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Identifier 000416843
a
Title Επίδραση της χημειοθεραπείας με η χωρίς τον αντιαγγειογενετικό παράγοντα Bevacizumab στα ανοσοκατασταλτικά κύτταρα σε ασθενείς με μη- μικροκυτταρικό καρκίνο του πνεύμονα
Alternative Title The effect of chemotherapy with or without the antiangiogenic agent Bevacizumab on the immunosuppressive cells in patients with non-small cell lung cancer.
Author Κοϊνης, Φίλιππος
Thesis advisor Κωτσάκης, Αθανάσιος
Αγγελάκη, Σοφία
Τζανάκης, Νικόλαος
Reviewer Σαμώνης, Γεώργιος
Χαμηλός, Γεώργιος
Κουτσόπουλος, Αναστάσιος
Abstract Introduction Lung cancer (LC) is among the most frequent type of malignancies, accounting for 13% of the newly diagnosed cases worldwide in 2012. Nonsmall- cell lung cancer (NSCLC) represents almost 85% of the total number of LC diagnoses and can be further classified into different subtypes based on the expression of certain molecular and histological features. Cancer-related Inflammation has been identified as a hallmark of cancer. Host immune system interacts with tumor cells in a complex and paradoxical process that has been shown to consist of three phases: elimination, equilibrium and escape. During the escape phase tumor cells have acquired the ability to evade immune surveillance by establishing an immunosuppressive tumor microenvironment and begin to grow uncontrollably. A wide range of cytokines released by either tumor cells or other cells in the tumor stroma appear to disrupt the balance between the antitumor immune response and immune tolerance and play a crucial role in the development of potent immunosuppressive mechanisms by inducing immunosuppressive cells, including myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Treg), in both the tumor microenvironment and the peripheral blood of cancer patients. Vascular endothelial growth factor (VEGF) is the principal mediator and key promoter of tumor angiogenesis. Recent data suggest a multifaceted inhibitory effect of VEGF on the development of a potent antitumor immune response. Mechanisms underlying the immunosuppressive capacity of VEGF have been found to include inhibition of maturation of dendritic cells, promotion of MDSCs migration and selective accumulation in tumor sites and induction of Treg proliferation and enhancement of their suppressive functionality. Taking into consideration the proposed link between VEGF and immunosuppression, it can be hypothesized that, the monoclonal antibody against VEGF, bevacizumab may be useful in reversing tumor-induced immune tolerance. 118 There is a growing body of evidence that links MDSC-, Treg- and VEGFinduced immunosuppression to tumor development and progression. Accumulating data suggest that the presence of these cells in the peripheral blood and in the tumor microenvironment is significantly associated with the patients’ clinical outcome and survival. However, there is no consensus on the phenotypic and functional characterization of these cells in NSCLC patients. Furthermore, there are limited data regarding the effect of first-line chemotherapy, with or without anti-VEGF therapy, on the frequency and functionality of these cells in patients with NSCLC. The aim of this study was to identify, phenotypically and functionally characterize subpopulations of MDSCs and Treg in the peripheral blood of NSCLC patients, determine their frequency at diagnosis and evaluate their prognostic significance. Additionally, the methodology developed allowed the study of the effect of chemotherapy, with or without the addition of bevacizumab, on the functionality and the levels of circulating immunosuppressive cells (Treg/MDSCs) and the correlation of their changes with the clinical outcome. Patients and methods Peripheral blood was collected from newly diagnosed, treatment-naïve patients with stage IIIB/IV NSCLC before initiation of treatment, after the 3rd cycle of chemotherapy, as well as at the time of disease progression. Blood samples from aged- and sex-matched healthy volunteers were used as normal controls. Various subpopulations of MDSCs and Treg were identified and analyzed using flow cytometry. Their immunosuppressive activity was assessed by demonstrating their inhibitory effect on IFN-γ production by activated T-cells. Results Phenotypic characterization of MDSCs subpopulations with clinical significance and their prognostic role in treatment-naïve patients with NSCLC A total of 110 patients were enrolled in this study. The patients’ median age was 68 years. Approximately, 84.5% were men, 51.8% had adenocarcinoma and 74.5% had stage IV disease. Briefly, three new subpopulations of MDSCs were identified in patients with NSCLC: two monocytic [CD14+CD15−CD11b+CD33+HLA-DR−Lin− (CD15−M-MDSCs) and 119 CD14+CD15+CD11b+CD33+HLA-DR−Lin− (CD15+ M-MDSCs)] and one granulocytic [CD14−CD15+CD11b+CD33+HLA-DR−Lin− (G-MDSCs)]. The levels of these MDSCs subpopulations were significantly increased in the peripheral blood of the patients compared to healthy donors [(CD15+ MMDSCs, 3.5 ± 0.5% versus 0.5 ± 0.2%, p≤ 0.0001), (CD15− M-MDSCs, 5.2 ± 0.5% versus 3 ± 0.8%, p= 0.04) and (G-MDSCs, 2 ± 0.5% versus 0.1 ± 0.02%, p≤ 0.0001)]. However, the difference in the frequency of these subpopulations among patients with stage III disease and those with stage IV disease was not statistically significant. Patients who progressed after 3 cycles of treatment had higher M-MDSCs levels prior to initiation of treatment (CD14+CD15+HLADR −Lin−: 1.1 ± 0.3% and CD14+CD15−HLA-DR−Lin−: 5.5 ± 1.1%) compared to those with disease control (CR, PR and SD) (0.6 ± 0.07%, p=0.02 and 2.9 ± 0.3%, p=0.02, respectively). Moreover, MDSCs’ percentage within the “normal range” (levels within the 90% of the healthy controls) prior to initiation of therapy was associated with a longer progression-free survival (PFS) and overall survival (OS) compared to increased (>90% of the healthy controls) MDSCs’ levels (10.87 versus 5.3 months, p=0.005 and 12.9 versus 7.1 months, p=0.008, respectively). In the multivariate analysis, increased frequency of circulating CD15+ Μ-MDSCs before treatment initiation was identified as an independent prognostic factor for poor survival (PFS: HR = 2.41, 95% CI: 1.37–4.24, p=0.002 and OS: HR = 2.35, 95% CI: 1.25–4.41, p=0.008). Finally, it was demonstrated that these MDSCs subpopulations are functional and suppress the production of IFN-γ by activated CD3+ T cells. Effect of treatment on the MDSCs’ subpopulations in the peripheral blood of patients with NSCLC A total of 46 patients were included, 33 on the chemotherapy arm and 13 on the chemotherapy plus bevacizumab arm. The patients’ median age was 68 and 85% were male. After 3 cycles of treatment, 24% of the patients experienced PR, 67% SD and 9% PD. Overall, chemotherapy did not appear to have a uniform or significant impact on the levels or the functionality of the various subpopulations of circulating MDSCs. However, three cycles of bevacizumab-based chemotherapy resulted 120 in a statistically significant decrease in the levels of the G-MDSCs subpopulation compared to regimens that did not include bevacizumab (mean change in percentages of G-MDSCs with the bevacizumab-based versus non– bevacizumab-based regimens: –0.68% ± 0.34% versus 0.69% ± 0.38%, p=0.0086). Furthermore, after 3 cycles of treatment, patients experiencing PD had significantly increased levels of all three MDSCs subpopulations compared to the levels in patients that responded (CR+PR+SD) to treatment. Interestingly, patients with PD had a statistically significant increase in the percentage of CD15+ M-MDSCs compared to the pre-treatment levels (p=0.03). Phenotypic characterization of Treg subpopulations with clinical significance and their prognostic role in treatment-naïve patients with NSCLC A total of 156 patients with NSCLC were enrolled in this study. The patients’ median age was 62 years, 82.1% were male, 57.6% had adenocarcinoma and 82.1% had stage IV disease. Briefly, the levels of CD4+CD25+ Treg were significantly increased in the peripheral blood of the patients compared to healthy donors (24.81 ± 1% versus 14.67 ± 1.5%, respectively, p= 0.0002). Depending on the stage of activation and differentiation, three CD4+ Treg subtypes were further analyzed: naive, effector and terminal effector Treg. Patients with PD as best response to first line treatment had increased percentage of naive Treg (CD25highCD127-/lowCD152-FoxP3lowCD45RO–) prior to treatment initiation compared to those with disease control (CR, PR and SD) (3.17 ± 0.58% versus 0.81 ± 0.33%, respectively, p=0.003). Moreover, increased (>90% of the healthy controls) levels of naïve Treg at baseline were associated with significantly shorter OS compared to that of patients with Treg percentage within the normal range (levels within the 90% of the healthy controls) (OS: 18.37 versus 40.47 months, respectively, p=0.039). Similarly, patients with increased levels of effector Treg(CD25highCD127lowCD152+FoxP3+CD45RO+) before treatment initiation had significantly shorter PFS (6.8 versus 8.53 months, p=0.046) and OS (5 versus 15.37 months, p=0.037) relative to that of patients with Treg levels within the normal range. In contrast, patients 121 experiencing PD had significantly lower levels of terminal effector Treg (CD25highCD127-CD152+FoxP3+CD45RO+) at baseline compared to those with disease control (PD versus CR+PR+SD: 7.48 ± 1.27% versus 14.08 ± 2.74%, respectively, p=0.04). Moreover, increased levels of terminal effector Treg were associated with longer PFS (16.2 versus 7.5 months respectively, p=0.03) and OS (undefined versus 12.63 months, respectively, p=0.049). Multivariate analysis revealed that increased frequency of circulating naive Treg at baseline is an independent prognostic factor for poor OS (HR = 8.632, 95% CI: 2.226-33.468, p=0.002). Finally, it was found that, all the above described, Treg subpopulations, regardless of their activation and differentiation stage, are functional and suppress the production of IFN-γ by activated CD4+ T cells. Effect of treatment on the Treg subtypes in the peripheral blood of patients with NSCLC A total of 46 patients were included in this study, the same patients analyzed for the effect of treatment on MDSCs. Blood samples were obtained before initiation of first-line therapy and after 3 cycles of treatment. Overall, chemotherapy was not associated with a significant change in the levels of the CD4+CD25highCD127−CD152+FoxP3+ Treg, in the peripheral blood of the patients. However, after three cycles of bevacizumab-based chemotherapy, patients receiving bevacizumab had statistically significant decreased levels of CD4+CD25highCD127−CD152+FoxP3+ Treg compared to patients who did not receive bevacizumab (percentages after 3 cycles of treatment with bevacizumab-based versus non-bevacizumab-based regimens: 14.81 ± 4.615% versus 36.44 ± 4.506%, p=0.0058). In particular, regarding naive Treg, patients who received 3 cycles of bevacizumab-based treatment had statistically significantly lower cell counts compared to patients who did not receive bevacizumab (naive Treg percetnage after 3 cycles of treatment, bevacizumab versus non-bevacizumab based treatment: 0.6273 ± 0.345% versus 3.44 ± 1.076%, p=0.019). In contrast, non-bevacizumab based chemotherapy was associated with a statistically significant increase in the naive Treg levels in the peripheral blood of the patients (naive Treg pre- and 122 after 3 cycles of treatment without bevacizumab: 1.456 ± 0.353% versus 3.44 ± 1.076% respectively, p=0.045). There was no statistically significant difference in the levels of effector Treg between patients treated with or without the addition of bevacizumab (mean change in effector Treg percentage, bevacizumab based versus non-bevacizumab based regimens: 0.3818 ± 1.54% versus -1.59 ± 3.2%, p=0.49). Finally, after 3 cycles of treatment, patients treated with bevacizumab had decreased levels of terminal effector Treg compared to patients who did not receive bevacizumab (terminal effector Treg percentage after 3 cycles of treatment, bevacizumab based versus non-bevacizumab regimens: 7.3 ± 2.958% versus 15.72 ± 3.128%, p=0.059). Conclusions In conclusion, this study precisely defines, phenotypically and functionally, distinct MDSCs and Treg subpopulations with prognostic significance in the peripheral blood of patients with NSCLC. Quantification of their frequency in the peripheral blood has emerged as an independent prognostic factor for survival in patients with NSCLC, highlighting the potential clinical utility of the particular biomarker. The selective targeting of specific MDSCs and Treg subpopulations through bevacizumab-based treatment provides evidence for the immunomodulating effect of anti-angiogenic therapy and bevacizumab emerges as a promising agent in “taking the foot off the brake of immunosuppression”. Nevertheless, these experimental and clinical findings remain to be confirmed prospectively in future randomized clinical trials enrolling more patients with more homogeneity in the histological type, the disease burden and the treatment that they receive. At the same time, efforts should be mounted toward linking the levels of the circulating MDSCs and Treg with their frequency in the tumor microenvironment, where they exert their immunosuppressive action. Improving the understanding of the biology and the functionality of MDSCs and Treg is considered to be of paramount importance for the development of novel strategies to efficiently detect, quantify and, finally, target tumor-induced immunosuppression.
Language Greek
Subject Ανοσοκαταστολή
Issue date 2018-07-18
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/a/7/5/metadata-dlib-1535992185-293149-25337.tkl Bookmark and Share
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