Clinical significance of soluble CADM1 as a novel marker for adult T-cell leukemia/lymphoma

Abstract

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

Figure 1.

High expression of sCADM1 transcript in adult T-cell leukemia/lymphoma. (A) Schematic representation of the structure and domain organization of membrane- bound cell adhesion molecule 1 CADM1 (mbCADM1) and its soluble form (sCADM1). mbCADM1 contains an extracellular domain (exons 2-10) containing three immunoglobulin-like C2-type domains, a transmembrane domain (exon 11), and a cytoplasmic domain (exon 12). sCADM1 is generated by alternative splicing in which intron 7 is retained and an in-frame stop codon is found immediately downstream of the immunoglobulin-like domain. Arrows indicate the position and direction of primers used in RT-PCR reactions. The mbCADM1, sCADM1, and total CADM1 (tCADM1) transcripts were amplified using primers for exon 7 and exon 8, exon 6 and intron 7, and exon 4 and exon 5, respectively. Numbers indicate exon numbers of the CADM1 gene. (B) Quantitative RT-PCR analysis of tCADM1, mbCADM1, and sCADM1 expression in CD4⁺ T lymphocytes from healthy volunteers (n=5) and sorted CADM1⁺ cells from peripheral blood of patients with smoldering-type (n=6), chronic-type (n=6), and acute-type (n=7) ATLL. The data were normalized to β-actin gene expression and expressed relative to a healthy control sample. Mean ± standard deviation is shown, **P<0.01 (Mann-Whitney U test).

Figure 2.

Plasma sCADM1 levels in adult T-cell leukemia/lymphoma patients examined by AlphaLISA. The plasma soluble form of cell adhesion molecule 1 (sCADM1) levels in 34 healthy volunteers, 78 human T-cell leukemia virus type 1 (HTLV-1) carriers, 12 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 77 smoldering-type, 23 chronic-type, 13 lymphoma-type, and 43 acute-type ATLL patients who were previously untreated were measured by AlphaLISA using anti-CADM1 antibodies. The box and whisker plots show the 5^th, 25^th, 50^th (median), 75^th, and 95^th percentile values, with outliers marked by solid dots. *P<0.05, **P<0.01, ***P<0.001 versus healthy volunteers or HTLV-1 carriers (Kruskal-Wallis test/Dunn's multiple comparison test). Median and 5^th and 95^th percentile values are indicated at the top of each column. The dot line indicates the 95^th percentile of plasma sCADM1 in healthy subjects.

Figure 3.

Association between plasma sCADM1 levels and various prognostic indicators in adult T-cell leukemia/lymphoma. (A) Scatter plot of plasma soluable form of cell adhesion molecule 1 (sCADM1) concentrations versus serum interleukin-2 receptor α (sIL2Rα) concentrations in 34 healthy volunteers, 78 human T-cell leukemia virus type 1 (HTLV-1) carriers, 77 smoldering-type, 55 chronic-type, 34 lymphoma-type, and 69 acute-type adult T-cell leukemia/lymphoma (ATLL) patients, including those who were previously untreated and treated with chemotherapy. Spearman correlation coefficient values (r) and P-values are shown on each of the graphs. (B) Scatter plot of plasma sCADM1 concentrations versus serum lactate dehydrogenase (LDH) concentrations in 45 HTLV-1 carriers, 69 smolderingtype, 38 chronic-type, 18 lymphoma-type, and 60 acute-type ATLL patients, including those who were previously untreated and treated with chemotherapy. Spearman correlation coefficient values (r) and P-values are shown on each of the graphs. (C) Scatter plot of plasma sCADM1 concentrations versus proviral load (PVL) in 66 HTLV-1 carriers, 56 smoldering-type, 56 chronic-type, and 61 acute-type ATLL patients, including those who were previously untreated and treated with chemotherapy. Spearman correlation coefficient values (r) and P-values are shown on each of the graphs. (D) Scatter plot of plasma sCADM1 concentrations versus white blood cell (WBC) counts in 32 healthy volunteers, 71 HTLV-1 carriers, 74 smoldering-type, 57 chronic-type, 33 lymphoma-type, and 69 acute-type ATLL patients, including those who were previously untreated and treated with chemotherapy. Spearman correlation coefficient values (r) and P-values are shown on each of the graphs.

Figure 4.

Plasma sCADM1 levels are related to treatment efficacy in adult T-cell leukemia/lymphoma patients. (A) Soluble form of cell adhesion molecule 1 (sCADM1) concentrations in the plasma of 23 chronic-type, 13 lymphoma-type, and 43 acute-type adult T-cell leukemia/lymphoma (ATLL) patients who were previously untreated and of 39 chronic-type, 18 lymphoma-type, and 7 acute-type ATLL patients who were previously treated with chemotherapy were measured by AlphaLISA. *P<0.05, ***P<0.001 (Mann-Whitney U test). The dot line indicates the 95^th percentile of plasma sCADM1 in healthy subjects. Note that the pre-treatment and post-treatment samples were derived from different individuals. (B) Serum interleukin-2 receptor α (sIL2Rα) concentrations in the serum of 19 chronictype, 12 lymphoma-type, and 43 acute-type ATLL patients who were previously untreated and of 32 chronic-type, 16 lymphoma-type, and six acute-type ATLL patients who were previously treated with chemotherapy. *P<0.05, ***P<0.001 (Mann-Whitney U test). The dot line indicates the upper limit of normal serum sIL2Rα. The same samples as Figure 4A were used. Fifteen cases with missing values on sIL2Rα (one lymphoma-type and four chronic-type in the pre-treatment group and one acute-type, two lymphoma-type, and seven chronic-type in the post-treatment group) were excluded. (C) Lactate dehydrogenase (LDH) concentrations in the serum of 18 chronic-type, nine lymphoma-type, and 40 acute-type ATLL patients who were previously untreated and of 15 chronic-type, seven lymphoma-type, and five acute-type ATLL patients who were previously treated with chemotherapy. *P<0.05 (Mann-Whitney U test). The dot line indicates the upper limit of normal serum LDH. The same samples as Figure 4A were used. Forty-nine cases with missing values on LDH (three acute-type, four lymphoma-type, and five chronic-type in the pre-treatment group and two acute-type, 11 lymphoma-type, and 24 chronic-type in the post-treatment group) were excluded. (D) Human T-cell leukemia virus type 1 (HTLV-1) proviral load (PVL) in 15 chronic-type, 11 lymphoma-type, and 35 acute-type ATLL patients who were previously untreated and in 39 chronic-type, 18 lymphoma- type, and 7 acute-type ATLL patients who were previously treated with chemotherapy. **P<0.01 (Mann-Whitney U test). The same samples as Figure 4A were used. Eighteen cases with missing values on PVL (eight acute-type, two lymphoma-type, and eight chronic-type in the pre-treatment group) were excluded. (E) White blood cell counts (WBC) counts in 19 chronic-type, 11 lymphoma-type, and 43 acute-type ATLL patients who were previously untreated and in 33 chronic-type, 16 lymphoma-type, and 6 acute-type ATLL patients who were previously treated with chemotherapy. ***P<0.001 (Mann-Whitney U test). The dot line indicates the upper limit of normal for WBC counts. The same samples as Figure 4A were used. Thirteen cases with missing values on WBC counts (two lymphoma-type and four chronic- type in the pre-treatment group and one acute-type, two lymphoma-type, and four chronic-type in the post-treatment group) were excluded.

Figure 5.

Plasma sCADM1 is useful for monitoring disease progression in adult T-cell leukemia/lymphoma patients. Plasma soluble form of cell adhesion molecule 1 (sCADM1) concentrations along with various prognostic parameters including serum interleukin-2 receptor α (sIL2Rα ), lactate dehydrogenase (LDH), and white blood count (WBC) count were monitored in six patients with adult T-cell leukemia/lymphoma (ATLL) before and after acute crisis (day 0). The drug regimen is indicated at the top of the figure: VP-16, etoposide; LSG15, VCAP-AMP-VECP (vincristine, cyclophosphamide, doxorubicin, and prednisone [VCAP], doxorubicin, ranimustine, and prednisone [AMP], and vindesine, etoposide, carboplatin, and prednisone [VECP]); PSL: prednisolone; KW-0761: mogamulizumab; THP-COP: pirarubicin, cyclophosphamide, vincristine, and prednisolone.