Elsevier

Lung Cancer

Volume 124, October 2018, Pages 90-94
Lung Cancer

Automated chromogenic multiplexed immunohistochemistry assay for diagnosis and predictive biomarker testing in non-small cell lung cancer

https://doi.org/10.1016/j.lungcan.2018.07.037Get rights and content

Highlights

  • The multiplexed IHC panel for diagnosis and immunophenotyping (MIDI) contains TTF1, p40, PD-L1, and pan-Keratin antibodies.

  • The multiplexed IHC panel for molecular profiling (MIMP) contains ALK, ROS1 and BRAFV600E antibodies.

  • The assays showed no without antigenicity loss, steric interference or increased cross-reactivity.

  • The assays rely on standard antigen retrieval and automated staining protocols.

Abstract

Objectives

The current challenge in the management of non-small cell lung cancer (NSCLC) in pathology laboratories is to combine immunohistochemistry (IHC) and molecular approaches on increasingly smaller biopsies and the need to reserve a fair amount of tumor material for molecular analyses with increasingly larger panels. The latest lung cancer classification, especially in the setting of poorly differentiated tumors, requires an IHC workup to allow for accurate diagnosis and also to preserve as much tissue as possible for molecular testing. Thus, it is recommended to reduce use of the term NSCLC not otherwise specified as much as possible and classify tumors according to their specific histologic subtype. This implies limiting the number of tissue slides despite the existence of specific and sensitive biomarkers (ALK, ROS1, BRAF V600E, PD-L1) and the obligation to distinguish lung adenocarcinoma (TTF-1 positive) from squamous cell carcinoma (p40 positive).

Materials and Methods

Samples from 18 patients with NSCLC, previously characterized for histologic and genomic/immune features, were included. Two multiplexed IHC assays were developed, for diagnosis and immunophenotyping including TTF1, p40, PD-L1, and pan-Keratin antibodies, and for molecular profiling panel including ALK, ROS1 and BRAF V600E antibodies.

Results

We developed two sensitive multiplexed IHC assays to comprehensively characterize major NSCLC histotypes and FDA-cleared predictive biomarkers, without antigenicity loss, steric interference or increased cross-reactivity. The assays rely on standard antigen retrieval and automated staining protocols, limiting the need for validation strategies.

Conclusion

Our multiplexed IHC approach provides a unique sample-sparing tool to characterize limited tissue samples in lung oncology and making it an alternative method in the clinical setting for therapeutic decision making of advanced NSCLC, provided that validation in a larger population is performed.

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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