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Highlights • Current biomarkers have limited utility for management of germ-cell-tumours. • Limitations include secretion restricted to specific subtypes and long half-life. • Limitations can make interpretation and clinical decision-making challenging. • Circulating microRNAs show promise for management of these tumours. • We identify specific circulating microRNAs for the choriocarcinoma subtype., Germ-cell-tumours (GCTs) are heterogeneous and management is complex. The current conventional biomarkers, alpha-fetoprotein and human-chorionic-gonadotropin (HCG), have limited utility for diagnosis/follow-up as secretion is restricted to specific malignant-GCT subtypes and long half-life can make interpretation and clinical decision-making challenging. We sought to identify circulating microRNAs that reflected choriocarcinoma disease activity more accurately than HCG in a metastatic primary mediastinal nonseminomatous-GCT (PMNSGCT) case with elevated diagnostic serum HCG (>250,000 U/L), consistent with pure choriocarcinoma. We undertook comprehensive microRNA profiling (n = 754 microRNAs) using two 384-well TaqMan Low-Density-Array cards in 16 serum samples; 10 from PMNSGCT diagnosis/follow-up and six controls. Key findings underwent confirmatory qRT-PCR. We identified a serum panel of choriocarcinoma-specific ‘chromosome-19-microRNA-cluster’ (C19MC) microRNAs that were highly elevated at diagnosis but fell rapidly on treatment and normalised before the second full chemotherapy course. We also re-confirmed serum elevation of the previously identified malignant-GCT marker miR-371a-3p at diagnosis. These circulating microRNA markers reflected choriocarcinoma disease activity more accurately than serum HCG and real-time knowledge would have assisted clinical decision-making. With further study, these microRNA markers will facilitate future management of such patients and are likely to result in improved outcomes.

Background: Quantifying circulating nucleic acids is an important new approach to cancer diagnosis/monitoring. Methods: We compared the suitability of serum versus plasma for measuring miRNAs using qRT-PCR and assessed how preanalytic variables that can affect circulating tumor DNA (ctDNA) quantification in plasma also influence miRNA levels. Results: Across 62 blood-derived specimens, plasma samples in EDTA, Streck-DNA, and Streck-RNA tubes showed significantly higher Ct values for multiple housekeeping miRNAs, compared with serum samples. For the EDTA-plasma tubes, this difference was only seen when including the high-speed centrifugation protocol used to optimize ctDNA extraction. In plasma samples derived from blood stored at room temperature for up to 14 days (conditions that typically apply to samples processed for biobanking), levels of endogenous housekeeping miRNAs gradually increased, in parallel with the hemolysis marker hsa-miR-451a, consistent with release from blood cells/platelets. It was necessary to normalize levels of the housekeeping miRNAs to those of hsa-miR-451a, to obtain the stable values needed for referencing test miRNA levels. Conclusions: Our data indicate that plasma samples prepared for ctDNA extraction are suboptimal for miRNA quantification and require the incorporation of multiple data normalization steps. For prospective studies designed to measure both miRNAs and ctDNA, the most suitable approach would be to obtain both serum (for miRNAs) and plasma (for ctDNA). If only plasma can be collected, we recommend an initial low-speed centrifugation step, followed by aliquoting the supernatant into parallel samples, one for direct miRNA quantification, and the other for a further high-speed centrifugation step to optimize ctDNA retrieval. Impact: These recommendations will help “future-proof” clinical studies in which quantification of circulating miRNAs is a component. Cancer Epidemiol Biomarkers Prev; 27(2); 208–18. ©2017 AACR.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

INTRODUCTION: Patients presenting with diabetes insipidus (DI) and subtle pituitary-stalk thickening on MRI represent a management challenge. LCH and germinoma work-up (including serum/CSF AFP/HCG) is usually negative, given the poor sensitivity of these tests. Risks of neurosurgical biopsy in such cases often outweigh perceived benefits. Consequently, biopsy is often only performed for radiological progression. This suboptimal approach may cause diagnostic delay and inferior outcomes. Non-invasive diagnostic microRNA testing for malignant germ cell tumours (MGCTs) has shown substantial promise. Here, we highlight the potential for microRNAs to facilitate earlier diagnosis of CNS germinoma. METHODS: Using qRT-PCR, we quantified CSF miR-371a-3p levels at presentation in two patients (one 15yo male, PIT#1; one 12yo female, PIT#2), both with DI and 5mm pituitary-stalk thickening, and compared levels with those at diagnosis from six CNS-MGCT patients and two controls (one CNS neuroblastoma, one low-grade-glioma). RESULTS: One patient (PIT#1) had elevated CSF miR-371a-3p levels consistent with those seen in CNS-MGCT patients; the other (PIT#2) had levels similar to those seen in controls. Over a two year period of surveillance (2015-2017), and consistent with the abnormally elevated CSF-miR-371a-3p level, the PIT#1 case showed clear radiological progression and after further negative serum/CSF AFP/HCG work-up, biopsy confirmed germinoma. Conversely, the PIT#2 patient showed no change in pituitary-stalk thickening, but other radiological changes suggestive of neurodegenerative LCH. CONCLUSIONS: CSF microRNAs show potential for screening individuals presenting with DI and pituitary-stalk thickening to identify those who might benefit from early biopsy. Ultimately, completely non-invasive CNS-MGCT diagnosis may be feasible.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed “GAGGUA” was enriched in the 3′ untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention. Cancer Res; 73(15); 4872–84. ©2013 AACR.

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ∼15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state. Cancer Res; 70(10); 4081–91. ©2010 AACR.