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A.J. Hubers



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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-021 - Validation of DNA Hypermethylation Analysis in Sputum for the Diagnosis of Lung Cancer (ID 1774)

      09:30 - 16:30  |  Author(s): A.J. Hubers

      • Abstract

      Background
      Lung cancer has the highest mortality of all cancers worldwide with a 5 year survival rate of <15%. The prognosis improves dramatically when the disease is detected at an early stage, and when curative treatment is possible. Current (low dose CT) screening and diagnostic procedures are suboptimal with low specificity. Thus, novel detection methods for lung cancer as stand alone or in combination with other methods are needed. DNA hypermethylation of biomarkers in sputum have shown to distinguish lung cancer cases from cancer-free controls. The aim of the present study was to validate the usage of DNA hypermethylation of biomarkers in sputum samples of lung cancer patients and controls for lung cancer diagnosis, in comparison with sputum cytology.

      Methods
      We prospectively collected sputum of lung cancer patients and controls during 3-9 days in the Amsterdam and Nieuwegein area, The Netherlands. From this sputum bank, a learning set (n=80 lung cancer patients, n=91 controls) and validation set (n=173 lung cancer patients, n=164 controls) were randomly composed. DNA promoter hypermethylation of the following biomarkers was assessed by means of quantitative methylation specific PCR: RASSF1A, APC, cytoglobin, 3OST2, PRDM14, FAM19A4 and PHACTR3. Cut-off values for positive hypermethylation were calculated using Youden’s index. Sputum cytology analysis was performed for all sputum samples. McNemar’s test was used to compare the difference between sensitivity of hypermethylation and sputum cytology for lung cancer diagnosis. A two-sided p-value <0.05 was considered significant.

      Results
      RASSF1A was best able to distinguish cases from controls, with sensitivity of 37-41% and specificity of 91-97% in both learning and validation sets. In multivariate analysis, a panel of RASSF1A, 3OST2 and PRDM14 showed highest sensitivity of 82% [95% confidence interval (CI): 76 – 88%] with a specificity of 68% [95% CI: 61 – 74%] in the learning set, with consistent results in the validation set. Molecular analysis was superior (P<0.001) over sputum cytology (sensitivity of 15%). The sensitivity of the biomarker panel did not improve when it was combined with sputum cytology. There was no association observed between DNA hypermethylation and clinical parameters such as age, smoking status, tumor stage, and histology.

      Conclusion
      This study validates hypermethylation analysis in sputum for the diagnosis of lung cancer. RASSF1A hypermethylation showed high specificity and thereby can have an important role in lung cancer diagnosis in symptomatic patients. A panel of biomarkers RASSF1A, 3OST2 and PRDM14 showed high sensitivity, but relatively low specificity.

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    P3.21 - Poster Session 3 - Diagnosis and Staging (ID 171)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Prevention & Epidemiology
    • Presentations: 1
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      P3.21-007 - <em>EGFR</em> mutation analysis in sputum of lung cancer patients: a multicenter multitechnique study (ID 1782)

      09:30 - 16:30  |  Author(s): A.J. Hubers

      • Abstract

      Background
      Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in lung adenocarcinomas and are associated with a high response to EGFR tyrosine kinase inhibitors. EGFR mutations can be detected in tumour tissue, cytology specimens and blood from lung cancer patients. Thus far, EGFR mutation analysis has not been systematically demonstrated for sputum samples. The aim of the present study was to determine whether EGFR mutation analysis is feasible on sputum samples, employing different assays in a multicenter study.

      Methods
      Sputum samples were collected from 10 lung cancer patients with confirmed EGFR mutation in their tumour tissue, 10 lung cancer patients without evidence of an EGFR mutation, and 10 patients with chronic obstructive pulmonary disease (COPD). DNA was isolated from the sputum and used for mutation analysis by Cycleave PCR, COLD-PCR, PangaeaBiotech SL technology (PST), and High Resolution Melting, respectively. Targeted resequencing (TruSeq Amplicon Cancer Panel) and droplet digital PCR were additionally performed on the 10 samples with EGFR mutation.

      Results
      Dependent on the assay, EGFR mutations could be detected in 30-50% of the sputum samples of patients with EGFR mutations (Table). The different techniques revealed consistent results, with slightly higher sensitivity for PST. Neither the lung cancer patients without EGFR mutation nor the COPD controls tested positive for EGFR mutations in their sputum samples, indicating high clinical specificity of all assays.

      Subject Gender Age (years) Tumour stage EGFR mutation status of tumour tissue[1] EGFR mutation analysis on sputum specimens[2]
      Cycleave PCR COLD-PCR PST[3] HRM-sequencing Cytology[4]
      A F 72 IV Del E746-A750 0 0 0 0 0
      B M 66 I Del E746-A750 0 2 0 0 0
      C[6] F 78 IV Del E746-A750 1 1 1 1 2
      D F 46 III Del E746-A750 0 0 1 0 0
      E[6] M 54 IV Del E746-A750 1 1 1 1 0
      F F 49 III Del E746-A750 & c.2369C>T [p.T790M] 0 0 0 0 0
      G F 54 IV Del E746-A750 & c.2369C>T [p.T790M] 0 0 1[5] 0 1
      H F 73 IV c.2753T>G [p.L858R] 0 0 0 0 0
      I F 61 IV c.2753T>G [p.L858R] 0 0 0 0 0
      J[6] M 60 IV Del E746-A750 1 1 1 1 2
      [1 ]del E746-A750= deletion exon 19 [2] mutation identified: 0=no, 1=yes, 2=dubious [3] exclusively del19 and L858R were assessed [4] tumour cells: 0=no, 1=yes, 2=in related sample of same patient [5 ]only del19 detected [6 ]TSACP and ddPCR both tested EGFR mutation (del19) positive.

      Conclusion
      EGFR mutations can be detected in sputum samples from patients with EGFR-mutated non-small cell lung cancer, which may replace biopsy procedure for some patients.