Development and validation of a new high-throughput method to investigate the clonality of HTLV-1-infected cells based on provirus integration sites

Firouzi, Sanaz; López, Yosvany; Suzuki, Yutaka; Nakai, Kenta; Sugano, Sumio; Yamochi, Tadanori; Watanabe, Toshiki

doi:10.1186/gm568

Cited by 58 publications

(69 citation statements)

References 60 publications

(104 reference statements)

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“…To overcome this limitation, Firouzi et al introduced oligomer tags of 8 nucleotides that marked sheared DNA before amplification, which enabled the discrimination of .60 000 cells, dramatically expanding the ability to quantify the number of cells in individual clones. 118 The results of these analyses provided firm evidence for the hypothesis that HTLV-1 carriers harbored a large number of HTLV-1-infected and immortalized clones and that each clone originated from a single infected T cell ( Figure 5). The size of cell populations belonging to specific clones (ie, clone sizes) also varied greatly, and some clones had populations large enough to be detected as clonal expansion by conventional techniques, such as Southern blot hybridization and inverse PCR.…”

Section: Clonal Growth and Identification Of Htlv-1-infected T Cells mentioning

confidence: 72%

Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1–infected T cells

Watanabe

2017

Blood

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Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) that develops through a multistep carcinogenesis process involving 5 or more genetic events. We provide a comprehensive overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of genetic abnormalities in ATL that include alterations highly enriched in genes for T-cell receptor–NF-κB signaling such as PLCG1, PRKCB, and CARD11 and gain-of function mutations in CCR4 and CCR7. Conversely, the epigenetic landscape of ATL can be summarized as polycomb repressive complex 2 hyperactivation with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27 methyltransferase enhancer of zeste 2 was shown to be induced by HTLV-1 Tax and NF-κB. Furthermore, provirus integration site analysis with high-throughput sequencing enabled the analysis of clonal composition and cell number of each clone in vivo, whereas multicolor flow cytometric analysis with CD7 and cell adhesion molecule 1 enabled the identification of HTLV-1–infected CD4+ T cells in vivo. Sorted immortalized but untransformed cells displayed epigenetic changes closely overlapping those observed in terminally transformed ATL cells, suggesting that epigenetic abnormalities are likely earlier events in leukemogenesis. These new findings broaden the scope of conceptualization of the molecular mechanisms of leukemogenesis, dissecting them into immortalization and clonal progression. These recent findings also open a new direction of drug development for ATL prevention and treatment because epigenetic marks can be reprogrammed. Mechanisms underlying initial immortalization and progressive accumulation of these abnormalities remain to be elucidated.

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Section: Clonal Growth and Identification Of Htlv-1-infected T Cells mentioning

confidence: 72%

Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1–infected T cells

Watanabe

2017

Blood

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“…These kinds of references can be easily prepared, because the absolute gene copy number is determined from the dilution rate of TLOm1. TL-Om1 cells were also used as a control in a deep-sequencing-based method for the quantification of the clone size of HTLV-1-infected cells in HTLV-1 carrier or ATL patients (28).…”

Section: Discussionmentioning

confidence: 99%

Identification of TL-Om1, an Adult T-Cell Leukemia (ATL) Cell Line, as Reference Material for Quantitative PCR for Human T-Lymphotropic Virus 1

et al. 2015

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Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TLOm1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR. Human T-lymphotropic virus 1 (HTLV-1) was the first retrovirus to be found in humans (1, 2). HTLV-1 is a cause of adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and HTLV-1-associated uveitis (3). Areas where HTLV-1 is endemic are distributed across several different regions, including southern Japan, the Caribbean, South America, and tropical Africa (4, 5). A recent report has shown that the area affected by this infection has expanded from the southern part of Japan to the entire country, particularly the Tokyo metropolitan area (6). Diagnostic tests for HTLV-1 infection are performed mainly with serological assays, such as enzyme-linked immunoabsorbent assay, particle agglutination assay, and Western blotting. Recently, another diagnostic test has been developed. Quantitation of integrated proviral DNA in peripheral blood (proviral load [PVL]) can be performed by quantitative PCR (qPCR) as a risk assessment for ATL or HAM/TSP (7,8).A few studies reported that several samples were positive for viral DNA when tested by PCR even though those samples had been found seroindeterminate for HTLV-1 when tested by Western blotting (9, 10). Their results suggest that HTLV-1 qPCR could be used as an additional test to confirm infection in seroindeterminate samples.Although many laboratories have developed qPCR methods for HTLV-1 detection in Japan, a wide variety of testing methods are used. For example, the target region, primers and probes, and internal control (IC) genes vary among the laboratories (8,(11)(12)(13)(14)(15). These variations lead to significa...

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“…Furthermore, these techniques could detect proliferation of either monoclonal or oligoclonal HTLV-1-infected cells, even in asymptomatic HTLV-1 carriers. More recently, next-generation sequencing (NGS) technology has been used to characterize the clonality of HTLV-1-infected cells in blood samples from carriers and ATL patients, [21][22][23][24][25] and analyses using these new methods have clearly demonstrated the polyclonal compositions of infected cells in vivo.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we accessed accumulated biomaterials and clinical information from the JSPFAD and conducted a clonality analysis of longitudinal samples from 20 carriers and ATL patients. Our analysis was based on the Tag-NGS system, 22 our new technique for the quantitative characterization of integration sites that measures the number of cells belonging to a specific clone.…”

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confidence: 99%

Clonality of HTLV-1–infected T cells as a risk indicator for development and progression of adult T-cell leukemia

Firouzi¹,

Farmanbar

Nakai

et al. 2017

Blood Advances

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Development and validation of a new high-throughput method to investigate the clonality of HTLV-1-infected cells based on provirus integration sites

Cited by 58 publications

References 60 publications

Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1–infected T cells

Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1–infected T cells

Identification of TL-Om1, an Adult T-Cell Leukemia (ATL) Cell Line, as Reference Material for Quantitative PCR for Human T-Lymphotropic Virus 1

Clonality of HTLV-1–infected T cells as a risk indicator for development and progression of adult T-cell leukemia

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