John Garratt

Implementation of a Canadian external quality assurance program for breast cancer biomarkers: an initiative of Canadian Quality Control in immunohistochemistry (cIQc) and Canadian Association of Pathologists (CAP) National Standards Committee/Immunohistochemistry.

Immunohistochemistry results for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are used to guide breast carcinoma patient management and it is essential to monitor these tests in external quality assurance (EQA) programs. Canadian Immunohistochemistry Quality Control is a web-based program with novel approach to EQA. Canadian Immunohistochemistry Quality Control RUN2 included tissue microarray slides with 38 samples tested by 18 immunohistochemical laboratories. Deidentified results were posted for viewing at www.ciqc.ca including all used protocols matched with scanned slides for virtual microscopy and garrattograms. Sensitivity, specificity, Kendall W test (concordance between laboratories), and κ statistics (agreement with designated reference values) were calculated. Kappa values were within the target range (>0.8, or “near perfect” agreement) for 85% results. Kendall coefficient was 0.942 for estrogen receptor, 0.930 for progesterone receptor, and 0.958 for human epidermal growth factor receptor 2. The anonymous participation, quick feedback, and unrestricted full access in EQA results provides rapid insight into technical or interpretive deficiencies, allowing appropriate corrective action to be taken whereas the use of tissue microarrays enables meaningful statistical analysis.

Standardization of Negative Controls in Diagnostic Immunohistochemistry: Recommendations From the International Ad Hoc Expert Panel

Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized. As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide “best practice recommendations” for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on “fit-for-use” principles.

HER2/neu Testing in Gastric Cancer by Immunohistochemistry: Assessment of Interlaboratory Variation

CONTEXT Immunohistochemical (IHC) testing for HER2/neu is becoming the standard of care for guiding adjuvant treatment of gastric carcinoma with trastuzumab. OBJECTIVE To assess interlaboratory variation in IHC staining and interpretation across multiple laboratories. DESIGN A tissue microarray consisting of 45 cores from 28 gastric cancers was distributed to 37 laboratories for HER2/neu assessment. The IHC results were compared against expert scores at an academic institution and correlated with in situ hybridization results from the originating specimen. Interlaboratory agreement was calculated using Cohen κ statistic. RESULTS The survey demonstrated several variations in IHC methods, including the primary antibodies in use. There was excellent agreement among laboratories in HER2/neu(+) (IHC 3(+)) cases (κ = 0.80 ± 0.01) and very good agreement among laboratories in HER2/neu(-) (IHC 0 or 1(+)) cases (κ = 0.58 ± 0.01). Less agreement was observed among laboratories when scoring equivocal (IHC 2(+)) cases (κ = 0.22 ± 0.01). Sensitivity and specificity of HER2/neu IHC were 99% and 100%, respectively, when measured against expert review and consensus score as a reference standard. CONCLUSIONS There is substantial interlaboratory agreement in the interpretation of HER2/neu IHC despite variability in protocols. Although HER2/neu IHC is a highly sensitive and specific test, primary antibody selection may significantly affect IHC results. Furthermore, gastric tumors require a unique scoring system and expertise in interpretation. Intratumoral heterogeneity has a significant effect on HER2/neu scoring by IHC. Ongoing quality assurance exercises among laboratories will help ensure optimized HER2/neu testing.

Inappropriate calibration and optimisation of pan-keratin (pan-CK) and low molecular weight keratin (LMWCK) immunohistochemistry tests: Canadian Immunohistochemistry Quality Control (CIQC) experience

Aims Pan-cytokeratin (pan-CK) and low molecular weight cytokeratin (LMWCK) tests are the most common immunohistochemistry (IHC) tests used to support evidence of epithelial differentiation. Canadian Immunohistochemistry Quality Control (CIQC), a new provider of proficiency testing for Canadian clinical IHC laboratories, has evaluated the performance of Canadian IHC laboratories in two proficiency testing challenges for both pan-CK and LMWCK. Methods CIQC has designed a 70-sample tissue microarray (TMA) for challenge 1 and a 30-sample TMA for challenge 2. There were 13 participants in challenge 1, and 62 in challenge 2. All results were evaluated and scored by CIQC assessors and compared with reference laboratory results. Results Participating laboratories often produced false-negative results that ranged from 20% to 80%. False-positive results were also detected. About half of participating clinical laboratories have inappropriately calibrated IHC tests for pan-CK and LMWCK, which are the most commonly used markers for demonstration of epithelial differentiation. The great majority of laboratories were not aware of the problem with calibration of pan-CK and LMWCK tests because of inappropriate selection of external positive controls and samples for optimisation of these tests. Benign liver and kidney are the most important tissues to include as positive controls for both pan-CK and LMWCK. Conclusions Participation in external quality assurance is important for peer comparison and proper calibration of IHC tests, which is also helpful for appropriate selection of positive control material and material for optimisation of the tests.

Utility of a CEA, CD15, calretinin, and CK5/6 panel for distinguishing between mesotheliomas and pulmonary adenocarcinomas in clinical practice.

Most reports on antibodies that claimed to separate mesothelioma from pulmonary adenocarcinoma originated from academic centers or specialized immunohistochemistry laboratories, but little is known about how such stains perform in general practice laboratories. The Canadian Immunohistochemistry Quality Control program circulates tissue array slides to laboratories across Canada; these are stained and then interpreted by the local laboratory and by a set of experienced reviewers. For Canadian Immunohistochemistry Quality Control run 16, tissue array slides from 16 pulmonary adenocarcinomas and 6 mesotheliomas were stained in 36 different laboratories for CEA, CD15, CK5/6, and calretinin. A total of 736 results (cores) were interpretable. If 3 of 4 staining results concordant with the diagnosis was accepted as definitive, 166/192 (86.4%) mesothelioma cores and 461/544 (84.7%) adenocarcinoma cores were correctly diagnosed. However, if 4 of 4 concordant markers were required, then 93/192 (48.4%) mesothelioma cores and 265/544 (48.7%) adenocarcinoma cores were correctly diagnosed. Only 3/192 (1.6%) mesothelioma cores were incorrectly classified as carcinomas and 8/544 (1.5%) of adenocarcinoma cores incorrectly classified as mesotheliomas on the basis of the immunoprofile (ie, 3 of 4 or 4 of 4 marker results were discordant with the diagnosis). We conclude that, in a study based on results from nonspecialized laboratories, the combination of CEA, CD15, calretinin, and CK5/6, used as a panel, has a very low false-positive rate when separating pulmonary adenocarcinomas from mesotheliomas; however, single negative or incorrect results are common, therefore the panel is only useful diagnostically if 3 of 4 correct results are deemed acceptable for diagnosis.

Improved breast cancer biomarker detection through a simple, high frequency, low cost external proficiency testing program

Aims: We describe a simple, low cost, high frequency immunohistochemistry external proficiency testing program, and show how its use can lead to improved breast cancer biomarker detection. Methods: Over a 30 month period in British Columbia, Canada, we used tissue microarray slides to follow the performance of twelve clinical laboratories in nine separate external proficiency testing runs. Sensitivity for detection of oestrogen receptor (ER), progesterone receptor (PR), and HER2 were calculated for each laboratory, biomarker, and run. Results: Mean sensitivities for detection of ER, PR, and HER2 were 97.1%, 84.8%, and 90.7%, respectively. HER2 sensitivity improved over time, from 87.0% to 92.9% (p = 0.04), with a trend towards improvement seen for PR (81.9–88.1%, p = 0.13). ER sensitivities were high throughout the test period. Improvements occurred without mandating any specific laboratory changes. Conclusions: This simple, low cost, high frequency external proficiency testing program is highly sustainable and can be implemented in any multi‐institutional group or region.

Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee for High Complexity Testing/Immunohistochemistry: guidelines for the preparation, release, and storage of unstained archived diagnostic tissue sections for immunohistochemistry.

OBJECTIVES Formalin-fixed, paraffin-embedded unstained archived diagnostic tissue sections are frequently exchanged between clinical laboratories for immunohistochemical staining. The manner in which such sections are prepared represents a type of preanalytical variable that must be taken into account given the growing importance of immunohistochemical assays, especially predictive and prognostic tests, in personalized medicine. METHODS Recommendations were derived from review of the literature and expert consensus of the Canadian Association of Pathologists-Association canadienne des pathologists National Standards Committee for High Complexity Testing/Immunohistochemistry. RESULTS Relevant considerations include the type of glass slide on which to mount the unstained sections; the thickness of the tissue sections; the time from slide preparation to testing; the environment, particularly the temperature at which the unstained sections will be maintained prior to testing; the inclusion of on-slide positive control tissue where possible; and whether patient identifier(s) should be included on slide labels. CONCLUSIONS Clear communication between requesting and releasing laboratories will facilitate the proper preparation of unstained sections and also ensure that applicable privacy considerations are addressed.

Developing ALK Immunohistochemistry and In Situ Hybridization Proficiency Testing for Non-Small Cell Lung Cancer in Canada: Canadian Immunohistochemistry Quality Control Challenges and Successes.

Intrachromosomal rearrangements involving the ALK gene are found in 3% to 5% of non–small cell lung cancers. Crizotinib is a tyrosine kinase inhibitor that has been shown to prolong progression-free survival in patients with advanced non–small cell lung cancer harboring ALK gene rearrangements. In Canada, ALK immunohistochemistry (IHC) is used as a screening test before confirmation by fluorescence in situ hybridization (FISH). Canadian Immunohistochemistry Quality Control (CIQC) provides ALK (Lung Cancer) proficiency testing (PT) for Canadian IHC laboratories. Samples included 32 previously characterized cases (IHC and FISH) either from the Canadian ALK (CALK) project or from CIQC reference laboratories. The same design was used for both runs. A total of 20 laboratories participated in Run 1 and 22 in Run 2. Some laboratories participated in the anticipation of future need and used the PT exercise as a part of test development and validation. Results of the IHC testing were first self-reported using the CIQC TMA Scorer and then evaluated by expert assessment. FISH results were self-reported only. Participants also reported details about IHC and FISH protocols. The &kgr;-values were calculated, for which values >0.80 were used as acceptable results, respectively. The pass rate between the 2 runs and between different primary antibodies were compared. Six of the 22 protocols (27%) in Run 1 and 15 of the 22 (68%) protocols in Run 2 passed the CIQC PT criteria for IHC testing. The increase in the pass rate for Run 2 was significant (P=0.03, Wilcoxon signed-rank test). All reported FISH results were correct. CALK laboratories had significantly higher &kgr;-values than non-CALK laboratories (P=0.002, t test). PT for IHC for rare diseases such as ALK-positive lung cancer is feasible, but challenging. The academic nature of the CIQC program and collaboration on a national level facilitated the development of appropriate PT samples. Participating laboratories made use of the PT exercise either to confirm that their testing was properly calibrated or to improve their protocols, which was confirmed by the achievement of significantly better results in Run 2. They also used CIQC’s PT program for new test development and optimization.

Canadian external quality assurance program for breast cancer biomarkers.

To the Editor: We wish to respond to the letter from Dr Flynn and Ms Schirripa regarding our recent report on our continuing nationwide efforts to improve immunohistochemical testing, including breast cancer biomarker testing, in Canada. They state unequivocally that there were misrepresentations in our paper, but do not indicate what they are. We would like to emphasize here that our paper does not evaluate or comment on the work of the Quality Management Program, Laboratory Services (their employer) at all, but rather on our own academic program. The methods and results of the Quality Management Program, Laboratory Services testing are not available to us and therefore could not be included in our discussion. As these alleged misrepresentations were not specifically identified, we are denied the opportunity to effectively respond. We cannot dismiss this failure to identify the alleged misrepresentations as an oversight, as it seems that Dr Flynn and Ms Schirrapa have chosen to use the forum provided by the ‘‘Letters to the Editor’’ to promote their program in vague general terms and create doubt about our program through allegations of ‘‘misrepresentations.’’ The Canadian Immunohistochemistry Quality Control provides the opportunity for Canadian laboratories to participate in their own national external quality assurance program for diagnostic immunohistochemistry and we are saddened that we are perceived as competition rather than collaborators of our Canadian colleagues, as we all have to face the difficult task of helping to raise standards of practice in this challenging area of clinical laboratory practice.

Standardization of Positive Controls in Diagnostic Immunohistochemistry: Recommendations From the International Ad Hoc Expert Committee

performed H&E staining (Figure a and b was from identical area). 5 areas of normallooking lung parenchyma for each slide were randomly selected. The number of alveolar sac surrounded by string-like framework (FW) was counted. Area ratios of Pores of Kohn to each alveolar sac membrane surrounded by framework (PK) were calculated with images from the spots of interest (Figure c). Image J was used for stack construction and measurement of images (Figure d). Clinical data including pulmonary function test of patients was collected. Comparison of clinical data was performed with t test. Multilevel linear mixed model was used to evaluate differences of FW and PK and associations with clinical data. P values <.05 were considered significant.