A paclitaxel-hyaluronan conjugate (ONCOFID-P-B™) in patients with BCG-unresponsive carcinoma in situ of the bladder: a dynamic assessment of the tumor microenvironment.

BACKGROUND: The intravesical instillation of the paclitaxel-hyaluronan conjugate ONCOFID-P-B™ in patients with bacillus Calmette-Guérin (BCG)-unresponsive bladder carcinoma in situ (CIS; NCT04798703 phase I study), induced 75 and 40% of complete response (CR) after 12 weeks of intensive phase and 12 months of maintenance phase, respectively. The aim of this study was to provide a detailed description of the tumor microenvironment (TME) of ONCOFID-P-B™-treated BCG-unresponsive bladder CIS patients enrolled in the NCT04798703 phase I study, in order to identify predictive biomarkers of response. METHODS: The composition and spatial interactions of tumor-infiltrating immune cells and the expression of the most relevant hyaluronic acid (HA) receptors on cancer cells, were analyzed in biopsies from the 20 patients enrolled in the NCT04798703 phase I study collected before starting ONCOFID-P-B™ therapy (baseline), and after the intensive and the maintenance phases. Clinical data were correlated with cell densities, cell distribution and cell interactions. Associations between immune populations or HA receptors expression and outcome were analyzed using univariate Cox regression and log-rank analysis. RESULTS: In baseline biopsies, patients achieving CR after the intensive phase had a lower density of intra-tumoral CD8+ cytotoxic T lymphocytes (CTL), but also fewer interactions between CTL and macrophages or T-regulatory cells, as compared to non-responders (NR). NR expressed higher levels of the HA receptors CD44v6, ICAM-1 and RHAMM. The intra-tumoral macrophage density was positively correlated with the expression of the pro-metastatic and aggressive variant CD44v6, and the combined score of intra-tumoral macrophage density and CD44v6 expression had an AUC of 0.85 (95% CI 0.68-1.00) for patient response prediction. CONCLUSIONS: The clinical response to ONCOFID-P-B™ in bladder CIS likely relies on several components of the TME, and the combined evaluation of intra-tumoral macrophages density and CD44v6 expression is a potentially new predictive biomarker for patient response. Overall, our data allow to advance a potential rationale for combinatorial treatments targeting the immune infiltrate such as immune checkpoint inhibitors, to make bladder CIS more responsive to ONCOFID-P-B™ treatment.

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion.

ABSTRACT: Circular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL.

MiR-146a-5p enrichment in small-extracellular vesicles of relapsed pediatric ALCL patients promotes macrophages infiltration and differentiation.

Anaplastic large cell lymphoma (ALCL) is a CD30-positive lymphoma accounting for 20% of all pediatric T-cell lymphomas. Current first line treatment can cure most of ALCL patients but 10-30% of them are resistant or relapse. In this context, liquid biopsy has the potential to help clinicians in disease screening and treatment response monitoring. Small-RNA-sequencing analysis performed on plasma small-extracellular vesicles (s-EVs) from 20 pediatric anaplastic lymphoma kinase positive (ALK + ) ALCL patients at diagnosis revealed a specific miRNAs cargo in relapsed patients compared to non-relapsed, with seven miRNAs enriched in s-EVs of relapsed patients. MiR-146a-5p and miR-378a-3p showed a negative prognostic impact both in univariate and multivariate analysis, possibly representing, together with let-7 g-5p, a miRNA panel for the early identification of high-risk patients. Among them, miR-146a-5p is known to modulate tumor supporting-M2 macrophages differentiation, but the role of these cells in pediatric ALK + ALCL is still unknown. To elucidate the role of miR-146a-5p and M2 macrophages in pediatric ALCL disease, THP-1-derived macrophages were treated with s-EVs from ALK + ALCL cell lines, showing increased miR-146a-5p intracellular expression, migrating capability and M2-markers CD163 and Arginase-1 upregulation. In turn, conditioned media from M2 macrophages or miR-146a-5p-transfected THP-1 increased ALCL cells' aggressive features and were enriched in interleukin-8. Overall, these data suggest a role of miR-146a-5p in promoting macrophage infiltration and M2-like polarization in ALCL. Our findings incite further investigation on the role of M2 macrophages in ALCL aggressiveness and dissemination, also considering the novel treatment options targeting tumor associated macrophages.

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision.

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation.

CircFBXW7 in patients with T-cell ALL: depletion sustains MYC and NOTCH activation and leukemia cell viability.

Circular RNAs (circRNAs) are emerging as new players in leukemogenic mechanisms. In patients with T-cell Acute Lymphoblastic Leukemia (T-ALL), the recent report of a remarkable dysregulation of circRNAs incited further functional investigation. Here we focus on circFBXW7, highly expressed in T-cells, with a notably high abundance of the circular compared to linear transcript of FBXW7. Two T-ALL patient cohorts profiled with RNA-seq were analyzed in comparison with five populations of developing thymocytes as normal counterpart, quantifying circRNA and gene expression. CircFBXW7 expression was very heterogeneous in T-ALL patients allowing their stratification in two groups with low and high expression of this circRNA, not correlated with FBXW7 mutation status and T-ALL molecular subgroups. With a loss-of-function study in T-ALL in vitro, we demonstrate that circFBXW7 depletion increases leukemic cell viability and proliferation. Microarray profiling highlighted the effect of the circFBXW7 silencing on gene expression, with activation of pro-proliferative pathways, supporting a tumor suppressor role of circFBXW7 in T-ALL. Further, MYC and intracellular NOTCH1 protein levels, as well as expression of MYC target and NOTCH signaling genes were elevated after circFBXW7 depletion, suggesting an inhibitory role of circFBXW7 in these oncogenic axes. Plus, low circFBXW7 levels were associated with a particular gene expression profile in T-ALL patients, which was remarkably mirrored by the effects of circFBXW7 loss-of-function in vitro. CircFBXW7 depletion notably emerges as a new factor enhancing a proliferative phenotype and the activation of the MYC signaling pathway, key players in this aggressive malignancy.

Systematic benchmarking of statistical methods to assess differential expression of circular RNAs.

Circular RNAs (circRNAs) are covalently closed transcripts involved in critical regulatory axes, cancer pathways and disease mechanisms. CircRNA expression measured with RNA-seq has particular characteristics that might hamper the performance of standard biostatistical differential expression assessment methods (DEMs). We compared 38 DEM pipelines configured to fit circRNA expression data's statistical properties, including bulk RNA-seq, single-cell RNA-seq (scRNA-seq) and metagenomics DEMs. The DEMs performed poorly on data sets of typical size. Widely used DEMs, such as DESeq2, edgeR and Limma-Voom, gave scarce results, unreliable predictions or even contravened the expected behaviour with some parameter configurations. Limma-Voom achieved the most consistent performance throughout different benchmark data sets and, as well as SAMseq, reasonably balanced false discovery rate (FDR) and recall rate. Interestingly, a few scRNA-seq DEMs obtained results comparable with the best-performing bulk RNA-seq tools. Almost all DEMs' performance improved when increasing the number of replicates. CircRNA expression studies require careful design, choice of DEM and DEM configuration. This analysis can guide scientists in selecting the appropriate tools to investigate circRNA differential expression with RNA-seq experiments.

Discovery of fusion circular RNAs in leukemia with KMT2A::AFF1 rearrangements by the new software CircFusion.

Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

Detecting differentially expressed circular RNAs from multiple quantification methods using a generalized linear mixed model.

Finding differentially expressed circular RNAs (circRNAs) is instrumental to understanding the molecular basis of phenotypic variation between conditions linked to circRNA-involving mechanisms. To date, several methods have been developed to identify circRNAs, and combining multiple tools is becoming an established approach to improve the detection rate and robustness of results in circRNA studies. However, when using a consensus strategy, it is unclear how circRNA expression estimates should be considered and integrated into downstream analysis, such as differential expression assessment. This work presents a novel solution to test circRNA differential expression using quantifications of multiple algorithms simultaneously. Our approach analyzes multiple tools' circRNA abundance count data within a single framework by leveraging generalized linear mixed models (GLMM), which account for the sample correlation structure within and between the quantification tools. We compared the GLMM approach with three widely used differential expression models, showing its higher sensitivity in detecting and efficiently ranking significant differentially expressed circRNAs. Our strategy is the first to consider combined estimates of multiple circRNA quantification methods, and we propose it as a powerful model to improve circRNA differential expression analysis.

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.

CRAFT: a bioinformatics software for custom prediction of circular RNA functions.

Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.

Clinical significance of circulating tumor cells and cell-free DNA in pediatric rhabdomyosarcoma.

Liquid biopsy analysis represents a powerful and noninvasive tool to uncover biomarkers for disseminated disease assessment and longitudinal monitoring of patients. Herein, we explored the value of circulating and disseminated tumor cells (CTC and DTC, respectively) and cell-free DNA (cfDNA) in pediatric rhabdomyosarcoma (RMS). Peripheral blood and bone marrow samples were analyzed to detect and enumerate CTC and DTC, respectively. We used the epithelial cellular adhesion molecule (EpCAM)-based CellSearch platform coupled with an automatic device to collect both EpCAM-positive and EpCAM-low/negative CTCs. The standard assay was implemented, including the mesenchymal marker desmin. For selected cases, we molecularly profiled primary tumors and liquid biopsy biomarkers using whole-exome sequencing and droplet digital PCR, respectively. RMS patients with metastatic disease had a significantly higher number of CTCs compared to those with localized disease, whereas DTCs were detected independently of disease presentation. The use of the desmin marker remarkably increased the identification of CTCs and DTCs in RMS samples. Of note, CTC clusters were detected in RMS patients with disseminated disease. Further, cfDNA and CTC molecular features closely reflected the molecular makeup of primary tumors and informed of disease course.

Sensitive, reliable and robust circRNA detection from RNA-seq with CirComPara2.

Circular RNAs (circRNAs) are a large class of covalently closed RNA molecules originating by a process called back-splicing. CircRNAs are emerging as functional RNAs involved in the regulation of biological processes as well as in disease and cancer mechanisms. Current computational methods for circRNA identification from RNA-seq experiments are characterized by low discovery rates and performance dependent on the analysed data set. We developed CirComPara2 (https://github.com/egaffo/CirComPara2), a new automated computational pipeline for circRNA discovery and quantification, which consistently achieves high recall rates without losing precision by combining multiple circRNA detection methods. In our benchmark analysis, CirComPara2 outperformed state-of-the-art circRNA discovery tools and proved to be a reliable and robust method for comprehensive transcriptome characterization.

CircIMPACT: An R Package to Explore Circular RNA Impact on Gene Expression and Pathways.

Circular RNAs (circRNAs) are transcripts generated by back-splicing. CircRNAs might regulate cellular processes by different mechanisms, including interaction with miRNAs and RNA-binding proteins. CircRNAs are pleiotropic molecules whose dysregulation has been linked to human diseases and can drive cancer by impacting gene expression and signaling pathways. The detection of circRNAs aberrantly expressed in disease conditions calls for the investigation of their functions. Here, we propose CircIMPACT, a bioinformatics tool for the integrative analysis of circRNA and gene expression data to facilitate the identification and visualization of the genes whose expression varies according to circRNA expression changes. This tool can highlight regulatory axes potentially governed by circRNAs, which can be prioritized for further experimental study. The usefulness of CircIMPACT is exemplified by a case study analysis of bladder cancer RNA-seq data. The link between circHIPK3 and heparanase (HPSE) expression, due to the circHIPK3-miR558-HPSE regulatory axis previously determined by experimental studies on cell lines, was successfully detected. CircIMPACT is freely available at GitHub.

Bioinformatic Pipelines to Analyze lncRNAs RNAseq Data.

RNA-sequencing could be nowadays considered the gold standard to study the coding and noncoding transcriptome. The great advantage of high-throughput sequencing in the characterization and quantification of long noncoding RNA (lncRNA) resides in its capability to capture the complexity of lncRNA transcripts configuration patterns, even in the presence of several alternative isoforms, with superior accuracy and discovery power compared to other technologies such as microarrays or PCR-based methods. In this chapter, we provide a protocol for lncRNA analysis using through high-throughput sequencing, indicating the main difficulties in the annotation pipeline and showing how an accurate evaluation of the procedure can help to minimize biased observations.

Bioinformatic Analysis of Circular RNA Expression.

Circular RNAs (circRNAs) are stable RNA molecules generated by backsplicing that play regulatory functions through interaction with other RNA and proteins, as well as by encoding peptides. Dysregulation of circRNA expression can drive cancer development and progression with different mechanisms. CircRNAs are currently regarded as extremely attractive molecules in cancer research for the identification of new and possibly targetable disease regulatory networks and for the development of biomarkers for cancer diagnosis, prognosis definition, and monitoring. Using specific experimental and computational protocols, circRNAs can be identified through RNA-seq by spotting the reads spanning backsplice junctions, which are specific to circular molecules. In this chapter, we report a state-of-the-art computational protocol for a genome-wide analysis of circRNAs from RNA-seq data, which considers circRNA detection, quantification, and differential expression testing. Finally, we indicate how to determine circular transcript sequences and the resources for an in silico functional characterization of circRNAs.

Low miR-214-5p Expression Correlates With Aggressive Subtypes of Pediatric ALCL With Non-Common Histology.

The unsatisfactory cure rate of relapsing ALK-positive Anaplastic Large-Cell Lymphoma (ALCL) of childhood calls for the identification of new prognostic markers. Here, the small RNA landscape of pediatric ALK-positive ALCL was defined by RNA sequencing. Overall, 121 miRNAs were significantly dysregulated in ALCL compared to non-neoplastic lymph nodes. The most up-regulated miRNA was miR-21-5p, whereas miR-19a-3p and miR-214-5p were reduced in ALCL. Characterization of miRNA expression in cases that relapsed after first line therapy disclosed a significant association between miR-214-5p down-regulation and aggressive non-common histology. Our results suggest that miR-214-5p level may help to refine the prognostic stratification of pediatric ALK-positive ALCL.

MicroRNA-497/195 is tumor suppressive and cooperates with CDKN2A/B in pediatric acute lymphoblastic leukemia.

We previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In a search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes. We found high expression of miR-497 and miR-195 (hereafter miR-497/195) in patient-derived xenograft samples with slow engraftment derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression, and we identified cyclin-dependent kinase 4 (CDK4)- and cyclin-D3 (CCND3)-mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195-mediated cell cycle regulation was underscored by finding (in an additional independent series of patient samples) that high expression of miR-497/195 together with a full sequence for CDKN2A and CDKN2B (CDKN2A/B) was associated with excellent outcome, whereas deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as a new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.