MONITORING METHOD FOR ADULT T-CELL LEUKEMIA/LYMPHOMA (ATL)

publication date: 2018-10-11; filing date: 2017-04-06The present invention refers to a method for preparing a linear PCR product from genomic DNA derived from cells of a host subject infected with an retrovirus or a subject suffering from a disease associated with said retrovirus, wherein the PCR product contains a target sequence comprising an integration site of the retrovirus in the host genomic DNA of the cells, said integration site comprising at least the terminal end of 3'-LTR or 5'-LTR sequence of the retrovirus and the adjacent host genomic DNA sequence, wherein the PCR product comprises a first terminus and a second terminus and sequences in the following order: sequences specific for the first terminus, a sequence comprising at least6 consecutive random nucleotides followed by a linker sequence, host genomic DNA sequence, at least the terminal endof 3'-LTR or 5' -LTR sequence of the retrovirus, sequences specific for the second terminus; wherein the PCR product is prepared by specific steps. The present invention also refers to a method for determining and longitudinally monitor the dominant leukemic T lymphocyte clone in subjects suffering from Adult T-cell leukemia/lymphoma (ATL), wherein a linear PCR product is prepared by the method according to the first aspect of the present invention, said PCR product is subjected to multiplex sequencing thereby determining all insertion sites and all shearing sites, the shearing sites are correlated to the respective insertion site, followed by counting the number of different shear sites for each insertion site representing a specific T lymphocyte clone, removing any PCR duplicate from consideration by eliminating reads that have the same insertion site and the same random tag, and determining the abundance of each specific T lymphocyte clone therefrom

Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq

In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1–3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs

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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Improving the bioinformatics analysis of HTS clonality data in virus-induced leukemia

Proviral integration into the host genome is one of the main hallmarks of infection by oncogenic retroviruses. This event creates a life-long signature, each infected cell being characterized by a specific integration site (IS). Monitoring of the clonal architecture over time (clone: population of cells sharing an identical IS) has significantly contributed to a better understanding of HIV persistence, gene therapy vector mediated treatment and deltaretrovirus-induced leukemia. Our lab recently developed an optimized high-throughput sequencing (HTS) based clonality method. It enables the identification of proviral integration sites genome-wide while simultaneously quantifying the abundance of the corresponding clones. The method is superior to any of the previously available protocols, mainly in terms of sensitivity, cost-effectiveness and hands-on time, making it suitable for routine clinical observation of infected individuals. Using this method, we recently showed that longitudinal monitoring of the dominant leukemic clone in patients infected by Human T-cell Leukemia Virus-1 (HTLV-1) better predicts therapeutic response (Artesi et al, Leukemia, 2017). We applied the method to biological samples isolated from HTLV-1 infected patients and Bovine Leukemia Virus (BLV) infected animals (bovine and sheep). This resulted in the generation of an unprecedented volume of raw sequence data. In this study we developed a novel clonality analysis pipeline that better exploits the potential of the method, improving previously published protocols

Investigating non-coding viral transcripts in Bovine Leukemia Virus induced leukemia

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of infected individuals develop leukemia/lymphoma following a long period of asymptomatic infection (~7 years in cattle, several decades in human). Experimental infection of sheep with BLV results in a reduced latency period (2 years on average), making for an attractive cancer model. A further advantage of the BLV system is that it is possible to infect sheep via injection of a cloned provirus, facilitating the mutation of specific parts of the viral genome to examine the function of viral products in vivo. Like HTLV-1, the BLV mRNAs/proteins are transcribed from the viral 5’ long terminal repeat (LTR), a region rich in regulatory elements. It was previously believed that the BLV provirus was transcriptionally silent in tumors, however we identified a cluster of five abundantly expressed non-canonical RNA polymerase III dependent microRNAs (miRNAs) encoded by BLV (Rosewick et al., PNAS 2013). In addition, using RNA sequencing we recently discovered viral antisense transcripts originating in the 3' Long Terminal Repeat (LTR) of the BLV provirus (Durkin et al., Retrovirology 2016) . While 5'LTR dependent transcription is absent in malignant cells, both the viral miRNAs and the antisense transcripts are expressed in all BLV induced leukemic and pre-leukemic samples examined to date, pointing to a vital role in the life cycle of the virus and a critical function in cellular transformation.Investigating non-coding viral transcripts in Bovine Leukemia Virus induced leukemi

Identification and characterization of novel bovine leukemia virus (BLV) antisense transcripts in leukemic and pre-leukemic clones

The deltaretrovirus Bovine Leukemia Virus (BLV) is 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, produces 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. Like the case in HTLV-1 the 5’LTR BLV provirus is transcriptionally silent in tumors, however the provirus is not entirely quiescent, constitutively express the BLV microRNAs in tumors. Using RNA-seq, we found that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ~600bp (AS1-S) and a less abundant longer transcript of ~2200bp (AS1-L). Alternative splicing also creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV

Somatic Structural And Numerical Aberrations In Bovine Leukemia Virus Induced Tumors

Bovine Leukemia Virus (BLV) is a deltaretrovirus that integrates into B-cells producing a lifelong infection in cattle. Like its close relative Human T-cell leukemia virus-1 (HTLV-1), BLV induces an aggressive leukemia/lymphoma in about ~5% of infected individuals. While not a natural host it is possible to infect sheep with BLV and in contrast to cattle, all infected sheep develop tumors at an accelerated rate (~18 months). Historically research into both viruses has primarily focused on their transcripts/proteins. However secondary events are likely to be important as only a subset of infected individuals, following many decades of infection, develop a tumor. At the current time little is known about the landscape of somatic changes in BLV induced tumors. To examine gross numerical and structural variants (SVs) we assayed 12 bovine tumors on the BovineSNP50 Illumina BeadChip as well as 22 ovine tumors on the OvineSNP50 Illumina BeadChip. We also carried out whole genome sequencing (~30X) on 4 ovine tumors with matched normal tissue. Initial examination of the tumors revealed frequent aneuploidy, with orthologous regions of the genome involved in both species. Focal SVs identified included an amplification (>4 copies) of the terminus of BTA16 in three tumors (contains PTPRC & miR-181), while the tumor suppressor CDKN2A on OAR2 was deleted in multiple ovine tumors. For the 4 sequenced tumors multiple time points over the course of infection were available allowing us to determine when these SVs arose via nested PCR. Interestingly we observed that the SVs involving well know cancer driver genes generally appear many months prior to tumor development. These preliminary results indicate that tumors induced by HTLV-1 and BLV display somatic structural changes that impinge on overlapping sets of genes and point to the emergence of SVs affecting cancer driver genes in the preleukemic clone, well before the clone undergoes rapid expansion

Structural And Numerical Somatic Changes In BLV Induced Tumors

Background Bovine Leukemia Virus (BLV) is a deltaretrovirus that integrates into B-cells producing a lifelong infection in cattle. Like its close relative Human T-cell leukemia virus-1 (HTLV-1) BLV induces an aggressive leukemia/lymphoma in about ~5% of infected individuals. While not a natural host it is possible to infect sheep with BLV and in contrast to cattle, all infected sheep develop tumors at an accelerated rate (~18 months). Historically research into both viruses has primarily focused on their transcripts/proteins. However secondary events are likely to be important as only a subset of infected individuals, following many decades of infection, develop a neoplasm. Recent work in HTLV-1 induced adult T cell leukemia/lymphoma (ATL) identified a large number of somatic changes associated with malignancy. At the current time little is known about the landscape of somatic changes in BLV induced tumors. Methods To examine gross numerical and structural aberrations in BLV induced tumors we assayed 12 bovine tumors on the BovineSNP50 Illumina BeadChip as well as 22 ovine tumors on the OvineSNP50 Illumina BeadChip. The resultant data was examined with penCNV in combination with visual inspection of the Log R ratios and B allele frequencies. Results The tumors from both species showed frequent aneuploidy with the whole or a large part of chromosomes BTA5, BTA10, BTA14 and BTA24 duplicated in >50% of the bovine tumors. In the ovine tumors chromosomes OAR5, OAR7, OAR9 and OAR16 were frequently duplicated. It is interesting to note that BTA14 is orthologous to OAR9 and both are orthologous to HSA8q, a part of the human genome frequently duplicated in ATLs and other leukemias. In addition a number of focal structural variants were observed. In cattle the terminus of BTA16, which includes the CD45 gene and miR-181 was amplified (>4 copies) in three tumors. In sheep, mirroring observations in ATL, the CDKN2A gene was deleted in multiple tumors. Conclusion These preliminary results indicate that tumors induced by HTLV-1 and BLV display somatic structural changes that impinge on overlapping sets of genes. Secondarily, it appears that in the case of BLV despite the much shorter incubation periods in sheep, the resultant tumors in both the natural and the experimental host display evidence of substantial genome instability

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