Assessment of Predictive Biomarkers in Cancer Tissues using Micro-Immunohistochemistry followed by DNA Sequencing
A.L. Leblond (1), D. Machiraju (1), R. Casanova (1), E. Bianchi (1), M. Rechsteiner (1), P. Wild (1), A. Jones (2), D. Heintze (2), A.T. Ciftlik (2), A. Soltermann (1)
1 Department of Pathology and Molecular Pathology, University Hospital, Zurich, Switzerland
2 Lunaphore Technologies SA, EPFL Innovation Park, Lausanne, Switzerland
Background: Limited access to cancer tissues strikes the need to combine on the same clinical specimen immunohistochemistry (IHC) and molecular profiling. Micro-IHC using the Microfluidic Tissue Processor (MTP) allows a fast and reliable staining that could be used for diagnostic evaluation on formalin-fixed paraffin-embedded (FFPE) sections of cancer tissues. This study aims at combining on a unique FFPE tissue section MTP-based micro-IHC and molecular analysis.
Methods: FFPE tissue sections from BRAF V600E mutated cancers were immunohistochemically stained for pan-cytokeratin and BRAF V600E mutation using MTP at room temperature. Positively stained cells were scored, dissected, processed for DNA extraction and next generation sequencing (NGS).
Results: MTP-based micro-IHC was comparable to automated platform-based IHC. MTP-stained area displayed precise borders confining positive cells in a 17x17mm2 zone and gradient of positive- towards negative- stained area across 38.5±8 micrometers. Titration curve experiments by MTP indicated that H score evolution fitted to an exponential model and strong immunoreactivity required 4-minute incubation time with primary antibody. Sufficient amounts of DNA (from 4.34±1ng /cell block to 152±27ng /surgical specimen) allowed to detect BRAF V600E mutation by NGS.
Conclusion: MTP-based micro-IHC is a relevant approach to combine on a unique FFPE tissue section minute range IHC and NGS.