A 14-year-old girl with premature ovarian insufficiency but with a positive pregnancy test.

OBJECTIVES: Childhood cancer survivors are at risk for premature ovarian insufficiency, especially after treatment with alkylating agents. The objective of this report is to highlight a case in which this phenomenon caused a false-positive pregnancy test. CASE PRESENTATION: A workup was performed in a 14-year-old girl with a positive pregnancy test. She was diagnosed with stage IV neuroblastoma of the left adrenal gland at the age of 4 years. She received extensive treatment, including alkylating agents, and had been diagnosed with premature ovarian insufficiency. An LH/hCG suppression test was performed using high dose 17 bèta-estradiol: hCG levels normalized. CONCLUSIONS: The pregnancy test was false-positive due to production of low amounts of hCG by the pituitary gland as a result of high LH concentrations following premature ovarian insufficiency. It may be helpful to perform the LH/hCG suppression test to prove pituitary origin of the hCG overproduction.

Analysis of a mouse germ cell tumor model establishes pluripotency-associated miRNAs as conserved serum biomarkers for germ cell cancer detection.

Malignant testicular germ cells tumors (TGCTs) are the most common solid cancers in young men. Current TGCT diagnostics include conventional serum protein markers, but these lack the sensitivity and specificity to serve as accurate markers across all TGCT subtypes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs and informative biomarkers for several diseases. In humans, miRNAs of the miR-371-373 cluster are detectable in the serum of patients with malignant TGCTs and outperform existing serum protein markers for both initial diagnosis and subsequent disease monitoring. We previously developed a genetically engineered mouse model featuring malignant mixed TGCTs consisting of pluripotent embryonal carcinoma (EC) and differentiated teratoma that, like the corresponding human malignancies, originate in utero and are highly chemosensitive. Here, we report that miRNAs in the mouse miR-290-295 cluster, homologs of the human miR-371-373 cluster, were detectable in serum from mice with malignant TGCTs but not from tumor-free control mice or mice with benign teratomas. miR-291-293 were expressed and secreted specifically by pluripotent EC cells, and expression was lost following differentiation induced by the drug thioridazine. Notably, miR-291-293 levels were significantly higher in the serum of pregnant dams carrying tumor-bearing fetuses compared to that of control dams. These findings reveal that expression of the miR-290-295 and miR-371-373 clusters in mice and humans, respectively, is a conserved feature of malignant TGCTs, further validating the mouse model as representative of the human disease. These data also highlight the potential of serum miR-371-373 assays to improve patient outcomes through early TGCT detection, possibly even prenatally.

Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT): Patient Characteristics, Treatment, and Outcome-A Systematic Review.

BACKGROUND: Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare aggressive ovarian malignancy mainly affecting children, adolescents, and young adults. Since the discovery of mutations in the SMARCA4 gene in 2014, SCCOHT has become the subject of extensive investigation. However, international uniform treatment guidelines for SCCOHT are lacking and the outcome remains poor. The aim of this systematic review is to generate an overview of all reported patients with SCCOHT from 1990 onwards, describing the clinical presentation, genetic characteristics, treatment, and outcome. METHODS: A systematic search was performed in the databases Embase, Medline, Web of Science, and Cochrane for studies that focus on SCCOHT. Patient characteristics and treatment data were extracted from the included studies. Survival was estimated using Kaplan-Meier's methodology. To assess the difference between survival, the log-rank test was used. To quantify the effect of the FIGO stage, the Cox proportional hazard regression model was estimated. The chi-squared test was used to study the association between the FIGO stage and the surgical procedures. RESULTS: Sixty-seven studies describing a total of 306 patients were included. The median patient age was 25 years (range 1-60 years). The patients mostly presented with non-specific symptoms such as abdominal pain and sometimes showed hypercalcemia and elevated CA-125. A great diversity in the diagnostic work-up and therapeutic approaches was reported. The chemotherapy regimens were very diverse, all containing a platinum-based (cisplatin or carboplatin) backbone. Survival was strongly associated with the FIGO stage at diagnosis. CONCLUSIONS: SCCOHT is a rare and aggressive ovarian cancer, with a poor prognosis, and information on adequate treatment for this cancer is lacking. The testing of mutations in SMARCA4 is crucial for an accurate diagnosis and may lead to new treatment options. Harmonization and international collaboration to obtain high-quality data on diagnostic investigations, treatment, and outcome are warranted to be able to develop international treatment guidelines to improve the survival chances of young women with SCCOHT.

Integrated genomic analysis reveals aberrations in WNT signaling in germ cell tumors of childhood and adolescence.

Germ cell tumors (GCTs) are neoplasms of the testis, ovary and extragonadal sites that occur in infants, children, adolescents and adults. Post-pubertal (type II) malignant GCTs may present as seminoma, non-seminoma or mixed histologies. In contrast, pre-pubertal (type I) GCTs are limited to (benign) teratoma and (malignant) yolk sac tumor (YST). Epidemiologic and molecular data have shown that pre- and post-pubertal GCTs arise by distinct mechanisms. Dedicated studies of the genomic landscape of type I and II GCT in children and adolescents are lacking. Here we present an integrated genomic analysis of extracranial GCTs across the age spectrum from 0-24 years. Activation of the WNT pathway by somatic mutation, copy-number alteration, and differential promoter methylation is a prominent feature of GCTs in children, adolescents and young adults, and is associated with poor clinical outcomes. Significantly, we find that small molecule WNT inhibitors can suppress GCT cells both in vitro and in vivo. These results highlight the importance of WNT pathway signaling in GCTs across all ages and provide a foundation for future efforts to develop targeted therapies for these cancers.

Undetectable anti-Mullerian hormone and inhibin B do not preclude the presence of germ cell tumours in 45,X/46,XY or 46,XY gonadal dysgenesis.

OBJECTIVE: Individuals with 45,X/46,XY or 46,XY gonadal dysgenesis are at increased risk of germ cell malignancies. Therefore, prophylactic bilateral gonadectomy is advised in girls and considered in boys with atypical genitalia for undescended, macroscopically abnormal gonads. However, severely dysgenetic gonads may not contain germ cells rendering gonadectomy unnecessary. Therefore, we investigate if undetectable preoperative serum anti-Müllerian hormone (AMH) and inhibin B can predict the absence of germ cells, (pre)malignant or otherwise. DESIGN, PATIENTS AND MEASUREMENTS: Individuals who had undergone bilateral gonadal biopsy and/or gonadectomy because of suspected gonadal dysgenesis in 1999-2019 were included in this retrospective study if preoperative AMH and/or inhibin B were available. Histological material was reviewed by an experienced pathologist. Haematoxylin and eosin and immunohistochemical stainings for SOX9, OCT4, TSPY and SCF (KITL) were used. RESULTS: Thirteen males and 16 females were included, 20 with 46,XY and 9 with 45,X/46,XY DSD. Three females had dysgerminoma alongside gonadoblastoma; two gonadoblastoma, one germ cell neoplasia in situ (GCNIS) and three males had pre-GCNIS and/or pre-gonadoblastoma. Gonadoblastoma and/or dysgerminoma were present in 3/11 individuals with undetectable AMH and inhibin B, one of whom also had non-(pre)malignant germ cells. Of the other 18, in whom AMH and/or inhibin B were detectable, only one had no germ cells. CONCLUSIONS: Undetectable serum AMH and inhibin B cannot reliably predict the absence of germ cells and germ cell tumours in individuals with 45,X/46,XY or 46,XY gonadal dysgenesis. This information should help in counselling about prophylactic gonadectomy, taking into account both the germ cell cancer risk and potential for gonadal function.

Teratoma Assay for Testing Pluripotency and Malignancy of Stem Cells: Insufficient Reporting and Uptake of Animal-Free Methods-A Systematic Review.

Pluripotency describes the ability of stem cells to differentiate into derivatives of the three germ layers. In reporting new human pluripotent stem cell lines, their clonal derivatives or the safety of differentiated derivatives for transplantation, assessment of pluripotency is essential. Historically, the ability to form teratomas in vivo containing different somatic cell types following injection into immunodeficient mice has been regarded as functional evidence of pluripotency. In addition, the teratomas formed can be analyzed for the presence of malignant cells. However, use of this assay has been subject to scrutiny for ethical reasons on animal use and due to the lack of standardization in how it is used, therefore questioning its accuracy. In vitro alternatives for assessing pluripotency have been developed such as ScoreCard and PluriTest. However, it is unknown whether this has resulted in reduced use of the teratoma assay. Here, we systematically reviewed how the teratoma assay was reported in publications between 1998 (when the first human embryonic stem cell line was described) and 2021. Our analysis of >400 publications showed that in contrast to expectations, reporting of the teratoma assay has not improved: methods are not yet standardized, and malignancy was examined in only a relatively small percentage of assays. In addition, its use has not decreased since the implementation of the ARRIVE guidelines on reduction of animal use (2010) or the introduction of ScoreCard (2015) and PluriTest (2011). The teratoma assay is still the preferred method to assess the presence of undifferentiated cells in a differentiated cell product for transplantation since the in vitro assays alone are not generally accepted by the regulatory authorities for safety assessment. This highlights the remaining need for an in vitro assay to test malignancy of stem cells.

Expression of Intermediate Filaments in the Developing Testis and Testicular Germ Cell Cancer.

Cytokeratin and desmin expression have been associated with Sertoli cell maturity and the development of testicular germ cell cancer (TGCC). Thus, the present study aimed to characterize the expression of these intermediate filaments in normal testis development and TGCC. Cytokeratin and desmin were determined by immunohistochemistry and immunofluorescence in human fetal, and adult testis and tissue from patients with pre-invasive germ cell neoplasia in-situ (GCNIS) or invasive TGCC. Desmin was expressed in Sertoli cells of the human fetal testis, and the proportion of desmin expressing Sertoli cells was significantly reduced in the second trimester, compared with the first trimester (31.14% vs. 6.74%, p = 0.0016). Additionally, Desmin was expressed in the majority of Sertoli cells in the adult testis and TGCC samples. Cytokeratin was detected in Sertoli cells of human fetal testis but was not expressed in Sertoli cells of human adult testis. In patients with TGCC, cytokeratin was not expressed in Sertoli cells in tubules with active spermatogenesis but was detected in Sertoli cells in tubules containing GCNIS cells in patients with both pre-invasive and invasive TGCC. In conclusion, desmin was not associated with Sertoli cell maturation or progression to TGCC. However, cytokeratin appeared to be an indicator of impaired Sertoli cell maturation.

Familial Male-limited Precocious Puberty (FMPP) and Testicular Germ Cell Tumors.

OBJECTIVE: The purpose of this study is to report development of a malignant testicular germ cell tumor (GCT) in 2 young adult males with familial male-limited precocious puberty (FMPP) because of LHCGR pathogenic variants in 2 families. Secondarily, to study the possible relation between FMPP and testicular tumors and to investigate whether FMPP might predispose to development of malignant testicular tumors in adulthood a literature review is conducted. METHODS: Data on 6 cases in 2 families are obtained from the available medical records. In addition, a database search is performed in Cochrane, PubMed, and Embase for studies that report on a possible link between FMPP and testicular tumors. RESULTS: The characteristics of 6 males with FMPP based on activating LH receptor (LHCGR) germline pathogenic variants are described, as are details of the testicular GCTs. Furthermore, a literature review identified 4 more patients with signs of FMPP and a (precursor of) testicular GCT in adolescence or adulthood (age 15-35 years). Additionally, 12 patients with signs of precocious puberty and, simultaneously, occurrence of a Leydig cell adenoma or Leydig cell hyperplasia are reported. CONCLUSION: There is a strong suggestion that FMPP might increase the risk of development of testicular GCTs in early adulthood compared with the risk in the general population. Therefore, prolonged patient monitoring from mid-pubertal age onward including instruction for self-examination and periodic testicular ultrasound investigation in patients with a germline LHCGR pathogenic variant might contribute to early detection and thus early treatment of testicular GCT.

Humanised Mice and Immunodeficient Mice (NSG) Are Equally Sensitive for Prediction of Stem Cell Malignancy in the Teratoma Assay.

The use of human pluripotent stem cells (hPSCs) in regenerative medicine has great potential. However, it is important to exclude that these cells can undergo malignant transformation, which could lead to the development of malignant tumours. This property of hPSCs is currently being tested using the teratoma assay, through which cells are injected into immunodeficient mice. Transplantation of stem cells in immunocompromised recipient animals certainly has a much higher incidence of tumour formation. On the other hand, the results obtained in immunodeficient mice could indicate a risk of tumour formation that is practically not present in the human immunocompetent recipient. The presence of a humanised immune system might be more representative of the human situation; therefore, we investigated if the demonstrated malignant features of chosen and well-characterised stem cell lines could be retrieved and if new features could arise in a humanised mouse model. Hu-CD34NSGTM (HIS) mice were compared side by side with immunocompromised mice (NSG) after injection of a set of benign (LU07) and malignant (LU07+dox and 2102Ep) cell lines. Analysis of the tumour development, histological composition, pathology evaluation, and malignancy-associated miRNA expression levels, both in tumour and plasma samples, revealed no differences among mouse groups. This indicates that the HIS mouse model is comparable to, but not more sensitive than, the NSG immunodeficient model for studying the malignancy of stem cells. Since in vivo teratoma assay is cumbersome, in vitro methods for the detection of malignancy are urgently needed.

Chromosome 3p25.3 Gain Is Associated With Cisplatin Resistance and Is an Independent Predictor of Poor Outcome in Male Malignant Germ Cell Tumors.

PURPOSE: Cisplatin is the main systemic treatment modality for male type II germ cell tumors (GCTs). Although generally very effective, 5%-10% of patients suffer from cisplatin-resistant disease. Identification of the driving mechanisms of resistance will enable improved risk stratification and development of alternative treatments. METHODS: We developed and characterized cisplatin-resistant GCT cell line models and compared their molecular characteristics with patient samples with cisplatin resistance and/or a poor clinical outcome. Subsequently, the association between the overlapping genetic features and clinical data was assessed. Finally, we used Cox regression to determine the prognostic relevance of these features within the currently used risk classification. RESULTS: Gain of chromosome 3p25.3 was detected in all cisplatin-resistant cell lines, and copy number of this region correlated with the level of resistance (R = 0.96, P = 1.5e-04). Gain of this region was detected at low frequencies in primary tumors and at higher frequencies in relapsed and/or cisplatin-resistant tumors. Chromosome 3p25.3 gain was associated with shorter progression-free survival and overall survival, with the strongest association observed in nonseminomas excluding pure teratomas. 3p25.3 gain was more frequently observed in tumors with yolk sac tumor histology and predicted adverse outcome independent of the International Germ Cell Cancer Collaborative Group risk classification and the presence of TP53/MDM2 alterations. CONCLUSION: On the basis of both in vitro analyses and clinical data, we found 3p25.3 to be strongly associated with cisplatin resistance and poor clinical outcome in male type II GCTs. Using genomic profiling, 3p25.3 status could help to improve risk stratification in male patients with type II GCT. Further characterization of this locus and underlying mechanisms of resistance is warranted to guide development of novel treatment approaches for cisplatin-resistant disease.

The Occurrence of MET Ectodomain Shedding in Oral Cancer and Its Potential Impact on the Use of Targeted Therapies.

The receptor tyrosine kinase MET has gained attention as a therapeutic target. Although MET immunoreactivity is associated with progressive disease, use of targeted therapies has not yet led to major survival benefits. A possible explanation is the lack of companion diagnostics (CDx) that account for proteolytic processing. During presenilin-regulated intramembrane proteolysis, MET's ectodomain is shed into the extracellular space, which is followed by γ-secretase-mediated cleavage of the residual membranous C-terminal fragment. The resulting intracellular fragment is degraded by the proteasome, leading to downregulation of MET signaling. Conversely, a membrane-bound MET fragment lacking the ectodomain (MET-EC-) can confer malignant potential. Use of C- and N-terminal MET monoclonal antibodies (moAbs) has illustrated that MET-EC- occurs in transmembranous C-terminal MET-positive oral squamous cell carcinoma (OSCC). Here, we propose that ectodomain shedding, resulting from G-protein-coupled receptor transactivation of epidermal growth factor receptor signaling, and/or overexpression of ADAM10/17 and/or MET, stabilizes and possibly activates MET-EC- in OSCC. As MET-EC- is associated with poor prognosis in OSCC, it potentially has impact on the use of targeted therapies. Therefore, MET-EC- should be incorporated in the design of CDx to improve patient stratification and ultimately prolong survival. Hence, MET-EC- requires further investigation seen its oncogenic and predictive properties.

The Role of TP53 in Cisplatin Resistance in Mediastinal and Testicular Germ Cell Tumors.

Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations (testicular versus mediastinal/extragonadal), with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line (hemizygously mutated TP53, mutant TP53 mediastinal GCT). The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.

Paclitaxel, Ifosfamide, and Cisplatin in Patients with Poor-prognosis Disseminated Nonseminomatous Germ Cell Tumors with Unfavorable Serum Tumor Marker Decline After First Cycle of Chemotherapy. The GCT-SK-003 Phase II Trial.

BACKGROUND: Germ cell tumors represent highly curable disease even in metastatic stage. However, poor-risk patients with an unfavorable serum tumor marker (STM) decline after the first cycle of chemotherapy represent a subgroup with dismal prognosis, with approximately 50% cure rate using bleomycin, etoposide, and cisplatin (BEP). OBJECTIVE: The aim of this study was to determine the efficacy and safety of paclitaxel, ifosfamide, and cisplatin (TIP) in this patient population. DESIGN SETTING AND PARTICIPANTS: This was an open-labeled, nonrandomized, single-center phase II trial to study the efficacy and toxicity of TIP in the first-line treatment of germ cell tumor patients with an unfavorable decline of STMs. Nineteen patients with a poor prognosis according to the International Germ Cell Cancer Collaboration Group classification and an unfavorable STM decline after the first cycle of chemotherapy were included in this phase II study (NCT02414685). The treatment regimen consisted of paclitaxel 250 mg/m2 on day 1, ifosfamide 1200 mg/m2 on days 1-5, and cisplatin 20 mg/m2 on days 1-5, totally for four cycles. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was complete response (CR) rate. An optimal Simon two-stage design was used with a type I error of 5% and study power of 80%. If fewer than six CRs to study therapy have been observed among the first 19 patients, the study was to be terminated. RESULTS AND LIMITATIONS: A CR was achieved in four (21.1%) patients; therefore, the study was terminated in the first stage. A favorable response rate (CR or partial remission with negative tumor markers) was observed in 14 (78.9%) patients. At a median follow-up period of 35.2 mo (range, 5.6-62.1 mo), ten (52.6%) patients experienced disease progression and eight patients (42.1%) died. The 2-yr progression-free and overall survival was 41.2% (95% confidence interval [CI] 16.8-65.7) and 72.7% (95% CI 48.9-96.4), respectively. TIP was well tolerated, and no unexpected toxicity was observed. No informative biomarkers, including miR-371a-3p was identified. CONCLUSIONS: Treatment modification from the BEP to the TIP regimen in patients with an unfavorable STM decline after the first cycle of chemotherapy was not associated with improved outcome, and four cycles of BEP remain the standard treatment option in this patient population. PATIENT SUMMARY: Poor-risk patients with an unfavorable serum tumor marker decline after the first cycle of chemotherapy represent a subgroup with dismal prognosis, with an approximately 50% cure rate using bleomycin, etoposide, and cisplatin (BEP). Treatment modification from the BEP regimen to the paclitaxel, ifosfamide, and cisplatin regimen in patients with an unfavorable serum tumor marker decline after the first cycle of chemotherapy was not associated with improved outcome, and four cycles of BEP remain the standard treatment option in this patient population.

Combining Hypermethylated RASSF1A Detection Using ddPCR with miR-371a-3p Testing: An Improved Panel of Liquid Biopsy Biomarkers for Testicular Germ Cell Tumor Patients.

The classical serum tumor markers used routinely in the management of testicular germ cell tumor (TGCT) patients-alpha fetoprotein (AFP) and human chorionic gonadotropin (HCG)-show important limitations. miR-371a-3p is the most recent promising biomarker for TGCTs, but it is not sufficiently informative for detection of teratoma, which is therapeutically relevant. We aimed to test the feasibility of hypermethylated RASSF1A (RASSF1AM) detected in circulating cell-free DNA as a non-invasive diagnostic marker of testicular germ cell tumors, combined with miR-371a-3p. A total of 109 serum samples of patients and 29 sera of healthy young adult males were included, along with representative cell lines and tumor tissue samples. We describe a novel droplet digital polymerase chain reaction (ddPCR) method for quantitatively assessing RASSF1AM in liquid biopsies. Both miR-371a-3p (sensitivity = 85.7%) and RASSF1AM (sensitivity = 86.7%) outperformed the combination of AFP and HCG (sensitivity = 65.5%) for TGCT diagnosis. RASSF1AM detected 88% of teratomas. In this representative cohort, 14 cases were negative for miR-371a-3p, all of which were detected by RASSF1AM, resulting in a combined sensitivity of 100%. We have described a highly sensitive and specific panel of biomarkers for TGCT patients, to be validated in the context of patient follow-up and detection of minimal residual disease.

Recurrence of a Mediastinal Germ-Cell Tumor as a Somatic-Type Malignancy: A Complex Case Report.

Background and case: An adolescent male presented with a second mediastinal tumor 1.5 years after treatment of a proven malignant germ-cell tumor in that location. The differential diagnosis included a recurrent germ-cell tumor or a non-germ cell malignancy. Serum tumor markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) were negative. The first biopsy was not informative, and the second biopsy gave a broad differential diagnosis including secondary non-germ cell malignancy using histology and immunohistochemistry. DNA methylation profiling, RNA sequencing, and targeted microRNA371a-3p profiling was subsequently performed, without a supportive result. After resection of the tumor the definitive diagnosis yielded two secondary non-germ cell malignancies in the form of a leiomyosarcoma and a solitary neuro endocrine carcinoma (NEC). In spite of the differences between the molecular profiles of the initial germ-cell tumor, the leiomyosarcoma and large-cell NEC are clonally related, as determined by the presence of identical chromosomal breakpoints. The copy number profiles suggest an initial polyploidization step, followed by various independent chromosomal gains and losses. This case demonstrates that germ-cell tumors must be evaluated carefully, including molecularly, in which the non-germ cell malignancy is negative for miR-371a-3p, both in tissue as well as in serum, in contrast to the primary tumor. We conclude that the patient presented with a primary type II mediastinal GCT and, a year and a half later, followed by a leiomyosarcoma and a large-cell NEC presenting as two secondary somatic-type malignancies clonally related to the original GCT. Conclusions: Malignant germ-cell tumors are known to recur as a somatic-type malignancy in very rare cases. This case report illustrates the challenges faced in defining the nature and clonality of the secondary somatic-type malignancies.

Comparative Analyses of Liquid-Biopsy MicroRNA371a-3p Isolation Protocols for Serum and Plasma.

MicroRNAs (miRNAs) are short, non-coding RNAs involved in translation regulation. Dysregulation has been identified in cancer cells. miRNAs can be secreted and detectable in body fluids; therefore, they are potential liquid-biopsy biomarkers. The miR-371a-3 cluster members are an example, monitoring the presence of malignant germ cell tumors based on patient serum/plasma analyses. However, a large variety of isolation techniques on sample types (serum vs. plasma) are reported, hampering interstudy comparisons. Therefore, we analyzed the impact of using the miRNeasy Serum/Plasma Kit (cell-free total RNA purification) Qiagen extraction kit and the TaqMan anti-miRNA bead-capture procedure of ThermoFisher for miRNA isolation. Ten normal male matched serum and plasma samples and seventeen testicular germ cell tumor patient serum samples were investigated. The Qiagen kit requires a higher input volume (200 µL vs. 50 µL), resulting in higher sensitivity. Serum and plasma comparison demonstrated high similarity in miRNA levels. Titration experiments showed that the bead-capture procedure is superior in cases of lower starting volumes (<100 µL). This study highlights the strengths and limitations of two different isolation protocols, relevant for in vivo analysis with small starting volumes. In summary, miRNA detection levels results varied little between plasma and serum, whereas for low volumes the bead capture isolation method is preferable.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors.

BACKGROUND: Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors. METHODS: Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo. RESULTS: We demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11. CONCLUSIONS: VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

Treatment and Survival of Malignant Extracranial Germ Cell Tumours in the Paediatric Population: A Systematic Review and Meta-Analysis.

OBJECTIVE: This systematic review and meta-analysis was performed to explore overall survival (OS) and event free survival (EFS) rates internationally over the past two decades and to define specific subgroups with inferior outcomes which may demand different treatment strategies. METHODS: The search focused on malignant extracranial germ cell tumours (GCTs) in the paediatric population. The initial database search identified 12,556 articles; 32 articles were finally included in this review, comprising a total of 5095 patients. RESULTS: The studies were heterogeneous, varying from single institution reports to large prospective trials. Older studies, describing eras where non-platinum-based chemotherapy regimens were used, showed clearly worse outcomes. Survival for stage I-II gonadal disease is excellent. On the other hand, patients with an initial alpha-fetoprotein (AFP) > 10,000 ng/mL or kU/L, age > 11 years and stage IV disease confer a survival disadvantage. For testicular disease in particular, lymphovascular invasion and certain histopathological subtypes, such as embryonal carcinoma (EC) and mixed malignant GCTs, survival is poorer. Survival data for sacrococcygeal and mediastinal GCTs show a heterogeneous distribution across studies in this review, independent of year of publication. Patients > 12 years presenting with a mediastinal GCT pose a subpopulation which fares worse than GCTs in other locations or age groups. This is independent of AFP levels, stage of disease or treatment protocol, and these patients may demand a different treatment strategy. CONCLUSIONS: This review describes the heterogeneous nature of GCTs in different anatomical locations, impacting on stage at presentation, treatment modalities used and survival data. Despite this heterogeneity, in line with the current developmental biology-based classification system, subpopulations can be defined which have an inferior EFS and OS and where future research and more individualised treatment would help to improve survival.

Mechanisms of TP53 Pathway Inactivation in Embryonic and Somatic Cells-Relevance for Understanding (Germ Cell) Tumorigenesis.

The P53 pathway is the most important cellular pathway to maintain genomic and cellular integrity, both in embryonic and non-embryonic cells. Stress signals induce its activation, initiating autophagy or cell cycle arrest to enable DNA repair. The persistence of these signals causes either senescence or apoptosis. Over 50% of all solid tumors harbor mutations in TP53 that inactivate the pathway. The remaining cancers are suggested to harbor mutations in genes that regulate the P53 pathway such as its inhibitors Mouse Double Minute 2 and 4 (MDM2 and MDM4, respectively). Many reviews have already been dedicated to P53, MDM2, and MDM4, while this review additionally focuses on the other factors that can deregulate P53 signaling. We discuss that P14ARF (ARF) functions as a negative regulator of MDM2, explaining the frequent loss of ARF detected in cancers. The long non-coding RNA Antisense Non-coding RNA in the INK4 Locus (ANRIL) is encoded on the same locus as ARF, inhibiting ARF expression, thus contributing to the process of tumorigenesis. Mutations in tripartite motif (TRIM) proteins deregulate P53 signaling through their ubiquitin ligase activity. Several microRNAs (miRNAs) inactivate the P53 pathway through inhibition of translation. CCCTC-binding factor (CTCF) maintains an open chromatin structure at the TP53 locus, explaining its inactivation of CTCF during tumorigenesis. P21, a downstream effector of P53, has been found to be deregulated in different tumor types. This review provides a comprehensive overview of these factors that are known to deregulate the P53 pathway in both somatic and embryonic cells, as well as their malignant counterparts (i.e., somatic and germ cell tumors). It provides insights into which aspects still need to be unraveled to grasp their contribution to tumorigenesis, putatively leading to novel targets for effective cancer therapies.