Background: Angiogenesis is the process by which new blood vessels develop from existing vasculature and is a critical step in all tumours to facilitate growth beyond a few millimetres. As this process is largely inactive in physiological circumstances in adults, it represents an attractive therapeutic target in oncology. Drugs that target the angiogenic process are classified as anti-angiogenic agents. The first anti-angiogenic drug to be approved by the FDA was bevacizumab; a recombinant humanized monoclonal antibody against VEGF. Randomised studies in colorectal cancer (and other solid malignancies) have reported prolonged progression free survival and overall survival for bevacizumab. However, standard radiological criteria, Response Evaluation Criteria In Solid Tumours (RECIST), although widely employed to assess response to therapy in clinical trials, are generally insensitive to the predominantly cytostatic effects of anti-angiogenic and other targeted therapies. Alternative methods of predicting or assessing early response to such agents are needed, particularly given the cost and toxicity implications of such treatments. However, biomarkers to aid selection of patients for anti-angiogenic therapies, including bevacizumab, remain elusive. Purpose: To investigate Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI), Diffusion Weighted Imaging (DWI) and circulating angiocytokines, measured using an ELISA multiplex, as prognostic markers in patients with metastatic colorectal cancer treated with bevacizumab and chemotherapy. Results: Seventy patients were treated. DCE-MRI and DWI parameters showed good reproducibility with coefficient of variation between 3.7 to 23% for parameters. The median progression free survival, the primary end point of the trial, was 9.3 months. The overall response rate was 44%. The clinical variables which were significant for progression free survival on univariate analysis were: performance status (p=0.005), CEA (p=0.04) and serum LDH (p=0.005). Biomarkers which were significant for progression free survival on univariate analysis were serum VEGF-A (p=0.02), serum HGF (p=0.005), sVEGFR-2 (p=0.02). In each case, low values of the biomarker were associated with improved outcome. Multivariate analysis identified Ktrans (p=0.015), performance status (p=0.008) and serum HGF (p=0.003) as the most significant predictors of progression free survival. A prolonged progression free survival was associated with a good ECOG performance status, high Ktrans and low serum HGF.Conclusions: Whilst these results are encouraging, future work is required to establish whether HGF and Ktrans are prognostic markers for metastatic colorectal cancer and their precise role in the prediction of patients likely to benefit from treatment with bevacizumab.

BACKGROUND: Many different ocular diseases produce a common symptom of macular edema - a leakage of fluid into the retina. In addition to the presence of this structural aberration, functionally, the retina’s capacity to effectively conduct electrochemical signals will be impaired. The impediment can be demonstrated by a decreased electrical response measured via electroretinography (ERG) and visualized as a waveform with quantifiable amplitude. Macular edema and its associated effects on retinal structure and function are resultant of abnormal blood vessel growth, or angiogenesis. The process of angiogenesis involves a pathway of multiple growth factors and signaling molecules, including vascular endothelial growth factor (VEGF). Modern day treatments to help resolve macular edema target VEGF in order to inhibit pathological angiogenesis; two such anti-VEGF medications are aflibercept, or Eylea, and bevacizumab, also known as Avastin. This retrospective cohort study aims to compare the outcomes of patients treated with either Avastin or Eylea, and to observe what structural or functional changes occur in each sample. METHODS: This study included twelve eyes of 8 eligible patients that were injected with intravitreal Avastin for diabetic macular edema (DME), clinically significant macular edema (CSME), proliferative diabetic retinopathy, or non-proliferative diabetic retinopathy (NPDR), and had resolved macular edema. Five eyes of 6 eligible patients that were injected with intravitreal Eylea for NPDR, neovascular age-related macular degeneration (AMD), cystoid macular edema (CME), central retinal vein occlusion (CRVO), or DME, and had resolved edema were also included in the study. Patient data was randomly screened and collected via the MDIntelleSys (MDI) electronic medical record system at Fromer Eye Centers, New York. No personally identifiable information was collected in this study. The parameters used for comparison of the two anti-VEGF medications were the length of treatment, frequency of injection, change in ERG magnitude between subsequent scans, and net change in ERG magnitude between the baseline scan and the most recent scan obtained during the period of the study. The means of each parameter were determined to be the best measure of central tendency to summarize the data. Welch’s t-tests were conducted at a significance level of α= 0.05 between the parameters of each group to determine the significance of the differences in the means obtained. RESULTS: There was no significant difference found between the mean number of injections, time until recovery, change in magnitude, and net change in magnitude for both Avastin-treated patients and those treated with Eylea. CONCLUSION: The results of this study support the conclusion that both aflibercept and bevacizumab are comparably effective anti-VEGF treatments, in both relieving macular edema and restoring function in retinal cells. The similar outcomes observed in each treatment group offer insight into the versatility of anti-VEGF treatment and provide physicians with the flexibility of pursuing alternative medication options for their patients. Further study into the structural and functional effects of various anti-VEGF medications is needed to account for variables such as age, sex, race, or other possibly confounding factors. An inclusion of other quantifiable data such as visual acuity would also benefit this investigation. Furthermore, this study is limited by its focus solely on anti-VEGF medication; this subject of discussion would benefit from an experimental comparison between anti-VEGF treatments and other allopathic interventions. Recent studies have suggested alternatives to anti-VEGF altogether such as intravitreal triamcinolone acetonide and intravitreal steroids like dexamethasone, marketed as Ozurdex.

Les glioblastomes sont les tumeurs cérébrales les plus fréquentes avec une incidence en France de l'ordre de 4 nouveaux cas par an et pour 100 000 habitants (2400/ an). Le traitement standard pharmacologique des glioblastomes nouvellement diagnostiqués consiste en première ligne en une administration de témozolomide (75 mg/m2/j) pendant la radiothérapie, suivie d’une consolidation de 6 cycles. Cependant, malgré ce traitement, la médiane de survie n’est que de 15 mois et de 3 à 9 mois pour les rechutes. De nouvelles approches thérapeutiques sont donc indispensables. Parmi elles, ont été évalués le recours à d’autres chimiothérapies (irinotecan) et à l’inhibition de l’angiogénèse. L'angiogenèse est en effet un processus critique dans la progression GBM. L'inhibition de l'angiogenèse, induisant une réduction des vaisseaux sanguins, permet une diminution de l’apport des nutriments et d'oxygène à la tumeur. De manière générale, l’efficacité des traitements du glioblastome est soumise, dans un premier temps, à leur passage intra-cérébral au travers de la barrière hémato-encéphalique (BHE). L’objectif de notre travail était d’une part d’étudier l’impact de l’expression du transporteur d’efflux ABCB1 sur la distribution cérébrale du témozolomide (TMZ) et de l’irinotecan (CPT-11), et d’autre part, d’évaluer le rôle du bevacizumab (BVZ)(inhibiteur de l’angiogénèse) dans la modulation du passage intra-cérébral et intra-tumoral du TMZ et du CPT-11. A l'aide d'une étude pharmacocinétique comparative chez les souris CF1 mdr1a (+/+) et les souris CF1 mdr1a (-/-), nous avons mis en évidence un efflux actif du TMZ, du CPT-11 et de son métabolite actif le SN-38 du cerveau vers le plasma, impliquant ABCB1 au niveau de la BHE. Nous avons également démontré in vivo que le TMZ s'accumule dans la tumeur cérébrale et que le prétraitement par BVZ augmente la distribution tumorale de TMZ. En revanche, le BVZ n’a montré aucun effet sur la distribution cérébrale ou tumorale du CPT-11. Le BVZ apparaitrait donc comme un moyen intéressant d’augmenter la distribution intratumorale du TMZ. Dans ce même objectif, une collaboration initiée dans le cadre de cette thèse a permis de mettre en évidence l’intérêt de l’utilisation d’ultrasons dans l’ouverture de la BHE et l’amélioration de la distribution cérébrale des médicaments. / Glioblastomas are the most common brain tumors occurring in France with an incidence of 4 new cases per year per 100 000 population (2400/year). The gold standard pharmacological treatment of newly diagnosed glioblastoma relies on temozolomide administration (75 mg/m2/d) concomitant to radiotherapy, followed by six cycles consolidation. However, despite this treatment, the median survival is only 15 months and relapse occurs within 3 to 9 months. New therapeutic approaches are needed. Among them, other chemotherapies (irinotecan) and inhibition of angiogenesis were explored. Angiogenesis is a critical process in GBM progression. Inhibition of angiogenesis, inducing a reduction of the blood vessels, reduces supply of nutrients and oxygen to the tumor. The effectiveness of GBM treatment is subjected to intra-brain diffusion through the blood-brain barrier. The objective of this study was firstly to study the impact of efflux transporter ABCB1 brain expression on temozolomide (TMZ) and irinotecan (CPT-11) brain distribution, and secondly, to assess the role of bevacizumab (BVZ)(angiogenesis inhibitor) in the modulation of TMZ and CPT-11 brain and tumor distribution. Using a comparative pharmacokinetic study in CF1 mdr1a (+/+) and CF1 mdr1a (-/-) mice, we demonstrated an active efflux of TMZ, CPT-11 and its active metabolite SN-38 from the brain to the plasma involving ABCB1. We also demonstrated in vivo that TMZ accumulates in brain tumor and BVZ pretreatment increased TMZ tumor distribution. However no effect of BVZ on CPT-11 brain or tumor distribution was evidenced. Therefore BVZ would appear to be an interesting way to increase TMZ tumor distribution. The same objective was pursued through a different approach using ultrasound unfocused to open the BBB (Carthera collaboration).

Actuellement, il n’existe pas de techniques fiables pour la détection de marqueurs du cancer ou le suivi de l’administration de médicaments tels que les anticorps monoclonaux. Cette détection est essentielle pour réaliser un diagnostic précoce et ainsi améliorer le pronostic de survie des patients, et d’autre part adapter la posologie et un traitement personnalisé à chaque patient. L’objectif majeur de ce travail a été de concevoir et de développer des systèmes dits biocapteurs répondant à ces problématiques : d’une part, la détection et la quantification de l’activité des métalloprotéinases matricielles (MMP) en tant que nouvel outil de diagnostic de la progression et du développement tumoral; et d’autre part, le dosage d’un anticorps anti-VEGF, le bevacizumab, impliqué dans les biothérapies antiangiogéniques. La quantification de l’activité métalloprotéasique a été rendue possible grâce à la conception d’un biocapteur basé sur un édifice supramoléculaire. Celui-ci est constitué de substrats peptidiques fluorogéniques des MMP et d’une surface fonctionnalisée par des cyclodextrines. Une détection par fluorescence a alors permis d’évaluer l’efficacité et la spécificité de ce système à quantifier l’activité des MMP in vitro et ex vivo en conditions de biopsie tumorale. D’autre part, nous avons conçu des biocapteurs basés sur un mime peptidique du VEGF, permettant le dosage du bevacizumab. L’utilisation de ce mime en remplacement du VEGF humain a été démontrée, et plusieurs systèmes supramoléculaires fonctionnalisés par ce peptide ont été synthétisés, en vue de la conception d’une plateforme de détection régénérable des interactions peptide/protéine par transduction acoustique. / Nowadays, there are no reliable techniques for detecting biomarkers of cancer or for monitoring therapeutic drugs such as monoclonal antibodies in blood samples. This detection is essential in order to highlight the early onset of a disease prior to the appearance of clinical symptoms and therefore ensure a greater therapeutic effect, but also to provide a personalized treatment for each patient. The major goal of this thesis work was to design and develop plateforms of detection called biosensors answering these problematics: on one hand, detection and monitoring of matrix metalloproteinases (MMP) activities as a new tool for the diagnosis of tumoral progression, and on the other hand, quantification of an anti-VEGF antibody named bevacizumab, involved in antiangiogenic therapies. Monitoring of MMP activities was made possible by the design of a biosensor based on a supramolecular assembly between fluorogenic susbtrates of MMP and cyclodextrins functionalized surface. Fluorescence detection has enabled to evaluate the efficacity and specificity of this system to quantify MMP activities in vitro and ex vivo in tumoral biopsy conditions. On the other hand, we have designed biosensors based on a cyclopeptide mimicking human VEGF for the monitoring of bevacizumab. The ability of this peptide to substitute the human protein has been demonstrated and several supramolecular assemblies functionalized by this peptide have been synthesized in order to create a regenerable biosensor screening peptide/protein interactions by acoustic transduction.

<p>Tumorsko pupljenje (TP) u karcinomu je morfolo&scaron;ki fenomen koji predstavlja pojavu pojedinačnih ili malih grupa dediferentovanih tumorskih ćelija koje se na invazivnom frontu karcinoma odvajaju od glavne tumorske mase. Kod metastatskog kolorektalnog karcinoma (KRK) definitivno ne možemo odrediti pravi doprinos TP. Cilj je bio da se ispita terapijski patohistolo&scaron;ki odgovor na primenjeni hemioterapijski režim, prognostički i nezavisni negativni značaj TP , kao i korelacija TP i terapijskog odgovora histolo&scaron;ke regresije kod R0 reseciranih sinhronih i metahronih jetrenih metastaza KRK, koji su primali polihemioterapije po protokolu Folfox 4, sa i bez VEGF inhibitora &ndash; bevacizumaba (AV).&nbsp; Studija je prospektivno &ndash; retrospektivna i obuhvata 77 bolesnika oba pola, uzrasta preko 18 godina, sa patohistolo&scaron;ki verifikovanim jetrenim metastazama KRK, koji su operisani u Institutu za onkologiju Vojvodine u periodu od 1. maja 2007. do 1. juna 2017. godine. Od ukupno 120 bolesnika, njih 77 je ispunjavalo sledeće kriterijume: da je histolo&scaron;ki dokazan metastatski adenokarcinom kolorektuma sa R0 resekcijom i da su preoperativno dobijali HT sa biolo&scaron;kom terapijom ili bez nje. Bolesnike smo podelili u dve grupe: KRK &ndash; sinhrona metastatska bolest i KRK &ndash; metahrona metastatska bolest. Nakon selekcije bolesnika, rađena je mikroskopska analiza rutinskih histolo&scaron;kih i imunohistohemijskih preparata i određivana je gustina TP, histolo&scaron;ka regresija prema mTRG bodovanju komparirala se sa radiolo&scaron;kim odgovorom po RECIST-u. Događaji od interesa u kliničkom toku bolesti jesu progresija nakon hirur&scaron;kog zahvata jetrenih metastaza i ukupno preživljavanje u periodu od 24 meseca. Nema statistički značajne patohistolo&scaron;ke razlike u učestalosti lo&scaron;ijeg terapijskog odgovora (mTRG 3 &ndash; 5) u odnosu na bolji terapijski odgovor (mTRG 1, 2) između bolesnika sa sinhronom i metahronom metastatskom bole&scaron;ću KRK, koji su lečeni hemioterapijskim protokolom Folfox4: 13 (76,5%) vs. 13 (72,2%); p = 0,774. Kod bolesnika sa sinhronim metastazama KRK, lečenih hemioterapijskim protokolom Folfox 4, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine, i to kod bolesnika sa malom u odnosu na one sa velikom gustinom TP: 10 (90,9%) vs. 5 (55,6%); p = 0,049. Kod tih bolesnika, lečenih hemioterapijskim protokolom Folfox4/AV, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine, i to kod bolesnika sa malom u odnosu na one sa velikom gustinom TP: 9 (100%) vs. 6 (33,3%); p = 0,048. Kod bolesnika sa metahronim metastazama KRK lečenih hemioterapijskim protokolom Folfox4, sa i bez AV, nema statistički značajne razlike u učestalosti preživljavanja tokom dve godine u odnosu na gustinu TP. Kod bolesnika sa sinhronim i metahronim metastazama KRK nema statistički značajne razlike u učestalosti lo&scaron;ijeg histolo&scaron;kog odgovora na terapiju (mTRG 3 &ndash; 5) kod onih sa malom u odnosu na one sa velikom gustinom (TP): (8 (50%) vs. 15 (78,9%); p = 0,072 i TP: 8 (80%) vs. 13 (72,2%); p = 0,649). Kod bolesnika sa sinhronim metastazama KRK lečenih hemioterapijskim protokolom Folfox4, sa i bez AV, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine u odnosu na gustinu TP. Takođe, kod tih bolesnika velika gustina TP je nezavistan negativan faktor prognoze u odnosu na date terapijske režime, &scaron;to se vidi u preživljavanju tokom dve godine.</p> / <p>Tumor budding (TB) in cancer is a morphological phenomenon representing the appearance of single or small groups of dedifferentiated tumor cells that separate from the main tumor mass on the invasive front of cancer. In metastatic colorectal cancer (MCC), the true contribution of TB cannot be determined. The aim was to investigate the therapeutic pathohistological response to the applied chemotherapy, the prognostic and independent negative significance of TB, as well as the correlation of TB and the therapeutic response of histological regression in R0 resectable synchronous and metachronous liver metastases of MCC receiving polychemotherapy according to the Folfox 4 protocol, with and without VEGF inhibitors - bevacizumab (AV). The research was conducted as a prospective &ndash; retrospective study that included 77 patients of both sex, over 18 years of age, with pathohistologically verified MCC liver metastases, who underwent surgery at the Institute of Oncology of Vojvodina from 1st May 2007 until 1st June 2017. From 120 patients, 77 patients met the following criteria: they had histologically proven metastatic colorectal adenocarcinoma with R0 resection and also received preoperative chemotherapy with or without biological therapy. The patients were divided into two groups: MCC - synchronous metastatic disease and MCC - metachronous metastatic disease. After the patient selection, microscopic analysis of routine histological and immunohistochemical preparations was performed, the density of TB was determined, and the histological regression according to mTRG scoring was compared with a radiologic response according to the RECIST. The events of interest in the clinical course of the disease were the progression of hepatic metastases after surgery and overall survival during 24 months. There is no statistically significant pathohistological difference in the incidence of worse therapeutic response (mTRG 3 - 5) compared to the better therapeutic response (mTRG 1, 2) between patients with synchronous and metachronous MCC who were treated with the Folfox4 chemotherapy protocol: 13 (76.5%) vs. 13 (72.2%); p = 0.774. In patients with synchronous MCC metastases treated with the Folfox 4 chemotherapy protocol, there was a statistically significant difference in the survival rates during two years particularly in patients with low versus high TB density: 10 (90.9%) vs. 5 (55.6%); p = 0.049. In those patients who were treated with the Folfox4 / AV chemotherapy protocol, there was a statistically significant difference in survival rates during two years particularly in patients with low TB density in reference to those with high: 9 (100%) vs. 6 (33.3%); p = 0.048. In patients with metachronous MCC metastases who were treated with the Folfox4 chemotherapy protocol, with and without AV, there was no statistically significant difference in survival rate during two years when referring to the TB density. In patients with synchronous and metachronous metastases, MCC has no statistically significant difference in the incidence of worse histological response to therapy (mTRG 3 - 5) in patients with low TB density versus the ones with high density (TB): (8 (50%) vs. 15 (78.9%); p = 0.072 and TP: 8 (80%) vs. 13 (72.2%); p = 0.649). In patients with synchronous MCC metastases who were treated with the Folfox4 chemotherapy protocol, with and without AV, there is a statistically significant difference in survival rates during a two-year follow up when referring to the TB density. Also, the high density of TB is an independent negative prognostic factor in these patients in reference to the given therapeutic regimens, as seen in the two-year survival rate.</p>

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-20T15:09:52Z No. of bitstreams: 2 Dissertação - Tatiana Vieira de Brito Marquez - 2010.pdf: 2722177 bytes, checksum: d3f571bcc9c3e44cf51b9d4539e6191d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-20T15:10:12Z (GMT) No. of bitstreams: 2 Dissertação - Tatiana Vieira de Brito Marquez - 2010.pdf: 2722177 bytes, checksum: d3f571bcc9c3e44cf51b9d4539e6191d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-20T15:10:12Z (GMT). No. of bitstreams: 2 Dissertação - Tatiana Vieira de Brito Marquez - 2010.pdf: 2722177 bytes, checksum: d3f571bcc9c3e44cf51b9d4539e6191d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2010-07-06 / OBJECTIVES: To compare the response of bevacizumab and the response of the laser photocoagulation with indirect binocular ophthalmoscope (IBO) in infants with retinopathy of prematurity (ROP) stages II or III. METHODS: Data collection was performed in the neonatal intensive care unit of the Hospital Materno Infantil, from May to November 2009. Preterm infants with ROP stages II or III, with or without the plus disease were included. They were divided into two groups: on group I, an intra-vitreous injection of bevacizumab was performed and on group II, laser photocoagulation with IBO was performed. RESULTS: In group I, from the 20 eyes that underwent the application of bevacizumab, there was total regression of ROP with one application in 14 eyes, being 4 eyes with complete regression with two doses of bevacizumab, and 2 eyes with total regression after two applications of bevacizumab and one application of laser. On group II, from the 20 eyes treated with laser photocoagulation with IBO, there was total regression of ROP with laser therapy in 14 eyes and there was total regression with two laser sessions in 6 eyes. CONCLUSION: Bevacizumab is effective if compared to laser photocoagulation in ROP stages II and III. / OBJETIVOS: Comparar a resposta terapêutica do bevacizumabe intravítreo com à resposta da fotocoagulação a laser com oftalmoscópio binocular indireto, em prematuros portadores de retinopatia da prematuridade estágios II ou III. MÉTODOS: A coleta de dados foi realizada na UTI neonatal do Hospital Materno Infantil, no período de maio a novembro de 2009. Foram incluídos os prematuros com retinopatia da prematuridade graus II ou III, com ou sem doença plus. Estes foram divididos em dois grupos: no grupo I foi realizada a injeção intra-vitrea de bevacizumabe, no grupo II foi realizada a fotocoagulação a laser com oftalmoscópio binocular indireto. RESULTADOS: No grupo I, dos 20 olhos incluídos, houve regressão total da retinopatia da prematuridade com uma aplicação de bevacizumabe em 14 olhos, regressão total com duas aplicações em 4 olhos e regressão total após duas aplicações de bevacizumabe e uma aplicação de laser em 2 olhos. No grupo II, dos 20 olhos tratados com fotocoagulação a laser com oftalmoscópio binocular indireto, houve regressão total da retinopatia da prematuridade com uma sessão em 14 olhos e houve regressão total com duas sessões em 6 olhos. CONCLUSÃO: O bevacizumabe é tão eficiente quanto a fotocoagulação a laser na retinopatia da prematuridade nos estágios II e III.

Le VEGF est une cible validée pour le traitement du cancer colorectal métastatique avec le bevacizumab (Avastin) et l’aflibercept (Zaltrap) qui sont approuvés en première ligne et seconde ligne de traitement. Malgré les efforts intenses, il n’existe pas de biomarqueurs prédictifs pour identifier les patients qui pourraient répondre ou non aux therapies anti-VEGF. Récemment, différents sous-groupes de cancers colorectaux ont été identifies se basant sur l’expression de genes, incluant le groupe CMS4, un sous-groupe de phénotype mésenchymateux, angiogénique et de mauvais prognostic. Nous voulons établir si le phenotype mésenchymateux est prédictif pour la réponse aux agents anti-VEGF. Les cellules de cancer colorectal ont été étudiées in vitro et in vivo pour l’expression des marqueurs épithéliaux (E-cadherine, gamma-catenine, cytokeratine 18) et mésenchymateux (vimentine, N-cadherine, fibronectine) par qRT-PCR and western-blot et leur distribution par E-cadherine et beta-catenine pour immunocytochimie. Cinq modèles de cancer colorectal ont été rangés selon un phénotype épithélial prononcé (HT-29 et DLD-1), intermédiaire (HCT-116) à mésenchymateux (HCT-116/5-FU, LS174T) et les capacités d’inhibition de la croissance par le bevacizumab et l’aflibercept ont été établi. La sécrétion de VEGF a été déterminé par ELISA et la densité microvasculaire a été caractérisé par immunohistochimie quantitative. Le phénotype mésenchymateux est associé à une grande densité vasculaire mais pas aux ligands VEGF. Deux modèles de xénogreffes (DLD-1, HCT-116/5-FU) sont sensibles au bevacizumab et à l’aflibercept, deux modèles sont plus sensibles à l’aflibercept qu’au bevacizumab et un modèle est résistant aux deux molécules. L’aflibercept est plus efficace que le bevacizumab sur l’ensemble des modèles. Le phénotype mésenchymateux n’est pas prédictif pour la réponse aux agents ciblant le VEGF, ni positivement, ni négativement. / VEGF is a validated target for treatment of metastatic colorectal cancer (mCRC) with bevacizumab (avastin) and aflibercept (zaltrap) being approved for first and second line treatment, respectively. Despite intense efforts, no predictive biomarkers are available to identify patients likely to respond, or not, to VEGF-targeted therapies. Recently, different subtypes of CRC have been identified based on gene expression analysis including CMS4, a molecular subgroup with a mesenchymal phenotype, prominent angiogenesis and poor prognosis. We here wish to establish if the mesenchymal phenotype is predictive for the response to VEGF-targeted agents. CRC cell lines were examined for expression of epithelial (E-cadherin, gamma-catenin, cytokeratin 18) and mesenchymal (vimentin, N-cadherin, fibronectin) markers in vitro and in vivo by qRT-PCR and Western blot analysis and for the cellular distribution of E-cadherin and beta-catenin by ICC. Five CRC models were selected ranging from pronounced epithelial (HT-29, DLD-1), intermediate (HCT-116) to mesenchymal (HCT-116/5-FU, LS174T) and the tumor growth inhibitory activity of bevacizumab and aflibercept was established. VEGF-secretion was determined by ELISA and the microvascular density was characterized by quantitative IHC analysis. The mesenchymal phenotype was associated with higher microvascular density, but not with the expression of VEGF ligands. Two CRC xenograft models (DLD-1, HCT-116/5-FU) were sensitive to both bevacizumab and aflibercept, two models were more sensitive to aflibercept, compared to bevacizumab (HT-29, HCT-116), and one model (LS174T) was resistant to both agents. Aflibercept was more potent than bevacizumab in all CRC models. The mesenchymal phenotype was not predictive for the response to VEGF-targeted agents, neither positively nor negatively.

Free analyte is measured to be able to understand the pharmacological effects of a drug in the body, the binding to its ligand, and the effective drug level among other things. Thereby, it is important with correct measurements of free analyte, although it is not that easy to achieve since overestimations can occur. In this project, several immunoassays were developed for the bioanalytical methods Gyrolab and ELISA to measure free analyte, where the biotherapeutics Avastin® and Lucentis®, and the ligand VEGF were used as analytes. One difference between the methods is the short contact time of just a few seconds for Gyrolab compared to the sample incubation time of a couple of hours for ELISA. One difference between the antibodies is that Lucentis is an affinity-matured Fab region, and therefore, has a stronger affinity to VEGF compared to Avastin. When free Avastin was measured, the difference was significant, with ELISA estimating higher concentrations compared to Gyrolab. However, this was not the case for all assays. In some cases, this was probably due to differences between the methods that could not be seen. Otherwise, the results with no difference between the methods, when measuring free analyte with Lucentis as the drug, were expected due to the stronger affinity and longer halftime of dissociation. However, the difference with the longer sample incubation time for ELISA compared to the short contact time for Gyrolab seems to influence the measurement of free analyte, depending on the affinity of the components being measured.

Retinale Venenverschlüsse kann man in Astvenenverschlüsse und Zentralvenenverschlüsse unterteilen. Beide Erkrankungen werden primär mit intravitrealen Injektionen behandelt, wie den Anti-VEGF-Antikörpern oder dexametasonhaltigen Implantaten. Ziel dieser Arbeit ist die Evaluation der Therapiewirksamkeit von Anti-VEGF- Injektionen bei Patienten mit geringem Visus von ≤ 0,2 bei Behandlungsbeginn anhand von OCT und Visus von 150 Patienten im Behandlungsverlauf von bis zu zwölf Monaten. Die Gruppe der Patienten mit Baseline-Visus >0,2 stellt dabei die Vergleichsgruppe dar. In der Patientengruppe A mit Visus ≤ 0,2 bei ZVV stellte sich ein Visusanstieg von 0,10 auf 0,20 ± 0,18 nach zwölf Monaten ein. Im Patientenkollektiv B mit Visus >0,2 und ZVV stellte sich eine Visusbesserung von 0,43 auf 0,61 ± 0,27 ein. In der Patientengruppe A mit Visus ≤ 0,2 bei VAV stellte sich ein Visusanstieg von 0,11 auf 0,27 ± 0,07 nach zwölf Monaten ein. Im Patientenkollektiv mit Visus > 0,2 und VAV stellte sich eine Visusbesserung von 0,52 auf 0,68 ± 0,18 ein. Damit ist bei allen Gruppen eine signifikante Visusverbesserung nachweisbar, wobei es ersichtlich ist, dass der bessere initiale Visus auch einen besseren Endvisus determiniert. Bei der Patientengruppe mit Anfangsvisus ≤ 0,2 konnte signifikant eine größere absolute Netzhautdickenabnahme bei stets höherer Netzhautdicke als in der Referenzgruppe nachgewiesen werden.:Inhaltsverzeichnis 1 Einleitung..........................................................................................................................1 1.1 Netzhautanatomie.....................................................................................................1 1.1.1 Blutversorgung der Netzhaut...............................................................................2 1.1.2 Venenverschlüsse der Retina..............................................................................3 1.2 Makulaödem..............................................................................................................7 1.3 Der molekulare Signalweg beim Makulaödem......................................................8 1.4 Diagnostik................................................................................................................10 1.4.1 Symptome.........................................................................................................10 1.4.2 Sehschärfenprüfung..........................................................................................10 1.4.3 .Ophthalmoskopie..............................................................................................11 1.4.4 Bildgebende Diagnostik.....................................................................................12 1.4.4.1 Fluoreszeinangiographie............................................................................12 1.4.4.2 Optische Kohärenz-Tomografie.................................................................13 1.5 Therapie der Venenverschlüsse............................................................................15 1.5.1 Intravitreale Injektionen.....................................................................................15 1.5.1.1 VEGF-Antikörper........................................................................................16 Bevacizumab.....................................................................................................17 Ranibizumab......................................................................................................17 Aflibercept..........................................................................................................17 1.5.1.2 Therapieschemata von intravitrealen Anti-VEGF-Injektionen....................18 1.5.1.3 Kortikosteroide...........................................................................................19 1.5.2 Laserkoagulation...............................................................................................20 Fokale Laserkoagulation....................................................................................20 Periphere Laserkoagulation...............................................................................21 1.6 Prognose.................................................................................................................21 2 Arbeitshypothese und Fragestellung..........................................................................23 3 Materialien und Methoden.............................................................................................24 3.1 Behandlungsablauf................................................................................................24 3.2 Patientenkollektiv und Statistik............................................................................26 4 Ergebnisse......................................................................................................................27 4.1 Patientenkollektiv...................................................................................................27 Demografie des Patientenkollektivs...................................................................30 4.2 Art der Behandlung................................................................................................35 4.3 Laserkoagulation....................................................................................................40 4.4 Patientengruppe Lucentis und ZVV......................................................................41 4.5 Patientengruppe Lucentis und VAV......................................................................49 5 Diskussion......................................................................................................................54 6 Zusammenfassung der Arbeit......................................................................................59

Angiogeneseinhibitoren haben innerhalb weniger Jahre die Medizin revolutioniert. Auf dem Gebiet der Augenheilkunde ist es v.a. das Bevacizumab (Avastin ®), welches bei verschiedenen Krankheitsbildern eingesetzt wird, um den Verlust an Sehschärfe zu bekämpfen, der u.a. durch eine Vergrößerung der Netzhautdicke zustande kommt. Diese Arbeit untersucht die Veränderungen von Visus und Netzhautdicke (Makulaödem) an Patienten mit diabetischer Retinopathie (DR) und retinalem Venenverschluss (RVO). Verschiedene Studien zeigen, dass nicht alle Patienten von dieser neuen Therapieform profitieren. Mit dem Ziel herauszufinden, welche Patienten auf die Bevacizumabbehandlung ansprechen, werden Einflussfaktoren analysiert, die mit einer Visusverbesserung und einer Verringerung der Netzhautdicke in Verbindung gebracht werden können. Zu diesen Einflussfaktoren zählen: der Untersuchungszeitpunkt, die Art und Ausprägung der Erkrankung und der Visus vor einer Behandlung. Ein zusätzlich untersuchter Einflussfaktor bei Patienten mit RVO ist die Verschlussdauer. Die Arbeit weist nach, dass sowohl Patienten mit DR als auch Patienten mit RVO auf die Behandlung ansprechen. Subgruppenanalysen können diese Ergebnisse auch für die Entitäten nicht proliferative DR (NPDR), Astvenen- (BRVO) und Zentralvenenverschluss (CRVO) belegen. Die Analyse der Einflussfaktoren zeigt, dass Patienten mit einer geringen Sehschärfe vor der Behandlung größere Verbesserungen der Sehschärfe nach der Behandlung aufweisen als Patienten mit initial guter Sehschärfe. Die Verschlussdauer hingegen erweist sich nur bedingt als signifikanter Einflussfaktor auf die Bevacizumabtherapie. Die Ergebnise dieser Arbeit können eine Entscheidungshilfe für den Einsatz der Bevacizumabbehandlung bei Patienten mit DR und RVO darstellen.