| Objective: The purposes of this study are to detect the correlation between PD-L1 expression and p-AKT expression, and to evaluate the relationship between their expression and prognosis in DLBCL, and then to explore the potential novel mechanism that whether PD-1/PD-L1 could directly active the intracellular PI3K/AKT/m TOR signal pathway in DLBCL tumor cells, apart from evading host immunity. Finally we hope to explore a novel therapeutic approach for DLBCL and to improve the prognosis of patients in the future.Methods: Immunohistochemistry(IHC) was applied to assess the expression of PD-L1 and p-AKT in DLBCL patients as well as allthe completely preserved data about pathology and clinic was collected. The statistical software SPSS17.0was selected to analyze the correlation between the expression of PD-L1 and p-AKT and outcome in DLBCL. In vitro, we selected five DLBCL cell lines and detected total PD-L1 and membrane PD-L1(m PD-L1) proteins using Western blotting and flow cytometry, respectively. Stimulating with human recombinant PD-1/Fc protein for 0, 24 h and 48 h, DLBCL cells were collected to detect the variation of expression level of total and phosphorylated PDK1(Ser241), AKT(Ser473), AKT(Thr308) and m TOR(Ser2448) using Western blotting.Results: We collected the tissues and the completely clinic data of 100 patients, who were diagnosed with DLBCL from January 2008 to December 2011 in Tianjin Medical University Cancer Institute and Hospital. The median follow-up duration was 52.4 months(ranging from 1.5 to 89.1 months) and the deadline of follow-up was in June 2015. The results of IHC showed that both PD-L1 and p-AKT were overexpressed in 54%(54/100) and 48%(48/100) DLBCL cases, respectively. The co-expression of PD-L1 and p-AKT was in 32% DLBCL cases. Spearman analysis showed that PD-L1 expression was correlated with p-AKT expression(R=0.244, χ2=5.962; P=0.017). Among those clinical characteristics including gender, age, pathology subtype, IPI scores, clinical stages and chemotherapy regimens, we found that PD-L1 expression was higher in non-GCB subtype than GCB subtype(χ2=5.260, P=0.026), and p-AKT expression was higher on patients with >60-year ages than patients with ≤60-year ages(χ2=5.702, P=0.027). Kaplan-Meier analysis showed that 3-year OS and 5-year OS of DLBCL patients with positive expression of PD-L1 or p-AKT were poorer than those patients with negative expression of PD-L1 or p-AKT. The difference was statistically significant(P < 0.05). Among those patients with both negative expression of PD-L1 and p-AKT, single positive expression of PD-L1 and p-AKT, 3-year OS and 5-year OS of DLBCL patients with co-expression of PD-L1 and p-AKT were the worst(P < 0.05). Univariate analysis demonstrated that age(χ2=5.963, P=0.015), pathological subtype(χ2=4.823, P=0.028), clinical stages(χ2=9.470, P=0.003), IPI grades(χ2=7.645, P=0.006), chemotherapy regimens(χ2=5.825, P=0.016), PD-L1 expression(χ2=8.945, P=0.003), p-AKT expression(χ2=9.246, P=0.002), co-expression of PD-L1 and p-AKT(χ2=13.992, P<0.001) were associated with poor prognosis of DLBCL patients. Multivariate cox analysis showed that clinical stages [HR(95%CI)=3.726(1.273–10.902), P=0.016], chemotherapy regimens [HR(95%CI) =3.564(1.378–9.217), P=0.009]; PD-L1 expression [HR(95%CI)=4.740(1.097–20.477), P=0.037] and p-AKT expression [HR(95%CI)=6.2205(1.244–30.949), P=0.026] were the independent prognostic factors negatively impacting survival. All those five DLBCL cell lines were overexpressed PD-L1 and membrane PD-L1(m PD-L1). After stimulating DLBCL cell lines with human recombinant PD-1/Fc protein for 24 h and 48 h, the phosphorylation levels of PDK1(Ser241), AKT(Ser473), AKT(Thr308) and m TOR(Ser2448) were up-regulated in different DLBCL cell lines.Conclusion: 1. PD-L1 and p-AKT were overexpressed in DLBCL patients. PD-L1 expression was correlation with pathological subtype and p-AKT expression was correlation with ages in DLBCL patients. 2. PD-L1 expression was positive correlation with p-AKT expression in DLBCL. 3. Three years and 5 years OS of DLBCL patients with PD-L1 or p-AKT positive expression were worse than those patients with PD-L1 or p-AKT negative expression.Among those patients with both negative expression of PD-L1 and p-AKT, single positive expression of PD-L1 or p-AKT, 3 years and 5 years OS of DLBCL patients with co-expression of PD-L1 and p-AKT were the worst. The differences were statistically significant. 4. All the characteristics including age, pathological subtype, clinical stages, IPI scores, chemotherapy regimens, PD-L1 expression, p-AKT expression and co-expression of PD-L1 and p-AKT were associated with poor prognosis of DLBCL patients. But clinical stages, chemotherapy regimens, PD-L1 and p-AKT expression were the independent prognostic factors negatively impacting survival. 5.PD-1/PD-L1 pathway could directly active the intracellular PI3K/AKT/m TOR signaling in DLBCL. |
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