title: A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours.
abstract: 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.
authors.affiliation: Department of Paediatric Haematology and Oncology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK.|Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.|Department of Paediatrics, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Murray
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.|AstraZeneca, Cambridge Science Park, Cambridge CB4 0FZ, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Bell
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.
authors.forename: Katie L
authors.initials: KL
authors.lastname: Raby
authors.affiliation: Department of Pathology, Erasmus MC University Medical Center Rotterdam, Cancer Institute, Rotterdam, Netherlands.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Rijlaarsdam
authors.affiliation: Department of Pathology, Erasmus MC University Medical Center Rotterdam, Cancer Institute, Rotterdam, Netherlands.
authors.forename: Ad J M
authors.initials: AJ
authors.lastname: Gillis
authors.affiliation: Department of Pathology, Erasmus MC University Medical Center Rotterdam, Cancer Institute, Rotterdam, Netherlands.
authors.forename: Leendert H J
authors.initials: LH
authors.lastname: Looijenga
authors.affiliation: AstraZeneca, Cambridge Science Park, Cambridge CB4 0FZ, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Brown
authors.affiliation: AstraZeneca, Cambridge Science Park, Cambridge CB4 0FZ, UK.
authors.forename: Benoit
authors.initials: B
authors.lastname: Destenaves
authors.affiliation: Department of Paediatric Haematology and Oncology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK.
authors.forename: James C
authors.initials: JC
authors.lastname: Nicholson
authors.affiliation: Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.|Department of Histopathology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Coleman
mesh_terms: D000293:Adolescent; D014408:Biomarkers, Tumor / Q000097:blood* / Q000134:cerebrospinal fluid; D018236:Carcinoma, Embryonal / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis; D016543:Central Nervous System Neoplasms / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D002648:Child; D002675:Child, Preschool; D031954:Choriocarcinoma, Non-gestational / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis; D006063:Chorionic Gonadotropin / Q000097:blood / Q000134:cerebrospinal fluid; D018240:Endodermal Sinus Tumor / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis; D005260:Female; D018237:Germinoma / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000134:cerebrospinal fluid; D009364:Neoplasm Recurrence, Local / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D009373:Neoplasms, Germ Cell and Embryonal / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D010051:Ovarian Neoplasms / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D012445:Sacrococcygeal Region; D012680:Sensitivity and Specificity; D013736:Testicular Neoplasms / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D000509:alpha-Fetoproteins / Q000134:cerebrospinal fluid / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D006063:Chorionic Gonadotropin; C568081:MIRN367 microRNA, human; C554834:MIRN371 microRNA, human; C561021:MIRN372 microRNA, human; C527114:MIRN373 microRNA, human; D035683:MicroRNAs; D000509:alpha-Fetoproteins