CD38 Expression Levels on Myeloma Cells and the Frequency of Circulating CD38-Positive Treg Cells Are Associated with the Response to Daratumumab in Multiple Myeloma
Authors: Kitadate Akihiro , Kobayashi Hiroki , Abe Yoshiaki , Narita Kentaro , Miura Daisuke , Takeuchi Masami , Matsue Kosei 
Published: October 2019

Abstract
Background: CD38 is highly expressed on plasma cells and is a good target for multiple myeloma (MM) therapies. Daratumumab (DARA), a humanized antibody to CD38, has emerged as a promising treatment for MM. DARA targets CD38-expressing myeloma and non-plasma cells, including CD38-positive (CD38+) regulatory T (Treg) cells. To investigate the mechanism of action of DARA, we analyzed baseline CD38 expression levels and lymphocyte subsets, including CD38+ Treg cells, before and after DARA treatment.
Methods: This study comprised 32 patients with relapsed/refractory MM, who were treated with DARA at our Center and were followed up for more than one cycle of the drug. Peripheral blood and bone marrow samples were analyzed before and during treatment. Using flow cytometric analysis, CD38 expression (mean fluorescence intensity (MFI)) and lymphocyte subsets (CD4/CD8 T cells, natural killer (NK) cells, and Treg cells) were evaluated. Treg cells were identified as a fraction of the CD4+CD25highCD127dim population.
Results: The median age of the patients was 78 years (range, 53-92 years). Twenty-two patients (69%) had received more than three prior therapies. Patients received a median of four prior lines of therapy (range, 2-11 prior therapies). All patients were refractory to their last line of therapy, and 89% were refractory to both proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs). Eight patients (25%) received a PI-based DARA-containing regimen, 21 (66%) received an IMiD-based DARA-containing regimen, and 3 (9%) received other DARA-containing regimens. Twenty patients (63%) had a partial response (PR) or better (responders), whereas 12 patients (37%) did not respond to the drug (non-responders).
The pretreatment levels of CD38 MFI were significantly higher in responders than in non-responders (p < 0.0005). Before treatment, the absolute number of CD38+ Treg cells was significantly higher in responders (median, 12.3 cells/µL; range, 5.1-37.3 cells/µL) than in non-responders (median, 4.5 cells/μL; range, 1.1-12.8 cells/µL, p = 0.001), but absolute Treg cell numbers were not associated with DARA response. CD38+ Treg cells were depleted in all treated patients, but CD38-negative Treg cell numbers remained relatively stable after DARA treatment. The absolute CD8+ T-cell numbers were significantly increased after DARA treatment (236.1 ± 146.2 vs. 388.6 ± 183.4/μL, p = 0.002). In addition, the absolute HLA-DR+ activated T-cell numbers were also significantly increased (p = 0.002). However, the increased numbers of CD8+ and HLA-DR+ T cells were not associated with clinical responses. The numbers of CD56+ NK cells were immediately depleted in all treated patients. As a control, we also examined the changes in lymphocyte subsets in patients treated with elotuzumab, the first approved monoclonal antibody for MM treatment. However, there was no change in CD38+ Treg and total Treg cell numbers.
Next, to investigate the role of CD38+ Treg cells in disease progression, we analyzed the frequencies of Treg cells in healthy volunteers and in patients with monoclonal gammopathy of undetermined significance, smoldering MM, newly diagnosed MM, and relapsed MM. The CD38+ Treg cells were significantly higher in the relapsed MM group than in the other disease or control groups, suggesting that CD38+ Treg cells may play an important role in the progression of MM.
Finally, we investigated the CD38 expression and frequency of CD38+ Treg cells in durable responders. Among 20 responders, 13 patients showed a long-lasting response (>6 months) or deepening response (defined as durable responders). The absolute number of CD38+ Treg cells was significantly higher in the durable responders (p = 0.009). On the other hand, the CD38 MFI of myeloma cells was not associated with a durable response.
Conclusions: Pretreatment levels of the CD38 MFI could be a predictive marker for the early response to DARA treatment. Moreover, the frequency of CD38+ Treg cells present before treatment could also serve as a durable-response marker. These results suggest that the depletion of CD38+ Treg cells could contribute to some immunological mechanism of DARA. This study provides evidence to support multiple mechanisms of action for DARA, including antibody-dependent cell-mediated cytotoxicity and immunomodulatory effects.

Disclosures
No relevant conflicts of interest to declare.