Survival of diffuse large B-cell lymphoma (DLBCL) patients has improved by inclusion of rituximab. Refractory/recurrent disease caused by treatment resistance is, however, a major problem. Determinants of rituximab sensitivity are not fully understood, but effect of rituximab are enhanced by antagonizing cell surface receptor CXCR4. In a two-step strategy, we tested the hypothesis that prognostic value of CXCR4 in DLBCL relates to rituximab treatment, due to a hampering effect of CXCR4 on the response of DLBCL cells to rituximab. First, by investigating the prognostic impact of CXCR4 mRNA expression separately for CHOP (n=181) and R-CHOP (n=233) cohorts and, second, by assessing the interaction between CXCR4 and rituximab in DLBCL cell lines. High CXCR4 expression level was significantly associated with poor outcome only for R-CHOP-treated patients, independent of IPI score, CD20 expression, ABC/GCB and B-cell-associated gene signature (BAGS) classifications. s. For responsive cell lines, inverse correlation was observed between rituximab sensitivity and CXCR4 surface expression, rituximab induced upregulation of surface-expressed CXCR4, and growth-inhibitory effect of rituximab increased by plerixafor, supporting negative impact of CXCR4 on rituximab function. In conclusion, CXCR4 is a promising independent prognostic marker for R-CHOP-treated DLBCL patients, possibly due to inverse correlation between CXCR4 expression and rituximab sensitivity.

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease with variable clinical outcome, accounting for at least 25-30 % of adult non-Hodgkin lymphomas. Approximately one third of DLBCL patients are not cured by the currently used treatment regimen, R-CHOP. Hence, new treatment strategies are needed. Antagonizing the CXCR4 receptor might be promising since the CXCR4-CXCL12 axis is implicated in several aspects of tumor pathogenesis as well as in protection from chemotherapeutic response. In Burkitt lymphoma, the CXCR4 antagonist plerixafor has already been shown to enhance the therapeutic effect of rituximab, the immunotherapeutic agent of R-CHOP; but this is yet to be confirmed for DLBCL. We, therefore, investigated the effect of plerixafor on DLBCL cellular response to rituximab.MethodsIn this in vitro study, human DLBCL cell lines were treated with rituximab and/or plerixafor, concomitantly or in sequence. The trypan blue exclusion method and MTS-based assays were used to evaluate cellular proliferation, whereas flow cytometry was used for assessment of apoptosis status and CXCR4 surface expression level. Linear mixed effects models were used to assess statistical significance.ResultsWe observed that simultaneous addition of plerixafor and rituximab resulted in a significant decrease in DLBCL cellular proliferation, compared to monotherapeutic response. The effect was dose-dependent, and concomitant administration was observed to be superior to sequential drug administration. Accordingly, the fraction of apoptotic/dead cells significantly increased following addition of plerixafor to rituximab treatment. Furthermore, exposure of DLBCL cells to plerixafor resulted in a significant decrease in CXCR4 fluorescence intensity.ConclusionsBased on our results, implying that the anti-proliferative/pro-apoptotic effect of rituximab on DLBCL cells can be synergistically enhanced by the CXCR4 antagonist plerixafor, addition of plerixafor to the R-CHOP regimen can be suggested to improve treatment outcome for DLBCL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40364-016-0067-2) contains supplementary material, which is available to authorized users.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common form of B-cell-derived non-Hodgkin lymphoma, with a varying response and long-term outcome following therapy. The anti-CD20 monoclonal antibody rituximab has improved the survival outcome of DLBCL patients significantly. However, refractory and recurrent disease are major clinical problems due to drug-specific molecular resistance in this heterogeneous disease and patients with early relapse after rituximab-containing first-line therapy have a poor prognosis. Both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity are considered to be involved in the rituximab-mediated depletion of B-cells; however, the precise mechanism of action for rituximab in the patients is not known, and little is known about determinants of rituximab resistance in the treatment of DLBCL. CXCR4 is a seven transmembrane domain receptor that is associated with heterodimeric G proteins. Since the apoptotic effect of rituximab is enhanced using an antagonist against CXCR4 in Burkitt lymphoma (1) we speculate that similar mechanisms may exist in DLBCL. Thus, if CXCR4 is a central player in determining the resistance of DLBCL cells to rituximab induced complement-dependent cytotoxicity the expression level of CXCR4 therefore also impacts the prognosis of DLBCL patients. Methods: In a two-step strategy, we have challenged this hypothesis; first, by clinical data analysis of the prognostic impact of CXCR4 gene expression in clinical DLBCL datasets and, secondly, by laboratory analysis in a preclinical model of 14 DLBCL cell lines, studying, in vitro, the effect of CXCR4 in rituximab-mediated complement-dependent cytotoxicity. Results: Our results document that the normal expression pattern of centroblasts having higher CXCR4 membrane expression levels than centrocytes, is maintained in the malignant condition of DLBCL when individual patient samples at time of diagnosis are associated to normal B-cell subset phenotypes using the expression based "B-cell associated gene signature" (BAGS) classification system (2). The prognostic impact of CXCR4 gene expression is significant in R-CHOP but not in CHOP treated patients and independent of both the international prognostic index (IPI) scoring system as well as the (BAGS)-defined classification. Experimental in vitro studies of rituximab-induced complement-dependent cytotoxicity in systematic dose response screens of 14 CD20+ DLBCL cell lines using growth inhibition as out-read parameter resulted in very heterogeneous responses ranging from fully resistance to hypersensitivity. An inverse correlation between rituximab sensitivity and the level of gene- as well as membrane-located CXCR4 expression was documented, but exclusively in sensitive cell lines. In addition, rituximab induced membrane-located CXCR4 expression in a complement-independent manner in DLBCL cell lines. Combining rituximab and the CXCR4-antagonist plerixafor increased the cytotoxic effect of rituximab in cell lines supporting that CXCR4 has negative impact on the function of rituximab. Conclusion: This study supports that CXCR4 expression is a pathogenic variable, reflecting the reminiscent state of normal B-cell differentiation of the malignant DLBCL cells. In addition, CXCR4 provides additional and independent prognostic information in DLBCL patients treated with R-CHOP, and plays a role in rituximab-mediated CDC sensitivity in DLBCL cell lines. Disclosures No relevant conflicts of interest to declare.

BackgroundThe concept of precision medicine in cancer includes individual molecular studies to predict clinical outcomes. In the present N = 1 case we retrospectively have analysed lymphoma tissue by exome sequencing and global gene expression in a patient with unexpected long-term remission following relaps. The goals were to phenotype the diagnostic and relapsed lymphoma tissue and evaluate its pattern. Furthermore, to identify mutations available for targeted therapy and expression of genes to predict specific drug effects by resistance gene signatures (REGS) for R-CHOP as described at http://www.hemaclass.org. We expected that such a study could generate therapeutic information and a frame for future individual evaluation of molecular resistance detected at clinical relapse.Case presentationThe patient was diagnosed with a transformed high-grade non-Hodgkin lymphoma stage III and treated with conventional R-CHOP [rituximab (R), cyclophosphamide (C), doxorubicin (H), vincristine (O) and prednisone (P)]. Unfortunately, she suffered from severe toxicity but recovered during the following 6 months’ remission until biopsy-verified relapse. The patient refused second-line combination chemotherapy, but accepted 3 months’ palliation with R and chlorambucil. Unexpectedly, she obtained continuous complete remission and is at present >9 years after primary diagnosis. Molecular studies and data evaluation by principal component analysis, mutation screening and copy number variations of the primary and relapsed tumor, identified a pattern of branched lymphoma evolution, most likely diverging from an in situ follicular lymphoma. Accordingly, the primary diagnosed transformed lymphoma was classified as a diffuse large B cell lymphoma (DLBCL) of the GCB/centrocytic subtype by “cell of origin BAGS” assignment and R sensitive and C, H, O and P resistant by “drug specific REGS” assignment. The relapsed DLBCL was classified as NC/memory subtype and R, C, H sensitive but O and P resistant.ConclusionsThorough analysis of the tumor DNA and RNA documented a branched evolution of the two clinical diagnosed tFL, most likely transformed from an unknown in situ lymphoma. Classification of the malignant tissue for drug-specific resistance did not explain the unexpected long-term remission and potential cure. However, it is tempting to consider the anti-CD20 immunotherapy as the curative intervention in the two independent tumors of this case.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-016-0063-0) contains supplementary material, which is available to authorized users.