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L.G. Horvath



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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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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): L.G. Horvath

      • 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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      P2.06-011 - Phosphorylated-Akt expression is a prognostic marker in early stage non-small cell lung cancer (NSCLC) (ID 1151)

      09:30 - 09:30  |  Author(s): L.G. Horvath

      • Abstract

      Background
      The 5-year survival for stage IB non-small cell lung cancer (NSCLC) is only 55%, but the benefit of adjuvant chemotherapy in this setting remains equivocal. Numerous prognostic markers have been examined, but none to date have moved into clinical practice. There is an urgent need to identify novel molecular markers that can select high risk patients, who may potentially benefit from adjuvant chemotherapy.

      Methods
      We identified 471 consecutive patients with stage IB primary NSCLC according to the American Joint Commission on Cancer, (AJCC) 6[th] edition tumour-node-metastasis staging system, who underwent surgical resection between 1990 and 2008. Patients who received neoadjuvant or adjuvant treatments were excluded. Pathology reports were reviewed and pathologic characteristics were extracted. Expression of phosphorylated Akt (pAkt) in both cytoplasmic and nuclear locations was assessed by immunohistochemistry, and clinicopathologic factors were analyzed against 10-year overall survival using Kaplan-Meier and Cox proportional hazards model.

      Results
      455 (96.6%) cancers were adequate for pAkt immunohistochemical analysis. The prevalence of pAkt expression in the cytoplasm and nucleus of the cancers was 60.7% and 43.7% respectively. Patients, whose cancers expressed higher levels of pAkt in the cytoplasm, had a trend towards longer overall survival than those with lower levels of cytoplasmic pAkt (p=0.06). Conversely, patients whose cancers expressed higher levels of pAkt in the nucleus had a poorer prognosis than those with lower levels of nuclear pAkt expression (p=0.02). Combined low cytoplasmic/high nuclear expression of pAkt was an independent predictor of overall survival [HR=2.86 (95% CI:1.35-6.04); p=0.006] when modeled with age [HR= 1.05 (95% CI: 1.03-1.07); p<0.001], extent of operation [HR= 2.11 (95% CI: 1.48-3.01); p<0.001], visceral pleural invasion [HR=1.63 (95% CI: 1.24-2.15); p<0.001], gender, tumour size, histopathologic type and grade (p>0.05).

      Conclusion
      Levels of expression of pAkt in the cytoplasm and nucleus and visceral pleural invasion are independent prognostic factors that can help to select patients with high risk disease, who may potentially benefit from adjuvant chemotherapy.

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    P3.18 - Poster Session 3 - Pathology (ID 177)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P3.18-003 - ROS1 Gene Rearrangements in Non-Small Cell Lung Carcinoma - A New Genetic Target that can be Identified by Immunohistochemistry and FISH (ID 1482)

      09:30 - 09:30  |  Author(s): L.G. Horvath

      • Abstract

      Background
      Targeted therapies aimed at specific molecular genetic alterations are revolutionizing cancer treatment, particularly in non-small cell lung cancer (NSCLC). ROS1 is an oncogene that encodes a transmembrane tyrosine kinase receptor that has high homology with the intracellular kinase domain of ALK. Driver mutations involving translocation of the ROS1 gene have recently been identified in NSCLC and show promise as a target for tyrosine kinase inhibitors. In this study we aimed to: (1) Investigate the incidence and clinicopathological features of NSCLCs harbouring ROS1 rearrangements in an Australian population. (2) Investigate the accuracy of immunohistochemistry (IHC) compared to FISH at identifying tumours with ROS1 rearrangements.

      Methods
      We tested for ROS1 translocations using both a FISH breakapart probe (Zytovision and Abbott Molecular) (≥15% cells with split signals or single green 3' signal considered positive for rearrangement), and immunohistochemistry (D4D6 clone, Cell Signaling Technology). Testing was undertaken on both (1) A retrospective cohort of 316 early stage lung adenocarcinomas in tissue microarrays. (2) A prospective cohort of 42 NSCLC, selected on clinical grounds for mutation testing (eg EGFR/KRAS/ALK negative samples and young age or never/light smoker).

      Results
      In the retrospective cohort, only 1 case was positive for ROS1 gene rearrangement by FISH (0.3% incidence). ROS1 IHC identified positive staining in 7 (2.0%) cases, including the FISH+ case. ROS1 IHC had a sensitivity of 100% and specificity of 98% for identifying ROS1 gene rearrangements. In the prospective cohort of 42 cases, 4 cases with ROS1 gene rearrangement were identified by FISH and all 4 cases showed positive ROS1 immunohistochemical staining. Of the total 5 cases with ROS1 gene rearrangement, all occurred in adenocarcinomas from female patients with an age range of 33-81 years (mean 58). Four of the five patients were non-smokers and two were of Asian ethnicity. All 5 cases were negative for ALK rearrangements and in the 4 cases where EGFR status was known, they were all wild type.

      Conclusion
      ROS1 gene rearrangements occur in a very small percentage of lung adenocarcinomas with distinctive clinicopathological features and appear to be mutually exclusive with other driver mutations in the small number of positive cases available for evaluation. Screening with IHC may be a suitable method of reducing the number of cases requiring FISH testing.