NordiQCEQA module
for lung cancer markers
Søren Nielsen
Director, NordiQC
Aalborg University Hospital, Denmark1
1. NordiQC review of selected markers used for
lung cancer diagnosis in pathology
2. Focus on protocol and technical challenges
3. Addressing choice of control material
Agenda:
NordiQC – EQA data on IHC for lung cancer
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NordiQC
• International academic IHC proficiency testing program
• Founded 2003 by Nordic pathologists
• Independent non-profit organisation
• Institute of Pathology, Aalborg University Hospital, DK
• General module: 3 runs/year• 15-18 different markers
• Breast cancer IHC module: 2 runs/y• 3-5 different markers (HER2, ER, PR,..)
• HER-2 ISH module: 2 runs/year• BRISH, FISH (breast cancer)
• Companion module
• PD-L1 KEYTRUDA & PD-L1 TECENTRIQ
www.nordiqc.org
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IHC – Potential in lung cancer pathology
Primary or secondary
TTF1, Calretinin, CDX2, GATA3, ….
Predictive
ALK, PD-L1, ROS1….
NSCLC or SCLC
CGA, SYP, CD56….
Adenocarcinoma or squamous
TTF1, Napsin A, CK5, p40….
NordiQC – EQA data on IHC for lung cancer
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Markers tested in NordiQC for lung cancer
Purpose Last run Pass rate No of labs
TTF1Lung vs non-lungAdenocarcinoma vs squam. Run 46, 2016 67% 272
Napsin A Lung vs non-lung Run 44, 2015 78% 162
Calretinin Lung vs mesothelioma Run 52, 2018 72% 269
CEA Lung vs mesothelioma Run 54, 2018 63% 272
CGA NSCLC vs SCLC Run 53, 2018 76% 296
SYP NSCLC vs SCLC Run 52, 2018 75% 308
CD56 NSCLC vs SCLC Run 37, 2013 81% 196
p40 Adeocarcinoma vs squam. Run 48, 2016 74% 188
CK5 Adeocarcinoma vs squam. Run 55, 2019 44% 263
ALK Predictive for Crizotinib Run 51, 2017 61% 189
PD-L1 Predictive for Crizotinib Run C5, 2019 80% 176
• Selection of clone
• Careful calibration of Ab or protocol for RTU
• Use of efficient HIER preferable at akaline pH
• Use of sensitive detection systems (3-step)
• Selection of right ”performance controls”
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General observations
NordiQC – EQA data on IHC for lung cancer
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NordiQC – EQA data on IHC for lung cancer
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Markers tested in NordiQC for lung cancer
Successful clones Less succesful clones
TTF1 mAb SPT24, rmAb SP141 mAb 8G7G3/1
Napsin A mAb IP64, MRQ-60 pAbs 760-4446, 352A-7x
Calretinin mAb DAK-Calret1, rmAb SP65, pAb 18-0211 rmAb SP13
CEA mAb CEA31, COL1 mAb II7, TF3H8-1
CGA mAb LK2H10, (pAb A0430) mAb DAK-A3, 5H4
SYP mAb 27G12, DAK-SYNAP, rmAb MRQ-40 mAb SY38
CD56 mAb CD564, 123C3 (non-VMS), rmAb MRQ-42 mAb 123C3 on VMS
p40 mAb BC28, rmAb ZR8 pAbs….
CK5 mAb XM26, rmAb BSR55, EP24 (SP27) mAb D5/16 B4 (rmAb SP27)
ALK mAb OTI1A4, rmAb D5F3 mAb ALK1
PD-L1 mAb 22C3 as CDx kit, rmAb SP263 as CDx kit mAb 22C3, E1L3N as LDT
NordiQC – EQA data on IHC for lung cancer
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Recommendable tissue controls in NordiQC for lung cancer
Critical positive tissue controls Negative tissue controls
TTF1 LE: lung terminal bronchi NE: liver, colon/appendix
Napsin A LE: kidney NE: colon/appendix
Calretinin LE: adrenal gland NE: colon/appendix
CEA LE: colon/appendix NE: liver, tonsil
CGA LE: colon/appendix NE: liver, tonsil
SYP LE: colon/appendix NE: liver, tonsil
CD56 LE: tonsil NE: liver, tonsil
p40 LE: placenta NE: liver, tonsil
CK5 LE: pancreas NE: liver, colon/appendix
ALK LE: colon/appendix NE: liver, colon/appendix
PD-L1 LE: tonsil NE: tonsil
LE: Low expression NE: No expression
IHC – Biomarker controls
CD56: Optimal Insufficient…….
Tissues/cells with only high expression will not identify:
1. A poorly calibrated IHC assay2. A reduced sensitivity in an optimally calibrated IHC assay
If an IHC test is used to demonstrate the target antigen beingexpressed at different levels, the controls must reflect this !
NordiQC – EQA data on IHC for lung cancer
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Focus;
Technical performance
Level of analytical sensitivity- Unknown primary
Level of analytical specificity
NordiQC – EQA data on IHC for lung cancer
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Clone…..
NordiQC – EQA data on IHC for lung cancer
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Clone…..
Control /Calibration
Consequence…
NordiQC – EQA data on IHC for lung cancer
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NordiQC – EQA data on IHC for lung cancer
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Focus;
Technical performance
Level of analytical sensitivityLevel of analytical selectivity
NordiQC – EQA data on IHC for lung cancer
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Clone…..
pAbs are no-go
NordiQC – EQA data on IHC for lung cancer
1.Technical quality: p40 – ”the good, the bad and the ugly”
NordiQC – EQA data on IHC for lung cancer
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Placenta;level of analytical sensitivity
Tonsil;Level of analytical specificity
NordiQC – EQA data on IHC for lung cancer
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Focus;
Technical performance
Level of analytical sensitivityLevel of analytical selectivity
An IHC assay can have one or more purposes and it is crucial to secure the need is fulfilled
IHC for CK5
1. To differentiate prostate gland hyperplasia / PIN from prostate adenocarcinoma
2. Identify squamous cell differentiation in lung carcinomas
3. .....
Prostate sample Lung sample
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E Torlakovic et al. AIMM 2017;25:4-11
Evolution of Quality Assurance for Clinical
Immunohistochemistry in the Era of
Precision Medicine: Part 1
Same protocol applied for different
purposes and meeting the requirements
(source; www.nordiqc.org)
Identification of purpose of the test
NordiQC – EQA data on IHC for lung cancer
NordiQC – EQA data on IHC for lung cancer
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XM26 versus D5/16 B4
XM26 seems to win…
Clone
HIER buffer – High pH
Detection kit – 3-step
XM26 or SP27…
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27Lung squam. cell
Lung TMA
Lung ad carc
Lung squam
Lung large cell
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27Lung squam. cell
Lung TMA
Lung ad carc
Lung squam
Lung large cell
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27Lung squam cell carc.
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27Lung adenocarc.
Lung TMA
Lung ad carc
Lung squam
Lung large cell
NordiQC – EQA data on IHC for lung cancer
mAb XM26 rmAb SP27TMA NeoplasiaGastric ad. carc.
Internal NordiQC data;14/62 lung adenocarcinomas pos by SP27 and neg by p40 / CK5 XM26
NordiQC – EQA data on IHC for lung cancer
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Focus;
Technical performance
Level of analytical sensitivityLevel of analytical selectivity
???
NordiQC – EQA data on IHC for lung cancer
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IHC – Biomarker controls
Examples for 17 markers
Generel expected patterns
High expression(Right antibody)
Low expression(Appropriate sensitivity)
No expression(Appropriate specificity)
Which tissueWhich cellsWhich extensionWhich intensity
NordiQC – EQA data on IHC for lung cancer
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Focus;
Technical performance
Level of analytical sensitivity
Is the test calibrated for the purpose?
Two main diagnostic areasincluded
An IHC assay can have one or more purposes and it is crucial to secure the need is fulfilled
IHC for ALK
1. To identify anaplastic large cell cell lymphoma
2. To identify lung adenocarcinoma with ALK mutation
3. .....
ALCL Lung ad. carc + EML-ALK mutation
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E Torlakovic et al. AIMM 2017;25:4-11
Evolution of Quality Assurance for Clinical
Immunohistochemistry in the Era of
Precision Medicine: Part 1
Same protocol applied for different
purposes not meeting the requirements
(source; www.nordiqc.org)
Identification of purpose of the test
NordiQC – EQA data on IHC for lung cancer
NordiQC – EQA data on IHC for lung cancer
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NordiQC – EQA data on IHC for lung cancer
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NordiQC – EQA data on IHC for lung cancer
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NordiQC Companion module
PD-L1
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What is the ”right level” of IHC quality staining? – Goals and tools….
NordiQC focus for IHC;
1. Technical quality
2. Calibrated for clinical and diagnostic use
Reference Standard Method
(method to determine accuracy)
Reference Standard Material
(material with relevant and criticalexpression level of the target analyte)
Patient Samples
(analytical sensitivity / specificity)
Reference Standard Readout
(cut-off’s and cells in focus)
Purpose of IHC assay
NordiQC standard fundamentals for EQA results and lab guidance
NordiQC standard fundamentals for EQA results and lab guidance
Reference Standard Method
mAb OTI1A4 (”Aalborg LDT)
ALK SureFISH
Reference Standard Material
Colon, appendix, tonsil
Patient Samples
Lung carc; Pos (FISH pos)
Lung carc; Neg (FISH neg)
ALCL; Pos (FISH pos)
Merkel carc; Pos (FISH neg)
Reference Standard Readout
Pos; weak - strong cytoplasmic reac.
Neg; no cytoplasmic reac.
Purpose of IHC test;
ALK IHC expression in lungcarcinoma to guide treatment with
Crizotinip, Ceretinip etc
NordiQC design set-up ALK Lung IHC – general module
Results obtained to identify best practice methods and reference materials
1. Start-up methods for laboratory validation / verification2. Confirmatory assessment for already validated methods
How to set-up an EQA program for PD-L1?
• Many purposes• 1’ line treatment in NSCLC, 2’ line treatment in NSCLC, 1 and/or 2’ line treatment in….
• KEYTRUDA, OPDIVO, TECENTRIQ, IMFINZI…
• Many Reference standard methods – CDx assays used in clinical trials• 22C3 SK006 (Dako), 28-8 SK005 Dako, SP142 Roche, SP263 Roche….
• Many Reference standard read-out / scoring methods• 50% TPS, 25% TPS, 1% TPS, CPS ≥20, CPS ≥10, CPS ≥1, IC1%, IC5%...
• Limited data on reference standard materials• Tonsil, placenta, tumours….
Reference Standard Method
22C3 CDx IHC assay, SK006
Reference Standard Material
Tonsil, placenta, cell lines
Patient Samples
NSCLC TPS; <1%
NSCLC TPS; ≥1-49%
NSCLC TPS; ≥50%
Reference Standard Readout
TPS neg; <1% tumour cells pos
TPS low; ≥1-49% tumour cells pos
TPS high ≥50% tumour cells pos
Purpose of IHC test;
PD-L1 expression in NSCLC for stratification with KEYTRUDA
treatment using present cut-offsfor 1’ and 2’ line
NordiQC main focus for PD-L1 IHC EQA initiative for NSCLC in 2017
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NordiQC EQA PD-L1 NSCLC – Reference methods + reference material
Reference Standards
Patient samples
Reference Method
Reference read-out
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NordiQC PD-L1 C1 results - performance against reference
0%
20%
40%
60%
80%
100%
CDx LDT
PD-L1 IHC assay performance
Optimal Good Borderline Poor
Modified from Table 1 NordiQC run C1
The three CDx assays 28-8 SK005, 22C3 SK006 and SP263 790-4905 most successful
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0%
20%
40%
60%
80%
100%
CDx LDT
PD-L1 IHC assay performance
Optimal Good Borderline Poor
Modified from Table 3 NordiQC run C2
CDxs most successful and highest proportion of optimal marks – LDT’s inferior but
superior to data from C1
NordiQC PD-L1 C2 results - performance against reference
0
10
20
30
40
50
60
70
80
90
100
C1 C2 C3 C4 C5
PD-L1 IHC Pass rates - NordiQC EQA
Overall CDx-all LDT CDx-22C3
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NordiQC PD-L1 IHC performance data – runs C1 to C5 – NSCLC
www.nordiqc.org
N=68 N=145 N=146 N=163 N=176
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www.nordiqc.org
False NegativePD-L1 status change
High to Low / Low to Neg
False PositivePD-L1 status change
Negative to Low / Low to High
Other cause”Technical”
C1 (50% insuff.) 68% 15% 17%
C2 (16% insuff.) 68% 23% 5%
C3 (9% insuff.) 100% - -
C4 (14% insuff.) 74% 4% 22%
C5 (20% insuff.) 59% 18% 23%
C1-C5 average 22% insufficient
NordiQC PD-L1 IHC performance data – runs C1 to C5Characteristics of insufficient results
74% 12% 14%
Which Companion Diagnostic assay (CDx)? How to calibrate Laboratory Developed Tests (LDT)? How to prove successful IHC test?
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Examples of insufficient results in NordiQC runs C1 – C5
SK006 TPS ≥50% SK006 TPS 1-49% SK006 TPS <1%PD-L1 IHC TPS 1-49% PD-L1 IHC TPS <1% PD-L1 IHC TPS 1-49%
”FN” ”FN” ”FP”
Reference standard mat.ICAPCs*
Test performance characteristics (critical) Source
ALK Lung Colon / Appendix A weak to moderate staining reaction in axons NordiQC
CD117Colon / Appendix
A weak to moderate staining in Cajal cells NordiQC
Estrogen receptor Tonsil + Uterine cervixA weak to moderate staining in folliculardendritic cells
NordiQC
HER2 BC HER2 2+ (with amp)An equivocal complete weak to moderate moderate staining in >10% of TCs
NordiQC
PD-L1…. TonsilA weak to moderate staining in germinalcentre T-cells and macrophages
NordiQC
Critical controls – Reference standard materials / ICAPCs* - predictive IHC
*Immunohistochemical critical assay performance controls - ICAPCs
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1. E Thunissen et al.
Lung cancer 113 (2017) 102-105
PD-L1 IHC in NSCLC with a global
and methodological perspective
2. E Thunissen et al.
Arch Pathol Lab Med. 2017-0106-SA
doi: 10.5858/arpa.
Immunohistochemistry for pulmonary biomarkers
1. “For selection of the samples… samples with a highepitope concentration will reach the maximum level ofstaining and are likely to be positive in any PD-L1Assay. This notion may also hold for placentaas an external positive control.”
2. “The optimal IHC positive control has anintensity performance at or above the lower limit ofdetection and is defined by an observed positive reaction(staining) in a tissue/cellular element that is known toexpress low levels of the evaluated marker.”
Critical versus non-critical samples for internal QC
Accurate
InaccurateNordiQC run C1
1. Identify best practice protocols - Abs
2. Set-up internal and EQA programs to
support and monitor quality and
development of new tests (e.g. ROS1)
3. Identification of reliable reference materials
What do we need for IHC precision testing in lung cancer.. ??
NordiQC – EQA data on IHC for lung cancer
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