摘要
[背景和目的]
在泌尿系肿瘤中,膀胱肿瘤是最常见的恶性肿瘤,在全球发病率呈增高趋势。然而用于膀胱癌诊断、治疗、复发监测和治疗相关并发症的费用高昂,给患者造成重大的经济负担；并且膀胱癌5年复发率高达50%～90%,约35%非肌层浸润性膀胱癌会随着复发其分期、分级也相应提高,其中约10%～20%进展为肌层浸润性癌,需行根治性膀胱全切和尿流改道术,且可能出现尿失禁、排尿困难和性功能障碍等并发症,给患者社会活动和生活质量带来严重影响。因此,早期诊断和有效预测膀胱癌复发风险是目前临床迫切需要解决的问题。
目前预测膀胱癌复发风险的肿瘤标记物多为基因产物,以蛋白质为主；其余的为一些酶类。这些肿瘤标记物多为大分子,易受到酶类的破坏,不易保存,多需血液样本,取样不方便,特别对于预后复发分级低的膀胱癌检出率较低且费用高。修饰核苷在RNA中存在,由转录后多种特定修饰酶同多聚核苷分子相作用而形成；同时其具有稳固的分子结构,不容易发生反应和改变形态,并且分子质量比较小；通过尿液即可取样储存较容易；其排放水平在已报道的文献中发现多种肿瘤中均比较高,即可识别的肿瘤谱系广等优势。尿液修饰核苷作为非常有潜力的肿瘤标记物已发现对多种肿瘤的诊断、疗效了解、病情监测以及预后起到了积极作用。我们前期通过检测膀胱癌患者和正常人1-甲基腺苷(m1A)、N4-乙酰胞苷(ac4C)、腺嘌呤核苷(A)、06-甲基鸟苷(06-MeG)、5'-脱氧-5'-甲基硫代腺苷(MTA)、1-甲基次黄苷(1-MeI)和1-甲基鸟苷(1-MeG)这7种修饰核苷的水平差异,筛选出4种核苷水平明显高于常人,分别是1-甲基腺苷(m1A)、N4-乙酰胞苷(ac4C)、O6-甲基鸟苷(06-MeG)、1-甲基次黄苷(1-MeI)；而mlA联合1-MeI检测具有极高的灵敏度(92.45%)和特异性(87.50%),可作为BUCC首选肿瘤标记物。因此尿液修饰核苷可作为具有广阔的临床应用前景的肿瘤标记物。
本研究通过检测膀胱癌术后患者不同临床时期2种尿液修饰核苷(m1A.1-MEI)水平及其与正常人水平的差异,以及不同的病理分级、分期术后患者2种尿液修饰核苷水平的差异,探讨2种尿液修饰核苷检测在膀胱尿路上皮细胞癌预后监测中的应用价值,进而探索一种用于监测膀胱癌患者预后的新方法。
[方法]
选取病理证实为膀胱尿路上皮细胞癌(bladder urothelial cell carcinoma, BUCC)患者85例。临床分期Tis～T155例,T2～T430例；组织学分级G127例、G240例、G318例。膀胱肿瘤电切术后随访1年,其中浸润性癌患者因自身耐受因素或患者坚持选择而采取电切术。每3个月复查一次。术后第3个月均未出现复发。术后6个月复发20例,术后9个月复发18例,术后12个月复发19例,共计57例设为复发组。28例术后1年内未复发者设为未复发组。复发组Tis-T135例,T2～T422例；未复组分别为20例、8例。健康对照组50例。采取高效液相色谱/电喷雾-四极杆-飞行时间质谱技术(HPLC/ESI-Q-TOF-MS)检测各组尿液修饰核苷(MIA,1-MeI)水平。统计学比较尿液修饰核苷水平与膀胱癌生物学行为的关系。
[结果]
1.手术前后不同时间之间2种尿液修饰核苷水平差异有统计学意义(m1A:F=13.707,P<0.05,1-MeI:F=46.760,P<0.05)。其中未复发组中术后3个月2种尿液修饰核苷水平(m1A:3.24±0.40,1-MeI:5.73±0.67)明显低于术前(m1A:4.34±0.98,1-MeI:14.22±4.05P<0.005)。复发组中术后3个月2种尿液修饰核苷水平(m1A:3.31±0.33,1-MeI:5.67±0.55)与术前水平(m1A:4.32±1.19,1-MeI:14.31±4.12P<0.005)相比也明显降低。
2.未复发组中术后3、6、9、12个月水平(MIA:3.24±0.40、3.26±0.24、3.38±0.3、3.27±0.27;1-MEI:5.73±0.67.5.84±1.10.5.66±0.73.5.78±0.66)分别与术前相比,均明显降低(P<0.005),但术后各时间点的水平相互比较无变化(P>0.005),术后总体水平变化趋势为降低且维持在较低状况且较稳定。
3.复发组中术后6、9、12个月水平(MIA:4.04±0.48.4.11±0.47.4.09±0.53,1-MEI:11.46±1.34、12.14±1.22、12.33±1.27)分别与术前比较,无明显变化(P>0.005),与术后3个月水平相比均明显升高(P<0.005),术后总体水平变化趋势为先降低后上升并维持在高水平状况并接近术前水平。
4.术前时间点,未复发组和复发组水平均高于对照组(m1A:2.91±0.84,1-MeI:5.56±1.25P<0.01).在术后6、9、12个月各时间点均以复发组水平为最高(P<0.01)。
5.G1、G2、G3级之间2种尿液修饰核苷水平(MIA:5.10±1.93.4.72±1.71、5.26±2.13;1-MEI:16.52±8.11.14.22±7.65.13.43±6.57)比较无差异(P>0.01)。Tis-T1与T2-T4临床分期之间2种尿液修饰核苷水平(MIA:4.77±1.43、4.82±1.38;1-MEI:15.13±5.87.16.32±6.41)比较同样无差异(P>0.01)。Tis-T1中复发组水平(m1A:5.92±1.28,1-MeI:20.01±8.53)高于未复发组(mlA:4.02±1.22,1-MeI:11.21±6.45P<0.05), T2-T4中亦如此。
[结论]
1. MIA.1-MEI,2种尿液修饰核苷水平检测可对治疗疗效进行评估以及对病情变化进行监测。
2. MIA.1-MEI,2种尿液修饰核苷水平检测可对膀胱癌患者预后复发风险进行判断。
Background and objective
Bladder cancer is the most common urothelial tumors. It is in the increasing trend in recent years in the world. However, it caused heavy economic burden to patients, such as in the diagnose、 remedy and recurrence surveillance and curing the complication.The researches indicated that the recurrence rate of bladder cancer was up to50%-90%in5years.About35%non-muscle invasive bladder cancer patients' grade and periodization will be raised when it recurrenced,and about10%-20%patients could develop to muscle invasive bladder cancer.These patients must be operated by radical cystectomy and urinary diversion surgery, and it would appear some complications such as uroclepsia、dysury、sexual dysfunction and so on. The patients'social activities and the quality of life will be seriousiy impacted impacted. Therefore, early diagnose and forecast the recurrence of bladder cancer effectively is a urgent problem in clinical.
At present, the major tumor markers which predict recurrence of bladder cancer risk are the gene products, and the main is protein,and the rest are some enzymes.These tumor markers are mostly macromolecules and vulnerable to be destructed by enzymes, they can not be kept well and need more blood samples, and sampling is not convenient, and especially for the bladder cancer of prognosis of low recurrence classification,the detection rate is low and the cost is very high.The modified nucleosides are in the RNA, which is formed by various specific modification enzymes after transfer acting on poly-1-nucleosides. The modified nucleosides also have many superiority, such as small molecular weight、stable structure and it is hard to react and modifying shape,and samples are easy to save and can be collected in urine、easy to collect and no damage、the tumors pedigree which is detected is popular and so on. Modified nucleosides as a tumor marker which have great potential are discovered playing a positive role in many aspectes such as the diagnose of cancer、monitor of illness and acquaintance the curative effect.Though detected the change of levels of m1A、ac4c、A、06-MeG、MTA、1-MEI、1-MEG in bladder cancer patients and the normal of our early study, we found that four modified nucleosides'levels were higher than the normal's, They were m1A、ac4、06-MEG、1-MEI. The detection of M1A uniting1-MeI had high sensitivity (92.45%) and specificity (87.50%), and they could be the first select umor markers for BUCC.Therefor, urine modified nucleosides as a tumor markers have a broad prospects in clinical practice.
This research is designed to detected the change of different levels of two modified nucleosides(M1A、1-MEI) in bladder cancer patients who were after the surgery and the normal at the different clinical stages and the patients who were after the surgery were in different pathology grades and clinical stages, and in order to find out the value of the detection in bladder cancer monitoring and provide a new method of detection in prognosis of patients with bladder cancer.
Methods
We enrolled85patients with bladder urothelial cell carcinoma (BUCC) confirmed by pathological examination.The85patients were fulfilled one year follow-up visit after TUR-BT and reviewed every three months.The muscle invasive cancer patients who were poor of the body condition or insisted on choosing this operation were operated by TUR-BT.The85patients did not relapse in the third month after operation.In the sixth monthafter operation20examples relapsed.18examples and19examples relapsed respective in the ninth month and the twelfth month after operation. Patients with recurrent add up to57examples as a recurrence group. The remaining28cases did not relapse in one year after operation as a not recurrence group. In85examples clinical stage55examples are Tis-T1, while30examples are T2-T4. In85examples pathology grade27examples are G1,40examples G2and18examples G3. In Tis-T1recurrence group are35examples, while a not recurrence group20examples. In T2-T4recurrence group are22examples,while a not recurrence group8examples.The50examples in the ordinary person are a control group. High-performance liquid chromatography/electrospray ionization-quadrupole-time-of-flight mass spectromerry was used to measure the levels of change of two urine modified nucleosides (M1A、1-MeI) which the patients with bladder cancer had different pathology grades、clinical stages,before or after operation and recurrence or not recurrence.
Results
1. The levels of change of two urine modified nucleosides in different time before and after operation have statistical significance (m1A:F=13.707,P<0.05,1-MeI:F=46.760,P<0.05). The levels of the third month after operation in not recurrence group(m1A:3.24±0.40,1-MeI:5.73±0.67)significantly lower than before operation (m1A:4.34±0.98,1-MeI:14.22±4.05P<0.005).The levels of the third month after operation in recurrence group (m1A:3.31±0.33,1-MeI:5.67±0.55) also significantly lower than before operation(m1A:4.32±1.19,1-MeI:14.31±4.12P<0.005).
2. The levels of the third month、the sixth month、the ninth month、the twelfth month after operation in not recurrence group(M1A:3.24±0.40、3.26±0.24、3.38±0.31、3.27±0.27;1-MEI:5.73±0.67、5.84±1.10、5.66±0.73、5.78±0.66) significantly lower than before operation. But there were no changes at different points in time after operation (P>0.005). The trend of total levels change after operation peformed fall and maintained in a low condition.
3. The levels of the sixth month、the ninth month、the twelfth monthafter operation inrecurrence group (MIA:4.04±0.48、4.11±0.47、4.09±0.53,1-MEI:11.46±1.34、12.14±1.22、12.33±1.27) had no significantly change (P>0.005).The levels compared with the third monthafter operation inrecurrence groupsignificantly higher than it.The trend of total levels change after operation peformed first decreased and then increased and maintained at a high level of status and close to the preoperative level.
4. Preoperative time points, without recurrence and relapse group was higher than the control (mlA:2.91±0.84,1-MeI:5.56±1.25P<0.01).The levels of the sixth month、the ninth month、the twelfth monthafter operation at all time points are recurrent group was the highest.
5. The levels of change of G1、G2、G3grade (MIA:5.10±1.93、4.72±1.71、5.26±2.13;1-MEI:16.52±8.11、14.22、7.65、13.43、6.57) had no statistical significance (P>0.01). The levels of change ofTis-T1and T2-T4clinical stage (MIA:4.77±1.43、4.82±1.38;1-MEI:15.13±5.87、16.32±6.41)also had no statistical significance(P>0.01).The levels in recurrence group in Tis-T1(mlA:5.92±1.28,1-MeI:20.01±8.53) were higher than the levels in not recurrence group (mlA:4.02±1.22,1-MeI:11.21±6.45P<0.05), which is the same in T2-T4.
Conclusion
1. The levels of change of M1A and1-MEI, that two kinds of urine modified nucleoside levels in the assessment of treatment efficacy as well as monitoring the condition changes.
2. Two kinds of urinary modified nucleoside levels'detection can judge the prognosis of recurrence risk for the bladder cancer patients.
引文
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