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S. Nakahata et al.
recurrence of ATLL occurred shortly after allo-HSCT. These results suggest that mea-surement of plasma sCADM1 lev- els may be useful in monitoring response to chemotherapy as well as in predicting recurrence of ATLL after allo-HSCT.
In order to further evaluate the relationship between plas- ma sCADM1 concentration and treatment response or the leukemic cell burden during the clinical course of ATLL patients, six ATLL patients were followed over a period of 45 to 1,219 days during treatment to assess changes in sCADM1 levels along with WBC, LDH, and sIL2R in the peripheral blood (Online Supplementary Table S4, Figure 5). In all of the patients except one (Pt #6), the levels of sCADM1 and sIL2R showed similar changes during the observation period, and importantly, increases in sCADM1 appears to correlate with poor outcome (Pt #1 and #3). While changes in LDH levels were related to changes in lev- els of sCADM1 in four patients (Pt #1, #2, #3, and #5), WBC counts did not appear to correlate with the levels of sCADM1 and sIL2R (Figure 5). Moreover, the sCADM1 level was also evaluated during the asymptomatic period and ATLL disease progression in each patient. In most cases, changes in the levels of sCADM1 from the asympto- matic state to ATLL stages showed a similar tendency to those observed in PVL (Online Supplementary Figure S11-12). In one case (MK1006), we observed a marked increase of sCADM1 from the smoldering to the lymphoma-type, which coincided with an increase in sIL2R (from 2,080 U/mL to 36,300 U/mL), whereas PVL was not increased in the peripheral blood (Online Supplementary Figure S12).
As a few HTLV-1 carriers had an elevated plasma sCADM1 concentration compared to healthy subjects (Figure 2), we assessed whether sCADM1 levels are a pre- dictive marker for the development of ATLL. Because PVL>4% is a known predictor for the development of ATLL,36 we compared the sCADM1 concentrations of asymptomatic HTLV-1 carriers with PVL>4% and PVL<4%, along with those who later progressed to ATLL (Online Supplementary Figure S13). There were no diffe- rences in plasma sCADM1 levels between the high and low PVL groups and no association was found between sCADM1 level and PVL in the univariate analysis (Online Supplementary Figure S13, Online Supplementary Table S5), although a slightly higher median sCADM1 concentration was observed in HTLV-1 carriers who later developed ATLL compared to the other groups (Online Supplementary Figure S13). Thus, these data suggest that while measurement of plasma sCADM1 alone may not be used to predict the pro- gression of HTLV-1 carriers to indolent ATLL, it may be helpful for monitoring disease progression to an aggressive state, leukemic cell burden, and/or treatment response in ATLL patients. Finally, in order to assess the diagnostic effi- ciency of the measurement of plasma sCADM1, we exam- ined receiver operating characteristics (ROC) in patients with different subtypes of ATLL and in healthy controls. The area under the ROC curve of sCADM1 was 0.94 for acute and lymphoma-type, 0.74 for chronic-type, and 0.66 for smoldering-type ATLL (Table 2), suggesting that sCADM1 is a promising functional biomarker for the diag- nosis of aggressive ATLL.
Discussion
In this study, we used AlphaLISA technology to mea- sure sCADM1 concentrations in the peripheral blood.
Although ELISA is a well-known technique to measure the plasma concentration of target proteins, we found that detection sensitivity of plasma sCADM1 in our AlphaLISA system was enhanced ~10-fold compared to a convention- al ELISA method (data not shown). By using this system, we successfully determined the range of plasma sCADM1 concentrations in healthy subjects and cut-off points for plasma sCADM1 between healthy and ATLL patients.
The disease progression in ATLL patients may involve the accumulation of genetic alterations and clonal expan- sion of ATLL cells. Although recent studies have identified somatic mutations that act as drivers during ATLL pro- gression,37,38 the application of this genetic information towards diagnosing patients is not easy, because the genomic abnormalities of ATLL cells are very complicated. In addition, clonality assessment is currently high-cost and time-consuming. Therefore, the development of effective methods for predicting disease progression is urgently needed.
CADM1 is transcriptionally upregulated in HTLV-1- infected T cells and ATLL cells in almost all cases,19,21,39,40 and in this study we found that the same CADM1 pro- moter appears to activate the expression of both sCADM1 and the membrane-bound form of CADM1 transcripts in ATLL. On the other hand, plasma sCADM1 levels were drastically increased in acute stage ATLL compared to chronic stage ATLL (median 1,066.7 ng/mL vs. 204.0 ng/mL, respectively), whereas the median percentages of CD4+CADM1+ T cells were 78.8% and 43.1% in acute and chronic-type ATLL, respectively (Online Supplementary Figure S8). Moreover, we observed increases in plasma sCADM1 levels during follow-ups with patients, which were accompanied by poor patient outcomes, whereas WBC counts were not changed (Figure 5). As multivariate analysis of several blood biomarkers for ATLL identified sCADM1 as the only independent serum marker for aggressive ATLL with significant differences (Table 1), plasma sCADM1 may be a potential risk factor of the aggressiveness of ATLL cells. In addition, while sIL2Rα is secreted not only by leukemia cells but also during various inflammatory responses,15,41 plasma sCADM1 was not increased in HAM/TSP patients or in a patient with GvHD after allo-HSCT for ATLL. Therefore, combined sCADM1 and sIL2Rα measurements may become a promising method for more accurately diagnosing ATLL develop- ment in HAM/TSP patients, or discriminating ATLL relapse and GvHD following allo-HSCT for ATLL. In the current clinical setting, assessment of treatment efficacy in ATLL is based on serum biochemical parameters such as sIL2Rα, LDH, and calcium levels, and clinical findings including computed tomography and positron emission tomography examination results, however, these tests are unable to determine the depth of response to treatment, which can be evaluated by detection of MRD. Given that sCADM1 seems to be a specific biomarker for ATLL, the measurement of plasma sCADM1 may be a useful in measuring the depth of response to therapy, and a change in the sCADM1 plasma level may become an important cli-nical criteria for not only determining the transplant adap-tability, but also determining the choice of consoli- dation or maintenance treatments and their periods.
It has been reported that sCADM1 can bind to the extra- cellular domain of CADM1 through an interaction between their Ig domains.27 CADM1 contains a PSD95/Dlg/ZO-1 (PDZ) domain that interacts with T lymphoma invasion
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haematologica | 2021; 106(2)