MATADOR: A prospective, randomised, phase III biomarker trial in breast cancer patients

A prospective, randomised, phase III biomarker trial (ISRCTN61893718) that run in 29 hospitals in the Netherlands. 664 Patients with an indication for adjuvant chemotherapy and a pT1-3, pN0-3 tumour were randomised 1:1 to 6 cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 2 weeks [dose dense (dd) AC] or docetaxel 75 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide 500 mg/m2 every 3 weeks (docetaxel-doxorubicin-cyclophosphamide; TAC) by means of the automated ALEA system (FormsVision BV) using Pocock minimization technique. Randomization was performed centrally at the Netherlands Cancer Institute. G-CSF (pegfilgrastim) was given to all patients. Radiotherapy and endocrine therapy were given according to the contemporary Dutch guidelines. Formalin-fixed paraffin-embedded (FFPE) material was collected between 2004 and 2012. RNA was isolated from FFPE tissue with a tumor cell percentage of at least 40% using the AllPrep DNA/RNA Mini Kit according to the manufacturer's instructions (Qiagen). Quantification and purity were measured using the NanoDrop 2,000 spectrophotometer and the 2,100 Bioanalyzer (Agilent Technologies). Libraries of cDNA were constructed with the TruSeq RNA Access Library Prep Kit (Illumina) and single-end sequenced using the HiSeq 2,500 (Illumina). Bulk mRNA sequencing data were analyzed using the RSEM package and the reads were aligned to the hg38 reference genome using STAR with default parameters. The immunohistochemistry biomarkers are describe in Opdam TheBreast 2026 with 10year follow-up (data till 2025). Immunohistochemistry was performed on a BenchMark Ultra autostainer (Ventana Medical Systems) for: ER Roche Diagnostics 5278406001 PR Roche Diagnostics 5278392001 HER2 Roche Diagnostics 5278368001 AR Cell Marque clone SP107 Ki67 Dako #M7240 clone MIB-1 ABCB1 Cell signal 13978 clone E1Y7S BCL2 Dako M0887 clone 124 GSTP1 Abnova MAB9663 clone LW29 MAPT USBiological 2B2.100 T1029 Thioredoxin Cell signaling cs2429_c63c6 TUBB Santa Cruz 5274 clone D-10 TUBB3 Abcam14545 TUJ1 Cyclin D1 RM-9104-S Thermo clone SP4 EZH2 Leica Clone 6A10 EZH2-L-CE pH2AX Cell Signaling #2577 P53 M7001 Dako clone DO-7本研究为一项前瞻性、随机III期生物标志物试验（ISRCTN61893718），在荷兰29家医院开展。入组的664例符合辅助化疗指征且肿瘤分期为pT1-3、pN0-3的患者，通过荷兰癌症研究所中心化的自动化ALEA系统（FormsVision BV），采用Pocock最小化法以1:1比例随机分配至两组：一组接受每2周1次的阿霉素60 mg/m²联合环磷酰胺600 mg/m²方案，共6个周期（剂量密集型（dd）AC方案）；另一组接受每3周1次的多西他赛75 mg/m²、阿霉素50 mg/m²联合环磷酰胺500 mg/m²方案（多西他赛-阿霉素-环磷酰胺，TAC方案）。所有患者均予粒细胞集落刺激因子（pegfilgrastim，培非格司亭）治疗，并根据当时荷兰临床指南接受放疗与内分泌治疗。 2004年至2012年间收集福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）组织样本。使用Qiagen公司的AllPrep DNA/RNA Mini试剂盒，按照制造商说明书从肿瘤细胞占比≥40%的FFPE组织中提取RNA。采用NanoDrop 2000分光光度计与Agilent 2100生物分析仪（Agilent Technologies）对RNA进行定量与纯度检测。使用Illumina的TruSeq RNA Access文库制备试剂盒构建cDNA文库，并通过Illumina HiSeq 2500测序平台进行单端测序。采用RSEM软件包分析批量mRNA测序数据，使用STAR软件以默认参数将测序reads比对至hg38参考基因组。 本研究的免疫组化生物标志物相关信息已发表于Opdam于《The Breast》2026年刊发的研究，随访时长达10年（数据截止至2025年）。在BenchMark Ultra全自动染色机（Ventana Medical Systems）上完成以下指标的免疫组化检测： 雌激素受体（ER）：罗氏诊断（Roche Diagnostics），货号5278406001 孕激素受体（PR）：罗氏诊断，货号5278392001 人表皮生长因子受体2（HER2）：罗氏诊断，货号5278368001 雄激素受体（AR）：Cell Marque，克隆号SP107 Ki67：Dako，货号#M7240，克隆号MIB-1 ABCB1：Cell Signaling，货号13978，克隆号E1Y7S BCL2：Dako，货号M0887，克隆号124 GSTP1：Abnova，货号MAB9663，克隆号LW29 MAPT：USBiological，货号2B2.100 T1029 硫氧还蛋白：Cell Signaling，货号cs2429_c63c6 TUBB：Santa Cruz，货号5274，克隆号D-10 TUBB3：Abcam，货号14545，克隆号TUJ1 细胞周期蛋白D1：Thermo，货号RM-9104-S，克隆号SP4 EZH2：Leica，克隆号6A10，货号EZH2-L-CE 磷酸化组蛋白H2AX（pH2AX）：Cell Signaling，货号#2577 P53：Dako，货号M7001，克隆号DO-7