| Background:To date, radical excision, including the primary focus and involved regional lymph nodes, is the only potentially curative treatment for patients with colon carcinoma. A comprehensive project based on a surgical resection was commonly accepted by most gergeons for their patients. However, the outcome of colom cancer has been poor, up-to-date. Approximately 50 per cent of patirnts with colon carcinoma who have received surgical resection will died in five years. The presence or absence of nodal metastases is the single most important prognostic factor in resectable colon carcinoma and currently is the primary indication for adjuvant systemic therapy. Despite the more favorable prognosis of patients with localized cancer and no regional lymph nodes metastases demonstrated by routine histopathologic examination, up to 30 per cent of the patients will develop recurrent disease, despite apparent surgical cure. It is likely that some of these clinical failures are in part due to pathologic understaging of the tumor at the time of resection, because of occult metastasis in regional lymph nodes, bone marrow and peripheral blood, missed by routine histopathologic technology. The 5-year survival was 91 per cent approximately in patients of colon carcinoma with negative lymph nodes examinated by reverse transcriptase polymerase chain reactions(RT-PCR), compared with 50 per cent 5-year survival for patients with negative lymph nodes examinated by routine histopathology, whereas demonstrated positive by RT-PCR and immunohistochemistry(IHC) finally. Numerous studies have demonstrated micrometastases in colorectal carcinoma lymph node specimens via IHC, multilevel sectioning, monoclonal antibody techniques, and RT-PCR. However, because the resection apecimen usually contains 15 to 37 lymph nodes, even may search for 295 lymph nodes per patients, the routine use of these ultrastaging techniques on all excised lymph nodes would be time consuming and expensive. There is not clinical feasibility at all.According to physiological anatomy, the lymph draining of colon carcinoma follows a standing pathway. Sentinel lymph node(SLN) can be identifided the initial lymph node to receive metastatic cells from a primary tumor that drains via lymphatics. It is the regional lymph node most likely to contain early metastases.With the present biological investigation, the mutation of ongenes and, or anti-ongenes are the important factors which may result in malignant change, but the genetic polymorphism contribute to the manifestation pathological phenotypes and susceptibility of patients to malignant disorders. Fibroblast growth factor receptor-4 (FGFR4) was expressed obviously in the developing embryonic tissues and the malignant tissues, such as breast cnacer, pancreatic carcinoma and renal carcinoma, whereas FGFR4 protein was not expressed in the normal colonic mucosa. A novel allele of FGFR4 gene was identified in patients with breast cancer and normal blood donors. This single nucleotide polymorphisms(SNPs) are associated with aggressive disease progression and therefore represent a gene alteration that predisposes the breast cancer patients to poor clinical outcome. Relationship between the SNPs of FGFR4 gene and the micrometastases of colon carcinoma was not demonstrated thoroughly.Objective:(1) To investigate whether the technique of SLN mapping may be used for colon carcinoma. Whether the SLN status is the representative of regional lymph nodes. To investigate the clinical significance of SLN mapping during the operation.(2) To study the difference of metastasis and micrometastasis of colon carcinoma between SLNs and regional lymph nodes. To study the clinical importance of micrometastasis in lymph nodes.(3) To investigate the distinctive expression of FGFR4 protein between colon tumor and normal colonic mucosa, and the correlation between lymph node metastasis and FGFR4 protein expression.(4) To screen the allele polymorphism of No.1217 base of FGFR4 gene in patients with colon carcinoma and normal donor, and to search the relationship among FGFR4 gene SNPs and lymph node micrometastasis.Methods:Sixty-eight patients with primary colon carcinoma, range from 34 to 86 years old(average 60.8 years old, 43 male and 25 female), underwent colectomy with curative intent were studied prospectively. After mobilization of the colon tumor and mesentery, 5ml patent blue dye of 1% concentration was injected subserosally around the tumor. The first nodes highlighted with green dye among 5min were identified as SLNs. Additional nodes were identified after the operation from radically resected specimens including marked SLN. All lymph nodes in the colon carcinoma specimen were examined by conventional haematoxylin and eosin (HE) staining to detecte metastases. Micrometastases of lymph nodes were demonstrated by carcinoembryonic antigen immunohistochemistry (CEA-IHC) and cytokeratin-20 immunohistochemistry (CK20-IHC). FGFR4 protein expression in carcinoma and normal colonic mucosa tissues were investigated by western blot. The allele polymorphism of the No. 1217 base of FGFR4 gene in patients and normal donor were screened by real-time PCR (TaqMan PCR) quantiication Results:1. 1 to 4 SLNs were identified in 65 of 68 patients (96%), 3 patients had no SLNs detected because of the technique and anatomy. Overall, 158 SLNs were retrieved from the operative specimens, with a mean of 2.4 SLNs per patient65 patients( 158/65). SLNs were of 13 percent among the total 1,213 lymph nodes(17.8 per patient).2. By the conventional HE staining, 21.5 percent(34/158) of SLNs were positive. 27.7 percent( 18/65) of colon carcinoma patients with pymph node metastases determined by SLN. The skip SLN metastasis rate was 3.0 percent(2/65) when examined by routine HE staining.3. The metastasis rate in non-SLNs was 5.4 percent(57/l,055) determined by routine HE examination.4. Metastasis rate in SLNs was higher obviously than in non-SLNs and in total regional lymph nodes(/)<0.001). The accuracy positive rate of SLNs for predicting positive metastatic disease was 78.3 percent( 18/23), and the false-negative rate of SLNs was 10.6 percent(5/47).5. Micrometastases were found by CEA-IHC in 50 SLNs among 124 negative SLNs diagnosed by HE staining. Micrometastatic rate was 54.0 percent(27/50) in the conventional negative patients. IHC staining was more sensitive than routine HE staining(P<0.001).6. Micrometastatic rate in non-SLNs was 2.1 percent(21/998), and the false-negative rate of patient was 8.7%(2/23) according to the SLN status diagnosed by CEA-IHC. There were 2 patents which micrometastases were happened only in non-SLN(2.9 percent, 2/68).7. FGFR4 protein was expressed in 32 patients among the 68 patients(47.1 percent). 55.3 percent of the patients with lymph node metastases expressed FGFR4 protein in tumor tissues via 28.6 percent of patients without lymph node metastases(/)=0.041). No FGFR4 protein was expressed in normal colonic mucosa.8. A 1217 C—T transition of FGFRA gene exist in human being. Among 30 normal donor, the No. 1217 base shows C homozygote in 13(43.3 percent), C/T heterozygote shows in 14 (46.7 percent) and T homozygote shows in 3 patients(10.0 percent). The No. 1217 base shows C homozygote in 22 patients among 55 patients(41.5 percent), C/T heterozygote shows in 27 patients(50.9 percent) and T homozygote shows in 4 patients(7.6 percent). There was no a statistically significant of the FGFRA gene SNPs between patients and normal donor.9. Patients with C/T heterozygote or T homozygote will probably develop to lymph nodes and distant metastases(P=0.008, P=O.O33). Patients with C/T heterozygote had a 100 percent(4/4) rate of lymph node metases and a 25.5 percent(l/4) of distnt metastases. Patients with T homozygote shows 81.5 percent(22/27) and 11.1 percent(3/27), respectively. Patients with C homozygote shows 50.0 percent( 11/22) and 4.5 percent(l/22), respectively. There was a statistically significant correlation between FGFRA gene SNPs and metastases of patients with colon carcinoma.Conclusions:1. The technique of SLN mapping with patent blue dye is simple, feasible and accurate in colon carcinoma. It is worth to applicate in clinic. Metastases was seen more frequently in SLNs than in regional lymph nodes, with representativeness for regional lymph nodes.2. Because of the finding of intraoperative SLN mapping, the margin of radical resection will be more reasonable. Focused examination of SLNs will obviously decrease the clinic intensity of patholoists.3. Metastases can be found effectually by the means of IHC. The identification of lymph SLN micrometastases may upstage some patients from Dukes A/B to B/C disease, who may then benefit from further adjuvant chemotherapy. Micrometastases in SLNs.There may be a potential prognostic significance of SLN micrometastases detecting.4. FGFR4 protein expressed in some colon tumor tissues, and the expression was associated with lymph node metastases.5. A SNPs of 1217 C—T transition of FGFR4 gene exist in human being. This SNPs may correlation with the tumor node metastasis, local infiltration and distant metastases of patients with colon carcinoma. Patients with C/T heterozygote or T homozygote will probably develop to lymph nodes and distant metastases compared with C homozygote. |
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