In the last decade, methods to investigate the immune status of the tumor microenvironment (TME) and predict immune checkpoint blockade efficacy have been intensively developed as immunoscores or immune signatures. However, since the immunological gene signature is a complex and time-consuming approach, a simpler scoring system using numerical data is needed to evaluate a large number of tumors. We previously established a TME immune-type classification system based on PD-L1 and CD8B gene expression, and 5032 cancer patients were classified into 4 types. Based on the expression levels of immune response-associated genes in each type, these genes were assigned scores ranging 1 to 4, representing a spectrum from immunosuppressive to immunostimulating genes. We herein calculated tumor immune status scoring algorithm (TIMMUSCORA) scores based on the expression data of 300 immune response-associated genes in 5,013 pancancer patients, and investigated the relationship of these scores with the prognosis and other clinicopathological features of cancer patients. Rectal cancers with higher scores than the cut-off value of 0 showed a good prognosis, which was closely associated with the immune response-associated genes PDCD1, GZMB, TNFRSF10C, EBI3, and IRF1. A correlation analysis of the rectal cancer cohort suggested that high TIMMUSCORA scores correlated with high TMB value and the consensus molecular subtyping 1 status, while low scores correlated with WNT gene expression. Therefore, the TIMMUSCORA system has potential in evaluations of the immune status of the TME and the prognosis of solid cancers.

Chromosomal instability (CIN) is associated with immunosuppression in the tumor microenvironment (TME), resulting in cancer progression, metastasis, and resistance to immune checkpoint blockade therapy. We previously established a tumor immune status scoring algorithm (TIMMUSCORA) in which the immune status in the TME is evaluated numerically from activation to suppression. In the present study, we clarified the relationships between structural variation-related parameters and immunological features by applying TIMMUSCORA to solid cancers. Whole genome sequencing (WGS) and gene expression profiling (GEP) data were obtained from 394 cancer patients, and CIN-related parameters, such as the tumor mutation burden (TMB), structural variant (SV), microsatellite instability (MSI) score, ploidy, homologous recombination deficiency score, and chromothripsis (CT) and whole genome duplication (WGD) scores, were assessed. The TIMMUSCORA system demonstrated that most CIN-related parameters contributed to the low TIMMUSCORA score implicating an immunosuppressive state. Comparisons of differentially expressed genes between WGD- or CT-positive and -negative tumors showed the down-regulation of B cell markers, the down-regulation of myeloid cell markers, and the up-regulation of the NKG2D gene. In addition, the following novel observations were verified; (1) TP53 and EGFR mutation events can be associated with WGD and low TIMMUSCORA scores, and (2) NK cell activation and cancer–testis antigen gene up-regulation might be associated with CT. These results suggest that TIMMUSCORA might be useful tool evaluating immune status of CIN-harboring tumors. In future, the specific mechanism for CIN-associated immunosuppression in the tumor can be explored and clarified.

Background/Aim: Immune checkpoint blockade has achieved great success as a targeted immunotherapy for solid cancers. However, small molecules that inhibit programmed death 1/programmed death ligand 1 (PD-1/PDL1) binding are still being developed and have several advantages, such as high bioavailability. Previously, we reported a novel PD-1/PD-L1-inhibiting small compound, SCL-1, which showed potent antitumor effects on PD-L1+ tumors. These effects were dependent on CD8+ T-cell infiltration and PD-L1 expression on tumors. The present study investigated the in vivo antitumor activity of SCL-1 in various mouse syngeneic tumor models. Materials and Methods: Twelve syngeneic mice models of tumors, such as colon, breast, bladder, kidney, pancreatic, non-small cell lung cancers, melanoma, and lymphomas, were used for in vivo experiments. Tumor mutation burden (TMB) was analyzed by whole exome sequencing (WES) using reference DNA from mouse blood. The proportion of CD8+ T-cells infiltrating tumors before and after treatment was assessed using flow cytometry and immunohistochemistry (IHC). Results: SCL-1 had a markedly greater antitumor effect (11 sensitive tumors and 1 resistant tumor among the 12 tumor types) than the anti-mouse PD-1 antibody (8 sensitive tumors and 4 resistant tumors). In addition, the tumor growth inhibition rate (%) was more closely associated with TMB in the SCL-1 group than in the anti-PD-1 antibody group. Furthermore, in vivo experiments using PD-L1 gene knockout and lymphocyte- depletion technologies demonstrated that the antitumor activity of SCL-1 was dependent on CD8+ T-cell infiltration and PD-L1 expression in tumors. Conclusion: SCL-1 has great potential as an oral immunotherapy that targets immune checkpoint molecules in cancer treatment.

BACKGROUND/AIM:Recently, neoantigen (NA) profiling has been intensively performed for the development of novel immunotherapy. We previously reported a melanoma case with a high tumor mutation burden that achieved complete remission after anti-programmed death-1 therapy. We herein revisited the same case, characterized the NA profiles of other metastatic lesions using in silico algorithms and in vitro CTL assays, and investigated the immunological status, including tumor-infiltrating lymphocytes and the T cell receptor (TCR) repertoire profile, in metastatic sites. MATERIALS AND METHODS:NA candidates obtained from whole-exome sequencing were applied to the HLA-binding prediction algorithm, NetMHCpan4.1. HLA-A*2402-restricted sequence candidates with a strong binding capacity (<50 nM) and elution affinity (<1%) were selected and evaluated for synthetic peptide candidates. The immunological status in metastatic sites was characterized using gene expression profiling, immunohistochemistry, and a TCR repertoire analysis. RESULTS:The genomic analysis revealed that all metastatic sites, such as costal, intra-muscular, and brain lesions, had >1,500 SNVs, and 12 driver mutations were common to all sites. New driver mutations were identified in intra-muscular (KMT2C: p.P3292S) and brain (JAK1: p.S404P) metastases and a functional analysis of these mutations revealed that JAK1 mutation exhibited a promoting effect on invasion activity. CTL assays using synthetic NA peptides identified more NA epitopes in brain metastasis. CONCLUSION:These results might suggest that the heterogeneity of driver gene mutations is unremarkable, while immunological response is variable in metastatic sites. As a result, the genomic and immunological investigation has provided a very valuable and informative suggestion regarding better cancer therapy decisions.

Quickly identifying driver gene mutations in solid cancers is important. However, next-generation sequencing (NGS)-based mutation detection methods are time-consuming and expensive. Peptide nucleic acid (PNA) probe-based mutant mRNA detection systems are quick and inexpensive. We previously demonstrated that epidermal growth factor receptor (EGFR)-mutations were efficiently visualized in formalin-fixed paraffin-embedded (FFPE) specimens from transplanted non-small cell lung cancer (NSCLC) tumors using an EGFR mutation-specific PNA:DNA probe. Herein, the efficiency of PNA:DNA probes in detecting EGFR-mutations in FFPE specimens from patients with NSCLC and the colocalization of EGFR-mutations with tumor-infiltrating lymphocyte (TIL) status were determined. The EGFR mutation L858R-specific PNA:DNA probe detected heterogeneously localized mutations with a sensitivity similar to detection with the anti-L858R antibody. TIL analysis of L858R-mutated tumors revealed that CD8+PD-1+ T cells and CD68+ macrophages were scarce in tumors, but in the cytokeratin-positive intra-tumoral regions, CD4+, FoxP3+, and CD204+ cells tended to be more abundant in the L858R-positive tumor area than in the negative area. Thus, PNA:DNA probes specific for EGFR-mutations can detect areas with heterogeneous EGFR mutants in whole cancer tissues and can be used to evaluate the mutation-associated TIL status in EGFR-mutant cancer tissues.

Although triple-negative breast cancers are still challenging to treat, the development of novel neoadjuvant chemotherapy combined with immune checkpoint antibodies is promising. Our group developed the small compound-based anti-PD-1/PD-L1 inhibitor SCL-1 and reported its potent anti-tumor effects on various syngeneic mouse tumors. We herein investigated the efficacy of SCL-1 using an in vivo humanized NOG mouse system. We established a humanized mouse system using double major histocompatibility complex-knockout NOG mice transplanted with MDA-MB231 breast cancer cells and HLA-matched human PBMCs. Tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry and real-time PCR. An RNA-sequencing analysis (RNA-seq) of SCL-1-treated MDA-MB231 tumors was performed to identify differentially expressed genes. Orally administered SCL-1 exerted potent anti-tumor effects with > 50% reduction in tumor sizes, which were dependent on PD-L1 expression and T-cell infiltration. Its effects were significantly stronger than those of nivolumab or atezolizumab. A TIL analysis revealed effector CD8+ T cells expressing cytotoxic markers and exhausted markers as well as increases in NK cells and B cells. RNA-seq showed the up-regulated expression of tumor-specific long non-coding (lnc) RNAs in SCL-1-treated tumor tissues, some of which exhibited high HLA-binding activity. SCL-1 exerted strong tumor growth inhibitory effects that were mediated by effector T-cell induction inside tumors and the up-regulated expression of lncRNAs as neoantigens leading to CTL activation. The up-regulated expression of lncRNAs in SCL-1-treated MDA-MB231 tumors is a novel result and may be one of the mechanisms responsible for the anti-tumor efficacy of SCL-1.

BACKGROUND/AIM:Advances in whole-genome sequencing have revealed that many RNA genes have important functions in various cellular processes and long non-coding RNAs (lncRNAs) have been attracting increasing attention. In the present study, we characterized a large number of lncRNAs from cancer patients and classified them based on their relationships with immune response-associated genes and cancer prognosis. MATERIALS AND METHODS:435 lncRNAs were identified as differentially expressed genes from 5,013 cancer patients using the limma package and were narrowed down to 80 lncRNAs commonly expressed in more than 4 cancer types. The relationships of these lncRNAs with immune response-associated genes were examined using Pearson's correlation and the tumor immune scoring system. Through a receiver operating characteristic (ROC) analysis of highly scored lncRNAs with the tumor-infiltrating lymphocyte (TIL) status using QuanTIseq, a cancer prognostic analysis was conducted using a multivariate Cox proportional hazards model. RESULTS:In a correlation analysis of 80 lncRNA genes and immune response-associated genes, lncRNA genes were scored and classified as high, intermediate, and low immune status score groups based on the Tumor Immune Status Scoring Algorithm (TIMMUSCORA). The ROC analysis of lncRNA genes with a high score (>200) revealed high sensitivity for the identification of TILs, such as CD8+ T cells and B cells. Furthermore, 11 of 80 lncRNA genes were identified as prognostic genes. CONCLUSION:Twelve lncRNA genes with a high immune status score were identified as immunogenic and CD8+ T cell infiltration-associated genes, with two being indicators of a positive cancer prognosis. These results indicate the potential of 12 lncRNA genes with a high immune status score as immunogenic biomarkers associated with a positive immune response and prognosis.

Local recurrence after surgical resection is a major clinical challenge in patients with advanced head and neck cancer (HNC). We previously reported using transfer of a flap treated with dendritic cells (DCs) (“immuno-flap”) as a novel cancer immunotherapy in a rat model of HNC. However, several problems, such as the improvement of DC yield and the verification of DC tracking, remained to be solved. The initial DC production protocol was revisited, and the specific mechanism of the antitumor effect associated with functional DCs was investigated. The yield and the purity of potent DCs were improved, and an in vivo tracking experiment revealed that, when injected into the skin flap, the DCs migrated to local lymph nodes within 24 h. In an in vivo long-term protection model in which DCs were injected into the skin flap weekly for 4 weeks, no strong antitumor effect on HNC tumor progression was observed, but a remarkable pathological effect was verified. Moreover, an enzyme-linked immunospot assay demonstrated that more CD8+ T cells positive for PD-1, CD40LIG, and TNF-α expression and producing interferon γ specific to SCC-158 tumor cells were identified in tumors and spleens from rats treated using immuno-flaps. These results indicate that immuno-flap transfer to prevent local recurrence in patients with HNC has potential for clinical application given the improved yield and quality of antitumor DCs.

In drug discovery research, it is important to identify target proteins of bioactive small-molecule compounds and analyse their functions. In this study, we examined whether target membrane proteins could be captured by compounds that bind to membrane proteins on the cell surface. For this purpose, we performed affinity purification using the compound-immobilized nanomagnetic beads. Affinity purification with nanomagnetic beads is known to be effective for determining the protein binding partners of small molecules. However, most previous studies have targeted proteins in the cytoplasm. As a model compound, we chose BMS-1166 (a representative small-molecule compound from Bristol Myers Squibb), a PD-1/PD-L1 immune checkpoint inhibitor that binds to PD- L1 and promotes PD-L1 dimerization. BMS-1166-immobilized beads were manufactured and incubated with extracts of cells with high PD-L1 protein expression. The bound protein was confirmed by western blotting and proteomic analysis to be PD-L1. BMS-1166-immobilized nano-magnetic beads were able to specifically bind and capture the membrane protein PD-L1. In addition, high-purity protein could be obtained from cell extracts in a single step. This is the first report of the purification of a membrane protein to high purity with nanobeads. Nanomagnetic beads with immobilized compounds are an effective tool for identifying the protein binding partners of small molecules, especially when the targets are membrane proteins.

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.

Survival benefit in humanized NOG-dKO mice compared with humanized control NOG mice.

High expression levels of phospho-STAT3 and PD-L1 proteins in the SCC-3 cell line

Engraftment of human PBMCs in NOG-dKO mice after transplantation.

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.

Engraftment of human PBMCs in humanized NOG-dKO mice treated with anti-PD-1 antibody.

Statistical analysis of iFN-γ-based CTL assay and NK cell assay.

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.

Flow cytometry analysis of TIL from control or anti-PD-1 antibody-treated SCC-3 tumors.

Effect od anti-PD-1 antibody on SCC3 and U87 tumor growth in vivo.