Charles J. Robbins, BS; Mengi He, BS; Nay Chan, PhD; Revekka Khaimova, BS; Katherine Bates, BS; Ioannis P. Trontzas, MD; Liam Scott, BS; Myrto Moutafi, MD, PhD; Chandra B. Coleman, BS; Salilsha Hill, BS; Daniel C. Liebler, PhD; Regan Fulton, MD, PhD; and David L. Rimm, MD, PhD; doi: https://doi.org/10.1200/PO-25-00128

Purpose

Accurate quantification of human epidermal growth factor receptor 2 (HER2) and trophoblast cell-surface antigen 2 (TROP2) expression could aid in identifying patients with cancer likely to benefit from emerging HER2 and TROP2 antibody-drug conjugate (ADC) therapies or potentially help oncologists choose which drug to use first, on the basis of the level of the ADC target in the tumor. We developed a standardized multiplex quantitative immunofluorescence (QIF) assay to simultaneously measure HER2 and TROP2 protein levels in cancer tissue.

Materials and Methods

A multiplex QIF assay was optimized on tissue microarrays (TMAs) by selecting optimal antibody clones and concentrations to achieve maximal signal-to-noise ratios. We create and release Qymia, a QuPath extension to enable simultaneous molecular compartmentalization and fluorescence quantification in TMAs and whole-slide images. Calibration curves, generated from cell line microarrays with HER2/TROP2 measured by mass spectrometry, were used to convert QIF signal into protein concentrations (attomoles/mm2). The validated assay was applied to a serial collection of 323 breast cancer specimens in TMA format to characterize HER2 and TROP2 expression distributions.

Results

The assay demonstrated linearity across a wide dynamic range of biomarker expression with strong interassay and interoperator reproducibility. Application to 323 breast cancer TMA specimens revealed a weak inverse correlation between HER2 and TROP2 (r = –0.17; P = .001). HER2 was detectable in approximately 85% of TMA cores, including 51% of triple-negative breast cancer TMA cores. TROP2 was detectable in over 96% of specimens across all subtypes.

Conclusion

This multiplex immunofluorescence assay provides an approach to accurately and precisely measure HER2/TROP2 levels within breast cancer tissue and compare relative levels of target expression in a breast cancer tissue population. This assay is now ready for studies to assess clinical validity and utility.

Emily Deutschman, Regan Fulton, Callum M. Sloss; Evaluation of Laboratory-Derived Immunohisochemical Assays for Folate Receptor α Expression in Epithelial Ovarian Cancer and Comparison With a Companion Diagnostic. Arch Pathol Lab Med 2025; doi: https://doi.org/10.5858/arpa.2024-0210-OA

Context

The VENTANA FOLR1 (FOLR1-2.1) RxDx (FOLR1 CDx) assay, developed by Roche Tissue Diagnostics, is a Food and Drug Administration–approved immunohistochemical assay intended for use in the assessment of folate receptor α (FRα) expression in formalin-fixed, paraffin-embedded epithelial ovarian, fallopian tube, and primary peritoneal tumor specimens. No published reports have compared the performance of other available FRα antibodies with the approved FOLR1 CDx.

Objective

To assess the performance of research FRα laboratory-developed tests compared with the FOLR1 CDx.

Design

The performance of 6 FRα-targeting antibodies was compared with the approved FOLR1 CDx in normal fallopian tube specimens. Two antibodies were selected for further assessment and compared with the FOLR1 CDx in ovarian tumor specimens.

Results

Of the 6 antibodies tested, 4 displayed a lack of specific membrane staining and/or high background, whereas 2 antibodies, produced by Leica Biosystems and Biocare Medical, respectively, exhibited specific and sensitive FRα staining. When assessed for their ability to correctly identify FRα-positive samples (per the FOLR1 CDx label, ≥75% of viable tumor cells with moderate and/or strong membranous staining intensity), both assays overpredicted FRα positivity compared with the FOLR1 CDx in archival ovarian tumor samples.

Conclusions

These data highlight the need for caution in antibody selection when developing immunohistochemistry-based assays, as some antibodies failed to cleanly and specifically identify FRα expression. We identified 2 antibodies appropriate for further investigation; however, as developed, these antibodies may overselect patients for treatment with FRα-targeted therapies.

Moutafi M, Robbins CJ, Yaghoobi V, Fernandez AI, Martinez-Morilla S, Xirou V, Bai Y, Song Y, Gaule P, Krueger J, Bloom K, Hill S, Liebler DC, Fulton R, Rimm DL. Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer. Lab Invest. 2022 May 20. doi: 10.1038/s41374-022-00804-9. Epub ahead of print. PMID: 35595825.

Abstract

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUATM method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the

levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Aung, T.N., Acs, B., Warrell, J. et al. A new tool for technical standardization of the Ki67 immunohistochemical assay. Mod Pathol 34, 1261–1270 (2021).  https://doi.org/10.1038/s41379-021-00745-6

Abstract

Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67+) with Sf9 (Spodoptera frugiperda) (Ki67-) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.

Fulton, Regan. (2021). Getting a Grip on Ki-67. Applied Immunohistochemistry & Molecular Morphology : AIMM. 29. 83-85.  10.1097/PAI.0000000000000908. 

Tretiakova, M., Fulton, R., Kocherginsky, M. et al. Concordance study of PD-L1 expression in primary and metastatic bladder carcinomas: comparison of four commonly used antibodies and RNA expression. Mod Pathol 31, 623–632 (2018). https://doi.org/10.1038/modpathol.2017.188

Abstract

Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of correlation between expression and clinical outcomes and immunotherapy response may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72–0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86–0.94). Manual scores were highly concordant with automated Aperio scores (0.94–0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.

Cheung, Carol & D'Arrigo, Corrado & Dietel, Manfred & Francis, Glenn & Fulton, Regan & Gilks, C. & Hall, Jacqueline & Hornick, Jason & Ibrahim, Merdol & Marchetti, Antonio & Miller, Keith & Krieken, Han & Nielsen, Søren & Swanson, Paul & Taylor, Clive & Vyberg, Mogens & Zhou, Xiaoge & Torlakovic, Emina. (2016). Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 4. Applied Immunohistochemistry & Molecular Morphology. 25. 1. 10.1097/PAI.0000000000000469. 

Abstract

The numbers of diagnostic, prognostic, and predictive immunohistochemistry (IHC) tests are increasing; the im- plementation and validation of new IHC tests, revalidation of existing tests, as well as the on-going need for daily quality as- surance monitoring present significant challenges to clinical laboratories. There is a need for proper quality tools, specifically tissue tools that will enable laboratories to successfully carry out these processes. This paper clarifies, through the lens of labo- ratory tissue tools, how validation, verification, and revalidation of IHC tests can be performed in order to develop and maintain high quality “fit-for-purpose” IHC testing in the era of precision medicine. This is the final part of the 4-part series “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

Sheffield BS, Fulton R, Kalloger SE, Milne K, Geller G, Jones M, Jacquemont C, Zachara S, Zhao E, Pleasance E, Laskin J, Jones SJ, Marra MA, Yip S, Nelson BH, Gown AM, Ho C, Ionescu DN. Investigation of PD-L1 Biomarker Testing Methods for PD-1 Axis Inhibition in Non-squamous Non-small Cell Lung Cancer. J Histochem Cytochem. 2016 Oct;64(10):587-600. doi: 10.1369/0022155416665338. Epub 2016 Sep 2. PMID: 27591097; PMCID: PMC5037503.

Abstract

Inhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rapidly being incorporated into the treatment of non–small cell lung cancers (NSCLCs). Despite clear benefits to certain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD1-axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA ISH. Additional cases with whole genome and transcriptome data were assessed for molecular correlates of PD-L1 overexpression. Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results. There is significant discordance between the PD-L1 status of primary tumors and lymph node metastases. RNA ISH may be a useful adjunct to complement PD-L1 IHC testing. 

Fitzgibbons PL, Bradley LA, Fatheree LA, Alsabeh R, Fulton RS, Goldsmith JD, Haas TS, Karabakhtsian RG, Loykasek PA, Marolt MJ, Shen SS, Smith AT, Swanson PE; College of American Pathologists Pathology and Laboratory Quality Center. Principles of analytic validation of immunohistochemical assays: Guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Arch Pathol Lab Med. 2014 Nov;138(11):1432-43. doi: 10.5858/arpa.2013-0610-CP.  Epub 2014 Mar 19. PMID: 24646069.

Abstract

Context: Laboratories must validate all assays before they can be used to test patient specimens, but currently there are no evidence-based guidelines regarding validation of immunohistochemical assays. Objective: To develop recommendations for initial analytic validation and revalidation of immunohistochemical assays. Design: The College of American Pathologists Pathology and Laboratory Quality Center convened a panel of pathologists and histotechnologists with expertise in immunohistochemistry to develop validation recommendations. A systematic evidence review was conducted to address key questions. Electronic searches identified 1463 publications, of which 126 met inclusion criteria and were extracted. Individual publications were graded for quality, and the key question findings for strength of evidence. Recommendations were derived from strength of evidence, open comment feedback, and expert panel consensus. Results: Fourteen guideline statements were established to help pathology laboratories comply with validation and revalidation requirements for immunohistochemical assays. Conclusions: Laboratories must document successful analytic validation of all immunohistochemical tests before applying to patient specimens. The parameters for cases included in validation sets, including number, expression levels, fixative and processing methods, should take into account intended use and should be sufficient to ensure that the test accurately measures the analyte of interest in specimens tested in that laboratory. Recommendations are also provided for confirming assay performance when there are changes in test methods, reagents, or equipment.