ESR02 – Eirini Christodoulou – based in Leiden

Project Title: Using functional studies to explore potential high penetrance melanoma susceptibility genes identified using whole exome and genome sequencing.
PhD Award Expected: Autumn 2020

A journey in scientific research is my dream career pathway! My passion to study Biology developed when my mother was diagnosed with Breast Cancer. During my Undergraduate course at Swansea University I was fascinated by the multi-factorial and complex mode of Cancer Development and undertook my research project in Cancer Chemo-prevention. I then pursued my Master’s degree at The University of Manchester in Cancer Research where I completed two research placements enhancing my laboratory techniques and scientific critique. I also organized my own vacation work experience at Larnaca General Hospital and Karaiskakio Foundation where I became familiar with diagnosis and processing of clinical samples. A highlight of my University life is being the president of Swansea University Dance Society with more than 400 members. Our contribution to charity events was a success with our donations to different organizations and our end-of-year show being dedicated to the Cyprus Anticancer Society, a charity organization dear to me and my mother. I am a young, passionate researcher ready to undertake my PhD project in Melanoma Genetics and I am highly excited to join MELGEN training network and collaborate with fellow scientists! My future goals are to complete my PhD successfully, contribute to the success of MELGEN and discover my opportunities as a Post-Doc!

Research Summary

Cutaneous Melanoma (CM) develops from malignant transformation of melanocytes, the pigment producing cells colonizing our skin. CM is one of the deadliest types of skin cancer due to its high metastatic propensity. There is an estimated 232 100 CM cases diagnosed and about 55 500 (24%) reported deaths annually worldwide [1].

A family history of melanoma has a significant role in determining an individual’s risk of developing the disease. About 10-12% of reported CM cases occur in familial kindreds (Figure 1) therefore, familial (or hereditary) melanoma is arbitrarily defined by the clustering of at least two or more melanomas in first degree relatives [2]. Back in the 90s, scientists have revealed CDKN2A as the first gene responsible for familial clustering of melanoma [3]. A year later, a specific founder mutation has been identified in Dutch melanoma families, known as the p16-Leiden mutation [4]. CDKN2A is the most common melanoma susceptibility gene known today (Figure 1) [5]. Carriers with germline CDKN2A mutations have 70% risk of developing melanoma [6] and an additional 15-20% risk of developing pancreatic carcinoma (PC) [7]. Interestingly, clinical studies suggest variability in occurrence of melanoma and PC within families, indicating that modifying factors may contribute to the risk of developing these two tumor types in p16-Leiden carriers [7]. Determination of genetic risk factors that modulate the risk of PC in p16-Leiden families would therefore allow for a better identification of patients at increased risk that might benefit from personalized clinical management. One of the aims of this project, was to identify genetic factors, responsible for PC risk in p16-Leiden families. We investigated a variable genomic region (SNP) within a high-cancer risk locus as a modifying genetic risk factor for PC and melanoma in p16-Leiden families [8]. We did not find a significant association of PC and melanoma risk with SNP variant presence as determined by statistical analysis [8]. Therefore, genetic modifiers for the prediction of PC and melanoma in p16-Leiden carriers remain to be determined.

Figure 1: Summary of currently identified melanoma susceptibility genes. The first pie chart on the left represents two settings of CM where 90% of cases are found in the general population (sporadic) and 10% report a family history of melanoma (familial). Zooming into the familial setting, several melanoma susceptibility genes have been identified thus far with CDKN2A accounting for most families (40%) and several other genes, each responsible for about <1% of families. There is still a proportion of unknown genetic variants in occurrence of hereditary melanoma. Furthermore, there is probably also a proportion of families (20%) explained by polygenic risk factors; an effect of multiple genes but also environmental risk factors.

Moreover, even though scientific studies provide evidence for CDKN2A bi-allelic loss to be an important event in the transition to invasive melanoma in sporadic cases, there is no evidence about functional inactivation of this tumor suppressor gene in the progression stages of hereditary melanoma [9]. An additional project aim, was to investigate CDKN2A inactivation through loss-of-heterozygosity (LOH) in patients carrying a germline CDKN2A mutation. Application of sensitive digital PCR (dPCR) technology allowed for precise quantification for CDKN2A LOH in primary melanoma and common melanocytic nevi from p16-Leiden mutation carriers. Remarkably, we found CDKN2A LOH to be an early event in the development of familial melanoma.

Since the identification of CDKN2A, other genes have been reported as melanoma susceptibility genes including CDK4 [10], BAP1 [11], MITF [12], TERT [13], POT1 [14], ACD, TERF2IP [15], GOLM1 [16], EBF3 [17] and POLE [18] .These genes account for about 10% of germline variation, however, still about 50% requires investigation (Figure 1). Herein, we aimed to elucidate the genetic basis of familial melanoma and discover novel melanoma predisposition genes by application of whole exome sequencing (WES) analysis. We have identified a novel co-segregating variant, in the checkpoint regulatory gene, NEK11 (Figure 2). We showed somatic loss of the wildtype allele in the melanoma tumor of a NEK11 mutation carrier and demonstrated loss-of-function through protein instability. Therefore, our findings support NEK11 as a candidate melanoma susceptibility gene [19]. This work is absolutely essential to improve genetic testing and counselling of familial melanoma kindreds.

Figure 2: Co-segregation of NEK11 alteration in a Dutch melanoma family. Co-segregation of NEK11 alteration was confirmed by analyzing germline DNA from all family members.

In addition to susceptibility genes, a second class of genes is relevant for the biology and treatment of cancer [20]. These are fitness genes that could serve as therapeutic targets and their identification seems mandatory since relapse is a common event upon initial treatment of CM. In a collaboration with the Welcome Trust Sanger Institute (UK), we aimed to apply CRISPR-Cas9 screening technology and data to identify genetic vulnerabilities in melanoma (Figure 3). Previously reported melanoma fitness genes such as SOX10, BRAF and MITF were among the most significant hits, suggesting that results from this screen analysis are specific for melanoma phenotypes. Remarkably, genetic inhibition of novel fitness genes that are negative regulators of cell growth had a pronounced effect on decreased proliferation in cell-lines, pointing towards possible targets for melanoma therapy.

Figure 3: Schematic overview of a CRISPR-CAS9 knockout screen. The cell-lines of interest (different cancer lineage) are infected by the human library that knocks out all known genes. The endpoint of depleted and enriched genes is read using Next Generation Sequencing technology at days 0 and 14 (or 21) after infection. Those genes that are depleted compared to the initial abundance depict genes essential for cell growth whereas genes that are enriched indicate genes that may serve as possible tumor suppressors.

Collectively, through application of novel genomic techniques in this PhD project, we hope to have explored in detail the genetic mechanisms in familial and sporadic melanoma.

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Christodoulou E, Visser M, Potjer TP, van der Stoep N, Rodriguez-Girondo M, van Doorn R, Gruis N. Assessing a single SNP located at TERT/CLPTM1L multi-cancer risk region as a genetic modifier for risk of pancreatic cancer and melanoma in Dutch CDKN2A mutation carriers. Familial Cancer 2019; 18: 439-444.  Available at:

Christodoulou E, van Doorn R, Visser M, Teunisse A, Versluis M, van der Velden P, Hayward NK, Jochemsen A, Gruis N. NEK11 as a candidate high-penetrance melanoma susceptibility gene. J Med Genet. 2020 Mar;57(3):203-210. Available at: