Breast cancer: Antibody selection, protocol optimzation controls and EQA
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1 Breast cancer: Antibody selection, protocol optimzation controls and EQA Workshop in Diagnostic Immunohistochemistry Oud St. Jan/ Old St. John Brugge (Bruges), Belgium June 13th 15nd 2018 Rasmus Røge, MD, NordiQC scheme organizer With compliments to Søren Nielsen
2 Invasion? Subclassification (lobular/ductal) Metastasis Treatment? IHC markers in Breast Cancer Smooth muscle myosin Actin p63 E-cadherin GATA3 Mammaglobin GCDFP-15 ER HER2 PR CK5
3 Invasion? Subclassification (lobular/ductal) Metastasis Treatment? IHC markers in Breast Cancer Smooth muscle myosin Actin p63 E-cadherin GATA3 Mammaglobin GCDFP-15 ER HER2 PR CK5
4 Participants in NordiQC General Breast cancer Her2 ISH Run 2018: General 359, Breast cancer module 460, HER2-ISH 231, Companion module 187
5 Estrogen receptor (ER) Data obtained in run B25, 2018
6 Main focus of assessment: Appropriate technical quality (signal-to-noise, good morphology etc.) Appropriate analytical sensitivity and specificity indicated by concordance of ER status and proportion of positive cells in the included tumours to references Breast cancer module assessment setup (B25)
7 Uterine cervix Tonsil Carcinoma (High) Carcinoma (Low) Carcinoma (Neg)
8 ER: Overall performance
9 ER: Protocol parameters 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%
10 ER: Development in Ab clones
11 ER: Pass rate influenced by participation New participants Old participants Run B10, % (n=61) 71% (n=134) Run B15, % (n=54) 86% (n=208) Run B19, % (n=86) 73% (n=259) Run B25, % (n=38) 93% (n=326)
12 ER: Typical challenges 85% Weak / False negative 10% False positive 5% Impaired morphology, etc Sufficient Insufficient Too low titre (EP1, SP1 conc.) Insufficient HIER, 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
13 Concentrated format: Overall protocol parameters HIER alk. ph 2- & 3-step kits ER: Selection of primary Ab and format Carefully calibration of primary Ab
14 ER: Selection of primary Ab and format
15 ER: Basic protocol for optimal staining Retrieval Titre Detection RTU Detection mab 1D5 HIER High 1: & 3-step Dako 2- & 3-step mab 6F11* HIER Ci, High 1: & 3-step Leica 3-step rmab EP1 HIER High 1: & 3-step Dako 2- & 3-step rmab SP1 HIER High 1: & 3-step Ventana 2- & 3-step * Efficient HIER, high conc., 3-step pol. & low stringent washing can give aberrant nuclear staining Not seen on Ventana stainer, rarely on Autostainer and most commonly on Bond stainer.
16 ER: Controls
17 Progesteron receptor (PR) Data obtained in run B24, 2018
18 Main focus of assessment: Appropriate technical quality (signal-to-noise, good morphology etc.) Appropriate analytical sensitivity and specificity indicated by concordance of PR status and proportion of positive cells in the included tumours to references Breast cancer module assessment setup (B24)
19 Uterine cervix Can vary. Tonsil Carcinoma (High) Carcinoma (Low) Carcinoma (Neg)
20 PR: Overall performance
21 PR: Typical challenges 75% Weak / False negative 20% False positive 5% Impaired morphology, etc Tonsil Sufficient Insufficient Too low titre (16, PgR636) Insufficient HIER Clone SP2 and 1E2. 1E2 mainly by off-label protocol (ext. sensitivity) Clone 1A6, Biotin-based kits, HIER in pressure cooker
22 PR: Selection of primary Ab and format
23 PR: 1E2 RTU False-positive staining (B18-24) Tonsil Carcinoma (Neg) Tonsil 1E2 Carcinoma (Neg) 1E2 Typically related to reduced HIER time and/or increased incubation time of primary Ab
24 PR: Basic protocol for optimal staining Retrieval Titre Detection RTU Detection mab 16 HIER High 1: & 3-step Leica 3-step mab PGR636* HIER (High) 1: & 3-step Dako 3-step mab PGR1294 HIER (High) 1: & 3-step Dako 2-step rmab 1E2** HIER High - - Ventana 2-step * mab clone PGR636 has shown to be less successful on Ventana BenchMark Ultra ** rmab clone 1E2, RTU might provide aberrant false pos. result by 3-step protocols, reduced HIER and prolonged Ab incubation time compared to Ventana guidelines
25 PR: Controls
26 HER-2 IHC Data obtained in run B25, 2018
27 Main focus of assessment: Appropriate technical quality (signal-to-noise, good morphology etc.) Appropriate analytical sensitivity and specificity indicated by concordance of HER2 status to IHC reference slides and FISH status in all the included tumours. Breast cancer module assessment setup (B25)
28 HER2 IHC: Results B25
29 > Optimal Amplified, 3+ Amplified, 2+ Unamplified, 2+ Unamplified, 0 False negative Poor Amplified, 1+ Amplified, 0 Unamplified, 0 Unamplified, 0
30 > Optimal Amplified, 3+ Amplified, 2+ Unamplified, 2+ Unamplified, 0 False positive Poor Amplified, 3+ Amplified, 3+ Unamplified, 3+ Unamplified, 1+
31 Typical causes for insufficient results in the NordiQC HER2 IHC breast module FDA / CE-IVD HER2 IHC kits PATHWAY, Ventana: Too short HIER (<24 min) and/or too short incubation of primary Ab (<12 min) HercepTest, Dako: Too short HIER (<40 min) and/or too short incubation of primary & secondary Ab (<30 min) Oracle, Leica: No single or combination of causes have been identified Laboratory developed assays Inappropriate titre of primary Ab Less successful primary Ab Insufficient HIER
32 HER2 IHC: FDA-/CD-IVD versus LD assays
33 HER2 IHC: Controls Histology: 3+ tumour 2+ tumour Cell lines: Applicable for DIA & ref data comparing run-to-run
34 Histocyte cell lines HER2 stained with: PATHWAY IHC Cell line 1 3+ Cell line 2 2+ Cell line 3 1+ Cell line 4 0
35 HER-2 ISH Data obtained in run H13, 2018
36 HER2 ISH module assessment setup (H13)
37 HER2 ISH: BRISH results H13
38 INFORM HER2 Dual ISH, Ventana HER2 Black Chr17 Red ZytoDot 2C, ZytoVision HER2 Green Chr17 Red HER2 ISH: Optimal results Unamplified Amplified
39 HER2 Black Chr17 Red INFORM HER2 Dual ISH, Ventana Excessive protelysis HER2 ISH: Technically insufficients results Neg areas >25% Silver precipitates
40 Tycial causes for insufficient BRISH HER2 results INFORM HER2 Dual ISH, Ventana Excessive proteolysis (> 16 min) HIER in CC1 DuoCISH pharmdx, Dako Insufficient proteolysis Inappropriate handling of chromogen ZytoDot 2C, ZytoVision Excessive proteolysis However, in most insufficient results no single cause (or combination) could be identified
41 Development of pass rate in the NordiQC HER2 ISH module
42 HER2 Gene-Protein-Assay (Roche): HER2 IHC + DDISH ( ) Pass rates H9: 86% (n=7) H10: 75% (n=12) H11: 50% (n=14) H12: 94% (n=17) H13: 100% (n=17) 42
43 Pass rates for ER, PR and HER2 IHC have improved due to robust clones and high quality IHC systems. Conclusions CE-IVD labelled RTU assays / systems show superior performance compared to laboratory developed assays. HER2 BRISH (DDISH/SISH/CISH) results have not been improved significantly.
Optimization of antibodies, selection, protocols and controls Breast tumours
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