External qualitiy assurance in molecular testing of NSCLC · External qualitiy assurance in molecular testing of NSCLC ... presentation: Murray S. Lungscape ... Murray S. Lungscape

External qualitiy assurance inmolecular testing of NSCLC

Ulrike Gruber-MoesenbacherInstitute of Pathology

Universitary Teaching Hospital Feldkirch, AustriaWorking Group Pulmopath of the Austrian Society of Pathology (ÖGPath)

Bled, May 14th 2014

Why quality assurance in moleculartesting?

• standardization and• reproducibility of processes for

• decentral testing• timely availability of

– required analyses– for all therapeutic facilities

How to achieve reproducible results?

• internal laboratory validation for biomarker testing– immunohistochemistry:– In situ hybridization– molecular tests

» mRNA expression» DNA mutation

– use• standardized protocols• validated approaches• consistent „common“ positive control materials

• external quality assurance– external audits – certification/accreditation– interlaboratory ring trials– EQA schemes

modified from: Qualitätssicherungs-Initiative "QuIP" der Deutschen Gesellschaft für Pathologie und des Bundesverband Deutscher Pathologen zur diagnostischen Immunhistochemie und Molekularpathologie andpresentation: Murray S. Lungscape - A Project by ETOP. presentation. Barcelona; 2011 Nov 18 2011

standards and references in moleculartesting







EQA schemes xgen & tiss France x xQiP Germany xItaly xSpain xAustrian Pulmo Group x

French CQ Network

education.ign.fr

LeuvenReference platform : Nijmegen laboratory (H van Krieken, M M.Ligtenberg)

AFAQAPCurie

Partner Platforms

Joint partner

Reference Platform

EGFRKRAS

28 labelled andlicensed platforms48 molecularlaboratories- Oncohematology- Solid tumors

The organization of the Italian scientificsocieties

Permanent joint Board of the Italian Association of Medical Oncology (AIOM) andthe Italian Society of Pathology (SIAPEC-IAP) for the «Molecular characterizationof tumors for therapeutic purpose»

AIOM SIAPEC

Coordinators Carmine Pinto Claudio Clemente

Members Vincenzo Adamo Antonio Marchetti

Andrea Ardizzoni Oscar Nappi

Nicola Normanno Anna Sapino

Giampaolo Tortora Gian Luigi Taddei

AimsOrganize workinggroups on specifictopics

Outline guidelines

EQA programs

Training

ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORIFONDAZIONE G. Pascale – NAPOLISC Biologia Cellulare e Bioterapie

CENTRO RICERCHE ONCOLOGICHEMERCOGLIANO (AV)

Laboratorio di Farmacogenomica

KRAS, EGFR, BRAF

(ALK)

Dr. Iosu Solawww.seap.es

EQA

Kras Control Case 3-4

•Global..

•Pan-EU EQA•somatic EGFR mutation

testing•Susana Benelloch, Fiona Blackhall, Rachel Butler, Fortunato

Ciardiello, Zanda Deans, Manfred Dietel, Martin Filipits, Keith Kerr,Fred Hirsch, Samuel Murray, Nicola Normano, Simon Patton, SanjayPopat, Rolf Stahel, Miguel Taron, Erik Thunnissen, Andrew Wallace

•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).

organization of EQA schemes

van Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecular pathology. Ann Oncol. 2013 Apr 23.

ESP Lung EQA program• What is the ESP Lung EQA program?• The European Society of Pathology (ESP) established a European EQA program for testing lung

biomarker mutations in NSCLC.This program aims to ensure optimal accuracy and proficiency in mutation testing across all countriesor institutions in the world.

• A European pilot ESP Lung EQA scheme will be run in 2012 with two rounds. The first will start inMarch and contains only ALK testing, while the second by the end of 2012 will consist of a combinedEGFR, ALK en KRAS analysis. The practical organization of this European EQA program is done incollaboration with a European working group lead by Dr. E. Thunnissen, and the Biomedical QualityAssurance Research Unit of the KU Leuven lead by Prof. Dr. E. Dequeker.The ESP Lung EQA programworks in close contact with Prof. Dr. H. van Krieken, executive board member of the ESP, and Prof. Dr.G. Bevilacqua, chair of the ESP molecular working group.

• The aim of the first round of the ESP Lung EQA scheme is to evaluate the reliability of the analysis ofImmunohistochemistry (IHC) and/or in-situ hybridization (ISH) ALK test including reporting, as wellas interpretation of digitized FISH images.

• The second round is designed to evaluate EGFR, KRAS and ALK biomarker testing in NSCLC.You may choose to particpate in one or more of the following tests: EGFR mutation, KRAS mutation,ALK FISH, ALK IHC, and/or ALK RT-PCR.For ALK rearrangement analysis (FISH, IHC and/or RT-PCR), one TMA with cores from formalin-fixedand paraffin-embedded material will be designed and blank histological sections on microscope slideswill be provided. One of the distributed slides is for H&E. Twelve different samples will be present onthe TMA slides. In addition, for ALK FISH participants, 4 digital cases will be available online forinterpretation of digitized FISH images.For the IHC and the digital ALK FISH rearrangement this scheme is in collaboration with UKNEQAS.For EGFR and KRAS mutation analysis, a separate set of formalin-fixed and paraffin-embeddedsamples will be sent.

• Laboratories performing adequate in this EQA round will be published on the ESP website.

ESP EQA CoordinationUniversity of Leuven - Department ofPublic HealthBiomedical Quality Assurance ResearchUnitHerestraat 49, box 602, 3000 Leuven,BelgiumFax: +32 (0)16 34 79 49E-mail: [email protected]: http://lung.eqascheme.orgLogistics Scheme coordinatorProf. Dr. Els DequekerTel: +32 (0)16 34 58 81ESP Lung coordinatorDr. Erik ThunnissenTel: +31 (0)20 4444 048Scheme assistant coordinatorLien TembuyserTel: +32 (0)16 33 01 43

ESP LUNG EQA Scheme 2014• Part I: EGFR mutation analysis – registration open•• ESP Lung External Quality Assessment Scheme 2014:

The program aims to ensure optimal accuracy and proficiency in biomarker testing in non-small cell lung cancer (EGFR, ALK and ROS1 testing). Allcountries can participate in the scheme.

•• Our experience from the previous two pilot rounds has enabled us to optimize the set-up and the samples of the new scheme, which is designed to

evaluate EGFR, ALK and ROS1 testing in NSCLC. The ESP Lung EQA program of 2014 is divided in two parts:• - Part I : EGFR mutation analysis• - Part II: ALK and ROS1 testing•• Set up of Part I• For EGFRmutation analysis, 5 FFPE cell line samples and 4 resection specimens will be provided on glass slides.• All of the EQA samples have been thoroughly validated and a sufficient amount of material will be foreseen to perform the analysis.• The laboratory needs to test the EQA samples using routine protocols and submit results within 2 weeks after arrival of the samples. It will be requested

to issue written reports for several cases.•• Registration and sample distribution• Samples will be distributed at the end of May – beginning of June 2014• Starting today, the registration for the EGFR subscheme is open. The deadline for registration is May 16, 2014. You will receive an email later on to

confirm the exact sending date for the EGFR scheme.• A maximum of 150 laboratories can participate, registrations will close as soon as 150 registrations are made.• If you wish to participate to the EGFR subscheme, please login and register online: http://lung.eqascheme.org.•• There will be a separate registration phase for the ALK and ROS1 subschemes, which will open in a few months. You will receive an email invitation to

inform you when registration is possible. Participation in several subschemes will be possible: ALK FISH, ALK FISH Digital, ALK IHC, ALK RT-PCR, ROS1FISH, and ROS1 IHC.

•• Dr. E. Thunnissen from the University VUMC Amsterdam will be the scheme organiser. A team of medical and technical experts supports validation and

evaluation of this scheme. Prof. Dr. E. Dequeker and her team from the KU Leuven will be the scheme coordinator.







.

Initiative for quality assurance of theGerman Society of Pathology andAssociation of German Pathologists fordiagnostic immunhistochemistry andmolecular pathology

•a panel of 2 – 3 reference institutes prepares•material for ring trials providing a•pool of test material and performs•calibration of tests.•organisation of the trials is offered by alogistic corporation

The Austrian way

• tu felix Austria nube– multifunctional expensive organization for EQA– standardized „official“ external assessment

versus

– easily accessible– interlaboratory comparison of– routine procedures and reports

Austrian EGFR ring trialdesign

• multinational interlaboratory ring trial– 19 participants

• 9 guest labs• 10 Austrian labs

• 4 distributions per year– 3 unstained slides from 3 resection-specimen with or without EGFR-mutation

• number of samples needed for reliable evaluation: 10 per yearvan Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecular pathology. Ann Oncol. 2013 Apr 23

• form with questions to be filled in– turnaround time– technical indicators– simulation of definitive reports– histological diagnosis of tumor

• confirmation of participation edited by AG Pulmopathology of theAustrian Society of Pathology

• offer to discuss problems

LaboratoryaddressLab IDResponsible MDResponsible technicianTele-mailDate receivedDate of reportEGFR

Case A Case B Case C Comment for QA/questionsPercentage of tumor cells within section(as in regular report)how was percentage calculated?Tumor cell enrichment – yes/no : methodof enrichment (e.g. macrodissection byneedle, scratching)Method of DNA extractionMethod of measurement of DNAscratched from 1 or 2 slides / volume ofeluate (µl)Amount of DNA extracted (as in regularreport)Quality of DNAEvaluation of DNA-qualityMethod of sequencing (as in regular report)What types of mutations can be detectedby your method? Sensitivity of yourmethod, if known?Result (as in regular report)Interpretation of result as in regular reportAdditional comments to oncologydepartment or recommendationsHistologic diagnosis of case incl. patternsand percentage, as done in regular reports

Clinicians and patients want:clinically relevant, reliable, standardized, comparable reports

EQA addresses• reliability

– quantity and quality of tumor and DNA– tumor typing

• clinical relevance– timelyness

• standardisation– methods

• comparability– reporting

• licencing/accreditation– QA as requirement for licencing and billing

reliability

• pathology– tissue sampling

• pathologists can recommend techniques of tissuesamplig

– tumor typing• pathologists type the tumors

– dissection• pathologists dissect the reliable portion of tumors

reliability –tissue conservation - fixation

• Cytology– spread up to 4 slides ,– fixation with alcoholic spray immediately– rinse the needle with 1 ml NaCl

• Histology– fixation in 4% (10%) neutral- buffered formalin– time of fixation

• 6 – 12h for small bisopsies,• 8 – 18h for resection specimen• maximum 3 days

– time of resection should nearly be– start of fixation and should be– indicated in the requisition form

reliabilitytissue sampling –cell counts

– cytology• exfoliative• FNA with/without US

– cellblock facitilitates series

– cells for DNA extraction• 200 – 400• 1 – 25% tumor cells of all cells depending

on technique

– histologyreachablequantity

FNA 21-g FNA19-g transbron-chial biopsy

CT-guidedFNA

Wegde-resection

cells ≥ 100 ≥ 150 ≥ 300 ≥ 500 more

no. biopsies 4 4 4-5 2-3 1

reliabilityimportance of tissue selection

Without microdissectionthe error rate can be up to

8%* due only toinadequate samples.

* independent of any technical problemsWeichert W et al. J Mol Diagn;12:35-42Murray S. Pan-EU EQA somatic EGFR mutation testing;2010

tumour cell percentage – concordance?LabID MD A-II/13

lepid.ACB-II/13acin.AC

C-II/13CB

MD A-I/14acin.AC

B-I/14acin.AC

C-I/14acin.AC

01 n 40% 85% 10-15% y 90% 90% 80%

02 y 15% 50% 80% y 50% 50% 50%

04 y 20% 50% 80% n 50 70 70

05 y 50% 70% 50% y 80% 80% 70%

06 n 40% 80% 90% n 50% 60% 60%

07 n 60% 100% 40% y 70% 93% 85%

08 y 30% 90% 95% y 35% 60% 60%

09 y,nn 20% 60% 60% y 50% 60% 75%

10 y 50% 70% 30% y 50% 50% 50%

11 y 50% 70% 60% y 70-80% 80% 80%

12 y 30% 75% 40% y 80% 80% 70%

13 y 50% 80% 70% y 70% 80% 90%

14 y 30% 70% 15% y 60% 60% 60%

15 y 25% 90% 50% n 70% 65% 85%

16 y,nn 60-70% 80% 80% y 80% 80% 80%

18 n 10-20% 70-80% ne y,nn 75-80% 70-75% 80-85%

19 n 10% 50% 30% n 70% 90% 70%

21 y 45-55% 60-70% 75-85% y 30% 50-60% 50-60%

22 y 80% 80% 90%

tumor cell percentage

• depends on– person counting

• subjectivity: relation neoplastic/ non-neoplastic cells– tumor-type and stroma-reaction

• lepidic: more discrepancy – what is tumor?• relation: tumor –stroma/inflammation

– macrodissection• no consequent increase by MD

• preanalytical phase should not be scored in EQA tests– before adequate guidelines have been publishedvan Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecular pathology Ann Oncol. 2013

Apr 23

tumor cell percentage• consequences QA:

– discrepancies in tumor cell counts cause questions– tumor cell counts necessary?

• measurement of minimal amount of tumor content is stillbased on tumor cell percentage

• discrimination steps (10 – 25%?)• minimum tumor cell percentage 5%, 20%

– standards should be discussed for• tumor cell content

– how many areas?– how many cells?– how many counters?– dissection necessary?

from report form LabID10 I/2014

Diagnosis of case as done in regular reports

Case A Case B Case CID01 enterische Variante eines pulmonalen

Adenocarcinoms mit azinäen Anteilen mitComedonekrosen (? neuroednokriner Anteil?)

prädominant azionäres + fokal solidespulmonales Adenocarcinom G3

prädominant azinäres Adenocrcinom G2

ID0 NSCLC NSCLC NSCLC

ID04 Adenokarzinom (50%solid, 50%papillär) Adenokarzinom predominant azinär70%,( je 10% solide lepidisch,papillär)

Adenokarzinom (40%lepidisch,40%papillär,20%solid)

ID05 EGFR mutant adenocarcinoma EGFR mutatn adenocarcinoma EGFR wild type adenocarcinomaID06 adenocarcinoma solid type acinar adenocarcinoma with solid

componentstubular adenocarcinoma

ID07 adenocc. adenocc. adenocc.ID08 pulmonary adenocarcinoma

acinar patternpulmonary adenocarcinomaacinar and solid pattern

pulmonary adenocarcinomadominant acinar, solide and lepidic pattern

ID09 Gering diff. , prädominant acinäresAdenocarcinom mit soliden (20%) Anteilen,Das Vorliegen einer

Gering diff., prädominant acinäresAdenocarcinom, mit soliden (30%) undlepidischen (10%) Anteilen

Gering diff., prädominant acinäres Adenokarzinom mitsoliden (25%), mikropapillären (5%) und lepidischen(30%) Anteilen.

ID10 Moderately differentiated mixedadenocarcinoma (acinar 50%, micropapillary5%, solid 30%, papillary 5%, cribriform 10%)

Poorly differentiated mixedadenocarcinoma (40% acinar, 40%papillary, 20% solid)

Moderately differentiated mixed adenocarcinoma (40%acinar, 40% papillary, 20% solid)

ID11 Teils tubulär, teils solid bis kribriform mitKomedonekrose, G2

Adenokarzinom teils tubulär (50%), teilssolid (<10%) mit lepidischem Wachstum,G3

Adenokarzinom mixed type, teils tubuloazinär (50%),teilweise lepidisch (50%), fokal mikropapillär, G3

ID 12 - - -ID13 Adeno Ca, G3

prädominant solide, cribriform Adeno Ca, G2 Adeno Caazinär

ID14 adenocarcinoma III, mixed type (cribriform,glandular)

adenocarcinoma III mixed type(glandular, solid)

adenocarcinoma II mixed type (glandular,micropapillary, solid)

ID15 Adenocarcinoma, predominantly acinar (acinar,lepidic)

Adenocarcinoma, predominantly acinar Adenocarcinoma, predominantly papillary (papillary,solid)

ID16 Adenoca. präd acinär, G2 Adenoca, prädom. acinär , 10% solide,G3

Adenoca präd. papillär, G2

ID18 Invasive predominantly acinaryadenocarcinoma (enteric? – approx. 55-60%) +solid alveolar NSCLC with „neuroendocrine“morphology (approx. 40-45%).On the basis of HE histology (IHC necessary)we prefere the Dx. of a primary NSCLC(theoretically Dif Dx. of a MANEC MTS)

Invasive predominantly acinaryadenocarcinoma, with presence ofcribriform, solid, clear cell, giant cell andpapillary structures, as well as with smallfoci resembling high grade foetaladenocarcinoma (subnuclear vacuoles)

Invasive predominantly lepidic adenocarcinoma,with presence of micro-papillary and alveolar spacefilling structures, but also with presence of sporadicyolk-sac like intracytoplasmatic globules, morula-likesquamoid proliferates and lipoblast-like forms

See the description of the cases –in all three cases the IHC analysisis necessary for a correcttyping/subtyping of thecarcinomas

ID19 Adenocarcinoma, predominant acinar pattern(acinar 60%, solid 40%)

Adenocarcnoma, predominant acinarpattern (acinar 40%, papillary 20%, solid20%, lepidic 20%)

Adenocarcinoma, predominant papillary pattern(papillary 40%, solid 30%, lepidic 30%)

ID20 Adenocarcinoma pracipuae acinosum (70%)partim papillare (30%).

Adenocarcinoma praecipuae papillare(80%) partim acinosum (15%),micropapillare (5%).

Adenocarcinoma praecipuae papillare (80%) partimacinosum et micropapillare (3%).

ID21 Fetal type adenocarcinoma Adenocarcinoma with acinar pattern(10% clear cells, 5% solid pattern,5% lipid pattern, <5% micropapilarpattern)

Adenocarcinoma with papillary pattern(15% micropapillarypattern, 10% acinar pattern, 5%lipid pattern)

ID22 - - -

reliability: tumor typing







Clinical relevancetime to report

• „reflex“- testing during diagnosis• no delay sending slides or DNA-extract to

molecular lab, if in institute of pathology• no loss of information between different labs• EGFR, KRAS possible in 2 – 7 working days• ALK possible in additional 1-7 working days

depending on workload

time to report5 rounds of Austrian RT EGFR 2013/2014

RT-# participants/correct dates

min # WD max # WD average

I/2013 April 16/13 3 19 7,7

II/2013 August 18/13 2 17 6,5

III/2013October

19/17 3 10 6,1

IV/2013December

19/13 3 9 5,1

I/2014 April 19/15 3 12 6,5







methods – standardizationEQA samples

• declaration of– type of mutations detected and– sensitivity of test

• EQA provider– should adhere to

• DNA content at the upper limit of available techniquesand

• types of mutations detected by most availabletechniques in terms of scoring

van Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecular pathology. AnnOncol. 2013 Apr 23

standardizationEGFR Testing Methods

Sequencing ARMS (DxS)Pyro-

sequencingFragmentanalysis SSCP/DHPLC HRM

Limit ofdetection 10–20% 1% 5–10% 5% 10–20% 10–20%

DNA required 10–100ng 100–500ng 10–100ng 10–100ng 10–100ng 10–100ng

% EGFRmutations >99% >90% >95%

Ex20 insertions& Ex19 dels >90% >90%

Mut charact-erisation Yes No Yes No No No

CE-marked No Yes No No No No

Murray S. Pan-EU EQA somatic EGFR mutation testing; 2010.

methods: types of mutation, sensitivityWhat types of mutations can be detected by your method? Sensitivity of your method, if known?

ID01 41 Mutationen in exons 18-21ID02 LaCOBAS EGFR Mutation TestID04 41 different mutations in Exon 18,19,20,21 ( 5% of mutated tumor cells in wildtyp background)ID05 41 variants by sensitivity of 5%ID06 mutation rate above 5%ID07 exon, 19, exon 21ID08 18 – 21 exon anyID09 29 mutations detected by the TheraScreen EGFR29 Mutation Kit. Sensitivity according to the manufacturer: 15%ID10 Exon 18: Mutations on Codon 719: G719X (X=S, C, A, D)

Exon 19: all Deletions within Codon 746-750Exon 20: Mutation Codon 768 (S768I), Insertion Codon 770, 771, 774; Mutation Codon 790 (T790M)Exon 21: Mutation Codon 858 and 861 (L858R, L861Q)5% tumor cells should be tested in minimum to get reliable results

ID11 Exon 18: Punktmutationen im Codon 719Exon 19: sämtliche derzeit in Version 1.0/ Oktober 2011 beschriebene DeletionenExon 20: Punktmutationen Codon 790 (T790M) und 768 (S768I) InsertionenExon 21: Mutationen Codon 858 (L858R)

Id 12 all types (del, ins, sub, dup)ID13 Point mutations codons 719, 768, 790, 858-861; Deletions Exon 19,

Sensitivity 5-10% mt in wildtypeID14 19 deletions in exon 19, T790M,L858R. L861Q, G719X, S763I, 3 insertions in exon 20; sensitivity 1-10% mutated DNAID15 Exon 18: G719X (G719A, G719S, G719C)

Exon 19: deletions and complex mutationsExon 20: S768I, T790M and insertionsExon 21: L858R

ID16 T790M, Deletionen Exon 19, S768I, L858R, G719X, Insertionen Exon 20ID18 41 specific mutations in exons 18, 19, 20 and 21 of the EGFR gene, e.g. deletions (e.g. exon 19 del), insertions (e.g. exon 20 ins) or point mutations (e.g. S768I, L858R,

T790M or G719X) can be detected.Sensitivity: >5% mutant copies of FFPE DNA in a background of wild type DNA

ID19 by our method 3 point mutations can be detected in exon 18 (G719A, G719C, G719S), 29 deletions and complex mutations in exon 19, 2 point mutations (S768I, T790M)and 5 insertions in exon 20 and L858R in exon 21. overall 41 mutations

ID20 Cobas EGFR Mutation Test detects 41specific mutations (substitutions, insertions, deletions) in exons 18-21 of the EGFR gene. Test sensitivity, according to themanufacturer: 5% of mutant allele content in the wild-type background

ID21 T790MExon 19 Deletions - detects 19 deletions, but does not distinguish between themL858RL861QS768IG719X - detects G719A, G719S and G719C, but does not distinguish between themExon 20 Insertions - detects 2319-2320 insCAC and 2310-2311 insGGT, but does not distinguish between themExon 20 Insertion - detects 2307-2308 insGCCAGCGTG (ins9)The limit of detection varies, according to the manufacturer between 0,1-1% diluted in the wild-type genomic DNA. The exact sensitivity has not been locally validated inorder to establish the minimum proportion and number of cancer cells needed for mutation detection as recommended by ACP/IASLC/AMP guidelines.

ID22 41 specific mutations in exons 18, 19, 20, 21 (insertions + deletions), >5% mutant copies of FFPET DNA in a background of wt DNA Cobas EGFR Mutation Test (Roche)







comparabilityclear reports – EQA samples

• pathologist should offer integrated reports, including– typing of tumor and– correlated molecular changes– with interpretation of the effects of mutations

• EQA should score the postanalytical phase with respectto– patient identification– report layout and content and– biological and clincal interpretation based on the

predefined elements

van Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecularpathology. Ann Oncol. 2013 Apr 23

comparability

clear Clinical Report• Four categories

– Sensitising/Resistancemutation detected Fully interpret the mutation

– No mutation detected– Sample failed

– Unclassified variantidentified Test genomic DNA

•Minimum reporting recommendations?

report – interpretation – comparability?interpretation of result as in regular report

ID01 - - -ID02 -ID04 Im vorliegenden Untersuchungs-material findet

sich eine Doppelmutation.Aktivierende Mutation im Exon 21 (L858R) undResistenzmutation im Exon 20 (T790M)

Im vorliegenden Untersuchungsmaterial findetsich eine aktivierende Mutation im Exon 21 (L858R)

Im vorliegenden Untersuchungsmaterial ist eine Mutationdes EGFR Gens nicht nachweisbar

ID05 double classical EGFR mutation detected classical EGFR mutation detected wild type EGFR detectedID06 activating mutation L858R is neutralized by the

resistance mutation of T790MNo indication for tyrosine kinase inhibitor

activating mutationindication for tyrosine kinas inhibitor therapy

wild typeno indication for tyrosine kinase inhibitor

ID07 activating EGFR mutationdetected (L858R), 25%

activating EGFR mutationdetected (L858R), 40%

activating EGFR mutation of codon 19 and 21not detected

ID08 EGFR resistence mutation inexon 20 and activating mutation inexon 21

EGFR activating mutation inexon 21

No mutation found in exon 18-21

ID09 - - -ID10 2 muations: L858R is an activating and T790M

a resistance mutationactivating mutation activating mutation

ID11 Aktivierende Mutation und Resistenzmutation Aktivierende Mutation Keine Mutation im EGFR GenID 12 - - -ID13 Doppelmutation Aktivierende Mutation Molekulargenetische Analyse für EGFR: Keine Mutation

bzw. Deletion im EGFR-GenID14 - - -ID15 Sample is positive for activating L858R

mutation and also for inactivating T790Mmutation.

Sample is positive for activating L858Rmutation

Sample is negative for EGFR gene mutation.

ID16 - - -ID18 Mutation L858R is considered to represent an

activating EGFR mutation sensitive to TKIsMutation T790M is considered to associatedwith resistance to EGFR TKIs

Mutation L858R is considered to represent anactivating EGFR mutation sensitive to TKIs

Negative result – without the mutation (WT/WT) in theexamined exons 18, 19, 20 and 21

ID19 EGFR positive result may confer TKI sensitivitybesides concurrent resistance mutationresistance mutation

EGFR positive result confers TKI sensitivity EGFR negative, no benefit with TKI treatment

ID20 The sample tested exhibits the mutation L858Rsensitizing to EGFR tyrosinekinase inhibitors(EGFR-TKI) and the mutation T790M conferringresistance to EGFR-TKI therapy.

The sample tested does not exhibit themutation sensitizing to EGFR tyrosine kinaseinhibitors (EGFR-TKI)

The sample tested exhibits the mutation sensitizing toEGFR tyrosine kinase inhibitors (EGFR-TKI).

ID21 Presence of thec.2369C>T p.T790Mmutation on the exon 20 of the EGFR gene andc.2573T>G p.L858R mutation in the exon 21 ofthe EGFR gene .

Presence of the c.2573T>G p.L858Rmutationin the exon 21 of the EGFR gene

Presence of an 3 nucleotide insertion in the exon 20 ofthe EGFR gene

ID22 - - -

report – additional comments – comparable?Additional comments to oncology department or recommendations

ID01 -ID02 -ID04ID05 coexistance of a classical sensitising and resistance

mutant clones: early resistance expectedeligible for EGFR TKI therapy: sensitising mutation not eligible for TKI therapy

ID06 - - -ID07 - - -ID08 need an oncoteam decision, (biologically long

response not expected with TKI)response to therapy with TKIexpected

response to therapy with TKI to be not expected

ID09 Metastase (in erster Linie GI) muß ausgeschlossenwerden

no KRAS mutation detected

ID10 ususally we do not give recommendations tooncologists; but we know from the literature that theprogression free survival is likely to be shorter when aresistance mutation ist present prior to EGFR-targetedtherapy; therapy with Gefitinib an dERlotinib istprobably not going to work. In this case, Afatinibshould be chosen

no comment Insertions in exon 20 are a subset of activating EGFR mutationsprimarily known for their reported association with de novoresistance to TKIs. To date, however, few studies have focused onthis subset.

ID11 Keine TKI Therapie wegen Resistenzmutation TKI Therapie sinnvoll Keine TherapieID 12 Derzeit keine unterstützende Datenlage zum Einsatz

von IRESSA (T790M = Resistenzmutation), eineverminderte Wirksamkeit von IRESSA® kann somitnicht ausgeschlossen werden

Datenlage unterstützt den Einsatz von IRESSA Derzeit keine unterstützende Datenlage zum Einsatz vonIRESSA®

ID13 Resistenzmutation – geringe TKI-Sensitivität zuerwarten TKI-Sensitivität zu erwarten Keine TKI-Sensitivität zu erwarten

ID14 -ID15 Presence of the activating L858R mutation is

associated with EGFR TKI sensitivity. Presence of theinactivating T790M mutation is associated with EGFRTKI resistance and can be secondary, followingtherapy.Reference:Travis W. D. et al. Proc Am Thorac Soc. 2011Sep;8(5):381-5.

Presence of the activating L858R mutation isassociated with EGFR TKI sensitivity: EGFR TKItherapy is recommended.Reference:Travis W. D. et al. Proc Am Thorac Soc. 2011Sep;8(5):381-5.

Good response to EGFR TKI therapy is not expected.Travis W. D. et al. Proc Am Thorac Soc. 2011 Sep;8(5):381-5.

ID16 - -ID18 In relation to the finding of EGFR examination, the

analyses of other genes genes (e.g. ALK, etc.) mightbe necessary

Analysis of other genes (e.g. ALK, etc.) seems to beunnecessary.

Because of histologic diagnosis of adenocarcinoma, analysis ofother genes (e.g. ALK, etc.) seems to be necessary..

ID19 - - -ID20 EGFR-TKI therapy is NOT recommended. EGFR-TKI therapy is NOT recommended. EGFR-TKI therapy is recommended.ID21 Usually the presence of the L858R mutation of the

EGFR gene confers sensitivity to the EGFR tyrosine-kinase inhibitors.The coexistence of a T790M mutation if the patienthas been pretreated generate resistance to firstgeneration EGFR tyrosine-kinase inhibitors.In non pretreated patiens in case of the coexistence ofL858R and T790M mutations there is insufficient datato support the use of first line tyrosin kinase inhibitors.

Presence of the L858R mutation of the EGFR geneconfers sensitivity to the EGFR tyrosine-kinaseinhibitors

The technique does not distinguish between the two insertions2319-2320 insCAC and 2310-2311 insGGT. This coding variantpredicts a sensitivity to EGFR tyrosine-kinase, but this is lower andless predictive than Exon 19 deletions and exon 21 - L858R.

ID22 - - -







licencing/ billing

...can be charged if the test is perfomedaccording to guidelines for qualityassurance edited by the federalassociation „KassenärztlicheBundesvereinigung“...

consequence of poor performance• unsuccessful performance in molecular testing

have no established consequence in Europe, canlead to– withdrawal of test by the lab or– measures by either

• professional organizations or the• appropriate governmental regulatory agency

• EQA organizers can– help and support for improvements by providing

• reference material• methodological advice• qualitiy management and• revieweing corrective and preventive action plans

van Krieken JH, Siebers AG, Normanno N. European Consensus Conference for external quality assessment in molecular pathology. Ann Oncol. 2013 Apr 23

conclusion• EQA

– assists to standardize and to control– suggests how to control– helps for comparability of resuslts– helps the lab to be reliable

• what do patients/oncologists want?– quality of molecular tests

• clinically relevant samples• in a short time• valid results• clearly reported

modified from: Murray_EGFR-EQA_2010

Documents

External qualitiy assurance in molecular testing of NSCLC · External qualitiy assurance in molecular testing of NSCLC ... presentation: Murray S. Lungscape ... Murray S. Lungscape