通过精确划分局部癌化区域以及可靠的生物标志物来确定最佳安全切除边界 (EM)，从而能够预测和及时评估患者对免疫治疗的反应，显着影响黑色素瘤的有效治疗。在这项研究中，沿着诊断-转移-治疗-细胞MRD轴进行优化的双相“免疫荧光染色与荧光原位杂交相结合”（iFISH），以纵向共同检测全谱完整的CD31-肿瘤细胞。 TC)、CD31 非整倍体肿瘤内皮细胞 (TEC)、活的和坏死的 CTC 以及在切除的原发肿瘤未切片标本中表达 PD-L1/Ki67/p16/Vimentin 的循环 TEC (CTEC)、EM、解剖的前哨淋巴结 (SLN)和早期黑色素瘤患者的外周血。在常规安全电镜（2cm）处检测到大量PD-L1非整倍体TC/TEC，首次定量表明现场癌化电镜的存在。与原发灶中的 TC/TEC 和血液中的 CTC/CTEC 中的高度异质性 PD-L1 表达和 Chr8 非整倍性相反，SLN 和 EM 中几乎所有 TC/TEC 都是同质的 PD-L1 单倍体细胞。动态监测和细胞 MRD 评估显示，与 PD-L1 CTC 对免疫检查点抑制剂（ICI-抗 PD-1）有反应相比，多倍体（≥五倍体 8）PD-L1 和 Ki67 CTEC 对 ICI 致敏具有耐药性T细胞。在治疗应激的淋巴和血行转移级联中，iFISH 组织和液体活检 TC/TEC/CTC/CTEC 的分层表型和核型分析能够对最佳安全 EM 进行适当的重新指定，对细分的靶细胞的分布进行深入表征和绘图帮助说明其转移相关性，最终改善肿瘤进展、转移、治疗耐药和复发的风险分层和临床干预。版权所有 © 2024。由 Elsevier B.V. 出版。

An optimum safety excision margin (EM) delineated by precise demarcation of field cancerization along with reliable biomarkers that enable predicting and timely evaluating patients' response to immunotherapy, significantly impact effective management of melanoma. In this study, optimized biphasic "immunofluorescence-staining integrated with fluorescence-in situ-hybridization" (iFISH) was conducted along the diagnosis-metastasis-treatment-cellular MRD axis to longitudinally co-detect a full spectrum of intact CD31- tumor cells (TCs), CD31+ aneuploid tumor endothelial cells (TECs), viable and necrotic CTCs and circulating TECs (CTECs) expressing PD-L1/Ki67/p16/Vimentin in unsliced specimens of the resected primary tumor, EM, dissected sentinel lymph nodes (SLNs) and peripheral blood in an early-stage melanoma patient. Numerous PD-L1+ aneuploid TCs/TECs were detected at the conventional safety EM (2 cm), quantitatively indicating existence of the field cancerized EM for the first time. Contrary to highly heterogeneous PD-L1 expression and Chr8 aneuploidy in TCs/TECs in the primary lesions and CTCs/CTECs in blood, almost all TCs/TECs in SLNs and EM were homogeneously PD-L1+ haploid cells. Dynamic monitoring and cellular MRD assessment revealed that in contrast to PD-L1+ CTCs being responsive to the immune checkpoint inhibitor (ICI-anti-PD-1), multiploid (≥pentasomy 8) PD-L1+ and Ki67+ CTECs were resistant to ICI-sensitized T cells. In therapeutically stressed lymphatic and hematogenous metastatic cascades, stratified phenotypic and karyotypic profiling of iFISH tissue and liquid biopsied TCs/TECs/CTCs/CTECs enables appropriate re-specification of the optimal safety EM, in-depth characterization and mapping of distribution of subcategorized target cells to help illustrate their metastatic relevance, ultimately improving risk stratification and clinical intervention of tumor progression, metastases, therapy resistance and relapse.Copyright © 2024. Published by Elsevier B.V.