In this thesis Prognoslic factors of early recurrence of colorectal liver metastases after surgical therapy the autor characterizes the epidemidemiology, diagnostics and treatment of colorectal liver metastases (CLM) in relation to biological activity of tumour and the possibilities of determination. Contemporary the author introduces to problematics of tumour markers, which determine CLM and to clinical prognostic factors of CLM.

In this thesis Prognoslic factors of early recurrence of colorectal liver metastases after surgical therapy the autor characterizes the epidemidemiology, diagnostics and treatment of colorectal liver metastases (CLM) in relation to biological activity of tumour and the possibilities of determination. Contemporary the author introduces to problematics of tumour markers, which determine CLM and to clinical prognostic factors of CLM.

Laser thermal ablation (LTA) is an in situ ablative technique that induces heat destruction of liver tumours. Despite increasing clinical use of LTA, reports of long-term outcomes and limitation of treatment in specific cohorts of patients with liver tumours are lacking. In addition, the mechanisms of action of therapy have not been fully elucidated. This study highlights the long-term clinical results and limitations of LTA in the treatment of a cohort of patients with unresectable colorectal liver metastases and examines the mechanisms of action of thermal ablative injury in a murine model.

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality. About 50 % of patients with CRC will develop subsequent liver metastases (CLM). The survival for untreated CLM is only a few months and liver resection provides the only chance for a lasting cure. It is therefore essential to detect CLM early, enabling successful surgical resection and achieving a long-term cure. There are no optimal tumour markers for CRC or CLM. The best marker available is Carcinoembryonic Antigen (CEA), a marker found elevated in about 50-60% of patients with CLM, but also in many other conditions. The main focus of cancer research has been on the malignant cancer cell. However, a tumour consists of more than cancer cells. A major part of all solid tumours is made up by the stroma. The tumour stroma is defined as the non-malignant cells of a tumour such as fibroblasts, the cells of the vascular and immune systems as well as the extracellular matrix (ECM). The basement membrane (BM) is a specialized form of the ECM in which type IV collagen is the major protein component. All epithelial cells need a contact to the BM and the definition of an invasive cancer is the degradation of the BM and the spread of cancer cells beyond this structure. Different metastatic growth patterns of CLM have previously been described, namely the desmoplastic, pushing and replacement type of CLM. These differ in their stromal reaction in the border, which separates the tumour from the normal liver. In this thesis the tumour stroma of CRC and CLM is studied with a special emphasis on stromal collagens. The aim is to investigate whether stromal collagens/ circulating type IV collagen can be used as tumour markers for CRC and CLM, and to compare this to the conventional marker CEA. The circulating type IV collagen level is also measured in liver metastases from other primary tumours than CRC. Furthermore, the differences between the stroma of a primary CRC that metastasizes to the liver when compared to a CRC that never spreads are analysed. Additionally, the metastatic growth pattern of CLM is studied in relation to the primary tumour, stromal components and survival. We also sought out to find whether CRC cell lines possess the trait to produce ECM proteins endogenously, and in response to a normal liver stroma in a novel organotypic model for CLM. Methods: Expression patterns of type I, III and IV collagen were studied by immunofluorescence (IF), chemical staining and immunohistochemistry (IHC) in normal colorectal tissue, normal liver, CRC, CLM, benign liver lesions and in liver metastases of other origin than CRC. Circulating plasma levels of type IV collagen were analysed in healthy controls, patients with CRC (T stage I-III) and in patients with CLM. Samples were analysed at the time of diagnosis, during and after oncological and surgical treatment and at the time of relapsing or progressive disease. Additionally, circulating levels were analysed in patients with benign liver lesions and in liver metastases of other origin than CRC. The metastatic growth pattern of CLM was classified according to earlier descriptions. CRC cell lines were studied regarding their production of type IV collagen. The growth, invasiveness and stromal production in CRC cell lines were also investigated in a new organotypic model for CLM using human liver specimens. Results: Circulating type IV collagen levels are increased in patients with CLM and other epithelial-derived liver metastases, and is found normal in patients with primary CRC (stage I-III), with liver metastases from tumours of non-epithelial origin, benign liver lesions and in healthy controls. The type IV collagen levels in patients with CLM reflect the tumour burden in the liver, decreases in response to therapy and is found increased in progressive or relapsing disease. The combination of circulating type IV collagen and CEA increased the sensitivity and specificity for detecting CLM. Livermetastatic CRC displayed an increased stromal production when compared to non-metastatic CRC, with an increased type IV collagen expression in the direct vicinity of the CRC cells. The earlier described growth patterns of CLM were verified, with the pushing type of CLM associated with a short survival and poor outcome. Furthermore, CRC cell lines possess the trait of endogenously producing type IV collagen. The novel organotypic liver model revealed that CRC cell lines grown in the context of normal liver stroma, devoid of other cells, does not elicit a desmoplastic reaction. Conclusion: Circulating type IV collagen is a promising tumour marker for CLM, where the levels reflect the hepatic tumour burden and can detect disease relapse after liver surgery. The combination of the tumour markers CEA and type IV collagen is superior to CEA alone. The stromal composition of primary CRC predicts the risk of subsequent CLM and the metastatic growth pattern of CLM is related to survival.

Laser thermal ablation (LTA) is an in situ ablative technique that induces heat destruction of liver tumours. Despite increasing clinical use of LTA, reports of long-term outcomes and limitation of treatment in specific cohorts of patients with liver tumours are lacking. In addition, the mechanisms of action of therapy have not been fully elucidated. This study highlights the long-term clinical results and limitations of LTA in the treatment of a cohort of patients with unresectable colorectal liver metastases and examines the mechanisms of action of thermal ablative injury in a murine model.

Liver metastases are the most common cause of death in colorectal cancer patients. The standard of care and potential for cure for colorectal liver metastases is resection, but often times disease it too extensive for this treatment. Over the years, cancer research has made way for advances in treating progressive disease through immunotherapy. By genetically modifying an individual’s immune system using virally transduced chimeric antigen receptor T cells (CAR-T), patients are better able to receive exquisitely specific T cells to target specific tumors. Furthermore, selective delivery strategies may enhance efficacy while limiting detrimental, systemic adverse effects. Not only this, CAR-Ts have also lead to complete remission in some liquid tumors while maintaining the potential for remission in solid tumors as well. This literature review takes readers through the emergence of the different generations of CAR-T and the various studies including clinical trials that have demonstrated the safety and efficacy of CAR-T. The second portion of this paper will outline the design for a phase II clinical trial using intrahepatic CAR-T therapy in addition to selective internal radiation therapy (SIRT) for refractory CEA+ colorectal liver metastases. Benefits and limitations of using these therapies are further discussed.

Despite progress in our understanding of the molecular drivers that propagate the overall process of metastasis, the adaptation of specific organs upon these molecular interactions for metastatic entry remains poorly understood. This is particularly true for liver metastases, the liver being a common site for metastatic disease, and metastatic hepatic tumors are more prominent than primary hepatocellular or biliary tumors. Liver metastases most commonly arise from colorectal cancer than any other cancer and constitute one of the most detrimental outcomes of cancer, characterized by poor prognosis, high mortality, and no effective therapies available other than surgical interventions. Since interactions between tumour cells and the tumour microenvironment play an important part in the engraftment, survival, and progression of the metastases, the discovery of new drivers of liver metastasis with the potential to become therapeutic and preventive targets is required to advance the care of liver metastasis patients as well as cancer patients at risk of metastatic spread to the liver. The alteration of the physical structure of the tissue is extremely important in the progression of malignant diseases, such as cancer metastasis, as it directly affects the extravasation and colonization of tumour cells. The major hurdles in liver metastasis research, stem not only from our insufficient understanding of the molecular mechanisms directing and mediating metastasis particularly to the liver but also from the limited number of pre-clinical models available that mimic human disease and enable the study of the complex interactions between tumor cells and the liver microenvironment. The liver metastatic process underlies the acquisition of key adaptations by tumor-derived factors and is determined by both tumour-intrinsic properties and the crosstalk between tumour cells and stromal cells in the liver. A normal functioning and structurally intact extracellular matrix (ECM) constitute a hostile “soil” for seeding tumor cells to colonize. Eventually, it is the ability of tumor cells to remodel the liver microenvironment and create a supportive niche for metastatic tumor cell survival and outgrowth that determines successful metastatic colonization. Among tumour-secreted factors, which are recognized as major contributors to the formation of pre-metastatic and metastatic niches, tumor-derived extracellular vesicles (EVs) have recently arisen as crucial players in cell-to-cell communication and in the remodeling of distant microenvironments that favor organ-specific metastasis. Therefore, we sought to determine the role of tumor-derived EVs in the modulation of the liver microenvironment and their specific contribution to supporting metastatic colonization of the liver. The preliminary step to this process was to establish a model system to identify EV-associated targets and their effect on the ECM remodelling. Immunohistochemical analyses of primary colon tumour (CRC) and secondary liver metastases (CRC liver MET) tissue samples from patients with CRC revealed higher stromal TIMP1 levels in CRC liver MET than in CRC. The elevated stromal TIMP1 signature in the invasive front was associated with poor progression-free survival in patients with CRC liver MET. Our characterisation of the CRC tumour-derived EVs showed TIMP1 enrichment in the EVs (TIMP1EV) compared to its parental cell. Using cultures of primary liver fibroblasts, we could demonstrate that TIMP1 enrichment in the CRC-EVs was associated with regulation of TIMP1 levels in the EV-conditioned liver fibroblasts. Using our optimized ex vivo 3D ECM remodelling assay, we observed that pre-conditioning the liver fibroblasts with EVs from CRC cells promotes ECM remodelling. In accordance with our cell line model, we showed that serum-derived TIMP1EV from CRC patients promotes ECM remodelling. Moreover, high serum TIMP1EV expression in CRC liver MET patients was significantly associated with poor overall survival. In addition, our data also indicated that the determination of EV-associated TIMP1 is superior for non-invasive diagnosis than the analysis of soluble TIMP1 from total serum. Finally, we showed that HSP90AA is constitutively bound to TIMP1EV and that targeting HSP90AA leads to TIMP1 downregulation and inhibits ECM-mediated remodelling. This study defining the contribution of extravesicular TIMP1 to liver metastasis brings a novel insight into the molecular mechanisms through which tumor-secreted factors packaged via EVs promote remodelling of the liver microenvironment. The clinical significance of overexpression of extravesicular TIMP1 in patients with colorectal liver metastases highlights its potential as a prognostic biomarker and therapeutic target. With further clinical studies, Heparin and HSP90 inhibitors targeting the EV mediated TIMP1 regulation could be a putative treatment strategy to treat colorectal liver metastases.:Table of Contents Abbreviations v 1. Introduction 1 1.1 Colorectal cancer 1 1.1.1. Incidence and mortality 1 1.1.1. Tumor staging 2 1.1.1. Pattern of distant metastases in colorectal cancer 5 1.2 Colorectal liver metastases 6 1.2.1 Current evaluation and treatment strategies for colorectal liver metastases 7 1.2.2 The liver metastasis cascade - a multi-step process 10 1.3 Tumor microenvironment 12 1.3.1 Tumour-stroma interactions 15 1.3.2 ECM remodelling and its role in CRC tumor progression 17 1.4 Extracellular vesicles 21 1.4.1 EV types 21 1.4.2 Biogenesis and secretion of EVs 22 1.4.3 Molecular composition of EVs 24 1.4.4 Biological functions of EVs 26 1.4.5 EVs in Tumor microenvironment 28 1.4.6 EVs in Tumor-fibroblast communication 29 1.4.7 Role of EVs in colorectal cancer 31 1.5 Tissue inhibitor of metalloproteinases (TIMP1) 35 1.5.1 TIMP1 in cancer 37 2. Background and Research Aims 39 3. Material and Methods 40 3.1 Material 40 3.1.1 Devices 40 3.1.2 Additional material and equipment 42 3.1.3 Fine chemicals 43 3.1.4 Biochemicals 45 3.1.5 Primary antibodies 46 3.1.6 Secondary antibodies 47 3.1.7 Nucleic acids 47 3.1.8 Consumables 50 3.1.9 Softwares 51 3.2 Methods 52 3.2.1 Patients 52 3.2.2 Immunohistochemistry 52 3.2.3 Hematoxylin eosin staining 54 3.2.4 Cell lines 54 3.2.5 Primary liver fibroblast cell lines 54 3.2.6 Passaging and freezing of cells 55 3.2.7 Revival of frozen cells 55 3.2.8 Cell counting 56 3.2.9 EV Isolation from CRC cell lines 56 3.2.10 Isolation of serum-derived EVs from liquid biopsies 56 3.2.11 Characterisation of EVs 57 3.2.12 Treatment of Fibroblasts with EVs 58 3.2.13 Stimulation of PFs with recombinant TIMP1 59 3.2.14 RNA isolation 59 3.2.15 cDNA synthesis 59 3.2.16 Quantitative Real-Time PCR (qRT-PCR) 60 3.2.17 Protein quantification 61 3.2.18 Immunoblotting and co-immunoprecipitation 61 3.2.19 ELISA 62 3.2.20 TIMP1 Knock-Out (KO) and Over-Expression (OE) 62 3.2.21 17 AAG and HSP90AA antibody treatment 63 3.2.22 3D ECM-remodelling assay 63 3.2.23 PKH staining 65 3.2.24 In vivo experiments 65 3.2.25 DAPI staining 66 3.2.26 Tissue explant model 66 3.2.27 Statistical analysis and reproducibility 67 4. Results 68 4.1 Identification of TIMP1 as target molecule 68 4.1.1 Identification of TIMP1 as a target through data mining 68 4.1.2 Localization pattern of TIMP1 in CRC and CRC liver MET 70 4.1.3 Invasion front-specific overexpression of TIMP1 in the stroma of patients with CRC liver MET is associated with poor progression-free survival (PFS) 72 4.2 Model system to study CRC-EV mediated ECM remodelling 73 4.2.1 Investigating the role of CRC- derived EVs in the evolution of colorectal liver metastases 73 4.2.2 Characterizsation of isolated EVs from the CRC cell lines 74 4.2.3 TIMP1 enrichment in EVs derived from CRC cell lines 75 4.2.4 CRC-derived TIMP1EV regulates TIMP1 levels in recipient fibroblasts 76 4.2.5 TIMP1EV mediated TIMP1 upregulation in the recipient fibroblast is an EV-mediated effect 79 4.2.6 Recombinant TIMP-1 induces TIMP1 levels in recipient pFs in a time- and concentration-dependent manner 81 4.2.7 Alteration of TIMP1 levels in HCT 116 cells translates into EVs but does not affect EV packaging. 83 4.2.8 TIMP1EV levels in CRC EVs determine TIMP1 levels in recipient fibroblasts 85 4.2.9 EV-mediated TIMP1 upregulation in pFs induces ECM remodelling 86 4.2.10 TIMP1 levels in the PFs influence the extent of ECM remodelling 88 4.3 Clinical significance of TIMP1EV 89 4.3.1 TIMP1 enriched in serum-derived EVs of CRC patients compared to healthy controls 89 4.3.2 Serum derived TIMP1EV from CRC patients regulate TIMP1 levels in primary liver fibroblasts 91 4.3.3 Serum derived TIMP1EV from CRC patients promote ECM remodelling 93 4.3.4 TIMP1EV exhibits superior stratification power compared to soluble TIMP1 in liquid biopsies 93 4.3.5 TIMP1EV is a non-invasive independent prognostic marker in colorectal liver metastases 94 4.4 Targeting TIMP1EV mediated ECM remodelling 97 4.4.1 TIMP1EV binds to HSP90AA 97 4.4.2 HSP90 inhibition interferes with TIMP1 protein stabilisation 99 4.4.3 17AAG attenuates TIMP1EV-mediated ECM remodelling 101 4.5 EVs derived from murine CRC cell lines regulate TIMP1 levels in recipient fibroblasts 104 4.6 Increased homing of CRC EVs to the liver compared to other organs 106 4.7 TIMPEV regulates TIMP1 levels in liver tissues 108 5. Discussion 112 5.1 TIMP1 Localization and its significance in liver metastases 112 5.2 Model system to study the role of CRC-EVs in liver metastasis 113 5.3 In-vitro model to study the pro-metastatic effects of TIMP1EV 114 5.4 Serum-derived extravesicular TIMP1 and its pro-metastatic functions underlying remodeling of the extracellular matrix 116 5.5 Clinical significance of TIMP1EV in colorectal liver metastases 117 5.6 Scope of HSP90 inhibitors in the prevention and treatment of CRC liver metastases...……………………………………………………………………………………..118 6. Future perspectives and concluding remarks 120 7. Graphical summary of the findings 122 Zusammenfassung 123 Summary 125 List of figures 127 List of Tables 129 References 130 Acknowledgements 163 Appendix 165

La résection hépatique est le seul traitement curatif des tumeurs malignes du foie et l’insuffisance hépatique est la première cause de morbi-mortalité après hépatectomie. L’amélioration du traitement des tumeurs du foie inclut le dévelopement de statégies capables de prévenir ou de traiter cette complication. Sur une série prospective récente de 232 hépatectomies (avec 0,8 % de mortalité à 3 mois) pour métastases hépatiques de cancer colorectal (MHCCR), une insuffisance hépatique était présente dans 7 % des cas d’hépatectomies majeures et était le facteur de plus mauvais pronostic pour la survie à 2 ans. Notre objectif a été d’évaluer de nouvelles approches thérapeutiques pour leur capacité à corriger l’insuffisance hépatique post-opératoire après hépatectomie élargie pour MHCCR. Un moyen mécanique de modulation pneumatique de l’hémodynamique portale et un moyen pharmacologique d’utilisation d’un facteur de survie des hépatocytes, la protéine recombinante HIP/PAP, ont été testés respectivement chez le porc et le rat. Nous montrons qu‘après hépatectomie (PHX) de 70 % sur foie normal, l’injection systémique de protéine HIP/PAP en péri-opératoire stimulait la régénération hépatique et améliorait la fonction hépatique chez le rat. En présence de MHCCR en place depuis 7 jours, la protéine HIP/PAP n’aggravait pas la maladie métastatique, et semblait au contraire diminuer la croissance tumorale après hépatectomie. In vitro, HIP/PAP n’augmentait pas la croissance tumorale de lignées cellulaires transformées dérivées de cancer du colon. Chez le porc, une sténose portale pneumatique pendant et après PHX majeure laissant en place moins de 0,5% du poids corporel entraînait une diminution de la pression portale intra-hépatique sans modifier le débit comparé au groupe sans anneau. Cette modulation de la pression était associée à une diminution significative de la bilirubine sérique et des lésions histologiques du foie restant au 7ème jour après chirurgie. Au total, il serait possible grâce à la protéine HIP/PAP et à l’anneau portal de corriger l’insuffisance hépatique post-hépatectomie en protégeant les cellules du foie de la mort et du stress secondaires aux modifications hémodynamiques et biochimiques. La question de leur association pour diminuer l’incidence de l’insuffisance hépatique reste entière. / Liver resection is the only curative treatment of tumoral liver malignancies and post-operative liver insufficiency is the 1st cause of post-operative mortality. Tumor liver treatment improvements must be associated to innovatives methods to prevent and cure this complication. On a recent prospective study including 232 hepatectomies (with a 3-month post-operative mortality of 0.8 %) for colorectal liver metastases (CRLM), post-operative liver insufficiency was present after 7 % of major hepatectomies and was the worst 2-year survival prognostic factor. To prevent this complication, we have evaluated a perioperative systemic injection of a recombinant anti-oxydant protein (HIP/PAP) before a major hepatectomy for CRLM in rats. In vitro then in model of implanted CLRM since 7 days, we have showed that HIP/PAP did not increased tumoral growth. After 70 % hepatectomy, perioperative systemic HIP/PAP injection improved liver function. After 70 % hepatectomy, HIP/PAP seemed decrease tumoral growth of implanted CRLM. On pig, we have assessed the consequences of a portal vein stenosis with pneumatic ring during and after a major hepatectomy that conserved a liver volume < 0.5 % of body weight. With this method, we have showed that the portal stenosis decreased intra-hepatic portal pressure wihout modify the portal flow by comparison with pigs without ring. This device was associated with a significant diminution of bilirubin plasmatic concentration and liver remnant histological lesions at sacrifice on post-operative day 7. Overall, chemical and physical methods and moreover their combination should allowed to decrease post-operative liver insufficicency.

In recent years laparoscopic liver surgery established itself into today’s standard of care regarding surgical liver treatment. It was a long way for minimally invasive liver resection to develop and popularize as it was accompanied by initial reservations and concerns. Some of these already had been clarified while other questions still remain and require further investigation in the complex field of laparoscopic liver surgery. Initial concerns with respect to oncological inferiority and technical inapplicability in contrast to open surgery treatment could have been disproved within the framework of retrospective studies. In contribution to that, the aim of the study was to compare the surgical results and postoperative outcomes of consecutive laparoscopic liver resections (LLR) and open liver resections (OLR) at the high-volume liver tumor center of Leipzig university hospital. Since common classification systems for open liver surgery cannot be applied for LLR, the introduction of specific difficulty scoring systems for LLR helps to assess and classify the complexity of minimal invasive liver resection. With an increase in experience, modification of hybrid surgery and the application of novel visualization techniques such as indocyanine green (ICG) staining or hyperspectral imaging (HSI), more challenging procedures were accomplished, that initially would have been contraindicated for the laparoscopic approach (e.g. perihilar cholangiocarcinoma (pCCA) requiring biliary reconstruction). During the years 2018 and 2019 42% of all liver resections were approached laparoscopically at the Leipzig University hospital. A retrospective data analysis of n=231 patients undergoing LLR or OLR for the years 2018 and 2019 was performed and previously determined variables were collected. As a primary outcome measure, the short-term surgical and postoperative outcome of patients receiving LLR (=LLR group) compared to the patient cohort being treated by open resection (=OLR group) was evaluated. All liver resections were executed or assisted by the same two surgeons. Prior to surgery, every case was reviewed in a multidisciplinary tumor-board meeting and primarily assessed for possible minimal invasive approach. Analysis for patient demographics, pathologic diagnosis, radiologic findings and peri- and intraoperative surgical data was carried out. For LLRs intraoperatively, ICG counter perfusion staining was used in anatomic liver resection and direct ICG tumor staining was employed for tumor demarcation. With respect to classification, the extent of OLR was graded according to the Brisbane 2000 terminology in minor and major resections, whereas LLRs were categorized by means of difficulty (in accordance with Ban et al. and Di Fabio et al.). For measurement of surgical complication and assessment of morbidity, the Clavien-Dindo classification was applied. OLR was performed in n=124 (57%) and LLR in n=93 (43%). From all minimally invasive treated patients, 79% were operated totally laparoscopic and 16% were laparoscopic-hand-assisted due to infeasible lesions in the posterosuperior segments 7, 8 and 4a. In 5 cases a conversion to open surgery was necessary because of inaccessibility, tumor infiltration or morbid obesity. 28% of patients had previous upper abdominal surgery, whereof 36% in the OLR group and 19% in the LLR group. Regarding patient demographics, the mean age was significantly higher in OLR and the sex ratio was in favor of men for both groups. Malignant tumor lesions comprised 77%, while 24% were benign lesions. In both groups this larger number of malignant oncologic operation remained valid. The most common benign indications comprised focal nodular hyperplasia (FNH) and liver adenomas. It was shown that patients with CCA and Colorectal liver metastases (CRLM) were predominantly treated by open surgery, while patients with HCC diagnosis received LLR to a greater extent. Concerning the type of liver resection, non-anatomical resections were the most frequent in the cohort with 47%, thereof 55% LLR and 40% OLR. Followed second most by anatomic right and left hemihepatectomies and third most by left lateral resections, which were predominantly performed in laparoscopic technique. On the other hand, extended resections and trisectionectomies were predominantly operated by OLR. Radical lymphadenectomy was performed to a greater extent during OLR. Results showed that the mean operative time was longer for OLR (341 minutes in median) compared to LLR (273 minutes in median). Also the mean length of hospital stay was shorter for LLR patients, as well as abdominal drains were placed to lesser extent in LLR compared to OLR. In regard to R0-resection, R0-rates were higher in LLR with 98% vs. 86% in OLR. Thereby being highest for CRLM resections, followed by HCC and CCA. Putting all liver resections into classification systems, it was found that of all open procedures, 52% had major and 48% underwent minor resection according to Brisbane 2000. From the LLR group, in accordance with Di Fabio et al. 39% were classified as laparoscopic major hepatectomies, comprising 44% laparoscopic traditional major hepatectomies (LTMH) and 56% laparoscopic posterosuperior major hepatectomies (LPMH), which were technically challenging. The difficulty index stated by Ban et al. was classified as low for 8% of all performed LLRs, intermediate for 45% and of high difficulty in even 47%. Relating to morbidity (=Clavien-Dindo 3b or greater), patients with LLR had significantly lower morbidity compared to OLR. The same applies for in-hospital mortality. Our data show that despite the high number of complex and high-difficulty-classified liver resections that were performed, morbidity and mortality rates were low. As mentioned before, R0 resection rate in the LLR group was better than in the OLR group, however, this was not a case matched study, so a direct comparison is not valid. But still the study could demonstrate that the high number of LLRs being performed at the Leipzig University hospital, did not impair R0-resection rates. With an overall hospital mortality rate of 5.9% in the cohort, good results were achieved. Particularly the low rate of 1% in the LLR group speaks for itself and confirms that the development of a minimal invasive liver resection program should be on the right track. The majority of patients in the LLR and OLR group received an oncologic resection, what also resembles the global attitude that minimally invasive techniques are not reserved for selected tumor entities. Still it should be emphasized, the indication for a liver resection should not be loosened just due to minimal invasive accessibility, especially in benign liver lesions. Nevertheless, in the study the majority of benign lesions was operated by LLR. A few patients diagnosed with CCA received LLR. Thereof predominantly iCCA cases were indicated for a minimal invasive approach without biliary duct reconstruction and satisfying short-term outcomes over OLR could be obtained. However, only one case of pCCA which required Roux-Y bile duct reconstruction was treated with LLR in the study group, so if laparoscopic surgery is capable to replace the open approach in terms of treatment strategies for pCCA remains questionable. Patients with CRLM represent the centerpiece of our study population, still only 13% received LLR. The main reason of applying OLR was the high tumor load requiring future liver remnant augmentation strategies. As liver resection is confirmed to be the approach of choice for patients with HCC in cirrhosis, it is not surprising that HCC diagnosis accounted for the major part of LLRS in our collective.:Vorbemerkung und Bibliographie, 3 Abkürzungsverzeichnis, 4 Einführung, 5 - 1. Development of minimal invasive liver surgery, 5 - 2. Prior concerns of LLR, 6 - 3. Benefits of laparoscopic surgery, 6 3.1 General advantages of minimal invasive surgery, 6 3.2 Specific benefits of applying LLR, 7 - 4. Indications for LLR, 7 4.1 Benign liver lesions, 8 4.2 Malignant liver lesions, 8 4.3 Liver transplantation, 9 - 5. Technical supplement, 9 5.1 Hybrid and hand-assisted techniques, 10 - 6. Classification systems, 11 6.1 Difficulty scoring, 11 6.2 Clavien-Dindo Classification ,12 - 7. Limitations of LLR, 12 - 8. Aim of the study, 13 Publikation, 14 Zusammenfassung, 26 Literaturverzeichnis, 30 Darstellung des eignen Beitrags, 34 Selbstständigkeitserklärung, 35