CK19与CEA在非小细胞肺癌外周血微转移中的对比研究

目的检测非小细胞肺癌（NSCLC）患者外周血细胞角蛋白（CK19）及癌胚抗原（CEA）的表达,探讨对检测NSCLC微转移的可行性。方法应用RT—PCR技术检测48例NSCLC患者、15例肺良性疾病（BLD）患者和10例健康人外周血CK19及CEA mRNA的表达。结果NSCLC患者外周血中CK19及CEA mRNA的阳性表达率分别为68．75％、58．33％,在BLD组中表达率分别为13．33％、6．67％,而对照组外周血中均无表达,NSCLC组CK19及CEA表达均高于BLD组及对照组（P〈0．05）。外周血CK19及CEA的表达与临床分期及淋巴结转移密切相关,与病理类型及分化程度无关。结论CK19与CEA均可作为检测NSCLC患者外周血微转移较为合适的分子标志物,联合检测有助于提高检测的阳性率。     

基因检测胃癌区域淋巴结微转移的实验研究

目的　探讨 RT- PCR检测胃癌区域淋巴结微转移及其意义。方法　以病理组织学和 RT- PCR扩增角蛋白 1 9(K1 9)对 1 8例胃癌患者的癌组织标本及 78个淋巴结进行检测。结果　 1 8例胃癌癌组织均有 K1 9m RNA表达 ,78个淋巴结中 ,组织学阳性 1 6个 ,而 RT- PCR阳性达 2 6个。结论　 RT- PCR扩增 K1 9m RNA检测胃癌区域淋巴结微转移是一种敏感、特异的检测方法 ,结合淋巴结微转移的情况 ,对胃癌患者手术淋巴结清扫范围有一定的参考价值 ,有助于更精确的分期及对预后的判断。     

CEA mRNA在乳腺癌患者外周血中的表达及临床意义

目的以套式RT—PCR方法检测癌胚抗原（CEA）mRNA在乳腺癌患者外周血中的表达，并探讨其在乳腺癌诊断与治疗中的意义。方法收集乳腺癌患者52例（乳腺癌组）、乳腺增生性病变患者40例（增生组）、健康查体者20例（对照组），采集外周血样本。分别用套式RT—PCR法检测其外周血中CEAmRNA的表达。结果乳腺癌组外周血CEAmRNA阳性率为40．4％，增生组及对照组分别为5．0%和0，两两比较均有显著差异（P均〈0．01）；乳腺癌组TNMⅢ期、Ⅳ期患者CEAmRNA阳性表达率明显高于Ⅰ期、Ⅱ期患者，外周血CEAmRNA阳性表达与淋巴结转移密切相关。结论外周血CEAmRNA表达可作为乳腺癌转移的一个辅助监测指标。     

Detection of circulating gastric cancer cells in peripheral blood using real time quantitative RT-PCR

BACKGROUND/AIMS: Detection of occult cancer cells in peripheral blood or bone marrow has recently received a great deal of attention regarding the prediction of postoperative recurrence of the cancer, and for novel strategies of adjuvant therapy. This study addresses the detection of circulating tumor cells in peripheral blood in patients with gastric cancer using Quantitative RT-PCR. METHODOLOGY: Common mRNA targets for RT-PCR for detection of small numbers of cancer cells in gastric cancer are CK18, CK19, CK20, and CEA. Ten milliliter of peripheral venous blood was taken from 14 healthy Japanese volunteers and 101 patients with gastric cancer. Samples were analyzed using real-time TaqMan technology and a Model 7700-sequence system. The group of gastric cancer patients included 69 individuals with curative disease on preoperative diagnosis and 32 individuals with a non-curative operation or recurrence of the disease. RESULTS: The number of CK19 and CK20 mRNA copies was significantly increased in patients with a non-curative operation or recurrence of gastric cancer (CK19; p=0.0087, CK20; p=0.0022) compared with healthy volunteers. Cut-off levels of CK19 or CK20 copy numbers were determined by the maximum value of healthy volunteers. For CK19, there were 61 (88.4%) negative cases and 8 (11.6%) positive cases in 69 individuals with curative gastric cancer. There was a significant difference in tumor stages between CK19 positive and negative patients with curative disease on preoperative diagnosis. For CK20, there were 59 (85.5%) negative cases and 10 (15.5%) positive cases. There was no statistical difference between CK20 positive and negative cases for all clinicopathological factors. On postoperative day 14, there was a significant difference between positive and negative cases regarding tumor size, tumor stage, and lymph node metastasis for CK19, and tumor stage and lymph node metastasis for CK20. Five-year survival rates of patients with CK19 positive or negative cases were 50.0% or 79.0%, respectively (p=0.0347). While, for CK20, 5-year survival rates for positive cases was 51.9%, and for negative cases 78.9% (p=0.0490). CONCLUSIONS: Micrometastases of gastric cancer can be detected in circulating peripheral blood using quantitative real-time RT-PCR. CK19 is a better marker than CK18, CK20 and CEA, and could be clinically useful to estimate prognosis or to make a postoperative strategy of adjuvant treatment.     

The potential of cytokeratin 20 and mucin 2 mRNA as metastasis markers in regional lymph nodes of colon cancer patients investigated by quantitative RT-PCR

Purpose  The presence of regional lymph node metastases is one of the most important prognostic factors in colon cancer. Nevertheless, up to 30% of the lymph node negative patients experience disease recurrence. Possibly, this patient group may be identified by more sensitive techniques than routine histopathological examination of the lymph nodes. Methods  In the present study, we have evaluated the detection of colon cancer lymph node metastases by real-time RT-PCR quantitation of the epithelial-specific cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNAs. Results  Both assays were able to detect dilutions of tumor cells down to one tumor cell in 10⁶ normal lymphocytes. CK20 and MUC2 mRNA were quantitated in 52 normal lymph nodes from 12 patients undergoing surgery for benign bowel diseases and in 144 primary colon tumors. The median tumor level of both markers were more than 10⁴-fold higher than the highest level in normal lymph nodes, indicating that the markers had a potential for metastasis detection in a clinical context. We applied the assays to 61 lymph nodes with known metastases detected by routine staining. Elevated CK20 or MUC2 mRNA levels were detected in 57 (95%) of the 61 LNs. Conclusions  Thus, CK20 and MUC2 quantitation by real-time RT-PCR seems to be a promising, sensitive tool to detect metastases in regional lymph nodes from colon cancer patients.     

Risk Factors for Occult Lymph Node Metastasis of Colorectal Cancer Invading the Submucosa and Indications for Endoscopic Mucosal Resection

Purpose Although risk factors for histologically overt lymph node metastasis in patients with early-stage colorectal cancer have been clarified, the risk factors for occult lymph node metastasis are not clear. This study was designed to clarify risk factors for lymph node metastasis, including occult metastasis, in patients with colorectal cancer invading the submucosa and to determine the criteria for endoscopic resection of early colorectal cancer. Methods The risk factors for lymph node metastasis, including occult metastasis, were analyzed in 86 cases of surgically resected colorectal cancer invading the submucosa. The lymph nodes were assessed by immunohistochemistry with cytokeratin antibody CAM5.2. Results The frequencies of overt and occult metastasis to the lymph nodes were 13 percent (11/86) and 13 percent (10/75), respectively. Multivariate analysis showed vascular invasion (P = 0.001) and tumor budding (P = 0.003) to be independent risk factors for lymph node metastasis, including occult metastasis. For tumors with submucosal invasion ≤1,000 μm, no lymph node metastasis was found. The frequencies of lymph node metastasis for tumors with submucosal invasion of 1,000 to 2,000 μm and >2,000 μm were 21 and 37 percent, respectively. In considering combinations of risk factors, there was no lymph node metastasis in tumors having neither vascular invasion nor tumor budding and submucosal invasion of ≤3,000 μm. Conclusions Vascular invasion, tumor budding, and the degree of submucosal invasion were significant risk factors for lymph node metastasis, including occult metastasis. These three factors can be used in combination to identify patients requiring additional surgery after endoscopic resection. Supported in part by a Grant-in-Aid for Scientific Research (no. 15390401) from the Japanese Ministry of Education, Science, and Culture. Presented at the Congress of Japan Surgery Society, Tokyo, Japan, March 29 to 31, 2006. Reprints are not available.     

Tumor micrometastases in mesorectal lymph nodes and their clinical significance in patients with rectal caner

AIM: To investigate the number, size, and status of lymph nodes within the mesorectum and to explore the prognostic significance of lymph node micrometastases in patients with rectal cancer. METHODS: Thirty-one patients with rectal cancer undergone total mesorectal excision between October 2001 and October 2002 were included. Mesorectal nodes retrieved from the resected specimens were detected with a combination of haematoxylin and eosin (HE) staining and immunohistochemistry (IHC). The relations between lymph node metastases, micrometastases and postoperative recurrence were analyzed. RESULTS: A total of 548 lymph nodes were harvested, with 17.7+/-8.2 nodes per case. The average number of metastatic nodes in HE-positive patients and micrometastatic nodes in IHC-positive patients was 5.2+/-5.1 per case and 2.2+/-1.3 per case, respectively. The mean size of all nodes and metastatic nodes was 4.1+/-1.8 mm and 5.2+/-1.7 mm in diameter, respectively. The mean size of micrometastatic nodes was 3.9+/-1.4 mm in diameter. The size of the majority of mesorectal nodes (66.8%), metastatic nodes (52.6%), and micrometastatic nodes (79.5%) was less than 5 mm in diameter. During a median follow-up period of 24.6+/-4.7 mo, 5 patients (16.7%) had recurrence, of them 2 died and 3 survived. Another case died of tumor unrelated cause and was excluded. All 5 recurrent cases had 3 or more nodes involved, and one of them developed only lymph node micrometastases. The mean number of both metastatic and micrometastatic nodes per case differed significantly between the recurrent and non-recurrent groups (P<0.01 and P = 0.01, respectively). CONCLUSION: The majority of lymph nodes, metastatic, and micrometastatic lymph nodes within the mesorectum are smaller than 5 mm in diameter. The nodal status and the number of lymph nodes involved with tumor metastases and micrometastases are related to the rapid postoperative recurrence.     

Detection of micrometastases and skip metastases with ex vivo sentinel node mapping in carcinoma of the colon and rectum

Background The debate over sentinel lymph node mapping (SLNM) and focused pathologic examination to detect micrometastases in patients with colorectal cancer (CRC) continues. We present in this paper our experience with SLNM for CRCs to improve staging. In addition, we have detailed the mapping procedure on an anatomical basis to define skip metastasis. Materials and methods Forty-seven patients underwent ex vivo SLNM. Immediately after resection, 1 ml of patent blue VF was injected submucosally around the tumor. Lymph nodes harvested from the first 15 patients were mapped in a standard fashion as the blue-stained nodes (SLNs), and the others (non-SLNs) were dissected away. In the remaining 32 patients, the lymph nodes were also mapped separately in relation to their anatomic location and described as epicolic-paracolic, intermediate, and principal. The blue-stained nodes (SLNs) and non-SLNs, negative by hematoxylin and eosin stain, were further stained with cytokeratin immunohistochemical analysis and carcinoembryonic antigen. Results A total of 873 histologically confirmed LNs were examined with a mean of 18.6±8.1 nodes per patient. In 46 of 47 patients (97.8%), SLNs were identified. Immunohistochemical staining revealed micrometastases in the lymph nodes of four patients, which were negative by conventional methods. Anatomical skip metastases were noted in 4 of 32 patients studied (12.5%). Conclusion Ex vivo SLNM in CRCs is a feasible technique with a high SLN identification rate. Results of anatomical mapping of lymph nodes correlates with the limited literature, suggesting that occult skip metastases can occur in the apical lymph node group and may occur outside the resected area.     

癌胚抗原在结直肠癌淋巴结微转移检测中的应用

目的 探讨Ⅰ和Ⅱ期结直肠癌术后病理因素及淋巴结微转移对术后5年无瘤生存率的影响.方法 Ⅰ和Ⅱ期结直肠癌患者共126例,均行结直肠癌根治术.每例结直肠癌患者的淋巴结数平均为16枚(10～28枚),用癌胚抗原(CEA)指标对所有淋巴结进行免疫组化染色.统计分析临床病理因素及微转移对术后5年无瘤生存率的影响.结果 术后平均随访64.11(64～106)个月.淋巴管侵犯和肿瘤侵袭深度与淋巴结的CEA表达呈正相关,而其他临床病理因素与淋巴结CEA表达无明显相关性.10项临床病理因素对5年无瘤生存率的影响差异均无统计学意义(P＞0.05).淋巴结CEA表达阴性、孤立肿瘤细胞巢和微转移患者的5年无瘤生存率分别为75.4%、68.2%和46.2%.孤立肿瘤细胞巢患者与CEA阴性患者5年无瘤生存率比较差异无统计学意义(P=0.245).微转移患者与CEA阴性患者比较,前者5年无瘤生存率明显较低(P=0.003).结论 对于Ⅰ和Ⅱ期结直肠癌,若淋巴结中检测到微转移,其预后较差,术后复发率较高,应予以积极的术后辅助化学治疗.     

CK-19检测在乳腺癌前哨淋巴结微转移诊断中的价值

目的　探讨角蛋白- 19(Keratin- 19,CK- 19)检测在乳腺癌前哨淋巴结(SL N)微转移诊断中的价值。方法　采用亚甲蓝生物染色的方法对4 4例 、期乳腺癌患者的SL N进行活检,均行冰冻病理切片、石蜡切片(SE染色)检查;采用逆转录聚合酶链反应(RT- PCR)技术进行CK- 19检测。结果　SL N冰冻病理切片、石蜡切片示癌转移率分别为15 .91% (7/ 4 4 )、18.18% (8/ 4 4 ) ;CK- 19检测的阳性表达率为34.0 9% (15 / 4 4 )。其中,冰冻和石蜡切片阳性的SL N中CK- 19均阳性表达。CK- 19在SL N中的阳性表达率明显高于其冰冻及石蜡病理切片阳性率(P<0 .0 5 )。结论　CK- 19检测能提高乳腺癌SL N微转移的检出率,降低其假阴性率。     

Routine modified D2 lymphadenectomy performance in pT1-T2N0 gastric cancer

AIM: To evaluate routine modified D2 lymphadenectomy in gastric cancer, based on immunohistochemically detected skip micrometastases in level Ⅱ lymph nodes.METHODS: Among 95 gastric cancer patients who were routinely submitted to curative modified D2 lymphadenectomy, from January 2004 to December 2008, 32 were classified as pN0. All level Ⅰ lymph nodes of these 32 patients were submitted to immunohistochemistry for micrometastases detection.Patients in whom micrometastases were detected in the level Ⅰ lymph node stations ( n = 4) were excluded from further analysis. The level Ⅱ lymph nodes of the remaining 28 patients were studied immunohistochemically for micrometastases detection and constitute the material of the present study.RESULTS: Skip micrometastases in the level Ⅱ lymph nodes were detected in 14% (4 out of 28) of the patients.The incidence was further increased to 17% (4 out of 24) in the subgroup of T12 gastric cancer patients. All micrometastases were detected in the No. 7 lymph node station. Thus, the disease was upstaged from stage ⅠA to ⅠB in one patient and from stage ⅠB to Ⅱ in three patients.CONCLUSION: In gastric cancer, true R0 resection may not be achieved without modified D2 lymphadenectomy.Until D2+/D3 lymphadenectomy becomes standard,modified D2 lymphadenectomy should be performed routinely.     

Nomogram to Predict Occult N2 Lymph Nodes Metastases in Patients With Squamous Nonsmall Cell Lung Cancer

For nonsmall cell lung cancer (NSCLC) patients without distant metastases, occult involvement of N2 lymph nodes would be of the utmost importance in determining both treatment and survival. The key to optimal treatment strategies relied on accurate diagnosis, in particular accurate clinical tumor staging. Patients with clinical N0 or N1 staging preoperatively had a sizeable risk to have occult N2 lymph nodes metastases.From November 2004 to March 2007, the entire database in a tertiary hospital of all patients with a pathologic diagnosis of squamous NSCLC underwent anatomical pulmonary resection and systematic mediastinal lymph node dissection were retrospectively collected and reviewed. A nomogram was developed on the basis of a multivariable logistic regression model with a combination of all potential variables. In order to surmount the potential of overestimating predictive performance, both bootstrapping for internal validation and an independent external validation set were employed.A nomogram incorporating the significant risk factors was created to predict the probability of occult N2 lymph nodes metastases. The calibration plot for the probability of occult N2 lymph nodes metastases showed an optimal agreement between the predicted probabilities by nomogram and actual observed probabilities. An objective and accurate nomogram predictive model for occult N2 lymph nodes metastases was drawn up and validated internally and externally in patients with squamous NSCLC.The nomogram model, as a robust tool in predicting occult N2 lymph nodes involvement, could be involved in a cost-effective application of specific diagnostic and therapeutic strategies.     

K-19 mRNA RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer

AIM: To investigate the value and prospect of RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer. METHODS: Histopathology was used and K19 mRNA expression was detected by RT-PCR in tumor tissues and lymph nodes from gastric cancer patients undergoing radical resection of gastric carcinoma. RESULTS: K19 mRNA was expressed in all tumor specimens of 30 cases; of the 126 lymph nodes, 26 were histopathologically positive (20.6%), and 42 positive (33.3%) by RT-PCR. Amplification fragments of 460 and 540 bp were shown in all the tumor tissues and metastatic lymph nodes after K19 andβ-actin RT-PCR, while only a 540 bp fragment appeared in the lymph nodes of non-tumor patients. CONCLUSION: K19 mRNA RT-PCR is sensitive and specific in testing micrometastasis in regional lymph nodes of gastric cancer, and it is superior to routine histopathology.     

Lack of influence of cytokeratin-positive mini micrometastases in "Negative Node" patients with colorectal cancer: findings from the national surgical adjuvant breast and bowel projects protocols R-01 and C-01

PURPOSE: Results of the few extant reports concerning the clinical significance of so-called "occult micrometastases" of lymph nodes of patients with Dukes A and B colorectal cancer have been variable. We examined the presumably negative nodes of a larger cohort of such patients who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trials R-01 and C-01 for the influence of what we preferably designate as nodal mini micrometastases on parameters of survival. METHODS: Mini micrometastases were detected by immunohistochemical staining of the original lymph node sections with anticytokeratin A1/A3 in a total of 241 Dukes A and B patients with rectal and 158 with colonic cancers. Their frequency, as well as that of nuclear and histologic grades, and an estimation of their relationship to relative risks were correlated with overall and recurrence-free survival by univariate and multivariate analyses. RESULTS: Nodal mini micrometastases were detected in 73 of 399 (18.3 percent) patients of this cohort. They failed to exhibit any significant relationship to overall or recurrence-free survival. No association between the assessments of tumor differentiation and mini micrometastases was found. Nuclear and histologic grades also failed to further discriminate overall or recurrence-free survival in patients with A or B stages of colonic or rectal cancers in this cohort. CONCLUSION: The immunohistochemical demonstration of nodal mini micrometastases failed to discriminate high- and low-risk groups of patients with colorectal cancer who were designated as being node-negative after routine pathologic examination.