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Background The recently identified classical and basal-like molecular subtypes of pancreatic cancer impact on overall survival (OS). However, the added value of routine subtyping in both clinical practice and randomized trials is still unclear, as most studies do not consider clinicopathological parameters. This study examined the clinical prognostic value of molecular subtyping in patients with resected pancreatic cancer. Methods Subtypes were determined on fresh-frozen resected pancreatic cancer samples from three Dutch centres using the Purity Independent Subtyping of Tumours classification. Patient, treatment, and histopathological variables were compared between subtypes. The prognostic value of subtyping in (simulated) pre- and postoperative settings was assessed using Kaplan–Meier and Cox regression analyses. Results Of 199 patients with resected pancreatic cancer, 164 (82.4 per cent) were classified as the classical and 35 (17.6 per cent) as the basal-like subtype. Patients with a basal-like subtype had worse OS (11 versus 16 months (HR 1.49, 95 per cent c.i. 1.03 to 2.15; P = 0.035)) than patients with a classical subtype. In multivariable Cox regression analysis, including only clinical variables, the basal-like subtype was a statistically significant predictor for poor OS (HR 1.61, 95 per cent c.i. 1.11 to 2.34; P = 0.013). When histopathological variables were added to this model, the prognostic value of subtyping decreased (HR 1.49, 95 per cent c.i. 1.01 to 2.19; P = 0.045). Conclusion The basal-like subtype was associated with worse OS in patients with resected pancreatic cancer. Adding molecular classification to inform on tumor biology may be used in patient stratification.

BOEKBESPREKING

Hodgkin lymphoma and testicular cancer survivors have an increased risk of developing second primary gastrointestinal malignancies. Whether the pathogenesis of the gastrointestinal malignancies in cancer survivors differs from cancer in the general population is unknown. This thesis investigated the pathogenesis of gastrointestinal malignancies in Hodgkin lymphoma and testicular cancer survivors. Furthermore, the effectiveness of colorectal cancer surveillance is investigated in these cancer survivors. Additionally, this thesis will focus on mismatch repair deficiency, as identification of Lynch syndrome could have implications for the patients and implications for treatment choice.

Melanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.In this thesis, we aim to explore the degree to which epigenetics play a role in melanoma, namely, inherited and acquired epigenetic alterations in melanoma susceptibility and development.

We set out the get a better understanding of the role of Bone Morphogenetic Protein(BMP) signalling in normal intestine and in carcinogenesis. The BMP pathway isknown to be a major player in the development of colorectal cancer (CRC). CRC isone of the leading causes of cancer-related deaths in the western world. Althoughsurvival and recurrence of CRC have improved, 5-year survival is low at only 65%(https://seer.cancer.gov – US data). Improving our understanding of the molecularpathways involved in CRC will potentially allow earlier detection, better predictionand personalized therapy. To briefly summarise the research we have done, we startedby investigating the function of BMP in the normal intestine. We then went on tostudy the role of BMP signalling in carcinogenesis, mainly the role of non canonicalBMP signalling in the development of metastasis. We ended with a focus on patientsby explaining how we can improve estimation of prognosis using expression levelsof several BMP components and how targeting the BMP pathway can be used forpersonalized treatment of patients.

A family history of breast cancer is one of the most important risk factors for the disease. Over the last decades many genetic loci associated with breast cancer risk have been discovered. In spite of this, only approximately half of the familial relative risk (FRR) for breast cancer can be explained by the currently known genetic risk factor. In this thesis we have explored the role of rare genetic variants in familial breast cancer with the help of next generation sequencing. Through this approach we have not been able to identify any novel high-risk breast cancer susceptibility alleles. Although there are likely still several extremely rare risk alleles to be discovered and the presence of high-risk alleles outside of protein-coding regions cannot be excluded, it seems presently unlikely that these will explain a substantial proportion of familial breast cancer. Both our work and that of others has suggested that most non BRCA1/2 familial breast cancer cases are likely explained by a combination of low-, and moderate-risk susceptibility alleles.

Potential relevant biomarkers can be found at different levels in tumour developmentand disease progression. This thesis is divided into three overarching parts. Colorectalcancer was studied from a population-based perspective (part I) to a molecular level,detailed as protein expression (part II) and (epi)genetics (part III), as indicated in Figure 2.In part I the use of adjuvant chemotherapy in patients with locally advanced rectalcancer, who underwent resection after preoperative (chemo)radiotherapy, wasevaluated in a meta-analysis based on individual patient data. Since four randomizedcontrolled trials individually did not end the ongoing debate about the role of adjuvantchemotherapy 14,68-70. In part II the ability by tumour cells to evade the immunerecognition was studied, especially the role of the non-classical HLA class I moleculeHLA-G was studied in detail. In part III, an epigenetic biomarker, LINE-1 methylationlevel, was studied in a dedicated stage II colon cohort. In addition, an establishedgenetic biomarker for colon cancer, MSI, was studied in a large rectal cancer cohort.

In this thesis we describe the latest developments in the field of advanced thyroid cancer. Several clinical trials with sorafenib and everolimus were performed. The relation between clinical outcome and mutational status was analyzed. Furthermore, the pharmacokinetics of everolimus in patients with advanced thyroid cancer was described.

Colorectal cancer is one of the most frequently diagnosed cancers in the Western world. Both hereditary and genetic factors play a role in its etiology. In approximately 3% of colorectal cancer cases the underlying cause is a hereditary cancer predisposition syndrome called Lynch syndrome. This thesis focuses on an important subset of Lynch syndrome patients, namely those carrying a mutation in the mismatch repair gene PMS2. Relatively little was known about PMS2-associated Lynch syndrome compared to Lynch syndrome caused by other genes. We provide evidence that PMS2 carriers should be considered a separate entity among Lynch patients. First off, PMS2 carriers have a lower penetrance for colorectal and endometrial cancer. Moreover, they are not at increased risk of other Lynch-associated cancers, such as ovarian cancer. The reason for relatively low colorectal cancer penetrance was investigated by analyzing the somatic mutation spectrum of these tumors. This indicated that PMS2 carriers may not develop cancer from so-called mismatch repair deficient crypts. Lastly the effect of lifestyle and single-nucleotide-polymorphisms (SNPs) was investigated which revealed no significant influence on colorectal cancer risk. The results of the studies described in this thesis have resulted in reconsideration of surveillance guidelines of PMS2-associated Lynch syndrome patients.

The generally poor prognosis of patients with epithelial ovarian cancer patients treated with curative intent, calls for additional treatment modalities and possible success might lie in a combination of chemotherapy and immunotherapy. This thesis focuses on the interaction of chemotherapy with the immune system and describes new combined chemo-immunotherapy treatment strategies. This thesis has explored new strategies, immune-modulation of the IL-6 pathway and a vaccine against p53, to enhance immune surveillance and to disable tumour immune evasion in ovarian cancer patients. The future challenge for immunotherapy against ovarian cancer is a tailored combinatorial approach to test the rationale of potentially synergistic therapies that can induce efficient antitumour immunity and prolong patients’ survival.

Onderzoek naar de klinische karakteristieken van patiënten met paragangliomen. Hierbij ligt de nadruk op mutaties in de SDH-genen. Onderdeel van dit research project in een nationale multicenter studie waaraan de academische centra in Nederland deelnemen, om zo alle SDHB mutatie dragers te kunnen bundelen. Zo kan de genotype-fenotype relatie van deze zeldzame mutatie worden onderzocht in een cohort van ongeveer 200 mutatiedragers. Een ander onderdeel van dit PhD research project is het onderzoeken of SDH-mutaties ook geassocieerd zijn met ándere tumoren. Het blijkt namelijk dat SDH-mutatiedragers niet alleen paragangliomen ontwikkelen, maar bijvoorbeeld ook renaalcelcarcinoom of schildklierkanker. Tevens wordt de behandeling van maligne paragangliomen en feochromocytomen met MIBG danwel chemotherapie in beeld gebracht door middel van een systematische review en meta-analyse betreffende deze twee therapieën.

Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While this complexity is an analytical challenge, it also offers a wide range of opportunities for biomarkers and treatment targets. This thesis deals with the analysis of colorectal cancer (CRC)-associated glycomic changes. Current knowledge on CRC-associated glycan changes and their biological role have been reviewed in Chapter 1. In Chapters 2, 3, and 6, we developed novel, high-end methodologies for the glycomic analysis of tissues and cell lines to be able to expand our knowledge on cancer glycomics and to overcome some limitations of current techniques. By applying these new methods, this thesis also covers the characterization of changes in glycosylation in CRC tissues as well as cell lines, thereby contributing to the understanding of CRC biology while identifying cancer-specific signatures underlying CRC development. These signatures can be further explored as potential markers to improve patient care. Additionally, in Chapter 5, we extended our research to pancreatic duct adenocarcinoma and characterized the N-glycome of PDAC cells with different metastatic potential and of a normal pancreatic duct cell line.

Both copy number losses and homozygosity of chromosome 7 are extremely rare events in many tumor types, indicating that the retention of both the maternal and paternal copies of chromosome 7 is essential for the tumor cells. This thesis compiles our research into the driving force that is behind the retention of heterozygosity on chromosome 7. The retention of heterozygosity on chromosome 7, we hypothesised, is due to the presence of a set of mono-allelically expressed genes on this chromosome, which are essential for tumor cell survival; so called cell survival genes. Loss of either copy of chromosome 7 would result in a complete loss of expression of those cell survival genes which are exclusively expressed from that particular allele. We have identified 6 imprinted cell survival genes on chromosome 7, that play a role in chromosome 7 retention in thyroid cancer. Additionally, we have analysed allele specific expression on chromosome 7 in a set of novel low passage colorectal cancer cell lines. We also report the extensive characterization, as well as the transcriptome and methylome profiling of these cell lines.

In this thesis, we studied some of the genetic and molecular alterations that accompany the tumorigenesis of mismatch repair (MMR) and MUTYH deficient colorectal cancers, with focus on immune escape mechanisms. We discovered that loss of Human Leukocyte Antigen (HLA) class I expression was more frequent in those tumors than in the remaining spectrum of colorectal cancers (CRCs), and that distinct genetic mechanisms explained HLA class I abrogation in sporadic and hereditary mismatch repair deficient tumors. We then explored a potential association between the magnitude and quality of lymphocytic infiltration with the tumors__ HLA class I phenotypes and clinicopathological stages in a cohort of Lynch CRCs. Higher lymphocytic infiltration, particularly of cytotoxic lymphocytes, was observed in cancers presenting HLA class I defects and in tumors diagnosed at earlier stages, thus, supporting the role of the immune system in selecting immune evasive tumor traits and in counteracting cancer progression. Finally, we functionally characterized one of the most common genetic alterations that occur in MMR deficient CRCs. We demonstrated that TGFbeta signalling is active in these tumors and that TGFbeta activation occurs in a TGFBR2-dependent manner, despite the presence of truncating biallelic mutations in its gene in the majority of MMR deficient tumors.

Pathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel molecular insights into alterations in normal cellular function have led to the identification of targets for innovative therapies. Testing for biomarkers combined with molecular pathology has created the potential for __personalized medicine__ and improved diagnosis, treatment and prognosis. New technologies for molecular analysis in molecular tumor diagnostics and research must be developed and implemented to keep pace with the latest insights, resulting in a constant cycle of change. Such translational research can only progress if patient material can be accessed from the archives for further study. The resulting new insights and strategies will eventually be implemented in patient care. This thesis describes three important issues in this cycle of change with a focus on molecular pathology. Tissue preparation, method development and data analysis.

This thesis describes molecular methods to distinguish separate colon tumour entities. Furthermore, it shows that distinct immune escape mechanisms, in particular distinct mechanisms of corrupting the HLA system, are operational in subsets of colon tumours. The apparent necessity of some colon tumours to circumvent the immune system might underscore the potential of immune based therapy approaches. Alternatively, it may suggest that such therapies will only lead to selection of tumour cells with HLA alterations, limiting the value of these approaches. In general, the identification of distinct tumour types to be targeted by tailor-made therapy is essential studying the success of any applied strategy.

Differentiated thyroid carcinoma (DTC) has favorable prognosis with high cure rate under treatment of thyroidectomy followed by radiotherapy. However, therapeutic efficiency decreases when dedifferentiation occurs. One of the purposes in this study is to use pharmaceuticals intervention to promote therapeutic responsiveness in thyroid carcinomas. Lithium compound and retinoid derivatives have been applied in thyroid cancer patients and in normal and thyroid cancer cell lines. The results indicate that Lithium compound doesn__t influence either iodine uptake in vivo and in vitro conditions or iodine efflux in cell lines observed. Retinoid, Bexarotene, slightly increases iodine uptake in thyroid cancer patients but does not reach the therapeutic level. In attempt to find efficient diagnostic markers, 156 patient samples including subsets of thyroid carcinomas and benign thyroid disorders have been investigated using immunohistochemistry in tissue micro-array sections. The results indicate that combination of protein expression of NIS, FN and Gal-3 may distinguish benign tumors from malignant thyroid carcinomas with high accuracy. As thyroid cancer patients have high survival rate, efficient and sensitive screening tool is crucial for follow up in DTC. In this study, thyroglobulin (Tg) of over 600 patients has been studies at unicenter with uniformed treatment. The results prove that multiple tests of Tg at fix time points may predict DTC prognosis and death.