[Abstract] Objective To investigate the N0 stage colorectal cancer lymph node micrometastases and its clinical significance. Cytokeratin 20 (cytokeratin, CK20) mRNA expression method with fluorescence quantitative polymerase chain reaction (FQ  PCR) detection of 45 routine radical mastectomy N0 of 453 patients with colorectal cancer lymph node detection of micrometastasis. 453 lymph nodes in 45 cases N0 patients with colorectal cancer, 20 patients (44.4%) 46 (10.2%) micrometastases in lymph detected. Micrometastases and independent of patient age, gender, tumor size, degree of differentiation, depth of invasion and tumor related (χ 2 = 5.445, P <0.05). 20 cases of micrometastasis in patients with and 25 patients without micrometastases in patients with an average follow-up time of 19.6 and 21.4 months, respectively. 20 cases micrometastases seven cases occurred in patients with recurrence and metastasis, 5 deaths; without micrometastases in 25 cases, only 1 case of death due to recurrence and metastasis (χ 2 = 7.305, P <0.05). Micro metastasis and without micrometastases group survival rates were 75.0% (15/20) and 96.0% (24/25), the difference between the two groups of patients was statistically significant (χ 2 = 4.240, P <0.05). Conclusions of CK20 mRNA FQ  PCR detection the N0 of colorectal cancer lymph node micrometastases in a sensitive and specific method to determine prognosis and develop a reasonable treatment plan to provide a theoretical basis of accurate clinical staging.

Key words colorectal tumors; lymph node metastasis; micrometastasis; keratin; fluorescence quantitative polymerase chain reaction

 Detection of Micrometastasis and Its Clinical Significance in Lymph Nodes from N0 Colorectal Cancers by FQ  PCR

    CHEN Wei  xiang1, FEI Shao  hua1, ZHU Lei 2, WU Jian 1, DING Jun 1, TAO Xiang1

    1. Department of Pathology, Gongli Hospital, Shanghai 200135, China; 2.Department of Molecular Pathology, Cancer Hospital, Fudan UniversityAbstract: Objective To study the detection of lymph node micrometastasis in N0 colorectal cancer patients and its clinical significance in these patients.Methods In the study, 453 lymph nodes obtained from 45cases of N0 colorectal cancer going curative operation were examined, using fluorescent quantity polymerase chain reaction (FQ  PCR) assay to detect cytokeratin 20 (CK20) mRNA expression for identifying micrometastasis.Results Micrometastasis was detected in 46 lymph nodes (10.2%, 46/453) of 20 cases (44.4%, 20/45). The presence of micrometastasis was correlated with invasion depth of primary tumor, but was not related to sex, age, size and differentiation of primary tumor. The mean follow  up time in the positive groups (20 cases) with micrometastasis and the negative groups (25 cases) without micrometastasis were 19.6 and 22.4 months respectively, but there were 7 cases post  operative recurrence, metastases and 5 cases died in positive groups (20 cases) with micrometastasis, only 1 case died because of recurrence or metastases in negative groups (25 cases) without micrometastasis (χ2 = 7.305, P <0.05). The disease  free survival of positive and negative groups was 75.0 % (15/20) and 96.0% (24/25), respectively. The two groups have significant difference (χ2 = 4.240, P <0.05). Conclusion CK20 FQ  PCR assay is a sensitive and specific method to detect lymph node micrometastasis in the patients with N0 colorectal cancer. The detection of CK20 mRNA in lymph nodes may contribute to improve the accuracy of clinical staging, determine patients’ prognosis and provide information for rational adjuvant therapy.

    Key words: Colorectal neoplasms; Lymphatic metastases; Micrometastasis; Cytokeratin; Fluorescent quantity Polymerase Chain Reaction (FQ  PCR)

Colorectal cancer is one of the common malignant tumors in China. Lymph node metastasis in colorectal cancer metastasis, decided that one of the most important indicators of prognosis of colorectal cancer. However, routine HE staining of patients with colorectal cancer without lymph node metastasis, “N0, 15.1% to 46.1% in the five years after surgery died of local recurrence or distant metastasis, prompting the presence of the lymphatic system or systemic micrometastases [1]. Cytokeratin 20 (CK20) is not expressed on all lymphocytes [2], and often limited in the gastrointestinal epithelial cells, with more strict the epithelial tissue specificity [2,3]. In this study, the use of the CK20 this feature, detected 45 cases of N0 patients with colorectal cancer to lymph nodes of CK20 expression by fluorescence quantitative PCR (FQ  PCR), to detect micrometastasis of recurrence in patients with colorectal cancer clinician judgment, transfer, prognosis and provide the basis for a reasonable treatment of perioperative.

    1 Materials and methods

    1.1 pathological data and specimen collection

    1.1.1 experimental group collected Gong Li, Shanghai Hospital from June 2004 to March 2007 were treated part colorectal underwent radical tumor resection specimens, all specimens were enrolled after conventional histopathological diagnosis of N0 . Collected specimens of 45 cases, including cancer 17 cases, colon cancer, 28 cases; 23 males and 22 females; aged 26 to 76 years, with a median age of 61 years old. According to the TNM classification standard developed by the International Union Against Cancer in 1997, Phase I of the 15 cases (T1-2 N0M0), Phase II, 30 cases (T3 N0 M0). Tumor-infiltrating this group the depth by AJCC standard, colon cancer is divided into: T1 ~ T4 rectal cancer is divided into: T1 ~ T3; classified as adenocarcinoma, mucinous adenocarcinoma histological type of tumor histological classification according to WHO standard signet ring cell carcinoma. Where adenocarcinoma divided into adenocarcinoma with mucus differentiated and pure adenocarcinoma two depending on the presence or absence of mucus. Immediately collected after surgical resection of the bowel and mesentery lymph node 453, an average of 10 per case. Lymph node each costing one divided into two, half frozen in liquid nitrogen and placed in the refrigerator freezer spare  80 ℃; half the number with 10% formalin-fixed, paraffin-embedded, and sent for pathological examination.

    1.1.2 The negative control group underwent benign intestinal disease (colonic adenomas, one cases of rectal polyps) 14 lymph nodes, lymph node also each chop, split in two, half for RNA extraction, the other half of the number pathological examination.

    1.1.3 The positive control group 2 patients with colorectal cancer in patients with stage Ⅲ 8 HE staining positive lymph nodes as a positive control.

    1.1.4 internal reference control to control by the housekeeping gene β-actin as an internal reference.

    1.2 fluorescence quantitative PCR detection of CK20 mRNA

    1.2.1 kit primer CK20 mRNA quantitative PCR kit (Shanghai Jiusheng medical supplies)

    1.2.2 Total RNA was extracted using Trizol extraction method.

    1.2.3 FQ  PCR detection of the extracted RNA samples for reverse transcription reaction; preparing the positive template standard gradient; samples of total RNA as a template, the detection system of the sample was prepared for reverse transcription reaction, take 5 μl of the RT product; Preparation Good positive on the standards and the samples tested at the same time, the instrument for PE7700 fluorescence analyzer, in strict accordance with the kit instructions operation, reaction conditions: 94 ° C for 5 min, then at 94 ° C for 30 sec, 62 ° C for 30 sec, 72 ° C for 30 sec. 40 cycle. After the reaction, calculated by computer software analysis quantitative results. Quantitative unit is the number of gene copies / ml.

    1.2.4 result judgment according to the standard curve to obtain the concentration (the number of gene copies / ml) of the test specimens. Concentration> 1 × 103 copies / ml for the positive results: the concentration of <1 × 103 copy number / ml was negative.

    1.3 statistical methods

    All data entry computer Epidata 3.02, SPSS 11.5 statistical package carried χ2 test and Fisher exact test for categorical data.

    2 Results

    2.1 lymph nodes of patients with colorectal cancer samples and a sample of the results of the control group

    453 lymph nodes in 45 cases N0 patients with colorectal cancer, 20 cases (44.4%) 46 (10.2%) lymph node FQ  PCR result is positive, that presence of micrometastases (LNMM) N0 upgraded to stage N1. Positive samples concentration of 1.333 × 103 ~ 2.188 × 108 copy number / ml. 2 cases of benign lesions of the gastrointestinal tract 14 lymph nodes FQ  PCR results were negative; eight and two cases of patients with stage Ⅲ HE staining positive lymph nodes FQ  PCR results were positive.

    CK20 expression rate of 2.2 in patients with colorectal cancer lymph node with clinical pathological factors

    45 cases N0 patients with colorectal cancer micrometastases in patients positive rate with sex, age, tumor size, histological type, tumor differentiation, and tumor intestinal circumference and other violations no correlation with the depth of tumor invasion , are shown in Table 1.

    2.3 of colon cancer in patients with rectal cancer in patients with lymph node CK20 mRNA expression differences

    A total of 302 lymph nodes in 28 patients with N0 stage colon cancer patients, 15 cases (53.57%) 35 (11.59%) lymph node micrometastases detected. 151 lymph nodes in 17 cases N0 rectal cancer patients, 5 cases (29.41%) 11 (7.25%) lymph node micrometastases detected. Patients with colon cancer than rectal cancer patients more prone to lymph node micrometastasis but FQ  PCR results showed no significant difference (χ2 = 2.501, P = 0.114).

    2.4 follow-up data

    (1) the relationship between tumor recurrence and metastasis: All patients were followed up, the longest follow-up time of 42 months, the shortest in April, with an average of 20.3 months. 20 cases of micrometastasis in patients with no micrometastases in patients with an average follow-up time of 19.6 and 21.4 months, respectively, but the 20 patients with lymph node seven cases CK20mRNA positive expression in tumor recurrence or metastasis (after 8 to 26 months), metastasis and recurrence rate in 25 cases up to 35%, negative expression in 25 cases one cases of metastasis, metastasis and recurrence rate of only 4%, and the difference was statistically significant (χ 2 = 7.305, P <0.05).

    (2) and survival in patients with relations: N0 LNMM survival rate of patients, 75.0% (15/20) below no LNMM by 96.0% (24/25), the two groups of the survival rate difference was statistically significant ( χ2 = 4.240, P <0.05). Table 1 clinicopathological characteristics of colorectal cancer patients with lymph node CK20 mRNA expression between

    3 Discussion

    Colorectal cancer is one of the most common malignant tumor. For nearly 20 years, radical resection of colorectal continue to progress, but does not significantly improve the prognosis of patients. Approximately 50% of patients died of of intraperitoneal local or regional recurrence and metastasis. While the cancer cells in the peritoneal cavity, lymph nodes, bone marrow and peripheral blood micrometastasis are important factors that lead to colorectal cancer recurrence and metastasis. At present most of the domestic and foreign scholars RT  PCR detection of lymph node micrometastases, especially in the sentinel lymph node micrometastasis [4]; of peripheral blood of patients with malignant tumors of epithelial FQ  PCR detection of tumor markers detect micrometastases [5]. Lymph nodes of patients with colorectal cancer for routine pathological FQ  PCR without lymph node metastasis, “micrometastasis detection applications marker CK20 micrometastases in lymph nodes as the experiment. Experimental results show that 44.4% (20/45) of patients, 10.2% (46/453) of lymph node micrometastases, below the bright red designed [6] FQ  PCR detection of colorectal cancer was found in 19/31 (61.29%) patients peripheral blood micrometastases. In this study, the range reported in the literature with RT  PCR detection of lymph node micrometastasis [7,8] between 21% to 100% of N0 patients with colorectal cancer. This indicates that the FQ  PCR detection of lymph node micrometastasis reliable diagnostic method. Also part of N0 colorectal cancer patients with lymph node that cancer cells have the conditions of lymph node metastasis. Response to the lymph nodes in the presence of CK20 mRNA-positive cells were targeted early treatment [9].

    The results of this study show that, independent of lymph node micrometastasis and the patient’s age, sex, tumor location, tumor size and tumor histology is closely related with the depth of tumor invasion, especially pT3 patients, lymph node micrometastasis rate of 56.7% (17 / 30). Therefore, we recommend pT3 colorectal conventional lymph node micrometastasis detection, for early detection of micrometastases and given a reasonable and timely treatment.

    Currently, lymph node micrometastasis detection impact on the prognosis of different reports. The results of this study: the survival of lymph node micrometastasis group is less than no micro-metastasis, micrometastasis and prognosis, and lymph node metastasis in patients with good prognosis. But does not rule out that the results of this study and the study sample size, short follow-up time and other reasons related. Greenson et al [9] reported that long-term follow-up (6 months to 10 years), micrometastasis-positive patients with high recurrence rate, as early recurrence and short survival micrometastasis-negative patients with relatively good prognosis. Micrometastasis and prognosis of colorectal cancer is worthwhile to continue in-depth research, we study the long-term results are still under follow-up observation.