NordiQC. Seminal results during 15 years.. Søren Nielsen Scheme Manager NordiQC Aalborg University Hospital, Denmark

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1 Seminal results during 15 years.. Søren Nielsen Scheme Manager NordiQC Aalborg University Hospital, Denmark

2 Now and the After 55 Years 2

3 IHC Quality Problem 9 nordic labs 2001 Run 2 CK-LMW 3

4 The biomarker protocol trap Caution: not for faint-hearted lab personnel!!!!! Decalcification Preparation Tissue Type, Dimension, Laser resection, De-differentiation With 3 choices for 5 variables in each phase = > 4 million protocols. Controls Quantification Reporting Fixation Time, Type, Volume Preanalytic Postanalytic Section Thickness Storage Drying Pre-treatment Manual Stainer Visualization Sensitivity, Specificity Primary antibody Clone, Dilution Buffer, Time, Temp Analytic Interpretation Localization Positive/Negative - cut-off level Development Sensitivity, Localization

5 Perspective 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 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 (lung) 5

6 EQA Program Aim of NordiQC: The aim of Nordic immunohistochemical Quality Control (NordiQC) is to promote the quality of immunohistochemistry and expand its clinical use by arranging schemes for immunohistochemical proficiency testing and providing examples of recommended protocols, tissue controls and other relevant information including descriptions of epitopes and technical protocol parameters.

7 Topics: Have we seen an improvement of IHC test quality? Has it been possible to identify recommendable IHC protocols? Has it been possible to identify recommendable IHC controls? 7

8 EQA Program Class / Type I IHC result and score is related to 1. Technical quality Class / Type II 2. Calibration level can the test be applied for the intended purpose? (Input on analytical sensitivity / specificity of the IHC method)

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12 Parameters affecting the IHC analytical test quality: 1. The quality of the antibodies available 1. Signal-to-noise 2. Affinity 3. Specificity 2. The flexibility and consistency of the IHC protocol backbone 1. Instrumentation fully automated 2. Broad spectrum of ancilliary reagents HIER, detection. 3. The calibration of the IHC assay to Fit-For-Purpose 1. One or multiple purposes. 2. Defined and accepted purpose 12

13 Parameters affecting the IHC analytical test quality: 1. The quality of the antibodies available 1. Signal-to-noise 2. Affinity 3. Specificity 2. The flexibility and consistency of the IHC protocol backbone 1. Instrumentation fully automated 2. Broad spectrum of ancilliary reagents HIER, detection. 3. The calibration of the IHC assay to Fit-For-Purpose 1. One or multiple purposes. 2. Defined and accepted purpose 13

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15 Ab use Run SP4 / EP12 DCS6 / P2D11F11 5% (3/57) 86% (49/57) Run % (251/257) 2% (5/257

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17 New Abs / clones out-perform previous Gold-standards YR145 and EP10 were most succesful. YR145 used as reference Ab by NordiQC. Same scoring criteria and same tissue composition all 5 runs - GIST vs Desmoid tumour - Appendix as pos/neg control

18 Too many choices.. Too many bad choices. Just focusing on primary Ab clone; Good choice: B22.1/B EP17 - EP17/30 Pass rate 93% (56/60 participants) Bad choice: CAM5.2 Pass rate 29% (10/35 participants)

19 Parameters affecting the IHC analytical test quality: 1. The quality of the antibodies available 1. Signal-to-noise 2. Affinity 3. Specificity 2. The flexibility and consistency of the IHC protocol backbone 1. Instrumentation fully automated 2. Broad spectrum of ancilliary reagents HIER, detection. 3. The calibration of the IHC assay to Fit-For-Purpose 1. One or multiple purposes. 2. Defined and accepted purpose 19

20 Estrogen receptor; Pass rate influenced by protocol harmonization and availability of fully automated IHC systems 2003 B B23 Ready-To-Use format 21% 81% HIER by in-house buffer 88% 5% HIER by high ph 70% 94% Polymer/multimer kit 56% 97% Fully automated system 6% 78%

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22 Antibody clone choice depends on IHC platform for laboratory developed assays Semi-automated versus Fully-automated Adjustment period (where to add Omnis) Semi-automated versus Fully-automated

23 Antibody clone choice depends on IHC platform for laboratory developed assays Melan A mab clone A103 AS48 Link versus OMNIS Similar settings Left Right

24 An antibody clone is like a child and a biological being requiring attention Breast ductal carcinoma Renal clear cell carcinoma CK LMW mab B22.1/B23.1 or DC10 VMS Ultra - OptiView CK LMW mab 5D3

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28 Parameters affecting the IHC test quality: 1. The quality of the antibodies available 1. Signal-to-noise 2. Affinity 3. Specificity 2. The flexibility and consistency of the IHC protocol backbone 1. Instrumentation fully automated 2. Broad spectrum of ancilliary reagents HIER, detection. 3. The calibration of the IHC assay to Fit-For-Purpose 1. One or multiple purposes. 2. Defined and accepted purpose 28

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30 Clone (20% ALK1 ) Detection system CALIBRATION PURPOSE OF TEST

31 100% pass rate ALK in ALCL 67% pass rate ALK in lung cancer

32 Class / Type I 2. Calibration level can the test be applied for the intended purpose? Class / Type I 1. Esophagus; positive tissue control with CK HMW, primary subtypes 5 & Liver; positive tissue control with CK LMW, primary subtypes 8 & SCLC; neoplasia with moderate level CKs 4. Lung ad. carc; neoplasia with high level CKs Range seen in diagnostics 5. RCC; neoplasia with low level CKs

33 Lung squam. cell carc. Lung ad. carc. SCLC RCC CK-PAN - mab AE1/AE3 Prot. 1 SP54 CK-PAN - mab AE1/AE3 Prot. 2SP54

34 Lung squam.. Esophagus Lung ad. carc. Liver SCLC RCC CK-PAN - mab AE1/AE3 Prot. 1 SP54 CK-PAN - mab AE1/AE3 Prot. 2SP54

35 CD56: Optimal Insufficient. Tissues/cells with only high expression will not identify: 1. A poorly calibrated IHC assay 2. A reduced sensitivity in an optimally calibrated IHC assay If an IHC test is used to demonstrate the target antigen being expressed at different levels, the controls must reflect this!

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37 External controls icapcs; IHC critical assay performance controls

38 Examples for 17 markers Generel expected patterns High expression (Right antibody) Low expression (Appropriate sensitivity) No expression (Appropriate specificity) Which tissue Which cells Which extension Which intensity

39 Parameters affecting the IHC test quality: 1. The quality of the antibodies available 1. Signal-to-noise 2. Affinity 3. Specificity 2. The flexibility and consistency of the IHC protocol backbone 1. Instrumentation fully automated 2. Broad spectrum of ancilliary reagents HIER, detection. 3. The calibration of the IHC assay to Fit-For-Purpose 1. One or multiple purposes. 2. Defined and accepted purpose 39

40 RTU system Ab, protocol, reagents & platform

41 EQA Program Analysis: Concentrated antibodies; Clones Titre range Epitope retrieval Platform performance Ready-To-Use antibodies; Protocol Plug-and-play Modification need

42 EQA Program Analysis: Concentrated antibodies; Clones Titre range Epitope retrieval Platform performance Ready-To-Use antibodies; Protocol Plug-and-play Modification need

43 EQA Program Analysis: Concentrated antibodies; Clones Titre range Epitope retrieval Platform performance Ready-To-Use antibodies; Protocol Plug-and-play Modification need

44 NordiQC seminal results on the analytical part of IHC indicate; 1. Identification of purpose of IHC assay must be identified. 2. Both LDT and RTU systems will be the basis for IHC testing, however RTU systems will be most widely used. 3. Selection of antibody clone depends on the IHC system. 4. Identification and use of reliable tissue / cell line controls with defined performance characteristics are essential to monitor correct calibration and precision testing. 5. External quality control is still needed and must be developed together with industry (IHC vendors, DIA, KOL, etc ). 44

45 Thanks to Staff: Jesper Lund Lauridsen Lise Emanuelsen Lilli Malaca Guldager Maria Lund Nielsen External data analysts: Michael Bzorek Ole Nielsen Assessors: Ari Ristimäki (FI) Jan Klos (NO) Michael Bzorek Ole Nielsen Viktoria Gaspar (S) Assia Bassarova (NO) Päivi Heikkilä (FI) Vibeke Jensen Viktoria Gaspar (S) Anne-Vibeke Lænkholm Michael Bzorek Ole Nielsen Andreas Scheel (D) Birgit Guldhammer General module Breast module HER2 ISH module Companion dx module Mogens Vyberg, Rasmus Røge, Søren Nielsen

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