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C. Murone



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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): C. Murone

      • 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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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-039 - Expression of Mucin 1 in non-small cell lung cancer: Relationship between immunohistochemistry, tumour characteristics and survival (ID 2765)

      09:30 - 16:30  |  Author(s): C. Murone

      • Abstract

      Background
      Mucin 1 (MUC1), a glycoprotein highly expressed in many malignancies, is being explored as an antigen for immunotherapy. How best to measure MUC1 expression in non-small cell lung cancer (NSCLC) and its prognostic value in NSCLC are under discussion.

      Methods
      Tissue microarrays (TMAs) were constructed using triplicate 1mm cores of formalin-fixed paraffin-embedded tumour and stained with 214D4 (recognizes protein core) and MA695 (recognizes carbohydrate epitope) anti-MUC1 antibodies. TMAs were assessed for polarization, cytoplasmic and membranous staining intensity (scored 0–3) and proportion cells positive (0–100%; scored 0–5), averaged for multiple cores. A composite score (intensity x cells positive) was derived (range 0–15).

      Results
      TMAs from 518 patients were analyzed: 69% male, 95% Caucasian, 7% never-smoking; 49% adenocarcinoma, 35% squamous cell, 7% large cell; 43% stage I NSCLC, 33% stage II, 23% stage III. Immunohistochemistry staining intensity, proportion positive cells and depolarization were very similar between antibodies, with high concordance in composite score (R2=0.71, p<0.0001). Polarization was discordant in 8%. Similar scores were seen for N0, N1 and N2 when assessed by either antibody. For 77 cases with paired primary/N2 nodal tissue, mean 214D4 composite scores were 10.7 and 10.1 and MA695 scores 9.5 and 9.4, respectively. Discordant staining in primary but not node was seen in 5.2% and 10.4% with 214D4 and MA695, respectively. For 27 cases with neoadjuvant chemotherapy vs no chemotherapy, mean 214D4 scores were 10.2 vs 10.1 and MA695 9.3 vs 9.6, respectively. Higher scores with each antibody trended toward improved survival (non-significant). Polarization was associated with improved survival (whole cohort) with 214D4 (80.6 vs 47.8 months; HR 1.37 [95%CI 1.078–1.742], p=0.01 log rank test) and MA695 (95.8 vs 45.7 months; HR 1.48 [95%CI 1.159–1.878], p=0.002). Polarization with 214D4 was strongly associated with improved survival for adenocarcinoma (HR 1.92 [95%CI 1.385–2.668], p<0.0001) but not for non-adenocarcinoma. Similarly, polarization with MA695 conferred a survival advantage in adenocarcinoma (HR 1.68 [95%CI 1.225–2.311], p=0.001) but not non-adenocarcinoma cases. Data on MUC1 immunohistochemistry and circulating soluble MUC1 will be presented.

      214D4 MA695
      No staining 3.5% 6.2%
      Mean intensity
      - Cytoplasmic 1.8 1.7
      - Membranous 2.1 2.2
      Mean cells positive 3.9 3.6
      Mean composite score 10.1 9.6
      - Adenocarcinoma 13.1 12.0
      - Squamous cell 7.1 7.0
      - Large cell 7.2 7.7
      Depolarization 66.7% 62.4%

      Conclusion
      Over 93% were MUC1 immunohistochemistry positive, with higher scores in adenocarcinoma. Composite scores for the two antibodies were highly correlated and depolarization largely concordant. MUC1 expression was generally maintained in paired primary/nodal tumour and was similar across nodal stages and following neoadjuvant chemotherapy. Polarization was associated with improved survival in adenocarcinoma. Further investigation is needed to determine which antibodies best predict outcomes.

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    P3.10 - Poster Session 3 - Chemotherapy (ID 210)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.10-034 - Is loss of MGMT a therapeutic target in lung cancer? (ID 2118)

      09:30 - 16:30  |  Author(s): C. Murone

      • Abstract

      Background
      MGMT is a DNA repair protein which removes alkylating DNA adducts from the O[6] position of guanine. Expression of MGMT is often silenced by promoter methylation in human cancers. MGMT methylation is a predictive marker for prolonged survival in glioblastoma patients treated with an alkylating agent, temozolomide. As MGMT methylation has been found in lung cancers, there is an increasing interest on the clinical utility of temozomolide in the treatment of human cancers. However, it is essential to use appropriate quantitative or semi-quantitative method methods to definitively establish the methylation status of the tumour.

      Methods
      We critically assessed MGMT methylation status in 6 lung cancer cell lines and 56 lung tumours using three different methodologies. We first assessed the MGMT methylation pattern using methylation sensitive – high resolution melting (MS-HRM). The methylation status at each CpG dinucleotide was assessed bisulfite pyrosequencing of methylated clones. The level of MGMT methylation was quantified using quantitative methylation specific PCR.

      Results
      MGMT methylation was found in 3 lung cancer cell lines by MS-HRM. The melting profiles of all methylated samples were indicative of heterogeneous methylation pattern by melting curve analysis. To examine the methylation status at each CpG sites of individual template, two MGMT methylated lung cell lines (H1666 and H69) were further tested by limiting dilution analysis and bisulfite pyrosequencing. The number and site of methylated CpG dinucleotides greatly varied in each template, confirming the heterogeneous methylation pattern in both cell lines. In 56 lung tumours, heterogeneous MGMT methylation was detected in seven samples (13%) by MS-HRM. The level of MGMT methylation was then estimated. 17 lung tumours, including the 7 MS-HRM positives and 10 additional tumours, were positive. However, the methylated level in all of the methylated samples was low, ranging from below 1% (12 samples) and up to 12%.

      Conclusion
      The level of MGMT promoter in lung cancer is difficult to estimate. Ideally clonal analysis should be used to estimate the proportion of methylated alleles. Alternatively, methylation profiling using MS-HRM followed by pyrosequencing can be used to identify tumours showing significant levels of methylation. If MGMT methylation is found only in a small proportion of tumour cells, it is unlikely to be a useful target for therapy. Overcalling of MGMT methylated tumours may provide the explanation for the lack of survival benefit with temozolomide treatment in MGMT-methylated lung cancer patients in a recent phase II clinical trial (NCT00423150). This indicates that incorporation of immunohistochemistry for the MGMT protein should also be part of the assessment of the MGMT status of lung cancer.