京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23826号 / 人健博第97号 / 新制||人健||7(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 精山 明敏, 教授 椎名 毅, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Background: The standard of care for mediastinal staging during endobronchial ultrasound (EBUS) is Systematic Sampling (SS) where a minimum of 3 lymph node (LN) stations are biopsied, even if they appear normal on imaging. When LNs appear normal on PET and CT, the Canada Lymph Node Score can also identify if they appear normal on EBUS. For these Triple Normal LNs, the pretest probability of malignancy is < 6%, and routine biopsy may not be required. This preliminary study introduced Selective Targeted Sampling (STS), which omits biopsy of Triple Normal LNs and compared it firsthand to SS. Methods: A prospective, feasibility RCT was conducted to determine whether the progression of a definitive trial was warranted. Primary outcomes and their progression criterium were recruitment rate (70% acceptable minimum); procedure length (no overlap between sampling methods’ 95%CIs); and missed nodal metastasis (overlap between sampling methods’ diagnostic accuracy 95%CIs and crossing of the null for the percent difference in diagnosis). cN0-N1 NSCLC patients undergoing EBUS were randomized to the STS or SS arm. Patients in the STS arm were then crossed over to the SS arm to receive standard of care. Wilson’s CI method and McNemar’s test of paired proportions were used for statistical comparison. Surgical pathology was the reference standard. Results: Thirty-eight patients met the eligibility criteria, and all were recruited (100%; 95%CI: 90.82 to 100.00%). The median procedure lengths, in minutes, for STS and SS were 3.07 (95%CI: 2.33 to 5.52) and 19.07 (95%CI: 15.34 to 20.05) respectively. STS had a diagnostic accuracy of 100% (95%CI: 74.65% to 100.00%), whereas SS was 93.75% (95%CI: 67.71% to 99.67%) with the inclusion of inconclusive results. Percent difference in diagnosis between sampling method was 5.35% (95%CI: -0.54% to 11.25%). Conclusion: With the progression criteria successfully met, a subsequent multicentered, non-inferiority crossover trial comparing STS to SS is warranted. / Thesis / Master of Science (MSc) / Before deciding on treatment for patients with lung cancer, a critical step in the investigations is finding out whether the lymph nodes in the chest contain cancer. This is best done with a needle that biopsies those lymph nodes through the walls of the airway, known as endobronchial ultrasound transbronchial needle aspiration. Guidelines require that every lymph node in the chest be biopsied through a process called Systematic Sampling. However, new research has suggested that some lymph nodes may not need a biopsy. These lymph nodes are ones with a very low chance of cancer, based on their imaging tests. In this study, Selective Targeted Sampling was introduced whereby lymph nodes that appeared normal were not initially biopsied. The study followed a feasibility design, which proved sufficient patient interest, adequate safety and possible benefits in pursuing a larger trial comparing Selective Targeted Sampling to Systematic Sampling.

Είναι ευρέως αποδεκτό και καλά τεκμηριωμένο ότι ο υποδοχέας του επιδερμικού αυξητικού παράγοντα (epidermal growth factor receptor, EGFR) ελέγχει σημαντικές λειτουργίες των καρκινικών κυττάρων, όπως τον πολλαπλασιασμό και την απόπτωση, αλλά και διαδικασίες όπου συμμετέχουν περισσότεροι του ενός τύποι κυττάρων, όπως τη διήθηση και την αγγειογένεση. Μεταξύ των τύπων καρκίνου, στην ανάπτυξη των οποίων συμμετέχει ο EGFR, είναι και ο μη μικροκυτταρικός καρκίνος του πνεύμονα (ΜΜΚΠ). Πολύ πρόσφατα δεδομένα δείχνουν ότι ένα άλλο μόριο που εμπλέκεται στην ανάπτυξη του καρκίνου του πνεύμονα είναι το Notch. Ο ρόλος του είναι περίπλοκος και διττός: Έχει προταθεί ότι το Notch επάγει την ανάπτυξη του ΜΜΚΠ και αναστέλλει την ανάπτυξη του μικροκυτταρικού καρκίνου του πνεύμονα (ΜΚΠ). Επιπλέον, έχει βρεθεί ότι το μονοπάτι μεταγωγής σήματος του Notch επηρεάζει, αλλά και επηρεάζεται από άλλα μόρια. Στην παρούσα μεταπτυχιακή εργασία διερευνήθηκε ο ρόλος του EGFR και του Notch στην ανάπτυξη κυττάρων ΜΜΚΠ χρησιμοποιώντας τον προσδέτη του EGFR, EGF και τον αναστολέα της γ-σεκρετάσης DAPT. Για τη διεξαγωγή των πειραμάτων χρησιμοποιήθηκαν οι ανθρώπινες καρκινικές κυτταρικές σειρές ΜΜΚΠ Η23, Α549, Η661 και ΗCC827. Οι κυτταρικές σειρές Η23, Α549 και Η661 εκφράζουν τον αγρίου τύπου (wild type, wt) EGFR και η κυτταρική σειρά HCC827 εκφράζει EGFR που φέρει τη μετάλλαξη (mutation) (DE746- A750). Αρχικά με ανάλυση κατά western μελετήθηκε το προφίλ των κυττάρων ως προς τα επίπεδα έκφρασης του ενδοκυττάριου τμήματος του Notch (Notch Intracellular Domain, NICD). Βρέθηκε ότι τα κύτταρα Η23 εκφράζουν τα υψηλότερα επίπεδα Notch ICD, τα κύτταρα Η661 και HCC827 μέτρια επίπεδα και τα κύτταρα Α549 τα χαμηλότερα. Στη συνέχεια με τη μέθοδο του ΜΤΤ έγινε έλεγχος του DAPT στον πολλαπλασιασμό των κυττάρων και βρέθηκε ότι τα κύτταρα Η661 είχαν τη μεγαλύτερη αναστολή, παρόμοια συμπεριφορά έδειξαν και τα Α549. Τα κύτταρα Η23 εμφάνισαν μικρότερη ανταπόκριση σε σχέση με τα Η661 ενώ τα κύτταρα HCC827 εμφανίστηκαν ανθεκτικά στο DAPT. Η ανασταλτική δράση του DAPT στα κύτταρα Η661 συνοδεύτηκε με επαγωγή της απόπτωσης η οποία προσδιορίστηκε με τη μέθοδο αννεξίνης V καθώς και με επαγωγή της αυτοφαγίας η οποία ανιχνεύτηκε κάνοντας ανάλυση κατά western για τα πρωτεϊνικά επίπεδα της beclin-1. Περαιτέρω τα κύτταρα ενεργοποιήθηκαν με EGF και εν συνεχεία προστέθηκε DAPT. Παρατηρήθηκε ότι στα κύτταρα Η23 η προσθήκη του EGF δεν επέτρεψε να δράσει ανασταλτικά το DAPT ενώ στα Η661 εν μέρει ο EGF αντέστρεψε την ανασταλτική δράση του DAPT. Επιλέγοντας τις κυτταρικές σειρές Η23 και Η661, μελετήθηκε η δράση του DAPT και του EGF στα επίπεδα του Notch ICD. Παρατηρήθηκε ότι στα κύτταρα Η23, το DAPT μείωσε με χρονοεξαρτώμενο τρόπο τα πρωτεϊνικά επίπεδα του Notch ICD μέχρι και 6 ώρες μετά την προσθήκη του στα κύτταρα ενώ 24 ώρες μετά το φαινόμενο αντιστράφηκε. Η προσθήκη του EGF δεν επηρέασε τα επίπεδα του Notch ICD σε καμία από τις χρονικές στιγμές που μελετήθηκαν. Στα Η661 κύτταρα το DAPT προκάλεσε χρονοεξαρτώμενη μείωση των επιπέδων Notch ICD η οποία διήρκησε μέχρι και 24 ώρες μετά τη προσθήκη του DAPT. Ο EGF όπως και προηγουμένως δεν επηρέασε τα επίπεδα του Notch ICD. Παρατηρώντας ότι στα Η661 το DAPT ασκεί δράση με μεγαλύτερη διάρκεια σε σχέση με τα κύτταρα Η23, τα κύτταρα Η661 ενεργοποιήθηκαν με EGF και στη συνέχεια προστέθηκε το DAPT προκειμένου να δούμε τη δράση του συνδυασμού στα επίπεδα του Notch ICD. Βρέθηκε ότι ο EGF αντέστρεψε την μείωση των Notch ICD επιπέδων που προκαλεί μόνο του το DAPT. Τα αποτελέσματα αναδεικνύουν ότι τα μονοπάτια του EGFR και του Notch, συνηγορούν προς την ίδια κατεύθυνση για τη μείωση του όγκου και αυτό υποδηλώνει έναν ελκυστικό δρόμο συνδυαστικών προσεγγίσεων για τη θεραπεία του ΜΜΚΠ, που μπορεί να ενισχύσει τη δράση των ανασταλτικών παραγόντων του EGFR σε όγκους. Συμπερασματικά, θα μπορούσαμε να υποθέσουμε ότι στο ΜΜΚΠ: α) τα δύο μονοπάτια EGFR και Notch συνεπικουρούν για την ανάπτυξη του όγκου, β) η αναστολή του Notch είναι πιο αποτελεσματική σε κύτταρα με ενδιάμεσα επίπεδα ενεργού Notch 1, προκαλώντας τόσο απόπτωση όσο και αυτοφαγία, και γ) η μετάλλαξη του EGFR προσφέρει αντίσταση στη δράση αναστολέα της γ-σεκρετάσης. / It is widely accepted and well established that the epidermal growth factor receptor (EGFR) controls important processes of tumor cells, such as proliferation and apoptosis, but also processes involving more than one type of cells such as invasion and angiogenesis. It has been found that the EGFR has an important role in the development of several types of cancer including non-small cell lung cancer (NSCLC). Very recent data indicate that another molecule, which is involved in the development of lung cancer, is Notch. Its role is complicated and is under investigation. It is suspected that Notch has a growth promoting function in NSCLC, whereas exerts an inhibitory effect in small cell lung cancer (SCLC). Furthermore it has been found that the signaling pathway of Notch can affect/ can be affected by other molecules. This thesis investigated the role of EGFR and Notch in cell growth of NSCLC cells using the ligand of EGFR, EGF and gamma-secretase inhibitor, DAPT. To conduct the experiments the human NSCLC cell lines H23, A549, H661 and HCC827 were used. The cell lines H23, A549 and H661 express the wild type (wt) EGFR and the cell line HCC827 expresses EGFR bearing the mutation (mt) DE746-A750. Initially, we studied the profile of NSCLC cells regarding the protein levels of Notch intracellular domain (Notch ICD) using western blot analysis. It was found that H23 cells express the higher levels Notch ICD, H661 and HCC827 cells express intermediate levels and A549 cells express the lowest levels of Notch ICD. The next step was the evaluation of DAPT effect in cell proliferation using the MTT assay. We found that DAPT caused the greatest inhibition to H661 and A549 cells. DAPT was less effective to H23 cells while had no effect to HCC827 cells. The inhibitory effect of DAPT in H661 cells was in line with the induction of apoptosis and autophagy, as was detected using annexin V assay and western blot analysis for beclin-1, respectively. Furthermore, cells were stimulated with EGF and subsequently DAPT was added. We found that the stimulatory effect of EGF was not reversed by DAPT in H23 cells. However a partial reverse of EGF stimulation was observed in H661 cells. The next step was to study the effect of DAPT and EGF at Notch ICD protein levels, in H23 and H661 cells. We found that DAPT reduced the protein levels of Notch ICD in H23 cells, with a time-dependent manner, up to 6 hours after DAPT addition and this effect reversed 24 hour later. The addition of EGF did not affect the levels of Notch ICD at any time point tested. In H661 cells, DAPT caused a time-dependent reduction of Notch ICD protein levels up to 24 hours after DAPT addition to cells. EGF as previously, did not affect the levels of Notch ICD in these cells. Since DAPT was more effective to H661 cells, these cells stimulated with EGF and then DAPT was added in order to study the effect of the combination at the levels of Notch ICD. We found that EGF reversed the decrease of Notch ICD protein levels caused by DAPT alone. These results indicate that the pathways of EGFR and Notch might act with a synergistic fashion and this could be an attractive approach for the treatment of NSCLC. Summarizing our results, we might assume that in NSCLC: a) both pathways of EGFR and Notch exert a significant role in tumor growth, b) the inhibition of Notch is more effective in cells with intermediate levels of activated Notch 1, causing both apoptosis and autophagy, and c) the EGFR mutation confers resistance to the effect of γ- secretase inhibitor.

Les inhibiteurs de tyrosine kinase (ITK) du récepteur à l’Epidermal Growth Factor (EGFR) constituent un nouveau traitement du cancer bronchique non à petites cellules (CBNPC), particulièrement efficace chez les patients porteurs d’une mutation d’EGFR (EGFR M+). Néanmoins, certains de ces patients peuvent avoir une résistance primaire à ces traitements, et les autres développent inéluctablement une rechute correspondant au phénomène de résistance acquise. L’objectif de ce travail était d’étudier les mécanismes de résistances primaire et acquise chez les patients avec CBNPC EGFR M+.Nous avons montré que le statut mutationnel de KRAS était le même dans la tumeur primitive et les métastases dans la majorité des cas de CBNPC. Dans quelques cas cependant, le statut différait entre tumeur primitive et métastase, ce qui soulève la question d’une dissociation de la réponse aux ITK, les mutations de KRAS étant associées à une résistance primaire au traitement.Nous avons par ailleurs mis en évidence la fréquente inactivation de la voie p53, soit par mutation de TP53 soit par diminution d’expression de p14arf, dans les tumeurs EGFR M+, qui pourrait être une des causes des variations de réponse aux ITK chez les patients EGFR M+. Enfin, nous avons montré que la résistance à deux ITK de nouvelle génération, PF299804 et WZ4002, passait par un phénomène en deux étapes impliquant d’une part l’activation de la voie IGF1R, via la diminution de l’expression d’IGFBP3, et d’autre part celle de la voie des MAP kinases. Ces travaux pourraient déboucher sur des stratégies thérapeutiques innovantes chez les patients présentant une résistance acquise aux ITK de nouvelle génération. / Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKI) provide clinical efficacy in Non Small Cell Lung Cancer (NSCLC) patients, especially in the presence of an EGFR mutation. However, some EGFR mutated patients display primary resistance to EGFR TKI, and the others will ultimately develop acquired resistance. We focused our work on mechanisms of primary and acquired resistance in EGFR mutated NSCLC.We first showed that KRAS mutational status is the same in primary NSCLC and matched metastases in most of the cases. However, in some patients, we found a discordant KRAS status between primary tumors and metastases, which could translate into a discordant response to EGFR TKI, since KRAS mutations are associated with resistance to EGFR TKI.We also showed that EGFR mutated tumors are associated in most of the cases with an inactivation of the p53 pathway, either through a TP53 mutation or through loss of expression of p14arf, which could account for some of the variations observed in the response to TKI in EGFR mutated patients.Last, we showed that acquired resistance to two new generation EGFR TKI, PF299804 and WZ4002, occurred through a multistep process involving activation of the IGF1R pathway through downregulation of IGFBP3 and activation of the MAP kinase pathway. These results provide new insights into the treatment of NSCLC in EGFR mutated patients with acquired resistance to new generation TKI.

Introduction : L'installation de caméras de Tomographie à Emission de Positons (TEP) a eu lieu en 2003 dans l'inter région Grand-Est de la France. Plusieurs études ont montré ses performances diagnostiques et ses bénéfices en évitant: des interventions de chirurgie exploratrice devenue inutile (thoracotomie aux séquelles douloureuses, laparotomie exploratrice), et certains examens complémentaires d'imagerie ou d'exploration fonctionnelle inutile. Objectifs : 1) évaluer le changement des stratégies diagnostiques du Nodule Pulmonaire Isolé (NPI) et du Cancer du Poumon Non à Petites Cellules (CPNPC) induit par l'implantation de la TEP dans l'inter - région Grand - Est de la France ; 2) mesurer l'impact du changement sur la qualité de vie des patients atteints d'un NPI et d'un CPNPC ; et 3) étudier le rôle pronostique de la QV sur la survie des patients atteint d'un CPNPC.Méthodes : Etude prospective multicentrique constituant 2 cohortes « Avant (2001 - 2002) » et « Après (2004 - 2005) » de l'implantation de la TEP. Ont été recueillis : les caractéristiques sociodémographiques des patients, clinique du NPI et du CPNPC, les examens diagnostiques et traitements réalisés jusqu'à 6 mois après la fin du bilan. Deux auto-questionnaires de QV (SF36 et QLQ C-30) ont été distribués à 3 et 6 mois après la fin du bilan diagnostique.Résultats : 1) Nous avons observé une diminution du nombre d'examens réalisés au cours du bilan diagnostique d'un NPI de 4 examens en phase avant à 3 en moyenne en phase après. Néanmoins, la fréquence des examens invasifs jugés évitables a posteriori pratiqués chez les patients ayant un NPI bénin n'était pas différente (47% phase avant versus 49% phase après). Un an après son installation, la TEP a été utilisée chez 11% des patients. 2) Les patients atteints d'un NPI et quelque soit sa nature, maligne ou bénigne, ont eu un score moyen de QV plus bas (-8 à -32 points, p<0.001) que celui mesuré dans la population générale française indépendamment du sexe et de l'âge. Les scores QV des patients ayant un NPI bénin se révélaient être statistiquement plus élevés que ceux des patients ayant un NPI malin, notamment dans les dimensions « fonctionnement social », « rôle physique » et « rôle émotionnel » (+10, +14 et +18 points respectivement, p=0,02 à 0,04).3) Les bons scores en fonctionnement physique en SF-36 (HR=0.78 ; IC=0.68 - 0.90 ; p<0.001), et en la limitation due à l?état physique en QLQ-C30 (HR=0.53 ; IC=0.59 - 0.89 ; p=0.003) indiquant une bonne perception des patients sur leurs fonctionnement physique sont liés à une meilleure survie. Les symptômes élevés de constipation (HR=1.18 ; IC=1.005-1.38 ; p=0.04) en QLQ-C30, par contre, est lié à une mauvaise survie. Le sexe féminin est lié à une meilleure survie (HR=0.55 ; IC=0,33 - 0,94 ; p=0,04). Cependant, le stade III et IV du CPNPC est associé à une mauvaise probabilité de survie (HR=1,72 ; IC=1,16 - 2,57 ; p=0,007).Discussion/conclusion : Les stratégies diagnostiques du NPI ont changé. L'impact de cette maladie, mesuré de façon subjective par la qualité de vie (QV) des patients peut indiquer certains domaines essentiels à améliorer dans sa prise en charge car une meilleure QV en oncologie broncho-pulmonaire est associée à une meilleure survie / Background: The Positron Emission Tomography was installed in 2003 in the North-eastern region of France. Previous studies showed its diagnostic performance and its benefices in avoiding: surgical exploratory interventions proved to be unnecessary a posteriori (for example, thoracotomy or laparotomy with painful consequences), and certain complementary imaging tests or unnecessary functional explorations. Objectives : 1) to evaluate changes in diagnostic strategies of Solitary Pulmonary Nodule (SPN) and Non Small Cell Lung Cancer (NSCLC) induced by the implantation of PET in the Northeastern region of France ; 2) to mesure the impact of these changes on the health related quality of life (HRQoL) of patients with SPN and NSCLC; and 3) to study the prognostic role of the HRQoL on the survival of patients with NSCLC.Methodes: An observational, prospective, multi-center design was applied. Two cohorts « Before (2001 - 2002) - After (2004 - 2005) » the PET implantation in the regions were settled up. Data collected included: patients' socio-demographic and clinical characteristics, the diagnostic tests and then treatments that were carried out during 6 months after the end of the diagnostic process. Two HRQoL questionnaires (the generic questionnaire SF-36 and the cancer specific one QLQ-C30) were distributed at 3 and 6 months after the end of the diagnostic process.Results:1) The number of diagnostic tests of patients with SPN decreased significantly from a mean of 4 in the before-PET to 3 tests in the after-PET period. Meanwhile, there was not any difference of the frequency of invasive tests considered unnecessary aposteriori for patients with benign SPN (47% before-PET versus 49% after-PET period). One year after its installation, the PET was used in 11 % of diagnostic strategies. In 7.7% of cases it was used after the bronchoscopy. 2) A comparison of the QoL with that of the French general population revealed that patients with SPN, whatever the diagnosis, benign or malignant, had worse mean scores (-8 to -32 points, p<0.001) compared to the general population with similar age and sex. A difference of 24, 30 and 32 points were observed in the "physical functioning", "emotional role" and "physical role" (p<0.001). The scores of patients with benign nodule were higher than those of malignant, especially on "social functioning", "physical role", and "emotional role" (+10, +14 and +18 points respectively, p=0.02 to 0.04). 3) Good scores on "physical functioning" of SF-36 (HR=0.78; CI=0.68 - 0.90; p<0.001) as well as "role functioning" of QLQ-C30 (HR=0.53; CI=0.59 - 0.89; p=0.003) were related to a better survival. Higher symptoms of constipation (HR=1.18; CI=1.005-1.38; p=0.04) in QLQ-C30 were associated to a worse one. While being a woman was associated to a better survival (HR=0.55 ; CI=0,33 - 0,94 ; p=0,04), the stage III and IV of the NSCLC was related to a lower one (HR=1,72 ; CI=1,16 ? 2,57 ; p=0,007) .Conclusion: The diagnostic strategies of SPN changed after PET was available for medical practice. This study showed the negative impact of SPN to patients' HRQoL. It indicates the domains in which health practitioners could interfere in order to improve the management of these diseases, because this study confirmed previous studies in pulmonary oncology, that patients' QoL is related to the survival

O câncer de pulmão é um dos mais incidentes e letais, e, assim, a busca de novos fármacos é necessária. Atualmente o desenvolvimento de fármacos conta com abordagens computacionais que otimizam este processo. Dado que a fosfatidiletanolamina desempenha importantes papeis fisiológicos e uma das enzimas envolvidas na sua síntese, a CTP:fosfoetanolamina-citidilil-transferase (Pcyt2) é frequentemente superexpressa em células de câncer de pulmão, no presente trabalho, foram avaliados o potencial terapêutico de CHY-1, um análogo da miltefosina desenvolvido como inibidor da enzima Pcyt2, e os mecanismos inerentes à sua atividade antitumoral. O CHY-1 apresentou citotoxicidade superior ao seu protótipo e a outro inibidor da enzima Pcyt2, a meclizina. Além disso, as células malignas foram mais sensíveis ao CHY-1 do que as células não-tumorigênicas. Em conclusão, o presente trabalho evidencia o potencial do CHY-1 como um inibidor da enzima Pcyt2 e candidato a fármaco com atividade preferencial para câncer de pulmão. / Lung cancer is one of the most incident and lethal cancers, thus, the pursuit for new drugs is necessary. Nowadays, new drugs development has computational tools that improves this process. Once that phosphatidylethanolamine plays several important physiological roles and one of the enzymes of its production pathway, CTP:phosphoethanolamine cytidylyltransferase (Pcyt2), is usually overexpressed in lung cancer cells, therefore, this study aimed was to evaluate the antitumor effects of CHY-1, a miltefosine analogue developed as an inhibitor of Pcyt2 enzyme, and to investigate the mechanisms related to its antitumor action. CHY-1 was more cytotoxicity than its prototype, miltefosine, and was more cytotoxic than another inhibitor Pcyt2 enzyme, meclizine. Morevover, malignant cells were more sensitive to CHY-1 effects than non-tumorigenic cells. In conclusion, this work presents CHY-1 as an inhibitor of Pcyt2 enzyme and new candidate a drug with preferential activity on NSCLC cells.

Das Bronchialkarzinom ist die häufigste Todesursache bei den Krebserkrankungen und weist eine schlechte Prognose auf. Die Behandlung besteht aus einer Chemotherapie mit platinbasierten Medikamenten, doch der Erfolg ist unbefriedigend. In den letzten Jahren wurden zielgerichtete Therapien gegen Proteine wie den EGFR entwickelt. Klinische Studien zeigten, dass nur Subpopulationen von den Medikamenten Erlotinib und Cetuximab profitieren. Eine bessere (Vor-)Selektion der Patienten ist wünschenswert, um unnötige Behandlungen zu vermeiden. Für diese Analysen bedarf es relevanter präklinischer Modelle. Im Rahmen dieser Arbeit wurden 25 Xenograftmodelle des Lungenkarzinoms vergleichend charakterisiert. Ein Schwerpunkt bestand im Vergleich der Xenografts mit ihren Patiententumoren. Die Analyse der Histologie, der Proliferationsmarker als auch der Genexpressionsprofile fand übereinstimmende Ergebnisse in den Patiententumoren und ihren abgeleiteten Xenografts. Mit Hilfe von mRNA-, Protein- und SNP-Profilen ressistenzassoziierter Marker der Chemotherapie konnte die Bedeutung der Modelle zur Charakterisierung von prädiktiven und prognostischen Markern aufklärt werden. Diese Arbeit untersuchte auch Marker der anti-EGFR-Therapien. mRNA- und Proteinprofile der ERBB-Rezeptoren sowie der Liganden wurden erstellt und stimmten mit publizierten klinischen Daten überein. Genexpressionsstudien in Erlotinib Respondern und Non-Respondern zur Therapieoptimierung identifizierten den Wachstumsfaktor VEGFA als Ziel für eine Kombinationsbehandlung mit dem Angiogeneseinhibitor Bevacizumab. Die Kombination von Bevacizumab mit Erlotinib führte zu einem reduzierten Tumorwachstum. Die Ergebnisse dieser Arbeit machten deutlich, dass die individuellen Tumoreigenschaften in den patientenabgleiteten Xenografts auf Gen- und Proteinebene erhalten bleiben und diese als Modelle zur Markeranalyse sowie zur Therapieoptimierung eingesetzt werden können. / Lung cancer is still one of the most frequent cancers worldwide. The treatment option is classical chemotherapy that is based upon the combination of platin-based drugs. But no further improvement seems to be possible. For some years targeted drugs against single proteins like the EGFR were developed. The clinical trials showed that only subpopulations of patients benefit from the treatment. A better selection of patients to avoid treatment would be helpful. Therefore, pre-clinical models that are suitable for analysis and that represent clinical populations of patients are required. In this work 25 patient derived xenografts from lung cancer were intensely studied. First, the xenografts were compared with their corresponding patient tumor. The analysis of the histology and the expression of proliferation and epithelial or mesenchymal markers showed concordance of the patient tumor and the derived xenograft. The gene expression profiles were also maintained. Further analysis should elucidate the relevance of the xenografts as models for the characterisation and validation of predictive and prognostic markers. SNP, mRNA and protein expression profiles of resistance markers for chemotherapy were generated and showed similarities with clinical data. As marker for the anti-EGFR targeted therapies the ERBB receptors and the ligands of the EGFR were analysed. The mRNA and protein expression profiles resemble clinical data sets. An optimisation of the therapy should be achieved with gene expression studies. The vascular endothelial growth factor A was identified for a combination treatment with the anti-angiogenic drug bevacizumab in erlotinib resistant tumors. The combination of erlotinib and bevacizumab reduced the tumor growth in selected models. In summary, the analysis could show that the individual characteristics of the patient tumor were maintained in the xenograft. The models are a reliable tool for studies designed to improve treatment strategies.

Die antiangioneogenetischen Effekte in-vitro und in-vivo in Kombination mit der selektiven Inhibition der TNFalpha Produktion durch Thalidomid läßt dieses Medikament als geeignete Therapie für Krankheitszustände erscheinen, bei denen die TNFalpha Toxizität eine pathogenetische Rolle spielt, die Immunität jedoch intakt bleiben muß. Ziel dieser Monographie waren Untersuchungen der Wirkungen von Thalidomid in vitro, in vivo und klinische Studien. In einer prospektiv-randomisierten, doppel-blind Studie an 70 Patienten mit kleinzelligem Bronchialkarzinom konnte die Verlängerung der Überlebenszeit durch die additive Therapie mit Thalidomid in Kombination mit einer Standardpolychemotherapie und Strahlentherapie nachgewiesen werden. Vor dem additiven Einsatz des Angioneogenesehemmers Thalidomid in der Chirurgie muß unbedingt sichergestellt sein, daß die Therapie mit Thalidomid nicht zu einer erhöhten postoperativen Morbidität und Letalität führt. Durch in-vitro Untersuchungen konnte eine Proliferationshemmung von Kaninchenendothelzellen durch metabolisiertes Thalidomid bewiesen werden. In einer randomisierten Studie an New Zealand White Kaninchen konnte gezeigt werden, daß die intraperitoneale Gabe von Thalidomid den Berstungsdruck von Kolonanastomosen im Kaninchenmodell im Vergleich zu einer Kontrollgruppe nicht erniedrigt. Darüberhinaus zeigte sich in diesem Tiermodell, daß die Rate an postoperativen Verwachsungen durch die intraperitoneale Gabe von Thalidomid signifikant vermindert wird. Das Medikament könnte eine Rolle in der additiven Therapie von Patienten mit einem kleinzelligen Bronchialkarzinom spielen. Darüberhinaus wird die Heilung von Kolonanastomosen nicht durch die intraperitoneale Gabe von Thalidomid negativ beeinflusst. Somit könnte ein Einsatz in der perioperativen Therapie bei der Resektion gastrointestinaler Karzinome in der Zukunft erwogen werden. / The proven antiangiogenic effects in vivo and in vitro in combination with a selective inhibition of the tnf alpha production seem to predestine thalidomide as an agens for diseases with a pathological elevated tnf alpha level. The theses of this monography were in vitro, in vivo and clinical effects of thalidomide. In a prospective randomized placebo controlled study on 70 patients with proven small cell lung cancer the additive treatment with oral thalidomide lead to a significant prolonged survival in combination with radio chemotherapy. Considering the treatment of thalidomide in combination with surgical therapy of patients it is essential to prove that this treatment does not lead to a higher postoperative morbidity of the patients. In an in vitro assay we could prove the antiproliferative effect of metabolized thalidomide in rabbit endothelial cells. We were than able to demonstrate that intraperitoneal administered thalidomide does not impair anastomotic healing of colonic anastomoses in a New Zealand white rabbit model compared to a control group. In addition to this significant less postoperative adhesions were found in the thalidomide group. In conclusion did the additive treatment with thalidomide in patients with small cell lung cancer lead to a prolonged survival. The antiangiogenic treatment with thalidomide in a perioperative setting does not impair the healing of colonic anastomoses in a rabbit model and may be possible in patients undergoing gastrointestinal resections in the future.

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Excision Repair Cross-Complementation group 1 (ERCC1) est une enzyme de réparation de l’ADN fréquemment déficiente dans le cancer bronchique non-à-petites cellules. Bien qu’une expression faible d’ERCC1 soit prédictive de réponse aux sels de platine, l’efficacité des chimiothérapies à base de platine est limitée par leur toxicité et l’apparition de résistance, justifiant la nécessité de stratégies thérapeutiques alternatives. Par ailleurs, l’absence de test compagnon diagnostic permettant d’évaluer la fonctionnalité d’ERCC1 dans la pratique clinique empêche actuellement toute thérapie personnalisée basée sur le statut ERCC1.Afin d’identifier de nouvelles stratégies thérapeutiques pour les tumeurs ERCC1-déficientes en exploitant le concept de létalité synthétique, des screens à haut-débit , utilisant des composés pharmaceutiques ou par ARN interférence, ont été réalisés dans un modèle isogénique de CBNPC déficient en ERCC1. Cette approche a permis d’identifier plusieurs inhibiteurs de poly(ADP-ribose) polymerase 1 et 2 (PARP1/2), tels l’opalarib (AZD2281), le niraparib (MK-24827) et BMN 673 comme sélectifs pour les cellules ERCC1-déficientes. Les mécanismes sous-tendant cette sensibilité sélective ont été étudiés, et les résultats suivants ont été mis en évidence : (i) les cellules ERCC1-déficientes présentent un blocage prolongé en phase G2/M après exposition à l’olaparib ; (ii) l’isoforme 202 d’ERCC1, dont le rôle a été récemment mis en évidence dans la résistance aux sels de platine, module également la sensibilité aux inhibiteurs de PARP ; (iii) la déficience en ERCC1 est épistatique avec les défauts de recombinaison homologue (RH), malgré une capacité normale des cellules ERCC1-déficientes à former des foyers RAD51 ; ceci suggère qu’ERCC1 pourrait intervenir dans la réparation d’une lésion de l’ADN induite par l’inhibiteur de PARP1/2 en amont de l’invasion du brin d’ADN lors de la RH ; (iv) l’inhibition de l’expression de PARP1 par ARN interférence permet de restaurer la résistance aux inhibiteurs de PARP1/2, dans les cellules ERCC1-déficientes uniquement. Ces résultats suggèrent que les inhibiteurs de PARP1/2 pourraient représenter une nouvelle stratégie thérapeutique chez les patients dont la tumeur est déficiente en ERCC1 et un essai clinique va être mis en place pour évaluer cette hypothèse.Afin d’explorer la présence de biomarqueurs de la fonctionnalité d’ERCC1, quatre approches ont été entreprises en parallèle dans le modèle isogénique de CBNPC déficient en ERCC1: (i) irradiation aux UV, afin d’évaluer la voie NER (Nucleotide Excision Repair); (ii) séquençage d’exome, dans le but de rechercher une signature génomique (ADN) ; (iii) analyse du transcriptome cellulaire, pour identifier des modifications d’expression d’ARN ; et (iv) SILAC (Stable Isotope Labeling by Amino acids in Cell culture) afin de comparer le protéome des cellules ERCC1-déficientes et ERCC1-proficientes. Ces approches ont permis d’identifier une potentielle signature génomique, ainsi que de biomarqueurs d’activité – guanine deaminase (GDA) et nicotinamide phosphoribosyltransferase (NAMPT). De plus amples validations et investigations mécanistiques de ces observations préliminaires sont actuellement requises. / Excision Repair Cross-Complementation group 1 (ERCC1) is a DNA repair enzyme that is frequently deficient in non-small cell lung cancer (NSCLC). Although low ERCC1 expression correlates with platinum sensitivity, the clinical effectiveness of platinum therapy is limited - mainly by toxicities and occurrence of resistance - highlighting the need for alternative treatment strategies. In addition, the lack of a reliable assay evaluating ERCC1 functionality in the clinical setting currently precludes personalising therapy based on ERCC1 status. To discover new synthetic lethality-based therapeutic strategies for ERCC1-defective tumours, high-throughput drug and siRNA screens in an isogenic NSCLC model of ERCC1 deficiency were performed. This approach identified multiple clinical poly(ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors such as olaparib (AZD-2281), niraparib (MK-4827) and BMN 673 as being selective for ERCC1 deficiency. The mechanism underlying ERCC1-selective effects was dissected by studying molecular biomarkers of tumour cell response, and revealed that: (i) ERCC1-deficient cells displayed a significant delay in double-strand break repair associated with a profound and prolonged G2/M arrest following PARP1/2 inhibitor treatment; (ii) ERCC1 isoform 202, which has recently been shown to mediate platinum sensitivity, also modulated PARP1/2 sensitivity; (iii) ERCC1-deficiency was epistatic with homologous recombination deficiency, although ERCC1-deficient cells did not display a defect in RAD51 foci formation. This suggests that ERCC1 might be required to process PARP1/2 inhibitor induced DNA lesions prior to DNA strand invasion; and (iv) PARP1 silencing restored PARP1/2 inhibitor resistance in ERCC1-deficient cells but had no effect in ERCC1-proficient cells, supporting the hypothesis that PARP1 might be required for the ERCC1 selectivity of PARP1/2 inhibitors. This study indicated that PARP1/2 inhibitors as a monotherapy could represent a novel therapeutic strategy for NSCLC patients with ERCC1-deficient tumours, and a clinical protocol is being written to evaluate this hypothesis.To investigate whether a surrogate biomarker of ERCC1 functionality could be developed, four parallel approaches were undertaken in the ERCC1-isogenic NSCLC model: (i) UV irradiation, to evaluate the Nucleotide Excision Repair (NER) pathway; (ii) whole exome sequencing, to look for an ERCC1-associated genomic scar at the DNA level; (iii) transcriptomic analysis, to investigate changes at the RNA expression level; and (iv) SILAC (Stable Isotope Labeling by Amino acids in Cell culture) analysis, to compare proteomic profiles between ERCC1-proficient and ERCC1-deficient cells. These approaches allowed the identification of putative genomic signature and potential metabolic surrogate biomarkers - guanine deaminase (GDA) and nicotinamide phosphoribosyltransferase (NAMPT). Further validation and mechanistic investigations of these latter preliminary observations are warranted.