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W.A. Cooper



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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): W.A. Cooper

      • 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): W.A. Cooper

      • 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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    P3.11 - Poster Session 3 - NSCLC Novel Therapies (ID 211)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.11-046 - ROS1 overexpression by immunohistochemistry in non-small cell lung cancer: clinical characteristics, natural history and potential new therapeutic target based on two Australian cases (ID 3081)

      09:30 - 09:30  |  Author(s): W.A. Cooper

      • Abstract

      Background
      Recent years have seen worldwide interest in the study of driver mutations in lung cancer, in particular epidermal growth factor receptor mutation (EGFR) and anaplastic lymphoma kinase gene rearrangement (ALK). ROS1 gene rearrangement is a recently identified driver mutation and potential therapeutic target for crizotinib and similar agents. However little is known of the natural history of patients with ROS1, and moreover the diagnostic value of immunohistochemistry (IHC) compared to fluorescent in-situ hybridization (FISH).

      Methods
      12 patients from a single Australian tertiary institution with advanced non-small cell lung cancer (NSCLC) were screened for ROS1 overexpression and gene rearrangement. Selection was based on negative testing for EGFR and ALK, and unusually long natural history.

      Results
      We report 2 patients with ROS1 overexpressed advanced NSCLC, their unique characteristics, long natural history and the use of IHC as a complementary method to FISH in identifying these patients. Mr GL was a 62 year-old Caucasian man and lifelong non-smoker who presented with an incidental 19mm subpleural left lower lobe lung nodule found on computed tomography (CT) when he was treated for pneumonia in 2008. He was monitored with CT for his pulmonary nodule and pre-existing interstitial lung disease. In 2011, CT and subsequent positron emission tomography (PET) showed new regional lymphadenopathy and widespread sclerotic bone disease with the pulmonary nodule unchanged in size but moderately glucose avid. Axillary and supraclavicular lymph node biopsies confirmed metastatic adenocarcinoma consistent with a lung primary. EGFR and ALK testing was negative. He received induction and maintenance chemotherapy until disease progression in 2013. His original biopsy tested negative for ROS1 rearrangement by FISH but stained strongly positive for ROS1 overexpression by IHC using the Epitomics rabbit monoclonal antibody (D4D6) with diffuse cytoplasmic positivity. He was commenced on crizotinib, achieving and maintaining stable disease after three months. Mrs MM was a 54 year-old Caucasian woman and lifelong non-smoker who presented with an incidental 26mm right lower lobe lung nodule found on CT when she presented with left sided chest pain in 2009. PET and endobronchial biopsy of mediastinal lymph nodes confirmed stage IIIA lung adenocarcinoma. EGFR and ALK testing was negative. She received neo-adjuvant chemotherapy, followed by right lower lobectomy and post-operative radiotherapy. In 2010 she developed right supraclavicular lymph node recurrence and achieved radiological complete response after radiotherapy. In 2011 she developed another isolated nodal recurrence in the right supraclavicular fossa, which was surgically resected and confirmed adenocarcinoma. It stained strongly positive for ROS1 overexpression by IHC and positive for ROS1 rearrangement by FISH. In 2013, PET found an isolated hepatic metastasis. She was commenced on crizotinib with plans for re-staging and consideration for liver directed therapy. Clinical progress of the patients will be updated and presented.

      Conclusion
      Our cases of ROS1 overexpressed NSCLC illustrate unique patient characteristics of never-smoking status, adenocarcinoma histology, negative testing for EGFR and ALK, and an unusually long natural history. Our cases highlight the need for greater understanding of the predictive value of ROS1 overexpression by IHC as opposed to FISH alone for targeted therapy.