Subpar reporting of pre-analytical variables in RNA-focused blood plasma studies.

Extracellular RNA (cell-free RNA; exRNA) from blood-derived liquid biopsies is an appealing, minimally invasive source of disease biomarkers. As pre-analytical variables strongly influence exRNA measurements, their reporting is essential for meaningful interpretation and replication of results. The aim of this review was to chart to what extent pre-analytical variables are documented, to pinpoint shortcomings and to improve future reporting. In total, 200 blood plasma exRNA studies published in 2018 or 2023 were reviewed for annotation of 22 variables associated with blood collection, plasma preparation, and RNA purification. Our results show that pre-analytical variables are poorly documented, with only three out of 22 variables described in over half of the publications. The percentage of variables reported ranged from 4.6% to 54.6% (mean 24.84%) in 2023 and from 4.6% to 57.1% (mean 28.60%) in 2018. Recommendations and guidelines (i.e., BRISQ, ASCO-CAP, BloodPAC, PPMPT, and CEN standards) have currently not resulted in improved reporting. In conclusion, our results highlight the lack of reporting pre-analytical variables in exRNA studies and advocate for a consistent use of available standards, endorsed by funders and journals.

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles.

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

Long noncoding RNA expression profiling in cancer: Challenges and opportunities.

In recent years, technological advances in transcriptome profiling revealed that the repertoire of human RNA molecules is more diverse and extended than originally thought. This diversity and complexity mainly derive from a large ensemble of noncoding RNAs. Because of their key roles in cellular processes important for normal development and physiology, disruption of noncoding RNA expression is intrinsically linked to human disease, including cancer. Therefore, studying the noncoding portion of the transcriptome offers the prospect of identifying novel therapeutic and diagnostic targets. Although evidence of the relevance of noncoding RNAs in cancer is accumulating, we still face many challenges when it comes to accurately profiling their expression levels. Some of these challenges are inherent to the technologies employed, whereas others are associated with characteristics of the noncoding RNAs themselves. In this review, we discuss the challenges related to long noncoding RNA expression profiling, highlight how cancer long noncoding RNAs provide new opportunities for cancer diagnosis and treatment, and reflect on future developments.

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients.

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of noninvasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in 2 independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis, and overall survival revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential abundance analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume. These findings were validated in longitudinal serum samples from metastatic neuroblastoma patients, where the 9 miRNAs were associated with disease burden and treatment response.

Detecting long non-coding RNA biomarkers in prostate cancer liquid biopsies: Hype or hope?

Prostate cancer is a heterogeneous malignancy, with clinical courses widely differing between indolent and aggressive lethal disease. This heterogeneity calls for a more personalized approach towards diagnosis, prognosis, treatment decision, monitoring and follow-up of patients. In this review, we discuss the possibilities and drawbacks of detecting RNA biomarkers in biological fluids to improve disease-specific survival and quality of life. In particular, we examine literature on long non-coding RNAs in blood and urine of prostate cancer patients. We thereby specifically focus on the need for standard operation procedures on many different levels, analytical validation, clinical validation, and assessment of clinical utility. We argue that thorough multi-step validation of putative biomarkers is necessary for successful translation into clinical prostate cancer care. Our recommendations may also prove useful to biomarker research in other cancers.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Unique long non-coding RNA expression signature in ETV6/RUNX1-driven B-cell precursor acute lymphoblastic leukemia.

Overwhelming evidence indicates that long non-coding RNAs have essential roles in tumorigenesis. Nevertheless, their role in the molecular pathogenesis of pediatric B-cell precursor acute lymphoblastic leukemia has not been extensively explored. Here, we conducted a comprehensive analysis of the long non-coding RNA transcriptome in ETV6/RUNX1-positive BCP-ALL, one of the most frequent subtypes of pediatric leukemia. First, we used primary leukemia patient samples to identify an ETV6/RUNX1 specific expression signature consisting of 596 lncRNA transcripts. Next, integration of this lncRNA signature with RNA sequencing of BCP-ALL cell lines and lncRNA profiling of an in vitro model system of ETV6/RUNX1 knockdown, revealed that lnc-NKX2-3-1, lnc-TIMM21-5, lnc-ASTN1-1 and lnc-RTN4R-1 are truly regulated by the oncogenic fusion protein. Moreover, sustained inactivation of lnc-RTN4R-1 and lnc-NKX2-3-1 in ETV6/RUNX1 positive cells caused profound changes in gene expression. All together, our study defined a unique lncRNA expression signature associated with ETV6/RUNX1-positive BCP-ALL and identified lnc-RTN4R-1 and lnc-NKX2-3-1 as lncRNAs that might be functionally implicated in the biology of this prevalent subtype of human leukemia.

LIN28B overexpression defines a novel fetal-like subgroup of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B overexpression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy 7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMML.

MYCN-driven regulatory mechanisms controlling LIN28B in neuroblastoma.

LIN28B has been identified as an oncogene in various tumor entities, including neuroblastoma, a childhood cancer that originates from neural crest-derived cells, and is characterized by amplification of the MYCN oncogene. Recently, elevated LIN28B expression levels were shown to contribute to neuroblastoma tumorigenesis via let-7 dependent de-repression of MYCN. However, additional insight in the regulation of LIN28B in neuroblastoma is lacking. Therefore, we have performed a comprehensive analysis of the regulation of LIN28B in neuroblastoma, with a specific focus on the contribution of miRNAs. We show that MYCN regulates LIN28B expression in neuroblastoma tumors via two distinct parallel mechanisms. First, through an unbiased LIN28B-3'UTR reporter screen, we found that miR-26a-5p and miR-26b-5p regulate LIN28B expression. Next, we demonstrated that MYCN indirectly affects the expression of miR-26a-5p, and hence regulates LIN28B, therefore establishing an MYCN-miR-26a-5p-LIN28B regulatory axis. Second, we provide evidence that MYCN regulates LIN28B expression via interaction with the LIN28B promoter, establishing a direct MYCN-LIN28B regulatory axis. We believe that these findings mark LIN28B as an important effector of the MYCN oncogenic phenotype and underline the importance of MYCN-regulated miRNAs in establishing the MYCN-driven oncogenic process.

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728.