Multi-Omic Single-Cell Dissection of Leukemic T-Cell Lymphoma Following CAR T-Cell Therapy [Spatial Transcriptomics]

A 63-year-old male who received ciltacabtagene autoleucel (cilta-cel) CAR-T cells and the GPRC5DxCD3 bispecific talquetamab for early relapse of his multiple myeloma (MM) developed a leukemic peripheral T-cell lymphoma (PTCL) with cutaneous and intestinal involvement. Longitudinal single-cell RNA and T-cell receptor sequencing of peripheral blood and bone marrow revealed two hyperexpanded CAR-carrying, exhausted effector-memory T-cell clones with marked immunophenotypic as well as transcriptional alterations and different susceptibilities towards treatment with dexamethasone. Spatial transcriptomes of skin lesions confirmed the aberrant CAR-expressing T cells. Whole genome sequencing revealed three distinct integration sites, within the introns of ZGPAT, KPNA4, and polycomb-associated non-coding RNAs. Pre/post-CAR-T whole-genome analyses implicated clonal outgrowth of a TET2-mutated precursor propelled by additional sub-clone specific LOH and other secondary mechanisms Overall design: Two tissue sections of a formalin-fixed paraffin-embedded (FFPE) skin sample, from a patient treated with CAR-T cells, of the left dorsal region. Three custom probe sets were developed to detect CAR-positive (cilta-cel) spots. To demonstrate the specificity of the cilta-cel custom probes, one slide with a skin sample was subjected to the Visium Spatial Protocol WITHOUT custom probes, another slide with a skin sample was subjected to the Visium Spatial Protocol WITH custom probes and an extramedullary disease sample (sample excision from the lumbar vertebral body) from a patient not treated with CAR-T cells was subjected to the Visium Spatial Protocol WITH custom probes.

一名63岁男性在接受西达基奥仑赛（ciltacabtagene autoleucel, cilta-cel）CAR-T细胞与GPRC5DxCD3双特异性抗体塔法他莫（talquetamab）治疗多发性骨髓瘤（multiple myeloma, MM）早期复发后，出现伴皮肤及肠道受累的白血病性外周T细胞淋巴瘤（peripheral T-cell lymphoma, PTCL）。对患者外周血与骨髓开展纵向单细胞RNA测序及T细胞受体测序后，发现两个过度扩增的携带CAR的耗竭型效应记忆T细胞克隆，二者存在显著的免疫表型与转录组改变，且对地塞米松治疗的敏感性存在差异。皮肤病变的空间转录组分析证实了异常CAR表达T细胞的存在。全基因组测序结果显示存在三个不同的整合位点，分别位于ZGPAT、KPNA4基因内含子及多梳相关非编码RNA区域。CAR-T治疗前后的全基因组分析提示，TET2突变前体细胞的克隆扩增由额外的亚克隆特异性杂合性缺失（loss of heterozygosity, LOH）及其他次级机制所驱动。

实验整体设计：本研究纳入2份来自接受CAR-T治疗患者左背部区域的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）皮肤样本组织切片。设计了3组定制探针用于检测CAR阳性（cilta-cel）斑点。为验证cilta-cel定制探针的特异性，设置如下对照实验：1份皮肤样本切片仅执行Visium空间转录组实验方案（Visium Spatial Protocol）且未添加定制探针；1份皮肤样本切片执行Visium空间转录组实验方案（Visium Spatial Protocol）并添加定制探针；另使用1份未接受CAR-T治疗患者的髓外疾病样本（腰椎体切除样本）执行Visium空间转录组实验方案（Visium Spatial Protocol）并添加定制探针。