data_sheet_1_Changes in the TCRβ Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report.PDF

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.

异种细胞治疗性疫苗CSF-470在一项随机性的II/III期临床试验（CASVAC-0401，NCT01729663）中，对于IIB-IIC-III期皮肤黑色素瘤患者而言，其在远处无病生存期方面相较于中等剂量的IFNα2b显示出显著的益处。在完成2年的CSF-470免疫方案后，患者编号006出现了多个肺部和一处皮下黑色素瘤转移灶；后者已行切除。在本报告中，我们对疫苗接种过程中血液和肿瘤组织中免疫细胞群体的变化进行了分析，特别关注T细胞库。免疫组化检测显示，相较于原发肿瘤，转移灶中浸润的CD8+、CD4+和CD20+淋巴细胞显著增加。淋巴细胞紧附于含有Granzyme-B颗粒的死亡肿瘤细胞。全外显子测序评估表明，肿瘤突变负荷为中度至高度，其中BRAFV600E为主要致癌驱动因素。突变特征表现为二嘧啶位置的大数量突变，这是黑色素瘤的典型特征。通过RNA-Seq分析，针对皮下转移灶的相关肿瘤和免疫相关基因进行了研究，揭示了典型黑色素瘤抗原和增殖相关肿瘤基因的表达。同时检测到了刺激性和抑制性免疫转录本，以及T细胞效应功能活跃的证据。在免疫方案结束时，外周血监测显示CD4+和CD8+细胞数量增加。通过CDR3-T细胞受体β（TCRβ）测序，在整个免疫过程中观察到外周T细胞免疫库中新克隆的产生和寡克隆性的增加。血液中检测到克隆的选择性扩增和新出现克隆的增多，同时其他克隆数量减少。在肿瘤浸润淋巴细胞中，占主导地位的克隆（50%）既包括新出现也包括预先存在的，这些克隆在CSF-470免疫后随血液扩增。这些克隆在时间上持续存在，因为在完成免疫2年后，转移灶中仍检测到51%的克隆存在于血液中。这是首次报告使用CSF-470疫苗免疫黑色素瘤患者的TCRβ库发生调节。免疫后，外周免疫群体以及肿瘤细胞组中观察到的变化表明，疫苗可以诱导一种抗肿瘤适应性免疫库，该免疫库能够达到肿瘤病灶，并至少在血液中持续2年。