Nivolumab receptor occupancy on effector regulatory T cells predicts clinical benefit.

Immune checkpoint inhibitor discovery represents a turning point in cancer treatment. However, the response rates of solid tumors remain ~10%-30%; consequently, prognostic and immune-related adverse event (irAE) predictors are being explored. The programmed cell death protein 1 (PD-1) receptor occupancy (RO) of PD-1 inhibitors depends on the number of peripheral blood lymphocytes and their PD-1 expression levels, suggesting that the RO may be related to efficacy and adverse events. As PD-1 inhibition affects each T-cell subset differently, the RO of each cell population must be characterized. However, relevant data have not been reported, and the prognostic relevance of this parameter is not known. In this study, we aimed to clarify the association between the nivolumab RO in each T-cell population and patient prognosis and reveal the development of irAEs in nivolumab-treated patients. Thirty-two patients were included in the study, and the mean follow-up period was 364 days. The nivolumab RO on effector regulatory T cells (eTregs) was significantly lower in the group that presented clinical benefits, and a significant negative association was observed between PD-1 occupancy on eTregs and all-cause mortality. The results suggest that the nivolumab RO on eTregs may be a prognostic factor in PD-1 inhibitor therapy, implying that the inhibition of PD-1/PD-ligand 1 (PD-L1) signaling on eTregs may attenuate antitumor effects.

Methionine restriction of glioma does not induce MGMT and greatly improves temozolomide efficacy in an orthotopic nude-mouse model: A potential curable approach to a clinically-incurable disease.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.

Butyricimonas is a key gut microbiome component for predicting postoperative recurrence of esophageal cancer.

BACKGROUND: Recently, intestinal bacteria have attracted attention as factors affecting the prognosis of patients with cancer. However, the intestinal microbiome is composed of several hundred types of bacteria, necessitating the development of an analytical method that can allow the use of this information as a highly accurate biomarker. In this study, we investigated whether the preoperative intestinal bacterial profile in patients with esophageal cancer who underwent surgery after preoperative chemotherapy could be used as a biomarker of postoperative recurrence of esophageal cancer. METHODS: We determined the gut microbiome of the patients using 16S rRNA metagenome sequencing, followed by statistical analysis. Simultaneously, we performed a machine learning analysis using a random forest model with hyperparameter tuning and compared the data obtained. RESULTS: Statistical and machine learning analyses revealed two common bacterial genera, Butyricimonas and Actinomyces, which were abundant in cases with recurrent esophageal cancer. Butyricimonas primarily produces butyrate, whereas Actinomyces are oral bacteria whose function in the gut is unknown. CONCLUSION: Our results indicate that Butyricimonas spp. may be a biomarker of postoperative recurrence of esophageal cancer. Although the extent of the involvement of these bacteria in immune regulation remains unknown, future research should investigate their presence in other pathological conditions. Such research could potentially lead to a better understanding of the immunological impact of these bacteria on patients with cancer and their application as biomarkers.

Randomized phase I/II study of vascular endothelial growth factor receptor peptide vaccines for patients with hepatocellular carcinoma.

AIM: We evaluated the safety and efficacy of vascular endothelial growth factor receptor (VEGFR)-targeted peptide vaccines for the immunization of patients with unresectable hepatocellular carcinoma (HCC) who had responded to transarterial chemoembolization. METHODS: Twenty-two patients were randomized 1:1 to receive VEGFR-targeted peptides or placebo. The primary end-point was the safety assessment of the immunization. The secondary end-points were evaluation of immunological responses and clinical outcomes. RESULTS: No severe adverse events were induced by the study agents. Among the 12 patients in the vaccine group, a VEGFR1-specific cytotoxic T lymphocyte (CTL) response was induced in eight (66.7%) patients and a VEGFR2-specific CTL response was induced in 10 (83.3%). The median progression-free survival (PFS) and overall survival (OS) rates were 4.8 and 52.0 months, respectively, in the vaccine group, and 2.7 and 21.8 months, respectively, in the placebo group. No statistically significant differences were found between the two groups (PFS p = 0.925, OS p = 0.190). When divided into two groups according to immunoreactivity, the median PFS of patients with and without a strong immune response to VEGFR1 were 7.4 and 2.7 months, and that to VEGFR2 were 10.6 and 2.7 months, respectively; there were significant differences according to the immune response. CONCLUSIONS: Immunotherapy with peptide vaccines targeting VEGFR1 and VEGFR2 was well tolerated with no serious adverse events. It also effectively induced peptide-specific CTLs in patients with unresectable HCC.

Superiority of [11C]methionine over [18F]deoxyglucose for PET Imaging of Multiple Cancer Types Due to the Methionine Addiction of Cancer.

Positron emission tomography (PET) is widely used to detect cancers. The usual isotope for PET imaging of cancer is [18F]deoxyglucose. The premise of using [18F]deoxyglucose is that cancers are addicted to glucose (The Warburg effect). However, cancers are more severely addicted to methionine (The Hoffman effect). [11C]methionine PET (MET-PET) has been effectively used for the detection of glioblastoma and other cancers in the brain, and in comparison, MET-PET has been shown to be more sensitive and accurate than [18F]deoxyglucose PET (FDG-PET). However, MET-PET has been limited to cancers in the brain. The present report describes the first applications of MET-PET to cancers of multiple organs, including rectal, bladder, lung, and kidney. The results in each case show that MET-PET is superior to FDG-PET due to the methionine addiction of cancer and suggest that the broad application of MET-PET should be undertaken for cancer detection.

Baseline soluble MICA levels act as a predictive biomarker for the efficacy of regorafenib treatment in colorectal cancer.

BACKGROUND: To evaluate the effect of regorafenib on soluble MHC class I polypeptide-related sequence A (MICA) (sMICA) level in vitro. In addition, we clinically examined whether its plasma levels were associated with regorafenib activity in terms of progression-free survival (PFS) in patients with CRC. METHODS: Human CRC cell line HCT116 and HT29 cells were treated with regorafenib and its pharmacologically active metabolites, M2 or M5 at the same concentrations as those in sera of patients. We also examined the sMICA levels and the area under the plasma concentration-time curve of regorafenib, M2 and M5. RESULTS: Regorafenib, M2, and M5 significantly suppressed shedding of MICA in human CRC cells without toxicity. This resulted in the reduced production of sMICA. In the clinical examination, patients with CRC who showed long median PFS (3.7 months) had significantly lower sMICA levels than those with shorter median PFS (1.2 months) (p = 0.045). CONCLUSIONS: MICA is an attractive agent for manipulating the immunological control of CRC and baseline sMICA levels could be a predictive biomarker for the efficacy of regorafenib treatment.

Pharmacokinetics of gefitinib in elderly patients with EGFR-mutated advanced non-small cell lung cancer: a prospective study.

BACKGROUND: Gefitinib is recommended as a first-line treatment option for elderly patients with non-small cell lung cancer (NSCLC). Because no pharmacokinetics of gefitinib have been examined, we prospectively assessed the pharmacokinetics of gefitinib in patients with epidermal growth factor receptor gene-mutated advanced NSCLC who were 75 years or older. METHODS: Gefitinib was orally administered once daily at a dose of 250 mg. The concentrations of gefitinib and its major metabolite O-desmethyl gefitinib in plasma were measured by high-performance liquid chromatography. The area under the plasma concentration-time curve from time 0 to 48 h (AUC0-48) was calculated. Polymorphisms in CYP3A5, CYP2D6, ABCG2, ABCB1, and OATP1B1 were analyzed by direct sequencing. RESULTS: Eighteen patients with a median age of 80.5 years (range, 75-89) with adequate liver and kidney functions were examined. AUC0-48 values of gefitinib and O-desmethyl gefitinib in this population were 9.49 ± 3.5 and 10.6 ± 14 µM h, respectively. Compared to the gefitinib pharmacokinetics observed in a previous phase I study in Japan, systemic exposure to gefitinib in elderly patients was slightly higher than that in younger patients. Three patients experienced grade 3 diarrhea, increases in alanine aminotransferase, and aspartate aminotransferase levels 30 days after starting gefitinib treatment. The CYP2D6 genotype was associated with CYP2D6-mediated metabolism of gefitinib to O-desmethyl gefitinib. CONCLUSIONS: We demonstrated for the first time the systemic exposure to gefitinib in elderly patients with NSCLC. TRIAL REGISTRATION: The study was registered with the University Hospital Medical Information Network-Clinical Trials Registry Japan (UMIN000026409) on November 8, 2013.

The Sympathetic Nervous System Contributes to the Establishment of Pre-Metastatic Pulmonary Microenvironments.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Hemorrhagic Colitis Caused by Everolimus in a Patient with Nonfunctional Pancreatic Neuroendocrine Neoplasms: A Case Report.

A 60-year-old woman was diagnosed with nonfunctional pancreatic neuroendocrine neoplasm with multiple liver metastases and was administered everolimus. Due to persistent epigastric pain and diarrhea, a colonoscopy was performed on the 14th day after the start of everolimus administration, which revealed small bleeding ulcers in the ileocecal region, transverse colon, and rectum. These adverse effects were attributed to the everolimus; it was immediately discontinued, and the patient's clinical symptoms and imaging findings improved. We concurred that the administration of calcium channel blockers resulted in the inhibition of everolimus metabolism and the disease onset. The everolimus was discontinued. There was no subsequent recurrence of hemorrhagic colitis.

A high number of PD-L1+ CD14+ monocytes in peripheral blood is correlated with shorter survival in patients receiving immune checkpoint inhibitors.

PURPOSE: Targeting of anti-programmed cell death protein-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) is a standard therapeutic strategy for various cancers. The aim of the present study was to investigate the prognostic effect of pretreatment PD-L1 expression levels in peripheral blood mononuclear cell (PBMC) subsets for patients with several cancer types receiving anti-PD-1 blockade therapies. PATIENTS AND METHODS: Thirty-two patients undergoing anti-PD-L1 blockade therapy, including 15 with non-small cell lung cancer, 14 with gastric cancer, 1 with melanoma, 1 with parotid cancer, and 1 with bladder cancer, were recruited for the present study. PD-L1 expression levels in CD3+, CD4+, CD8+, CD45RA+ and CCR7+ T cells; CD20+ B cells; CD14+ and CD16+ monocytes were measured via flow cytometry before treatment. The percentages of PD-L1+ cells in respective PBMC subsets were compared with respect to different clinicopathological conditions and the association with overall survival (OS) was assessed. RESULTS: The percentages of PD-L1+ with CD3+, CD4+ and CD8+ T cells including naïve and memory T cell subsets, or CD20+ B cells during pretreatment were not markedly correlated with the OS of patients (p > 0.05); however, the percentage of the PD-L1+ CD14+ monocyte subset was significantly correlated with OS (p = 0.0426). CONCLUSION: Increase in pretreatment expression levels of PD-L1 on CD14+ monocytes is associated with the OS of patients treated with immune checkpoint inhibitors. Further evaluation of large sample size and each specific cancer type might clarify the predictive role of PBMC in patients.

Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

[Gut Microbiota and Tumor Immune Microenvironment].

Gut microbiota plays a major role in the cancer microenvironment associated with immune checkpoint blockade. Recent developments in gut microbiota research have benefited from technological innovations by next-generation sequencers, and the research has exploded. Therefore, we considered gut microbiota from the viewpoint of antitumor and adverse events of immune checkpoint inhibitors.

Characterization of tumor-infiltrating immune cells in relation to microbiota in colorectal cancers.

BACKGROUND: Several articles have recently reported that certain colon microbiota can improve the efficacy of cancer immunotherapy. To develop new treatment strategies, including immunotherapy for colorectal cancer (CRC), we evaluated the correlations between subpopulations of tumor-infiltrating immune cells (TIICs) and intestinal microbiota in CRC. METHODS: Fresh surgically resected specimens, formalin-fixed paraffin-embedded whole tissue samples, and stool samples were collected. TIICs including Tregs, Th17 cells and tumor-associated macrophages (TAMs) in the surgically resected specimens were analyzed using flow cytometry. FOXp3, CD8, CD163, and phosphorylated-STAT1-positive TIICs in the whole tissue samples were analyzed using IHC, and intestinal microbiota in the stool samples was analyzed using 16S metagenome sequencing. TIICs subpopulations in the normal mucosa and tumor samples were evaluated, and the correlations between the TIIC subpopulations and intestinal microbiota were analyzed. RESULTS: FOXp3lowCD45RA+ Tregs were significantly reduced (p = 0.02), FOXp3lowCD45RA- Tregs were significantly increased (p = 0.006), and M1 TAMs were significantly reduced in the tumor samples (p = 0.03). Bacteroides (phylum Bacteroidetes) and Faecalibacterium (phylum Firmicutes) were increased in the patients with high numbers of Tregs and clearly high distribution of FOXp3highCD45RA- Tregs, which are the effector Tregs. Faecalibacterium, Ruminococcaceae, Eubacterium (phylum Firmicutes), and Bacteroides were increased in patients with a high distribution of M1 TAMs. CONCLUSIONS: The findings of the present study indicate that immune responses to tumors are suppressed in the tumor microenvironment of CRC depending on the increment of Tregs and the reduction of M1 TAMs and that intestinal microbiota might be involved in immunosuppression.

Lack of correlation between the costs of anticancer drugs and clinical benefits in Japan.

Both overall survival (OS) and progression-free survival (PFS) are primary endpoints of phase III studies of new anticancer drugs. Medical care expenditures, especially oncology drug prices, are rapidly increasing; however, the impact of oncology drug prices on OS and PFS is unclear. We analyzed the relationship between oncology drug prices and clinical outcomes in Japan. The costs of a full course or 1 year of treatment were estimated on the basis of the latest National Health Insurance Drug Price Standards, and the relationship between costs and improvements in OS or PFS obtained with each drug were analyzed. Cost-effectiveness was compared between new-class drugs and next-in-class drugs. We then developed a simple model for estimating the costs required to prolong OS and PFS by 1 day and used this model to compare cost-effectiveness. Drug costs were not significantly related to treatment outcomes in terms of PFS or OS. There was no significant difference in the median cost between novel drugs and the next-in-class drugs (P = 0.39). The oncology drug cost required to prolong PFS by 1 day was more expensive than the drug cost required for prolong OS by 1 day. Prices of oncology drugs should be decided on the basis of actual clinical benefits for cancer patients, and the drug price evaluation process should be disclosed in Japan.

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.

A first-in-human study investigating biodistribution, safety and recommended dose of a new radiolabeled MAb targeting FZD10 in metastatic synovial sarcoma patients.

BACKGROUND: Synovial Sarcomas (SS) are rare tumors occurring predominantly in adolescent and young adults with a dismal prognosis in advanced phases. We report a first-in-human phase I of monoclonal antibody (OTSA-101) targeting FZD10, overexpressed in most SS but not present in normal tissues, labelled with radioisotopes and used as a molecular vehicle to specifically deliver radiation to FZD10 expressing SS lesions. METHODS: Patients with progressive advanced SS were included. In the first step of this trial, OTSA-101 in vivo bio-distribution and lesions uptake were evaluated by repeated whole body planar and SPECT-CT scintigraphies from H1 till H144 after IV injection of 187 MBq of 111In-OTSA-101. A 2D dosimetry study also evaluated the liver absorbed dose when using 90Y-OTSA-101. In the second step, those patients with significant tumor uptake were randomized between 370 MBq (Arm A) and 1110 MBq (Arm B) of 90Y-OTSA-101 for radionuclide therapy. RESULTS: From January 2012 to June 2015, 20 pts. (median age 43 years [21-67]) with advanced SS were enrolled. Even though 111In-OTSA-101 liver uptake appeared to be intense, estimated absorbed liver dose was less than 20 Gy for each patient. Tracer intensity was greater than mediastinum in 10 patients consistent with sufficient tumor uptake to proceed to treatment with 90Y-OTSA-101: 8 were randomized (Arm A: 3 patients and Arm B: 5 patients) and 2 were not randomized due to worsening PS. The most common Grade ≥ 3 AEs were reversible hematological disorders, which were more frequent in Arm B. No objective response was observed. Best response was stable disease in 3/8 patients lasting up to 21 weeks for 1 patient. CONCLUSIONS: Radioimmunotherapy targeting FZD10 is feasible in SS patients as all patients presented at least one lesion with 111In-OTSA-101 uptake. Tumor uptake was heterogeneous but sufficient to select 50% of pts. for 90Y-OTSA-101 treatment. The recommended activity for further clinical investigations is 1110 MBq of 90Y-OTSA-101. However, because of hematological toxicity, less energetic particle emitter radioisopotes such as Lutetium 177 may be a better option to wider the therapeutic index. TRIAL REGISTRATION: The study was registered on the NCT01469975 website with a registration code NCT01469975 on November the third, 2011.

Phase I clinical trial of a five-peptide cancer vaccine combined with cyclophosphamide in advanced solid tumors.

We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.

Randomized phase II/III clinical trial of elpamotide for patients with advanced pancreatic cancer: PEGASUS-PC Study.

Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo-controlled, double-blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide + gemcitabine) or Placebo group (placebo + gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington-Fleming test was applied to the statistical analysis in this study to evaluate the time-lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n = 100; Placebo group, n = 53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington-Fleming P-value, 0.918; log-rank P-value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486-1.557). Median survival time was 8.36 months (95% CI, 7.46-10.18) for the Active group and 8.54 months (95% CI, 7.33-10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival.