5‐6‐2015

1

External quality assesment in Europe

Predictive markers as game changer

Han van Krieken, 

Nijmegen, the Netherlands

Prognosis

• Differentiation/grade

• Extend (TNM)

• Additional features

• Angioinvasion

• Protein expression

Prediction

Concentrate therapeutic interventions on patients likely 

to benefitEfficacy

.

EfficiencySpare expense in patients

not likely to benefit

Predictivebiomarkers

Spare potential sideeffects in patientsnot likely to benefit

Safety

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2

Prediction

• Dependant on tumor type

• The same gene alteration has different effects in different tumors

External quality assessment more important than ever

• The result of a single test determines clinical management

• Proces/Technology more complicated

• Rapid changes in scope

QA is a key part of Good Laboratory Practice, GLP

Test Facility Organization and Personnel

FacilitiesApparatus, materials and reagents

Quality Assesmentprogramme

Components of GLP:

Test systems Test & reference items

Standard operating procedures

Performance of the study

Reporting of study results

Storage and retention of records and materials

g

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3

Validation and verification of diagnostic tests

New diagnostic test

e.g. • Laboratory developed methods

• Existing tests used outside of approved scope

Existing, approved test

e.g. • Commercial CE-IVD kits

• Approved tests with defined performance specifications

VALIDATIONVALIDATION

Define performance specification: accuracy, limitations, controls

VERIFICATION

Compare performance with existing specifications

Test implementation

Continual validation

Performance monitoring and audits

ESP Quality Assurance Programme

2008: With support from Amgen, multidisciplinary group formsand makes proposal for European QA Programme

2012: The multidisciplinary group establishes guidelines toharmonize EQA schemesin molecular pathology

EV0313

2014: June–Sept online registration open for 2014−2015 ESP Colon EQA scheme

2009: First European KRAS EQA scheme pilot tests in 11 expert centres

Regional EQA roll‐out approves 55 centres

2010−12: Annual KRAS EQA schemes award 296 approvals in32 countries

2013: ESP Colon EQA scheme expands to include KRAS, NRAS and BRAFtesting

2015: June–Sept results of2014−2015 ESP Colon EQA scheme

EGFR Belongs to the ErbB Family of Cell Surface Receptors

ErbB1

(EGFR)ErbB2

(HER2/neu)

ErbB3

(HER3)

ErbB4

(HER4)

Inhibition of EGFR dimerisation and downstream

signalling that includes RAS proteins

EGFREGFREGFR EGFR HomodimerHomodimer

RASRASGTPGTP RASRAS

GDPGDP

EGFEGF

TGFTGF‐‐αα PanitumumabPanitumumab

EGFREGFR

Yang XD, et al. Cancer Res 1999; 59:1236-43; Foon KA, et al. Int J Radiat Oncol Biol Phys 2004; 58:984-90;

Hecht JR, et al. Proc Am Soc Clin Oncol 2004; 22:A3511; Crawford J, et al. Proc Am Soc Clin Oncol 2004; 22:A7083.©2007 Amgen Inc. All rights reserved©2007 Amgen Inc. All rights reserved

GDPGDPRAFRAF

MEKMEK

ERKERK

ElkElkMycMyc

JNKJNK

JunJun

JNKKJNKK

PAKPAK

NckNck

RacRac

PLCPLCγγ

PPKCKC

PPTENTEN

PPI3KI3K

S6KS6K

AKTAKT

mTORmTOR

ProliferationProliferationAntiAnti‐‐apoptosisapoptosisAngiogenesisAngiogenesisSurvivalSurvivalMetastasisMetastasis

FosFos

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KRAS Is Important in Growth and Cell Division—Mutations Can Cause Cancer

• Ras proteins are GTPases• Ras family members include: 

KRAS, NRAS, and HRAS

• Normal cycle occurs between a

GDPinactive

GDPinactive Normal: 

– Growth

– Proliferation

– Differentiation

• Normal cycle occurs between a GDP‐bound (inactive) and a GTP‐

bound (active) form of Ras

• Specific mutations in the KRASgene result in a constitutively 

active protein

GTP

activeGTP

active

GDP

GTP

inactive

active

*

GDP

GTP

inactive

active

*Schubbert S, et al. Nature Rev Cancer. 2007;7:295‐308.

ABNORMAL: 

– Growth

– Proliferation

– Differentiation

Cet + FOLFIRI(n=178)

FOLFIRI(n=189)

No. events 130 154

Median OS 28.4 20.2

(95% CI) (24.7–31.6) (17.0–24.5)

HR 0.69 (0.54–0.88)

p=0.0024

OS: KRAS wt vs. RAS wt

KRAS wt (exon 2) RAS wt

Cet + FOLFIRI(n=316)

FOLFIRI(n=350)

No. events 242 288

Median OS 23.5 20.0

(95% CI) (21.2–26.3) (17.4–21.7)

HR 0.796 (0.670–0.946)

p=0.0093

Data on file

Cetuximab + FOLFIRI

FOLFIRICetuximab + FOLFIRI

FOLFIRI

EPAR cetuximab, june 2014

Implications for current practice in mCRC

Expanded RAS analysis of 1st‐line mCRC patients improved the benefit/risk profile of EGFR‐targetedimproved the benefit/risk profile of EGFR targeted 

therapy

There are many different RAS testing methods

IVDmethods

Laboratory based methodsNon‐commercial “in‐house” methods

Commercial test kits

Conform to Directive 98/97/EC and carry CE marking

RUOmethods

Not validated for use in patient IVD

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5

Laboratory based RAS testing methods

Gel electrophoresis Sequencing

• Temporal temperature gradient • Denaturing gradient• Constant denaturant capillary

electrophoresis SSCP

• Sanger sequencing

• Pyrosequencing

15 ‐ 20%3

5%3

Allele-specific PCR Other• SSCP

• Allele-specific amplification based on primer design(ARMS, REMS, FLAG, RFLP)

• Ligation detection reaction (LDR, LDR spFRET)

• Coamplification at lower denaturation temperature (COLD)

• Hybridization assays

• HRMA (screening)

Limit of detection

0.1 ‐ 1%2

~10%3

Conventional ‘Sanger’ sequencing

PCR

Sequence

KRAS TP53BRAFEGFR

PB1 PB2

Next generation sequencing

PCR

KRASTP53

BRAFEGFR

PCR

Sequence

PB3

PB4

Dias nummer 19

PB1 KRAS & NRAS?Peter Burdon; 09-09-2013

PB2 Commnet: suggest to indicate number of amplicons to be sequenced for each genePeter Burdon; 09-09-2013

Dias nummer 20

PB3 KRAS & NRAS?Peter Burdon; 09-09-2013

PB4 List total number of actionable amplicons?Peter Burdon; 09-09-2013

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6

OncoNetwork Consortium: 8 experienced labs

in colon and lung cancer diagnosis

Prof. Orla SheilsTrinity College Dublin, Ireland

Dr. Marjolijn Ligtenberg & Dr. Bastiaan TopsRadboud University

Nijmegen Medical CentreThe Netherlands

Prof. Ian CreeWarwick Medical School United Kingdom

Dr. Cristoph Noppen & Dr. Henriette Kurth

VIOLLIER AG Basle Switzerland

Dr. Nicola NormannoCentro Ricerche Oncologiche

Mercogliano, Italy

Prof. Pierre Laurent PuigUniversité Paris Descartes, France

Dr. Ludovic LacroixInstitut Gustave Roussy Paris, France

Prof. Aldo ScarpaARC-NET University of

Verona Italy

VIOLLIER AG Basle, Switzerland

Alain RicoRosella Petraroli

RAS testing

• A tumour sample is sent to the laboratory for analysis• The test can use fresh, frozen or paraffin-embedded tissue

• A pathologist confirms that the tissue is cancerous and selects test materialse ec s es a e a

• A sample of DNA is prepared for the RAS test

• The polymerase chain reaction (PCR) is used to amplify the DNA sample and test for RAS mutation status

The ESP Colon EQA scheme

ESP/European QA Council 

European QA Coordinator for KRAS testing

Regional QA programmes

Centralised QAprogramme

OECD Guidelines for Quality Assurance in Molecular Genetic Testing. http://www.oecd.org (accessed 9/10/2013);

van Krieken JH, et al. Ann Oncol 2013;24:1958–63; van Krieken JH, et al. Virchows Arch 2008;453:417–31.

Participating laboratories

programmes programme

10

ESP EQA schemes for CRC: overview

Scheme GenesNumberof labs

Number of countries

% of labsreported genotypes correctly

KRAS EQA 2009 KRAS 61 9 69%

KRAS EQA 2010 KRAS 76 14 67%

KRAS EQA 2011 KRAS 124 27 72%

Slide courtesy of ESP EQA Group. *only KRAS mandatory24

Q

KRAS EQA 2012 KRAS 105 26 71%

RAS EQA 2013 KRAS/NRAS/BRAF* 131 25 73%

Numericalscoring system

1 point: correct genotype or in case mutation was not screened andidentified as WT

0.5 points: no result given due to low DNA quality or technical failures (since 2012)

0 points: incorrect genotype

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• 125 laboratories submitted RAS genotype results, 105 also performed BRAF testing (optional):

• 40 labs (32%) made at least one major genotyping error: 

• 20 laboratories made 1 major genotyping error (16%), whereas 2 or more 

major genotyping errors were made by 20 laboratories (16%)

ESP Colon EQA 2014: results 

major genotyping errors were made by 20 laboratories (16%) 

• 12 labs (9.60%) reported one or more technical error(s):

• 9 laboratories (7.20%) reported one test failure and 3 laboratories (2.40%) 

reported a test failure for multiple samples; 2 laboratories (1.60%) made a 

combination of genotyping errors and technical errors.

• 82 labs (65.60%) participated successfully (no major genotyping error and a score on technical evaluation of ≥18/20)

Slide courtesy of ESP EQA Group.

ESP EQA schemes for CRC: overview

Scheme GenesNumberof labs

Number of countries

% of labsreported genotypes correctly

KRAS EQA 2009 KRAS 61 9 69%

KRAS EQA 2010 KRAS 76 14 67%

Slide courtesy of ESP EQA Group. *only KRAS mandatory

**KRAS and NRAS mandatory26

KRAS EQA 2011 KRAS 124 27 72%

KRAS EQA 2012 KRAS 105 26 71%

Colon EQA 2013 KRAS/NRAS/BRAF* 131 25 73%

Colon EQA 2014‐2015

KRAS/NRAS/BRAF** 125 30 66%

Results most recent colon EQA scheme (2014‐

2015)

Type of error Number of labs 

% (n=125)

1 major genotypingerror*

20 16%

≥2 major genotyping errors*

20 16%

1 technical failure 9 7.20%

27

≥2 technical failures 3 2.40%

Combination of genotyping and technical errors

2 1.60%

Nomenclature error 33 26.6%

• average genotyping score is 18.06/20 (90.28%). • 82 laboratories (65.60%) participated successfully (no major genotyping error 

and a score on technical evaluation of ≥18/20)

• 46 laboratories (36.80%) obtained the maximum score (20/20)

*false positive, false negative, wrong mutation, samples switched, mutation not screened

Country (n=number of participating 

laboratories)

Average genotype 

score

/20

Average 

genotype score

%

Number of labs 

successful 

% of labs 

successful

The Netherlands (n=22) 18.07 90.34% 17 77.27%

Belgium (n=13) 18.12 90.58% 10 76.92%

Austria (n=9) 18.94 94.72% 7 77.78%

France (n=8) 17.06 85.31% 3 37.50%

Denmark (n=8) 18.88 94.38% 6 75.00%

Spain (n=6) 18.42 92.08% 2 33.33%

Sweden (n=6) 18.17 90.83% 4 66.67%

Italy (n=6) 18.58 92.92% 4 66.67%

RESULTS MOST RECENT COLON EQA SCHEME (2014)

prof dr E Dequeker 28

Italy (n 6)

Czech republic (n=5) 19.80 99.00% 5 100.00%

Turkey (n=4) 14.88 74.38% 1 25.00%

Switzerland (n=4) 18.88 94.38% 3 75.00%

Germany (n=4) 19.63 98.13% 4 100.00%

Norway (n=4) 13.38 66.88% 0 0.00%

Slovakia (n=3), Portugal (n=3), Hungary 

(n=3), Poland (n=3), Greece (n=2), Israel 

(n=2), Japan (n=1), Australia (n=1), 

United Kingdom (n=1), Romania (n=1), 

Brazil (n=1), Singapore (n=1), Colombia 

(n=1), Finland (n=1), Bulgaria (n=1), 

Slovenia (n=1)

17.71 88.54% 16 61.54%

TOTAL (n=125) 18.06 90.28% 82 65.60%

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NSCLC EQA Scheme Genes Number of l b t i

Number of t i

NSCLC EQA scheme

2 pilot rounds in 2012

2014: new biomarker ROS1 included

Increasing nr of partipants and nr of participating countries

prof dr E Dequeker 29

laboratories countries

Pilot ‐ Lung EQA 2012 a ALK 68 22

Pilot ‐ Lung EQA 2012 b ALK, EGFR, KRAS 149 26

Lung EQA 2014, part I EGFR 144 29

Lung EQA 2014, part II ALK, ROS1 139 28

Lung EQA 2015, ALK scheme ALK 157 32

Lung EQA 2015, ROS1 scheme

ROS1 84 19

Lung EQA 2015, EGFR scheme

EGFR 111 25

Scheme Subscheme Number of labs % of labs successful

Pilot Lung EQA 2012 a

ALK FISH 54 72%

ALK FISH Digital* 67 82%

ALK IHC 29 52%

Pilot Lung EQA 2012 b

ALK FISH 104 68%

ALK FISH Digital* 106 74%

ALK RT‐PCR 8 educational

ALK IHC 58 64%

KRAS 92 educational

ESP EQA Schemes for NSCLC: results

prof dr E Dequeker 30

EGFR 107 educational

Lung EQA 2014, part I

EGFR 144 61%

Lung EQA 2014, part II

ALK IHC 96 70%

ALK FISH 116 69%

ALK FISH Digital* 81 educational

ALK RT‐PCR 6 educational

ROS1 IHC 31 90%

ROS1 FISH 56 64%

*In collaboration with UKNEQAS

ESP Lung EQA Schemes: results from 2 pilot rounds in 2012

Subscheme Error rate round 1* Error rate round 2*

Digital ALK 

FISH

3,6%

(7 out of 

195)

0,5% due to FP 1,1 %

(4 out of 

366)

0,8% due to FP

3,1% due to FN 0,3% due to FN

7,3% 0,5% due to FP 5,2% 4,0% due to FP

prof dr E Dequeker 31

ALK FISH TMA (14 out of 

193)

(22 out of 

423)6,7% due to FN 1,2% due to FN

ALK IHC

13,0%

(30 out of 

230)

7,4% due to FP 8,2%

(44 out of 

540)

5,0% due to FP

5,6% due to FN 3,2% due to FN

*Error rate = (number of FP + number of FN)/total number of informative resultsFP, false positives; FN, false negatives

• Website• kras.eqascheme.org

• lung.eqascheme.org

• E‐mail• colon.eqa@kuleuven.be

l @k l b

More information

• lung.eqa@kuleuven.be

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Concern!

• Very high level of reliability is needed because of the no harm principle

• Not all laboratories have taken up full RAS testing

• Potential detrimental effect

• Data presented at ECCO

The sensitivity issue

• Technical sensitivity

• Neoplastic cell content

• Clinical relevant sensitivity

RAS testing sensitivity: Comparison between studies

• RASmutation frequency in CRYSTAL is in line with that of other trials applying a 

threshold of 5%

Study Starting material Methodology Cut‐off (%)RAS assessable 

(n)New RAS mt (n)

Mutation rate (%)

CRYSTAL Extracted DNA BEAMing 5 430 63 15

OPUS Extracted DNA BEAMing 5 118 31 26

Macrodissected specimen

*Exact cut‐off not known, but assumed from technology used Data on fileNGS, next‐generation sequencing

FIRE IIIMacrodissected specimen (>200 cells, >30% tumor content)

Pyrosequencing 5* 407 65 16

PRIMETumor slides (macrodissected if tumor content <50%)

Sanger sequencing,WAVE‐based technology

5–10* 620 108 17

PEAKTumor slides (macrodissected if tumor content <50%)

Sanger sequencing,WAVE‐based technology

5–10* 221 51 23

20020408Tumor slides (not further specified)

NGS 1–5* 165 29 18`

CAPRITumor slides (macrodissectedif tumor content <50%)

NGS 2% 182 29 16

CAPRI‐GIOM Trial – NGS sequencing29 additional mutations found in 182 

KRAS exon 2 wild‐type cases:

• Possibly different histology sections

used for new and original analysis

• Limit of detection NGS (2%) vs. SANGER 

SEQ (5‐10%) 

All dditi l KRAS 2 All additional KRAS exon 2 

mutations found with NGS were

confirmed with SANGER Sequencing

or therascreen KRAS RGQ kit

Ciardiello, F. , Normanno, N. et al. (2014). Annals of Oncology. doi:10.1093/annonc/mdu230

underperformance of routine practice underscores need of EQA program and guidelines for RAS‐testing

CONSISTENCY: 84.1%

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Reproducibility RAS‐testing in NL

CONSISTENCY 97 6%

166 DNA samples from 17 Dutch centers retested at the Radboudumc:

• Only 4 samples were discordant:

• In 3 samples a mutation in RAS was found that was not reported but not tested for

• In 1 sample a mutation was reported that was not found by the Radboudumc

wildtypeKRAS

exon 2KRAS

exon 3KRAS

exon 4NRAS

exon 2NRAS

exon 3NRAS

exon 4 Total

wildtype 87 1 1 1 90

KRAS exon 2 1 58 59

KRAS exon 3 7 7

KRAS exon 4 5 5

NRAS exon 2 1 1

NRAS exon 3 4 4

NRAS exon 4 0 0

Total (%) 88 (53%) 58 (35%) 7 (4%) 6 (4%) 2 (1%) 5 (3%) 0 (0%) 166

Pa

rtic

ipa

tin

g

Ce

nte

r

Radboudumc

CONSISTENCY: 97.6%

Conclusion

• Full RAS testing is mandatory

• Full Ras testing requires in house, non commercial methods

• EQA is important

• Rapid uptake of RAS testing in the Netherlands

• High reliability of RAS testing in the Netherlands

Conclusion

• EQA is important

• High quality testing available in each country

• Collaboration is the way to go• European network for EQA

• Diagnostic versus predicitve?

• Molecular versus protein based?