Prognostic Analysis Of HBV Infection In DLBCL And The Role Of T Cell-mediated Immunity

Part OneThe impact of hepatitis B virus (HBV) infection on clinical outcomes of diffuse large B cell lymphoma (DLBCL) patients under immune-chemotherapyBackgrounds and Purpose Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma in China. A positive association between persistent, hepatitis B surface antigen (HBsAg)-positive hepatitis B virus (HBV) infection and DLBCL may exist. In addition, HBV infection in DLBCL patients is a common complication, as there has been an increased awareness of reactivation of occult HBV infection after chemotherapy. However, the clinical relevance of HBV infection with respect to DLBCL disease stages and patient survival remains unclear. In this multiple-center clinical study, we retrospectively analyze the efficacy of rituximab-combined chemotherapy in DLBCL patients with a diverse spectrum of serological profiles related to HBV infection. The main objective of the current study was to analyze the clinical features and to evaluate the prognostic factors of HBV infection in DLBCL patients, figure out the major risk factors related to poor outcomes with multiple regression analysis. Patients and Methods Three hundred and eighty four newly diagnosed patients consecutively registered in the Shanghai Lymphoma Research Group (including 6 tertiary hospitals in Shanghai) between March 1998 and December 2008 were included in this retrospective study. All patients registered in our group had received at least four cycles of first-line CHOP-or R-CHOP-like regimens and had at least a follow-up of 3 months. Patients in both groups were divided into two subgroups by HBsAg subsequently. Progression-free survival (PFS) and overall survival (OS) of the patients have or have not received rituximab were analyzed separately. A Cox regression model was used to ascertain the prediction value of the serum HBV marker in survivals.Results Most of baseline characteristics of patients did not differ significantly between different subgroups concerning the treatment and different HBV infection statuses. Patients in the CHOP group got a median PFS of 83.6 months and a median OS of 86.5 months. PFS/OS did not differ significantly between the HBsAg-positive patients and the HBsAg-negative patients. In the R-CHOP group, the HBsAg-negative patients had advantages over the HBsAg-positive patients in both PFS and OS. We conducted a multivariate analysis by entering into a Cox regression model to verify this kind of relationship. After adjusting for age, parameters of International Prognostic Index(IPI scores), elevated serum lactate dehydrogenase (LDH) level, extronodal cites≥2, Ann Arbor stage Ⅲ-Ⅳ, a positive HBsAg was still an independent risk factor in both PFS and OS. The relative risk (RR) of a positive HBsAg was 2.681 (p=0.022) in PFS and 3.344 (p=0.006) in OS.Conclusions HBV infection status doesn’t have a notable impact on the survival of DLBCL patients who received CHOP regimen. But in selected population that undertaking R-CHOP treatment, HBV naive or resolved HBV-infected patients hold their survival advantage when compared to consistent HBV-infected patients. HBsAg is the major serological marker of HBV infection, which served as an independent risk factor of DLBCL survival in the era of combined immuno-chemotherapy.Part TwoT cell-mediated immune response to chemotherapy in DLBCL patients with HBsAg-positive hepatitis B virus (HBV) infectionBackgrounds and Purpose The etiologic roles of virus infection in lymphoma development and patients’outcomes involve an activation of tumor-related genes secondary to integration of host genome, or inactivation of tumor suppressor genes, or immune status changes in the host cells that resulted with a decreased immune surveillance capability. Variations in the patterns of cell mediated immunity in patients with chronic HBV infection, or in patients with hepatic cirrhosis secondary to hepatitis have been verified by a number of studies. In the case of DLBCL, the lymphomagenic mechanism of HBV infection may attribute to the latter one. Since the clinical relevance of HBV infection with respect to DLBCL disease stages and immune patterns of T lymphocyte subsets during chemotherapy remains unclear, in this part (Part Two), we tried to identify the characteristics of T cell mediated immunity in DLBCL patients with HBsAg-positive HBV infection, thereafter, to explore the possible cell-mediated immune mechanisms of HBsAg positive HBV infection on the survival of DLBCL.Patients and Methods ① A total of 294 newly diagnosed DLBCL patients were enrolled in this cohort study. Four-color flow cytometric method was used to enumerate the absolute number of CD3+, CD4+, CD8+ T lymphocytes and the CD4+/CD8+ ratio in peripheral blood samples, at the onset of disease,2-4 months,4-6 months and 6-12 months after the initiation of chemotherapy, individually, ②A small size case-control study was conducted in 63 newly diagnosed DLBCL patients, who received R-CHOP chemotherapy,21 with HBsAg positive HBV infection and 42 were HBsAg negative. Both groups were matched by age, gender, disease stage and International Prognostic Index (IPI) score. The values of overall survival (OS) and progression-free survival (PFS) in both group were recorded and absolute count of circulating CD3+, CD4+, CD8+ T lymphocytes and the CD4+/CD8+ ratio were measured before chemotherapy and after 4 cycles of R-CHOP chemotherapy. ③ Ten cases of HBsAg positive DLBCL patients and 20 cases of HBsAg negative DLBCL were enrolled in a sub-study, after matching for indicators of gender, disease stage and IPI score. Twenty healthy volunteers were also enrolled as healthy control group. The number of CD4+CD25+CD127low T lymphocytes, regulatory T lymphocyte (Treg) and CD8+CD28" suppressor T cells (Ts) were counted both before the initiation of chemotherapy and 4 cycles of chemotherapy.④ Serum level of tumor necrosis factor-α (TNF-α), soluble interleukin-2 receptor (sIL-2r), interleukin-6 (IL-6) and interleukin-10 (IL-10) were analyzed among 67 DLBCL patients, of whom 43 achieved remission after chemotherapy (10 were HBsAg positive,33 were HBsAg negative),24 were newly diagnosed DLBCL. Another 20 healthy volunteer served as the control group.Results ①The count of CD4+ lymphocytes was lower in HBsAg positive group during the first 6 months after the initiation of chemotherapy, compared with that in the HBsAg negative group. From the middle stage to the late stage of chemotherapy (4-6 months after chemotherapy), the CD4+/CD8+ ratio in peripheral blood samples was significantly lower in HBsAg positive group than that in the HBsAg negative group. ②Case-control studied revealed that of patients who were newly diagnosed as DLBCL and received R-CHOP regimen, a positive serological test of hepatitis B surface antigen was still an indepent risk factor of poor OS(RR=4.082). But a positive HBsAg lost its predictive value of PFS when concerning about the count of CD8+ T lymphocytes. HBsAg-negative patients had advantages over the HBsAg-positive patients in both PFS and OS. In the middle of chemotherapy, the count of CD4+T lymphocyte and the CD4+/CD8+ ratio in peripheral blood samples were both lower in the HBsAg positive group.③ The number of circulating Treg and Ts was higher in DLBCL patients compared with the healthy control, no matter before R-CHOP regimen or after chemotherapy. The count of peripheral Treg in HBsAg positive DLBCL patients was even higher during R-CHOP chemotherapy. ④ Compared with the healthy control, serum level of TNF-a and sIL-2r were both higher in DLBCL patients, either at the beginning of chemotherapy or during their remission. There was an overall reduction in serum level of TNF-a and sIL-2r when patients achieved remission, but HBsAg positive DLBCL patients had a higher sIL-2r level compared with the HBsAg negative.Conclusion For newly diagnosed DLBCL patients who received R-CHOP regimen, positive serological test of hepatitis B surface antigen was an important risk factor of poor survival partly associated with the impairment of T cell immunity. The dynamic nature of cell mediated immune response was characterized as a low counts of CD4+ T lymphocyte during the first several cycles of chemotherapy followed by a decreased circulating CD4+/CD8+ ratio and expansion of Treg in the middle of chemotherapy. Of patients achieving DLBCL remission, the HBsAg positive had a higher serum level of sIL-2r than that in the HBsAg negative, which might help to explain how the increased counts of Treg in the peripheral blood sample from HBsAg positive DLBCL patients negative affected the patients’ survival.Part ThreeGene expression profile of Peripheral T cell receptor Vβ in HBsAg-positive DLBCL PatientsBackgrounds and Purpose Clonal periphral T-cell proliferation and skewed usage of T cell receptor (TCR) could happened in the setting of DLBCL, as a host immune adaptive response to tumor specific antigen or tumor associated antigen. Meanwhile there was an adaptive change of TCR repertoire secondary to antigen modification in virus infection diseases. When DLBCL was concurrent with HBV infection, the profiles peripheral TCR repertoire remained unclear among HBV infection subgroup, non-HBV infection group or HBV infection alone. Using quantitative real-time Polymerase chain reaction (RT-PCR) technique, we analyzed the gene expression profile of TCR Vβ in DLBCL patients (both HBsAg positive and HBsAg negative), while peripheral TCR Vβ repertoire established based on blood sample from healthy volunteers served as the healthy control. Difference in gene expression profiles between DLBCL with HBV infection and hepatic cirrhosis secondary to HBV infection were also analyzed, in order to figure out the mechanism of HBV infection on the outcomes of DLBCL.Methods ①A method using SYBR Green Quantitative RT-PCR was established to test the diversity of TCR Vβ. To each of the 24 TCR Vβsubfamilies, relative expression was presented as the percentage of that Vβ subfamily gene abundance to the whole Vβ gene abundance. The normal range of peripheral TCR Vβ spectrum was established with the blood sample of 10 healthy volunteer. A Vβ subfamily with a value beyond three standard deviations to the mean of the healthy control was considered as skewed usage of TCR Vβ. ② One case of large granular T lymphocyte leukemia and one case of T-cell prolymphocytic leukemia were enrolled, peripheral blood samples from these two patients were analyzed to profiling the TCR Vβ repertoire.③ We compared the TCR VβP repertoire profiling among 9 cases of hepatic cirrhosis secondary to HBV infection,15 cases of HBsAg positive DLBCL and 20 HBsAg negative one. To those identified as skewed usage of TCR, further analysis was performed to test the degree and frequency of TCR Vβskewness.Results ①Skewed usage of T cell receptor VP were present in both cases of clonal expansion of T cell hematologic malignancies, using SYBR Green Quantitative real time PCR, which consistent with the results from flow Cytometry. ②Skewed usage of T cell receptor Vβ were identified in all hepatic cirrhosis (9/9), all HBsAg negative DLBCL patients (20/20) and most of the HBsAg positive patients (14/15). The frequent usage Vβ repertoire in those patients was Vβ6, Vβ7, Vβ11, Vβ12 or Vβ21. No significant difference in the expression of TCR Vβ families among three gorups was observed, ③A profound higher usage of certain VP subfamily with a relative expression beyond ten standard deviations compared to the healthy control was much prevalent in HBsAg positive DLBCL patients, compared to HBsAg negative patients (8/15 vs 4/20, P<0.05). Hepatic cirrhosis patients had a similar phenomenon probably associated with clonal deviation (P=0.837).Conclusion Based on our established methods using SYBR Green Quantitative real time PCR, we found Skewed usage of T cell receptor Vβ were present in most DLBCL patients. HBsAg positive DLBCL patients had a much higher usage of Vβ subfamily, compared to those HBsAg negative, which suggested that a significant skewed TCR Vβ family occurred in patients with HBV infection. The impact of skewed usage of T cell receptor Vβ on patients’survival need further studies.