Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-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. METHODS: We 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. RESULTS: MiR-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. CONCLUSION: Targeting 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.

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

Anti-oncostatin M antibody inhibits the pro-malignant effects of oncostatin M receptor overexpression in squamous cell carcinoma.

The oncostatin M (OSM) receptor (OSMR) shows frequent gene copy number gains and overexpression in cervical squamous cell carcinomas (SCCs), associated with adverse clinical outcomes. In SCC cells that overexpress OSMR, the major ligand OSM induces multiple pro-malignant effects, including invasion, secretion of angiogenic factors, and metastasis. Here, we demonstrate, for the first time, that OSMR overexpression in SCC cells activates cell-autonomous feed-forward signalling, via further expression of OSMR and OSM and sustained STAT3 activation, despite expression of the negative regulator suppressor of cytokine signalling 3 (SOCS3). The pro-malignant effects associated with OSMR overexpression are critically mediated by JAK-STAT3 activation, which is induced by exogenous OSM and also by autocrine OSM-OSMR interactions. Importantly, specific inhibition of OSM-OSMR interactions by neutralizing antibodies significantly inhibits STAT3 activation and feed-forward signalling, leading to reduced invasion, angiogenesis, and metastasis. Our findings are supported by data from 1254 clinical SCC samples, in which OSMR levels correlated with multiple cognate genes, including OSM, STAT3, and downstream targets. These data strongly support the development of OSM-OSMR-blocking antibodies as biologically targeted therapies against SCCs of the cervix and other anatomical sites. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression of the oncostatin-M receptor in cervical squamous cell carcinoma is associated with epithelial-mesenchymal transition and poor overall survival.

BACKGROUND: Copy-number gain of the oncostatin-M receptor (OSMR) occurs frequently in cervical squamous cell carcinoma (SCC) and is associated with adverse clinical outcome. We previously showed that OSMR overexpression renders cervical SCC cells more sensitive to the major ligand oncostatin-M (OSM), which increases migration and invasion in vitro. We hypothesised that a major contribution to this phenotype would come from epithelial-mesenchymal transition (EMT). METHODS: We performed a comprehensive integrated study, involving in vitro cell line studies, in vivo animal models and numerous clinical samples from a variety of anatomical sites. RESULTS: In independent sets of cervical, head/neck and lung SCC tissues, OSMR expression levels correlated with multiple EMT-associated phenotypic markers and transcription factors. OSM treatment of OSMR overexpressing cervical SCC cells produced consistent EMT changes and increased tumour sphere formation in suspension culture. In a mouse model, OSMR overexpressing SCC cells treated with OSM showed significant increases in lung colonisation. The biological effects of exogenous OSM were mirrored by highly significant adverse overall survival in cervical SCCs with OSMR overexpression (N=251). CONCLUSIONS: OSM:OSMR interactions are able to induce EMT, increased cancer stem cell-like properties and enhanced lung colonisation in SCC cells. These changes are likely to contribute to the highly significant adverse outcome associated with OSMR overexpression in cervical SCCs.

Virus transcript levels and cell growth rates after naturally occurring HPV16 integration events in basal cervical keratinocytes.

Cervical carcinogenesis is characterized by a clonal selection process in which the high-risk human papillomavirus (HRHPV) genome usually changes from the extra-chromosomal (episomal) state seen in productive infections to DNA that is integrated into host chromosomes. However, it is not clear whether all HRHPV integration events provide cells with a selective growth advantage compared with the episome-containing cells from which they originate. It is also unclear whether selection of cells containing a particular integrant from a mixed population simply reflects the highest levels of virus oncogene expression or has additional determinants. These early events in cervical carcinogenesis cannot readily be addressed by cross-sectional studies of clinical samples. We used the W12 model system to generate a panel of cervical squamous cell clones that were derived from an identical background under non-competitive conditions and differed only by the genomic site of HPV16 integration. Compared with the 'baseline' episome-containing cells from which they were isolated, only 9/17 clones (53%) showed significantly greater growth rates and only 7/17 (41%) showed significantly greater expression of the major virus oncogenes E7/E6. There were significant variations in levels of HPV16 transcription per DNA template, changes that were associated with histone modifications in the integrated virus chromatin. Cell growth rates showed only weak and non-significant associations with protein and mRNA levels for E7, E6, and the mean E7/E6 values. We conclude that HPV16 integration in basal cervical cells does not necessarily lead to increased levels of virus oncogenes, or to a competitive growth advantage, when compared with the initiating episome-containing cells.

Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Oncostatin M receptor (OSMR) is commonly over-expressed in advanced cervical squamous cell carcinoma (SCC), producing a significantly worse clinical outcome. Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness. Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of cervical and oral SCC. TGM2 depletion in cervical SCC cells abrogated OSM-induced migration on fibronectin-coated surfaces and invasiveness through extracellular matrix, while ectopic expression of TGM2 increased cell motility and invasiveness. Confocal microscopy and co-immunoprecipitation assays showed that TGM2 interacted with integrin-α5ß1 in the presence of fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5ß1 and fibronectin were also over-expressed in cervical and oral SCC, where levels correlated with those of OSMR and TGM2. This combined tissue and in vitro study demonstrates for the first time that stimulation of over-expressed OSMR in cervical SCC cells activates TGM2/integrin-α5ß1 interactions and induces pro-malignant changes. We conclude that an OSMR/TGM2/integrin-α5ß1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease.

BACKGROUND & AIMS: Models of non-alcohol-related fatty liver disease (NAFLD) reveal features of accelerated ageing, such as impaired regeneration, and an increased risk of hepatocellular carcinoma. The relation between accelerated ageing, disease progression and clinical outcome has not been previously investigated and is the subject of the current study. METHODS: Liver sections from 70 patients with NAFLD (105 biopsies) and 60 controls were studied for telomere length, nuclear area, DNA damage and cell cycle phase markers, using quantitative fluorescent in situ hybridization and immunohistochemistry. RESULTS: Hepatocyte telomeres were shorter in NAFLD than controls (p <0.0001). Hepatocytes in NAFLD demonstrated lack of cell cycle progression beyond G1/S phase and high-level expression of p21, the universal cell cycle inhibitor (p=0.001). γ-H(2)AX expression increased with steatosis (p=0.01), indicating DNA damage, and was associated with shorter hepatocyte telomeres (p <0.0001). Hepatocyte p21 expression correlated with fibrosis stage and diabetes mellitus, independently (p <0.001 and p=0.002, respectively). Further analysis revealed that an adverse liver-related outcome was strongly associated with higher hepatocyte p21 expression and greater hepatocyte nuclear area (p=0.02 and p=0.006), but not with telomere length. In paired biopsies, changes in hepatocyte p21 expression and nuclear area mirrored changes in fibrosis stage (p=0.01 and p=0.006, respectively). CONCLUSIONS: These findings are consistent with hepatocyte senescence and permanent cell cycle arrest in NAFLD. Hepatocyte senescence correlated closely with fibrosis stage, diabetes mellitus, and clinical outcome. Hepatocyte p21 expression could be used as a prognostic marker and for stratification in clinical studies.

Sustained telomere length in hepatocytes and cholangiocytes with increasing age in normal liver.

UNLABELLED: Telomeres, a validated biomarker of aging, comprise multiple nucleotide repeats capping chromosomes that shorten with each cell cycle until a critical length is achieved, precipitating cell senescence. Only two previous studies focused on the effect of aging in "normal" liver tissue, but these studies were compromised by small sample size, limited age range, tissue derived from individuals with an increased risk of senescence, and the use of liver homogenates. We developed a robust large-volume, four-color quantitative fluorescent in situ hybridization technique to measure telomere length in large numbers of hepatocytes, Kupffer cells, hepatic stellate cells, CD4-positive and CD8-positive lymphocytes, and cholangiocytes. Following validation against the gold standard (Southern blotting), the technique was applied to normal archived paraffin-embedded liver tissue obtained following reperfusion of implanted donor liver. We studied 73 highly selected donors aged 5-79 years with a short medical illness preceding death and no history of liver disease, reperfusion injury, or steatosis and normal graft function 1-year posttransplantation. Cholangiocytes had significantly longer telomeres compared with all other intrahepatic lineages over a wide age range (P < 0.05). Age-related telomere attrition was restricted to sinusoidal cells (i.e., Kupffer cells [P = 0.0054] and stellate cells [P = 0.0001]). Cholangiocytes and hepatocytes showed no age-related telomere shortening. CONCLUSION: In normal liver and over a broad age range, cholangiocytes have longer telomeres than all other intrahepatic lineages. Age-related telomere length decline is restricted to Kupffer cells and stellate cells.

Small non-coding RNA sequencing reveals global dysregulation of piwi-interacting RNA (piRNA) expression in gonadal malignant germ cell tumours.

BACKGROUND: Analyses of small non-coding RNA (ncRNA) expression in malignant germ cell tumours (GCTs) have focused on microRNAs (miRNAs). As GCTs all arise from primordial germ cells, and piwi-interacting RNAs (piRNAs) have important roles in maintaining germline integrity via transposon silencing, we hypothesised that malignant GCTs are characterised by fundamental piRNA dysregulation. AIMS: We undertook global small ncRNA sequencing in malignant GCTs, in order to describe small ncRNA expression changes for both miRNAs and piRNAs. MATERIALS AND METHODS: We performed small ncRNA next generation sequencing on a representative panel of 47 samples, comprising malignant GCT (n = 31) and control (n = 16) tissues/cell lines. Following quality control and normalisation, filtered count reads were used for differential miRNA and piRNA expression analyses via DESeq2. Predicted mRNA targets for piRNAs were identified and utilised for pathway enrichment analyses. RESULTS: Overall, miRNAs and piRNAs comprised 21.9% and 43.0% of small ncRNA species, respectively. There were 749 differentially expressed miRNAs in malignant GCTs, of which 536 (72%) were over-expressed and 213 (28%) under-expressed. The top-ranking over-expressed miRNAs were exclusively from the miR-371∼373 and miR-302/367 clusters. The most significantly under-expressed miRNAs were miR-100-5p, miR-214-3p, miR-125b-5p and let-7 family members, including miR-202-3p. There were 1,121 differentially expressed piRNAs in malignant GCTs, of which 167 (15%) were over-expressed and 954 (85%) under-expressed. Of note, of the top-20 differentially expressed piRNAs, 16 were over-expressed, of which piR-hsa-2506793 was both top-ranking and most abundant. Mobile element (ME; i.e., transposon)-associated piRNAs comprised 166 (15%) of the 1,121 differentially expressed piRNAs, of which 165 (>99%) were down-regulated. The remaining 955 (85%) non-ME-associated piRNAs may have wider cellular roles. To explore this, predicted mRNA targets of differentially expressed piRNAs identified putative involvement in cancer-associated pathways. CONCLUSION: This study confirms previous miRNA observations, giving credence to our novel demonstration of global piRNA dysregulation in gonadal malignant GCTs, through both ME and non-ME-associated pathways, which likely contributes to GCT pathogenesis.

Evaluation of miR-371a-3p to predict viable germ cell tumor in patients with pure seminoma receiving retroperitoneal lymph node dissection.

INTRODUCTION AND OBJECTIVE: Conventional serum tumor markers (STMs) for testicular germ cell tumors (GCTs) offer limited performance with particularly poor sensitivity in cases of minimal residual disease and pure seminoma. While growing evidence has indicated miR-371a-3p to be a superior biomarker, its utility in detecting pure seminoma at recurrence has not been extensively explored. This study's objective was to explore miR-371a-3p's utility in detecting metastatic pure seminoma at retroperitoneal lymph node dissection (RPLND). METHODS: RNA was isolated from patient serum samples collected pre-RPLND. Fifteen patients were assigned to our 'benign' (n = 6) or 'seminoma' (n = 9) group based on pathological confirmation of viable seminoma. Five of the patients received chemotherapy before RPLND (PC-RPLND), and 10 were chemotherapy naïve. MiR-371a-3p expression was quantified via RT-quantitative polymerase chain reaction. The Cq values were statistically evaluated to obtain performance measurements. RESULTS: Median relative expression of miR-371a-3p was higher in the Seminoma group than the Benign, but this difference was not statistically significant (Rq = 3705 and 241, respectively, p = 0.2844). Of the 10 chemotherapy naïve patients, nine had viable seminoma at RPLND, and seven had elevated miR-371a-3p expression. Among the five postchemotherapy (PC) patients, zero had viable GCT at RPLND, and two had elevated miR-371a-3p expression. The primary RPLND group presented 78% sensitivity and 100% specificity. Specificity in the PC-RPLND group was 60%. An optimal Rq threshold of 28.62 was determined by Youden's J statistic, yielding 78% sensitivity and 67% specificity. Receiver operating characteristic analysis provided an AUC of 0.704 (95% CI: 0.43-0.98, p = 0.1949). Despite modest performance, miR-371a-3p exhibited improved sensitivity and specificity compared with conventional STMs. CONCLUSIONS: MiR-371a-3p outperformed STMs in the primary RPLND settings. However, miR-371a-3p was not a robust predictor of pathology in the PC setting. These results suggest that pure seminoma at RPLND is a clinical context, wherein the miRNA assay may require further refinement.

Refining the serum miR-371a-3p test for viable germ cell tumor detection: identification and definition of an indeterminate range.

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) GCT pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to a) utilize threshold-based approaches using raw Cq values, b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and c) to re-run any sample with an indeterminate result.

Refining the serum miR-371a-3p test for viable germ cell tumor detection.

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (GCT) pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to (a) utilize threshold-based approaches using raw Cq values, (b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and (c) to re-run any sample with an indeterminate result.

A Circulating MicroRNA Panel for Malignant Germ Cell Tumor Diagnosis and Monitoring.

Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs (miRNAs) in in the circulation presents specific challenges. Here, we describe an optimized research protocol to assess serum sample quality and quantify levels of a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p) that enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop primers. A multiplexed preamplification stage is then employed to increase the sensitivity of the final quantification step, which is performed using standard singleplex Taqman qPCR methodology.

Circulating microRNAs as biomarkers to assist the management of the malignant germ-cell-tumour subtype choriocarcinoma.

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.

A Multi-institutional Pooled Analysis Demonstrates That Circulating miR-371a-3p Alone is Sufficient for Testicular Malignant Germ Cell Tumor Diagnosis.

BACKGROUND: Circulating microRNAs have clear potential for improving malignant germ-cell-tumor (MGCT) diagnosis. Here, we address the central issue of whether measurement of a single microRNA is sufficient for detecting testicular MGCTs, or whether there is added benefit in quantifying other members of the 4-microRNA panel previously identified (miR-371a-3p/miR-372-3p/miR-373-3p and miR-367-3p). PATIENTS AND METHODS: We performed a pooled analysis of available published raw data where all 4 panel miRNAs had been assessed using pre-amplification PCR technology (4 studies; total 329 patients). Two studies using identical methodology (and identical normalization using endogenous miR-30b-5p) were used in the discovery phase (n = 51 patients: 17 MGCT, 34 controls). The 2 other studies (n = 278 patients: 140 MGCT, 138 controls), which assessed the same test panel but with different normalization approaches (endogenous miR-93-5p, exogenous cel-miR-39-3p), were used for the validation phase. We derived sensitivity, specificity, positive- and negative-predictive-values (PPV/NPV) for the detection thresholds that maximised the Youden Index (YI). RESULTS: In the discovery-phase, the YI was 0.97 for miR-371a-3p (sensitivity = 1, specificity = 0.97), 0.71 (miR-367-3p), 0.68 (miR-372-3p), and 0.50 (miR-373-3p). These findings were confirmed in the validation-phase, with YI of 0.75 for miR-371a-3p (sensitivity = 0.90, specificity 0.85), 0.55 (miR-367-3p), 0.47 (miR-372-3p), and 0.51 (miR-373-3p). Importantly, no combination of markers added additional diagnostic benefit to miR-371a-3p alone, in either the discovery or the validation phase. CONCLUSION: Quantifying circulating miR-371a-3p alone is sufficient for testicular MGCT diagnosis. PCR measurement of this single miRNA marker will be more cost-effective and easier to interpret, facilitating future incorporation into routine clinical practice.

The Alpha-1 Antitrypsin Polymer Load Correlates With Hepatocyte Senescence, Fibrosis Stage and Liver-Related Mortality.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is an important, inherited cause of chronic liver disease. Marked variation in fibrosis stages in patients with homozygous deficiency and those factors that determine whether heterozygous carriers develop liver fibrosis, remain unexplained. Murine studies implicate polymerized alpha-1 antitrypsin (AAT) within hepatocytes as pathogenic. AIMS AND METHODS: The relationship between the quantity of polymerized AAT within hepatocytes (polymer load), stage of hepatic fibrosis and liver-related clinical outcomes (death, evolution to hepatocellular carcinoma, or need for liver transplantation) were investigated using liver tissue from 92 patients at first presentation with either homozygous or heterozygous AATD. Further tissue-based studies were undertaken to determine if polymerized AAT was associated with failure of cell cycle progression, accelerated aging or hepatocyte senescence by immunohistochemical analysis. RESULTS: The AAT polymer load correlated closely with hepatic fibrosis stage and long-term clinical outcome, independent of homozygous or heterozygous status. AAT polymers within hepatocytes correlated closely with failure of cell cycle progression assessed using cell cycle phase markers, accelerated aging manifest as shortened telomeres and other markers consistent with hepatocyte senescence manifest as the presence of nuclear p21 expression and enlarged nuclei. The proportion of p21 positive hepatocytes or hepatocytes with enlarged nuclei correlated with hepatic fibrosis stage and the long-term clinical outcome. CONCLUSION: These data suggest that accumulation of AAT polymers within hepatocytes drives senescence. Quantitation of both the AAT polymer load or hepatocyte senescence markers correlated with hepatic fibrosis stage and the long-term clinical outcome. Either or both could be considered markers of disease severity and treatment response in clinical trials.

Clinical utility of circulating miR-371a-3p for the management of patients with intracranial malignant germ cell tumors.

BACKGROUND: The current biomarkers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) have limited sensitivity/specificity for diagnosing malignant germ cell tumors (GCTs) and "marker-negative" patients require histological confirmation for diagnosis. However, GCTs at intracranial sites are surgically relatively inaccessible and biopsy carries risks. MicroRNAs from the miR-371~373 and miR-302/367 clusters are over-expressed in all malignant GCTs and, in particular, miR-371a-3p shows elevated serum levels at diagnosis for testicular disease. METHODS: Using our robust preamplified qRT-PCR methodology, we quantified miR-371a-3p levels in serum and cerebrospinal fluid (CSF) in a series of 4 representative clinical cases, 3 with intracranial malignant GCT and 1 with Langerhans cell histiocytosis (LCH), compared with appropriate control cases. RESULTS: Serum and/or CSF miR-371a-3p levels distinguished those with intracranial malignant GCTs from LCH and, if known in real time, could have helped clinical management. The benefits would have included (1) the only confirmatory evidence of an intracranial malignant GCT in 1 case, supporting clinical decision making; (2) early detection of intracranial malignant GCT in another, where an elevated CSF miR-371a-3p level preceded the histologically confirmed diagnosis by 2 years; and (3) confirmation of an intracranial malignant GCT relapse with an elevated serum miR-371a-3p level, where serum and CSF AFP and HCG levels were below thresholds for such a diagnosis. CONCLUSIONS: This series highlights the potential for microRNA quantification to assist the noninvasive diagnosis, prognostication, and management for patients with intracranial malignant GCTs. Serum and CSF should be collected routinely as part of future studies to facilitate the extension of these findings to larger patient cohorts.