table_6_Changes in the TCRβ Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report.xlsx

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB–IIC–III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor β (TCRβ) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRβ repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

同种异体治疗性疫苗CSF-470在一项随机II/III期临床试验（CASVAC-0401，NCT01729663）中，针对IIB、IIC至III期皮肤黑色素瘤患者，相较于中剂量干扰素α2b，可显著提升其无远处转移生存期。在完成2年的CSF-470免疫接种方案后，编号006的患者出现多处肺部及1处皮下黑色素瘤转移灶，后续对该转移灶进行了切除手术。本报告分析了接种过程中患者血液及肿瘤组织内免疫细胞群的变化，重点聚焦T细胞受体库（T-cell repertoire）。免疫组织化学检测显示，相较于原发肿瘤，转移灶内浸润的CD8+、CD4+及CD20+淋巴细胞数量显著增多。淋巴细胞紧密黏附于含有颗粒酶B（Granzyme-B）颗粒的濒死肿瘤细胞。全外显子测序评估结果表明，该肿瘤的肿瘤突变负荷为中至高水平，BRAFV600E为主要致癌驱动突变。突变特征显示存在大量双嘧啶位点突变，这是黑色素瘤的典型特征。通过RNA测序（RNA-Seq）分析皮下转移灶的相关肿瘤及免疫基因，结果检测到典型黑色素瘤抗原及增殖相关肿瘤基因的表达，同时还检测到刺激性与抑制性免疫转录本，以及T细胞效应功能活化的相关证据。外周血监测结果显示，免疫接种方案结束时，患者外周血中CD4+及CD8+细胞数量有所升高。通过CDR3-T细胞受体β（TCRβ）测序分析发现，在整个免疫接种过程中，外周T细胞受体库中出现了新克隆的生成，且寡克隆性程度升高。在血液中检测到一种克隆漂移现象：部分预先存在的克隆及新出现的克隆发生扩增，同时其他克隆的占比降低。在肿瘤浸润淋巴细胞中，占比50%的优势克隆均为在CSF-470免疫接种后于血液中扩增的新克隆与预先存在的克隆。这些克隆随时间持续存在：完成免疫接种2年后，转移灶中51%的克隆仍可在患者血液中被检测到。本报告是首项针对接受CSF-470疫苗接种的黑色素瘤患者，其TCRβ受体库调控情况的研究报道。免疫接种后，外周免疫细胞群及肿瘤微环境中观察到的变化表明，该疫苗可诱导抗肿瘤适应性免疫受体库，该受体库可抵达肿瘤病灶，并至少在血液中持续存在2年。