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Christophe Dooms

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    ES07 - Thoracic Ultrasonography: Diagnosis and Staging (ID 10)

    • Event: WCLC 2019
    • Type: Educational Session
    • Track: Interventional Diagnostics/Pulmonology
    • Presentations: 1
    • Now Available
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      ES07.04 - Predictive Molecular Testing on Small Biopsy Samples (Now Available) (ID 3189)

      13:30 - 15:00  |  Presenting Author(s): Christophe Dooms

      • Abstract
      • Presentation
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      According to international guidelines for molecular testing as updated in 2018, any lung cancer sample (tissue biopsy or cytology) with adenocarcinoma or not-otherwise specified (NOS) histology and adequate tumor cellularity should be tested for routinely treatable mutations with a turnaround time of no more than 10 working days.1,2 Single gene testing or restricted hotspot testing methods were developed to screen for EGFR p.Leu858Arg mutations or deletions with the exon 19 in advanced stage non-small cell lung cancer. Next-generation sequencing (NGS) platforms have facilitated multigene mutational profiling using small amounts of nanograms (ng) of DNA, making the NGS technology attractive for and applicable to small biopsy and cytology specimens. NGS and especially targeted NGS panels were rapidly validated for small biopsy samples and implemented in diagnostic laboratories as they focus on hotspot regions and frequently altered genes with direct and known consequence on therapy.3,4 Compared to sequential single-gene testing, targeted NGS is considerably faster and more cost-effective.5

      There are however several barriers to universal broad biomarker testing. Despite most tests can be run on only small biopsy and cytology specimen, in real world testing rates are far from 100% (even for EGFR, ALK and ROS1 testing) and up to 25% of samples lack sufficient tumor material in small biopsies. Molecular testing accuracy depends on multiple factors that include overall tumor cellularity, method of fixation, tumor fraction of the sample, and the analytical sensitivity of the molecular testing platform used for the analysis. Pre-analytical strategies to improve testing success are: (1) work with dedicated interventionalists (e.g. radiology, pulmonology) to get sufficient tissue as one single pass is not enough, (2) work with dedicated pathologists to enhance quality control and reflex testing, and (3) consider ROSE.

      In a reference center with dedicated interventionalists, a dropout rate of 3.4% was observed either due to quantitatively insufficient tumor material or inadequate nucleic acid quality based on a series of 3,000 consecutive lung cancer cases that were sequenced.6 In a randomized trial comparing two different needle sizes and a turn-around team (interventional pulmonologist, pathologist, molecular biologist) using standard operating procedures, several conclusions could be drawn given a successful NGS testing rate for all clinically relevant genes in 96% of samples.7 Four needle passes were needed to obtain adequate material for molecular analysis. A tissue core was reported present in almost 70% of specimens for both needle types. Less than 3% of samples had tumor cellularity of <10%, and there was no significant difference in tumor cellularity between 19G and 22G needles. Both the tumor surface area measured and the amount of DNA extracted from the selected cell block were larger for the 19G compared to the 22G specimen, with a median tumor surface area of 4.91 mm2 vs 2.35 mm2 and median DNA extracted of 1150 ng vs 818 ng, respectively.

      There is a paucity of articles outlining best practice guidelines for immunocytochemistry. With proper optimization and rigorous quality control, high-quality staining can be achieved on cellblock and non-cellblock preparations.8 Cytology preparations that are non-formalin-fixed provide the best alternative source of well-preserved DNA.

      Cytology specimens allow for rapid on-site adequacy assessment (ROSE), which can ensure the collection of adequate and sufficient material for ancillary studies including immunohistochemistry and molecular testing. Although there are no universally accepted criteria for EBUS-TBNA lymph node adequacy, structured semi-quantitative scoring schemes for ROSE and diagnostic category assignments have been proposed. The decision of whether or not to provide ROSE for EBUS-TBNA procedures is largely institution dependent. Validation studies are essential with correct implementation of these pre-analytical factors for any molecular testing in cytologic samples.

      In conclusion, a single NGS panel test covering all clinical relevant markers is most tissue and cost efficient. Strategies to obtain a higher rate of successful testing, even on only small biopsy and cytology specimens should be considered whenever needed: reflex testing, dedicated interventionalists, and strong communication with all team members.


      1. Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018 Mar;20(2):129-159.

      2. Kalemkerian GP, Narula N, Kennedy EB, et al. Molecular Testing Guideline for the Selection of Patients With Lung Cancer for Treatment With Targeted Tyrosine Kinase Inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology. Clinical Practice Guideline Update. J Clin Oncol. 2018 Mar 20;36(9):911-919.

      3. Bennett N, Farah C. Next-generation sequencing in clinical oncology: next steps towards clinical validation. Cancers 2014;6:2296-2312.

      4. Le Mercier M, De Nève N, Blanchard O, et al. Clinical application of targeted next generation sequencing for lung cancer patients. Belgian J Med Oncol 2015;27:2-8.

      5. Pennell N, Mutebi A, Zhou Z, et al. Economic impact of next-generation sequencing vs sequential single-gene testing modalities to detect genomic alterations in metastatic non-small cell lung cancer using a decision analytic model. J Clin Oncol 2018;36(15_suppl):9031-9031

      6. Volckmar AL, Leichsenring J, Kirchner M, et al. Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases. Int J Cancer. 2019 Jan 17. doi: 10.1002/ijc.32133. [Epub ahead of print]

      7. Dooms C, Vander Borght S, Yserbyt J, et al. A Randomized Clinical Trial of Flex 19G Needles versus 22G Needles for Endobronchial Ultrasonography in Suspected Lung Cancer. Respiration. 2018;96(3):275-282.

      8. Jain D, Nambirajan A, Borczuk A, et al; IASLC Pathology Committee. Immunocytochemistry for predictive biomarker testing in lung cancer cytology. Cancer Cytopathol. 2019 May;127(5):325-339.

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