Randomized, Phase II study of pemetrexed plus bevacizumab versus pemetrexed alone after treatment with cisplatin, pemetrexed, and bevacizumab in advanced non-squamous, non-small cell lung cancer: TORG (thoracic oncology research group) 1321.

INTRODUCTION: Cisplatin plus pemetrexed followed by pemetrexed is an efficacious platinum combination regimen for advanced non-squamous, non-small cell lung cancer (NSCLC). Data regarding the addition of bevacizumab, especially in maintenance treatment, are insufficient. METHODS: Eligibility criteria included: no prior chemotherapy; advanced, non-squamous, NSCLC; performance status ≤1; and epidermal growth factor receptor mutation-negative. Patients (N = 108) received induction chemotherapy with cisplatin, pemetrexed, and bevacizumab every 3 weeks for four cycles, and tumor response was needed to confirm four-week response duration. Patients with at least stable disease were randomized to pemetrexed/bevacizumab or pemetrexed alone. The primary endpoint was progression-free survival (PFS) after induction chemotherapy. Myeloid-derived suppressor cell (MDSC) counts of peripheral blood samples were also analyzed. RESULTS: Thirty-five patients each were randomized to the pemetrexed/bevacizumab group and the pemetrexed alone group. PFS was significantly better in the pemetrexed/bevacizumab group than in the pemetrexed alone group (7.0 vs. 5.4 months, hazard ratio: 0.56 [0.34-0.93], log-rank p = 0.023). In patients with partial response to induction therapy, median overall survival was 23.3 months in the pemetrexed alone group and 29.6 months in the pemetrexed/bevacizumab group (log-rank p = 0.077). Pretreatment monocytic (M)-MDSC counts tended to be greater in the pemetrexed/bevacizumab group with poor PFS than in those with good PFS (p = 0.0724). CONCLUSIONS: Addition of bevacizumab to pemetrexed as maintenance therapy prolonged PFS in patients with untreated, advanced, non-squamous NSCLC. Furthermore, an early response to induction therapy and pretreatment M-MDSC counts may be related to the survival benefit of the addition of bevacizumab to the combination of cisplatin and pemetrexed.

Ruxolitinib for hematopoietic cell transplantation-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe complication after allogeneic hematopoietic cell transplantation (HCT) and can cause graft failure or multi-organ failure. Here, we report two children with refractory HCT-associated HLH treated with ruxolitinib. In the first patient, ruxolitinib resolved fever, cytopenia and hyperferritinemia. In another patient, although severe hepatic failure, which developed and worsened before the administration of ruxolitinib, was irreversible, rapid improvement in fever, leukopenia and hyperferritinemia was observed. Of note, multiplex cytokine profiling showed amelioration of cytokine storm in both patients. Ruxolitinib may be an encouraging option for HCT-associated HLH.

Identification of Novel HLA Class II-Restricted Neoantigens Derived from Driver Mutations.

Neoantigens derived from tumor-specific genetic mutations might be suitable targets for cancer immunotherapy because of their high immunogenicity. In the current study, we evaluated the immunogenicity of 10 driver mutations that are frequently expressed in various cancers using peripheral blood mononuclear cells from healthy donors (n = 25). Of the 10 synthetic peptides (27-mer) derived from these mutations, the six peptides from KRAS-G12D, KRAS-G12R, KRAS-G13D, NRAS-Q61R, PIK3CA-H1047R, and C-Kit-D816V induced T cell responses, suggesting that frequent driver mutations are not always less immunogenic. In particular, immune responses to PIK3CA-H1047R, C-Kit-D816V, KRAS-G13D, and NRAS-Q61R were observed in more than 10% of the donors. All six peptides induced human leukocyte antigen (HLA) class II-restricted CD4⁺ T cell responses; notably, PIK3CA-H1047R contained at least two different CD4⁺ T cell epitopes restricted to different HLA class II alleles. In addition, PIK3CA-H1047R and C-Kit-D816V induced antigen-specific CD8⁺ T cells as well, indicating that they might contain both HLA class I- and class II-restricted epitopes. Since the identified neoantigens might be shared by patients with various types of cancers and are not easily lost due to immune escape, they have the potential to be promising off-the-shelf cancer immunotherapy targets in patients with the corresponding mutations.

Association between soluble immune mediators and tumor responses in patients with nonsmall cell lung cancer treated with anti-PD-1 inhibitor.

Although programmed death (PD)-1 immune checkpoint therapies target the immune system, the relationship between inflammatory factors and the clinical outcome of anti-PD-1 therapy for nonsmall cell lung cancer (NSCLC) is not fully understood. Here we examined the association between soluble immune mediators and the outcome of treatment with PD-1 inhibitors in patients with advanced/recurrent NSCLC. In two independent cohorts, we assessed the levels of 88 different soluble immune mediators in peripheral blood before and after anti-PD-1 treatment, and evaluated their associations with clinical outcomes. In the training cohort, the plasma levels of chitinase 3-like-1 and GM-CSF before treatment (p = 0.006 and p = 0.005, respectively) and changes in the plasma levels of CXCL2, VEGF, IFNα2, and MMP2 after treatment (p < 0.001, p = 0.019, p = 0.019, and p = 0.012, respectively) were significantly correlated with PFS. The change in the plasma CXCL2 level was also significantly associated with treatment-related AEs (p = 0.017). In the validation cohort, however, only the changes in the plasma levels of CXCL2 and MMP2 after treatment were associated with PFS (p = 0.003 and p = 0.006, respectively), and these changes were maintained during the course of anti-PD-1 therapy in patients who showed better clinical outcomes, even in those with tumor pseudoprogression. Since CXCL2 and MMP2 can be easily measured by minimally invasive blood sampling, they could be useful for monitoring of clinical outcomes in NSCLC patients receiving PD-1 inhibitor therapy.

[Translational Research in Immuno-Oncology - Comprehensive Analysis of Phenotypes and Functions of Various Immune Cells Using the Colorectal Cancer Surgical Sample].

Colorectal cancer(CRC)is one of the increasing cancers, and development of novel treatments is imperative for advanced CRC. In recent years, immune checkpoint inhibitors have demonstrated impressive clinical efficacy in various types of cancers, but only limited clinical responses were reported in CRC. It has been reported that such poor therapeutic effects might be possibly due to immune suppressive mechanisms other than immune checkpoints in the CRC. Therefore, comprehensive grasp of the immune environment is considered to be of significance for CRC patients. Our study aims to comprehensively assess the phenotypes and functions of various immune cells isolated from various tissues, including primary tumors and related lymph nodes, in primary CRC patients. Our preliminary results by flow cytometry analysis suggest that the immune suppression, particularly in regional lymph nodes, may be related to tumor progression. Further studies remain to be performed to more thoroughly elucidate immune suppressive mechanisms, and thereby to develop a novel immunotherapeutic strategy in the CRC.

Interleukin-6/STAT3 signaling as a promising target to improve the efficacy of cancer immunotherapy.

Overcoming the immunosuppressive state in tumor microenvironments is a critical issue for improving the efficacy of cancer immunotherapy. Interleukin (IL)-6, a pleiotropic cytokine, is highly produced in the tumor-bearing host. Previous studies have indicated that IL-6 suppresses the antigen presentation ability of dendritic cells (DC) through activation of signal transducer and activator of transcription 3 (STAT3). Thus, we focused on the precise effect of the IL-6/STAT3 signaling cascade on human DC and the subsequent induction of antitumor T cell immune responses. Tumor-infiltrating CD11b+ CD11c+ cells isolated from colorectal cancer tissues showed strong induction of the IL-6 gene, downregulated surface expression of human leukocyte antigen (HLA)-DR, and an attenuated T cell-stimulating ability compared with those from peripheral blood mononuclear cells, suggesting that the tumor microenvironment suppresses antitumor effector cells. In vitro experiments revealed that IL-6-mediated STAT3 activation reduced surface expression of HLA-DR on CD14+ monocyte-derived DC. Moreover, we confirmed that cyclooxygenase 2, lysosome protease and arginase activities were involved in the IL-6-mediated downregulation of the surface expression levels of HLA class II on human DC. These findings suggest that IL-6-mediated STAT3 activation in the tumor microenvironment inhibits functional maturation of DC to activate effector T cells, blocking introduction of antitumor immunity in cancers. Therefore, we propose in this review that blockade of the IL-6/STAT3 signaling pathway and target molecules in DC may be a promising strategy to improve the efficacy of immunotherapies for cancer patients.

Current status and future prospects of peptide-based cancer vaccines.

Cancer immunotherapy has attracted attention worldwide owing to the recent development of immune checkpoint inhibitors. However, these therapies have shown limited efficacy, and further advancements are needed before these modalities can progress to widespread use. Immune checkpoint inhibitors are a type of nonspecific cancer immunotherapy, and antitumor effects are only observed when cancer-specific T cells are found within the nonspecifically activated T-cell group. In order to facilitate the development of potent immunotherapies, selective enhancement of cancer-specific T cells is essential. In this report, we discuss current and future perspectives, including the latest clinical trials of cancer-specific immunotherapies, particularly cancer peptide vaccines.

Immune Responses to Epidermal Growth Factor Receptor (EGFR) and Their Application for Cancer Treatment.

Epidermal growth factor receptor (EGFR) is a prototypic cell-surface receptor belonging to the ErbB/HER onocogene family. Overexpression or somatic mutations of EGFR have been reported to play an important role in tumorigenesis in various types of epithelial cancers. Therefore, targeting of EGFR with specific blocking antibodies or inhibitors have been developing for treatment for EGFR-associated tumors. Immune responses to HER2, another molecule of the ErbB/HER onocogene family, have been well studied, but only limited information on the immune responses to EGFR in cancer has been currently available. In this review, we have summarized the available data and discussed potential clinical importance of the anti-EGFR immune responses and EGFR-mediated immune regulation in cancer. Several lines of evidence suggest that cellular and humoral immune responses to EGFR might be useful as a marker and/or target for cancer therapy against EGFR-associated tumors. In addition, recent studies suggest the critical roles of EGFR-mediated signaling in regulation of expression of an immune checkpoint molecule, programmed death-ligand 1 (PD-L1) in tumor cells. Further studies are warranted to clarify the impact of the anti-EGFR immune responses and EGFR-mediated immunomodulation for clinical application for cancer treatment.

IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4(+) T cells.

Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4(+) T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b(+)CD11c(+) cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b(+)CD11c(+) cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b(+)CD11c(+) cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4(+) T and CD8(+) T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy.

IL-11 induces differentiation of myeloid-derived suppressor cells through activation of STAT3 signalling pathway.

Myeloid-derived suppressor cells (MDSCs) are immune negative regulators in the tumour microenvironment. Interleukin (IL)-11, a member of IL-6 family cytokines, functions through the unique receptor IL-11 receptor α coupled with the common signal transducer gp130. IL-11-gp130 signalling causes activation of the JAK/STAT3 pathway. IL-11 is highly upregulated in many types of cancers and one of the most important cytokines during tumourigenesis and metastasis. However, the precise effect of IL-11 on differentiation into MDSCs is still unknown. Here, we found that CD11b+CD14+ monocytic MDSCs were generated from peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence of IL-11. IL-11-conditioned PBMCs induced higher expression of immunosuppressive molecules such as arginase-1. A reduction of T-cell proliferation was observed when MDSCs generated in the presence of IL-11 were co-cultured with CD3/CD28-stimulated, autologous T cells of healthy donors. Culture of normal PBMCs with IL-11 led to STAT3 phosphorylation and differentiation into MDSCs via STAT3 activation. We confirmed expressions of both IL-11 and phosphorylated STAT3 in tumour tissues of colorectal cancer patients. These findings suggest that monocytic MDSCs may be induced by IL-11 in the tumour microenvironment. Thus, IL-11-mediated regulation in functional differentiation of MDSCs may serve as a possible target for cancer immunotherapy.

Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine.

We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4(+) Th1 cells and CD8(+) Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.

Identification of a meiosis-specific protein, MEIOB, as a novel cancer/testis antigen and its augmented expression in demethylated cancer cells.

Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen. Indeed, the MEIOB gene is expressed only in the testis and not in other normal tissues. The mRNA expression of MEIOB was greatly enhanced in several lung cancer cell lines after the treatment with DAC. Furthermore, we identified a variety of helper epitopes of the MEIOB antigen, which were recognized by MEIOB antigen-specific T cells in a HLA-restriction manner. Finally, we demonstrated that IFN-γ production of MEIOB peptide-specific helper T cells in response to HLA-matched cancer cells was greatly augmented by treatment with DAC and IFN-γ. Taken together, these findings show DAC to be a promising tool for finding novel CT antigens and for developing a future novel combination cancer vaccine chemotherapy.

Establishment of a stable T lymphoma cell line transduced with HLA-A*24:02-restricted WT1-specific TCR genes and its application to antigen-specific immunomonitoring.

Wilms' tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A*24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epitopes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A*24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A*24:02-restricted WT1-specific CTLs. Here we induced several WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vß usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer patients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.

First clinical trial of cancer vaccine therapy with artificially synthesized helper/ killer-hybrid epitope long peptide of MAGE-A4 cancer antigen.

A patient with pulmonary metastasis of colon cancer was treated with artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP) of MAGE-A4 cancer antigen. The patient was vaccinated with MAGE-A4-H/K-HELP combined with OK432 and Montanide ISA-51. There were no severe side-effects except for a skin reaction at the injection site. MAGE-A4-H/K-HELP induced MAGE-A4-specific Th1 and Tc1 immune responses and the production of MAGE-A4-specific complement-fixing IgG antibodies. Tumor growth and carcinoembryonic antigen tumor marker were significantly decreased in the final diagnosis. This is the first report that artificially synthesized MAGE-A4-H/K-HELP induces Th1-dependent cellular and humoral immune responses in a human cancer patient.