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M. Martorell



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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-025 - Evaluation of NGS and RT-PCR Methods for ALK Assessment in European NSCLC Patients: Results from the ETOP Lungscape Project (ID 5001)

      14:30 - 15:45  |  Author(s): M. Martorell

      • Abstract

      Background:
      The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%, depending on population and detection method. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This is one of the first studies comparing all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.

      Methods:
      96 cases from the ETOP Lungscape iBiobank (N=2709) selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK) were examined by FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014), central RT-PCR and NGS. H-score 120 is used as cutoff for IHC+. For both RT-PCR and NGS, RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers were used covering the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used, allowing simultaneous sequencing of 70 ALK, RET and ROS1 specific fusion transcripts associated with NSCLC, as well as novel ALK translocations using 5’-3’ ALK gene expression ‘Imbalance Assay’.

      Results:
      NGS provided results for 90 cases, while RT-PCR for 77. Overall, 70 cases have results for all 4 methods, with fully concordant 60 (85.7%) cases (49 ALK-, 11 ALK+). Before employing the ‘Imbalance Assay', in 5 of the remaining 10 cases, NGS differs from the other methods (3 NGS-, 2 NGS+), while in the other 5, NGS agrees with RT-PCR in all, IHC in 2, and FISH in 1. Using the concordant result of at least two of the three methods as true negative/positive, the specificity and sensitivity of the fourth is 96/94/100/96% and 94/94/89/72% for IHC/FISH/RT-PCR/NGS, respectively (incorporating imbalance: NGS sensitivity=83%). Imbalance scores are presented here for 18 NGS- cases: 9 ‘NGS-/FISH+/IHC+’, 9 ‘NGS-/FISH-/IHC-‘. Among the ‘NGS-/FISH+/IHC+’, there is strong evidence of imbalance in 4 cases (score’s range: 0.0144-0.0555), uncertain in 5 (range: 0.0030-0.0087), and no evidence (scores≤0.0004) in the 9 negative cases.

      Conclusion:
      NGS is a useful screening tool for ALK rearrangement status, superior to RT-PCR when RNA yield is limited. When using NGS, it is critically important to integrate the 5’-3’ imbalance assay and to confirm with one or more additional methods in the ‘imbalance’ cases. Data further highlight the possibility of missing actionable rearrangements when only one screening methodology is available.

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    P1.05 - Poster Session with Presenters Present (ID 457)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Early Stage NSCLC
    • Presentations: 1
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      P1.05-013 - Lung Tumorspheres as a Platform for Testing New Therapeutic Strategies in Non-Small Cell Lung Cancer (ID 4848)

      14:30 - 15:45  |  Author(s): M. Martorell

      • Abstract

      Background:
      Resistance to treatment is one of the causes influencing the high mortality of lung cancer. This feature seems to be linked to a subpopulation known as Cancer Stem Cells (CSCs), which are able to grow as spheroids (suspension culture). The aim of the study was to obtain tumorspheres from lung cancer cell lines and to use them as an in vitro platform for drug screening.

      Methods:
      Cells from lung cancer cell lines (A549, H1650, PC9, H460 and H358) were grown in monolayer and as spheroids. Cultured cells were used: (i) to compare the cytotoxic effect of anticancer drugs in adherent vs lung-tumorspheres (ii) to perform a high-throughput screening with a commercial chemical library (Prestwick) and (iii) to analyze the citotoxicity of specific inhibitors of Wnt, Hedgehog and Notch pathways. Briefly, cells were plated at the desired density in 200 μl of medium in 96-well plates and compounds were added at 4 different concentrations (n=3). Cell viability was measured after 48 and 72h, using MTS Assay. Cell viability was normalized to the respective mock-treated control cells and presented as percentage of control.

      Results:
      Cells cultured in serum-free conditions were able to form spheroids, such as stem-like cells. Under these culture conditions, classical anticancer drugs (cisplatin, paclitaxel, vinorelbine and pemetrexed) exhibited mild or null cytotoxic effects on A549, H1650, PC9, H460 and H358 spheroids. Moreover, we performed a high-throughput screening with Prestwick library and remarkably, three compounds reduced the number of viable cancer cells. As regards ‘stemness’ inhibitors, Wnt (IWP2 and XAV939) and Hedgehog inhibitors (Vismodegib) show high activity against tumorspheres (p<0.05), suggesting them as possible therapeutic strategies in NSCLC

      Conclusion:
      Our data suggest that lung-tumorspheres showed resistance to classical anticancer drugs, strengthening its possible use as a short-term culture platform for a simple, and cost- effective screening to investigate novel therapeutic approaches. In this setting, some compounds were identified as promising therapeutic agents on lung tumorspheres, but confirmatory data are still necessary. This project was supported by [RD12/0036/0025] from RTICC, SEOM 2012, [PI12-02838 and PI15-00753] from ISCIII