The role of DNA modifying enzymes in malignant testicular germ cell tumors

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MESTRADO EM ONCOLOGIA

ESPECIALIZAÇÃO EM ONCOLOGIA LABORATORIAL

The role of DNA modifying enzymes in

malignant testicular germ cell tumors

Rita Castro Guimarães

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2019

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IN STI TU T O D E CIÊ N C IA S B IO MÉ DI CA S A BE L S A LA Z A R

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RITA MANUELA MARQUES CASTRO GUIMARÃES

The Role of DNA Modifying Enzymes in Malignant Testicular Germ Cell Tumors Dissertação de candidatura ao grau de Mestre em Oncologia – Especialização em Oncologia Laboratorial submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto

Orientador – Professor Doutor Rui Manuel Ferreira Henrique

Professor Catedrático Convidado Departamento de Patologia e Imunologia Molecular do Instituto de Ciências Biomédicas Abel Salazar da

Universidade do Porto;

Assistente Graduado do Serviço de Anatomia Patológica do Instituto Português de Oncologia do Porto;

Investigador Sénior do Grupo de Epigenética e Biologia do Cancro no Centro de Investigação Instituto Português de Oncologia do Porto.

Coorientadora – Professora Doutora Carmen de Lurdes Fonseca Jerónimo Professora Associada Convidada com Agregação no Departamento de Patologia e Imunologia Molecular do Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto;

Investigadora Auxiliar e Coordenadora do Grupo de Epigenética e Biologia do Cancro no Centro de Investigação Instituto Português de Oncologia do Porto.

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“A dream does not become reality through magic; It takes sweat, determination and hard work”.

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AGRADECIMENTOS

A vida é feita de esforço, empenho e dedicação, neste momento chega ao fim mais uma etapa da minha vida, repleta de desafios e resiliência, mas com uma grande conquista. Contudo, nenhuma verdadeira conquista se obtém sozinho. Assim, ao longo de todo o meu percurso, só tenho a agradecer a todos por estarem a meu lado.

Desde já, o meu profundo agradecimento ao meu orientador, Professor Doutor Rui Henrique e à minha coorientadora, Professora Doutora Carmen Jerónimo por me terem dado oportunidade de fazer parte do Grupo de Epigenética e Biologia do Cancro (GEBC) do Instituto Português de Oncologia do Porto.

Ao Professor Doutor Rui Henrique por todos os conselhos, ensinamentos e dúvidas esclarecidas. Obrigada pela disponibilidade, pela paciência e pelas palavras de ânimo quando os resultados não eram os melhores.

À Professora Carmen Jerónimo, na qualidade de Diretora do Mestrado em Oncologia, muito obrigada por me ter aceitado neste Mestrado e muito obrigada pelas críticas e ideias sugeridas durante a execução da prática da dissertação.

Ao Professor Doutor Manuel Teixeira, Diretor do departamento de Genética e do Centro de Investigação do Instituto Português de Oncologia (IPO) do Porto, por ter autorizado o desenvolvimento do meu projeto no Centro de Investigação.

À Técnica Coordenadora do Serviço de Anatomia Patológica, Dra. Maria do Amparo Diegues pela disponibilidade em conciliar o meu horário de trabalho com as aulas e a prática do meu projeto.

Ao Dr. João Lobo por todo o apoio prestado, que potenciou o meu crescimento pessoal e profissional. Agradeço toda a dedicação e empenho depositados na prática deste projeto, desde o início até ao fim. Pelo seu árduo trabalho, pela sua persistência, sabedoria e conhecimento sobre os tumores de células germinativas do testículo, bem como ao nível da epigenética, muitas das minhas dúvidas, incertezas e hesitações foram esclarecidas. Foi um grande pilar na execução deste projeto. Muito obrigada por acreditares em mim.

Ao Serviço de Anatomia Patológica do IPO Porto, do qual me orgulho de fazer parte, em particular às minhas colegas Mariana Ferreira, Sofia Paulino, Isa Carneiro e Ana Teresa Martins, pelas palavras nos momentos mais difíceis e pela troca de experiências. À Paula Lopes e Renata Vieira, que com a sua experiência e conhecimentos ao nível da imunohistoquímica me ajudaram na resolução de alguns problemas na execução da técnica e me foram sugerindo várias opções, estando sempre presentes nos momentos menos bons.

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sugestões e disponibilidade para esclarecimento de dúvidas relativas à análise estatística deste estudo.

À Vera Gonçalves que me foi orientando no percurso deste projeto, que nunca se recusou a ajudar-me e a esclarecer-me quando as minhas dúvidas “existenciais” surgiam. A ela devo a minha aprendizagem a pipetar, e a orientação nos laboratórios do GEBC, esteve sempre ao meu lado quando os meus primeiros resultados começaram a surgir. Obrigada pelos teus ensinamentos, pela tua força e coragem nos momentos mais difíceis.

À Sara Reis que foi incansável. Obrigada por me teres ajudado nas otimizações dos anticorpos, nas tomadas de decisões e na execução de praticamente toda a imunohistoquímica. Nunca desististe de mim, da minha capacidade prática e nunca te recusaste a ajudar-me mesmo quando as minhas decisões não eram as melhores e falhavam de seguida

Às minhas colegas do GEBC: à Rita Oliveira por estar ao mau lado e me dar “uma mão” na execução de uma parte da imunhistoquímica e pelo apoio nas aulas e no estudo de algumas Unidades Curriculares. À Cláudia Lima por estar presente e sempre que precisava de ajuda na utilização do material dos laboratórios do GEBC nunca se recusou a prestar.

Aos restantes membros do GEBC, que nunca me viraram as costas e me orientavam quando me sentia “perdida” nos laboratórios.

Aos meus colegas do Mestrado em Oncologia, que nunca se esqueceram de mim quando precisávamos de tomar decisões ou de esclarecimentos de algo relacionado com a Dissertação.

Ao Miguel por estar sempre a meu lado. Pelo tempo dispensado, pela paciência que teve quando eu chegava a casa e os resultados obtidos não eram os melhores. Esteve sempre pronto a animar-me, a dar-me uma palavra amiga e incentivar-me a nunca desistir.

À Debora e ao Vítor Hugo, “os do costume” por me ouvirem e tentarem acalmar-me nos momentos de maior stress e por estarem sempre prontos a ajudar-me a ultrapassar os obstáculos encontrados.

E por último, mas não menos importante, aos meus pais, por nunca desistirem de mim e me incentivarem a correr sempre atrás dos meus sonhos. À minha irmã e ao meu cunhado que estão sempre presentes em todas as etapas da minha vida. Obrigado por fazerem parte de mim.

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RESUMO

INTRODUÇÃO: Os tumores de células germinativas do testículo (TGCT) são uma família complexa de neoplasias, afectando maioritariamente os jovens adultos caucasianos e representam 1% de todos os tumores malignos no sexo masculino. Estão divididos em dois grupos principais: os tumores relacionados com a neoplasia de células germinativas in situ (GCNIS) e os não derivados desta lesão. Os tumores relacionados com GCNIS incluem dois grandes grupos: os seminomas (SE) e os tumores não seminomatosos (NS). Estes últimos, podem ainda dividir-se em subtipos principais: carcinoma embrionário (CE), coriocarcinoma (CC), tumor do saco vitelino (SV) do tipo pós-pubertário e teratoma (TE) do tipo pós-pubertário. Todos estes subtipos podem ainda surgir combinados entre si (e mesmo com o subtipo SE), constituindo um tumor de células germinativas misto. Esta heterogeneidade morfológica e clínica não parece ser explicada por alterações genéticas apenas (dado o número muito reduzido de mutações nestes tumores e a presença de uma anomalia citogenética universal – o ganho do cromossoma 12p), o que leva à procura de alterações epigenéticas que possam estar envolvidas nesta heterogeneidade. Assim, diferentes padrões de metilação e a melhor compreensão destes fenómenos pode ser promissor para auxiliar na distinção entre diferentes tipos de TGCTs, no diagnóstico precoce, e num seguimento e terapêutica mais adequados.

OBJECTIVOS: O objetivo deste projeto é avaliar os níveis de expressão (a nível do transcrito e proteico) de três enzimas (DNMT3A, DNMT3B e TET2) em tecido tumoral dos diferentes tipos de TGCT e em linhas celulares representativas das várias entidades. Posteriormente, pretende-se avaliar a possibilidade de associação da expressão destas três enzimas com parâmetros clinico-patológicos.

MATERIAIS E MÉTODOS: Numa primeira fase, extraímos o RNA e procedemos à síntese de cDNA. A expressão relativa das três enzimas foi determinada por Real-time quantitative

Polymerase Chain Reaction (RT-qPCR). A expressão proteica destas enzimas foi

determinada através de imunohistoquímica. Quatro linhas celulares foram também estudadas, representativas dos diferentes subtipos tumorais. As amostras de tecido tumoral corresponderam a tecidos fixados em formol e posteriormente parafinados, provenientes de uma coorte de 119 doentes com TGCTs, bem caracterizados clínica e histopatologicamente.

RESULTADOS: Globalmente, verificaram-se diferenças significativas nos níveis de expressão das três enzimas entre os grandes subtipos SE e NS (a nível do transcrito e proteico) e entre as várias linhas celulares. A expressão de DNMT3A foi significativamente maior nos NS em comparação com SE, e este achado foi confirmado por imunohistoquímica. A expressão desta enzima foi significativamente superior nos SE

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comparativamente com EC, mostrando diferenças entre estes dois importantes subtipos, com agressividade distinta. Observou-se uma maior expressão relativa de DNMT3B nos SE em comparação com NS, ao contrário do reportado na literatura. Contudo, comparando SE com EC, a expressão foi significativamente superior no EC. A expressão de TET2 foi significativamente superior nos SE em comparação com NS, enquanto grupo, e individualmente, comparando com EC. Não se verificaram associações significativas entre a expressão destas enzimas e o estadio da doença.

CONCLUSÕES E PERSPECTIVAS FUTURAS: A expressão das enzimas epigenéticas que estabelecem e removem metilação do DNA varia consoante o subtipo tumoral. A maior expressão da desmetiláse TET2 nos SE é compatível com o seu genoma francamente hipometilado, como descrito. Já a maior expressão da metiltransferase DNMT3A nos NS também está de acordo com maior metilação global encontrada nestes tumores. Um melhor conhecimento da expressão destas enzimas epigenéticas nos TGCT pode permitir melhorar a terapêutica destes doentes, dado existirem inibidores específicos das mesmas, alguns já utilizados na Clínica.

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vii ABSTRACT

INTRODUCTION: Testicular germ cell tumors (TGCT) are a complex family of neoplasms, mostly affecting young Caucasian adults and represent 1% of all malignant tumors in males. They are divided into two main groups: tumors related to germ cell neoplasia in situ (GCNIS) and those unrelated to it. GCNIS-related TGCTs are the most common and include two major groups: seminomas (SE) and nonseminomatous tumors (NST). The latter can be further divided into major subtypes: embryonic carcinoma (EC), choriocarcinoma (CH), postpubertal type yolk-sac tumor (YST) and postpubertal type teratoma tumor (TE). All of those subtypes can still be combined with each other (even with the SE subtype), resulting in a mixed germ cell tumor. This morphological and clinical heterogeneity does not seem to be explained by genetic changes alone (given the very small number of mutations in these

tumors and the presence of universal cytogenetic abnormality – the emergence of

chromosome 12p), which leads to the search for epigenetic changes that may be involved in this heterogeneity. Thus, different methylation patterns and better understanding of these phenomena may be promising to assist in discriminating among different types of TGCTs, in allowing for early diagnosis, appropriate follow-up and therapy.

OBJECTIVES: The aim of this project is to evaluate the expression levels (at a transcript and protein level) of three enzymes (DNMT3A, DNMT3B and TET2) in tumor tissue of the different types of TGCTs and in representative cell lines of the various entities. Subsequently, we intend to evaluate the possibility of associating the expression of these three enzymes to clinical-pathological parameters.

MATERIALS AND METHODS: Firstly, we extracted RNA and then we synthesized cDNA. The relative expression of the three enzymes was determined by Real-time quantitative

Polymerase Chain Reaction (RT-qPCR). The protein expression of these enzymes was

determined by immunohistochemistry. Four cell lines were also assessed, representing the different tumor subtypes. Tumor tissue samples corresponded to formalin-fixed and later paraffin-embedded tissues from a cohort of 119 TGCT patients, clinically and histopathologically well characterized.

RESULTS: Globally, significant differences in expression levels of the three enzymes were found between SE and NST subtypes (at the transcript and protein levels) and among the various cell lines. DNMT3A expression was significantly higher in NST compared to SE, and this finding was confirmed by immunohistochemistry. The expression of this enzyme was significantly increased in SE compared to EC, disclosing differences between these two important subtypes, with distinct aggressiveness. There was higher relative expression of DNMT3B in SE compared to NST, contrarily to previous publications. However, when comparing SE to EC, the expression was significantly higher in EC. TET2 expression was significantly overexpression in SE compared to NST as a group and individually compared

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to EC. There were no significant associations between the expression of these enzymes and disease stage.

FUTURE CONCLUSIONS AND PERSPECTIVES: The expression of epigenetic enzymes that establish and remove DNA methylation varies depending on the tumor subtype. Higher expression of TET2 demethylase in SE is compatible with its hypomethylated genome, as described previously. The higher expression of DNMT3A methyltransferase in NST is also related to the higher overall methylation found in these tumors. A better understanding of the expression of these epigenetic enzymes in TGCT may improve the therapy of these patients, as there are specific inhibitors of these enzymes, some already used in the Clinics.

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FIGURE INDEX

Figure 1: Worldwide incidence of testicular germ cell tumors in 2018 (From Globocan 2018). ... 2 Figure 2: Worldwide mortality of testicular germ cell in 2018 (From Globocan 2018). ... 3 Figure 3: Differentiative relations between TGCTs (adapted from Chieffi, P. [1]). ... 6 Figure 4: DNA methylation in cells. When there is transfer of a carbon group assigned by SAM to give 5'-carbon cytosine, the CpG islands are methylated and there is no gene transcription. It is a mechanism mediated by DNMTs. ...19 Figure 5: DNA demethylation in cells. When the methyl group (added a hydroxyl group) is removed from the 5'-carbon cytosine, the CpG islands become unmethylated and there is transcription of the gene. It is a mechanism mediated by TET proteins. ...19 Figure 6: Representative graph of mRNA expression in representative cell lines of TGCTs. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Ordinary one-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001). ...32 Figure 7: Representative graph of mRNA expression in SE and NST of TGCTs. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U Test, ****p<0.0001). ...33 Figure 8: Representative graph of mRNA expression in Pure SE and NST. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U Test, ***p<0.001). ...34 Figure 9: Representative graph of mRNA expression in Pure SE and SE in the context of mixed tumors. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U Test). ...35 Figure 10: Representative graph of mRNA expression in subtypes of TGCTs. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U test, **p<0.01; ****p<0.0001, all p-values are adjusted to multiple comparisons). ...36 Figure 11: Representative graph of mRNA expression in SE and EC. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U Test, *p<0.05; ***p<0.001, ****p<0.0001)...37 Figure 12: Representative graph of mRNA expression in stage I and II/III. (A) DNMT3A, (B) DNMT3B, (C) TET2 (Mann-Whitney U Test) ...37 Figure 13: Representative graph and immunostaining of protein expression in SE and NST. (A) Immunoexpression intensity of DNMT3A, (B) Percentage of stained cells in DNMT3A, (C) Strong DNMT3A nuclear immunoexpression in SE (40x), (D) Strong DNMT3A nuclear immunoexpression in CH (40x), (E) Immunoexpression intensity of DNMT3B, (F) Percentage of stained cells in DNMT3B, (G) Strong DNMT3B nuclear immunoexpression in SE (40x), (H) Strong DNMT3B nuclear immunoexpression in EC (40x), (I) Immunoexpression intensity of TET2, (J) Percentage of stained cells in TET2, (K) Strong

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TET2 nuclear immunoexpression in SE (40x), (L) Strong TET2 nuclear immunoexpression in YST (40x), (Chi-square test, *p<0.05, **p<0.01, ***p<0.001). ...40 Figure 14: Representative graph of protein expression in pure SE and NST. (A) Immunoexpression intensity of DNMT3A, (B) Percentage of stained cells in DNMT3A, (C) Immunoexpression intensity of DNMT3B, (D) Percentage of stained cells in DNMT3B, (E) Immunoexpression intensity of TET2, (F) Percentage of stained cells in TET2 (Chi-square test, *p<0.05, ***p<0.001). ...41 Figure 15: Representative graph of protein expression in pure SE and SE in the context of mixed tumors. (A) Immunoexpression intensity of DNMT3A, (B) Percentage of stained cells in DNMT3A, (C) Immunoexpression intensity of DNMT3B, (D) Percentage of stained cells in DNMT3B, (E) Immunoexpression intensity of TET2, (F) Percentage of stained cells in TET2 (Chi-square test, *p<0.05, ***p<0.001). ...42 Figure 16: Representative graph of protein expression in subtypes of TGCT. (A) Immunoexpression intensity of DNMT3A, (B) Percentage of stained cells in DNMT3A, (C) Immunoexpression intensity of DNMT3B, (D) Percentage of stained cells in DNMT3B, (E) Immunoexpression intensity of TET2, (F) Percentage of stained cells in TET2 (Chi-square test; *p<0.05, **p<0.01. All p-values are adjusted to multiple comparisons). ...44 Figure 17: Representative graph of protein expression in SE and EC. (A) Immunoexpression intensity of DNMT3A, (B) Percentage of stained cells in DNMT3A, (C) Immunoexpression intensity of DNMT3B, (D) Percentage of stained cells in DNMT3B, (E) Immunoexpression intensity of TET2, (F) Percentage of stained cells in TET2 (Chi-square test, **p<0.01, ***p<0.001) ...45

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TABLE INDEX

Table 1: Classification of testicular germ cell tumors, according to WHO 2016 [2]. ... 4 Table 2: Clinical TNM classification of testicular germ cell tumors (adapted from [2, 61, 63] ). ...10 Table 3: Pathological TNM classification of testicular germ cell tumors (adapted from [2, 61, 63]). ...11 Table 4: Staging grouping of testicular germ cell tumors (adapted from [2, 61, 63]). ...12 Table 5: Prognostic-based staging system for metastatic germ cell cancer (International Germ Cell Cancer) (adapted from [61, 66, 67]). ...13 Table 6: Summary of the different therapeutic schemes for treatment of testicular germ cell tumors (adapted from [61, 66, 67]). ...14 Table 7: Cell lines of (testicular) germ cells tumors and their general characteristics (adapted from [77, 78, 80-82]). ...16 Table 8: DNMTs e TETs enzymes, biological function, own alteration and alteration on the disease (adapted from [3, 116, 117, 126, 127, 142]). ...22 Table 9: Summary of most relevant publications regarding the role of DNA modifying enzymes involved in testicular germ cell tumors (adapted from [3]). ...23 Table 10: Clinical and pathological characteristics of TGCT patients included in this study. ...31 Table 11: Mann-Whitney U test corrected for multiple comparisons (Bonferroni´s method) of DNMT3A, DNMT3B and TET2 among different cells lines. ...33 Table 12: Mann-Whitney U test analysis of DNMT3A, DNMT3B and TET2 in different subtypes combinations of TGCTs. All p-values are adjusted to multiple comparisons. ... iii Table 13: Qui-square and Fisher’s exact test analysis from immunoexpression intensity of DNMT3A, DNMT3B and TET2 in subtypes of TGCT. All p-values are adjusted to multiple comparisons. ... iv Table 14: Qui-square and Fisher’s exact test analysis from percentage of stained cell of DNMT3A, DNMT3B and TET2 in subtypes of TGCT. All p-values are adjusted to multiple comparisons. ... iv

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LIST OF ABREVIATONS

μg Microgram μL Microliter µm Micrometer 5mC 5-methylcytosine ºC Celsius degree % Percentage

ADCA-DN Autosomal dominant cerebellar ataxia, deafness, and

narcolepsy

AFP Alpha-fetoprotein

AITL Angioimmunoblastic T-cell lymphoma

AJCC American Joint Committee on Cancer

AML Acute myeloid leukemia

AP-2ϒ Transcription factor activator protein 2ϒ

AZFc Azoospermia factor c

BEP Cisplatin, etoposide; bleomycin

C Cytosine

cDNA Complementary DNA

CH Choriocarcinoma

CH3 Methyl group

cm Centimeter

CMML Chronic myelomonocytic leukemia

CpG Cytosine-phosphate-Guanine

CRIPTO (TDGF1) Teratocarcinoma-derived growth factor 1

CT Computerized tomography

DNA Deoxyribonucleic acid

DNMT DNA methyltransferases

EC Embryonal carcinoma

EP Etoposide, cisplatin

FFPE Fixed in formalin and embedded in paraffin

G Guanine

GATA3 GATA binding protein 3

GCNIS Tumor related to germ cell neoplasia in situ

GEBC Epigenetic group and cancer biology

H&E Hematoxylin and Eosin

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i12p Isochromosome 12p

ICF Immunodeficiency, centromeric instability, and facial

dysmorphism

IGCCCG International Collaborative Group on Germ Cell

Cancer

GCT Germ cells tumor

IGD In greatest dimension

IPO PORTO Portuguese Oncology Institute of Porto

IU/ml International unit/mililiter

KIT KIT proto-oncogene receptor tyrosine kinase

LDH Lactate dehydrogenase

LIN28 Protein humane LIN28

lncRNA Long non-coding RNA

M Distant metastases

MBD Methylation binding domains

MDS Myelodysplastic syndromes

mg Milligram

MGCT Mixed germ cell tumors

miRNA MicroRNA

ml Milliliter

MPN Myeloproliferative neoplasms

MRI Magnetic resonance imaging

mRNA Messenger RNA

N Regional lymph nodes

ng Nanogram

NANOG Nanog homeobox

ncRNA Non-coding proteins

NPVM Non-pulmonary visceral metastases

NST Non-seminomatous tumors

PDE11A Phosphodiesterase 11A

PET Positron emission tomography

PFS Progression free survival

PLAP placental alkaline phosphatase

POU5F1 (OCT3/4) Pou class 5 homeobox 1

RNA Ribonucleic acid

RPLND Retroperitoneal lymph node dissection

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S Serum markers

SALL4 Spalt like transcription factor 4

SAM S-adenosyl-L-methionine

SE Seminoma

sncRNA Small non-coding RNA

SOX2 SRY-box 2

SOX17 SRY-box 17

T Primary tumor

T-ALL T-cell acute lymphoblastic leukemia

TDS Testicular dysgenesis syndrome

TE Postpubertal-type teratoma

TET ten-eleven translocation proteins

TGCT Testicular germ cells tumor

TSG Tumor suppressor gene

UICC Union for Cancer Control

ULN Upper limit of normal

US Ultrasonography

VIP Etoposide, cisplatin, ifosfamide

WHO World Health Organization

YST Postpubertal-type yolk-sac tumor

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1. TESTICULAR GERM CELL TUMORS

Testicular germ cells tumors (TGCTs) are a very complex tumor model and comprise a very heterogeneous group of tumors, which makes the diagnosis, therapeutics and prognosis establishment difficult. Over the years, several TGCT classification systems have emerged. However, in the most recent edition of World Health Organization (WHO) in 2016, it was approved that TGCT subtypes are categorized into two major groups: tumors related to germ cell neoplasia in situ (GCNIS) and the unrelated ones [2]. Importantly, tumors not derived from GCNIS are very rare and thus the epidemiological approach and risk factors mainly cover GCNIS-related cancers, as these constitute more than 95% of testicular neoplasms [2, 3].

1.1. Epidemiology

TGCTs are uncommon tumors as they constitute only 1% of male cancers worldwide. However, they are the most frequent in young Caucasian men aged between 15 and 44 years in developed countries [4-6]. Worldwide, there is a geographic variation of the incidence of these neoplasms depending on ethnicities and lifestyles. Thus, there is an increase in incidence in more developed countries with an industrialized lifestyle, such as Europe and North America, and a decrease in incidence in less developed countries with black ethnic groups (Figure 1) [5].

Regarding mortality, the TGCTs have a high rate of cure [5, 7]. However, there is still an appreciable mortality rate in the least developed countries due to the several genetic, social and environmental factors (Figure 2).

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1.2. Risk Factors

Currently, there are several risk factors that can contribute to TGCTs, mainly at the genetic and environmental levels – comprising the so-called “environmental model” of this disease [2, 8, 9]. We can name the following according to WHO:

- Personal history: It was described that patients with TGCTs in one testis have a

25-fold increased risk of developing a contralateral tumor, with bilateral germ cell tumors comprising 2% of all testis tumors [10].

- Cryptorchidism: History of an undescended testicle or cryptorchidism increases

the risk of developing a testis tumor by up to 2 to 8-fold if left surgically uncorrected or if corrected only after puberty [11].

- Family history: It represents one of the strongest known risk factors for TGCTs,

as proven by Hemminki et al., in which the overall familial risks were 3.78 and 8.78 for son-father and brothers’ relations, respectively [12-14].

- Perinatal factors: Maternal bleeding, birth order, number of siblings, inguinal

hernia, twinning, low birth weight have all been associated with TGCTs [8-10, 15].

- Environmental factors: The main substances with a potential association with

testicular cancer include diethylstilbestrol exposure in utero, organochlorines pesticides exposure, polychlorinated biphenyls, polyvinyl chlorides and phthalates; firefighting, aircraft maintenance, leather and metal workers are also in risk. Maternal smoking and marijuana intake have also been associated with increased risk of TGCTs development [10, 15].

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- Genetic susceptibility: Part of genetic susceptibility can be explained by the

presence of the gr / gr microdeletion (removal of part of the AZFc region) on the Y chromosome [16]. This is due to the presence of a low penetrance susceptibility allele, visible in cases of male infertility and which increases the risk of developing sporadic or familial TGCTs in 2 to 3-fold [17, 18]. We can also verify mutations in the PDE11A gene, as reported by Horvath et al., in 19% of the families studied. Somatic genetic alterations have been reported in TGCTs such as activating mutations in KIT and KRAS oncogenes and pathognomonic gain of chromosome 12p as isochromosome 12p (i12p) [19-23].

Nevertheless, other risk factors have been described, namely infertility; subfertile patients have an increased risk of TGCTs in 1.6-2.8-fold, as do patients with testicular microlithiasis [24].

Overall, it is a combination of both genetic and environmental factors that results in the so-called testicular dysgenesis syndrome (TDS) [9, 25, 26]. TDS increases the risk of TGCTs and comprises, among others, cryptorchidism, testicular atrophy and inguinal hernia [25, 26].

1.3. Testicular Germ Cell Tumors Subtypes

As mentioned above, TGCTs are divided into two major groups depending on their tumorigenesis: those related and those unrelated to GCNIS [2]. Within these groups we can subdivide the TGCTs as demonstrated in table 1.

Table 1: Classification of testicular germ cell tumors, according to WHO 2016 [2].

Classification Tumor Subtype

Testicular germ cells tumors related to germ cells neoplasia in situ

Non-invasive germ cell neoplasia

Germ cell neoplasia in situ (GCNIS)

Specific forms of intratubular germ cell neoplasia

Invasive postpubertal type TGCTs

Seminoma

Nonseminomatous tumor: Embryonic Carcinoma Nonseminomatous tumor: Yolk-sac tumor Nonseminomatous tumor: Choriocarcinoma Nonseminomatous tumor: Teratoma

Nonseminomatous tumor: Mixed germ cell tumors Testicular germ cells

tumors unrelated to germ cells neoplasia in

situ

Prepubertal-type TGCTs

Yolk-sac tumor Teratoma

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1.3.1. Testicular germ cell tumors related to germ cell

neoplasia in situ

These represent the largest part of TGCTs and may be subdivided into two biologically and clinically diverse groups: seminomas (SEs) and nonseminomatous tumors (NSTs) [15]. NSTs can be further subdivided according to their differentiation into stem-cell like Embryonal Carcinoma (EC); postpubertal type yolk-sac tumor (YST) and Choriocarcinoma (CH), both representing the extra-embryonic strains; and postpubertal type teratoma (TE), representing the somatic lineage (Figure 3) [15, 27].

These tumors have common characteristics: they are postpubertal type tumors, usually presenting after adolescence; they are associated with an anomalous testicular background; they have a malignant clinical behavior; and, in almost all cases, they are cytogenetically similar, that is, they exhibit an alteration in the chromosome 12p, becoming a i12p [2, 15, 27].

Throughout this dissertation only the GCNIS related TGCTs will be approached.

1.3.1.1. Germ Cell Neoplasia in situ

GCNIS is originated from gonocytes whose maturation during fetal life has been discontinued or performed in an abnormal manner. These cells remain latent until puberty [15, 27]. Polyploidization constitutes the initial step for GCNIS formation, which only gains invasive potential after the hormonal changes that occur at puberty, progressing then to the default pathway and originating SE or NST [7, 26, 28]. An alteration from default pathway or regaining pluripotency (a phenomenon known as ‘reprogramming)’ can then lead to emergence of an EC cell and originate NST components [29, 30].

This form is asymptomatic and is often incidentally diagnosed, as in the case of biopsies during infertility treatments [28, 31].

GCNIS is a precursor lesion of TGCTs (50% of all cases will progress to an invasive TGCTs within 5 years and 70% of cases will progress within 7 years) [26, 28]. In most cases it is found in the testicular parenchyma adjacent to the tumor [26]. Therefore, we can say that TGCTs can derive from a non-invasive form, becoming an invasive one. It remains to be proven that all GCNIS will become invasive tumors [7, 8, 32].

Sexual developmental disorders and testicular dysgenesis syndrome may be associated with a higher risk of developing GCNIS [33, 34].

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GCNIS cells, such as pluripotent cells, express various tumors markers that may aid in their identification, such as the OCT3 / 4 (POU5F1), placental alkaline phosphatase (PLAP), AP-2ϒ, NANOG, LIN28 and podoplanin proteins [35-40].

At the epigenetic level, we can see changes in GCNIS cells such as global hypomethylation of deoxyribonucleic acid (DNA), overexpression of miR-371-373 and histone modifications [6, 41].

1.3.1.2. Seminoma

SE are the most common subtype of TGCTs. They are also the less aggressive, the most sensitive to chemotherapy and hence the forms with better prognosis [26]. These tumors represent about 50% of all TGCTs cases, with a mean age at diagnosis of 35 years [2, 26, 30]. In most cases it occurs in postpubertal type testicles, being very rare in prepubertal type children and in older men (> 70 years) [42, 43].

The presence of SE may be associated with history of cryptorchidism and immunodeficiency disorders [2]. In many cases there can be SE presence in combination with other NST subtypes resulting in mixed tumors, and it is possible to find any combination of NST and SE [2, 7]. Pr ol ifer ati on R ep ro gr am m in g

Embryonal Carcinoma Seminoma

Postpubertal type yolk-sac tumor Choriocarcinoma Postpubertal Type Teratoma

Germ Cell Neoplasia in situ

Figure 3: Differentiative relations between TGCTs (adapted from Chieffi, P. [1]).

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In the case of SE, the profile of immunohistochemical markers resembles GCNIS cells, in which we find positivity for OCT3 / 4, PLAP and podoplanin proteins. We can also verify positivity for SALL4, SOX17 and CD117 and negativity for CD30, glypican 3, SOX2, alpha-fetoprotein (AFP) and human β-gonadotrophin (β-HCG) [2, 42, 44]. At the level of the serum tumor markers [AFP, β-hCG and lactate dehydrogenase (LDH)], there are no significant increases in the initial progression of the disease (with the exception of minor rising in β-hCG when the SE is admixed with syncytiotrophoblastic cells) [2, 6, 7, 29].

Regarding epigenetics, SE cells contain permissive histone modifications and DNA hypomethylation, facilitating chromatin accessibility [2].

1.3.1.3. Embryonal Carcinoma

This TGCT is the most frequent NST subtype and it is found in young adults between 25 and 35 years old [42].

EC is composed of germ cells similar to embryonic stem cells and pluripotent cells [2]. This subtype occurs mostly in the context of a mixed germ cell tumor (as opposed to its pure form). Even though less frequent (about 16%), its pure form is more aggressive, highly malignant, with a rapid growth and early leading to metastasis [42, 45-47]. Morphologically, in this tumor subtype we can see expression of OCT3 / 4, CD30, SOX2, SALL4, cytokeratins and negativity for SOX17, glypican 3, KIT (or focal positivity), podoplanin, β-hCG, carcinoembryonic antigen and epithelial membrane antigen [2, 29, 42, 44, 45].

The pattern of serum markers in these tumors is also not elevated (AFP, β-hCG and LDH) in a pure form, but it usually shows some positivity when they are combined with other subtypes, in a mixed pattern. The prognosis depends on the disease clinical and pathological stage and the proportion of EC cells in mixed tumors (indeed, a recent study has demonstrated the negative impact on prognosis of an amount of EC superior to 50% of the tumor mass) [2, 7].

1.3.1.4. Postpubertal type yolk-sac tumor

Patients diagnosed with YST usually have ages between 15 and 40 years. This is characterized by the presence of germ cells that differentiates to resemble extraembryonic structures, including yolk sac, allantoid and extra-embryonic mesenchyme [2].

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Pure YST are extremely rare, with almost all forms occurring in the context of mixed tumors (and these components are frequently overlooked and hard to diagnose) [15, 48]. Immunohistochemically, this tumor shows positivity for PLAP, α1-antitrypsin, KIT, SALL4, cytokeratins, AFP and glypican 3 and negativity for β-hCG, OCT3 / 4 and CD30 [2, 42, 49, 50].

In this case, we can state that serum levels may raise the suspicion for this entity, as about 95% of individuals with this tumor subtype show the presence of elevated levels of AFP in the blood [42, 44].

1.3.1.5. Choriocarcinoma

CH is a very rare TGCT subtype (being almost always part of a mixed tumor) that only accounts for 1% of all TGCT [15, 51, 52]. This tumor is found in patients between 20 and 39 years old [52].

This tumor is characterized by the presence of cells similar to the trophoblastic cells of the extra-embryonic chorion (cytotrophoblastic, intermediate trophoblastic and syncytiotrophoblastic cells) [2].

It is a subtype with a high propensity for metastatic disease. Early dissemination and hemorrhagic complications are common [51-53]. In these cases, individuals do not respond well to chemotherapy and about 79% of patients after orchidectomy and treatment die within 3 years [52].

In these patients we can often observe specific hormone-related symptoms, such as gynecomastia and hyperthyroidism [42].

CH cells express β-hCG, SALL4, GATA3, glypican 3, α-inhibin and human placental lactogen. In addition, very high levels of blood β-hCG are evident [44].

1.3.1.6. Postpubertal Type Teratoma

TE is a tumor characterized by the presence of tissues of one or more germinal layers (endoderm, mesoderm and ectoderm), and may include well-differentiated mature tissues or immature embryonic-like tissues [2]. The presence of TE is found more frequently in mixed components (47%) than in the pure form (from 2.7% to 7%), and is frequent in metastases [48, 54].

These tumors are diagnosed in young adults and about one-third are metastatic when diagnosed [54]. The prognosis of this disease, either recurrent or metastatic after treatment, is more favorable in comparison to other subtypes of metastatic TGCTs

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(hence the need for documenting non-teratomatous viable tumor in metastatic resections) [55].

1.3.1.7. Mixed Germ Cell Tumors

As noted, mixed germ cell tumors (MGCTs) are very common and these show the presence of two or more components of malignant TGCTs. In this case, the mean age of the individuals at diagnosis is 30 years of age [2, 42].

All combinations are possible, however in the case of a SE component, the diagnosis is occurred generally at a later stage [42, 48]. The EC subtype is most frequently found in MGCTs, followed by postpubertal type TE, postpubertal type YST, SE and CH [44]. In MGCTs, when we observe the presence of high serum levels of β-hCG, it indicates the presence of CH foci or syncytiotrophoblastic cells, whereas when we observe elevated serum levels of AFP we suspect of the presence of YST foci [42, 48].

Histologically, MGCTs with SE and NST components are similar to their pure forms, but their proportions have important clinical relevance and should be treated according to the worst prognostic component (hence the importance of documenting aggressive components such as CH or EC) [42, 56].

1.4. Diagnosis and Staging

The first approach to individuals with TGCTs is determinant. In patients presenting with signs or symptoms of a testicular mass a scrotal ultrasonography (US) is performed. This procedure is sensitive in detecting this type of tumor, but has poor specificity [57]. The is then subjected to computerized tomography (CT) and to measurement of serum tumor markers (AFP, β-HCG and LDH) in the blood [58]. The latter must be carefully analyzed as the levels of sensitivity and specificity low and they can lead to diagnosis errors due to the presence of some of these markers in other tumor types or even in non-tumoral conditions. In fact, only 60% of individuals with TGCTs present elevated serum markers at the time of diagnosis [59, 60].

Initially, the clinical staging of the disease is performed, through thoracic and abdominopelvic CT, by the evaluation of the serum markers and the extent of the disease [61]. In the latter, we must consider the involvement and size of regional and distant lymph nodes and the presence of visceral metastases. In these cases, we can use magnetic resonance imaging (MRI), positron emission tomography (PET) and bone scintigraphy when necessary [62]. Thus, the presence or absence of regional lymph

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nodes (N), the presence or absence of distant metastases (M), and the status of serum markers (S) can be established before orchidectomy (Table 2) [63].

Table 2: Clinical TNM classification of testicular germ cell tumors (adapted from [2, 61, 63] ). Clinical TNM Classification (cTNM)

T - Primary tumor

Except for pTis and T4, for while radical orchiectomy is not always necessary for classification purposes, the extent of the primary tumor is classified after radical orchiectomy (pathologic stage).

Otherwise, TX is used if radical orchiectomy has not been performed. N - Regional lymph nodes

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Metastasis with a lymph node mass ≤ 2 cm IGD or multiple lymph nodes, none < 2 cm IGD

N2 Metastasis with a lymph node mass > 2 cm but not > 5 cm IGD or multiple lymph nodes with any one mass > 2 cm but not > 5 cm IGD.

N3 Metastasis with a lymph node mass > 5 cm IGD M - Distant metastasis

M0 No distant metastasis

M1 Distant metastasis

M1a Non-retroperitoneal nodal or pulmonary metastases

M1b Non-pulmonary visceral metastases site

S - Serum tumor markers (only pre-orchiectomy)

SX Serum markers studies not available or not performed

S0 Serum markers levels within normal limits

S1 LDH <1.5 x ULN and β-hCG <5000 mlU/mL and AFP <1000 ng/mL

S2 LDH 1.5-10 x ULN or β-hCG 5000-50 000 mlU/mL or AFP 1000- 10 000 ng/mL

S3 LDH >10 x ULN or β-hCG >50 000 mlU/mL or AFP >10 000 ng/mL

IGD: In greatest dimension; LDH: Lactate dehydrogenase; β-hCG: Beta-human chorionic gonadotropin; AFP: Alpha-fetoprotein; ULN: Upper limit of normal for the LDH assay.

Following the surgery (radical orchiectomy), staging can be refined – the pathological

staging (Table 3), which is based on the histopathological evaluation of the material received from the surgery [63]. According to the American Joint Committee on Cancer (AJCC) and the International Union for Cancer Control (UICC), both clinical and pathological stages are based on the TNM system in order to correctly stage and group the stages of the disease (Table 4). Besides the important changes introduced in the

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2016 WHO Classification scheme for these tumors, the newest 8th_{edition of the AJCC}

staging system also introduced some relevant changes (namely in the T categories, including a subdivision of pT1 SEs into pT1a and pT1b, based on size).

Table 3: Pathological TNM classification of testicular germ cell tumors (adapted from [2, 61, 63]). Pathological TNM classification (pTNM)

T - Primary tumor

TX Primary tumor cannot assessed

T0 No evidence of primary tumor (e.g. histological scar in testis)

Tis Germ cell neoplasia in situ

T1 Tumor limited to testis (including rete testis invasion) without lymphovascular invasion

T1a* Tumor smaller than 3 cm in size

T1b* Tumor 3 cm or larger in size

T2

Tumor limited to testis (including rete testis invasion) with lymphovascular invasion or tumor invading hilar soft tissue or epididymis or penetrating visceral mesothelial layer covering the external surface of tunica albuginea with or without lymphovascular invasion

T3 Tumor invades spermatic cord with or without lymphovascular invasion T4 Tumor invades scrotum with or without lymphovascular invasion N - Regional lymph nodes

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Metastasis with a lymph node mass ≤ 2 cm IGD and ≤ 5 positive nodes, none > 2 cm IGD

N2 Metastasis with a lymph node mass > 2 cm but not > 5cm IGD; or > 5 nodes positive, none > 5 cm IGD; or evidence of extranodal tumor extension

N3 Metastasis with lymph node mass > 5 cm IGD M - Distant metastasis

M0 No distant metastasis

M1 Distant metastasis

M1a Non-retroperitoneal nodal or pulmonary metastases

M1b Non-pulmonary visceral metastases site

S - Serum tumor markers (only post-orchiectomy)

SX Serum markers studies not available or not performed

S0 Serum markers levels within normal limits

S1 LDH <1.5 x ULN and β-hCG <5000 mlU/mL and AFP <1000 ng/mL

S2 LDH 1.5-10 x ULN or β-hCG 5000-50 000 mlU/mL or AFP 1000- 10 000 ng/mL S3 LDH >10 x ULN or β-hCG >50 000 mlU/mL or AFP >10 000 ng/mL

IGD: In greatest dimension; LDH: Lactate dehydrogenase; β-hCG: Beta-human chorionic gonadotropin; AFP: Alpha-fetoprotein; ULN: Upper limit of normal for the LDH assay.

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Table 4: Staging grouping of testicular germ cell tumors (adapted from [2, 61, 63]).

T N M S Stage group PTis N0 M0 S0, SX Stage 0 pT1-4 N0 M0 SX Stage I pT1 N0 M0 S0 Stage IA pT2 N0 MO S0 Stage IB pT3 N0 M0 S0 pT4 N0 M0 S0 Any pT/TX N0 M0 S1-3 Stage IS Any pT/TX N1-3 M0 SX Stage II Any pT/TX N1 M0 S0 Stage IIA Any pT/TX N1 M0 S1 Any pT/TX N2 M0 S0 Stage IIB Any pT/TX N2 M0 S1 Any pT/TX N3 M0 S0 Stage IIC Any pT/TX N3 M0 S1

Any pT/TX Any N M1 SX Stage III

Any pT/TX Any N M1a S0

Stage IIIA

Any pT/TX N1-3 M0 S2

Stage IIIB

Any pT/TX N1-3 M0 S3

Stage IIIC Any pT/TX Any N M1a S3

Any pT/TX Any N M1b Any S

Anatomopathological evaluation of both primary tumor and resection of metastases is decisive because it dictates the subsequent treatment and prognosis of the disease [2, 63].

1.5. Prognosis and Therapeutic Approaches

TGCTs are highly curable solid tumors [46, 64]. Survival rate of patients suffering from these tumors exceed 80% even in the case of disseminated disease, and mortality has steadily declined owing to advances in multimodal treatments. Globally, the 5-year overall survival rate for localized diseases is of 99.2%, for regional diseases it is of 96% and for disseminated diseases it is of 73.1% [7, 65].

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prognosis score based on advanced disease status, primary tumor location, histology, metastases, and serum tumor markers immediately prior to chemotherapy administration to assist in further treatment of the patients (Table 5) [61, 64, 66-68].

Table 5: Prognostic-based staging system for metastatic germ cell cancer (International Germ Cell Cancer) (adapted from [61, 66, 67]).

Prognosis group

Good

Intermediate

Poor

Seminoma (90% of cases) Nonseminoma (56% of cases) Seminoma (10% of cases) Nonseminoma (28% of cases) Seminoma Nonseminoma (16% of cases)

5-year PFS: 82% 5-year PFS: 89% 5-year PFS: 67% 5-year PFS: 75% 5-year PFS: 41% 5-year overall

survival: 86%

5-Year overall survival: 92%

5-year overall survival: 72%

All of the following criteria:

No patients classified as poor prognosis

All of the following criteria:

● Any primary site ●

Testis/retroperitoneal primary

● Any primary site ●

Testis/retroperitoneal primary

● Mediastinal primary

● No NPVM ● No NPVM ● NPVM ● No NPVM ● NPVM

● Normal AFP ● AFP < 1000 ng/mL ● Normal AFP ● AFP 1000 - 10 000 ng/mL or ● AFP > 10 000 ng/mL or ● Any β-Hcg ● β-hCG < 5000 IU/L (1000 ng/mL) ● Any β-hCG ● β-hCG 5000 - 50 000 IU/L or ● β-hCG > 50 000 IU/L (10 000 ng/mL) or ● Any LDH ● LDH < 1.5 x ULN ● Any LDH ● LDH 1.5 - 10 x ULN ● LDH > 10 x ULN

PFS: Progression free survival; NPVM: Nonpulmonary visceral metastases; AFP: Alpha-fetoprotein; LDH: Lactate dehydrogenase; β-hCG: Beta-human chorionic gonadotropin; ULN: Upper limit of normal range.

Currently, 80% of individuals with stage I TCGT (>80% SE and >60% NST) benefit from surgery at the time of diagnosis [69]. Individuals with SE are more likely to relapse and to invade the rete testis if the tumor is larger than 4cm [70, 71]. In NSTs, the presence of blood vessels or lymphatic invasion is a predictor of metastasis [72]. In stages II and III, there are also high survival rate in the cases that undergo orchidectomy followed by adjuvant treatment [65].

Therefore, in the case of a primary TGCT, the treatment is inguinal radical orchiectomy, as it provides diagnostic and staging information, followed by active surveillance in stages I in the SE; or adjuvant treatments, such as chemotherapy with cisplatin (isolated

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or combined with radiation therapy) or retroperitoneal lymph node dissection (RPLND) according to the remaining different stages of the disease [61, 73, 74].

Chemotherapy is often used to cure TGCTs when it spreads out of the testicle or to decrease the risk of relapse. Nowadays, the administration of cisplatin is widely accepted by the community [67, 73]. It can be used either alone or combined in cycles, depending on the stage of the disease (Table 6). Although cisplatin is highly effective in advanced disease, it is cytotoxic and about 20% of individuals have a poor prognosis because of primary or acquired resistance to this drug [61, 63, 66, 67, 73]. Thus, research on new therapeutic options is urgently needed.

Table 6: Summary of the different therapeutic schemes for treatment of testicular germ cell tumors (adapted from [61, 66, 67]).

Schemes therapeutic

Seminoma Non-seminoma

Clinical stage I

Active surveillance Active surveillance

Adjuvant chemotherapy Adjuvant chemotherapy

* 1 cycle of Carboplatin AUC x 7 * 1 cycle of BEP Adjuvant radiotherapy Risk-adapted treatment

(Not recommended for young patients Low risk (no vascular invasion): High risk (vascular invasion)

* Surveillance (standard) * Adjuvant chemotherapy, 1 cycle BEP (standard)

* Adjuvant chemotherapy, 1

cycle BEP * Nerve-sparing RPLND

* Nerve-sparing RPLND * Surveillance Risk-adapted treatment

RPLND (Low or high risk of metastatic disease)

Clinical stage II A/B

Radiotherapy (standard) Radiotherapy to paraaortic and ipsilateral iliac field and additional boost to the enlarged lymph nodes if necessary.

Chemotherapy Chemotherapy

* 3 cycles of BEP or 4 cycles of EP * 3 cycles of BEP or 4 cycles of EP

Clinical stage IIC/III Good prognosis Chemotherapy Good prognosis Chemotherapy * 3 cycles of BEP or 4 cycles of EP

* 3 cycles of BEP or 4 cycles of EP

Intermediate prognosis

Chemotherapy

Intermediate prognosis Chemotherapy * 4 cycles of BEP or VIP * 4 cycles of BEP Poor prognosis Chemotherapy * 4 cycles of BEP

AUC: Area under the curve; BEP: Cisplatin, etoposide; bleomycin; EP: Etoposide, cisplatin; RPLND: Retroperitoneal lymph node dissection; VIP: Etoposide, cisplatin, ifosfamide.

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1.6. Cell lines of testicular germ cell tumors

Tumor cell lines constitute a set of biological models, representing the diversity, heterogeneity and drug resistance of certain tumors. These are used for cancer screening, in particular for assessing tumorigenesis and the efficiency of drugs given to a particular tumor [75].

They are similar to the cells of origin and try to represent the same characteristics of the tumor cells, like the activity of the telomerase, the expression of stem cells markers, as well as other genetic alterations [75, 76]. These in vitro cultured cells are induced, developed, proliferated and killed under suitable conditions and are similar to those in in

vivo conditions. The cell lines used throughout this dissertation are NCCIT, NT2, 2101EP

and TCam2 (Table 7) [77-81].

NCCIT is a cell line derived from extra-gonadal germ cells of a mixed tumor with EC and TE component [78]. NT2 is a pluripotent cell line derived from pure EC [77, 78]. 2101ET is a nullipotent cell line, which is derived from teratocarcinoma [78]. These lines have a strain resistant to cisplatin [77, 78]. Finally, TCam2 is a cell line compatible with SE cells and with a high hypomethylation content [80, 81].

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Table 7: Cell lines of (testicular) germ cells tumors and their general characteristics (adapted from [77, 78, 80-82]).

CELL LINES

TCam2 NT2 2101EP NCCIT

TGCTs

subtype SE cell line NST cell line

Derivation Derived from a testicular seminoma Derivative of testicular EC

Derived from a testicular tumor classified as Teratocarcinoma

Derived from a germ cell tumor extra-gonadal, mediastinal, mixed of EC and TE Genetic and epigenetic characteristics

● It introduces the i12p

Pluripotent and p53 wild type

Nullipotent and p53 wild

type P53 mutated

● Hypomethylation and analysis of imprinting genes compatible with Seminoma

● Genome wide mRNA and miRNA expression profiling compatible with Seminoma (expressing the miRs complex 371-373; expressing mRNA and protein OCT3 / 4 and NANOG) ● IHQ for several Seminoma-compatible markers (PLAP +, KIT +, SCF +, CD30-, SOX2-)

Resistance to

Cisplatin It is not resistant to Cisplatin

It has a Cisplatin resistant form (Ntera-2-R) It has a Cisplatin-resistant form (2102EP-R) It has a Cisplatin-resistant form (NCCIT-R) Other features

Used to study the transition between SE of good prognosis vs NST; It can be used to study different forms of QT, and RT and different treatment sensibility

Used to test new compounds in sensitive line vs resistant to treatment with cisplatin and to study viability, angiogenesis, proliferation.

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2. EPIGENETICS

Waddington, in 1940, was the first describing “Epigenetics” as “The interactions of genes with their environment, which bring the phenotype into being”. Later, in 2000, Baylin describes it as being “Potentially reversible heritable change in the pattern of gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene.” Currently accepted by all, epigenetics is the study of hereditary and reversible changes in gene expression without altering DNA sequences [83, 84].

The study of epigenetic mechanisms underlying tumorigenesis as well as the search for new therapeutic approaches for the treatment of cancer has been a challenge for researchers [85].

2.1. Epigenetic Mechanisms

Epigenetic mechanisms act by modifying the accessibility of chromatin for the regulation

of transcription, causing repression or expression of the gene [86, 87].

These mechanisms have an essential role in normal human physiology, where they participate in embryonic development, cell and tissue differentiation, immune response, but also in pathological processes, such as cancer [88-90]. In the latter, the mechanisms interact through the inactivation of tumor suppressor genes or oncogenes activation [91]. Thus, there are several and complex epigenetic mechanisms, including: histone post-translational modifications and chromatin remodeling, non-coding ribonucleic acid (RNAs) regulation and DNA methylation [3, 87].

2.1.1. Histone post-translational modifications or

chromatin remodeling

Histones are dynamic proteins with roles in DNA-packaging, thus acting on regulation of chromatin [92]. The basic chromatin unit, as described in 1991, is the nucleosome, which consists of an octamer formed by two copies of each histone core: H2A, H2B, H3 and H4, which have several variants, and approximately 147 base pairs of DNA. H2A and H2B present sequences specific to each species, while H3 and H4 are the most studied proteins [3, 93, 94].

Histones undergo a series of post-translational modifications. These are chemical changes made after the translation of proteins that aim to increase the variability of the characteristics and functions of proteins, such as acetylation, methylation,

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phosphorylation, ubiquitination, among others [3, 95]. All these modifications, which are introduced by several families of enzymes, constitute the so-called "Histone Code", which is read and recognized by additional proteins in order to regulate gene expression and repair DNA damage [3, 92, 95]. A failure in any of these regulatory mechanisms or proteins may be crucial for the development of neoplastic cells [96].

2.1.2. Non-coding RNA

The complexity of the human genome is challenging and still unknown. About 98.5% of our genome consists of RNA sequences that do non-coding proteins (ncRNA) [97]. However, these ncRNAs produce structural, catalytic and RNA regrowth transcripts that play an important role in the normal development and progression of disease [98]. The ncRNAs can be divided into two broad categories according to their size: the small ncRNAs (sncRNA) (<200 bp) and the large ncRNAs (lncRNA) (> 200 bp). Both ncRNAs include different regions, where different functions are performed [99, 100]. MicroRNAs (miRNAs), which are part of the sncRNA family, are currently the most studied and about 60% of the genes are regulated by them. A single miRNA can regulate dozens of transcripts at the same time and a transcript can be regulated by several miRNAs [99-102]. Therefore, deregulation of the miRNA in processes such as the cell cycle, differentiation, proliferation and apoptosis can lead to the formation and presence of neoplastic cells [101, 102].

2.1.3. DNA methylation

According to Feinberg and Vogelstein, in 1983, “loss of DNA methylation at CpG

dinucleotides was the first epigenetic abnormality to be identified in cancer cells”. Currently, DNA methylation is the most studied epigenetic mechanism and it occurs mainly in the cytosine (C) residues that precede the guanine bases (G) [85, 91, 103]. We find mostly these structures grouped in small dense stretches of DNA, called the CpG islands, located in several sites that present specific regions for protein binding, called methylation binding domains (MBD) [103-106]. These can be present in CpG islands of the promoter regions or first exons, in CpG island shores, in CpG island coding regions and in CpG island repetitive sequences [85, 107]. The CpG islands occupy about 50-60% of the promoter region and may have a size equal to or greater than 200bp [108]. Briefly, methylation consists on the covalent addition of a methyl (CH3) group [accessible via S-adenosyl-L-methionine (SAM)] to the 5' carbon cytosine, originating

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methylcytosine (5mC) [91, 109]. Generally, CpG islands are unmethylated, keeping the chromatin accessible for genetic transcription. In contrast, when the CpG islands are methylated, there is inhibition of gene expression and, therefore, no transcription of messenger RNA (mRNA) during tissue development (Figure 4) [108].

This process is dynamic and regulated by the group of enzymes called DNA methyltransferases (DNMTs) that are responsible for establishing and maintaining the methylation patterns (Table 8) [3, 91, 109].

Currently, studies have demonstrated the occurrence of global and active loss of methylation of the paternal genome during embryogenesis, unlike the maternal genome, which is passively demethylated due to DNA replication during cell divisions [3, 110-115]. DNA demethylation may be, then, an active mechanism or a passive mechanism, or a combination of both [115-117]. Active demethylation involves oxidation of 5mC to 5hmC mediated by TET proteins (ten-eleven translocation), and subsequent BER (Base Excision Repair) targeting (Figuare 5) (Table 8) [116, 118-120]. On the other hand, passive demethylation consists of the gradual loss of methylation at the beginning stage of the embryo development due to lack of DNMT reduction during DNA replication [116, 121].

Several studies have shown that deregulation of DNA methylation patterns is involved in

CH₃

SAMCH

3

SAH DNMTs

Cytosine 5’ Methyl-Cytosine

Gene

CpG islands Transcription OFF

CH 3 5’ Methyl-Cytosine TET s 5’ Hydroxymethyl-Cytosine OH α-KG succinate O₂

Gene

CpG islands Transcription ON Figure 4: DNA methylation in cells. When there is transfer of a carbon group assigned by SAM to give 5'-carbon cytosine, the CpG islands are methylated and there is no gene transcription. It is a mechanism mediated by DNMTs.

Figure 5: DNA demethylation in cells. When the methyl group (added a hydroxyl group) is removed from the 5'-carbon cytosine, the CpG islands become unmethylated and there is transcription of the gene. It is a mechanism mediated by TET proteins.

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the formation and development of cancer, as well as in the formation of metastases [91, 108]. About 20-60% of tumor cells are characterized by the overall loss of methylation when compared to the non-tumor cells [108]. Thus, we can identify two patterns of methylation in cancer:

- Global Hypomethylation of DNA – It occurs particularly in repetitive sequences,

retrotransposons, coding regions and introns. Loss of methylation can lead to chromosomal instability, reactivation of genes involved in cancer, activation of oncogenes, silencing of chromosome X and loss of imprinting [85, 122, 123].

- Localized hypermethylation of the specific CpG islands - As it is the case of promoter hypermethylation of tumor suppressor genes (TSGs). TSGs are involved in various biological functions, such as the cell cycle, DNA repair mechanisms, apoptosis and angiogenesis. Excessive methylation in the TSG promoter results in its inadequate inactivation, which can lead to the initiation and progression of cancer [3, 85, 103]. The existence of mutations in TETs may be associated with the phenotype of DNA hypermethylation [3, 91, 108, 116].

2.2. Epigenetic Alterations in Testicular Germ Cell Tumors

DNA methylation is involved in fundamental biological processes, such as cell cycle, cell division and proliferation, metabolism, pluripotency, genomic imprinting and DNA repair, and globally regulate the transcription of many genes [3, 29, 124, 125]. The phenotypic diversity and behavior of TGCTs may be the result of epigenetic dysregulation because they mainly share a unifying cytogenetic background and show very few mutations [3, 29, 126]. This deregulation can provide key information for the standard diagnosis and also give rise to biomarkers for subtyping and prognostic purposes [3, 127]. Several studies have reported dysregulation of many DNA modifying enzymes like DNMTs e TETs as well as differences in methylation patterns of various promoter genes between the two major TGCT subtypes (SE and NST) (Table 8) [3, 128-135].

The genes that give rise to pluripotent cells are believed to be associated with hypomethylation [3]. Generalized hypomethylation of DNA is a characteristic of fetal germ cells, but after birth it is modified to hypermethylation in male germ cells [3, 25, 136-138].

In fact, changes in methylation pattern between SE and NST are notorious. SEs show almost no methylation in the CpG islands, while NSTs show methylation of the same islands comparable to other solid tumors [29, 139-141]. This leads us to believe that SEs have an increase in transcriptional activity unlike NSTs. Smiraglia et al. proposed a

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model in which SEs undergo a global demethylation process, while NSTs undergo a de

novo methylation process [29, 141].

In fact, more undifferentiated TGCTs (SE, GCNIS) are more hypomethylated, whereas more differentiated TGCTs (TE, YST and CH) show a higher degree of methylation. EC shows an intermediate pattern [29, 139-141].

In a recent in silico analysis of a publicly available database Lobo et al. put in evidence changes in DNMT3A, DNMT3B and TET2 between SE and NST samples. That is, the first two are decreased in SEs when compared to EC, while the latter is comparatively decreased in EC (Table 9) [3].

Different methylation levels with different expression of DNA modifying enzymes in different subtypes of TGCTs may allow for a more precise and accurate diagnosis, prognosis and treatment of germ cells tumors, since the methylation pattern is different in the several stages of embryonic development [3, 29, 126].

The role of DNA modifying enzymes in malignant testicular germ cell tumors