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B. Hegedus



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    P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.05-012 - An inducible transition cell model reflecting epithelioid versus sarcomatoid differentiation of Malignant Pleural Mesothelioma (ID 1892)

      09:30 - 09:30  |  Author(s): B. Hegedus

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related malignancy characterized by frequent resistance to chemo- and radiotherapy. Signals induced by Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) have been identified as important drivers for malignant growth in several tumor entities including thoracic malignancies. We have recently demonstrated that FGFR signals stimulate growth and migration in MPM cell models, whereas inhibition of FGFR1 reduced tumor growth and had an antagonistic effect on malignant behavior of MPM cells. In several cell models of biphasic MPM, FGF2 induced phenotypical changes reminiscent of epithelial-mesenchymal-transition (EMT). In this study we analyzed these FGF-induced morphological and functional alterations and the associated signal transduction mechanisms in more detail.

      Methods
      Cells were stimulated with recombinant FGF2 and analyzed by microscopy and ImageJ software. The specific inhibitors PD166866, UO126, MK2206, LY294002 and SB431542 were used to block FGFR1, MEK, AKT, PI3K and TGFbeta receptors, respectively. Alterations in gene expression were determined via whole-genome expression arrays and further evaluated by immunofluorescence. Downstream signaling was investigated by immunoblotting.

      Results
      In M38K and SPC212 cells FGF2 induced morphological alterations that were characterized by a more spindle-shaped appearance and reduced contacts with adjacent cells. This correlated with increased cell migration. With respect to signal transduction, the effects of FGF2 could be blocked by inhibition of FGFR1 or MEK, whereas inhibition of AKT, PI3K or receptors of the TGFbeta/activin family had no effect. Expression arrays of both cell models indicated regulation of several matrix metalloproteinases (MMP1, MMP4), the integrin subunit ITGA6 and the two TGFbeta family-related proteins INHBB and Smad7. Since the phenotypical changes were similar to EMT, genes previously connected to EMT were analyzed. Whereas slug/SNAI2 and ZEB1 were increased, other mesenchymal genes such as vimentin or N-cadherin were already expressed at high levels in the untreated cells, likely due to the mesodermal origin of mesothelial cells. In M38K, E-cadherin was decreased whereas in the more “fibroblastoid-type” SPC212 E-cadherin was generally expressed at very low levels.

      Conclusion
      Our data suggest that FGF2 induces morphological changes that result in a more sarcomatoid cell morphology and expression of some markers connected to EMT. These effects depend on the MAPK pathway and are connected to more aggressive cell behavior, paralleling the higher aggressiveness and worse prognosis of the sarcomatoid and biphasic compared to the epithelioid histological subtype of MPM.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P3.06-009 - Paradoxical increase in Ki67 with neoadjuvant chemotherapy in NSCLC (ID 1143)

      09:30 - 09:30  |  Author(s): B. Hegedus

      • Abstract

      Background
      Neoadjuvant chemotherapy is used to help downstage cancer, and is widely used in locally-advanced breast cancer. Studies of Ki67 proliferation index in breast cancer have been fairly extensively evaluated. Comparison of core and surgical specimens in breast cancers without exposure to chemo- or radiotherapy revealed technical variation of up to 20% in the Ki67 index score. In non-small cell lung cancer (NSCLC), however, little is known about the association of rate of change of Ki67 after neoadjuvant chemotherapy with radiographic response and clinical outcomes. We surveyed NSCLC from patients treated with neoadjuvant chemotherapy.

      Methods
      NSCLC patients treated with neoadjuvant chemotherapy were identified from 3 Hungarian hospitals and 1 community hospital in the United States. Matched pre-chemotherapy and post-surgical resection formalin-fixed, paraffin-embedded (FFPE) tumor specimens were collected and the Ki67 index was scored under an IRB exemption. We set an absolute difference of 20% between pre-chemotherapy and post-resection Ki67 scores as “meaningful” to avoid possible technical variation reported in the literature. Radiographic response to neoadjuvant chemotherapy by RECIST 1.0 criteria was also measured. Fisher’s exact test was used to measure the relationship between gender, histology, and type of chemo with “meaningful” Ki67 index. Logistic regression was used to test the relationship between Ki67 index decline rate and outcome subgroup (response/no response). Decline rate was defined as a ratio of decrease of Ki67 to its level at baseline.

      Results
      63 matched cases were identified. 46 cases were analyzable for pre-chemotherapy and post-operative Ki67, and chemotherapy regimen; 40 cases also had response criteria by RECIST. Of the 46 cases, the median patient age was 59 years (range 40-77), 23 were men, and 30 of 34 had a smoking history. There were 24 adenocarcinomas and 22 squamous cell carcinomas. Stages I, II, III, and IV were 2, 9, 31, and 4; respectively. All but two patients received a platinum-doublet, with 24 containing gemcitabine. 5 patients also received neoadjuvant radiotherapy. The mean Ki67 index scores were 35% (range 1-100%) pre-chemotherapy and 32% (0-100%) post-resection. 9 patients (19.6%) had a paradoxical “meaningful” increase in Ki67 index after neoadjuvant chemotherapy. Of the 40 patients with RECIST response data, there was 1 complete response, 34 partial responses, 4 stable diseases, and 1 disease progression. There were no statistically significant differences between gender, histology subtype, or type of platinum doublet administered associated with this paradoxical increase. There was no statistically significant difference in Ki67 decline rate between responders and nonresponders.

      Conclusion
      In this cohort of patients, there was a paradoxical “meaningful” increase in Ki67 index after neoadjuvant chemotherapy in ~20% of patients, without a clear association between histology or platinum doublet administered. For patients receiving neoadjuvant chemotherapy, the Ki67 index decline rate was not associated with radiographic response. Approximately 1/5 of NSCLC may have selection of tumor cells for a higher proliferative index when undergoing neoadjuvant platinum-based chemotherapy. The effect of this on progression-free and overall survival is being evaluated.

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      P3.06-046 - The tumor suppressor integrin-alpha 7 is frequently downregulated in malignant pleural mesothelioma: biological and prognostic consequences (ID 3071)

      09:30 - 09:30  |  Author(s): B. Hegedus

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a malignancy characterized by therapy resistance and poor outcome. The high mortality of MPM is largely due to its invasive growth, local recurrence and locoregional spread. The identification of molecular changes that lead to this phenotype is indispensable. ITGA7 is a laminin binding receptor and has been identified as a novel tumor suppressor based on its frequent mutation in other malignancies. However, the alterations of ITGA7 have not yet been studied in MPM.

      Methods
      ITGA7 mRNA levels of normal mesothelial and MPM cells were measured by q-RT-PCR. ITGA7 promoter silencing in mesothelioma cell cultures was quantified by methylation-specific PCR analysis and by sequencing. Migratory activity of MPM cells has been investigated by 2D videomicroscopy. In vitro cell proliferation and adhesion assays were performed on siRNA transfected cells to demonstrate the biological consequence of decreased ITGA7 expression. ITGA7 expression in MPM tissue specimens was analysed by immunohistochemistry (IHC) and correlated to clinical outcome data.

      Results
      ITGA7 is highly expressed by normal mesothelial cells while decreased in MPM cells. In most MPM cells the expression of ITGA7 was reduced through promoter hypermethylation. ITGA7 promoter is hypermethylated in a number of tested MPM cell cultures (n=13) and the ratio of promoter methylation inversely correlates with ITGA7 expression. MPM cells with high in vitro migratory activity demonstrated a significantly lower ITGA7 expression when compared to slow migrating MPM cells. Proliferation of normal mesothelial cells is inhibited by laminin and this inhibitory effect is abolished by downregulation of ITGA7 expression via siRNA transfection. Adhesion of normal mesothelial and MPM cells is enhanced by laminin, however, it is not decreased by downregulation of ITGA7. The clinical significance of ITGA7 protein expression was investigated by IHC in a cohort of 89 MPM surgical specimens. Importantly, patients with high ITGA7 expression had significantly longer median overall survival (448 days) than patients with low expression (247 days, log rank test: p=0,0281).

      Conclusion
      ITGA7 is a novel tumor suppressor in MPM and its expression is down-regulated in MPM cells by promoter hypermethylation. Moreover, low ITGA7 expression in tumor cells influences clinical outcome as a negative prognostic factor in human MPM.