External Quality Assessment of Breast Marker Analysis. NordiQC data

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1 External Quality Assessment of Breast Marker Analysis NordiQC data Søren Nielsen Scheme Manager NordiQC Aalborg University Hospital, Denmark Aalborg

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3 Markers assessed in NordiQC Predictive markers Diagnostic markers Estrogen receptor Progesteron receptor HER2 IHC HER2 ISH (Ki67) GATA3 GCDFP15 Mammaglobin CK high mol. weight. Myosin heavy chain p63

4 App. 600 laboratories in total > 60 countries Generel Breast HER-2 ISH

5 Focus: Appropriate technical quality; signal-to-noise, morphology etc Appropriate analytical sensitivity and specificity indicated by concordance of ER status in the included tumours to reference

6 Uterine cervix Tonsil Breast carc. high Breast carc. low Breast carc. negative

7 Estrogen receptor; 14 runs slides assessed / protocols analyzed Sufficient Optimal 49% (n=1.111 slides) Good 29% (n=665 slides) Insufficient Borderline 13% (n=286 slides) Poor 9% (n=205 slides) 85% Weak / False negative 10% False positive 5% Poor signal/noise, imp. morph., etc

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9 Estrogen receptor; Pass rate influenced by participation New participants Old participants Run 10, % (n=61) 71% (n=134) Run B15, % (n=54) 86% (n=208) Run B19, % (n=86) 73% (n=259)

10 Estrogen receptor; Pass rate influenced by protocol harmonization B B15 Titre range / average titre 1: / 1:125 1: / 1:90 HIER by in-house buffer 88% 6% HIER by high ph 70% 94% Polymer/multimer kit 56% 93% Fully automated system 6% 59%

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12 HIER alk. ph 2- & 3-step kits Carefully calib. Uterine cervix all epithelial cells

13 Estrogen receptor; 85% Weak / False negative 10% False positive 5% Impaired morphology, etc Suf. Ins. Too low titre (SP1, EP1) Insufficient HIER (all clones) Clone 1D5 Clone 6F11 by HIER at high ph, 3-step pol. (not observed on VMS) Clone 1D5 at high titre, Biotin-based kits, HIER in pressure cooker

14 Focus: Appropriate technical quality; signal-to-noise, morphology etc Appropriate analytical sensitivity and specificity indicated by concordance of PR status in the included tumours to reference

15 Uterine cervix Breast carc. high Breast carc. low Breast carc. negative

16 Progesterone receptor; 8 runs slides assessed / protocols analyzed Sufficient Optimal 59% (n=798 slides) Good 22% (n=302 slides) Insufficient Borderline 10% (n=135 slides) Poor 9% (n=122 slides) 75% Weak / False negative 20% False positive 5% Poor signal/noise, imp. morph., etc

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19 HIER alk. ph 2- & 3-step kits Carefully calib. Uterine cervix all columnar epith. cells, and majority of basal squam. epith. cells (can be neg. in some pts).

20 Progesterone receptor; 75% Weak / False negative 20% False positive 5% Impaired morphology, etc Suf. Ins. Too low titre, Insufficient HIER Clone SP2 and 1E2. 1E2 mainly by off-label protocol (ext. sensitivity) Clone 1A6, Biotin-based kits, HIER in pressure cooker

21 Focus: Appropriate technical quality; signal-to-noise, morphology etc Appropriate analytical sensitivity and specificity indicated by concordance to FISH status in the included tumours.

22 NordiQC Internal QC of Test Material IHC status determined by: PATHWAY, HercepTest & Oracle Potential Donor tissue HER2 IHC status ISH status Selected Donor tissue HER2 HER2 0/1+ Un-amp. HER2 0/1+ Un-amp. HER2 2+ Un-amp. HER2 2+ Low level amp. IHC status Level 1 HER2 3+ High level amp. 4-6 identical TMA IHC status Level 2 IHC status Level 3 Slides circulated Thanks to the companies providing HER2 IHC kits for validation

23 NordiQC Internal QC of Test Material HercepTest PATHWAY Oracle Donor tissue validated by IHC using HercepTest, PATHWAY & Oracle Status confirmed by ISH

24 NordiQC Internal QC of Test Material TMA no. 1 HercepTest TMA no. 1 PATHWAY TMA no. 1 Oracle Each TMA (no: 1-6) validated by IHC using HercepTest, PATHWAY & Oracle

25 NordiQC Internal QC of Test Material TMA no. 1 Slide 1 Slide 50 Slide 100 Each TMA (no: 1-5) validated throughout the block by IHC using HercepTest, PATHWAY & Oracle 2+ Subsequently unstained slides are distributed to the laboratories

26 > CK Optimal Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0 Poor 10-20% HER2+ found in 2+ cat. Ampl. 3+ Ampl. 1+ Unampl. 1+ Unampl. 0 26

27 > CK Optimal Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0 Poor Ampl. 3+ Ampl. 2+ Unampl. 3+ Unampl. 1 27

28 Material processed according to ASCO/CAP

29 Pass rate of 19 HER2 IHC assessments in NordiQC App 90 % of insuff. results are FN and seen both by FDA / CE-IVD kits and laboratory developed assays. FP results have virtually only been seen by laboratory developed assays. 29

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31 CK7 ISH: ISH: Optimal Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl. 0 ISH: Good Increased need for additional ISH Ampl. 3+ Ampl. 2+ Unampl. 2+ Unampl

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34 Technically optimal results in the NordiQC HER2 ISH breast module: INFORM HER2 Dual ISH, Ventana ZytoDot 2C, ZytoVision HER2 black chr17 red HER2 green chr17 red U A 34

35 Typical causes for insufficient results in the NordiQC HER2 ISH breast module: FDA / CE-IVD HER2 BRISH (CISH/DDISH/etc) kits: INFORM HER2 Dual ISH, Ventana: Excessive proteolysis (>16M), HIER in CC1. DuoCISH pharmdx, Dako: Insufficient proteolysis, inappropriate handling of chromogen. ZytoDot 2C, ZytoVision: Excessive proteolysis. In 90% of insufficient results, no single or combination of causes could be identified 35

36 90% of ins. results used approriate protocol. 36

37 Technically insufficient results in the NordiQC HER2 ISH breast module: INFORM HER2 Dual ISH, Ventana HER2 black chr17 red Excessive protelysis U Neg areas >25% Silver precip. U 37

38 4 central parametres to adress for accurate HER2 IHC/ISH test 1. Pre-analytics mainly time-to and time-in NBF fixation 2. Analytical method used robustness and calibration 3. Control material precision of information 4. Interpretation experience, consistency, cut-off value

39 Lab 1; scored 1+ Lab 2; scored as 1+

40 Lab 1; scored 3+ Lab 2; scored as 3+

41 Lab 1; scored 2+ Lab 2; scored as 2+

42 What is faint? Visible at 40X? What is weak? Visible at 10X, 20X? Up to 20-40% HER2 IHC tests are reflexed to ISH due to expanded criteria for 2+ (internal data)

43 Pass rate of 19 HER2 IHC assessments in NordiQC From a technical point of view, it might be critical to change from a robust and relatively simple IHC assay to a less robust and complex test and simultanously more expensive test 43

44 Conclusions: 1. Pass rates for ER, PR and HER2 IHC are improved. Robust clones, high quality IHC systems. 2. FDA / CE-IVD labelled RTU assays / systems have shown superior performance compared to laboratory developed assays. 3. HER2 BRISH (DDISH/SISH/CISH) results have not been improved.

45 Assessors in NordiQC breast module and HER2 ISH Breast module Vibeke Jensen, DK Päivi Heikkilä, Fi Viktoria Gapar, SE Rasmus Røge, DK Søren Nielsen, DK (Assia Bassarova, NO) HER2 ISH Anne-Vibeke Lænkholm, DK Ole Nielsen, DK Michael Bzorek, DK Søren Nielsen, DK

46 See you later