Yasuto Akiyama

Objective: To evaluate the impact of CYP2A6 genetic polymorphisms on survival outcomes in patients with pancreatic ductal adenocarcinoma (PDAC) receiving perioperative S-1 therapy. Background: S-1, an oral fluoropyrimidine containing tegafur, is widely used for perioperative treatment of PDAC in Japan. Tegafur requires metabolic activation to 5-fluorouracil, primarily by CYP2A6 . Given the high prevalence of reduced-function CYP2A6 variants in East Asian populations, their clinical significance in PDAC remains unclear. Methods: This retrospective study included 145 patients with PDAC who underwent curative resection and received adjuvant S-1, including 78 patients who had received S-1–containing neoadjuvant therapy. Germline CYP2A6 variants were identified using whole-exome sequencing of peripheral blood samples. Decreased-function alleles (*4, *7, *9) and the rare nonsense allele *48 were evaluated. Patients were classified into poor, intermediate, and normal metabolizer groups. Survival outcomes were analyzed using Kaplan-Meier and Cox regression analyses. Results: Decreased-function CYP2A6 genotypes were identified in 82 patients (56.6%), including 6 poor metabolizers (4.1%). These patients had a higher frequency of lymph node metastasis than those with normal genotypes (78.0% vs. 52.4%, P =0.001). Recurrence-free survival (RFS) was significantly shorter (median, 19.2 vs. 32.3 mo; P =0.040), whereas overall survival (OS) was shorter but not statistically significant (47.9 vs. 62.2 mo; P =0.104). In multivariable analysis, CYP2A6 poor metabolizer status was independently associated with OS (HR, 3.17; 95% CI, 1.06–9.46; P =0.039). Among the 78 patients who received S-1–containing neoadjuvant therapy, both OS and RFS were significantly worse in those with decreased-function genotypes. Conclusions: Decreased CYP2A6 functional genotypes were associated with poorer oncological outcomes in patients with PDAC receiving perioperative S-1 therapy, suggesting that the CYP2A6 genotype may serve as a biomarker for optimizing treatment strategies.

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.

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.

Whole‐genome analyses have revealed that large‐scale structural variations (SVs) such as whole‐genome duplication (WGD) occur early in the development of many cancers. However, the diversity of chromosomal abnormalities within tumors before and after WGD remains poorly understood. Here, we analyzed various types of Japanese tumor genomes via whole‐genome sequencing and examined the diversity of WGD by focusing on large SVs at the chromosomal level. WGD was detected in 52% of cases, while the frequency of chromothripsis (CT) was 20%. Although aneuploidy via deletion of chromosome arms was common in many cancers, in rare ovarian cancers, all chromosomes were near‐haploidy before WGD. Minor allele analysis revealed that many non‐mutated ohnolog genes drifted down chromosome arms after WGD and returned to normal ploidy, but only 17p, including TP53, which is also an ohnolog, underwent loss of heterozygosity due to arm deletion before WGD in most cancers. TP53 mutations were frequently detected in WGD and CT‐positive tumors, and these SVs strongly correlated with homologous recombination deficiency scores. Furthermore, these tumors had many mutations that continued to generate neoantigens and resulted in worse survival outcomes. Diversity analysis of tumors with WGD will provide a new perspective on structural abnormalities in tumor genomes.

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: 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 (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.

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/PD-L1) 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.