Soluble and Membrane P-Glycoprotein Expression in Lymphocytes from Diffuse Large B Cell Non-Hodgkin's Lymphoma Patients Treated with R-CHOP

Abstract

Introduction Diffuse large B cell lymphoma (DLBC) accounts for 25% of all Non-Hodgkin's Lymphoma (NHL) in the United States. Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) has been the first-line therapy for patients with DLBC-NHL since the early 2000s. Nearly 40% relapse in the following 2-years after complete response and 15% are refractory to first-line therapy. Despite many efforts, other chemotherapy combinations have failed to improve the response rate of NHL patients. Mechanisms of resistance to cytotoxic drugs are vast and include augmented drug efflux mediated through ATP-dependent pumpsas P-glycoprotein (P-gp), which has been related to lesser efficacy. This is due to an over-expression of the glycoprotein with the consequent increase of the drug's efflux from the cytosol. Almost all components of the R-CHOP therapy are P-gp substratesand thereby are affected by P-gp activity. Objectives Analyze serum, plasmatic levels, and membrane expression of P-gp in lymphocytes from patients with DLBC-NHL. Methods A case-control study was conducted to measure serum and plasmatic levels of P-gp, and membrane P-gp expression at the end of treatment on DLBC-NHL patients at “Hospital Civil de Guadalajara Fray Antonio Alcalde,” diagnosed from April to September 2022. Inclusion criteria were age ≥ 18 years, without cancer history and without P-gp inductors or inhibitors. Exclusion criteria were pregnancy, hepatitis, autoimmune disease, Richter syndrome and infection 15 days before blood draw. Using whole blood samples, we identified T and NK cells expressing membrane P-gp by flow cytometry on Attune® NxT (Life Technologies) using the following antibodies and fluorochrome conjugates: CD3-APC/Cy7, CD4-AlexaFlour488, CD8-APC, CD56-BrilliantViolet, CD243-PE. Data was analyzed with FlowJo v10 software. Serum and plasmatic P-gp measurement were done using the Human Permeability Glycoprotein (P-gp) ELISA kit (MyBioSource). Statistical Analysis Proportion comparison between groups was made using Chi-Square or Fisher exact tests. A quantitative variable comparison was done using the U Mann-Witney test. Correlation analysis was made using the Spearman correlation coefficient. Statistical significance was calculated using the p<0.05 two-tailed test. All statistical analysis was made using the software R version 4.1.2 and ggpurb/ggplot2 package. Results Only 15 patients and 30 control individuals were included. Group characteristics and comparisons are summarized in table 1. Regarding the DLBC-NHL group, 27% patients were classified as Lugano I, II and IV stages respectively and 20% as Lugano III stage. Nearly 27% of the patients received radiotherapy as consolidation therapy. Also, 40% of patients showed lymphopenia with a median of 1.23 x10 3/μL (IQR 0.94 - 1.34). Serum and plasmatic P-gp concentrations were similar between groups (p>0.05). The CD8+, CD4+, and CD56+ lymphocyte percentage with membrane P-gp expression comparison between the DLBC-NHL group and the control group presented no significant differences (p>0.05). However, the P-gp median fluorescence index (MFI) in CD8+, CD4+, and CD56+ lymphocytes was higher in the control group than DLBC-NHL group (p<0.05), as displayed in figure 1. Discussion When analyzing only the DLBC-NHL group, we identified a negative correlation between serum P-gp concentration and its expression in NK cell membrane and a positive correlation with membrane expression in CD4+ and CD8+ lymphocytes. Kato et al. reported that P-gp can be secreted through exosomes in patients with drug resistance. Lettau et al. found that CD4+ and CD8+ T lymphocytes have higher exosome expulsion compared to NK cells. These could be related to our finding of negative correlation between serum P-gp and membrane P-gp expression in NK lymphocytes. Ghetie et al. found that Rituximab-an anti-CD20 monoclonal antibody-inhibits P-gp efflux function and therefore makes multi-drug-resistant cell cultures chemosensitive again. B-cells affected by rituximab may influence T-cell P-gp expression and this may be the reason why MFI was lower on DLBC-NHL group than control group. This study helps to expand the understanding of P-gp's role in DLBC-NHL patients as well as R-CHOP therapy effects on the expression of the P-gp in circulating T-cells.