FIELD The invention is in the field of oncology. More specifically, the invention relates to methods for typing and treating germ cell tumors.

1. Introduction

Over the past years we have witnessed a substantial increase in the number of publications focusing on liquid biopsies. These are particularly useful in the context of cancer, as non-invasive means of diagnosis and follow-up [Dominguez- Vigil ET AL., 2018. Oncotarget 9: 2912-2922] MicroRNAs are among the various liquid biopsy-based molecular biomarkers showing promise in this field. They are involved in the post-transcriptional regulation of the functionality of genes, and are crucial modulators of several biological processes, including embryonic and germ cell development [Eini et al., 2013. Int J Dev Biol 57: 319-332] One of the advantages of microRNAs as liquid biopsy-based biomarkers relates to their relative stability in body fluids. Moreover, they can be easily detected and quantified in a cost-beneficial manner, with high sensitivity and specificity [Anfossi et al., 2018. Nat Rev Clin Oncol 15: 541-563.3]

Germ cell tumors (GCTs) are very diverse, comprising various histological subtypes (the most common being seminomas [SEs] and the several non-seminoma [NS] subtypes), anatomical distributions (both gonadal - testicular and ovarian - and extragonadal tumors, along the midline of the body) and afflicting a wide range of age groups (pediatric, young-adults and even old-adults) [Lobo et al., 2018. Hum Pathol 82: 113-124] Their most fascinating characteristic is that they are developmental cancers: each tumor entity resembles a phase of embryonic and germ cell development and recapitulates the epigenetic pattern of the respective originating cell [Lobo et al., 2019. Int J Mol Sci 20: 258; Stevenson and Lowrance, 2015. Urol Clin North Am 42: 269-275] The main variant based on epidemiological characteristics are the so-called Type II testicular germ cell tumors (TGCTs), also known as germ cell neoplasia in situ (GCNIS)-related GCTs of the testis. They are the most common neoplasms among young-adult men in Western civilization, but are also amongst the most curable solid cancers, which could make one assume there is not much more to improve in this field [Curreri et al., 2015. Urol Oncol 33: 392-398] However, precisely because of this, both patients and clinicians face novel unexpected challenges, including risk of overtreatment, exposing patients to unnecessary long-term side effects of chemo- and radiotherapy; and also insecurity about stratification of patients to different follow-up and treatment protocols [Ostrowski and Walsh, 2015. Urol Clin North Am 42: 409-420; Honecker et a , 2018. Ann Oncol 29: 1658-1686; Jacobsen and Honecker, 2015. Andrology 3: 111- 121; Hamilton et a , 2019. J Clin Oncol 37: 1919-1926] The existing biomarkers used nowadays in the clinic (alpha fetoprotein [AFP], human chorionic gonadotropin subunit 6 [h-HCG] and lactate dehydrogenase [LDH]) are informative, but show limited utility in daily practice to respond to all these issues; therefore, better biomarkers for the disease are needed [Nicholson et a , 2019. Cancer Epidemiol 59: 15-21; Costa et a , 2017. Epigenomics 9: 155-169;

Dieckmann et a , 2019. Biomed Res Int 2019: 5030349]

From the biological perspective over these neoplasms, microRNAs emerge as promising biomarkers [Murray and Coleman, 2012. Nat Rev Urol 9: 298-300] In fact, a set of (embryonic) microRNAs (including the miR-371/373 cluster and the miR-367) have proved their value in the past years as biomarkers of (T)GCTs in a multitude of studies with various designs [Murray et a , 2011. Am J Clin Pathol 135: 119-125; Rijlaarsdam et a , 2015. Andrology 3: 85-91; Murray et a , 2016. Br J Cancer 114: 151-162; Dieckmann et a , 2017. Fur Urol 71: 213-220; Radtke et a , 2018. Urol Int 100: 470-475.]. Particularly remarkable is the miR-371a-3p, outperforming classical serum markers in various clinical contexts [Henrique and Jeronimo, 2017. Fur Urol 71: 221-222; Singla et a , 2019. Fur Urol 76: 541-542; Nappi and Nichols, 2019. Urol Clin North Am 46: 449-457] It is related to all different histological elements of (T)GCTs, except mature teratoma, for which a proven informative biomarker is lacking so far. This specific microRNA profile in (T)GCTs might also be exploited for treatment purposes, targeting overexpressed oncogenic microRNAs and/or replenishing underexpressed tumor suppressor microRNAs [Nappi and Nichols, 2019. Urol Clin North Am 46: 449-457], or even to be used for suicide gene activation [Murray and Coleman, 2019. J Clin Oncol 37: 1432-1435]

For any biomarker to be introduced in the clinic, appropriate technical issues should be considered. Specifically, for microRNAs, a standardized pipeline with appropriate quality control and normalization is crucial to obtain reliable results [van Agthoven et al., 2017. Cell Oncol 40: 379-388; Radtke et al., 2017. J Cancer Res Clin Oncol 143: 2383-2392] Another important issue relates to hemolysis content in blood samples, as it can interfere with the detection levels of certain microRNAs [Bedel et al., 2017. Stem Cells Transl Med 6: 382-393; Shah et al.,

2016. PLoS One 11: e0153200], as well as the choice of analysis of serum or plasma. However, the real impact of these matters on specific assays and the best way to approach them is still debatable.

MicroRNAs can be secreted from tumor cells in various ways [Myklebust et al., 2019. Frontiers in Genetics 10: 463] In spite of the data on the putative impact in a clinical setting, microRNA synthesis and secretion dynamics in (T)GCTs are still largely unknown, and a proper characterization of these processes in (T)GCTs has not yet been tackled. Since these representative models reflect to some extent the biology of these tumors, complementing such in vitro data with further data derived from in vivo pre-clinical models could be extremely valuable to identify the most informative microRNAs for clinical application.

The aim of this work is to investigate in detail the dynamics of microRNA synthesis and secretion in (T)GCT cell lines, correlating with patterns observed in mouse models, achieving a reliable combined in vitro and in vivo model for identifying the most promising candidate microRNAs. In addition, the impact of pre-analytical variables (hemolysis, choosing serum vs. plasma) on microRNA quantification is investigated. Moreover, the potential role of miR-375 in a liquid biopsy setting, confirming or disproving preliminary data reported on tumor tissues, is performed. This setup will be informative for other disease processes as well.

2. Brief description of the invention

The invention is based on a combined in vitro and in vivo identification model to predict relevant microRNAs in germ cell tumors (GCT), taking into account various pre-analytical variables. The model is informative to identify relevant microRNAs, especially in a liquid biopsy setting.

In a recent integrated analysis, Shen et al. [Shen et al., 2018. Cell Rep 23: 3392-3406] suggested that miR-375 is overexpressed in tissue samples from teratoma and yolk sac tumor (and mixed tumors containing these subtypes). This finding could be useful, particularly in the context of primary diagnoses in cases of pediatric GCT, as well as in the metastatic context after chemotherapy in both pediatric and adult patients. The reason is that detection of residual teratoma is clinically important and challenging. However, this data has not been validated so far. The invention provides a method of typing an individual for the presence of a germ cell tumor, comprising providing a sample of said individual, preferably a RNA sample, quantifying an expression level of hsa-miR-885-5p in said sample, comparing the quantified expression level of miR-885-5p in said sample to a level of expression of miR-885-5p in a control, and typing said individual for the presence of a germ cell tumor based on the relative expression level of miR-885-5p in said sample of said individual.

A preferred method further comprises quantifying an expression level of miR- 371a-3p in said sample and typing said individual for the presence of a germ cell tumor based on the relative expression levels of miR-885-5p and miR-371a-3p. A preferred method may further comprise quantifying expression levels of miR-448 and miR-197-3p in said sample, and typing said individual for the presence of a germ cell tumor based on the relative expression levels of miR-885-5p, miR-448 and miR-197-3p, or on the relative expression levels of miR-885-5p, miR-371a-3p, miR- 448 and miR-197-3p. A method of the invention comprises isolating RNA from said sample and determining individual miR expression levels in the isolated RNA. Preferably, individual miR expression levels are determined following bead-based microRNA capture.

In an embodiment, miR expression levels are determined by reverse transcriptase- quantitative amplification, preferably by reverse transcriptase- quantitative polymerase chain reaction.

In an embodiment, miR expression levels are determined by contacting a nucleic acid with a peptide nucleic acid (PNA) oligomer capable of hybridising to a portion of the miR sequence, and integrating a modified base with the nucleic acid/PNA duplex that is complementary to a nucleobase of the miR nucleic acid. characterised by means of the detectable tag of the PNA monomer. The integration of a specific modified base may be determined by mass spectrometry, or, for example, by integration of a labeled modified base such as a fluorescently labeled modified base. In an embodiment, said miR expression levels are determined by sequencing, preferably next generation sequencing.

The sample in methods of the invention comprises germ cell tumor cells. Said sample preferably is a biopsy, such as a liquid biopsy. Said sample preferably is or comprises blood plasma.

Upregulation of miR-885-5p, preferably of miR-885-5p, miR-885-5p and miR- 371a-3p, and non-upregulation of miR-371a-3p, when compared to the control, is indicative for the presence of a teratoma, especially a mature teratoma, in methods of typing according to the invention.

Upregulation of miR-371a-3p and non-upregulation of miR-885-5p are indicative for the presence of an embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminoma-like tumor, pure or mixed, in methods of typing according to the invention.

The invention further provides a method of treating a patient suffering from a germ cell tumor comprising typing an individual for the presence of a germ cell tumor by a method according to the invention, treating a patient that is typed as having upregulation of miR-885-5p, preferably upregulation of miR-885-5p and non-upregulation of miR-371a-3p, by surgery, and treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non-upregulation of miR-885-5p, with a platinum compound and at least one alkaloid.

Said platinum compound preferably is carboplatin or cisplatin. Said at least one alkaloid preferably comprises a taxane, etoposide, or a combination thereof.

A preferred method of treating according to the invention comprises treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non-upregulation of miR-885-5p, with bleomycin, etoposide and cisplatin.

The invention further provides a method of treating a patient suffering from a germ cell tumor comprising typing an individual for the presence of a germ cell tumor by a method of typing according to the invention, treating a patient that is typed as having upregulation of miR-885-5p, preferably upregulation of miR-885- 5p and non-upregulation of miR-371a-3p, by down-modulation of miR-885-5p expression, and treating a patient that is typed as having upregulation of miR- 371a-3p, preferably upregulation of miR-371a-3p and non-upregulation of miR-885- 5p, with down-modulation of miR-371a-3p expression.

The invention further provides a method of treating a patient suffering from an embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminoma-like tumor, pure or mixed germ cell tumor, comprising altering a differentiation stage of said tumor by upregulation of miR-885-5p in said tumor, thereby inducing differentiation of said tumor into a teratoma, thereby enabling treatment of said patient, for example, by surgery. 3. Figure legends

Figure 1. MicroRNA expression levels in cell lines and matched conditioned media. (A-B) Waterfall plots showing Ct values for microRNAs in cell lines (A) and matched conditioned media (B) (2102Ep, NCCIT, NT2 and TCam-2). Targets miR- 371a-3p, miR-372-3p and miR-367 are highlighted in color (orange, red and green, respectively); (C) Venn diagram showing detected microRNAs in cells (left panel) and matched conditioned media (middle panel), and microRNAs specifically secreted by each cell line after eliminating the microRNAs already present in the fetal calf serum (i.e., negative control, right panel). Target miR-371a-3p is highlighted by a star. Numbers outside the Venn diagram (not included in any ellipse) refer to microRNAs not detected in any sample. Abbreviations: FCS: fetal calf serum.

Figure 2. Effect of cell proliferation, multiple freezing/thawing and exosomal isolation in microRNAs detection in cell media. (A) Cell proliferation (right) and effect on miR-371a-3p Ct values for TCam-2 and NCCIT media. The asterisk indicates the time of refreshment of medium; (B) miR-371a-3p Ct values for conditioned medium of TCam-2 according to different incubation temperatures and freezing/thawing; (C) miR-371a-3p Ct values in TCam-2 cell medium after microRNA bead-based isolation or after exosomal fraction isolation by different methods. Absence of cel-miR-39-3p supports that exosome isolation step was successful. High Ct values for cel-miR-39-3p were depicted in bead isolation (but still lower than in exosome isolation) because of low concentration of the microRNA. Abbreviations: AmiR - A-beads microRNA isolation; Ex AmiR - total exosome isolation prior to A-beads microRNA isolation; Ex Kit - total exosome isolation prior to RNA isolation with total exosome RNA isolation kit; Ex 63 - exosome isolation using Dynabeads coated with anti-CD63 antibodies.

Figure 3. Prediction of tumor specific microRNAs able to be detected in liquid biopsy samples. MicroRNA expression in mice tumor xenografts vs. matched endpoint plasma samples. Ct values for mouse human tumor xenografts with malignant (A) and benign (B) histology vs. matched endpoint plasma samples from the same mice. Notice that in (A) the microRNAs already shown to be informative (cluster 371/373 and microRNA-367) are detected both in xenografts and plasma samples. Figure 4. Validation of candidate microRNAs for teratoma histology in a post chemotherapy retroperitoneal lymph node dissection series. (A-C) Relative levels of miR-885-5p, miR-448 and miR-197-3p in serum of patients with teratomas and healthy males; (D-F) Relative levels of miR-885-5p, miR-448 and miR-197-3p in sera of patients with the various histological tumor types and healthy males; (G-H) ROC curves for the discrimination among teratoma patients and healthy male individuals. Because controls were included in the comparison, the reference sample was the one showing the highest Ct value in these analyses. Abbreviations: AUC - area under the curve; CT - chemotherapy; ROC - receiving operator characteristic; RPFND - retroperitoneal lymph node dissection; TE - teratoma. Figure 5. Relative microRNA expression levels after orchiectomy. (A) Box plots showing relative miR-371a-3p, miR-375, miR-372-3p, miR-373-3p and miR- 367 expression levels in serum samples of clinical stage I testicular germ cell tumor patients (n=12) at different time points after orchiectomy. (B) Fine graphs showing relative miR-371a-3p, miR-375, miR-372-3p, miR-373-3p and miR-367 levels in 3 patients with multiple serum samples collected within the first 24 h after orchiectomy. The horizontal dashed line depicts the 50% reduction level (bottom right panel) for miR-371a-3p; (C) Scatter plots showing the correlation between relative miR-371a-3p, miR-372-3p and miR-373-3p levels and primary tumor size in patients with clinical stage I testicular germ cell tumor (n=12). Figure 6. miR-375 and miR-371a-3p as serum biomarkers for TGCTs in the context of post-chemotherapy RPFND. Boxplots showing: relative post chemotherapy miR-371a-3p (A) and miR-375 (D) expression levels in TGCT patients with different histological subtypes (fibrosis/necrosis, teratoma, viable tumor) at RPFND; relative miR-371a-3p (B) and miR-375 (E) expression levels in different time points: pre-chemotherapy, post-chemotherapy and post-RPLND; and relative pre-chemotherapy miR-371a-3p (C) and miR-375 (F) expression levels according to disease stage (I/II vs III). Abbreviations: CT - chemotherapy; RPLND - retroperitoneal lymph node dissection. Figure 7. miR-375 and miR-371a-3p as serum biomarkers for GCTs with teratoma/yolk sac tumor histologies. Boxplots showing relative levels of miR-371a- 3p (A-B) and miR-375 (C-D) among different histological subtypes and controls within the same age range. Because controls were included in the comparison, the reference sample was the one showing the highest Ct value in these analyses. Figure 8. The effect of hemolysis on microRNA levels. miR-23a/451a ratio (A) and Ct-values of the spike-in ath-miR-159a (B), normalizer hsa-miR-30b-5p (C), and target assay hsa-miR-372a-3p (D) according to hemolysis visual scoring and the pre-determined miR-23a/451a ratio cutoff (9.15).

Figure 9. Ct values for microRNAs in matched serum and plasma samples from normal males. Ct values of normalizer hsa-miR-30b-5p (A), spike-in ath-miR- 159a (B), and target microRNAs hsa-miR-371a-3p (C) and hsa-miR-375 (D) in matched serum and plasma samples from normal blood donors (n=66); Ct values of hemolysis controls hsa-miR-23a-3p (E) and hsa-miR-451a (F), and respective miR- 23a/451a ratio (G) in matched serum and plasma samples from normal blood donors (n=ll).

Figure 10. Systematic overview of the (combined in vitro and in vivo) identification model for microRNAs in liquid biopsies (left panel) MicroRNAs are secreted by tumor cells and released into the bloodstream, therefore they could possibly be detected in liquid biopsies; (upper panel) In order to predict which microRNAs are informative in liquid biopsies, a combined in vitro and in vivo identification model was set up. In vitro: cell lines + conditioned media. In vivo: xenograft mice + plasma samples. Human samples: patient cohort studies in serum/plasma/CSF; (middle panel) Methods used for identifying microRNAs. Appropriate negative controls should be used depending on the context: fetal calf serum, normal plasma/serum/CSF samples and normal mice. Bead-captured-based microRNA-isolation assures good results and is less troubled by detection issues such as hemolysis. High throughput strategies followed by targeted-assay validation are warranted. Analysis and quality control steps are crucial to assure reproducible results; (bottom panel) In parallel, there are several detection issues to take into account when identifying microRNAs. MicroRNA levels could be influenced by hemolysis, as they are released from ruptured erythrocytes. Differences between Ct values of specific target assays exist between serum and plasma, so mixed cohorts are troublesome. MicroRNAs should have a steady decrease after surgery rather than a fluctuating expression level over time. Exosomes are a major means of microRNAs secretion. Temperature could also possibly influence the dynamics of microRNA secretion, although it seems to be a rather stable process. 4. Detailed description of the invention

Definitions

The term “germ cell tumor”, as is used herein, refers an abnormal and excessive growth of germ cells. Germ-cell tumors can be cancerous or benign. Germ cells tumors normally occur inside an ovary or testis, but may originate outside the gonads. A germ cell tumor can be a teratoma, a yolk sac tumor, a choriocarcinoma, an embryonal carcinoma, a seminoma, or a mixed germ cell tumor such as a teratocarcinoma.

The term “teratoma”, as is used herein, refers to a type of germ cell tumor that comprises different types of cells originating from all three germinal cell layers. A teratoma may comprise mature cells, immature cells, or a mixture of mature and immature cells. A teratoma may be benign, or malignant.

The term “mature teratoma”, as is used herein, refers to a type of germ cell tumor comprising mature cells originating from all three germinal cell layers. A mature teratoma generally is benign. The term “yolk sac tumor”, as is used herein, refers to a type of germ cell tumor that shows endodermal differentiation, including derivatives thereof such as intestine, liver, lung. A yolk sac tumor may comprise mature cells, immature cells, or a mixture of mature and immature cells. A yolk sac tumor usually is malignant.

The term “choriocarcinoma”, as is used herein, refers to a type of germ cell tumor is a trophoblastic tumor comprising proliferation of syncytiotrophoblast, cytotrophoblast and intermediate trophoblast. A choriocarcinoma is malignant.

The term “embryonal carcinoma”, as is used herein, refers to a type of germ cell tumor comprising primitive epithelial tumor cells. An embryonal carcinoma often presents in the testes of adolescent boys. An embryonal carcinoma usually is malignant.

The term “seminoma”, as is used herein, refers to a type of germ cell tumor comprising undifferentiated cells. A seminoma may be termed seminoma in testis, dysgerminoma in ovary, and germinoma in the brain. A seminoma is always malignant.

The term “mixed germ cell tumor”, as is used herein, refers to a type of germ cell tumor comprising two or more different types of germ cell tumors, such as a teratoma and embryonal carcinoma, and a seminoma, yolk sac tumor and teratoma. These tumors typically occur in adults aged 20-50 years, with an average age at diagnosis of about 30 years.

The term “microRNA”, as is used herein, refers to small non-coding RNA molecules of about 20-25 nucleotides in length. Said microRNAs may be partially complementary to one or more messenger RNA (mRNA) molecules. Their apparent function is to modulate gene expression by a variety of mechanisms including breakdown of a mRNA, prevention of translation of a mRNA, or stabilization of a mRNA. The miRBase, hosted by the Sanger Institute, provides individual miR nomenclature, sequence data, annotation and target prediction information.

The nomenclature of miRs is standardized. The prefix "miR" is followed by a dash and an unique number. A capitalized "miR-" refers to the mature form of the miR, while an uncapitalized "mir-" refers to the pre-miR and the pri-miR. miRs with nearly identical sequences except for one or two nucleotides are annotated with an additional lower case letter, for example, miR-124a and miR-124b. miRs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts, termed pri-miRs. The primary transcript is cleaved by a ribonuclease enzyme to produce an approximately 70-nt stem-loop precursor miR (pre-miR), which is further cleaved by a cytoplasmic Dicer ribonuclease to generate the mature miR and antisense miR star (miR*) products. When it can not be determined which sequence is predominantly expressed, the microRNAs are indicated as -5p (from the 5' arm) or -3p (from the 3' arm). Pre-miRs, pri-miRs and genes that result in identical mature miRs but that are located at different places in the genome are indicated with an additional dash-number suffix. For example, the human pre-miRs hsa-mir- 194-1 and hsa-mir- 194-2 result in an identical mature miR (hsa-miR-194) but are derived from genes located in different genome regions.

The term “miR-371a-3p”, as is used herein, refers to a specific miR molecule that is expressed from a gene on human chromosome 19. Within a region of 1.1 kb, two further miR genes are located, miR-372 (MI0000780) and miR-373. Said genes are expressed in a similar, though not identical, fashion as “miR-371a-3p”. Hence, in some instances, upregulation or non-upregulation of miR-371a-3p refers to upregulation or non-upregulation of miR-371a-3p, miR-372 and miR-373.

The term “liquid biopsy”, as is used herein, refers to any liquid portion of the body such as milk, blood, synovial fluid, urine, cerebrospinal fluid, bronchiolar lavage fluid, extracellular fluid including lymphatic fluid and transcellular fluid, tear fluid, and/or vitreous humor.

The term “blood”, as is used herein, includes reference to blood serum and blood plasma. The terms “blood serum” and “blood plasma” both refer to blood components without cells, whereby blood serum also excludes clotting factors such as fibrinogen. As is known to a person skilled in the art blood may, for example, be centrifuged to remove cellular components. The thus obtained plasma may be coagulated followed by, for example, centrifugation to remove clotting factors, resulting in serum. The term “platinum compound”, as is used herein, refers to a alkylating, platinum based chemotherapy agent used to treat tumors. Said platinum compound includes, for example, cisplatin (azane;dichloroplatinum), carboplatin (azanide;cyclobutane-l,l-dicarboxylic acid;platinum(2+)), nedaplatin (azanide;2- hydroxyacetic acid;platinum(2+)), oxaliplatin ([(lR,2R)-2- azanidylcyclohexyl]azanide;oxalate;platinum(4+)), and satraplatin (acetic acid;azane;cyclohexanamine;dichloroplatinum).

The term “alkaloid”, as is used herein, refers to a group of organic compounds that contain predominantly basic nitrogen atoms. An alkaloid preferably includes a taxane, docetaxel, paclitaxel, a peptide antibiotic such as bleomycin and actinomycin, etoposide, vincristine, vinblastine and vinorelbine.

Methods of typing

Germ cell tumors are neoplasms that occur in children, teens and adults. Germ cell tumors are rare, about 900 children and adolescents are diagnosed in the Netherlands each year. They make up about 4% of all cancers in children and adolescents. Germ cell tumors most commonly appear in the gonads, but may also appear at ectopic sites of the body.

The invention provides a method of tying an individual for the presence of a germ cell tumor, comprising providing a sample of said individual, quantifying an expression level of miR-885-5p in said sample, comparing the quantified expression level of miR-885-5p in said sample to a level of expression of miR-885-5p in a control, and typing said individual for the presence of a germ cell tumor based on the relative expression level of miR-885-5p in said sample of said individual. Said sample comprises germ cell tumor cells and may be isolated from the tumor mass, for example as a biopsy, or may be isolated as a liquid biopsy.

A preferred sample is or comprises a liquid biopsy, preferably blood, more preferably plasma.

A sample can be freshly prepared at the moment of isolating, or is cooled and stored below 0 °C after isolation. Said sample preferably is stored at -70°C until further assaying as is described herein below.

Alternatively, said sample is stored under conditions that preserve the quality of nucleic acid such as DNA and/or RNA. Examples of such preservative conditions are the addition of RNase inhibitors such as RNAsin (Pharmingen), RNasecure (Ambion), RNAlater (Assuragen; US06204375), and/or the addition of non-aquous solutions such as an universal methanol/polyethylene glycol-based fixative and/or a Tissue -Tek VIP® Fixative (Sakura Finetek USA Inc.).

Further assaying of a sample comprises extracting or isolating RNA from said sample. Methods for isolating RNA, preferably total RNA, are known in the art and include guanidinium- acid-phenol extraction methods such as TRIzol and TRI reagent (ThermoFisher Scientific); silica technology, for example using glass fiber filters such as RNeasy (Qiagen); density gradient centrifugation using cesium chloride or cesium trifluoroacetate; magnetic bead technology such as Dynabeads (ThermoFisher Scientific); and/or lithium chloride/urea based extraction (Auffray and Rougeon, 1980. Eur J Biochem 107: 303-314). miR expression levels may be determined following bead-based microRNA capture. For example, magnetite nanoparticles carrying a complementary sequence of a specific miR on their surface, such as antisense miR-885-5p, may be used to extract and isolate miR-885-5p from a sample. Said antisense-coated particles may comprise antisense nucleotide molecules, or antisense nucleotide mimetics such as locked nucleic acid (LNA) or peptide nucleic acid (PNA) molecules. Commercially available antisense miR-beads may be obtained from, for example, Luminex and DestiNA Genomics).

Methods to determine expression levels of microRNAs including at least miR- 885-5p are known to a skilled person and include, but are not limited to, Northern blotting, quantitative PCR, microarray analysis and RNA sequencing.

It is preferred that RNA expression levels of at least miR-885-5p and other miR sequences are determined simultaneously. Simultaneous analyses can be performed, for example, by multiplex qPCR, RNA sequencing procedures, and microarray analysis.

Microarray-based analysis involves the use of selected biomolecules that are immobilized on a solid surface, an array. A microarray usually comprises nucleic acid molecules, termed probes, which are able to hybridize to gene expression products. The probes are exposed to labeled sample nucleic acid, hybridized, and the abundance of gene expression products in the sample that are complementary to a probe is determined. The probes on a microarray may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA. The probes may also comprise DNA and/or RNA analogues such as, for example, nucleotide analogues or peptide nucleic acid molecules (PNA), or combinations thereof. The sequences may also be in vitro synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.

In the context of the invention, a probe is to be specific for a microRNA such as miR-885-5p. A probe is specific when it comprises a continuous stretch of nucleotides that are complementary to a nucleotide sequence of a microRNA such as miR-885-5p, or a cDNA product thereof. A probe can also be specific when it comprises a continuous stretch of nucleotides that are partially complementary to a nucleotide sequence of a gene expression product of said gene, or a cDNA product thereof. Partially means that a maximum of 5% from the nucleotides in a continuous stretch of at least 20 nucleotides differ from the corresponding nucleotide sequence of a gene expression product of said gene. The term complementary is known in the art and refers to a sequence that is related by base pairing rules to the sequence that is to be detected. It is preferred that the sequence of the probe is carefully designed to minimize nonspecific hybridization to said probe.

It is preferred that the probe is, or mimics, a single stranded nucleic acid molecule. The length of said complementary continuous stretch of nucleotides can vary between 15 bases and several kilo bases, and is preferably between 18 bases and 1 kilobase, more preferred between 20 and 100 bases, and most preferred between about 20 and about 60 nucleotides. Said between about 20 and about 60 nucleotides are preferably identical to a nucleotide sequence of a microRNA, or a cDNA product thereof. To determine the RNA expression level by micro- arraying, the gene expression products in the sample are preferably labeled, either directly or indirectly, and contacted with probes on the array under conditions that favor duplex formation between a probe and a complementary molecule in the labeled gene expression product sample. The amount of label that remains associated with a probe after washing of the microarray can be determined and is used as a measure for the gene expression level of a nucleic acid molecule that is complementary to said probe.

The determined RNA expression level can be normalized for differences in the total amounts of nucleic acid expression products between two separate samples by comparing the level of expression of one or more genes that are known not to differ in expression level between samples.

Another preferred method for determining RNA expression levels is by sequencing, preferably next- generation sequencing (NGS), of RNA samples. High throughput sequencing techniques for sequencing RNA have been developed. NGS platforms, including Illumina® sequencing; Roche 454 pyrosequencing®, ion torrent and ion proton sequencing, and ABI SOLiD® sequencing, allow sequencing in parallel.

Such high throughput sequencing techniques include, for example, sequencing-by-synthesis. Sequencing-by-synthesis or cycle sequencing can be accomplished by stepwise addition of nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in U.S. Patent No. 7,427,673; U.S. Patent No. 7,414,116; WO 04/018497; WO 91/06678; WO 07/123744; and U.S. Patent No. 7,057,026, all of which are incorporated herein by reference. Alternatively, pyrosequencing techniques may be employed. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi et ah, Analytical Biochemistry 242(l):84-9 (1996); Ronaghi, M. Genome Res. 11(1):3-11 (2001); Ronaghi, M. et ah, Science 281:5375, 363 (1998); U.S. Patent No. 6,210,891 ; U.S. Patent No.

6,258,568 ; and U.S. Patent No. 6,274,320, which are all incorporated herein by reference. In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons. Sequencing techniques also include sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are inter alia described in U.S. Patent No 6,969,488 ; U.S. Patent No. 6,172,218; and U.S. Patent No. 6,306,597. Other sequencing techniques include, for example, fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).

Sequencing techniques can be performed by directly sequencing RNA, or by sequencing a RNA-to-cDNA converted nucleic acid library. Most protocols for sequencing RNA samples employ a sample preparation method that converts the RNA in the sample into a double-stranded cDNA format prior to sequencing. Conversion of RNA into cDNA and/or cRNA using a reverse-transcriptase enzyme such as M-MLV reverse-transcriptase from Moloney murine leukemia virus, or AMV reverse-transcriptase from avian myeloblastosis virus, is known to a person skilled in the art.

Real-time PCR, also called quantitative PCR (qPCR), is a technique which is used to amplify and simultaneously quantify a template nucleic acid molecule such as a microRNA. The detection of the amplification product can in principle be accomplished by any suitable method known in the art. The amplified products may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.

Alternatively, the amplified product may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels which maybe associated with nucleotide bases include, for example, fluorescein, cyanine dye and BrdUrd.

When using for example Scorpion primers or a probe-based detection system, a primer or the probe is preferably labelled with a detectable label, preferably a fluorescent label. Preferred labels for use in this invention comprise fluorescent labels, preferably selected from Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO-TEC GmbH, Siegen, Germany), YakimaYellow (Epoch Biosciences Inc, Bothell, WA, USA), Cal610 (BioSearch Technologies, Petaluma, CA, USA), Cal635 (BioSearch Technologies, Petaluma, CA, USA), FAM (Thermo Fisher Scientific Inc., Waltham, MA USA), TFT (Thermo Fisher Scientific Inc., Waltham, MA USA), HEX ((Thermo Fisher Scientific Inc., Waltham, MA USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, Cy7 (Thermo Fisher Scientific Inc., Waltham, MA USA), Alexa dyes (Thermo Fisher Scientific Inc., Waltham, MA USA), Tamra (Thermo Fisher Scientific Inc., Waltham, MA USA), ROX (Thermo Fisher Scientific Inc., Waltham, MA USA), JOE (Thermo Fisher Scientific Inc., Waltham, MA USA), fluorescein isothiocyanate (FITC, Thermo Fisher Scientific Inc., Waltham, MAUSA), and tetramethylrhodamine (TRITC, Thermo Fisher Scientific Inc., Waltham, MA USA). A probe is preferably labeled at the 5’ end with a detectable label, preferably a fluorescent label.

A primer such as a Scorpion primer, or a probe preferably has a fluorescent label at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Quenchers, for example tetramethylrhodamine TAMRA, dihydrocyclopyrroloindole tripeptide minor groove binder, are known in the art.

Preferred quenchers are Black Hole Quencher®- 1 (BHQ1) and BHQ2, which are available from Biosearch Technologies, Petaluma, CA, USA). The BHQ1 dark quencher has strong absorption from 480 nm to 580 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as FAM, TET, CAF Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes. The BHQ2 dark quencher has strong absorption from 599 nm to 670 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes. BHQ1 and BHQ2 may quench fluorescence by both FRET and static quenching mechanisms.

A method of quantifying an expression level of a microRNA such as miR-885- 5p in a sample, may comprise the steps of (a) contacting a nucleic acid with a peptide nucleic acid (PNA) oligomer capable of hybridising to a portion of the nucleic acid and lacking a nucleobase complementary to a nucleobase of the nucleic acid, to form a nucleic acid/PNA duplex; and (b) contacting the nucleic acid/PNA duplex with modified bases according to the first aspect of the invention; wherein the modified nucleobase which integrates with the nucleic acid/PNA duplex is complementary to the nucleobase of the nucleic acid, the nucleotide being characterized by means of the detectable tag of the PNA monomer.

One of skill in the art will appreciate that the part of the PNA oligomer which lacks a base complementary to a nucleobase of the nucleic acid sequence, may present a moiety, for example a secondary amine, capable of reacting reversibly with a moiety of the modified bases described above. As such, upon contact with the nucleic acid/PNA duplex, a modified base which is complementary (or matched) to a nucleobase of the nucleic acid, may be incorporated into the nucleic acid/PNA duplex by the formation of, for example: (i) a reversible iminium species between the secondary amine of the PNA oligomer and the reacting moiety (aldehyde group) of the modified nucleobase and/or (ii) the formation of hydrogen bonds between the modified nucleobase and the nucleobase of the nucleic acid.

In an embodiment, the method may comprise the further step of trapping the base integrated with the nucleic acid/PNA duplex and complementary to (i.e. paired with) the nucleotide of the nucleic acid. For example, the reversible reaction between the secondary amine of the PNA oligomer and the group capable of reversible covalent reactions of the modified nucleobase may be stopped. For example, iminium species may be reduced to give rise to stable tertiary amines using reducing agents such as sodium cyanoborohydride.

It is to be understood that the phrase "capable of hybridising to a portion of the nucleic acid sequence" should be taken to mean that the PNA oligomer is complementary to, or shares a certain degree of homology with, a portion of the nucleic acid sequence.

The skilled artisan will readily understand that by means of the detectable tag present on each of the modified nucleobases contacted with the nucleic acid/PNA duplex, it may be possible to detect which modified base has been incorporated into the nucleic acid/PNA duplex. As such, characterization of the nucleotide of the nucleic acid may easily be achieved. For example, if the modified nucleobase found to have integrated with the nucleic acid/PNA duplex comprises a tag which indicates that it comprises a thymine nucleobase, in accordance with standard complementary base pairing, the nucleotide of the nucleic acid must comprise an adenine nucleobase.

In methods of typing according to the invention, the control preferably is a sample, such as an RNA sample, isolated from a tissue of a healthy individual, or isolated from a tumor, preferably a germ cell tumor. Said germ cell tumor preferably has been identified as a pure or mixed embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminomadike tumor.

Said control may comprise an RNA sample from a relevant cell line or mixture of cell lines. The RNA from a cell line or cell line mixture can be produced in-house or obtained from a commercial source such as, for example, Stratagene Human Reference RNA.

Even more preferably, said control is a pooled RNA sample that is isolated from tissue comprising germ cell tumor cells from multiple individuals, such as from more than 2 individuals, more than 10 individuals, more than 20 individuals, more than 30 individuals, more than 40 individuals, more than 50 individuals. Typing of a sample can be performed in various ways. In one method, a coefficient is determined that is a measure of a similarity or dissimilarity of a sample with a previously established reference miR expression level. Said previously established reference miR expression level may be specific for a certain tumor type, e.g. a teratoma, preferably a mature teratoma. Such a reference expression level can be referred to as a profile template. Typing of a sample can be based on its (dis)similarity to a single profile template or based on multiple profile templates, such as a teratoma and a seminoma.

A number of different coefficients can be used for determining a correlation between the miR expression level in a sample from an individual and a profile template. Preferred methods are parametric methods which assume a normal distribution of the data. One of these methods is the Pearson product-moment correlation coefficient, which is obtained by dividing the covariance of the two variables by the product of their standard deviations. Preferred methods comprise cosine-angle, un-centered correlation and, more preferred, cosine correlation (Fan et al„ Conf Proc IEEE Eng Med Biol Soc. 5:4810-3 (2005)).

Said correlation with a profile template is used to produce an overall similarity score for expression levels of microRNAs that are used. A simil rity score is a measure of the average correlation of gene expression levels of a set of microRNAs in a sample from an individual patient and a profile template. Said similarity score can, but does not need to be, a numerical value between +1, indicative of a high correlation between the gene expression level of the set of genes in a sample of said cancer patient and said profile template, and -1, which is indicative of an inverse correlation. If a similarity threshold value is employed, it is preferably set at a value at which an acceptable number of individual patients with a teratoma would score as false negatives, and an acceptable number of individual patients without a teratoma would score as false positives. A similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.

In methods of the invention, an expression level of miR-885-5p and miR-371a- 3p is determined, preferably of miR-885-5p, miR-448, miR-197-3p, and miR-371a- 3p, more preferably of miR-885-5p, miR-448, miR-197-3p, miR-371a-3p, miR-372, and miR-373.

In case the sample is or comprises blood, serum and/or plasma, methods of typing according to the invention may additionally comprise determining miR-23a and miR-451a expression levels, whereby a miR-23a/miR-451a ratio discriminates between samples with no hemolysis from those with evidence of hemolysis. More specifically, a low miR-23a/451a ratio is obtained in samples with a lower hemolysis score.

In methods of typing according to the invention, upregulation of miR-885-5p, preferably of miR-885-5p, miR-885-5p and miR-371a-3p, and non-upregulation of miR-371a-3p, when compared to the control, is indicative for the presence of a teratoma, especially a mature teratoma. In methods of typing according to the invention, upregulation of miR-371a-3p and non-upregulation of miR-885-5p, preferably of miR-885-5p, miR-885-5p and miR-371a-3p, are indicative for the presence of a, mixed or pure, yolk sac tumor, choriocarcinoma, embryonal carcinoma, or seminoma.

The methods of typing according to the invention may further be accompanied by determining levels of a-fetoprotein (AFP), human chorionic gonadotrophin (hCG) and its b-subunit (hCGb ), lactate dehydrogenase (LDH), placental alkaline phosphatase (PLAP), or a combination thereof, in a liquid biopsy, preferably in blood, serum or plasma. For example, serum elevation of AFP and HCG may be indicative for the presence of yolk sac tumor and choriocarcinoma, respectively.

The presence of one or more of these markers provides a highly sensitive and specific indicator of the presence of certain histologic components. Testing for serum tumor markers prior to definitive treatment may help in confirming the presence of a specific germ cell tumor. Furthermore, serial assay of these markers is useful for monitoring the response to chemotherapy and for subsequent post treatment follow-up.

Methods of treating

In an aspect, the invention provides a platinum compound and at least one alkaloid for use as a medicament for treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non- upregulation of miR-885-5p, according to the methods of the invention.

Further provided is an use of a platinum compound and at least one alkaloid for the preparation of a medicament for treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non- upregulation of miR-885-5p, according to the methods of the invention.

The invention further provides a method of treating a patient suffering from a germ cell tumor comprising typing an individual for the presence of a germ cell tumor by the method of typing according to the invention, treating a patient that is typed as having upregulation of miR-885-5p, preferably upregulation of miR-885- 5p and non-upregulation of miR-371a-3p, by surgery, and treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a- 3p and non-upregulation of miR-885-5p, with a platinum compound and at least one alkaloid.

Treatment by surgery refers to a physical procedure in which tumor- associated cells are removed from the body. Said procedure comprises making incisions in tissue, often skin or tissue aligning internal cavities in a human patient. When the patient is treated by surgery, a part of a tumor, preferably a complete tumor comprising substantially all tumor cells, may be excised from the patient, together with a small margin of healthy cells surrounding the tumor.

Further treatment methods can be performed before or after surgery, being neoadjuvant or adjuvant therapy, respectively. Neoadjuvant therapy is used to reduce the initial tumor size to facilitate subsequent surgery and providing surgery a higher chance of completely excising the tumor. Adjuvant therapy is applied after surgery, and is usually provided when all detectable tumor cells are removed, but when a statistical risk of relapse remains, due to the presence of undetectable tumor cells.

When a patient is further treated with chemotherapy, it is often applied as neoadjuvant therapy, adjuvant therapy or both therapies. Targeted therapy, immunotherapy, and hormone therapy are preferred above chemotherapy whenever possible, as it may result in less side effects. The main side effects of chemotherapy are due to the chemotherapy drugs affecting healthy fast growing cells as well as tumor cells. Targeted therapy is possible whenever a marker is known to which a target agent is available, such as monoclonal antibodies in the case of immunotherapy.

When a patient is further treated with radiation therapy, said radiation therapy is often applied at the beginning of a treatment, thus before surgery and before, during or after neoadjuvant therapy. Alternatively, radiation therapy can be applied after surgery to prevent tumor reoccurrence. Radiation therapy may be intended for curative, adjuvant, neoadjuvant or palliative therapy. Radiation therapy may be applied before or after treating the human patient with a stroma- targeting agent.

A preferred platinum compound is selected from the group consisting of carboplatin and cisplatin (which may be administered at a dosage of 40-100 mg/m ² every 1 to 4 weeks). Said at least one alkaloid preferably comprises a taxane such as paclitaxel, docetaxel, cabazitaxel, a peptide antibiotic, etoposide, or a combination thereof.

A preferred treatment of a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non-upregulation of miR-885-5p, comprises bleomycin (3-[[2-[2-[2-[[(2S,3R)-2-[[(2S,3S,4R)-4-[[(2S,3R)-2- [[6-amino-2-[(lS)-3-amino-l-[[(2S)-2,3-diamino-3-oxopropyl]a mino]-3-oxopropyl]-5- methylpyrimidine-4-carbonyl]amino]-3-[(2R,3S,4S,5S,6S)-3-[(2 R,3S,4S,5R,6R)-4- carbamoyloxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4, 5-dihydroxy-6- (hydroxymethyl)oxan-2-yl]oxy-3-(lH-imidazol-5-yl)propanoyl] amino] -3-hydroxy-2- methylpentanoyl] amino] -3-hydroxybutanoyl] amino]ethyl] - 1, 3-thiazol-4-yl] -1,3- thiazole-4-carbonyl]amino]propyl-dimethylsulfanium), etoposide (5S,5aR,8aR,9R)- 5 - [ [(2R, 4aR, 6R, 7R, 8R, 8aS) - 7, 8- dihy droxy-2-methyl- 4, 4a, 6,7,8,8a- hexahydropyrano[3,2-d][l,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5 -dimethoxyphenyl)- 5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5-f] [l,3]benzodioxol-8-one) and cisplatin.

The invention further provides a method of treating a patient suffering from a germ cell tumor comprising typing an individual for the presence of a germ cell tumor by the method according to the invention, treating a patient that is typed as having upregulation of miR-885-5p, preferably upregulation of miR-885-5p and non-upregulation of miR-371a-3p, by down-modulation of miR-885-5p expression, and treating a patient that is typed as having upregulation of miR-371a-3p, preferably upregulation of miR-371a-3p and non-upregulation of miR-885-5p, with down-modulation of miR-371a-3p expression.

Alteration of expression of a miR gene, for example down-modulation or silencing of miR-885-5p expression, or of miR-371a-3p expression, may be accomplished, for example, by expression of zinc finger protein (ZFP) transcription factors attached to a “gene repression” domain in order to down-regulate (repress) the expression of a hsa-miR-885-5p encoding gene in vivo, preferably in said germ cell tumor cells; by introducing inactivating alterations in the gene encoding miR- 885-5p in vivo, preferably by alteration, including deletion of part or all, of the gene. Said alteration may be accomplished, for example, by TALEN, zinc finger nuclease or CRISPR-CAS mediated alteration.

For this, a nuclease may be targeted to at least one specific position on said hsa-miR-885-5p encoding gene, preferably at least at two specific positions. Said targeting may be mediated by the TALE DNA binding domains, or by CRISPR single chimeric guide RNA sequences. The nuclease, a FOK1 nuclease in the case of a TALEN, and a CAS protein or CAS-related protein, preferably a CAS9 protein, for CRISPR mediated double stranded breaks in the genomic DNA of the gene encoding the hsa-miR-885-5p encoding gene. The introduction of DNA double stranded breaks increases the efficiency of gene editing via homologous recombination, in the presence of suitable donor DNA to alter at least one amino acid residue in a region that encodes a conserved, extracellular domain of a gene encoding a hsa-miR-885-5p encoding gene.

Zinc finger proteins are DNA-binding motifs and consist 5 of modular zinc finger domains that are coupled to a nuclease. Each domain can be engineered to recognize a specific DNA triplet in the region of the gene that encodes a conserved, extracellular domain of said receptor. A combination of three or more domains results in the recognition of a sequence that is specific for a hsa-miR-885-5p encoding gene. Expressing said coupled zinc finger protein-nuclease in a relevant cell, preferably a germ cell tumor cell, in the presence of a recombinant nucleic acid molecule comprising at least part of the hsa-miR-885-5p encoding gene comprising a deletion or alteration, will result in alteration of the gene encoding the hsa-miR- 885-5p encoding gene.

Similarly, synthetic transcription factor DNA binding domains (DBDs) can be programmed to recognize specific DNA motifs. Such transcription activator-like effector (TALE) DNA binding domains (DBD) preferably contain a number, from 7 to 34, highly homologous direct repeats, each consisting of 33-35 amino acids. Specificity is contained in the two amino acid residues in positions 12 and 13 of each repeat. Since the DNA:protein binding code of said two amino acid residues has been deciphered, it is possible to design TALEs that bind any desired target DNA sequence by engineering an appropriate DBD. Typically, the TALEs are designed to recognize 15 to 20 DNA base-pairs, balancing specificity with potential off targeting (Boettcher and McManus, 2015. Mol Cell 58: 575-585). A TALE, preferably at least two TALEs specific for a gene encoding a hsa-miR-885-5p encoding gene, may than be coupled to a nuclease, for example Cast). Expressing said coupled TALE-nucleases in a relevant cell, in the presence of a recombinant nucleic acid molecule comprising at least at least part of the hsa-miR-885-5p encoding gene comprising a deletion or alteration, will result in alteration of the hsa-miR-885-5p encoding gene. A preferred site-specific recombinase is CRISPR associated protein 9 (Cas 9). Cas9 is a RNA-guided DNA endonuclease enzyme that can cleave any sequence that is complementary to the nucleotide sequence in a CRISPR-comprising guide RNA. The target specificity of this system originates from the gRNA:DNA complementarity, and is not dependent on modifications to the protein itself, like in TALE and Zinc-finger proteins.

Alternatively, or in addition, alteration of expression of a miR gene, for example down-modulation or silencing of miR-885-5p expression or of miR-371a-3p expression, may be accomplished by expression of an antisense oligodeoxynucleotide (asODN), such as an siRNA, in said germ cell tumor cells.

Expression of a site-specific recombinase, an asODN and/or a siRNA molecule may be provided by generating an expression cassette encoding the site-specific recombinase, asODN and/or siRNA molecule. Said expression cassette preferably comprises a polymerase II or polymerase III enhancer/promoter. A preferred polymerase III enhancer/promoter is selected from the U6 and HI promoter. Said expression cassette preferably is provided in a vector, preferably a viral vector that is able to transduce germ cell tumor cells. Said viral vector preferably is a recombinant adeno-associated viral vector, a herpes simplex virus-based vector, or a lentivir us -based vector such as a human immunodeficiency virus-based vector. Said viral vector most preferably is a herpes simplex virus-based vector.

Further described herein is a method of treating a patient suffering from an embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminoma-like tumor, pure or mixed germ cell tumor, comprising altering a differentiation stage of said tumor by upregulation of miR-885-5p in said tumor, thereby inducing differentiation of said tumor into a teratoma, preferably a mature teratoma. Said teratoma may enable treatment using surgery.

MiR-885-5p in an embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminoma-like tumor, pure or mixed germ cell tumor, may be upregulated by expression of miR-885-5p by providing said tumor with an expression cassette encoding said miRNA molecule. Said expression cassette preferably comprises a polymerase II or polymerase III enhancer/promoter. A preferred polymerase III enhancer/promoter is selected from the U6 and HI promoter. An especially preferred promoter is the promoter of miR-371a-3p, which is active in embryonal carcinoma, yolk sac tumor, choriocarcinoma, or a seminoma-like tumor, pure or mixed germ cell tumor cells, but not in teratoma cells. Said expression cassette preferably is provided in a vector, preferably a viral vector that is able to transduce germ cell tumor cells. Said viral vector preferably is a recombinant adeno- associated viral vector, a herpes simplex virus-based vector, or a lentivirus-based vector such as a human immunodeficiency virus-based vector. Said viral vector most preferably is a herpes simplex virus-based vector.

Table 1. MicroRNA sequences 5. Examples

Example 1

Materials and methods Statistical analyses

Detailed statistical analyses performed are described under each section (see below). Data was tabulated using Microsoft Excel 2016 and analyzed using

GraphPad Prism 6 and IBM SPSS Statistics version 24. Heatmaps of microRNA data were generated in R using the “pheatmap” clustering software package, using default settings. Venn diagrams were designed using Interactive Venn [Heberle et al., 2015. BMC Bioinformatics 16: 169] Statistical significance was set at p<0.05. MicroRNA isolation, quantification and quality control

For liquid biopsy-based studies (including conditioned media), microRNAs were isolated (from 50m1 samples) by the ampTSmiR test (magnetic bead-based isolation) using the KingFisher Flex System (ThermoFisher), followed by cDNA synthesis, p re -amplification step (12 cycles) and real-time quantitative polymerase chain reaction (RT-qPCR), of which the pipeline has been extensively reported by us before [Gillis et al., 2013. Mol Oncol 7: 1083-1092; van Agthoven and Looijenga, 2017. Oncotarget 8: 58037-58049] A non-human microRNA spike-in (ath-miR- 159a; ThermoFisher) was added in a fixed amount to the samples (2pL of a 1 nM stock solution) for quality control of RNA isolation and cDNA synthesis. All samples included in the study (except those used specifically for exploring the hemolysis effect - see below) were visually inspected for hemolysis, and none with obvious pink discoloration was present. No samples had to be excluded due to poor microRNA recovery, based on recovery of the spike-in ath-miR-159a (variation in Ct values within ± 2 Ct after p re -amplification). Ct values were normalized to the endogenous reference miR-30b-5p. MicroRNA levels were relatively quantified according to the 2-AACT method (after normalization to housekeeping miR-30b-5p and to the average ACt of the control/normal male samples included) and plotted in log2 format for readability. To assure quality control, RT-qPCR efficiency and inter-plate comparability, serial dilutions (1:8) of cDNA from SE-like cell line TCam-2 [de Jong et a , 2008. Genes Chromosomes Cancer 47: 185-196] were included for each assay tested. A no template control was included for every assay in the cDNA synthesis, pre-amplification steps and RT-qPCR. RT-qPCR was run in QuantStudio 12K Flex Real-Time PCR System (ThermoFisher).

MicroRNA profiling.

For all four cell lines (TCam-2, NCCIT, NT2 and 2102Ep, see below), matched conditioned media, fetal calf serum, mouse xenografts, sera/plasma samples and cerebral spinal fluid (CSF) samples, microRNA profiling were performed on bead- captured microRNAs (as described above). Samples were reverse transcribed using Megaplex Primer Pool A and B, followed by a pre-amplification step of 12 cycles (using Megaplex PreAmp Primer Pool and TaqMan PreAmp Master Mix). The product was loaded on the matching TaqMan Low-Density Array (TLDA) Cards A+B. All reagents were purchased from Thermo Fisher/Life Technologies (Bleiswijk, NL). For the CSF samples only card A was run; individuals had the following age and gender: 44, male; 43, male; 42, male; and 54, female. TaqMan microRNA array output data (sds files) were uploaded in the ThermoFisher Cloud App (available at thermofisher.com/mysso/loginDisplay) and analyzed using defined threshold settings for each individual microRNA. Cq values were exported and filtered for poor amplification performance; for consistency we will use Ct when discussing filtered Cq values. To determine whether the microRNA isolation method could impact on our results throughout the experiments and several datasets, TLDA cards using cDNA obtained from total RNA extraction were compared to TLDA cards using cDNA obtained after microRNA bead-capture, for each of the four cell lines. Additionally, to determine the effects of pre- amplification on comparisons between cells and matched media, the Ct values from the TLDA cards for the 2102Ep cell line with and without pre-amplification step were compared.

Cell lines were cultured as previously described [Gillis et al., 2011. Int J Androl 34: e 160- 174] In brief, TCam-2, NT2 and 2102EP were cultured in RPMI 1640 medium with glutamax, and NCCIT using DMEM (high glucose) glutamax, in both cases with 10% fetal calf serum (HyClone, Perbio, Utah, U.S.A). In all experiments, fetal calf serum was used as a negative control. The identification of the cell lines used was determined before based on genome wide copy number variations [de Jong et al., 2008. Genes Chromosomes Cancer 47: 185-196] To determine whether the amount of the miR-371/373 cluster and miR-367 is balanced between cells and matched media in each cell line, microRNA profiling using TLDA cards was performed and waterfall plots were built using the raw Ct values of the cells. The same procedure was used for the respective media, using the order of Ct values of cells as reference. Finally, delta Ct (ACt) was calculated. To further investigate the stability of the secretion process and how active secretion could be affected by several stressing (metabolic) conditions, miR-371a-3p levels were assessed in medium of TCam-2 and NCCIT cells with different proliferation rate (cells grown over 192 hours, with medium sampling and cell counting over several time points) and with different conditioned medium incubation temperatures (room temperature for 27 hours; on ice for 27 hours; incubated at 37°C with 5% C02; frozen and thawed five times and ten times). For quantification, both regular cell count (confluent, in a Biirker counting chamber) and EVQuant methodology (extracellular vesicles [EVs] per mL, a technology recently developed by the Erasmus MC Rotterdam as a novel, practical and low- cost approach for EVs quantification) were used [Hartjes et al., 2019. Bioengineering 6: 43]

To test whether the microRNAs are packed into exosomes, 50 mΐ conditioned media of TCam-2 cells was subjected to four experimental conditions: direct microRNA bead-based capture, as described above (AmiR); and total exosome isolation (using Total Exosome Isolation Reagent, ThermoFisher) to reach a pre enriched exosome suspension, followed by either microRNA bead-based capture (Ex AmiR), Total Exosome RNA & Protein Isolation Kit (Ex Kit) or immunoprecipitation with superparamagnetic beads coated with CD63 antibodies (Ex 63, Exosome - Dynabeads ® Human CD63 Isolation/Detection, ThermoFisher). For quality control of microRNA purification and cDNA synthesis, the non-human miR spike-in ath-miR-159a was used (added during all microRNA purifications in a standard concentration of 0.2 pL per sample from a 1 nM stock solution). Dependent on the method of microRNA purification, the spike in was added to lysis buffer (ABC beads) or to lxPBS (Exosome RNA isolation). To determine the efficiency of exosome isolation, C. elegans microRNA cel-miR-39-3p was used (added to cell medium prior to exosome isolation, in a standard concentration of 0.2 pL per sample from a 1 nM stock solution). Spike-ins, hsa-miR-30b-5p and hsa- 371a-3p were quantified in all four situations using RT-qPCR as described above. Non-detection of cel-miR-39-3p in quantitative analysis was used as proof of successful exosome isolation.

In order to extend the reach of our microRNA-identification model, we used a mouse xenograft model already described by us (which contains both benign and malignant teratoma samples as determined by teratoma assay); for details about the origin of animals and related information please refer to [Hartjes et ah, 2019. Bioengineering 6: 7] Briefly, the aforementioned (T)GCT cell lines and also human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (IPS) were injected subcutaneously into immunodeficient mice and tumor xenografts grew until a maximum size of 2cm ³ (endpoint), after which they were collected for histological evaluation and microRNA isolation. Endpoint mouse EDTA plasma samples were also obtained (data not shown). As negative controls, a mixture of normal mouse tissues (n=2) and plasma from normal mice (n=3) were used. The microRNA isolation using magnetic beads and TLDA-based microRNA profiling was subsequently performed in all samples as previously described [Salvatori et a , 2018. Stem Cell Reports 11: 1493-1505] Relevant microRNAs were then validated by targeted analyses as previously stated. The performance of these microRNAs in discriminating teratoma from control patients was assessed through receiving operating characteristic (ROC) curve construction. Youden’s method [Massolt et al., 2018. PLoS One 13 :e0194259; Youden, 1950. Cancer 3: 32-35] was used to achieve a cut-off to maximize the sensitivity and specificity. In addition, area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were ascertained.

Hemolysis analyses and heparin contamination The “miR-23a/451a ratio”, reported by Shah et al [Shah et al., 2016. PLoS

One 11: e0153200] as an accurate measure of hemolysis in serum samples, has not been validated in the same experimental conditions as ours (i.e. after bead-based microRNA capture followed by cDNA synthesis and pre-amplification of the purified microRNAs), hence the same cutoffs described by the authors may not apply to our work. So, we set out to explore and validate another more appropriate methodology of assessing this issue in our experimental conditions. A total of 775 serum samples (follow-up samples from TGCT patients provided by University Medical Center Groningen (UMCG) reported recently [Schisterman et al., 2005. Epidemiology 16: 73-81] and pooled blood bank-derived sera provided by Sanquin, Amsterdam) were included and hemolysis was scored from 0 to 5, based on visual inspection (pink discoloration), as described before [Bedel et al., 2017. Stem Cells Transl Med 6: 382-393] After bead-based microRNA capture, cDNA synthesis and p re -amplification of the product, RT-qPCR for hsa-miR-23a-3p, mmu-miR-451a, ath-miR-159a, hsa-miR-30b-5p and hsa-miR-372-3p was performed, and the miR- 23a/451a ratio was calculated. ROC curve analysis assessed the performance of the ratio in discriminating hemolysis presence, and Youden’s index [Massolt et al., 2018. PLoS One 13 :e0194259] was used to achieve the optimal cutoff. Mann- Whitney U-test and Kruskal- Wallis test were employed as appropriate for assessing differences among score groups. For exploring the impact of heparin contamination in microRNA quantification, a subset of graft preservation fluids (n=8) from patients undergoing kidney transplantation (described in [Roest et al., 2019. Transplantation 103: 329- 335]) that were and shown to be contaminated with heparin (n=4) was selected, and subsequently quantified after microRNA isolation using miRNEAsy spin columns or after bead capture followed by p re -amplification. Ct values were determined and compared with or without heparinase 1 treatment.

Serum vs. plasma analyses

Fifty pairs of matched serum and EDTA plasma samples from normal male blood donors (controls) were included in the study (obtained from Sanquin, Amsterdam). After microRNA isolation (performed as described above), RT-qPCR for ath-miR-159a, hsa-miR-30b-5p and hsa-miR-371a-3p was performed. Moreover, an additional set of 11 pairs of serum/plasma samples were included, for which hsa-miR-23a-3p, mmu-miR-451a, ath-miR-159a, hsa-miR-30b-5p, hsa-miR-371a-3p and hsa-miR-375 were also profiled. The Wilcoxon matched-pairs signed rank test was used for assessing differences among Ct values of specific targets between serum/plasma samples of matched individuals.

MicroRNAs decrease after orchiectomy

A cohort of 12 clinical stage I TGCT patients (selected from a previous work [Leao et a , 2018. J Urol 200: 126-135]) and five normal male blood donors

(controls) was included in the study. The microRNA isolation and RT-qPCR for ath- miR-159a, hsa-miR-30b-5p, hsa-miR-371a-3p, hsa-miR-372-3p, hsa-miR-373-3p, hsa-miR-367 and hsa-miR-375 was performed. Inter-group differences were compared using the Mann-Whitney U test or Kruskal Wallis tests, as appropriate. For paired variables (microRNA levels along different time points) the Friedman test was employed. Dunn’s test for multiple comparisons was used and all reported p-values are two-tailed and adjusted to this. Spearman’s correlation test was used to correlate two continuous variables. In patients with multiple samples within 24h after orchiectomy, data over time was plotted as percent of the preoperative microRNA levels, and serum half-life was estimated.

MicroRNA-375 analyses

In order to confirm or disprove the proposed value of miR-375 as a biomarker of GCTs, namely related to the teratoma and yolk sac tumor histological subtypes [O'Brien et ah, 2018. Front Endocrinol 9: 402], a cohort of 113 serum samples from 36 patients undergoing chemotherapy followed by retroperitoneal lymph-node dissection (RPLND) was selected (from a previous cohort already reported by us [van Agthoven and Looijenga, 2017. Oncotarget 8: 58037-58049]), and additionally 12 normal male blood donors were included. Samples were collected in three time points: pre-chemotherapy; post-chemotherapy and pre-RPLND; and post-RPLND. Additionally, a second cohort of sera samples from 26 patients in pediatric/young- adult age range (including both type I and type II tumors with teratoma and yolk sac histologies plus one ovarian dysgerminoma) and 10 sera samples from pediatric individuals with no neoplastic conditions and within the same age range were included. RT-qPCR for both the targets hsa-miR-371a-3p and hsa-miR-375 was performed as previously described and the same quality control measures were pursued. Statistical analysis was performed as detailed in the above section.

Ethics approval

Use of patient samples remaining after diagnosis was approved for research by the Medical Ethical Committee of the EMC (the Netherlands), permit no. 02.981. This included permission to use the secondary samples without further consent. Samples were used according to the “Code for Proper Secondary Use of Human Tissue in The Netherlands” developed by the Dutch Federation of Medical Scientific Societies (FMWV, version, 2002; update 2011). Animal experiments were approved by the Dutch Central Commission for Animal experimentation (Centrale Commissie voor Dierproeven). The study was conducted in accordance with the Declaration of Helsinki.

Results MicroRNA in vitro dynamics

Firstly, to investigate the potential impact of the methodology applied on microRNA isolation (after exclusion of inappropriate curves and cases with no evidence of microRNAs in cells) we investigated the results after either bead-based captured microRNAs or non-bead-based extraction from total RNA. The results show very minor changes in Ct values among the matched samples using the two methods, demonstrating that the bead-capture process is not saturated, so comparisons between the various datasets are informative (data not shown). Adso, the variability among samples with and without the pre-amplification step was minor, with a median of 10.25 Ct difference among samples (data not shown), so that comparisons are not influenced by the pre-amplification step used.

We then first included all profiled microRNAs and looked specifically at the patterns of expression of the panel known to be relevant in GCTs (miR-371/373 cluster and miR-367) based on multiple independent studies. The TLDA data was analyzed and the targets miR-371a-3p, miR-372-3p and miR-367 were specifically highlighted (Figure 1A-B). These three microRNAs were indeed among the highest expressed in TCam-2, 2102EP and NCCIT (both in cells and respective media). Specifically, for NT2, only miR-367 was amongst the highest expressed (again both in cells and respective media). Out of the 768 microRNAs profiled, 477, 389, 468 and 536 were not detected in TCam-2, NT2, NCCIT and 2102EP cell lines, respectively. Only 180 microRNAs were detected in all four cell lines. A similar pattern was found for the matched media; 552, 576, 616 and 577 microRNAs were not in the detection range for the TCam-2, NT2, NCCIT and 2102EP media, respectively, and only 112 were detected in all four media (Figure 1C, left and middle panel).

Hence, we then focused only on microRNAs in the detection range (Ct values <34) for each cell line. There is not a direct proportional association between microRNA amounts/content in matched cells and media, meaning that some microRNAs are indeed selectively secreted.

In order to identify microRNAs specifically secreted by each ced line, analysis of the fetal calf serum (negative control) was also taken into account. Indeed, the number of microRNAs detectable in conditioned media of the several cell lines is substantially lower after exclusion of those already present in the fetal calf serum (Figure 1C, right panel), resulting from nonhuman specificity of the assays used. In the end, only 15%, 12%, 7% and 12% of the 768 profiled microRNAs fulfill these criteria for TCam-2, NT2, NCCIT and 2102Ep, respectively.

In line with that, considering the microRNAs detectable in cells, 101/291 (34.7%), 81/379 (21.4%), 48/300 (16.0%) and 67/232 (28.9%) microRNAs were found to be specifically secreted by TCam-2, NT2, NCCIT and 2102EP cell lines, respectively (Ct <34 in media and >34 in fetal calf serum). Likewise, 125/291 (43.0%), 213/379 (57.0%), 182/300 (60.7%) and 103/232 (44.4%) microRNAs were demonstrated not to be secreted into conditioned media of TCam-2, NT2, NCCIT and 2102EP, respectively (Ct > 34 in media and in fetal calf serum). Importantly, in most of the cases, the miR-371/373 cluster and miR-367 were found to be present in the final list of specifically secreted microRNAs (Table 2).

Secretion of miR-371a-3p is minimally influenced by ced count:

Levels of miR-371a-3p were assessed in TCam-2 and NCCIT conditioned medium when cells were at various phases of culture density. The Ct values of this microRNA remained stable in TCam-2 and NCCIT with different proliferation indexes, with only slight fluctuations upon medium change (at 74 and/or 146 hours). Ct values after 192 hours of culturing were the same (for TCam-2) and only slightly lower (for NCCIT) when compared to the first time point (27 hours), despite a continuous growing activity of cells in culture (Figure 2A). This suggests secretion is not/minimally influenced by cell count. Furthermore, when comparing both TCam-2 and NCCIT cell lines, the amount of extracellular vesicles as determined by EVQuant methodology influences more the miR-371a-3p Ct values than the amount of cells; despite NCCIT showing greater amount of cells in the considered timepoint, the number of EVs is lower, rendering higher Ct values for miR-371a-3p. The opposite scenario is observed for TCam-2 (Table 3). Also, lowering incubation temperature or even multiple freezing/thawing did not influence miR-371a-3p levels in TCam-2 conditioned medium, underscoring the stability of the microRNA after the secretion process (Figure 2B). Secretion of miR-371a-3p by TCam-2 cells seems to occur via exosomes:

Table 2. Ct-values for miR-371a-3p, miR-372-3p, miR-373-3p and miR-367 in cell lines and matched conditioned media.

Results of the experiments evaluating whether the specific microRNAs are placed into exosomes are summarized in Figure 2C. Calibrator ath-miR-159a and normalizer hsa-miR-30b-5p were similar in all fractions, as expected. Exosomal isolation from TCam-2 medium succeeded, proven by undetectable cel-miR-39-3p for all exosome isolation methods. The calibrated Ct-value for miR-371a-3p after total microRNA isolation (AmiR) was similar to the Ct-value after exosomal miR isolation using the Total Exosome RNA and Protein Isolation Kit (Ex Kit) and to a lesser extent after using the paramagnetic beads (Ex AmiR). Secretion of miR- 371a-3p in TCam-2 is therefore predominantly related to the exosomal fraction.

After immunoprecipitation with CD63+ Dynabeads® (Ex 63), the Ct-value for miR- 371a-3p was higher, indicating a selection of the total fraction of exosomes using this method. Table 3. Relation between cell count, extracellular vesicles amount and miR-371a- 3p.

MicroRNAs as biomarkers using an in vivo mouse model Using a similar methodology as in the in vitro analyses, normal mice samples

(both tissue mixtures and plasma) were used as negative controls. A total of 173 and 176 microRNAs were in the detection range in the two normal mouse tissue samples (157 shared by both), and hence were discarded from the analysis. In plasma samples, a total of 231, 220 and 230 microRNAs were detected in the normal mouse plasma samples (190 shared by all three), and were also discarded from the analysis.

Then we aimed at uncovering the human microRNAs that were able to be identified specifically in the liquid biopsy context. We focused on the mouse human tumor xenografts with malignant histology (derived from injection with Lu07dox, TCam-2 and 2102Ep cells), representing the human GCTs closely. Among the microRNAs consistently detected in all these xenografts (n=44), a cluster of microRNAs are found to be specifically detectable in the matched endpoint plasma samples; among these we find the miR-371/373 cluster and miR-367 (Figure 3A). Additionally, we studied the xenografts with benign histology (i.e., teratoma, derived from injection with H9, H9dox, H9hybrid and Lu07). Only 25 microRNAs were consistently detected in all these xenografts and their detection in matched endpoint plasma samples was infrequent. However, the miR-885-5p was detected in all matched plasmas, and also miR-448 and miR-197-3p were detected in at least 50% of plasma samples, making them candidates for detecting mature/benign teratoma in liquid biopsies (Figure 3B).

Focusing on cell lines and respective conditioned media, miR-885-5p was detected in NT2 cells and respective medium, while miR-448 was found in NCCIT cells only and not in the conditioned matched medium.

MicroRNA targeted analyses of potential candidates confirm the value of the in vivo model:

Given our data on microRNA profiling indicating miR-885-5p, miR-448 and miR-197-3p as possible candidate biomarkers of (benign) mature teratoma, and given the relevance of discovering such a microRNA for the field, these microRNAs were selected for further validation by targeted analyses, using a cohort of post chemotherapy (but pre- RPLND) serum samples and six normal male sera.

Indeed, relative serum levels of both miR-885-5p and miR-448 were significantly higher in teratoma patients when compared to healthy males (p=0.0046 and p=0.0140, respectively), even after adjusting for multiple comparisons (adjusted p-values of 0.0176 and 0.0291, respectively). The same tendency was observed for miR-197-3p, although not reaching statistical significance (p=0.0763) (Figure 4A-F). Relative levels of miR-885-5p and miR-448 allowed for discrimination of teratoma from control patients with an AUC of 0.89 and 0.84, depicting good performance parameters, further demonstrating the value of our model (Figure 4G and H). Moreover, when defining “a positive test” when at least one of these two microRNAs is above the defined cutoff, discrimination performance increases, showing sensitivity of 93.3% and accuracy of 90.5%. Performance parameters of these two microRNAs are depicted in Table 4. The relative levels of these microRNAs were, however, not significantly different among patients with only fibrosis/necrosis, teratoma and with viable tumor (non- ter atom a) histological compositions, limiting their value in this specific clinical setting (Figure 4 D-F).

Validation and proof of concept: miR-371a-3p and miR-375 as candidate biomarkers of germ cell tumors in liquid biopsy setting

There was a significant decrease of relative levels of miR-371a-3p and miR- 372-3p after orchiectomy, seen at 48h for miR-372-3p (p=0.0029) and already at 24h for miR-371a-3p (p=0.0066 for 24h and p=0.0029 for 48h after orchiectomy, respectively). An apparent continuous decrease is also seen for miR-373-3p, although it did not reach statistical significance. No significant differences were observed for miR-367 nor miR-375 (Figure 5A).

For the three patients with multiple samples collected within 24h after the orchiectomy, a continuous steady decrease of microRNA relative levels was clear only for miR-371a-3p, with the remaining microRNAs showing fluctuations in expression levels over time. The halfdife of miR-371a-3p in these patients was <4h (Figure 5B).

Table 4. Performance parameters for miR-885-5p and miR-448 in discriminating teratoma from normal male individuals

Context Sensitivity Specificity PPV NPV Accuracy

(%) (%) (%) (%) (%) miR-885-5p 86.7 83.3 92.9 71. 1 85.7 miR-448 ^. 80.0 ^. 83.3 ^. 100 ^. 62 5 ^. 81.0 ^.

Both miR-885-5p and miR-448 above 73.3 83.3 91.7 55.6 76.2 defined cutoff Either miR-885-5p or miR-448 above 93.3 83.3 93.3 83.3 90.5 defined cutoff

Abbreviations: PPV - positive predictive value; NPV - negative predictive value

Patients’ age at diagnosis was not significantly correlated with any microRNA relative expression levels. For miR-371a-3p and miR-372-3p there was a significant, strong positive correlation between tumor size and relative expression levels (rs=0.75 and rs=0.8, p=0.025 and p=0.014, respectively). A similar correlation was also found for miR-373-3p relative expression levels, although it did not reach significance (rs=0.617, p=0.086) (Figure 5C). miR-371a-3p relative expression levels were positively correlated with miR-372-3p and miR-373-3p levels (rs=0.817 and rs=0.800, p=0.011 and p=0.014, respectively). There were no significant differences between the preoperative levels of all microRNAs among SE and NS samples.

Analysis of TLDA data suggests miR-371a-3p, but not miR-375, as a specific biomarker in liquid biopsy setting: When analyzing TLDA data referring to miR-375, and comparing with the miR-371a-3p, distinct profiles are observed: while miR-371a-3p is found to be secreted into the media of all four (T)GCT cell lines, miR-375 is only detected in media of NT2 and TCam-2 (data not shown). Also, in our mouse xenograft model dataset, miR-371a-3p is clearly detected in mouse endpoint plasma exclusively in cases of malignant histology and not in controls or benign teratoma, whereas miR- 375 is detected in all situations as well as in controls (data not shown). Also, while miR-371a-3p was not detected in any of the 16 normal male serum samples, miR- 375 was detected in 11/16 of these samples.

Validation studies in patient- derived data confirm the clinical utility of miR-371a- 3p, but not miR-375, as liquid biopsy biomarker for GCTs (types I and II)

Post-chemotherapy miR-371a-3p relative levels were significantly higher in patients with viable tumor (non-teratoma) at RPLND when compared to those presenting with teratoma only or fibrosis/necrosis (adjusted p-values of 0.0228 and 0.0348, respectively), independently replicating our previous observations on the series [23] However, no significant variation was observed for miR-375 relative levels. Also, the pre-chemotherapy miR-371a-3p relative levels were significantly higher than those after chemotherapy and after RPLND (adjusted p-values <0.0001 for both), while for miR-375 no significant changes were noted. Relative levels of miR-371a-3p in the pre-chemotherapy period were associated with tumor burden, being significantly higher in stage III disease (p=0.0009) (Figure 6A-C), while again no significant variation was seen for miR-375 (Figure 6D-F). Importantly, in all these three circumstances, miR-375 levels did not differ significantly from the ones observed in normal male controls (data not shown).

Additionally, pre-chemotherapy levels of miR-371a-3p significantly and positively correlated with the size of the metastatic mass, the serum b-HCG and the serum LDH before treatment (rs=0.50, p=0.002; rs=0.44, p=0.008; and rs=0.69, p<0.001, respectively); the same tendency was found for AFP levels although it did not reach significance (rs=0.319, p=0.062). None of these correlations or tendencies was found for miR-375. Both microRNAs levels did not correlate significantly with patients’ age (p=0.117 and p=0.207).

A description of the diagnoses of each individual included in the cohort of teratoma and yolk sac tumor cases is known. Confirming previous findings of the limited use of miR-371a-3p in the context of teratoma-predominant tumors [van Agthoven et a , 2017. Cell Oncol 40: 379-388; Radtke et a , 2018. Urol Int 100: 470-475], there were no significant differences between relative levels of miR-371a- 3p among normal male sera and sera corresponding to patients with pure teratoma (Figure 7A). Relative levels of this microRNA were, however, higher when considering the pure yolk sac tumors and the dysgerminoma (Figure 7B). For miR- 375, the relative levels were the same among all tested sera samples, including all histological subtypes and controls (Figure 7C-D). Technical considerations when working with liquid biopsy-based biomarkers

Regarding presence of hemolysis, visual inspection demonstrated a distribution of hemolysis score in the cohort under evaluation as follows: 710 samples with score 0, 30 with score 1, 18 with score 2, 12 with score 3, two with score 4, and three with score 5. The miR-23a-3p was stable in all samples, with no significant differences among cases with or without hemolysis (p=0.421). Also, the miR-23a/451a ratio was significantly lower in samples with lower hemolysis scores (pO.001, Figure 8A).

By ROC curve analysis, the miR-23a/451a ratio was able to discriminate samples with no hemolysis (score 0) from those with evidence of hemolysis (scores 1-5) with an AUC of 0.812. The optimal cutoff for discriminating these groups of samples in our work was a ratio of 9.15, allowing for a sensitivity of 77% and specificity of 80% in the discrimination (data not shown). Samples were then categorized in respect to hemolysis presence based on this cutoff.

We then set out to assess the impact of hemolysis on specific assays, scored by the two different methods (visual inspection and our predetermined cutoff). There was no significant impact of hemolysis (determined by either method) on the microRNA isolation procedure itself, as there were no significant differences in the Ct values of spike-in ath-miR-159a (Figure 8B). There were also no significant differences in Ct values of the normalizer miR-30b-5p between samples with hemolysis scores 0 vs scores 1-5. miR-30b-5p Ct values were significantly different among score groups and miR-23a/451a ratio groups, but the magnitude of this difference was minor and, importantly, at the expense of samples containing severe hemolysis (scores 4-5, for which Ct values were lower), as illustrated in Figure 8C. Finally, there was no significant impact of hemolysis (determined by either method) in the Ct values of the specific target assay miR-372a-3p (Figure 8D).

As a proof of concept, considering the RPLND patient cohort included in our study, visual inspection showed only samples with hemolysis scores 0-1 (data not shown). Of the 144 samples, 26 (18%) showed a miR-23a/451a ratio above the defined cutoff. However, hemolysis (defined by the aforementioned cutoff) showed no significant impact on Ct values of either the spike-in ath-miR-159a, the normalizer hsa-miR-30b-5p or the target assays hsa-miR-371a-3p or hsa-miR-375 (data not shown).

Regarding fluid samples containing heparin contamination (kidney graft preservation solution), as another example of possible detection interference, we demonstrate that Ct values of both spiked-in synthetic microRNAs cel-miR-54-3p and cel-miR-39-3p, as well as the endogenous microRNAs hsa-miR-21-5p and hsa- miR-505-3p, clearly are not affected by heparin in case of the bead-capture procedure. In case RNA is isolated by commonly used spin column procedure, clear interference by heparin contamination is observed, which can be corrected by treatment with 6 IU heparinase 1 during cDNA synthesis (data not shown). Significant differences in microRNA levels are found among serum and plasma samples:

When comparing matched serum and EDTA plasma samples from normal male blood donors, a significant difference in the Ct values of the normalizer miR- 30b-5p was depicted (pO.OOOl) (Figure 9A). Plasma samples showed significantly lower Ct values when compared to matched serum samples. No significant differences were found for the spike-in ath-miR-159a, despite some variation (Figure 9B). Additionally, Ct values of miR-371a-3p were significantly higher in plasma samples when compared to serum (p=0.0048) (Figure 9C). On the contrary, plasma samples showed significantly lower Ct values of miR-375 when compared to matched serum samples (p=0.0137) (Figure 9D).

No plasma samples were considered to have hemolysis by the cutoff defined by us, with only one serum sample being above the cutoff. Plasma samples showed significantly lower Ct values for miR-23a and miR-451a when compared to serum samples (p=0.001 and p=0.0029, respectively) (Figure 9E-F); however, no significant differences in the miR 23a/451a ratio among matched serum and plasma samples were depicted (Figure 9G).

MicroRNA profiles of control serum and cerebral spinal fluid samples are distinct:

Although normal serum and plasma have been investigated for microRNA profiling, this has not been performed for normal CSF samples so far. Therefore, microRNA profiling was performed on four control CSF samples (TFDA card A), i.e., without neoplastic or inflammatory disease. Additionally, 16 normal male sera samples were also subjected to the same microRNA profiling (TFDA cards A+B). Of the 384 microRNAs investigated in the CSF samples, 307 (80%) were not detected in any of the samples. Only 16 microRNAs were consistently detected in all four samples (data not shown). In contrast, of the 384 (plate A) and 764 (plates A+B) microRNAs investigated in the sera samples of healthy males, only 131 (34%) and 369 (48%) were absent from all samples, respectively. Thirty-eight and 50 microRNAs (10% and 7%) were detected in all 16 sera samples, respectively (data not shown). The commonly used normalizer in serum samples miR-30b-5p was barely detected in 2/4 CSF samples (Ct values of 33.4 and 33.9), indicating its inappropriate ness for normalization purposes.