Multidrug resistance (MDR) in small cell lung cancer is one of the major causes of failures of chemotherapy. MDR is a means of protection of tumor cells against chemotherapeutic drugs. Although the molecular basis of MDR is not fully understood, genes involved in apoptosis may be mutated. Recent finding of a link between over-expression of an apoptotic gene, cyclooxygenase 1 (cox 1), and MDR suggests that cox 1 is involved in the development of MDR phenotype. This research was an attempt to observe whether up-regulation of cox 1 contributes to the MDR phenotype in small cell lung cancer cells. This research ultimately may provide a mechanism to reverse the abberant up-regulation of apoptosis genes associated with multidrug resistance to either eliminate or control reproduction of cancer cells. Real time RT PCR was used to confirm the up-regulation of cox 1 in cultured MDR resistant small cell lung cancer cells (GLC4). The up-regulated cox 1 expression was down-regulated using RNA interference technology (RNAi) by transfection with an anti-cox 1 siRNA. More than 90% transfection of cells was confirmed using confocal microscopy. Down-regulation of cox 1 was validated as the protein expression significantly decreased (P=0.004) from multidrug resistant small cell lung cancer transfected cells compared to multidrug resistant nontransfected cells. There was decrease level of expression of cox 1 in multidrug resistant cells after the knockdown with siRNA specific to cox 1. The decreased level of cox 1 expression and, therefore, Cox 1 production increased the rate of apoptosis in small cell lung cancer cells as indicated by its sensitivity to the doxorubicin. / Department of Biology

The gene, cox-1, is over-expressed in cultured GLC4 small cell lung cancer cells concurrent with the development of multi-drug resistance (MDR) as a result of the use of the chemotherapeutic agent used to combat the cancer, doxorubicin. Prevention of MDR has been a tremendous challenge in cancer research and this research is concerned with abolishment of MDR as a cancer survival strategy. RNA-mediated interference technology (RNAi) was employed using siRNA to decrease cox-1 expression and temporarily restore the susceptibility of the cells to doxorubicin. GLC4 cells are of three types: S (sensitive cells never exposed to doxorubicin); ADR (MDR cells cultured in doxorubicin), and; REV (revertant cells previously cultured in presence of doxorubicin but no longer). REV and ADR cells were transfected with cox-1 siRNA. After 24 h, 1x106cells were used for RNA isolation and 1 μg of RNA was used for RT-PCR to assess down-regulation of cox-1 RNA. RT-PCR results indicated that cox-1 RNA was down-regulated to basal levels seen before exposure to doxorubicin. Ct values for GLC4/ADR and cox-1 down-regulated GLC4/ADR cells were 23 and 34, respectively. The result indicated abundant levels and moderate levels of cox-1 mRNA in the ADR cells and the transfected ADR cells respectively. The relative expression level of cox-1 mRNA was 33% higher in the non-transfected GLCR/ADR cells as compared to the transfected GLCR/ADR cells as shown by the curve. Two hundred thousand cells were used for hemacytometer cell counts in the presence of trypan blue to assess cell viability. cox-1 down-regulation in ADR cells resulted in a significantly higher percentage of non-viable cells (25.4%) as compared to its non-transfected control (20.5%) using a Student’s t-test (*P <0.05). Similarly, fluorescence microscopy confirmed that apoptosis was significantly increased in the ADR cells treated with doxorubicin and cox-1 siRNA simultaneously (69.4%) as compared to its non-transfected control (56.7%) (*= P <0.01). A Western blot analysis performed by Fernando Cuadrado indicated that siRNA transfection decreased the expression of COX-1 by 66% in GLC4/ ADR cells as compared to the non-transfected control using densitometry. However, no conclusive results were obtained using flow cytometry as the flow cytometer was incapable of analyzing the mixed cell population (adherent and suspension) which is a characteristic of this cell line, GLC4. Thus, we have clearly demonstrated that MDR cancer cells can be altered temporarily to become susceptible to doxorubicin, a potentially important finding for the treatment of cancer patients. / Department of Biology

No description available.

Recent studies have suggested significant variation in radiotherapy schedules used to treat advanced NSCLC, both between different centres as well as between countries. In this study, treatment methodologies have been explored using management plans proposed by radiation oncologists when given general questions and theoretical case histories for patients with advanced NSCLC. Methods and Materials The survey was conducted by sending a questionnaire to twenty four radiotherapy centres in Europe. The questionnaire was composed of two sections. The first section concerned reasons for starting radiotherapy, parameters that influence the choice of total dose and fractionation for radiotherapy and the kind of equipment that is used. The second section examines five case histories and asked the responders about the management of these five theoretical patients also regarding the radiotherapy techniques proposed and the aim of treatment (radical or palliative). Furthermore, trials comparing different regimens of palliative radiotherapy in patients with NSCLC were compared. Nineteen trials were reviewed. There were important differences in the doses of radiotherapy investigated, the patient characteristics and the outcome measures. Results In the first part responders (70% of the centres) suggested as the most important factors that influence the choice of total dose and fractionation for radiotherapy, distant metastases, performance status of the patient, lung function and size of the primary tumour. The most common reasons for starting the treatment is not only symptom relief, but also cure and prolongation of life. In the second part, more than 95% of the responders replied that they would give radiotherapy in each of these cases. The median total doses proposed where 20Gy/5fractions/1week or 30Gy/10fractions/2weeks for cases A and D (equivalent dose for fractionation 2Gy per fraction=23 and 33Gy) and 60-68Gy/30fractions/6weeks or 68Gy/34fractions/7weeks for cases B, C and E. For case E, 20% of the responders suggested Stereotactic Body Radiotherapy with 63Gy in 3 Fractions. The total dose and number of fractions of radiotherapy could be related to the perceived aims and expectations of treatment e.g. those aiming to extent life would give significantly higher total doses in a larger number of fractions, whereas those aiming to relieve symptoms would give significantly lower total doses. For the review to the literature there is no strong evidence that any regimen gives greater palliation. Higher dose regimens give more acute toxicity, especially oesophagitis. There is evidence for a modest increase in survival (5% at 1 year and 3% at 2 years) in patients with better performance status (PS) given higher dose radiotherapy. Some regimens are associated with an increased risk of radiation myelitis. Conclusions This survey demonstrates a range of treatment strategies for advanced and inoperable NSCLC within Europe. There are a number of factors that influence the perceived aims of treatment and treatment planning. These factors should be taken into account when evaluating the effectiveness of different irradiation techniques, especially in the determination of radiobiological parameters and dose-response relations. The majority of patients should be treated with short courses of palliative radiotherapy, of 1 or 2 fractions. Care should be taken with the dose to the spinal cord. The use of high dose palliative regimens should be considered for and discussed with selected patients with good performance status. More research is needed into reducing the acute toxicity of large fraction regimens and into the role of radical compared to high dose palliative radiotherapy. In the future, large trials comparing different RT regimens may be difficult to set up because of the increasing use of systemic chemotherapy. Trials looking at how best to integrate these two modalities, particularly in good PS patients need to be carried out. / Πρόσφατες μελέτες έχουν αναδείξει σημαντική ποικιλία στα ακτινοθεραπευτικά σχήματα που χρησιμοποιούνται στην ακτινοθεραπεία του μη μικροκυτταρικού καρκίνου του πνεύμονα προχωρημένου σταδίου. Στη συγκεκριμένη μελέτη θεραπευτικές μεθοδολογίες έχουν διερευνηθεί χρησιμοποιώντας τεχνικές που προτείνονται από ογκολόγους ακτινοθεραπευτές . Υλικά και Μέθοδοι: Η μελέτη αποτελείται από δυο μέρη. Στο πρώτο ένα ερωτηματολόγιο εστάλη σε είκοσι τέσσερα ακτινοθεραπευτικά κέντρα στην Ευρώπη .Το ερωτηματολόγιο αποτελούνταν από δυο τμήματα. Στο πρώτο ζητούνταν οι λόγοι για τους οποίους γίνεται έναρξη της ακτινοθεραπείας, οι παράμετροι που επηρεάζουν την επιλογή για τη συνολική δόση και τις συνεδρίες για την θεραπεία και τον εξοπλισμό που χρησιμοποιούν. Στο δεύτερο τμήμα παρουσιαστήκαν πέντε θεωρητικά κλινικά περιστατικά και ζητήθηκε η αντιμετώπιση αυτών των θεωρητικών ασθενών. Στο δεύτερο μέρος της μελέτης πραγματοποιήθηκε ανασκόπηση στη βιβλιογραφία και σύγκριση των αποτελεσμάτων κλινικών δοκιμών που έχουν πραγματοποιηθεί στο παρελθόν. Αποτελέσματα: Στο ερωτηματολόγιο απάντησαν το εβδομήντα τοις εκατό των κέντρων στα όποια εστάλη. Στο πρώτο μέρος ως οι πιο σημαντικοί παρόντες που επηρεάζουν την επιλογή της τελικής δόσης και τις συνεδρίες οριστήκαν οι παρουσία απομακρυσμένων μεταστάσεων, η κλινική εικόνα του ασθενούς, η πνευμονική λειτουργία και το μέγεθος του πρωτογενούς όγκου. Οι σημαντικότεροι λόγοι για έναρξη θεραπείας είναι ανακούφιση από τα συμπτώματα καθώς και επιμήκυνση της ζωής. Στο δεύτερο μέρος ενενήντα πέντε τοις εκατό των κέντρων απάντησαν ότι θα πραγματοποιούσαν ακτινοθεραπεία και στους πέντε αυτούς ασθενείς. Η επιλογή της συνολικής δόσης και συνεδρίων επηρεάζεται από την θεώρηση της θεραπείας ως παρηγορική ή θεραπευτική. Τα κέντρα που είχαν στόχο την επιμήκυνση της ζωής έδιναν μεγαλύτερες δόσεις και περισσότερες συνεδρίες εν αντιθέσει με τα κέντρα που είχαν στόχο την υποχώρηση των συμπτωμάτων που έδιναν μικρότερης δόσεις σε λιγότερες συνεδρίες. Στο δεύτερο μέρος υπολογιστήκαν οι σχετικές βιολογικές δραστικότητες από τα δεδομένα της βιβλιογραφίας καθώς και ο παράγοντας πολλαπλασιασμού του όγκου και κατασκευάστηκαν καμπύλες δόσης απόκρισης. Συμπεράσματα: Η μελέτη αποδεικνύει την ύπαρξη ποικιλίας στις τεχνικές που χρησιμοποιούνται στη θεραπεία προχωρημένου και ανεγχείρητου μη μικροκυτταρικού καρκίνου του πνεύμονα. Αυτοί οι παράγοντες πρέπει να συνυπολογίζονται όταν εκτιμάται η αποτελεσματικότητα διαφορετικών ακτινοθεραπευτικών τεχνικών, κυρίως στο προσδιορισμό ακτινολογικών παραμέτρων και σχέσεων δόσης –απόκρισης.

Access to bevacizumab, an important component of oncology treatment regimens, may be limited. This survey of oncologists in the US (n = 150), Europe (n = 230), and emerging markets (EM: Brazil, Mexico, and Turkey; n = 130) examined use of and barriers to accessing bevacizumab as treatment of advanced solid tumors. We also assessed the likelihood that physicians would prescribe a bevacizumab biosimilar, if available. Bevacizumab was frequently used as early-line therapy in metastatic colorectal cancer, metastatic non-squamous non-small-cell lung cancer, and metastatic ovarian cancer (all markets), and as a second-line therapy in glioblastoma multiforme (US, EM). A greater percentage of EM-based physicians cited access-related issues as a barrier to prescribing bevacizumab versus US and EU physicians. Lack of reimbursement and high out-of-pocket costs were cited as predominant barriers to prescribing and common reasons for reducing the number of planned cycles. Overall, similar to 50% of physicians reported they "definitely" or "probably" would prescribe a bevacizumab biosimilar, if available. Efficacy and safety data in specific tumor types and lower cost were factors cited that would increase likelihood to prescribe a bevacizumab biosimilar. A lower cost bevacizumab biosimilar could address the unmet needs of patients and physicians worldwide, and may have the greatest impact on patient outcomes in EM.

The use of stereotactic body radiation therapy employing few large fractions of radiation dose for the treatment of non-small cell lung cancer has been proven very successful, high values of tumour control probability (TCP) being clinically achieved. In spite of the success of the fractionation schedules currently used, there is a tendency towards reducing the number of fractions for economical and practical reasons, and also for maximizing the comfort of the patients. It is therefore the main aim of this thesis to investigate the impact of a severely reduced number of fractions on the tumour control probability for tumours that contain hypoxic areas. The impact on TCP of other factors such as hypoxic fraction, distribution of the oxygen partial pressure and location of the hypoxic volume within the tumour were also investigated. The effect of tumour motion due to breathing was included and evaluated using Cone Beam Computed Tomography (CBCT) data from patients imaged with internal markers in the liver and pancreas. The results clearly showed that in the presence of hypoxia, TCP is seriously compromised if there is not enough time for reoxygenation between fractions. A reduction in the number of fractions of just one fraction may require an increase of several Gy per fraction to obtain a similar TCP. The diaphragmatic tumour motion range showed little influence on TCP provided that the PTV encompassed all tumour positions. The dose delivered to the PTV margin was found not to be the only factor that is significant for local control, the average dose correlated better with TCP. The agreement of the results of this work with clinical results also serve as a strong indicator that inter-fraction reoxygenation is an important process in real-life patients treated with stereotactic body radiotherapy.

Abstract Reactive nitrogen species (RNS) and reactive oxygen species (ROS) have been linked with the pathogenesis of lung malignancies and chronic obstructive pulmonary disease (COPD). In vitro studies indicated that mesothelioma and lung carcinoma cell lines synthesize nitric oxide synthases (NOS) mRNA. The Comet-assay indicated that asbestos fibers caused DNA single -strand breaks in mesothelial cells, and this effect was enhanced by glutathione depletion. The use of FPG in the Comet assay indicated that the asbestos induced DNA strand breaks were oxidant mediated. In vivo non-neoplastic pleura was mostly negative for inducible NOS (iNOS), while inflamed pleura was positive. The immunohistochemical expression of iNOS was detected in 74% and 96% of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often intense iNOS immunoreactivity compared to the sarcomatoid subtype. Normal mesothelial cells showed occasional positivity for endothelial NOS (eNOS), but reactive mesothelial cells were strongly stained. eNOS was found in 89% of mesotheliomas. Vascular endothelial growth factor (VEGF) was identified in 47%, a VEGF receptor FLK1 in 69% and the VEGF receptor, FLT1, in 71% of mesotheliomas. FLK1 or FLT1 immunoreactivities were more often seen in epithelioid and biphasic mesotheliomas than in sarcomatoid mesotheliomas. In lung samples of non-smokers, smokers and COPD patients, the levels of nitrotyrosine were higher in alveolar macrophages of smokers and COPD patients than in the non-smokers and in the alveolar epithelium of smokers and COPD patients than in the non-smokers. The iNOS expression was weak in the bronchial and alveolar epithelium in all groups but eNOS was most prominently expressed in alveolar macrophages while neuronal NOS (nNOS) was negative in all of the major cell types of the lung. Bronchial metaplasia-dysplasia-sequence was clearly positive for iNOS, nNOS and nitrotyrosine. Thus, smoking can cause protein nitration also in normal lung. Prominent iNOS and nNOS immunoreactivity in metaplasia-dysplasia-lesions suggests a divergent role of NOSs in carcinogenesis and destruction of alveolar epithelium in emphysematous lung. In lung cancer samples, iNOS was detected in 40% cases, while 89% and 81% cases were positive for eNOS and nNOS, respectively. Intense eNOS staining was seen more often in adenocarcinomas than in squamous cells carcinomas, and iNOS immunoreactivity was seen more often in grade I-II tumors than in grade III tumors. The patients with tumors showing high expression of iNOS, eNOS and nNOS, exhibited better survival, but this was not an independent prognostic factor.

A common co-pathology of large lung tumors located near the central airways is collapse of portions of lung due to blockage of airflow by the tumor. Not only does the lung volume decrease as collapse occurs, but fluid from capillaries also fills the space no longer occupied by air, greatly altering tissue appearance. During radiotherapy, typically administered to the patient over multiple weeks, the tumor can dramatically shrink in response to the treatment, restoring airflow to the lung sections which were collapsed when therapy began. While return of normal lung function is a positive development, the change in anatomy presents problems for future radiation sessions since the treatment was planned on lung geometry which is no longer accurate. The treatment must be adapted to the new lung state so that the radiation continues to accurately target the tumor while safely avoiding healthy tissue. However, to account for the dose delivered previously, correspondences of anatomy between the former image when the lung was collapsed and the re-expanded lung in a current image must be obtained. This process, known as deformable image registration, is performed by registration software. Most registration algorithms assume that identical anatomy is contained in the images and that intensities of corresponding image elements are similar; both assumptions are untrue when collapsed lung re-expands. This work was to develop an algorithm which accurately registers images in the presence of lung expansion. The lung registration method matched CT images of patients aided by vessel enhancement and information of individual lobe boundaries. The algorithm was tested on eighteen patients with lung collapse using physician-specified correspondences to measure registration error. The image registration algorithm developed in this work which was designed for challenging lung patients resulted in accuracy comparable to that of other methods when large lung changes are absent.

Small cell lung cancer (SCLC) is an extremely aggressive disease characterized by early metastasis and acquired resistance to therapy. SCLC is distinguished by its neuroendocrine (NE) component; the role of which is not fully understood in metastasis and response to therapy. Patients respond exceptionally well to first round chemotherapy; however, relapse with therapy-resistant tumours is virtually inevitable. Hypoxic regions within tumours can contribute towards metastasis and therapy resistance, highlighting hypoxia-targeted therapy as a novel approach for improving treatment for SCLC patients. Tumours are highly phenotypically heterogeneous, raising debate over the roles played by each cell type. Analysis of NE and epithelial markers in SCLC cell lines highlighted this inter-tumour heterogeneity. Further heterogeneity is displayed in SCLC xenograft tumours that show areas of dual epithelial and NE marker expression as well as regions negative for both markers. Irradiating xenograft tumours enhanced heterogeneity of the NE marker, pro-opiomelanocortin (POMC), which is ectopically secreted by a subset of SCLC tumours. Examining changes in marker expression post-therapy could provide vital information regarding transitions that can serve to guide therapy. SCLC is a highly metastatic disease. The role of the NE phenotype in human SCLC is not fully understood, but is considered essential for metastasis in murine models. Sub-cutaneous, intravenous and intra-splenic injection were carried out and resulted in no metastasis, spontaneous tumour generation and peripheral liver tumour growth, respectively. POMC expression was present and extremely heterogeneous within the liver, suggesting that NE properties are maintained in metastases; however, further work is necessary to develop a more consistent metastatic model that can be used to assess responses to therapy in a more clinically relevant setting. SCLC tumours proliferate rapidly and outgrow their nutrient and oxygen supplies, resulting in hypoxic conditions. Here, carbonic anhydrase IX (CA IX) becomes up-regulated in order to maintain pH levels suitable for survival. The specific CA IX inhibitor, S4, induces hypoxia-specific cell death in vitro and impairs tumour growth in vivo. This response is further accentuated by combining S4 with single or repeated cisplatin doses. Combination treatment reduced gene expression of S-phase kinase-associated protein (Skp2), associated with cisplatin resistance. CA IX inhibition combined with cisplatin chemotherapy therefore presents a novel treatment for SCLC tumours that could reduce therapy resistance. In summary, heterogeneity is extremely important when choosing treatment options for SCLC and must be considered when basing treatment on single biopsies. NE and epithelial markers are present within sub-cutaneous and liver tumours; however, a reliable multi-organ metastatic model is necessary to fully appreciate the role of these markers in the spread of SCLC. Hypoxic regions within sub-cutaneous xenograft tumours upregulate CA IX. Inhibition of this enzyme resulted in impaired tumour growth, particularly when used together with cisplatin. Combining CA IX inhibition with cisplatin presents a much-needed novel therapy for SCLC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22005号 / 医博第4519号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM