| IntroductionThe rectal cancer is one of the most familiar malignant tumors in mankind. It is the third among the whole body cancer in our country. Metastases and recurrence are the important characteristics of the malignant tumor, and are also the main reasons of the death of patients with carcinoma. About 30% -40% patients who had accepted therapy by radical operation were found metastases or recurrence in 5 years. Clinically whether chemotherapy should be need and who need chemotherapy among the patients in Dukes'A and B stage after surgery operation are being discussed, so positive cancer cell detected in peripheral blood and lymph node around the tumor may be the standard which should be chosen. Cytokeratin 20 (CK20) was found by Moll et al in 1990. It is a kind of polypep-tide whose expression is largely restricted to gastric and intestinal epithelium. Unlike other cytokeratins used to detect micro - metastases, it has stricter particularity of epithelium tissue. Normally, CK20 can be found in gastric and intestinal epithelium, urothelium and Merkel cell, and this transcript is not present in endothelial, blood cell, smooth muscle, lymphoid cells or other cell types normally found in lymph nodes. It is expressed in almost all colorectal cancers and this expression remains stable in metastases; proliferation and recurrence. Its characteristic of distribution makes itself to be a kind of excellent mark of tumor. So CK20 becomes a focus in the field of tumor molecular biology.In the present study, we used Fluorescence quantitative polymerase chainreaction (FQ-PCR) to detect expression of CK20 mRNA in peripheral blood and lymph node for rectal cancer patients. From this we can analyze the relations a-mong the expression of CK20 in peripheral blood, Dukes'stage and the type of histology. Also we detected the lymph nodes around the tumor by CK20FQ-PCR and routine histological technology, compared the rates of metastases obtained from the two methods, discussed whether the former is of the value of predicting metastases of rectal cancer and judging the situation of patient after surgical operation.Materials and methodsThe cases were obtained from The Forth Common Surgery Ward of The Second Affiliated Hospital of Chinese Medical University from Sep, 2003 to Dec, 2004. We collected the peripheral blood of 47 patients with rectal cancer, 10 patients with non-cancerous intestinal diseases and ten healthy volunteers as normal controls, and the lymph nodes around the tumor of patients with rectal cancer . During 47 cases of rectal cancer samples, the maximal age is 87 years old, the minimal age is 36 years old, the mean age is 62 years old; male: 29 cases, female: 18 cases; Dukes 'A stage: 15 cases, Dukes' B stage: 16 cases, Dukes' C stage; 13 cases , Dukes' D stage; 3 cases; high differentiation adenocarcino-ma; 23 cases, middle differentiation adenocarcinoma: 18 cases, low differentiation adenocarcinoma: 3 cases, mucinous adenocarcinoma: 3 cases. The chemotherapy and radiotherapy were not used before surgical operation in all cases , with the systimic lymph node expurgation in the operation. It is approved by pathology post-operation. The clinical stages of tumor are defined by improved Dukes' stage.1. sample preparing:Collected 5 ml the peripheral blood of 47 patients with rectal cancer and 10 patients with non-cancerous intestinal diseases before their surgical operation, and ten healthy volunteers as normal controls, taked it into tubes with anti-agglutination by hparin( hparin 2mg /ml, 0.5ml ) , fixed, used the liquid of Separating lymphoid cell to obtain WBC, frozen in - 80℃ for FQ-PCR preparation.Lymph nodes were harvested from freshly resented specimens and labeled. One-half of each node was kept for routine histopathology, while the other half was frozen in - 80℃ for FQ-PCR preparation. The Trizol method was used to extract total RNA.2. Synthesis of the primer and prode:base-sequence of CK20mRNA and prodeGenes for: Base sequenceUpstream primer 5 '-GGA AGT CGA TGG CCT ACA CAA -3' Downstream primer 5'-GGC CTG GAG CAG CAT CAA-3' prode . 5'-FAM-GATCTGGGCAACACTGTCAATGTGGAG-TAMRA-3'It was composed by ZhongShan Gene Research Center3. Detected by. FQ-PCR:Total RNA samples were used to cDNA synthesis; prepared the normal grads of positive template; took 5ul production of cDNA synthesis to be the template, which was used to preparate the detective system of samples; positive template and detective samples were put in the machine together. The condition of reaction was: 93℃ 2min, 93℃ 30min,55℃ lmin, total was 40 cycles. After the reaction, the software in computer can analyze the quantitative production. Its unit is copy/ul.4. Statistic analyze;Use SPSS12.0 software to do Nonparametric test and x~2 test, P <0.05 was considered to be significance.ResultsNone of the ten patients with non-cancerous intestinal diseases or the ten healthy volunteers was positive for CK20 mRNA . It had statistical difference from the group of rectal cancer (72.34% , 34/47) (x~2 = 37.434,P <0.01). The expressing quantity of CK20 in peripheral venous blood from 47 patients with rectal cancer had positive correlation with Dukes ' stage of rectal cancer(r = 0. 680, P < 0.01). The positive-rate of CK20 in stage C + D (93. 75% , 15/16)washigherthanstageA + B(61.29% ,19/31) ( x~2 =6.571,P< 0.05). And in each pathologic type, the positive-rate of CK20 was-, high-differentiation adenocarcinoma 65.22% (15/23) , middle differentiation adenocarcino-ma 72.22% ( 13/18 ) , low differentiation adenocarcinoma and mutinous carcin-omal00% (6/6) , three above have no difference of statistics ( x2 =4.442,P > 0.05 ). Analyzing the expressing quantity of CK20 in peripheral venous blood and doing Mann-Whitney Test among the Dukes' stages, stage A has no difference of statistics from stage B ( Z = - 0. 977, P = 0. 358 > 0. 05 ) ; stage C is higherthanstageB(Z = -3.798,P <0. 001 ) , stage D is higher than stage C (Z= -2.354,P=0.014<0.05).155 surgically removed lymph nodes from 47 cases of rectal cancer were examined by CK20 FQ-PCR assay. Results were compared with that by routine pathology. The metastasis in lymph nodes were detected in 16 cases (34. 04% , 16/47) and 31cases(65.96% ,31/47) (x2 =9.744,P<0.01)by routine histology and CK20FQ-PCR respectively . The rates of metastasis in lymph node-s detected by routine histology and CK20 FQ-PCR were 12. 90% (20/155) and 38.06% (59/155) respectively ( X2 =26.739,P <0. 01) . In every Dukes' stage the rate of metastasis in lymph nodes detected by routine histology was lower than by CK20 FQ-PCR.DiscussionCK20 is a recently identified polypeptide whose expression is largely restricted to gastric and intestinal epithelium. Unlike other cytokeratins used to detect micrometastases, this transcript is not present in endothelial and lymphoid cells or other cell types normally found in lymph nodes. It is expressed in almost all rectal cancer, and this expression keeps stable in lymph node metastases.FQ-PCR technique was devised by PE Company of American in 1995. It is operated easily ,rapidly, detennine result objectively, well repeated, the range of quantity is wide(0 ~ 1014copy/L) , need no gradiently dilution of sample, high sensitivity ,high specificity, high truth, utilizes the completely blocking op-erating system, overcome the deficiency such as be contaminated easily and occur false-positive like the traditional PCR technique. So FQ-PCR technique is a method of simplify, easily operated and clinical significance, and is a advanced technology that can be used in gene diagnostic, assessment of therapy effect, renovating technique and clinical research.None of the ten patients with non-cancerous intestinal diseases or the ten healthy volunteers was positive for CK20 mRNA, it is shown that CK20 has high specificity. In some of Dukes' stage A and B, which were determinated no me-tastases by routine examinations, CK20 was positive in the peripheral blood of rectal cancer patients, it is manifested that tumor cells had been in peripheral blood of earlier rectal cancer and was the condition of metastases from blood. So some of the patients in these stages should need chemotherapy or radiotherapy. The expressing quantity of CK20 in peripheral blood of rectal cancer patients has positive correlation with Dukes' stage, and increases with the development of disease. It was higher in stages C + D than in A + B. This conclusion was coincidence with Mr. SunNianxu et al. The quantity of CK20 in peripheral blood has no difference of statistics among the pathologic types, it was coincidence with Mr. HuangPeilin et al. All above show that detection of CK20mRNA can help to judge the malignant degree and prognosis of disease, and be a kind of basis for instructing assist therapy of rectal cancer patients before or after surgery, judging recurrence and monitoring the effect of chemotherapy.155 surgically removed lymph nodes from 47 cases of rectal cancer were examined by CK20 FQ-PCR assay. Results were compared with that by routine pathology. The metastasis rate in lymph nodes detected by routine histology was lower than by CK20FQ-PCR. This conclusion can be reached in every Dukes' stage either. It shows that detecting metastases of tumor in lymph nodes by CK20FQ-PCR is more sensitive than by routine histology, and earlier rectal cancer in Dukes'A and B stage may have the micro-metastases in lymph nodes. So judging the stage of tumor only through routine histology is not enough. And CK20FQ-PCR may make it complete.FQ-PCR had been used in many fields of medical research, especially in genetics for diagnosis chromosome disease and monogenic disease, and researc-... |
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