Specialespecifikt kursus i Patologisk Anatomi 2018

Diagnostiske metoder Immunhistokemi

1. External Quality Assurance

Prof. Mogens VybergNordiQCInstitute of PathologyAalborg, Denmark

IMMUNOHISTOCHEMISTRY IN CANCER DIAGNOSTICS

• IHC - ancillary test, mostly used in the analysis and classification of tumours

• Antibody panels with diagnoses based on staining patterns (‘algorithms’)

• Relatively organ/tissue restricted transcription factors, has improved the accuracy of tumour diagnoses

• TTF-1, CDX2, PAX8, GATA3, p40 . . .

• IHC as a rapid and inexpensive surrogate for molecular studies: Mutations may give rise to• Occurrence of mutation-specific proteins

• BRAF, ALK, IDH1 …• Overexpression of proteins

• HER2, p53 …• Loss of proteins

• MMRPs, SMAD4, E-cad, INI1, ATRX …• Protein accumulations in an abnormal

cell compartment• Beta-catenin

• Predictive markers make IHC a stand-alone key to correct targeted therapy

• ER, PR, HER2, Ki67, PD-L1 …

IMMUNOHISTOCHEMISTRY IN CANCER DIAGNOSTICS

”Next generation immunohistochemistry”

Diagnostic utility of IHC may be hampered by• Preanalytical issues• (e.g., poor, short or delayed fixation; decalcification)

• Analytic issues:• Less successful or too dilute antibody clones/RTUs• Insufficient epitope retrieval• Insensitive visualization systems• Platform problems

• Post-analytical issues • (e.g. interpretation, reporting, image analysis)

IMMUNOHISTOCHEMISTRY IN CANCER DIAGNOSIS

• International organization for quality assurance of IHC• Founded 2003 by Nordic pathologists • Independent, scientific, not-for-profit organisation • Institute of Pathology, Aalborg University Hospital, DK

• General module: 3 runs/year• 15-18 different marker challenges

• Breast cancer IHC module: 2 runs/y• HER-2, ER/PR, Ki67/E-Cad …

• HER-2 ISH module: 2 runs/year• BRISH, FISH

• Companion module 2017-• PD-L1 / Lung cancer ….

Nordic Immunohistochemical Quality Control

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100

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800

2003 2005 2007 2009 2011 2013 2015

Nordic immunohistochemical Quality Control

NordiQC Participants

Nordic labs

2015-18

WWW.NORDIQC.ORGFREE ACCESS

Free PMC Article

Nordic immunohistochemical Quality Control

Serial sections stained for Estrogen receptor

Lab. A Lab. B

Optimally processed ductalbreast carcinoma tissue

Serial sections stained for Estrogen receptor

Lab. A Lab. B

False neg.

High expressor

Low expressor

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Serial sections stained for Estrogen receptorUterine cervix

Lab. A Lab. B

False neg.

Tonsil

Controls

Uterine cervix

Tonsil

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Serial sections stained for Estrogen receptor

Clone 6F11 in 15/37 labsClone SP1/EP1/1D5 in 225 labs

False pos.(mRNA=0)

ExternalQualityAssurance !

Uterine cervix Uterine cervix

Tonsil Tonsil

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Control

correct false negative

External Quality Assurance – ER

Craig Allred

“Through the inquiry, the public learned that between 1997 and 2005 nearly 400 of about 1,000 breast cancer patients receivedincorrect test results of the ER status of their breast tumors.”

External Quality Assessment

Suboptimal IHC assays may be due to:• Preanalytical issues

• Fixation too short, too late, decalcification too soon…• Analytical issues:

• Less successful / too dilute antibody clones/RTUs• Insufficient epitope retrieval• Insensitive visualization systems• Platform problems

• Post-analytical issues • Interpretation criteria, interobserver variation …

The challenge of IHC

Should beidentified

with proper controls

h

Lung+

Uroth

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WW

W.N

ORDIQ

C.ORG

Participant site

Alpha-methylacyl-CoA racemase CyclinD1 MLH1

Alpha-smooth muscle actin Cytokeratin 5 MSH2

Anaplastic lymphoma kinase Cytokeratin 7 MSH6

B-cell specific activator protein Cytokeratin 19 Multiple myeloma oncogene 1

bcl-2protein Cytokeratin 20 Myosin, smooth muscle heavy chain

bcl-6protein Cytokeratin, high molecular weight Napsin A

Calretinin Cytokeratin, low molecular weight Neurofilament protein

Cancer antigen 125 Cytokeratin, pan- Octamer transcription factor-3/4

Carcinoembryonic antigen Desmin p16ink4a

CD3 Detected on GIST-1 p40

CD4 E-cadherin p53

CD5 Epithelial cell adhesion molecule p57

CD8 Epithelial membrane antigen p63

CD10 Estrogen receptor alpha Paired box gene-2 protein

CD14 Factor VIII related antigen Paired box gene-8 protein

CD15 GATA3 Placental alkaline phosphatase

CD19 Glial fibrillary acidic protein PMS2

CD20 Glypican 3 Podoplanin

CD23 Gross cystic disease fluid protein-15 Prostate specific acid phosphatase

CD30 HER-2 Prostate specific antigen

CD31 Hepatocyte antigen Prostein

CD34 Human chorionic gonadotropin Progesterone receptor

CD45 Immunoglobulin kappa S-100 protein beta

CD56 Immunoglobulin lambda Sal-like protein 4

CD68 Immunoglobubin M SOX10

CD79a Ki-67 Synaptophysin

CD99 Mammaglobin Terminal deoxynucl. transferase

CD117 Melan-A Vimentin

Chromogranin Melanosoma specific antigen Wilm's tumour-1 protein

~100 IHCmarkers

in NordiQC

RunsTested 1-15 times

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WW

W.N

ORDIQ

C.ORG

Participant site

Multi-tissue FFPE blocks10% NBF 24-48 h (ASCO/CAP guidelines …)• Normal and clinically relevant tumour tissues • Different levels of antigen expression

• high, moderate, low, none

Test material

2 unstained slides for each marker send to the participants1 stained slide returned for central assessment

Open website

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PDF file e-mailed to the individual participants with assessment marks, and – if suboptimal –explanations and recommendations

Nordic Immunohistochemical Quality Control

Assessing the immunohistochemical assay quality Based on “standard” processed circulated tissues

Identifying optimal and insufficient results Correlated to antibodies, protocols and stainer platforms

Publishing general results Website: www.nordiqc.org Scientific journals

Giving directions for improvement Individually tailored recommendations

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Selected NordiQC publications

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Selected publications

AIMM 2015, 23:1

AIMM 2014, 22:241

AIMM 2016-17

NordiQC

Score Criteria: Staining reaction considered …Optimal … Perfect or close to perfect in all of the included

tissue cores

Good … Fully acceptable in all of the included tissue cores. However, the protocol may be optimized to ensure the best staining intensity and signal-to-noise ratio

Borderline … Insufficient because of, e.g., a generally too weak staining or a false negative staining of one of the included tissues, or a minor false positive staining reaction

Poor … Very insufficient because of, e.g., false negative staining of several of the included tissues, or a major false positive staining reaction

35%

33%

21%11%

OptimalGoodBorderlinePoor

NordiQC assessment results 2003 – 2014

General module ~ 20,000 slides ( ~100.000 core sections)

Insufficient 32%

58%21%

9% 12%

OptimalGoodBorderlinePoor

NordiQC assessment results 2003 – 2014

Breast cancer module ~ 9,000 slides (~35,000 core sections)

Insufficient 21%

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

Protocol recommendations

Typical protocol providing an optimal result

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352 labs advised in 6 challenges with repeated tests

(CGA, Calr, CD5, CD15, CD23, CK-LMW)

No. Improved %

Positive 227 167 74

Negative 125 22 18

Results of NordiQC recommendations

NordiQC EQA: Estrogen Receptor 2003-11

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10

20

30

40

50

60

70

80

90

100

8 10 13 B1 B3 B5 B7 B8 B10 B11 B13 B15 B17PASS RATE (%)

45%

87%

0

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20

30

40

50

60

70

80

90

100

8 10 13 B1 B3 B5 B7 B8 B10 B11 B13 B15 B17PASS RATE (%)

70

281122

141197

Number of participants

NordiQC EQA: Estrogen Receptor 2003-11

Estrogen receptorPass rate (optimal + good) by participant status

New participants ’Old’ participants

Run 10, 2004 57% 71%

Run B15, 2010 70% 86%

Run B19, 2015 51% 73%

Average 59% 77%

NordiQC EQA: Estrogen Receptor

Estrogen receptorChanges in protocol standardization 2003 - 2013

2003B8

2013B15

Titre range / average 1:10-1.000 / 1:125 1:10-400 / 1:90

HIER buffer by vendor 6% 88%

HIER by high pH 70% 94%

Polymer/multimer kit 56% 93%

Fully automated system 6% 59%

NordiQC EQA: Estrogen Receptor

2003 2005 2007 2009 2011 2013 2015

Estrogen receptorAntibody clone selection

SP1

EP16F111D5

NordiQC EQA: Estrogen Receptor

• Appropriate technical quality• Signal-to-noise, morphology etc.

• Appropriate analytical sensitivity and specificity:• Concordance with reference

NordiQC EQA: Estrogen Receptor

• Appropriate technical quality• Signal-to-noise, morphology etc.

• Appropriate analytical sensitivity and specificity:• Concordance with reference

NordiQC EQA: Estrogen Receptor

Uterine cervix

Breast carc. high Breast carc. low Breast carc. neg.

Tonsil

NordiQC EQA: Estrogen Receptor

EQA of breast markers - run B19: HER2

• Appropriate technical quality• Signal-to-noise, morphology etc.

• Appropriate analytical sensitivity and specificity:• Concordance with reference

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46

NordiQC runs for HER2 IHCCK7

Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0

Optim

al

Ampl. 3+ Ampl. 1+ Unampl. 1+ Unampl. 0

Poor

47

47

NordiQC runs for HER2 IHCCK7

Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0

Optim

al

Ampl. 3+ Ampl. 2+ Unampl. 3+ Unampl. 1

Poor

14%insuff.

HER-2 staining results in 17 runs

HER-2 staining: Approved vs. lab developed IVD

Approved IVD (n=1145)

Lab devel. IVD(n=558)

FN FP FN FP

NordiQCB6-B14

127 (11%)

0 141 (25%)

28(5%)

NordiQC -Roche

Collaboration

Roche – NordiQC joint venture

”Normalized” to the American breast cancer population: ~ 300 patients per year with approved IVD~ 700 patients per year with lab developed tests

Roche – NordiQC joint venture

”Normalized” to the American breast cancer population

HER-2 staining: Approved vs. lab developed IVD

Every $1 saved by laboratoriesusing cheaper reagents potentially results in ~ $6 additional costs to the health care system

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

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Poor antibodies (few examples)

Antigen Clone High expressor

Low expressor

Non expressor

CD5 CD5/54/F6 √ FN –CD23 MHM6 √ FN –CD31 1A10 (√) FN –CD31 SP38 (√) FN –CD138 5F7 (√) FN –CDX2 SP54 (√) FN FPCDX2 CDX2-88 √ FN FPCEA TF-3H8-1 √ √ FPCGA DAK. A3 √ FN –CK20 PW31 √ (√) –PR SP2 √ √ FPSYP SY38 √ FN –

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Poor antibodies: CD5

CD5 N Sufficient* Optimal*4C7 conc 145 74% 49%SP19 conc 11 91% 46%CD5/54/F6 conc 28 4% 0%

* With optimal protocol settings

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Poor antibodies: CD5

SP19 + optimal protocol CD5/54/F6

TonsilB-C

LL

TP FN

FNTP

Optimal (16%)

Poor antibodies: CD31

JC70A 1A10

Optimal (16%)

Poor antibodies: CD31

JC70A 1A10

Haemangiosarcoma

Poor antibodies – MLH1

MLH1 clone ES05 MLH1 clone EPR3894

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

CD5 Run 24 N Sufficient* Optimal*SP19 conc 11 91% 46%SP19 RTU Dako 3 100% 100%SP19 RTU VMS 14 79% 14%

Poor RTU formats: CD5

* With optimal protocol settings

FN

CD5 Run 34 N Sufficient* Optimal*SP19 RTU VMS 33 97% 97%

Poor RTU formats: CGA

LK2H10 REF pAb RTU Comp.1

mAb LK2H10 RTU Comp.3mAb LK2H10 RTU Comp.2

Medullary carcinoma

Poor RTU formats: CGA

Small cell carcinoma

LK2H10 REF pAb RTU Comp.1

mAb LK2H10 RTU Comp.3mAb LK2H10 RTU Comp.2

High Expressors

Low Expressor

Non-expressor

Optimal Insufficient

Chromogranin A

Critical Assay Performance Control: Peripheral nerves

Poor RTU formats: CGA

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

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Platform dependent antibodies

Antigen Clone XT / Ultraautomated

Bond-maxautomated

Autostainersemiautomated

CD4 1F6 FN Weak √

SP35 √ √ √CD56 123C3 FN Weak √

MRQ-42 √ ? √CD79a JCB117 Weak √ √

SP18 √ √ √BSAP/Pax5 24 FN Weak √

SP34 √ √ √BCL6 PG-B6p FN Weak √

GI191E/A8 √ √ √SYP 27G12 Weak √ √

MRQ-40 √ √ √

Hodgkin lymphoma NS

clone SP34RTU VMS/CM

clone 24RTU VMS/CM

Platform dependent antibodies: PAX5

x200 x200

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

70

Inappropriate antibody dilution – CD79a

JCB117 appropriate JCB117 too dilute

Plasmacytom

a

Inappropriate antibody dilution – Ig light chains

~1:300 ~1:3.000 ~1:30.000

IgK: Dako pAb A0191

Inappropriate antibody dilution – Ig light chains

239 IgK tests, 12 Abs: 12% optimalDako pAb A0191: 17% optimal+TRS/Ci 3.000-16.000: 29 % optimal

All other Abs: 0% optimal

Alternative: FLOW cytometry

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

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Inappropriate retrieval

AE1/AE3 + HIER AE1/AE3 + proteolysis

LiverR

CC

FNTP

FNTP

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Misleading datasheets

Giving false negative results when only LMW-CKs are present

Misleading datasheets

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IHC - NordiQC 2014

AE1/AE3 : Optimal results only obtained by HIER in NordiQC runs

Dako: RTU – HIER Conc: Proteolysis or HIERLeica: RTU – Proteolysis Conc: HIERThermo: Conc: HIER Quanto – Proteolysis UltraVision…………AE1/AE3/PCK26: Optimal results mainly obtained by HIER+protelysis in NordiQC runs

VMS: RTU - Proteolysis

Misleading data sheets + Wrong control material used

By 17th October 2014

Improved datasheets

NordiQC run 41 2014 – ECAD 271 labs

False positive: EP700Y

Fra: Galloway, Mary [mailto:Mary.Galloway@fda.hhs.gov] Sendt: 13. november 2014 01:14Til: Søren Nielsen / Region NordjyllandEmne: RE: Changes Made to Package Inserts

Sören,Thanks for identifying and alerting us to the issues with anti-E-cadherin (36) and anti-Pan Keratin. The package inserts are now changed (see links below).I hope we can continue to learn of any future staining problems you may uncover.Much appreciated!Mary

RCC

FNTP

Misleading datasheets

Giving false negative results in low expressing cells and tumours

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Major causes of insufficient stains in ~ 9,000 slides

Less successful antibodies (17%)

poor antibodies, less robust antibodies, poorly calibrated RTUs

stainer platform dependent antibodies

Insufficiently calibrated antibody dilutions (20%)

Insufficient or erroneous epitope retrieval (27%)

Error-prone or less sensitive visualization systems (19%)

Other (17%)heat induced or proteolysis induced impaired morphology

drying out phenomena

stainer platform dependant protocol issues

excessive counterstaining impairing interpretation

NordiQC general results 2003 – 2014

NordiQC run 41/42 2014 - MMR

MMR MLH1 mAb clone ES05, 1:20 LeicaUltraView + Amplification OptiView + Amplification (Tyr.)

1’ generation 3-step multimer, VMS 2’ generation 3-step multimer, VMS

NordiQC run 41 2014 – PMS2 131 labs

NO

mutation

Optimal: 47% Insufficient: 15%

NordiQC run 41 2014 – PMS2 131 labs

Mutation

Optimal: 47% Insufficient: 15%

NordiQC run 41 2014 – PMS2 131 labs

Mutation

Optimal: 47% Insufficient: 15%

•Too dilute Ab•Insufficient HIER•Insensitive viz system

PD-L1

>10%

Companion

PD-L1

Tonsil

NSCLCTPS >50%Immuno-therapy: 1. line

NSCLCTPS 1%?Immuno-therapy2. lineor none

22C3 RTU 22C3 LDT

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Tailored recommendations

Replace less successful antibodies (conc./RTU)

Calibrate the antibody concentration

Use HIER (instead of proteolysis or no retrieval)

Increase HIER time / temperature / buffer pH For 95% of epitopes pH 8-9 is preferable to pH 6

Use a non-biotin based viz. system

Use FDA approved kits instead of home-brews

. . . . .

Improve the internal QC: Identify the right controls Select well defined low expressor cells/tissues

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External Quality Assurance (EQA)

Provides objective evidence of lab performance

Identifies methodological errors

Provides directions for improvements & controls

The results of the NordiQC work indicate that

Improvement of IHC is strongly needed

EQA schemes, industry and KOL must align - describing the requirements for optimal IHC performance.

Conclusion

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Collaboration between Companies and EQA schemes

• Define expression patterns for markers

• Identify best controls and CSQIs

• Implement these in guide lines and package inserts

Companies

• Discontinue poor antibodies

• Guide laboratories

• platform dependent clones

• Amend inappropriate package inserts.

Conclusion

Almost 1/3 of all IHC stains produced by NordiQC participants are still insufficient ! New labs New antibodies, techniques, platforms Increasing demands

How many IHC stains produced by labs not participating in an EQA scheme are insufficient ?How many scientific publications are based oninsufficient IHC stains ?What are the consequences for the patients ?

Perspective