EQA of gynecological cancer markers The NordiQC experience. Rasmus Røge Scheme Organizer, NordiQC Aalborg University Hospital

Transcription

1 EQA of gynecological cancer markers The NordiQC experience Rasmus Røge Scheme Organizer, NordiQC Aalborg University Hospital

2 Markers WT1 Discussed by prof. Mogens Vyberg prev. ER Discussion by Søren Nielsen: Friday 12/6 PAX8 p16 p57 Ca-125

3 Data Data obtained between 2005 and 2015 Core group members: Michael Bzorek Viktoria Gaspar Jan Klos Jesper Lund Lauridsen Ole Nielsen Søren Nielsen Ari Ristimäki Rasmus Røge Tomas Seidal Mogens Vyberg

4 PAX8 Paired Box gene-8 protein Nuclear transcription factor of the paried box family expressed during development of thyroid, kidney and Müllerian tract Normal tissue: Nuclear staining of Thyroid, kidney, fallopian tube, uterus Neoplasms: 99% of high-grade ovarian serous carcinoma, 98% of endometrial adenocarcinomas 71% of non-serous ovarian epithelial neoplasms, 91% of thyroid, 90% of renal cell carcinomas, Most squamous lesions of cervix negative

5 PAX8 NordiQC results Run 34, 2012 Run 42, 2014 Participants (n) Pass rate 64% 71% Tissue Fallopian tube Tonsil Renal clear cell carcinoma Kidney Lung adenocarcinoma Serous ovarian carcinoma Run 42, 2014

6 PAX8 NordiQC results

7 PAX8 NordiQC results Run 42, 2014 Optimal Good Borderline Poor Concentrated 39% 32% 22% 6% RTU-format 22% 46% 26% 7% Main causes of insufficient Weak or false-negative staining Less succesful perfomance of the mab MRQ-50 on the Ventana Benchmark Platform Use of low-sensitive detection systems Use of too short efficient HIER time Too low concentration of the primary antibody False positive staining Too high concentration of the primary antibody

8 MRQ-50 clone (1:200), HIER in alkaline buffer, 2-step polymer visulization system, LabVision autostainer MRQ-50 clone (Ventana RTU), HIER in alkaline buffer, 2-step polymer visulization system, Ventana BenchMark ultra PAX8 NordiQC results

9 PAX8 MRQ-50 Concentrated on Ventana: Only 1 optimal slide (using highsensitive detection system (Optiview) and 64 min HIER) RTU on Ventana: Only 6% optimal slides (2/36), both using increased HIER-time and a 3-step polymer/multimer-based detection system

10 MRQ-50 clone (1:200), HIER in alkaline buffer, 2-step polymer visulization system, LabVision autostainer MRQ-50 clone (Ventana RTU), HIER in alkaline buffer, 2-step polymer visulization system, Ventana BenchMark ultra PAX8 Controls

11 PAX8 Cross-reactivity Due to cross-reaction with B-cell specific activator protein (PAX5 / BSAP) Seen in mab raised against the N-terminal of the protein Some clones, BC12 and rmab ZR-1, do not exhibit this pattern

12 p16 Cyclin-dependent kinase inhibitor 2A / p16ink4a Slows down cell cycle by prohitibiting progression from G1 to S phase Mutations involved in wide range of cancers Overexpression is related to HPV-infection in many tissues Positive in cervical dysplasia and carcinomas Also important in oro-pharyngeal cancers

13 p16 Only evaluated once in 2009 Tissue block for staining consisted of Tonsil Uterine cervix Uterine cervical CIN3 Uterine cervical squamous cell carcinoma

14 p16 NordiQC results

15 p16 Frequent causes of insufficient staining: Less succesful antibodies rmab EP4353Y Insufficient HIER Too low concentration of the primary antibody

16 p16 mab E6H4 + CINtec -kit mab EP4353Y

17 p16 Controls mab E6H4 + CINtec -kit mab EP4353Y

18 p16 NordiQC results

19 p57 Cyclin-dependent kinase inhibitor 1C / Kip2 Involved in cell regulation Only maternal allelle is expressed Therefore, partial/incomplete moles have preserved p57 expression, while complete moles are p57 negative

20 p57 Only assessed once (2014) Tissue block for staining consisted of Tonsil Normal placenta 2 x Partial / Incomplete mole 2 x Complete mole

21 p57 NordiQC results

22 p57 Frequent causes of insufficient staining: Too low concentration of the primary antibody Too high concentration of the primary antibody (resulting in false-positives) Insufficient heat induced epitope retrieval (HIER)

23 p57 Partial mole mab Kp10, HIER in alkaline buffer, 3-step multimer based detection system (OptiView, Ventana) mab 25B2 (low titer), HIER in alkaline buffer, 2-step multimer based detection system (UltraView, Ventana)

24 p57 Controls mab Kp10, HIER in alkaline buffer, 3-step multimer based detection system (OptiView, Ventana) mab 25B2 (low titer), HIER in alkaline buffer, 2-step multimer based detection system (UltraView, Ventana) The protocol must be calibrated to give a moderate to strong nuclear staining reaction in the majority of cytotrophoblastic and intermediate trophoblastic cells, while an at least weak to moderate nuclear staining reaction must be seen in the majority of villous stromal cells. p57-report, Run 41, NordiQC

25 Ca-125 Membrane mucin-like glycoprotein Also known as MUC16 Tumor biomarker elevated, among others, in ovarian cancer but also in some benign conditions

26 Ca-125 Run 15, 2005 Run 29, 2010 Participants (n) Pass rate 67% 82% Tissue block for staining Appendix Fallopian tube 2 x Sereous ovarian carcinoma grade II &III

27 Ca-125 results

28 Ca-125 Frequent causes of insufficient staining: Too low concentration of the primary Ab Omission of HIER Insufficient HIER too short efficient HIER time HIER in citrate ph 6.0 Endogenous biotin

29 Ca-125 mab M11 with HIER OV185:1 too diluted

30 Ca-125 Controls mab M11 with HIER OV185:1 too diluted Normal salpinx is a recommendable positive control: Virtually all the epithelial cells must show a moderate to strong, distinct membranous staining. Ca-125-report, Run 29, NordiQC

31