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

前列腺癌的皮肤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改变。此外，全身治疗本身可以改变转移性疾病的分子景观。因此，基于组织的生物标志物评估骨转移而不是原发性肿瘤的分子变化，以预测预后或对靶向治疗的反应可能对这些患者有益。在这个拟议的研究项目中，我们将应用基因表达测定法来稳健地检测差异表达的候选基因和途径调控模式，因为FFPE材料通常是唯一可用的材料，所以存档福尔马林固定，石蜡包埋（FFPE）的前列腺癌患者骨转移。通过我们的前期工作，已经鉴定出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（NanoString nCounter®），并且可以定制。基因表达与临床数据的相关性随后将鉴定一组具有预后和/或预测潜力的基因。该基因组将在骨转移瘤组织中进行回顾性和前瞻性验证。此外，将检测来自循环肿瘤细胞的RNA，以评价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。将使用两种前列腺癌细胞系（VCaP/PC-3）进行功能验证，这两种细胞系均来源于前列腺癌骨转移。对于基因组中的5-7个过表达基因，我们将使用siRNA进行瞬时敲减以评估过表达的生物学后果。在这个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全身治疗的反应的认识。它将为晚期前列腺癌患者提供转化获益。我们期望，我们的基因表达测定可以单独或与传统的临床或基于组织的参数结合作为生物标志物。此外，我们将表明，强大的基因表达分析是可行的，从FFPE组织骨转移，从而促进进一步研究其他肿瘤类型，容易发生骨转移。

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.