Part I Analysis Of Clinical Features And Prognostic Factors Of Chronic Lymphocytic Leukemia Part II The PD-1/PD-L1 Pathway Inhibitor Reverses The Immunosuppressive State Of Chronic Lymphocytic Leukemia

Objective:To investigate the clinical and laboratory characteristics and survival of Chinese patients with chronic lymphocytic leukemia(CLL),to evaluate the natural history of patients with CLL and a 17p deletion(17p-)and to identify the predictive factors within this subgroup,we performed this study.Meanwhile,the established clinical staging systems(Rai/Binet)of CLL cannot accurately predict the appropriate treatment of patients in the earlier stages.In order to predict the time to first treatment(TTFT)in patients of early stages,we evaluated the prognostic role of conventional markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system,the CLL prognostic index(CLL-PI).Moreover,we also evaluated the response rate and long-term therapeutic efficacy of Rituximab(R)for patients with chronic lymphocytic leukemia.Methods:503 patients with CLL admitted in our hospital from Oct 1998 to Feb 2015 were retrospectively analyzed.Baseline characteristics were compared using Chi-square test and Kaplan-Meier methodology was undertaken for survival analyses.Taking advantage of a population of 406 untreated Chinese patients with CLL at early and advanced stage of disease,we identified the strongest prognostic markers of TTFT and,subsequently,in a cohort of 173 patients who had complete data for all 3 variables,we integrated the data of traditional staging system,cytogenetic aberrations and mutational status of immunoglobulin heavy chain variable region(IGHV)in CLL-PI.And the clinical data of CLL patients receiving FC ± R(with or without R)regimen or CHOP/CVP ± R regimen in our hospital were analyzed retrospectively.Therapeutic efficacy and survival of patients treated with different regimens were evaluated and compared.Results:(a)Clinical and laboratory features:The median age was 58(26-86)years with male/female ratio of 1.99.196 cases of the patients were at the clinical stage of Binet A(40.5%),followed by Binet C(30.1%)and Binet B(29.3%).21.1%had anemia at diagnosis,while 26.5%had an elevated level of lactate dehydrogenase(LDH)and the expression of CD38 were detected among 29.1%of the patients.Clonal abnormalities were observed using fluorescence in situ hybridization(FISH)analysis.Those involving 13q deletion were the most frequent(47.3%),followed by IgH translocation(22.4%),trisomy 12(21.2%)and 17p-(14.5%).The mutational status of IGHV was determined among 230 cases,165 cases of which were found to be with mutated status(71.7%).The most frequently encountered gene was V4-34(12.4%).The median progression-free survival(PFS)was 89.0 months(95%CI 75.0-103.0),while the median overall survival(OS)was 129.0 months(95%CI 106.9-151.1);(b)The overall response rate(ORR)in patients with a 17p-was 56.9%,and patients with a high percentage of 17p-(defined as more than 25%of cells harbouring a 17p-)had a lower ORR.The median OS in patients with a 17p-was 78.0 months,which was significantly shorter than the OS in patients without this genetic abnormality(median 162.0 months,P<0.001).Within the subgroup with a 17p-,PFS was significantly shorter in patients at Binet stage B-C and patients with elevated LDH,B symptoms,unmutated IGHV and a high percentage of 17p-;(c)Based on multivariate Cox regression analysis,three independent factors for TTFT were identified:advanced clinical stage,17p-and unmutated IGHV.Applying weighted grading of these independent factors,a CLL-PI was constructed based on regression parameters,which could categorize four different risk groups[low risk(score 0),intermediate low(score 1),intermediate high(score 2)and high risk(score 3-6)]with significantly different TTFT(median TTFT of NR,65.0 months,36.0 months and 19.0 months,respectively,P<0.001);(d)Among 72(43.6%)patients treated with R,the complete remission(CR)rate and the ORR were significantly higher than the rates among patients treated without R(38.9%vs.21.5%,P=0.015;83.3%vs.60.2%,P=0.001).Patients who achieved CR had a significant prolonged PFS and OS(P<0.01).The median PFS and OS for patients treated with R were 53.0(95%CI 27.0-79.0)months and 112.0(95%CI 81.1-142.9)months,which were longer than the PFS(median 28.0,95%CI 18.3-37.7)and the OS(median 89.0,95%CI 72.0-106.0)of patients treated without R,but the results were not statistically significant.According to the cytogenetic features,patients were grouped into high-risk subgroup(with 17p-or 11q-)and non-high-risk subgroup.And in the high-risk subgroup,the ORR was significantly better if patients were treated with R;in the non-high-risk subgroup,the PFS was significantly prolonged in patients treated with R(P=0.05).Conclusions:These findings suggest that this cohort of Chinese CLL patients were younger at diagnosis and had similar OS compared with patients in the western world,which,to some extent,could reflects the characteristics of CLL patients in China.Patients with a 17p-CLL have a variable prognosis that might be predicted using simple clinical and laboratory characteristics.Meanwhile,this study developed a weighted,integrated CLL-PI prognostic system of CLL patients which combines the critical genetic prognostic markers with traditional clinical stage.This novel modified PI system could be used to discriminate among groups and may help predict the TTFT and prognosis of patients with CLL.The chemoimmunotherapy with R results in higher CR rate and ORR among patients with CLL,which is significantly better than traditional chemotherapy.Among patients without high risk cytogenetic abnormalities,the addition of R can prolonged PFS.Those patients who achieve CR have longer survival.Objective:In the microenvironment of chronic lymphocytic leukemia(CLL),a variety of cell populations,including mesenchymal stromal cells(MSC)and monocytes,contributes to the proliferation of CLL cells and CLL patients display T-cell dysfunction and have an increased susceptibility towards infections.Programmed death-1 is a receptor involved in tumor-mediated immunosupression and supporting tumor proliferation through binding with its ligand,PD-L1.To investigate the expression of PD-1/PD-L1 pathway in CLL microenvironment and the impact of blockade of PD-1/PD-L1 on accessory and immune cell function as well as CLL cell growth,we performed this study.Methods:Surface expression of PD-1 on immune effector cells and PD-L1 expression on CD 19+ CLL cells and CD11b+ monocytes were determined in peripheral blood from newly diagnosed(ND),refractory and relapsed(R/R)CLL patients versus healthy donor(HD).Magnetic-beads based cell sorting was used to purify the cell subsets.We define the impact of blockade of PD-1/PD-L1,alone or in combination with lenalidomide(Len),on autologous anti-tumor response and tumor cell growth.Results:The CD4+/CD8+ T cell raio is significantly decreased compared to HD(1.53± 1.34 vs.3.55±5.11,P=0.018).We defined inverted CD4+/CD8+ ratio as the value was less than one.13 cases(32.5%)had an inverted CD4+/CD8+ ratio.T-cell subpopulations were analyzed by flow cytometry.CD4+ and CD8+ subset cells were stained with CD45RA and CCR7 in order to quantify the relative number of naive(TNaive),central memory(Tcm),effector memory(Tem)and terminally differentiated effector cells(Teff).Cumulative data indicated marked differences in the composition of both CD4+ and CD8+ T-cell subsets.The most significant differences in the CD4+compartment concerned the decrease of TNaive and the relative increase of Tem populations(P<0.001).Similar results were found in the CD8+ compartment(P<0.001).TNaive subpopulations were significantly decreased and Tem increased in R/R patients compared to ND patients(P<0.001)and Tem subset in Binet B-C patients significantly increased compared with Binet A patients(P=0.026).Meanwhile,the relative number of regulatory T cells(Tregs)in CLL patients significantly increased compared to HD(P=0.0077).Both ND and R/R patients CLL cells have increased surface PD-1 expression compared with HD.In CLL patients,the expression level of PD-1 was significantly higher than HD,both on CD4+ T cells(31.7±15.4%vs.17.0±8.0%,P<0.001)and on CD8+ T cells(18.8±13.4%vs.10.6±7.2%,P<0.001).Notably,PD-1 expression also increased on Tregs in CLL patients compared HD(24.7±15.2%vs.14.5±7.0%,P<0.001).When determining surface PD-1 expression in the different subsets we found that the molecule was expressed by each subpopulation:In CLL,the PD-1 expression on CD4+ subpopulations was higher than in HDs:TNaive:7.5 ± 10.7%vs.1.2±0.8%;Tcm:22.3±15.0%vs.13.6±8.2%;Tem:43.0±15.1%vs.34.1±9.1%Teff:21.8 ± 17.8%vs.11.1 ±10.4%,P<0.01).There is also a significant increase in PD-1 expression on CLL cells compared to CD19+ cells from HD(P<0.01).However,the PD-L1 expression on CLL cells did not reach statistical significance.Besides,monocytes from CLL patients displayed higher expression of PD-L1(P=0.0034),and the concentration of soluble PD-L1 in the plasma was also higher detected by ELISA(P=0.0195).When co-cultured with autologous monocytes,the expression of PD-L1 on CLL cells was induced while the expression of PD-1 was down-regulated.Importantly,PD-1/PD-L1 blockade abrogates monocytes-induced CLL cell growth,and combined blockade of PD-1/PD-L1 with lenalidomide(Len)further inhibits monocytes-induced tumor growth.These effects are associated with induction of intracellular expression of IFN-y and granzyme B in effector cells.Finally,lenalidomide with PD-1/PD-L1 blockade promote anti-tumor immunity of effector cells(P<0.05).Conclusions:These findings suggest that checkpoint signaling plays an important role in providing the tumor-promoting,immune-suppressive microenvironment in CLL,and that PD-1/PD-L1 blockade induces anti-CLL immune response that can be enhanced by lenalidomide,providing the framework for clinical evaluation of combination therapy.