NordiQC EQA module - OLV Z

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….


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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”

•

General observations


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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


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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 !


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Focus;

Technical performance

Level of analytical sensitivity- Unknown primary

Level of analytical specificity


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Clone…..


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Clone…..

Control /Calibration

Consequence…


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Focus;


Level of analytical sensitivityLevel of analytical selectivity


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Clone…..

pAbs are no-go


1.Technical quality: p40 – ”the good, the bad and the ugly”


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Placenta;level of analytical sensitivity

Tonsil;Level of analytical specificity


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Focus;



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



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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…


mAb XM26 rmAb SP27


mAb XM26 rmAb SP27Lung squam. cell

Lung TMA

Lung ad carc

Lung squam

Lung large cell


mAb XM26 rmAb SP27Lung squam cell carc.


mAb XM26 rmAb SP27Lung adenocarc.

Lung TMA

Lung ad carc

Lung squam

Lung large cell


mAb XM26 rmAb SP27TMA NeoplasiaGastric ad. carc.

Internal NordiQC data;14/62 lung adenocarcinomas pos by SP27 and neg by p40 / CK5 XM26


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Focus;



???


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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


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Focus;


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



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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


mAb OTI1A4 (”Aalborg LDT)

ALK SureFISH


Colon, appendix, tonsil

Patient Samples

Lung carc; Pos (FISH pos)

Lung carc; Neg (FISH neg)

ALCL; Pos (FISH pos)

Merkel carc; Pos (FISH neg)


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….


22C3 CDx IHC assay, SK006


Tonsil, placenta, cell lines

Patient Samples

NSCLC TPS; <1%

NSCLC TPS; ≥1-49%

NSCLC TPS; ≥50%


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

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10

20

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40

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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.. ??


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NordiQC EQA module - OLV Z

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