Kobayashi Daiki, Kosumi Takuya, Lam Queenie Lai Kwan, Fujita Shigeharu, Hijikata Yasuki, Takeda Kaori, Narita Tomoya, Yamashita Naomi, Richard Guilhem, De Groot Anne S, Yamashita Naohide
Division of General Internal Medicine, Department of Medicine, Tokyo Medical University Ibaraki Medical Center, Ibaraki, Japan.
Fujita Health University, Toyoake, Japan.
Front Immunol. 2025 Jul 11;16:1609116. doi: 10.3389/fimmu.2025.1609116. eCollection 2025.
Immune checkpoint inhibitors show limited efficacy in tumors with low tumor mutational burden, partly due to insufficient neoantigen presentation.
We developed a novel approach for neoantigen identification using circulating tumor cells (CTCs) isolated via leukapheresis and flow cytometry. Peripheral blood mononuclear cells (PBMCs) were collected from 11 stage IV cancer patients and 2 healthy volunteers. CTCs were enriched by depleting CD45 hematopoietic cells and selecting CD45Vimentin cells, which were confirmed cytologically to contain malignant cells. Hematopoietic lineage analysis showed that over 50% of the CTC fraction consisted of non-hematopoietic cells. DNA extracted from both the CTC and normal hematopoietic fractions underwent exome sequencing. Neoantigens were identified using the Ancer bioinformatics platform.
In representative patients with gastric and salivary gland cancers, 94,636 and 46,423 CTCs were isolated, respectively. DNA yields were sufficient for exome sequencing without amplification or extensive cell culture. A total of 102 (patient with gastric cancer) and 108 (patient with salivary gland cancer) neoantigens were identified in each subject, including high-ranking T-cell epitopes derived from single nucleotide variants and frameshift mutations. According to the same procedures we could successfully identify a large number of neoantigens from the CTCs of all stage IV cancer patients. This confirms the feasibility of identifying individual patient-specific neoantigens from CTCs without requiring tumor biopsies.
This is the first study to demonstrate successful neoantigen identification using non-amplified CTCs isolated by apheresis and flow cytometry. The approach provides a minimally invasive, scalable alternative for neoantigen discovery and may better capture tumor heterogeneity compared to single-site biopsies. This method holds promise for enabling rapid, personalized immunotherapy strategies, including peptide vaccines, dendritic cell vaccines, and mRNA-based treatments.
免疫检查点抑制剂在肿瘤突变负担低的肿瘤中疗效有限，部分原因是新抗原呈递不足。
我们开发了一种使用通过白细胞分离术和流式细胞术分离的循环肿瘤细胞（CTC）进行新抗原鉴定的新方法。从11名IV期癌症患者和2名健康志愿者中收集外周血单核细胞（PBMC）。通过去除CD45造血细胞并选择CD45Vimentin细胞来富集CTC，经细胞学确认这些细胞含有恶性细胞。造血谱系分析表明，超过50%的CTC部分由非造血细胞组成。从CTC和正常造血部分提取的DNA进行外显子组测序。使用Ancer生物信息学平台鉴定新抗原。
在具有代表性的胃癌和唾液腺癌患者中，分别分离出94,636个和46,423个CTC。DNA产量足以进行外显子组测序，无需扩增或广泛的细胞培养。在每个受试者中分别鉴定出102个（胃癌患者）和108个（唾液腺癌患者）新抗原，包括源自单核苷酸变异和移码突变的高级别T细胞表位。按照相同程序，我们能够从所有IV期癌症患者的CTC中成功鉴定出大量新抗原。这证实了无需肿瘤活检即可从CTC中鉴定个体患者特异性新抗原的可行性。
这是第一项证明使用通过白细胞分离术和流式细胞术分离的未扩增CTC成功鉴定新抗原的研究。该方法为新抗原发现提供了一种微创、可扩展的替代方法，与单部位活检相比，可能更好地捕捉肿瘤异质性。该方法有望实现快速、个性化的免疫治疗策略，包括肽疫苗、树突状细胞疫苗和基于mRNA的治疗。
