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S.A. O'Toole



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    MS21 - Practical Problems in Lung Cancer Diagnosis - Application of the 2011 Adenocarcinoma Classification (ID 38)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Pathology
    • Presentations: 1
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      MS21.2 - Molecular Diagnosis in Cytology and its Place in the New Classification (ID 557)

      14:22 - 14:39  |  Author(s): S.A. O'Toole

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      Abstract
      There has been very little improvement in outcome from lung cancer over the last two decades but the identification of actionable mutations and structural rearrangements in subsets of patients with lung adenocarcinoma holds hope for the near future, particularly if these agents successfully move into the adjuvant setting. Detection of key molecular targets is central to this new understanding of lung adenocarcinoma and targeted therapy but poses significant challenges for implementation into routine clinical diagnostics. The importance of molecular testing in lung adenocarcinoma has been emphasized not only in the updated classification of adenocarcinoma but also in the recently released molecular testing guidelines for selection of lung cancer patients for EGFR and ALK Tyrosine Kinase Inhibitors produced by the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology this year with a central recommendation that tissue should be prioritized for EGFR and ALK testing. Recent molecular and genomic studies of lung adenocarcinoma in particular has resulted in the identification of other low incidence, novel driver mutations including structural rearrangements in ROS1 and RET-KIF5B as well as recognition of point mutations in BRAF and HER2 among others. It is apparent that “profiling” lung cancers for a range of important and potentially treatable driver mutations may offer significant advantages such as cost effective, rapid identification of actionable changes and efficient triage for clinical trials of novel agents. However performing molecular testing in lung cancer can be challenging given the majority of patients present with inoperable disease. This means histological and molecular diagnosis is generally performed on small biopsies including cytological specimens often with very limited material for testing. Furthermore much of this tissue has undergone formalin fixation and paraffin processing with subsequent DNA cross-linkage and fragmentation. There are additional problems of contamination with non-malignant tissue elements including stroma and inflammatory cells. There are also time pressures with the need for rapid results with current recommendations for results to be available within 10 working to allow appropriate triage for therapy. It is important to direct testing to appropriate clinical groups likely to benefit. While there are strong demongraphic associations of actionable mutations in lung adenocarcinoma, including non-smoking status, younger age, female sex and Asian ethnicity, these criteria are insufficiently robust to exclude patients without these characteristics from testing. Current recommendations in limited specimens are that molecular testing for EGFR or ALK gene changes be primarily undertaken in adenocarcinoma or cases with a component of adenocarcinoma. Fortunately cytology is emerging as a robust method for classification of lung cancer and these specimens are increasingly utilized for mutation testing. Large cell or histologically undifferentiated carcinomas with features suggestive of adenocarcinoma differentiation eg TTF-1 expression are also suitable for molecular testing. Molecular testing of limited biopsies may also be considered in cases showing squamous or small cell histology guided by clinical features such as ethnic background and non-smoking status among others. There is good concordance between primary and metastatic sites for EGFR mutational status and specimens from either site are acceptable for testing with choice based on morphological assessment of optimal specimens for molecular testing There are a wide variety of molecular techniques available to assess for the presence of key driver mutations in lung adenocarcinoma, each with their own limitations and advantages, but there is no perfect technology that fulfills all clinical and laboratory needs, especially on the limited material usually available in lung cancer mutation testing. Virtually all techniques for EGFR testing depend on PCR amplification, which is a major issue where limited DNA template is present raising the possibility of both false negative (due to sensitivity issues) and false positive results (eg due to amplification of formalin artifacts). In our own practice we have found that standard methods for DNA quantification such as spectrophotometry significantly overestimates the amount of DNA available for testing in comparison to more specific methods such as DNA fluorometry or estimates of amplifiable DNA copy number. We currently perform routine diagnostic mutation testing via multigene mutation profiling using a commercial panel, Oncocarta v1.0, on the massARRAY Sequenom platform in combination with fragment analysis for EGFR exon 19 and 20 insertions and deletions. This allows simultaneous determination of key mutations in EGFR and KRAS status as well as identifying rare but potentially actionable changes in BRAF, PIK3CA and HER2. In parallel, we perform immunohistochemistry for ALK and ROS1 to allow rapid triage for FISH testing if mutation profiling is negative. However not all cases have sufficient material for this approach and we are currently validating a new custom panel which can be performed reliably with less DNA. While cytological specimens are problematic in the generally small amount of tumour material available for testing, they have the advantage of often containing a relatively pure population of tumour cells, with a marked reduction in stromal contamination especially in FNAB specimen. Earlier studies suggested cytological specimen were less preferred to small tissue biopsies but a number of more recent publications have highlighted the suitability of these specimens. While the most recent recommendations suggest that cell block specimens allowing pretest morphological assessment are preferred to fresh smears, fresh material offers generally better quality and quantity of DNA. FISH testing for ALK gene rearrangement on cytological specimens is feasible and increasingly widely performed. Generally FISH testing requires less cellular material than mutation testing and the direct visualization of the molecular assay in tumor cells gives greater confidence that a negative result is far less likely to reflect problems with sensitivity as for EGFR testing. Expert morphological assessment is critical to ensure malignant cells are being assessed in this setting. In summary, cytology specimens are a commonly tested lung cancer diagnostic specimen and offer a number of advantages over more invasive small biopsies. Expert cytological assessment of specimens should be undertaken prior to molecular testing to maximize the quality and accuracy of testing.

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