Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Breast Cancer Res Treat. 2013 Jan;137(1):45-56. doi: 10.1007/s10549-012-2279-y. Epub 2012 Nov 6.
Disseminated tumor cells (DTCs) detected in the bone marrow (BM) of breast cancer patients identify women at high risk of recurrence. DTCs are traditionally detected by immunocytochemical staining for cytokeratins or single gene expression measurements, which limit both specificity and sensitivity. We evaluated the Nanostring nCounter™ platform for multi-marker, gene expression-based detection and classification of DTCs in the BM of breast cancer patients. Candidate genes exhibiting tumor cell-specific expression were identified from microarray datasets and validated by qRT-PCR analysis in non-malignant human BM and identical samples spiked with predefined numbers of molecularly diverse breast tumor cell lines. Thirty-eight validated transcripts were designed for the nCounter™ platform and a subset of these transcripts was technically validated against qRT-PCR measurements using identical spiked BM controls. Bilateral iliac crest BM aspirates were collected and analyzed from twenty breast cancer patients, prior to neoadjuvant therapy, using the full 38-gene nCounter™ code set. Tumor cell-specific gene expression by nCounter™ was detected with a sensitivity of one cancer cell per 1 × 10(6) nucleated BM cells after optimization. Measurements were quantitative, log linear over a 20-fold range, and correlated with qRT-PCR measurements. Using the nCounter™ 38-gene panel, 6 of 8 patients (75 %) who developed metastatic disease had detectable expression of at least one transcript. Notably, three of these patients had detectable expression of ERBB2 in their BM, despite the fact that their corresponding primary tumors were HER2/ERBB2 negative and therefore did not receive trastuzumab therapy. Four of these patients also expressed the PTCH1 receptor, a newly recognized therapeutic target based on hedgehog signaling pathway inhibition. The presumptive detection and classification of DTCs in the BM of breast cancer patients, based on sensitive and quantitative multi-marker detection of gene expression using the nCounter™ platform, provide an opportunity to both predict early distant recurrence and, more importantly, identify opportunities for preventing the spread of disease based on the expression of unique, therapeutically actionable gene targets. This study demonstrates the application of a new technology for multiplexed gene expression-based detection of DTCs in the BM of breast cancer patients and identifies at least two therapeutically targetable genes that are frequently expressed in the BM of patients who develop metastatic disease.
在乳腺癌患者的骨髓（BM）中检测到的弥散性肿瘤细胞（DTCs）可识别出高复发风险的女性。传统上，通过免疫细胞化学染色检测细胞角蛋白或单基因表达测量来检测 DTCs，这限制了特异性和敏感性。我们评估了 Nanostring nCounter™平台在乳腺癌患者 BM 中基于多标记物、基因表达的 DTC 检测和分类。从微阵列数据集中鉴定出具有肿瘤细胞特异性表达的候选基因，并通过非恶性人类 BM 和用预定数量的分子上多样化的乳腺癌细胞系掺入的相同样本的 qRT-PCR 分析进行验证。设计了 38 个用于 nCounter™平台的验证转录本，并针对相同的 BM 对照物使用双边髂嵴 BM 抽吸物，在新辅助治疗前从 20 名乳腺癌患者中收集和分析，使用完整的 38 个基因 nCounter™代码集。通过优化，使用 nCounter™检测到每 1×10(6)个有核 BM 细胞中有一个癌细胞的肿瘤细胞特异性基因表达的灵敏度。测量结果是定量的，对数线性范围为 20 倍，与 qRT-PCR 测量结果相关。使用 nCounter™38 基因谱，8 名发生转移疾病的患者中有 6 名（75%）可检测到至少一种转录本的表达。值得注意的是，尽管他们相应的原发性肿瘤是 HER2/ERBB2 阴性，因此没有接受曲妥珠单抗治疗，但其中 3 名患者的 BM 中可检测到 ERBB2 的表达。这些患者中的 4 名还表达了 PTCH1 受体，这是基于 hedgehog 信号通路抑制的新发现的治疗靶点。基于 nCounter™平台对基因表达的敏感和定量多标记物检测，在乳腺癌患者的 BM 中推定检测和分类 DTCs，为预测早期远处复发提供了机会，更重要的是，为基于表达独特的、可治疗的靶基因来预防疾病的传播提供了机会。本研究展示了一种新技术在乳腺癌患者 BM 中基于多标记物的 DTC 检测中的应用，并确定了至少两个在发生转移疾病的患者 BM 中经常表达的治疗靶标基因。
