Novel preclinical models, therapies and biomarkers for testicular cancer Rosas Plaza, Fernanda

Novel preclinical models, therapies and biomarkers for testicular cancer Rosas Plaza, Fernanda

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10.33612/diss.119056452

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Rosas Plaza, F. (2020). Novel preclinical models, therapies and biomarkers for testicular cancer.

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NOVEL PRECLINICAL MODELS, THERAPIES AND BIOMARKERS FOR TESTICULAR CANCER

PhD thesis

Cover and layer-out design: Merit González Printing of this thesis was supported by:

- Stichting Werkgroep Interne Oncologie - The Graduate School of Medical Sciences - University Library

© Copyright 2020, F. X. Rosas Plaza

All rights reserved. No part of this thesis may be reproduced, stored or

transmitted in any form without permission by the author.

to obtain the degree of PhD at the University of Groningen

on the authority of the

Rector Magnificus Prof. C. Wijmenga and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on Monday 9 March 2020 at 14:30

by

born on 31 May 1987 in Mexico City, Mexico

PhD thesis

Fernanda Ximena Rosas Plaza

therapies and biomarkers

for testicular cancer

Prof. S. de Jong Prof. J. A. Gietema

Prof. M. A. T. M. van Vugt Assessment committee Prof. W.N. Keith

Prof. A. van den Berg

Prof. F.A.E. Kruyt

Table of contents

Chapter 1 Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6 Chapter 7

General Introduction and outline of thesis 7 miR-371a-3p, miR-373-3p and miR-367-3p as serum biomarkers in metastatic testicular germ cell cancers before, during and after chemotherapy 29 Cells, 2019, 8, 1221

Dual mTORC1/2 inhibition sensitizes testicular cancer

models to cisplatin treatment 55

Molecular Cancer Therapeutics, 2020, 19: 590-601 Establishment and characterization of testicular cancer patient-derived xenograft models for preclinical

evaluation of novel therapeutic strategies 81 In preparation

AFP levels do not predict response to cisplatin in

testicular cancer patient-derived xenograft models 105 Summary, general discussion and future perspectives 115 Nederlandse samenvatting (Summary in Dutch) 129

Acknowledgements 135

List of publications 138

Chapter 1

General introduction and outline of the thesis

9

Clinical and pathological characteristics of testicular cancer

Testicular cancer (TC) accounts for approximately 1% of all cancers in men worldwide, while it is also the most common solid tumor among young men (15-40 years)

. Incidence of TC varies widely across different regions, with an age- standardized rate of 8.7% in Western Europe compared to 0.2% in Central Africa

. Incidence rates of TC have increased since the 1960s in Northern European countries, affecting age groups older than 15 years. No causative mechanism for this increase had been identified so far

.

Well known risk factors associated with the development of TC include cryptorchidism, a family history of TC and a previous diagnosis of TC

. Other recently described risk factors are low birth weight, low gestational age at birth, inguinal hernia, high intake of dairy products and ‘heavy’ cannabis use

. Germ cell tumor (GCT) is the predominant histology of TC patients (95%), divided into seminomas and non-seminomas, of which seminomas are slightly more common than non-seminomas

. GCTs arise from gonocytes that failed to differentiate into spermatogonias, and the pluripotency of these cells allow them to generate highly diverse histological tumors. Seminomas are blocked in the earliest differentiation state whereas non-seminomas consist of diverse histological subtypes depending on the degree of differentiation: embryonal carcinoma (undifferentiated), choriocarcinoma, yolk sac tumor or teratoma (well-differentiated)

. Mixed GCTs are common and may appear in any form.

Diagnosis of TC is performed with clinical evaluation, ultrasound examination of the testis and determination of serum tumor markers. These tumor biomarkers include LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and ß-HCG (ß-human chorionic gonadotropin), and assist in the diagnosis, risk stratification and follow-up of TC patients

. Current risk stratification was established in 1997 by the International Germ Cell Consensus Classification (IGCCC) and constitutes a prognostic staging system for patients with disseminated disease (Table 1)

.

Overall cure rate of TC is good, mostly resulting from the high sensitivity of TCs

to the chemotherapeutic cisplatin in a metastatic setting. Relative 5-year overall

survival of seminoma and non-seminoma patients is 98% and 93%, respectively

.

Patients with stage I disease have more than 99% overall survival regardless of

tumor type. However, patients with metastatic disease have different survival

outcomes depending on the tumor type and prognosis group. The IGCCC

classification stratifies patients into good, intermediate and poor prognosis groups

Table1. Risk stratification for metastatic patients

Non-seminoma Seminoma

Risk group RMH

Stage Spread and tumor markers Survival Spread and tumor markers Survival Good

prognosis

II-IV • Testis or retroperitoneal primary

• No metastasis to organs other than the lung and/

or lymph nodes

• Tumor markers level normal or at least 1 tumor marker above normal:

• LFD < 1.5x ULN

• beta-hCG < 5000 mIU/mL

• AFP < 1000 ng/mL

56% of patients 5-year PFS: 89%

5-year OS: 92%

• Any primary site

• No metastasis to organs other than lungs and/or lymph nodes

• Normal AFP

• Any beta-hCG and LDH

90% of patients 5-year PFS: 82%

5-year OS: 86%

Intermediate prognosis

II-IV • Testis or retroperitoneal primary

• No metastasis to organs other than the lungs and/

or lymph nodes

• At least 1 tumor marker substantially above normal:

• LDH 1.5 - 1.0x ULN beta-hCG ≥ 5000 ≤ 50000 mIU/mL

• AFP ≥ 1000 ≤ 10000 ng/mL

28% of patients 5-year PFS: 75%

5-year OS: 80%

Any primary site Metastasis to organs other than the lungs and/or lymph nodes Normal AFP Any beta-hCG and LDH

10% of patients 5-year PFS: 67%

5-year OS: 72%

Poor prognosis

II-IV • Mediastinal primary with or without metastases

• Metastasis to organs other than the lungs and/or lymph nodes

• At least 1 or more tumor maker levels highly elevated:

• LDH > 10x ULN

• beta-hCG > 50000 mIU/mL

• AFP > 10000 ng/mL

16% of patients 5-year PFS: 41%

5-year OS: 48%

No poor prognosis patients

No poor prognosis patients

RMH: Royal Marsden Hospital, LDH: lactate dehydrogenase, hCG: human chorionic gonadotropin, AFP: alpha- fetoprotein, ULN: upper limit of normal, PFS: progression free survival, OS: overall survival

which are determined based on clinical features: the location of the metastasis

and levels of different tumor biomarkers. Even though recent studies have

shown improvement of the initially reported survival rates

, general consensus

states that the good prognosis group has an 86% and 92% 5-year overall survival

for seminoma and non-seminoma patients respectively. Intermediate prognosis

TC patients have 72% and 80% 5-year overall survival for seminoma and non-

seminoma tumors, respectively, and the poor prognosis group including only

non- seminoma patients have a 48% 5-year overall survival

.

11

Standard treatment of TC

Stage I or localized disease is treated with orchiectomy, while metastatic patients are given cisplatin based chemotherapy after orchiectomy is performed.

Patients with disseminated disease are treated with three or four courses of BEP regimen including bleomycin, etoposide and cisplatin. Approximately 20% of patients with disseminated disease will need second-line treatment as a consequence of relapse or refractory disease

. Different salvage treatments have been reported with long term remissions ranging from 23% using VeIP/

VIP (cisplatin, ifosfamide and etoposide/vinblastine)

to 63% and 73% using TIP (paclitaxel, ifosfamide and cisplatin)

or GIP (gemcitabine, ifosfamide and cisplatin)

respectively. High-dose chemotherapy initially showed promising results, however more recent clinical trials have not demonstrated superior efficacy over standard salvage treatments

.

Genetic alterations in TC

Few susceptibility loci associated with the development of TC have been identified. Independent genome wide association studies found that genetic predisposition of TC was associated to variants influencing the KITLG/KIT pathway. Variants within the 12q22 region, comprising the KITLG gene, were associated with an elevated risk of TC

. KITLG gene encodes the ligand for the receptor tyrosine kinase c-KIT. Chromosome 5 was also found to have a TC- associated variant affecting the SPRY4 gene. SPRY4 functions as an inhibitor of mitogen-activated protein kinase (MAPK) pathway, which is downstream of the KIT pathway. Both susceptibility loci were associated with seminomas and non-seminomas, suggesting that despite their diverse histology they share a common biological origin, further supported by the frequent observation of mixed GCTs.

TCs are highly aneuploid and frequently show large scale copy number

gains and losses. Copy number gains on chromosomes 7, 8, 21, 22 and X are

commonly observed as well as losses on chromosomes 4, 5, 11, 13 and 18

.

However the most common anomaly is the presence of a 12p isochromosome

(i(12p)) affecting more than 80% of TCs

. This region of the small arm of

chromosome 12 contains the oncogene KRAS, as well as stem cell related genes

including NANOG and STELLAR. Of note, pre-malignant lesions do not contain

i(12p)

suggesting that it is not involved in the development of the precursor

lesion. Importantly, amplifications of the KIT gene were identified in 21% of

seminomas and 9% of non-seminomas. Copy number gains of the KIT gene,

located on chromosome 4, were associated with an increased expression of the

protein

.

Despite the high aneuploidy of TCs, the somatic mutation rate is low. Whole exome sequencing revealed that TC tumors have a mutation rate of 0.51 somatic mutations per Mb, while lung cancers carry 8.0 mutations/Mb, and melanomas 11.0 mutations/Mb

. KIT was the most significantly mutated gene in TC, observed predominantly in seminomas

. The proportion of KIT hotspot mutations in seminomas ranges from 18%

to 31.3%

. Studies investigating mutations in K-RAS and N-RAS specifically, have found that incidence of RAS mutations is on average 20%-30% in both seminomas and non-seminomas

. Available data on which tumor type has a higher incidence of RAS mutations is non-conclusive. Mutations in cell division cycle 27 gene (CDC27) were recently described in 11.9% of TC patients and few PIK3CA and AKT1 mutations have been reported as well

. Accordingly, pathway analysis of somatic mutations identified that genes involved in metabolism were the most frequently mutated (93%), with the PI3K/AKT pathway as one of the most affected pathways (54%) in TC

.

Relevance of cisplatin and apoptosis induction in TC

Since the introduction of cisplatin-based chemotherapy in the 1970s, curing rates of metastatic TC improved drastically

. Even though cisplatin has been used in the clinic for a long time and is a curative drug in some types of pediatric cancer and young adult cancers in addition to TC, the mechanisms behind the excellent sensitivity in these tumor types are still elusive. TP53 is one of the most commonly mutated genes in cancer, however it is rarely altered in TC before treatment

. The presence of wild type (WT) TP53 is thought to be one of the key factors involved in response to chemotherapy in these patients. The TP53 gene encodes the p53 protein, a transcription factor involved in many cellular processes including cell cycle arrest, DNA repair, apoptosis, autophagy and metabolism

.

Cellular stress in response to cisplatin treatment has been widely studied. Once

cisplatin enters the cell, it interacts with DNA. Cisplatin generates different forms

of DNA adducts, which are predominantly intrastrand cross-links and secondly

interstrand cross-links, protein-DNA cross-links and DNA monoadducts

.

The cisplatin-DNA cross-links disrupt the structure of the DNA which can be

recognized by DNA repair proteins. Cisplatin-induced intrastrand crosslinks are

mostly repaired by the nucleotide excision repair (NER) pathway

. However,

human cells deal poorly with this type of DNA damage due to the binding of high

mobility groups (HMG) proteins to the DNA adducts, blocking their repair

.

In addition, some essential proteins involved in NER including ERCC1, XPF

and XPA have low expression levels in TC, contributing to the high levels of

sensitivity to cisplatin treatment

. Furthermore, the cytotoxicity of cisplatin can

partially be explained by to the formation of DNA double strand breaks (DSBs),

13

during the processing of DNA interstrand cross-links in replicating cells

. The DNA damage response (DDR) machinery is activated in response to DSBs and as a consequence leads to activation of the ataxia telangiectasia mutated (ATM) kinase. ATM subsequently phosphorylates p53 as well as the main p53 negative regulator, mouse double minute homolog (MDM2). Both replication stress and DNA damage can stabilize p53, for example, by promoting p53 phosphorylation and blocking the degradation regulated by the p53 inhibitor MDM2. The phosphorylated form of MDM2 is unable to ubiquitinate p53, allowing its full activation

. As a result of DNA damage, p53 induces cell cycle arrest and apoptosis. The upregulation of p21/CDKN1A, encoded by the CDKN1A gene, is crucial for the induction of cell cycle arrest in G1 phase. Apoptosis, on the other hand, can be triggered by the p53-mediated transcription of pro-apoptotic BCL-2 family members including PUMA and PMAIP1 (NOXA)

. Activation of the DDR induced by cisplatin in TC, leads to a rapid induction of apoptosis with a major role for p53. Contributing to the hypersensitivity of TCs to apoptosis is the low mutation rate of TP53, as mentioned before

. Of note, no correlation was found between p53 expression levels and cisplatin sensitivity.

Accumulation of cisplatin and the amount of cisplatin bound to DNA in TC cells are not clearly correlated with cisplatin sensitivity, which is more related to the lack of repair of cisplatin-damaged DNA in TC cells

. For TC it has been described that cisplatin-induced p53 activation can induce apoptosis via both the extrinsic apoptotic pathway mediated by the FAS death receptor

and the intrinsic apoptotic pathway via the BH3-only family members: p53 upregulated modulator of apoptosis (PUMA) and Noxa

. FAS receptor activation results in cleavage of procaspase-8, leading to cleavage of the pro- apoptotic BH3 interacting-domain death agonist (BID). The cleaved BID fragment (tBID) targets the mitochondria and induces the oligomerization of BCL-2- associated X protein (BAX) and BCL-2-antagonist/killer 1 (BAK) in the outer mitochondrial membrane. Mitochondrial leakage leads to the release of cytochrome C and cleavage of procaspase-9 into fully active caspase-9

. Activation of the intrinsic apoptotic pathway is regulated by molecular interactions between members of the B cell lymphoma 2 (BCL-2) family of apoptosis regulating proteins, which can be either pro- or anti-apoptotic. The BH3-only proteins promote BAX and BAK activation, by inhibiting the anti-apoptotic Bcl-2 proteins and, in some instances, by directly engaging with BAX and BAK

. The anti-apoptotic Bcl-2 members prevent apoptosis initiation by sequestering pro-apoptotic proteins.

Caspase 8 and 9 activation will lead to cleavage of executioner caspases (-3, -6 and -7) that will orchestrate the degradation of the cell

.

Even though most TC tumors retain WT TP53, some posttranscriptional

modifications are able to repress p53 pro-apoptotic activity. For example,

carboxyl- terminal lysine methylation on p53 was shown to inhibit PUMA and

CDKN1A expression in teratocarcinoma cells

. Methyltransferase knockdown

and expression of p53 mutants that cannot be methylated restored PUMA and CDKN1A expression. Although effects on apoptosis by PUMA or CDKN1A downregulation and restoration were not evaluated, their involvement in response to cisplatin has been reported in TC

and other cancer types

. However, whether cells enter cycle arrest or undergoes apoptosis as a consequence of CDKN1A or PUMA upregulation, respectively, remains unclear and might be context dependent

.

Cisplatin resistance in TC

Genomic alterations affecting the MDM2/p53 axis have been described in chemo- resistant TC. A large study of 180 TC tumors using whole exome sequencing data (WES) described that most of the samples with MDM2/p53 alterations were found in resistant tumors and the majority were post-treatment specimens

. Moreover, chemo-resistant tumors exhibited a trend towards higher positivity for both p53 and MDM2 compared to sensitive specimens, suggesting increased importance of MDM2 mediated inhibition of p53 activity

. This hypothesis is supported by functional studies using cisplatin-sensitive and -resistant TC cell lines indicating that the interaction between p53 and MDM2 requires higher doses of cisplatin to be disrupted in resistant cell lines

. More genomic alterations in PIK3CA, AKT1 and MTOR are found in resistant tumors, although not statistically significant. KRAS mutations are also overrepresented in resistant tumors. These data are in line with similar reports that compared sensitive versus resistant tumors and found a small number of PIK3CA, AKT1 and KRAS mutations in resistant tumors only

.

The PI3K/Akt/mTOR pathway has been described to be involved in response to cisplatin using mutational analyses, as mentioned earlier, but also in several functional studies. Akt kinase hyperactivation was seen in resistant TC cells by putative PDGFRß overexpression

(Figure 1). PDGFRß inhibition with Pazopanib resulted in sensitization to cisplatin treatment in vitro and was an effective treatment in combination with the HER2 inhibitor lapatinib to treat resistant TC models in vivo

. PI3K and Akt inhibition were tested in combination with cisplatin in resistant TC cell lines showing apoptosis induction compared to cisplatin treatment alone

.

As described above excellent sensitivity of TC tumors to cisplatin treatment is partly caused by a diminished capacity to repair cisplatin-DNA adducts.

Consequently, cisplatin resistance may arise from an enhanced ability to resolve

DNA damage, or by increased coping with unrepaired lesions. Overexpression

of HMGB1, which can inhibit NER by binding to the DNA adducts, has been

reported in several tumor types and was associated with poor prognosis

.

In addition, increased expression of ERCC1 in ovarian cancer cell lines was

15

associated with cisplatin resistance

. Currently there is no data available suggesting that changes to ERCC1 or HMG proteins such as posttranslational modifications are involved in cisplatin resistance of TC. Reduced HR activity may contribute to cisplatin sensitivity in TC. One report has compared cisplatin sensitive and resistant TC cell lines, showing that cisplatin sensitivity correlated with the level of proficiency of TC cells to repair interstrand cross-links. Despite differences in HR proficiency between cisplatin sensitive and resistant cell lines, all TC cell lines proved more HR deficient compared to a non-TC cisplatin resistant cell line

. More research should be conducted to determine if and how DNA damage repair pathways are involved in cisplatin resistance of TC.

Figure 1. Pathways involved in cisplatin sensitivity

Once cisplatin enters TC cells it induces different types of DNA damage. This leads to the initiation of DNA damage response (DDR), which is mainly orchestrated by p53. DNA damage induced stabilization of p53 results in p53-mediated transcription of different proteins, i.e. MDM2, its main negative regulator, and the pro-apoptotic proteins PUMA and NOXA. High expression of these pro-apoptotic proteins activates the intrinsic apoptotic pathway. After oligomerization of BAX and BAK in the outer mitochondrial membrane cytochrome C is released from the mitochondria of as a result caspase 9 is activated. The extrinsic apoptotic pathway can be initiated by p53-mediated increase and activation of the FAS receptor, which induces procaspase-8 cleavage. Growth factor stimuli provide pro-survival signals to the cell through the PI3k/

AKT/mTOR pathway by inducing protein synthesis and inhibiting pro-survival proteins. The Ras/Raf/ERK pathway also provides pro-survival signals to the cell. Red line indicates inhibition, black line indicates stimulation.

FAS ligand

FASR

Proliferation Survival

Translational control Protein synthesis

Polyubiquitination and proteasomal degradation Cell cycle arrest DNA repair Metabolism

P P

Ras

PRaf

PI3K

PTEN

p53

BAD

PUMA mTORC2

mTORC1

4e-BP1

PDK1 PIP2

MEK 1/2 P

ERK 1/2 P

P P

PAKTP PPIP3

P P

UbUb

p53P MDM2P

DDR

Cisplatin Hypoxia

Retinoic acid Noxa

Oct4 Cytochrome C

Cytochrome C

BAX BAK

BAX BAK

Apoptosis BCL2,

BCLxL MCL1 BID tBID

Caspase 8Pro

Active Caspase 8

Caspase 9

Caspase 3/7 FADD

P S6P

Novel targeted therapies in TC

Clinical trials using targeted drugs are scarce. The susceptibility of TC towards inhibition of the PI3K/AKT/mTORC pathway in preclinical studies pushed forward two clinical trials in patients with refractory disease or relapse using everolimus

and pazopanib

as single agents. Efficacy of the mTORC1 inhibitor everolimus was minimal, while treatment with the multitargeted tyrosine kinase (TK) inhibitor pazopanib achieved limited tumor activity. Two other small clinical trials tested the efficacy of TK inhibition alone in heavily treated TC patients.

Imatinib was used to treat 6 KIT-positive patients with imatinib showing poor activity with 5 out of 6 patients developing progressive disease

. Five patients refractory to first-line therapy were included in a phase II study in which the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib was tested

. Only one of these patients responded to treatment. Finally, a recent article studied the response of refractory TC to immune checkpoint inhibition.

Twelve patients were treated with the antibody against PD-1, pembrolizumab, with no partial or complete responses observed

.

Another interesting pathway as a clinical target for treatment of TC is the DDR pathway. TC tumors have been shown to be deficient for interstrand DNA cross- link repair, leading to formation of DSBs

. Normally, DSBs are repaired by HR, a DNA repair mechanism that is impaired in TC cells

. Unresolved DSBs, however, provide a therapeutic opportunity as HR-deficient tumors are highly sensitive to PARP1 inhibitors

. Expression of PARP1 was high in TC tumors and not in normal testis tissue, but did not show a correlation with clinical variables

. Treatment of TC cells with the PARP inhibitor olaparib sensitized cells to cisplatin treatment, in particular cisplatin-resistant cell lines

. Currently, two phase II trials are evaluating the potential of PARP inhibition in relapsed or refractory TC, either as single agent (NCT02533765, active, not recruiting) or combined with gemcitabine and carboplatin (NCT02860819, active and recruiting).

Novel circulating biomarkers in TC

Serum tumor markers LDH, AFP and ß-HCG are used to follow clinical response

to chemotherapy in patients with metastatic disease. However, only ~60% of TC

patients show elevated tumor markers at start of treatment

. 90% of patients

with non-seminomatous tumors are positive for AFP or ß-HCG, while 30% of

seminoma patients are positive for ß-HCG

. Of note, AFP can be produced by

other malignancies as well, including hepatocellular carcinoma and chronic

liver diseases, e.g. cirrhosis. Other tumor types can also secrete ß-HCG, like

pancreatic, biliary and gastric cancers

. Independent of tumor type, LDH is

elevated in 40- 60% of cases; although LDH is the least specific marker of all

17

three

. The IGCCC stratification helps identifying patients that are at higher risk of dying of the disease. However, a large subset of patients classified as intermediate and poor prognosis will be cured with chemotherapy. Tumor markers that more accurately predict disease outcome are therefore needed.

Only a few novel circulating biomarkers have been studied in TC: the circulating cytokeratin 18

, the demethylated promoter regions of the long non-coding RNA X inactive transcript (XIST) gene

and the non-coding microRNA cluster miR- 371

. Circulating full-length and caspase-cleaved cytokeratin 18 (CK18) are regarded as markers of cell death in patients treated with chemotherapy. CK18 levels were evaluated in 34 patients during the course of chemotherapy to study their correlation with treatment response. Even though CK18 levels rose after start of each chemotherapy course, the study lacked enough patients with poor clinical outcome to confirm the utility of this marker to predict clinical response.

Demethylated promotor regions of XIST have been evaluated in a small study using plasma DNA of TC patients and healthy donors. 55% of non-seminoma and 71% of seminoma patients’ plasma was positive for unmethylated XIST, while 0% of the healthy donors resulted positive. Despite the high specificity of this tumor maker, sensitivity was relatively low. In addition, no studies confirming the applicability of demethylated XIST have been published. Finally, several studies have shown the potential utility of members of the miR-371 cluster (miR-371a-3p, miR-372-3p and miR-373-3p) and the pluripotency associated miR-367-3p

in diagnosis

and follow-up of TC patients. Levels of these miRs in blood have been shown to predict the presence of viable disease

or detect relapses after chemotherapy

. As such, these miRs are recognized as putative tumor markers for diagnosis and follow-up of TC patients.

Novel preclinical models in TC

Despite the fact that TC is a complex and heterogeneous disease, TC models including cell lines and xenografts are scarce. Only 20 human and mouse cell lines

and less than 10 xenograft models

have been reported. Available cell lines and xenografts almost exclusively cover the embryonal carcinoma subtype highlighting the obvious need for more models representing the other histological subtypes.

Patient derived xenografts (PDX) are used more frequently in current cancer

research due to several advantages over cell line-based xenografts. Some highly

valuable features of PDX models are maintenance of tumor heterogeneity, high

similarity to human tumors

and prediction of drug response to treatment

.

Use of TC PDX models in cancer research has been described

. So far, only

14 orthotopically established TC PDX models

, and 12 subcutaneous TC PDX

models

have been reported. However, only one systematic report is available

of their establishment

. The aforementioned study described the establishment

of 14 non-seminomatous PDX models, specifically from the choriocarcinoma, embryonal carcinoma and yolk sac subtypes, as well as mixed tumors with yolk sac, teratoma and embryonal carcinoma components. Orthotopic implantation of the tumor pieces was more successful than subcutaneous implantation.

These PDX models were used to study cisplatin resistance and to test novel combinatorial strategies using a glucosylceramide synthase (GCS) inhibitor, DL- threo-PDMP, and a multi-targeted receptor tyrosine kinase inhibitor, sunitinib, in combination with cisplatin

.

Other promising 3D models in cancer research are organoids. Testicular organoids have been developed mostly from non-cancerous human adult tissue with the limitation of testicular histological organization being hardly recognized

. One study that used seminoma tumors to develop hanging-drop cultures reported that tumor morphology was preserved, however, it was conserved for only 7 days, along with proliferative capacity

. Sensitivity to chemotherapeutic drugs like cisplatin was tested in human organoids derived from non-cancerous tissue showing decreased viability compared to untreated organoids

. However, toxicity studies using other anticancer drugs and combinatorial strategies on cancerous TC organoids are lacking.

Thus, even though TC is a highly curable disease there are some therapeutic challenges left to tackle. Tumor marker evaluation before and during treatment is a highly valuable tool, yet, the available tumor markers, AFP, ß-HCG and LDH have several limitations in terms of sensitivity and specificity. This is mostly detrimental for follow-up of patients. Another issue affecting TC patient’s survival is cisplatin resistance, since there are no other therapeutic options for these men that relapse and do not respond to second line treatment or those that develop refractory disease. Finally, one of the most reliable cancer models that researches have are PDX models, but few TC models are available. By increasing the amount of models and the biological knowledge of these models, we could test novel therapies and study cisplatin resistance in depth.

Therefore, the aim of this thesis is to tackle these therapeutic challenges. Clinical

use of miR levels as tumor markers was evaluated in TC patients in order to

improve diagnosis, stratification and follow-up of TC patients. Additionally, we

explored cisplatin resistance in TC and developed in vivo models to test novel

combinatorial therapies identified in the laboratory.

19

In chapter 2 we explored the behavior of three microRNAs, miR-371a-3p, miR-373- 3p and miR-367-3p, in TC patients with metastatic disease receiving chemotherapy. Recent studies have shown the above mentioned miRs are upregulated specifically in TC patients compared to other cancer patients. Relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TC patients using the ampTSmiR test. Two cohorts were included:

a prospectively included and a retrospectively selected cohort were studied.

In total, 109 patients were included in the study. Blood samples were drawn at start of treatment and during follow-up at week 1, 3, 6, 8 and after 3-6 months.

The last evaluated sample was taken after 1 year of completing chemotherapy.

We studied the behavior of miR levels during and after chemotherapy and it’s correlation with classical tumor markers, treatment and poor outcome.

In chapter 3 we aimed to find novel combinatorial therapies to sensitize TC cells lines towards cisplatin in order to provide new treatment options for TC patients that do not respond to cisplatin-based chemotherapy. It has been shown that PI3K/Akt downregulation sensitizes resistant TC cell lines and PDX models to cisplatin

. Additionally, hyperactivation of the PI3K/AKT/mTORC pathway was linked to cisplatin resistance showing resistant sublines had higher levels of p-AKT473 compared to their sensitive parental cells

. In this chapter we studied the activation of kinases, including receptor tyrosine kinases, and downstream substrates in five cisplatin-sensitive or resistant TC cell lines using phospho-kinase arrays and western blotting. We tested inhibitors of most active kinases in the PI3K/AKT/mTORC pathway using apoptosis assays and survival assays to evaluate the most effective treatment in TC cell lines. Additionally, two TC patient-derived xenografts (PDX) from a chemo-sensitive and a -resistant patient, were treated with cisplatin in the absence or presence of kinase inhibitor.

In chapter 4 we evaluated the feasibility to use tumor samples obtained via orchiectomy from TC patients operated at the University Medical Center Groningen to develop subcutaneous TC PDX models between March 2016 and June 2018, we obtained 10 tumor samples from which one tumor sample was diagnosed as a precancerous lesion, eight were non seminomas and one was pure seminoma. We could establish 3 TC PDX models by subcutaneous implantation of solid tumor pieces in NOD scid gamma (NSG) mice. PDX models and matched patient tumors were characterized using immunohistochemistry to assess proliferation (Ki-67), human vs mouse stroma (Cyclophilin A) and caspase activity (cleaved caspase-3) as well as copy number variation and RNA- sequencing analysis to evaluate tumor evolution over several passages. PDX models were treated with cisplatin and other targeted drugs to assess their sensitivity towards them.

OUTLINE OF THE THESIS

In chapter 5, we investigated whether PDX models can be used as suitable models to study two well-known TC biomarkers: AFP and ß-HCG. These markers are used in diagnosis, and follow-up of TC patients. However, not all tumors from TC patients secrete these proteins. In addition, chemotherapy treatment results in normalized marker levels in some initially marker-positive TC patients, although viable tumor cells are still present. Since PDX tumors consist of human tumor cells and mouse normal cells, these models allow studying tumor cell specific secretion of these markers. We selected three PDX models, derived from three patients that were AFP positive and two of them having detectable ß-HCG levels as well. We related the expression of these markers in the PDX models before and after treatment with cisplatin with tumor volume using immunohistochemistry and ELISA.

In chapter 6, we summarized and analyzed the findings of this thesis in light of

current knowledge, and finally provide future perspectives.

21

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Chapter 2

MiR-371a-3p, miR-373-3p and miR-367-3p as serum biomarkers in metastatic testicular germ cell cancers before, during and after chemotherapy

Ximena Rosas Plaza, Ton van Agthoven, Coby Meijer, Marcel A. T. M. van Vugt, Steven de Jong, Jourik A. Gietema and Leendert H. J. Looijenga.

Cells, 2019, 8, 1221

Background: LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and ß-HCG (human chorionic gonadotropin) are used in diagnosis, and follow-up of Testicular Germ Cell Cancer (TGCC) patients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p and miR-367-3p and clinical features in metastatic TGCC.

Methods: Relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TGCC patients. A prospectively included and a retrospectively selected cohort were studied (total patient number=109). Blood samples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) levels were determined using the ampTSmiR test.

Results: At start of chemotherapy, miR-371a-3p, miR-373-3p and miR-367-3p levels were positively correlated to LDH. Median level of these miRs was higher in patients who developed a relapse after complete biochemical remission (n=34) than in those who had complete durable remission (n=60). Higher levels of miR-367-3p were found in patients with refractory disease (n=15) compared to those who had complete response. miR levels decreased during the first week of chemotherapy in patients with complete response and stayed below threshold after one year of treatment.

Conclusion: High miR levels at start of chemotherapy are associated with worse clinical outcome and can assist in early diagnosing of relapses.

Keywords: testicular germ cell cancer, microRNA, serum biomarkers, ß-HCG, AFP, LDH.

Abstract