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S. Nakahata et al.
ImmunoAssay Buffer (25 mM HEPES, pH 7.4, 0.1% Casein, 0.5% Triton X-100, 1 mg/mL Dextran-500, and 0.05% Proclin-300). The reaction mixture was incubated at 23oC for 1 hour (h) and the chemical emission was read on an EnSpire Alpha microplate rea- der (PerkinElmer Waltham, MA). The concentrations of sCADM1 were obtained from a standard curve generated by recombinant CADM1 protein (Sino Biological, Beijing, China) using four-para- meter logistic curve fitting, and multiplied by the dilution factor. All measurements were done in duplicate.
Additional methods are provided in the Online Supplementary Appendix.
Results
The soluble form of CADM1 mRNA is efficiently expressed in ATLL
As sCADM1 can be generated through alternative spli- cing by an intron retention event27,28 or ectodomain shed- ding of CADM1 protein,29-31 the mRNA expression of a soluble splice variant of CADM1 and the level of CADM1 shedding were initially determined in ATLL-related cell lines and leukemia cells from patients with acute-type ATLL by RT-PCR and western blotting, respectively. Because the sCADM1 transcript contains the 118-bp sequence of intron 7 (Figure 1A),27 we used PCR primers located in exon 6 and intron 7 of CADM1 to amplify the sCADM1 transcript. Semiquantitative RT-PCR showed that sCADM1 mRNA, along with the membrane-bound form of CADM1 (mbCADM1) mRNA, was highly expressed in all of the HTLV-1-positive ATLL-related cell lines (Online Supplementary Figure S1A). We then per- formed quantitative RT-PCR analysis with purified CD4+CADM1+ leukemic cell populations from various types of ATLL patients.21 A significantly higher abundance of sCADM1 mRNA was observed in all ATLL samples tested, including smoldering, chronic, and acute-type ATLL, compared with CD4+ T cells from healthy volun- teers (Figure 1B). Similarly, the levels of mbCADM1 or total CADM1 (tCADM1) were significantly higher in all subtypes of ATLL than controls (Figure 1B). In order to quantify CADM1 shedding in ATLL, we analyzed the le- vels of two membrane-associated C-terminal fragments (αCTF, 18 kDa and CTF, 35 kDa), which are generated by the proteolytic cleavage of CADM1 by ADAM and unidentified proteases, respectively.29-31 Although the CTF fragment of CADM1 was detected in ATLL-related cell lines, very weak or no signal was found in leukemia cell samples from ATLL patients (Online Supplementary Figure S2A-B). Additionally, CADM1 undergoes alternative spli- cing between exons 7 and 11 (Figure 1A) and the inclusion of exon 9 confers shedding susceptibility to CADM1.31 RT- PCR analysis revealed that the majority of the mbCADM1 mRNA skipped both exons 9 and 10 (Online Supplementary Figure S1A-B). Therefore, these results suggest that upre- gulated expression of sCADM1 mRNA, which is generat- ed by retention of intron 7, might be the main cause of the increased level of sCADM1 protein in ATLL.
Plasma sCADM1 levels increase with disease progression in ATLL
In order to investigate the clinical utility of sCADM1 in the diagnosis of ATLL patients, we developed a highly sen- sitive method for the measurement of plasma sCADM1 with the use of the AlphaLISA technology, which is based
on energy transfer from a streptavidin donor bead to an AlphaLISA acceptor bead in close proximity.32,33 The donor and the acceptor beads were conjugated with biotinylated anti-CADM1 (3E1) and anti-CADM1 (103-189) antibodies, respectively (see Methods). Using recombinant human CADM1 protein, the assay system was capable of detect- ing sCADM1 in the plasma at concentrations as low as ~0.2 ng/mL (Online Supplementary Figure S3). First, we determined the sCADM1 levels in the peripheral blood of healthy volunteers, HTLV-1 carriers, and patients with HAM/TSP and various types of ATLL who had not been previously treated. The median values for plasma and serum sCADM1 from healthy volunteers were 181.3 ng/mL and 173.5 ng/mL with 5th-95th percentile ranges of 142.7-234.0 ng/mL and 131.7-215.5 ng/mL, respectively (Online Supplementary Figure S4A). There were neither a sig- nificant differences between the sCADM1 levels of males and females, nor amongst the age groups (Online Supplementary Figure S4B-C). In addition, the majority of HTLV-1 carriers and HAM/TSP patients had sCADM1 lev- els within the standard reference range (Figure 2). The median plasma sCADM1 concentration was slightly high- er in smoldering-type ATLL patients (210.2 ng/mL) than in healthy volunteers (181.3 ng/mL), and it was elevated in chronic-type ATLL patients, with a median peak value of 267.1 ng/mL (Figure 2). The median plasma sCADM1 level was 1055.0 ng/mL (range: 173.6-6,931.0 ng/mL) in acute- type ATLL patients, more than 5-fold higher than in the control group (Figure 2 and Online Supplementary Figure S5A). In addition, 2 of 10 patients with the unfavorable chronic-type ATLL35 had high levels of plasma sCADM1 (Online Supplementary Figure S6).
Next, we determined the association between plasma sCADM1 concentrations and other clinical and/or bio- chemical parameters in ATLL patients (Online Supplementary Figure S5A-E). Among these subjects, serum concentrations of sIL2R were higher in patients with all subtypes of ATLL compared to healthy volunteers, and concentrations increased with disease progression to acute-type or lymphoma-type ATLL (Online Supplementary Figure S5B). Importantly, while the patients with HAM/TSP showed significantly higher sIL2R levels com- pared with healthy volunteers, as reported previously,9 plasma sCADM1 levels were in the healthy range (Online Supplementary Figure S5A-B), confirming that sCADM1 is a specific marker for ATLL. Most patients with acute-type or lymphoma-type ATLL had elevated serum lactate dehy- drogenase (LDH) levels (Online Supplementary Figure S5C). HTLV-1 proviral load (PVL) in the peripheral blood increased in smoldering-type ATLL patients and was elevated in chronic-type and acute-type ATLL, and was similar to white blood cell (WBC) concentrations (Online Supplementary Figure S5D-E). Serum sIL2R levels strongly correlated with plasma sCADM1 levels (r=0.66, P<0.001) (Figure 3A), and moderate correlations were found between plasma sCADM1 and LDH levels (r=0.52, P<0.001) (Figure 3B) as well as plasma sCADM1 and PVL levels in the peripheral blood (r=0.47, P<0.001) (Figure 3C). The correlation between plasma sCADM1 and PVL levels was higher than that between plasma sCADM1 and the levels of oligoclonality of HTLV-1-infected clones (Online Supplementary Figure S7A-B). There was a weak correlation between WBC counts and plasma sCADM1 levels (r=0.37, P<0.001) (Figure 3D). Additionally, the increase in plasma concentrations of sCADM1 was much higher than the
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