Why Cytology for Molecular Testing? Pros and Cons

  • Lukas BubendorfEmail author


In this era of precision medicine, molecular testing of tumor specimens has become indispensible in order to seek opportunities for optimized and personalized treatment in every cancer patient. Since most pivotal trials leading to the approval of targeted drugs rely on tissue specimens for predictive marker testing, biomarker testing on tissue biopsy specimens has long been considered the gold standard. In the real diagnostic world, however, malignant tumors are often diagnosed by cytology alone. This necessitates translating protocols for biomarker testing for the use in cytology, which comes with advantages and challenges. There is a general consensus that the quality of DNA obtained from ethanol-based cytological specimens is excellent allowing for highly accurate mutation analysis. Enrichment of tumor cells by laser capture microdissection is a powerful and robust method to rescue tumor cytological specimens with a low tumor cell proportion for DNA-based analysis. The fact that cells on smears, cytospins, or liquid-based cytology (LBC) preparations are intact makes them also an ideal substrate for the analysis of chromosome and gene copy numbers or gene rearrangements by fluorescence in situ hybridization. In contrast, the great variability of preparation, fixation, and staining methods in cytology is a hurdle toward standardized protocols for predictive immunohistochemistry. This can be partly overcome by cell blocks that can be processed like formalin-fixed and paraffin-embedded tissue specimens for biomarker testing. Although there is need for more standardization in protocols of molecular testing on cytological specimens, the utility of cytology for biomarker testing is now undisputed.


Molecular testing Cytology FNA Mutation analysis Nuclear truncation Cell block Fish Laser microdissection DNA quality 


  1. 1.
    Kerr KM, Bubendorf L, Edelman MJ, Marchetti A, Mok T, Novello S, et al. Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer. Ann Oncol. 2014;25(9):1681–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Roy-Chowdhuri S, Aisner DL, Allen TC, Beasley MB, Borczuk A, Cagle PT, et al. Biomarker testing in lung carcinoma cytology specimens: a perspective from members of the pulmonary pathology society. Arch Pathol Lab Med. 2016. Apr 15 (Epub ahead of print).Google Scholar
  3. 3.
    Bubendorf L, Lantuejoul S, de Langen AJ, Thunnissen E. Nonsmall cell lung carcinoma: diagnostic difficulties in small biopsies and cytological specimens: number 2 in the series “Pathology for the clinician” edited by Peter Dorfmuller and Alberto Cavazza. Eur Respir Rev. 2017;26(144). Scholar
  4. 4.
    da Cunha Santos G. Standardizing preanalytical variables for molecular cytopathology. Cancer Cytopathol. 2013;121(7):341–3.CrossRefPubMedGoogle Scholar
  5. 5.
    Moelans CB, Oostenrijk D, Moons MJ, van Diest PJ. Formaldehyde substitute fixatives: effects on nucleic acid preservation. J Clin Pathol. 2011;64(11):960–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Cheng L, Zhang S, MacLennan GT, Williamson SR, Davidson DD, Wang M, et al. Laser-assisted microdissection in translational research: theory, technical considerations, and future applications. Appl Immunohistochem Mol Morphol. 2013;21(1):31–47.PubMedGoogle Scholar
  7. 7.
    Bubendorf L, Savic S, Ruiz C. Molecular techniques. In: Bibbo M, Wilbur DC, editors. Comprehensive cytopathology. 4th ed. Philadelphia: Elsevier Saunders; 2015. p. 912–25.Google Scholar
  8. 8.
    Sauter G, Moch H, Moore D, Carroll P, Kerschmann R, Chew K, et al. Heterogeneity of erbB-2 gene amplification in bladder cancer. Cancer Res. 1993;53(10 Suppl):2199–203.PubMedGoogle Scholar
  9. 9.
    Sauter G, Feichter G, Torhorst J, Moch H, Novotna H, Wagner U, et al. Fluorescence in situ hybridization for detecting erbB-2 amplification in breast tumor fine needle aspiration biopsies. Acta Cytol. 1996;40(2):164–73.CrossRefPubMedGoogle Scholar
  10. 10.
    Savic S, Bubendorf L. Common fluorescence in situ hybridization applications in cytology. Arch Pathol Lab Med. 2016;140(12):1323–30.CrossRefPubMedGoogle Scholar
  11. 11.
    Zlobec I, Raineri I, Schneider S, Schoenegg R, Grilli B, Herzog M, et al. Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens. Lung Cancer. 2010;68(2):192–7.CrossRefPubMedGoogle Scholar
  12. 12.
    Vlajnic T, Somaini G, Savic S, Barascud A, Grilli B, Herzog M, et al. Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology. Cancer Cytopathol. 2014;122(8):627–34.CrossRefPubMedGoogle Scholar
  13. 13.
    Zhou F, Moreira AL. Lung carcinoma predictive biomarker testing by immunoperoxidase stains in cytology and small biopsy specimens: advantages and limitations. Arch Pathol Lab Med. 2016;140(12):1331–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Lee GD, Lee SE, Oh DY, Yu DB, Jeong HM, Kim J, et al. MET Exon 14 skipping mutations in lung adenocarcinoma: clinicopathologic implications and prognostic values. J Thorac Oncol. 2017;12(8):1233–46.CrossRefPubMedGoogle Scholar
  15. 15.
    Bubendorf L, Buttner R, Al-Dayel F, Dietel M, Elmberger G, Kerr K, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch. 2016;469(5):489–503.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bubendorf L, Lantuejoul S, Yatabe Y. Analysis in cytology. In: Tsao MS, Hirsch FR, Yatabe Y, editors. IASLC Atlas of ALK and ROS1 testing in lung cancer. 2nd ed. North Fort Myers: International Association for the Study of Lung Cancer, Editorial Rx Press; 2016.Google Scholar
  17. 17.
    Pisapia P, Lozano MD, Vigliar E, Bellevicine C, Pepe F, Malapelle U, et al. ALK and ROS1 testing on lung cancer cytologic samples: perspectives. Cancer. 2017;125(11):817–30.Google Scholar
  18. 18.
    Savic S, Bode B, Diebold J, Tosoni I, Barascud A, Baschiera B, et al. Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol. 2013;8(8):1004–11.CrossRefPubMedGoogle Scholar
  19. 19.
    van der Wekken AJ, Pelgrim R, t Hart N, Werner N, Mastik MF, Hendriks L, et al. Dichotomous ALK-IHC is a better predictor for ALK inhibition outcome than traditional ALK-FISH in advanced non-small cell lung cancer. Clin Cancer Res. 2017;23(15):4251–8.CrossRefPubMedGoogle Scholar
  20. 20.
    Wang W, Tang Y, Li J, Jiang L, Jiang Y, Su X. Detection of ALK rearrangements in malignant pleural effusion cell blocks from patients with advanced non-small cell lung cancer: a comparison of Ventana immunohistochemistry and fluorescence in situ hybridization. Cancer Cytopathol. 2015;123(2):117–22.CrossRefPubMedGoogle Scholar
  21. 21.
    Skov BG, Skov T. Paired comparison of PD-L1 expression on cytologic and histologic specimens from malignancies in the lung assessed with PD-L1 IHC 28-8pharmDx and PD-L1 IHC 22C3pharmDx. Appl Immunohistochem Mol Morphol. 2017;25(7):453–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Thunnissen E, Yatabe Y, Lantuejoul S, Bubendorf L. Immunohistochemistry for PD-L1. In: Tsao MS, Kerr KM, Dacic S, Yatabe Y, Hirsch FR, editors. IASLC Atlas for PD-L1 immunohistochemistry testing in lung cancer. North Fort Myers: International Association for the Study of Lung Cancer, Editorial Rx Press; 2017.Google Scholar
  23. 23.
    da Cunha Santos G, Saieg MA. Preanalytic specimen triage: smears, cell blocks, cytospin preparations, transport media, and cytobanking. Cancer. 2017;125(S6):455–64.Google Scholar
  24. 24.
    Knoepp SM, Roh MH. Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques. Cancer Cytopathol. 2013;121(3):120–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Balassanian R, Wool GD, Ono JC, Olejnik-Nave J, Mah MM, Sweeney BJ, et al. A superior method for cell block preparation for fine-needle aspiration biopsies. Cancer Cytopathol. 2016;124(7):508–18.CrossRefPubMedGoogle Scholar
  26. 26.
    Crapanzano JP, Heymann JJ, Monaco S, Nassar A, Saqi A. The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine. CytoJournal. 2014;11:7.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Jain D, Mathur SR, Iyer VK. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies. Cytopathology. 2014;25(6):356–71.PubMedGoogle Scholar
  28. 28.
    Lindsey KG, Houser PM, Shotsberger-Gray W, Chajewski OS, Yang J. Young investigator challenge: a novel, simple method for cell block preparation, implementation, and use over 2 years. Cancer. 2016;124(12):885–92.Google Scholar
  29. 29.
    Tian SK, Killian JK, Rekhtman N, Benayed R, Middha S, Ladanyi M, et al. Optimizing workflows and processing of cytologic samples for comprehensive analysis by next-generation sequencing: memorial Sloan Kettering cancer center experience. Arch Pathol Lab Med. 2016.Google Scholar
  30. 30.
    van Hemel BM, Suurmeijer AJ. Effective application of the methanol-based PreservCyt fixative and the cellient automated cell block processor to diagnostic cytopathology, immunocytochemistry, and molecular biology. Diagn Cytopathol. 2013;41(8):734–41.CrossRefPubMedGoogle Scholar

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of PathologyUniversity Hospital BaselBaselSwitzerland

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