Martin A. Rijlaarsdam

Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: A proof of principle

Germ cell cancers (GCC) are the most frequent malignancy in young Caucasian males. GCC can consist of seminomas (SE) and non‐seminomas (malignant NS: embryonal carcinoma (EC), yolk sac tumor (YS), choriocarcinoma (CH) and teratoma (TE)). Current serum‐markers used for diagnosis and follow‐up (AFP, hCG) are predominantly related to YS and CH and marker positivity can vary during disease. Therefore, stable markers consistently identifying more GCC components, specifically the stem cell components SE and EC, are of interest. Expression of the embryonic stem cell miR‐371‐3 and miR‐302/367 clusters in SE/EC/YS suggest possible application of these micro‐RNAs as GCC tumor‐markers. The TSmiR protocol constitutes a complete, quality‐controlled pipeline for the detection of miRs in serum, based on magnetic bead‐based purification and qPCR quantification. As a proof of principle, TSmiR was applied to five independent serum sample series including 80 GCCs, 47 controls, 11 matched pre/post orchidectomy samples and 12 no‐GCC testicular masses. GCC serum samples showed a consistent, significant (p < 0.0064) increase of miR‐371/372/373/367 levels. Analogous, serum levels returned to baseline after orchidectomy (stage‐I disease). Moreover, there was a trend toward higher miR levels in patients with metastasis. These results imply suitability for diagnosis and follow‐up. TSmiR showed an overall sensitivity of 98%, clearly outperforming the traditional serum markers AFP/hCG (36%/57%, sensitivityAFP = 3%/45%; sensitivityhCG = 62%/66%, SE/NS). TSmiR misclassified one tumor as a control. Serum AFP/hCG and TSmiR combined identified all T samples correctly. In conclusion, TSmiR constitutes a highly sensitive and reproducible serum test for GCC patients, suitable to be prospectively tested for diagnostic and follow‐up purposes.

Identification of known and novel germ cell cancer‐specific (embryonic) miRs in serum by high‐throughput profiling

microRNAs (miRs) are short non‐coding RNA molecules (≈21 nucleotides) involved in regulation of translation. As such they are crucial for normal cell development and differentiation as well as cellular maintenance. Dysregulation of miRs has been reported in various diseases, including cancer. Interestingly, miRs can be informative as tumor classifiers and disease biomarkers. Recent studies demonstrated the presence of miRs in body fluids like serum, thus providing a putative non‐invasive tool to study and monitor disease state. Earlier targeted studies by several independent groups identified specific embryonic miRs as characteristic for germ cell tumors (GCT) (miR‐371‐2‐3 & miR‐302/367 clusters). This study reports a high‐throughput miR profiling (≈750 miRs) approach on serum from testicular germ cell tumor patients (14 seminoma and 10 non‐seminoma) and controls (n = 11), aiming at independent identification of miRs as candidate biomarkers for testicular GCT. A magnetic bead capture system was used to isolate miRs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling. The previously identified miRs 371 and 372 were confirmed to be specifically elevated in serum from germ cell tumor patients. In addition, several novels miRs were identified that were discriminative between germ cell cancer and controls: miR‐511, ‐26b, ‐769, ‐23a, ‐106b, ‐365, ‐598, ‐340, and let‐7a. In conclusion, this study validates the power of the embryonic miRs 371 and 372 in detecting malignant GCT (SE and NS) based on serum miR levels and identifies several potential novel miR targets.

An oncofetal and developmental perspective on testicular germ cell cancer

Germ cell tumors (GCTs) represent a diverse group of tumors presumably originating from (early fetal) developing germ cells. Most frequent are the testicular germ cell cancers (TGCC). Overall, TGCC is the most frequent malignancy in Caucasian males (20-40 years) and remains an important cause of (treatment related) mortality in these young men. The strong association between the phenotype of TGCC stem cell components and their totipotent ancestor (fetal primordial germ cell or gonocyte) makes these tumors highly relevant from an onco-fetal point of view. This review subsequently discusses the evidence for the early embryonic origin of TGCCs, followed by an overview of the crucial association between TGCC pathogenesis, genetics, environmental exposure and the (fetal) testicular micro-environment (genvironment). This culminates in an evaluation of three genvironmentally modulated hallmarks of TGCC directly related to the oncofetal pathogenesis of TGCC: (1) maintenance of pluripotency, (2) cell cycle control/cisplatin sensitivity and (3) regulation of proliferation/migration/apoptosis by KIT-KITL mediated receptor tyrosine kinase signaling. Briefly, TGCC exhibit identifiable stem cell components (seminoma and embryonal carcinoma) and progenitors that show large and consistent similarities to primordial/embryonic germ cells, their presumed totipotent cells of origin. TGCC pathogenesis depends crucially on a complex interaction of genetic and (micro-)environmental, i.e. genvironmental risk factors that have only been partly elucidated despite significant effort. TGCC stem cell components also show a high degree of similarity with embryonic stem/germ cells (ES) in the regulation of pluripotency and cell cycle control, directly related to their exquisite sensitivity to DNA damaging agents (e.g. cisplatin). Of note, (ES specific) micro-RNAs play a pivotal role in the crossover between cell cycle control, pluripotency and chemosensitivity. Moreover, multiple consistent observations reported TGCC to be associated with KIT-KITL mediated receptor tyrosine kinase signaling, a pathway crucially implicated in proliferation, migration and survival during embryogenesis including germ cell development. In conclusion, TGCCs are a fascinating model for onco-fetal developmental processes especially with regard to studying cell cycle control, pluripotency maintenance and KIT-KITL signaling. The knowledge presented here contributes to better understanding of the molecular characteristics of TGCC pathogenesis, translating to identification of at risk individuals and enhanced quality of care for TGCC patients (diagnosis, treatment and follow-up).

A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours.

Background:The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR–371–373 and miR–302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups.Methods:We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT–PCR profiling for miR–371–373 and miR–302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients.Results:The exogenous non-human spike-in cel–miR–39–3p and the endogenous housekeeper miR–30b–5p were optimal for obtaining robust serum and CSF qRT–PCR quantification. A four-serum miRNA panel (miR–371a–3p, miR–372–3p, miR–373–3p and miR–367–3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples.Conclusions:The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs.

Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).

Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines

Background Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. Results This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. Conclusion The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.

DMRforPairs: identifying Differentially Methylated Regions between unique samples using array based methylation profiles

BackgroundArray based methylation profiling is a cost-effective solution to study the association between genome methylation and human disease & development. Available tools to analyze the Illumina Infinium HumanMethylation450 BeadChip focus on comparing methylation levels per locus. Other tools combine multiple probes into a range, identifying differential methylated regions (DMRs). These tools all require groups of samples to compare. However, comparison of unique, individual samples is essential in situations where larger sample sizes are not possible.ResultsDMRforPairs was designed to compare regional methylation status between unique samples. It identifies probe dense genomic regions and quantifies/tests their (difference in) methylation level between the samples. As a proof of concept, DMRforPairs is applied to public data from four human cell lines: two lymphoblastoid cell lines from healthy individuals and the cancer cell lines A431 and MCF7 (including 2 technical replicates each). DMRforPairs identified an increasing number of DMRs related to the sample phenotype when biological similarity of the samples decreased. DMRs identified by DMRforPairs were related to the biological origin of the cell lines.ConclusionTo our knowledge, DMRforPairs is the first tool to identify and visualize relevant and significant differentially methylated regions between unique samples.

miMsg: a target enrichment algorithm for predicted miR–mRNA interactions based on relative ranking of matched expression data

MOTIVATION Algorithms predicting microRNA (miR)-mRNA interactions generate high numbers of possible interactions, many of which might be non-existent or irrelevant in a certain biological context. It is desirable to develop a transparent, user-friendly, unbiased tool to enrich miR-mRNA predictions. RESULTS The miMsg algorithm uses matched miR/mRNA expression data to enrich miR-mRNA predictions. It grades interactions by the number, magnitude and significance of misplacements in the combined ranking profiles of miR/mRNA expression assessed over multiple biological samples. miMsg requires minimal user input and makes no statistical assumptions. It identified 921 out of 56 262 interactions as top scoring and significant in an actual germ cell cancer dataset. Twenty-eight miR-mRNA pairs were deemed of highest interest based on ranking by miMsg and supported by current knowledge about validated interactions and biological function. To conclude, miMsg is an effective algorithm to reduce a high number of predicted interactions to a small set of high confidence interactions for further study. AVAILABILITY AND IMPLEMENTATION Matlab source code and datasets available at www.martinrijlaarsdam.nl/mimsg . SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Abstract 5225: MicroRNA profiling in serum samples from donors with germ cell cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNAs are short non-coding RNA molecules (∼21 bases) that have been identified as important regulators of gene expression at the translational and transcriptional level. They are known to play a crucial role in cell development, differentiation, and disease. Dysregulation of miRNAs has been linked to cancer development and tumor progression. In addition, miRNAs have been identified as tumor classifiers and disease biomarkers. Recent studies have shown that miRNAs are present in body fluids (serum, saliva, semen, urine) thus providing a non-invasive tool to study and monitor disease states. Earlier research studies identified specific miRNAs as characteristic for germ cell tumors, such as testicular cancer (miR-372, miR-373). miRNA Profiling (∼760 miRNAs) was performed to identify additional miRNAs as candidate biomarkers in serum samples for testicular cancer (seminoma and non-seminoma types), from normal and cancer tissue in parallel with their matched serum samples collected from patients with testicular cancer. For this research study we used a miRNA specific bead capture system to isolate miRNAs from serum and TaqMan® Array Card platform for profiling. We will present results from this research study comparing the miRNA expression patterns in matched normal, cancer tissue, and serum. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Kathy Y. Lee, Sunali Patel, Kathleen Hayashibara, Shirley Chu, Ad J. M Gillis, Martin Rijlaarsdam, Lambert C.J Dorssers, Leendert H. J Looijenga. MicroRNA profiling in serum samples from donors with germ cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2014-5225

Abstract 3394: In-depth gene expression profiling of seminomatous testicular germ cell tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In depth Whole Transcriptome Analysis by RNA-Seq was used to compare the expression profile of testicular germ cell tumors with the histology of seminoma and normal testis. From this analysis we identified a large number of genes that showed differential expression with statistically high significance, (p 2X FC). The list included genes related to stem cell pleuripotency (NANOG, POU5F1), proliferation (KRAS, CCND2), a number of non-coding RNAs (snoRNAs, miRNA precursors, XIST), and testicular cancer related genes (LDHB, AKAP4). From this list we identified a subset of genes for validation and further screening. We used the OpenArray® RT-qPCR platform to quantitatively screen up to 224 coding and non-coding genes using samples from different testicular germ cell tumors (seminoma and non-seminoma) and normal tissue. Results from these screening experiments will be presented. For research use only. Not for diagnostic procedures. Citation Format: Sunali Patel, Shirley Chu, Lovorka Degoricija, Kathy Y. Lee, Ad J.M Gillis, Martin Rijlaarsdam, Lambert C.J Dorssers, Leendert H.J Looijenga. In-depth gene expression profiling of seminomatous testicular germ cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3394. doi:10.1158/1538-7445.AM2014-3394