Post on 03-Apr-2018
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
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
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: lung.eqa@med.kuleuven.beWebsite: 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.
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
.
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
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
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
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
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)
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
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 - - -
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
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