京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21300号 / 医博第4389号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 小川 修, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Structured abstract Hypothesis Cancer stem cells (CSCs) are subpopulations of cells which could contribute to tumor growth, metastasis formation and chemoresistance. CSCs can be detected by surface markers assessed by immunohistochemistry methods. A typical surface marker for CSCs is CD44 (standard form). We assumed, that CD44(s) could serve as a prognostic factor and marker of chemoresistance in patients with epithelial ovarian cancer. The aim of study 1. To recruit group of patients with histologically verified epithelial ovarian carcinoma. 2. To evaluate prognostic significance of known prognostic factors in our series of patients. 3. To assess the expression of CD44 in specimens of primary tumors and specimens of implantation metastasis using immnunohistochemistry and analyze their correlation. 4. To evaluate the expression of CD44 in relation to known prognostic factors. To analyze the significance of CD44 expression evaluation for overall survival, disease-free interval and chemoresistance. To find CD44 positivity cut-off by using statistical methods Materials and Methods A retrospective study was performed on 87 patients with histologically verified EOC. All patients were tested for primary tumor specimens, 48 of them were tested with regard to both specimens of primary tumor and implantation...

Structured abstract Study objectives: Ovarian carcinoma 1/ comparison of sensitivities among monitored markers CA 125, HE4, CA 19-9, CEA, TK, TPS, MonoTotal 2/ comparison of false positivity of markers CA 125 and HE4 3/ use of CA 125, HE4 and ROMA index in the diagnostics of ovarian carcinoma 4/ use of CA 125 and HE4 in the follow-up of ovarian cancer Endometrial carcinoma 1/ feasibility of use of biomarkers CA125 and HE4 in patients with endometrial cancer in pre- operative management Study design: Retrospective data analysis Settings: Department of Obstetrics and Gynecology, Medical Faculty and Teaching Hospital in Pilsen Patients and Methods: Ovarian cancer 1/ Sensitivity of markers CA 125, HE4, CA 19-9, CEA, TK, TPS, and MonoTotal was assessed in 266 patients - 19 with ovarian cancer and 247 with benign disorders. 2/ False positivity of markers CA125 and HE4 was evaluated in a total of 390 patients with benign diagnoses - 60 women with endometriosis, 70 pregnant patients, 67 patients with ascites, 60 with pleural effusion, 25 with cardiac failure , 80 with renal insufficiency and 28 with hepatic failure. 3/ As a part of this objective we evaluated 552 patients with abnormal pelvic abnormality - 30 women had a histologically confirmed malignant ovarian tumor. Other 522 women had a benign condition. The...

Das Ovarialkarzinom weist von allen gynäkologischen Tumorerkrankungen die höchste Sterblichkeit auf. Die Therapie umfasst eine radikale Operation sowie eine adjuvante, platin-basierte Chemotherapie. Darüber hinaus kommen seit einigen Jahren zielgerichtete Therapien wie der antiangiogenetische Antikörper Bevacizumab oder PARP-Inhibitoren bei Patientinnen mit einer homologen Rekombinationsdefizienz (HRD) zum Einsatz. Trotz dieser neuen Ansätze weisen vor allem Patientinnen mit einem fortgeschrittenen Ovarialkarzinom eine schlechte Prognose auf. Für Bevacizumab konnte zwar eine Verlängerung des progressionsfreien Überlebens nachgewiesen werden, es bleibt jedoch unklar, welche Patientinnen von einer Therapie mit Bevacizumab profitieren. Die Suche nach einem prädiktiven Biomarker für das Ansprechen auf Bevacizumab war Gegenstand verschiedener Studien. Auch wenn einige vielversprechende Ansätze publiziert wurden, konnte bislang keiner dieser Ansätze in der klinischen Praxis etabliert werden. In meiner Arbeit habe ich das molekulare Ziel von Bevacizumab, VEGF‑A, untersucht. VEGF‑A besteht aus verschiedenen Isoformen mit unterschiedlichen pro- und antiangiogenetischen Eigenschaften. Das Gleichgewicht zwischen pro- und antiangiogenetischen VEGF‑A-Isoformen scheint direkt mit der angiogenetischen Aktivität eines Tumor assoziiert zu sein. Daher war mein Ziel, die klinische Relevanz der Isoform VEGF‑A165b in Hinblick auf ihre prognostische Aussagekraft sowie ihr Potenzial als prädiktiver Marker für das Ansprechen auf Bevacizumab zu untersuchen. Die VEGF‑A165b-Expression wurde hierfür mittels Immunhistochemie in formalin-fixiertem, in Paraffin eingebettetem Gewebe von 413 Patientinnen untersucht, welches in Form eines Tissue Microarrays vorlag. Die Patientinnen waren Teil der deutschen Kohorte der internationalen, multizentrischen ICON7-Studie und wurden entweder mit der Standardchemotherapie oder mit der Standardchemotherapie und zusätzlich Bevacizumab behandelt. Die Ergebnisse der Immunhistochemie wurden mittels Lichtmikroskopie beurteilt. Mittels Kaplan-Meier- sowie Cox-Analysen wurde die Assoziation zwischen der VEGF‑A165b-Expressions und dem Ansprechen auf Bevacizumab untersucht. Die Kaplan-Meier- sowie die univariate Cox-Analyse zeigte keinen Unterschied im PFS sowie im Gesamtüberleben (OS) zwischen der Gruppe der Patientinnen mit einer niedrigen und der Gruppe der Patientinnen mit einer hohen VEGF‑A165b-Expression. Dies galt sowohl für die Gesamtkohorte als auch für die beiden Therapiegruppen bei getrennter Analyse. Somit zeigte sich die VEGF‑A165b-Expression als nicht prognostisch relevant in der untersuchten Kohorte. Anschließend erfolgte eine getrennte Analyse der Patientinnen mit einer niedrigen sowie der Patientinnen mit einer hohen VEGF‑A165b-Expression. Für jede der beiden Gruppen wurde das PFS und das OS zwischen den beiden Behandlungsarmen (Standardchemotherapie mit oder ohne Bevacizumab) verglichen. Für die Gruppe der Patientinnen mit einer hohen VEGF‑A165b-Expression konnte in der Kaplan-Meier- sowie der univariaten Cox-Analyse kein signifikanter Einfluss von Bevacizumab auf das PFS (HR: 0,759; 95%KI = 0,530 – 1,089; p = 0,134) oder das OS (HR: 0,898; 95%KI = 0,597 – 1,350; p = 0,606) nachgewiesen werden. Für die Gruppe der Patientinnen mit einer niedrigen VEGF‑A165b-Expression konnte in der univariaten Cox-Analyse eine signifikante Verbesserung sowohl des PFS (HR: 0,727; 95%KI = 0,538 – 0,984; p = 0,039) als auch des OS (HR: 0,662; 95%KI = 0,458 – 0,958; p = 0,029) unter Therapie mit Bevacizumab nachgewiesen werden. In der multivariaten Cox-Analyse erwies sich dieser Effekt sowohl für das PFS (HR: 0,610; 95%KI = 0,446 – 0,834; p = 0,002) als auch für das OS (HR: 0,527; 95%KI = 0,359 – 0,775; p = 0,001) als unabhängig von den etablierten prognostischen Faktoren. Damit zeigt meine Arbeit erstmals, dass die immunhistochemisch detektierte VEGF‑A165b-Expression prädiktiv ist für ein Ansprechen auf Bevacizumab bei Patientinnen mit einem Ovarialkarzinom. Die Ergebnisse stammen aus der retrospektiven Analyse eines umfangreichen Patientinnenkollektivs, welches Teil der internationalen ICON7-Studie war, und sind von hoher klinisch-translationaler Relevanz. Da der immunhistochemische Nachweis von VEGF‑A165b leicht in die pathologische Routinediagnostik zu integrieren ist, könnte dieser neue prädiktive Biomarker bei der Entscheidung helfen, ob eine Behandlung mit Bevacizumab für am Ovarialkarzinom erkrankte Patientinnen infrage kommt. Da der aktuelle Therapiestandard für HRD-positive Patientinnen eine Kombination von Bevacizumab mit einem PARP-Inhibitor vorsieht, ist eine prospektive Validierung der Ergebnisse in einem so behandelten Kollektiv notwendig, um die Relevanz der VEGF‑A165b-Expression als prädiktiver Marker für das Ansprechen auf diese Kombinationstherapie zu evaluieren. / Among female malignancies, ovarian cancer has the highest mortality rate. Therapy comprises radical tumor debulking, followed by adjuvant platinum-based chemotherapy. In recent years, targeted treatment approaches have been integrated into the standard treatment of ovarian cancer, such as the addition of the anti-angiogenic antibody bevacizumab or the addition of PARP inhibitors in patients with homologous repair deficiency (HRD). However, most patients with advanced ovarian cancer continue to face a poor prognosis. Although an improvement in progression-free survival (PFS) has been shown for bevacizumab, it remains hard to predict which patients can profit from an addition of bevacizumab to standard chemotherapy. Several study groups have been looking for a predictive marker for bevacizumab response in ovarian cancer patients. While promising approaches have been suggested, none of these have been implemented into clinical practice. In my thesis, I focussed on the molecular target of bevacizumab, VEGF‑A, which consists of several isoforms with pro- or antiangiogenic properties. It has been proposed that the balance of pro- and antiangiogenic VEGF‑A isoforms is directly linked to the angiogenic activity of a tumor. Therefore, the objective of my thesis was to investigate the clinical relevance of the VEGF‑A165b isoform in ovarian cancer patients with regard to i) its prognostic relevance and ii) its potential to predict response to bevacizumab. Expression of VEGF‑A165b was detected by immunohistochemistry using formalin-fixed paraffin-embedded tissue from 413 patients, arranged in a tissue microarray. The patients were participants in the German contribution to the ICON7 multicenter phase III trial and were each treated with standard platinum-based chemotherapy either with or without bevacizumab. Staining results were evaluated using optical microscopy. Kaplan-Meier analysis and Cox regression analysis were performed in order to explore the association between response to bevacizumab and VEGF‑A165b expression. Kaplan-Meier and univariate Cox regression analysis did not show a difference in PFS and overall survival (OS) between the VEGF‑A165b-low- and the VEGF‑A165b-high-expressing group. This was the case for the overall cohort as well as for both treatment arm groups separately. Thus, VEGF‑A165b expression in itself did not show a prognostic relevance in the study cohort. Subsequently, patients were stratified in a VEGF‑A165b-low- and a VEGF‑A165b-high-expressing group. For each group, PFS and OS were compared between the two treatment arms: standard platinum-based chemotherapy with or without bevacizumab respectively. For VEGF‑A165b-high-expressing patients, Kaplan-Meier and univariate Cox regression analyses did not show a significant effect of bevacizumab on PFS (HR: 0.759; 95%CI = 0.530 – 1.089; p = 0.134) or OS (HR: 0.898; 95%CI = 0.597 – 1.350; p = 0.606). However, for the VEGF‑A165b-low-expressing group, univariate Cox regression analysis showed a significant improvement in PFS (HR: 0.727; 95%CI = 0.538 – 0.984; p = 0.039) and OS (HR: 0.662; 95%CI = 0.458 – 0.958; p = 0.029) under bevacizumab. This effect was independent of established risk factors for both PFS (HR: 0.610; 95%CI = 0.446 – 0.834; p = 0.002) and OS (HR: 0.527; 95%CI = 0.359 – 0.775; p = 0.001). Therefore, the results of my thesis suggest for the first time that VEGF‑A165b protein expression as detected by immunohistochemisty is predictive for response to bevacizumab treatment in ovarian cancer patients. These findings were obtained from a retrospective analysis of a comprehensive patient cohort from an international clinical trial (ICON7) and are of high clinical-translational relevance. Since VEGF‑A165b detection is possible by standard immunohistochemistry, it can be envisioned that this novel predictive biomarker may guide bevacizumab related treatment decisions in ovarian cancer patients. Given the retrospective nature of my approach, a prospective validation of my results will be imperative to determine whether VEGF‑A165b expression also predicts response to combined treatment with beacizumab and PARP inhibitors, which is the new treatment standard for HRD-positive ovarian cancer patients.

High-grade serous ovarian cancer (HGSOC) is the most aggressive subtype of ovarian cancer, and its heterogeneity poses a challenge for the discovery of reliable diagnostic biomarkers, therapeutic targets, and predicting treatment response, particularly to immunotherapy. The current standard diagnostic and treatment options are inadequate, resulting in late diagnosis and poor prognosis. To improve our understanding of the immunophenotype of tumours, potentially enhancing diagnostic and treatment capabilities, the aim of the present study was to develop a stringent workflow for studying the immune microenvironment of HGSOC tumours. We utilized publicly available single-cell RNA sequencing data and literature to identify genes enriched in certain cell types of HGSOC tumours, followed by validation using immunofluorescent-based multiplex protein profiling. A 9-plex immunofluorescence workflow was developed using the Opal™ system, and quantitative image analysis was performed to evaluate the expression of PD-L1, CD8A, FoxP3, CD163, KRT7, PDGFRB, and CD79A in large tissue sections of ovarian cancer. Each of these markers are specific to different cell types, and by staining the multiplex marker panel together with new markers with little or no literature linked to HGSOC we can gain novel insights on the immune microenvironment of HGSOC. In this project, for a proof of concept, we focused on two proteins; GZMK and SLAMF7. The optimized multiplex panel developed as part of this project will be used to identify cell-type-specific markers that may play a crucial role in the immune microenvironment of HGSOC, which could lead to better immunophenotype stratification of patients and a more optimal immunotherapy response. Moreover, the panel could also be used to study markers of less well-known immune cell types, further improving our understanding of HGSOC. Overall, this project has the potential to significantly contribute to the development of reliable diagnostic biomarkers and therapeutic targets for HGSOC, ultimately improving patient outcomes.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and urgently requires new therapies. Oncolytic viruses (OV) are a strong contender. OVs interact with immune components of the TME, which can be altered due to specific genetic mutations. The present study evaluates the impact of specific tumour mutations on the response to carboplatin, the current standard of care, and VSV∆M51, a promising OV candidate. After a study of genetically diverse models, constitutive KRas activation enhanced VSV∆M51 replication in-vitro and sensitivity in syngeneic in-vivo models. VSV∆M51 prolonged survival in syngeneic tumour- bearing mice with KRas, Trp53 and Pten mutations, including one tumour model that did not respond to carboplatin. Response to VSV∆M51 in-vivo was associated with activation of CD4+ and CD8+ T lymphocytes in the peritoneal TME. In summary, VSV∆M51-based immunotherapy has shown promise in diverse murine models of EOC bearing clinically relevant mutations.