Multiomic Analysis of CD38 Expression Levels in 209 Untreated and Daratumumab-Resistant Patients

Abstract

The anti-CD38 antibody Daratumumab (Dara) is widely used in first-line as well as in relapsed/refractory multiple myeloma (MM). Primary or secondary resistance eventually occurs in all treated patients but the underlying mechanisms are poorly understood: Genetic alterations that lead to loss of function affecting epitopes on CD38, low baseline expression of CD38 or the selection of CD38dim cells were associated with suboptimal response to treatment. Interestingly, drugs such as all-trans retinoic acid, panobinostat or DNMT inhibitors increase Dara efficiency through reversible upregulation of CD38. Overall, three cohorts with overall 209 patients were examined for this dataset. We conducted flow cytometry in 21 therapy naïve and 19 Dara-refractory patients using a Dara epitope-specific antibody. All untreated and 11 of the resistant patients stained positive for CD38 MM, suggesting CD38 independent resistance mechanisms. In eight patients, CD38 expression was not detected; however, in five of them, additional staining with a CD38 multiepitope antibody confirmed CD38 persistence, reflecting epitope blockade through Dara or epitope mutation that interferes with the binding of the antibody. Finally, in three out of 19 refractory patients, CD38 could not be detected with both antibodies. To quantify CD38 receptor density on a molecular level, we established super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in the same patients. dSTORM uses fluorophore-conjugated antibodies and provides an unprecedented molecular 20 nm resolution for individual cells. We observed intra-patient heterogeneity of the CD38 receptor density with a mean expression of 35.15 ± 13.42 receptors/µm² (range 3.61-67.24) in the Dara naïve patients. Notably, in the refractory group, we found a significant decline (p=0.03) with a mean of 22.7 ± 18.26 receptors/µm² (range 17.07-63.77). In two of the three patients with no CD38 detectable, CD38 persistence could be assessed by dSTORM below the detection rate of flow cytometry. One patient remained negative for CD38, suggesting true loss of the receptor, causing Dara resistance in this patient. Next, we confirmed downregulation of CD38 gene expression using qPCR, and significantly reduced expression in the Dara refractory cohort (p=0.01). Of note, correlation between gene and receptor expression was moderate (p=0.49, r=0.57), leaving room for epigenetic regulation or posttranscriptional modification. To interrogate whether epigenetic regulation of CD38 via DNA methylation may underlie differential CD38 receptor expression, we performed deep bisulfite sequencing of MM cells and PBMCs obtained from 41 patients from our institution. CD38 on chromosome 4 contains several regulatory regions: One promoter, two adjacent promoter flanking regions and 11 enhancer regions described in ENSEMBL genome browser (GRCh38.13). The CD38 promoter was fully unmethylated (mean methylation: 0.31% ± 0.2%, range 0.2%-0.9%), but variable methylation of a CD38 promoter flanking region upstream of the promoter was detectable with 30.08% ± 17.8% (range 0.9%-61.5%) in the MM cells compared to 59.6% ± 3.9% (range 49.0%-66.7%) in the PBMCs from the same patients (p <0.001). This highlights phenotypic plasticity and suggests that DNA methylation may play a regulatory role in CD38 expression. We are currently investigating the methylome of all 14 regulatory regions in 38 Dara resistant patients (18 of them with longitudinal sampling before and after Dara therapy), 33 Dara responsive patients and 52 plasma cells obtained from healthy donors. Finally, in a cohort of 127 patients from our institution with WGS data available we screened for genetic alterations with potential impact on the CD38 receptor expression. We found monoallelic CD38 deletions in 18 patients, with one patient having an additional 11kb deletion conferring to an exon 3 loss. Exon-6 skipping, as recently described, was not detectable in the cohort. In conclusion, genetic alterations affecting the CD38 locus and true loss of the CD38 receptor are rare events and do not explain Dara resistance in most affected patients. dSTORM imaging provides additional insights in the CD38 receptor downregulation in MM patients treated with anti-CD38 moAb therapy. Regulatory mechanisms such as epigenetic regulation need to be further explored.