RNA sequencing of periprostatic fat after a 6 month deep androgen deprivation therapy

Androgen deprivation therapy treated patients (n=11) were recruited from an open label neoadjuvant phase II study in which patients with high-risk disease received a ‘supercastration’ regimen consisting of degarelix 240/80 mg subcutaneously every four weeks; abiraterone acetate 500 mg orally daily titrating upwards every two weeks by 250 mg to a final dose of 1000 mg daily; bicalutamide 50 mg orally daily; and prednisolone 5 mg orally twice daily for a total of 6 months (Australian New Zealand Clinical Trials Registry 12612000772842). Untreated patients with similar pre-treatment characteristics were obtained from a prospective prostatectomy biorepository22,23. Prior to ligation of the dorsal venous complex and prostate pedicles, the anterior prostate was defatted and the specimen was removed immediately, placed in a sterile container and transferred on ice for long-term storage in the vapour phase of liquid nitrogen. A total of 50–100 µg of adipose tissue was separated from fresh frozen samples stored at −160°C. RNA was isolated using the Qiagen RNeasy Lipid Tissue Mini Kit and eluted in 35 µL nuclease-free water. 0.5–1 µg of total RNA was used as the input for cDNA library synthesis using TruSeq RNA Sample Prep Kit v2 (Illumina), and libraries were constructed according to manufacturer’s instructions. Samples were sequenced on a HiSeq 2500 (Illumina) using 101 base paired-end chemistry, aiming for 50 million mapped paired-end reads per sample.

在开放标签的新辅助II期临床试验中，招募了接受去雄激素疗法治疗的11名患者（n=11）。该试验中，高风险疾病患者接受了由度他瑞林240/80毫克皮下注射，每四周一次；阿比特龙醋酸盐500毫克口服，每两周剂量上调250毫克，直至每日1000毫克；比卡鲁胺50毫克口服，每日一次；以及泼尼松龙5毫克口服，每日两次，持续6个月的治疗方案（澳大利亚新西兰临床试验注册号12612000772842）。从具有相似治疗前特征的预期性前列腺切除术生物库中获取了未接受治疗的患者。在结扎背静脉丛和前列腺蒂之前，将前前列腺去脂并立即取出样本，放入无菌容器中，在液氮的蒸汽相中进行长期储存。从储存于-160°C的新鲜冷冻样本中分离出总脂肪组织50-100微克。使用Qiagen RNeasy Lipid Tissue Mini Kit提取RNA，并以35微升无核酸酶水洗脱。0.5-1微克的RNA总量用作cDNA文库合成的输入，使用Illumina的TruSeq RNA Sample Prep Kit v2进行文库构建，并按照制造商的说明进行操作。样品在HiSeq 2500（Illumina）上使用101碱基配对端化学进行测序，每个样本的目标是50百万个映射配对端读数。