Immunocytochemistry. Results - Summary Graphs - Pass Rates Best Methods - Selected Images. Run 100. Assessment Dates: 3 rd - 22 nd January 2013

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1 Run 1 IN THIS ISSUE Short Report In-house Tissue Survey Page 2-3 Immunocytochemistry Modules Immunocytochemistry Results - Summary Graphs - Pass Rates Best Methods - Selected Images Assessment Dates: 3 rd - 22 nd January 213 General Pathology: CD31/CD34 & CD Breast Pathology: PR Breast Pathology: HER2 IHC Gastric: HER2 IHC 3-37 Lymphoid Pathology: CD3 & CD Neuropathology: Synaptophysin & -Amyloid Cytology: CD45 & Cytokeratin Alimentary Tract: GIST: CD117 & DOG In-situ Hybridisation Modules Breast: HER2 ISH Interpretive 7-73 Breast HER2 ISH Technical 74-8 Commercial sponsors in this Issue Cover Photos: Top image: Breast Hormonal Receptor Module; Optimally stained UK NEQAS ICC distributed samples stained with PgR 636 clone. (A) high (B) Low and (C) negative PR- expressing tumours. Bottom Images: Breast hormonal receptor module; Good in-house control showing (left) High- (right) low PR-expressing tumour samples. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent

2 General Information Data shown in this article is collated from UK NEQAS ICC & ISH assessments and is presented and described as collected, and does not ether endorse nor denounce any particular product or method and is provided as a guide to highlight optimal and sub-optimal staining methodologies. UK NEQAS ICC & ISH does not endorse any of the products featured by the commercial sponsors and are placed at the discretion of UK NEQAS ICC & ISH. Furthermore, commercial companies featured do not have any input or influence over the content, including results that are shown. For further information of the UK NEQAS ICC & ISH scheme, general EQA enquiries, slide returns and advertising opportunities please contact: Dr Merdol Ibrahim, Scheme Manager UK NEQAS ICC & ISH Suite 4/12 Hamilton House Mabledon Place London WC1H 9BB, UK Tel: +44 (2) merdol.ibrahim@ucl.ac.uk For enquiries concerning training issues, meetings, or courses, please contact: Mr Keith Miller, Scheme Director UK NEQAS ICC & ISH Suite 4/12 Hamilton House Mabledon Place London WC1H 9BB, UK Tel: +44 (2) k.miller@ucl.ac.uk Director Mr Keith Miller (k.miller@ucl.ac.uk) Manager Dr Merdol Ibrahim (merdol.ibrahim@ucl.ac.uk) Deputy Director Mr Andrew Dodson (a.r.dodson@liverpool.ac.uk) Assistant Manager Ms Suzanne Parry (s.parry@ucl.ac.uk) Office Manager Mrs Ailin Rhodes (a.rhodes@ucl.ac.uk) Quality Manager Mr Neil Bilbe (n.bilbe@ucl.ac.uk) Clerical Assistant Mrs Clara Lynch (clara.lynch@ucl.ac.uk) ASSESSORS United Kingdom Mr C Abbott, Bristol Dr N Atkey, Sheffield Dr M Arends, Cambridge Dr M Ashton-Key, Southampton Mrs J Bell, Nottingham Mr N Bilbe, London Mr D Blythe, Leeds Mr J Brown, London Dr L Carson, Aberdeen Ms E Clark, Surrey Mrs A Clayton, Preston Mrs A Cramer, Manchester Dr S Di Palma, Surrey Mr A Dodson, Liverpool Mrs G Donald, Maidstone Mr R Fincham, Cambridge Mr D Fish, Reading Mrs S Forrest, Liverpool Dr I Frayling, Cardiff, Wales Ms J Freeman, London Dr C Gillett, London Ms J Gorst, Bucks Mr J Gregory, Birmingham Prof A Hanby, Leeds Mr N Hand, Nottingham Ms L Happerfield, Cambridge Dr R Hunt, Stockport Dr M Ibrahim, London Mr P Jackson, Leeds Prof B Jasani, Cardiff Mrs N Johnson, Cambridge Ms S Jordan, London Dr J Joseph, Preston Mrs M Judd, Southampton Ms Kathryn Kennedy, Belfast Mrs J MacMillan, Glasgow Mr C Marsh, Newcastle Dr P Maxwell, Belfast Dr G King, Aberdeen Mrs H McBride, Belfast Mr J McGloin, London Dr S McQuaid, Belfast Mr K Miller, London Ms J Moorhead, London Dr M Morgan, Cardiff Ms P Jones, London Ms A Newman, London Mrs L Necus, Kettering Dr G Orchard, London Ms S Parry, London Ms A Patterson, Belfast Dr S Pinder, London Dr M Pitt, Preston Mrs F Rae, Edinburgh Dr A Riley, Stirling Mr G Rock, Birmingham Mr J Ronan, Nottingham Dr J Starczynski, Birmingham Mrs C Thomas, Preston Mr P Thompson, Leeds Mr A Watson, Newcastle Mr P Wencyk, Nottingham Mrs H White, Maidstone Mrs J Williams, Portsmouth Ms S Wozniak, Cardiff Australia Mrs J Brincat, Victoria Canada Mrs J Tunnicliffe, Vancouver, Prof. J Bartlett, Toronto Denmark Mr J Askaa, Copenhagen Dr E Baslev, Herlev Dr B Rasmussen, Roskilde Germany Dr Iris Nagelmeier, Kassel Hungary Dr T Krenacs, Szeged Ireland Prof E Kaye, Dublin Mr K McAllister, Dublin Dr T O Grady, Dublin Ms Yvonne Connolly, Dublin Dr Hilary Magee, Dublin Portugal Dr J Cabecadas, Lisbon Dr M Franco, Lisbon Dr F Schmitt, Porto Mr A Ferrero, Lisbon Mrs T Periera, Lisbon Mr R Roque, Lisbon Mr J Matos, Lisbon Slovenia Dr M Flezar, Ljubljana Mrs I Kirbis, Ljubljana South Africa Mrs R Van Wijk, Cape town Sweden Dr G Elmberger, Stockholm Switzerland Dr Pierre-Andre Diener, St Gallen Journal layout and design prepared by UK NEQAS ICC & ISH UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent

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4 Participants' In-house Controls - A Short Survey Neil Bilbe, Merdol Ibrahim, Suzanne Parry and Keith Miller Introduction The use and subsequent assessment of participants in-house control material has often been the subject of much debate. The choice of material/tissue and its appropriateness can also have an influence on the score that the lab is awarded during the assessment. Although in-house slides are not part of the poor performance monitoring, participants are reminded that their results from in-house slide assessments can or could be used to gauge performance. The annual User Survey sent out in May 212 contained an extra section at the end, where participants were asked 4 questions about the use of in-house controls along with a section to allow participants to add any comments. Module trends were also analysed. tissue type or pathological entity differs from the overall trends. N.B. Most labs subscribe to several different modules (average of 3+) so if the protocols for using negative controls varies from one module/pathology to another, this may not be evident from a single response. The following graphs are therefore produced as a guide: 1. The use of negative controls; response of Yes by Module: Approximately 165 responses were received, which accounts for about one third of total participant numbers, which nevertheless gives an idea of the current trends used by laboratories. Results 1. Do you use negative controls when staining? 2. Placing the control section on the same slides as the test section by Module: 2. Do you place the control section on the same slide as the test? 3. Do you use the same control procedures for UK NEQAS ICC & ISH sections as your own? 3. Using the same control procedures on NEQAS slides as day to day cases by Module: 4. Do you try and use the same tissue type as the UK NEQAS ICC & ISH section/module? Using the cross tabulation facility to gauge how individual modules have responded, may help to see if a particular 2

5 Participants' In-house Controls - A Short Survey 4. Trying to use the same tissue as the material sent by NEQAS by Module: Summary and conclusions Negative controls is used by about half of the labs (48%). The only significant response was the low use by neuropathology labs (36%). The majority of labs (51%) use a composite slide with both the test and control section/material on, although the cytology labs returned the lowest response (45%), which is understandable if you have a smear or large cytospin etc. As expected almost all labs (87%) use the same controls for NEQAS slides as their own surgical cases. Half the labs (48%) use the same tissue type as the NEQAS section/module, with no significant trend by module. Participants Comments: It is not always possible to have the same type of control tissue. Methods optimised for our processing. Difficult to use cytospin preparation controls for the Cytology module. For some antigens in the Neuro module, our routine positive control tissue is 'non-neuro' (e.g. Ki-67, cytokeratin). We run negatives when working up antibodies and validating new control material, once satisfied we no longer use them. We have altered our in house breast composite blocks to have non DCIS tumour, as per NEQAS. Positive controls are placed on the test slide where possible i.e. if space allows. Tumour control tissue is always run on a separate slide. Positive controls run with NEQAS sections tend to be the in-house sections submitted for that antibody. We have a very limited choice of control tissue being a small routine lab. It's often impossible to get cytology in-house controls so we have to resort to a paraffin equivalent Cytology might have a paraffin cell block, so will need different retrieval. Capacity is an issue, we are currently only running negatives with breast and cytology specimens. Not always the same type of tumour available. 3

6 The General Pathology Module Run 1 Julie Williams and Andrew Dodson Gold Standard Second Antibody Antigens Assessed: CD31/CD34 CD3 Tissue Sections circulated: Normal Appendix & GIST Tonsil Number of Registered Participants: 36 Number of Participants This Run 355 (99%) Introduction Endothelial Markers - Gold Antibody CD31 Also called Platelet Endothelial Cell Molecule-1 (PECAM-1) is a transmembrane glycoprotein found at endothelial cell junctions which is thought to play a role in angiogenesis, wound healing and thrombosis. It is expressed on endothelial cells in a wide range of tissues including sinusoidal cells of liver, lymph nodes and spleen. It is also found on megakaryocytes, platelets and other haematopoietic cells including B-lymphocytes particularly in the follicular mantle zone. Its main diagnostic use is in the identification of endothelial tumours as it is said to be a more sensitive marker than either CD34 or Von Willebrand Factor (DeYoung et al). It has been shown to be a good marker of endothelial differentiation. Staining is predominately in the cell membrane but some weaker staining of the cytoplasm can be seen. CD34 is a transmembrane glycoprotein which is expressed on immature haematopoietic stem/progenitor cells, capillary endothelial cells and embryonic fibroblasts. It can also be found in splenic marginal zones, dendritic interstitial cells around vessels, nerves, hair follicles, muscle cells and sweat glands in various tissues. CD34 labels capillaries in most tissues but may be absent in large veins and arteries and is negative in the sinus endothelium of placenta and spleen. CD34 is an excellent indicator of vascular differentiation, regardless of the tumour grade, therefore it is a good marker for vascular tumours (Cerilli et al, Leong). Due to the comparatively wide range of cell types stained by CD34 it is recommended that this antibody is used with a panel of endothelial makers including CD31 and Von Willebrand Factor. There are a number of clones available for CD34, of which, QBEnd/1 is the most popular. Staining with CD34 is restricted to the cell membrane. Features Of Optimal Immunostaining CD31 Strong staining of the endothelial cells in the blood vessels and lymphatic vessels throughout the appendix Strong staining of the T-cells in the base of the lamina propria and weaker staining of the B-cells in the follicular mantle with HIER pre-treatment. Minimal background staining. CD34 Strong staining of the endothelial cells in the blood vessels and lymphatic vessels throughout the appendix Strong staining on the dendritic interstitial cells in the lamina propria with HIER pre-treatment Minimal background staining. Features Of Sub-optimal Immunostaining CD31 and CD34 Weak or negative staining of the endothelial cells and other elements in the appendix. Non-specific nuclear staining possibly caused by overpretreatment. Inappropriate staining of, for example, epithelium and lymphocytes, probably due to over-pretreatment References 1. DeYoung BR, Wick MR, Fitzgibbon JF et al. CD31: an immunospecific mark for endothelial differentiation in human neoplasms. Applied Immunohistochem.1993; 1: Cerilli LA and Wick MR. Immunohistology of soft tissue and osseous neoplasms. In: Diagnostic Immunohistochemistry. Dabbs DJ. (Editor). Churchill Livingstone, Philadelphia 22; Leong AS-Y, Cooper K, Leong FJW-M. CD34. In: Manual of Diagnostic Antibodies for Immunohistology (1st Ed.). Oxford University Press, Oxford 1999; Leong AS-Y, Cooper K, Leong FJW-M. Factor VIII RA (Von Willebrand factor). In: Manual of Diagnostic Antibodies for Immunohistology (1st Ed.).Oxford University Press, Oxford 1999; 167. CD3 - Secondary Antibody CD3 antigen is a complex of closely related polypeptide chains found on the surface membrane of T-lymphocytes in close physical association with the T-cell receptor. CD3 appears in the cytoplasm early in T-cell development and on the surface at the late-thymocyte stage, persisting throughout normal T-cell maturation (Wood et al). The most common use of the CD3 antibody is in lymphoma diagnosis as it is present in the majority of mature T-cell neoplasms (9%) although occasionally tumours lose the antigen during the neoplastic process e.g. anaplastic large cell lymphomas. It is not found in B-cell or nonlymphoid malignancies. CD3 labels T-lymphocytes in reactive tonsil sections, most of which are found in the peri-follicular areas, but some are also found in the germinal centres and mantle zones of secondary lymphoid follicles, in the stratified squamous epithelium and the loose connective tissue. Staining with CD3 is confined to the cytoplasm and/or cell membrane. Features Of Optimal Immunostaining Normal Tonsil Strong and crisp, well-localised staining in the cell membranes of T-cells. Clean background with no staining of the B-cells (most noticeably seen in the germinal centres, in which isolated T- cells should be stained and the B-cells should be unstained). Features Of Sub-optimal Immunostaining Weak, uneven, partially missing, or poorly localised, diffuse staining in the membranes. Excessive background staining, possibly due to over pretreatment. Inappropriate staining of other cells including epithelial cells. References 1. Joel F, Leong W-M Leong S-Y. Essential markers in malignant lymphoma: A diagnostic approach. J Histotech 22; 25(4): Wood KM, Pallesen G, Ralfkiaer E, et al. Heterogeneity of CD3 antigen expression in T-cell lymphoma. Histopathol 1993;22: UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 4

7 RUN 1 GENERAL PATHOLOGY Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 1. Good demonstration of CD31 in the UK NEQAS appendix section, showing strong staining of the endothelial cells and vessels and also the mantle zone B and T cells. Section stained with the Dako JC/7A antibody, 1:2, on the Ventana XT, CC1 standard and Ultraview kit. Fig 2. Optimal demonstration of CD31 in the UK NEQAS GIST section. The tumour is negative for CD31 but the endothelial cells are strongly stained as expected and the background stroma is clean. Section stained with the Dako JC/7A antibody, 1:2, on the Ventana XT, CC1 standard and Ultraview kit. Fig 3. Optimal demonstration of CD34 in the UK NEQAS distributed appendix section, showing strong distinct staining of the vessels and endothelial cells. Section stained with the Dako QBEND antibody, 1:5, using the Dako PT Link, autostainer and FLEX kit. Fig 4. Good demonstration of CD34 in UK NEQAS GIST section, showing intense, well localised, predominantly membranous and cytoplasmic staining. Section stained with the Dako QBEND antibody, 1:5, using the Dako PT Link, autostainer and FLEX kit. Fig 5. Borderline pass for the demonstration of CD34 in the UK NEQAS appendix section. The staining is weak with fewer endothelial cells and vessels staining than expected (compare to Fig 3). No methodology details were provided. Fig 6. Sub-optimal demonstration of CD34 in the UK NEQAS GIST section. Although the tumour is positive as expected, the staining is weak (compare to Fig 4). No methodology details were provided. 5

8 RUN 1 GENERAL PATHOLOGY Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 7. Sub-optimal demonstration of CD31 in the UK NEQAS GIST. The section shows non-specific inappropriate nuclear staining of the tumour cells. Section stained with the Dako JC/7A antibody, 1:1, using the Dako PT Link, autostainer and FLEX kit. Fig 8. Good example of an in house placenta control stained with CD34, demonstrating the strong and crisp staining of the endothelial cells. Section stained with the Ventana QBend 1 pre-diluted antibody on the Ventana XT, CC1 standard and Ultraview kit. Fig 9. Optimal demonstration of CD3 in the UK NEQAS tonsil section, showing distinct staining of virtually all the peripheral and germinal centre T-cells. The strong membranous staining is shown to better advantage in the high power insert (B). Section stained with the Dako F antibody, 1:2, using the Dako PT Link, autostainer and FLEX kit. Fig1. Borderline pass staining of CD3 in the UK NEQAS tonsil section (compare to Fig 9). Although the T-cells are demonstrated the staining is very weak and diffuse. Section stained with the Dako F antibody, 1:2, on the Leica Bond III, ER2 and Refine kit. Fig 11. Poor demonstration of CD3 in the UK NEQAS tonsil section. The staining is diffuse and the section appears to have been over pre-treated. Section stained with the Leica PS1 antibody, 1:25, on the Ventana XT, CC1 standard and iview kit. Fig 12. Borderline pass staining of CD3 in the UK NEQAS tonsil section. Although the peripheral and germinal T-cells show strong distinct membranous staining, the background staining is excessive. Section stained with the Dako polyclonal antibody, 1:1, using the Dako PT Link, autostainer and FLEX kit. 6

9 Run 1 GENERAL PATHOLOGY Module GRAPHICAL REPRESENTATION OF PASS RATES 14 RUN 1A CD31 / CD34 on NEQAS Sections Individual 4 = (%) 18 RUN 1B CD31 / CD34 on in-house Sections Individual 4 = (%) no. of returns = (%) 6 = (%) 7 = (%) 8 = 7 (2%) 9 = 4 (1%) 1 = 6 (2%) 11 = 17 (5%) 12 = 28 (8%) 13 = 2 (6%) 14 = 21 (6%) 15 = 43 (12%) 16 = 137 (39%) 17 = 43 (12%) no. of returns = (%) 6 = (%) 7 = (%) 8 = 1 (%) 9 = 3 (1%) 1 = 2 (1%) 11 = 5 (1%) 12 = 5 (1%) 13 = 6 (2%) 14 = 9 (3%) 15 = 21 (6%) 16 = 175 (5%) 17 = 71 (2%) = 19 (5%) 19 = 8 (2%) 2 = 2 (1%) = 35 (1%) 19 = 1 (3%) 2 = 8 (2%) Summary Summary 16-2 = 29 (59%) 16-2 = 299 (85%) = 84 (24%) = 36 (1%) 1-12 = 51 (14%) 1-12 = 12 (3%) - 9 = 11 (3%) - 9 = 4 (1%) Median = 14. Median = 14. no. of returns RUN 1C CD3 on NEQAS Sections Individual 4 = 1 (%) 5 = (%) 6 = (%) 7 = (%) 8 = 2 (1%) 9 = 5 (1%) 1 = 1 (%) 11 = 5 (1%) 12 = 14 (4%) 13 = 21 (6%) 14 = 15 (4%) 15 = 33 (9%) 16 = 138 (4%) 17 = 59 (17%) 18 = 25 (7%) 19 = 21 (6%) 2 = 9 (3%) no. of returns RUN 1D CD3 on in-house Sections Individual 4 = (%) 5 = 1 (%) 6 = (%) 7 = (%) 8 = 3 (1%) 9 = 6 (2%) 1 = 2 (1%) 11 = 5 (1%) 12 = 17 (5%) 13 = 1 (3%) 14 = 11 (3%) 15 = 21 (6%) 16 = 168 (49%) 17 = 55 (16%) 18 = 24 (7%) 19 = 11 (3%) 2 = 11 (3%) Summary Summary 16-2 = 252 (72%) 16-2 = 269 (78%) = 69 (2%) = 42 (12%) 1-12 = 2 (6%) 1-12 = 24 (7%) - 9 = 8 (2%) - 9 = 1 (3%) Median = 13.5 Median =

10 Run 1 GENERAL PATHOLOGY Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE GENERAL PATHOLOGY MODULE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score >12/2) on UK NEQAS sections. General Pathology Run: 1 General Pathology Run: 1 Primary Antibody : CD31 / CD34 Antibody Details N % Abcam CD31 ab9498 (JC/7A) 1 Beckman Coulter CD34 IM786 (QBend1) 2 5 BioGenex AM236 5M (QBend1) CD Cell Marque CD31 131M/ (JC7) 2 1 Cell Marque CD34 134M/76-262/CMC33 (QBend) 4 1 Dako CD31 RTU FLEX IR61 (JC7A) 4 75 Dako CD34 RTU FLEX IR632 (QBend1) 7 86 Dako CD34 RTU N1632 (QBend1) 8 1 Dako M823 (JC/7A) CD Dako M7165 (QBend1) CD Immunotech CD (QBend1) 1 LabVision CD34 MS363P (QBend1) 2 5 Lecia/Novocastra CD31 NCL-CD-31-1A1 (1A1) Leica/Novocastra CD31 RTU PA25 (1A1) 2 5 Leica/Novocastra CD34 NCL-END (QBend) Leica/Novocastra CD34 RTU PA212 (QBend) 15 1 Other 5 1 Serotec CD34 MCA 547 (QBend1) 3 1 SKYBIO CD34 (QBend1) 1 1 Vector CD34 VP C345 (QBend1) 8 88 Ventana CD (JC/7A) 3 67 Ventana CD (JC7) 6 67 Ventana CD (QBend1) Primary Antibody : CD3 Antibody Details N % Abcam (SP7) ab Cell Marque (R. poly) 3A /7 4 1 Dako (F7.2.38) M Dako (R. polyclonal) A Dako FLEX RTU (R. poly) IR Labvision/Neomarkers (SP7) Leica (LN1) NCL-L-CD Leica (PS1) NCL-CD3-PS Leica (PS1) NCL-L-CD3-PS Leica (PS1) RTU CD3-PS1 5 1 Leica (UCHT1) NCL-CD3 3 1 Leica Bond RTU (LN1) PA Other Vector (LN1) VPC Ventana Confirm (2GV6) General Pathology Run: 1 General Pathology Run: 1 CD3 CD31 / CD34 CD3 CD31 / CD34 Heat Mediated Retrieval N % N % Biocare Decloaking Chamber Dako Pascal Dako PTLink Lab vision PT Module Leica Bond III ER Leica Bond III ER Leica BondMax ER Leica BondMax ER Microwave Oven NOT APPLICABLE 7 71 Other Pressure Cooker Pressure Cooker in Microwave Oven Steamer Ventana Benk CC1 (Extended) Ventana Benk CC1 (Mild) Ventana Benk CC1 (Standard) Ventana Benk CC1# (8mins) 3 1 Ventana Benk CC2 (mild) 1 1 Ventana Benk ULTRA CC1 (Exten.) Ventana Benk ULTRA CC1 (Mild) Ventana Benk ULTRA CC1 (Stan.) Ventana Benk ULTRA CC1# (8mins) 1 1 Ventana Benk ULTRA CC2 (Mild) 1 1 Ventana Benk ULTRA CC2 (Stan.) 1 1 Ventana Benk XT CC1 (Extended) Ventana Benk XT CC1 (Mild) Ventana Benk XT CC1 (Standard) Ventana Benk XT CC1# (8mins) 2 5 Ventana Benk XT CC2# (8mins) 1 Water bath 68 OC Water bath OC Enzyme Mediated Retrieval N % N % AS PER KIT Dako Proteinase K (S32) 1 1 NOT APPLICABLE VBS Bond Enzyme Ventana Protease Ventana Protease 1 (76-218) 2 5 8

11 Run 1 GENERAL PATHOLOGY Module General Pathology Run: 1 General Pathology Run: 1 CD3 CD31 / CD34 CD3 CD31 / CD34 Detection N % N % A Menerini Polymer (MP-XCP) AS PER KIT Biocare polymer (M4U534) BioGenex SS Polymer (QD 42-YIKE) 1 1 BioGenex SS Polymer (QD 43-XAKE) Dako EnVision FLEX ( K8/1) Dako EnVision FLEX+ ( K82/12) Dako Envision HRP/DAB ( K57) Dako Envision+ HRP mouse K44/5/6/7 1 1 Dako Envision+ HRP rabbit K48/9/1/ Dako rb-a-mo Ig (E354) 1 1 Dako REAL HRP/DAB (K51 ) LabVision UltraVision LP HRP (TS 125 HD) LabVision UltraVision ONE Polymer ( TL-12/5-HDJ/T Leica Bond Polymer Define (DS9713) Leica Bond Polymer Refine (DS98) MenaPath X-Cell Plus (MP-XCP) None NOT APPLICABLE Novocastra Novolink PDS (RE7-14/15/28/29-K) Other Power Vision DPVB999 HRP Vector Elite ABC Kit (PK-72) Vector ImmPRESS Universal (MP-75) Ventana iview system (76-91) Ventana OptiView Kit (76-7) Ventana UltraView Kit (76-5) Chromogen N % N % A. Menarini Liquid Stable DAB kit 2 5 AS PER KIT BioGenex DAB (QD43) BioGenex Liquid DAB (HK153-5K) BioGenex liquid DBA (HK-124-7K) Dako DAB Liquid (K3465) Dako DAB+ Liquid (K3468) Dako DAB+ REAL Detection (K51) 1 1 Dako EnVision Plus kits Dako FLEX DAB Dako REAL EnVision K57 DAB Dako REAL K51 DAB LabVision (TA-125-HD) Leica Bond Polymer Refine kit (DS98) menapath xcell kit DAB (MP-86) Other Sigma DAB (D56 5) 1 1 Sigma DAB (D5637) Ventana DAB Ventana iview Ventana Ultraview DAB Vision BioSystems Bond X DAB 2 1 General Pathology Run: 1 CD3 CD31 / CD34 Automation N % N % BioGenex GenoMX 6i BioGenex Optimax Dako Autostainer Dako Autostainer Link Dako Autostainer plus Dako Autostainer Plus Link LabVision Autostainer Leica Bond Max Leica Bond-III Menarini - Intellipath FLX None (Manual) Other Shandon Sequenza Ventana Benchmark Ventana Benchmark ULTRA Ventana Benchmark XT BEST METHODS - Gold Standard Antibody A selection from just a few of the best methods employed by participants CD31 / CD34 - Method 1 Participant scored 19/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Vector CD34 VP C345 (QBend1), 3 Mins, ambient ºC Dilution 1: 5 Automation: Dako Autostainer Link 48 Dako FLEX+ kit Main Buffer: Dako FLEX wash buffer Dako PTLink, Buffer: DAKO target retrieval high ph, PH: 9 Chromogen: Dako FLEX DAB, ambie ºC., Time 1: 5 Mins, Time 2: 5 Mins Detection: Dako EnVision FLEX+ ( K82/12), 2 Mins, ambient ºC Prediluted 9

12 Run 1 GENERAL PATHOLOGY Module CD31 / CD34 - Method 2 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Automation: Main Buffer: Dako M7165 (QBend1) CD34, 4 Mins BioGenex GenoMX 6i BioGenex SS Link-Label Optimax Wash Buffer Pressure Cooker, Buffer: citrate/edta, PH: 6 Chromogen: Detection: BioGenex liquid DBA (HK-124-7K), Time 1: 1 Mins, Time 2: 1 Mins BioGenex SS Polymer (QD 42-YIKE), 2 Mins CD31 / CD34 - Method 3 Participant scored 19/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Automation: Dako M823 (JC/7A) CD31, 32 Mins, 37 ºC Dilution 1: 2 Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Chromogen: Detection: Ventana reaction buffer (95-3) Ventana Benk XT CC1 (Standard) NOT APPLICABLE Ventana Ultraview DAB Ventana UltraView Kit (76-5) Prediluted CD31 / CD34 - Method 4 Participant scored 18/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Lecia/Novocastra CD31 NCL-CD-31-1A1 (1A1), 3 Mins, RT ºC Dilution 1: 25 Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Chromogen: Detection: Bond Wash Buffer (AR959) Leica BondMax ER2 NOT APPLICABLE Leica Bond Polymer Refine kit (DS98), RT ºC., Time 1: 1 Mins Leica Bond Polymer Refine (DS98), 8 Mins, RT ºC Prediluted BEST METHODS - Secondary Antibody A selection from just a few of the best methods employed by participants CD3 - Method 1 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Leica Bond RTU (LN1) PA553 Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: AS PER KIT Leica BondMax ER2 NOT APPLICABLE Chromogen: AS PER KIT Detection: AS PER KIT 1

13 Run 1 GENERAL PATHOLOGY Module CD3 - Method 2 Participant scored 2/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Dako (F7.2.38) M7254, 3 Mins, RT ºC Dilution 1: 2 Automation: Dako Autostainer Link 48 Dako FLEX kit Main Buffer: Dako FLEX wash buffer Dako PTLink, Buffer: Flex Target Retrieval Solution High ph Chromogen: Dako FLEX DAB, RT ºC., Time 1: 5 Mins, Time 2: 5 Mins Detection: Dako EnVision FLEX+ ( K82/12), 3 Mins, RT ºC Prediluted CD3 - Method 3 Participant scored 2/2 (UK NEQAS Slide) and 18/2 (In House slide) using this method. Primary Antibody: Ventana Confirm (2GV6) , 32 Mins, 37 ºC Prediluted Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk ULTRA CC1 (Stan.) Chromogen: Ventana Ultraview DAB Detection: CD3 - Method 4 Participant scored 19/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Vector (LN1) VPC316, 3 Mins, 21 ºC Dilution 1: 5 Automation: Dako Autostainer Link 48 Dako FLEX+ kit Main Buffer: AS PER KIT Dako PTLink, Buffer: Flex High ph, PH: 9 Chromogen: Dako FLEX DAB, 21 ºC., Time 1: 5 Mins, Time 2: 5 Mins Detection: Dako EnVision FLEX ( K8/1), 15 Mins, 21 ºC Prediluted 11

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15 The Breast Hormonal Receptor Module Run 1 Merdol Ibrahim & Suzanne Parry Antigen Assessed: Tissue Sections circulated: Number of Registered Participants: 333 Number of Participants This Run 315 (95%) Progesterone Receptors (PR) Sections from a composite block (see table below) Circulated Tissue The table below shows the staining characteristics of the tissue sections circulated during Run 1, which composed of three infiltrating ductal carcinomas (IDCs) with differing levels of receptor expression. The staining of the tumours were characterised using the Novocastra/Leica 16(A), Ventana (1E2) (A&B) and Dako PgR636 (A&B) antibody clones Tissue Sections % positivity Staining Intensity Allred / Quick Score A. IDC >95% High 8 B. IDC* 1-5% Medium-High 3-6 C. IDC % Negative Cell Lines** A 4-6% High 6-7 B 11-33% High 5-6 C % Negative * Some of the tissue blocks showed variability in Low-medium PR expression and this was taken into consideration during the assessments **Cell lines were also included on the slide but were not included in the actual assessment procedure. Cell lines are being validated for future use alongside tissue samples as a sensitivity/specificity gauge General Guideline Used in The Assessment of Slides SCORE STAINING PATTERN Slide not returned by Participant. 1 or 2 No staining or staining of considerably fewer nuclei than expected in one or more of the distributed tissue sections, or inappropriate staining of nuclei in cells not expected to stain. 3 Staining of 1% or greater of tumour nuclei in each of the positive tumour sections, though substantially less than expected to stain, or staining is weaker than expected. 4 or 5 Demonstration of the expected proportion of nuclei stained in the invasive tumours, with roughly the expected staining intensity. Marks are also deducted when correct clinical interpretation of staining may be hindered due to factors such as: - False positive / false negative / non-specific or inappropriate staining - Excessive cytoplasmic or diffuse nuclear staining - Excessively strong or weak haematoxylin counterstain - Excessive Antigen retrieval resulting in morphological damage - Poor quality/inadequate choice of in-house control tissue ( poor/inadequate fixation, damaged cell morphology, over retrieval etc.) In-House Tissue Recommendations & Assessment Participants in-house control tissue MUST consist of composite breast tissue (cell line controls are an acceptable alternative), placed onto a single slide as outlined below: i. >8% tumour positivity with high intensity (Allred/Quick score 7-8) ii. 3-7% tumour positivity with low-moderate intensity (Allred/Quick score 3-6) iii. Negative tumour, with normal positively stained glands (Allred/Quick score ) Participants NOT using a composite control are assessed a maximum score of a 'borderline' pass (1-12/2). UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 13

16 The Breast Hormonal Receptor Module Run 1 Introduction Expression of the steroid hormone receptors, oestrogen receptor-alpha (ER-α) and progesterone receptor (PR, types A and B) in breast epithelial cells is important in the development and function of the normal breast (Anderson). They also play a key-role in proliferative and neoplastic diseases of the breast (Cui et al.). When examined by immunocytochemical staining, approximately 75% of primary breast cancers express ER-α, and of those approximately 5% co-express PR. Hormone receptor status is a good predictor of response to antioestrogen based treatments such as Tamoxifen and aromatase-inhibitors (Fisher et al). PR expression is under the control of ER-α, and because of this it has been proposed that PR expression in a tumour indicates the presence of functional ER-α (Cui et al); moreover, it potentially defines a subpopulation of patients with superior response to tamoxifen (Osborne et al); conversely, there is evidence that ER-α positive tumours that are PR negative are best treated with aromatase-inhibitors in preference to the ER-antagonists (Cui et al.). Finally, patients whose breast tumours are ER-α negative but PR positive respond equally well to anti-oestrogen based treatments as do those whose tumours are ER-α positive (Ciocca and Elledge). All these factors lead to the conclusion that correct PR status is becoming increasing important. Correct staining protocols and validated staining techniques are therefore vital to avoid false ER and/or PR staining (see Immunocytochemistry journal Run number 99; Rhodes et al. and Ibrahim et al.,), which can have a direct impact on patient treatment regime PR Cell Lines Progesterone receptor expressing cell lines were also incorporated alongside tissue sections for this assessment. The cell lines were not used as part of the assessment procedure, but data was collated on the sensitivity and specificity of cell lines used for PR-staining alongside the breast tissue sections. The cell lines were kindly prepared and donated by Dako and consisted of; a) CAMA-1 (human breast carcinoma) PR positive cell line, b) HT-29 (human colon cancer) PR negative cell lines, and c) a mixture of CAMA-1 and HT-29 to provide a low PR-positive cell line. NEQAS tissue samples were tested by staining every 5th section from the relevant tissue blocks using the Leica 1A6 clone. This made sure that any heterogeneity in the tissue samples did not result in a participant being penalised. Furthermore, samples were also tested by the relevant commercial companies to further verify the expected level of staining and included Leica (clone 1A6), Dako (clone pgr 636) and Ventana (clone 1E2). Assessment Results Features of Optimal Immunostaining Staining of the expected proportion of invasive tumour nuclei with the anticipated staining intensity Intense nuclear staining of the appropriate distribution in normal glands Cytoplasmic staining not excessive No background staining of connective tissues or inappropriately localised staining Features of Sub-optimal Immunostaining False positive/negative staining Relatively weak nuclear staining of the receptor positive tumours Excessive cytoplasmic staining Excessive background staining of connective tissue elements Inappropriate staining of some cells e.g. lymphocytes, fibroblasts NEQAS Slide Results The overall pass rate on NEQAS sections was 57% (scores of 13-2/2) for participants as a whole, with quite a high failure rate of 26%. Analysis of data showed that the main reason for failure was due to false-positive staining with the Ventana 1E2 clone. Only 12/99 (12%) participants using the Ventana 1E2 clone achieved acceptable scores of >13/2. Of the 87 labs that did fail using the 1E2 clone 8 (92%) did so due to unacceptable PR staining in the NEQAS distributed negative tumour. UK NEQAS stained samples received directly from Ventana showed the expected PR-negative staining, and the high and low expressing samples also showed the expected range of PR-positivity. It was noted that the slide stained by Ventana was used in conjunction with an UltraBlock step (Antibody diluent # ) for 8 minutes. A retrospective survey of 1E2 users (n=56) showed that 45 (8%) did not use an UltraBlock step and 11 (2%) did use an UltraBlock step. Of the 11 UltraBlock users only 3 (27%) participants achieved the expected level of staining, whereas the remaining 8 (73%) still showed PR staining in the UK NEQAS distributed PR-negative sample. The initial data analyses is therefore inconclusive as to the effectiveness of using an UltraBlock step. Although false-positive staining appeared to be the main reason for failure for this assessment, there were also cases of false-negative staining (See fig 8), especially in the UK NEQS distributed low-mid PR-expressing sample (sample B), which were mainly due to insufficient retrieval protocols. It is noteworthy that the NEQAS slides were also tested for ER and HER2 expression and the distributed negative case was found to be ER-negative (using 6F11 clone) and HER2-negative (using Roche 4B5). Further Considerations on the 1E2 Antibody Clone There was no apparent non-specific staining on the UK NEQAS PR-negative tumour with the 1E2 clone, but in some cases the UK NEQAS colon adenocarcinoma cell line was also shown to have unexpected nuclear PRstaining (see fig 7). Is the 1E2 clone more sensitive or is it recognising another epitope that the other, mouse monoclonal antibodies do not? UK NEQAS has on previous assessments observed false positive staining with the 1E2 clone but not to the extent as shown in this current assessment, so it is impossible to comment on the extent of this apparent false-positive staining in the diagnostic setting. Good Laboratory Practice and UK NEQAS Breast Biomarker Audit Data Laboratories should continually carry out audits on breast biomarker rates to internally monitor aberrant change in rates. Introduction of a new antibody into laboratory practice should involve the validation of the new antibody prior to UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 14

17 The Breast Hormonal Receptor Module Run 1 clinical testing. At least 1 cases (multiple TMA s on a single slide are also acceptable) should be validated against cases which have previously been stained and interpreted. Laboratories should also consider sending samples to a known reference laboratory to corroborate their findings. New automated systems do not necessarily have the same staining protocols (antibody incubation, retrieval times etc) so again should undergo a revalidation process with samples of known expression. Laboratories should make a note of antibody batch numbers and if issues do arise then the commercial company distributing/producing the antibody in question should be made aware as soon as possible. New antibody batches should be quality controlled before applying to clinical cases for example by running on control tissue samples of known breast hormonal receptor expression. UK NEQAS audit of breast biomarkers shows that from between (Ibrahim et al., in prep) of 19,994 cases audited; ER-/PR+/HER2 cases averaged 1% (n=29) and the ER-/PR+/HER2+ averaged.4% (n=659). In the UK the average ER and PR positivity rates were 82.2% and 66.5%, respectively. However individual testing rates will differ due to numerous factors such as patient age, tumour grade/type, ethnicity etc. In-House Tissue Results Assessors scored in-house slides based on the staining quality, readability of the samples, the correct choice of tissue, and good morphological preservation. Participants were given a maximum score of 12/2 if the in-house control samples did not comprise of a composite breast tissue section with the following levels of ER expression: i) a high expressing tumour (Allred=7-8) ii) a mid expressing tumour (Allred=3-6) and iii) a negative tumour (Allred=). The presence of normal staining glands is also encouraged. For this assessment participant in-house control material showed an overall pass rate of 73%, with 22% achieving borderline scores and 4% having unacceptable scores. The main reason for failure was due to UK NEQAS assessors highlighting to participants the conflicting observation of selfassessment scores compared to the observed staining seen by the UK NEQAS assessors. For example Fig 12 shows a sample which was interpreted by the participant as PRnegative but which showed PR-positive staining. It is therefore important to feedback such observation to the participant concerned, so that they can take steps to see whether there is either an interpretation or staining issue. samples showing high, low-mid and negative breast hormonal receptor expression. Participants not submitting the recommended control tissue are scored a maximum of 12/2. Assessors also score samples based on quality of tissue and morphological integrity References 1. Jennet M. Harvey, Gary M. Clark, C. Kent Osborne, and D. Craig Allred (1999) Estrogen Receptor Status by Immunohistochemistry Is Superior to the Ligand-Binding Assay for Predicting Response to Adjuvant Endocrine Therapy in Breast Cancer J Clin Oncol 17: Robin Leake, Diana Barnes, Sarah Pinder, Ian Ellis, Liz Anderson, Tom Anderson, Ruth Adamson, Tony Rhodes, Keith Miller and Rosemary Walker (2) J. Clin. Pathol. 2: Anderson E. The role of oestrogen and progesterone receptors in human mammary development and tumorigenesis. Breast Cancer Res. 22; 4: Fisher B, Costantino J, Redmond C, Poisson R, Bowman D, Couture J, Dimitrov NV, Wolmark N, Wickerham DL, Fisher ER, et al. (1989) A randomized clinical trial evaluating tamoxifen in the treatment of patients with node-negative breast cancer who have estrogenreceptor-positive tumors. N Engl J Med 1989: Osborne CK, Schiff R, Arpino G, et al. Endocrine responsiveness: understanding how progesterone receptor can be used to select endocrine therapy. Breasst 25; 14: Ciocca DR and Elledge R. Molecular markers for predicting response to tamoxifen in breast cancer patients. Endocrine. 2;13: Rhodes A, Jasani B, Balaton AJ, Barnes DM, Anderson E, Bobrow LG, Miller KD (21). Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays. Am J Clin Pathol. 21 Jan;115(1): Ibrahim M, Dodson A, Barnett S, Fish D, Jasani B, Miller K. (28) Potential for falsepositive staining with a rabbit monoclonal antibody to progesterone receptor (SP2): findings of the UK National External Quality Assessment Scheme for Immunocytochemistry and FISH highlight the need for correct validation of antibodies on introduction to the laboratory. Am J Clin Pathol. 129: Acknowledgments We would like to thank Dako, Ventana and Leica for agreeing to stain the UK NEQAS Breast hormonal receptor samples with their respective assays. Difference in Scoring Criteria Between UK NEQAS & In-house Tissue Samples Participants should be cautious when comparing their UK NEQAS score directly to that of in-house control samples, as the two samples are scored on different factors: UK NEQAS distributed slides are scored against the expected levels of staining as defined by 1) initial patient diagnosis for PR 2) re-tested by staining every 5th section from the relevant tissue blocks using the Leica 1A6 clone 3) Samples are also tested by the relevant commercial companies to further verify the expected level of staining and included Leica (clone 1A6), Dako (clone pgr 636) and Ventana (clone 1E2). In-house tissue samples are scored based on whether a participant has submitted slides with breast tumour UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 15

18 RUN 1 BREAST STEROID HORMONE RECEPTOR Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 1. (A,B,C) Optimally stained UK NEQAS ICC distributed samples stained with Ventana 1E2 using Ventana UltraBlock. (A) high (B) Low and (C) negative PR expressing tumours showing the expected levels of staining. Note that PR expression in sample B varied depending on the block used and scoring baseline was adjusted accordingly. Fig 2. (A,B,C) Optimally stained UK NEQAS ICC distributed samples stained with Leica ready to use PR, clone 16. (A) high (B) Low and (C) negative PR expressing tumours showing the expected levels of staining. Note that PR expression in sample B varied depending on the block used and scoring baseline was adjusted accordingly. Fig 3. (A,B,C) Optimally stained UK NEQAS ICC distributed samples stained with Dako PgR 636 clone. (A) high (B) Low and (C) negative PR expressing tumours showing the expected levels of staining. Note that PR expression in sample B varied depending on the block used and scoring baseline was adjusted accordingly Fig 4. Expected level of staining in the UK NEQAS ICC distributed cell line (Note: cell lines were not assessed). Cell lines showed PR expression with Allred scores of (A) between 4-6 (B) 3-4 and (C) o (negative). Samples stained with the Ventana 1E2 clone using Ventana UltraBlock. Fig 5. Higher than expected PR staining in the UK NEQAS ICC distributed low expressing sample (sample B). Note the unexpected spurious staining indicated by the black arrows in the majority of tumour cells. Fig 6. (a) Unacceptable staining in the UK NEQAS ICC distributed PR-negative sample, showing what appear to be an Allred score of 4-5. (B) Colon adenocarcinoma cell line from the same slide showing acceptable staining with the cell lines remaining negative for PR expression. Sample stained with Ventana 1E2 clone. 16

19 RUN 1 BREAST STEROID HORMONE RECEPTOR Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 7. (a) Unacceptable staining in the UK NEQAS ICC distributed PR-negative sample, showing unacceptable PR-positive staining of tumour cells and excessive cytoplasmic staining. (B) Colon adenocarcinoma cell line expressing false positive PR staining along with morphological damage. Sample stained with Ventana 1E2 with CC1 (mild) antigen retrieval. Fig 8. (A,B) Low PR-expressing UK NEQAS ICC distributed PR samples both stained with the Leica 16 clone, with (A) showing the expected level of staining and (B) showing false-negative staining. (A) Retrieved using a Biocare Decloaking Chamber and (B) using a Ventana Benchmark CC1 standard. 9. Good in-house TMA control tissue showing >9% PR positive tumour cells of high intensity. Section stained using the Dako PgR 636 clone with Dako PT link antigen retrieval. 1. Good in-house TMA control tissue showing PR positive tumour cells with an overall Allred score of 5-6. Section stained using the Dako PgR 636 clone with Dako PT link antigen retrieval. 11. Good PR-negative in-house control. Section stained using the Dako PgR 636 clone with Dako PT link antigen retrieval. 12. (A,B) Unacceptable staining in in-house control samples that were submitted as being negative for PR. It is difficult to confirm whether the interpretation was incorrect or the staining was false-positive. Both participants did however also had unacceptable PR-staining in the UK NEQAS distributed PR-negative sample. Both slides stained with the Ventana 1E2 clone. 17

20 Run 1 BREAST STEROID HORMONE RECEPTOR Module GRAPHICAL REPRESENTATION OF PASS RATES 7 RUN 1E Progesterone on NEQAS Sections ALL PARTICIPANTS Individual 4 = (%) 5 = (%) 28 RUN 1E Progesterone on NEQAS Sections UK PARTICIPANTS Individual 4 = (%) 5 = (%) 6 6 = (%) 7 = 2 (1%) 24 6 = (%) 7 = (%) no. of returns = 62 (2%) 9 = 18 (6%) 1 = 15 (5%) 11 = 8 (3%) 12 = 28 (9%) 13 = 18 (6%) 14 = 27 (9%) 15 = 29 (9%) 16 = 25 (8%) no. of returns = 27 (17%) 9 = 5 (3%) 1 = 11 (7%) 11 = 2 (1%) 12 = 15 (1%) 13 = 7 (4%) 14 = 17 (11%) 15 = 16 (1%) 16 = 2 (13%) 1 17 = 53 (17%) 18 = 16 (5%) 4 17 = 2 (13%) 18 = 5 (3%) = 11 (3%) 2 = 3 (1%) = 8 (5%) 2 = 3 (2%) Summary Summary 16-2 = 18 (34%) 16-2 = 56 (36%) = 74 (23%) = 4 (26%) 1-12 = 51 (16%) 1-12 = 28 (18%) - 9 = 82 (26%) - 9 = 32 (21%) Median = 13.5 Median = RUN 1F Progesterone on in-house Sections ALL PARTICIPANTS Individual 4 = (%) 5 = 1 (%) 6 = (%) 7 = (%) 8 = 5 (2%) RUN 1F Progesterone on in-house Sections UK PARTICIPANTS Individual 4 = (%) 5 = 1 (1%) 6 = (%) 7 = (%) 8 = (%) no. of returns = 7 (2%) 1 = 9 (3%) 11 = 2 (6%) 12 = 4 (13%) 13 = 27 (9%) 14 = 17 (5%) 15 = 22 (7%) 16 = 57 (18%) no. of returns = (%) 1 = 1 (1%) 11 = 11 (7%) 12 = 14 (9%) 13 = 2 (13%) 14 = 9 (6%) 15 = 17 (11%) 16 = 4 (26%) 1 17 = 58 (19%) 18 = 3 (1%) 5 17 = 25 (16%) 18 = 11 (7%) = 13 (4%) 2 = 4 (1%) = 5 (3%) 2 = 2 (1%) Summary Summary 16-2 = 162 (52%) 16-2 = 83 (53%) = 66 (21%) = 46 (29%) 1-12 = 69 (22%) 1-12 = 26 (17%) - 9 = 13 (4%) - 9 = 1 (1%) Median = 13.5 Median =

21 Run 1 BREAST STEROID HORMONE RECEPTOR Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE BREAST STEROID HORMONE RECEPTOR MODULE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score >12/2) on UK NEQAS sections. Breast Steroid Hormone Receptor Run: 1 Breast Steroid Hormone Receptor Run: 1 Primary Antibody : Progesterone Antibody Details N % Biogenex AM172 (1A6) 1 Dako IR68 (PgR 636) (A&B) 1 9 Dako IS68 (PgR 636) (A&B) 2 1 Dako K194 (PgR 1294 (b)) 2 1 Dako M3569 (PgR 636) (A&B) Dako N163 RTU (PgR 636) (A&B) 3 67 Novocastra NCL-L-PGR-312 (16) (A) Novocastra NCL-L-PGR-AB (16+SAN27) (A&B) 1 9 Novocastra NCL-L-PGR/2 (1A6) (A&B) 3 67 Novocastra NCL-PGR (1A6) (A&B) 3 1 Novocastra NCL-PGR-312 (16) (A) Novocastra NCL-PGR-AB (16+SAN27) 4 1 Novocastra PA312 (16) (A) 8 88 Novocastra RTU-PGR (1A6) (A&B) 1 1 Novocastra RTU-PGR-312 (16) (A) 6 83 Other 1 6 Serotec MCA 18 (1A6) (A&B) 1 Vector Labs VP-P975 (1A6) (A&B) 2 1 Vector Labs VP-P976 (16) (A) 2 1 Vector Labs VP-P977 (16+SAN27) (A&B) 2 5 Ventana PgR (16) (A) 2 Ventana (1E2) (A&B) 7 11 Ventana (1E2) (A&B) Ventana (SP2) (A&B) 3 33 Zytomed Sytems SA-RBK-2 (SP42) (A&B) 1 1 Automation Progesterone N % BioGenex GenoMX 6i 2 5 BioGenex Optimax 1 1 Dako Autostainer 6 83 Dako Autostainer Link Dako Autostainer plus 8 88 Dako Autostainer Plus Link 6 83 LabVision Autostainer 5 6 Leica Bond Max Leica Bond-III Menarini - Intellipath FLX 4 75 None (Manual) 6 33 Shandon Sequenza 4 1 Ventana Benchmark 8 13 Ventana Benchmark ULTRA Ventana Benchmark XT Ventana Discovery 1 Ventana NexES 1 Breast Steroid Hormone Receptor Run: 1 Breast Steroid Hormone Receptor Run: 1 Heat Mediated Retrieval Progesterone N % Biocare Decloaking Chamber 4 5 Dako Pascal 2 1 Dako PTLink Lab vision PT Module 4 75 Leica Bond III ER Leica Bond III ER Leica BondMax ER Leica BondMax ER Microwave Oven 8 75 Other 4 75 Pressure Cooker 9 56 Ventana Benk CC1 (Extended) 5 2 Ventana Benk CC1 (Mild) 2 1 Ventana Benk CC1 (Standard) Ventana Benk ULTRA CC1 (Mild) Ventana Benk ULTRA CC1 (Stan.) Ventana Benk ULTRA CC2 (Mild) 1 Ventana Benk XT CC1 (Extended) 5 2 Ventana Benk XT CC1 (Mild) 14 7 Ventana Benk XT CC1 (Standard) Water bath OC 3 33 Enzyme Mediated Retrieval Progesterone N % AS PER KIT 4 5 Dako Protease (S219) 1 1 NOT APPLICABLE VBS Bond Enzyme VBS Bond Enzyme

22 Run 1 BREAST STEROID HORMONE RECEPTOR Module Breast Steroid Hormone Receptor Run: 1 Breast Steroid Hormone Receptor Run: 1 Detection Progesterone N % A Menerini Polymer (MP-XCP) 1 1 AS PER KIT 1 8 BioGenex SS Polymer (QD 43-XAKE) 1 1 Dako EnVision FLEX ( K8/1) 7 86 Dako EnVision FLEX+ ( K82/12) Dako Envision HRP/DAB ( K57) 7 86 Dako Envision+ HRP mouse K44/5/6/7 2 1 Dako rb-a-mo Ig (E354) 1 Dako REAL HRP/DAB (K51 ) 1 1 LabVision UltraVision LP HRP (TL 125 HLJ) 1 1 LabVision UltraVision LP HRP (TS 125 HD) 1 LabVision UltraVision ONE Polymer ( TL-12/5-HDJ/T 1 Leica Bond Polymer Refine (DS98) MenaPath X-Cell Plus (MP-XCP) 4 5 None 1 1 NOT APPLICABLE 2 1 Novocastra Novolink PDS (RE7-14/15/28/29-K) 3 1 Other Power Vision DPVB999 HRP 1 Vector ImmPRESS Universal (MP-75) 1 Ventana iview system (76-91) Ventana OptiView Kit (76-7) 4 Ventana UltraView Kit (76-5) Chromogen Progesterone N % A. Menarini Liquid Stable DAB kit 2 5 AS PER KIT BioGenex DAB (QD43) 1 1 BioGenex liquid DBA (HK-124-7K) 1 Dako DAB K Dako DAB Liquid (K3466) 1 1 DAKO DAB+ 1 1 Dako DAB+ Liquid (K3468) 2 1 Dako DAB+ REAL Detection (K51) 1 Dako EnVision Plus kits 3 1 Dako FLEX DAB 39 9 Dako REAL EnVision K57 DAB 6 1 Dako REAL K51 DAB 1 1 Leica Bond Polymer Refine kit (DS98) menapath xcell kit DAB (MP-86) 5 6 Other 12 5 Sigma DAB (D5637) 2 Ventana DAB 6 83 Ventana iview Ventana Ultraview DAB Vision BioSystems Bond X DAB 4 1 BEST METHODS A selection from just a few of the best methods employed by participants Progesterone - Method 1 Participant scored 18/2 (UK NEQAS Slide) and 15/2 (In House slide) using this method. Primary Antibody: Vector Labs VP-P976 (16) (A), 15 Mins, rt ºC Dilution 1: 1/1 Automation: Leica Bond-III Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) Leica BondMax ER2 NOT APPLICABLE Chromogen: Leica Bond Polymer Refine kit (DS98), rt ºC., Time 2: 1 Mins Detection: Leica Bond Polymer Refine (DS98), 8 Mins, rt ºC Prediluted Progesterone - Method 2 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Novocastra NCL-PGR-312 (16) (A), 3 Mins, 23 ºC Dilution 1: 1:35 Automation: Dako Autostainer Link 48 Dako FLEX+ kit Main Buffer: Dako FLEX wash buffer, PH: 7.6 Dako PTLink, Buffer: ENVISION TR SOLN HIGH ph, PH: 9 Chromogen: Dako FLEX DAB, 23 ºC., Time 1: 5 Mins, Time 2: 5 Mins Detection: Dako EnVision FLEX+ ( K82/12), 35 Mins, 23 ºC Prediluted Progesterone - Method 3 Participant scored 2/2 (UK NEQAS Slide) and 18/2 (In House slide) using this method. Primary Antibody: Dako M3569 (PgR 636) (A&B), 3 Mins, 25 ºC Dilution 1: 8 Automation: Dako Autostainer Plus Link Dako FLEX+ kit Main Buffer: Dako FLEX wash buffer Dako PTLink, Buffer: high ph NOT APPLICABLE Chromogen: Dako FLEX DAB, 25 ºC., Time 1: 5 Mins, Time 2: 5 Mins Detection: Dako EnVision FLEX+ ( K82/12), 15 Mins, 25 ºC Prediluted 2

23 Run 1 BREAST STEROID HORMONE RECEPTOR Module Progesterone - Method 4 Participant scored 17/2 (UK NEQAS Slide) and 13/2 (In House slide) using this method. Primary Antibody: Ventana (1E2) (A&B), 12 Mins, 37 ºC Prediluted Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk ULTRA CC1 (Mild) Chromogen: Ventana Ultraview DAB, 37 ºC., Time 1: 8 Mins, Time 2: 8 Mins Detection: Ventana UltraView Kit (76-5), 8 Mins, 37 ºC Prediluted NOTE: The above method also incorporated Ventanan UltraBlock (Antibody diluent - option1) for 8 minutes 21

24 The Breast HER2 ICC Module Run 1 Keith Miller, Suzanne Parry and Merdol Ibrahim Antigen Assessed: HER2 Sections Circulated: Composite cell lines (see table below) Number of Registered Participants: 354 Number of Participants This Run 316 (89%) Specific Guideline Used in The Assessment of Slides: The immunohistochemical results were evaluated by UK NEQAS assessors scoring independently using an adapted method initially devised by the Clinical Trials Assay. Due to the nature of cell lines, overall percentage staining criteria can not be accurately applied when scoring cell lines. Communication with Leica Microsystems, with whom UK NEQAS ICC & ISH developed the cell lines, indicate that the expected level of membrane staining for a given cell line may range from 3-9% from the viable cell line population. For this reason when cutting sections, every 5th section is stained as a reference point to gauge the expected level of staining throughout the cell block/s. The table below demonstrates the staining patterns looked for in cell lines and in-house tissue sections during assessments. Score Assessment of Cell Line Staining Pattern Assessment of In-House Tissue Sections 3+ Strong complete cell membrane staining Strong complete cell membrane staining in >3% of tumour cells 2+ Weak to moderate complete cell membrane staining Weak to moderate complete cell membrane staining in >1% of tumour cells 1+ Faint barely perceptible incomplete membrane staining Faint barely perceptible incomplete membrane in >1% of cells staining Negative No staining in the control cell line No staining in the control cell line UK NEQAS Specific Scoring Algorithm: UK NEQAS ICC & ISH devised an EQA specific algorithm for scoring the cell lines so as to provide participants additional technical feedback. As well as taking into account the expected range (3-9% see above) of cell line membrane staining, the acceptable staining levels of each of the cell lines are as follows: Table below illustrates the expected level of staining for each of the circulated breast carcinoma cell lines along with the UK NEQAS scoring criteria used in assessments ONLY. Cell line Score Acceptable Level/s of Staining During Assessments Description of Staining Pattern Used By the Assessors SK-BR only The 3+ cell line has a wide threshold of complete membrane staining showing strong staining. Only this level of membrane staining is deemed acceptable for this cell line. MDA-MB or 1+/2+ or 2+/3+ or 2+/1+ or 3+/2+ i) 1+/2+ or 2+/1+: Staining is slightly weaker than expected with membrane showing more 1+ compared to 2+ (1+/2+) or 2+ membrane staining is present but also showing 1+ staining (2+/1+). ii) 2+/3+ or 3+/2+: Staining is slightly weaker than expected with membrane showing more 2+ compared to 3+ (2+/3+) or 3+ membrane staining is present but also showing 2+ staining (3+/2+). MDA-MB or /1+ or 1+/ i) /1+: Staining is more towards the weaker end of 1+ staining but still acceptable. ii) 1+/: Staining is more towards the weaker end of 1+ staining but still acceptable. MDA-MB-231 Negative or /1+ or 1+/ /1+ or 1+/ = Cells are starting to show very weak membrane staining U /Uninterpretable Assessors may also give a score of 'U' which indicates that the cell lines were 'uninterpretable due to the reasons set out below. Borderline Pass: A score of U/x e.g. U/3+ or U/2+ or U/1+ or U/ indicates a borderline uninterpretable scores indicating that the staining is just about readable and further improvements are required. Cell Line Positioning: Cell lines are positioned on microscope slides, as illustrated below. Any variation in this pattern is indicated on distribution of the slides UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 22

25 The Breast HER2 ICC Module Run 1 Validation of Distributed Cell Lines IHC Validation The NEQAS cell line sections were quality controlled prior to sending out using the Leica Oracle, Ventana Pathway 4B5 and the Dako Herceptest as the gold standards. ISH Validation of Distributed Samples The NEQAS cell lines were also assessed for gene expression using FISH (Abbott Vysis). The table below shows the HER2 FISH ratio ranges provided by the UK NEQAS reference centres for Run 24 of the HER2 ISH assessments: Cell line Score HER2 gene:ch17 ratio range HER2 status by FISH SK-BR Amplified MDA-MB Borderline: Amplified to Not Amplified MDA-MB Not amplified MDA-MB-231 negative Not amplified Introduction HER2 immunohistochemistry has been routinely used as a predictive marker in breast cancer for more than 1 years. Patients with HER2 positive tumours generally have a poor overall prognosis. However, patients with metastatic disease showed an improved rate of survival when the humanized monoclonal antibody Trastuzumab (Herceptin) was given alone or added with chemotherapy treatment (Slamon et al., 1998). Herceptin, in the metastatic setting was made available in the UK in 2 by the UK National Institute for Clinical Excellence (NICE). In 25, Herceptin in the adjuvant setting was also shown to be effective in primary breast cancers by reducing the risk of recurrence and mortality (see articles by Wolff et al., (26) and Walker et al., (28) for further commentary and citations), and in 26 NICE approved primary breast screening for HER2. In the UK alone this has resulted in approximately 4, breast cancers per year being tested for HER2. With so many more cases now being tested worldwide, it is imperative that correct protocols and methodologies are followed and adhered to. The UK NEQAS HER2 IHC module assesses the technical quality of staining achieved by laboratories and provides feedback to laboratories to help improve the analytical phase of patient diagnosis. Two recommended HER2 testing guidelines are: 1. The ASCO/ CAP guidelines by Wolff et al., (26) and 2. UK updated guidelines by Walker et al., (28). Both provide invaluable guidelines covering interpretation, tissue fixation and antibody validation. The article by Walker et. al., also provides guidelines on the minimum number of tests per year, (25 for HER2 IHC and 1 HER2 ISH) that labs should be testing in order to provide a robust HER2 IHC service. Participants who continue to have difficulty in producing acceptable staining results should be aware that the members of staff at UK NEQAS are always willing to assist any laboratory that is struggling to meet the required standard of HER2 immunostaining, and would be happy to receive request for assistance via in the first instance (see front of journal for contact details). In-House Control Tissue Recommendations Correct choice of in-house control tissue and good morphological preservation is paramount to gauge the sensitivity of the HER2 test which can have an impact on clinical interpretation. UK NEQAS ICC & ISH therefore recommends the following: In-house control tissue (or cell lines) must include 3+,2+ and 1+/ invasive breast cancer cases. However, it has become quite apparent that as patient tumour size and respective biopsies become smaller laboratories may be having difficulty finding appropriate invasive control material. It is therefore acceptable, if laboratories are having problems in finding appropriate invasive control material, to submit DCIS tissue showing differing levels of membrane staining as an acceptable alternative. Laboratories must indicate on their datasheet which component of the tumour they have scored otherwise the invasive component (if present) will be assessed. Important: The Ventana OptiView detection system has not been validated for use with the 4B5 HER2 for IHC. Any laboratory using this system for breast HER2 testing should be aware that they are doing so off label usage. References 1. Slamon D, Leyland-Jones B, Shak S, et al. Addition of Herceptin (humanised anti-her2 antibody) to first line chemotherapy for (HER2+/MBC) markedly increases anticancer activity: a randomised, multinational controlled phase III trial. Proc ASCO 1998;17:98a. 2. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al: Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353: , Bartlett JM, Ibrahim M, Jasani B, et al. External quality assurance of HER2 FISH testing: results of a UK NEQAS pilot scheme. J Clin Pathol 27 6 (7): Walker RA, Bartlett JM, Dowsett M, Ellis IO, Hanby AM, Jasani B, Miller K, Pinder SE. HER2 testing in the UK: further update to recommendations. J Clin Pathol (7): Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 27;25:1 28. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 23

26 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining F, -! " & # #& staining, which is completely circumferential and minimal cytoplasmic staining. (A) Stained with the Leica Oracle kit (B) Ventana 4B5 and (C) Dako Hercept Test. F! " # membrane staining in the majority of cells, with less intensive staining than that seen with the 3 " $ %& ' ( 3 ) ' * & + F. 3 /,, + & clumps show partial membrane staining, which is discernible from the brush border staining. (A) Stained with the Leica Oracle kit (B) Ventana 4B5 and (C) Dako Hercept Test. F / - " morphology damage due to excessive antigen retrieval: Home-brew method. (B) Shows excessive cytoplasmic staining, again due to excessive antigen retrieval: Ventana Optiview detection was used, which is currently not recommended for HER2 staining. F / + & 3 #& & & / 3 s # & " # 3 " ' ) & m 3m!,1,22! 4+ ' 3 F$5 3 ' F 6 / sections shows cell morphology damage caused by inappropriate and excessive antigen retrieval. (A) Stained with the Ventana 4B5 and Optiview kit (not recommended for HER2 staining). (B) Stained with a home-brew method and pressure cooker antigen retrieval. 24

27 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining 789 [; <=>??@AB>CD@ D@E@D GH IB>8=8=9 GH BJ@ <K LMNOP?@DD D8=@ Q\OSQRSV[] WV^Y; WOY PJG_I?GcAD@B@ c@cc`>=@ IB>8=8=9 >=a cg`@ `@A`@I@=B>B8E@ GH > T^ staining. (B) Shows excessive counterstain, making it difficult to interpret any membrane staining. (A) stained using a home-brew method and (B) the Ventana 4B :; <=>??@AB>CD@ D@E@D GH IB>8=8=9 GH BJ@ <K LMNOP =@9>B8E@?@DD D8=@ QROSQRSTUV WXYZ showing artefactual and non-specific cytoplasmic staining caused by excessive antigen retrieval. Section stained with a home-brew method using the Novocastra CB11 antibody and the Biocare MACH 4 polymer detection kit. 789 l; GH >= f8= JGgI@h U^?G=B`GD IB>8=@a _8BJ BJ@ \>ig j@`?@ab k@ibz IJG_8=9 intense, crisp membrane staining. 789 VX; GH >= f8= JGgI@h T^?G=B`GD IB>8=@a _8BJ BJ@ \>ig j@`?@ab k@ibz IJG_8=9 weak to moderate membrane staining. 789 VV; GH >= f8= JGgI@h V^?G=B`GD IB>8=@a _8BJ BJ@ \>ig j@`?@ab k@ib; kj@ staining is weak and partially membranous. Fig 12. Good example of an in house negative control stained with the Dako Hercept Test. 25

28 Run 1 BREAST HER2 ICC Module GRAPHICAL REPRESENTATION OF PASS RATES Run 1 HER2 on NEQAS Cell Lines ALL PARTICIPANTS Run 1 HER2 on NEQAS Cell Lines UK PARTICIPANTS No. of Returns 12 8 No. of Returns Acceptable BorderLine Inappropriate Acceptable BorderLine Inappropriate Acceptable = 18 (57%) participants BorderLine = 37 (12%) participants Inappropriate = 11 (32%) participants Acceptable = 69 (86%) participants BorderLine = 6 (8%) participants Inappropriate = 5 (6%) participants Run 1 HER2 IHC on in-house tissue sections ALL PARTICIPANTS Run 1 HER2 IHC on in-house tissue sections UK PARTICIPANTS No. of Returns 8 6 No. of Returns Acceptable BorderLine Inappropriate Acceptable BorderLine Inappropriate Acceptable = 128 (42%) participants BorderLine = 6 (2%) participants Inappropriate = 114 (38%) participants Acceptable = 43 (54%) participants BorderLine = 23 (29%) participants Inappropriate = 14 (18%) participants For Inhouse assessor make a combined decision as to the acceptability based upon the following: a) In-house control tissue must include 3+,2+ and 1+/ invasive breast cancer cases (not DCIS). b) Tissue fixation, preservation of morphology, antigen retrieval, counterstain etc is also be taken into consideration when scoring. 26

29 Run 1 BREAST HER2 ICC Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE BREAST HER2 ICC MODULE The following tables record the number of participants (N) using each primary antibody. The percentage (%) refers to the proportion of these participants achieving 'Acceptable' or 'Borderline' scores. Breast HER2 ICC Run: 1 Breast HER2 ICC Run: 1 Antibody Details N % Biocare CME 342 A,B (EP145Y) 3 Cell Marque CMA 61 (CB11) 1 1 Dako A485 C-erB-2 (poly) Dako HercepTest K524 (poly) 9 67 Dako HercepTest K527 (poly) 7 1 Dako Link HercepTest SK1 (poly) Invitrogen (CB11) 1 Labvision / Neomarkers RM-913 (SP3) 7 Leica Oracle HER2 Bond IHC (CB11) Neomarkers MS-73-P 1 Novocastra NCL-L-CB11 (CB11) 1 1 Novocastra NCL-L-CBE356 (1A7) 2 1 Other 7 43 Vector CB11 VP-C Ventana Confirm 79/ (4B5) 5 9 Ventana pathway (CB11) 1 1 Ventana Pathway 79-1 (4B5) 12 1 Ventana Pathway (4B5) Automation BioGenex GenoMX 6i Dako Autostainer Dako Autostainer Link 48 Dako Autostainer plus Dako Autostainer Plus Link LabVision Autostainer Leica Bond Max Leica Bond-III None (Manual) Other Shandon Sequenza Ventana Benchmark Ventana Benchmark ULTRA Ventana Benchmark XT Ventana NexES N % Breast HER2 ICC Run: 1 Breast HER2 ICC Run: 1 Heat Mediated Retrieval Biocare Decloaking Chamber Dako Pascal Dako PTLink Lab vision PT Module Leica Bond III ER1 Leica Bond III ER2 Leica BondMax ER1 Leica BondMax ER2 Microwave Oven NOT APPLICABLE Other Pressure Cooker Steamer Ventana Benk CC1 (8mins) Ventana Benk CC1 (Extended) Ventana Benk CC1 (Mild) Ventana Benk CC1 (Standard) Ventana Benk ULTRA CC1 (Stan.) Ventana Benk ULTRA CC2 (Mild) Ventana Benk XT CC1 (8mins) Ventana Benk XT CC1 (Mild) Ventana Benk XT CC1 (Standard) Ventana ULTRA CC1 (Mild) Water bath 68 OC Water bath OC N % Enzyme Mediated Retrieval AS PER KIT Dako Protease (S219) NOT APPLICABLE Other VBS Bond Enzyme 1 Ventana Protease 1 (76-218) N %

30 Run 1 BREAST HER2 ICC Module Breast HER2 ICC Run: 1 Breast HER2 ICC Run: 1 Detection AS PER KIT Biocare SLAB (STU HRP 7H,L1) Dako HerCep Test (K524) Dako EnVision FLEX ( K8/1) Dako EnVision FLEX+ ( K82/12) Dako Envision HRP/DAB ( K57) Dako Envision+ HRP rabbit K48/9/1/11 Dako gt-a-mo biotin (E433) Dako HerCep Test Autor (K527) Dako HerCep Test Autor (SK1) Dako rb-a-mo Ig (E354) LabVision UltraVision LP HRP (TS 125 HD) LabVision UltraVision ONE Polymer ( TL-12/5-HDJ/T Leica Bond Polymer Refine (DS98) None NOT APPLICABLE Novocastra Novolink PDS (RE7-14/15/28/29-K) Other Ventana iview system (76-91) Ventana OptiView Kit (76-7) Ventana UltraView Kit (76-5) N % Chromogen A. Menarini Liquid Stable DAB kit AS PER KIT BioGenex liquid DBA (HK-124-7K) Dako DAB K3468 Dako DAB Liquid (K3465) Dako DAB Liquid (K3466) DAKO DAB+ Dako DAB+ Liquid (K3468) Dako DAB+ REAL Detection (K51) Dako EnVision Plus kits Dako FLEX DAB Dako REAL EnVision K57 DAB Leica Bond Polymer Refine kit (DS98) Other Sigma DAB (D5637) Ventana DAB Ventana iview Ventana Ultraview DAB Vision BioSystems Bond X DAB Zymed DAB N % BEST METHODS A selection from just a few of the best methods employed by participants 1 HER2 IHC - Method 1 Participant scored Acceptable (UK NEQAS Slide) using this method. Primary Antibody: Leica Oracle HER2 Bond IHC (CB11), 3 Mins, RT ºC Prediluted Automation: Main Buffer: Leica Bond-III Leica BondMAx Refine KIT Bond Wash Buffer (AR959) Leica Bond III ER1, PH: 9 Chromogen: Detection: Leica Bond Polymer Refine kit (DS98), RT ºC., Time 1: 1 Mins Leica Bond Polymer Refine (DS98), 1 Mins, RT ºC Prediluted 1 HER2 IHC - Method 2 Participant scored Acceptable (UK NEQAS Slide) using this method. Primary Antibody: Ventana Pathway 79-1 (4B5), 12 Mins Prediluted Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana ULTRA CC1 (Mild) Chromogen: Ventana Ultraview DAB Detection: Ventana UltraView Kit (76-5) Prediluted 1 HER2 IHC - Method 3 Participant scored Acceptable (UK NEQAS Slide) using this method. Primary Antibody: Dako Link HercepTest SK1 (poly), 3 Mins Prediluted Automation: Dako Autostainer Plus Link Dako FLEX kit Main Buffer: Dako FLEX wash buffer Dako PTLink, Buffer: Dako citrate buffer Chromogen: Dako FLEX DAB, Time 1: 1 Mins, Time 2: 1 Mins Detection: Dako HerCep Test Autor (SK1), 3 Mins Prediluted 28

31 Run 1 BREAST HER2 ICC Module 1 HER2 IHC - Method 4 Participant scored Acceptable (UK NEQAS Slide) using this method. Primary Antibody: Dako HercepTest K524 (poly) Automation: None (Manual) Other Main Buffer: AS PER KIT Water bath OC Chromogen: AS PER KIT Detection: Dako HerCep Test (K524) 29

32 The Gastric HER2 IHC (Pilot Module) Run 1 Merdol Ibrahim, Suzanne Parry, Jane Bell, Bharat Jasani, Phillipe Taniere and Iris Nagelmeier Antigen Assessed: Tissue Sections circulated: Number of Registered Participants 141 HER2 IHC Number of Participants This Run 119 (84%) Composite slide consisting of: A. 2+ Intestinal gastric carcinoma B. 3+ Intestinal gastric carcinoma C. 1+ or 2+ Intestinal gastric carcinoma depending on the tissue section received by the laboratory D. or 1+ Intestinal gastric carcinoma depending on the tissue section received by the laboratory Table: 1 Gastric HER2 ICC Membrane Scoring Guidelines by Hoffman et al., (28) and Rüschoff et al., (21) Score Surgical / resections (As used in this assessment) Biopsies (negative) No staining in < 1% of tumour cells No staining in any of the tumour cells 1+ (negative) 2+ (equivocal*) 3+ (positive) Faint barely perceptible incomplete membrane staining in >1% of cells staining Weak/ moderate complete, basolateral or lateral membrane reactivity in > 1% of tumour cells Strong complete, basolateral or lateral membrane reactivity in > 1% of tumour cells Tumour cells with faint barely perceptible incomplete membrane staining, irrespective of percentage of tumour cells stained Tumour cells with weak/ moderate complete, basolateral or lateral membrane reactivity irrespective of % tumour cells stained Tumour cells with strong, basolateral or lateral membrane reactivity irrespective of percentage of tumour cells stained * Equivocal cases should be refluxed to ISH testing. Note: in the UK, NICE guidelines recommend that only 3+ cases are put forward for Trastuzumab treatment, see Validation of Distributed Samples IHC Validation of Distributed Samples The NEQAS tissue sections were quality controlled prior to sending out, using the Ventana pathway 4B5, Dako Herceptest and Leica Oracle systems. Important: The Leica Oracle HER2 IHC System is not currently supported for gastric HER2 IHC in the UK and any laboratory using the kit for gastric purposes should be aware that they are doing so off label usage and would need to fully validate the Leica Oracle system prior to diagnostic use. ISH Validation of Distributed Samples The tissue was also assessed for gene expression using Leica HER2 FISH, Dako IQFISH and Ventana/Roche DDISH. Section Label From slide Label End Staining Pattern with Ventana 4B5 Table 2: HER2 status by ISH* A 2+ Amplified B 3+ Amplified C 1+ or 2+ * Non-Amplified D or 1+ * Non-Amplified * Due to the heterogeneity of the tissue, both scores were deemed acceptable according to the section received by the laboratory UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 3

33 The HER2 IHC Gastric Pilot Module Run 1 Assessment Procedure Assessment is carried out around a multi-header microscope with each slide being assessed by 4 independent assessors and a microscope lead. Each of the NEQAS samples (A-D) (as well as in-house samples where submitted) are individually assessed, with each assessor providing membrane interpretation (see table 3), and an individual score out of 5 (see table 4) based interpretability of membrane staining and technical feedback. The four assessors scores are then combined to give an overall score out of 2 and it s interpretation (see table 4). Table 3: UK NEQAS Specific Membrane Scoring Criteria UK NEQAS ICC & ISH uses an EQA specific scoring criteria when scoring the tissue sections, so as to provide participants additional technical feedback, which is illustrated below. Expected membrane staining Scoring criteria used by UK NEQAS ICC & ISH 3+ i) 3+: as expected Ii) 2+/3+ or 3+/2+: Staining is slightly weaker than expected with membrane showing more 2+ compared to 3+ (2+/3+) or 3+ membrane staining is present but also showing 2+ staining (3+/2+). 2+ i) 1+/2+ or 2+/1+: Staining is slightly weaker than expected with membrane showing more 1+ compared to 2+ (1+/2+) or 2+ membrane staining is present but also showing 1+ staining (2+/1+). ii) 2+/3+ or 3+/2+: Staining is slightly weaker than expected with membrane showing more 2+ compared to 3+ (2+/3+) or 3+ membrane staining is present but also showing 2+ staining (3+/2+). 1+ i) /1+: Staining is more towards the weaker end of 1+ staining but still acceptable. ii) 1+/: Staining is more towards the weaker end of 1+ staining but still acceptable. Neg. /1+ or 1+/ = Staining starting to show very weak membrane staining U = Uninterpretable: Assessors may also give a score of 'U' which indicates that the cell lines / tissue sections were 'uninterpretable due to the reasons set out below. U/x = Borderline interpretable. A score of U/x e.g. U/3+ or U/2+ or U/1+ or U/ indicates that the staining is just about readable and further improvements are required. Any other membrane score other that assigned for each of the expected scores are deemed as unacceptable Individual Assessor Score Table 4: Numeric Scoring Criteria used to Calculate the Overall Pass Mark Combined Assessor Slide not submitted for assessment 1 & = Unacceptable = Borderline 4 & = Acceptable 16-2 = Acceptable/ Excellent Standard Overall Score Interpretation Overall the samples are of unacceptable quality for clinical interpretation and technical improvements need to be made. Marks may have been deducted due to: False positive / negative membrane staining Excessive cytoplasmic staining Excessive morphological damage Excessive staining of normal glands Overall the samples are borderline interpretable indicating that technical improvements need to be made. Marks may have been deducted due to: Weaker / stronger than expected membrane staining Some cytoplasmic staining Morphological damage Overall the samples show acceptable membrane staining and are suitable for interpretation. Further comments are also provided on individual participant reports. Other factors which may lead to a low score include: excessive/ insufficient haematoxylin staining; poor quality of in-house control tissue; poor/inadequate choice of control tissue; poor/inadequate fixation; excessive antigen retrieval etc. Introduction Immunohistochemical testing of HER2 status is now routinely used in breast cancer testing and is recognised as a prognostic and predictive marker, generally used alongside breast hormonal receptor markers ER/PR. Patients who are HER2 positive (IHC 3+ and IHC 2+/ISH+ ) have been shown to benefit from Herceptin (Trastuzumab) therapy and increased overall survival rate. More recently the Trastuzumab for Gastric Cancer (ToGA) study, which investigated Trastuzumab in HER2 positive advanced gastric cancer (Bang et al., 21) showed overall median survival of nearly 3 months. Similar to breast cancer, the ToGA trial showed an increased benefit from Trastuzumab treatment for patients showing 3+ IHC and IHC2+/FISH+ expression. The initial development of the HER2 scoring criteria was developed as a precursor to the ToGA trial (Hoffman et al., 28) with the study group modifying the breast HER2 IHC scoring algorithm to compensate for the incomplete membrane staining and greater tumour heterogeneity seen in gastric cancers. A different scoring system was also established for re-section and core biopsies as illustrated in table 1. A more recent article by Rüschoff et al., (21) has validated the scoring procedure further with a detailed approach to stepwise HER2 IHC scoring in gastric cancers. The article also compared the Ventana 4B5 assay with the Herceptest and indicated a tendency towards higher sensitivity for 4B5 detecting positive UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 31

34 The HER2 IHC Gastric Pilot Module Run 1 HER2 amplification but There was no difference between both test platforms with respect to the ISH-negative cases with the one equivocal case testing negative at some sites and as positive at others. Rüschoff and colleagues also used both fluorescent (Dako pharmdx or PathVysion Vysis) and chromogen (Ventana BDISH) to confirm their IHC findings. Assessment Results Features Of Acceptable Staining (figs 1-4, 11) Membrane staining of the invasive tumour with the expected expression level Cytoplasmic staining not excessive No background staining of stromal tissues or inappropriately localised staining Features Of Unacceptable Staining (figs 7-1, 12) Weaker than expected membrane staining of the invasive tumour Lower or higher than the expected expression level of staining False positive or negative membrane staining Excessive cytoplasmic staining Excessive background staining or inappropriately localised staining Excessive morphological damage Excessive staining of normal glands Additional Comments a. The Ventana 4B5 did show some epithelial staining (intestinal metaplasia) (not illustrated). Participants were not scored down as the assessment team were aware that this is one of the staining characteristics of the Ventana 4B5 antibody and did not detract from the actual staining of the gastric cancer b. The Leica Oracle HER2 IHC System is not currently supported for gastric HER2 IHC and any laboratory using the kit for gastric purposes should be aware that they are doing so off label usage and would need to fully validate the Leica Oracle system prior to diagnostic use. Pass Rates Of the 119 participants that took part in this assessment, results from the NEQAS distributed samples showed an overall acceptable pass rate of 55% (65/119), with 25% of participants scoring a borderline pass and an unacceptable rate of 2%. There was a 7% drop in unacceptable rates from Run 99. The main reasons for unacceptable scores were mostly due to under-staining of the 2+ and 1+ tumour sections, excessive cytoplasmic staining, excessive counterstain, and false positive or non-specific staining. Of the 119 participants submitting NEQAS slides, 11 (85%) also submitted in-house control material, of which 5% showed acceptable staining, 43% were borderline, and 8% received an unacceptable score. The main reasons for unacceptable scores for in-house material were due to excessive cytoplasmic staining, excessive counterstain, or poor tissue quality/fixation. In some instances participants selfassessment on interpretation was discordant with that of the NEQAS assessment panel. In a clinical setting, misinterpretation could potentially lead to a higher or lower number of patients being referred for Herceptin therapy. Methodologies Of those participants who submitted methodology data, the main methodology employed was the Ventana 4B5 antibody with 67% (67/11) of participants using this assay, and showed an overall acceptable rate of 75% (5/67). The second most popular method was a home-brew method using the Dako polyclonal antibody (17%, n=17) which had a low pass rate of only 6%. The Dako polyclonal was noted to be used with a full range of retrieval methods, including water bath, microwave oven and pressure cooker. The methodologies employed and their pass rates are further illustrated in the technical parameter tables. It is important to note that because of the small number of users for several differing methodologies it is not possible to say whether such methods/antibodies are suitable for gastric IHC in the context of the UK NEQAS gastric assessment. Control Tissue and Recommendations In-house control slides were also requested for this module, however, it was left an open choice for laboratories to submit either gastric or breast control material. For this run 11/119 laboratories submitted an in-house control, with the majority of these being composite control slides with 3+, 2+ and 1+/ membrane staining. Gastric controls would be the preferred tissue choice as inhouse controls for this module, but it is quite understandable that laboratories may find it difficult to source good quality controls, therefore laboratories were not penalised if they had submitted breast carcinomas. Whether gastric or breast tissue is used, laboratories should still submit in-house controls showing 3+, 2+ and 1+/ levels of membrane expression. It is also acceptable to submit a heterogeneous in-house gastric tumour control with areas of 3+ and areas of 2+ membrane expression as long as the participant indicates the areas of expected membrane staining. Important: The Ventana OptiView detection system has not been validated for use with the 4B5 HER2 for IHC. Any laboratory using this system for gastric HER2 testing should be aware that they are doing so off label usage. References: 1. Hofmann M, Stoss O, Shi D et al., Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology (7): Rüschoff J, Dietel M, Baretton G etc al., HER2 diagnostics in gastric cancerguideline validation and development of standardized immunohistochemical testing. Virchows Arch (3): Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK; ToGA Trial Investigators. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2- positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet (9742): Acknowledgments We would like to thank Dako, Ventana and Leica for agreeing to stain samples from the UK NEQAS distributed gastric HER2 ICC slides. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 32

35 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining F 9 '*- -:-! " #$ %&'( )*+ ),-.' 145 Stained with Ventana 4B5 with UltraView detection and (B) Dako HercepTest kit. F! " #$ %&'( )*+ ),-./ " 8 complete and intense membrane staining. Stained with Ventana 4B5 with UltraView detection and (B) Dako HercepTest kit. Fig 3. Acceptable range of staining of the UK NEQAS distributed negative gastric tissue sample (sample C ). Due to heterogeneity of tissue both image examples (A & B) were deemed *- 1'5 ( 8" " a ;/<6 8"" " 8,- 94,,*) 1/5 ( 8" " s= >) 8"" -, ) :)? ,,*) F ; '*- )!! " #$ %&'( )*+ 94 ) +,- 1,-.s5 *): (,- + (A) Ventana 4B5 and (B) Dako HercepTest kit. F < >C +!,-.'./ " a sse(> 1'5 (,-.' 145 8,-! 1) 3;w G ;H;5 1/5,-./ ,-! 1) I9J5 >C status was also determined using Leica and Dako FISH which also showed the expected amplified status for both samples A and B. Fig 6. HER2 status of samples C and D stained using the Ventana DDISH. (A) Sample C 1:5 8 G,-! 1) ) 99 G 9<;5 1/5,-.s G,-! 1) ) 99n G 9<;5 >C ), + K Dako FISH which also showed the expected amplified status for both samples C and D. 33

36 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining Fig 7. Unacceptable staining of sample A ; 2+ UK NEQAS ICC tumour section (compare with fig 1). Example shows morphological damage and uniterpretable membrane staining. Stained with the Ventana 4B5 using CC1 mild retrieval, but which appears to have been retrieved for longer or the slide may have been baked prior to staining, which is not recommended. Fig 8. Unacceptable staining of sample A ; 2+ UK NEQAS ICC gastric tumour section (compare with fig 1). There is very little membrane staining and tissue appears very disrupted. Stained with the Dako HercepTest kit using water bath retrieval. Fig 9. Unacceptable staining of sample B ; 3+ UK NEQAS ICC gastric tumour section (compare with fig 2). This sample was deemed uniterpretable due to excessive cytoplasmic staining and morphological damage. Stained with the Dako polyclonal antibody using a pressure cooker for retrieval. Fig 1. Unacceptable staining of sample 'C'; negative UK NEQAS ICC gastric tumour section (compare with fig 3). The sample show morphological damage and excessive haematoxylin. Stained with the Ventana 4B5 using CC1 mild retrieval. Fig 11. Good example of an in house composite gastric HER2 control. (A) 3+: Intense, crisp membrane staining (B) 2+: weak to moderate membrane staining, and (C) negative tumour remains unstained. Sections stained with Ventana 4B5, using CC1 (mild) retrieval and UltraView detection system. Fig 12. Unacceptable example of an in house (A) 2+ and (B) negative gastric HER2 controls. Both samples were deemed as uniterpretable; due to poor tissue quality and preservation. Sections stained with Ventana 4B5, using CC1 (standard) retrieval. 34

37 Run 1 35 RMN OPPQT QUWXY[\ ]^R_ `]b ct N^def fg\x[ctw Individual h i h jklm 35 RMN OPPQ QUWXY[\ ]^R_ `]b ct [T} c~wg fg\x[ctw Individual k i p jplm 3 o i p jplm q i r jrlm 3 h i r jrlm o i v jvlm 25 t i h jklm u i vh jvrlm 25 q i p jplm t i p jplm no. of returns 2 15 x i p jplm vp i h jklm vv i t jqlm vr i vx jvqlm vk i r jrlm no. of returns 2 15 u i o jolm x i p jplm vp i t jtlm vv i v jvlm vr i ko jkolm 1 vh i r jrlm vo i vv jxlm 1 vk i r jrlm vh i k jklm 5 vq i vr jvplm vt i kk jrulm 5 vo i r jrlm vq i rh jrhlm vu i o jhlm vx i p jplm rp i p jplm y z {y {z 19 2 vt i t jtlm vu i k jklm vx i q jqlm rp i k jklm Summary Summary vq rp i op jhrlm vq rp i hk jhklm vk vo i vo jvklm vk vo i t jtlm vp vr i kp jrolm vp vr i hk jhklm p x i rh jrplm p x i u julm ƒ i vr pp ƒ i vk op ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score > ˆ Š Œ Ž Primary Antibody N % 6 2 Dako Link HercepTest SK1 (poly) 4 3 Leica Oracle HER2 Bond IHC (CB11) Other ˆ Š Œ Ž Automation QUWXY[\ HER2 IHC N % 3 33 Dako Autostainer plus 6 Dako Autostainer Plus Link 2 1 Leica Bond Max Leica Bond-III 2 5 None (Manual) 14 Ventana Benchmark 3 1 Ventana Benchmark ULTRA

38 Run 1 ª «±²³ µµ Heat Mediated Retrieval š œ žÿ IHC N % Dako Pascal 1 Dako PTLink 4 75 Lab vision PT Module 2 Leica Bond III ER1 1 1 Leica BondMax ER1 6 Microwave Oven 5 Other 2 5 Pressure Cooker 5 2 Ventana Benk CC1 (Mild) 15 Ventana Benk CC1 (Standard) 2 1 Ventana Benk CC2 (mild) 1 1 Ventana Benk ULTRA CC1 (Stan.) 2 5 Ventana Benk ULTRA CC2 (Mild) ª «±²³ µµ Detection š œ žÿ HER2 IHC N % AS PER KIT Dako HerCep Test Autor (SK1) Other ª «±²³ µµ š œ žÿ HER2 IHC Enzyme Retrieval N % AS PER KIT 2 5 BioGenex Protease 1 NOT APPLICABLE ª «±²³ µµ š œ žÿ HER2 IHC Chromogen N % AS PER KIT LabVision DAB 1 4 Other Ventana DAB 2 5 Ventana iview 4 ¹ Ventana Ultraview DAB 61 º BEST METHODS A selection from just a few of the best methods employed by participants Primary Antibody: Automation: Main Buffer: Chromogen: Ventana iview Kit AS PER KIT AS PER KIT Detection: 36

39 Run 1 Participant scored 17/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Dako Link HercepTest SK1 (poly) Automation: Dako Autostainer Plus Link AS PER KIT Main Buffer: AS PER KIT Dako PTLink AS PER KIT Chromogen: AS PER KIT Detection: AS PER KIT Participant scored 17/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Automation: Dako Autostainer Link 48 AS PER KIT Main Buffer: Dako PTLink, Buffer: HERCEPTEST EPITOPE RETRIEVAL Chromogen: Detection: Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Ventana UltraView DAB Ventana reaction buffer (95-3) Ventana Ultraview DAB 37

40 The Lymphoma Module Run 1 David Blythe and Suzanne Parry Gold Standard Antigens Assessed: CD3 CD5 Second Antibody Tissue Sections circulated: Hodgkin's Lymphoma Reactive Tonsil & Mantle Cell Lymphoma Number of Registered Participants: 231 Number of Participants This Run 226 (98%) Introduction Gold Standard: CD3 CD3 is a transmembrane cytokine receptor and plays a role in regulating the function, differentiation and/or proliferation of normal lymphoid cells¹. Malignant cells in Hodgkin lymphoma are termed Hodgkin (mono-nuclear) or Reed-Sternberg (multi-nuclear) cells, although they can also be referred to as Hodgkin Reed- Sternberg (HRS) cells. The origin of HRS cells became clear when techniques were established to isolate single HRS cells from biopsy specimens and analyse them for rearranged immunoglobulin genes by single cell PCR. It is now known that these cells represent clonal populations of transformed germinal centre B-cells in both classical Hodgkin lymphoma (CHL) and nodular lymphocyte predominant lymphoma (NLPHL), although in rare cases they can be derived from T- cells. In summary, in CHL, the HRS cells are positive with CD3, often positive with CD15 (in approximately 8% of cases) and generally negative for B-cell antigens such as CD2 or CD79a (approximately 3% of CHL cases will show some staining with these markers). In NLPHL, the HRS cells (also known as L&H cells or popcorn cells) are negative for both CD3 and CD15 and are generally positive for B-cell antigens. CD3 staining is found in CHLs, anaplastic large cell lymphomas, germ cell tumours and in a varying proportion of activated T- and B-cells. The staining pattern of CD3 in CHL is very similar to that of CD15 but without the granulocyte positivity. Some plasma cells will also stain with this antibody. Features of Optimal Immunostaining In a CHL there should be: Membrane staining (granular) in most HRS. Golgi staining of some HRS. Clean background. Features of Sub-optimal Immunostaining Weak, uneven or negative staining of the HRS. Excessive background staining, or non-specific staining of cell types or components not expected to stain. Tissue destruction due to harsh heat induced antigen retrieval. References 1. de Bruin PC, et al. CD3 expression in normal and neoplastic lymphoid t issue: biological aspects and clinical implications (review). Leukaemia 1995;9: Second Antigen: CD5 CD5 is a 67-kDa transmembrane glycoprotein, which is involved in B- and T-cell receptor signal transduction (Taylor). Normal cell types which are positive for this antigen include thymocytes, the majority of T-cells and a small number of B- cells (B-1 lymphocytes). This marker is useful in a variety of diagnostic situations (Taylor; Bishop; Dabbs): B-cell Lymphomas Positive in the vast majority (>9%) of B-cell chronic lymphocytic leukaemia and B-cell small lymphocytic lymphomas Positive in the majority of mantel cell lymphomas Negative in almost all other low grade B-cell lymphomas e.g. Follicular lymphoma T-cell Lymphomas Positive in most (approximately 85%) of T-cell acute lymphoblastic leukaemia and lymphoblastic lymphomas Distinguishes T-cell lymphoma (CD5 +ve) from extranodal T/NK cell lymphoma (CD5 -ve) Thymic Carcinoma Positive in most thymic carcinomas (6-1%), distinguishing thymic carcinomas from pulmonary carcinomas which are usually CD5 ve. Features of Optimal Immunostaining Reactive Tonsil The majority of T-cells in the inter-follicular areas should show strong, predominantly membranous staining. Scattered T-cells in the germinal centres should be demonstrated. Scattered B-cells in the mantle zones should be stained, but these may be more weakly positive. Clean background. Mantle Cell Lymphoma Strong predominantly membranous staining of the tumour cells. Darker staining of any normal scattered T-cells within the tumour. Clean background. Features of Sub-optimal Immunostaining Weak staining of cells expected to stain, especially T-cells in the inter-follicular areas. Poor localisation of staining to cell membranes. Disruption of normal cellular detail, due to excessive or incorrect antigen retrieval. References 1. Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidise-labelled antibody. J Clin Pathol 19 74; 27: Bishop PW. Immunohistochemistry vade mecum. mmunohistochemistry.info/ 3. Dabbs DJ. Immunohistology of metastatic carcinomas of unknown primary. In: Dabbs DJ (ed) Diagnostic immunohistochemistry (end ed). 26. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 38

41 RUN 1 LYMPHOMA Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 1. Optimal demonstration of CD3 in the UK NEQAS Hodgkin s lymphoma section. The strong membrane and paranuclear staining of the Hodgkin s cells is shown to better advantage in the high power insert (B). Section stained with the Dako Ber-H2 antibody, 1:4, using the hijk lm opqjr itukvuipqxy iqz {o } zxux~upkq jpu Fig 2. Good demonstration of CD3 in the UK NEQAS Hodgkin s lymphoma section, showing intense membranous staining of the Hodgkin s cells. Shown to better advantage in the high power insert (B). Section stained with the Dako Ber-H2 antibody, 1:4, using the Leica BondMax ER2 and Refine detection kit. Fig 3. Sub-optimal demonstration of CD3 in the UK NEQAS Hodgkin s lymphoma section. (Compare to Figs 1&2). Although the expected Hodgkin s cell s are demonstrated, the staining is weak. Stained with the Leica 1G12 antibody, 1:2. Pre-treated in the microwave, and using the Labvision autostainer and Ultravision detection kit. Fig 4. Sub-optimal CD3 staining in the UK NEQAS Hodgkin s lymphoma section. Although the expected cells are demonstrated, the section shows excessive background staining. Section stained with the Dako Ber-H2 antibody, 1:4, on the Leica BondMax, ER2 for 15 minutes. Fig 5. Sub-optimal CD3 staining in the UK NEQAS Hodgkin s lymphoma section. Staining of the Hodgkin s cells is difficult to interpret due to non-specific and inappropriate staining of lymphocytes, most likely caused by excessive antigen retrieval. Sections stained with the Leica 1G12 RTU antibody, on the Bond Max, ER2 for 3 minutes with the Refine kit. Fig 6. Good example of an in house control stained for CD3, demonstrating the intense membranous staining and dot-like reactions seen in the Hodgkin s lymphoma. Section stained pu u x xquiqi ƒxy yx zpˆtuxz iqup kzšr tvpq u x xquiqi ƒxq~ Œiyj }m Ž vuiqziyz and Ultraview kit 39

42 RUN 1 LYMPHOMA Module Selected Images showing Optimal and Sub-optimal Immunostaining š œ ž Ÿ œ œ ª«Ÿ œ ž œÿ œ ± ž in the inter-follicular areas and scattered T-cells in the germinal centres show strong, Ÿ šœ ²Ÿ œ³ž ž «œ ž š œ œ ž Ÿ µ ²œš ¹ œ the BondMax ER2 amd Refine detection kit. Fig 8. Good demonstration of CD5 in the UK NEQAS distributed mantle cell lymphoma. The strong membranous staining of the neoplastic cells is shown to better advantage in the high power insert (B). Section stained with Novocastra 4C7 antibody, 1:25, using the Dako PT Link, autostainer and Flex kit. Fig 9. Poor demonstration of CD5 in the UK NEQAS reactive tonsil. Staining of the T-cells is º š š ³ž» œ Ÿ œ ¼ «œ ž š ½ ² ž œ «¾ ²œš ¹ Ÿ ± Ÿ š Ÿ² š ž³ À š ³ž ºœ Á ª½ š œ º œ «œ  ¾ ½Ã ³ œž Ÿ Fig 1. Sub-optimal demonstration of CD5 in the UK NEQAS mantle cell lymphoma. Staining of the T-cells is weak and diffuse. Shown more clearly in the high power insert (B). Fig 11. Sub-optimal demonstration of CD5 in the UK NEQAS reactive tonsil. Not only is the staining weak, but the number of T-cells expressed is much lower than expected (compare to ¼ «œ ž š œ œ ž Ÿ µ ²œš ¹ÄÄ œ Ÿ Å Ÿº mild and Ultraview kit. Fig 12. Good example of an in house appendix control stained with CD5. Staining of the T-cells in the inter-follicular areas and scattered in the germinal centres is strong and distinct, and the background is clean. 4

43 Run 1 LYMPHOMA Module 6 ÆÇÈ ÉÊÊË ÌÍÎÊ ÏÐ ÈÑÒÓÔ ÔÕÖ ØÏÐÙ Individual Ú Û Ü ÝÜÞß 5 RUN 1M CD3 on in-house Sections Individual Ú Û Ü ÝÜÞß à Û Ü ÝÜÞß à Û Ü ÝÜÞß 5 á Û Ü ÝÜÞß â Û Ü ÝÜÞß 4 á Û Ü ÝÜÞß â Û ç ÝåÞß no. of returns ã Û ä ÝåÞß æ Û Ü ÝÜÞß åü Û ä ÝåÞß åå Û à ÝçÞß åç Û åú ÝáÞß åä Û çä ÝåÜÞß åú Û äá ÝåáÞß no. of returns 3 2 ã Û Ü ÝÜÞß æ Û ä ÝåÞß åü Û ä ÝåÞß åå Û åç ÝàÞß åç Û ää ÝåàÞß åä Û ää ÝåàÞß åú Û çæ ÝåäÞß 1 åà Û äú ÝåàÞß åá Û àú ÝçÚÞß 1 åà Û äà ÝåáÞß åá Û Úá ÝçåÞß åâ Û äü ÝåäÞß åâ Û çç ÝåÜÞß åã Û åâ ÝãÞß åæ Û à ÝçÞß çü Û ç ÝåÞß è é êè êé 19 2 åã Û à ÝçÞß åæ Û Ü ÝÜÞß çü Û Ü ÝÜÞß Summary Summary åá ë çü Û åüã ÝÚãÞß åá ë çü Û âä ÝääÞß åä ë åà Û æä ÝÚåÞß åä ë åà Û æâ ÝÚäÞß åü ë åç Û çç ÝåÜÞß åü ë åç Û Úã ÝççÞß Ü ë æ Û ä ÝåÞß Ü ë æ Û à ÝçÞß ìíîïðñ Û åúòàü ìíîïðñ Û åäòüü 5 ÆÇÈ ÉÊÊÈ ÌÍó ÏÐ ÈÑÒÓÔ ÔÕÖ ØÏÐÙ Individual Ú Û ç ÝåÞß èô ÆÇÈ ÉÊÊõ ÌÍó ÏÐ ØÐö ÏøÙÕ ÔÕÖ ØÏÐÙ Individual Ú Û ç ÝåÞß à Û Ü ÝÜÞß à Û Ü ÝÜÞß 4 á Û Ü ÝÜÞß â Û ç ÝåÞß 6 á Û ç ÝåÞß â Û Ü ÝÜÞß no. of returns 3 2 ã Û åå ÝàÞß æ Û Ú ÝçÞß åü Û å ÝÜÞß åå Û Ú ÝçÞß åç Û äå ÝåÚÞß åä Û åú ÝáÞß no. of returns ã Û ç ÝåÞß æ Û ä ÝåÞß åü Û ç ÝåÞß åå Û Ú ÝçÞß åç Û åã ÝãÞß åä Û çá ÝåçÞß åú Û çú ÝååÞß åà Û äü ÝåäÞß 2 åú Û çá ÝåçÞß åà Û ÚÚ ÝçÜÞß 1 åá Û Úâ ÝçåÞß åâ Û çú ÝååÞß 1 åá Û áâ ÝäÜÞß åâ Û åã ÝãÞß åã Û çå ÝæÞß åã Û ä ÝåÞß è é êè êé 19 2 åæ Û ã ÝÚÞß çü Û Ü ÝÜÞß è é êè êé 19 2 åæ Û ä ÝåÞß çü Û Ü ÝÜÞß Summary Summary åá ë çü Û åüü ÝÚàÞß åá ë çü Û æå ÝÚåÞß åä ë åà Û áã ÝäÜÞß åä ë åà Û æá ÝÚÚÞß åü ë åç Û äá ÝåáÞß åü ë åç Û çú ÝååÞß Ü ë æ Û åæ ÝæÞß Ü ë æ Û æ ÝÚÞß ìíîïðñ Û åçòàü ìíîïðñ Û åçòàü 41

44 Run 1 LYMPHOMA Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE LYMPHOMA MODULE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score > Lymphoma Run: 1 Lymphoma Run: 1 Primary Antibody : CD3 Antibody Details N % ùúûüýþýÿ B 1 Cell Marque 13M (Ber-H2) 6 Dû ùý 1 Dû ùý Dû ùý 4 1 lþ ýû 1 lþ ýû ù 2 1 Lýú ûû!" ùûþ# $ B ü 5 1 Lýú ûû!" ùûþ# $ B % &' 11 1 Lýú ûû!" 'L 'D ü 5 6 Lýú ûû!" 'L 'D ù 5 Lýú ûû!" 'L L 'D ü 12 Lýú ûû!" 'L L 'D % &' 16 1 Lýú ûû!" $ 'D ü 4 1 Other 4 () Výþ"þ % % ùý *( + Z,ý# ùý 1 1 Primary Antibody : CD5 Antibody Details N % ùúûý ' %%ù ' 1 1 ùúû-ýþýÿ $ $' ' 1 'ýll C.ý ÿ ' 4 25 Dû % 5 Dû $ /L Lúþ ' 4 () Dû $ B."û l.! ' 1 1 Dako M3633 (SP19) 2 5 Dû ' 5 Dû % 'D / 1 Lú!úûþ % ' 1 Labvision RM-9119 (SP19) 3 33 Lýú ùûþ# $ B ' ýû ý! % ' 1 1 ûû!" ùûþ# B ' ( 1 ûû!" 'L 'D L / 1 ûû!" 'L 'D ' ' 52 (( ûû!" 'L L 'D ' ' 69 (( Other 2 5 Spring Bio. M319 (SP19) 2 5 Vý"û V ' ' 15 1( Výþ"þ % 1 9 Výþ"þ 'D % % Lymphoma Run: 1 Lymphoma Run: 1 CD3 234 CD3 234 Heat Mediated Retrieval N % N % Biocare Decloaking Chamber ( Dako PTLink +( 35 (( Lab vision PT Module Leica Bond III ER Leica Bond III ER Leica BondMax ER1 5 ( 6 5 Leica BondMax ER Microwave Oven 6 33 ( 14 Other ( 1 6 1( Pressure Cooker 4 () 4 25 Pressure Cooker in Microwave Oven 1 1 Steamer 1 1 Ventana Benk CC1 (Extended) ( 1 Ventana Benk CC1 (Mild) Ventana Benk CC1 (Standard) 5( (1 Ventana Benk CC2 (mild) 1 1 Ventana Benk CC2 (Standard) 1 1 Ventana Benk ULTRA CC1 (Exten.) Ventana Benk ULTRA CC1 (Stan.) Výþ"þ ùýþ '' ÿ"ýþ#ý# Výþ"þ ùýþ '' úl# Výþ"þ ùýþ '' "þ## ( Výþ"þ $L B '' úþ! Ventana ULTRA CC1 (Mild) 4 () 4 5 W"ý " % % 6' 2 2 Enzyme Mediated Retrieval N % N % AS PER KIT Dako Protease (S219) 2 5 NOT APPLICABLE Other

45 Run 1 LYMPHOMA Module Lymphoma Run: 1 Lymphoma Run: 1 CD3 789 CD3 789 Detection N % N % A :;<;=><>?@EFG;= H:?IJK?M AS PER KIT N>@O;<;P QQ?@EFG;= HRS TUXIY[\]M 1 1 N>@O;<;P QQ?@EFG;= HRS T^XIJA\]M Dako EnVision FLEX ( K8/1) S_`@ ]<a>b>@< cd]je H \fxxughum ij S_`@ ]<k>b>@< mn?gsan H \oxxpm S_`@ ]<k>b>@<e mn? G@qb; \TXXTgogrgp Dako rb-a-mo Ig (E354) S_`@ n]ad mn?gsan H\oXXh M 1 1 LabVision UltraVision LP HRP (TS 125 HD) 1 1 d_sa>b>@< teu=_a>b>@< vw]?@efg;= H xdixhugoimsygx Leica Bond Intense R Detection (DS9263) 1 d;>z_ N@<{?@EFG;= n; ><; HSQ}fXXM ~ i ~ ii :;<_?_u JIK;EE?Eqb H:?IJK?M ~ NOT APPLICABLE Other 9 i Power Vision DPVB999 HRP 1 1 a;zu@= [GG?n]QQ t<>k;=b_e H:?IpoXXM a;zu@= t<>k;=b_e N>@u A<u> G@g=s [ƒo HNAIhTXXM 1 1 a;<u_<_ >a>; bfbu;g HprXIX}hM 9 ~i 9 ~ a;<u_<_ v u>a>; \>u HprXIpXXM a;<u_<_ teu=_a>; \>u HprXIoXXM Chromogen N % N % A. Menarini Liquid Stable DAB kit 1 1 AS PER KIT i ~i BioGenex DAB (QD43) 1 1 BioGenex Liquid DAB (HK153-5K) 1 1 N>@O;<;P E> q>{ SNA Hm\IhUTIp\M 1 1 SA\v SANe S_`@ SANe d> q>{ H\^TrfM Dako EnVision Plus kits S_`@ cd]j SAN ~ S_`@ n]ad ]<a>b>@< \oxxp SAN Dako REAL K51 DAB 1 1 LabVision (TA-125-HD) d;>z_ N@<{?@EFG;= n; ><; `>u HSQ}fXXM ~i 9 ~ˆ i G;<_ _u Pz;EE `>u SAN H:?IfrXM Other ~ Q>ƒG_ SAN HSor^pM 2 2 Ventana DAB Ventana iview i ~ i 63 Ventana Ultraview DAB 55 i 62 i a>b>@< N>@QFbu;Gb N@<{ J SAN 1 Lymphoma Run: 1 CD3 789 Automation N % N % N>@O;<;P O;<@:J rxxx> Dako Autostainer S_`@ Aqu@bu_><;= d><` Tf 31 1 ji ~ Dako Autostainer plus 4 25 ~ ~ Dako Autostainer Plus Link ~ i 4 5 LabVision Autostainer 4 ~ 4 5 Leica Bond Max 55 i 54 i d;>z_ N@<{ J 1 1 Leica Bond-III 34 ii 29 ~ :;<_=><> I [<u;ee> _u cdj None (Manual) 4 ~ 3 Other 3 ~ 3 33 Q _<{@< Q; q;<š_ Ventana Benchmark ~ Ventana Benchmark ULTRA ji a;<u_<_ N;<z G_=` Jx 46 i 49 ~ A selection from just a few of the best methods employed by participants CD3 - Method 1 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Ventana UltraView DAB Ventana reaction buffer (95-3) 43

46 Run 1 LYMPHOMA Module CD3 - Method 2 Participant scored 2/2 (UK NEQAS Slide) and 15/2 (In House slide) using this method. Primary Antibody: Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Chromogen: Detection: CD3 - Method 3 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Ventana reaction buffer (95-3) CD3 - Method 4 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: BEST METHODS - Secondary Antibody Primary Antibody: Automation: Main Buffer: Chromogen: Detection: 44

47 Run 1 LYMPHOMA Module Participant scored 19/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Automation: Dako Autostainer Link 48 Main Buffer: Chromogen: Detection: Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Ventana iview Kit Ventana reaction buffer (95-3) Ventana Benk CC1 (Standard) Ventana iview 45

48 The Neuropathology Module Run 1 Neil Bilbe and Merdol Ibrahim Gold Standard Second Antibody Antigens Assessed: Synaptophysin Β-Amyloid Tissue Sections circulated: Number of Registered Participants: 67 Normal Cerebellum & Dysembryoplastic Neuroepithelial Tumour (DNET) Number of Participants This Run Synaptophysin 67 (1%) Β-Amyloid 49 (73%) Cerebral Cortex/Alzheimer's Summary Graphs - Pass Rate Tables Best Method - Selected Images UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 46

49 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining Fig 1. Optimal demonstration of synaptophysin in the UK NEQAS normal brain section. The staining is intense and punctate, with a variable distribution in the fibrillary background. Section stained with the Novocastra 27G12 antibody, 1:5, on the Ventana Ultra Benchmark, CC1 mild and Optiview kit. Fig 2. High power image showing good synaptophysin staining in the UK NEQAS dysembryoplastic/neuroepithelial tumour (DNET) section, showing strong staining of the fibrillary elements. Section stained with the Novocastra 27G12 antibody, 1:5, on the Ventana Benchmark XT, CC1 standard and Ultraview kit. Fig 3. Sub-optimal very weak synaptophysin staining in the UK NEQAS distributed normal brain Œ Ž Œ Ž š œœ Ž Ž Œ ž ŽŸ ŽŸŒ œ Ž ŽŸŒ ªŒ Ž «Œ Ÿ Ž Ž ± Œž Ž Fig 4. Sub-optimal demonstration of synaptophysin in the UK NEQAS DNET section. The staining is weak and the level of elements demonstrated is lower than expected (compare to fig 2). Section stained with the Novocastra 27G12 antibody (dilution not specified), using the Dako PT link, autostainer and Flex detection kit. Fig 5. Poor demonstration of synaptophysin in the UK NEQAS DNET section. The background staining is excessive. This is most likely caused by the antibody dilution being too concentrated. Section stained with the Vector SP11 antibody, 1:1, using the Dako PT link, autostainer and Flex kit. ² ³ Œµ Œ Ÿ Œ Ž Ž Ÿ ŸŒ Ÿ ž strong cytoplasmic staining with a clean background. Section stained with the Novocastra 27G12 antibody, 1:1 on the Bond III ER1 and Refine kit. 47

50 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining ¹º» ç½ èéåºâçì ÀÁ ÃÄÅÆÇź Ã È ÉÊÇÂËÌ ºÀ à ÅÍÁ ÎÏ ÐÑÒÓÔ ÓÌÕÍÁºÂÁÆÖÄ ÁÆÁØÆÇÌ ÆÅÁÙ section, showing strong cytoplamsic staining of the amyloid plaques with a clean background. ÔÁ ź à ÄÅǺÃÁÀ ܺÅÍ ÅÍÁ ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãäåá à ÅÍÁ æáãåçãç êáã ÍÂÇÆÚ ëìá ííâ extended and Ultrview detection kit. ¹º» ¼½ ¾ À ÀÁ ÃÄÅÆÇź Ã È ÉÊÇÂËÌ ºÀ à ÅÍÁ ÎÏ ÐÑÒÓÔ ÓÌÕÍÁºÂÁÆÖÄ ÁÆÁØÆÇÌ ÆÅÁÙ ÄÁ ź à using the APAAP detection technique. The amyloid plaques are strongly stained and the ØÇ Ú»Æ ÛÃÀ ºÄ ÌÁÇý ÔÅǺÃÁÀ ܺÅÍ ÅÍÁ ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãäåá à ÅÍÁ æáãåçãç ÎÌÅÆÇ Benchmark. ¹º» ð½ ÔÛØÊ éåºâçì ÀÁ ÃÄÅÆÇź Ã È ÉÊÇÂËÌ ºÀ à ÅÍÁ ÎÏ ÐÑÒÓÔ ÓÌÕÍÁºÂÁÆÖÄ ÁÆÁØÆÇÌ ÆÅÁÙ section, showing very weak staining of the amyloid plaques (compare to fig 7). Section stained ܺÅÍ ÅÍÁ ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãäåá à éæáêåæáçåâáãå à ÅÍÁ ÝÇÚ ÇÛÅ ÄÅǺÃÁÆá ¹ÌÁ٠ںŽ ¹º» âå½ ÎÃÇ ÁéÅÇØÌÁ ÉÊÇÂËÌ ºÀ ÄÅǺúû à ÅÍÁ ÎÏ ÐÑÒÓÔ ÓÌÕÍÁºÂÁÆÖÄ ÁÆÁØÆÇÌ ÆÅÁÙ ÄÁ ź à (compare to fig 7). The amyloid plaques are not staining as expected. Section stained with the ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãîåá à éæáêåæáçåâáãå à ÅÍÁ æáãåçãç êáã ÍÂÇÆÚ ëì ÇÃÀ ÎÌÅÆÇïºÁÜ ÚºÅ½ ¹º» ââ½ ÔÛØÊ éåºâçì ÀÁ ÃÄÅÆÇź Ã È ÉÊÇÂËÌ ºÀ à ÅÍÁ ÎÏ ÐÑÒÓÔ ÓÌÕÍÁºÂÁÆÖÄ ÁÆÁØÆÇÌ ÆÅÁÙ½ The section shows non-specific inappropriate staining in the background. Sections stained with ÅÍÁ ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãâååá à éæáêåæáçåâáãå à ÅÍÁ óáº Ç ê ÃÀôÇÙ ÇÃÀ õáèºãá ÀÁÅÁ ź à kit. ¹º» âñ½ ¾ À ÁÙÇÂéÌÁ È Çà òºã Í ÛÄÁÖ ÓÌÕÍÁºÂÁÆÖÄ ØÆǺà ÃÅÆ Ì ÄÅǺÃÁÀ ܺÅÍ ÉÊÇÂËÌ ºÀ½ ìíá cytoplamsic staining is strong and distinct and the background is clean. Section stained with ÅÍÁ ÝÇÚ Þ¹ßàÝ ÇÃÅºØ ÀËá âãâååá à éæáêåæáçåâáãå à ÅÍÁ ÝÇÚ ÇÛÅ ÄÅǺÃÁÆ ÜºÅÍ ¹ÌÁÙ ÀÁÅÁ ź à kit. 48

51 Run 1 14 ö ø ùúúû üýþÿr R ý þ þ ø ü ü þ Individual 4 2 RUN 1H Synaptophysin on in-house Sections Individual no. of returns no. of returns Summary Summary M 15 M ö ø ùúú ÿÿý þ ø ü ü þ Individual 4 16 ö ø ùúú! ÿÿý þ þ " ü þ Individual no. of returns no. of returns Summary Summary M 1 M 4 49

52 Run 1 ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score > Neuropathology Run: 1 Neuropathology Run: 1 Primary Antibody : Synaptophysin Antibody Details N % BioGenex AM 6 (clone SNP ) 1 Biogenex MU 363 UC 2 1 C#$$ %&'()# **+,-./ : ;<=$>? 1 C#$$ %&'()# **+@-A/A2A+A. ;%@B-/:? 1 1 Dako A1 (polyclonal) 1 DAKO FLEX IR776 (SY38) 2 1 Dako M776 (clone SY38 ) 2 55 Dako N1566 (rb poly) 1 Lab Vision RM 9111 SO 1 NeoMarkers SP Novocastra Bond RTU PA299 (rb poly) 1 1 Novocastra NCL-L-SYNAP-299 (27G12) Novocastra NCL-SYNAP-299 (27G12) 9 67 Other 1 Thermo Sci/Pierce PA (Poly) 1 1 Vector VP-RM9 (SP11) 1 Ventana (MRQ-4) 1 Ventana CONFIRM (SP-11) 8 5 Primary Antibody : Beta-amyloid Antibody Details N % Chemicon/Millipore MAB1561 (4G8) 2 1 Dako M Novocastra NCL-B AMYLOID Other 1 1 Sigma A Neuropathology Run: 1 Neuropathology Run: 1 Beta-amyloid Synaptophysin Beta-amyloid Synaptophysin Heat Mediated Retrieval N % N % Biocare Decloaking Chamber 1 1 Dako PTLink Leica Bond III ER Leica Bond III ER Leica BondMax ER Leica BondMax ER Microwave Oven NOT APPLICABLE Other Pressure Cooker Ventana Benk CC1 (8mins) 1 Ventana Benk CC1 (Extended) 1 1 Ventana Benk CC1 (Mild) 1 1 Ventana Benk CC1 (Standard) 4 25 Ventana Benk ULTRA CC1 (Exten.) 2 5 Ventana Benk ULTRA CC1 (Stan.) 6 67 Ventana Benk XT CC1 (Mild) 4 25 Ventana Benk XT CC1 (Standard) Ventana ULTRA CC1 (Mild) Enzyme Mediated Retrieval N % N % AS PER KIT 1 1 NOT APPLICABLE Other

53 Run 1 Neuropathology Run: 1 Neuropathology Run: 1 Beta-amyloid Synaptophysi n Detection N % N % AS PER KIT Biocare polymer (M4U534) 1 1 DEFGHIHJ KK LFNOPHQ STU VWXYZ[\]^ 1 1 DEFGHIHJ KK LFNOPHQ STU V_XY`a\]^ 1 1 Dako EnVision FLEX ( K8/1) UbcF ]IdEeEFI fg]`h S \ixxwjkw^ 4 1 l ml UbcF ]InEeEFI opljuad S \qxxr^ 2 1 gbsdeeefi tnuqbdeeefi vw] LFNOPHQ S xgyxkwjqyouyjx 1 ghezb DFI{ LFNOPHQ UH EIH SUK}rk_^ 1 1 ghezb DFI{ LFNOPHQ ph EIH SUK}iXX^ 15 ~l 19 l HIbLbu `Y HNN LNƒe S LY` L^ 1 1 None 2 5 NOT APPLICABLE 1 1 wfnfzbeuqb wfnfneic LUK Sp]rYkVXjkqXjWiXjW}XY\^ 1 1 Other Vector Elite ABC (PK-61) 1 1 Vector Elite Universal ABC (PK-62) 1 1 dhiubib EdEH eoeuhp Sr XYX}k^ dhiubib v uedeh \Eu Sr XYrXX^ 2 1 dhiubib tnuqbdeh \Eu Sr XYqXX^ ~ ml Chromogen Beta-amyloid Synaptophysin N % N % AS PER KIT ~ BioGenex DAB (QD43) 1 1 BioGenex Liquid DAB (HK153-5K) 1 1 UbcF UaDh ge ƒe{ S\_V i^ 1 UbcF UaDh p]ag UHuHzuEFI S\qXXk^ 1 Dako EnVision Plus kits UbcF fg]` UaD 5 ~ˆ l l UbcF p]ag ]IdEeEFI \qxxr UaD LabVision DAB 1 ghezb DFI{ LFNOPHQ ph EIH ceu SUK}iXX^ 16 ~~ 19 l PHIb bu JzHNN ceu UaD S LYi X^ 1 1 Other 3 Šl 2 5 KE Pb UaD SUq _r^ 1 1 Ventana DAB 1 1 Ventana iview Ventana Ultraview DAB ~ ml deeefi DEFKOeuHPe DFI{ ` UaD 1 1 Neuropathology Run: 1 Beta-amyloid Synaptophysin Automation N % N % DEFGHIHJ GHIF ` XXXE UbcF aƒufeubeihq geic Vi l~ Dako Autostainer plus Dako Autostainer Plus Link LabVision Autostainer 1 1 Leica Bond Max lm Leica Bond-III ~ ~~ 9 l~ HIbQEIE Y [IuHNNE bu fg` 1 1 None (Manual) l ~Š 2 Other K bi{fi KH ƒhiœb 1 Ventana Benchmark Ventana Benchmark ULTRA 4 lm 12 m~ dhiubib DHIz PbQc `x A selection from just a few of the best methods employed by participants Synaptophysin - Method 1 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: BioGenex SS Polymer-HRP BioGenex DAB (QD43), Time 1: 1 Mins, Time 2: 1 Mins Synaptophysin - Method 2 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Leica Bond Max AS PER KIT AS PER KIT Leica BondMax ER1 AS PER KIT AS PER KIT 51

54 Run 1 Synaptophysin - Method 3 Participant scored 2/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Novocastra NCL-L-SYNAP-299 (27G12), 36 Mins, 21 ºC Dilution 1: 1 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk XT CC1 (Mild) NOT APPLICABLE Chromogen: Ventana Ultraview DAB Detection: Ventana UltraView Kit (76-5) Prediluted Synaptophysin - Method 4 Participant scored 2/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Biogenex MU 363 UC, 32 Mins, 37 ºC Dilution 1: 1:5 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3), PH: 7.4 Ventana Benk XT CC1 (Standard) NOT APPLICABLE Chromogen: Ventana Ultraview DAB, RT ºC., Time 1: 8 Mins, Time 2: 4 Mins Detection: Ventana UltraView Kit (76-5), 8 Mins, 37 ºC Prediluted BEST METHODS - Secondary Antibody A selection from just a few of the best methods employed by participants Beta-amyloid - Method 1 Participant scored 19/2 (UK NEQAS Slide) and 13/2 (In House slide) using this method. Primary Antibody: Novocastra NCL-B AMYLOID, 3 Mins, 2 ºC Dilution 1: 1 Automation: None (Manual) ABC Main Buffer: Tris Buffered Saline (TBS), PH: 7.6 Microwave Oven NOT APPLICABLE Chromogen: Sigma DAB (D5637), PH: 7.6, 2 ºC., Time 1: 1 Mins Detection: Vector Elite Universal ABC (PK-62) Beta-amyloid - Method 2 Participant scored 19/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Novocastra NCL-B AMYLOID, 15 Mins, 25 ºC Dilution 1: 1 Automation: Leica Bond-III Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) NOT APPLICABLE, Buffer: Formic Acid Neat Chromogen: Leica Bond Polymer Refine kit (DS98) Detection: Leica Bond Polymer Refine (DS98), 8 Mins, 25 ºC 52

55 Run 1 Beta-amyloid - Method 3 Participant scored 2/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana ULTRA CC1 (Mild) Other Chromogen: Ventana Ultraview DAB Detection: Beta-amyloid - Method 4 Primary Antibody: Automation: Main Buffer: Chromogen: Detection: Leica Bond Max Leica BondMAx Refine KIT Other NOT APPLICABLE AS PER KIT AS PER KIT 53

56 The Cytology Module Run 1 Neil Bilbe and Merdol Ibrahim Gold Standard Second Antibody Antigens Assessed: CD45 Cytokeratin Tissue Sections circulated: Number of Registered Participants: 73 Number of Participants This Run 72 (99%) Effusion containing lymphocytes, macrophages, erythrocytes and mesothelial cells Effusion containing carcinoma cells, resected tonsil cells, mesothelial cells and lymphocytes Summary Graphs - Pass Rate Tables Best Method - Selected Images UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 54

57 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining œ ª œ «ž œ ±² ³ µ ž œ «demonstrates localised membrane staining in the tumour cells and discernible nuclear ¹ œ œ œ ž œ º» ¼ «œ Ž ½ ¾ ž œºœ ž œ Phosphatase - Anti-Alkaline Phosphatase (APAAP). Section stained with Dako clones Ž š œ Ž ž ŸŸŸ œ œ À Á ½  ž œ œ ±² ³ µ ž œ µ¹œ few of the lymphocytes remain unstained for CD45. Section stained with Dako clones Ž š œ Ž ž à  œ Fig 4. Poor demonstration of CD45 on the UK NEQAS cytospin. The staining is excessive with œ œ œ œ ««œ œ ž œ ½ Ž œä ž Å ÆÆ š œ Ž žã » ž» œ ½œ Fig 5. Unacceptable demonstration of CD45 on the UK NEQAS cytospin. There is extensive morphological damage due to the use of heat retrieval, making interpretation difficult. Section œ ž œ ½ Ž œä ž Å ÆÆ º Ž ½ ±«œ CC1 (mild) pre-treatment. œ Ç ž œ ¹ ««Ä«½ ««ž œ «««œ ž Ä staining in the tumour cells and a good level of counterstain. Section stained with the Dako Ž œä ž žœ«¹ ž ½ ¹ œ 55

58 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining ÈÉÊ ËÌ ÍÎÏÉÐÑÒ ÓÔÐÕÖ ÏØÑÏÉÕÖ ÕÙ ÚÛ ÕÖ ÏÜÔ ÝÛ Þßàáâ ãäïõ ÎÉÖÌ åüôøô É ÏØÕÖÊ ãäïõîòñ ÐÉã staining of tumour cells with a good level of counter satin. Section stained with the Dako æþèççèé ÓÉÒêÏÔÓ çëìíí ÕÖ Ñ îôöïñöñ ïôöãüðñøð ñå òéïü ÖÕ ÎØÔóÏØÔÑÏÐÔÖÏÌ Fig 8. Borderline acceptable demonstration of CK on the UK NEQAS cytospin. The staining is becoming too strong and there is inappropriate staining of non-tumour cells. Section stained with Becton Dickinson CAM5.2 on a Leica Bond III. No other staining details were provided. Fig 9. Borderline acceptable demonstration of CK on the UK NEQAS cytospin stained using Alkaline Phosphatase - Anti-Alkaline Phosphatase (APAAP). The staining is becoming too strong and there is inappropriate staining of non-tumour cells. Section stained with Becton Dickinson CAM5.2 on a Leica Bond III. No other staining details were provided Fig 1. Optimal in-house cell block control from a metastatic carcinoma stained for cytokeratin. âôãïéõö ÏÑÉÖÔÓ ê ÉÖÊ ôñðõ æþèççèé ÓÉÒêÏÔÓ çëçíí ÕÖ Ñ îôöïñöñ ïôöãüðñøð ñå òéïü ÖÕ pre-treatment. Fig 11. Optimal in-house cell block from a pleural effusion stained for cyokeratin. Section ÏÑÉÖÔÓ ê ÉÖÊ ôñðõ æþèççèé ÓÉÒêÏÔÓ çëõíí ÕÖ Ñ îôöïñöñ ïôöãüðñøð ñå òéïü ÖÕ ÎØÔóÏØÔÑÏÐÔÖÏÌ 56

59 Run 1 CYTOLOGY Module GRAPHICAL REPRESENTATION OF PASS RATES 28 RUN 1R CD45 / LCA on NEQAS Sections Individual 4 = (%) 5 = (%) 35 RUN 1S CD45 / LCA on in-house Sections Individual 4 = (%) 5 = (%) 24 6 = (%) 7 = (%) 3 6 = (%) 7 = (%) no. of returns = 1 (1%) 9 = (%) 1 = 2 (3%) 11 = 5 (7%) 12 = 4 (6%) 13 = 7 (1%) 14 = 5 (7%) no. of returns = (%) 9 = 1 (1%) 1 = (%) 11 = 1 (1%) 12 = 13 (19%) 13 = 2 (3%) 14 = 1 (1%) 8 15 = 12 (17%) 16 = 27 (38%) 1 15 = 5 (7%) 16 = 35 (5%) 4 17 = 4 (6%) 18 = (%) 5 17 = 7 (1%) 18 = 1 (1%) = 2 (3%) 2 = 3 (4%) = 2 (3%) 2 = 2 (3%) Summary Summary 16-2 = 36 (5%) 16-2 = 47 (67%) = 24 (33%) = 8 (11%) 1-12 = 11 (15%) 1-12 = 14 (2%) - 9 = 1 (1%) - 9 = 1 (1%) Median = 14. Median = RUN 1T Cytokeratin (CK) on NEQAS Sections Individual 4 = (%) 24 RUN 1U Cytokeratin (CK) on in-house Sections Individual 4 = (%) 5 = (%) 5 = (%) 16 6 = (%) 7 = (%) 2 6 = (%) 7 = (%) 8 = (%) 8 = 1 (1%) no. of returns = 1 (1%) 1 = (%) 11 = 3 (4%) 12 = 7 (1%) 13 = 2 (3%) 14 = 6 (8%) 15 = 16 (22%) no. of returns = (%) 1 = (%) 11 = 1 (1%) 12 = 8 (11%) 13 = 3 (4%) 14 = 5 (7%) 15 = 5 (7%) 4 16 = 2 (28%) 17 = 8 (11%) 4 16 = 24 (34%) 17 = 9 (13%) 18 = 4 (6%) 18 = 8 (11%) = 4 (6%) 2 = 1 (1%) = 3 (4%) 2 = 3 (4%) Summary Summary 16-2 = 37 (51%) 16-2 = 47 (67%) = 24 (33%) = 13 (19%) 1-12 = 1 (14%) 1-12 = 9 (13%) - 9 = 1 (1%) - 9 = 1 (1%) Median = 15. Median =

60 Run 1 CYTOLOGY Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE CYTOLOGY MODULE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score >12/2) on UK NEQAS sections. Cytology Run: 1 Cytology Run: 1 Primary Antibody : CD45 / LCA Antibody Details N % Dako M742 (clone UCHL1) CD45RO 1 1 Dako M71 (clones 2B11+PD7/26) Dako M833 (clone PD7/26) CD45RB 1 1 Dako N Dako RTU FLEX LINK IR751 (2B11 + PD7/26) 2 1 Leica/Novocastra NCL-L-LCA (X16/99) 2 1 Leica/Novocastra NCL-LCA-RP (RP2/18 + 2/22) 2 5 Leica/Novocastra RTU-LCA-RP (RP2/18 + 2/22) 1 1 Other 1 1 Ventana CONFIRM (RP2/18) 7 86 Primary Antibody : Cytokeratin (CK) Antibody Details N % Becton Dickinson (CAM5.2) 3 67 BioGenex MU71-UC (clones AE1/AE3) 2 1 Biomedicals BMA-T Chemicon MAB 3412 (AE1/AE3) 1 1 D Biosystems MOB-19 (AE1/AE3) 1 1 Dako FLEX RTU IR53 (AE1/AE3) 2 1 Dako M821(MNF116) Dako M3515 (AE1/AE3) Dako N159 (AE1/AE3) 2 1 ImmunoBS MM-112 (CK cocktail) 1 1 Leica/Novacastra NCL- CK5/6/8/18 (Multi 5D3/LP34) 1 1 Leica/Novacastra NCL- L-CK5/6/8/18 (Multi 5D3/LP Leica/Novocastra NCL-L-AE1/AE Leica/Novocastra RTU PA99 (AE1/AE3) 1 1 Other 6 1 Ventana AE1/AE3/PCK Cytology Run: 1 Cytology Run: 1 Primary Antibody : CD45 / LCA Primary Antibody : Cytokeratin (CK) Antigen Retrieval N % Antigen Retrieval N % YES YES NO NO Breakdown of participants reporting YES N Breakdown of participants reporting YES N Heat Mediated Heat Mediated Enzyme Enzyme Both 16 Both 14 Not Specified Not Specified Cytology Run: 1 Cytology Run: 1 Heat Mediated Retrieval Heat Mediated Retrieval Cytology Run: 1 Cytology Run: 1 Enzyme Mediated Retrieval Enzyme Mediated Retrieval 58

61 Run 1 CYTOLOGY Module Cytology Run: 1 Cytology Run: 1 Detection CD45 / LCA Cytokeratin (CK) N % N % AS PER KIT BioGenex HRP (HK 519-6K) 1 1 BioGenex SS Polymer (QD 43-XAKE) Dako EnVision FLEX ( K8/1) Dako EnVision FLEX+ ( K82/12) Dako Envision HRP/DAB ( K57) Dako Envision+ HRP mouse K44/5/6/ Dako REAL ( K55) 1 LabVision UltraVision LP HRP (TS 125 HD) Leica Bond Polymer Refine (DS98) None 1 1 NOT APPLICABLE Other Power Vision DPVB999 HRP Ventana iview system (76-91) Ventana OptiView Kit (76-7) Ventana UltraView Kit (76-5) Chromogen CD45 / LCA Cytokeratin (CK) N % N % AS PER KIT BioGenex DAB (QD43) BioGenex Liquid DAB (HK153-5K) DAKO DAB Dako DAB+ Liquid (K3468) Dako FLEX DAB Dako REAL EnVision K57 DAB 1 1 Dako REAL K55 Alkaline phosphatase 1 1 Leica Bond Polymer Refine kit (DS98) Other Sigma DAB (D4168) Sigma DAB (D5637) Ventana DAB Ventana iview Ventana Ultraview DAB Cytology Run: 1 Automation CD45 / LCA Cytokeratin (CK) N % N % BioGenex GenoMX 6i BioGenex Optimax Dako Autostainer Dako Autostainer Link Dako Autostainer plus Dako Autostainer Plus Link LabVision Autostainer Leica Bond Max Leica Bond-III None (Manual) Ventana Benchmark Ventana Benchmark ULTRA Ventana Benchmark XT

62 Run 1 CYTOLOGY Module BEST METHODS - Gold Standard Antibody A selection from just a few of the best methods employed by participants CD45 / LCA - Method 1 Participant scored 2/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Ventana CONFIRM (RP2/18), 32 Mins, 37 ºC Prediluted Automation: Ventana Benchmark XT Ventana iview Kit Main Buffer: Ventana reaction buffer (95-3), PH: 7.6 Ventana Benk XT CC1 (Mild), Buffer: CC1, PH: 7.8 Chromogen: Ventana iview, Time 1: 8 Mins Detection: Ventana iview system (76-91), 8 Mins Prediluted CD45 / LCA - Method 1 Participant scored 17/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Ventana CONFIRM (RP2/18), 16 Mins, 37 ºC Prediluted Automation: Ventana Benchmark ULTRA AS PER KIT Main Buffer: Ventana reaction buffer (95-3) NOT APPLICABLE NOT APPLICABLE Chromogen: Ventana DAB Detection: Ventana OptiView Kit (76-7) Prediluted CD45 / LCA - Method 1 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Leica/Novocastra NCL-L-LCA (X16/99),.5 Mins, 2 ºC Dilution 1: 4 Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) Leica BondMax ER1 Chromogen: Leica Bond Polymer Refine kit (DS98) Detection: CD45 / LCA - Method 1 Participant scored 19/2 (UK NEQAS Slide) and 18/2 (In House slide) using this method. Primary Antibody: Dako M71 (clones 2B11+PD7/26), 3 Mins, RT ºC Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) Leica BondMax ER1 Chromogen: Leica Bond Polymer Refine kit (DS98) Detection: Leica Bond Polymer Refine (DS98), RT ºC 6

63 Run 1 CYTOLOGY Module BEST METHODS - Secondary Antibody A selection from just a few of the best methods employed by participants Cytokeratin (CK) - Method 1 Participant scored 2/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Dako M821(MNF116), 32 Mins, 37 ºC Dilution 1: 1:1 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Protease 1 (76-218), 37 ºC. Digestion Time NEQAS: 8 Mins. In-House: 8 Mins Chromogen: AS PER KIT Detection: AS PER KIT Cytokeratin (CK) - Method 1 Participant scored 19/2 (UK NEQAS Slide) and 18/2 (In House slide) using this method. Primary Antibody: Dako M3515 (AE1/AE3), 32 Mins Dilution 1: 1:1 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk XT CC1 (8mins) Ventana Protease Digestion Time In-House: 8 Mins Chromogen: Ventana Ultraview DAB Detection: Ventana UltraView Kit (76-5) Cytokeratin (CK) - Method 1 Participant scored 18/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Leica/Novacastra NCL- L-CK5/6/8/18 (Multi 5D3/LP34 Dilution 1: 25 Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Chromogen: Detection: Bond Wash Buffer (AR959) Leica BondMax ER1 NOT APPLICABLE Leica Bond Polymer Refine kit (DS98) Leica Bond Polymer Refine (DS98) Prediluted Cytokeratin (CK) - Method 1 Participant scored 19/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Ventana AE1/AE3/PCK26, 28 Mins, 37 ºC Prediluted Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) NOT APPLICABLE Chromogen: Ventana Ultraview DAB, 37 ºC., Time 1: 8 Mins Detection: Ventana UltraView Kit (76-5), 8 Mins, 37 ºC Prediluted 61

64 The Alimentary Tract Module: CD117 & DOG-1 Run 1 Andrew Dodson and Suzanne Parry First Antibody Antigens Assessed: CD117 DOG-1 Tissue Sections circulated: Number of Registered Participants: 15 GIST, Appendix & Desmoid Second Antibody GIST, Appendix & Desmoid Number of Participants This Run CD (98%) DOG-1 77 (73%) Gold Standard - CD117 CD117 (c-kit) is a transmembrane tyrosine kinase growth factor receptor involved in cell differentiation. It is expressed by melanocytes, mast cells and interstitial cells of Cajal, and is also expressed in gastrointestinal stromal tumors (GIST). Until recently, patients with GIST faced a limited choice of treatment regimes, which included radiotherapy and surgery with limited success. However, Glivec (Imatinib), which was originally developed for chronic myeloid leukemia (CML), has proved effective in the targeted treatment of GISTs by directly inhibiting the action of CD117. Tissue Distribution Circulated tissue comprised of GIST, appendix and a desmoid tumour. Participants were requested to stain the samples using their routine protocol, whether with or without heat mediated antigen retrieval. Features of Optimal Immunostaining (see Figs 1-3) Good localisation of CD117 to cells of the GIST Good localisation of CD117 to mast cells Good localisation of CD117 to interstitial cells of Cajal No staining of desmoid tumour Features of sub-optimal Immunostaining (see Figs 4-5) Weak and/or patchy staining of the tumour cells of the GIST Little or no staining of the mast cells Excessive background / non specific staining Staining of the desmoid tumour Second Antibody - DOG1 Discovered on GIST 1 (DOG-1) antibody was initially described in 24 4 and has now started to be recognized as a more specific marker of GISTs than CD117 4,5,6. A recent study has shown DOG-1 sensitivity of 99% and found to be strong and easier to interpret than CD The groups also showed that seven CD117 negative GIST cases were found to be DOG-1 positive, and six of the tumours had PDGFRA mutations. Furthermore, DOG-1 is not expressed in Kaposi sarcomas of the GI tract which have been shown to stain for CD In more cases, combined CD117 and DOG-1 immunohistochemistry has been suggested as a sufficient panel to confirm diagnosis, with CD117 and DOG-1 negative cases tested further with a panel of antibodies including SMA, desmin, S1 and molecular analysis, should be considered 6. Tissue Distribution Circulated tissue were from sequential sections used for the CD117 distribution as show above and comprised of GIST, appendix and a desmoid tumour. Features of Optimal Immunostaining (see Figs 7-8) Good localisation of DOG-1 to cells of the GIST Good localisation of DOG-1- to interstitial cells of Cajal No staining of desmoid tumour Features of Sub-optimal Immunostaining (see Figs 9-1) Weak and/or patchy staining of the tumour cells of the GIST Excessive background / non specific staining Staining of the desmoid tumour Staining of the mast cells (Note: Mast cells are not expected to stain with DOG-1) References 1. Cordless et al., Biology of Gastrointestinal Stromal Tumours. J Clin Oncol 24, 22(18): Blay et al., Consensus meeting for the management of gastrointestinal stromal tumors. Report of the GIST Consensus Conference of 2-21 March 24, under the auspices of ESMO. Ann Oncol 25 6: Parfitt JR, Rodriguez-Justo M, Feakins R, Novelli MR (28) Gastrointestinal Kaposi's sarcoma: CD117 expression and the potential for misdiagnosis as gastrointestinal stromal tumour. Histopathology 28, 52: West RB, Corless CL, Chen X et al. The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am. J. Pathol. 24; 65; Espinosa I, Lee CH, Kim MK et al. A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors. Am. J. Surg. Pathol. 28; 32; Novelli M, Rossi S, Rodriguez-Justo M, Taniere P, Seddon B, Toffolatti L, Sartor C, Hogendoorn PC, Sciot R, Van Glabbeke M, Verweij J, Blay JY, Hohenberger P, Flanagan A, Dei Tos AP. DOG1 and CD117 are the antibodies of choice in the diagnosis of gastrointestinal stromal tumours. Histopathology 21, 57(2): Parfitt JR, Rodriguez-Justo M, Feakins R, Novelli MR. Gastrointestinal Kaposi s sarcoma: CD117 expression and the potential for misdiagnosis as gastrointestinal stromal tumour. Histopathology 28; 52; UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 62

65 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining Fig 1. Optimal demonstration of CD117 in the UK NEQAS distributed GIST section, showing intense well localised predominantly membranous and cytoplasmic staining. Section stained with the Dako polyclonal antibody, 1:35, using the Dako PT link, autostainer and FLEX detection kit. Fig 2. Good demonstration of mast cells with CD117 in the UK NEQAS ICC distributed appendix. Section stained with the Dako polyclonal antibody, 1:4, on the Ventana XT, CC1 standard and Ultraview kit. Fig 3. Good demonstration of the (A) cells of Cajal in the muscularis propria of the appendix, and the expected demonstration of (B) mast cells in the UK NEQAS ICC distributed desmoid ö øùúûü öùýúü þ ÿúùs s ö øú úø öùýúüúü úö öù ûü ýüþ þúöùúüøù ýüþ ùs ýø ûüþ remains unstained and clean. Fú ûû þ ûüöù ýù þ úü ùs ö øùúûü û ý ÿúùs Fú Although staining is present, it is very weak. Section stained with the Dako polyclonal antibody, 1 üû ù ýù üù ûü ùs! üùýüý " üøsý # ýüþ ù ý$ú ÿ úù Fú % û ùúý & ööúûü úü ùs þúöù úù þ þ öûúþ ö øùúûü ûù only are the expected mast cells staining, but there is also non-specific false-positive staining in the tumour cells. Section stained with the Novocastra T595 pre-diluted antibody, on the BondMax, ER2 and Refine kit. Fig 6. Excellent in-house control section. The mast cells in the bowel and the GIST tumour show strong specific staining and the background is clean. Stained with Dako polyclonal ýüùúûþ 1a öúü ùs ýû úü ýùûöùýúü ýüþ F'# þ ù øùúûü úù 63

66 RUN 1 Selected Images showing Optimal and Sub-optimal Immunostaining Fig 7.Optimal demonstration of DOG-1 in the UK NEQAS distributed GIST section, showing intense well localised cytoplasmic and membranous staining. Section stained with the Leica K9 antibody, 1:1, using the Dako PT link, autostainer and FLEX detection kit. Fig 8. Expected DOG-1 expression in the UK NEQAS ICC distributed desmoid section. The tumour and mast cells remain unstained with a clean background. Section stained with the Abcam DOG-1 antibody, 1:2, using the Dako PT link, autostainer and FLEX detection kit. Fig 9. Sub-optimal DOG-1 expression in the UK NEQAS ICC distributed desmoid. The section shows non-specific and false-positive staining in the tumour. Section stained with the Leica K9 ()*+,-./ )7 *89 :(;- <= >+); (5*-6*(+)9? ;+*E Fig 1. Poor demonstration of DOG-1 in the UK NEQAS ICC distributed appendix. The section shows excessive background and non-specific staining. Stained with the Leica K9 antibody, 1:25, using the Leica BondMax ER2 and Refine detection kit. Fig 11. Excellent example of an in-house control section, showing good demonstration of a :GHI2J HKL= (>-)7 M+*8 D9>>6 -N O(P(> +) *89 (.P(D9)* )-?Q(> *+6659E L*(+)9. M+*8 *89 A9+D( RS antibody, 1:1 on the Ventana ULTRA, CC1 mild and Ultraview kit. 64

67 Run 1 ALIMENTARY TRACT PATHOLOGY Module GRAPHICAL REPRESENTATION OF PASS RATES 24 RUN 1V CD117 on NEQAS Sections Individual 4 = (%) 24 TUV WXXY Z[WW\ ]^ _^`b]cde fegh_]^d Individual 4 = (%) 2 5 = (%) 6 = (%) 2 5 = (%) 6 = (%) 7 = 1 (1%) 7 = (%) no. of returns = 2 (2%) 9 = 2 (2%) 1 = 1 (1%) 11 = 2 (2%) 12 = 1 (1%) 13 = 6 (6%) 14 = 7 (7%) no. of returns = (%) 9 = 3 (3%) 1 = (%) 11 = (%) 12 = 1 (1%) 13 = 5 (5%) 14 = 5 (5%) 15 = 11 (11%) 15 = 8 (8%) 4 16 = 21 (2%) 17 = 14 (14%) 4 16 = 23 (22%) 17 = 19 (18%) = 15 (15%) 19 = 7 (7%) 2 = 4 (4%) = 17 (17%) 19 = 11 (11%) 2 = 2 (2%) Summary Summary 16-2 = 61 (59%) 16-2 = 72 (7%) = 24 (23%) = 18 (17%) 1-12 = 13 (13%) 1-12 = 1 (1%) - 9 = 5 (5%) - 9 = 3 (3%) Median = 13.5 Median = 15.5 no. of returns RUN 1Vb DOG1 on NEQAS Sections Individual 4 = (%) 5 = (%) 6 = (%) 7 = (%) 8 = 1 (1%) 9 = (%) 1 = 1 (1%) 11 = 1 (1%) 12 = 4 (5%) 13 = 1 (1%) 14 = 2 (3%) 15 = 9 (12%) 16 = 5 (6%) 17 = 12 (16%) 18 = 9 (12%) 19 = 13 (17%) 2 = 19 (25%) no. of returns TUV WXXYi [jkw ]^ _^`b]cde fegh_]^d Individual 4 = 1 (1%) 5 = (%) 6 = (%) 7 = (%) 8 = 3 (4%) 9 = (%) 1 = (%) 11 = (%) 12 = 2 (3%) 13 = 2 (3%) 14 = 6 (8%) 15 = 8 (1%) 16 = 15 (19%) 17 = 17 (22%) 18 = 7 (9%) 19 = 11 (14%) 2 = 5 (6%) Summary Summary 16-2 = 58 (75%) 16-2 = 55 (71%) = 12 (16%) = 16 (21%) 1-12 = 6 (8%) 1-12 = 2 (3%) - 9 = 1 (1%) - 9 = 4 (5%) Median = 14.5 Median =

68 Run 1 ALIMENTARY TRACT PATHOLOGY Module ANTIBODIES AND OTHER TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE ALIMENTARY TRACT PATHOLOGY MODULE The following tables record the number of participants (N) using each primary antibody and the percentage (%) of these participants achieving acceptable staining (a score >12/2) on UK NEQAS sections. Alimentary Tract Pathology Run: 1 Alimentary Tract Pathology Run: 1 Primary Antibody : CD117 Antibody Details N % Cell Marque 117R/S-xx (YR145) 2 1 Dako A452 (rb poly) Epitomics AC-29 (EP1) 1 Novocastra NCL-CD117 (T595) 1 1 Novocastra NCL-L-CD117 (T595) 4 5 Novocastra RTU-CD117 (T595) 1 Ventana (rb poly) 1 1 Ventana (9.7) 5 8 Primary Antibody : DOG1 Antibody Details N % Abcam TMEM16A (ab53212) 1 Cell Marque 244R-14/15/16 (SP31) 1 1 Cell Marque 244R-17/18 (SP31) 2 1 Leica NCL-L-DOG-1 (K9) Leica PA219 (K9) 7 1 Menarini MP-385-CM1/1 1 1 Other 1 1 Spring Biosciences M331 (rb poly) 1 1 Ventana (SP31) Alimentary Tract Pathology Run: 1 Alimentary Tract Pathology Run: 1 lmnno DOG1 lmnno DOG1 Heat Mediated Retrieval N % N % Dako PTLink Lab vision PT Module 1 1 Leica Bond III ER Leica Bond III ER Leica BondMax ER Leica BondMax ER NOT APPLICABLE 9 44 Other Pressure Cooker Pressure Cooker in Microwave Oven 1 1 Ventana Benk CC1 (Mild) Ventana Benk CC1 (Standard) Ventana Benk CC1# (8mins) 1 1 Ventana Benk CC2 (mild) 1 1 Ventana Benk CC2 (Standard) 1 1 Ventana Benk ULTRA CC1 (Exten.) Ventana Benk ULTRA CC1 (Mild) Ventana Benk ULTRA CC1 (Stan.) Ventana Benk ULTRA CC1# (8mins) 1 1 Ventana Benk XT CC1 (Mild) Ventana Benk XT CC1 (Standard) Ventana Benk XT CC1# (8mins) 1 1 Water bath OC 1 1 Enzyme Mediated Retrieval N % N % AS PER KIT 1 1 NOT APPLICABLE

69 Run 1 ALIMENTARY TRACT PATHOLOGY Module Alimentary Tract Pathology Run: 1 Alimentary Tract Pathology Run: 1 pqrrt DOG1 pqrrt DOG1 Detection N % N % AS PER KIT BioGenex SS Polymer (QD 42-YIKE) 1 1 BioGenex SS Polymer (QD 43-XAKE) 1 1 Dako EnVision FLEX ( K8/1) 2 5 Dako EnVision FLEX+ ( K82/12) Dako Envision HRP/DAB ( K57) Leica Bond Polymer Refine (DS98) MenaPath X-Cell Plus (MP-XCP) None 1 1 NOT APPLICABLE 1 1 Other Power Vision DPVB999 HRP Ventana iview system (76-91) Ventana OptiView Kit (76-7) Ventana UltraView Kit (76-5) Chromogen N % N % AS PER KIT BioGenex Liquid DAB (HK153-5K) 1 1 BioGenex liquid DBA (HK-124-7K) 1 1 Dako EnVision Plus kits Dako FLEX DAB Dako REAL EnVision K57 DAB Leica Bond Polymer Refine kit (DS98) menapath xcell kit DAB (MP-86) Other Sigma DAB (D5637) Ventana DAB Ventana iview Ventana Ultraview DAB Alimentary Tract Pathology Run: 1 pqrrt DOG1 Automation N % N % BioGenex GenoMX 6i Dako Autostainer 1 Dako Autostainer Link Dako Autostainer plus Dako Autostainer Plus Link Leica Bond Max Leica Bond-III Menarini - Intellipath FLX None (Manual) Other Ventana Benchmark Ventana Benchmark ULTRA Ventana Benchmark XT BEST METHODS - Gold Standard Antibody A selection from just a few of the best methods employed by participants Participant scored 2/2 (UK NEQAS Slide) and 16/2 (In House slide) using this method. Primary Antibody: Dako A452 (rb poly), 3 Mins, 23 ºC Dilution 1: 35 Automation: Dako Autostainer Link 48 Dako FLEX+ kit Main Buffer: Dako FLEX wash buffer, PH: 7.6 Dako PTLink, Buffer: Target retrieval sol, high ph, PH: 9 Chromogen: Dako FLEX DAB, 23 ºC., Time 1: 6 Mins, Time 2: 6 Mins Detection: Other, 15 Mins, 23 ºC Prediluted Participant scored 18/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Novocastra NCL-L-CD117 (T595), 15 Mins, 21 ºC Prediluted Dilution 1: 1/1 Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) Leica BondMax ER2 NOT APPLICABLE Chromogen: Leica Bond Polymer Refine kit (DS98), 21 ºC., Time 1: 1 Mins Detection: Leica Bond Polymer Refine (DS98), 8 Mins, 21 ºC Prediluted 67

70 Run 1 ALIMENTARY TRACT PATHOLOGY Module Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Dako A452 (rb poly), 52 Mins, 37 ºC Dilution 1: 4 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk CC1 (Standard) Chromogen: Ventana Ultraview DAB Detection: Ventana UltraView Kit (76-5) Prediluted Participant scored 2/2 (UK NEQAS Slide) and 17/2 (In House slide) using this method. Primary Antibody: Cell Marque 117R/S-xx (YR145), 32 Mins, r.t. ºC Prediluted Dilution 1: 1:1 Automation: Ventana Benchmark ULTRA Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk XT CC1 (Standard) Chromogen: Ventana Ultraview DAB Detection: BEST METHODS - Secondary Antibody A selection from just a few of the best methods employed by participants DOG1 - Method 1 Participant scored 2/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Leica NCL-L-DOG-1 (K9), 32 Mins, 37 ºC Dilution 1: 4 Automation: Ventana Benchmark XT Ventana UltraView DAB Main Buffer: Ventana reaction buffer (95-3) Ventana Benk CC1 (Standard) NOT APPLICABLE Chromogen: AS PER KIT Detection: AS PER KIT DOG1 - Method 2 Participant scored 2/2 (UK NEQAS Slide) and 2/2 (In House slide) using this method. Primary Antibody: Leica NCL-L-DOG-1 (K9), 3 Mins, 2 ºC Dilution 1: 1 Automation: Dako Autostainer Plus Link Dako FLEX+ kit Main Buffer: Dako FLEX wash buffer, PH: 7.6 Dako PTLink, Buffer: HIGH PH TRS, PH: 9 NOT APPLICABLE Chromogen: Dako FLEX DAB, 2 ºC., Time 1: 1 Mins, Time 2: 1 Mins Detection: Dako EnVision FLEX+ ( K82/12), 15 Mins, 2 ºC Prediluted 68

71 Run 1 ALIMENTARY TRACT PATHOLOGY Module DOG1 - Method 3 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Ventana (SP31) , 37 ºC Prediluted Automation: Ventana Benchmark XT Other Main Buffer: Ventana reaction buffer (95-3) Ventana Benk XT CC1 (Standard), Buffer: CC1 NOT APPLICABLE Chromogen: Other, 37 ºC., Time 1: 8 Mins, Time 2: 8 Mins Detection: Ventana OptiView Kit (76-7), 8 Mins, 37 ºC Prediluted DOG1 - Method 4 Participant scored 2/2 (UK NEQAS Slide) and 19/2 (In House slide) using this method. Primary Antibody: Leica PA219 (K9), 8 Mins Automation: Leica Bond Max Leica BondMAx Refine KIT Main Buffer: Bond Wash Buffer (AR959) Leica BondMax ER2 Chromogen: AS PER KIT Detection: Leica Bond Polymer Refine (DS98), 15 Mins 69

72 The HER2 ISH Module - Interpretive Run 29 Merdol Ibrahim, Suzanne Parry and 9 Reference Centres Gene Assessed: Sections circulated: HER2 Number of Registered Participants: 177 Number of Participants This Run 15 (85%) Composite block containing 4 cell line pellets (see below) The Summary box below gives details of the HER2 phenotype and genotype of the cell lines circulated for these assessments, which comprised of formalin fixed and paraffin processed sections from a composite block of four human breast carcinoma cell lines. The cell lines are the same ones which are used in the HER2 IHC module. The sections were positioned on the microscope slides as indicated. Cell Line HER2 status by IHC 1 HER2 status by FISH 2 SK-BR-3 3+ Amplified MDA-MB Borderline: Amplified / Not Amplified MDA-MB Not Amplified MDA-MB-231 Negative Not Amplified 1. Scored using the Clinical Trials assay system 2. ISH status as assessed by the UK NEQAS ICC & FISH reference laboratories (Bartlett et al., 27) AN IMPORTANT NOTE ABOUT THE TYPES OF PREPARATIONS WHICH ARE ASSESSED IN THIS MODULE Although this module had previously been entitled HER2 FISH, the organizers would like to make it clear that the submission of results from slides stained using chromogenic in-situ hybridisation (CISH) and silver in-situhybridisation (SISH) are equally acceptable, and are welcomed. Copy number data produced from slides stained using this approach will be assessed in a similar way to that produced by single colour FISH users i.e. against the copy number data produced by the reference centres. Cell Line Positioning: Cell lines are positioned on microscope slides, as illustrated below. Any variation in this pattern is indicated on distribution of the slides Introduction After successful conclusion of the pilot phase we are pleased to announce that this is now a full UK NEQAS module. The results of the first pilot and a three year follow-up have now been published [1,2]. Furthermore, the scheme has allowed multiple ring studies to be undertaken and have allowed new methodologies, for example the Ventana silver ISH (SISH) [3], Kreatech FISH probes [4] and the Ventana Dual ISH (BDISH and DDISH) [5] to be analysed. With the growing importance of colourimetric assays, both chromogenic and silver based in situ detection systems, the scheme has changed from a FISH to an ISH based scheme, where all molecular in situ assays for HER2 gene copy/amplification determination are included. This move reflects an on-going development in chromogenic assay systems which it is envisaged will lead to a wider uptake of these assays within the diagnostic community. For UK laboratories carrying out diagnostic HER2 ISH testing participation is therefore mandatory under Clinical Pathology Accreditation (UK), and National Quality Assurance Advisory Panel (NQAAP) regulations. It should also be emphasised that the scheme remains very-much open to non-uk laboratories as an aid to monitoring and improving performance. Recommendations Diagnostic testing recommendations for HER2 ISH was originally published by Walker et at [6] but a more recent publication covering recommendations for both breast and gastric in-situ hybridisation methods is now available [7]. Assessment of Slides (rationale behind the adopted scoring system) Cell lines were selected to cover diagnostic intervals for the HER2 FISH test, where gene amplification is recognised if the ratio of HER2 signals to Chromosome 17 signals exceeds 2.. Previous evidence suggests that inter-observer error in scoring FISH signals can be controlled within 1% for both HER2 and other solid tissue analyses (Bartlett et al., 21, Bartlett, 25 for review). This results in a small population of cases (<2.% of cases, unpublished observations; FISH used as frontline test) where the result reported lies within a 1% range of the diagnostic interval, i.e. between 1.8 and 2.2. Whilst this compares favourably with the approximately 15% of cases reported as borderline by immunohistochemistry it highlights the need to rigorously control scoring approaches. Data suggest UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 7

73 The HER2 ISH Module - Interpretive Run 29 How the scoring system works Schematic representation of the scoring system; the example illustrated uses the Reference Centre set of HER2/Ch17 ratios obtained for the SK-BR3 (3+ Amplified) cell line at run 4. In this case the lowest ratio obtained by a Reference Centre was 3.19, and the highest 4.1; participants submitting ratios within this range were judged to have achieved an appropriate result (score = 3). The lower cut-off for acceptable ratios (score = 2) was calculated as 3.19 minus 1% of 3.19, that is 2.87; and the upper cut-off was calculated as 4.1 plus 1% of 4.1, that is Participants who submitted ratios out-with these 1% cut-offs were judged to have achieved an inappropriate and received a score of 1. Except in the case of the MDA-MB-453 cell line, misdiagnosis (amplified reported as non-amplified, and visa versa) resulted in a score of. Superscript notation and abbreviation used in figure: * = does not apply to results obtained for MDA-MB-453 (2+ Borderline: Amplified to Non-Amplified) cell line; RC = Reference Centre. that allowing variation to increase by 5% (to 15%) would double the number of equivocal cases (to 4%) and allowing an increase to 2% variation at the diagnostic interval would lead to a fourfold increase in equivocal cases (Bartlett unpublished observations). These observations underpin the approach taken in assessing participants results. For each cell line, participants scored 3 points if the result reported fell within the range observed by the reference laboratories. These reference laboratory results already reflect the acceptable observer variation of 1% for inter-observer variation in scoring discussed above since they are collated from seven different laboratories, each of which presents two scores (up to 14 observations per cell line per assessment). Thus results must fall within this range to be regarded as appropriate. For each cell line, participants scored 2 points if results lay within a further 1% of the range of results observed by the reference centres. These results reflect a band approximating to the 2% variation described above. Such results reflect acceptable performance with the potential for improvement. For results, which lay out with this second band of variation one point was given, representing inappropriate performance. For such specimens it is important to note that despite the correct diagnosis being given in many cases, a degree of variation >2% from the accepted result is indicative of a high degree of potential for error in diagnosis. In cases where results were not only deviating to this degree from the accepted result but were also misdiagnosed (e.g. where a non-amplified case is reported as amplified) a score of was recorded in recognition of the negative impact this has on the laboratories likely performance on diagnostic samples. Please note the following important rider: for the MDA-MB-453 cell line, where borderline results are often obtained and lie within the 2% margin-for-error described above, such results are NOT regarded as misdiagnoses for the purpose of this scheme. References 1. Bartlett JMS, Ibrahim M, Jasani B, et al. (27) External quality assurance of HER2 FISH testing: Results of a UK NEQAS pilot scheme. J Clin Pathol; 6: Bartlett JM, Ibrahim M, Jasani B, Morgan JM, Ellis I, Kay E, Connolly Y, Campbell F, O'Grady A, Barnett S, Miller K. (29) External quality assurance of HER2 FISH and ISH testing: three years of the UK national external quality assurance scheme. Am J Clin Pathol; 131: Bartlett JM, Campbell FM, Ibrahim M, Wencyk P, Ellis I, Kay E, Connolly Y, O'Grady A, Di Palma S, Starczynski J, Morgan JM, Jasani B, Miller K. (29) Chromogenic in situ hybridization: a multicenter study comparing silver in situ hybridization with FISH. Am J Clin Pathol; 132: Bartlett JM, Campbell FM, Ibrahim M, Thomas J, Wencyk P, Ellis I, Kay E, Connolly Y, O'Grady A, Barnett S, Starczynski J, Cunningham P, Miller K. (21) A UK NEQAS ICC and ISH multicentre study using the Kreatech Poseidon HER2 FISH probe: intersite variation can be rigorously controlled using FISH. Histopathology; 56: Bartlett JM, Campbell FM, Ibrahim M, O'Grady A, Kay E, Faulkes C, Collins N, Starczynski J, Morgan JM, Jasani B, Miller K. (211) A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay. Am J Clin Pathol; 135: Walker RA, Bartlett JM, Dowsett M, Ellis IO, Hanby AM, Jasani B, Miller K, Pinder SE. (28) HER2 testing in the UK: further update to recommendations. J Clin Pathol; Bartlett JM, Starczynski J, Atkey N, Kay E, O'Grady A, Gandy M, Ibrahim M, Jasani B, Ellis IO, Pinder SE, Walker RA. (211) HER2 testing in the UK: recommendations for breast and gastric in-situ hybridisation methods. J Clin Pathol; 64: UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 71

74 Run 29 ISH Module 6 uvw xy z{u x }~z ƒ ˆ Individual 4 = 3 (2%) 2 uvw xy z{u x }~z vš Œˆ Individual 4 = (%) 5 = 4 (3%) 5 = 1 (2%) 5 6 = 3 (2%) 7 = 7 (5%) 16 6 = 1 (2%) 7 = (%) no. of returns = 15 (1%) 9 = 11 (7%) 1 = 16 (11%) 11 = 34 (23%) 12 = 57 (38%) no. of returns = 1 (2%) 9 = (%) 1 = 6 (13%) 11 = 16 (36%) 12 = 2 (44%) 2 Summary Summary = 57 (38%) 9-11 = 61 (41%) 4-8 = 32 (21%) = 2 (44%) 9-11 = 22 (49%) 4-8 = 3 (7%) Median = Median = 11. uvw xy Ž Œ { Œ ƒ ˆ ƒ ˆ Œ ƒ ˆ % of Participants % Pass Rate Copy No.: Ventana Inform Copy No.: Zymed Invitrogen spot-light 5 1 Ratio China Ratio Dako Medical DuoCISH Technologies Kit 6 Ratio Dako Pharm Dx Ratio In house FISH Ratio Kreatech Probes Ratio Leica HER2 FISH TA9217 Ratio Other - FISH Ratio Pathvysion Vysis Kit 5 Ratio Ventana Dual ISH (BDISH) Ratio Ventana Inform Dual ISH (DDISH) 4 Ratio Ventana Inform SISH 2 Ratio Zytovision ZytoLight 1 uvw xy Ž Œ { Œ vš Œˆ ƒ ˆ Œ ƒ ˆ % of Participants % Pass Rate Copy No Ventana Inform 4 Ratio Dako Pharm Dx 7 Ratio Kreatech Probes 4 Ratio Leica HER2 FISH TA9217 Ratio Pathvysion Vysis Kit 7 Ratio Ventana Dual ISH (BDISH) Ratio Ventana Inform Dual ISH (DDISH) 2 2 Ratio Ventana Inform SISH Ratio Zytovision ZytoLight 1 72

75 Run 29 ISH Module š œ žÿ Ÿ ª «± ² ³ ²Ÿ «± Number of Participants % Pass Rate Copy No.: Ventana Inform Copy No.: Zymed µ Invitrogen spot-light 7 2 Ratio China Ratio Dako Medical DuoCISH Technologies Kit 1 Ratio Dako Pharm Dx 4 3 Ratio In house FISH Ratio Kreatech Probes 5 Ratio Leica HER2 FISH TA Ratio Other - FISH Ratio Pathvysion Vysis Kit 7 Ratio Ventana Dual ISH (BDISH) Ratio Ventana Inform Dual ISH (DDISH) 6 Ratio Ventana Inform SISH 3 Ratio Zytovision ZytoLight 2 1 š œ žÿ Ÿ š ²± ² ³ ²Ÿ «± Number of Participants % Pass Rate Copy No¹ Ventana Inform Ratio Dako Pharm Dx Ratio Kreatech Probes Ratio Leica HER2 FISH TA Ratio Pathvysion Vysis Kit 3 Ratio Ventana Dual ISH (BDISH) 13 Ratio Ventana Inform Dual ISH (DDISH) 1 1 Ratio Ventana Inform SISH Ratio Zytovision ZytoLight

76 The HER2 ISH Module - Technical (pilot) Run 29 Suzanne Parry, Merdol Ibrahim and 9 Reference Centres Gene Assessed: Sections circulated: Number of Participants Taking Part This Run HER2 ISH Composite slide consisting of 4 breast tumours of known HER2 expression (see table below) 135 (52 Chromogenic & 83 Fluorescent) The Summary Box below gives details of the HER2 phenotype and genotype of the cell lines circulated for these assessments, which comprised of formalin fixed and paraffin processed sections from a composite block of four human breast carcinoma cell lines. The cell lines are the same ones which are used in the HER2 IHC module. The sections were positioned on the microscope slides as indicated. Cell Line HER2 status by IHC 1 HER2 status by FISH 2 SK-BR-3 3+ Amplified MDA-MB Borderline: Amplified to Not Amplified MDA-MB Not Amplified MDA-MB-231 Negative Not Amplified 1. Scored using the Clinical Trials assay system 2. ISH status as assessed by the UK NEQAS ICC & FISH reference laboratories (Bartlett et al., 27) Cell Line Positioning: Cell lines are positioned on microscope slides, as illustrated below. Any variation in this pattern is indicated on distribution of the slides Assessment Procedure Chromogen ISH (CISH / SISH / BDISH / DDISH etc.) was assessed around a multi-header microscope with each slide being assessed by 4 independent assessors each providing overall scores out of 5 for the slide as a whole (not the individual cell lines). The scores are added together to give a score out of 2, along with an overall pass rate comment. Fluorescent ISH (FISH) was assessed by a single assessor from one of the UK NEQAS reference centres and given a score out of 2 for the slide as a whole (not the individual cell line), along with an overall pass rate comment. A summary of the assessment scoring criteria and it s interpretation is shown in the table on the next page Each of the UK NEQAS cell lines (A-D) (as well as in-house samples where submitted) are individually assessed for the quality of ISH staining. Assessors do not count the HER2/ CEP17 signals. The accuracy of signal enumeration is assessed in the 'interpretive' section of the HER2 ISH module. Important: If two separate slides are submitted with separate CEP17 and HER 2 copy numbers (e.g. Ventana/Roche SISH), then the assessors regard this as a single result, and as such, combined comments are placed in a single results column. Note that, Kit control slides are no longer requested for submission for this module. Results Summary CISH Results Selected images showing the acceptable and unacceptable levels of staining for the different methods are illustrated in figures 1-6. Overall there was a decrease in acceptable scores compared to the previous assessment, with participants showing an overall acceptable rate of 54% (28/52), which was down by 14% from the previous run. The borderline results increased to 27% compared to 18% from the previous assessment. And the unacceptable rates rose by 5% this time round to 19% compared to 14% from the previous assessment. The main CISH method of choice was the Ventana Inform Dual ISH with 38/57 (67%) participants using this kit. There was an improvement in pass rates on the in-house control material submitted by participants, with the data showing an overall increase in acceptable rate to 69% compared to 59% from the previous assessment. The unacceptable rate for this run was 7%, which was an improvement on the rate of 15% from the previous run. The data shows encouraging results, however, it is still apparent that many participants still need to optimise their methodology UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 74

77 The HER2 ISH Module - Technical (pilot) Run 29 Table below shows a summary of the assessment scoring criteria and interpretation. Scoring Interpretation Acceptable Individual Assessor 4-5/5 or Overall score >13/2 Borderline Individual Assessor 3/5 or Overall Score = 1-12/2 Unacceptable Individual Assessor 1-2/5 or Overall score <9/2 = Score = The cell line/s and/or in-house tissue show a good/very good standard of staining and are suitable for interpretive analyses. The cell line/s and/or in-house tissue is borderline interpretable with 2 out of the 4 assessors indicating that improvements can be made with respect for example: Excessive / Weak Nuclear staining Level of probe hybridisation Excessive background staining Also see assessor comments on your report The cell line/s and/or in-house tissue is unacceptable for interpretation with at least 3 out of the 4 assessors indicating that there are hybridisation issues, which could be due: Excessive / Weak Nuclear staining Poor probe hybridisation Missing Her2 copy no. / CEP 17 Excessive background staining Also see assessor comments on your report Slide Not submitted for assessment to allow for more accurate clinical interpretation. Note that although percentage pass rates are shown, the number of participants using certain methods are overall quite low, n=1 for some methods. FISH Results Selected images showing the acceptable and unacceptable levels of staining for the different methods are illustrated in figures Overall FISH pass rates showed a slight decrease from 63% in the previous assessment to 58% (48/83) in this run. The unacceptable rate also increased from 23% to 29%. Inhouse control material also showed a lower pass rate of 57% compared to 71% in the previous run. The in-house unacceptable scores increased slightly to 25% from 22%, as seen in the previous assessment. Again, we did not require kit controls and these were not scored for this run. Of the methods submitted the Pathvysion Vysis kit remains the method of choice, used by 65% (64/98)of participants. This time round the Vysis kit showed a slightly higher acceptable rate of 42% compared to the previous assessment of 4%. Equally, the unacceptable rate needs improving, and was much the same as the previous run at 48%. The next most popular method was the Dako PharmDX kit, which showed a much improved acceptable rate of 78%, compared to 43% seen in the previous run. However, the number of labs using this method is still quite low (n=9). Comparing CISH and FISH Methods It is advisable not to over-compare the CISH and FISH results as there may still be issues with labs submitting FISH slides which are not properly cover-slipped or fluorescence is inadequately preserved. IMPORTANT Recommendations for Returning FISH Slides for NEQAS Assessments a. Section should be mounted using a fluorescence antifade mounting media, which prevents loss/quenching of fluorescence e.g. Vectashield (Vector labs), Dako Fluorescence Mounting Medium (Dako), Fluoromount (SIGMA/Aldrich, VWR). Note: The Abbott Vysis kit already contains DAPI in phenylenediamine dihydrochloride, which is an anti fading reagent, but we have found that some laboratories also used the above mentioned mounting media. b. Seal the coverslip edges onto the slide using clear varnish or another sealing agent, which will stop the slide from drying out. c. Send back FISH slides as soon as you have finished your own interpretation. d. There is no need to send back slides packed in ice/dry ice. Please return in the slide mailer that is provided. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 75

78 The HER2 ISH Module - Technical (pilot) Run 29 How to Use this Report to Compare NEQAS Cell Line Technical & Interpretive Results: Once you have your results for both the Interpretive and Technical HER2 ISH modules, use both results to troubleshoot whether there are interpretive and/or technical issues with your ISH methodology. The table below shows a brief troubleshooting guide, which we hope will assistant you further. UK NEQAS ICC & ISH recommends that laboratories should continually monitor their performance to make sure that both technical and interpretive results are frequently audited. Interpretive Result Appropriate or Acceptable Appropriate or Acceptable Interpretive & Technical Troubleshooting Guide Technical Result (pre-pilot module) Acceptable The cell lines/tissue samples show a good standard of staining and have been interpreted correctly Appropriate or Acceptable Unacceptable Acceptable The cell lines/tissue samples show a good standard of staining BUT there appears to be issues with interpretation i.e. HER2 copy number and/or CEP17 overestimated/underestimated. Borderline Unacceptable Recommend that scoring/counting criteria is reviewed The cell lines/tissue samples are of borderline acceptability for staining quality. Recommend that technical method (kit/assay) is further optimised. Unacceptable Borderline The technical staining can be improved as this may be effecting interpretation. Recommend that technical method (kit/assay) is optimised (or where relevant a standardised kit is used as per instructions) as interpretation may be effected by the borderline quality of staining. The cell lines/tissue are unacceptable for interpretation and caution should be taken when interpreting from these slides. Recommend that technical method (kit/assay) is optimised (or where relevant a standardised kit is used as per instructions) as interpretation, although appears correct, may lead to incorrect interpretation of clinical cases Unacceptable Unacceptable The cell lines/tissue samples are unacceptable for technical staining and interpretation. Reporting from such cases is very likely to lead to incorrect interpretation of clinical cases. If there is persistent underperformance: - seek assistance from kit/assay manufacturer - seek assistance from UK NEQAS or colleagues - re-validate protocol (retrospectively and prospectively) - review scoring criteria - send clinical cases to a reference centre to confirm your results UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 76

79 BREAST HER2 ISH Module Selected Images showing Optimal and Sub-optimal Immunostaining º»¼ ¾ ÀÀÁÂÃÄÅÆÁ ÇÁÈÃÄÈÄ ÉÉÊËÌ»È ÃÍÁ ÎÏ ÐÑÒ Ë ÀÁÆÆ Æ»ÈÁÓÔ Õ Ö ãø ÄÚÂƻۻÁÜ ÕÓÄÚÂÆÁ Ö ä ÕÞÖ ½Ø Áåæ»ßÙÀÄÆ ÀÁÆÆ Æ»ÈÁÓ ÕËÄÚÂÆÁ ÞÖ¾ ÊÈ ÅÙÃÍ ÀÄÓÁÓ Ü»ÓûÈÀà ÌÑའӻ¼ÈÄÆÓ ÕÅÆÄÀáÖ ÄÈÜ ÝÍ â signals (red) are clearly demonstrated. º»¼ ½¾ ÀÀÁÂÃÄÅÆÁ ÇÁÈÃÄÈÄ ÉÉÊËÌ»È ÃÍÁ ÎÏ ÐÑÒ Ë ÀÁÆÆ Æ»ÈÁÓÔ Õ Ö Ø ÈÙÈ ÄÚÂƻۻÁÜ ÕÓÄÚÂÆÁ ÝÖ ÄÈÜ ÕÞÖ ÈÁ¼ÄûßÁ ÀÁÆÆ Æ»ÈÁ ÕÓÄÚÂÆÁ ÉÖ¾ ÊÈ ÅÙÃÍ ÀÄÓÁÓ Ü»ÓûÈÀà ÌÑའӻ¼ÈÄÆÓ ÕÅÆÄÀáÖ ÄÈÜ ÝÍ â signals (red) are clearly demonstrated with the expected level of copies per cell. º»¼ ã¾ ÀÀÁÂÃÄÅÆÁ ÇÁÈÃÄÈÄ ËÊËÌ ÌÑའÀÙÂç ÈÙ¾»È ÃÍÁ ÎÏ ÐÑÒ Ë ÀÁÆÆ Æ»ÈÁÓÔ Õ Ö ãø ÄÚÂƻۻÁÜ ÕÓÄÚÂÆÁ Öè ÕÞÖ ½Ø Áåæ»ßÙÀÄÆ ÕÓÄÚÂÆÁ ÞÖ ÄÈÜ ÕÝÖ Ø ÈÙÈ ÄÚÂƻۻÁÜ ÀÁÆÆ Æ»ÈÁ ÕËÄÚÂÆÁ ÝÖ¾ ÆÆ cases show strong distinct signals with the expected level of copies per cell. º»¼ é¾ ÞÙêÜÁêÆ»ÈÁ ÂÄÓÓ ÉÉÊËÌ ÓÃĻȻȼ»È ÃÍÁ ÎÏ ÐÑÒ Ë ÀÁÆÆ Æ»ÈÁÓÔ Õ Ö ãø ÄÚÂƻۻÁÜ ÕÓÄÚÂÆÁ Öè although the signals can be seen, the section has been over-developed and the interpretation»ó ÛæêÃÍÁê Í»ÈÜÁêÁÜ Åç ÈæÀÆÁÄê ÜæÓþ ÕÞÖ ½Ø Áåæ»ßÙÀÄÆ ÕÓÄÚÂÆÁ ÞÖ ÓÍÙëÓ ÁìÀÁÓÓ»ßÁ ÈÙÈíÓÂÁÀ»Û»À background and leaching of the probe. º»¼ î¾ ÑìÄÚÂÆÁÓ ÙÛ æèäààáâãäåæá ÓÃĻȻȼ»È ÃÍÁ ÎÏ ÐÑÒ Ë ½Ø Áåæ»ßÙÀÄÆ ÕÓÄÚÂÆÁ ÞÖ ÀÁÆÆ Æ»ÈÁÓÔ (A) Signals are difficult to read due to excessive red leaching and background staining (Dako Duo CISH method). (B) The HER2 copy no. is unreadable, and there is excessive silver deposit in the background (Invitrogen Spotlight method). Fig 6. Good example of an in house control stained with DDISH. The section consists of (A) amplified, (B) equivocal and (C&D) 2 non-amplified tissues. 77

80 BREAST HER2 ISH Module Selected Images showing Optimal and Sub-optimal Immunostaining Fig 7. Two examples of acceptable FISH in the UK NEQAS amplified 3+ amplified (sample A). (A) Stained with an in house method, showing strong HER2 (green) and Ch17 (red) signals. (B) Stained using the Vysis kit. In both cases distinct HER2 signals and Ch17 signals are clearly demonstrated. Fig 8. Acceptable FISH staining in the UK NEQAS cell lines: (A) 3+ amplified cell line (sample A) stained using the Dako PharmDx kit and (B) 2+ equivocal cell line (sample B) stained using the Vysis kit. Both show bright distinct HER2 and Ch17 signals present at the expected level. Fig 9. Acceptable FISH staining in the UK NEQAS cell lines: (A) 1+ non-amplified cell line (sample C) stained using the Vysis kit and (B) negative cell line (sample D) stained with the Kreatech probes. In both cases the HER2 and C17 signals show the expected level of copies per cell. Fig 1. Staining in the UK NEQAS 3+ amplified cell line (sample A). Although both the Ch17 (green) and HER2 (red) signals are bright and clearly demonstrated, there is excessive green signal. The slide also shows drying artefact around the cells. Stained with the Vysis kit Fig 11. Staining in the UK NEQAS distributed cell line. The HER2 and Ch17 signals are clearly demonstrated, but the cells show morphological damage. Slide stained with the Vysis kit. Fig 12. Unacceptable staining in the UK NEQAS 3+ amplified cell line. There is excessive background and fluorescence obscuring the nuclei, making interpretation impossible. Slide stained using the Vysis kit. 78

81 Run 29 Breast HER2 ISH Technical Module Chromogen ISH: Pass Rates and Methods Overall Pass Rates % % % 19% % 1 1 7% Acceptable (n=28) Borderline (n=14) Unacceptable (n=1) Acceptable (n=29) Borderline (n=1) Unacceptable (n=3) 1% 9% 8% 14 NEQAS Slides: Methods Used and Percentage Pass Rates % 6% 5% 4% Unacceptable Borderline Acceptable 3% 2% 1% % Dako DuoCISH (n=2) Ventana Dual ISH (BDISH) (n=7) Ventana Inform (n=3) Ventana Inform Dual ISH (DDISH) (n=38) Ventana Inform SISH (n=6) Zymed ï Invitrogenð spot-light (nñ1) 1% In-house Slides: Methods Used and Percentage Pass Rates 9% 8% % 6% 5% 4% 3% 2% 1% Unacceptable Borderline Acceptable % Dako DuoCISH (nñ2) Ventana Dual ISH (BDISH) (nñ7) Ventana Inform (nñ3) Ventana Inform Dual ISH (DDISH) (nñ38) Ventana Inform SISH (nñ6) Zymed ï Invitrogenð spot-light (nñ1) 79

82 Run 29 Breast HER2 ISH Technical Module Fluorscent ISH: Pass Rates and Methods FISH: Overall Pass Rates % 6 57% Acceptable (n=48) 13% Borderline (n=11) 29% Unacceptable (n=24) Acceptable (n=3) 19% Borderline (n=1) 25% Unacceptable (n=13) 1% NEQAS Slides: Methods Used and Percentage Pass Rates 9% 8% 7% 6% 5% 4% 3% Unacceptable Borderline Acceptable 2% 1% % 14 China Medical Technologies Kit (n=7) Dako Pharm Dx (n=9) In house FISH (n=4) Kreatech Probes (n=3) Leica HER2 FISH TA9217 (n=5) 33 Other - FISH (n=3) 42 Pathvysion Vysis Kit (n=64) 33 Zytovision ZytoLight (nò3) 1% 9% 8% 14 In-house Slides: Methods Used and Percentage Pass Rates 25 7% 6% 5% 4% 3% 2% 1% % 86 China Medical Technologies Kit (nò7) Dako Pharm Dx (nò9) 25 5 In house FISH (nò4) Kreatech Probes (nò3) 8 2 Leica HER2 FISH TA9217 (nò5) 1 Other - FISH (nò3) Pathvysion Vysis Kit (nò64) 33 Zytovision ZytoLight (nò3) Unacceptable Borderline Acceptable 8

83 UK NEQAS ICC & ISH Improving Immunocytochemistry for over 25 Years For advertising opportunities contact: UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent