Skip to main content
SpringerLink
Log in

RNA-in-situ-Hybridisierung: Technologie, Möglichkeiten und Anwendungsbereiche

RNA in situ hybridization: technology, potential, and fields of application

  • Schwerpunkt: In-situ-Hybridisierung
  • Published:
Der Pathologe Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Abb. 1
Abb. 2
Abb. 3
Abb. 4

Literatur

  1. www.acdbio.com, aufgerufen am 30. Mai 2020

  2. Alvarado MD, Prasad C, Rothney M et al (2015) A prospective comparison of the 21-gene recurrence score and the PAM50-based prosigna in estrogen receptor-positive early-stage breast cancer. Adv Ther 32:1237–1247. https://doi.org/10.1007/s12325-015-0269-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Anderson CM, Laeremans A, Wang XM et al (2018) Visualizing genetic variants, short targets, and point mutations in the morphological tissue context with an RNA in situ hybridization assay. J Vis Exp. https://doi.org/10.3791/58097

    Article  PubMed  PubMed Central  Google Scholar 

  4. Anderson CM, Zhang B, Miller M et al (2016) Fully automated RNAscope in situ hybridization assays for formalin-fixed paraffin-embedded cells and tissues. J Cell Biochem 117:2201–2208. https://doi.org/10.1002/jcb.25606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Antonarakis ES, Lu C, Luber B et al (2015) Androgen receptor splice variant 7 and efficacy of Taxane chemotherapy in patients with metastatic castration-resistant prostate cancer. JAMA Oncol 1:582–591. https://doi.org/10.1001/jamaoncol.2015.1341

    Article  PubMed  PubMed Central  Google Scholar 

  6. Baena-Del Valle JA, Zheng Q et al (2017) Rapid Loss of RNA Detection by In Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides. Am J Clin Pathol 148:398–415. https://doi.org/10.1093/AJCP/AQX094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Baker AM, Huang W, Wang XM et al (2017) Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution. Nat Commun. https://doi.org/10.1038/s41467-017-02295-5

    Article  PubMed  PubMed Central  Google Scholar 

  8. Baker M (2015) Reproducibility crisis: Blame it on the antibodies. Nature 521:274–276

    Article  CAS  Google Scholar 

  9. Bingham V, McIlreavey L, Greene C et al (2017) RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples. Oncotarget 8:93392–93403

    Article  Google Scholar 

  10. Bradbury A, Plückthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518:27–29. https://doi.org/10.1038/518027

    Article  CAS  PubMed  Google Scholar 

  11. Baumgart SJ, Nevedomskaya E, Haendler B (2019) Dysregulated transcriptional control in prostate cancer. Int J Mol Sci. https://doi.org/10.3390/ijms20122883

    Article  PubMed  PubMed Central  Google Scholar 

  12. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. Biotechniques 48:197–209. https://doi.org/10.2144/000113382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cassidy A, Jones J (2014) Developments in in situ hybridisation. Methods 70:39–45. https://doi.org/10.1016/j.ymeth.2014.04.006

    Article  CAS  PubMed  Google Scholar 

  14. Cejalvo JM, Jacob W, Fleitas Kanonnikoff T et al (2019) A phase Ib/II study of HER3-targeting lumretuzumab in combination with carboplatin and paclitaxel as first-line treatment in patients with advanced or metastatic squamous non-small cell lung cancer. ESMO Open. https://doi.org/10.1136/esmoopen-2019-000532

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chen CC, Wada K, Jarvis ED (2012) Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue. J Vis Exp. https://doi.org/10.3791/3764

    Article  PubMed  PubMed Central  Google Scholar 

  16. www.agilent.com aufgerufen am 30. Mai 2020

  17. Darby IA, Bisucci T, Desmoulière A et al (2006) In situ hybridization using cRNA probes: isotopic and nonisotopic detection methods. Methods Mol Biol 326:17–31. https://doi.org/10.1385/1-59745-007-3:17

    Article  CAS  PubMed  Google Scholar 

  18. Datta S, Malhotra L, Dickerson R et al (2015) Laser capture microdissection: big data from small samples. Histol Histopathol 30:1255–1269. https://doi.org/10.14670/HH-11-622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Del Re M, Biasco E, Crucitta S et al (2017) The detection of androgen receptor splice variant 7 in plasma-derived exosomal RNA strongly predicts resistance to hormonal therapy in metastatic prostate cancer patients. Eur Urol 71:680–687. https://doi.org/10.1016/j.eururo.2016.08.012

    Article  CAS  PubMed  Google Scholar 

  20. Felsberg J, Hentschel B, Kaulich K et al (2017) Epidermal growth factor receptor variant III (EGFRvIII) Positivity in EGFR-amplified glioblastomas: prognostic role and comparison between primary and recurrent tumors. Clin Cancer Res 23:6846–6855. https://doi.org/10.1158/1078-0432.CCR-17-0890

    Article  CAS  PubMed  Google Scholar 

  21. Ferreira HJ, Esteller M (2018) Non-coding RNAs, epigenetics, and cancer: tying it all together. Cancer Metastasis Rev 37:55–73. https://doi.org/10.1007/s10555-017-9715-8

    Article  CAS  PubMed  Google Scholar 

  22. Figueroa JM, Skog J, Akers J et al (2017) Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients. Neuro Oncol 19:1494–1502. https://doi.org/10.1093/neuonc/nox085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Journal 63:378–383

    CAS  Google Scholar 

  24. Hannouf MB, Zaric GS, Blanchette P et al (2020) Cost-effectiveness analysis of multigene expression profiling assays to guide adjuvant therapy decisions in women with invasive early-stage breast cancer. Pharmacogenomics J 20:27–46. https://doi.org/10.1038/s41397-019-0089-x

    Article  CAS  PubMed  Google Scholar 

  25. Kwa M, Makris A, Esteva FJ (2017) Clinical utility of gene-expression signatures in early stage breast cancer. Nat Rev Clin Oncol 14:595–610. https://doi.org/10.1038/nrclinonc.2017.74

    Article  CAS  PubMed  Google Scholar 

  26. www.leica.com, aufgerufen am 30. Mai 2020

  27. https://www.leicabiosystems.com/news-events/news-details/article/leica-biosystems-introduces-acds-rnascope-technology-for-use-on-bond-iii/News/detail/, abgerufen am 27. Juni 2020

  28. Lang G (2013) Histotechnik: Paraxislehrbuch für die Biomedizinische Analytik, 2. Aufl. Springer, Berlin Heidelberg

    Book  Google Scholar 

  29. Mahmood R, Mason I (2008) In-situ hybridization of radioactive riboprobes to RNA in tissue sections. Methods Mol Biol 461:675–686. https://doi.org/10.1007/978-1-60327-483-8_45

    Article  CAS  PubMed  Google Scholar 

  30. Mirghani H, Casiraghi O, Amen F et al (2015) Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice. Mod Pathol 28:1518–1527. https://doi.org/10.1038/modpathol.2015.113

    Article  CAS  PubMed  Google Scholar 

  31. O’Leary JJ, Chetty R, Graham AK et al (1996) In situ PCR: pathologist’s dream or nightmare? J Pathol 178:11–20

    Article  Google Scholar 

  32. Pardue ML, Gall JG (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc N A S 64:600–604

    Article  CAS  Google Scholar 

  33. Peng WX, Koirala P, Mo YY (2017) LncRNA-mediated regulation of cell signaling in cancer. Oncogene 36:5661–5667. https://doi.org/10.1038/onc.2017.184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Player AN, Shen LP, Kenny D et al (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49:603–611

    Article  CAS  Google Scholar 

  35. Prat A, Galván P, Jimenez B et al (2016) Prediction of response to neoadjuvant chemotherapy using core needle biopsy samples with the prosigna assay. Clin Cancer Res 22:560–566. https://doi.org/10.1158/1078-0432.CCR-15-0630

    Article  CAS  PubMed  Google Scholar 

  36. https://www.prnewswire.com/news-releases/fda-grants-merrimack-fast-track-designation-for-seribantumab-mm-121-in-non-small-cell-lung-cancer-300294237.html, abgerufen ab 30. Mai 2020

  37. Rakovitch E, Gray R, Baehner FL et al (2018) Refined estimates of local recurrence risks by DCIS score adjusting for clinicopathological features: a combined analysis of ECOG-ACRIN E5194 and Ontario DCIS cohort studies. Breast Cancer Res Treat 169:359–369. https://doi.org/10.1007/s10549-018-4693-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rakovitch E, Nofech-Mozes S, Hanna W et al (2015) A population-based validation study of the DCIS Score predicting recurrence risk in individuals treated by breast-conserving surgery alone. Breast Cancer Res Treat 152:389–398. https://doi.org/10.1007/s10549-015-3464-6

    Article  PubMed  PubMed Central  Google Scholar 

  39. Roe APA, Catherine J, Momin T et al (2019) RNA in situ hybridization for Epstein-Barr virus and cytomegalovirus: comparison with in situ hybridization and immunohistochemistry. Appl Immunohistochem Mol Morphol. https://doi.org/10.1097/PAI.0000000000000568

    Article  PubMed  Google Scholar 

  40. Rooper LM, Gandhi M, Bishop JA, Westra WH (2016) RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization. Oral Oncol 55:11–16. https://doi.org/10.1016/j.oraloncology.2016.02.008

    Article  CAS  PubMed  Google Scholar 

  41. https://www.the-scientist.com/sponsored-webinars/an-urgent-need-for-validating-and-characterizing-antibodies-37623, abgerufen am 16. Juni 2020

  42. Schoeberl B, Faber AC, Li D et al (2010) An ErbB3 antibody, MM-121, is active in cancers with ligand dependent activation. Cancer Res 70(6):2485–2494. https://doi.org/10.1158/0008-5472.CAN-09-3145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sestak I, Buus R, Cuzick J et al (2018) Comparison of the performance of 6 prognostic signatures for estrogen receptor-positive breast cancer: a secondary analysis of a randomized clinical trial. JAMA Oncol 4:545–553. https://doi.org/10.1001/jamaoncol.2017.5524

    Article  PubMed  PubMed Central  Google Scholar 

  44. Sfanos KS, Yegnasubramanian S, Nelson WG et al (2019) If this is true, what does it imply? How end-user antibody validation facilitates insights into biology and disease. Asian J Urol 6:10–25. https://doi.org/10.1016/j.ajur.2018.11.006

    Article  PubMed  Google Scholar 

  45. Simmons O, Bolanis EM, Wang J et al (2014) In situ hybridization (both radioactive and nonradioactive) and spatiotemporal gene expression analysis. Methods Mol Biol 1194:225–244. https://doi.org/10.1007/978-1-4939-1215-5_12

    Article  CAS  PubMed  Google Scholar 

  46. Soliman H, Flake DD, Magliocco A et al (2019) Predicting expected absolute chemotherapy treatment benefit in women with early-stage breast cancer using Endopredict, an integrated 12-gene Clinicomolecular assay. JCO Precis Oncol 3:1–10

    Google Scholar 

  47. Sotiriou C, Piccart MJ (2007) Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer 7:545–553

    Article  CAS  Google Scholar 

  48. Speel EJ, Hopman AH, Komminoth P (2006) Tyramide signal amplification for DNA and mRNA in situ hybridization. Methods Mol Biol 326:33–60. https://doi.org/10.1385/1-59745-007-3:33

    Article  CAS  PubMed  Google Scholar 

  49. Speel EJ, Saremaslani P, Roth J et al (1998) Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem Cell Biol 110(6):571–577. https://doi.org/10.1007/s004180050319

    Article  CAS  PubMed  Google Scholar 

  50. Storer PD, DeLucia T (2006) Semiquantitative in situ hybridization using radioactive probes to study gene expression in motoneuron populations. Methods Mol Biol 326:247–254. https://doi.org/10.1385/1-59745-007-3:247

    Article  CAS  PubMed  Google Scholar 

  51. Strell C, Hilscher MM, Laxman N et al (2019) Placing RNA in context and space—methods for spatially resolved transcriptomics. FEBS J 286:1468–1481. https://doi.org/10.1111/febs.14435

    Article  CAS  PubMed  Google Scholar 

  52. Taube JM, Akturk G, Angelo M et al (2020) The Society for Immunotherapy in Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother Cancer. https://doi.org/10.1136/jitc-2019-000155

    Article  PubMed  PubMed Central  Google Scholar 

  53. Thangarajah F, Eichler C, Fromme J et al (2019) The impact of EndoPredict ® on decision making with increasing oncological work experience: can overtreatment be avoided? Arch Gynecol Obstet 299:1437–1442. https://doi.org/10.1007/s00404-019-05097-w

    Article  CAS  PubMed  Google Scholar 

  54. Tonella L, Giannoccaro M, Alfieri S et al (2017) Gene expression signatures for head and neck cancer patient stratification: are results ready for clinical application? Curr Treat Options Oncol. https://doi.org/10.1007/s11864-017-0472-2

    Article  PubMed  Google Scholar 

  55. van den Bent MJ, Gao Y, Kerkhof M et al (2015) Changes in the EGFR amplification and EGFRvIII expression between paired primary and recurrent glioblastomas. Neuro Oncol 17:935–941. https://doi.org/10.1093/neuonc/nov013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. www.ventanamed.com, aufgerufen am 30. Mai 2020

  57. Wang F, Flanagan J, Su N et al (2012) RNAscope—A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14:22–29. https://doi.org/10.1016/j.jmoldx.2011.08.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Warford A (2016) In situ hybridisation: technologies and their application to understanding disease. Prog Histochem Cytochem 50:37–48. https://doi.org/10.1016/j.proghi.2015.12.001

    Article  PubMed  Google Scholar 

  59. Zielinski D (2019) Digitalisierung und Multiplex-IHC als prädiktive Biomarker für neue Immuntherapeutika : Neue Herausforderungen an die Immunhistochemie. Pathologe 40:256–263. https://doi.org/10.1007/s00292-019-0607-2

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Targos Molecular Pathology GmbH, Germaniastraße 7, 34119, Kassel, Deutschland

    Christina Schipper &  Dirk Zielinski

Authors
  1. Christina Schipper
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk Zielinski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dirk Zielinski.

Ethics declarations

Interessenkonflikt

C. Schipper und D. Zielinski geben an, dass kein Interessenkonflikt besteht.

Für diesen Beitrag wurden von den Autoren keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien.

Additional information

Schwerpunktherausgeber

H. U. Schildhaus, Essen

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00292-020-00839-z

Schlüsselwörter

Keywords

Search

Navigation