Objective: To elucidate the expression levels of key immune biomarkers, phosphate and tension homology deleted on chromosome ten (PTEN) and programmed cell death protein1(PD-1),of different immune tolerance pathway in classic Hodgkin's lymphoma (CHL) to further determine their clinical role and prognostic significance. Methods: The clinical features and prognostic factors of 56 CHL patients, who were admitted to the TianJin Medical University Cancer Institute from February 2003 to August 2013, were retrospectively analyzed. PTEN and PD-1 protein expression levels were analyzed by immunohistochemistry, Epstein-Barr virus encoded RNA (EBER) was performed by in situ hybridization assay. Correlations between the expression of biomarkers and clinicopathologic parameters were examined and survival analyses were performed. Results: This cohort of 56 CHL patients included 34 males and 22 females with a median age of 25 years (ranged from 7 to 71 years). In a univariate analysis, age≥45, IPS score >2, EBER positive, high expression of PTEN protein conferred inferior 5-year OS and 5-year PFS; In a multivariate model, age≥45, IPS score >2, EBER positive, high expression of PTEN protein were identified as the independent adverse prognostic factors for CHL. Conclusions: This study suggested for the first time that PTEN was independent prognostic immune biomarkers in CHL, which provided the novel therapeutic strategy of immune therapy for CHL.
Adolescent ; Adult ; Aged ; Child ; Female ; Hodgkin Disease ; Humans ; Male ; Middle Aged ; PTEN Phosphohydrolase/analysis* ; Prognosis ; Programmed Cell Death 1 Receptor/analysis* ; Retrospective Studies ; Young Adult

Objective: To explore the mechanism of ibrutinib on drug resistance diffuse large B-cell lymphoma (DLBCL) cells. Methods: DLBCL cell line was cultured with mesenchymal stem cells (MSC) , and DLBCL cells which migrated and adhered to MSC under microscope was counted. The secretion of CXCL12 by MSC were measured by ELISA. The expression of CXCR4 on DLBCL cells were measured by flow cytometry, HBL-1 cells were transfected with a CXCR4-lentivector. An Annexin Ⅴ-binding assay was used to detect the induction of apoptosis. Clonogenic growth of DLBCL cells was evaluated on MethoCult media. Ibrutinib was injected into NOD/SCID mice, tumor growth was assessed via caliper measurements every 3 days. Results: MSC promoted migration and adhesion of DLBCL cells to MSC. Ibrutinib inhibited migration and adhesion of DLBCL cells to MSC in a dose-dependent manner (P<0.05) . CXCL12 secreted by MSC and CXCR4 expressed on DLBCL cells could induce each other, which upgraded the levels of secretion and expression. Ibrutinib could inhibit the secretion of CXCL12 (SUDHL10: 660 pg/ml vs 1 400 pg/ml, P=0.004; HBL-1: 720 pg/ml vs 1 490 pg/ml, P=0.018; DLBCL:850 pg/ml vs 1 450 pg/ml, P=0.004) and expression of CXCR4 (P<0.05) . When co-cultured with MSC, the ratio of HBL-1 cells apoptosis in the group of control, mitoxantrone, ibrutinib, mitoxantrone+ibrutinib were respectively 15.1%, 17.5%, 23.5%, 58.7%. After transfected with a CXCR4-lentivector and overexpressed CXCR4, the ratios of HBL-1 cells apoptosis were 14.2%, 16.1%, 22.5%, 38.3% respectively. The ratio of DLBCL cells apoptosis induced by mitoxantrone was lower when co-cultured with MSC (P<0.05) . But with the addition of ibrutinib, the ratio of apoptosis was increaed and it was similar to cultivation without MSC, which suggested ibrutinib could inhibit drug-resistance induced by MSC. But after transfected with a CXCR4-lentivector, the overexpression of CXCR4 was detected and the ratio of apoptosis was significantly lower when co-cultured with MSC which demonstrated that ibrutinib inhibited drug-resistance by inhibiting the expression of CXCR4. MSC enhanced lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. The number of colonies of control, MSC, Ibrutinib, MSC+Ibrutinib were 113±5, 205±4, 62±9, 123±3 (2.5×10³/well, ±s) , respectively. The tumor volume of NOD/SCID mice were respectively 6 500, 17 000, 4 000, 10 000 mm³. Ibrutinib inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vitro. Conclusion Ibrutinib targeted the CXCL12/CXCR4 axis, inhibited the expression of CXCR4 and inhibited MSC-mediated drug resistance. Ibrutinib also inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. These results provided a scientific rationality for relapsed/refractory DLBCL treatment with ibrutinib.