The Clinical And Experimental Study Of Mononuclear Phagocyte System In Diffuse Large B Cell Lymphoma

BackgroundDiffuse large B cell lymphoma(DLBCL), the most common aggressive non-Hodgkin lymphoma(NHL), is potentially curable in more than 50% of cases.Even introduction of rituximab, approximately one-third of patients will relapse or have refractory disease that leads to death. High-dose therapy(HDT) and autologous stem cell transplantation(ASCT), the standard treatment for salvage chemosensitive relapsed/refractory disease, but only benefits 30% of these patients. So the novel treatment strategies for relapsed/refractory patients with DLBCL are essential.Mononuclear phagocytic system(MPS) mainly includes monocytes, macrophages and dendritic cells. MPS as an important component of innate immunity, not only plays crucial roles in defence against invading infectious pathogens, tissue homeostasis and immune surveillance, but also as its differentiation or function abnormalities involves in cardiovascular disease, chronic obstructive pulmonary disease(COPD), tuberculosis and autoimmune disease. In recent years, MPS is the focus of attention because of its close relationship with the onset and development of tumor. Monocytes are heterogeneous and play important roles in phagocytosis, inflammatory, antigen presenting function, immune regulation. Human monocytes according to the expression levels of CD14 and CD16 can be divided into three subsets: classical subsets(CD14++ CD16-), intermediate subset(CD14++ CD16+) and non-classical subset(CD14+CD16++). Nowadays, more clinical evidence suggests CD16+monocytes(include CD14++CD16+ monocytes and CD14+CD16++ monocytes)closely related to some tumors. In addition, different monocyte populations involve in the onset and development of tumor, such as CD14+HLA-DR-/low monocytescontribute to systemic immunosuppression by inhibiting the host antitumor response,tyrosine kinase receptor Tie-2-expressing monocytes(TEMs) involve in tumor angiogenesis and CCR2+ inflammatory monocytes play an important role in promoting tumor invasion and metastasis. Macrophages are versatile cells that are capable of displaying different functional activities, some of which are antagonistic: they can be immunostimulatory or immunesuppressive, and either promote or restrain inflammation. This functional plasticity is regulated by local cues to which the macrophages respond. Two distinct states of polarized activation by different factors for macrophages in tumor microenvironment have been recognized: classically activated macrophages(M1) which have antitumor effect and alternatively activated macrophages(M2) which have tumor promoting function. Tumor-associated macrophages(TAMs), the majority similar to M2 macrophages in phenotype and function, involve in tumor growth, invasion, metastasis, angiogenesis and have a powerful immunosuppressive function. It is for these reasons that cells of MPS have become attractive targets to manipulate for cancer therapy. However, the clinical and experimental study of MPS in DLBCL is laking.ObjectiveTo comprehensively analyse on the significance and value of absolute monocyte count(AMC) in predicting clinical prognosis of DLBCL, test peripheral blood monocyte subsets, related cytokines and gene level detection, and evaluate TAMs,related chemokines and their receptors. To further preliminarily explore their clinical mechanisms.Methods1. Retrospectively analysis of relationships between clinical prognosis and AMC at diagnosis, AMC at the time of first relapse in 244 newly diagnosed, 163 relapsed/primary refractory DLBCL, respectively, and the function of AMC in prediting recurrence risk in 220 follow-up DLBCL.2. Peripheral blood samples were collected from 32 newly diagnosed, 29 remission, 21 relapsed DLBCL patients and 30 healthy controls. Detection of distributions of monocyte subsets, CD14+ HLA-DR-/low monocytes, CCR2+ inflammatory monocytes, Tie-2+ monocytes by flow cytometry; concentration of Arginase-1(Arg-1),Monocyte chemotactic factor-1(MCP-1), Angiopoietin-2(Ang-2) in plasma by enzyme-linked immunosorbent assay(ELASA), mRNA expression levels of CCR2 and Tie-2 by Real time PCR.3. We examined CD68, CD163, MCP-1, CCR2 and phosphorylation of signal transducer andactivator of transcription 3(pSTAT3) expressions in 221 DLBCL tissue sections by immunohistochemistry, and the prognostic value were investigated.Results1. Newly diagnosed DLBCL patients with higher AMC at diagnosishad lower complete remission(CR) rate, overall survival(OS) rate and progression free survival(PFS) rate compared with those with lower AMC, respectively.Multivariate Cox proportional hazard model showed high AMC at diagnosis was an independent adverse prognostic factor for DLBCL. Combined with International Prognostic Index(IPI), AMC could provide additional prognostic information for DLBCL. In low, intermediate risk groups according to IPI score,DLBCL patients could be further stratified by AMC at diagnosis, and among these patients, about 20% had shorter PFS and OS. Similar results were obtained when low, intermediate risk patients treated with immunotherapy(combined rituximab) were risk-stratified by AMC. Also, high AMC at the time of first relapse was an adverse prognostic factor for OS and PFS in relapsed/primary refractory DLBCL received second-line therapy or HDT followed with ASCT. Relapsed/primary refractory DLBCL patients with higher AMC at the time of first relapse had lower response rate, PFS and OS rate compared with those with lower AMC, respectively. When combined with international prognostic index at relapse(saaIPI), relapsed/primary refractory DLBCL patients identified by saaIPI as low, intermediate risk could be further stratified by AMC at the time of first relapse, and about 25% among them had shorter survival. Similar results were obtained in low, intermediate risk patients treated with rituximab containing salvage therapy. Finally, high AMC at follow-up was a marker to assess risk of DLBCL relapse, and the sensitivity and specificity for AMC were54.0% and 66.7%. DLBCL with higher follow-up AMC had higher recurrence rate, relative risk rate and cumulative hazard rate compared with lower AMC, respectively. Combined with lactate dehydrogenase(LDH)level at follow-up, wecould get the same results regardless of LDH level.2. The proportion of CD14+ monocytes(% PBMCs), intermediate monocytea(%CD14+ monocytes), CD14+HLA-DR-/low monocytes(% CD14+ monocytes), CCR2+monocytes(% PBMCs) and Tie-2+ monocytes(% CD14+ monocytes) was all higher in newly diagnosed DLBCL patients than those in healthy controls, respectively. In different subgroups of DLBCL patients, the above described monocytes were higher in relapsed group than those in newly diagnose group, and were lower in remission group than those in newly diagnosed group. With IPI score increased, the above described monocytes were all increased. After immunotherapy(combined rituximab), in remission patients the above described monocytes were all lower at the time of remission than those at the time of newly diagnosis, in relapsed patients they were higher at the time of relapse than those at the time of newly diagnosis. Comparing the data of newly diagnosed DLBCL patients and healthy controls, using receiver operating characteristic curves(ROC) analysis, we found that the increased monocytepopulation could be a maker for early diagnosis of DLBCL. The concentration of related cytokines Arg-1, MCP-1 and Ang-2 in plasma was positive correlated with CD14+ HLA-DR-/low monocytes(% CD14+ monocytes), CCR2+ monocytes(%PBMCs) and Tie-2+ monocytes(% CD14+ monocytes), respectively. The expression levels of CCR2 and Tie-2 m RNA were in line with corresponding flow cytometry results.3. A significantly positive correlation was detected between CD68 and CD163 density by Spearman correlation test analysis. Similarly, AMC showed a positive correlation with CD68 density and a positive correlation with CD163 density in DLBCL tissues,respectively. High CD163 expression is an independent adverse prognostic factor for DLBCL. According to CD68 and CD163 expression, low, intermediate risk DLBCL patients could be further stratified, and the frequencies of CD68 and CD163 expression levels increased gradually accompanied with IPI score. MCP-1 positive staining was exclusively observed in cytoplasm of DLBCL cells. Expression of CCR2 in DLBCL cells was found in either membrane or cytoplasm. Positive staining of CCR2 was also found in vascular endothelial cells and macrophages in DLBCL specimens,respectively. And pSTAT3 positively expressed in the nucleus of DLBCL cells. A significantly positive correlation was detected between MCP-1 and CCR2 expression in DLBCL tumor cells by Spearman correlation test analysis. Similarly, pSTAT3 showed a positive correlation with MCP-1 expression and a positive correlation with CCR2 expression, respectively. High MCP-1, CCR2 and positive pSTAT3 expression were all independently adverse prognostic indicators for OS and PFS of DLBCL. In different IPI score groups, especially in low, intermediate risk group, the three all could be identified high-risk patients and provide more prognostic information.The frequencies of MCP-1, CCR2 and pSTAT3 expression levels increased gradually accompanied with IPI score, and interestingly, the combination of MCP-1 or CCR2 expression with IPI score had an advantage over IPI score alone in predicting prognosis by ROC curve analysis.Conclusion1. AMC index is an independent prognostic factor in newly diagnosed, relapsed/primary refractory DLBCL, and can provide effective information for monitoring recurrence when dynamic detection in follow-up.2. Peripheral blood intermediate subset monocytes, which can be used as a marker for early diagnosis, may play an important role in the onset and development of DLBCL.In addition, CD14+HLA-DR-/low monocytes, CCR2+ inflammatory monocytes and Tie-2+ monocytes may play crucial roles in systematic immunesuppressive function,angiogenesis and tumor invasion and metastasis, respectively.3. The increase in the number of TAMs is an independent adverse prognostic factor,and positively correlates with AMC. The MCP-1/CCR2 axis through the STAT3 signaling pathway may play an important role in DLBCL invasion and metastasis.