Standardization of Ki-67 Immunohistochemical Staining for Diagnosing Grade of Gastrointestinal Stromal Tumor (GIST) A report of the research regarding the establishment of pathological diagnostic criteria in order to standardize cancer diagnosis and treatment. Supported by a grant-in-aid for cancer research from the Ministry of Health, Labor and Welfare of Japan Articles Tadashi Hasegawa, MD, PhD Professor and Director Department of Surgical Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan Introduction Ki-67 is a molecule that can be easily detected in growing cells in order to gain an understanding of the rate at which the cells within a tumor are growing. Ki-67 immunohistochemical staining using proliferation markers, particularly MIB-1, a monoclonal antibody to the Ki-67 antigen, is useful for objective histopathological evaluation of tumor proliferation activity. It has also been recognized that staining of Ki-67 is essential for diagnosing tumor grade. The Ki-67 labeling index (LI), based on the proportion of tumor-positive cells, has usually been used as an indication for evaluation, and many reports have shown its clinical significance in a variety of cancers regardless of whether the tumor origin is epithelial or nonepithelial. Surveys have even been conducted to ascertain the present situation of the Ki-67 staining technique, and questionnaires have been sent to 33 pathological laboratories throughout Japan. They revealed that, in actuality, there were differences among the laboratories in terms of reaction time, antigen retrieval processing methods, the period from preparation of thin slices to staining, and so forth. In addition, 88% of the laboratories replied that the results of this staining are influenced by and vary with staining conditions, particularly the processing method for antigen retrieval. The surveys suggested the necessity of assessing the actual influence of differences in the processing method, on the results and the judgment as well as the necessity of standardization (Figure 1). Fact-finding surveys, including a slide survey using Ki-67 staining specimens, were also conducted. Interobserver and intraobserver variations in the Ki-67 LI were assessed with the cooperation of 12 pathological specialists in hematoxylin-eosin (HE) and Ki-67 stained specimens from 20 tumor lesion cases. This type of verification is believed to be necessary for staining of Ki-67 to be used as a reference for diagnosing or classifying histological subtypes. With this method, which was most frequently adopted by the aforementioned pathologists as a method of calculating the Ki-67 LI, the staining characteristics deemed moderate or strong were judged to be positive, and 100-500 tumor cells at sites with numerous positive cells were observed with 40x objective lenses. Interobserver variation in real-valued Ki-67 LI was quite remarkable, but when the concordance rate of the Ki-67 LI was determined by classifying the Ki-67 LI into less than 5% (<5%), 5%10%, and higher than 10% (10%) categories, the mean concordance rate for the 1st and 2nd determination categories was 78.3% and the Kappa (k) value was 0.664 showing some degree of concordance. (A Kappa statistic of 0.75 represents excellent concordance, 0.40-0.70 represents good to fair, and < 0.40 represents poor concordance). On the basis of these survey results, a study was conducted to accurately diagnose and grade Gastrointestinal Stromal Tumor (GIST). This study was conducted in part by research to establish a pathologic diagnostic criterion to standardize cancer diagnosis and treatment. The study was supported by a grant-in-aid for cancer research from the Ministry of Health, Labor and Welfare of Japan. Standardization of the Ki-67 staining method In the assessment of standardization of the Ki-67 staining with MIB-1, the National Cancer Center (NCC) method that has already been shown to be useful for estimating prognoses (1) (autoclave treatment using citrate buffer pH 6; see Figure 1-a), and a standardized (STD) method established in this study (water bath treatment using Tris-EDTA solution pH 9; see Figure 1-f) were used. Both the NCC and the STD methods were compared in terms of the correlation (equality of the staining results) in 52 GIST cases. The results of category-based evaluation (categories according to proportions of positive cells <5%, 5% and <10%, 10% and <20%, 20% and <30%, and 30%) showed the concordance rate for both methods to be 94.2% with a k value of 0.87 showing a high concordance rate (Table 1, Figure 2). The regression analysis of the Ki-67 LI values calculated from the positive cell count revealed that there was also a close correlation between these methods with a 0.94 correlation coefficient (Table 1, Figure 3). To confirm the reproducibility of the staining by the established STD method among laboratories, a similar assessment Connection 2008 | 50 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. 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