The Basic And Clinical Research Of Sentinel Lymph Node Mapping During Operation In Colon Cancer

Colon cancer is one of the most common cancers in our country. According to the data of the 2005 fourth nationwide seminar on colorectal operation, the incidence of colorectal cancer is increasing gradually and lies third in all of cancer, only second to lung cancer and stomach cancer. Clinical research indicates that lymph node metastasis is the most important factor affecting its prognosis. Due to absence of favorable evidence to survival, chemotherapy is commonly not applied toⅠandⅡstage colon cancer. However, there were twenty to thirty percent of patients with earlier cancer, which locates within intestinal wall and haD no metastasis, inevitably died of recurrence and distal metastasis though the standard radical operation was enforced on them. According to the clinical and basic research, the most possible factor is the no-check of metastasis, which degrades someⅢstage cancers toⅠorⅡstage cancers, thus chemotherapy was not used. Tumor staging is the most important survival factor, which is closely associated with lymph node metastasis and lymph node quantity. For accurate staging, it is impractical to routinely check all of the lymph nodes. There are more that sixty-nine percent of lymph node smaller than zero point five centimeter of diameter. Traditional surgical check is most likely to neglect lymph node with metastasis. Moreover, micrometastasis is very susceptible to be missed. All of these questions need a new method to find those occult metastases or micrometastases as far as possible. Sentinel lymph node mapping (SLNM) is the new method raised to try to resolve the above questions and now is gradually carried out in clinical. SLNM only needs to focus on some limited lymph nodes (one to four), so it can check them in detail and it can amend the tumor stating accordingly. The conception of SLN was raised in 1977 when Cabanas described the penial lymphatic drainage. It has been widely studied and applied clinically in the solid tumor until Morton successfully applied it to melanoma in 2000. The SLN is defined as the node that receive the tumor's primary lymphatic drainage at first and that is considered to be most likely to have metastasis or micrometastasis. According to the conception of the SLN, the tumor cell's metastasis starts from primary tumor, then goes through lymphatic channel sequentially to other site. Thus the site that receives the lymphatic drainage at first is most susceptible to have metastasis and then it can predicate metastasis-situation of regional lymph node. For the lymphatic drainage system of colon is relatively stable and the operation technique is relatively changeless, the SLN mapping to colon cancer is capable to be studied and so we can find its value in the digestive solid tumor and can apply it to the diagnosis and therapy of other solid tumor.The SLNM, as a micro-invasive technique, has a higher diagnosis rate of lymph node metastasis. Its clinical feasibility and accuracy has been validated in the breast cancer and melanoma. It has a good economy-time-effect radio. However the study of the SLNM in colon cancer started from 2000 and its value is still a controversy, which forces us to study its value deeply and try to find an more easily-operated technique and find a good evaluating-standard. Our topic is the basic study and clinical study on the feasibility of the SLNM in colon cancer, which includes three parts:①The clinical feasibility of the SLNM in colon cancer during operation and its clinical value;②The detection of micrometastasis in the SLN of colon cancer and its connection with patients' prognosis;③the association of SLN micrometastasis with pathologic character and related gene of colon cancer.PartⅠThe clinical feasibility of the sentinel lymph node mapping in colon cancer during operation and its clinical value;Objective: To apply sentinel lymph node mapping(SLNM)to colon cancer during operation with 1% methylence blue and study the its clinical feasibility and its clinical value.Methods: SLNM was implemented with 1% methylence blue during operation on eighty-six patients with colon cancer and the value of SLN was accordingly studied. SLN was checked by means of routine Hematoxylin-Eosin staining(HE) and immunohistochemistry staining(IHC).Results:①SLN was detected in 82 from 86 patients by SLNM with 1% methylence blue during operation and the success rate was 95.3% (82/86).There was no aberrant lymphatic drainage(ALD) to be found among 82 patients.②When only routine HE was applied to check lymph node of 82 patients, there found 35 patients with lymph node metastasis. When IHC was applied further to check SLN, there found 48 patients with lymph node metastasis. There were 3 patients who were diagnosed as lymph node metastasis by HE on none-SLN (NSLN) but diagnosed as no lymph node metastasis by IHC,so the false negative rate of SLN-check was 8.6%(3/35). There found 13 patients who were diagnosed as no lymph node metastasis by HE on SLN but diagnosed as lymph node metastasis by IHC, which upgraded 13 patients from stageⅠorⅡto stageⅢ. The diagnostic sensitivity, specificity and accuracy of SLN is 93.8%(45/48), 91.9%(34/37)and 96.3%(79/82)respectively.③There found 1377 lymph nodes in 82 patients, among them 182 were SLNs. There are 1 to 4 SLNs of one patient and 2.0±0.49 per patient. SLN accounts for 13.2% (182/1377) of total lymph nodes(TLN).④136 among 182 SLNs lied beside the intestinal wall. 35 SLNs were medial lymph nodes and 11 SLNs were central lymph nodes. There were 8 patients had skip metastasis(SM) and skip metastasis rate was 9.8% (8/82).The SLNs of skip metastasis accounted for 11.5% of the total SLNs.⑤When only routine HE was applied to check lymph nodes, there found 76 metastatic lymph nodes and metastatic rate of lymph node was 5.5%(76/1377). There found 43 metastatic SLNs and metastatic rate of SLN was 23.6%(43/182). Metastatic rate of NSLN was 2.8%(33/1195). Metastatic rate of SLN of 82 patients was significantly higher than that of the total lymph node(TLN) and NSLN(P<0.01).In 35 patients who had lymph node metastasis there found 76 metastatic lymph nodes. Among these, 43 were metastatic SLNs. The TLN metastatic rate of the patient who had lymph node metastasis detected only by the routine HE was 12.6%(76/602)while metastatic rate of SLN was 54.4%(43/79 )and metastatic rate of SLN was 6.3%(33/523). Metastatic rate of SLN was significant higher than that of the TLN and NSLN(P<0.01).⑥When the routine HE to lymph node and IHC to SLN of 82 patients, there found 107 metastatic lymph nodes. Among these, there were 74 SLNs. Metastatic rate of TLN was 2.8%(33/1195)metastatic rate of NSLN was 4.6%, while metastatic rate of SLN was 40.7%(74/182).The metastatic rate of SLN was significant higher than that of the TLN and NSLN(P<0.01). There found 107 metastatic lymph nodes in patients who had lymph node metastasis, among these were 74 SLNs. The metastatic rate of TLN was13.0%(107/820)and The metastatic rate of NSLN was 4.6%(33/711),while the metastatic rate of SLN was 67.9%(74/109). The metastatic rate of SLN was significant higher than that of TLN and NSLN(P<0.01).Conclusions:①SLNM during operation on colon cancer with 1% methylence blue was clinically safe and feasibly.②SLNM during operation can find skip metastasis, but we did not find ALD.③SLN can predict the metastatic status of lymph node of colon cancer.④SLN can upgrade some patients' tumor staging.PartⅡThe detection of micrometastasis in the SLN of colon cancer and its association with patients' prognosisObjective: To detect the micrometastasis in the SLN of colon cancer and analyze its association with patients' prognosisMethods:①SLN was detected by means of Envision immunohistochemistry to see if cytokeratin-20 protein was expressed.②Real-time quantitative reverse transcription polymerase chain reaction (real-time RT-PCR) method was established. LightCyler instrument was used for real-time RT-PCR. CK20mRNA expression level of patient's SLN was detected with GAPDH as inner comparison after operation.CK20mRNA expression index was calculated as CK20mRNA copy/ GAPDH copy×104.③All the patients were followed-up.④T test was used to compare the median. x2 test or Fisher test was used to compare the ratio. Comparison of the random datum of two groups was analyzed with Mann-Whitney test. Comparison of survival rate between two groups was analyzed with Log-rank test.Results:①When SLN was checked by means of IHC in those 47 patients who were diagnosed as no lymph node metastasis (pN0),there were 16 SLNs in which CK20 was positively expressed and 13 patients with CK20 positively expressed in SLN.②When SLN was checked by means of real-time RT-PCR, there found 25 patients of no lymph node metastasis and there were 31 SLNs in which CK20 mRNA was positively expressed and 25 patients with CK20 mRNA positively expressed in SLN. Among those 25 patients, there were 13 patients in whom CK20 is also checked out by IHC and there were 12 patients in which CK20 was only positively detected by real-time RT-PCR. The sensitivity of real-time RT-PCR was significant higher than that of IHC(P<0.01).CK20 expression index ranged from 37.2 to 521. 9 from those 13 patients with positive CK2O expression detected not only by IHC but also by real-time RT-PCR and the median of the CK20 expression index was 247.8. However CK20 expression index ranged from 6.5 to 134.8 in those 12 patients with positive CK2O expression only detected by real-time RT-PCR and the median was 57.8. CK20 expression index of patients with CK20 detected by IHC is significant higher than that of patients detected only by real-time RT-PCR(P<0.001).③Among 47 patients who were diagnosed as no lymph node metastasis by routine HE, there were 13 (13/47, 27.7%)patients who recurred or had metastasis and 7 (7/47, 14.9%)patients died. Among 13 patients who were diagnosed as SLN micrometastasis by IHC ,there were 5(5/13, 38.5%)patients who recurred or have metastasis and 3( 3/13,23.1% ) patients died. Among 12 patients who were diagnosed as SLN micrometastasis not by IHC but by real-time RT-PCR ,there were 5(4/12,33.3%)patients who recurred or had metastasis and 2(2/12, 16.6%)patients died. Among 22 patients who were diagnosed as no SLN micrometastasis by IHC and real-time RT-PCR ,there were 4(4/22,18.9%)patients who recurred or had metastasis and 2(2/22, 9.1%)patients died.The cumulative recurrence(metastasis) rate of patients with SLN micrometastasis was higher than that of patients without SLN micrometastasis(P<0.05).The cumulative survival rate of patients with SLN micrometastasis was lower than that of patients without SLN micrometastasis.Conclusions:①IHC and real-time RT-PCR are sensitive means to detect SLN micrometastasis.②Real-time RT-PCR is more sensitive than IHC to detect SLN micrometastasis.③SLN micrometastasis is closely associated with patients' prognosis. There is a higher recurrence rate and metastasis rate in the patients with SLN micrometastasis.④The detection of SLN micrometastasis is a clinically feasible technique, which can raise the positive rate of lymph node then upgrade the patient's pathological staging.PartⅢthe association of SLN micrometastasis with the pathologic character and related gene of colon cancer.Objective: to study the association of SLN micrometastasis with the tumor pathologic character and tumor related gene in colon cancer.Methods: SLN micrometastasis was detected in 47 patients who were checked as no lymph node metastasis(pN0). The related genes (VEFG,P53,nm23及COX-2) were checked by means of IHC.Results:①There were 25 patients with SLN micrometastasis among 47 patients who were detected as no lymph node metastasis(pN0).②SLN micrometastasis rate of the patient whose tumor diameter was no less than 2 centimeters was 57.9%, while that of the patient whose tumor diameter was less than 2 centimeters was 33.3%.The former was significant higher than the latter(P<0.05). SLN micrometastasis rates of the patients who had mass-type, ulcerated-type and infiltrating-type tumor were 33.3%, 52.6% and 63.2% respectively. The former two were significant higher than the latter one (P<0.05). SLN micrometastasis rates of the patients whose tumor cells were well, medially and poorly differentiated were 40.0%, 52.6% and 56.5% respectively. As the tumor cell gradually poorly differentiated, SLN micrometastasis rates of the patients gradually increased. But there was no statistical difference between their SLN micrometastasis rate(P>0.05).There was no SLN micrometastasis in patients whose tumor was classified as pT1. SLN micrometastasis rates of patients whose tumors were classified as pT2, pT3 and pT4 were 33.3%, 61.1% and 64.7% respectively. As the tumor infiltrated deeper into intestinal wall, the patient' SLN micrometastasis rate increased gradually(P < 0.05).③SLN micrometastasis rate of patient whose tumor negatively expressed VEGF was 32.0%, while that of patient whose tumor positively expressed VEGF was 77.3%. The latter was significantly higher than the former(P<0.01). SLN micrometastasis rate of patient whose tumor negatively expressed P53 was 47.8%, while that of patient whose tumor positively expressed P53 was 57.7%. The latter was higher than the former, but there is no statistical difference(P > 0.05). SLN micrometastasis rate of patient whose tumor negatively expressed nm23 was 60.0%, while that of patient whose tumor positively expressed nm23 was 41.2%. The former was significantly higher than the latter(P < 0.05). SLN micrometastasis rate of patient whose tumor negatively expressed COX-2 was 47.6%, while that of patient whose tumor positively expressed COX-2 was 57.7%. The latter was higher than the former, but there is no statistical difference(P>0.05).Conclusions:①Tumor pathologic character is associated with SLN micrometastasis. Among tumor pathologic characters, configuration, diameter and infiltrating depth are closely associated with SLN micrometastasis.②Some tumor genes are associated with SLN micrometastasis. Among tumor genes, VEGF and nm23 are closely associated with SLN micrometastasis.