Automated chromogenic multiplexed immunohistochemistry assay for diagnosis and predictive biomarker testing in non-small cell lung cancer
Introduction
Current standards of care of non-small cell lung cancer (NSCLC) patients assign therapeutic decisions on the basis of specific histologic and genomic/immune-based characteristics of the patient’s tumor [1]. NSCLC must be classified into different histotypes [e.g., lung adenocarcinoma (LADC) versus lung squamous cell carcinoma (LSCC)] as this determines eligibility for biomarker testing and then therapeutic strategies [2]. The current list of indispensable biomarkers associated to approved therapies worldwide include EGFR mutations, ALK and ROS1 rearrangements, the BRAF V600E mutation in LADC and PD-L1 expression in tumor cells in both LADC and LSCC [3]. However, as the amount of histologic and genomic/immune-based information required has grown recently, analysis of tumor tissue often rests on small tissue samples [4].
To address the increase in the essential clinical need for high-output diagnostic and predictive biomarker testing in NSCLC, we developed an automated brightfield multiplexed chromogen-based immunohistochemistry (mIHC) assay that could be readily integrated into the routine clinical setting.
Section snippets
Tumor samples
For this study, formalin-fixed paraffin-embedded samples from 18 patients with NSCLC, previously characterized for histologic [LADC, n = 3; LSCC, n = 3; or lung adenosquamous carcinoma, LASC, n = 3) and genomic/immune features (ALK, n = 3; ROS1, n = 3; BRAF V600E, n = 3; PD-L1, n = 3), were obtained from the Hospital-Integrated Biobank (BB-0033-00025, Pasteur Hospital, Nice) after IRB approval and informed written consent.
Multiplex immunohistochemistry
Two mIHC assays were developed: i) the mIHC for diagnosis and
Results
The staining pattern of individual DAB-stained slides was compared with the matched single colored chromogen slide and the mIHC slide (Fig. 1). Comparison of mIHC staining panels with single conventional IHC DAB staining showed equivalent sensitivity and specificity for histotypes and genomic/immune markers throughout the 18 NSCLC cases included in the study (κ scores = 1, respectively). In addition, κ scores for inter-pathologist agreement were 1 for all dichotomous markers. The intra-class
Discussion
To our knowledge, our group is the first to report the technical development and feasibility of two sensitive mIHC assays to comprehensively characterize NSCLC major histotypes using FDA-cleared predictive biomarkers.
The mIHC assays demonstrated no antigenicity loss, steric interference, and/or increased cross-reactivity. The assays rely on standard antigen retrieval and automated staining protocols, limiting the need for validation strategies, providing an alternative method in a clinical
Author contributions
Study concept and design: Ilie M, Hofman P.
Acquisition, analysis, and interpretation of data: Ilie M, Hamila M, Hofman V, Hofman P.
Drafting of the manuscript: Ilie M, Hofman P.
Critical revision of the manuscript for important intellectual content: All authors.
Obtained funding: Hofman P.
Administrative, technical, or material support: Beaulande M, Hamila M, Erb G.
Study supervision: Ilie M, Hofman P.
Funding
The study was supported by Université Côte d’Azur, Initiative of Excellence IDEX; Cancéropole PACA; “Conseil Départemental des Alpes-Maritimes”, France; “Région Provence Alpes-Côte d’Azur”, France. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Competing interests
MI has received honoraria from AstraZeneca and Boehringher-Ingelheim. PH has received honoraria from AstraZeneca, Roche, Bristol-Myers Squibb, and Merck & Co. Inc. MB and GE are employees of Roche Diagnostics France. There are no other competing financial interest or other conflicts.
Acknowledgements
None.
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