University of Groningen Genomic heterogeneity of clear cell renal cell carcinoma Ferronika, Paranita

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University of Groningen

Genomic heterogeneity of clear cell renal cell carcinoma

Ferronika, Paranita

DOI:

10.33612/diss.101437783

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Publication date: 2019

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Ferronika, P. (2019). Genomic heterogeneity of clear cell renal cell carcinoma. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.101437783

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Introduction

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11 Introduction

1. Clear cell renal cell carcinoma (ccRCC)

1. 1. Clinical and epidemiological aspects of ccRCC

Worldwide, kidney cancer is the 14th_{most common cancer, accounting for 3% of all sporadic}

cancers [1]. According to the 2016 World Health Organisation (WHO) classification, kidney cancer is classified into subtypes based on the following characteristics: predominant cytoplasmic and architectural features, anatomic location of tumours, correlation with a specific renal disease background, underlying molecular alterations and familial predisposition syndromes [2].

Renal cell cancer accounts for 90% of all kidney cancer, and clear cell renal cell carcinoma (ccRCC), characterized typically abundant clear cytoplasm due to lipid or glycogen deposition, is its most common subtype (75%) [2, 3]. The worldwide incidence of ccRCC is >337,000 new cases/year [4]. Although there are some hereditary tumour syndromes that feature ccRCC, including von Hippel-Lindau syndrome, most ccRCCs are sporadic.

The ratio between the incidence of localized and metastatic kidney cancer is 2:1 [5]. For patients with localized kidney cancer that is diagnosed early, and treated by total or partial nephrectomy, the prognosis is quite good: the five-year survival rate is up to 90% [6]. However, metastases will still manifest during follow-up in one third of these patients [5]. For patients with metastatic disease, which is treated with systemic therapy either in combination with primary tumour removal (cytoreductive nephrectomy) or not [3], five-year survival decreases to 12% [6].

Prognostic parameters have been defined that predict the clinical outcome of kidney cancer subtypes including ccRCC. The first prognostic assessment is based on the morphological feature: tumour grade. The latest tumour grade assessment is described in the 2016 International Society of Urological Pathology (ISUP)/WHO classification. It classifies the tumour into grade 1-4 based on nucleolar grade and the presence of the extreme form of tumour dedifferentiation [7]. The presence of independent sarcomatoid or rhabdoid dedifferentiation independently decreases overall survival of the kidney cancer patients [8, 9]. Other prognostic parameters used in pathological assessment of kidney cancer are the presence of tumour necrosis, the presence of microvascular invasion, and the tumour subtype itself [10]. These pathological prognostic parameters, in combination with other clinical parameters including tumour stage and size, are used to predict the cancer-specific survival of patients [11].

1.2. Frequent somatic alterations in sporadic ccRCC

Early genomic studies revealed large copy number variations in ccRCC, with loss of one copy of the short arm of chromosome 3 being the most characteristic/prominent [12-14]. Also, according to the more recent cancer genome atlas project (TCGA) database, the chromosomal alterations in ccRCC are dominated by the loss of the short arm of chromosome 3 in close to 100% of the cases

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[15]. Other frequent chromosomal alterations include loss of 14q (47%), 9p (32%), 9q (32%) and gain of 5q (60%) [15, 16]. The loss of chr9 or 14q has been found to correlate with a higher tumour grade, the presence of metastasis, and worsened cancer-specific and recurrence-free survivals [17, 18].

As far back as 1994, biallelic inactivation [14] had been used to identify VHL as a major tumour suppressor gene in several cases of sporadic ccRCC. With several hundred tumours now analysed with high- throughput sequencing, the Catalogue of Somatic Mutations in Cancer (COSMIC) and the TCGA now give a clear overview of the mutation frequencies and significant altered pathways in ccRCC [15, 19]. With a mutation frequency of 40-50%, VHL is still the most frequently mutated gene observed in ccRCC. VHL has an important role in the regulation of the two crucial hypoxia inducible factors, HIF-1α and HIF-2α. The list of top-six mutated genes in ccRCC is completed by

PBRM1 (38%), SETD2 (12%), BAP1 (9.5%), MTOR (8%), and KDM5C (5%) [15], which all, except MTOR,

are known as chromatin-modifying tumour suppressor genes [20-23]. Remarkably, the four most frequently mutated genes –VHL, PBRM1, SETD2 and BAP1 – are all located on the short arm of chromosome 3. Thus, a single event, e.g. loss of one copy of the short arm of chromosome 3, results in hemizygosity for four tumour suppressor genes. In addition, TP53 is among the most frequently mutated genes in ccRCC with a mutation frequency of 3% [15], but this is a relatively low mutation frequency compared to other cancers. ccRCC is thus characterized by the alteration of genes involved in epigenetic regulation (e.g. chromatin modification) and cellular metabolism regulation (e.g. cellular oxygen sensing) [24].

Some mutated genes have been correlated with clinical behaviour in ccRCC. Mutations in TP53, together with BAP1, were recently correlated with worsened survival in ccRCC patients [24, 25]. Based on the same studies, SETD2-inactivating mutations were suggested to correlate with a higher recurrence rate. Other genes worth mentioning because of their clinical significance are VHL and MTOR. The availability of targeted therapies aimed at inhibiting the VHL and mTOR signalling pathways makes screening for mutations in these genes, and other genes in these pathways, important [3, 26].

1.3. Tumour evolution and metastasis development in ccRCC

The mutational events that occur in the evolution of sporadic ccRCC have been the topic of many studies. As discussed above, the major event in the development of sporadic ccRCC is 3p loss or 3p uniparental disomy (copy-neutral loss of heterozygosity), which occurs in 94% of the cases [24]. This event, along with inactivation of the remaining allele of VHL by mutation or promoter methylation or by functional inactivation of other genes encoding proteins of the VHL-elongin BC protein complex (i.e. TCEB1, TCEB2, CUL2/5 and RBX1) contributes to 92% of the ccRCC cases. This biallelic inactivation of VHL disrupts the VHL/HIF pathway, which has an important role in the regulation of energy metabolism, angiogenesis, cell proliferation and apoptosis [27]. Von Hippel-Lindau disease is characterized by a germline mutation causing inactivation of one of the VHL copies [28]. This disease is clinically classified based on the presence of either pheochromocytoma, ccRCC, or both [29]. The mean age of onset of hereditary ccRCC in Von Hippel-Lindau patients is Chapter 1

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44 years [30], which contrasts with the mean age of onset of 62 years for sporadic ccRCC in the general population [28]. In both hereditary and sporadic ccRCC, additional mutations in other genes might contribute to tumour progression [24, 31]. These mutations might be present in all population of tumour cells or in a tumour subclone, as indicated by a low number of mutant reads frequency [31].

The development of metastatic cells is initiated by clonal expansion of competent cells from the primary tumour [32]. This process might involve multiple advantageous aberrations that are gained in specific subclones at different time points during evolution [33]. The metastatic-competent cells can originate from a major clone of the primary tumour or from rare subclones that arise at a later stage [34]. These metastatic clones can progress further through clonal evolution and develop multiple subclones within the metastatic site [32].

2. Personalized Medicine and Tumour Heterogeneity

Decades of research have, above all, taught us that cancer is complex. Each type of cancer shows variable behaviour among patients with respect to tumour progression, clinical outcome and response to therapy. Many research projects, including TCGA, have reported on the large variety of gene mutations and gene expression profiles in tumours. The ENCODE project [35] added another layer of information by focusing on the role of epigenetic changes. Personalized medicine aims to use the variations in cancer genetics and epigenetics to improve therapy and its outcome for individual patients. Although the number of studies in the field of personalized medicine is growing fast, development of this area faces further challenges. Genomic variation among tumour cells is not only present among patients, but also within an individual patient, a phenomenon now commonly referred to as intratumour heterogeneity. Intratumour heterogeneity was first recognized in the 19th_{century by Rudolf Virchow and other pathologists who showed variation in}

the morphologic features within one tumour (percentage of tumour cells with particular tumour grade distributed among tumour nest) [36]. Fluorescence in-situ hybridization–based studies have shown heterogeneity among tumour cell populations of primary, recurrent and metastatic bladder cancer with respect to chromosome numbers [37]. Flow cytometry has revealed variations in the DNA index in primary tumour and lymph node metastases of individual cervical cancer patients [38]. Such intratumour heterogeneity could challenge targeted therapy because the different individual tumour cells in one patient may respond differently to specific drugs. The existence of intratumour heterogeneity was further reinforced by multiregion sampling of ten cases of ccRCC [39, 40]. Subsequently, this intratumour heterogeneity was also observed in other cancers, including breast cancer, prostate cancer, colorectal cancer, non-small cell lung cancer and liver cancer [41-45].

Recently, it was found that certain morphologic features, such as rhabdoid differentiation, that can be present in a specific area within a primary ccRCC, may be associated with the mutational profile of that particular area [46]. In a few reported cases, intratumour heterogeneity has also been identified in metastatic ccRCC. Metastatic ccRCC itself is a lethal disease that develops in

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about one third of ccRCC cases. Metastasis in ccRCC is thought to be initiated by clonal expansion of rare subclones within the primary tumour. The identification of these subclones and their genomic profile is complex. It is still not clear how one could recognize the primary tumour subclone from which the metastatic tumour evolved. The identification of genomic profiles from distinct areas of the primary and metastatic tumours can describe genomic alterations that are crucial for the development of metastases. This may help to identify the genomic events that turn primary tumour cells into metastasis-prone cells.

A novel way to interrogate intratumour heterogeneity in high resolution is based on the analysis of single cells. Single-cell sequencing on whole-genome amplified single-cell DNA has recently been applied to study intratumoural heterogeneity in primary tumours of several cancers including breast cancer, kidney cancer, bladder cancer, colon cancer, essential thrombocythemia and glioblastoma [41, 47-52]. Single-cell sequencing enables the detection of genomic alterations in minor tumour subclones that may be missed by bulk sequencing due to dilution within a background of other tumour cells and the normal admixture within a tumour.

By applying these different analyses we hope to better understand how intratumour heterogeneity evolves. Identifying the genomic alterations that characterize the major and minor clones, the early and late events, and the primary tumour and metastases might help to stratify patients in a way that allows for more effective therapy. This may positively influence the clinical outcome of each individual patient.

3. The aim and outline of this thesis

Although ccRCC has been frequently studied, survival prognosis is still generally poor for this tumour type. Some genomic alterations, together with other clinical factors, have been shown to correlate with prognosis in ccRCC; however, their potential correlation with survival prognosis varies between different studies [25, 53]. Variation among studies also exists in the mutation frequency of the identified genes. This variation is influenced by factors such as ethnic background, patient selection, tumour cell content and sequencing platform [15, 54]. The influence of patient selection and tumour cell content might reflect the presence of intertumour and intratumour heterogeneity in ccRCC and the impact of these heterogeneities on clinical studies of ccRCC. With the work described in this thesis we aimed to contribute to the knowledge of mutational intertumour and intratumour heterogeneity, including that of metastases, and to that of the associations of these gene variants with clinical features. The ultimate aim is that these data can help further improve personalised cancer treatment. In a joint Groningen–Maastricht project, we studied the mutational spectra in 250 ccRCC cases with the goal of correlating these mutational patterns with patient characteristics and disease outcome. The first preliminary data on this project are described in chapter 2. In chapter 3, we studied intratumour heterogeneity in primary ccRCC from Indonesian and Dutch patients. Our objective here was to evaluate whether or not we could use a targeted-sequencing approach to identify intratumour heterogeneity and to see if the mutation pattern correlated with the histological tumour grade across tumour regions. In Chapter 1

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chapter 4, we took a detailed look at intratumour heterogeneity and the profiles of a range of metastases and used this to reconstruct the order of mutational events leading to metastases. Single-cell sequencing is a novel approach to study intratumour heterogeneity at an even more detailed level. In preparation for such studies in ccRCC, we had the opportunity, presented in chapter 5, to perform single-cell PCR-free low-coverage whole-genome sequencing in small cell lung carcinoma, focussing on copy number alterations.

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