PO.CL01.22 · 临床研究
用于实时监测循环肿瘤细胞上前列腺特异性膜抗原（PSMA）表达的标准化CELLSEARCH®检测的开发与验证
Development and validation of a standardized CELLSEARCH® assay for real-time monitoring of prostate-specific membrane antigen (PSMA) expression on circulating tumor cells
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摘要 Abstract
中文摘要
背景：
前列腺特异性膜抗原（PSMA）是一种关键生物标志物，其动态表达决定了新兴PSMA靶向放射性配体疗法的临床应用价值。一个主要的临床挑战仍在于缺乏一种标准化、高度敏感且微创的PSMA表达监测方法。我们通过开发并验证一种整合到CELLSEARCH®平台（唯一经FDA批准用于CTC计数的系统）中的新型PSMA特异性检测来应对这一挑战。
方法：
该PSMA检测利用CELLSEARCH®平台对EpCAM+ CTCs的免疫磁富集，随后用荧光标记的抗细胞角蛋白、抗CD45、DAPI和高亲和力抗PSMA抗体进行染色。使用掺入健康供者血液的PSMA阳性（LNCaP、22Rv1）和PSMA阴性（PC-3.9）细胞系进行分析验证，评估在CellSave™采血管中96小时样本保存时间内的检测敏感性、特异性和回收率。捕获的CTCs适用于平台成像及后续分子分析。
结果：
定制的PSMA检测表现出卓越的分析性能，满足所有预先设定的标准。掺入与回收的PSMA阳性LNCaP细胞之间的线性关系稳健，在所有受试供者中R²值均>0.999。PSMA阳性细胞的平均回收率百分比证实了高准确性，在10至1000个细胞的掺入水平下范围为83.3%至93.3%。检测精密度也得到证实，在10个细胞掺入水平下变异系数（CV）为6.97%。在一个初步临床队列（N=20）的前列腺癌（PCa）患者中，该检测成功检出了PSMA+ CTCs。随后对富集的患者样本进行正交流式细胞术分析和细胞分选，以独立确认PSMA蛋白表达。对分选细胞的下游分子分析证实了PCa特异性基因组改变的存在，为PSMA阳性CTC群体提供了分子学验证。
结论：
这种新型、经全面验证的PSMA CTC检测提供了一种高度特异且标准化的用于PSMA检测的液体活检工具。其稳健的分析性能确立了该平台在监测PSMA表达动态以指导转移性前列腺癌患者治疗决策方面的应用价值。
查看英文原文 English abstract
BACKGROUND:
Prostate-Specific Membrane Antigen (PSMA) is a critical biomarker whose dynamic expression dictates the clinical utility of emerging PSMA-targeted radioligand therapies. A major clinical challenge remains the lack of a standardized, highly sensitive, and minimally invasive method for PSMA expression monitoring. We addressed this by developing and validating a novel PSMA-specific assay integrated into the CELLSEARCH® platform, the only FDA-cleared system for CTC enumeration.
METHODS:
The PSMA assay utilizes the CELLSEARCH® platform's immunomagnetic enrichment of EpCAM+ CTCs, followed by staining with fluorescently labeled anti-cytokeratin, anti-CD45, DAPI, and a high-affinity anti-PSMA antibody. Analytical validation was conducted using PSMA-positive (LNCaP, 22Rv1) and PSMA-negative (PC-3.9) cell lines spiked into healthy donor blood, assessing assay sensitivity, specificity, and recovery over a 96-hour sample preservation time in CellSave™ tubes. Captured CTCs are suitable for both platform imaging and subsequent molecular analysis.
RESULTS:
The customized PSMA assay demonstrated exceptional analytical performance, meeting all pre-defined criteria. The linear relationship between spiked and recovered PSMA-positive LNCaP cells was robust, achieving R^2 values >0.999 across all tested donors. High accuracy was confirmed by mean percent recovery for PSMA-positive cells, which ranged from 83.3% to 93.3% for spike levels of 10 to 1000 cells. Assay precision was also confirmed, with a Coefficient of Variation (CV) of 6.97% at the 10-cell spike level. In a preliminary clinical cohort (N=20) of PCa patients, the assay successfully detected PSMA+ CTCs. Enriched patient samples were then subjected to orthogonal flow cytometry analysis and cell sorting to provide independent confirmation of PSMA protein expression. Downstream molecular analysis of the sorted cells confirmed the presence of PCa-specific genomic alterations, providing molecular verification of the PSMA-positive CTC population.
CONCLUSIONS:
This novel, fully validated PSMA CTC assay provides a highly specific, and standardized liquid biopsy tool for PSMA detection. Its robust analytical performance establishes the utility of the platform in monitoring PSMA expression dynamics to inform therapeutic decisions in metastatic prostate cancer patients.
利益披露 Disclosure
S. M. Jones,
Menarini Silicon Biosystems Employment.
T. Bui,
Menarini Silicon Biosystems Employment.
D. Gullipalli,
Menarini Silicon Biosystems Employment.
S. Powell,
Menarini Silicon Biosystems Employment.
M. LaCava,
Menarini Silicon Biosystems Employment.
Z. Simandi,
Menarini Silicon Biosystems, Inc. Employment.