A high-throughput detection method for the clonality of Human T-cell leukemia virus type-1-infected cells in vivo
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A high‑throughput detection method for the clonality of Human T‑cell leukemia virus type‑1‑infected cells in vivo Masumichi Saito1 · Hiroo Hasegawa2,3 · Shunsuke Yamauchi2 · So Nakagawa4,5 · Daisuke Sasaki2 · Naganori Nao6 · Michikazu Tanio1 · Yusaku Wada7 · Takahiro Matsudaira7 · Haruka Momose1 · Madoka Kuramitsu1 · Makoto Yamagishi8 · Makoto Nakashima8 · Shingo Nakahata9 · Hidekatsu Iha10 · Masao Ogata11 · Yoshitaka Imaizumi12 · Kaoru Uchimaru8 · Kazuhiro Morishita9 · Toshiki Watanabe13 · Yasushi Miyazaki12,14 · Katsunori Yanagihara2,3 Received: 16 March 2020 / Revised: 23 June 2020 / Accepted: 29 June 2020 © Japanese Society of Hematology 2020
Abstract Approximately 10–20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a highthroughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells. Keywords HTLV-1 · ATL · RAIS (rapid amplification of integration site)
Introduction Human T-cell leukemia virus type-1 (HTLV-1) primarily infects CD4-positive T-cells, and the provirus integrates into the host genome. A subset of the infected cells transforms to malignant T-cells after a long period, and clonal expansion stimulates the pathogenesis of adult T-cell leukemia/lymphoma (ATL). To assess the clonal expansion of Masumichi Saito, Hiroo Hasegawa and Shunsuke Yamauchi contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12185-020-02935-5) contains supplementary material, which is available to authorized users. * Masumichi Saito [email protected] * Hiroo Hasegawa hhase@nagasaki‑u.ac.jp Extended author information available on the last page of the article
HTLV-1-infected malignant cells, Southern blotting has long been routinely performed as a confirmatory diagnostic test for ATL in Japan. However, this method has several considerations, including the amount of patient sample (blood DNA) required, technical skill, sensitivity, cost, and time. Alternative method
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