Development and validation of a gene expression assay as prognostic and predictive biomarker in prostate cancer bone metastases

前列腺癌的骨转移是世界范围内的主要健康负担，严重影响患者的生活质量。它们具有与原发肿瘤明显不同的分子变化。此外，全身治疗本身可以改变转移性疾病的分子格局。因此，以组织为基础的生物标志物评估骨转移瘤中的分子变化而不是原发肿瘤中的分子变化来预测预后或靶向治疗的反应可能对这些患者有利。在这项拟议的研究项目中，我们将应用基因表达分析来强有力地检测前列腺癌患者存档的福尔马林固定、石蜡包埋(FFPE)骨转移中差异表达的候选基因和通路调控模式，因为FFPE材料通常是唯一可用的材料。通过前期工作，我们已经确定了96个候选基因。基因表达将使用高度敏感的新型阵列系统进行研究，该系统使用颜色编码的条形码来检测转录本(NanoStringnCounter®)，并且可以定制。基因表达与临床数据的相关性将随后确定一组具有预后和/或预测潜力的基因。该基因小组将在骨转移瘤组织中进行回顾性和前瞻性验证。此外，来自循环肿瘤细胞的RNA将被测试，以评估其作为无转移组织患者的非侵入性生物标志物(液体活检)的潜力。将使用两个前列腺癌细胞系(VCaP/PC-3)进行功能验证，这两个细胞系都来自前列腺癌的骨转移。对于基因面板中5-7个过度表达的基因，我们将使用siRNA进行瞬时敲除，以评估过度表达的生物学后果。在这项拟议的研究项目中产生的数据将增强我们对前列腺癌骨转移的转录组及其对系统治疗的反应的了解。它将为晚期前列腺癌患者提供翻译上的好处。我们期望，我们的基因表达分析可以单独或结合传统的临床或组织参数作为生物标记物。此外，我们将展示，从骨转移的FFPE组织中进行稳健的基因表达分析是可行的，从而促进对其他容易发生骨转移的肿瘤类型的进一步研究。

Skeletal metastases from prostate cancer are a major health burden worldwide and severely affect a patient’s quality of life. They harbor molecular alterations distinctly different from the primary tumor. Moreover, systemic treatment itself can change the molecular landscape of metastatic disease. Thus, a tissue-based biomarker evaluating molecular changes in bone metastases rather than in the primary tumor to predict prognosis or response to –targeted– therapy could be of benefit for these patients. In this proposed research project, we will apply a gene expression assay to robustly detect differentially expressed candidate genes and patterns of pathway regulation in archival formalin-fixed, paraffin embedded (FFPE) bone metastases from prostate cancer patients, because FFPE material is usually the only available material. Ninety-six candidate genes have already been identified through our preliminary work. Gene expression will be studied using a highly sensitive novel array system, that uses color-coded barcodes to detect transcripts (NanoString nCounter®) and can be custom-built. Correlation of gene expression with clinical data will subsequently identify a panel of genes with prognostic and/or predictive potential. This gene panel will undergo retrospective as well as prospective validation in tumor tissue from bone metastases. In addition, RNA from circulating tumor cells will be tested to evaluate its potential as non-invasive biomarker (liquid biopsy) for patients, for whom metastatic tissue is not available. Functional validation using two prostate cancer cell lines (VCaP/PC-3), both derived from a prostate cancer bone metastasis, will be performed. For 5-7 overexpressed genes in the gene panel, we will perform transient knockdown using siRNA to evaluate the biological consequence of overexpression. Data generated in this proposed research project will enhance our knowledge of the transcriptome of prostate cancer bone metastases and its response to systemic treatment. It will provide a translational benefit for patients with advanced prostate cancer. We expect, that our gene expression assay can act as a biomarker either on its own or in conjunction with traditional clinical or tissue-based parameters. In addition, we will show, that robust gene expression analysis is feasible from FFPE tissue from bone metastases thus facilitating further research in other tumor types that are prone to bone metastases.