Dulaimi Essel, Uzzo Robert G, Greenberg Richard E, Al-Saleem Tahseen, Cairns Paul
Departments of Surgical Oncology and Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Clin Cancer Res. 2004 Mar 15;10(6):1887-93. doi: 10.1158/1078-0432.ccr-03-0127.
Bladder cancer is potentially curable in the majority of cases; however, the prognosis for patients with advanced disease at presentation remains poor. Current noninvasive tests such as cytology lack sufficient sensitivity to detect low-grade, low-stage tumors. Silencing of tumor suppressor genes, such as p16(INK4a), VHL, and the mismatch repair gene hMLH1, has established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancers. It is also a promising new target for molecular detection in bodily fluids including urine, a readily accessible fluid known to contain bladder cancer cells. Methylation-specific PCR (MSP) can determine the presence or absence of methylation of a gene locus at a sensitivity level of up to 1 methylated allele in 1000 unmethylated alleles, appropriate for identifying cancer cell DNA in a bodily fluid.
We first determined the frequency of hypermethylation of the Rb tumor suppressor gene by bisulfite sequencing and of the p16(INK4a), p14(ARF), APC, and RASSF1A tumor suppressor genes by MSP in 45 bladder cancers. We then designed a panel optimal for diagnostic coverage composed of the APC, RASSF1A, and p14(ARF) tumor suppressor genes. This panel was tested for detection of hypermethylation in matched sediment DNA from urine specimens obtained before surgery from the same 45 bladder cancer patients (2 Tis, 16 Ta, 10 T1, and 17 T2-4) as well as normal and benign control DNAs.
Hypermethylation of at least one of three suppressor genes (APC, RASSF1A, and p14(ARF)) was found in all 45 tumor DNAs (100% diagnostic coverage). We detected gene hypermethylation in the matched urine DNA from 39 of 45 patients (87% sensitivity), including 16 cases that had negative cytology. No hypermethylation of APC, RASSF1A, or p14(ARF) was observed in normal transitional cell DNAs or in urine DNAs from normal healthy individuals and patients with inflammatory urinary disease (cystitis). Furthermore, an unmethylated gene in the tumor DNA was always found to be unmethylated in the matched urine DNA (100% specificity).
Promoter hypermethylation of tumor suppressor genes is common in bladder cancer and was found in all grades and stages of tumors examined. Hypermethylation was detected in the urine DNA from 39 of 45 (87%) patients, including cases of early-stage disease amenable to cure. MSP may enhance early detection of bladder cancer using a noninvasive urine test.
大多数情况下膀胱癌有潜在治愈可能；然而，就诊时患有晚期疾病的患者预后仍然较差。当前诸如细胞学检查等非侵入性检测方法对低级别、低分期肿瘤的检测灵敏度不足。肿瘤抑制基因如p16(INK4a)、VHL以及错配修复基因hMLH1的沉默已证实启动子高甲基化是人类癌症中肿瘤抑制基因失活的常见机制。它也是体液包括尿液中分子检测的一个有前景的新靶点，尿液是一种易于获取且已知含有膀胱癌细胞的液体。甲基化特异性PCR（MSP）能够以高达1000个未甲基化等位基因中有1个甲基化等位基因的灵敏度水平来确定基因位点甲基化的有无，适用于识别体液中的癌细胞DNA。
我们首先通过亚硫酸氢盐测序确定45例膀胱癌中Rb肿瘤抑制基因的高甲基化频率，并通过MSP确定p16(INK4a)、p14(ARF)、APC和RASSF1A肿瘤抑制基因的高甲基化频率。然后我们设计了一个由APC、RASSF1A和p14(ARF)肿瘤抑制基因组成的对诊断覆盖最优化的组合。对来自45例相同膀胱癌患者（2例Tis、16例Ta、10例T1和17例T2 - 4）术前获取的尿液标本的匹配沉淀物DNA以及正常和良性对照DNA进行该组合检测高甲基化情况。
在所有45例肿瘤DNA中均发现三种抑制基因（APC、RASSF1A和p14(ARF)）中至少一种发生高甲基化（诊断覆盖率100%）。我们在45例患者中来自39例患者的匹配尿液DNA中检测到基因高甲基化（灵敏度87%），包括16例细胞学检查为阴性的病例。在正常移行细胞DNA或正常健康个体以及炎性泌尿系统疾病（膀胱炎）患者的尿液DNA中未观察到APC、RASSF1A或p14(ARF)的高甲基化。此外，肿瘤DNA中未甲基化的基因在匹配的尿液DNA中也总是未甲基化（特异性100%）。
肿瘤抑制基因的启动子高甲基化在膀胱癌中很常见，在所检测的所有肿瘤分级和分期中均有发现。在45例患者中的39例（87%）尿液DNA中检测到高甲基化，包括可治愈的早期疾病病例。MSP可能通过非侵入性尿液检测提高膀胱癌的早期检测。
