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International Journal of Hematology https://doi.org/10.1007/s12185-020-02935-5

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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

AbstractApproximately 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 high-throughput 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 trans-forms to malignant T-cells after a long period, and clonal expansion stimulates the pathogenesis of adult T-cell leu-kemia/lymphoma (ATL). To assess the clonal expansion of

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 con-siderations, including the amount of patient sample (blood DNA) required, technical skill, sensitivity, cost, and time. Alternative methods, including inverse-PCR [1], adaptor-ligated PCR [2–4], and target capture method [5], have been developed thus far, each of which has its own advantages compared with Southern blotting. Nevertheless, none of these methods is currently clinically used, suggesting that there are still some concerns.

Currently, non-restrictive linear amplification-mediated PCR (nrLAM-PCR), a sensitive and cost-efficient high-throughput detection method of transgene integration sites in the host genome, has been developed and widely used for the safety assessment of gene therapy with retro- and lentivi-ral vectors [6]. However, studies have reported that defective HTLV-1 provirus, primarily lacking the 5ʹ region, including the 5ʹ long terminal repeat (LTR), is present in approximately 40% of ATL patients, and that single-stranded linker ligation

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/s1218 5-020-02935 -5) contains supplementary material, which is available to authorized users.

* Masumichi Saito saitomas@nih.go.jp

* Hiroo Hasegawa hhase@nagasaki-u.ac.jp

Extended author information available on the last page of the article

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to a single-stranded DNA (ssDNA) fragment in nrLAM-PCR is not as efficient as the ligation of double-stranded DNA frag-ments and is completed in a minimum time of 16 h [7, 8]. In the present study, we modified the nrLAM-PCR and developed rapid amplification of integration sites (RAIS) to assess the clonality of HTLV-1 and its potential for use as an alternative routine molecular confirmatory test for ATL diagnosis and a prediction tool for the onset of ATL.

Materials and methods

Collection and preparation of human samples

Peripheral blood lymphocytes and biopsies (skins, lymph nodes, and bone marrows) from asymptomatic carriers and ATL patients were harvested after obtaining informed con-sent at Oita University, University of Miyazaki, and Naga-saki University Hospital, and as a collaborative project of the Joint Study on Prognostic Factors of ATL Development (JSPFAD). This study was approved by the research eth-ics committee of the Oita University (198), University of Miyazaki {972(G)}, Nagasaki University (16072504), Uni-versity of Tokyo (17-118), and National Institute of Infec-tious diseases (1002).

The methods used herein are provided in the electronic supplementary materials (ESM).

Results and discussion

In RAIS, ssDNA, containing both HTLV-1 and host genome sequences, was synthesized with a biotinylated-HTLV-1 spe-cifically designed primer upstream of 3ʹ LTR of HTLV-1 sequence, and instead of single-stranded linker ligation in

nrLAM-PCR, polyA-tailing was performed to increase the sensitivity and shorten the duration of the method. RAIS is composed of seven steps lasting approximately 4 h for amplification of the HTLV-1-integrated fragments in specimens harboring various proviral loads (PVLs) up to 0.16–100% (Figs. S1 and S2A). In contrast, Southern blot-ting has limited the assessment of the clonality of specimens harboring more than 4% of PVL, lasting approximately 3 d (Fig. S2B). Furthermore, we compared the sensitivity of polyA-tailing on RAIS and single-stranded linker ligation on nr-LAM-PCR and confirmed that polyA-tailing on RAIS has a sensitivity approximately 100-fold that of the single-stranded linker ligation on nrLAM-PCR (Figs. S3A, S3B, and S3C). Consistent with the previously reported character-istics of nrLAM-PCR [4], the assessment of samples upon mixing two genomic DNAs (HTLV-1-infected cell lines: TL-Om1 and ED harboring each single copy of HTLV-1) [9, 10] at different percentages revealed that RAIS can linearly amplify HTLV-1-integrated multiple fragments with high reproducibility (Fig. S3D).

Since the performance of RAIS was robust in in vitro experiments, we first tested RAIS for HTLV-1 clonality in primary specimens obtained from three asymptomatic car-riers and four ATL patients harboring a wide range of PVLs (1.5–152.7%), and re-compared its sensitivity via South-ern blotting. As expected, RAIS revealed PVL-dependent amplification of HTLV-1-integrated fragments among all tested specimens (Fig. 1a, b); however, concurrent with pilot experiments, Southern blotting could not identify the clonal-ity in the carriers with low PVL (Fig. 1c). RAIS with Sanger sequencing analysis for the carriers detected a multiclonal pattern of the infected cells, showing unreadable Sanger sequencing spectra in the position adjacent to the HTLV-1 provirus, and next-generation sequencing (NGS) analysis confirmed the existence of multiclones (Fig. 1d). Concurrent with the results of Southern blotting with EcoRI-digested DNA fragments, RAIS with these sequencing analyses also identified monoclonal and multiclonal patterns of malignant cells in cases of smoldering and chronic type ATL (ATL115 and ATL140) and in cases of lymphoma type and acute type ATL (ATL198 and ATL153) (Fig. 1e, f).

For further detection, we performed RAIS for 293 ATL specimens (PVL: 0.9–249.5%, smoldering: 64, chronic: 75, lymphoma: 19, acute: 135) (Table S1). Consequently, HTLV-1-integrated fragments were amplified in all ATL specimens, and Sanger sequencing analysis revealed that approximately 70% and 30% of ATL patients displayed monoclonal and multiclonal patterns, showing similar sequence spectra in the first tested ATL specimens but not carrier specimens, respectively (Fig. 1g). Furthermore, the resulting lower fre-quency of the monoclonal pattern in smoldering type ATL rather than other ATL types supported a clonal progression model in ATL patients, as previously reported [11]. These

Fig. 1 Rapid amplification of the integration site (RAIS) revealed the clonality of HTLV-1-infected cells in asymptomatic carriers (ACs) and adult T-cell leukemia/lymphoma (ATL) patients. a Amplifica-tion of HTLV-1-integrated fragments with RAIS in three ACs and four ATL patients (ATL115: smoldering, ATL140: chronic, ATL198: lymphoma, ATL153: acute). b Correlation between HTLV-1 pro-viral loads (PVLs) and amplification rate of HTLV-1 3′long termi-nal repeat (LTR) in RAIS. HTLV-1 3′LTR was amplified via qPCR, using PCR products obtained in step 6 of RAIS. The amplification of HTLV-1 3′LTR in AC12 was set as one. The correlation coefficient was determined between HTLV-1 PVLs and the amplification rate of the HTLV-1 3′LTR. c Southern blotting for HTLV-1 clonality in three ACs. The genomic DNA of LMY2 cells (HTLV-1-infected cell line) was used as a control. d RAIS with Sanger sequencing and next-gen-eration sequencing (NGS) for HTLV-1 clonality in c. Red arrows in Sanger sequencing spectra show the position of HTLV-1 integration sites in each case. The variety and size of HTLV-1-infected clones are shown in each pie chart. d Southern blotting for HTLV-1 clonality in four ATL patients. The genomic DNA of ST1 cells (HTLV-1-infected cell line) was used as a control. f RAIS with Sanger sequencing and NGS for HTLV-1 clonality in e. g Frequency of ATL patients harbor-ing the monoclonal or multiclonal pattern of HTLV-1-infected cells

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results indicate that RAIS has high potential as an alterna-tive routine molecular confirmatory test for ATL diagnosis.

In our previous study, longitudinal analysis of the clonality of HTLV-1 carriers revealed that only the carri-ers exhibiting an oligoclonal or monoclonal pattern with largely expanded clones (> 10% of PVL before being diagnosed with ATL) subsequently progressed to ATL [12]. We assumed that RAIS could differentiate clonal patterns in the asymptomatic stage with a lower PVL. To validate our hypothesis, we performed RAIS for 47 car-rier samples harboring > 0.16% of PVL and performed both Sanger sequencing and NGS analysis. NGS analysis yielded 50,829 and an average of 1081 HTLV-1-infected clones among the carrier samples (Table S2). Although possible to fail in detecting HTLV-1-infected cells harbor-ing a previously found provirus lacking the 3ʹ LTR and potentially improvable in future studies [13, 14], we devel-oped a method to quantify clonality and classify it into three patterns: monoclonal, oligoclonal, and polyclonal patterns in carriers via Sanger sequencing analysis and EditR, a software that measures the signal peak area for each nucleotide (index) (Figs. 2a, S4A, S4B, and S4C) [15]. Sanger sequencing spectra in the monoclonal pat-tern were entirely readable, and the values of the signal peak area in the host genome sequence were compara-ble to those in the HTLV-1 genome sequence (ideally, a 1:1 ratio between the total signal peak area value of the HTLV-1 and host genome sequences in perfect monoclo-nality), whereas oligoclonal and polyclonal patterns were unreadable at a position adjacent to the HTLV-1 provirus and the values of the signal peak area in the host genome sequence gradually decreased along with the complexity

of HTLV-1 clonality. To estimate the approximate cut-off values of each clonality among the carriers, we included Sanger sequencing and NGS analysis for four ATL speci-mens presented in Fig. 1f, and defined the monoclonal pat-tern with a range of clonality value (Cv), ~ 0.74 < Cv < 1 (Cv 0.74: the median of the minimum and maximum Cv of monoclonal and oligoclonal patterns, respectively, Cv defined as 1 if Cv > 1.00). NGS analysis confirmed that the monoclonal pattern comprised more than 60% occupancy of the total sequence reads with a single clone (first clone), without the second clone of comparable size with the first clone (e.g., AC10, Cv: 0.77). The oligoclonal pattern, defined with ~ 0.27 < Cv < ~ 0.74 (Cv 0.27: the median of the minimum and maximum Cv of oligoclonal and poly-clonal patterns, respectively) in our carrier specimens dis-playing more than 70% occupancy of the total sequence reads, comprised two to four clones with comparable clone size upon NGS analysis (e.g., AC18, Cv: 0.40), and the polyclonal pattern was defined as a group not meet-ing the criteria for monoclonal and oligoclonal patterns (0 < Cv < ~ 0.27, e.g., AC38, Cv: 0.11). Using this classi-fication method, we identified 31, 11, and 5 carriers (66%, 23%, and 11%, respectively) with polyclonal, oligoclonal, and monoclonal patterns, respectively (Fig. 2b). Since the carriers exhibiting oligoclonal or monoclonal patterns and harboring a high PVL constituted a high-ATL-risk group [16], we performed a combination of PVL and clonality analysis but failed to detect any association among the clonal patterns with PVL in the carriers (Fig. 2c). Moreo-ver, we observed that a carrier, with a 2.8% PVL and an oligoclonal pattern (Cv: 0.47) at the asymptomatic stage, progressed to lymphoma type ATL in approximately 3 years. For ATL diagnosis, Southern blotting failed to detect HTLV-1 integration because of 1.6% PVL in periph-eral blood (Fig. 2d), whereas RAIS detected the infected cells with an oligoclonal pattern (Cv: 0.51) with changes in the sizes of the first and second clones (Fig. 2e). Thus, we highly recommend regular follow-ups and monitoring of the clonality and PVL for HTLV-1 carriers, especially those exhibiting oligoclonal or monoclonal patterns of infected cells with high Cv, as quantified by RAIS. We also suggest using RAIS to determine the clonality of malig-nant cells in limited biopsy materials, including a needle biopsy from the lymph node or a skin biopsy, which did not provide enough material for Southern blotting herein.

Finally, NGS-based detection and quantification methods for clonality of HTLV-1-infected cells have currently been developed and can precisely measure each clone size [3, 4, 14], but there are concerns of cost regarding these methods at present. Thus, we believe that RAIS with our quantifica-tion method using Sanger sequencing but not NGS will be useful for a routine clinical test, providing a novel strategy for (pre-) diagnosis of ATL patients.

Fig. 2 Rapid amplification of the integration site (RAIS) classified HTLV-1 clonality into monoclonal, oligoclonal, and polyclonal pat-terns in asymptomatic carriers (ACs). a Strategy to classify clonal-ity into three patterns: “monoclonal,” “oligoclonal,” and “polyclonal.” Each representative clonal pattern is shown with Sanger sequenc-ing spectra. Red arrows in the Sanger sequencing spectra indicate the position of HTLV-1 integration sites. Color variation among the three clonal patterns displays the complexity of HTLV-1 clonality in ACs. The variety and size of the top 250 HTLV-1-infected clones are shown. The dotted lines indicate the criteria to classify mono-clonal, oligoclonal, and polyclonal patterns in our specimens. Four ATL specimens (Fig. 1f) were used to classify HTLV-1 clonality in ACs, and they are highlighted in red. b Frequency of monoclonal, oligoclonal, and polyclonal patterns among 47 ACs. c Proviral loads (PVLs) of ACs showing monoclonal, oligoclonal, and polyclonal pat-tern. The p-value was determined using Student’s t-test with Prism 7 software, and the median PVL is shown in each clonal pattern. d Southern blotting for HTLV-1 clonality in an asymptomatic carrier, who progressed to ATL. The genomic DNA of LMY2 cells (HTLV-1-infected cell line) was used as a control. e RAIS for HTLV-1 clon-ality in d. Red arrows in the Sanger sequencing spectra indicate the position of HTLV-1 integration sites. The variety and size of HTLV-1-infected clones are shown in each pie chart, and identical clones before and after ATL progression are indicated with the same color

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Acknowledgements We are grateful to Dr. Masao Matsuoka for pro-viding the ED cell line and a collaborative project of the Joint Study on Prognostic Factors of ATL Development (JSPFAD) for providing DNA obtained from the peripheral blood lymphocytes from HTLV-1 carriers and ATL patients. We also would like to thank Editage (www.edita ge.com) for English language editing. This work was supported by grants from JSPS KAKENHI (JP17H03594: M.S., JP15K14388: M.S. and M.H., JP15K08647: H.H).

Author contributions MS, HH, SY, DS, YW, TM, and MK performed research. SN, NN, MT, and HH performed data analysis. MY, MN, SN, HI, MO, YI, KU, KM, TW, YM, and KY provided patient samples. MS wrote the manuscript. MS and HH supervised the study.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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Affiliations

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

1 Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan

2 Department of Laboratory Medicine, Nagasaki University Hospital, Nagasaki, Japan

3 Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

4 Biomedical Informatics Laboratory, Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan

A high-throughput detection method for the clonality of Human T-cell leukemia virus…

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5 Micro/Nano Technology Center, Tokai University, Kanagawa, Japan

6 Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan

7 Biotechnological Research Support Division, FASMAC Co., Ltd, Kanagawa, Japan

8 Graduate School of Frontier Sciences, Department of Computational Biology and Medical Sciences, The University of Tokyo, Tokyo, Japan

9 Division of Tumor and Cellular Biochemistry, Department of Medical Sciences, University of Miyazaki, Miyazaki, Japan

10 Department of Microbiology, Faculty of Medicine, Oita University, Oita, Japan

11 Department of Hematology, Oita University, Oita, Japan12 Department of Hematology, Nagasaki University Hospital,

Nagasaki, Japan13 The Institute of Medical Science Research Hospital

and Future Center Initiative, The University of Tokyo, Tokyo, Japan

14 Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan