Urologic Oncology Program and Uro-Oncology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Division of Cancer Biology and Therapeutics, Departments of Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Theranostics. 2019 Apr 13;9(10):2812-2826. doi: 10.7150/thno.34485. eCollection 2019.
: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. : We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity. : The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). : Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.
我们的目标是开发一种循环肿瘤细胞（CTC）-RNA 检测方法，用于鉴定具有临床相关性的 RNA 特征，以评估转移性去势抵抗性前列腺癌（mCRPC）患者中雄激素受体信号抑制剂（ARSI）的敏感性。
我们通过将热敏（TR）-NanoVelcro CTC 纯化系统与 NanoString nCounter 平台相结合，开发了 NanoVelcro CTC-RNA 检测方法，用于细胞纯化和 RNA 分析。基于经过充分验证的组织前列腺癌分类系统（PCS），我们专注于最具侵袭性和 ARSI 耐药性的 PCS 亚型，即 PCS1，用于 CTC 分析。我们应用了严格的生物信息学流程来开发 CTC-PCS1 面板，该面板包含前列腺癌（PCa）CTC 特异性 RNA 特征，在背景白细胞（WBC）中的表达最小。我们使用经过充分表征的 PCa 细胞系验证了 NanoVelcro CTC-RNA 检测方法和 CTC-PCS1 面板，以证明检测方法的灵敏度和动态范围，以及 PCS1 Z 分数（PCS1 亚型的可能性估计）识别 PCS1 亚型和 ARSI 耐药性的特异性。然后，我们从接受 ARSI 治疗的 23 名 PCa 患者中选择了 31 个血液样本进行检测。计算了每个样本的 PCS1 Z 分数，并与 ARSI 治疗敏感性进行了比较。
使用 PCa 细胞系样本进行的验证研究表明，NanoVelcro CTC-RNA 检测方法可以检测 CTC-PCS1 面板中的 RNA 转录物，具有高灵敏度和线性动态范围为 5-100 个细胞。我们还表明，CTC-PCS1 面板中的基因在 PCa 细胞系中高度表达，在背景 WBC 中低表达。使用模拟血液样本条件的人工 CTC 样本，我们进一步证明了 CTC-PCS1 面板在识别 PCS1 样样本方面具有高度特异性，并且高 PCS1 Z 分数与 ARSI 耐药样本相关。在患者血液中，与 ARSI 敏感样本（ARSI-S，n=17）相比，ARSI 耐药样本（ARSI-R，n=14）的 PCS1 Z 评分显著更高（秩和检验，P=0.003）。在对最初对 ARSI 敏感（ARSI-S）但后来发展为耐药（ARSI-R）的 8 名患者进行分析时，我们发现 PCS1 Z 评分从 ARSI-S 时到 ARSI-R 时增加（配对 T 检验，P=0.016）。
使用我们的新方法，我们开发了一种首创的 CTC-RNA 检测方法，并证明了将临床上相关的组织 RNA 分析（如 PCS）转化为 CTC 检测的可行性。这种方法允许以非侵入性的方式检测与临床耐药性相关的 RNA 表达，这可以促进患者特异性治疗选择和耐药性的早期检测，这是精准肿瘤学的目标。
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