The Impact Of Expression Of Costimulatory Molecules In Peripheral Blood T Cells Among The Patients With DLBCL

BackgroundDiffuse large B cell lymphoma(DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma (NHL) accounting for 36.2 percent of NHL cases. Although the survival rate in DLBCL has significantly improved with the introduction of Rituximab, a few patients are still refractory or endure an early relapse of disease. It is important to make a further study on the pathogenesis of DLBCL. T lymphocytes play an important role for immune system. The abnormal expression of costimulatory molecules in peripheral blood T cells from solid tumor patients always exits, and it is associated with the negative immune response to tumors. There are few researches on the expression of costimulatory molecules in T cells from DLBCL patients, and our study aims to explore the impact of expression of costimulatory molecules in peripheral blood T cells among the patients with DLBCL. There is important role of bone marrow infiltrationin in clinical staging of DLBCL patients, and it is necessary for us to explore three evaluation methods of 18F-FDG PET/CT in detecting bone marrow infiltration in patients with newly diagnosed diffuse large B-cell lymphoma.Objectives1. To investigate the expression of costimulatory molecules in peripheral blood T cells from DLBCL patients, and the impact of expression of costimulatory molecules in T cells on T cell receptor(TCR)proximal signaling molecular events.2. To explore the prognostic values of PD-1-positive cells in the tumor microenvironment among the DLBCL,3. To investigate the impact of Chidamide on the expression of PD-1 in T cells and TCR-proximal signaling molecular events.4. To explore three evaluation methods of 18F-FDG PET/CT in detecting bone marrow infiltration in patients with newly diagnosed diffuse large B-cell lymphoma.MethodsBlood samples were obtained from forty-five patients with newly diagnosed diffuse large B-cell lymphoma(DLBCL) and thirty-nine healthy volunteers from July 2014 to March 2015 in our hospital. The expression of costimulatory molecules (CD28, CTLA4, PD1)in T cells were tested by Flow Cytometry. Then, the mRNA levels of T cell receptor(TCR) proximal signaling molecular events (ZAP70, PI3K, NFAT) were detected by Real-Time quantitative polymerase chain reaction. Finally, the levels of serum interleukin-10 were tested by enzyme-linked immunosorbentassay. Sixty cases selection for this study involved the collection of formalin-fixed, paraffin-embedded tissue, which represented the initial diagnostic procedure from patients with DLBCL from April 2010 to December 2014.Immunohistochemicalstaining for PD-1 was performed on tissue sections.Then investigate the relationship between PD-1 expression and prognosis of DLBCL. Peripheral blood mononuclear cells(PBMCs) were obtained from seven patients with DLBCL. It were co-cultured with Chidamide(3μmol/L,72h), at the last, to investigate the impact of Chidamide on the expression of PD-1 in T cells and TCR-proximal signaling molecular events. Seventy-seven patients with newly diagnosed diffuse large B-cell lymphoma from July 2012 to June 2014 in our hospital were retrospectively analyzed. All patients received both 18F-FDG PET/CT scan and bone marrow biopsy in the region of the posterior iliac crests. There were three evaluation methods of 18F-FDG PET/CT to detect bone marrow infiltration, including visual comparison (the FDG uptake in bone marrow of iliac crests was higher than the normal liver tissue), the maximal standardized uptake values (SUVmax) in bone marrow of iliac crests was more than or equal to 2.5, the ratio of maximal standardized uptake values of iliac crests bone marrow to liver parenchyma intensity exceeded 1. All results were compared with the bone marrow biopsy.Results1. Compared with healthy controls,the mean fluorescence intensity(MFI) of PD1 expression on CD4+T cells and the proportion of PD1 expression on CD8+T cells were significantly up-regulated in untreated patients with DLBCL(P<0.001). The MFI of CTLA4 expression on CD4+T cells and CD8+T cells, and the proportion of CTLA4 expression on CD8+T cells were significantly higher(P<0.05).The proportion of CD28 expression on CD4+T cells were also higher(P<0.05). The mRNA levels of T cell receptor(TCR) proximal signaling molecular events (ZAP70, PI3K, NFAT) in DLBCL patients were lower than healthy controls(P<0.05), and the levels of serum IL-10 were significantly higher than controls(P<0.05).The MFI of PD1 expression on CD4+T cells were significantly higher in patients with high-intermediate risk DLBCL than patients with low-risk DLBCL,the mRNA levels of TCR-proximal signaling molecular events (ZAP70, PI3K, NFAT) in high-intermediate risk DLBCL were also significantly lower than low-risk DLBCL (P<0.05). However, there were no significant differences of the expression of costimulatory molecules and the mRNA levels of TCR-proximal signaling molecular events (ZAP70, PI3K, NFAT)between patients with different DLBCL subtypes.The MFI of PD1 expression greater than 468 or CTLA4 expression greater than 997 on CD4+T cells was a poorer prognostic factor.2. In DLBCL,the positive PD-1 expression had a poorer overall survival(OS) and progression free survival(PFS) rate when compared with negative PD-1 expression, and OS in patients with higher expression of PD-1 were lower than which in patients with lower expression of PD-1. So it is necessary for us to extend the follow-up period and then investigate the relationship between PD-1 expression and the prognosis of DLBCL.3. In DLBCL, the MFI of PD-1 expression on CD4+T cells significantly decreased in patients with Chidamide treatment than in untreated patients(P<0.05), and the mRNA levels of NFAT was significantly higher(P<0.05).4. Visual comparison of 18F-FDG PET/CT could be used to diagnose bone marrow infiltration, with the sensitivity of 100%, specificity of 80%, positive predictive value of 48%, negative predictive value was 100%. When we took the SUVmax of iliac crests as the diagnostic threshold, the sensitivity was 75%, specificity was 92.31%, positive predictive value was 64.29%, negative predictive value was 95.24%. the ratio of SUVmax had the best diagnostic efficiency, with the sensitivity of 100%, the specificity of 90.77%, positive predictive value of 66.67%, negative predictive value of 100%.Conclusions1. There were abnormal expression of costimulatory molecules in peripheral blood T cells from patients with DLBCL, and the mRNA levels of TCR-proximal signaling molecular events significantly decreased, the levels of serum IL-10 were significantly higher than healthy controls.2. In DLBCL,the positive PD-1 expression had a poorer overall survival(OS) and progression free survival(PFS) rate when compared with negative PD-1 expression, it is necessary for us to extend follow-up period and then investigate the relationship between PD-1 expression and the prognosis of DLBCL.3. In DLBCL, the MFI of PD-1 expression on CD4+T cells significantly decreased in patients with Chidamide treatment than in untreated patients, and the mRNA levels of NFAT was significantly higher.4. The ratio of SUVmax has perfect diagnostic value in detecting diffuse large B-cell lymphoatic bone marrow involvement.