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B. Mercorella



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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-009 - Simultaneous Profiling of Multigene Mutations for the Effective And Efficient Diagnosis of Non-Small Cell Lung Carcinoma (ID 1078)

      09:30 - 09:30  |  Author(s): B. Mercorella

      • Abstract

      Background
      Identification of actionable driver mutations in non-small cell lung carcinoma (NSCLC) has become increasingly important for the prioritisation of targeted therapies. Mutational analysis of formalin-fixed paraffin embedded (FFPE) tissues presents several challenges including generally limited and fragmented DNA, the need to identify a range of biologically significant mutations and a pressing need for a fast turn around time at a cost-effective way. Our aim was to determine optimal methods for quantification of DNA for mutational analysis in NSCLC and to develop a new custom assay that could perform multigene mutational analysis on the limited quantity of DNA available in the small NSCLC samples frequently submitted for testing.

      Methods
      DNA was extracted from FFPE tissues including cytology specimens. Spectrophotometry quantification was compared with Qubit 2 Fluorometer measurements and the Sequenom SampleID assay for accurate and meaningful assessment of extracted DNA for diagnostic mutational profiling. We have previously established a diagnostic protocol for somatic mutation profiling in NSCLC using a commercial DNA mass spectrometry kit (Oncocarta v1.0) and compared it with a new custom kit “OncoFocus” developed in collaboration with Sequenom. These assays utilise target amplicons of small sizes for efficient amplification in fragmented DNA and simultaneously profile a range of actionable mutations in EGFR, KRAS, BRAF and NRAS. Preliminary verification of the “OncoFocus” assay was performed in 27 NSCLC samples, 3 lung cancer cell lines and 2 control genomic DNA samples.

      Results
      We found spectrophotometry significantly overestimated DNA quantity particularly at low concentrations. We also studied the correlation of DNA quantities with estimated copies of DNA templates as determined by SampleID. The results suggested that a minimum of 300 ng DNA is needed to achieve the required 300 – 500 amplifiable genomic copies per reaction for the OncoCarta analysis, which remains difficult to achieve for many diagnostic NSCLC samples. We developed a more focused diagnostic panel “OncoFocus” which could be performed reliably with less DNA but which includes key actionable mutations in 159 hotspots in EGFR (n=109), KRAS (n=17), BRAF (n=15) and NRAS (n=18) requiring only 150 ng of DNA. Somatic mutations were identified in 23 samples and 3 cell lines including EGFR (n=22), KRAS (n=6) and BRAF (n=1). No false positive results were observed in 4 FFPE and 2 control samples. The whole process from the receipt of FFPE samples to issuing a report can be completed within 5 working days and the “OncoFocus” panel has increased our capacity per chip (iPLEX II) from 15 to 31 samples. The “OncoFocus” panel also results in decreased per sample testing costs.

      Conclusion
      The Qubit fluorometer is a more reliable and accurate method to quantify DNA derived from FFPE for mutational analysis than spectrophotometry. We also conclude that DNA mass spectrometric analysis using a new custom “OncoFocus” panel is an effective and efficient test that simultaneously detects 159 mutational hotspots, in the generally lower quantity of DNA obtained from routine FFPE NSCLC samples.

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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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      P2.18-009 - Mutation Testing in Non-Small Cell Lung Cancer - Suitability of Small Biopsy and Cytology Specimens (ID 1750)

      09:30 - 09:30  |  Author(s): B. Mercorella

      • Abstract

      Background
      Patients with non-small cell lung cancer (NSCLC) harboring sensitizing mutations in epidermal growth factor receptor (EGFR) benefit from treatment with EGFR tyrosine kinase inhibitors. As most patients with NSCLC present with advanced-stage disease and are not candidates for surgical resection, somatic mutation testing is often performed on small biopsy and cytology specimens. Compared to resection specimens, the suitability of these specimens is not well established. We aimed to explore the suitability of small biopsy and cytology specimens for mutation testing in NSCLC.

      Methods
      We undertook a retrospective review of NSCLC mutation testing cases performed at Royal Prince Alfred Hospital, Sydney, from March 2012 to May 2013. Mutation testing was requested by the treating physician. DNA was extracted from formalin-fixed, paraffin embedded tissue and a multiplex PCR assay (OncoCarta Panel v1.0) used to identify mutations in 19 oncogenes including EGFR, KRAS, and BRAF. The results were analyzed on the Sequenom MassArray platform. Fragment analysis was also undertaken to assess for exon 19 deletions.

      Results
      Mutation testing was undertaken on 151 NSCLC specimens, including 44 (29.1%) resection specimens (27 lung resection specimens and 17 metastatic site resections), 67 (44.4%) small biopsy specimens, and 40 (26.5%) cytology specimens. Overall, EGFR mutations were detected in 32/151 (21.2%) cases, KRAS mutations in 29/151 (19.2%) cases, and BRAF mutations in 3/151 (2%) cases. Mutations were detected in 25/44 (56.8%) resection specimens, principally lung resection specimens (19/27, 70.4%), 26/67 (38.8%) small biopsies and 13/40 (32.5%) cytology specimens. The mutation rate was significantly lower in small biopsies (p=0.006) and cytology specimens (p=0.002), compared to lung resection specimens. Specifically, EGFR mutations were identified in 13/44 (29.5%) resection specimens, again mainly in lung resection specimens (10/27, 37%), 9/67 (13.4%) small biopsies and 10/40 (25%) cytology specimens. Compared to lung resection specimens, the proportion of EGFR mutation positive cases was significantly lower in small biopsy (p=0.01), but not in cytology specimens (p=0.29). One paired cytology and lung resection specimen from a single patient was available and both specimens confirmed the presence of an L858R EGFR mutation.

      Conclusion
      Mutations, including EGFR mutations, were most frequently detected in lung resection specimens. Compared to lung resection specimens, the EGFR mutation rate was significantly lower in small biopsy, but not in cytology specimens. This suggests that cytology specimens are more likely to be adequate for mutation testing than small biopsies such as core and bronchial biopsies. However, we cannot exclude bias in this study from differing referral patterns which may affect these results. Careful assessment of DNA quality and quantity is important for all specimens, particularly small biopsy specimens, to reduce the risk of false positive or negative results.