Author of

• 11866

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

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11881

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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): A. Gurpide
    • 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.