Targeting VEGFR2 with Ramucirumab strongly impacts effector/ activated regulatory T cells and CD8+ T cells in the tumor microenvironment
Several studies have established a correlation between the VEGF–VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers.
We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry.
Within the tumor microenvironment, both PD-L1 expression and CD8+ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA−FOXP3highCD4+ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8+ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2+ eTreg cell proliferation, and this effect could be inhibited by RAM.
This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.
KeywordsRegulatory T cells PD-1 Ramucirumab VEGFR2 Gastric cancer
- eTreg cell
effector regulatory T cell
Human epidermal growth factor receptor type 2
Immune checkpoint blockade
Non-small cell lung cancer
Peripheral blood mononuclear cell
- Treg cell
Regulatory T cell
Vascular endothelial growth factor
Vascular endothelial growth factor receptor 2
Recent advances in cancer immunotherapy, including the development of immune checkpoint blockades (ICBs), such as those targeting PD-1, PD-L1, and CTLA-4, have resulted in a paradigm shift in cancer treatment. In fact, the U.S. Food and Drug Administration has granted approvals for ICB use across several tumor types [1, 2, 3, 4, 5, 6, 7, 8, 9]. The capacity of tumor cells to cultivate immune escape mechanisms is one of the leading processes involved in tumor appearance and growth ; ICB is known to target one of these escape mechanisms. However, to date, the therapeutic efficacy of ICB remains insufficient, necessitating the establishment of more effective therapies, including combination therapies [1, 2, 3, 4, 5, 6, 7, 8, 9].
Ramucirumab (RAM), a fully humanized IgG1 monoclonal anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody, has been shown to exhibit significant efficacy as an anti-angiogenic agent in several cancers, including non-small cell lung cancer (NSCLC), gastric cancer (GC), colorectal cancer, and bladder cancer [11, 12, 13, 14, 15]. Further, several studies have reported that activation of the VEGF–VEGFR2 axis correlates with an immunosuppressive microenvironment. VEGF-VEGFR2 activation can induce the accumulation of immature dendritic cells, myeloid-derived suppressor cells, and regulatory T cells (Treg cells) and can inhibit the migration of T lymphocytes; these effects can be reversed by anti-angiogenic reagents [16, 17, 18, 19, 20]. Hence, at present, several clinical trials are underway to examine the effects of combined immunotherapy and anti-angiogenic treatments in various cancer types [18, 21]. However, the impact of anti-angiogenic reagents on the tumor immune microenvironment in human clinical samples remains unclear. Thus, this study aimed at using immunohistochemistry (IHC) and flow cytometry to investigate pre- and post-treatment tumor tissues obtained from patients with advanced GC receiving RAM-containing therapies.
Patients and samples
We reviewed medical records and collected patient data regarding clinicopathological features and treatment history. The cutoff date for analysis was August 31, 2017. All responses were assessed according to the RECIST v1.1 . In addition, progression-free survival (PFS) was assessed as the time from treatment initiation to clinical or radiographic progression or death from any cause. Overall survival (OS) was defined as the period from treatment initiation to the time of death from any cause.
DNA and RNA were extracted from archived FFPE tumor samples and were subjected to the Oncomine™ Comprehensive Assay version 3 (Thermo Fisher Scientific, Waltham, MA) which allowed to detect gene mutations, copy number variants and fusions across multiple genes (Additional file 1: Table S1). The detected genomic variant data were classified according to whether genetic drivers of cancer including gain- and loss-of-function mutations or single nucleotide variants based on the Oncomine Knowledgebase.
Evaluation of mismatch repair (MMR) status
MMR status was examined with IHC with anti-mutLhomolog 1 (MLH1) mAb, anti-mutS homolog 2 (MSH2) mAb, anti-postmeiotic segregation increased 2 (PMS2) mAb, and anti-mutShomolog 6 (MSH6) mAb using archived FFPE tumor samples. Tumors were considered negative for MLH1, MSH2, PMS2, or MSH6 expression only if there was a complete absence of nuclear staining in the tumor cells, and normal epithelial cells and lymphocytes were employed as an internal control. Tumors lacking MLH1, MSH2, PMS2, or MSH6 expression were defined as MMR deficient, whereas tumors that maintained expression of all markers were considered MMR proficient.
Evaluation of Epstein-Barr virus (EBV) infection
Chromogenic in situ hybridization (ISH) for EBV-encoded RNA was performed with fluorescein-labeled oligonucleotide probes (INFORM EBER probe) with enzymatic digestion (ISH protease 3, Ventana, Tucson, AZ) and an iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system. Archived FFPE tumor samples were used for the analysis.
Immunohistochemistry for immune profiles
Biopsy specimens were formalin-fixed, paraffin-embedded, and sectioned onto slides for IHC, which was conducted using monoclonal antibodies against PD-L1 and CD8 using an automatic staining instrument (BenchMark ULTRA; Ventana). PD-L1 positivity was defined as staining in ≥1% in tumor cells or immune cells. CD8 staining intensity was assessed as follows: no staining (0), weak staining (1+), moderate staining (2+), and strong staining (3+). Two researchers (D. Kotani and T. Kuwata) independently evaluated the stained slides. Multiplexed fluorescent immunohistochemistry was performed by the TSA method using an Opal IHC kit (PerkinElmer, Waltham, MA) according to the manufacturer’s instructions. Multiplexed fluorescent labeled images of three randomly selected fields (669 × 500 micrometer) were captured with an automated imaging system (Vectra ver3.0, PerkinElmer).
Flow cytometry analysis
We washed cells in PBS with 2% fetal calf serum and then stained with mAbs specific for CD3, CD4, CD8, CD45RA, PD-1, LAG-3, ICOS, VEGFR2, or CTLA-4 as well as a fixable viability dye. In addition, intracellular Ki67 and FOXP3 staining was performed with an anti-FOXP3 mAb and FOXP3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. After washing, we analyzed all cells with the LSRFortessa instrument (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR). Antibody dilutions were performed per the manufacturer’s instructions.
Antibodies and reagents
Alexa Fluor 700–conjugated anti-CD3 (UCTH1) mAb, violet 500-conjugated anti-CD4 (RPA-T4) mAb, and Brilliant Violet 421 (BV421)–conjugated anti-PD-1 (EH12) mAb were obtained from BD Biosciences (Franklin Lakes, NJ). BV785-conjugated anti-CD8 (RPA-T8) mAb, BV711-conjugated anti-CD45RA (HI100) mAb, and APC-conjugated anti-CTLA-4 (L3D10) mAb were purchased from BioLegend (San Diego, CA). Phycoerythrin (PE)-conjugated anti-FOXP3 (Forkhead Box P3, 236A/E7) mAb, PE- and Cy7-conjugated anti-ICOS (3D12) mAbs, peridinin–chlorophyll–protein complex (PerCP)- and Cy5.5-conjugated anti-Ki67 (20Raj1) mAb, and eFluor 780-conjugated fixable viability dye were obtained from eBioscience (Santa Clara, CA). Fluorescein isothiocyanate (FITC)–conjugated anti-LAG-3 (17B4) mAb was obtained from ENZO Life Science (Farmingdale, NY). Anti-CD4 (4B12) mAb, anti-Ki67 (MIB-1) mAb, anti-MLH1 (ES05) mAb, anti-MSH2 (FE11) mAb, anti-PMS2 (EP51) mAb, and anti-MSH6 (EP49) mAb for IHC were obtained from Dako (Copenhagen, Denmark). Anti-PD-L1 (SP263) mAb and anti-CD8 (SP57) mAb for IHC were obtained from Ventana. Anti-CD3 (SP7) mAb and anti-FOXP3 (236A/E7) mAb for IHC was purchased from Abcam; RAM was obtained from Eli Lilly and Company (Indianapolis, IN); and VEGFA was obtained from Wako (Osaka, Japan). In addition, APC was conjugated to RAM to detect VEGFR2.
We used Welch’s or paired t-tests to analyze continuous variables. All survival curves were estimated using the Kaplan–Meier method and compared using the log-rank test. All statistical analyses were two-tailed and were performed using Prism version 7 software (GraphPad Software, Inc., La Jolla, CA). P < 0.05 was considered statistically significant.
A total of 20 patients (age, 46–79 years; median, 67 years; men, 13; women, 7) with advanced GC and with available pre- and post-treatment samples that were scheduled to receive RAM-containing chemotherapy were enrolled in this study (Fig. 1). All clinical characteristics are summarized in Additional file 2: Table S2 and Additional file 3: Table S3. In all, 11 patients had an intestinal subtype, and 9 patients had a diffuse histology; all exhibited a good performance status (PS, 0 or 1). A majority of patients (14/20) received RAM-containing therapy as a second-line therapy. Individual treatment regimens also comprised paclitaxel (10/20), nab-paclitaxel (2/20), irinotecan (5/20), or RAM monotherapy (3/20). A total of 6 patients (30%) attained a partial response (PR), 11 (55%) had stable disease (SD), and 3 (15%) had progressive disease (PD). The median PFS and OS were 110 and 324 days, respectively. These clinical data were consistent with those of a previous study . Typically, patients were administered 8 mg/kg RAM intravenously on days 1 and 15 of a 28-day cycle, and imaging was performed every 6–8 weeks to assess antitumor response. Pre-treatment biopsies were conducted within 2 weeks of initiation of RAM-containing therapies. Post-treatment biopsies were performed along with each imaging assessment if possible. The median number of times the biopsy was performed in each patient was 3 (range, 2–6).
Genomic alterations, MMR status, and EBV infection
A recent study has shown that ICB exhibits efficacy against patients with MMR deficient or EBV-positive GC from comprehensive genomic analyses . We then analyzed genomic alterations, MMR status and EBV infection and all data are summarized in Additional file 4: Figure S1 and Additional file 3: Table S3 although 3 patients could not be assessed for NGS due to insufficient samples. TP53 were frequently mutated (10/17) and ERBB2, MET, FGFR2, or KRAS, amplification, or a RHOA mutation were also identified, which was in line with a previous study . In contrast, all were MMR proficient GC and only one was EBV-positive GC (Additional file 2: Table S2 and Additional file 3: Table S3).
PD-L1 expression and CD8+ T-cell infiltration after RAM-containing therapies
Accumulation of effector Treg cells in TILs
Reduction of eTreg cells in TILs after RAM-containing therapies
Reduction of PD-1 expression by CD8+ T cells in TILs after RAM-containing therapies
Prognostic significance of eTreg frequency in patients receiving RAM-containing therapies
We then assessed the prognostic significance of eTreg cells and PD-1 expression by CD8+ T cells in pre-treatment TILs. Patients with PR had a significantly higher frequency of eTreg cells in TILs than those with PD (32.56% ± 12.11% vs. 14.83% ± 3.18%; P = 0.036; Fig. 7c). There was no significant difference in % of eTreg-cell reduction among patients with PR, SD, and PD (15.41% ± 12.31% vs. 1.21% ± 10.7% vs. 8.37% ± 1.61%; P = 0.063 and 0.27, respectively; Additional file 12: Figure S9). Further, patients with a high frequency of eTreg cells in pre-treatment TILs had a significantly longer PFS than those with a low frequency of eTreg cells (161 days vs. 76 days; P = 0.032; Fig. 7c). Conversely, no significant difference was observed in the OS of these two groups (324 days vs. 350 days; P = 0.71). Although not significant, patients with PR tended to have a lower PD-1 expression by CD8+ T cells in pre-treatment TILs than those with PD (46.0% ± 22.65% vs. 72.5% ± 8.97%; P = 0.067; Fig. 7d), but there was no significant difference in % of PD-1 reduction by CD8+ T cells among patients with PR, SD, and PD (17.43% ± 11.23% vs. 11.66% ± 18.28% vs. 27.37% ± 7.96; P = 0.46 and 0.20, respectively; Additional file 12: Figure S9), and no significant difference was observed in the PFS between patients with a high and low pre-treatment PD-1 expression by CD8+ T cells in TILs (118.5 days vs. 110 days; P = 0.72; Fig. 7d). These results suggest that patients with a high frequency of eTreg cells in TILs, which can be decreased by RAM, exhibit a favorable response to RAM-containing therapies.
Impact of anti-VEGFR2 blockade on eTreg cells in vitro
Our clinical data revealed that RAM significantly reduces Treg cells in the tumor microenvironment. To gain insight into the cellular mechanism of eTreg-cell reduction, we assessed VEGFR2 expression in various types of T cells. VEGFR2 was highly expressed by eTreg cells compared with other fractions, as previously reported (Additional file 13: Figure S10) . In addition, VEGFR2+ eTreg cells had higher Ki67 expression compared with VEGFR2− eTreg cells (Additional file 13: Figure S10), suggesting a significant proliferative capacity in VEGFR2+ eTreg cells. When PBMCs were treated with VEGFA, a VEGFR2 ligand, eTreg cells (and in particular VEFR2+ eTreg cells) proliferated (Additional file 13: Figure S10). Further, the eTreg cells that were increased by VEGFA could be decreased by RAM treatment (Additional file 13: Figure S10). In contrast to eTreg cells, VEGFR2+CD8+ T cells exhibited slightly higher PD-1 expression than VEGFR2−CD8+ T cells, and PD-1 expression by CD8+ T cells did not increase after VEGFA treatment (Additional file 13: Figure S10). We propose that VEGFR2 is highly expressed by eTreg cells and that VEGFA stimulates VEGFR2+ eTreg cell proliferation, which can be overcome by RAM.
Recent studies have demonstrated that cancer immunotherapies, particularly those that modulate immune co-inhibitory signals, can be beneficial for patients with various types of cancers [1, 2, 3, 4, 5, 6, 7]. However, limited clinical efficacies necessitate the development of better therapies, including the identification of biomarker(s) and the formulation of combinatorial therapies with improved clinical efficacies. Tumor PD-L1 expression correlates with favorable clinical responses to anti-PD-1/PD-L1 antibodies [6, 32]; however, CD8+ T-cell infiltration is also critical for tumor immune responses [33, 34]. Intratumoral PD-L1 expression can be induced either by constitutive oncogenic signaling pathways or chromosomal alterations (innate resistance) or through IFN-γ signaling stimulated by host defense mechanisms, such as CD8+ T cells in tumors (acquired resistance). This indicates a probable correlation between CD8+ T cells and PD-L1 expression. VEGF is related to the immunosuppressive microenvironment in that it inhibits T-cell proliferation and infiltration; this inhibition can be reversed by anti-angiogenic reagents, including RAM [35, 36]. Both CD8+ T-cell infiltration and PD-L1 expression were induced by RAM-containing therapies. In addition, TIL eTreg cells and PD-1 expression by CD8+ T cells are decreased by RAM-containing therapies. To the best of our knowledge, this is the first study to demonstrate that an anti-angiogenic reagent can cause an increase in CD8+ T-cell infiltration and PD-L1 expression and a decrease in eTreg cells and PD-1 expression by CD8+ T cells in the tumor microenvironment in humans. PD-1 expression by TIL CD8+ T cells was decreased by RAM-containing therapies, which is in line with findings in an animal model demonstrating the effects of VEGFA/VEGFR2 on the expression of IC molecules . Furthermore, this study also demonstrated that the pre-treatment TIL eTreg frequency could be a biomarker for stratifying clinical responders to RAM-containing therapies. Corroborating our results, a previous pre-clinical animal study reported the efficacy of combining anti-PD-1/PD-L1 blocking therapies and anti-angiogenic reagents . Thus, this strategy is a promising combination, and several clinical trials have been pursued with favorable outcomes .
While systemic chemotherapy is the primary treatment option for patients with unresectable advanced GC, which is the fifth leading cancer worldwide , their prognosis remains poor, with a median survival time of approximately 1 year in those receiving conventional therapy [38, 39, 40]. The combination of trastuzumab, an antibody targeting human epidermal growth factor receptor type 2 (HER2), and chemotherapy has reportedly increased survival in patients with HER2-positive gastric or gastro-esophageal junction cancer , although HER2-positive tumors only account for 7–17% of GCs [41, 42, 43]. Based on the findings of two pivotal global phase III trials, REGARD and RAINBOW, RAM has become one of the standard chemotherapies for GC, though the absolute improvements provided by this therapy remain limited [12, 13]. A recent phase III trial demonstrated a survival benefit in advanced GC patients receiving nivolumab after two or more previous lines of chemotherapy. This resulted in the approval of nivolumab for the treatment of GC in Japan , although its therapeutic efficacy remains unsatisfactory. Another study has shown that ICB exhibits efficacy against patients with MMR deficient or EBV-positive GC from comprehensive genomic analyses . In our cohort, however, only one patient had EBV-positive GC and no one had MMR deficient GC. Thus, the relationship between these factors and immune status warrants to study with a large patient cohort. In contrast, our study suggests that a combination strategy using anti-angiogenic reagents and ICB may be a promising therapeutic approach for GC. Furthermore, several studies have already demonstrated the efficacy of both anti-angiogenic reagents and ICB in several other types of cancer, including NSCLC, renal cell carcinoma, and bladder cancer [5, 6, 8, 9, 14, 15]. Moreover, similar combination strategies have already been tested in clinical trials targeting these cancer types [18, 21].
We employed flow cytometry to investigate the detailed immune profiles of hosts, where significant differences exist between PBMCs and TILs. The RAM-containing therapy-induced reduction in TIL eTreg cells and PD-1 expression by CD8+ T cells suggests the importance of analyzing TILs, in which T cells directly attack tumor cells. The decrease of eTreg cells and PD-1 expression by CD8+ T cells in TILs in some PR patients may be a reflection of reduced tumor burdens, whereas this tendency was also observed at PD state, suggesting that RAM-containing therapies can reduce eTreg cells and PD-1 expression by CD8+ T cells in TILs independently of clinical responses. eTreg cells are a highly immunosuppressive subset of CD4+ T cells characterized by high expression of the master regulatory transcription factor FOXP3 [44, 45, 46]. Our study demonstrates that a high eTreg-cell frequency in TILs correlates with a better response to RAM-containing therapies. Thus, TIL eTreg cell frequency could be a biomarker for stratifying clinical responders. Considering the role of Treg cells in promoting tumor progression by suppressing antitumor immunity, the RAM-induced reduction in Treg cells could be more important for clinical responses to RAM-containing therapies in patients with a high eTreg frequency (in whom tumor progression should be highly dependent on eTreg cells) than in patients with a low eTreg frequency. Indeed, patients with PR, although not significant, tended to have higher eTreg-cell reduction.
Myeloid cells including myeloid-derived suppressor cells in tumors has been implicated in suppression of antitumor immunity , and VEGF/VEGF receptor blockade has been shown to reduce myeloid-derived suppressor cells in tumors and blood in preclinical tumor models and human cancers . A recent trial has shown the improved clinical outcome associated with atezolizumab (anti-PD-L1 antibody) and bevacizumab (anti-VEGFA antibody) compared with atezolizumab monotherapy in the immune suppressed myeloid cell high subgroup, suggesting that the addition of bevacizumab to atezolizumab may overcome myeloid-cell mediated resistance . In our present study, however, these cells could not be analyzed due to the limited sample volume, these findings should be tested in the future studies.
A previous study reported that Treg cells express high levels of VEGFR2 and that RAM therapy could reduce the frequency of VEGFR2+ Treg cells, thus corroborating the results of our study . Another study reported that peripheral Treg-cell numbers were diminished by bevacizumab (anti-VEGFA antibody)-containing therapies in patients with colorectal cancer , although TILs were only assessed by IHC. In addition to IHC, we employed flow cytometry for TIL analyses. Flow cytometry has several advantages to explore the detailed molecular expression in TILs, although investigating the location of TILs depends on IHC. By combining flow cytometry and IHC, we were able to address the entire landscape of immunological status in tumor microenvironment even with tiny biopsy samples. We investigated both PBMCs and TILs and determined that the reduction in eTreg cells was more profound in TILs than in PBMCs. The inconsistent result with the previous study  could possibly be attributed to our definition of a Treg cell; while CD25+FOXP3+CD4+ T cells were defined as “Treg cells” in the previous study, we defined these more precisely as “eTreg cells” (CD45RA−FOXP3highCD4+ T cell). These eTreg cells have a suppressive function, while non-Treg CD45RA−FOXP3lowCD4+ cells, which produce inflammatory cytokines such as IFN-γ are included in the CD25+FOXP3+CD4+ T cell population. Thus, our reported “eTreg cell” frequency in PBMCs was lower than the previously reported “Treg cell” frequency in PBMCs.
We observed a significant difference in immune profiles between PBMCs and TILs and determined that PD-L1 expression and CD8+ T-cell infiltration were increased following RAM-containing therapy in patients with advanced GC. Based on more detailed immune profiles, both TIL eTreg cells and PD-1 expression by CD8+ T cells were decreased by RAM-containing therapies, an effect not observed in PBMCs. Further, high eTreg frequencies in TILs correlate with favorable responses to RAM-containing therapies. Thus, our study is the first to utilize human clinical samples to highlight the significance of TIL analyses and to propose RAM as an immuno-modulator in combination with ICB.
We thank Ms. Tomoka Takaku, Miyuki Nakai, Konomi Onagawa, Megumi Takemura, Chie Haijima, Megumi Hoshino, Kumiko Yoshida, and Miho Ozawa for their technical assistance.
This study was supported by Grants-in-Aid for Scientific Research [S grant no. 17H06162 (HN), Challenging Exploratory Research grant no. 16 K15551 (HN), Young Scientists no. 17 J09900 (Y. Togashi), and JSPS Research fellow no. 17 K18388 (Y. Togashi)] from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Project for Cancer Research by Therapeutic Evolution (P-CREATE) from Japan Agency for Medical Research and Development (AMED) (no. 16cm0106301h0002, HN), by the National Cancer Center Research and Development Fund (no. 28-A-7, HN), by the Naito Foundation (HN and Y. Togashi), by the Takeda Foundation (Y. Togashi), and by the SGH Foundation (Y. Togashi). This study was executed in part as a research program supported by Eli Lilly Japan KK.
Availability of data and materials
All data generated or analyzed in this study that are relevant to the results presented in this article are included in this article and its supplementary information files (Additional file). Other data that were not relevant to the results presented here are available from the corresponding author upon reasonable request.
Conception and design: YT, HN, and KS. Development of methodology: YT, YT, and DK. Acquisition of data: YT, YT, DK, TK, ES, AK, and KS. Analysis and interpretation of data: YT, YT, DK, ES, AK, HN and KS. Writing, review, and/or revision of the manuscript: YT, YT, DK, TK, ES, AK, TD, HW, HN, and KS. Study supervision: YT, TD, HW, HN, and KS. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Institutional Review Board of the National Cancer Center (2015–048 and 2016–029) and was conducted in accordance with international ethics guidelines, including the Declaration of Helsinki. We obtained written informed consent from all participants before sampling.
Consent for publication
No individual data were used in this study.
Y. Togashi received honoraria from Eli Lilly Japan KK. KS received honoraria and grants from Eli Lilly Japan KK. No other disclosures are reported.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 1.Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366(26):2443–54. https://doi.org/10.1056/NEJMoa1200690 PubMed PMID: 22658127; PubMed Central PMCID: PMCPMC3544539.CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366(26):2455–2465. Epub 2012/06/02. doi: https://doi.org/10.1056/NEJMoa1200694. PubMed PMID: 22658128; PubMed Central PMCID: PMCPMC3563263.CrossRefGoogle Scholar
- 3.Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363(8):711–723. Epub 2010/06/05. doi: https://doi.org/10.1056/NEJMoa1003466. PubMed PMID: 20525992; PubMed Central PMCID: PMCPMC3549297.CrossRefGoogle Scholar
- 4.Herbst RS, Baas P, Kim DW, Felip E, Pérez-Gracia JL, Han JY, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387(10027):1540–50. https://doi.org/10.1016/S0140-6736(15)01281-7 PubMed PMID: 26712084.CrossRefGoogle Scholar
- 5.Brahmer J, Reckamp KL, Baas P, Crinò L, Eberhardt WE, Poddubskaya E, et al. Nivolumab versus Docetaxel in advanced squamous-cell non-small-cell lung Cancer. N Engl J Med 2015;373(2):123–135. Epub 2015/05/31. doi: https://doi.org/10.1056/NEJMoa1504627. PubMed PMID: 26028407; PubMed Central PMCID: PMCPMC4681400.CrossRefGoogle Scholar
- 7.Kang YK, Boku N, Satoh T, Ryu MH, Chao Y, Kato K, et al. Nivolumab in patients with advanced gastric or gastro-oesophageal junction cancer refractory to, or intolerant of, at least two previous chemotherapy regimens (ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017. Epub 2017/10/05. doi: https://doi.org/10.1016/S0140-6736(17)31827-5. PubMed PMID: 28993052.CrossRefGoogle Scholar
- 8.Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, et al. Nivolumab versus Everolimus in advanced renal-cell carcinoma. N Engl J Med 2015;373(19):1803–1813. Epub 2015/09/25. doi: https://doi.org/10.1056/NEJMoa1510665. PubMed PMID: 26406148; PubMed Central PMCID: PMCPMC5719487.CrossRefGoogle Scholar
- 9.Bellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee JL, Fong L, et al. Pembrolizumab as second-line therapy for advanced Urothelial carcinoma. N Engl J Med 2017;376(11):1015–1026. Epub 2017/02/17. doi: https://doi.org/10.1056/NEJMoa1613683. PubMed PMID: 28212060; PubMed Central PMCID: PMCPMC5635424.CrossRefGoogle Scholar
- 12.Fuchs CS, Tomasek J, Yong CJ, Dumitru F, Passalacqua R, Goswami C, et al. Ramucirumab monotherapy for previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial. Lancet 2014;383(9911):31–39. Epub 2013/10/03. doi: https://doi.org/10.1016/S0140-6736(13)61719-5. PubMed PMID: 24094768.CrossRefGoogle Scholar
- 13.Wilke H, Muro K, Van Cutsem E, Oh SC, Bodoky G, Shimada Y, et al. Ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (RAINBOW): a double-blind, randomised phase 3 trial. Lancet Oncol 2014;15(11):1224–1235. Epub 2014/09/17. doi: https://doi.org/10.1016/S1470-2045(14)70420-6. PubMed PMID: 25240821.CrossRefGoogle Scholar
- 14.Garon EB, Ciuleanu TE, Arrieta O, Prabhash K, Syrigos KN, Goksel T, et al. Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL): a multicentre, double-blind, randomised phase 3 trial. Lancet 2014;384(9944):665–673. Epub 2014/06/02. doi: https://doi.org/10.1016/S0140-6736(14)60845-X. PubMed PMID: 24933332.CrossRefGoogle Scholar
- 15.Petrylak DP, de Wit R, Chi KN, Drakaki A, Sternberg CN, Nishiyama H, et al. Ramucirumab plus docetaxel versus placebo plus docetaxel in patients with locally advanced or metastatic urothelial carcinoma after platinum-based therapy (RANGE): a randomised, double-blind, phase 3 trial. Lancet 2017. Epub 2017/09/12. doi: https://doi.org/10.1016/S0140-6736(17)32365-6. PubMed PMID: 28916371.CrossRefGoogle Scholar
- 16.Terme M, Pernot S, Marcheteau E, Sandoval F, Benhamouda N, Colussi O, et al. VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res. 2013;73(2):539–549. Epub 2012/10/29. doi: https://doi.org/10.1158/0008-5472.CAN-12-2325. PubMed PMID: 23108136.CrossRefGoogle Scholar
- 17.Voron T, Colussi O, Marcheteau E, Pernot S, Nizard M, Pointet AL, et al. VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors. J Exp Med 2015;212(2):139–148. Epub 2015/01/19. doi: https://doi.org/10.1084/jem.20140559. PubMed PMID: 25601652; PubMed Central PMCID: PMCPMC4322048.CrossRefGoogle Scholar
- 18.Voron T, Marcheteau E, Pernot S, Colussi O, Tartour E, Taieb J, et al. Control of the immune response by pro-angiogenic factors. Front Oncol 2014;4:70. Epub 2014/04/02. doi: https://doi.org/10.3389/fonc.2014.00070. PubMed PMID: 24765614; PubMed Central PMCID: PMCPMC3980099.
- 19.Yuan J, Zhou J, Dong Z, Tandon S, Kuk D, Panageas KS, et al. Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab. Cancer Immunol Res. 2014;2(2):127–32. https://doi.org/10.1158/2326-6066.CIR-13-0163 PubMed PMID: 24778276; PubMed Central PMCID: PMCPMC3991109.CrossRefPubMedGoogle Scholar
- 20.Wallin JJ, Bendell JC, Funke R, Sznol M, Korski K, Jones S, et al. Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma. Nat Commun 2016;7:12624. Epub 2016/08/30. doi: https://doi.org/10.1038/ncomms12624. PubMed PMID: 27571927; PubMed Central PMCID: PMCPMC5013615.CrossRefGoogle Scholar
- 21.Atkins MB, Plimack ER, Puzanov I, Fishman MN, McDermott DF, Cho DC, et al. Axitinib in combination with pembrolizumab in patients with advanced renal cell cancer: a non-randomised, open-label, dose-finding, and dose-expansion phase 1b trial. Lancet Oncol 2018. Epub 2018/02/09. doi: https://doi.org/10.1016/S1470-2045(18)30081-0. PubMed PMID: 29439857.CrossRefGoogle Scholar
- 23.Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–247. Epub 2008/12/23. doi: https://doi.org/10.1016/j.ejca.2008.10.026. PubMed PMID: 19097774.CrossRefGoogle Scholar
- 24.Kim ST, Cristescu R, Bass AJ, Kim KM, Odegaard JI, Kim K, et al. Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer. Nat Med 2018. Epub 2018/07/16. doi: https://doi.org/10.1038/s41591-018-0101-z. PubMed PMID: 30013197.CrossRefGoogle Scholar
- 26.Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, et al. Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med. 2013;5(200):200ra116. https://doi.org/10.1126/scitranslmed.3006504 PubMed PMID: 23986400; PubMed Central PMCID: PMCPMC4136707.CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood 2007;110(8):2983–2990. Epub 2007/07/20. doi: https://doi.org/10.1182/blood-2007-06-094656. PubMed PMID: 17644734; PubMed Central PMCID: PMCPMC2018674.CrossRefGoogle Scholar
- 28.Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 2009;30(6):899–911. Epub 2009/05/21. doi: https://doi.org/10.1016/j.immuni.2009.03.019. PubMed PMID: 19464196.CrossRefGoogle Scholar
- 30.Nagase H, Takeoka T, Urakawa S, Morimoto-Okazawa A, Kawashima A, Iwahori K, et al. ICOS(+) Foxp3(+) TILs in gastric cancer are prognostic markers and effector regulatory T cells associated with Helicobacter pylori. Int J Cancer. 2017;140(3):686–695. Epub 2016/10/27. doi: https://doi.org/10.1002/ijc.30475. PubMed PMID: 27756099.CrossRefGoogle Scholar
- 33.Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568–71. https://doi.org/10.1038/nature13954 PubMed PMID: 25428505; PubMed Central PMCID: PMCPMC4246418.CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515(7528):563–7. https://doi.org/10.1038/nature14011 PubMed PMID: 25428504; PubMed Central PMCID: PMCPMC4836193.CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Bouzin C, Brouet A, De Vriese J, Dewever J, Feron O. Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy. J Immunol. 2007;178(3):1505–11 PubMed PMID: 17237399.CrossRefGoogle Scholar
- 38.Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 2010;376(9742):687–97. https://doi.org/10.1016/S0140-6736(10)61121-X PubMed PMID: 20728210.CrossRefPubMedPubMedCentralGoogle Scholar
- 39.Ohtsu A, Shah MA, Van Cutsem E, Rha SY, Sawaki A, Park SR, et al. Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a randomized, double-blind, placebo-controlled phase III study. J Clin Oncol. 2011;29(30):3968–76. https://doi.org/10.1200/JCO.2011.36.2236 PubMed PMID: 21844504.CrossRefPubMedGoogle Scholar
- 43.Tanner M, Hollmén M, Junttila TT, Kapanen AI, Tommola S, Soini Y, et al. Amplification of HER-2 in gastric carcinoma: association with topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Ann Oncol. 2005;16(2):273–8. https://doi.org/10.1093/annonc/mdi064 PubMed PMID: 15668283.CrossRefPubMedGoogle Scholar
- 44.Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155(3):1151–64 PubMed PMID: 7636184.PubMedGoogle Scholar
- 48.McDermott DF, Huseni MA, Atkins MB, Motzer RJ, Rini BI, Escudier B, et al. Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma. Nat Med 2018;24(6):749–757. Epub 2018/06/04. doi: https://doi.org/10.1038/s41591-018-0053-3. PubMed PMID: 29867230.CrossRefGoogle Scholar
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