Augmentation of IFN-γ+ CD8+ T cell responses correlates with survival of HCC patients on sorafenib therapy

Abstract

BACKGROUNDSorafenib has been shown to reduce the extent of immunosuppression in patients with hepatocellular carcinoma (HCC). The rationale of this investigation was to identify biomarkers that can predict treatment efficacy of sorafenib in HCC patients and to unravel the mechanism by which sorafenib impedes immune suppression mediated by distinct immunosuppressive cell subsets.METHODSWith informed consent, blood samples were collected from 30 patients with advanced HCC, at baseline and 2 time points after initiation of sorafenib treatment. The frequency of PD-1+ T cells, ERK2 phosphorylation on flt-3+ Tregs and MDSCs, and T effector cell function were quantified by using flow cytometry.RESULTSElevated levels of CD8+Ki67+ T cells producing IFN-γ were associated with improved progression-free survival and overall survival (OS). High frequencies of these T cells were correlated with significantly reduced risk of death over time. Patients with an increased pretreatment T effector/Treg ratio showed significant improvement in OS. ERK+flt-3+ Tregs and MDSCs were significantly decreased after sorafenib therapy. Increased numbers of baseline flt-3+p-ERK+ MDSCs were associated with survival benefit of patients.CONCLUSIONA high baseline CD4+ T effector/Treg ratio is a potential biomarker of prognostic significance in HCC. CD8+Ki67+ T cells producing IFN-γ are a key biomarker of response to sorafenib therapy resulting in survival benefit. The immune modulation resulted from sorafenib-mediated blockade of signaling through the VEGF/VEGFR/flt-3 pathway, affecting ERK phosphorylation. These insights may help identify patients who likely would benefit from VEGFR antagonism and inform efforts to improve the efficacy of sorafenib in combination with immunotherapy.TRIAL REGISTRATIONNCT02072486.FUNDINGNational Comprehensive Cancer Network Oncology Research Program from general research support provided by Bayer US LLC (NCCNSORA0002), National Cancer Institute grant P30CA016056, and pilot funds from Roswell Park Alliance Foundation.

Keywords: Hepatology; Immunology; Liver cancer; T cells.

Conflict of interest statement

Conflict of interest: RI received consulting fees from Bayer.

Figures

Figure 1. Description of cohorts for biomarker study.

We recruited 39 patients for the study. Radiographically, biochemically, or biopsy-proven, measurable HCC that was not previously treated with systemic therapy and was Child-Pugh Class A or B were the eligibility criteria. The observation period for each patient was the time from the start of therapy with sorafenib to withdrawal of consent, death, or the last visit. Of the 39, 30 patients met eligibility criteria. One pretreatment and 2 posttreatment blood samples were collected for the biomarker study. Patients were nonevaluable for the primary biomarker endpoint if they provided fewer than 2 biomarker samples or came off the study before response assessment. Of 30 eligible patients, 22 provided 3 blood samples for biomarker study and 8 provided 2 blood samples. Response, dosing, and toxicity information were collected in a database, and deidentified biomarker data were analyzed in a blind fashion. PFS, progression-free survival.

Figure 2. Reduction in immunosuppressive cell subsets after sorafenib therapy.

Frequencies of various immune T cell subsets were calculated on live CD3+CD4+ or CD3+CD8+ T cell populations and MDSCs are CD14−HLA–DR−CD11b+CD33+. Absolute number is cells per milliliter. (A) Representative histogram offset showing the frequency of CD4+Foxp3+ Tregs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (B) Frequency and (C) absolute numbers of CD4+Foxp3+ Tregs pretreatment and after 28–35 days and 160 days (n = 22). (D) Representative histogram offset showing frequency of CD11b+CD33+ MDSCs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (E) Frequency and (F) absolute numbers of MDSCs pretreatment and after 28–35 days and 160 days (n = 22). (G) Ratio of CD4+CD127+ T cells to CD4+Foxp3+ T cells (CD4+CD127+ T cells/CD4+Foxp3+ T cells) pretreatment and after 28–35 days and 160 days (n = 22). (H) Ratio of CD8+CD127+ T cells to CD4+Foxp3+ T cells pretreatment and after 28–35 days and 160 days (n = 22). (I) Representative histogram offset, showing frequency of Foxp3+flt-3+p-ERK+ Tregs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (J) Frequency and (K) absolute numbers of Foxp3+flt-3+p-ERK+ Tregs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment (n = 22). (L) Representative histogram offset, showing frequency of flt-3+p-ERK+ MDSCs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (M) Frequency and (N) absolute numbers of flt-3+p-ERK+ MDSCs pretreatment, 28–35 days, and 160 days (n = 22). Each symbol represents an individual HCC patient. Error bars represent mean ± standard deviation (SD). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; paired t test.

Figure 3. Decrease in T cell exhaustion markers after sorafenib treatment.

Immunophenotypic analysis of T cells was performed after stimulation of PBMCs in vitro using anti–CD3/CD28 at pretreatment and after 28–35 days and 160 days of sorafenib treatment by multicolor flow cytometry. Frequencies of various immune cell subsets were calculated on live CD3+CD4+ or CD3+CD8+ T cell populations, and absolute number is cells per milliliter. (A) Representative histogram offset showing frequency of Foxp3+CTLA-4+ Tregs measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (B) Frequency and (C) absolute numbers of Foxp3+CTLA-4+ Tregs pretreatment, 28–35 days, and 160 days (n = 22). (D) Representative histogram offset showing frequency of CD4+Ki67+PD-1+ T cells measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (E) Frequency and (F) absolute number of CD4+Ki67+PD-1+ T cells pretreatment, 28–35 days, and 160 days (n = 22). (G) Representative histogram offset showing frequency of CD8+Ki67+PD-1+ T cells measured at pretreatment and after 28–35 days and 160 days of sorafenib treatment. (H) Frequency and (I) absolute number of CD8+Ki67+PD-1+ T cells pretreatment, 28–35 days, and 160 days (n = 22). Each symbol represents an individual HCC patient. Error bars represent mean ± standard deviation (SD). ****P < 0.0001; ***P < 0.001; **P < 0.01; paired t test.

Figure 4. Effect of sorafenib treatment on Teffs.

PBMCs from patients with HCC were stimulated with anti–CD3/CD28 in vitro for 48 hours and immunophenotypic analysis was performed. Frequencies of various immune cell subsets were calculated on live CD3+CD4+ or CD3+CD8+ T cell populations, and absolute number is cells per milliliter. (A) Representative histogram offset showing frequency of CD4+Ki67+IFN-γ+ T cells measured at different time points of sorafenib treatment. (B) Frequencies and (C) absolute number of CD4+Ki67+IFN-γ+ T cells pretreatment, 28–35 days, and 160 days (n = 22). (D) Representative histogram offset showing frequency of CD8+IFN-γ+ T cells measured at different time points of sorafenib treatment. (E) Frequencies and (F) absolute number of CD8+Ki67+IFN-γ+ T cells pretreatment, 28–35 days, and 160 days (n = 22). (G) Representative histogram offset showing frequency of CD8+Ki67+GRB+ T cells measured at different time points of sorafenib treatment. (H) Frequencies and (I) absolute number of CD8+Ki67+GRB+ T cells pretreatment, 28–35 days, and 160 days (n = 22). Each symbol represents an individual HCC patient. Error bars represent mean ± standard deviation (SD). ****P < 0.0001; ***P < 0.001; **P < 0.01; paired t test.

Figure 5. Kaplan-Meier plots showing both the predictive immune correlates of response to and efficacy of sorafenib treatment in patients with HCC.

Plots A–C show the correlation between CD8+Ki67+IFN-γ+ T cells before sorafenib treatment (baseline values) and patient survival. The association between CD8+Ki67+IFN-γ+ T cells and OS or PFS was calculated as described in Methods. (A) Absolute numbers of baseline values of CD8+Ki67+IFN-γ+ T cells and OS. (B) Frequency of CD8+Ki67+IFN-γ+ T cells at baseline and PFS. (C) Absolute numbers of CD8+Ki67+IFN-γ+ T cells and PFS. (D) Log change in absolute numbers of CD8+Ki67+IFN-γ+ T cells (log of the ratio [post/pre] and PFS).

Figure 6. Kaplan-Meier plots showing the baseline immune correlates predictive of response to sorafenib treatment in patients with HCC.

Plots A–C show the correlation between immune parameters before sorafenib treatment and patient outcome. The association between immune markers and OS or PFS was calculated as described in Methods. (A) Ratio of CD4+CD127+ T cells/CD4+Foxp3+ Tregs and OS. (B) Absolute numbers of CD4+Foxp3+CTLA-4+ T cells and PFS. (C) Absolute numbers of flt-3+p-ERK+ MDSCs and PFS.