Tumor-associated lymphangiogenesis in conjunctival
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Aus der Klinik für Augenheilkunde mit Poliklinik der Friedrich-Alexander-Universität Erlangen-Nürnberg Direktor: Prof. Dr. F. E. Kruse Tumor-associated lymphangiogenesis in conjunctival malignant melanoma Inaugural-Dissertation zur Erlangung der Doktorwürde der Medizinischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg vorgelegt von Paul Zimmermann aus Münchberg Gedruckt mit Erlaubnis der Medizinischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Dekan: Prof. Dr. J. Schüttler Referent: Priv.-Doz. Dr. Claus Cursiefen Korreferent: Prof. Dr. F. E.Kruse Tag der mündlichen Prüfung: 27. Januar 2010 Widmung: Meine Dissertation widme ich meiner Tochter Paula-Greta. Tumor-associated lymphangiogenesis in conjunctival malignant melanoma - Inhaltsverzeichnis: Zusammenfassung in Englisch (abstract) Seite 1 Zusammenfassung Seite 2-3 Einleitung in Englisch (Introduction) Seite 4-5 Einleitung Seite 6-7 Titelblatt Seite 8 (Seite 1 Artikel) Zusammenfassung/ Abstract Seite 9 (Seite 2 Artikel) Einleitung Seite 10-11 (Seite 3-4 Artikel) Material und Methoden Seite 12-14 (Seite 5-7 Artikel) Patienten und Bindegewebsschnitte Seite 12 (Seite 5 Artikel) Lymphgefäßfärbung (LYVE-1 and podoplanin) Seite 12-13 (Seite 5-6 Artikel) Ki67 Färbung Seite 13 (Seite 6 Artikel) Mitomycin C Behandlung Seite 13 (Seite 6 Artikel) Mikroskopische und Computer-gestützte Lymphgefäßanalyse Seite 13-14 (Seite 6-7 Artikel) Funktionelle und statistische Analyse Seite 14 (Seite 7 Artikel) Resultate/Ergebnisse Seite 15-17 (Seite 8-10 Artikel) Patienten und histopathologische Charakteristika Seite 15 (Seite 8 Artikel) Maligne Melanome der Bindehaut bilden intra- und peritumoral Lymphgefäße aus Maligne Melanome der Bindehaut sind assoziiert mit intraund peritumoraler Lymphangiogenese Einfluss des Tumordurchmessers auf die Lymphgefäße Seite 15 (Seite 8 Artikel) Seite 16 (Seite 9 Artikel) Seite 16 (Seite 9 Artikel) Effekt der Mitomycin C Therapie auf die Lymphgefäße Seite 17 (Seite 10 Artikel) Analyse der Lymphgefäße in Abhängigkeit der Tumorlokalisation Diskussion Seite 17 (Seite 10 Artikel) Seite 18-20 (Seite 11-13 Artikel) Resümee/ Schlussfolgerung Seite 20 (Seite 13 Artikel) Competing interests Seite 20 (Seite 13 Artikel) Literaturverzeichnis Seite 21-22 (Seite 14-15 Artikel) Abbildungsverzeichnis mit Legenden Seite 23-25 (Seite 16-18 Artikel) Abbildung 1: Tumor-assozierte Lymphgefäße des Malignen Melanoms der Bindehaut Abbildung 2: Tumor induzierte Lymphangiogenese: Signifikant mehr proliferierende Lymphgefäße wurden im Seite 23 (Seite 16 Artikel) Seite 23-24 (Seite 16-17 Artikel) Tumor und in der direkten Umgebung als in der weiter entfernten Bindehaut (> 300 μm) gefunden Abbildung 3: Einfluss des Tumordurchmessers auf die Lymphgefäße Abbildung 4: Einfluss des topischen Mitomycin C auf die Lymphgefäße Abbildung 5: Analyse der Lymphgefäße in Abhängigkeit der Tumorlokalisation Seite 24 (Seite 17 Artikel) Seite 24 (Seite 17 Artikel) Seite 24-25 (Seite 17-18 Artikel) Abbildungen: Seite 26-30 (Seite 19-23 Artikel) Abbildung 1 Seite 26 (Seite 19 Artikel) Abbildung 2 Seite 27 (Seite 20 Artikel) Abbildung 3 Seite 28 (Seite 21 Artikel) Abbildung 4 Seite 29 (Seite 22 Artikel) Abbildung 5 Seite 30 (Seite 23 Artikel) Literaturverzeichnis Seite 31-33 Abkürzungsverzeichnis Seite 34 Verzeichnis der Vorveröffentlichungen Seite 35 Anhang Seite 36 Danksagung Seite 37 Lebenslauf Seite 38-39 P.S.: Die Seitenzahlen in Klammern beziehen sich auf die Seitenzahlen der Veröffentlichung des Orginalartikels im BJO (British Journal of Ophthalmology). 1 Abstract: Background: To evaluate whether tumor-associated lymphangiogenesis, i.e. the formation of new lymphatic vessels (LVs) induced by a tumor, occurs in and around conjunctival malignant melanoma (MM). Methods: Clinical files and conjunctival specimens of 20 patients with histologically diagnosed conjunctival MM were analyzed. Sections were stained with LYVE-1 and podoplanin antibodies as specific lymphatic endothelial markers and Ki67 as proliferation marker. The tumor area and the area covered by LV (LVA), the LV number (LVN), and the LV density (LVD) were measured within the tumor and in the peritumoral area in digital images of the specimen. The LV results were correlated with the histopathological characteristics, tumor location, recurrence rate, mitomycin C therapy and presence of metastases. Results: LVs were detected in all specimens within the tumor and peritumorally. Significantly more Ki67+ proliferating lymphatic endothelial cells were detected in the tumor and in the peritumoral tissue up to 300 μm compared to the surrounding normal conjunctiva (>300 μm distance). There was a slightly positive correlation between the tumor size and the LVN and LVA in the 50 μm zone adjacent to the tumor. We did not find significant correlations between LVs and histopathological and clinical characteristics (location, shape, relapses, metastases), possibly due to small sample sizes. Non-limbal tumors with involvement of tarsus or fornix showed a tendency of higher LVD compared to limbal tumors. Conclusion: Conjunctival MMs display tumor-associated LV within and around the tumor. The MM seems to induce lymphangiogenesis not only in the tumor, but also in its proximity. 2 Zusammenfassung: Hintergrund und Ziele: Maligne Melanome der Bindehaut sind mit Lymphangiogenese, i.e. die Ausbildung neuer Lymphgefäße, assoziiert. Ziel der Studie war es, herauszufinden, ob Tumor- assoziierte Lymphangiogenese im Tumor, als auch in der peritumoralen Region auftritt. Methoden (Patienten, Material und Untersuchungsmethoden): Der klinische Casus als auch die histologischen Schnitte der Bindehaut von 20 Patientinnen/-en der Augenklinik Nürnberg-Erlangen wurden analysiert. Die histologischen Schnitte wurden mit LYVE-1 und Podoplanin Antikörper als spezifische Lymphendothelmarker und mit Ki67 als Proliferationsmarker gefärbt. Die Tumorfläche, die Lymphgefäßfläche (LVA= Lymphatic Vessel Area), die Anzahl der Lymphgefäße (LVN= Lymphatic Vessel Number) und die Lymphgefäßdichte (LVD= Lymphatic Vessel Density) wurden im Tumor und in der Tumorumgebung anhand von digitalen Bildern ausgemessen. Die Ergebnisse wurden mit den histopathologischen Eigenschaften, der Tumorlokalisation, der Rezidivrate, der Mitomycin C Therapie und dem Auftreten von Metastasen korreliert. Ergebnisse: Lymphgefäße konnten in allen histologischen Schnitten sowohl im Tumor als auch in der Umgebung des Tumors nachgewiesen werden. Signifikant mehr Ki67 positive proliferierende Lymphendothelzellen konnten im Tumor und in der Tumornähe (<300µm) als in der normalen Bindehaut (>300 μm) nachgewiesen werden. Es zeigte sich eine tendenzielle positive Korrelation zwischen der Tumorgröße und der Fläche als auch der Anzahl der Lymphgefäße in der 50µm peritumoralen Zone. Wir konnten keine signifikanten Korrelationen zwischen den histopathologischen und den klinischen Charakteristika (Lokalisation, Wachstum, Rezidive und Metastasen) verifizieren, möglicherweise auch 3 aufgrund der geringen Patientenanzahl. Nicht am Limbus lokalisierte Tumoren, die in den Tarsus oder Fornix vordrangen, zeigten eine tendenziell höhere Lymphgefäßdichte als Tumoren am Limbus. Praktische Schlussfolgerung: Maligne Melanome der Bindehaut zeigten Tumor-assozierte Lymphangiogenese innerhalb und in der Umgebung des Tumors. Das Maligne Melanom der Bindehaut scheint nicht nur im Tumor, sondern auch in der näheren Umgebung Lymphangiogenese, i.e. die Lymphgefäßneubildung, zu induzieren. 4 Introduction: Malignant melanomas (MMs) of the conjunctiva are associated with significant morbidity and mortality due to high rates of recurrence and metastasis. The dissemination of the tumor is linked to regional lymph nodes with subsequent distant metastasis. Compared to cutaneous MM, conjunctival MM is rare. The annual age adjusted incidence rates (per million) vary from 0.15 in Asians to 0.5 in non-Hispanic Whites. Up to date, there are only few features recognized as prognostic factors for conjunctival MM: Tumor location, expansion, relapse, multifocal location, involvement of the surgical margins and tumor depth are known prognostic factors for metastatic disease. Histopathological characteristics seem not to be consistently associated with the clinical outcome. The primary treatment of conjunctival MM is surgical: Complete excision with tumor-cell free margins represents the therapy of choice, but can not be sufficiently performed in cases of diffuse growth. Topical mitomycin C as adjunct therapy has been established; cryotherapy, laser ablation, radiation treatment, and chemotherapy in case of metastasis represent additional treatment options for conjunctival MM. Conjunctival MMs are rich in blood vessels, which play a role in systemic hematogenous metastasis. However, the main route of metastasis of conjunctival MM is lymphogenic: Ultrasonic examination of the draining lymph nodes or even surgical removal of the sentinel lymph nodes has been recommended. Up to now, it was not known, whether conjunctival MMs also display significant tumor-associated lymphangiogenesis, i.e. whether the tumor induces formation of new lymphatic vessels. The extent of lymph node metastasis is supposed to be a major determinant for prognosis and staging of tumors and it has been shown that tumor-induced lymphangiogenesis is a strong risk factor for tumor metastasis in different human cancers. The importance of tumor-induced lymphangiogenesis for lymphogenic metastasis in cutaneous MM has been shown recently. 5 Purpose of this study was to analyze whether conjunctival MMs also display tumor-induced lymphangiogenesis, which may represent a possible new prognostic factor. We used specific lymphatic endothelial markers to analyze the presence of LVs in the tumor itself and in the adjacent tissue and correlated these data with the clinical outcome and histopathological characteristics of the tumors. 6 Einleitung: Maligne Melanome der Bindehaut sind mit einer signifikanten Morbidität und Mortalität assoziiert, respektive einer hohen Rezidiv- als auch Metastasenrate. Die Tumor-dissemination breitet sich zu regionären Lymphknoten aus und setzt anschließend Fernmetastasen. Im Vergleich zu kutanen malignen Melanomen ist das konjunktivale maligne Melanom selten. Die jährliche, altersadaptierte, Inzidenzrate (pro Million Einwohner) variiert zwischen 0,15 bei Asiaten und 0,50 bei der weißen, nicht hispanisch stämmigen, Bevölkerung. Aktuell werden konjunktivalen nur wenige malignen Charakteristika Melanomen Ausdehnung, Rezidivneigung, multifokale chirurgischen Absetzungsrandes, und als Prognosefaktoren anerkannt: Tumorlokalisation, Lokalisation, Tumortiefe bei Beteiligung sind des bekannt als Prognosefaktoren für Metastasierung. Histopathologische Faktoren scheinen nicht übereinstimmend mit dem klinischen Verlauf assoziiert zu sein. Der primäre Therapieansatz beim konjunktivalen malignen Melanom ist chirurgisch: Therapie der Wahl ist die komplette tumorzellfreie Resektion; diese kann aber bei diffusem Wachstumsmuster nicht suffizient durchgeführt werden. Als adjuvante Therapie wurde eine topische Mitomycin-C Therapie etabliert; Kryotherapie, Lasertherapie, Radiatio und Chemotherapie bei Metastasen stellen alternative Therapieoptionen für konjunktivale maligne Melanome dar. Konjunktivale maligne Melanome sind reich an Blutgefäßen, welche eine Rolle bei der hämatogenen Hauptmetastasierungs-Route Absiedlung beim spielen. konjunktivalen Dennoch ist die malignen Melanom lymphogen: Ultraschalluntersuchung der drainierenden Lymphknoten oder auch chirurgische Entfernung der Sentinel-Lymphknoten werden empfohlen. Bis heute ist es nicht bekannt, ob konjunktivale maligne Melanome signifikante tumor-assoziierte Lymphangiogenese zeigen, d.h. ob der Tumor die Ausbildung von neuen Lymphgefäßen induziert. Das Ausmaß der Lymphknotenmetastasierung ist generell ein wichtiges Kriterium bezüglich der Prognose und des Tumorstagings und es wurde gezeigt, dass Tumor-assoziierte Lymphangiogenese ein starker Risikofaktor für Metastasierung bei verschiedenen menschlichen Tumoren ist. Kürzlich wurde 7 die Bedeutung der Tumor-assoziierten Lymphangiogenese für die lymphogene Metastasierung bei kutanen malignen Melanomen gezeigt. Ziel der Studie war, zu analysieren, ob konjunktivale maligne Melanome tumorassoziierte Lymphangiogenese induzieren, welche einen neuen prognostischen Faktor repräsentieren könnten. Wir benutzten spezifische Lymphendothelmarker, um die Präsenz von Lymphgefäßen im Tumor und im umgebenden Gewebe zu analysieren, und korrelierten diese Daten mit dem klinischen Verlauf und den histopathologischen Eigenschaften der Tumoren. 8 Tumor-associated lymphangiogenesis in conjunctival malignant melanoma Keywords: Lymphangiogenesis, Malignant Melanoma, Conjunctiva Paul Zimmermann*1, Tina Dietrich*1,2, Felix Bock1, Folkert K. Horn1, Carmen HofmannRummelt1, Friedrich E. Kruse1, Claus Cursiefen1 *both authors contributed equally and should be regarded as first authors 1 Department of Ophthalmology, University Erlangen-Nürnberg, Germany 2 Department of Ophthalmology, University Medical Center Regensburg, Germany Support: Interdisciplinary Center for Clinical Research (IZKF) Erlangen (A9), SFB 649 TP B10 Abstract: 249 words, text: 2674 words The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://bjo.bmj.com/ifora/licence.pdf). Correspondence: Claus Cursiefen, MD, Dept. of Ophthalmology, University of ErlangenNürnberg, Schwabachanlage 6, 91054 Erlangen, Germany; Tel.: 00499131 85-34141; Fax: 00499131 85-36401; Email: [email protected] 9 Abstract Background: To evaluate whether tumor-associated lymphangiogenesis, i.e. the formation of new lymphatic vessels (LVs) induced by a tumor, occurs in and around conjunctival malignant melanoma (MM). Methods: Clinical files and conjunctival specimens of 20 patients with histologically diagnosed conjunctival MM were analyzed. Sections were stained with LYVE-1 and podoplanin antibodies as specific lymphatic endothelial markers and Ki67 as proliferation marker. The tumor area and the area covered by LV (LVA), the LV number (LVN), and the LV density (LVD) were measured within the tumor and in the peritumoral area in digital images of the specimen. The LV results were correlated with the histopathological characteristics, tumor location, recurrence rate, mitomycin C therapy and presence of metastases. Results: LVs were detected in all specimens within the tumor and peritumorally. Significantly more Ki67+ proliferating lymphatic endothelial cells were detected in the tumor and in the peritumoral tissue up to 300 µm compared to the surrounding normal conjunctiva (>300 µm distance). There was a slightly positive correlation between the tumor size and the LVN and LVA in the 50 µm zone adjacent to the tumor. We did not find significant correlations between LVs and histopathological and clinical characteristics (location, shape, relapses, metastases), possibly due to small sample sizes. Non-limbal tumors with involvement of tarsus or fornix showed a tendency of higher LVD compared to limbal tumors. Conclusion: Conjunctival MMs display tumor-associated LV within and around the tumor. The MM seems to induce lymphangiogenesis not only in the tumor, but also in its proximity. 10 Introduction Malignant melanomas (MMs) of the conjunctiva are associated with significant morbidity and mortality due to high rates of recurrence and metastasis1,2. The dissemination of the tumor is linked to regional lymph nodes with subsequent distant metastasis 3. Compared to cutaneous MM, conjunctival MM is rare. The annual ageadjusted incidence rates (per million) vary from 0.15 in Asians to 0.5 in non-Hispanic Whites4,5. Up to date, there are only few features recognized as prognostic factors for conjunctival MM: Tumor location, expansion, relapse, multifocal location, involvement of the surgical margins and tumor depth are known prognostic factors for metastatic disease6, 7. Histopathological characteristics seem not to be consistently associated with the clinical outcome7. The primary treatment of conjunctival MM is surgical: Complete excision with tumor-cell free margins represents the therapy of choice, but can not be sufficiently performed in cases of diffuse growth. Topical mitomycin C as adjunct therapy has been established8; cryotherapy, laser ablation, radiation treatment, and chemotherapy in case of metastasis represent additional treatment options for conjunctival MM. Conjunctival MMs are rich in blood vessels, which play a role in systemic hematogenous metastasis. However, the main route of metastasis of conjunctival MM is lymphogenic: Ultrasonic examination of the draining lymph nodes or even surgical removal of the sentinel lymph nodes has been recommended. Up to now, it was not known, whether conjunctival MMs also display significant tumor-associated lymphangiogenesis, i.e. whether the tumor induces formation of new lymphatic vessels. The extent of lymph node metastasis is supposed to be a major determinant for prognosis and staging of tumors9 and it has been shown that tumor-induced lymphangiogenesis is a strong risk factor for tumor metastasis in different human 11 cancers10; 9; 11-13; 3; 14. The importance of tumor-induced lymphangiogenesis for lymphogenic metastasis in cutaneous MM has been shown recently10. Purpose of this study was to analyze whether conjunctival MMs also display tumor-induced lymphangiogenesis, which may represent a possible new prognostic factor. We used specific lymphatic endothelial markers to analyze the presence of LVs in the tumor itself and in the adjacent tissue and correlated these data with the clinical outcome and histopathological characteristics of the tumors. 12 Material and Methods Patients and conjunctival sections Clinical files and histological sections of conjunctival MMs of 20 patients who were treated at the Department of Ophthalmology of the University Erlangen-Nürnberg, Germany, between 1987 and 2005 were retrospectively analyzed. The files were screened and the documented treatment and follow-up was taken into consideration. The clinical outcome of all patients was re-evaluated at the end of 2006 and again 2008 by interviewing the patients’ general practitioners for any new progress of the disease since the last visit, especially for systemic metastasis. Lymphatic vessel staining (LYVE-1 and podoplanin) For staining of LVs LYVE-1 served as specific marker for lymphatic vascular endothelium. The preparation of the histological sections of conjunctival MMs was performed as described previously15. Briefly, tissue was fixed in neutral buffered formalin, embedded in paraffin and cut in 4 µm sections. After deparaffinization and rehydration, sections were digested with proteinase K (Dako, Hamburg, Germany), incubated for 10 minutes with horseradish peroxidase (HRP). Sections of conjunctival MMs were incubated for 30 minutes with a rabbit polyclonal antibody against human LYVE-1 (1:100; Dako, Hamburg, Germany) and HRP-conjugated secondary antibody before development with 3-amino-9-ethylcarbazole (AEC+) substrate (red reaction product) or 3,3´-diaminobenzidine (DAB; brown product). Sections were counterstained with Mayer haemalaun (Chroma, Münster, Germany). Positive controls were performed on corneoscleral ring specimens and negative controls with control IgG. Since LYVE-1 is also expressed on tissue macrophages16; 17 only clearly identifiable vessels with an erythrocyte-free vessel lumen were counted as LV and specimens were double-stained with podoplanin as second lymphatic endothelial marker. 13 For podoplanin immunostaining polyclonal rabbit anti-human antibody against podoplanin (1:200, Dako, Hamburg, Germany) was used, followed by biotinylated goat anti-rabbit IgG for 30 minutes and detection by a streptavidin peroxidase complex (using DAB/AEC+ as the chromogen substrate). Positive controls were performed as described above. Ki67 staining Sections of paraffin embedded specimens were double-stained with LYVE-1 and monoclonal antibody against Ki67 (clone MIB-1, Dako, Hamburg, Germany) as a specific marker for proliferating cells. LVs with at least 5 endothelial cells with nuclear Ki67 positivity were considered to be Ki67 positive. Mitomycin C treatment The additional topical mitomycin C treatment of conjunctival MM by eye drops is standardized in our department as two 14 day cycles with mitomycin C 0.02 % eye drops five times a day with a 14-day break. Some patients were not treated with mitomycin C eye drops due to allergy or refusal. To analyze the potential antilymphangiogenic effect of mitomycin C therapy, tumor specimens of patients, who received mitomycin C treatment and had excisions later on during their clinical course (because of new suspect lesions) were compared to the specimens obtained before mitomycin C treatment. Microscopy and computer-assisted vessel analysis Histological sections of conjunctival MMs of 20 patients were taken into consideration. Sections were analyzed with a light microscope (BX51, Olympus Optical Co., Hamburg, Germany) and digital color images were taken with a 12-bit CCD camera (Color-View I, Olympus, Hamburg, Germany; 40x and 100x magnification). Analyses were performed using Cell^F (Olympus, Hamburg, Germany) and Image J analyzing program (available via http://rsb.info.nih.gov / ij/download.html). Morphometrical LV 14 analysis was performed for the area of the tumor, the adjacent 50 µm zone, the midperipheral zone (50 -200µm), the peripheral zone (200-300µm) and the conjunctiva more than 300 µm away from the tumor border (defined as normal conjunctiva). If the tumor adjacent area was not completely represented on the specimen, we evaluated the area as far as represented. The tumor size was measured as the area covered by the tumor in the histological section. We determined the following parameters: 1. the LV number (LVN), 2. the area covered by LVs (LVA), 3. the LV density (LVD), determined by measuring the LVN and dividing it by the tumor cross sectional area (mm2). Functional and statistical analysis To determine statistical significance, quantitative analyses of the LVA, LVN, and LVD in all analyzed areas (intratumoral, 50 µm, 50 - 200 µm, 200 - 300 µm and >300 µm peritumoral) were performed in a standardized procedure using the statistic program InStat 3 (GraphPad Software Inc, San Diego, California, USA). Analyses were performed using the non-parametric test for the Ki67 analysis and the Pearson rank correlation for the correlation of tumor area to LVN and LVA. 15 Results Patients and histopathological characteristics The median age of the patients in the study was 70.4 years (43-100 years). 9 women and 11 men were treated. The MM of 13 patients was based on primary acquired melanosis (PAM); in 7 patients the origin of the MM remained unclear. The primary treatment was surgical, 10 patients had an additional topical mitomycin C treatment. The primary tumor was located in the fornix (2 patients), the tarsus and the upper lid (5 patients) or at the limbus/epibulbar conjunctiva (7 patients). 6 patients showed a widely disseminated tumor, including some who have had primary excision outside of our department, so that the primary tumor location was not known. 10 patients showed a diffuse, 5 patients a nodular, and 5 a mixed growing type of the MM. The histopathological characteristics were: 13 tumors of mixed cell type, 5 tumors of spindle cell type, and 2 tumors of epitheloid cell type. 8 of the 20 patients showed more than 5 relapses during the clinical course. 5 patients suffered from metastasis: 1 patient was diagnosed for gastric metastasis 7 years after primary diagnosis, two patients for submandibular and neck spreading after 2 and 3 years, 1 patient for craniopharyngeal metastasis after 1 year and 1 patient had parotical metastasis after 14 years. Conjunctival MMs display intra- and peritumoral LVs Using LYVE-1 and podoplanin staining, we identified LVs in all included MM specimens, both within the tumor itself and in the adjacent tissue (Figure 1). There was a similar staining pattern for both lymphatic vascular endothelial markers in the conjunctival MM specimens. 16 Conjunctival MMs are associated with intra- and peritumoral lymphangiogenesis To examine whether conjunctival MMs induce formation of new LVs, Ki67 staining was performed to detect proliferating lymphatic endothelial cells in the conjunctival MMs and the adjacent conjunctival tissue. Immunostaining with Ki67 revealed significantly more proliferating lymphatic endothelial cells in the tumor and in the directly adjacent conjunctiva compared to the peripheral zones. Non-parametric tests were performed for each zone separately with the following results concerning the ratio of Ki67-positive LVs: 1. tumor vs. tumor adjacent conjunctiva (50 µm zone) p=0.063 (not significant), 2. tumor vs. mid-peripheral zone (50 -200 µm) p=0.021, 3. tumor vs. peripheral zone (200-300 µm) p=0.031, 4. tumor vs. distant, presumably normal conjunctiva >300 µm from the tumor border p=0.002 (Figure 2). The results support the hypothesis of tumorassociated active lymphangiogenesis in the tumor and its proximity. Influence of tumor cross-sectional area on LVs We analyzed whether the extent of lymphangiogenesis in and around conjunctival MM was correlated with the tumor area. Therefore analyses of the LV parameters LVA, LVN, LVD were performed in the tumor and in the tumor environment (50 µm zone, 50-200 µm zone, 200-300 µm zone). Intratumoral LVN, LVA and LVD were not positively correlated to the tumor cross-sectional area (results for LVN and LVA shown as table in Fig. 3). The LVN and LVA in the 50 µm area directly adjacent to the tumor are positively correlated to the tumor cross sectional area; the results for the LVN demonstrated as scatter plot in Fig. 3 (Pearson correlation coefficient r=0.64; p=0.002). There was a slightly inverse correlation for the LVD in the tumor with the tumor cross-sectional area (r=-0.257, p=0.237). 17 Effect of mitomycin C therapy on LVs We analyzed the potential effect of topical mitomycin C treatment on LV formation. Specimens of patients (n=4) who underwent topical mitomycin C treatment after tumor excision and had subsequent excisions for tumor recurrence were analyzed for LVD and LVN (Figure 4). In 3 patients the specimens represented all 5 zones; in one patient the specimens showed only tumor without surrounding conjunctiva because of diffuse tumor growth (therefore only the tumor zone was analyzed in this patient). Because of the limited number of patients, statistical analysis was restricted to descriptive analyses (Figure 4). Additional LV analysis (LVD, LVN, LVA) of specimens from mitomycin C treated patients compared with specimens from patients without mitomycin C treatment did not show statistically significant results. Analysis of LVs in dependence of tumor location Analyzing the location of the tumor, 7 patients had limbal/epibulbar tumors, 7 patients had a MM at the fornix or tarsus and 6 patients had a disseminated MM. The analyses of LV parameters (LVN, LVA, LVD) did not show statistical significant results in these small sample sizes. Because of the small number of patients, we performed only descriptive analyses as scatter plot. Fig. 5 demonstrates the LVD in dependence of the tumor location: In 4 of 7 patients with non-limbal MMs with involvement of the fornix or tarsus the LVD was higher than in all limbal tumor specimens (n=7). 18 Discussion This study on conjunctival MM shows for the first time that conjunctival – and not only cutaneous– MMs display tumor-associated lymphangiogenesis. In our study we found LVs in the tumor itself as well as in the peritumoral area. These erythrocyte-free LVs are stained with two new markers specific for lymphatic vascular endothelium, which are LYVE-1 and podoplanin. Most remarkably, the degree of actively proliferating, Ki67 positive lymphatic vascular endothelial cells is significantly higher within the MM and in the close vicinity of the tumor compared to normal, resting conjunctival LVs more distant from the tumor site. The ratio of Ki67 positive LVs is reduced with growing distance from the tumor border which supports the hypothesis of active tumor-induced formation of new LVs (tumor-associated lymphangiogenesis). It has been shown recently that lymphangiogenic growth factors, which are secreted by a primary tumor, can induce lymphangiogenesis20-23. In our small pilot study there was a no significant correlation between tumor size (two-dimensional) and LVN and LVA in the tumor. The analyses of the tumor environment revealed a higher LVN and LVA in the 50 µm zone directly adjacent to the tumor, possibly being an indicator for active lymphangiogenesis in the tumor surroundings. We found a slightly inverse correlation between LVD in the tumor and the tumor cross sectional area. On the one hand that finding might be related to the two-dimensional calculations of the tumor area based on cross sectional specimens of the tumor. On the other hand small tumors might show a higher LVD as a signal for their starting potency of dissemination, possibly related to high secretion rates of lymphangiogenic growth factors. Another possible aspect is a centrally developing necrosis in larger tumors which might reduce the rate of lymphangiogenesis and cause reduced LVD. Furthermore, some studies describe a high interstitial pressure within tumors that promotes LV collapse13; thus compressed LV in larger tumors might appear smaller than LV in smaller tumors. 19 We analyzed the putative impact of lymphangiogenesis on recurrence rate and metastasis, but did not find a significant correlation, possibly related to the small sample sizes. Larger (prospective) studies now will have to evaluate tumor-associated lymphangiogenesis as a putative risk factor for tumor metastasis. Tumor location is one of the clinically most important prognostic predictors of conjunctival MM: non-limbal tumors show a higher incidence of initial systemic metastasis and reduced survival rates5; 7. This fact might be due to facilitated access to blood vessels or to the draining LVs. Our descriptive analysis of the tumor location, i.e. limbal vs. palpebral/fornix vs. disseminated conjunctival in correlation with parameters of lymphangiogenesis showed a tendency of higher LVD in non-limbal palpebral/fornix tumors. We did not find significant differences concerning the lymphangiogenic parameters in correlation with the different growth patterns i.e. shape of conjunctival MM. Additional studies are necessary to elucidate these aspects. Topical mitomycin C treatment might provide an antilymphangiogenic effect as it has been suggested in several other studies on the treatment of conjunctival MM18; 8. We performed only descriptive analyses due to the small sample sizes of histological tumor specimens after mitomycin C treatment: There was a tendency of reduced LVD in specimens from patients after mitomycin C therapy compared to specimens before mitomycin C therapy, while LVN and LVA were not reduced. Nevertheless, other factors as fibrotic tissue-remodeling after surgical excision, the influence of comedications as topical steroids or other causes can not be ruled out and may be of significant influence. The prognostic importance of intra- and peritumoral lymphangiogenesis is becoming more established by a growing number of studies on several human cancers10; 9; 3. The identification of high risk patients is helpful in order to individually optimize screening and treatment guidelines. The extent of tumor-associated lymphangiogenesis in conjunctival MM may be one novel prognostic criterion besides other known or putative risk factors. Immunostaining for lymphatic markers as LYVE-1 20 and podoplanin in routine histological work-up of tumor samples might be warranted if future studies reveal a correlation between tumor-induced lymphangiogenesis and prognosis of the tumor in terms of metastasis and recurrence rate. The histological analysis of lymphangiogenesis parameters compared with sentinel lymph node biopsy as a mean of guiding treatment and follow-up is under discussion9; 12; 19. This study on tumor-associated lymphangiogenesis may pave the road to new targets for innovative therapeutic approaches and serve as a novel prognostic parameter in conjunctival MM. Much effort in anti-lymphangiogenic and anti-angiogenic research has been made to develop new therapeutic approaches to inhibit tumor spreading. Recently, different assays of selective inhibition of LV growth on the ocular surface have been shown24;25. In the future anti-lymphangiogenic treatment options might help to minimize the risk of metastasis in conjunctival MM. Conclusion MMs of the conjunctiva display LVs within and around the tumor. There is evidence for tumor-induced lymphangiogenesis, i.e. formation of newly formed LVs. These LVs may act as conduits for tumor metastasis. Lymphangiogenesis parameters as LVD and LVN may become useful novel prognostic indicators for conjunctival MM. Novel antilymphangiogenic therapeutic strategies may contribute to optimize the therapy of conjunctival MM in the future. Competing interest: None declared. 21 References 1. Kurli M, Finger PT. Melanocytic conjunctival tumors. Ophthalmol Clin North Am 2005;18: 15-24. 2. Brownstein S. Malignant melanoma of the conjunctiva. Cancer Control 2004;11: 310-6. 3. Shields JD, Borsetti M, Rigby H et al. Lymphatic density and metastatic spread in human malignant melanoma. Br J Cancer 2004;90: 693-700. 4. Hu DN, Yu G, McCormick SA et al. Population-based incidence of conjunctival melanoma in various races and ethnic groups and comparison with other melanomas. Am J Ophthalmol 2008;145: 418-23. 5. Tuomaala S, Eskelin S, Tarkkanen A et al. Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites. Invest Ophthalmol Vis Sci 2002;43: 3399-408. 6. Shields CL. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Trans Am Ophthalmol Soc 2000;98: 471-92. 7. Tuomaala S, Toivonen P, Al-Jamal R et al. Prognostic significance of histopathology of primary conjunctival melanoma in Caucasians. Curr Eye Res 2007;32: 939-52. 8. Pe'er J, Frucht-Pery J. The treatment of primary acquired melanosis (PAM) with atypia by topical Mitomycin C. Am J Ophthalmol 2005;139: 229-34. 9. Tobler NE, Detmar M. Tumor and lymph node lymphangiogenesis--impact on cancer metastasis. J Leukoc Biol 2006;80: 691-6. 10. Detmar M, Hirakawa S. The formation of lymphatic vessels and its importance in the setting of malignancy. J Exp Med 2002;196: 713-8. 11. Dadras SS, Paul T, Bertoncini J et al. Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am J Pathol 2003;162: 1951-60. 12. Dadras SS, Lange-Asschenfeldt B, Velasco P et al. Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes. Mod Pathol 2005;18: 1232-42. 13. Jackson DG, Prevo R, Clasper S et al. LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol 2001;22: 317-21. 14. Straume O, Jackson DG, Akslen LA. Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res 2003;9: 250-6. 22 15. Cursiefen C, Schlotzer-Schrehardt U, Kuchle M et al. Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin. Invest Ophthalmol Vis Sci 2002;43: 2127-35. 16. Maruyama K, Ii M, Cursiefen C et al. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest 2005;115: 2363-72. 17. Cursiefen C, Chen L, Borges LP et al. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 2004;113: 1040-50. 18. Khong JJ, Muecke J. Complications of mitomycin C therapy in 100 eyes with ocular surface neoplasia. Br J Ophthalmol 2006;90: 819-22. 19. Tuomaala S, Kivela T. Metastatic pattern and survival in disseminated conjunctival melanoma: implications for sentinel lymph node biopsy. Ophthalmology 2004;111: 816-21. 20. Hirakawa S, Kodama S, Kunstfeld R et al. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J Exp Med 2005;201: 1089-99. 21. Streit M, Detmar M. Angiogenesis, lymphangiogenesis, and melanoma metastasis. Oncogene 2003;22: 3172-9. 22. Skobe M, Hamberg LM, Hawighorst T et al. Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am J Pathol 2001;159: 893-903. 23. Harrell MI, Iritani BM, Ruddell A. Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis. Am J Pathol 2007;170: 774-86. 24. Bock F, Onderka J, Dietrich T et al. Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation. Graefes Arch Clin Exp Ophthalmol 2008;246: 115-9. 25. Dietrich T, Onderka J, Bock F et al. Inhibition of inflammatory lymphangiogenesis by integrin alpha5 blockade. Am J Pathol 2007;171: 361-72. 23 Figure legends: Figure 1: Tumor-associated lymphatic vessels in MMs of the conjunctiva a) Representative image of LV staining with LYVE-1 antibody as specific marker for lymphatic endothelium: LYVE-1 positive peritumoral LVs. b) Representative image of podoplanin stained LVs in the tumor adjacent conjunctiva. c) Intratumoral LYVE-1 positive LVs (magnification x100/x200). Area of primary acquired melanosis is marked with an asterisk. Arrows: The LYVE-1/podoplanin stained lymphatic vessels (▬►) Note that erythrocyte filled blood vessels are not stained with these lymphatic endothelial specific markers. Figure 2: Tumor-induced lymphangiogenesis: Significantly more proliferating LVs were found intratumorally and next to the tumor than in distant conjunctiva (> 300 µm). Representative images of conjunctival MM specimen stained with LYVE-1 and proliferation marker Ki67. Ki67 positive cells are marked (arrowhead). a) Representative image of Ki67 positivity in tumor-associated lymphatic endothelial cells; red: Ki-67; brown: LYVE-1 (magnification x1000) b) Significantly more proliferating LVs were found intratumorally as well as in the directly adjacent tumor environment than in the more distant conjunctiva. Paired t test was performed for each zone separately with the following results: 1. tumor vs. tumor adjacent conjunctiva (50 µm zone) p=0.063 (not significant), 2. tumor vs. mid-peripheral zone (50 -200 µm) p=0.021, 3. tumor vs. peripheral zone (200-300 µm) p=0.031, 4. tumor vs. conjunctiva >300 µm from the tumor border p=0.002; n=20, median is marked; circles mark 3 outliers. The 24 results support the hypothesis of tumor-associated active lymphangiogenesis in the proximity of the tumor. Figure 3: Influence of tumor cross-sectional area on LVs Analyses of the LV parameters LVA, LVN, LVD were performed in the tumor and in the tumor environment (50 µm zone, 50-200 µm zone, 200-300 µm zone). Intratumoral LVN, LVA and LVD were not positively correlated to the tumor cross-sectional area (a). There was a slightly inverse correlation for the LVD in the tumor with the tumor cross-sectional area (r=-0.257, p=0.237). The LVN in the 50 µm area directly adjacent to the tumor was positively correlated to the tumor cross sectional area (Pearson correlation coefficient r=0.64; p=0.002), demonstrated as scatter plot (b). Figure 4: Effect of topical mitomycin C on LVs Scatter plot diagram of the measured LVD in the tumor (0), in the adjacent 50 µm zone (50), in the 50-200 µm zone (200), in the 200-300 µm zone (300) before and after mitomycin C therapy. LVD before mitomycin C therapy is marked as ring, LVD after mitomycin C therapy is marked as asterisk. Sections of 4 patients were analyzed, in one patient there was no surrounding conjunctiva represented on the specimen and only the tumor area was analyzed. Because of the limited number of patients descriptive analyses were performed. Figure 5: Analysis of LVs in dependence of tumor location Descriptive scatter plot: In 4 of 7 patients with non-limbal MMs with involvement of the fornix or tarsus the LVD was higher than in all limbal/epibulbar tumor specimens (n=7). 25 7 patients had limbal/epibulbar MMs, 7 patients had a MM at the fornix or tarsus and 6 patients had a disseminated MM. The analyses of LV parameters (LVN, LVA, LVD) did not show statistical significant results in these small sample sizes. Figure 1: 26 Figure 2: 27 Figure 3: 28 Figure 4: 29 Figure 5: 30 31 Literaturverzeichnis: 1. Brownstein S. 2004. Malignant melanoma of the conjunctiva. Cancer Control; 11: 310-6.(2004) 2. Bock F, Onderka J, Dietrich T, Bachmann B, Pytowski B, CursiefenC. 2008. Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation. Graefes Arch Clin Exp Ophthalmol; 246: 115-9.(2008) 3. Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, D´Amore PA, Dana MR, Wiegand SJ, Streilein JW. 2004. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest; 113: 104050.(2004) 4. Cursiefen C, Schlotzer-Schrehardt U, Kuchle M,Sorokin L,BreitenederGeleff S, Atitalo K, Jackson D. 2002. Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE- 1 and podoplanin. Invest Ophthalmol Vis Sci; 43: 2127-35.(2002) 5. Dadras SS, Lange-Asschenfeldt B, Velasco P, Nguyen L, Vora A, Muzikansky A, Jahnke K, Hauschild A, Hirakawa S, Mihm MC, Detmar M. 2005. Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes. Mod Pathol; 18: 1232-42.(2005) 6. Dadras SS, Paul T, Bertoncini J, Brown LF, Muzikansky A, Jackson DG, Ellwanger U, Garbe C, Mihm MC, Detmar M. 2003. Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am J Pathol; 162: 1951-60.(2003) 7. Detmar M, Hirakawa S. 2002.The formation of lymphatic vessels and its importance in the setting of malignancy. J Exp Med; 196: 713-8.(2002) 8. Dietrich T, Onderka J, Bock F, Kruse FE, Vossmeyer D, Stragies R, Zahn G, Cursiefen C. 2007. Inhibition of inflammatory lymphangiogenesis by integrin alpha5 blockade. Am J Pathol; 171: 361- 72.(2007) 9. Harrell MI, Iritani BM, Ruddell A. 2007. Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis. Am J Pathol; 170: 774-86.(2007) 32 10. Hirakawa S, Kodama S, Kunstfeld R, Kajiya K, Brown LE, Detmar M. 2005. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J Exp Med; 201: 1089-99. (2005) 11. Hu DN, Yu G, McCormick SA, Finger PT. 2008. Population-based incidence of conjunctival melanoma in various races and ethnic groups and comparison with other melanomas. Am J Ophthalmol; 145: 418-23. (2008) 12. Jackson DG, Prevo R, Clasper S, Banerji. 2001. LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol; 22: 317-21. (2001) 13. Khong JJ, Muecke J. 2006. Complications of mitomycin C therapy in 100 eyes with ocular surface neoplasia. Br J Ophthalmol; 90: 819-22.(2006) 14. Kurli M, Finger PT. 2005. Melanocytic conjunctival tumors. Ophthalmol Clin North Am; 18: 15-24.(2005) 15. Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, Van Rooijen N, Takenaka H, D'Amore PA, Stein-Streilein J, Losordo DW, Streilein JW. 2005. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest; 115: 2363-72.(2005) 16. Pe'er J, Frucht-Pery J. 2005. The treatment of primary acquired melanosis (PAM) with atypia by topical Mitomycin C. Am J Ophthalmol; 139: 229-34.(2005) 17. Shields CL. 2000. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Trans Am Ophthalmol Soc; 98: 471-92.(2000) 18. Shields JD, Borsetti M, Rigby H, Harper SJ, Mortimer PS, Levick JR, Orlando A, Bates DO. 2004. Lymphatic density and metastatic spread in human malignant melanoma. Br J Cancer; 90: 693-700.(2004) 19. Skobe M, Hamberg LM, Hawighorst T, Schirner M, Wolf GL, Alitalo K, Detmar M. 2001. Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am J Pathol; 159: 893-903.(2001) 33 20. Straume O, Jackson DG, Akslen LA. 2003. Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma. Clin Cancer Res; 9: 250-6. (2003) 21. Streit M, Detmar M. 2003. Angiogenesis, lymphangiogenesis, and melanoma metastasis. Oncogene; 22: 3172-9.(2003) 22. Tobler NE, Detmar M. 2006. Tumor and lymph node lymphangiogenesisimpact on cancer metastasis. J Leukoc Biol; 80: 691-6.(2006) 23. Tuomaala S, Eskelin S, Tarkkanen A, Kivelä T. 2002. Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites. Invest Ophthalmol Vis Sci; 43: 3399-408.(2002) 24. Tuomaala S, Kivela T. 2004. Metastatic pattern and survival in disseminated conjunctival melanoma: implications for sentinel lymph node biopsy. Ophthalmology; 111: 816-21.(2004) 25. Tuomaala S, Toivonen P, Al-Jamal R, Kivelä T. 2007. Prognostic significance of histopathology of primary conjunctival melanoma in Caucasians. Curr Eye Res; 32: 939-52.(2007) 34 Abkürzungsverzeichnis: Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE1) Seite 9 (Seite 2 Artikel) Lymphatic vessels (LV) Seite 9 (Seite 2 Artikel) Malignant melanoma (MM) Seite 9 (Seite 2 Artikel) Area covered by LV (LVA) Seite 9 (Seite 2 Artikel) The LV number (LVN) Seite 9 (Seite 2 Artikel) The LV density (LVD) Seite 9 (Seite 2 Artikel) Horseradish peroxidase (HRP) Seite 12 (Seite 5 Artikel) 3-amino-9-ethylcarbazole (AEC+) Seite 12 (Seite 5 Artikel) 3,3´-diaminobenzidine (DAB) Seite 12 (Seite 5 Artikel) Primary acquired melanosis (PAM) Seite 15 (Seite 8 Artikel) Mitomycin C (MMC) Seite 29 (Seite 22 Artikel) Die Seitenzahlen in Klammern beziehen sich auf die Veröffentlichung im BJO (British Journal of Ophthalmology): Tumor-associated lymphangiogenesis in conjunctival malignant melanoma Paul Zimmermann, Tina Dietrich, Felix Bock, Folkert K. Horn, Carmen Hofmann-Rummelt, Friedrich E. Kruse, Claus Cursiefen 35 Verzeichnis der Vorveröffentlichungen: Zimmermann P, Dietrich T, Bock F, Horn FK, Hofmann-Rummelt C, Kruse FE, Cursiefen C. 2009. Tumor-associated lymphangiogenesis in conjunctival malignant melanoma. Accepted for online first in the British Journal of Ophthalmology on 24. April 2009. (BJOPHTHALMOL/2008/147355). Bock F, König Y, Dietrich T, Zimmermann P, Baier M, Cursiefen C. 2007. Inhibition of angiogenesis in the anterior chamber of the eye. Ophthalmologe; April; 104(4):336-44 (2007) 36 Anhang: Vergleichen Sie hierzu bitte die Dissertation: Abbildungsverzeichnis mit Legenden Seite 23-25 (Seite 16-18 Artikel) Abbildungen: Seite 26-30 (Seite 19-23 Artikel) Abbildung 1 Seite 26 (Seite 19 Artikel) Abbildung 2 Seite 27 (Seite 20 Artikel) Abbildung 3 Seite 28 (Seite 21 Artikel) Abbildung 4 Seite 29 (Seite 22 Artikel) Abbildung 5 Seite 30 (Seite 23 Artikel) 37 Danksagung: Für die unermüdliche Unterstützung bei der Erstellung meiner Dissertation in den letzten 4 Jahren danke ich zum einen meinem Doktorvater PD Dr.med. Claus Cursiefen und meiner Betreuerin Dr. med. Tina Dietrich und Dr. rer. nat. Felix Bock, die alle drei stets ein offenes Ohr für meine Probleme hatten und mich unermüdlich tatkräftig, sei es mit Rat oder Tat, unterstützten. Zum anderen danke ich meiner Familie, die mir dies alles ermöglichte, mir stets Mut zusprach und mich auf dem langen Weg durch Studium und Dissertation begleitete. Für finanzielle Unterstützung der Arbeit und des Projektes danke ich dem IZKF Erlangen (Interdisziplinäres Zentrum für klinische Forschung). 38 Curriculum vitae: Paul Zimmermann, geb. am 29.12.1981 Sohn von Rudolf Zimmermann und Gudrun Zimmermann, geb. Kern Geschwister: Felix Zimmermann, Lukas Zimmermann Kind: Paula-Greta Zimmermann, geb. 23.10.2008 1988-1992 Kreuzbergschule Münchberg September 1992 bis Juni 2001 neusprachlicher Zweig des Gymnasiums Münchberg mit den Fremdsprachen Englisch, Latein und Französisch Juni 2001 Abitur in den Leistungskursen Mathematik und Latein Juli 2001 bis März 2002 Wehrdienst bei der Bundeswehr in Roding und Weiden Beginn des Studiums der Humanmedizin Sommersemester 2002 in Erlangen März 2004 Physikum Famulaturen in der Inneren Medizin und Chirurgie im Kreiskrankenhaus Münchberg, am Lehrstuhl für Präventive und Rehabilitative Sportmedizin am OSP (Olympia Stützpunkt Bayern; Leistungsdiagnostik) bei Professor Halle in München September/Oktober 2005 Start der Promotion im Rahmen des IZKF (Interdisziplinäres Zentrum für klinische Forschung) an der Augenklinik Erlangen bei PD Dr. C. Cursiefen mit dem Titel „Tumor-associated Lymphangiogenesis in conjunctival malignant melanoma“ Posterpräsentation bei der Jahrestagung der Ophthalomologische Gesellschaft) September 2006 PJ in der Chirurgie im Lehrkrankenhaus Hof (Feb.2007-Juni2007) Posterpräsentation auf der ARVO (Association in Research in Vision and Ophthalmology) in Fort Lauderdale/Florida im Mai 2007 DOG (Deutsche 39 PJ in der Inneren Medizin im Stadtspital Waid in Zürich (Juni2007Sept.2007) PJ in der Augenklinik der Universität Erlangen-Nürnberg (Okt.2007Jan.2008) Staatsexamen Humanmedizin April/Juni 2008 Approbation Humanmedizin Juni 2008 Ab August 2008 Klinikum Bamberg Medizinische Klinik I
