Safety and efficacy of salvage endoscopic submucosal dissection for Barrett's neoplasia recurrence after radiofrequency ablation.

BACKGROUND AND STUDY AIMS: This study evaluated the safety and efficacy of salvage endoscopic submucosal dissection for Barrett's neoplasia recurrence after radiofrequency ablation. PATIENTS AND METHODS: Data from patients at sixteen centers were collected for a multicentric retrospective study. Patients who underwent at least one RFA treatment for Barrett's esophagus and thereafter underwent further esophageal ESD for neoplasia recurrence were included. RESULTS: Data from 56 patients treated by salvage ESD performed between April 2014 and November 2022 were collected. Immediate complications included one muscular tear (1.8%) treated with stent (Agree classification: grade IIIa), two patients had transmural perforations (4%) and five patients had muscular tears (9%) treated with clips and without clinical impact and not considered as adverse event. Seven patients (12.5%) developed strictures, treated by balloon dilation (grade IIIa). Histological analysis showed 36 adenocarcinomas, 17 high-grade dysplasia, and 3 low-grade dysplasia. En-bloc and R0 resection rates were 89% and 66%, respectively. Resections were curative in thirty-three patients (59%), non-curative in 22 patients (39%), including 11 "local risk" (19.5%) and 11 "high risk" resections (19.5%). At the end of follow-up with a median time of 14 [0-75] months after salvage ESD eventually associated with further endoscopic treatment (RFA, argon plasma coagulation, endoscopic mucosal resection, ESD), neoplasia remission ratio was 37/53 (70%) and the median remission time was 13 [1-75] months. CONCLUSION: In expert hands, salvage ESD is a safe and effective treatment for recurrence of Barrett's neoplasia after RFA treatment.

Endoscopic duodenal mucosal resurfacing for nonalcoholic steatohepatitis (NASH): a pilot study.

Background and study aims Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease worldwide with limited treatment options. Duodenal mucosal resurfacing (DMR) has been associated with improvement in glycaemic parameters and liver function tests (LFTs) in type 2 diabetes. This study aimed to assess the effect of DMR in patients with NASH. Patients and methods This was a single-center, open-label pilot study. Patients with definite, biopsy-proven NASH (nonalcoholic fatty liver disease activity score [NAS] ≥ 4) underwent a single DMR procedure followed by a 2-week postprocedural diet, without lifestyle intervention. The primary outcome was either resolution of NASH with no worsening of fibrosis or improvement in fibrosis (≥ 1 stage) with no worsening of NASH at 12 months. Secondary outcomes were changes in key histological parameters of NASH, surrogate markers of fibrosis, LFTs, and metabolic factors at 12 months. Results From 2017 to 2019, 14 patients underwent successful DMR, of whom 11 were included in the analysis. After 12 months, no resolution of NASH was observed, while three patients (27 %) had marginal improvement in fibrosis with no worsening of NASH. Serious adverse events related to the procedure were reported in two patients out of 14 (14 %). Neither weight loss nor improvement in NAS score, or in the other secondary outcomes, were observed at 12 months. Conclusions In this small and heterogenous study population, we found that DMR, in the absence of lifestyle intervention, did not induce NASH resolution and marginally improved liver fibrosis at 12 months.

Targeted deep sequencing reveals clonal and subclonal mutational signatures in Adult T-cell leukemia/lymphoma and defines an unfavorable indolent subtype.

Adult T-cell leukemia/lymphoma (ATL) carries a poor prognosis even in indolent subtypes. We performed targeted deep sequencing combined with mapping of HTLV-1 proviral integration sites of 61 ATL patients of African and Caribbean origin. This revealed mutations mainly affecting TCR/NF-kB (74%), T-cell trafficking (46%), immune escape (29%), and cell cycle (26%) related pathways, consistent with the genomic landscape previously reported in a large Japanese cohort. To examine the evolution of mutational signatures upon disease progression while tracking the viral integration architecture of the malignant clone, we carried out a longitudinal study of patients who either relapsed or progressed from an indolent to an aggressive subtype. Serial analysis of relapsing patients identified several patterns of clonal evolution. In progressing patients, the longitudinal study revealed NF-kB/NFAT mutations at progression that were present at a subclonal level at diagnosis (allelic frequency < 5%). Moreover, the presence in indolent subtypes of mutations affecting the TCR/NF-kB pathway, whether clonal or subclonal, was associated with significantly shorter time to progression and overall survival. Our observations reveal the clonal dynamics of ATL mutational signatures at relapse and during progression. Our study defines a new subgroup of indolent ATLs characterized by a mutational signature at high risk of transformation.

An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites.

The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integration within low complexity regions of the genome, providing a more accurate estimation of clone abundance. Finally, we recommend the application of a "clean-and-recover" step to clonality datasets generated from large cohorts and longitudinal studies. In summary, we report an optimized bioinformatics workflow for NGS clonality analysis and describe a new set of steps to guide the computational process. We demonstrate that the application of this protocol to the analysis of HTLV-1 and bovine leukemia virus (BLV) clonality datasets improves the quality of data processing and provides a more accurate definition of the clonal landscape in infected individuals. The optimized workflow and analysis recommendations can be implemented in the majority of bioinformatics pipelines developed to analyze LAM-PCR-based NGS clonality datasets.

HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation.

The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.

Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis.

Human T-cell leukaemia virus type-1 (HTLV-1) and bovine leukaemia virus (BLV) infect T- and B-lymphocytes, respectively, provoking a polyclonal expansion that will evolve into an aggressive monoclonal leukaemia in ∼5% of individuals following a protracted latency period. It is generally assumed that early oncogenic changes are largely dependent on virus-encoded products, especially TAX and HBZ, while progression to acute leukaemia/lymphoma involves somatic mutations, yet that both are independent of proviral integration site that has been found to be very variable between tumours. Here, we show that HTLV-1/BLV proviruses are integrated near cancer drivers which they affect either by provirus-dependent transcription termination or as a result of viral antisense RNA-dependent cis-perturbation. The same pattern is observed at polyclonal non-malignant stages, indicating that provirus-dependent host gene perturbation contributes to the initial selection of the multiple clones characterizing the asymptomatic stage, requiring additional alterations in the clone that will evolve into full-blown leukaemia/lymphoma.

Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs.

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5 % develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5'LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. RESULTS: Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5'RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. CONCLUSIONS: The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV.

Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma.

Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy, however, remains poorly understood, especially as viral mRNA/protein are tightly silenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the bovine leukemia virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of noncanonical RNA polymerase III (Pol III)-transcribed viral microRNAs in leukemic B cells in the complete absence of Pol II 5'-LTR-driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, bovine leukemia virus microRNAs represent ∼40% of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. Bovine leukemia virus microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute to deciphering the intricate perturbations that underlie malignant transformation.