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Several randomized studies and meta-analyses have shown that simultaneous radio- and chemotherapy prolongs survival in patients with unresectable squamous cell carcinoma of the head and neck as compared with conventional radiotherapy. We assessed the feasibility and effectiveness of simultaneous radiotherapy (35 x 2 Gy) and chemotherapy [cisplatinum 100 mg/m(2) or carboplatin (AUC 6) on days 1, 22 and 43] in daily clinical practice in a cohort of 87 patients treated at our institute between 1998 and 2002. Eighty patients completed radiotherapy according to schedule. Eighty patients received two courses of chemotherapy and 50 patients three courses. Nephrotoxity, bone marrow suppression and ototoxicity were the most frequent side-effects. Median weight loss was 8.5%. Median survival was 15 months and 44% of the patients were alive at 2 years. Patients receiving three courses of chemotherapy had a better survival than patients receiving two or less courses. Treatment with simultaneous radio- and chemotherapy for advanced head and neck cancer is a demanding, but feasible treatment in daily clinical practice. Survival seems to be comparable with the results achieved in patients selected for clinical trials.

The research described in this thesis focuses on cancer patients’ intentions, needs and preferences with regard to receiving unsolicited genetic information obtained by tumor DNA analysis. We combine qualitative research and quantitative research methods with a normative ethical reflection on the introduction of DNA sequencing into daily practice in clinical oncology. In precision medicine, somatic and germline DNA sequencing are essential to make genome-guided treatment decisions in cancer patients. However, it can also uncover unsolicited findings (UFs) in germline DNA that could have a substantial impact on the lives of patients and their relatives. It is therefore critical to understand cancer patients’ preferences concerning UFs derived from sequencing. Cancer patients may be particularly vulnerable (physical and emotional) at the moment they have to make decisions concerning NGS and possible unsolicited findings. Understanding what a cancer patient could experience during the process of disclosing unsolicited findings from NGS will help oncologists in daily practice to accompany patients in making informed decisions In one of our qualitative studies, four interrelated themes were recognized when cancer patients were asked about their preferences, intentions, and needs concerning the return of unsolicited findings generated during a NGS procedure: cancer patients’ need to control their lives (theme 1), associated family dynamics (theme 2), and dealing with both the cognitive complexity (theme 3) and emotional complexity (theme 4) of receiving the findings. These insights with regard to the four themes contribute to a better understanding of what cancer patients consider important, and what motivates and influences them when they make decisions on the disclosure of unsolicited findings. This thesis also shows that education and counseling are vital to enable cancer patients to make informed decisions. Even for highly educated people, this topic is rather difficult to understand. Thus, to make an informed, autonomous decision with regard to the return of unsolicited findings, understandable, personalized and accessible information (written or digital) and educational material must be available to empower cancer patients and their family members. Our quantitative study showed that the vast majority of cancer patients desires to receive all UFs of genome testing, although a substantial minority does not wish to receive non-actionable findings. Importantly, the interest in learning about four different categories of UFs (actionable, non-actionable, reproductive significance, unknown significance) is equally high among curative and advanced-stage patients. However, there is also a substantial group of patients who do not want to be informed. Presenting categories of genetic test results was found to be a useful tool in enabling cancer patients to make a well-informed decision about receiving unsolicited findings from NGS A binning approach to UFs allows patients to accept actionable findings and at the same time to refuse non-actionable or uncertain findings. Binning helps a considerable minority of patients who do not wish to know everything. Especially women would benefit from differentiating between categories of UFs along these lines.

Anti-cancer treatment has come a long way, and there probably is a long way to go before we can offer every patient a therapy that works, is safe, and affordable. Many paths may be pursued to ultimately achieve this goal, and precision oncology could be one of them: by matching treatment to specific characteristics of a patient, his or her tumor, and its micro-environment, higher response rates may be achieved whilst toxicity and unnecessary costs could be avoided. This thesis discusses several biomarker-based strategies to tailor treatment to specific tumor- or patient-characteristics, ultimately aimed at improving outcomes for individual patients. Part II is dedicated to tumor micro-environment, and how it may be employed to counteract chemotherapy resistance. It describes two prospective studies, both build on the observation that (A) mesenchymal stem cells (MSCs) mediate platinum-induced chemoresistance in mice, and that (B) indomethacin prevented this type of chemoresistance. Present research has demonstrated that translation to human cancer patients is safe and probably worthwhile, given that patients with advanced cancer have higher levels of circulating MSCs; especially once they have received systemic anti-cancer treatment. Part III focusses on biomarkers for targeted- and immunotherapy. It describes the set-up and first results of the Drug Rediscovery Protocol (DRUP), a nationwide, prospective, multi-drug and pan-tumor trial aimed at identifying new tumor type/molecular variant-combinations that are sensitive to approved anti-cancer drugs. Analysis of the first ~500 clinical case submissions showed that there are many patients (>50% in our dataset) whose molecular tumor profile can potentially be targeted by an already available anti-cancer drug, to which these patients would otherwise not have access. So far, one cohort (nivolumab for patients with MSI tumors) has completed accrual: clinical benefit was observed in twenty patients (67%), including eleven patients (37%) with an objective response. Yet, outside the context of clinical trials, most of these patients do currently not have access to nivolumab treatment. Negotiations with Dutch health authorities are therefore ongoing. Ideally, this will lead to nivolumab-reimbursement under the condition that further / confirmatory data will be gathered (“coverage with evidence development”). If successful, other DRUP cohorts will hopefully follow. Part IV discusses two aspects of precision oncology in daily practice. First, the relationship between somatic mutations and treatment consequences is complex and can’t be fully captured by most currently used gene panels. Adequate interpretation of extensive tumor sequencing, on the other hand, requires expertise from clinical, pathological, biological, genetical and technical perspective. A new type of tumor board is therefore rising: the multidisciplinary genetic- or Molecular Tumor Board (MTB). Three recommendations were formulated that may serve as a roadmap for successful MTB implementation. We feel that standardization of MTB practice would not only prevent variation in quality of care, but would also help to move the emerging field of tumor sequencing forward. Second, the need for cross-disciplinary evidence development is addressed: anti-cancer treatment options now range from systemic therapies, to interventional radiology, and so forth. Evidence directly comparing across these modalities, however, is scarce.

Tailoring treatment to the unique cancer of each individual patient is important to improve patient outcomes, facilitate drug development and increase cost-effectiveness in health care. This thesis describes several research projects focused on personalized medicine in cancer. The first part of this thesis is about biomarker research. In chapter 2, we aimed to discover why a patient with a PEC-tumor had a remarkable response to treatment with the mTOR inhibitor everolimus. Whereas we expected there to be a genetic aberration somewhere in the mTOR pathway, that had resulted in its activation, no such aberration was found using either DNA sequencing or copy number analysis. Chapter 3 describes a biomarker identification study to predict benefit from treatment with everolimus. Patients with any type of advanced solid malignancy were included, underwent a pre-treatment tumor biopsy, and were treated with everolimus. Using DNA sequencing, copy number analysis and immunohistochemistry we found that mutations or deletions in PTEN could represent a tumor type agnostic biomarker for treatment benefit from everolimus (P=0.046). In Chapter 4, we describe the TTP ratio as a clinical trial endpoint to measure clinical benefit of everolimus. The TTP ratio uses an intra-patient control that corrects for natural tumor growth rate, and is especially useful to measure benefit of compounds that induce a reduction in tumor growth rate, as opposed to tumor regression. Response according to TTP ratio correlated to Overall Survival, and was also able to differentiate which patients had a longer Overall Survival within the RECIST stable disease cohort. The second part of this thesis focused on patient-derived tumor organoids. In chapter 5 we describe the characteristics of the tumor organoid culture platform and highlight all major publications in this field. In chapter 6, we describe the culture from single 18 Gauge metastatic biopsy specimens (71% success rate). More important though, is that the genetic landscape of these biopsies was preserved in culture. The few genetic aberrations that were discordant between these samples did not include any aberrations known to be of influence for drug sensitivity or tumor evolution. Chapter 7 describes an initial screen, in which treatment outcome is compared of paired in vitro and patient data for 5-FU/capecitabine + oxaliplatin chemotherapy. Whereas in vitro drug sensitivity did not correlate to clinical outcome (P=0.679), this does not imply that patient-derived tumor organoids are not able to predict treatment response of patients. It means that the assay in its current format is not yet sufficient for large scale testing of the expanded cohort. These results are also not representative for other tumor types and treatments. Chapter 8 describes the study design of the SENSOR, where treatment is selected for each individual patient with metastatic colorectal cancer on the basis of a comprehensive drug screen on their own tumor organoids. The first results demonstrate that the integration of patient-derived tumor organoids in the stratification of patients for an experimental treatment is feasible. The efficacy of this approach is currently under investigation.

This thesis focuses on various strategies to deal with cancer drug resistance and improve treatment efficacy. Technological advances have enabled researchers to gain more insight in the immense molecular complexity of cancer and mechanisms of drug resistance. The ability to measure cancer-related molecular aberrations provides an opportunity to predict response to treatment (enabling personalized cancer medicine). In addition, improved understanding of mechanisms of resistance may lead to new rationales to test drugs or innovative treatment strategies with well-established anticancer drugs. This thesis describes several aspects of the concept of personalized cancer medicine as well as several innovative strategies to delay drug resistance.

The process of cell division is fundamental to all life. From bacteria to man, any new cell can only arise by division of a preexisting cell. The adult human body is a product of trillions of cell division events and cell division remains essential in most organs to replenish cells during life. All cells except those that give rise to spermatocytes and oocytes must divide in such a way that the two arising daughter cells inherit identical copies of the genetic material encoded in the DNA. This most basic function of cell division is achieved by first accurately duplicating the DNA of the individual chromosomes and then equally segregating the copies over the two arising daughter cells. This sequence of events can be repeated as necessary and is known as the cell cycle. Damage to the DNA endangers the faithful transmission of genetic material to daughter cells, as damaged DNA cannot be correctly copied and broken fragments of chromosomes cannot be segregated. The initiation of DNA duplication and chromosome segregation are therefore blocked by the presence of damaged DNA and this block must be relieved once the DNA has been repaired. The resumnption of the cell cycle after a DNA damage-induced arrest and the molecular signals that control this process are the subject of this thesis.

Sensing the cellular environment and responding accordingly is pivotal for tissue development and homeostasis. One cellular structure that functions almost exclusively as a sensory organelle is the nearly ubiquitously present primary cilium, that has been implicated in orchestrating cellular responses to kinetic, photonic, morphogenic, olfactory and mitogenic stimuli. Dysfunction of cilia is the etiology for a large diversity of human disease phenotypes that are collectively known as ciliopathies. The severity of these disease manifestations can range from simple tissue cysts, infertility and retinal degeneration to prenatal lethal multi-organ system failure. The expression of cilia is restricted to the quiescent stage of the cell cycle as the centrosome is required for both ciliogenesis and formation of the spindles in cell division, rendering these processes mutually exclusive. Consequently, dysfunctional cilia relieve a physical block for cell cycle progression and for this reason cilia have also been implicated in the field of cancer biology for several years. Intriguingly however, it is becoming clear that cilia appear to be both required and permissive for tumorigenesis depending on the tumor subtype and primary oncogenic lesion. The scope of this thesis is to converge aspects of cilia biology with their role in cancer biology. Microtubules form the core of the cilium and serve as platforms for intracellular transport, without this transport, many cellular processes are greatly disturbed. A large family of molecular motors drive this transport and specifically in cilia, specific motors generate and maintain the axoneme. Few other molecular motors have been implicated in ciliogenesis. We describe an in vitro kinesome screen (targeting molecular motors) using an siRNA library, analyzed cilia frequencies, and identified a number of novel kinesins that perturb ciliary frequency. The kidney is a major site for the development of diseases that are associated with dysfunctional cilia, e.g. renal cysts. Several tumor suppressor proteins were previously associated with both renal cyst and cancer development, and these proteins were shown to function in sustaining ciliogenesis and cilia signaling in renal cysts. We analyzed cilia frequency in a tissue microarray of a variety of renal tumor subtypes, these results suggest that also in, both, familial and sporadic renal tumors, cilia loss is a common event that thus possibly contributes to tumorigenesis. Birt-Hogg-Dubé syndrome is a rare cancer syndrome that also features renal cyst and cancer development, suggesting a putative ciliary function for the mutated gene FLCN. Our observations indeed establish that BHD can be considered a novel atypical ciliopathy as our molecular analysis indicate a role for the FLCN tumor suppressor in ciliogenesis and ciliary signaling. Previously, our lab identified and described mutant lrrc50 zebrafish as a model for the human ciliopathy Kartagener’s syndrome. Unexpectedly, heterozygous zebrafish were found to develop testicular tumors. Characterization of these tumors suggested analogy to the human seminoma and genetic testing for mutations in human LRRC50 revealed loss-of-function mutations in both familial and sporadic samples of seminoma. To understand the role of LRRC50 (in tumorigenesis), we describe molecular aspects and functions of the LRRC50 gene product

Colorectal cancer is the second leading cause of cancer related death in Europe. The overall survival rate of patients with colorectal cancer is greatly affected by the presence of liver metastases, which occurs in about 50% of patients. Radical resection of colorectal liver metastases means a possibility of cure, resulting in a 5 year survival of 30-60%. In case of irresectable metastases, radiofrequency ablation (RFA), using heat to destroy tumour tissue, is an alternative surgical treatment option. Survival rates vary widely after surgery and it can still not be predicted accurately which patient will experience a recurrence and which not. The identification of prognostic markers are needed to select patient that possibly benefit from surgery and/or additional therapies. This thesis describes factors associated with outcome of patients undergoing surgery for colorectal liver metastases. First we discuss factors associated with recurrence after RFA. We demonstrate that the presence of viable tumour cells attached to the RFA needle applicators is associated with an increased chance of local tumour progression. This problem might be overcome by ablating the needle track. Furthermore we show that tumours close to vessels can be ablated safe and effectively using a multipolar RFA device, where previous literature demonstrated a problem effectively ablating these tumours using a monopolar device. Second, factors associated with outcome after resection are examined. Preclinical research demonstrated that the protein CD95 upon binding with its ligand activates pathways involved with a more agressive phenotype of colorectal liver metastases. Testing levels of CD95L in blood of patients demonstrated that higher levels of CD95L were associated with poor prognosis of patients with colorectal liver metastases. We used genomics and proteomics to identify more factors associated with poor prognosis of which the protein Maspin was a factor identified by both platforms. Next we used immunohistochemistry to determine Maspin expression in biopsies of primary colorectal cancer. We discovered a stage specific role for Maspin being prognostic in stage III but not in stage II primary colorectal cancer. Both Maspin and CD95L might be used in the future to select patients for surgery. Gene expression was further used to find a gene expression profile able to predict disease free survival. Unfortunately this attempt failed, possibly due to the phenomenon of ‘overfitting’. We did however discover that the profile was greatly influenced by administration of neoadjuvant chemotherapy. Neoadjuvant chemotherapy upregulates genes involved in the immune system. These genes seemed to have a tumour stimulating role, generating the question whether this genes might play a role in chemotherapy resistance. This thesis furthermore describes the Hepatica study, a randomized controlled study examining the addition of a angiogenesis inhibitor (bevacizumab) to a CAPOX regime. This study demonstrates the addition of bevacizumab is safe and leads to an improved non significant DFS. In conclusion, this thesis makes a start finding factors affecting outcome after surgery of colorectal liver metastases. Insight in the mechanisms involved with recurrence might select patients that will benefit from surgery and eventually lead to new therapeutic targets.

In addition to their direct effects on tumor cells, certain anticancer therapies elicit a prosurvival response in benign tissues in the tumor microenvironment. This host response can be seen as an attempt of the body to diminish chemotherapy-induced damage in tissues crucial for functioning. Unfortunately, this acute reaction to damage can be exploited by the tumor to reduce therapy-induced damage in cancer cells. The host response can therefore interfere with the antitumor activity of a drug, and may provide an explanation for the limited success of certain anticancer agents. Targeting the host response will likely augment current antitumor therapies. In this thesis we have utilized preclinical and translational studies to show that the host response following chemotherapy exposure consists of changes in numerous cell types and cytokines. For example, we show that chemotherapy can activate cells in the pulmonary vascular bed to create a favorable niche for tumor cell colonization, a process involving upregulation of VEGFR-1 on pulmonary endothelial cells. When chemotherapy was combined with antibodies targeting VEGFR-1, this effect was reversed and the number of lung colonies was decreased. Furthermore, platinum-containing chemotherapeutics induce a host response in mesenchymal stem cells, leading to secretion of rare fatty acids (PIFAs) that can protect tumors against a broad spectrum of chemotherapeutic drugs. Inhibitors of COX-1 and thromboxane synthase could prevent PIFA formation. Addition of either of these inhibitors to chemotherapy blocked the release of PIFAs from MSCs, ameliorating the antitumor effects of the cytotoxic agent. Interestingly, these same PIFAs are found in fish oil, which can induce resistance to chemotherapy in mouse models as well. In healthy volunteers, rapid rises in PIFA plasma levels were found after intake of the recommended daily amount of fish oil. Our findings show that fish oil might not be just an innocent food supplement during chemotherapy. Acute responses of host tissues to antitumor agents are not exclusive to chemotherapy. A similar host response takes place after administration of ‘cytotoxic-like’ vascular disrupting agents (VDAs) in mice and men. Mobilization of pro-tumorigenic bone marrow-derived cells into the circulation has been shown, which promotes angiogenesis and indirectly also tumor growth. When the mobilization of these bone marrow-derived cells was blocked by co-administration of an agent targeting VEGFR-2 present on their cell membranes, tumor regrowth was prevented, resulting in enhanced tumor control. All of these factors may interfere with the antitumor activities of the drugs. The net effect of antitumor therapy will thus be determined by both the direct antitumor effects, as well as by the counteracting host response. Based on our preclinical results, combining conventional chemotherapy with agents blocking specific aspects of the systemic host response seems promising to enhance chemotherapy efficacy. By improving our understanding of the prosurvival effects of chemotherapy on the microenvironment, we aim to provide novel strategies to optimize antitumor therapy. Therefore, this thesis is an incentive to combine conventional antitumor therapies with novel ‘targeted agents’, and to develop new drugs targeting the tumor microenvironment.