rence seen in these trials is in the order of 50%. This is the major reason for all the excitement associated with this drug. It is thus important for us pathologists to get the testing right. The principal testing methods used for determination of HER2 status are immunohistochemistry for protein overexpression and in situ hybridization using either fluorescence (FISH) or a chromogen (CISH). The data available on testing modalities and efficacy of identifying patients most likely to respond to trastuzumab is relatively limited, although a lot of data is available comparing the two tests to one another and the variability of testing in different laboratories. Interpretation of the data from the metastatic pivotal trial is complicated by the fact that most of it was performed using clinical trials assay (CTA) that is no longer available. Additionally, both 2+ and 3+ level(?) staining was considered positive. The clinical trials assay used a combination of two monoclonal antibodies 4D5 (recombinant humanized monoclonal antibody - rhuMAb HER-2) and CB11 (mouse anti-human mAb) to perform the assay. The 4D5 antibody, which was humanized into the commercially available drug (Herceptin® or trastuzumab), performed poorly on paraffin-fixed tissue. For these reasons, a newer and better test was needed. In co-operation with the Dako Corporation, the HercepTestTM, which uses a polyclonal antibody, was developed. At the same time, an in situ hybridization assay (FISH) was also being developed. This FISH test was retrospectively applied to tissues from patients from the pivotal trial. However, here again the analysis gets complicated due to significant variation (~25%) between laboratories that did the analysis. It has often been stated by some proponents of FISH technology that the data submitted in the FDA docket contains analytical errors. More recent analysis of the data from the CTA suggests that IHC and FISH are equivalent in identifying patients likely to respond to trastuzumab. Poor concordance between laboratories is also a significant issue as highlighted by the data comparing results of local and central testing of patients entered into two of the US adjuvant trials. Initial data analysis in 2002 showed that there was a significant discrepancy (~18%) in the results between the local laboratories and the central laboratories in both the National Surgical Adjuvant Breast Project (NSABP) and North Central Cancer Treatment Group (NCCTG) trials. The NSABP investigators attempted to split the ~100 cases into those that came from large volume laboratories (arbitrarily defined as those analyzing more than 100 cases/ month) and small volume laboratories. Although the numbers here were very small, a clear trend was noted. The results of the central laboratory disagreed with large volume laboratories in only ~3% of cases. This indicated that the major source of discrepancy was inherent to small volume laboratorie. This, rightly or wrongly, lead to the assumption that large volume laboratories are better at performing HER2 testing. The end result of this analysis was that central testing was mandated as part of the clinical trials. Comparison of the local and central laboratory results from N-9831 was presented more recently by Dr. Edith Perez, Mayo Clinics Multidisciplinary Breast Clinic in Jacksonville, FL. This has confirmed the discrepant results seen in preliminary analyses. The rates of disagreement are approximately 12% for FISH analysis and approximately 18% for IHC analysis using Dako HercepTestTM. Importantly, in this study all the cases with discrepant results were sent to an independent third laboratory for confirmation of results. The discordance between the two central laboratories was less than 5%. An effort is being made to collect the original HER2 stained slides to analyze the causes of discrepancy. The high discrepancy seen in the initial analysis and confirmed in later analysis presumed to be due to inaccurate testing in small volume laboratories and was a cause of concern. The American Society of Clinical Oncology (ASCO) and College of American Pathology (CAP) convened a panel to generate guidelines/recommendations to minimize the error rates. The recommendations were published in the summer of 2007 and are going to be made an integral part of the CAP inspection checklist. This comprehensive document covers all aspects of analysis, including pre-analytical components such as fixatives used, duration of fixation, analytical components, including reagents/ kits used, criteria for positivity, and the interobserver variability between pathologists in a given lab doing the reading of the stained slides. The panel has made amendments to the definition of IHC 3+ category, 59 | Connection 2008 April 2008, VOL 11 Connection IHC STANDARDIZATION AROUND THE WORLD In this issue Q&A with Patho Contents APRIL 2008, VOL 11 2 3 4 Editorial George L. Kumar, PhD Featured Laboratory The Osamura Editorial Immunohistochemistry (IHC) Standardization Around the World George L. Kumar, PhD Managing Featured Laboratory The Osamura Laboratory Laboratory headed by Prof. Robert Yoshiyuki Osamura, MD, Q&A Ask the Experts: On Immunohistochemistry (IHC) Standardization Professor Leong is Medical Di time interval between removal of tissue and immersion in fixative, the temperature of tissue storage Connection: How would you like to standardize IHC in Australia? As discussed above, standardization Connection: Antigen retrieval is essential in immunohistochemistry (IHC) in order to restore epitope Connection: Is a universal image analysis system feasible? No, not until we standardize the all impo Q&A Fernando Soares, MD, PhD University of São Paulo, São Paulo, Brazil Dr. Fernando Soares is F Connection: In your opinion, what is the biggest hurdle for standardization? Probably education in l There are no standards in Latin America. We always follow the manufacturers protocol during our firs Q&A Bryan R. Hewlett, ART, MLT Consultant Technologist to the Anatomic Pathology EQA program of There is no standardization of AR steps circumstances, it would be impossible toand, undera the pre Connection: How would you rate European (UK NEQAS, NordiQC) and US IHC standards to Canadian IHC sta Q&A Prof. Chen Jie Prof. Cui Quancai Peking Union Mediacl College Hospital, Beijing, China Prof. Connection: According to Goldstein et al. Appl Immunohistochem Mol Morphol 2007;15:124­133 Immunohis Connection: Is it true that a particular histology feature may be better demonstrated by other fixat Connection: What constitutes standardization of image analysis as applied to immunohistochemistry (I Q&A Dr. Tanuja Manjanath Shet Dr. Vani Parmar Tata Memorial Hospital, Mumbai, India Tanuja Manj ... the biggest hurdle in India is suboptimal fixation and processing of tissues. Though I agree th Connection: Is it true that a particular histology feature may be better demonstrated by other fixat Connection: Can you comment on the internal and external quality control (EQC) procedures followed i Q&A Prof. Robert Yoshiyuki Osamura Department of Pathology, Tokai University School of Medicine Connection: In your opinion, what is the biggest hurdle for standardization? The biggest hurdle for the standardization of image ... appropriate for pre-screeninganalysis is of the staining quality. Connection: Why is standardization of image analysis in diagnostic pathology important? Because the Q&A James F. Happel, DLM (ASCP), HTL Technical Director of Surgical Pathology, Research and Deve Connection: United Kingdom National External Quality Assessment Service (UK NEQAS) helps to ensure t Connection: The American Society of Clinical Oncology (ASCO) and the College of American Pathologist would recommend that standardization Ibegin with identifying a reliable and trustworthy source ... Interview Immunohistochemistry for Oestrogen and Progesterone Receptors Dr. Andrew Lee Consultant H Connection: What is the difference between the H score and the Allred score? Which is better? What d Connection: Can you comment on the burden in the laboratory, if one changes from a current ER/PR ass Opinion & Interview IHC Standardization: A Dako Perspective Dr. Ole Rasmussen R&D Director, Connection: Dako has developed Readyto-Use Antibodies for in vitro diagnostic applications. How is t Articles UK NEQAS-ICC & ISH: Historical perspective, current role, future directions Andy Dodso UK NEQAS-ICC in the 1990s In his first year as Scheme Organiser, Keith oversaw the transition to sub The application could be argued to represent a field change in terms of the rigour with which the an Assessment teams consist of four assessors, who view slides around a multi-headed microscope and sco The archive which UK NEQAS holds, both in terms of stained slides and methodological data, must sure For Immunocytochemistry and FISH RESULT: RUN 80L SLIDE: NEQAS Laboratory No: XXX Mr. A. Scientist De Figure 6. Feedback on results has always been given high priority, and for many years this has been a b c d Figure 7. The antigen chosen by Gerry Reynolds for his very first assessment run was kap Bibliography Selected UK NEQAS-ICC & ISH papers. Ibrahim M, Dodson AR, Barnett S, Fish D, Jasani Articles Nordic Immunohistochemical Quality Control (NordiQC) An Organization for External Quality A parameters (i.e. results interpretation and reporting) (4, 5). In an EQA setting, by circulating ser CD79a (Fig. 2) Among 112 laboratories submitting stains in the latest run, most used mAb clone JCB11 References 1. Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of immuno-histochemic Fig. 2. CD79a A. Optimal CD79a staining of the tonsil using the monoclonal antibody (mAb) clone JCB Standardization of Ki-67 Immunohistochemical Staining for Diagnosing Grade of Gastrointestinal Strom was conducted in 49 GIST cases. The concordance rate for the evaluation results at three laboratorie CB pH6 a b c d TE pH9 e f g h Autoclave 121° C/10 min Water bath 95° C/40 min Microwave 50 Table 1. Correlation between NCC and STD methods R2=0.9483 Categories of proportion NCC Method 3 Opinions Importance of Standardization for Predictive Prognosis David J. Dabbs, MD Chief of Pathol is documented and serves as a surrogate marker for the initial exposure to formalin. Since the first Opinions The New Era for ER and PRIts time to Standardize! Dr. Ian Ellis, B.Med.Sci. BM, MS, FRCpa et al 2001). The main reason for false-negative results is due to inefficient heat-induced epitope ( Standardization of HER2 TestingInconsistency Raises Questions Opinions Sunil S. Badve, MD, FRCPath rence seen in these trials is in the order of 50%. This is the major reason for all the excitement a which now requires expression of HER2 by at least 30% of tumor cells (instead of 10%). It has also r IHC CONSENSUS MEETING, JANUARY 27 2008, SANTA BARBARA, CA, USA , IHC CONSENSUS MEETING, JANUARY 27, Richard Cartun, PhD, Sunil Badve, MD Jon Askaa, PhD Søren Nielsen, HT, CT, Mogens Vyberg, MD Elizab Dako Abstracts Abstracts presented at the 30th Annual San Antonio Breast Cancer Symposium December Dako Abstracts Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal Dako Abstracts Abstracts presented at the United States and Canadian Academy of Pathology (USCAP) A Dako Abstracts Metastatic Pancreatic Endocrine Tumors in the Liver Express KOC Briones AJ, Bourne P Dako Abstracts Merkel Cell Carcinomas Express K Homology Domain Containing Protein Overexpressed in Dako Abstracts Immunohistochemical Analysis of KOC/IMP3 in Malignant Pleural Mesothelioma Xu H, Sim Dako Abstracts KOC, TTF-1 and CDX2 Discriminate Small-Cell Carcinoma from Carcinoid and Pancreatic Dako Publications Publications Co-authored by Dako: In Press Li L, Xu H, Spaulding B, Cheng L, Si Dako Meetings 2007 - National Society for Histotechnology Meeting. Denver, CO NSH workshop attend New Premium Quality Concentrated Antibodies New Products Your current and future needs drive the c Targeting Colorectal Adenocarcinomas, Anti-TIMP-1 and Anti-Villin Monoclonal Mouse Anti-Human Tissue Dakos FLEX Ready-to-Use Concept Enhance performance of your laboratory by running FLEX Ready-to-Use 2008 Product Catalog Available Now! The catalog features more than 170 new products, including the A