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.