Canadian Immunohistochemistry Quality Control Research Articles

Abstract PS5-46: Introduction and clinical validation of metrology standards for immunohistochemistry (IHC); New tool for standardization of estrogen receptor (ER) IHC assay in breast cancer

Abstract Traceability of measurement to a higher order reference standard is a foundation of laboratory testing. There is as yet no method for creating reference standards for cellular proteins in situ in an analogous fashion as for soluble analytes. At present, IHC laboratories produce results for breast cancer hormone receptors without connection to a reference standard. Not surprisingly, high rates of testing variation as well as discrepancies among IHC laboratories have been reported. To address this need, we developed a system of measurement traceability using a linked fluorescein tag for creating reference standards for any cellular analyte and, as a first test, validate it for estrogen receptor (ER) testing. In this study, the newly developed ER standard defines and compares the thresholds separating “high positive”, “low positive”, and “negative” tests according to updated ASCO/CAP guidelines as detected by clinical IHC laboratories in a national external quality assessment survey. This reference standard utilizes NIST Standard Reference Material (SRM) 1934 as a universal IHC standard. We calculated ER concentration based on a linked fluorescence measurement traceable to NIST SRM 1934 as each ER is linked to a single fluorescein, and fluorescein concentration equals ER concentration. Each laboratory’s lowest detected ER concentration (i.e. “limit of detection”, LOD) was compared to their results with 80 tumor samples enriched for triple negative breast cases. For the Canadian Immunohistochemistry Quality Control (CIQC) ER proficiency testing run, calibrator sets with peptides for the SP1, EP1, and 6F11 epitopes were created. The various concentrations were pipetted onto histology slides used by CIQC to place its 80-case breast cancer tissue microarray. These slides were stained by participating laboratories using their routine ER IHC protocols and returned to the CIQC. For the purpose of this study, Histology Score and ASCO/CAP categorical scoring recommendation was used for the readout. Results with SP1 clone are reported here because it was employed by overwhelming majority of laboratories. A total of 3,038 readouts were included in the analysis. Most IHC laboratories had a LOD between 10,000 – 25,000 molecules ER per microbead. Highly sensitive ER assays (low LODs) detected more positive cases while those with poorly sensitive assay detected fewer. The LOD correlated with the percent positive cases (R2= -.767, p < 0.0001, Spearman Correlation) as well as with cumulative individual laboratory Histology Score (R2= -.612, p < 0.0001, Spearman Correlation). The tumor samples accounting for this difference were principally “low positive” (ASCO/CAP classification) while ER-high positive tumors were less affected. Although the concept of a non-quantitative IHC LOD has been introduced with critical assay performance controls (iCAPCs, e.g. germinal center cells in the tonsil for ER), there were no tools until now to actually measure LOD for ER IHC testing. With the calibrators introduced here, it is now possible to measure the LOD for ER IHC assays. Furthermore, our data show that ER low positive and some cases of ER high positive breast cancers are highly affected by the variability of analytic sensitivity between clinical IHC laboratories. These data argue for the urgent need to standardize testing to a defined analytic sensitivity range, which is now possible for the first time. Beyond inter-laboratory standardization, known measured LOD also enables: i) methodology transfer from clinical trials to clinical laboratories; ii) determination of optimal analytical sensitivity to produce assays with highest clinical and analytical validity, and iii) daily monitoring of analytical sensitivity for each tested IHC slide. Citation Format: Emina Emilia Torlakovic, Seshi R. Sompuram, Kodela Vani, Lili Wang, Anika K Schaedle, Paul C. DeRose, Steven A. Bogen. Introduction and clinical validation of metrology standards for immunohistochemistry (IHC); New tool for standardization of estrogen receptor (ER) IHC assay in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-46.

Hematoxylin and Eosin Counterstaining Protocol for Immunohistochemistry Interpretation and Diagnosis.

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

Diagnostic Accuracy in Fit-for-Purpose PD-L1 Testing

PD-L1 testing by immunohistochemistry (IHC) has presented significant challenges not only for clinical laboratories, but also for external quality assurance (EQA) entities that provide proficiency testing (PT) for clinical laboratories. Canadian Immunohistochemistry Quality Control (CIQC) has used educational runs to explore approaches to sample design and analysis of results that would enhance patient safety. As PT for predictive biomarkers requires modeling at every level (design of the run, assessment of the run, and reporting of "pass" or "fail") based on "fit-for-purpose" principles, CIQC has applied those principles to PD-L1 PT runs. Each laboratory received unstained slides with TMA tissue cores from 104 randomly selected primary NSCLC and tonsil tissues to test with their current PD-L1 assay. Diagnostic sensitivity and specificity were calculated against designated gold standards based on the "3D" approach (drug-disease-diagnostic assay). Depending on the selection of fit-for-purpose gold standards and also on the selection of what was considered fit-for-purpose cut-off points, great variation in the performance (accuracy) of both companion/complementary diagnostic assays and laboratory developed tests was seen. "Fit-for-purpose" in PT for PD-L1 testing entails that the purpose(s) of each PT run is declared a priori, that the PT program has selected/designated purpose-specific gold standard results for the PT challenge, and that the PT materials for the PT run are designed and constructed to enable calculations of diagnostic accuracy.

Modeling Canadian Quality Control Test Program for Steroid Hormone Receptors in Breast Cancer: Diagnostic Accuracy Study.

The Canadian Immunohistochemistry Quality Control program monitors clinical laboratory performance for estrogen receptor and progesterone receptor tests used in breast cancer treatment management in Canada. Current methods assess sensitivity and specificity at each time point, compared with a reference standard. We investigate alternative performance analysis methods to enhance the quality assessment. We used 3 methods of analysis: meta-analysis of sensitivity and specificity of each laboratory across all time points; sensitivity and specificity at each time point for each laboratory; and fitting models for repeated measurements to examine differences between laboratories adjusted by test and time point. Results show 88 laboratories participated in quality control at up to 13 time points using typically 37 to 54 histology samples. In meta-analysis across all time points no laboratories have sensitivity or specificity below 80%. Current methods, presenting sensitivity and specificity separately for each run, result in wide 95% confidence intervals, typically spanning 15% to 30%. Models of a single diagnostic outcome demonstrated that 82% to 100% of laboratories had no difference to reference standard for estrogen receptor and 75% to 100% for progesterone receptor, with the exception of 1 progesterone receptor run. Laboratories with significant differences to reference standard identified with Generalized Estimating Equation modeling also have reduced performance by meta-analysis across all time points. The Canadian Immunohistochemistry Quality Control program has a good design, and with this modeling approach has sufficient precision to measure performance at each time point and allow laboratories with a significantly lower performance to be targeted for advice.

Calibration and Optimization of p53, WT1, and Napsin A Immunohistochemistry Ancillary Tests for Histotyping of Ovarian Carcinoma: Canadian Immunohistochemistry Quality Control (CIQC) Experience.

The Canadian Immunohistochemistry Quality Control provides proficiency testing for immunohistochemistry in Canadian laboratories. Canadian Immunohistochemistry Quality Control Run 42 assessed WT1, Napsin A, and p53; commonly used markers for histotyping ovarian carcinomas. A 42-core tissue microarray, which included the 5 major histotypes of ovarian carcinomas with a subset having known TP53 mutational status, was used for this Canadian Immunohistochemistry Quality Control challenge. Participants included 43 laboratories for p53, 29 for WT1, and 26 for Napsin A. p53 was scored as aberrant if the staining was strong and diffuse or absent. Napsin A and WT1 were scored positive if any tumor cells stained. The reference p53 expression pattern was inferred by TP53 mutation type when available. For WT1, Napsin A, and cases lacking mutational data, the reference staining pattern was based on the majority staining result. The error rate for p53 was 8.8%. Most errors (84%) were due to weak staining. The sensitivity and specificity of aberrant p53 expression for an underlying TP53 mutation was 91.6% and 87.9%, respectively. The error rate for WT1 was 0.76% with all errors occurring in laboratories using the 6F-h2 clone. The average errors for laboratories using 6F-h2 were 2.4 compared with 0 for WT-49. The error rate for Napsin A was 4%. The average errors for laboratories using polyclonal Napsin A were 3 compared with 1.1 for monoclonal Napsin A. Weak p53 staining increases interpretative errors, primarily due to absence of staining in tumors with wild-type TP53. p53 immunohistochemistry correlates strongly with TP53 mutational status. Polyclonal Napsin A and 6F-h2 may lack specificity in comparison to monoclonal Napsin A and WT-49.

Academic and Nonacademic Laboratories Perform Equally on CIQC Immunohistochemistry Proficiency Testing

To test whether academic centers (ACs) are more successful than nonacademic centers (NACs) in immunohistochemistry (IHC) external quality assessment challenges in the Canadian Immunohistochemistry Quality Control (CIQC) program. Results of 9 CIQC challenges for breast cancer marker (BM) and various non-breast cancer marker (NBM) tests were examined. Success rates were compared between AC/NAC laboratories and those located in small or large cities. Performance was also correlated with annual IHC case volumes. There was no statistically significant difference in performance in any of the comparisons. However, overall performance on BM was significantly better (P < .0001, t test) than on NBM tests regardless of AC/NAC nature or city size. The mean failure rate on NBM was approximately twice that of BM tests. Our results suggest that recent emphasis on breast hormone IHC quality assurance has led to improved test quality.

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.

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

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

Development of an Evidence-Based Approach to External Quality Assurance for Breast Cancer Hormone Receptor Immunohistochemistry: Comparison of Reference Values

Abstract Context.—External quality assurance and proficiency testing programs for breast cancer predictive biomarkers are based largely on traditional ad hoc design; at present there is no universal consensus on definition of a standard reference value for samples used in external quality assurance programs. Objective.—To explore reference values for estrogen receptor and progesterone receptor immunohistochemistry in order to develop an evidence-based analytic platform for external quality assurance. Design.—There were 31 participating laboratories, 4 of which were previously designated as “expert” laboratories. Each participant tested a tissue microarray slide with 44 breast carcinomas for estrogen receptor and progesterone receptor and submitted it to the Canadian Immunohistochemistry Quality Control Program for analysis. Nuclear staining in 1% or more of the tumor cells was a positive score. Five methods for determining reference values were compared. Results.—All reference values showed 100% agreement...

The Laboratory Score/Reference Method Score Ratio (LSRSR) Is a Novel Tool for Monitoring Laboratory Performance in Immunohistochemistry Proficiency Testing of Hormone Receptors in Breast Cancer

Canadian Immunohistochemistry Quality Control (CIQC) operates an academic proficiency testing (PT) program using a traditional expert panel-based qualitative assessment system. The image analysis approach is increasingly considered for use in PT to follow demand for precision in immunohistochemical test calibration. CIQC introduces and explores the usefulness of a novel image analysis-based tool, the laboratory score/reference method score ratio (LSRSR) for PT. Two CIQC runs with 33 and 57 participants, respectively, were analyzed for interlaboratory concordance for estrogen receptor results using expert panel-based and LSRSR systems. Samples included tissue microarrays with 40 tissue cores each. The LSRSR was calculated from participants' and reference laboratory H scores measured by image analysis. We found lower concordance with reference method results for participating laboratories by LSRSR than those reported by the expert panel; although the expert panel observed those differences, it was not able to measure them without LSRSR. LSRSR may be useful in monitoring laboratory performance for quantitative immunohistochemical testing.

Development of an Evidence-Based Approach to External Quality Assurance for Breast Cancer Hormone Receptor Immunohistochemistry: Comparison of Reference Values

External quality assurance and proficiency testing programs for breast cancer predictive biomarkers are based largely on traditional ad hoc design; at present there is no universal consensus on definition of a standard reference value for samples used in external quality assurance programs. To explore reference values for estrogen receptor and progesterone receptor immunohistochemistry in order to develop an evidence-based analytic platform for external quality assurance. There were 31 participating laboratories, 4 of which were previously designated as "expert" laboratories. Each participant tested a tissue microarray slide with 44 breast carcinomas for estrogen receptor and progesterone receptor and submitted it to the Canadian Immunohistochemistry Quality Control Program for analysis. Nuclear staining in 1% or more of the tumor cells was a positive score. Five methods for determining reference values were compared. All reference values showed 100% agreement for estrogen receptor and progesterone receptor scores, when indeterminate results were excluded. Individual laboratory performance (agreement rates, test sensitivity, test specificity, positive predictive value, negative predictive value, and κ value) was very similar for all reference values. Identification of suboptimal performance by all methods was identical for 30 of 31 laboratories. Estrogen receptor assessment of 1 laboratory was discordant: agreement was less than 90% for 3 of 5 reference values and greater than 90% with the use of 2 other reference values. Various reference values provide equivalent laboratory rating. In addition to descriptive feedback, our approach allows calculation of technical test sensitivity and specificity, positive and negative predictive values, agreement rates, and κ values to guide corrective actions.

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

AimsPan-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...

Canadian Immunohistochemistry Quality Control (CIQC): An Academic Program Providing Proficiency Testing to Canadian Clinical Immunohistochemistry Laboratories

Canadian Immunohistochemistry Quality Control (CIQC): An Academic Program Providing Proficiency Testing to Canadian Clinical Immunohistochemistry Laboratories

Canadian Immunohistochemistry Quality Control: An Academic Program Providing Proficiency Testing to Canadian Clinical Immunohistochemistry Laboratories

Canadian Immunohistochemistry Quality Control: An Academic Program Providing Proficiency Testing to Canadian Clinical Immunohistochemistry Laboratories

Implementation of a Canadian External Quality Assurance Program for Breast Cancer Biomarkers

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 kappa 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.