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M. Walkiewicz



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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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      P1.06-034 - MET expression, copy number and oncogenic mutations in early stage NSCLC (ID 2424)

      09:30 - 16:30  |  Author(s): M. Walkiewicz

      • Abstract

      Background
      The MET receptor tyrosine kinase and its ligand are associated with the malignant phenotype. In non-small cell lung cancer (NSCLC) MET expression increases with disease stage and is involved in de novo and acquired resistance to tyrosine kinase inhibitors. Despite this, in early stage NSCLC, conflicting data series have reported MET expression and copy number to be prognostic in some studies but not others[1,2]. We investigated a large cohort of patients who underwent curative surgical resection at our institution to determine whether MET receptor or gene amplification was prognostic.

      Methods
      Tissue Microarrays (TMAs) were constructed using 1mm cores of FFPE primary NSCLC tissues in triplicate. TMAs were stained with the MET SP44 clone and a H-score calculated based on % cells stained and intensity; (%cellsx1)+(%cellsx2)+(%cellsx3) with a minimum of 0 and maximum of 300. The mean of triplicate values was calculated. MET gene amplification was detected using Ventana’s MET DNP probe with ultraView SISH DNP silver detection, performed on Ventana’s XT autostainer. DNA was isolated and subjected to mutational profiling using Sequenom’s LungCarta panel.

      Results
      Data for 508 patients, 352 (69%) male, were available for analysis including 329 pathological node negative (pN0), 67 pN1, 104 pN2 and 8 patients with resected primaries and solitary brain metastases (M1). Most patients were smokers with only 33 (6%) non-smokers. The median MET H-score was 100 and consistent across N0, N1 and N2 patients, although was higher in M1 patients. Median H-scores were significantly higher in adenocarcinoma compared to squamous cell carcinoma (140 vs 91.5, p<0.0001). Increased MET expression (H-score>100) was seen in 227 (45%) patients. High quality DNA was isolated in 443/508 (87%) of samples. The commonest mutations were in KRAS (21%), TP53 (10%), EGFR (5%), PIK3CA (4%) MET (3%) and NRF2 (3%). No mutation was found in 44% of samples. EGFR and KRAS mutations were associated with significantly higher MET expression, whereas TP53 was associated with significantly lower expression (Chi square p=0.0005). These differences may reflect the higher rates of adenocarcinoma in both EGFR and KRAS mutated tumours. Increased MET copy number by SISH was only observed in 6 samples. MET expression was not associated with cancer specific survival across all stages. In tumours harbouring mutations and in wild type tumours, there were no significant differences in survival according to MET expression.

      Conclusion
      Although increased MET expression was associated with both KRAS and EGFR mutations, it was not prognostic in this large cohort of resected NSCLC. MET expression may be both predictive and prognostic in advanced NSCLC, but its role in early stage NSCLC is unclear. References: 1. Dziadziuszko R, et al. Correlation between MET Gene Copy Number by Silver In Situ Hybridization and Protein Expression by Immunohistochemistry in Non-small Cell Lung Cancer. Journal of Thoracic Oncology. 2012 Feb;7(2):340–7. 2. Cappuzzo F, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. Journal of Clinical Oncology. 2009 Apr 1;27(10):1667–74.

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    P1.12 - Poster Session 1 - NSCLC Early Stage (ID 203)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P1.12-018 - Overall Survival and Smoking Status in Resectable Non-Small Cell Lung Cancer (ID 2662)

      09:30 - 16:30  |  Author(s): M. Walkiewicz

      • Abstract

      Background
      Although the carcinogenic effects of cigarette smoking are important in the pathogenesis of lung carcinoma, the impact of quantitative smoking history on survival in resectable tumours has not been well described. Using a comprehensive database in which smoking was quantitatively documented, we analysed the impact of increasing number of pack years of smoking on stage, histology, mutation status and overall survival in an Australian population. We focused on patients without nodal involvement (N0) as they were less likely to have received neoadjuvant or adjuvant therapy.

      Methods
      Data was extracted from a large single institution database containing information on patients who underwent curative resection of non-small cell lung cancer from 1992 to 2012. Cigarette smoking history was documented in pack years. DNA was isolated and analysed using Sequenom’s LungCarta panel which interrogates 214 mutations in 26 genes. Statistical analysis was performed using Chi-square tests and the Kaplan Meier method for survival.

      Results
      Information on pack year smoking status was available for 470 patients, 70% of whom were male. This included 311 (66%) pathological N0 (pN0), 64 (14%) pN1 and 95 (20%) pN2. Smoking history ranged between 0 (never smokers N=32, 7%) and 180 pack years, with a median of 45 and mean of 48. Patients were divided into quartiles based on their smoking history: never- and < 10 pack year smokers (N=43; 9%), 11-25 pack years (N=74; 16%), 26-50 pack years (N=180; 38%) and >50 pack years (N=173; 37%). Adequate DNA was isolated in 425 samples. Frequencies of mutations were as follows: KRAS 21%, TP53 10%, EGFR 5%, PIK3CA 4%; other mutations occurred at lower frequencies. In 44% no mutation was found. Increased pack year history of smoking was not associated with overall survival. In the pN0 wild type population, no association with smoking and survival was seen. In the pN0 mutation group (Figure 1) those with a <25 pack year history had significantly better overall survival than heavier smokers (HR 0.61, 95% CI 0.40-0.92). Figure 1: Overall Survival by smoking status in pN0 tumours with a mutationFigure 1

      Conclusion
      Smoking status was not associated with overall survival across the entire cohort. In patients whose tumour harboured a mutation, increased smoking was associated with a less favourable mutation profile including in KRAS, TP53 and PIK3CA. In patients with pN0 disease a significant difference in overall survival was observed favouring light smokers. The presence of mutations in association with heavy smoking negatively impacts overall survival.

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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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      P2.06-026 - Association of New York-Esophageal Antigen-1 (NY-ESO-1) promoter methylation and survival in stage III non-small cell lung cancer (ID 2370)

      09:30 - 16:30  |  Author(s): M. Walkiewicz

      • Abstract

      Background
      Cancer-Testis antigens (CTAs) are immunogenic molecules exclusively expressed in normal testes but with aberrant expression in up to 30% of NSCLC. NY-ESO-1, is a CTA whose expression is associated with poorer survival but improved response to chemotherapy.[1] The promoter regions for many CTA genes contain CpG islands which are predominantly methylated in most tissues. In cancer, hypomethylation of CTA gene promoters hsvr been shown to be determining factor for the frequent re-expression of these genes. Herein we sought to correlate NY-ESO-1 promoter methylation (PM) with protein expression in lung cancers and test whether methylation status can be used as a predictive and prognostic marker in NSCLC.

      Methods
      We reviewed the clinicopathological data for patients with pathological N2 NSCLC treated with surgical resection followed by either observation or adjuvant chemotherapy, if treated after 2004. Genomic DNA from formalin fixed paraffin embedded (FFPE) samples were purified and bisulfite converted according to the manufacturer’s (QIAGEN) instructions. Mutational status was determined in genomic DNA using Sequenom's Oncocarta panel v1.0. Tumour samples were cut and stained with NY-ESO-1 antibody (E978). Previously described protocols for methylation-specific NY-ESO-1 primers[2] and beta-actin[3] control probes were used. Using real-time quantitative methylation-specific polymerase chain reaction (qMS-PCR), the level of NY-ESO-1 methylation was determined and expressed as the ratio of methylated to unmethylated molecules (M/UM). We used a cut-off fold-difference of 5 (Log2, M/UM) to separate tumour samples into hypomethylated and hypermethylated groups. Survival estimates were obtained using the Kaplan-Meier method. Comparison across groups was performed using the Chi-squared or Fisher's test.

      Results
      NY-ESO-1 PM MS-PCR was successful in 92/100 (92%) of specimens. NY-ESO-1 was hypermethylated in 86% (79/92) of samples. Age, smoking history, gender, histology and oncogenic mutations were not associated with presence of NY-ESO-1 PM. NY-ESO-1 hypomethylation was inversely associated with protein expression (p<0.0001). In multivariate analysis, hypomethylation of the NY-ESO-1 promoter was an independent poorer prognostic marker in patients not treated with chemotherapy (HR 2.97, 95%CI 1.06-8.29, p=0.038), after adjusting for age, gender, stage IIIA/B and histology. Conversely, in patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 PM, suggesting hypomethylated tumours remain chemosensitive.

      Conclusion
      NY-ESO-1 hypomethylation was an independent adverse prognostic marker in patients not treated with chemotherapy. Given their poorer outcome, association with chemosensitivity and known immunogenicity, CTAs may make attractive targets for immunotherapy, demethylating agents and immune checkpoint inhibition. References 1.John et al. PLOS One, In press. 2.James et al. Oncogene 2006. 3.Eads et al. Cancer Res 2001. .