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BACKGROUND/AIM: Brain metastasis, a leading cause of cancer death, is a clinical challenge. Recently, genetic characterization of brain metastatic lesions based on next generation sequencing-based advanced technologies, such as single-cell RNA sequencing, has been performed to develop novel efficient therapies. The present study aimed to investigate brain-metastasis-specific biomarkers as well as relevant prognostic factors. PATIENTS AND METHODS: The genetic profiles and expression levels of immune response-associated genes and 820 cancer-associated genes were compared between primary cancer lesions and metastatic cancer lesions obtained from nine cancer patients at the Shizuoka Cancer Center. Cytokine and chemokine marker genes were analyzed via quantitative PCR. T-cell receptor (TCR) repertoire profiling was performed for the same patients. For survival analysis, survival data of 52 cancer patients with brain metastases were utilized. RESULTS: Comparison of driver mutation profiling between primary and metastatic lesions revealed shared core mutations in both lesions and a few new mutations in metastatic lesions. A high tumor mutation burden (TMB) was detected in metastatic lesions. Volcano plot analysis revealed specific features of the metastatic tumor microenvironment, such as cancer signaling promotion and immune suppression due to decreased immune cell infiltration. Survival analysis revealed that three genes, the TREML2 gene, the BTLA gene on activated microglia and the CERS2 gene on metastatic tumor, were potent prognostic factors. CONCLUSION: High TMB in metastatic lesions indicates potential benefit from immune checkpoint inhibitor usage for brain metastasis and TREML2 and BTLA are factors associated with poor prognosis. Activated microglia may be novel targets for the treatment of brain metastasis.
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Biomarcadores de Tumor , Neoplasias Encefálicas , Humanos , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Femenino , Masculino , Biomarcadores de Tumor/genética , Persona de Mediana Edad , Pronóstico , Anciano , Mutación , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND/AIM: Recently, inactivating somatic mutations of SWI/SNF chromatin-remodeling genes in cancers have been reported. However, few studies have been performed regarding the immunological analysis of the tumor microenvironment (TME) in chromatin remodeling complex gene-mutated tumors. In the present study, we identified cancer patients harboring various mammalian SWI/SNF complex mutations and investigated the immunological features in those mutated cancers. PATIENTS AND METHODS: Cancer patients harboring any type of chromatin remodeling complex gene mutation were selected and clinicopathological features were compared between chromatin remodeling complex gene expression-low and expression-high groups. Specifically, expression levels of immune response-associated genes and cancer-associated genes were compared between the SMARCA4 expression-low and expression-high groups using volcano plot analysis. RESULTS: Among cancers harboring PBRM1, SAMRACA4 and ARID2 gene mutations, T-cell marker and mature B-cell marker genes were up-regulated in the tumor. Specifically, T-cell effector genes (CD8B, CD40LG), central memory marker genes (CD27, CCR7) and mature B-cell marker genes (CD20, CD38, CD79 and IRF4) were up-regulated, and cancer-associated genes including MYB, MYC and AURKB genes were down-regulated in the SMARCA4 expression-low group. Remarkably, heatmap of gene expression and immunohistochemistry (IHC) data demonstrated that the tertiary lymphoid structure (TLS) gene signature of mature B cells was up-regulated in SMACA4 gene-mutated stomach cancers. CONCLUSION: These results suggest that immune tumor microenvironment status, such as mature B cell recruitment featuring the TLS gene signature and immune activation mediated by cancer signal down-regulation, might contribute to the classification of SMARCA4 gene-mutated tumors as immune checkpoint blockade therapy-sensitive target tumors.
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Neoplasias , Microambiente Tumoral , Animales , Humanos , Microambiente Tumoral/genética , Mutación , Neoplasias/genética , Mamíferos , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. MATERIALS AND METHODS: Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. RESULTS: The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. CONCLUSION: PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ácidos Nucleicos de Péptidos , Animales , Humanos , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN , Sondas de ADN/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones Desnudos , Mutación , Ácidos Nucleicos de Péptidos/genética , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND/AIM: Anti-programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) antibody is a successful treatment for patients with solid cancers; however, there are several disadvantages that need to be resolved. Oral small molecule anti-PD-1/PD-L1 inhibitors have been developed and have good bioavailability. MATERIALS AND METHODS: Potent anti-PD-1/PD-L1 inhibitor candidates from the Shizuoka small compound library were screened and investigated for their antitumor activities in vitro and in vivo using a humanized mouse model. A search for small compounds that inhibit PD-1/PD-L1 binding among 67,395 compounds through three rounds of screening procedures identified six compounds. RESULTS: The two compounds (SCL-1 and SCL-2), which have as a key chemical structure of triazolopyridazin backbone with a piperazine residue on the aromatic ring and 1,3-diphenyl pyrazoline with hydrazinylphthalazine were selected based on in vitro assays and absorption, distribution, metabolism, and excretion (ADME) scoring and subjected to in vivo experiments using a humanized NOG mouse model. SCL-1 and SCL-2 exhibited moderate inhibitory activities against PD-1/PD-L1 binding compared to an anti-PD-1 antibody, with SCL-1 exerting markedly weaker cytotoxic effects on target cells than the other compounds. In in vivo experiments, SCL-1 exerted significant antitumor effects on PD-L1+ SCC-3 tumors, which were dependent on CD8+ T cell infiltration and PD-L1 expression in tumors. A pharmacokinetic study revealed that it has good bioavailability and distribution as an oral reagent. CONCLUSION: SCL-1 is a novel small compound that inhibits PD-1/PD-L1 binding and exerts potent antitumor effects. Thus, it has potential as an oral reagent for cancer immunotherapy.
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Antígeno B7-H1 , Neoplasias , Ratones , Animales , Antígeno B7-H1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Puntos de Control Inmunológico , Ligandos , Modelos Animales de Enfermedad , PiperazinasRESUMEN
BACKGROUND/AIM: The recurrence rate of head and neck squamous cell carcinoma (HNSCC) remains high; thus the control of recurrence is a clinical problem to be challenged. To clarify the precise mechanism, specific immunological biomarkers responsible for recurrence were investigated. PATIENTS AND METHODS: The expression levels of immune response-associated and Shizuoka Cancer Center 820 cancer-associated genes, and genetic mutations from whole-exome sequencing were compared between HNSCC patients who developed recurrence (n=8) and HNSCC patients who did not develop recurrence (n=19) using a volcano plot analysis. Cytokine and epithelial-mesenchymal transition marker genes were analyzed using quantitative PCR. Tumor-infiltrating lymphocytes, immune checkpoint molecules, and human papilloma virus status were investigated using immunohistochemistry (IHC). RESULTS: Twenty-seven evaluable patients with HNSCCs received radiation therapy after surgery. Recurrence was identified in 8 patients. TP53 mutations tended to be higher in patients who developed recurrence than in those who did not develop recurrence (75% vs. 31.6%). Gene expression profiling showed the down-regulation of T cell activation genes (ICOS, CD69 and CD83) and the upregulation of the ERBB4, EGFR, VEGF, HIF1A, TGFB1, TWIST1, IL-8, and PAX7 genes, which suggested the activation of the TP53 mutation-TGF-ß1-PAX7 pathway and epithelial-mesenchymal transition. Additionally, IHC indicated a tendency toward a reduction in T cell accumulation and an increase in M2-type macrophage infiltration in tumors that recurred. CONCLUSION: A TP53 mutation-mediated immune-suppressive state in the tumor microenvironment and TGF-ß1-PAX7-mediated EMT might contribute to the promotion of recurrence in patients with HNSCC after postoperative radiotherapy.
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Neoplasias de Cabeza y Cuello , Factor de Crecimiento Transformador beta1 , Transición Epitelial-Mesenquimal/genética , Neoplasias de Cabeza y Cuello/genética , Humanos , Papillomaviridae , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND: Many reports demonstrate that a high tumor mutation burden (TMB-H) is closely associated with good prognosis of cancer. However, specific studies investigating the association of various TMB statuses with overall survival in patients with solid tumors are scarce. PATIENTS AND METHODS: In the present study, we investigated the association of TMB status with overall survival in 5,072 patients with cancer from the HOPE project and clarified the specific mechanism responsible for the good prognosis of the TMB-H group. All tumors were classified into one of four groups based on TMB: ultralow (UL), low (L), intermediate (I) and high (H). RESULTS: The TMB-H group had a better prognosis than the TMB-I and TMB-L groups, but not than the TMB-UL group. Analyzing the expression of 293 immune response-associated genes, 17 genes were up-regulated in the TMB-H group compared to the TMB-I and TNB-L groups, and two genes [CD274 and interferon-γ (IFNG)] were identified as good prognostic factors. Analysis of immune cell populations inside tumors demonstrated that the frequencies of exhausted CD8+ T-cells, activated effector CD8+ T-cells and natural killer cells were significantly higher in the TMB-H group. The T-cell receptor repertoire numbers and the diversity evenness score (DE50) were lower in the TMB-H group than in TMB-UL group; however, no association of the DE50 value with the binding or elution affinity of epitope peptides from neoantigens was found. CONCLUSION: One possible mechanism for the good prognosis of the TMB-UL group compared to the TMB-H group might be that the TMB-UL group features a balance between immunosuppression and immunostimulation.
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Linfocitos T CD8-positivos , Neoplasias , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/patología , Humanos , Mutación , Neoplasias/patología , PronósticoRESUMEN
BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.
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Antígenos CD8/genética , Neoplasias Colorrectales/genética , Receptor de Muerte Celular Programada 1/genética , Neoplasias Gástricas/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/farmacología , Antígenos CD8/aislamiento & purificación , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Humanos , Procesamiento de Imagen Asistido por Computador , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos Infiltrantes de Tumor/patología , Masculino , Polimorfismo de Nucleótido Simple/genética , Receptor de Muerte Celular Programada 1/aislamiento & purificación , Programas Informáticos , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapiaRESUMEN
Project High-tech Omics-based Patient Evaluation (HOPE), which used whole-exome sequencing and gene expression profiling, was launched in 2014. A total of ~2,000 patients were enrolled until March 2016, and the survival time was observed up to July 2019. In our previous study, a tumor microenvironment immune type classification based on the expression levels of the programmed death-ligand 1 (PD-L1) and CD8B genes was performed based on four types: A, adaptive immune resistance; B, intrinsic induction; C, immunological ignorance; and D, tolerance. Type A (PD-L1+ and CD8B+) exhibited upregulated features of T helper 1 antitumor responses. In the present study, survival time analysis at 5 years revealed that patients in type A had a better prognosis than those in other categories [5 year survival rate (%); A (80.5) vs. B (73.9), C (73.4) and D (72.6), P=0.0005]. Based on the expression data of 293 immune response-associated genes, 62 specific genes were upregulated in the type A group. Among these genes, 18 specific genes, such as activated effector T-cell markers (CD8/CD40LG/GZMB), effector memory T-cell markers (PD-1/CD27/ICOS), chemokine markers (CXCL9/CXCL10) and activated dendritic cell markers (CD80/CD274/SLAMF1), were significantly associated with a good prognosis using overall survival time analysis. Finally, multivariate Cox proportional hazard regression analyses of overall survival demonstrated that four genes (GZMB, HAVCR2, CXCL9 and CD40LG) were independent prognostic markers, and GZMB, CXCL9 and CD40LG may contribute to the survival benefit of patients in the immune type A group.
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BACKGROUND/AIM: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before. MATERIALS AND METHODS: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated. RESULTS: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes. CONCLUSION: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter.
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Citocinas/metabolismo , Ensayo de Immunospot Ligado a Enzimas/métodos , Neoplasias/inmunología , Glioblastoma/inmunología , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Melanoma/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BACKGROUND/AIM: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy. PATIENTS AND METHODS: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients. RESULTS: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients. CONCLUSION: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.
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Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Glioma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Vacunas contra el Cáncer/inmunología , Polaridad Celular/inmunología , Supervivencia sin Enfermedad , Femenino , Glioma/inmunología , Glioma/patología , Humanos , Interferón gamma/genética , Interleucina-12/genética , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/patología , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Linfocitos T/inmunología , Vacunación/métodosRESUMEN
The aim of the study was to investigate the effect of chemo-radiation on the genetic and immunological status of rectal cancer patients who were treated with preoperative chemoradiotherapy (CRT). The expression of immune response-associated genes was compared between rectal cancer patients treated (n = 9) and not-treated (n = 10) with preoperative CRT using volcano plot analysis. Apoptosis and epithelial-to-mesenchymal transition (EMT) marker genes were analysed by quantitative PCR (qPCR). Other markers associated with the tumor microenvironment (TME), such as tumor-infiltrating lymphocytes (TIL) and immune checkpoint molecules, were investigated using immunohistochemistry (IHC). The clinical responses of preoperative CRT for 9 rectal cancer patients were all rated as stable disease, while the pathological tumor regression score (TRG) revealed 6 cases of grade2 and 3 cases of grade1. According to the genetic signature of colon cancers, treated tumors belonged to consensus molecular subtype (CMS)4, while not-treated tumors had signatures of CMS2 or 3. CRT-treated tumors showed significant upregulation of EMT-associated genes, such as CDH2, TGF-beta and FGF, and cancer stem cell-associated genes. Additionally, qPCR and IHC demonstrated a suppressive immunological status derived from the upregulation of inflammatory cytokines (IL-6, IL-10 and TGF-beta) and immune checkpoint genes (B7-H3 and B7-H5) and from M2-type macrophage accumulation in the tumor. The induction of EMT and immune-suppressive status in the tumor after strong CRT treatment urges the development of a novel combined therapy that restores immune-suppression and inhibits EMT, ultimately leading to distant metastasis control.
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Quimioradioterapia , Cuidados Preoperatorios , Neoplasias del Recto/inmunología , Neoplasias del Recto/terapia , Anciano , Apoptosis/genética , Citocinas/genética , Citocinas/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Persona de Mediana Edad , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Recently, the first series of small molecule inhibitors of PD-1/PD-L1 were reported by Bristol-Myers Squibb (BMS), which were developed using a homogeneous time-resolved fluorescence (HTRF)-based screening investigation of the PD-1/PD-L1 interaction. Additional crystallographic and biophysical studies showed that these compounds inhibited the interaction of PD-1/PD-L1 by inducing the dimerization of PD-L1, in which each dimer binds one molecule of the stabilizer at its interface. However, the immunological mechanism of the antitumor effect of these compounds remains to be elucidated. In the present study, we focused on BMS-202 (a representative of the BMS compounds) and investigated its antitumor activity using in vitro and in vivo experiments. BMS-202 inhibited the proliferation of strongly PD-L1-positive SCC-3 cells (IC50 15 µM) and anti-CD3 antibody-activated Jurkat cells (IC50 10 µM) in vitro. Additionally, BMS-202 had no regulatory effect on the PD-1 or PD-L1 expression level on the cell surface of these cells. In an in vivo study using humanized MHC-double knockout (dKO) NOG mice, BMS-202 showed a clear antitumor effect compared with the controls; however, a direct cytotoxic effect was revealed to be involved in the antitumor mechanism, as there was no lymphocyte accumulation in the tumor site. These results suggest that the antitumor effect of BMS-202 might be partly mediated by a direct off-target cytotoxic effect in addition to the immune response-based mechanism. Also, the humanized dKO NOG mouse model used in this study was shown to be a useful tool for the screening of small molecule inhibitors of PD-1/PD-L1 binding that can inhibit tumor growth via an immune-response-mediated mechanism.
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Antineoplásicos/farmacología , Antígeno B7-H1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Antineoplásicos/química , Antígeno B7-H1/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Silenciamiento del Gen , Antígenos de Histocompatibilidad/genética , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Receptor de Muerte Celular Programada 1/genética , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recently, clinical studies using anti-immune checkpoint molecule antibodies have been successful in solid tumors, such as melanoma and non-small cell lung cancers. However, pancreatic cancers are still intractable and difficult to treat once recurrence or metastasis occurs; thus, novel combined use of immune checkpoint blockade (ICB) with molecular targeted drugs is considered a therapeutic option. Previously, we developed a novel humanized MHC-double knockout (dKO) NOG mouse model and demonstrated that an anti-PD-1 antibody or a STAT3 inhibitor showed anti-tumor effects through an immunological mechanism. In the current study, using a humanized mouse model, we aimed to develop a combination therapy with an anti-PD-1 antibody and a STAT3 inhibitor (STX-0119) for use in vivo against pancreatic cancer. In an in vitro investigation, STX-0119 showed weak to moderate cytotoxic activity against several pancreatic cancer cell lines, which exhibited activated pSTAT3 and weak PD-L1 expression. However, unexpectedly, an in vivo study indicated that the combination of the anti-PD-1 antibody with STX-0119 remarkably reduced the anti-tumor effect and TIL numbers despite the effective anti-tumor activity against pancreatic cancer was produced individually by STX-0119 and the anti-PD-1 antibody. These results suggested that the combination of an anti-PD-1 antibody with specific signal inhibiting drugs should be carefully evaluated to avoid unexpected side effects, and such studies might contribute to the development of an effective combination regimen of ICB with cancer-targeting drugs such as tyrosine kinase inhibitors (TKIs).
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Interacciones Farmacológicas , Genes MHC Clase I/genética , Genes MHC Clase II/genética , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/trasplante , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Noqueados , Oxadiazoles/farmacología , Oxadiazoles/uso terapéutico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Quinolinas/farmacología , Quinolinas/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Quimera por Trasplante/inmunología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recent advances in next-generation sequencing have enabled rapid and efficient evaluation of the mutational landscape of cancers. As a result, many cancer-specific neoantigens, which can generate antitumor cytotoxic T-cells inside tumors, have been identified. Previously, we reported a metastatic melanoma case with high tumor mutation burden, who obtained complete remission after anti-PD-1 therapy and surgical resection. The rib metastatic lesion, which was used for whole-exome sequencing and gene expression profiling in the HOPE project, showed upregulated expression of PD-L1 mRNA and a high single-nucleotide variants number of 2712. In the current study, we focused on a metastatic melanoma case and candidate epitopes among nonsynonymous mutant neoantigens of 1348 variants were investigated using a peptide-HLA binding algorithm, in vitro cytotoxic T-cell induction assay and HLA tetramer staining. Specifically, from mutant neoantigen data, a total of 21,066 9-mer mutant epitope candidates including a mutated amino acid anywhere in the sequence were applied to the NetMHC binding prediction algorithm. From in silico data, we identified the top 26 mutant epitopes with strong-binding capacity. A cytotoxic T-cell induction assay using 5 cancer patient-derived PBMCs revealed that the mutant ARMT1 peptide sequence (FYGKTILWF) with HLA-A*2402 restriction was an efficient neoantigen, which was detected at a frequency of approximately 0.04% in the HLA-A24 tetramer stain. The present success in identifying a novel mutant antigen epitope might be applied to clinical neoantigen screening in the context of an NGS-equipped medical facility for the development of the next-generation neoantigen cancer vaccines.
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Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Epítopos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Citocinas/biosíntesis , Epítopos/genética , Antígeno HLA-A24/genética , Antígeno HLA-A24/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/metabolismo , Melanoma/patología , Mutación , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T , Resultado del Tratamiento , Secuenciación del ExomaRESUMEN
In 2014, the Shizuoka Cancer Center launched project Hightech Omicsbased Patient Evaluation (HOPE), which features whole exome sequencing (WES) and gene expression profiling (GEP) of fresh surgical specimens from cancer patients. With the development of clinical trials of programmed death1 (PD1)/PDligand 1 (PDL1) blockade, PDL1 expression and a high tumor mutation burden become possible biomarkers that could be used to predict immune responses. In this study, based on WES and GEP data from 1,734 tumors from the HOPE project, we established a tumor microenvironment (TME) immunetype classification consisting of 4 types to evaluate the immunological status of cancer patients and analyze immunological pathways specific for immune types. Project HOPE was conducted in accordance with the Ethical Guidelines for Human Genome and Genetic Analysis Research with the approval of the Institutional Review Board. Based on the expression level of the PDL1 and CD8B genes, the immunological status was divided into 4 types as follows: A, PDL1+CD8B+; B, PDL1+CD8B; C, PDL1CD8B; and D, PDL1CD8B+. Type A, with PDL1+ and CD8B+, exhibited an upregulation of cytotoxic T lymphocyte (CTL) killingassociated genes, Tcell activation genes, antigenpresentation and dendritic cell (DC) maturation genes, and Tcellattracting chemokine genes, which promoted Th1 antitumor responses. By contrast, type C, with PDL1 and CD8B, exhibited a low expression of Tcellactivating genes and an upregulation of cancer driver gene signaling, which suggested an immunesuppressive status. With regard to hypermutator tumors, PDL1+ hypermutator cases exhibited a specific upregulation of the IL6 gene compared with the PDL1 cases. On the whole, our data indicate that the classification of the TME immune types may prove to be a useful tool for evaluating the immunological status and predicting antitumor responses and prognosis.
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Antígeno B7-H1/genética , Antígenos CD8/genética , Secuenciación del Exoma/métodos , Perfilación de la Expresión Génica/métodos , Neoplasias/inmunología , Microambiente Tumoral , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Interleucina-6/genética , Mutación , Neoplasias/genética , PronósticoRESUMEN
Project High-tech Omics-based Patient Evaluation (HOPE), including comprehensive whole-exome sequencing (WES) and gene expression profiling (GEP) using freshly resected tumor specimens, has been in progress since its implementation in 2014. Among a total of 1,685 cancer patients, 13 melanoma patients were registered in the HOPE Project and were characterized using multi-omics analyses. Among the 13 melanoma patients, 4 were deceased, and 9 were alive. The mean overall survival (OS) and relapsefree survival (RFS) times of the melanoma patients were 16.9 and 14.7 months, respectively. Previously, we developed an immune responseassociated gene list, which consisted of 164 genes in Project HOPE, for evaluating the immunological status. In the present study, the association of immune responseassociated gene expression with immunological parameters, such as programmed death-ligand 1 (PD-L1) and CD8 expression levels, single nucleotide variant (SNV) number, and Vogelstein driver gene mutation number, was investigated. With respect to PD-L1 expression, both immuno-suppression and immuno-stimulation-related genes were upregulated in PD-L1-positive melanomas. In contrast, regarding Vogelstein driver mutations, several T-cell activation-related genes were significantly downregulated in the high driver gene mutation group. In addition, many T-cell activation-related genes were upregulated in the CD8-positive melanomas. The correlation of immune response-associated gene expression with the survival time of the melanoma patients was investigated. Eight specific genes were commonly identified as genes that were significantly correlated for both the overall OS and RFS time, which could be possible prognostic factors for melanoma patients. These results revealed that an immune response-associated gene panel could be an informative tool for evaluating the immunological status prior to clinical immunotherapy in the upcoming era of genomic cancer medicine.
Asunto(s)
Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Interleucina-8/genética , Melanoma/genética , Mutación , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico/genética , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
STAT3 is considered to be a key molecule to mediating tumor-induced immunosuppression in various manners at tumor sites, by acting through immune-regulatory cytokines derived from the tumor cells. Specific anti-STAT3 inhibitors have been developed using nude mouse models transplanted with human tumor cells. However, mouse systems cannot accurately represent the human immune response induced by STAT3 inhibitors, and more humanized therapeutic model based on human immune cells and tumors are needed. In the present study, an immune-deficient NOG mouse with the deletion of both MHC-class I and class II genes, an MHC-double knockout mouse (dKO-NOG), was developed and used to establish humanized immunotherapeutic model. We investigated the immunological effect of the STAT3 inhibitor STX-0119 against TMZ-resistant (TMZ-R) U87 glioma tumors by using humanized dKO-NOG mice. We compared the anti-tumor effects of STX-0119 between the nude and humanized dKO-NOG mouse models. An in vivo study using the nude mouse model showed that STX-0119 inhibited the growth of TMZR U87 tumors, but accumulation of tumor-infiltrating lymphocytes (TILs) were not promoted compared with the control levels. In contrast, STX-0119 inhibited tumor growth more rapidly and strongly in humanized dKO-NOG mice than in nude mice, and a large amount of TILs, mainly consisting of CD8+ T cells and macrophages, were found in the tumors. These results suggest that STX-0119 has anti-tumor activity occurring through the promotion of TIL accumulation at the tumor site and that humanized dKO-NOG mouse system may be a powerful tool to evaluate the effects of STAT3 inhibitors on human systems.
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Glioblastoma/tratamiento farmacológico , Leucocitos Mononucleares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Oxadiazoles/uso terapéutico , Quinolinas/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Resistencia a Antineoplásicos , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Leucocitos Mononucleares/trasplante , Ratones , Ratones Noqueados , Ratones Desnudos , Oxadiazoles/farmacología , Quinolinas/farmacología , Temozolomida , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Project HOPE (High-tech Omics-based Patient Evaluation) has been in progress since 2014 and uses whole-exome sequencing (WES) and gene expression profiling (GEP). Among a total of 1,685 patients with cancer, 13 with melanoma were registered and characterized using multi-omics analyses to investigate specific biomarkers in responders to programmed cell death-1 (PD-1) blockade. MATERIALS AND METHODS: The patients with melanoma comprised of six males and seven females, and their mean age was 68 years. Five patients were treated with nivolumab, and two were responders. RESULTS: GEP analysis demonstrated that PD-L1 expression was positive in for cases, and melanoma-associated antigens and tumor signaling-associated genes were up-regulated in tumor compared with normal tissues. Additionally, WES analysis indicated more single nucleotide variants (SNVs) per melanoma tumor compared to other tumor types. Remarkably, a case of complete remission after nivolumab therapy showed high expression of PD-L1 protein and the highest number of SNVs. CONCLUSION: The novel approach used in Project HOPE might be an efficient tool that facilitates identifying specific biomarkers predictive of good responders to anti-PD-1 therapy.
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Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Melanoma/genética , Melanoma/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Japón , Masculino , Melanoma/tratamiento farmacológico , Persona de Mediana Edad , Mutación , Nivolumab , Polimorfismo de Nucleótido Simple , Inducción de Remisión , Análisis de Secuencia de ADN , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Resultado del TratamientoRESUMEN
PURPOSE: Humanized mouse models using NOD/Shi-scid-IL2rγnull (NOG) and NOD/LtSz-scid IL2rγnull (NSG) mouse are associated with several limitations, such as long incubation time for stem cell engraftment and the development of xenograft versus host disease in mice injected with peripheral blood mononuclear cells (PBMCs). To solve problems, we used humanized major histocompatibility class I- and class II-deficient NOG mice (referred to as NOG-dKO) to evaluate the antitumor effect of anti-programmed death-1 (PD-1) antibody. EXPERIMENTAL DESIGN: Humanized NOG-dKO mice, in which human PBMCs and human lymphoma cell line SCC-3, or glioblastoma cell line U87 were transplanted, were used as an immunotherapy model to investigate the effect of anti-PD-1 antibody. A biosimilar anti-PD-1 mAb generated in our laboratory was administered to humanized NOG-dKO mice transplanted with tumors. RESULTS: Within 4 weeks after transplantation, human CD45+ cells in antibody-treated mice constituted approximately 70% of spleen cells. The injection of anti-PD-1 antibody reduced by more 50% the size of SCC-3 and U87 tumors. In addition, induction of CTLs against SCC-3 cells and upregulation of natural killer cell activity was observed in the antibody-treated group. Tumor-infiltrating lymphocyte profiling showed that more exhausted marker (PD1+TIM3+LAG3+) positive T cells maintained in anti-PD-1 antibody-treated tumor. A greater number of CD8+ and granzyme-producing T cells infiltrated the tumor in mice treated with the anti-PD-1 antibody. CONCLUSIONS: These results suggest that NOG-dKO mice might serve as a good humanized immunotherapy model to evaluate the efficacy of anti-PD-1 antibody prior to the clinical treatment. Clin Cancer Res; 23(1); 149-58. ©2016 AACR.
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Antineoplásicos Inmunológicos/farmacología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias/genética , Neoplasias/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Inmunofenotipificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/trasplante , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Análisis de Supervivencia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
BACKGROUND: Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach to overcome resistance is needed. MATERIALS AND METHODS: The efficacy of a combination treatment of STAT3 inhibitor, STX-0119, with rapamycin was investigated against our established TMZ-resistant U87 cell line. RESULTS: The growth-inhibitory effect of the combination treatment was significant against the TMZ-R U87 cell line (IC50: 78 µM for STX-0119, 30.5 µM for rapamycin and 11.3 µM for combination of the two). Western blotting analysis demonstrated that the inhibitory effect of STX-0119 on S6 and 4E-BP1 activation through regulation of YKL-40 expression occurred in addition to the inhibitory effect of rapamycin against the mTOR pathway. CONCLUSION: These results suggest that the STAT3 pathway is associated with the mTOR downstream pathway mediated by YKL-40 protein, and the combination therapy of the STAT3 inhibitor and rapamycin could be worth developing as a novel therapeutic approach against TMZ-resistant relapsed gliomas.