Maldonado Leonel, Brait Mariana, Michailidi Christina, Munari Enrico, Driscoll Tina, Schultz Luciana, Bivalacqua Trinity, Schoenberg Mark, Sidransky David, Netto George J, Hoque Mohammad Obaidul
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA. These authors contributed equally to this work.
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.
Oncotarget. 2014 Jul 30;5(14):5218-33. doi: 10.18632/oncotarget.2129.
By a candidate gene approach, we analyzed the promoter methylation (PM) of 8 genes genes (ARF, TIMP3, RAR-β2, NID2, CCNA1, AIM1, CALCA and CCND2) by quantitative methylation specific PCR (QMSP) in DNA of 17 non-recurrent and 19 recurrent noninvasive low grade papillary urothelial cell carcinoma (LGPUCC) archival tissues. Among the genes tested, by establishing an empiric cutoff value, CCND2, CCNA1, NID2, and CALCA showed higher frequency of methylation in recurrent than in non-recurrent LGPUCC: CCND2 10/19 (53%) vs. 2/17 (12%) (p=0.014); CCNA1 11/19 (58%) vs. 4/17 (23.5%) (p=0.048); NID2 13/19 (68%) vs. 3/17 (18%) (p=0.003) and CALCA 10/19 (53%) vs. 4/17 (23.5%) (p=0.097), respectively. We further analyzed PM of CCND2, CCNA1, and CALCA in urine DNA from UCC patients including LGPUCC and controls. The frequency of CCND2, CCNA1 and CALCA was significantly higher (p<0.0001) in urine of UCC cases [ 38/148 (26%), 50/73 (68%) and 94/148 (63.5%) respectively] than controls [0/56 (0%), 10/60 (17%) and 16/56 (28.5%), respectively)]. Most importantly we found any one of the 3 markers methylation positive in 25 out of 30 (83%) cytology negative LGPUCC cases. We also explored the biological function of CCNA1 in UCC. Prospective confirmatory studies are needed to develop a reliable tool for prediction of recurrence using primary LGPUCC tissues and/or urine.
通过候选基因方法，我们采用定量甲基化特异性PCR（QMSP）分析了17例非复发性和19例复发性非侵袭性低级别乳头状尿路上皮细胞癌（LGPUCC）存档组织DNA中8个基因（ARF、TIMP3、RAR-β2、NID2、CCNA1、AIM1、CALCA和CCND2）的启动子甲基化（PM）情况。在所检测的基因中，通过设定一个经验性临界值，发现CCND2、CCNA1、NID2和CALCA在复发性LGPUCC中的甲基化频率高于非复发性LGPUCC：CCND2为10/19（53%）对2/17（12%）（p = 0.014）；CCNA1为11/19（58%）对4/17（23.5%）（p = 0.048）；NID2为13/19（68%）对3/17（18%）（p = 0.003）；CALCA为10/19（53%）对4/17（23.5%）（p = 0.097）。我们进一步分析了CCND2、CCNA1和CALCA在包括LGPUCC患者和对照者的尿DNA中的PM情况。UCC病例尿液中CCND2、CCNA1和CALCA的频率显著更高（p < 0.0001）[分别为38/148（26%）、50/73（68%）和94/148（63.5%）]，而对照者尿液中分别为0/56（0%）、10/60（17%）和16/56（28.5%）。最重要的是，我们发现在30例细胞学检查阴性的LGPUCC病例中有25例（83%）这3种标志物中的任何一种甲基化呈阳性。我们还探究了CCNA1在UCC中的生物学功能。需要进行前瞻性验证研究，以开发一种利用原发性LGPUCC组织和/或尿液来预测复发的可靠工具。
