young onset (<65 yrs) prostate Ca, also

laryngeal, gallbladder, bile duct, stomach (unknown magnitude)

Breast Cancer Ovarian Cancer

Standard Germline Panel (NGS)

BRCA1

BRCA2

P53

PTEN

CDH1

ATM

CHEK2

STK11

PALB2

52%

32%

16%

“BRCAness” (NGS)

BRCA1

BRCA2

Testing in Tumor to

identify both somatic and

germline mutations

Commercially available gene panels:

Quality ControlReproducibility/Precision

Clinical Validation

Multigene Predictors ProEx ™ Br No Predictive Ability

Mammostrat ® No Predictive Ability

ExagenBC ™ No Predictive Ability

Oncotype DX ™ YES

Breast Cancer Two-Gene Expression Ratio (H/I™) No Predictive Ability

Celera Metastasis Score ™ No Predictive Ability

The Breast BioClassifier No Predictive Ability

MammaPrint® No Predictive Ability

The Rotterdam Signiture No Predictive Ability

Invasiveness Gene Signature No Predictive Ability

NuvoSelect ™ YES

(response to preoperative Paclitaxel/5-FU/doxorubicin/cyclophosphamide; and response to endocrine therapy in ER-ve patients)

Cytochrome p450 CYP2D6 Genotyping YES

(resistance to tamoxifen)

Oncotype Dx Mammaprint Blueprint EndoPredict Prosigna

Company Genomic Health Agendia Agendia Myriad/Sividon Nanostring

Technologies

CE/FDA cleared No/No Yes/Yes No/No Yes/No Yes/Yes

Model Centralised Centralised Centralised Decentralised Decentralised

Genes 21 70 80 11 58

Patient character Pre-Post M

ER+

HER2-

Node +/-

Pre-Post M

ER+/-

Node -

all HR+

HER2-

Node +/-

Post M

HR+

Node +/-

Classification Low-Int-High Low-High Low-High Low-Int-High

Sample FFPE Frozen Frozen FFPE FFPE

Individual Risk Yes No No No Yes

Characteristics

Not CE-IVD? Are they Validated?

IOM Statement on: Turning a signature into a Diagnostic

The RIGHT way

- Independent sample sets for each

phase, discovery, training and

validation

The WRONG way

- use of a (same) sample set for

discovery/training and validation

Gene Signature vs One Gene?

HER2 Basal 1 Basal 2 Luminal 1 Luminal 2 Luminal 3

p53p53

Sotiriou PNAS 2003

IACR - PC

• Open Scheme – >50 participants

– genotype

– reporting

– interpretation

– Material Issues!!!!

– Sensitivity assessment with alternative EQA scheme(s)

c. Open phase

• Schemes will also incorporate assessment of pathologists criteria for

determination of %NCC and acceptability of material for analysis

Global EQA for gene Signatures

Thank you!!

Thank you!!

Global EQA for gene Signatures

Significant sample challenges

Close to cut offs

Across different

phylogenies

Breast Cancer

Genetic heterogeneity in familial Melanoma

Represent approximately 2% of all Melanoma patients

molecular testing the why,the when and which tests? · molecular testing the why,the when and ......

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