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The management of intracranial germ-cell tumours is complex because of varied clinical presentations, tumour sites, treatments and outcomes, and the need for multidisciplinary input. Participants of the 2013 Third International CNS Germ Cell Tumour Symposium (Cambridge, UK) agreed to undertake a multidisciplinary Delphi process to identify consensus in the clinical management of intracranial germ-cell tumours. 77 delegates from the symposium were selected as suitable experts in the field and were invited to participate in the Delphi survey, of which 64 (83%) responded to the invitation. Invited participants represented multiple disciplines from Asia, Australasia, Europe, and the Americas. 38 consensus statements encompassing aspects of intracranial germ-cell tumour work-up, staging, treatment, and follow-up were prepared. To achieve consensus, statements required at least 70% agreement from at least 60% of respondents. Overall, 34 (89%) of 38 statements met consensus criteria. This international Delphi approach has defined key areas of consensus that will help guide and streamline clinical management of patients with intracranial germ-cell tumours. Additionally, the Delphi approach identified areas of different understanding and clinical practice internationally in the management of these tumours, areas which should be the focus of future collaborative studies. Such efforts should translate into improved patient outcomes.

\"Opening with Vince Lombardi's last win as coach of the Packers in Super Bowl II and closing with Joe Namath's Super Bowl III guarantee, James Nicholson delivers an original portrait of a sensational closing decade in American culture. Controversies on the field and in the ring reflected broader political and social turmoil in the late-sixties United States. With one of the most contentious presidential elections in US history, the ongoing civil rights movement, and the Vietnam War all storming in the background, Nicholson charts a course through the oddly unsettled waters of American sports in 1968: the Masters golf tournament decided by the strict enforcement of an arcane rule to the detriment of a foreign player; the winner of the Kentucky Derby disqualified for a drug violation; Muhammad Ali waiting in sports exile while he appealed a criminal conviction for draft evasion; an unorthodox rendition of the national anthem at the World Series nearly overshadowing the game it preceded; and the silent gesture at the Mexico City Olympics made by Tommie Smith and John Carlos that shocked the nation\"-- Provided by publisher.

Management of paediatric extracranial germ-cell tumours carries a unique set of challenges. Germ-cell tumours are a heterogeneous group of neoplasms that present across a wide age range and vary in site, histology, and clinical behaviour. Patients with germ-cell tumours are managed by a diverse array of specialists. Thus, staging, risk stratification, and treatment approaches for germ-cell tumours have evolved disparately along several trajectories. Paediatric germ-cell tumours differ from the adolescent and adult disease in many ways, leading to complexities in applying age-appropriate, evidence-based care. Suboptimal outcomes remain for several groups of patients, including adolescents, and patients with extragonadal tumours, high tumour markers at diagnosis, or platinum-resistant disease. Survivors have significant long-term toxicities. The challenge moving forward will be to translate new insights from molecular studies and collaborative clinical data into improved patient outcomes. Future trials will be characterised by improved risk-stratification systems, biomarkers for response and toxic effects, rational reduction of therapy for low-risk patients and novel approaches for poor-risk patients, and improved international collaboration across paediatric and adult cooperative research groups.

Homogeneous and common objective disease assessments and standardised response criteria are important for better international clinical trials for CNS germ cell tumours. Currently, European protocols differ from those of North America (the USA and Canada) in terms of criteria to assess radiological disease response. An international working group of the European Society for Paediatric Oncology Brain Tumour Group and North American Children's Oncology Group was therefore established to review existing literature and current practices, identify major challenges regarding imaging assessment, and develop consensus recommendations for imaging response assessment for patients with CNS germ cell tumours. New clinical imaging standards were defined for the most common sites of CNS germ cell tumour and for the definition of locoregional extension. These new standards will allow the evaluation of response to therapy in patients with CNS germ cell tumours to be more consistent, and facilitate direct comparison of treatment outcomes across international studies.

MicroRNAs (miRNAs) are short, nonprotein‐coding RNAs, and their expression is dysregulated in malignant germ cell tumors (GCTs). Here, we investigated the causes and consequences of downregulated miR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p levels in malignant GCTs regardless of age, site, or subtype. Quantitative RT‐PCR was used to assess miR‐99a‐5p/miR‐100‐5p, miR‐125b‐5p, and associated gene expression in malignant GCT tissues/cell lines [seminoma (Sem), yolk sac tumor (YST), embryonal carcinoma (EC)]. Cells were treated with demethylating 5‐azacytidine and pyrosequencing was performed. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment was used to treat malignant GCT cells. Global messenger RNA (mRNA) targets of the replenished miRNAs were identified and Metascape used to study pathway effects. We found that expression levels of miR‐99a‐5p/miR‐100‐5p and miR‐125b‐5p, their respective pri‐miRNAs, and associated genes from chromosomes 11 and 21 (chr11/chr21) were downregulated and highly correlated in malignant GCT cells. Treatment with 5‐azacytidine caused upregulation of these miRNAs, with pyrosequencing revealing hypermethylation of their chr11/chr21 loci, likely contributing to miR‐100‐5p/miR‐125b‐5p downregulation. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment resulted in growth inhibition in Sem/YST cells, with miR‐100‐5p/miR‐125b‐5p mRNA targets enriched in downregulated genes, which were involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Knockdown of the miR‐100‐5p/miR‐125b‐5p target tripartite motif containing 71 (TRIM71kd) recapitulated miR‐100‐5p/miR‐125b‐5p replenishment, with growth inhibition and cell cycle disruption of Sem/YST/EC cells. Further, replenishment led to reduced lin‐28 homolog A (LIN28A) levels and concomitant increases in let‐7 (MIRLET7B) tumor suppressor miRNAs, creating a sustained reversion of cell phenotype. In summary, combination miR‐100‐5p/miR‐125b‐5p mimic replenishment or TRIM71kd caused growth inhibition in malignant GCT cells via cell cycle disruption. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy. MiR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p microRNAs are downregulated in malignant germ cell tumors (GCTs). Combination replenishment of these microRNAs using mimics resulted in growth inhibition in representative cell lines, with consequent downregulation of target genes involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy.

Background We hypothesised that differences in microRNA expression profiles contribute to the contrasting natural history and clinical outcome of the two most common types of malignant germ cell tumour (GCT), yolk sac tumours (YSTs) and germinomas. Results By direct comparison, using microarray data for paediatric GCT samples and published qRT-PCR data for adult samples, we identified microRNAs significantly up-regulated in YSTs (n = 29 paediatric, 26 adult, 11 overlapping) or germinomas (n = 37 paediatric). By Taqman qRT-PCR we confirmed differential expression of 15 of 16 selected microRNAs and further validated six of these (miR-302b, miR-375, miR-200b, miR-200c, miR-122, miR-205) in an independent sample set. Interestingly, the miR-302 cluster, which is over-expressed in all malignant GCTs, showed further over-expression in YSTs versus germinomas, representing six of the top eight microRNAs over-expressed in paediatric YSTs and seven of the top 11 in adult YSTs. To explain this observation, we used mRNA expression profiles of paediatric and adult malignant GCTs to identify 10 transcription factors (TFs) consistently over-expressed in YSTs versus germinomas, followed by linear regression to confirm associations between TF and miR-302 cluster expression levels. Using the sequence motif analysis environment iMotifs, we identified predicted binding sites for four of the 10 TFs (GATA6, GATA3, TCF7L2 and MAF) in the miR-302 cluster promoter region. Finally, we showed that miR-302 family over-expression in YST is likely to be functionally significant, as mRNAs down-regulated in YSTs were enriched for 3' untranslated region sequences complementary to the common seed of miR-302a~miR-302d. Such mRNAs included mediators of key cancer-associated processes, including tumour suppressor genes, apoptosis regulators and TFs. Conclusions Differential microRNA expression is likely to contribute to the relatively aggressive behaviour of YSTs and may enable future improvements in clinical diagnosis and/or treatment.

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.

BackgroundMiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence ‘AAGUGC’, determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally.MethodsWe targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays.ResultsMiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase.ConclusionTargeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Germ cell tumours (GCTs) are a heterogeneous group of rare neoplasms that present in different anatomical sites and across a wide spectrum of patient ages from birth through to adulthood. Once these strata are applied, cohort numbers become modest, hindering inferences regarding management and therapeutic advances. Moreover, patients with GCTs are treated by different medical professionals including paediatric oncologists, neuro-oncologists, medical oncologists, neurosurgeons, gynaecological oncologists, surgeons, and urologists. Silos of care have thus formed, further hampering knowledge dissemination between specialists. Dedicated biobank specimen collection is therefore critical to foster continuous growth in our understanding of similarities and differences by age, gender, and site, particularly for rare cancers such as GCTs. Here, the Malignant Germ Cell International Consortium provides a framework to create a sustainable, global research infrastructure that facilitates acquisition of tissue and liquid biopsies together with matched clinical data sets that reflect the diversity of GCTs. Such an effort would create an invaluable repository of clinical and biological data which can underpin international collaborations that span professional boundaries, translate into clinical practice, and ultimately impact patient outcomes.