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V. Relan



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    P1.14 - Poster Session 1 - Mesothelioma (ID 194)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Mesothelioma
    • Presentations: 1
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      P1.14-011 - Changes in expression of cancer drug resistance genes in mesothelioma cells exposed to carboplatin (ID 2610)

      09:30 - 09:30  |  Author(s): V. Relan

      • Abstract

      Background
      Background A proportion of patients with mesothelioma respond to chemotherapy consisting of pemetrexed and platinum, but tumour responsiveness most often becomes blunted after several cycles. To discover the mechanism of loss of sensitivity to chemotherapeutic agents, we compared expression of cancer drug resistance genes between platinum sensitive and resistant mesothelioma cells.

      Methods
      Mesothelioma cells generated from three chemo-naïve patients propagated in cell culture were exposed to increasing concentrations of carboplatin until in vitro resistance of at least one log10 concentration difference in IC50 was achieved in dose response cytotoxicity assays. For each individual, cells resistant to carboplatin at 8µg/ml and at 20µg/ml were generated. Control cells from each line were passaged in parallel in medium only. Cells were in log phase growth and culture medium was free of platinum for at least two weeks prior to extraction of RNA using Qiagen RNAeasy Mini kits. High quality RNA (assessed by denaturing gel electrophoresis) was then DNase treated and reverse transcribed using Qiagen RT² Profiler PCR Array reagents. Gene expression in control and platinum resistant cells was determined from the Cancer Drug Resistance PCR Array of (Catalogue Array PAHS-004Z) according to manufacturer’s instructions.

      Results
      SULT1E1 was overexpressed in one mesothelioma line resistant to carboplatin at 8µg/ml, and in two of three resistant to 20µg/ml carboplatin, in comparison with parallel passaged controls. One of three cell lines resistant to carboplatin at both the 8µg/ml and 20µg/ml level overexpressed ERBB3, and another resistant at 20µg/ml overexpressed PPARγ. Drug resistance genes displayed more aberrant expression in cells resistant to higher concentrations of carboplatin.

      Conclusion
      The increase in expression of these three genes in mesothelioma resistant to carboplatin suggests that they may be useful targets for circumvention of resistance, but their mechanistic role in development of platinum resistance requires demonstration. In particular, since PPARγ ligands (e.g. roglitazone) have been shown to sensitise cancer cells to chemotherapeutic agents, and are proposed as anticancer agents, it is possible that the functional effect of PPARγ upregulation is moderating rather than causal. Supported by Cancer Australia.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.01-011 - Heterogeneity in tumour content and necrosis in primary lung cancers: Implications for molecular analysis (ID 3326)

      09:30 - 09:30  |  Author(s): V. Relan

      • Abstract

      Background
      Lung adenocarcinoma (AC) and squamous cell carcinoma (SCC) tumours have a large variance in tumour cell content. This heterogeneity is a concern for genomic studies, as it is difficult to distinguish mutational differences between tumour and non-tumour if low percentage tumour is used for analysis. In addition to this, tumour samples are affected by the amount of necrosis present, as the overall number of viable cells is decreased. We assessed tumour and necrotic content in lung tumour specimens from AC and SCC patients and aimed to identify possible implications for the suitability of these samples in molecular characterisation studies using next generation sequencing technology.

      Methods
      Lung tissue specimens were collected during the period of 1990 to 2013 from patients at The Prince Charles Hospital who consented to donate their surgically resected lung tissues for research. Tissues were macroscopically dissected, snap frozen in liquid nitrogen and stored at -80°C. A tissue section was taken and stained with haematoxylin and eosin (H&E) for two pathologists to independently assess tumour cell and necrotic content. Tumour cell content (TC) in each specimen was scored as percentage of viable cells as seen on the H&E slide, where necrotic content (NC) was recorded as a percentage of the whole slide section. Statistics were calculated using SPSS v21 software. Tumour specimens screened for eligibility to The Cancer Genome Atlas sequencing project are presented here.

      Results
      Tumours from 62 AC and 104 SCC subjects were scored (specimen characteristics in Table 1). Scoring between the two pathologists was highly correlated, with a high intraclass reliability (0.94 and 0.96 for TC and NC respectively).

      Table 1: Clinical and Pathological Characteristics of Specimens
      AC SCC
      Number of Specimens 384 609
      Number of Males/Females 36/26 84/20
      Median Specimens per Subject 4 4
      Range of Specimens per Subject 1-25 1-27
      Median TC 35% 30%
      Range of TC 0-88% 0-90%
      Median NC 0% 6%
      Range of NC 0-90% 0-100%
      Median Age 62 yrs 68 yrs
      Range of Age 45-85 yrs 46-91 yrs
      Median Smoking Pack Years 40 56
      Range of Smoking Pack Years 0-115 0-158
      TC varied from 0-~90% for both subtypes. Comparing AC and SCC, the median TC was higher in AC than SCC (35% vs 30% respectively, p<0.05). NC varied from 0-~100%, but was generally low. The median NC was statistically significantly different between AC and SCC (0% and 6% respectively, p<0.001). TC was weakly correlated with NC (Spearman Rank r = 0.32, p<0.01). There were no clinically important correlations between smoking pack years, gender or age with TC and NC of specimens.

      Conclusion
      Lung AC and SCC specimens are heterogeneous in terms of TC and NC. Therefore, only a small proportion of resected lung cancer specimens meet the criteria required for massively parallel sequencing projects that require high quality tumour DNA and RNA (ie low NC) and relatively low stromal contamination (ie high TC).