Immunocytochemistry. Results - Summary Graphs - Pass Rates Best Methods - Selected Images. Run 97. Assessment Dates: 26th March - 13th April 2012

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1 Short Report Breast ER EQA Slide Storage Study Run 97 IN THIS ISSUE Page 4-5 Immunocytochemistry Results - Summary Graphs - Pass Rates Best Methods - Selected Images Immunocytochemistry Modules Assessment Dates: 26th March - 13th April 212 General Pathology: SMA & Endothelial Breast pathology: ER A B Breast pathology: HER2 IHC 23-3 Lymphoid pathology: BCL2 & Ki-67 (MIB1) Neuropathology: Ki-67 (MIB1) & NFP Cytology: Melanoma & Cytokeratin Alimentary Tract: HNPCC: MSH2 & MSH C D In-situ Hybridisation Modules Breast: HER2 ISH Interpretive Breast HER2 ISH Technical Sponsors in this Issue Cover Photo: Taken from the ISH technical pilot assessment module (A,B) FISH staining in the UK NEQAS cell lines (A) amplified (Cell line A; IHC 3+) and (B) non-amplified (Cell line C; IHC 1+), showing an acceptable level of staining. (C,D) DDISH staining in the UK NEQAS (C) cell line and (D) in-house control tissue, showing an acceptable level of staining. 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 suboptimal staining methodologies. 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 3/2 Hamilton House Mabledon Place London WC1H 9BB, UK Tel: (+44) merdol.ibrahim@ucl.ac.uk For enquiries concerning training issues, meetings, or courses, please contact: Mr Keith Miller, Scheme Director UK NEQAS ICC & ISH UCL-AD, 21 University Street, University College London London WC1E 6JJ, UK Tel: (+44) 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, Southampton 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 Dr D Faratian, Edinburgh 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 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 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 Prof. 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

3 Meetings & Announcements Molecular Pathology: Focussing on Patients October 12th 14th 212 A joint meeting provided by UK NEQAS for ICC & ISH and UK NEQAS for Molecular Genetics at The Beaumont Estate Hotel & Conference Centre Old Windsor, UK We are delighted to announce for the first time a joint meeting between two UK NEQAS organisations that cover a wide range of tissue diagnostic technologies, slide and non-slide based. Already some exciting speakers have agreed to present cutting-edge aspects of their work. These include both national and international experts. For further details please contact: Beverley Meeting Voice 25 Anchor Road Aldridge West Midlands WS9 8PT t: +44 () f: +44 () e: beverley@meetingvoice.co.uk * * More information will follow soon * * Advertising Opportunities We are now offering advertising opportunities in the Immunocytochemistry e-journal. For further information and pricing please contact Suzanne: s.parry@ucl.ac.uk Neil: n.bilbe@ucl.ac.uk or Tel: +44 () UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 1

4 An interactive web based resource Breast and Gastric HER2 Testing Resource What is HER2? Library Section Interactive HER2 Scoring Training Centre IHC and ISH Testing Methods Meet the Expert Lectures Featuring Prof. Dowsett (UK), Prof. Viale (Italy), Prof. Rüschoff (Germany) and other international opinion leaders Prescribing information appears on next page 2 RXUKHERC362b May 212

5 PRESCRIBING INFORMATION HERCEPTIN (trastuzumab) 15 mg powder for concentrate for solution for infusion Indications: Treatment of HER2 positive early breast cancer (EBC): (i) following surgery, chemotherapy (CT) (neo/adjuvant) and radiotherapy (RT) (if applicable). (ii) following adjuvant CT with doxorubicin and cyclophosphamide, in combination with paclitaxel or docetaxel. (iii) in combination with adjuvant CT consisting of docetaxel and carboplatin. (iv) for locally advanced (including inflammatory) disease or tumours > 2cm in diameter, in combination with neoadjuvant CT followed by adjuvant Herceptin. Treatment of HER2 positive metastatic breast cancer (MBC): (i) as monotherapy following at least 2 CT regimens for MBC. Prior CT to include at least an anthracycline and a taxane, unless unsuitable. Hormone receptor positive patients must have failed hormonal therapy, unless unsuitable. (ii) in combination with paclitaxel for patients who have not received CT for MBC and where anthracyclines are not suitable. (iii) in combination with docetaxel for patients who have not received CT for MBC (iv) in combination with an aromatase inhibitor for the treatment of postmenopausal patients with hormone-receptor positive MBC, not previously treated with Herceptin. Treatment of patients with HER2 positive metastatic adenocarcinoma of the stomach or gastro-oesophageal junction who have not received prior anti-cancer treatment for their metastatic disease. Dosage and Administration: Please refer to Herceptin Summary of Product Characteristics (SmPC) for full guidance. HER2 testing mandatory prior to Herceptin. In patients with EBC and MBC, tumours should have HER2 overexpression at 3+ level by immunohistochemistry (IHC) or HER2 gene amplification by fluorescence or chromogenic in situ hybridisation (FISH or CISH). Herceptin should only be used for in patients with metastatic gastric cancer (MGC) whose tumours have HER2 overexpression at 3+ level by immunohistochemistry (IHC) or IHC 2+ with confirmatory fluorescent in situ hybridisation (FISH) or silver-enhanced in situ hybridisation (SISH) positive results. Only accurate and validated assays should be used. Physicians experienced with cytotoxic CT should initiate treatment with Herceptin. Dose (EBC): (i) loading dose 8 mg/kg body weight; subsequent doses 6 mg/kg repeated at 3-weekly intervals; alternatively (ii) loading dose 4 mg/kg body weight; subsequent doses weekly 2 mg/kg concomitantly with paclitaxel following chemotherapy with doxorubicin and cyclophosphamide. Dose (MBC): (i) loading dose 8 mg/kg body weight; subsequent doses 6 mg/kg repeated at 3-weekly intervals; alternatively (ii) loading dose 4 mg/kg body weight; subsequent doses weekly 2 mg/kg. Dose (MGC): loading dose 8 mg/kg body weight; subsequent doses 6 mg/kg repeated at 3-weekly intervals. Patients with EBC should be treated for 1 year or until disease recurrence, whatever occurs first. In MBC and MGC, administer until disease progression. Initial loading dose should be administered as 9 minute IV infusion; if loading dose well tolerated subsequent doses can be administered as 3 minute IV infusion. Do not administer as an IV push or bolus. Observe for infusion-related symptoms for at least six hours following start of first infusion and for two hours for subsequent infusions. Interruption of infusion may help control symptoms; consider resuming when symptoms abate. Resuscitation equipment must be available. Contraindications: Hypersensitivity to trastuzumab, murine proteins or any excipients. Severe dyspnoea at rest due to complications of advanced malignancy or requiring oxygen therapy. Precautions: Please refer to the Herceptin SmPC for further information. HER2 testing must be performed in a specialised laboratory to ensure adequate validation of test. Congestive heart failure (CHF) observed in breast cancer patients receiving monotherapy or in combination with paclitaxel or docetaxel; particularly following anthracycline-containing regimen may be moderate to severe and has been fatal. Avoid concomitant use of anthracyclines in the adjuvant and metastatic settings and only use neoadjuvantly in CT-naïve patients in conjunction with low-dose anthracycline regimens. Neoadjuvant use, with concurrent anthracyclines, is not recommended for patients >65 years. Avoid anthracycline based therapy for up to 25 weeks after stopping Herceptin. Caution in patients with symptomatic CHF, history of hypertension or coronary artery disease and in EBC, in those patients with an LVEF of 55 % or less. Monitor cardiac function at baseline and during treatment e.g. every three months and as required in MBC. In addition, every 6 months for up to 24 months following discontinuation of treatment in EBC. Further monitoring recommended for patients who receive anthracycline containing CT; yearly up to 5 years from last administration, or longer if a continuous decrease of LVEF observed. Consider discontinuing treatment in patients with asymptomatic LVEF decreases or patients who develop clinically significant heart failure unless benefits outweigh risks. Most who developed CHF in clinical trials improved with appropriate treatment and continued Herceptin therapy without additional clinical cardiac events. Serious infusion related reactions (IRR) reported infrequently (see side effects and adverse reactions), majority within 2.5 hours of start of first infusion and very rarely more than 6 hours after the first infusion. Should IRR occur, discontinue or slow the rate of infusion and monitor patient until resolution. Majority of patients experienced resolution and subsequently received further infusions. Serious IRRs have been successfully treated with oxygen, beta-agonists and corticosteroids. Fatal outcome are rare and have occurred within hours and up to one week following the infusion. Severe pulmonary events reported rarely; occasionally fatal; may occur as part of IRR or with delayed onset; patients with dyspnoea at rest may be at increased risk of fatal IRR and/ or pulmonary events; these patients should not be treated with Herceptin. Caution should be exercised for pneumonitis, especially in patients being treated concomitantly with taxanes. Drug Interactions: Drug interaction studies not performed in humans. Pregnancy and Lactation: Avoid during pregnancy unless potential benefit outweighs risk. Oligohydramnios reported post-marketing in pregnant women, some associated with fatal pulmonary hypoplasia of the foetus. Women of childbearing potential should be advised to use effective contraception during Herceptin and for at least six months after last dose. Do not breast-feed during Herceptin and for six months after last dose. Side-effects and Adverse Reactions: Cardiotoxicity, IRR, haematotoxicity (neutropenia) and pulmonary adverse events are amongst the most serious and or common adverse reactions reported in association with the use of Herceptin alone or in combination with CT in pivotal clinical trials for breast and gastric cancer as well as in the post-marketing setting. For full listings please refer to the Herceptin SmPC. *Denotes adverse reactions that are reported largely in association with IRRs. +Denotes adverse reactions observed in combination therapy following anthracyclines and combined with taxanes. Very common reactions: febrile neutropenia, tremor*, dizziness, headache, change in blood pressure*, irregular heart beat*, palpitation*, cardiac flutter*, ejection fraction decreased+, wheezing*, dyspnoea (14%), diarrhoea, vomiting, nausea, lip swelling*, abdominal pain, erythema, rash, swelling face*, arthralgia, muscle tightness*, myalgia, asthenia, chest pain, chills, fatigue, influenza-like symptoms, infusion related reactions (majority of infusion-related reactions are mild to moderate in intensity and tend to occur earlier in treatment; reactions include, but are not limited to, chills, fever, rash, nausea and vomiting, dyspnoea and headache), pain, pyrexia, conjunctivitis, increased lacrimation, hot flush, cough, epistaxis, rhinorrhoea. Common reactions: Pneumonia (<1%), neutropenic sepsis, cystitis, herpes zoster, infection, influenza, nasopharyngitis, sinusitis, skin infection, rhinitis, URTI, UTI, erysipelas, cellulitis, anaemia, neutropenia, thrombocytopenia, leukopenia, hypersensitivity, weight change, anorexia, anxiety, depression, insomnia, abnormal thinking, peripheral neuropathy, paraesthesia, hypertonia, somnolence, dysgeusia, ataxia, dry eye, congestive cardiac failure (2%), supraventricular tachyarrhythmia*, cardiomyopathy, hypotension*, vasodilatation, asthma, lung disorder, pharyngitis, pancreatitis, dyspepsia, haemorrhoids, constipation, dry mouth, hepatitis, hepatocellular injury, liver tenderness, acne, alopecia, dry skin, ecchymosis, hyperhydrosis, maculopapular rash, nail disorder, pruritus, arthritis, back pain, bone pain, muscle spasms, neck pain, renal disorder, breast inflammation, peripheral oedema, malaise, mucosal inflammation, oedema, contusion. Legal Category: POM Presentation and Basic NHS Cost: Pack of one 15 mg single dose vial (reconstituted solution contains 21 mg/ml trastuzumab): 47.4 excluding VAT Marketing Authorisation Number: EU/1//145/1 Marketing Authorisation Holder: Roche Registration Limited, 6 Falcon Way, Shire Park, Welwyn Garden City, AL7 1TW, United Kingdom Herceptin is a registered trade mark RXUKMEDI99 Date of Preparation: April 212 Adverse events should be reported. Reporting forms and information can be found at Adverse events should also be reported to Roche Products Ltd. Please contact Roche UK Drug Safety Centre on: RXUKHERC362b May 212

6 Short Report: Breast ER EQA Slide Storage Study Suzanne Parry, Clara Lynch & Merdol Ibrahim Study Overview & Objectives It is recognised that the storage of unstained slides of paraffin sections can have a detectable negative effect on the intensity of immunohistochemistry staining for various antigens, and may even lead to false negative immunostaining of certain tumour markers. As UK NEQAS ICC & ISH distributes 1 s of slides every quarter, it was decided to carry out a short study on loss of antigenecity on slide samples which are distributed for external quality assessments. It is important that the sections sent out by UK NEQAS ICC & ISH are properly quality controlled so participants can reliably achieve optimal immunohistochemical staining. Oestrogen receptor (ER) is particularly susceptible to loss of antigenicity during storage; therefore, UK NEQAS ICC & ISH decided to perform a time-course study to evaluate the effect of slide storage on the intensity of ER immunohistochemical staining under varying conditions. Methods Paraffin-embedded breast tissue slides consisting of known negative, mid- and high-expressing tumours were stained with ER, 6F11 antibody clone after storage at 1, 2, 4, 8 and 12 weeks. At each time interval, batches of slides were stored in the following conditions: 1. Room temperature in plastic transport containers C in transport container. 3. Room temperature in vacuumed transport container C in vacuumed transport container. 5. Room temperature in transport container within an air tight vacuum bag C in transport container within sealed vacuum bag. The progressive decline in the ER intensity with time was seen with all slides, and from 8 weeks onwards false negativity became very apparent in all slides stored at room temperature. Although ER expression was detected, sections stored at 4 C showed very weak sub-optimal staining at 8 weeks; at 12 weeks sections stored at 4 C also began to show false negative results. Additionally, those sections stored in a vacuum within a transport box and in a sealed bag only, showed excessive non -specific background staining after a storage interval of 8 weeks onwards. Conclusion Slide storage particularly at room temperature results in substantial loss of ER reactivity, with some ER-positive cases becoming ER negative after 8 weeks of storage. Even at 4 C, storage of unstained slides for up to 12 weeks may lead to false-negative immunostaining for ER. Vacuum-packed slides show no benefit to staining intensity, and when stored for over 8 weeks, can actually produce nonspecific staining. Improved Slide Storage Procedures UK NEQAS ICC & ISH now routinely stores all cut unstained sections for all modules at 4 C prior to despatching to participants. All ER Gold standard sections are now routinely tested at the time of distribution, as well as the deadline date for receipt of slides from participants. We have found no difference in the immunostaining intensity between these two time intervals (approximately 4 weeks) so far. Additional sections were stained with ER after storage at 3 and 12 weeks under the following conditions: 7. Room temperature in sealed vacuum bag, without a transporter container C in sealed vacuum bag (only). The staining intensity of all slides were assessed by visual microscopic examination by carrying out a comparison with stained slides carried out on day 1 after the sections were cut. Results/Findings There was no significant difference in staining intensity seen after 1 week with all the storage conditions. After 2 weeks, the intensity of staining appeared slightly weaker in all sections stored at room temperature, but there was no difference in intensity between the sections stored with or without a vacuum at either room temperature or at 4 C. At the 4 week time interval, there was a marked decrease in intensity with all slides, but particularly more so with sections stored at room temperature, and most notably in the midexpressing tumour. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 4

7 Short Report: Breast ER EQA Slide Storage Study Selected Images Showing Slide Storage Results Fig 1. Changes in ER expression in mid-expressing ER tumour sections stored at room temperature 1 Week 2 Weeks 4 Weeks 8 Weeks In the mid-expressing tumour sections (Allred=4-6), slides stored at room temperature for (A) one week showed a minimal decrease in ER expression. However after the 2nd week (B) there was a drop in the number and intensity of ER stained cells. By the 4th week there was a dramatic drop in the number of cells staining for ER, with approximately only 1% of tumour cells showing ER expression. By the 8th week at room temperature, no tumour cells were stained. Fig 2. Changes in ER expression in high-expressing ER tumour sections stored at room temperature 1 Week 2 Weeks 4 Weeks 8 Weeks In the high-expressing tumour sections (Allred=8), slides stored at room temperature did not show a great change in expression when compared to the mid-expressing tumour (see fig 1). (A,B) From Week 1 to 2 the high-expressing tumour showed a decrease in ER intensity but the number of ER expressing tumour cells remained the same. (C,D) from week4 to 8, there was not only a decrease in ER expression intensity but there were also fewer cells staining, with the overall Allred score dropping from 8 to 5. Fig 3. Changes in ER expression in high- and mid-expressing tumour sections stored at 4 O C 2 Weeks 8 Weeks 2 Weeks 8 Weeks Storage of slides at 4 C helped to decrease the loss of ER antigenicity over time. (A) The high expressing tumour after 2 weeks still showed strong ER expression (compare to fig 2B), but expression levels still decreased after 8 weeks at 4 C (compare to fig 2D). (C,D) The mid expressing tumour sections continued to show the original expected ER levels (Allred 5-6) after 2 weeks storage at 4 C (compare to fig 1B), but (D) the expression level continued to decrease until after 8 weeks the ER levels were very low, although still >1 % positive. Fig 4. Changes in ER expression in high- and mid-expressing tumour sections stored at 4 o C in a vacuum bag 8 Weeks: RT + Vacuum 8 Weeks: 4 o C+ Vacuum Storage of slides in a vacuum bag at (A) room temperature and (B) 4 o C. Both showed the preservation of nuclear ER expression, but there was also a significant increase in cytoplasmic and non-specific staining, making some of the samples uninterpretable. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 5

8 The General Pathology Module Run 97 Julie Williams and Andrew Dodson Gold Standard Second Antibody Antigens Assessed: SMA Endothelial Markers Tissue Sections circulated: Appendix & Leiomyosarcoma Appendix Number of Registered Participants: 365 Introduction Smooth Muscle Actin (SMA) The alpha-isoform of smooth muscle actin ( SMA) belongs to a group of cytoplasmic actins, of which there are six major different isoforms. It is useful for the demonstration of myogenic differentiation. Antibodies to SMA label smooth muscle cells, which are found in vascular walls, intestinal muscularis, and muscularis propria and in the stroma of various tissues. It also reacts with myoepithelial cells in tissues such as breast and salivary glands. The main diagnostic use of SMA is in a panel of antibodies to demonstrate leiomyomas and leiomyosarcomas. It is also used to identify the loss of myoepithelial cells around the ducts in invasive breast carcinomas. Features of optimal immunostaining In appendix there should be: Intense staining of the smooth muscle layers in the muscularis propria Staining of the delicate fibres extending into the epithelial crypts of the appendix The smooth muscle of the numerous blood vessels permeating the lamina propria should also be intensely stained No background staining Features of sub-optimal immunostaining Strong background staining, particularly of connective tissue Non-specific staining of other cell types not expected to stain, e.g. lymphocytes and epithelial cells. This is often due to excessive pretreatment conditions Weak or patchy staining of the smooth muscle layers Endothelial markers 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 megakarocytes, platelets and other haematopoetic 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 haematopoetic 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. Von Willebrand Factor (VWF): also known as Factor VIII- Related Antigen is a large polymeric protein which is synthesized exclusively by endothelial cells (where it is found within the Weibel-Palade bodies), and megakaryocytes. The function of VWF is to act as a carrier for Factor VIII in plasma, protecting the circulating coagulation coenzyme from proteolytic degradation. It is found in endothelial cells of capillaries, lymphatic vessels, arteries, and veins, also in the endothelial cells in glomeruli and the sinusoids of liver and spleen where it displays a diffuse or slightly granular pattern in the cytoplasm. It is not a very sensitive marker for vascular tumours as it is seldom expressed in poorly differentiated vascular tumours (Leong); its main diagnostic use being in the identification of benign and borderline endothelial tumours, such as haemangioma variants and haemangioendotheliomas (Cerilli et al). 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 pretreatment. 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. VWF Strong staining of the endothelial cells in the blood vessels and lymphatic vessels Some background staining of the connective tissue is to be expected 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 over-pretreatment. Inappropriate staining of, for example, epithelium and lymphocytes, probably due to over-pretreatment VWF Weak or negative staining of the endothelial cells. Excessive background staining. References 1. DeYoung BR, Wick MR, Fitzgibbon JF et al. CD31: an immunospecific marker 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. UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 6

9 ΡΥΝ 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε Σελεχτεδ Ιµαγεσ σηοωινγ Οπτιµαλ ανδ Συβ οπτιµαλ Ιµµυνοσταινινγ Φιγ 1. Οπτιµαλ δεµονστρατιον οφ ΣΜΑ ιν τηε ΥΚ ΝΕΘΑΣ λειοµψοσαρχοµα τυµουρ σεχτιον, σηοωινγ ιντενσε ωελλ λοχαλισεδ χψτοπλασµιχ σταινινγ. Σεχτιον σταινεδ ωιτη ακο 1Α4 αντιβοδψ, 1:2, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ΧΧ1 στανδαρδ ανδ Υλτραϖιεω δετεχτιον κιτ. Φιγ 2. Οπτιµαλ δεµονστρατιον οφ ΣΜΑ ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον, σηοωινγ στρονγ σταινινγ οφ τηε σµοοτη µυσχλε λαψερσ ιν τηε µυσχυλαρισ προπρια ανδ µυσχυαλρισ µυχοσαε. Τηε δελιχατε φιβρεσ αρε νιχελψ δεµονστρατεδ ανδ τηε βαχκγρουνδ ισ χλεαν. Σεχτιον σταινεδ ωιτη ακο 1Α4 αντιβοδψ, 1:2, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ΧΧ1 στανδαρδ ανδ Υλτραϖιεω δετεχτιον κιτ. Φιγ 3. Συβ οπτιµαλ δεµονστρατιον οφ ΣΜΑ ιν τηε ΥΚ ΝΕΘΑΣ λειοµψοσαρχοµα τυµουρ. (Χοµπαρε το Φιγ1). Τηε σταινινγ ισ ωεακ ανδ πατχηψ τηρουγηουτ τηε σεχτιον. Σταινεδ ωιτη ακο 1Α4 αντιβοδψ, 1:5, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ωιτη νο πρε τρεατµεντ ανδ τηε ιςιεω δετεχτιον κιτ. Φιγ 4. Συβ οπτιµαλ ωεακ δεµονστρατιον οφ ΣΜΑ ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον (Χοµπαρε το Φιγ2). Σεχτιον σταινεδ ωιτη ςεντανα πρε διλυτεδ αντιβοδψ, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ΧΧ1 µιλδ ανδ ιςιεω δετεχτιον κιτ. Φιγ 5. Ποορ δεµονστρατιον οφ ΣΜΑ ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον, σηοωινγ νον σπεχιφιχ σταινινγ οφ λψµπηοχψτεσ ανδ επιτηελιαλ χελλσ. Σεχτιον σταινεδ ακο 1Α4 αντιβοδψ, 1:6, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ΧΧ1 στανδαρδ ανδ Υλτραϖιεω δετεχτιον κιτ. Φιγ 6. Γοοδ εξαµπλε οφ αν ιν ηουσε λειοµψολιποµα χοντρολ σταινεδ ωιτη ΣΜΑ, σηοωινγ στρονγ σπεχιφιχ σταινινγ ωιτη α χλεαν βαχκγρουνδ. Σεχτιον σταινεδ ωιτη ακο 1Α4 αντιβοδψ, 1:1, ον τηε ςεντανα Βενχηµαρκ ΞΤ, ΧΧ1 µιλδ ανδ ιςιεω δετεχτιον κιτ. Πριντεδ ατ 9:35 ον Μονδαψ, 23 Απριλ, 212 7

10 ΡΥΝ 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε Σελεχτεδ Ιµαγεσ σηοωινγ Οπτιµαλ ανδ Συβ οπτιµαλ Ιµµυνοσταινινγ Φιγ 7. Οπτιµαλ δεµονστρατιον οφ Χ 34 ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον, σηοωινγ γοοδ στρονγ σταινινγ οφ ϖεσσελσ ανδ ενδοτηελιαλ χελλσ. Σεχτιον σταινεδ ωιτη τηε ακο ΘΒΕνδ αντιβοδψ, 1:1, ακο ΠΤ Λινκ, αυτοσταινερ ανδ ΦΛΕΞ κιτ. Φιγ 8. Γοοδ δεµονστρατιον οφ Χ 31 ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον. Χ 31 νοτ ονλψ σταινσ ενδοτηελιαλ χελλσ ανδ ϖεσσελσ, βυτ αλσο µαντλε ζονε Β χελλσ ανδ Τ χελλσ ασ σεεν ιν τηισ ιµαγε. Σεχτιον σταινεδ ωιτη τηε ακο (ϑχ/7α) αντιβοδψ, 1:3, ον τηε ςεντανα Βενχηµαρκ Υλτρα, ΧΧ1 στανδαρδ ανδ Υλτραϖιεω δετεχτιον κιτ. Φιγ 9. Συβ οπτιµαλ δεµονστρατιον οφ Χ 34 ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον. Αλτηουγη τηε ενδοτηελιαλ χελλσ ανδ ϖεσσελσ αρε δεµονστρατεδ, τηε σταινινγ ισ ωεακ (Χοµπαρε το Φιγ 7). Σεχτιον σταινεδ ωιτη τηε ακο ΘΒΕνδ αντιβοδψ, 1:2, ακο ΠΤ Λινκ (ηιγη πη βυφφερ), αυτοσταινερ ανδ ΦΛΕΞ κιτ. Φιγ 1. Οπτιµαλ δεµονστρατιον οφ Φαχτορ ςιιι ιν τηε ΥΚ ΝΕΘΑΣ αππενδιξ σεχτιον. Νοτεσ τηατ Φαχτορ ςιιι δοεσ νοτ σταιν αλλ χελλ τψπεσ ασ δεµονστρατεδ ωιτη Χ 31 ανδ Χ 34. Σεχτιον σταινεδ ωιτη τηε ακο πολψχλοναλ αντιβοδψ, 1:4, ωιτη νο πρε τρεατµεντ ον τηε ςεντανα Βενχηµαρκ Υλτρα ανδ Υλτραϖιεω δετεχτιον κιτ. Φιγ 11. Γοοδ εξαµπλε οφ αν ιν ηουσε κιδνεψ χοντρολ σταινεδ ωιτη Χ 34, σηοωινγ σπεχιφιχ ενδοτηελιαλ ανδ στρονγ ποσιτιϖιτε χψτοπλασµιχ σταινινγ ιν τηε χελλσ οφ τηε γλοµερυλι. Σεχτιον σταινεδ ωιτη ακο ΘΒΕνδ αντιβοδψ, 1:2, ον τηε Λειχα ΒονδΜαξ, ΕΡ2 ανδ Ρεφινε δετεχτιον κιτ. Πριντεδ ατ 9:35 ον Μονδαψ, 23 Απριλ, 212 8

11 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε 12 ΡΥΝ 97Α Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) ον ΝΕΘΑΣ Σεχτιονσ Ινδιϖιδυαλ 4 = (%) 14 ΡΥΝ 97Β Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) ον ιν ηουσε Σεχτιονσ Ινδιϖιδυαλ 4 = (%) 1 5 = (%) 6 = (%) 7 = (%) 12 5 = (%) 6 = (%) 7 = (%) νο. οφ ρετυρνσ = 7 (2%) 9 = 5 (1%) 1 = 6 (2%) 11 = 8 (2%) 12 = 32 (9%) 13 = 3 (9%) 14 = 32 (9%) 15 = 63 (18%) νο. οφ ρετυρνσ = 4 (1%) 9 = 2 (1%) 1 = 5 (1%) 11 = 3 (1%) 12 = 22 (6%) 13 = 14 (4%) 14 = 35 (1%) 15 = 57 (16%) 2 16 = 18 (31%) 17 = 39 (11%) 2 16 = 134 (39%) 17 = 43 (12%) 18 = 14 (4%) 18 = 2 (6%) = 6 (2%) 2 = 2 (1%) = 6 (2%) 2 = 3 (1%) Συµµαρψ Συµµαρψ 16 2 = 169 (48%) 16 2 = 26 (59%) = 125 (36%) = 16 (3%) 1 12 = 46 (13%) 1 12 = 3 (9%) 9 = 12 (3%) 9 = 6 (2%) Μεδιαν = 14. Μεδιαν = ΡΥΝ 97Χ Ενδοτηελιαλ Μαρκερ ον ΝΕΘΑΣ Σεχτιονσ Ινδιϖιδυαλ 4 = 1 (%) 16 ΡΥΝ 97 Ενδοτηελιαλ Μαρκερ ον ιν ηουσε Σεχτιονσ Ινδιϖιδυαλ 4 = (%) 5 = (%) 6 = (%) 14 5 = (%) 6 = (%) 8 7 = (%) 8 = 6 (2%) 12 7 = (%) 8 = 6 (2%) νο. οφ ρετυρνσ = 6 (2%) 1 = 6 (2%) 11 = 13 (4%) 12 = 26 (7%) 13 = 3 (9%) 14 = 33 (9%) νο. οφ ρετυρνσ = 4 (1%) 1 = 1 (%) 11 = 3 (1%) 12 = 24 (7%) 13 = 32 (9%) 14 = 39 (11%) 2 15 = 52 (15%) 16 = 99 (28%) 17 = 5 (14%) 18 = 18 (5%) = 41 (12%) 16 = 144 (41%) 17 = 33 (9%) 18 = 11 (3%) = 1 (3%) 2 = 2 (1%) = 6 (2%) 2 = 4 (1%) Συµµαρψ Συµµαρψ 16 2 = 179 (51%) 16 2 = 198 (57%) = 115 (33%) = 112 (32%) 1 12 = 45 (13%) 1 12 = 28 (8%) 9 = 13 (4%) 9 = 1 (3%) Μεδιαν = 13.5 Μεδιαν = 14. Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ, 212 9

12 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε ΑΝΤΙΒΟ ΙΕΣ ΑΝ ΟΤΗΕΡ ΤΕΧΗΝΙΧΑΛ ΠΑΡΑΜΕΤΕΡΣ ΕΜΠΛΟΨΕ ΒΨ ΠΑΡΤΙΧΙΠΑΝΤΣ ΙΝ ΤΗΕ ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ ΜΟ ΥΛΕ Τηε φολλοωινγ ταβλεσ ρεχορδ τηε νυµβερ οφ παρτιχιπαντσ (Ν) υσινγ εαχη πριµαρψ αντιβοδψ. Τηε περχενταγε (%) ρεφερσ το τηε προπορτιον οφ τηεσε παρτιχιπαντσ αχηιεϖινγ αχχεπταβλε σταινινγ (α σχορε >12/2) ον ΥΚ ΝΕΘΑΣ σεχτιονσ. Γενεραλ Πατηολογψ Ρυν: 97 Γενεραλ Πατηολογψ Ρυν: 97 Πριµαρψ Αντιβοδψ : Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Αντιβοδψ εταιλσ Ν % Α. Μεναρινι ΜΠ 1Χ/Πξ (1Α4) 1 Α. Μεναρινι ΜΥ 128 ΥΧ (ΙΑ4) 2 5 Βιογενεξ ΑΜ128 5Μ/1Μ (1Α4) 2 5 Βιογενεξ ΜΥ128 ΥΧ (ΡΤΥ) (1Α4) 2 1 Χελλ Μαρθυε 21Μ (Χλονε ΗΗΦ35) 1 1 Χελλ Μαρθυε 22Μ (Χλονε 1Α4) 7 43 ακο Μ635 ΣΜΑ (Χλονε ΗΗΦ35) 1 1 ακο Μ851 ΣΜΑ (Χλονε 1Α4) ακο ΡΤΥ (Χλονε 1Ρ611) 8 1 Ινϖιτρογεν (1Α4) 1 1 Λειχα/Νοϖοχαστρα ΠΑ943 (ασµ 1) 8 88 Νοϖοχαστρα ΝΧΛ ΣΜΑ (ασµ 1) Νοϖοχαστρα ΡΤΥ ΣΜΑ (ασµ 1) 6 83 Οτηερ 6 83 Σιγµα (1Α4) Α Σιγµα ΙΜΜΗ2 1ΚΤ (1Α4) 1 1 Τηερµο Σχιεντιφιχ/Νεοµαρκερσ ΜΣ 113 Π (1Α4) 2 1 Τηερµο Σχιεντιφιχ/Νεοµαρκερσ ΜΣ 742 Σ (ΗΗΦ35) 1 1 ςεχτορ ςπ Σ281 ΣΜΑ (ασµ 1) 1 1 ςεντανα (1Α4) ςεντανα (ΗΗΦ35) 4 75 Πριµαρψ Αντιβοδψ : Ενδοτηελιαλ Μαρκερ Αντιβοδψ εταιλσ Ν % ΒιοΓενεξ ΑΜ236 5Μ ( ΘΒενδ1) Χ Χελλ Μαρθυε Χ Μ/ (ϑχ7) 2 1 Χελλ Μαρθυε Χ Μ/76 262/ΧΜΧ33 (ΘΒενδ/1) 2 1 ακο Α82 (πολψχλοναλ) ςωφ 3 1 ακο Χ 31 ΡΤΥ ΦΛΕΞ ΙΡ61 (ϑχ7α) 1 1 ακο Χ 34 ΡΤΥ ΦΛΕΞ ΙΡ632 (ΘΒενδ/1) 3 1 ακο Μ823( ϑχ/7α) Χ ακο Μ7165 ( ΘΒενδ1) Χ ακο ΡΤΥ Χ 34 Ν1632 (ΘΒενδ/1) 1 1 Ι Λαβσ ΒΠ64 (ϑχ/7α) Χ 31 1 Ιµµυνοτεχη 1185 ( ΘΒενδ1) Χ Λαβςισιον ΜΣ363Π ( ΘΒενδ1) Χ Λειχα/Νοϖοχαστρα Χ 31 ΝΧΛ Χ 31 1Α1 (1Α1) 7 86 Λειχα/Νοϖοχαστρα Χ 31 ΡΤΥ ΠΑ25 (1Α1) 6 1 Λειχα/Νοϖοχαστρα Χ 34 ΝΧΛ ΕΝ ( ΘΒενδ1) 72 9 Λειχα/Νοϖοχαστρα Χ 34 ΡΤΥ ΠΑ212 ( ΘΒενδ1) 3 1 Λειχα/Νοϖοχαστρα Φαχτορ ςιιι ΡΤΥ ΠΑ4 (36Β11) 2 1 Οτηερ 6 5 Σεροτεχ ΜΧΑ 547 ( ΘΒενδ1) Χ ΣΚΨΒΙΟ ( ΘΒενδ1) Χ ςεχτορ ςπ Χ345 ( ΘΒενδ1) Χ ςεντανα Χ ( ϑχ7α) 4 25 ςεντανα Χ (1Α1) 2 5 ςεντανα Χ ( ϑχ7) 3 67 ςεντανα Χ (ΘΒενδ/1) 25 8 Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ, 212 1

13 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε Γενεραλ Πατηολογψ Ρυν: 97 Γενεραλ Πατηολογψ Ρυν: 97 Ηεατ Μεδιατεδ Ρετριεϖαλ Ενδοτηελιαλ Μαρκερ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Ν % Ν % Βιοχαρε εχλοακινγ Χηαµβερ ακο Πασχαλ ακο ΠΤΛινκ Λαβ ϖισιον ΠΤ Μοδυλε Λειχα Βονδ ΙΙΙ ΕΡ Λειχα Βονδ ΙΙΙ ΕΡ Λειχα ΒονδΜαξ ΕΡ Λειχα ΒονδΜαξ ΕΡ Μιχροωαϖε Οϖεν ΝΟΤ ΑΠΠΛΙΧΑΒΛΕ Οτηερ Πρεσσυρε Χοοκερ Στεαµερ ςεντανα Βενκ ΧΧ1 (Εξτενδεδ) ςεντανα Βενκ ΧΧ1 (Μιλδ) ςεντανα Βενκ ΧΧ1 (Στανδαρδ) ςεντανα Βενκ ΧΧ1# (8µινσ) ςεντανα Βενκ ΥΛΤΡΑ ΧΧ1 (Εξτεν.) 2 1 ςεντανα Βενκ ΥΛΤΡΑ ΧΧ1 (Μιλδ) ςεντανα Βενκ ΥΛΤΡΑ ΧΧ1 (Σταν.) ςεντανα Βενκ ΥΛΤΡΑ ΧΧ1# (8µινσ) ςεντανα Βενκ ΞΤ ΧΧ1 (Εξτενδεδ) ςεντανα Βενκ ΞΤ ΧΧ1 (Μιλδ) ςεντανα Βενκ ΞΤ ΧΧ1 (Στανδαρδ) ςεντανα Βενκ ΞΤ ΧΧ1# (8µινσ) Ωατερ βατη 68 ΟΧ Ωατερ βατη ΟΧ Ενζψµε Μεδιατεδ Ρετριεϖαλ Ενδοτηελιαλ Μαρκερ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Ν % Ν % ΑΣ ΠΕΡ ΚΙΤ ακο Προτεινασε Κ (Σ32) 2 1 ΜΠ ΒιοΜεδιχαλσ Τρψπσιν ΝΟΤ ΑΠΠΛΙΧΑΒΛΕ Οτηερ Σιγµα χηψµοτρψπσιν (Χ4129) 2 1 Σιγµα Πεπσιν (Π7) 1 1 Τρψπσιν 1 ςβσ Βονδ Ενζψµε ςβσ Βονδ Ενζψµε ςεντανα Προτεασε ςεντανα Προτεασε 1 (76 218) Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ,

14 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε Γενεραλ Πατηολογψ Ρυν: 97 Γενεραλ Πατηολογψ Ρυν: 97 ετεχτιον Ενδοτηελιαλ Μαρκερ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Ν % Ν % Α Μενερινι Πολψµερ (ΜΠ ΞΧΠ) ΑΣ ΠΕΡ ΚΙΤ ΒιοΓενεξ ΣΣ Πολψµερ (Θ 42 ΨΙΚΕ) ΒιοΓενεξ ΣΣ Πολψµερ (Θ 43 ΞΑΚΕ) ακο Ενςισιον ΦΛΕΞ ( Κ8/1) ακο Ενςισιον ΦΛΕΞ+ ( Κ82/12) ακο Ενϖισιον ΗΡΠ/ ΑΒ ( Κ57) ακο Ενϖισιον+ ΗΡΠ µουσε Κ44/5/6/7 1 1 ακο ρβ α µο Ιγ (Ε354) ακο ΡΕΑΛ ΗΡΠ/ ΑΒ (Κ51 ) Λαβςισιον Υλτραςισιον ΛΠ ΗΡΠ (ΤΛ 125 ΗΛϑ) Λαβςισιον Υλτραςισιον ΛΠ ΗΡΠ (ΤΣ 125 Η ) Λαβςισιον Υλτραςισιον ΟΝΕ Πολψµερ ( ΤΛ 12/5 Η ϑ/τ Λειχα Βονδ Πολψµερ Ρεφινε ( Σ98) ΜεναΠατη Ξ Χελλ Πλυσ (ΜΠ ΞΧΠ) Νονε ΝΟΤ ΑΠΠΛΙΧΑΒΛΕ Νοϖοχαστρα Νοϖολινκ Π Σ (ΡΕ7 14/15/28/29 Κ) Οτηερ Ποωερ ςισιον ΠςΒ999 ΗΡΠ ςεχτορ Ελιτε ΑΒΧ Κιτ (ΠΚ 72) ςεχτορ Ελιτε Υνιϖερσαλ ΑΒΧ (ΠΚ 62) ςεχτορ ΙµµΠΡΕΣΣ Υνιϖερσαλ (ΜΠ 75) ςεντανα ιςιεω σψστεµ (76 91) ςεντανα Οπτιςιεω Κιτ (76 7) ςεντανα Υλτραςιεω Κιτ (76 5) Χηροµογεν Ενδοτηελιαλ Μαρκερ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Ν % Ν % Α. Μεναρινι Λιθυιδ Σταβλε ΑΒ κιτ ΑΣ ΠΕΡ ΚΙΤ ΒιοΓενεξ ΑΒ (Θ 43) ΒιοΓενεξ Λιθυιδ ΑΒ (ΗΚ153 5Κ) ακο ΑΒ Κ ακο ΑΒ Λιθυιδ (Κ3465) 1 1 ακο ΑΒ+ Λιθυιδ (Κ3468) ακο ΑΒ+ ΡΕΑΛ ετεχτιον (Κ51) ακο Ενςισιον Πλυσ κιτσ ακο ΦΛΕΞ ΑΒ ακο ΡΕΑΛ Ενςισιον Κ57 ΑΒ ακο ΡΕΑΛ Κ51 ΑΒ Λαβςισιον (ΤΑ 125 Η ) µεναπατη ξχελλ κιτ ΑΒ (ΜΠ 86) Οτηερ Σιγµα ΑΒ ( 5637) Σιγµα ΑΒ ( 595) ςβσ Βονδ Πολψµερ Ρεφινε κιτ ( Σ98) ςεντανα ΑΒ ςεντανα ιϖιεω ςεντανα Υλτραϖιεω ΑΒ ςισιον ΒιοΣψστεµσ Βονδ Ξ ΑΒ Γενεραλ Πατηολογψ Ρυν: 97 Αυτοµατιον Ενδοτηελιαλ Μαρκερ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Ν % Ν % ΒιοΓενεξ ΓενοΜΞ 6ι ΒιοΓενεξ Οπτιµαξ ακο Αυτοσταινερ ακο Αυτοσταινερ Λινκ ακο Αυτοσταινερ πλυσ ακο Αυτοσταινερ Πλυσ Λινκ ακο ΤεχηΜατε Λαβςισιον Αυτοσταινερ Λειχα Βονδ Μαξ Λειχα Βονδ Ξ Λειχα Βονδ ΙΙΙ Μεναρινι Ιντελλιπατη ΦΛΞ Νονε (Μανυαλ) Οτηερ Σηανδον Σεθυενζα ΤεχηΜατε 5Πλυσ 1 1 ςεντανα Βενχηµαρκ ςεντανα Βενχηµαρκ ΥΛΤΡΑ ςεντανα Βενχηµαρκ ΞΤ ςεντανα ΝεξΕΣ 1 1 Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ,

15 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε ΒΕΣΤ ΜΕΤΗΟ Σ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Μετηοδ 1 Παρτιχιπαντ σχορεδ 18/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 18/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ςεχτορ ςπ Σ281 ΣΜΑ (ασµ 1), 3 Μινσ, ΡΤ Χ ιλυτιον 1: 25 Αυτοµατιον: Λαβςισιον Αυτοσταινερ ΑΒΧ Μαιν Βυφφερ: ΤΒΣ + Τωεεν Λαβ ϖισιον ΠΤ Μοδυλε, Βυφφερ: Ε ΤΑ, ΠΗ: 8 Χηροµογεν: ακο ΑΒ+ Λιθυιδ (Κ3468), ΡΤ Χ., Τιµε 1: 5 Μινσ ετεχτιον: ςεχτορ Ελιτε ΑΒΧ Κιτ (ΠΚ 72), 3 Μινσ, ΡΤ Χ Πρεδιλυτεδ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Μετηοδ 2 Παρτιχιπαντ σχορεδ 2/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 19/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ακο Μ851 ΣΜΑ (Χλονε 1Α4), 4 Μινσ, 37 Χ ιλυτιον 1: 2 Αυτοµατιον: ςεντανα Βενχηµαρκ ΞΤ ςεντανα Υλτραςιεω ΑΒ Μαιν Βυφφερ: ςεντανα ρεαχτιον βυφφερ (95 3) ςεντανα Βενκ ΞΤ ΧΧ1 (Στανδαρδ) Χηροµογεν: ςεντανα Υλτραϖιεω ΑΒ ετεχτιον: ςεντανα Υλτραςιεω Κιτ (76 5) Πρεδιλυτεδ Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Μετηοδ 3 Παρτιχιπαντ σχορεδ 19/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 16/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ακο ΡΤΥ (Χλονε 1Ρ611) Αυτοµατιον: ακο Αυτοσταινερ Λινκ 48 ακο ΦΛΕΞ κιτ Μαιν Βυφφερ: ακο ΦΛΕΞ ωαση βυφφερ ακο ΠΤΛινκ ΝΟΤ ΑΠΠΛΙΧΑΒΛΕ Χηροµογεν: ακο Ενςισιον Πλυσ κιτσ ετεχτιον: ακο Ενςισιον ΦΛΕΞ+ ( Κ82/12) Σµοοτη Μυσχλε Αχτιν (ΣΜΑ) Μετηοδ 4 Παρτιχιπαντ σχορεδ 19/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 16/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ςεντανα (1Α4), 6 Μινσ, 36 Χ Πρεδιλυτεδ Αυτοµατιον: ςεντανα Βενχηµαρκ ΥΛΤΡΑ Οτηερ Μαιν Βυφφερ: ςεντανα ρεαχτιον βυφφερ (95 3) Οτηερ Χηροµογεν: Οτηερ ετεχτιον: ςεντανα Οπτιςιεω Κιτ (76 7) Πρεδιλυτεδ Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ,

16 Ρυν 97 ΓΕΝΕΡΑΛ ΠΑΤΗΟΛΟΓΨ Μοδυλε ΒΕΣΤ ΜΕΤΗΟ Σ Ενδοτηελιαλ Μαρκερ Μετηοδ 1 Παρτιχιπαντ σχορεδ 19/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 16/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ακο Μ7165 ( ΘΒενδ1) Χ 34, 3 Μινσ, ΡΤ Χ ιλυτιον 1: 1:5 Αυτοµατιον: ακο Αυτοσταινερ Λινκ 48 ακο ΦΛΕΞ+ κιτ Μαιν Βυφφερ: ακο ΦΛΕΞ ωαση βυφφερ, ΠΗ: 7.4 ακο ΠΤΛινκ, Βυφφερ: ΑΚΟ, ΠΗ: 9 Χηροµογεν: ακο ΦΛΕΞ ΑΒ, ΡΤ Χ., Τιµε 1: 5 Μινσ, Τιµε 2: 5 Μινσ ετεχτιον: ακο Ενςισιον ΦΛΕΞ+ ( Κ82/12), 15 Μινσ, ΡΤ Χ Πρεδιλυτεδ Ενδοτηελιαλ Μαρκερ Μετηοδ 2 Παρτιχιπαντ σχορεδ 2/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 18/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ακο Μ7165 ( ΘΒενδ1) Χ 34, 6 Μινσ, 2 Χ ιλυτιον 1: 75 Αυτοµατιον: ακο Αυτοσταινερ πλυσ Στ ΑΒΧ Μαιν Βυφφερ: ΤΒΣ + Τωεεν, ΠΗ: 7.6 Μιχροωαϖε Οϖεν, Βυφφερ: ςεχτορ αντιγεν υνµασκινγ σολυτιον, ΠΗ: 6 ΝΟΤ ΑΠΠΛΙΧΑΒΛΕ Χηροµογεν: ακο ΑΒ+ Λιθυιδ (Κ3468), 2 Χ., Τιµε 1: 5 Μινσ, Τιµε 2: 5 Μινσ ετεχτιον: ςεχτορ Ελιτε Υνιϖερσαλ ΑΒΧ (ΠΚ 62), 3 Μινσ, 2 Χ ιλυτιον 1: { Σ1.ΑΒ2_ ιλυτιον} Ενδοτηελιαλ Μαρκερ Μετηοδ 3 Παρτιχιπαντ σχορεδ 19/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 17/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: ακο Μ7165 ( ΘΒενδ1) Χ 34, 3 Μινσ, 25 Χ ιλυτιον 1: 75 Αυτοµατιον: Λειχα Βονδ Μαξ ςβσ ΒονδΜΑξ Ρεφινε ΚΙΤ Μαιν Βυφφερ: Βονδ Ωαση Βυφφερ (ΑΡ959), ΠΗ: 7.4 Λειχα ΒονδΜαξ ΕΡ2, ΠΗ: 8 Χηροµογεν: ςβσ Βονδ Πολψµερ Ρεφινε κιτ ( Σ98), 25 Χ., Τιµε 1: 3 Μινσ, Τιµε 2: 3 Μινσ ετεχτιον: Λειχα Βονδ Πολψµερ Ρεφινε ( Σ98), 8 Μινσ, 25 Χ Πρεδιλυτεδ Ενδοτηελιαλ Μαρκερ Μετηοδ 4 Παρτιχιπαντ σχορεδ 2/2 (ΥΚ ΝΕΘΑΣ Σλιδε) ανδ 18/2 (Ιν Ηουσε σλιδε) υσινγ τηισ µετηοδ. Πριµαρψ Αντιβοδψ: Λειχα/Νοϖοχαστρα Χ 31 ΡΤΥ ΠΑ25 (1Α1), 15 Μινσ, ροοµ Χ Πρεδιλυτεδ Αυτοµατιον: Λειχα Βονδ Μαξ ςβσ ΒονδΜΑξ Ρεφινε ΚΙΤ Μαιν Βυφφερ: Βονδ Ωαση Βυφφερ (ΑΡ959) Λειχα ΒονδΜαξ ΕΡ2 Χηροµογεν: ςβσ Βονδ Πολψµερ Ρεφινε κιτ ( Σ98) ετεχτιον: Λειχα Βονδ Πολψµερ Ρεφινε ( Σ98), 8 Μινσ, ροοµ Χ Πριντεδ ατ 9:36 ον Μονδαψ, 23 Απριλ,

17 The Breast Hormonal Receptor Module Run 97 Suzanne Parry and Merdol Ibrahim Antigens Assessed: Oestrogen Receptors (ER) Tissue Sections circulated: Number of Registered Participants: 332 Sections from a composite block (see table below) Circulated Tissue The table below left shows the staining characteristics of the tissue sections circulated during Run97, which composed of three infiltrating ductal carcinomas (IDCs) with differing levels of receptor expression, along with three ER control cell lines (see photograph below right). The staining of the tumours were characterised using the 6F11 antibody clone. Note: Only the breast tissue cases were used to gauge the participants overall performance. Cell lines were used for validation purposes only. Tissue Sections % positivity Staining Intensity Allred / Quick Score A. IDC 85-95% High 7-8 B. IDC 5-75% Medium to high 4-6 C. IDC % Negative* Cell Lines C B A A >8% High 7-8 B 3-35% High 6 C % Negative A B C General Guideline Used in The Assessment of Slides SCORE Slide not returned by Participant. STAINING PATTERN 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 4-6) iii. Negative tumour, with normal positively stained glands (Allred/Quick score <1) 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 15

18 The Breast Hormonal Receptor Module Run 97 Introduction Oestrogen receptor alpha (ER- ) plays a vital role in both the prognosis and predictive response of patients who may be considered for hormone therapy 3. Following the work of Harvey and colleagues 1, immunohistochemistry has now become the recognised gold standard for determining patient ER status. It is therefore crucial that not only the antibodies are correctly validated prior to patient-tissue use, but also proper control tissues are used to gauge the sensitivity of the test. Furthermore, in the UK, the NHS Breast Screening Programme ( recommends the Quick score (Allred) 1,2 to semi quantify the proportion and intensity of nuclear staining, thus further standardising the scoring criteria. ER Cell Lines For this assessment ER expressing cell lines were also incorporated alongside the tissue sections. The cell lines were however not used as part of the assessment procedure, but data was collected on their staining pattern in order to validate their usability for possible future NEQAS ER/PR assessments. The cell lines were kindly prepared and donated by Dako and consisted of; a) CAMA-1 (human breast carcinoma) ER positive cell line, b) HT-29 (human colon cancer) ER negative cell lines, and c) a mixture of CAMA-1 and HT-29 to provide a pseudo-mid expressing cell line. For the assessment of UK NEQAS sections the team of assessors gauge the sensitivity and specificity of participants submitted stained slides and score them according to the expected level of staining that can be achieved using the 6F11 and EP1 antibody clones on the same composite tissue sent out to the participants. Assessment Results Features of optimal immunostaining (figs 1-4) 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 (figs 5 & 6) 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 *It was also noted that in some of the distributed slides the negative tumour sample had cut through so there were no tumour cells present. This was taken into consideration during the assessments and participants were not penalised. The overall pass rate on NEQAS sections was 67% (scores of 13-2/2) for participants as a whole, with a failure rate of 13%. Once again the main reason for failure during this assessment was due to false positive staining of the ER negative tumour section (section C) (see fig. 5A). The false positive staining was also mirrored in the negative cell line (Fig 6A & B), which further indicated the false positive nature of the staining. False negative staining was evident in the 6F11 antibody clone, and especially within samples that had been retrieved using a high ph retrieval method such as Bond Max ER2 or a Tris-EDTA based method. Once again all participants are encouraged to properly validate their methodologies and use appropriate composite control material, showing high, medium and negative ER expression, so both the sensitivity and specificity of the ER IHC staining can be adequately monitored 4,5. The Dako EP1 antibody was also used more widely. The number of participants using it increased from 3 (run 96) to 12 in the current assessment, with a pass rate of 75%. Unlike the 6F11 clone, the EP1 clone is recommended to be used with a high ph antigen retrieval method. In-house Tissue Results Due to breakdown of automation machines of our host laboratory (UCL-AD), we were unable to stain the spare unstained sections that are normally requested. Assessors therefore scored in-house slides based on the quality/ readability of the samples. Participants were given a maximum score of 12/2 if the in-house control samples did not comprise of a composite breast tissue with the following levels of ER expression: i) a high expressing tumour (Allred=7-8) ii) a mid expressing tumour (Allred=4-6) and iii) a negative tumour (Allred=). The presence of normal staining glands is also encouraged. Furthermore, it has been decided that as of this run unstained in-house slides will no longer be requested and the assessing of the in-house samples will be based on the staining quality, correct choice of tissue and good morphological preservation. Participants submitted in-house control material showed an overall pass rate of 68%, comparable to that of the NEQAS tissue. The main reasons behind the borderline scores were mainly due to participants not submitting control slides with composite controls i.e. a mid-expressing or negative tumour, which accounted for 54% of the assessor comments during the assessment. 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: 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: Ibrahim M, Dodson A, Barnett S, Fish D, Jasani B, Miller K. (28) Potential for false-positive 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: Rhodes A, Jasani B, Balaton, et al. (21) Study of inter-laboratory 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: UK NEQAS ICC & ISH. No part of this document can be copied or used without prior written consent 16

19 ΡΥΝ 97 ΒΡΕΑΣΤ ΣΤΕΡΟΙ ΗΟΡΜΟΝΕ ΡΕΧΕΠΤΟΡ Μοδυλε Σελεχτεδ Ιµαγεσ σηοωινγ Οπτιµαλ ανδ Συβ οπτιµαλ Ιµµυνοσταινινγ Φιγ 1. Οϖερϖιεω οφ οπτιµαλλψ σταινεδ ΥΚ ΝΕΘΑΣ διστριβυτεδ σεχτιονσ, σηοωινγ Α) ηιγη εξπρεσσινγ τυµουρ Β) µιδ εξπρεσσινγ τυµουρ ανδ Χ) νεγατιϖε τυµουρ. Σεχτιον σταινεδ ωιτη ΣΠ1 χλονε, 1:1 φορ 32 µινυτεσ, υσινγ ςεντανα Βενχηµαρκ ΧΧ1 Μιλδ. Φιγ 2. Α σεχονδ χοµποσιτε τισσυε βλοχκ διστριβυτεδ βψ ΥΚ ΝΕΘΑΣ, αγαιν σηοωινγ τηε εξπεχτεδ λεϖελσ οφ σταινινγ Α) ηιγη εξπρεσσινγ τυµουρ Β) µιδ εξπρεσσινγ τυµουρ ανδ Χ) νεγατιϖε τυµουρ. Σεχτιον σταινεδ ωιτη ΕΠ1 χλονε, πρε διλυτεδ φορ 2 µινυτεσ, υσινγ ακο ΠΤ λινκ αντιγεν ρετριεϖαλ, υσινγ ακο ηιγη πη ρετριεϖαλ βυφφερ. Φιγ 3. ΥΚ ΝΕΘΑΣ διστριβυτεδ χοντρολ χελλ λινεσ σηοωινγ τηε εξπεχτεδ λεϖελσ οφ σταινινγ. Α) ηιγη εξπρεσσινγ χελλ λινε (Αλλρεδ 7 8) Β) Μιδ εξπρεσσινγ χελλ λινε σηοωινγ α µιξτυρε οφ ηιγη ανδ νεγατιϖε χελλ λινεσ (1:2) (Αλλρεδ 5 6) ανδ Χ) Νεγατιϖε χελλ λινε (σεε ιντροδυχτιον φορ φυρτηερ δεταιλσ). Σεχτιον σταινεδ υσινγ σαµε µετηοδ ασ ιν φιγ 2. Φιγ 4. Ανοτηερ γοοδ εξαµπλε οφ ΥΚ ΝΕΘΑΣ διστριβυτεδ χοντρολ χελλ λινεσ σηοωινγ Α) ηιγη εξπρεσσινγ χελλ λινε (Αλλρεδ 7 8) Β) Μιδ εξπρεσσινγ χελλ λινε σηοωινγ α µιξτυρε οφ ηιγη ανδ νεγατιϖε χελλ λινεσ (1:2) (Αλλρεδ 5 6) ανδ Χ) Νεγατιϖε χελλ λινε. Σεχτιον σταινεδ ωιτη 6Φ11 χλονε, 1:5 ωιτη 15 µινυτεσ ινχυβατιον υσινγ τηε Λειχα Βονδ ΙΙΙ ωιτη ΕΡ1 (λοω πη) ρετριεϖαλ Φιγ 5. Α) Υναχχεπταβλε φαλσε νεγατιϖε σταινινγ οφ ΝΕΘΑΣ διστριβυτεδ σαµπλε. Σεχτιον σταινεδ υσινγ τηε 6Φ11 χλονε, 1:6 φορ 3 µινυτεσ, υσινγ ακο ΠΤ λινκ ταργετ ρετριεϖαλ Β) Τοο στρονγ σταινινγ ιν τηε ΝΕΘΑΣ διστριβυτεδ µιδ εξπρεσσορ. Σεχτιον σταινεδ υσινγ τηε ςεντανα πρε διλυτεδ ΣΠ1 χλονε υσινγ ςεντανα Βενχηµαρκ ΧΧ1 στανδαρδ ρετριεϖαλ. Φιγ 6. Υναχχεπταβλε φαλσε ποσιτιϖε σταινινγ οφ ΥΚ ΝΕΘΑΣ διστριβυτεδ Α) νεγατιϖε τισσυε σεχτιον ανδ Β) αδϕοινινγ νεγατιϖε χελλ λινε φροµ τηε σαµε σλιδε. Βοτη σεχτιονσ σηοω φαλσε ποσιτιϖε σταινινγ ανδ µορπηολογιχαλ δαµαγε. Σεχτιον σταινεδ ωιτη ςεχτορ 6Φ11 χλονε, διλυτεδ 1:8 φορ 3 µινσ., υσινγ ακο ΠΤ λινκ ηιγη πη ταργετ ρετριεϖαλ σολυτιον. Πριντεδ ατ 9:33 ον Μονδαψ, 23 Απριλ,

20 Ρυν 97 ΒΡΕΑΣΤ ΣΤΕΡΟΙ ΗΟΡΜΟΝΕ ΡΕΧΕΠΤΟΡ Μοδυλε 6 ΡΥΝ 97Ε Οεστρογεν ρεχεπτορσ ον ΝΕΘΑΣ Σεχτιονσ ΑΛΛ ΠΑΡΤΙΧΙΠΑΝΤΣ Ινδιϖιδυαλ 4 = (%) 5 = (%) 35 ΡΥΝ 97Ε Οεστρογεν ρεχεπτορσ ον ΝΕΘΑΣ Σεχτιονσ ΥΚ ΠΑΡΤΙΧΙΠΑΝΤΣ Ινδιϖιδυαλ 4 = (%) 5 = (%) 5 6 = (%) 7 = 5 (2%) 3 6 = (%) 7 = (%) νο. οφ ρετυρνσ = 26 (8%) 9 = 12 (4%) 1 = 16 (5%) 11 = 15 (5%) 12 = 32 (1%) 13 = 17 (5%) 14 = 3 (9%) 15 = 4 (12%) 16 = 59 (18%) 17 = 48 (15%) 18 = 8 (2%) νο. οφ ρετυρνσ = 9 (5%) 9 = 9 (5%) 1 = 11 (7%) 11 = 6 (4%) 12 = 9 (5%) 13 = 11 (7%) 14 = 17 (1%) 15 = 23 (14%) 16 = 31 (19%) 17 = 23 (14%) 18 = 4 (2%) = 9 (3%) 2 = 5 (2%) = 7 (4%) 2 = 5 (3%) Συµµαρψ Συµµαρψ 16 2 = 129 (4%) 16 2 = 7 (42%) = 87 (27%) = 51 (31%) 1 12 = 63 (2%) 1 12 = 26 (16%) 9 = 43 (13%) 9 = 18 (11%) Μεδιαν = 13.5 Μεδιαν = ΡΥΝ 97Φ Οεστρογεν ρεχεπτορσ ον ιν ηουσε Σεχτιονσ ΑΛΛ ΠΑΡΤΙΧΙΠΑΝΤΣ Ινδιϖιδυαλ 4 = (%) 5 = (%) 35 ΡΥΝ 97Φ Οεστρογεν ρεχεπτορσ ον ιν ηουσε Σεχτιονσ ΥΚ ΠΑΡΤΙΧΙΠΑΝΤΣ Ινδιϖιδυαλ 4 = (%) 5 = (%) 7 6 = (%) 7 = (%) 3 6 = (%) 7 = (%) νο. οφ ρετυρνσ = 4 (1%) 9 = 4 (1%) 1 = 7 (2%) 11 = 12 (4%) 12 = 75 (24%) 13 = 31 (1%) 14 = 21 (7%) 15 = 24 (8%) 16 = 55 (17%) νο. οφ ρετυρνσ = 1 (1%) 9 = 2 (1%) 1 = 4 (2%) 11 = 4 (2%) 12 = 31 (19%) 13 = 18 (11%) 14 = 12 (7%) 15 = 12 (7%) 16 = 31 (19%) 1 17 = 45 (14%) 18 = 2 (6%) 5 17 = 22 (13%) 18 = 12 (7%) = 1 (3%) 2 = 9 (3%) = 8 (5%) 2 = 7 (4%) Συµµαρψ Συµµαρψ 16 2 = 139 (44%) 16 2 = 8 (49%) = 76 (24%) = 42 (26%) 1 12 = 94 (3%) 1 12 = 39 (24%) 9 = 8 (3%) 9 = 3 (2%) Μεδιαν = 14. Μεδιαν = 14. Πριντεδ ατ 9:34 ον Μονδαψ, 23 Απριλ,