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M. Montañana



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    MO26 - Anatomical Pathology II (ID 129)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO26.07 - Usefulness of cytological samples for the assessment of ALK rearrangements in NSCLC patients. (ID 2351)

      11:05 - 11:10  |  Author(s): M. Montañana

      • Abstract
      • Presentation
      • Slides

      Background
      ALK gene rearrangement defines a new molecular subtype of NSCLC with response to Crizotinib, (Xalkori®) a dual MET and ALK inhibitor. To date, determination of ALK gene rearrangements has been performed in biopsies and/or surgical specimens. However, advanced lung cancer is often diagnosed by FNA cytology obtained through minimally invasive procedures, and frequently cytological specimens are the only samples available. We assessed the feasibility of determining ALK gene rearrangements in cytological samples.

      Methods
      We studied prospectively 53 cytological samples from 53 NSCLC patients (30 M/23 F) for ALK gene rearrangements by FISH (Abbot dual colour break apart probe). Tumour samples were obtained by bronchoscopy -FNA in 26 cases (49.1%), EBUS-FNA in 7 (13.2%), EUS-FNA in 3 cases (5.7%), CT-FNA in 3 (5.7%), and direct FNA in 6 cases (11.3%). Two cavity fluids (3.8%), 4 imprints from surgical specimens (7.5%), and 2 cellblocks received for consultation (3.8%) were also studied. FISH was performed on Papanicolau stained smears in 15 cases (28.3%), non-stained ThinPrep in 28 cases (52.8%), cell block in 9 cases (17%), and 1 stained ThinPrep. All cases were tested for EGFR and KRAS mutations.

      Results
      Thirty-seven samples (69.8%) were adequate for FISH analysis. Three cases (8.1%) had ALK gene rearrangements: positive cases were non-smoker women with adenocarcinoma, two of them with signet ring cells. One case had a concurrent EGFR mutation in exon 21. FISH study was unsuccessful in 16 cases (30.2%): 10 from Papanicolau stained smears (62.5%), 5 from unstained ThinPrep (31.3%), and 1 from a cell block. Nineteen ThinPrep slides were adequate for FISH analysis (86.4%) as well as 8 out of 9 cell blocks. Correlation cytological / paraffin embedded samples was performed in 4 cases with a concordance rate of 100%.

      Conclusion
      Determination of ALK gene rearrangements in cytological specimens is feasible. It is mandatory an exquisite management and care of the samples to preserve quality. ThinPrep and cell blocks are the most suitable samples for FISH analysis, while Papanicolau stained smears provide poor results. Coexistence of ALK gene rearrangements and EGFR mutations was observed in one case, indicating that such alterations are not necessarily mutually exclusive.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-015 - EGFR and KRAS mutational analysis using small amounts of DNA from FNA and small CNB is feasible and reproducible using a commercial real time PCR method. Validation of this PCR method in cytological samples. (ID 2721)

      09:30 - 09:30  |  Author(s): M. Montañana

      • Abstract

      Background
      EGFR and KRAS mutations guide treatment selection in NSCLC patients. With 75% of newly diagnosed cases at advanced stages, mutational analysis is performed in small samples: core needle biopsies (CNB) and fine needle aspiration (FNA). Both the cobas EGFR and KRAS test are CE-IVD. No validation studies of the cobas tests have been performed using cytological smears but it is important to extend the benefits of molecular targeted therapy while preserving tissue for additional marker testing.

      Methods
      EGFR and KRAS mutation status were studied in 140 non-selected samples from NSCLC patients: 49 CNB, 91 FNA. DNA was extracted directly from one stained smear in FNA samples and one 5-micron section in CNB using the cobas DNA Sample Preparation Kit. All samples contained ≥ 50% tumor cells. DNA concentration and ratio (A260/280) were recorded. All cases were studied using the cobas EGFR and KRAS mutation tests. Moreover, 123 and 125 cases were analyzed respectively for EGFR and KRAS mutational status using Sanger sequencing.

      Results
      CNB diagnosis was: 29 SqCC, 17 AC, 1 BAC, 1 adenosquamous, 1 NSCLC-NOS. FNA diagnosis was: 64 AC, 13 SqCC, 3BAC, 2 LCC, 2 adenosquamous, 7 NSCLC-NOS. DNA concentrations from CNB were higher and significantly different than DNA from FNA (p<0.001,U Mann Whitney). DNA quality was similar between sample types. Mutational analysis is shown in Table 1. Mutation rate for EGFR was 15.9% and 8.2 % and for KRAS 37% and 16.7% in FNA and CNB, respectively, but should be considered within the context of tumor type. 8.6% and 36.4% of the cases were below the manufacturer’s recommendations of 2ng/µl and 4ng/µl for EGFR and KRAS testing, respectively. Invalid rates were 2.1% (3) for EGFR and 5.5% (9) for KRAS. These results may be due to low DNA concentration (EGFR) or technical performance (KRAS) that was resolved with later samples. Sequencing invalid results were 42.3% for EGFR and 0.8% for KRAS. Table 1.

      　 FNA CNB
      EGFR WT 74 45
      　 Exon 19 Del 8 2
      　 Exon 20 Ins 0 1
      　 L858R 5 1
      　 Exon 18 G719X 1 0
      　 Invalid 3 0
      KRAS WT 51 40
      　 12/13 Mutation 29 8
      　 61 Mutation 1 0
      　 Invalid* 8 1
      * By sequencing 6 WT and 3 mutated

      Conclusion
      In addition to FFPE samples, identification of EGFR and KRAS mutations in FNA and CNB samples using cobas EGFR and KRAS Mutation Tests is faster, easier to use, and reproducible. Although DNA concentrations were lower from FNA, DNA quality was similar to CNB and provided valid results. Sequencing had lower sensitivity and was more time-consuming. Careful sample management, especially for FNA, by the pathologist is critical to ensure quality and to optimize DNA yields.