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Introduction
cell burden and the disease progression or status of ATLL patients.
Methods
Patient samples
Peripheral blood samples were collected from HTLV-1 carriers (n=94), patients with smoldering-type (n=80), chronic-type (n=70), acute-type (n=71), and lymphoma-type (n=37) ATLL, patients with HAM/TSP (n=12), and healthy volunteers as con- trols (n=35) (Online Supplementary Table S1). These samples were obtained from Miyazaki University Hospital (Miyazaki, Japan), National Hospital Organization Miyakonojo Medical Center (Miyazaki, Japan), Imamura General Hospital (Kagoshima, Japan), National Hospital Organization Kumamoto Medical Center (Kumamoto, Japan), and the Group of Joint Study on Predisposing Factors of ATL Development (JSPFAD, Japan). Informed consent was obtained from all patients. This study was approved by the Institutional Review Board at the Faculty of Medicine, University of Miyazaki, in accordance with the Declaration of Helsinki. Diagnosis of ATLL was based on clinical features, hematological characteristics, serum antibodies against HTLV-1, and monoclonal integration of the HTLV-1 proviral genome. Plasma and serum samples were prepared by centrifugation and stored at -80°C until use. Peripheral blood mononuclear cells (PBMC) were isolated by Histopaque density gradient centrifugation (Sigma-Aldrich, Tokyo, Japan) according to the manufacturer’s protocol. The pro- cedures for purification of ATLL cells from patients by using anti- CADM1-antibody-coated magnetic beads have been described elsewhere.21 CD4+ T cells from healthy volunteers were purified from PBMC using microbeads (Miltenyi Biotec, Auburn, CA, USA). Purity of isolated CD4+ CADM1+ cell populations from ATLL patients was confirmed by flow cytometry.21
Cell lines
The HTLV-1-negative human T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT4, the cutaneous T-cell lym- phoma (CTCL) cell line HUT78, the HTLV-1-infected T-cell line MT2, and ATLL-derived cell lines (S1T and ST1) were maintained in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 50 mg/mL of penicillin/strepto- mycin in a 5% CO2 chamber at 37°C. The IL-2-dependent ATLL- derived cell lines KK1 and KOB were maintained in complete RPMI 1640 medium supplemented with 0.75 g/mL of recombi- nant human IL-2 (Peprotech, Rocky Hill, NJ, USA). The human osteosarcoma cell line Saos-2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) supplemented with 10% FBS and 50 μg/mL of penicillin/streptomycin. MOLT4 was obtained from the Fujisaki Cell Center, Hayashibara Biochemical Laboratories (Okayama, Japan). MT2 was kindly provided by Dr. H. Iha (Oita University, Japan). ST1, KOB, and KK1 were kindly provided by Dr. Y. Yamada (Nagasaki University, Japan). S1T was a kind gift from Dr. N. Arima (Kagoshima University, Japan). Saos-2 was obtained from the RIKEN BioResource Center (Tsukuba, Japan).
Measurement of sCADM1 concentrations in the blood by AlphaLISA
The anti-CADM1 antibody (103-109) was generated by phage- display technology.34 AlphaLISA was performed in 96-well microtiter plates containing 5 mL of plasma sample, 10 mL of biotinylated anti-CADM1 antibody (3E1, MBL, Nagoya, Japan) (0.1 nM), 10 L of 10 mg/mL anti-CADM1 antibody (103-109) -con- jugated AlphaLISA acceptor beads (10 mg/mL), and 25 mL of strep- tavidin-coated AlphaLISA donor beads (40 mg/mL) in AlphaLISA
Adult T-cell leukemia/lymphoma (ATLL) is a refractory CD4+ T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1).1-3 It is estimated that 15- 20 million people are currently infected with HTLV-1 worldwide, and a high prevalence of HTLV-1 infection can be found in many parts of the world, including southwest- ern Japan, Melanesia, South America, sub-Saharan Africa, the Caribbean, Romania, central parts of Australia, and the Middle East. ATLL is classified into four subtypes – acute, lymphoma, chronic, and smoldering type. Patients with indolent ATLL (chronic or smoldering) have a better prognosis and watchful waiting or combined zidovudine (AZT) and interferon-α (IFN-α) therapy is standard treat- ment for indolent disease. Patients with aggressive forms (acute and lymphoma) have a very poor prognosis due to the intrinsic chemotherapy resistance of malignant cells. Allogeneic hematopoietic stem-cell transplantation (allo-HSCT), mogamulizumab, an anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, or AZT/IFN therapy are important for the treatment of ATLL; howev- er, the prognosis is unfavorable in many cases.4-7
Serum levels of interleukin-2 receptor α (sIL-2Rα, sCD25) are known to reflect tumor burden due to the high expression levels of IL-2R on ATLL cells.8 However, sIL2R levels are also increased during an inflammatory response related to HTLV-1-associated myelopathy/tropi- cal spastic paraparesis (HAM/TSP),9 or graft-versus-host disease (GvHD)10,11 that is often seen after mogamulizum- ab and allo-HSCT treatment;12-14 consequently, ATLL reoc- currence and inflammatory responses may not be distin- guishable. The elevation of sIL2R levels has also been reported in various hematologic and solid malignan- cies.15,16 Therefore, the development of a specific and reli- able diagnostic marker for ATLL is of great clinical signifi- cance.
CADM1 was originally isolated as a tumor suppressor gene in non-small cell lung cancer, and functions in the cell-cell adhesion of endothelial cells.17,18 We found that CADM1 was ectopically and highly expressed in HTLV-1- infected T cells and ATLL cells, resulting in the enhanced adhesion of ATLL cells to promote the invasion of ATLL cells into various organs.19,20 As CADM1 is consistently and specifically expressed in HTLV-1-infected T cells and ATLL cells,21-23 but not in most of the non-ATLL lym- phomas,21,24-26 CADM1 is now thought to be the best cell surface marker for ATLL.
sCADM1 consists of the extracellular domain of CADM1, which is generated by alternative splicing of CADM1 pre-mRNA27,28 or shedding of CADM1 protein on the cell surface.29-31 We have previously shown that sCADM1 protein is detected in the serum of acute-type ATLL patients.21 In the present study, we developed a highly sensitive and efficient method for the measurement of sCADM1 using the Alpha linked immunosorbent assay (AlphaLISA) technology.32,33 sCADM1 levels were found to be increased in smoldering to acute type ATLL, which is highly correlated with various clinical parameters, inclu- ding serum sIL-2α levels. Furthermore, sCADM1 levels correlated with the leukemic cell burden in ATLL patients during the course of chemotherapy treatments. These results suggest that sCADM1 can be a specific biomarker for ATLL, and that a measurement of sCADM1 may become a useful tool for accurately predicting leukemic
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sCADM1 as a new diagnostic marker of ATLL
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