Brain metastases (BM) have a 90% mortality rate within 4-12 months of diagnosis because they are mainly treated palliatively. The Singh Lab uses patient-derived BM samples in clinically relevant xenograft models of BM that recapitulate BM progression as seen in human patients. The objectives of my thesis were twofold: to understand whether targeting pre-metastatic cells could intercept BM formation, and whether macro-metastatic BM tumors could respond to immunotherapy as an alternative to palliative care. To intercept BM formation, I mined a Connectivity Map analysis on pre-metastatic BM gene expression signatures and identified a key regulator of the pre-metastatic process. Pharmacological and genetic perturbation of this target attenuates BMIC proliferation in vitro and slows down the formation of BM in vivo, highlighting a targetable metabolic vulnerability in BM. This work has inspired a preclinical drug development program focused on creating inhibitors with enhanced brain permeation for clinical translation. To identify whether macro-metastatic tumors respond to immunotherapy, we used a chimeric antigen receptor (CAR) T-cell to target the glycoprotein CD133, whose expression correlates with disease progression, recurrence, chemo- and radio-resistance and overall poor prognosis. We saw significant impediment in tumor progression and significant survival advantage in tumor-bearing mice after a single dose. Together, I propose that prophylaxis should be the focus of BM research moving forward and that immunotherapies should be considered where BM diagnosis precedes the chance for interception. The development of a drug that can eradicate pre-metastatic cells has the potential to prevent BM in at-risk cancer patients, while the development of a CD133-targetting immunotherapy can dramatically improve the prognosis of patients who would otherwise be limited to palliative care. / Thesis / Doctor of Philosophy (PhD) / Brain metastases have a 90% mortality rate within one year of diagnosis because they are largely untreatable. The objectives of this research were twofold: to understand whether we can prevent BM, and to understand whether developed metastatic brain tumors respond to immunotherapy. We discovered that inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH) prevents the growth of brain metastases cells. This inspired an ongoing drug development program aiming to develop IMPDH inhibitors as drugs that prevent metastatic brain cancer. To identify whether metastatic brain tumors respond to immunotherapy, we used a CAR-T cell immunotherapy to target the glycoprotein CD133 and found that tumor growth was delayed or completely blocked after a single dose. Based on these results, we propose that therapeutics that prevent the formation of brain metastases are a promising strategy to combat this deadly disease. Furthermore, we have shown that immunotherapies are a viable strategy for the treatment of brain metastases.

Tumour necrosis factor receptor associated factors (TRAFs) play a key role in signal transduction in mammalian cells. Several members of the TRAF family have been identified but only TRAF2 and TRAF6 are implicated in the regulation of both osteoclastic bone resorption and breast cancer. Here I studied the role of TRAF2 and TRAF6 in breast cancer induced osteoclastogenesis and osteolysis. I observed that TRAF2, but not TRAF6, is highly expressed in a highly metastatic bone-tropic clone of the human MDA-MB-231-BT (MDA-231-BT) breast cancer cells when compared to parental MDA-MB-231 (MDA-231) cells. Targeted knockdown of TRAF2, but not TRAF6, in both parental MDA-231 and bone-tropic MDA-231-BT breast cancer cells by siRNAs markedly reduced cell migration and significantly reduced the ability of these cells and their conditioned medium to induce osteoclast formation in RANKL stimulated bone marrow cultures. Encouraged by these data, I generated stable parental MDA-231 and bone-tropic MDA-231-BT breast cancer cell lines overexpressing TRAF2 using a retroviral approach. Then, I went on to show that overexpression of TRAF2 in parental MDA-231 cell line significantly stimulated directed cell migration and 3D invasion in vitro. Bone-tropic MDA-231-BT breast cancer cells over expressing TRAF2 or their conditioned medium were significantly effective in enhancing RANKL induced osteoclast formation in vitro. Mechanistic studies in parental MDA-231 and bone-tropic MDA-231-BT breast cancer cells revealed that over-expression of TRAF2 enhanced cell migration and osteoclastogenesis via a mechanism that involves the activation of the breast cancer oncogene IKKepsilon (IKKε) coupled with significant increase in levels of Vascular endothelial growth factor (VEGF). Ex vivo studies in human MDA-231-mouse calvaria organ co-cultures showed that conditioned medium obtained from MDA- 231 cells enhanced calvarial osteolysis. In vivo studies showed that overexpression of TRAF2 in the human breast cancer cells MDA-231 enhanced tumour incidence and tumour volume after orthotopic injection and exacerbated osteolysis after supracalvarial injection of conditioned medium from these cells. In conclusion, our studies showed that the TRAF2/IKK/VEGF axis in breast cancer cells regulates breast cancer cell motility in vitro, osteoclastogenesis in vitro and osteolysis ex vivo and in vivo. However, the role of TRAF2 in bone metastasis associated with breast cancer will require further in vivo investigation.

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.

BACKGROUND: The gold standard treatment for colorectal cancer liver metastases (CRCLM) is surgical resection. Unfortunately, the majority of patients with colorectal hepatic metastases are not candidates for resection. In recent years, several alternatives have emerged for patients whom are not resection candidates including modern systemic chemotherapy, targeted biologic treatments, regional therapies and local tumor ablation options. Microwave ablation (MWA) therapy is one such treatment alternative, based on thermal tissue ablation. This modality in concert with the most recent published literature on its use for patients with CRCLM will be reviewed in this paper. LITERATURE REVIEW FINDINGS: A structured review of the literature on ablative technologies was performed. In recent years, there has been an evolution from radiofrequency ablation (RFA) to microwave ablation therapy for the treatment of CRCLM. RFA has several limitations to its use and MWA theoretically avoids such limitations making it the currently preferable treatment option. There are limited publications comparing the use of RFA to MWA and limited publications on the use of microwave ablation for CRCLM. This paper will focus on the most recent data on MWA for CRCLM. This data can then be compared to the already published data on RFA. PROPOSED METHODS: Given the relative novel status for MWA as a treatment option for CRCLM, a potential disadvantage for its use is the perceived lack of knowledge across the medical professional spectrum. In an effort to expand the knowledge of MWA, the proposed outcomes for this study include creating a curriculum to be offered as a CME course focused for Primary Care Providers (PCPs) to provide a basis of clinical familiarity for its use. This effort will familiarize providers who may have patients diagnosed with CRCLM and also allow them to initiate the conversation about this therapy with their patients who may be candidates for this treatment. CONCLUSIONS: MWA therapy is a safe and effective treatment modality for CRCLM. Due to this new development in treating liver lesions originating from colorectal cancer, it’s imperative for providers to become familiar with these new technologies especially considering the high incidence of CRCLM. Therefore, a curriculum for PCPs will allow for a better understanding of this new technology and foster better provider-patient relationships.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24883号 / 医博第5017号 / 新制||医||1068(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 中島 貴子, 教授 石見 拓, 教授 中山 健夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No / The purpose of our study was to compare the effectiveness of 3.2 mm, 5 mm and 7.5 mm slice thicknesses in the detection and characterisation of liver lesions found on CT in patients with known or suspected malignant disease. 110 patients underwent portal phase imaging using four-slice MDCT. Two blinded observers independently read hard copy images at each slice thickness. The size and location of each lesion detected was recorded by each observer on a diagram of liver segmental anatomy. Each lesion was characterised as benign, malignant or indeterminate in nature. A diagnostic confidence score was allocated for each lesion on a scale of 1-4. The pathology or behaviour of lesions was assessed using surgery with intra-operative ultrasound (IOUS) and histology, or interval imaging with MRI, CT, or sonography. 294 lesions were detected, 64 (22%) of which were malignant. Both observers detected significantly more lesions on the 3.2 mm versus 7.5 mm slice thickness (p < 0.0001). Both observers detected more malignant lesions on 3.2 mm and 5 mm slice thicknesses versus 7.5 mm. As slice thickness decreased there was a significant increase in the sensitivity of malignant lesion detection for observer 1 (p < 0.001) and borderline significance for observer 2 (p = 0.07). As slice thickness decreased the proportion of lesions characterised as indeterminate by both observers fell. With thinner slices, both detection and characterisation of liver lesions were improved. A slice thickness no greater than 5 mm should be used to maximise both detection and correct characterisation of liver lesions.

Conclusion: Surgery should be considered whenever possible. This means that the patient has to be in good clinical condition (Karnofsky performance score > 70), the extracerebral metastases should be stable, the number of cerebral lesions should not exceed more than 3 seedings, and the age of the patient should be below 70 years. Since brain metastases are usually well circumscribed, complete extirpation seems to be possible. Postoperative MRI should be demanded in order to confirm complete extirpation. Additional radiotherapy is indicated in case of subtotal resection of a single lesion and in multiple lesions. In single brain metastasis a prospective randomized trial is necessary to prove whether conventional radiotherapy is essential after surgery in the primary treatment of the tumors or can be delayed until cerebral lesions recur. Radiosurgery is an alternative to surgery in the treatment of metastasis. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Bone metastases are a frequent clinical complication associated with cancer. The aim of this PhD thesis was to set up a model system for the study of tumour cell – bone cell interactions in vitro, ex vivo and in vivo and to use this system to test the efficacy of a novel therapeutic agent for the treatment of osteolytic bone disease. Co-culture or conditioned medium studies using human or mouse cancer cell lines were used to develop an in vitro model system of tumour cell – bone cell interactions. This showed that osteolytic tumour cells enhance osteoclast formation, fusion and resorption through the production of various factors that act directly on osteoclasts and their precursors. And in addition, that osteolytic tumour cells also enhance osteoclastogenesis indirectly via increasing the production of RANKL in osteoblasts. Other effects on osteoblasts included reductions in differentiation, migration and adhesion. Successful ex vivo and in vivo models for the study of tumour – induced osteolysis were created using adapted organ cultures and intratibial injection techniques respectively. IGF-1 and its receptor are known to play important roles in both bone metabolism and breast cancer. Therefore a study of the effects of IGF-1 receptor inhibition on tumour cell – bone cell interactions was performed. In vitro studies showed that the novel IGF-1 receptor tyrosine kinase inhibitor PQIP significantly inhibited IGF-1 and breast cancer enhanced osteoclast formation. Western blot analysis suggested this may be due to the inhibition of both IGF-1 and cancer conditioned medium induced PI3k/Akt activation. Moreover, treatment of osteoblasts with PQIP inhibited cancer cell conditioned medium induced increases in RANKL production. Ex vivo studies using human MDA-MB-231 – mouse calvarial organ co-cultures demonstrated that MDA-MB-231 cells caused osteolysis and this was completely prevented by PQIP without affecting cancer cell viability. Furthermore, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following mouse 4T1 breast cancer cell intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in breast cancer-induced osteoclast number and activity. Consistent with the significant inhibition of osteoblast differentiation, spreading, migration and bone nodule formation observed in vitro, PQIP also inhibited osteoblast number and bone formation in vivo. No inhibition of in vivo tumour volume was observed. These findings clearly suggest that oral PQIP treatment reduced the rate of cancer associated bone turnover. In conclusion, this thesis successfully demonstrates a model system for investigating tumour cell-bone cell interactions in vitro, ex vivo and in vivo. Using this model system I showed that pharmacologic inhibition of IGF-1 receptor kinase activity using PQIP inhibits osteoclast and osteoblast changes induced by breast cancer cells in vitro and in vivo and prevents osteolysis ex vivo and in vivo. This indicates that PQIP and its novel derivatives which are now in advanced clinical development may be of value in the treatment of osteolytic bone disease associated with breast cancer.