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S. Klebe



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    MS10 - Beyond Lung Cancer - The Pathology of Intra-Thoracic Mimics (ID 27)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Pathology
    • Presentations: 1
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      MS10.3 - Pleural Neoplasms (ID 503)

      14:00 - 15:30  |  Author(s): S. Klebe

      • Abstract
      • Presentation
      • Slides

      Abstract
      Pleural neoplasms may be difficult to diagnose because they must be distinguished from metastatic malignancy involving the pleura, and from benign reactive processes causing pleural thickening. In contrast to primary lung neoplasms, primary pleural neo­plasms are uncommon, with secondary involvement being more frequent. A correct diagnosis is important, so that appropriate therapy can be delivered. Also, the diagnosis may affect the pateint’s prospects for compensation. The most common primary pleural neoplasm is mesothelioma, but compared to lung tumours even mesotheliomas are relatively rare. Mesotheliomas exhibit a wide variety of histologic patterns, which may be confused with other neo­plasms. Here we consider those other pleural lesions that must be differen­tiated from mesothelioma. Secondary Malignant Neoplasms Affecting the Pleura Secondary neoplasms represent the most common malignancy affecting the pleura, with lung carcinoma being the most common, followed by metastatic breast cancer, malignant lymphoma, (including Hodgkin and non-Hodgkin malignant lymphomas). Metastatic carcinomas of ovarian or gastric origin, malignant melanoma and sarcomas account for only a small percentage of cancer-associated pleural disease (about 5%). Some of these may show diffuse infiltration of the pleura in a pattern indistinguishable from pleural malignant mesothelioma macroscopically and radiologically. The majority of such so-called pseudomesotheliomatous neoplasms originate from the lung, but metastases from kidney, thyroid gland, larynx, stomach and cutaneous malignant melanoma as well as various sarcomas, including malignant phyllodes tumor, have also been described. Renal cell carcinoma (RCC) and amelanotic malignant melanoma may also metastasize to the pleura. RCCs with a sarcomatoid pattern can present a diagnostic problem. Labelling for RCC-related markers such as CD10 can also be seen in mesotheliomas and in sarcomatoid RCCs, the other RCC markers may be negative: correlation with imaging studies is imperative. Rare cases of sarcoma metastatic to the pleura may mascerade as mesothelioma and comprehensive clinical history is of highest imprtance. Other Neoplasms Arising in the Pleura Thymoma Affecting the Pleura Thymoma may spread into the pleura from an anterior mediastinal thymoma, but primary pleural thymomas are described and can present as localised masses or with diffuse pleural thickening. The concept of primary pleural thymoma has become accepted, but only about 25–30 cases have been reported to date. Spindle Cell Neoplasms Synovial sarcoma of the pleura Both biphasic and monophasic synovial sarcomas (SySa) are characterized by a distinctive t(X;18) chromosomal translocation and the production of the resultant alternative fusion genes, SYT-SSX1 or SYT-SSX2. SySa are well recognised as primary intrathoracic neoplasms in the pleura, where they can be confused with biphasic or sarcomatoid mesothelioma, carcinosarcoma /spindle cell carcinoma, or a biphasic pulmonary blastoma. Clinical features, immunohistochemistry and electron microscopsy are of limited usefulness and it is the detection of the t(X;18) chromosomal translocation and expression of the resultant SYT-SSX1 or SYT-SSX2 that is diagnostic of SySa. This can be done by FISH, but PCR is more sensitive and we consider molecular analysis of t(X;18) to be mandatory for discrimination between a genuine pleural SySa versus a biphasic or monophasic mesothelioma with histological appearances that mimic SySa. Solitary Fibrous Tumors (SFTs) Of Pleura Solitary fibrous tumors (SFTs) are uncommon localized spindle-cell fibroblastoid neoplasms. They most commonly arise in relation to the visceral pleura (~80%) or the parietal pleura, but they can occur in the mediastinum, lung parenchyma or related to pericardium or diaphragm. Terms such as submesothelial fibroma, fibrous mesothelioma and localized fibrous tumor have been used as synonyms in the past. Localized fibrous tumor might be the best term, because multiple simultaneous tumours have been described but 'SFT' is well established. Thoracic SFTs can vary greatly in size abd have a peak incidence between the 4[th] and 6[th] decade. SFTs are often incidental findings in asymptomatic patients, and the radiologic appearances may suggest the diagnosis, but a biopsy is always required. Symptoms can be related to the size and site of the tumor with compression of surrounding tissues. The histology ranges from the 'patternless pattern' of Stout to 'herringbone', cellular, myxoid and hemangiopericytic or angiofibromatoid areas. The mitotic index may be useful to predict malignant behaviour. Desmoid Tumors of the Pleura Desmoid tumors in the region of the chest wall are well recognized and can impinge upon the parietal pleura, but primary desmoid tumors of the pleura and lung are extremely rare. Benign and Malignant Nerve Sheath Tumors Neoplasms that have histologic and immunohistochemical features of nerve sheath tumors can occur as primary tumours in the pleural cavity. Benign ones typically show Verocay bodies and Antoni A and B areas. When malignant, these cells can cause major diagnostic confusion. Immunohistochemical staining with neural markers such as S100 protein is helpful to confirm a neurogenic origin. Inflammatory Myofibroblastic Tumors Inflammatory pseudotumors (plasma cell granuloma; inflammatory myofibroblastic tumor) may occasionally involve the pleura. They consist of of a proliferation of spindle cells with varying numbers of inflammatory cells, with prominene of plasma cells. Current thinking favours the concept that these are neoplastic lesions with the capacity in some cases for multicentricity, angioinvasion and metastasis (especially in older patients in whom IHC for anaplastic lymphoma kinsae (ALK) is negative). Epithelioid Hemangioendothelioma of the Pleura Epithelioid hemangioendothelioma is a malignant angioformative neoplasm, where the neoplastic endothelial cells are epithelioid and sometimes quite bland in appearance. The pattern in H&E-stained sections may be virtually indistinguishable from mesothelioma, and both may label for thrombomodulin and in some cases, cytokeratins. Labelling for endothelial markers such as CD31, CD34 or factor VIII-RAG aids in the diagnosis. Pleuropulmonary Blastoma Pleuropulmonary blastomas are rare in the pleura and mostly occur in early childhood. Tumours consist of primitive cells underneath an epithelium with a cambium layer-like appearance as seen in sarcoma botryoides. Rhabdomyoblasts may be found and anaplastic sarcomatous elements, such as embrynal rhabdomyosarcoma, fibrous sarcoma, chondrosarcoma and undifferentiated sarcoma, may be present. Pleural Lymphomas Primary pleural lymphomas are rare, with primary effusion lymphoma (PEL) and pyothorax-associated lymphoma being the most common. In our experience, most cases of pleural lymphoma represent secondary spread in cases of previously-diagnosed extrapleural lymphoma.

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    MTE06 - Molecular Pathology Approach for Early Clinical Stage Mesothelioma: Cytological Diagnosis, and Reactive Mesothelial Hyperplasia (ID 50)

    • Event: WCLC 2013
    • Type: Meet the Expert (ticketed session)
    • Track: Mesothelioma
    • Presentations: 1
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      MTE06.1 - Molecular Pathology Approach for Early Clinical Stage Mesothelioma: Cytological Diagnosis, and Reactive Mesothelial Hyperplasia (ID 599)

      07:00 - 08:00  |  Author(s): S. Klebe

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-006 - Targeted delivery of RRM1-specific siRNA leads to tumour growth inhibition in malignant pleural mesothelioma (ID 1508)

      09:30 - 16:30  |  Author(s): S. Klebe

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy with poor prognosis. MPM is typically recalcitrant to treatment and new therapies are urgently needed. Multiple genes involved in proliferation and metabolic activity are upregulated in MPM and these represent attractive targets for an siRNA-based therapeutic intervention.

      Methods
      We carried out an RNAi-based screen of 40 target genes previously shown to be upregulated in MPM to identify candidate genes with roles in cell growth and survival in MPM cell lines. Effects of target gene silencing were measured using standard in vitro proliferation assays. Lead candidates were further assessed with siRNA dose response experiments. The specificity of siRNA-mediated growth inhibition was confirmed by assessing gene knockdown by real-time qPCR and Western blotting. The effects of the most potent siRNAs on xenograft tumour growth were assessed in vivo by delivery using EGFR-targeted, siRNA-loaded, minicells.

      Results
      All 40 genes were effectively silenced, and for 6 genes (PLK1, CDK1, NDC80, RRM1, RRM2 and BIRC5) knockdown with 2 independent siRNAs resulted in significant growth inhibition over time in multiple cell lines. Dose response experiments revealed that siRNAs specific for RRM1 and RRM2 were the most effective at inhibiting growth with IC50 values in the low nanomolar range. Intravenous administration of RRM1 siRNA packaged in minicells targeted with EGFR-specific antibodies (2x10[9] minicells per dose, 4 times per week for 3 weeks) led to consistent and dose-dependent inhibition of MPM tumor growth compared with treatment with an inactive siRNA. Reducing the dose and number of administrations did not reduce growth inhibition; as little as 1x10[9] minicells administered once a week were sufficient to completely inhibit MPM tumour growth.

      Conclusion
      RRM1 is an attractive target for siRNA-based inhibition, and siRNA delivery with EGFR-targeted minicells represents a novel therapeutic approach for MPM.

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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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      P2.05-011 - Characterisation of chemo-resistant syngeneic orthotopic rat pre-clinical models of mesothelioma. (ID 1718)

      09:30 - 16:30  |  Author(s): S. Klebe

      • Abstract

      Background
      Malignant mesothelioma is an aggressive cancer with a low response to current therapies and consequently a poor prognosis. There is an urgent need to identify novel and more effective treatments to improve survival and quality of life for patients with mesothelioma. While a number of novel therapeutic agents have recently been examined in clinical trials, to date none of these has resulted in changes to standard management. This is due in part to a lack of robustness and relevance of the pre-clinical models used to assess new treatments. Therefore validated and biologically relevant pre-clinical models that demonstrate the clinical behaviour and drug sensitivity of mesothelioma, as well as typical resistance mechanisms, are necessary to improve the discovery of new treatments. Here we describe the generation and evaluation of chemotherapy resistant pre-clinical models of mesothelioma derived from the previously utilized syngeneic II-45 rat mesothelioma model.

      Methods
      Cell lines resistant to the current standard of care agents: cisplatin, pemetrexed gemcitabine, vinorelbine, and cisplatin and pemetrexed in combination, were generated by 15 rounds of exposure to the respective agent. Normal rat mesothelial cells (4/4 RM.4), the parental II-45 and the 5 resulting chemo-resistant mesothelioma cell lines were characterised for resistance to these and other agents. Tumours arising from syngeneic pleural engraftment of these cell lines were assessed by size, morphology, immunohistochemical markers of human malignancy, chromosomal changes and expression of genes involved in drug resistance and metabolism. Engrafted rats were assessed for survival, and circulating haematological, cytokine and biomarker profiles were generated and compared.

      Results
      Five different II-45 cell lines with approximately 2-fold resistance to the chemotherapeutic agent they were repeatedly exposed to, were established (cisplatin, p < 0.05; pemetrexed, gemcitabine, vinorelbine, p < 0.001). Cross resistance to other classes of anti-cancer agents also developed indicating potential multi-drug resistant phenotypes. Tumours derived from both the parental and chemo-resistant cell lines were immunohistochemically indistinct from human mesothelioma with positive labeling for WT1, calretinin, HBME-1, cytokeratin and popoplanin and negative labeling for two carcinoma related markers TTF-1 and CD15. Homozygous deletion of p16[INK4A]/p14[ARF], and increased expression of several members of the ATP-binding cassette transporter superfamily and Androgen receptor (AR) were demonstrated, consistent with findings in human mesothelioma. Corresponding with biomarkers studies in human disease, increased levels of osteopontin (p < 0.01), mesothelin (p < 0.01), vascular endothelial growth factor (p < 0.001) and neutrophil to lymphocyte ratio were identified relative to control bloods. Further, the acquisition of chemo-resistance resulted in changes to tumour morphology with tumours ranging from essentially epithelioid in the gemcitabine resistant tumours to entirely sarcomatoid in the combination (cisplatin and pemetrexed) resistant tumours. Overall survival was also affected by the acquisition of resistance with rats with pemetrexed resistant tumours having decreased survival.

      Conclusion
      These models display many features corresponding with the human disease, and thus provide powerful and robust pre-clinical platforms for in vivo mesothelioma studies.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-017 - Long non coding RNAs (lncRNAs) are dysregulated in malignant pleural mesothelioma (MPM) (ID 1524)

      09:30 - 16:30  |  Author(s): S. Klebe

      • Abstract

      Background
      Malignant Pleural Mesothelioma (MPM) is an aggressive disease, often diagnosed at an advanced stage. It is characterized by a long latency period and prior asbestos exposure. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates rarely exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) plays an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. The aims of this study were to characterize the expression and function of these lncRNAs in MPM.

      Methods
      To identify novel lncRNAs involved in MPM, microarray profiling was performed on five cell lines - the immortalized normal mesothelial cell line (MeT-5A) and four MPM lines (two epithelioid H28 and H226 and two biphasic MM05 and MSTO) using Invitrogen’s NCode lncRNA microarrays. These allow simultaneous assessment of mRNA and lncRNA content. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological (>3-fold difference) significance. Expression of high priority candidates were technically validated using RT-qPCR, and biologically validated in three independent test sets. Pathway analyses were performed to interrogate the relationship between lncRNA and mRNA expression. Cell proliferation and colony formation assays were used to investigate lncRNA function.

      Results
      Microarray profiling and real-time qPCR validation identified 9 lncRNA candidates with significant differential expression in MPM compared with normal mesothelial cells Validation in three independent test sets by RT-qPCR analysis demonstrated consistent up-regulation of four of these lncRNAs. Receiver Operating Curve analysis showed that two of these candidates were able to separate benign pleura and MPM with high sensitivity and specificity. In addition, high expression of AK054908 was associated with nodal metastases with lower levels of AK130275 and AF268386 observed in patients receiving induction chemotherapy. Cases with higher EF177379 levels also demonstrated a trend to improved survival. The majority of mRNAs co-expressed with candidate lncRNAs were associated with cellular and metabolic processes including cell cycle, cell death and apoptosis. In functional studies, siRNA knockdown of AK130275 showed suppression of cell growth and colony formation in MPM cells with moderate changes observed following knockdown of EF177379.

      Conclusion
      To our knowledge this is the first systematic study of lncRNA expression profiles in MPM. We have found that lncRNA expression profiles can distinguish malignant mesothelium from normal pleural tissue, and that some lncRNAs are associated with nodal metastasis and long term survival. We also demonstrate that lncRNAs have potential prognostic and diagnostic utility with functional roles in regulating cell growth. Further work is required to evaluate whether these lncRNAs are capable of differentiating mesothelioma from lung cancer and benign asbestos-related diseases, and to reveal their specific functions in MPM pathogenesis.

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    P2.14 - Poster Session 2 - Mesothelioma (ID 196)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Mesothelioma
    • Presentations: 1
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      P2.14-008 - Effect of Aquaporin 1 Modulation in Malignant Pleural Mesothelioma: Inhibition of Cell Proliferation and Colony Formation in vitro and Tumour Growth in vivo using a Heterotopic Mouse Model (ID 2615)

      09:30 - 16:30  |  Author(s): S. Klebe

      • Abstract

      Background
      Aquaporin 1 (AQP1) has been shown to be an independent prognostic marker for survival in malignant pleural mesothelioma (MPM). AQP1 is a trans-membrane protein normally expressed in mesothelial cells and is part of a family of proteins involved in fluid homeostasis, cell proliferation and motility. AQPs have been implicated in various aspects of tumour development. The aim of the current study was to determine the functional role of AQP1 in MPM, using in vitro and in vivo models.

      Methods
      Primary MPM cells obtained from patient’s pleural effusions and the MPM cell line NCI-H226 were subjected to a specific pharmacological AQP1 blocker and AQP1 siRNA knockdown and examined for proliferation and colony formation using MTS and anchorage independent assays. Levels of AQP1 expression were determined by immunohistochemistry and RT-PCR. The influence of AQP1 on tumour growth in vivo was studied using a heterotopic mouse model. Briefly, 5 x 10[6] NCI-H226 cells were injected subcutaneously in the hind flank of BALB/C nude mice and allowed to grow to 100 mm[3] before daily intra-tumour injections of AQP1 blocker. Tumour size was measured daily.

      Results
      AQP1 expression correlates with cell proliferation in primary MPM cells (R[2] = 0.69). Blockade of AQP1 with pharmacological blocker or siRNA knockdown in MPM cells was shown to significantly decrease cell proliferation, both in NCI-H226 (p < 0.05) and primary MPM cells expressing AQP1 (p < 0.05). Primary MPM, where more than 20% of tumour cells expressed AQP1, showed greater reduction in proliferation compared to cells having AQP1 expression <20%. Application of AQP1 blocker decreased both the number and size of colonies formed after NCI-226 cells were subjected to anchorage independent growth (p < 0.05). In a pilot study using the heterotopic mouse model the size of tumours was decreased after 5 days using 20 μM of AQP1 blocker compared to an untreated control (n=6 for each group).

      Conclusion
      Results indicate that AQP1 plays a functional role in MPM. Modulation of AQP1 decreases cell proliferation and colony formation as well as the growth of MPM tumours in a heterotopic mouse model. Larger scale animal studies are required to further understand the role of AQP1 in MPM and the potential clinical implications of an AQP1 blocker in the treatment of MPM