Zusammenfassung
Signifikante Verbesserungen in der Technik der RNA-in-situ-Hybridisierung (RNA-ISH) in den zurückliegenden 5 Jahrzehnten haben neue Anwendungsfelder als attraktive Erweiterung zum diagnostischen Standardportfolio eröffnet. Im Gegensatz zu früheren Anwendungen sind aktuelle bDNA-Methoden höchst sensitiv und erlauben den Nachweis einzelner Moleküle am formalinfixierten und in Paraffin eingebetteten Gewebe ohne zusätzlichen Aufwand im Labor, da die Tests auf gängigen Laborautomaten laufen und im Lichtmikroskop ausgewertet werden können. Gegenüber molekularen Methoden wie RT-PCR oder Whole-genome-Analysen bleibt bei RNA-ISH die Gewebemorphologie erhalten und bietet so den entscheidenden Vorteil der Lokalisation der Zielzellen im Gewebe. Dieser Vorteil zeigt sich besonders bei sekretierten Proteinen und der Expression der Zielsequenz in mehreren Zelltypen. Erste klinische Studien haben RNA-ISH bereits erfolgreich zur Patientenselektion eingesetzt mit dem Ziel der Entwicklung zum „companion diagnostic“.
Neben dem Einsatz als Komplementärverfahren bei Entwicklung neuer Immunhistochemie(IHC)-Protokolle und als Ergänzung oder Alternative zur Immunhistochemie im Routineportfolio kommen durch die spezifische Nachweismöglichkeit von nichtcodierenden RNA-Spezies sowie Mutations- und Splicevarianten der RNA-in-situ-Hybridisierung eine besondere Bedeutung dort zu, wo geeignete Alternativen fehlen. Die insgesamt komplexere Anwendung erfordert neben der Entwicklung standardisierter Vorgehensweisen die Einbindung des Pathologen bei der Etablierung neuer Anwendungen und bei der Befundung in der Routinediagnostik.
Der vorliegende Artikel spannt den Bogen über die technische Entwicklung der RNA-in-situ-Hybridisierung bis hin zu aktuellen Anwendungsmöglichkeiten und berücksichtigt dabei die Erfahrungen der Autoren mit der Anwendung der Methodik in einem klinischen Auftragsforschungslabor.
Abstract
Significant improvements in the technology of RNA in situ hybridization (RNA-ISH) in the past five decades have opened up novel fields of its application as a valuable and an attractive adjunct to the portfolio of pathologist’s daily routine diagnostic practice.
In contrast to the former methodology, the current bDNA-based technology is not only easier to handle but also considerably more sensitive, enabling single-target molecule detection in formalin-fixed and paraffin-embedded tissue specimens without significant effort by both the lab and the evaluating pathologist, as assays can be run on standard automated staining devices and evaluated by light microscopy. Compared to molecular methods like RT-PCR and whole-genome analysis, RNA-ISH maintains tissue integrity thus offering the invaluable advantage of localization of target cells especially in relation to secreted proteins and expression of the target sequence in multiple cell types. The first clinical trials implementing RNA-ISH for patient stratification and selection are in progress and already led to the first drug approvals based on its use as a CDx test.
In addition to its role as a complementary method for the establishment of novel IHC procedures or as an addition or replacement to IHC in the standard routine portfolio, RNA-ISH has gained special importance for its capacity to detect noncoding RNA species or mutation or splice variants, where no alternative procedures are available. This more complex application requires development of standardized procedures and involvement of the pathologist during assay establishment and for routine specimen evaluation.
The present article reviews the development of RNA-ISH from its early uses to its current applications in research and diagnostics based on the authors’ considerable experience of applying it as tool in a biopharmaceutical research organization.
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Schipper, C., Zielinski, D. RNA-in-situ-Hybridisierung: Technologie, Möglichkeiten und Anwendungsbereiche. Pathologe 41, 563–573 (2020). https://doi.org/10.1007/s00292-020-00839-z
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DOI: https://doi.org/10.1007/s00292-020-00839-z