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P. Pauwels



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    MINI 24 - Epidemiology, Early Detection, Biology (ID 140)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
    • Presentations: 1
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      MINI24.11 - Expression of PD-1 and Its Ligands in Human Malignant Pleural Mesothelioma (ID 1676)

      16:45 - 18:15  |  Author(s): P. Pauwels

      • Abstract
      • Presentation
      • Slides

      Background:
      The discovery of immune checkpoint receptors as cytotoxic T lymphocyte antigen-4 (CTLA-4) and more recently programmed death-1 (PD-1) introduced a new era in cancer immunotherapy. Immune checkpoints are responsible for controlling and inactivating the immune system in order to avoid autoimmunity and prevent tissue damage. PD-1 is expressed primarily on activated effector T lymphocytes. Its natural ligands are programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). Expression of PD-L1/PD-L2 on tumor cells or in stroma impairs effector T lymphocyte activity within the tumor microenvironment. Trials with antibodies that block the ligand-immune checkpoint interaction have shown promising results in several cancer types.Data on few mesothelioma patients suggest that blocking immune checkpoints could offer new opportunities for treatment of this very aggressive tumor.. We investigated PD-1, PD-L1 and PD-L2 expression in MPM. Furthermore the effect of interferon-gamma (IFNg), an important cytokine for immune-mediated tumor control, on their expression pattern was analyzed.

      Methods:
      Flow cytometry and immunohistochemistry (IHC) were used for the expression of PD-1, PD-L1 and PD-L2 on human primary MPM and T cells and on MPM cell lines that cover the three major histological subtypes of of MPM, i.e. epitheloid (M28, H2795, H2818), sarcomatoid (VAMT-1, H2731, H-Meso-1) and mixed (NKI04, MSTO-211H) mesothelioma cells. The effect of stimulation with IFNg on expression of PD-1 and its ligands was measured.

      Results:
      PD-1 surface expression was found on T cells and not on MPM tumor cells, corresponding to literature showing that PD-1 is only expressed on T cells, B cells and macrophages. Different expression patterns were observed regarding PD-L1 and PD-L2. Flow cytometry showed significant PD-L1 expression on all the epitheloid and sarcomatoid mesothelioma cell lines. Two out of three cell lines tested positive for PD-L2, both for the epitheloid and the sarcomatoid subtype. The mixed cell lines were negative for PD-1 and its ligands. Following IFNg stimulation, PD-L1 and PD-L2 expression was induced or upregulated on all cell lines. Primary MPM cells showed variable expression of PD-L1. IHC data for PD-1 and PD-L1 expression correspond to the flow cytometry results.

      Conclusion:
      Taken together, these data on PD-1, PD-L1 and PD-L2 expression on human MPM cells and T cells support further investigation of the expression profile of the immune checkpoint PD-1 and its ligands in MPM patients samples. We are currently performing this using multicolor flow cytometry and IHC.

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    P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 3
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      P2.04-027 - Erlotinib Induce miRNA Alterations in T790M EGFR Mutated NSCLC: Preclinical Study (ID 2483)

      09:30 - 17:00  |  Author(s): P. Pauwels

      • Abstract
      • Slides

      Background:
      Lung cancer is one of the leading causes of cancer-related deaths worldwide. In the most common type, non-small cell lung cancer (NSCLC), an array of oncogenic driver mutations affecting growth factor receptors and signaling pathways, such as EGFR, KRAS, c-Met or ALK translocation has driven the development of directed-drug therapies with tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Nevertheless, the appearance of resistance mechanisms, such as c-Met amplification or de novo mutations in EGFR, decreases the effectiveness of these therapies. Therefore, we analyzed the levels of miRNA which have proved to be involved in disease progression (miR-30b-5p,195-5p,221-3p) in NSCLC cells that are resistant (H1975) or sensitive (HCC827) against first generation TKI (erlotinib) treatment, to assess whether they are markers of response to the treatment.

      Methods:
      The H1975 cell line (EGFR mutations T790M/L858R) was cultured in RPMI 1640 medium, supplied with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco). The sulforhodamine B assay was performed to assess cell proliferation. The cells were treated during 72h with 10µM erlotinib (SelleckChem) or DMSO. After collection, cells were lysed and RNA was extracted through commercial kit (RNA Mini Spin, GE Healthcare). The miRNAs profile analysis was performed through TaqMan Real-Time PCR and miRNA RNU-48 was used as endogenous control. Data was processed according to the formula 2[-ΔΔct]. Control values (H1975 + vehicle) are used as baseline and results are shown in logarithmic scale (Image).

      Results:
      Cells treated with 10µM erlotinib show an increased expression of miR-30b-5p, miR-195-5p compared to control values. On the other hand, we detected that the expression of the miR-221-3p was strongly down-regulated in respect the control values.

      Conclusion:
      H1975 carries the T790M mutation in EGFR, associated in NSCLC with appearance of resistance to TKIs treatment. However, we observed after treatment an increase of onco-suppressor miR-30b-5p and decrease of the oncogenic mir-221-3p, which are reported to correlate with good prognosis (Garofalo 2013, Zhong 2014). Moreover, mir-195-5p, another miRNA related with onco-suppression, is also upregulated, which has been reported to correlate with good response (Liu, 2015). Overall, our data suggests that erlotinib treatment alters miRNA in an EGFR mutated cell line. Further analysis of miRNA in exosomes produced by H1975, and comparison with HCC827 exosomal and cellular miRNA levels is currently undergoing in our group to confirm their value as response to treatment biomarkers.Figure 1



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      P2.04-092 - Sequencing of Actionable EGFR Mutations from PtDNA in NSCLC: A Feasibility Study of Non-Invasive Analysis of Sensitivity to TKI (ID 984)

      09:30 - 17:00  |  Author(s): P. Pauwels

      • Abstract

      Background:
      In ~10 % of Caucasian patients with non-small cell lung cancer (NSCLC), the presence of an activating epidermal growth factor receptor (EGFR)-mutation has resulted in a more favourable prognosis by its exquisite sensitivity to a targeted treatment with tyrosine kinase inhibitors (TKI’s). However, in up to 20% of those patients, the presence of "driver" alterations cannot reliably be established due to an inadequate diagnostic sample and/or impossibility to re-biopsy a patient. Activating EGFR-mutations can be accurately detected in plasma with a high concordance to matched tumour tissue with allele-specific PCR assays of plasma tumor DNA (ptDNA). This ‘liquid biopsy’ can replace response to treatment, allow detection of early relapse in the follow-up and estimate prognosis. The aim of this study is to assess the feasibility of detecting ptDNA in patients with EGFR mutations, to assess the concordance of ptDNA levels to mutations in matched tissue samples and to correlate ptDNA levels with clinical response or relapse after start of TKI-treatment.

      Methods:
      Tumour tissue samples of 20 patients (10 with/10 without activating EGFR-mutations), was assessed for the presence of the respective mutation in matched ptDNA, obtained either at presentation or during follow up. DNA was extracted from aliquots (1ml) of plasma with the use of QIamp circulating nucleic acid kit (Qiagen). The target DNA is amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas EGFR Mutation Test kit. The concordance was estimated with Bayesian variables.

      Results:
      26 tissue and 34 plasma samples were collected from 20 Caucasian patients with median age of 67 years (54 to 84), 60% female, 30% non-smokers, 100% adenocarcinomas and 95% histologically or cytologically confirmed stage IV NSCLC. The median number of samples per patient was 3 (2 - 5). In the tissue samples 6 patients had an exon 19 deletion, 1 had exon 18 mutation, 2 had exon 20 mutation and in 1 patient a simultaneous exon 19 deletion and T790M mutation. The Bayesian characteristics of ptDNA determination are as follows:

      A: at sample level ( n = 34) B: at study population level (n = 20)
      1. Prevalence of mutation 8.8% 10%
      2. Sensitivity 12.5% 20%
      3. Specificity 100% 100%
      4. PPV 100% 100%
      5. NPV 32.2% 55%
      6. Accuracy 38% 60%
      7. Concordance 11.5% 7.7%


      Conclusion:
      Detection of ptDNA in EGFR-positive patients is feasible. However, ptDNA mutation testing by cobas[R] 4800_Blood Test was not reliable due to the low analytical sensitivity and the heterogeneity of the patient population. Further studies with other methods as next-generation sequencing or digital droplet PCR are warranted.

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      P2.04-094 - Exosomal MiRNA Analysis of Non-Small Cell Lung Cancer (NSCLC) Liquid Biopsies. Mirror of the Disease Status?: Proof of Concept Study (ID 1395)

      09:30 - 17:00  |  Author(s): P. Pauwels

      • Abstract
      • Slides

      Background:
      The discovery of the alterations in EGFR, c-Met or ALK in NSCLC has driven the development of targeted-drug therapy using tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is needed. The exosomes extracted from blood samples could be non-invasive and regularly updated biomarkers. Here we analyze selected exosomal miRNAs to evaluate its biomarker potential in NSCLC.

      Methods:
      1 ml of serum/plasma sample from 11 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. Exosome were isolated through commercial-kit or D~2~O/sucrose density-gradient ultracentrifugation. After exosome characterization (Western-Blot, Transmission Electron Microscopy) a panel of miRNAs (30b-5p, 30c-5p, 103,195,221-3p,222-3p), correlated with NSCLC disease (Garofalo et al, 2013), was analyzed. The miRNAs profile analysis was performed through TaqMan Real-Time PCR and mir-1228-3p was used as endogenous control. The data was processed according to the formula 2[-ΔΔct]. Control values are used as baseline and results are shown in logarithmic scale (figure).

      Results:
      Patients without molecular alterations (WMA): The levels of miR-30b-5p/30c-5p/103/221-3p/222-3p were down-regulated relative to the healthy controls. The patient with docetaxel treatment (P2) has an increased down-regulation of these miRNAs compared to the non-treated patient (P1). Patient with BRAF G464V: We observed an increased up-regulation of miR-30b-5p/30c-5p/103/221-3p/222-3p just after stopping Erlotinib treatment (P4a) compared with one month after the treatment (P4b). Patients with C-Met 3+ over-expression: We detected an increase of expression of miR-30b-5p/30c-5p and a decrease of expression of mir221-3p/222-3p in a patient treated with Crizotinib (P6) compared to a non-treated patient (P5). Patients with EGFR (exon 19 del): We observed a decrease of expression of mir221-3p/222-3p in a patient treated with Afatinib beyond progression (P8) compared to a non-treated patient (P7). Patients with ALK t(2p23): We detected a decrease of expression of miR-30b-5p/30c-5p/103 compared to healthy controls. No differences between treated (P9-P11) and non-treated (P3) patients were observed. Nevertheless, mir221-3p/222-3p differs significantly between patients treated with Ceritinib one week (P10) and one month (P11) after the treatment was started.

      Conclusion:
      This panel of exosomal miRNAs derived from patients with varying mutations is responsive to different treatments. The down-regulation of miR-30b-5p/30c-5p in exosomes of patients with WMA and ALK t(2p23) mutations, mirrored the reported low levels of these miRNAs in NSCLC tissue (Zhong et al.,2014). Follow-up analysis to correlate clinical progression and exosome miRNAs profile is currently ongoing. Figure 1



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    P3.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 208)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 1
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      P3.01-022 - A Prospective Multicenter Study for ALK IHC+ Metastasized NSCLC (ID 2566)

      09:30 - 17:00  |  Author(s): P. Pauwels

      • Abstract
      • Slides

      Background:
      Pulmonary adenocarcinomas may harbor driver mutations, that sensitize tumors to drugs that specifically target the genetic alteration. Metastasized NSCLC with an EML4-ALK translocation are sensitive to a range of tyrosine kinase inhibitors, of which crizotinib is most extensively studied. ALK-positive NSCLC was determined in a phase III trial with fluorescence in situ hybridisation (ALK FISH+). ALK immunohistochemistry (IHC) seems to run parallel with ALK FISH positivity. However discrepant cases occur, which include ALK IHC+ FISH-. The aim of this study is to collect cases with ALK IHC+ and compare within this group response to crizotinib treatment of ALK FISH+ cases with ALK FISH- cases.

      Methods:
      A prospective multicenter investigator initiated research study was started in Europe. This study is supported by Pfizer. Cases diagnosed with ALK IHC+ lung cancer (5A4 or D5F3) treated with crizotinib are collected centrally. Slides are submitted centrally for validation of ALK IHC (with ETOP and Ventana protocol), ALK FISH (with Vysis probes) and DNA analysis.

      Results:
      The study started on April 1 2014 and is still open. Currently 10 centers are actively participating. 1443 cases have been examined with ALK IHC of which 39 (2.7%) recorded positive. 24 cases have been submitted to the database. The validation process is still ongoing. The fraction of ALK IHC+ FISH- cases is low. Two cases with ALK IHC+ FISH- metastastatic NSCLC responded to crizotinib treatment. In two cases ALK positivity could not be confirmed (ALK IHC- and ALK FISH-). These patients had progressive disease following crizotinib treatment.

      Conclusion:
      A clinically relevant question what the effect of ALK inhibitor treatment is on metastatic NSCLC ALK IHC+ FISH- compared to ALK IHC+ FISH+ is examined. Other centers with interested collaborating physicians are invited to participate.

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