Mechanistic role and prognostic value of CD20-positive microvesicles in rituximab-treated diffuse large B-cell lymphoma Free

Background

The addition of rituximab, an anti-CD20 monoclonal antibody, to the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen has significantly improved outcomes in patients with diffuse large B-cell lymphoma (DLBCL). However, treatment failure due to rituximab resistance remains a major clinical challenge. Tumor-derived microvesicles (MVs), known to mediate immune modulation and therapy resistance, may contribute to this phenomenon. Given that CD20 is expressed on DLBCL cells, we hypothesized that CD20-positive MVs shed from tumor cells may act as decoys, binding rituximab and attenuating its efficacy. This study aimed to investigate the functional role of CD20-positive MVs and evaluate their potential as prognostic biomarkers in DLBCL.

Methods

MVs were isolated from culture supernatants of primary tumor cells from four DLBCL patients. CD20 expression on MVs was confirmed by flow cytometry. Functional assays using OCI-Ly1 cells and a T cell/Nalm6 co-culture system evaluated the effects of CD20-positive MVs on tumor proliferation, rituximab response, and T cell-mediated cytotoxicity. Clinically, serum-derived MVs were analyzed in a training cohort (n=26) and a validation cohort (n=90) of newly diagnosed DLBCL patients treated with R-CHOP. Patients were stratified into high and low CD20-positive MV groups based on median expression levels.

Results

In vitro, OCI-Ly1 cells exposed to CD20-positive MVs exhibited enhanced proliferation compared to untreated control cells, and CD20-positive MVs impaired rituximab-mediated growth inhibition in OCI-Ly1 cells, suggesting that these vesicles reduce therapeutic efficacy by acting as decoys for rituximab. Furthermore, CD20-positive MVs suppressed T cell-mediated cytotoxicity in a GFP-expressing Nalm6 killing assay. Thus, Nalm6 cells showed increased survival when exposed to patient-derived MVs compared to the untreated control, while the combination of rituximab and MVs significantly increased cell viability compared to both untreated and rituximab-only conditions. In the training cohort, the median concentration of CD20-positive MVs was 3.66%, and patients were stratified into high and low CD20-positive MV groups based on this median value. The response to R-CHOP was complete response (n = 20), partial response (n = 3) and progressive disease (n = 3). All three patients with progressive disease to R-CHOP belonged to the high CD20-positive MV group. The high CD20-positive MV group exhibited significantly shorter progression-free survival (PFS) and overall survival (OS) compared to the low group. Clinical and laboratory characteristics were comparable between the two groups, thus, there was no significant difference. In contrast, when patients were stratified according to the median value of CD20-positive exosomes in serum, no significant differences in PFS or OS were observed. In the validation cohort, patients were stratified into high and low CD20-positive MV groups based on the median value (3.52%). Among those with progressive disease, 9 patients (20.0%, 9/45) were in the high CD20-positive MV group, which was higher than the 2 patients (4.4%, 2/45) in the low CD20-positive MV group, showing marginal statistical significance (P = 0.05). Survival analysis demonstrated that the high CD20-positive MV group had significantly worse PFS and OS. Baseline clinical and laboratory characteristics did not significantly differ between the high and low MV groups. In contrast, stratification based on CD20-positive exosome levels did not show any significant correlation with PFS or OS.

Conclusions:

Our findings reveal a novel mechanism of resistance to rituximab in DLBCL involving tumor-derived CD20-positive MVs. These MVs reduce the therapeutic efficacy of rituximab by sequestering the antibody and impairing T cell cytotoxicity, thereby promoting immune evasion and disease progression. CD20-positive MV levels in serum may serve as a valuable biomarker for predicting treatment response and prognosis in DLBCL. Further research is warranted to elucidate the molecular mechanisms and therapeutic implications.