Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy

Background: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. Methods: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). Results: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. Conclusion: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT

Low T-cell Receptor Diversity, High Somatic Mutation Burden, and High Neoantigen Load as Predictors of Clinical Outcome in Muscle-invasive Bladder Cancer

AbstractBackgroundThe success of cancer immunotherapies has highlighted the potent ability of local adaptive immune responses to eradicate cancer cells by targeting neoantigens generated by somatic alterations. However, how these factors interact to drive the natural history of muscle-invasive bladder cancer (MIBC) is not well understood.ObjectiveTo investigate the role of immune regulation in MIBC disease progression, we performed massively parallel T-cell receptor (TCR) sequencing of tumor-infiltrating T cells (TILs), in silico neoantigen prediction from exome sequences, and expression analysis of immune-related genes.Design, setting, and participantsWe analyzed 38 MIBC tissues from patients who underwent definitive surgery with a minimum clinical follow-up of 2 yr.Outcome measurements and statistical analysisRecurrence-free survival (RFS) was determined. TCR diversity was quantified using Simpson's diversity index. The main analyses involved the Mann-Whitney U test, Kaplan-Meier survival analysis, and Cox proportional hazards models.Results and limitationsLow TCRβ chain diversity, correlating with oligoclonal TIL expansion, was significantly correlated with longer RFS, even after adjustment for pathologic tumor stage, node status, and receipt of adjuvant chemotherapy (hazard ratio 2.67, 95% confidence interval 1.08–6.60; p=0.03). Patients with both a high number of neoantigens and low TCRβ diversity had longer RFS compared to those with fewer neoantigens and high TCR diversity (median RFS 275 vs 30 wk; p=0.03). Higher expression of immune cytolytic genes was associated with nonrecurrence among patients with low TCR diversity or fewer neoantigens. Limitations include the sample size and the inability to distinguish CD8+ and CD4+ T cells using TCR sequencing.ConclusionsThese findings are the first to show that detailed tumor immune-genome analysis at definitive surgery can identify molecular patterns of antitumor immune response contributing to better clinical outcomes in MIBC.Patient summaryWe discovered that clonal expansion of certain T cells in tumor tissue, possibly targeting cancer-specific antigens, contributes to prevention of bladder cancer recurrence

Biomonitoring of Urinary Cotinine Concentrations Associated with Plasma Levels of Nicotine Metabolites after Daily Cigarette Smoking in a Male Japanese Population

Human biomonitoring of plasma and urinary levels of nicotine, cotinine, and 3′-hydroxycotinine was conducted after daily cigarette smoking in a population of 92 male Japanese smokers with a mean age of 37 years who had smoked an average of 23 cigarettes per day for 16 years. Members of the population were genotyped for the nicotine-metabolizing enzyme cytochrome P450 2A6 (CYP2A6). The mean levels of nicotine, the levels of its metabolites cotinine and 3′-hydroxycotinine, and the sum of these three levels in subjects one hour after smoking the first cigarette on the sampling day were 20.1, 158, 27.7, and 198 ng/mL in plasma and 846, 1,020, 1,010, and 2,870 ng/mL in urine under daily smoking conditions. Plasma levels of 3′-hydroxycotinine and urinary levels of nicotine and 3′-hydroxycotinine were dependent on the CYP2A6 phenotype group, which was estimated from the CYP2A6 genotypes of the subjects, including those with whole gene deletion. Plasma cotinine levels were significantly correlated with the number of cigarettes smoked on the day before sampling (r = 0.71), the average number of cigarettes smoked daily (r = 0.58), and the Brinkman index (daily cigarettes × years, r = 0.48) under the present conditions. The sum of nicotine, cotinine, and 3′-hydroxycotinine concentrations in plasma showed a similar relationship to that of the plasma cotinine levels. Urinary concentrations of cotinine and the sum of nicotine metabolite concentrations also showed significant correlations with the plasma levels and the previous day’s and average cigarette consumption. The numbers of cigarettes smoked per day by two subjects with self-reported light smoking habits were predicted by measuring the urinary cotinine concentrations and using linear regression equations derived from above-mentioned data. These results indicate that biomonitoring of the urinary cotinine concentration is a good, easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers independent of genetic polymorphism of CYP2A6

Significant effect of polymorphisms in CYP2D6 and ABCC2 on clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients

Purpose The clinical efficacy of tamoxifen is suspected to be influenced by the activity of drug-metabolizing enzymes and transporters involved in the formation, metabolism, and elimination of its active forms. We investigated relationships of polymorphisms in transporter genes and CYP2D6 to clinical outcome of patients receiving tamoxifen. Patients and Methods We studied 282 patients with hormone receptor–positive, invasive breast cancer receiving tamoxifen monotherapy, including 67 patients who have been previously reported. We investigated the effects of allelic variants of CYP2D6 and haplotype-tagging single nucleotide polymorphisms (tag-SNPs) of ABCB1, ABCC2, and ABCG2 on recurrence-free survival using the Kaplan-Meier method and Cox regression analysis. Plasma concentrations of tamoxifen metabolites were measured in 98 patients receiving tamoxifen 20 mg/d. Results CYP2D6 variants were significantly associated with shorter recurrence-free survival (P = .000036; hazard ratio [HR] = 9.52; 95% CI, 2.79 to 32.45 in patients with two variant alleles v patients without variant alleles). Among 51 tag-SNPs in transporter genes, a significant association was found at rs3740065 in ABCC2 (P = .00017; HR = 10.64; 95% CI, 1.44 to 78.88 in patients with AA v GG genotypes). The number of risk alleles of CYP2D6 and ABCC2 showed cumulative effects on recurrence-free survival (P = .000000055). Patients carrying four risk alleles had 45.25-fold higher risk compared with patients with ≤ one risk allele. CYP2D6 variants were associated with lower plasma levels of endoxifen and 4-hydroxytamoxifen (P = .0000043 and .00052), whereas no significant difference was found among ABCC2 genotype groups. Conclusion Our results suggest that polymorphisms in CYP2D6 and ABCC2 are important predictors for the prognosis of patients with breast cancer treated with tamoxifen

トリプル ネガティブ ニュウガン ニオケル プロテアソーム カンレン インシ PAG1 ニヨル シンキ ゾウショク キコウ ノ カイメイ

Triple negative breast cancer （TNBC） is considered to be one of the most aggressive subtypes of all breast cancers. To identify novel potential therapeutic targets and clarify pathophysiological features for TNBC, we conducted Meta-gene profiling analysis based on gene-expression profiling of TNBC cases purified by lasermicrobeam microdissection, and found that proteasome-associated genes （PAGs） were commonly upregulated in various pathways including cell cycle regulation in TNBC. Depletion of PAGs with RNAi caused the upregulation of p27 and p21 proteins in MDA-MB-231 and HCC1937 cells, respectively, resulting in growth inhibition. Interestingly, immunocytochmical staining revealed that PAG1 was observed in the nucleoli and/or cytoplasm （n-PAG1 and c-PAG1） in TNBC cell line and clinical specimens. Immunohistochemical staining of 100 TNBCs showed that high level of n-PAG1 was significantly associated with poor disease free and overall survival of TNBC patients. These results indicate that n-PAG1 plays a critical role in nucleus during cell cycle progression and might be a novel prognostic indicator or an attractive molecular target of TNBC

Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3

Immunotherapies, including immune checkpoint blockades, play a critically important role in cancer treatments. For immunotherapies, neoantigens, which are generated by somatic mutations in cancer cells, are thought to be good targets due to their tumor specificity. Because neoantigens are unique in individual cancers, it is challenging to develop personalized immunotherapy targeting neoantigens. In this study, we screened "shared neoantigens", which are specific types of neoantigens derived from mutations observed commonly in a subset of cancer patients. Using exome sequencing data in the Cancer Genome Atlas (TCGA), we predicted shared neoantigen peptides and performed in vitro screening of shared neoantigen-reactive CD8+ T cells using peripheral blood from healthy donors. We examined the functional activity of neoantigen-specific T cell receptors (TCRs) by generating TCR-engineered T cells. Among the predicted shared neoantigens from TCGA data, we found that the mutated FGFR3Y373C peptide induced antigen-specific CD8+ T cells from the donor with HLA-A*02:06 via an ELISPOT assay. Subsequently, we obtained FGFR3Y373C-specific CD8+ T cell clones and identified two different sets of TCRs specifically reactive to FGFR3Y373C. We found that the TCR-engineered T cells expressing FGFR3Y373C-specific TCRs recognized the mutated FGFR3Y373C peptide but not the corresponding wild-type peptide. These two FGFR3Y373C-specific TCR-engineered T cells showed cytotoxic activity against mutated FGFR3Y373C-loaded cells. These results imply the possibility of strategies of immunotherapies targeting shared neoantigens, including cancer vaccines and TCR-engineered T cell therapies

Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3

Immunotherapies, including immune checkpoint blockades, play a critically important role in cancer treatments. For immunotherapies, neoantigens, which are generated by somatic mutations in cancer cells, are thought to be good targets due to their tumor specificity. Because neoantigens are unique in individual cancers, it is challenging to develop personalized immunotherapy targeting neoantigens. In this study, we screened "shared neoantigens", which are specific types of neoantigens derived from mutations observed commonly in a subset of cancer patients. Using exome sequencing data in the Cancer Genome Atlas (TCGA), we predicted shared neoantigen peptides and performed in vitro screening of shared neoantigen-reactive CD8+ T cells using peripheral blood from healthy donors. We examined the functional activity of neoantigen-specific T cell receptors (TCRs) by generating TCR-engineered T cells. Among the predicted shared neoantigens from TCGA data, we found that the mutated FGFR3Y373C peptide induced antigen-specific CD8+ T cells from the donor with HLA-A*02:06 via an ELISPOT assay. Subsequently, we obtained FGFR3Y373C-specific CD8+ T cell clones and identified two different sets of TCRs specifically reactive to FGFR3Y373C. We found that the TCR-engineered T cells expressing FGFR3Y373C-specific TCRs recognized the mutated FGFR3Y373C peptide but not the corresponding wild-type peptide. These two FGFR3Y373C-specific TCR-engineered T cells showed cytotoxic activity against mutated FGFR3Y373C-loaded cells. These results imply the possibility of strategies of immunotherapies targeting shared neoantigens, including cancer vaccines and TCR-engineered T cell therapies