Key Points• Response to the CD38-targeting antibody daratumumab is significantly associated with CD38 expression levels on the tumor cells.• Resistance to daratumumab is accompanied by increased expression of complementinhibitory proteins.The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow-localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS). (Blood. 2016;128(7):959-970)

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

Key Points• REP is an active combination in MM patients refractory to lenalidomide.• REP is an all-oral and generally well-tolerated regimen.The prognosis of multiple myeloma (MM) patients who become refractory to lenalidomide and bortezomib is very poor, indicating the need for new therapeutic strategies for these patients. Next to the development of new drugs, the strategy of combining agents with synergistic activity may also result in clinical benefit for patients with advanced myeloma. We have previously shown in a retrospective analysis that lenalidomide combined with continuous low-dose cyclophosphamide and prednisone (REP) had remarkable activity in heavily pretreated, lenalidomide-refractory MM patients. To evaluate this combination prospectively, we initiated a phase 1/2 study to determine the optimal dose and to assess its efficacy and safety in lenalidomide-refractory MM patients. The maximum tolerated dose (MTD) was defined as 25 mg lenalidomide (days 1-21/28 days), combined with continuous cyclophosphamide (50 mg/d) and prednisone (20 mg/d). At the MTD (n 5 67 patients), the overall response rate was 67%, and at least minimal response was achieved in 83% of the patients. Median progression-free survival and overall survival were 12.1 and 29.0 months, respectively. Similar results were achieved in the subset of patients with lenalidomide-and bortezomib-refractory disease as well as in patients with high-risk cytogenetic abnormalities, defined as t(4;14), t(14;16), del(17p), and/or ampl(1q) as assessed by fluorescence in situ hybridization. Neutropenia (22%) and thrombocytopenia (22%) were the most common grade 3-4 hematologic adverse events. Infections (21%) were the most common grade 3-5 nonhematologic adverse events. In conclusion, the addition of continuous low-dose oral cyclophosphamide to lenalidomide and prednisone offers a new therapeutic perspective for multidrug refractory MM patients. This trial was registered at www.clinicaltrials.gov as

We recently showed that the outcome of multiple myeloma (MM) patients treated in the REPEAT study (evaluation of lenalidomide combined with low-dose cyclophosphamide and prednisone (REP) in lenalidomide-refractory MM) was markedly better than what has been described with cyclophosphamide-prednisone alone. The outcome with REP was not associated with plasma cell Cereblon expression levels, suggesting that the effect of REP treatment may involve mechanisms independent of plasma cell Cereblon-mediated direct anti-tumor activity. We therefore hypothesized that immunomodulatory effects contribute to the anti-MM activity of REP treatment, rather than plasma cell Cereblon-mediated effects. Consequently, we now characterized the effect of REP treatment on immune cell subsets in peripheral blood samples collected on day 1 and 14 of cycle 1, as well as on day 1 of cycle 2. We observed a significant mid-cycle decrease in the Cereblon substrate proteins Ikaros and Aiolos in diverse lymphocyte subsets, which was paralleled by an increase in T-cell activation. These effects were restored to baseline at day one of the second cycle, one week after lenalidomide interruption. In vitro, lenalidomide enhanced peripheral blood mononuclear cell-mediated killing of both lenalidomide-sensitive and lenalidomide-resistant MM cells in a co-culture system. These results indicate that the Cereblon-mediated immunomodulatory properties of lenalidomide are maintained in lenalidomide-refractory MM patients and may contribute to immune-mediated killing of MM cells. Therefore, combining lenalidomide with other drugs can have potent effects through immunomodulation, even in patients considered to be lenalidomide-refractory.

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