摘要
研究背景
结直肠癌在世界范围内都是最常见的恶性肿瘤之一。在欧洲，结直肠癌无论是从发病率还是死亡率而言都是第二常见的恶性肿瘤。在亚洲包括我国，结直肠癌的发病数近几十年上升了2～4倍。虽然结直肠癌的治疗方法不断改进，但患者的生存率仍无明显改善。远处器官(肝、肺等)的血行转移是影响患者预后最常见和最严重的问题之一。因此，早期检测血行转移并推断患者复发的危险性对于结直肠癌的治疗是至关重要的。血循环中游离肿瘤细胞(circulating tumor cells，CTCs)的存在是肿瘤发生血行转移的基础。分子生物学技术的进步尤其是逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction，RT-PCR)技术的应用，已经使血液中少许肿瘤细胞的检出成为可能。细胞角蛋白20(cytokeratin，CK20)的表达具有严格的上皮组织特异性，在非上皮来源的正常血液中不表达。因此，一旦在结直肠癌患者外周血中检出有CK20 mRNA的阳性表达，即可推断有恶性肿瘤细胞的异位存在。然而，结直肠癌患者无论是在术前还是术中外周血CK20 mRNA表达的生物学意义仍不清楚，其表达的相关因素及其与患者预后的关系尚不明确。Ki-67是一种人类核增殖抗原，可以反映细胞的增殖状态，对于判断肿瘤预后有一定的价值。
研究目的
探讨结直肠癌患者围手术期外周血CK20 mRNA表达与临床病理特征以及预后的关系，同时了解外周血CK20 mRNA与相应肿瘤组织CK20及Ki-67表达之间的关系，评价外周血CK20 mRNA表达的临床意义，初步探讨其是否可以作为结直肠癌血行转移的预测指标。
研究方法
建立CK20 mRNA巢式RT-PCR法，应用健康人外周血和结直肠癌HCT-116细胞系评价其检测敏感性。
选择57例于2000年4月至2001年3月间在我院手术的散发性结直肠癌患者设为实验组，分别于手术前和术中切除肿瘤以后各抽取10 ml外周血，并收集肿瘤标本，详细登记患者的临床病理资料，术后对患者进行长期随访直至患者死亡或手术后5年底为止。
应用巢式RT-PCR法分别检测实验组患者术前和术中外周血CK20 mRNA的表达，并应用免疫组化EnVision~(TM)法检测相应肿瘤组织CK20和Ki-67的表达。将实验结果与临床病理资料以及长期随访结果进行统计学分析。
本研究还选择同一时期收入我院的15例结直肠良性病变患者以及10例健康献血者设为对照组，各抽取10 ml外周血，检测CK20 mRNA的表达。
研究结果
每1ml健康人外周血中混入5个以上HCT-116结直肠癌细胞，CK20 mRNA巢式RT-PCR法就可以检测到它的存在。
实验组57例散发性结直肠癌患者中，男性35例，女性22例，平均年龄(61.0±14.3)岁；结肠癌24例(其中近段结肠癌11例，远段结肠癌13例)，直肠癌33例。
实验组围手术期外周血CK20 mRNA的总检出率(术前和／或术中检测阳性的例数／总例数)为59.6％(34／57)。术前阳性率为42.1％(24／57)，术中增至56.1％(32／57)，差异有显著性(McNemar检验，P=0.039)。而结直肠良性病变对照组和健康对照组中均无一例外周血中检测到CK20 mRNA。Ⅰ、Ⅱ、Ⅲ、Ⅳ期患者围手术期外周血CK20 mRNA的阳性率依次为33.3％(2／6)、47.4％(9／19)、63.6％(14／22)、90.0％(9／10)，呈逐渐升高的趋势(线性趋势检验，P=0.010)。而且，与未发生转移(Ⅰ、Ⅱ期)的患者相比，已发生淋巴结或血行转移(Ⅲ、Ⅳ期)患者的外周血CK20 mRNA的阳性率显著增高(χ~2检验，P=0.033)。而不同肿瘤部位、肿瘤大小、组织学类型和分化程度的结直肠癌患者外周血CK20 mRNA的表达均无显著性差异(P值分别为0.411、0.620、0.449、0.388)。外周血CK20 mRNA的表达与CEA、CA50、CA19-9、CA242等血清肿瘤标志物水平亦无显著性相关(P值均＞0.05)。
本组结直肠癌肿瘤组织中CK20和Ki-67的阳性表达率分别为86.0％(49／57)和80.7％(46／57)。结直肠癌肿瘤组织CK20的阳性反应主要定位于细胞浆和细胞膜，少数细胞核亦着色；Ki-67的阳性反应主要定位于细胞核。肿瘤组织中CK20和Ki-67的表达越强，相应患者外周血CK20 mRNA的阳性率越高(P=0.042，P=0.001)。
实验组的随访率为96.5％(55／57)，其中适合生存分析者53例。外周血CK20mRNA阳性和阴性患者的5年生存率分别为43.8％(14／32)、90.5％(19／21)，二者有非常显著性差异(P=0.001)。外周血CK20 mRNA的表达与结直肠癌患者预后显著相关(Kaplan-Meier生存曲线，Log-rank单因素检验)。外周血CK20mRNA阳性患者的总生存时间(overall survival，OS)和无进展生存时间(progression-free survival，PFS)均显著短于阴性患者(P值分别为0.001和0.009)。并且，临床分期(TNM分期)也与结直肠癌患者预后显著相关。分期越晚，患者的OS和PFS越短(P＜O.001，P=0.020)。而且，Ⅲ期及以上分期患者的OS和PFS均显著短于Ⅱ期及以下分期患者(P＜0.001，P=0.007)。不同TNM分期和外周血CK20 mRNA表达情况没有对患者预后产生显著的交互作用(多因素方差分析，P=0.090)。结直肠癌肿瘤组织Ki-67的表达越强患者的OS越短(P=0.010)，但肿瘤组织Ki-67的表达与患者的PFS却无明显相关(P=0.598)。COX回归模型分析表明，TNM分期是影响结直肠癌患者预后的独立因素(P=0.018)，而外周血CK20 mRNA和肿瘤组织Ki-67的表达未进入最终模型(P值分别为0.284、0.425)。
结论
1．CK20 mRNA巢式RT-PCR法检测结直肠癌患者外周血中游离肿瘤细胞(CTCs)具有较高的敏感性和特异性。
2．结直肠癌患者术中外周血CK20 mRNA的检出率明显高于术前，提示手术操作本身可能增加肿瘤细胞播散入血的机会。
3．结直肠癌患者外周血CK20 mRNA表达与TNM分期呈正相关，分期越晚外周血CK20 mRNA的阳性率越高。
4．结直肠癌肿瘤组织CK20和Ki-67的表达越强，相应患者外周血CK20mRNA的阳性率越高，提示外周血CK20 mRNA的表达与原发肿瘤细胞的分化和增殖状态密切相关。
5．外周血CK20 mRNA阳性表达的结直肠癌患者预后较差。基因检测外周血中CTCs的确切的预后预测价值尚需大规模和长期的临床研究予以证实。
BACKGROUND
Colorectal cancer is one of the world's most common malignancies. In Europe, itis the second most common cancer both in terms of incidence and mortality. ManyAsian countries, including China, have experienced an increase of two to four times inthe incidence of colorectal cancer during the past few decades. Despite improvedtherapeutic modalities, the survival rate of the patients with colorectal cancer is notimproved profoundly. Circular metastasis in distal organs such as liver and lung is oneof the most common and serious problems influencing prognosis. Detecting circularmetastasis early and identifying the patients at risk of recurrence are essential tomanaging colorectal cancer. The presence of circulating tumor cells (CTCs) in theblood is the basis of circular metastasis. Advances in molecular biological technology,especially using reverse transcription-polymerase chain reaction (RT-PCR) method,have made it possible to detect even a few tumor cells in the blood. Cytokeratin 20(CK20) expression is restricted primarily to epithelial tissue. There is no CK20expression in normal blood other than epithelial tissue. As a result, if the expression ofCK20 mRNA is detected in peripheral blood of patients with colorectal cancer, atopicpresence of CTCs in peripheral blood can be inferred. However, the biologicalsignificance of the expression of CK20 mRNA in peripheral blood of patients withcolorectal cancer, both in the preoperative period and at the time of surgery, remainsunclear. Its correlating factors and the relationship between expression of CK20mRNA in peripheral blood and prognosis are not clear. Ki-67 is a human nuclearantigen associated with cell proliferation. Its prognostic value for malignancy hasbeen known.
OBJECTIVE
To investigate the relationship between expression of CK20 mRNA inperioperative peripheral blood of patients with colorectal cancer andclinicopathological features and prognosis. And to study the relationship betweenexpression of CK20 mRNA in peripheral blood and expression of CK20, Ki-67 in thecorresponding tumor tissue. Therefore to identify the clinical significance of CK20mRNA expression in peripheral blood of patients with colorectal cancer. And initiallyexamining the usefulness of CK20 mRNA as a predictor for circular metastasis ofcolorectal cancer.
METHODS
Establishing CK20 mRNA nested RT-PCR method and its detecting sensitivitywas than estimated by using healthy volunteer's peripheral blood samples andHCT-116 colorectal cancer cells.
Fifty-seven patients with sporadic colorectal cancer undergoing operation in ourhospital between April 2000 and March 2001 were selected as experimental group.Each 10 ml peripheral blood of patients was collected both in the preoperative periodand at the time of surgery after resection of tumor. The tumor tissue samples were alsoobtained postoperatively. Their clinicopathological features were registered in detail.Patients received long-term follow-up till death or 5 years after operation.
Using nested RT-PCR method to detect expression of CK20 mRNA inpreoperative as well as intraoperative peripheral blood of patients in experimentalgroup. And expression of CK20 and Ki-67 in the corresponding tumor tissue wasexamined using the EnVison~(TM) immunohistochemical method. The experimental datawas correlated with clinicopathological features and long-term follow-up data.
Fifteen patients with colorectal benign diseases admitted in our hospital duringthe same period, as well as 10 healthy volunteers were also selected as control groupin this study. Each 10 ml peripheral blood was collected from them to detect theexpression of CK20 mRNA.
RESULTS
The expression of CK20 mRNA could be successfully detected by nestedRT-PCR method when 1 ml healthy volunteer's peripheral blood mixed with morethan 5 HCT-116 colorectal cancer cells.
Of 57 patients with sporadic colorectal cancer in experimental group, 35patients were male, 22 patients were female, with an average age of 61.0±14.3 years.There were colon cancer in 24 (proximal colon cancer in 11, distal colon cancer in 13),and rectal cancer in 33.
The overall detection rate of CK20 mRNA in perioperative peripheral blood ofpatient in experimental group was 59.6％(34/57). CK20 mRNA was detected in 24 of57 (42.1％) preoperative peripheral blood samples, while in 32 of 57 (56.1％)intraoperative peripheral blood samples (McNemar test, P=0.039). None of peripheralblood samples from control group (patients with colorectal benign disease or healthyvolunteers) showed detectable CK20 mRNA.
In patients with tumor stagingⅠ,Ⅱ,ⅢandⅣ, the positive rate of CK20mRNA expression in perioperative peripheral blood was 33.3％(2/6), 47.4％(9/19),63.6％(14/22) and 90.0％(9/10), respectively. This result demonstrated thatexpression of CK20 mRNA in peripheral blood was significantly associated withadvanced tumor stage (Spearman correlation, P=0.010). Moreover, the rate of CK20mRNA expression in peripheral blood of patients with tumor stagingⅢ,Ⅳwassignificantly higher than that of patients with tumor stagingⅠ,Ⅱ(x~2 test, P=0.033).There was no significant relationship between expression of CK20 mRNA inperipheral blood and primary tumor location, tumor size, histological pattern, degreeof differentiation (P=0.411, 0.620, 0.449, 0.388, respectively). There was also nosignificant relationship between expression of CK20 mRNA in peripheral blood andserum levels of tumor markers including CEA, CA50, CA19-9, CA242 (P＞0.05).
The positive rate of CK20 and Ki-67 expression in tumor tissue of colorectalcancer was 86.0％(49/57) and 80.7％(46/57). The positive reaction of CK20 in tumortissue of colorectal cancer was mainly located in cytoplasm and cell membrane, and a small number of nuclei were also stained. The positive reaction of Ki-67 was mainlylocated in the tumor nuclei. With the increase of expressive intensity of CK20 andKi-67 in tumor tissue, the positive rate of CK20 mRNA expression in peripheral bloodwas increased accordingly (P=0.042, P=0.001).
The follow-up rate of patients in experimental group was 96.5％(55/57).Fifty-three patients were suited for survival analysis. The 5-year survival rate ofpatients with positive and negative expression of CK20 mRNA in peripheral bloodwas 43.8％(14/32) and 90.5％(19/21) (P=0.001). There was significant relationshipbetween prognosis of patients with colorectal cancer and expression of CK20 mRNAin peripheral blood (Kaplan-Meier survival curves, Log-rank univariate test). Theoverall survival (OS) and progression-free survival (PFS) of patients with CK20mRNA positive peripheral blood were significantly shorter than that of markergene-negative patients(P=0.001, P=0.009). Similarly, there was significantrelationship between prognosis of patients with colorectal cancer and clinical stage(TNM stage). Advanced tumor stage was significantly associated with shorter OS andPFS (P＜0.001, P=0.020). Moreover, the OS and PFS of patients with tumor stagingⅢ,Ⅳwere significantly shorter than that of patients with tumor stagingⅠ,Ⅱ(P＜0.001, P=0.007). There was no significant interaction to influence prognosis ofpatients between TNM stage and expression of CK20 mRNA in peripheral blood(multivariate mean square analysis, P=0.090). More expressive intensity of Ki-67 intumor tissue of colorectal cancer was significantly associated with shorter OS ofpatients (P=0.010), while there was no significant relationship between PFS ofpatients and expression of Ki-67 in tumor tissue (P=0.598). The COX regressionmodel analysis showed TNM stage was an independent predictor to the prognosis ofpatients with colorectal cancer (P=0.018), while expression of CK20 mRNA inperipheral blood and expression of Ki-67 in tumor tissue failed(P=0.284, P=0.425).
CONCLUSIONS
1. Detection of CK20 mRNA by nested RT-PCR to determine the presence ofcirculating tumor cells (CTCs) in peripheral blood of patients with colorectal cancer has high sensitivity and specificity.
2. The detection rate of CK20 mRNA in intraoperative peripheral blood issignificantly higher than that of CK20 mRNA in preoperative peripheral blood. Itseems that surgical manipulation may increase the incidence of hematogenousspreading of colorectal cancer cells.
3. A direct correlation is demonstrated between expression of CK20 mRNA inperipheral blood and TNM stage of colorectal cancer. The expression of CK20 mRNAin peripheral blood is correlated with advanced stage.
4. With the increase of expressive intensity of CK20 and Ki-67 in tumor tissueof colorectal cancer, the positive rate of CK20 mRNA expression in peripheral bloodwas increased accordingly. It seems that expression of CK20 mRNA in peripheralblood is closely related to the differentiation and proliferation of primary colorectalcancer cells.
5. The patients whose peripheral blood is CK20 mRNA positive show asignificantly poorer prognosis than patients who are marker gene negative. Largescale and long-term clinical studies are needed to confirm the prognostic value ofgenetically detecting CTCs in the peripheral blood.
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