1. Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation
- Author
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Diego Gabriel Dupouy, Markus Rechsteiner, Amy Jones, Alex Soltermann, Anne-Laure Leblond, Saska Brajkovic, University of Zurich, and Leblond, Anne-Laure
- Subjects
Proto-Oncogene Proteins B-raf ,Lung Neoplasms ,Skin Neoplasms ,DNA Mutational Analysis ,Microfluidics ,Adenocarcinoma of Lung ,610 Medicine & health ,DNA sequencing ,03 medical and health sciences ,0302 clinical medicine ,10049 Institute of Pathology and Molecular Pathology ,medicine ,Biomarkers, Tumor ,Humans ,Thyroid Neoplasms ,neoplasms ,Carcinoma, Renal Cell ,Melanoma ,biology ,High-Throughput Nucleotide Sequencing ,General Medicine ,medicine.disease ,Primary and secondary antibodies ,Molecular biology ,Immunohistochemistry ,Kidney Neoplasms ,Staining ,BRAF V600E ,2734 Pathology and Forensic Medicine ,Tissue sections ,Thyroid Cancer, Papillary ,030220 oncology & carcinogenesis ,Mutation (genetic algorithm) ,Colonic Neoplasms ,Mutation ,biology.protein ,030215 immunology - Abstract
OBJECTIVES Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas. METHODS For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS. RESULTS After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification. CONCLUSIONS Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality.
- Published
- 2019