The prognostic significance of isolated tumor cells in the lymph nodes of gastric cancer patients

Background  

The clinical significance of isolated tumor cells (ITC) detected immunohistochemically in the lymph nodes of gastric cancer patients is controversial. The aim of this study was to examine the prognostic impact of ITC in patients with gastric cancer.     

绿色荧光蛋白基因标记的胃癌细胞系的建立

目的 建立能连续传代稳定高表达绿色荧光蛋白（GFP）的胃癌细胞株,探讨胃癌的生长与转移特征。方法 利用脂质体将携带GFP-cDNA的真核表达载体质粒转染胃癌细胞株GC9811-P,经过G418筛选和克隆化培养,用荧光显微镜及流式细胞仪检测转染的EGFP基因在癌细胞体内外的表达,MTT比色法观察细胞生长,Western blot检测转染细胞和未转染细胞肿瘤相关抗原的表达。结果 携带EGFP的真核表达载体能有效地转染胃癌细胞GC9811-P,转染的细胞能稳定、高效和持久地表达EGFP,与未转染细胞比较,他们的生物学特性未改变（P〉0. 05）。在裸鼠皮下肿瘤中,EGFP也能稳定、高效的表达。结论 胃癌GC9811P-GFP的建立为研究肿瘤侵袭和转移的发生机制提供了理想的细胞株。     

Chemosensitivity of peritoneal micrometastases as evaluated using a green fluorescence protein (GFP)-tagged human gastric cancer cell line

The Chemosensitivity of micrometastases in the peritoneal cavity to a 5-fluorouracil derivative (TS-1) was examined with a micrometastasis model featuring a human gastric cancer cell line tagged with the green fluorescence protein ( GFP ) gene in nude mice. Peritoneal metastases on the omentum and mesentery could be specifically visualized even when minute or dormant and also externally monitored noninvasively under illumination with blue light from 1 day after intraperitoneal (i.p.) injection of tumor cells. Metastatic deposits formed after i.p. injection of 2×10⁶ tumor cells were significantly reduced by TS-1 in a dose-dependent manner (15–20 mg/kg), when it was orally administered from day 1 post-injection for 4 weeks (early administration). No such inhibition was evident after injection of 1×10⁷ tumor cells. When 2×10⁶ tumor cells given injection, the ascites-free period in TS-1-treated mice was significantly longer than in their untreated counterparts. Survival of TS-1-treated mice (5/15) was also significantly higher than the zero rate in controls (0/15), with 4 out of 5 surviving mice being free from peritoneal metastasis and the exception having only a few dormant metastases. In contrast, when TS-1 was administered starting from day 7 post-injection for 4 weeks (late administration), the survival and ascites-free period of the TS-1-treated mice were not significantly influenced. The results indicate that the Chemosensitivity of peritoneal metastases to TS-1 is dependent on the number of i.p. tumor cells and the timing of drug administration. Peritoneal micrometastases at an early stage are most susceptible and can be effectively eliminated by oral administration of an anti-cancer agent, which leads to the longer survival and better quality of life (QOL) of the mice. (Cancer Sci 2003; 94: 112–118)     

Paraaortic lymph node micrometastasis and tumor cell microinvolvement in advanced gastric carcinoma

Background. Paraaortic lymph node dissection in advanced gastric carcinoma is controversial. The purpose of this study was to investigate the incidence and significance of micrometastasis (MM) or tumor cell microinvolvement (TCM) in these critical lymph nodes. Methods. A total of 2339 lymph nodes, including 390 paraaortic nodes, obtained from 47 patients with advanced gastric carcinoma were examined immunohistochemically, using cytokeratin antibody. Results. Lymph node metastasis was found in 95 of the 390 paraaortic nodes of 14 patients by routine histological examination. MM or TCM was immunohistochemically detected in 45 of the 295 negative paraaortic lymph nodes from 15 of 33 patients (MM, n = 5; TCM, n = 10). The 5-year-survival rate in the paraaortic node-negative group and cytokeratin-positive group was significantly higher that that of the hematoxilin and eosin-positive group. The total number of lymph node metastases by hematoxylin and eosin staining and the pathological lymph node compartments, by cytokeratin-positive nodes, were prognostic factors by multivariate analysis. Conclusions. We demonstrated a high rate of MM or TCM in the paraaortic lymph nodes and suggest that such harbored metastases are related to the prognosis of patients with advanced gastric carcinoma. On the basis of this study, a multi-institutional study should be considered. Received for publication on June 7, 1999; accepted on Sept. 30, 1999     

Assessment of lymph node micrometastasis in early gastric cancer in relation to sentinel nodes

Background The development of endoscopic resection and reduced surgical procedures has progressed in recent years. Lymph node micrometastases can be cited as one of the problems with reduced operations. In this study, we investigated clinicopathological findings and sentinel lymph nodes (SNs) for associations with micrometastases. We discuss the indications for endoscopic mucosal resection (EMR), reduced surgery, and sentinel node navigation surgery (SNNS) based on the results. Methods Immunostaining with anti-cytokeratin antibodies was used as the method of exploring for micrometastases. Comparisons and assessments were made in regard to the presence or absence of micrometastases and various clinicopathological factors. Results The relationship between the clinicopathological factors and micrometastases was investigated in 120 patients with pT1pN0 gastric cancer. Significant differences in depth of invasion (mucosal [m] versus submucosal [sm]) and histological type (differentiated versus undifferentiated) were observed in both univariate analysis and multivariate analysis. Micrometastases were observed in 32% of the sm cancers, and they were observed in group 2 lymph nodes (no. 7) in 8%. They tended to be more common in the undifferentiated type. The micrometastatic lymph nodes were restricted to blue nodes (BNs) and lymph nodes within the dye flow area of patent blue (used intraoperatively explore for SNs). Conclusion It is considered that the indications for current EMR and reduced surgery in early gastric cancer are valid from the standpoint of micrometastases. But if the SNNS that has been studied in recent years is introduced, the lymphatic basin dissection method seems valid only if the case is s-pN0 early cancer.     

乳腺癌前哨淋巴结微转移的检测与临床意义

前哨淋巴结(SLN)活检能够准确评估区域淋巴结状态,为乳腺癌患者提供精确的分期,同时也减轻了淋巴结阴性患者的手术并发症.规范前哨淋巴结病理检测手段,充分利用分子生物学检测方法,有助于准确判断微转移.但前哨淋巴结的微转移在乳腺癌预后判断、治疗决策中的意义尚存争议.     

Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line.

Initial arrest, attachment, extravasation and subsequent extravascular growth of tumor cells in the secondary organs are believed to be crucial events for hematogenous metastasis, but the actual processes in living animals remain unclear. For the present study, we established green fluorescent protein (GFP)-expressing rat tongue carcinoma cell lines (RSC3) that permit real-time analysis of micrometastasis formation in combination with intravital video microscopy (IVVM). With this system, GFP-expressing metastatic (LM-EGFP) and non-metastatic (E2-EGFP) cell lines could be visualized at the cellular level in live mice for more than 1 month. Real-time IVVM analysis of liver metastases after intraportal injection of cells via a mesenteric vein revealed that both LM-EGFP and E2-EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evidence of a "rolling"-like movement along endothelial cell surfaces was observed. Quantitative analysis of GFP-positive foci showed that E2-EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a substantial number of LM-EGFP cells remained in the liver, probably due to stable attachment to the sinusoidal wall. Confocal laser scanning microscopic study in combination with laminin immunohistochemistry revealed that only LM-EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by subsinusoidal basement membrane. Our results suggest that micrometastasis formation by LM-EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attachment to subsinusoidal basement membrane and subsequent intravascular growth before extravasation. The difference in metastatic potential between the 2 lines may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study may be a powerful tool for analyzing targets for various anti-metastatic agents in the sequential process of metastasis.     

MAGE基因在检测非小细胞肺癌淋巴结微转移中的应用

背景与目的MAGE基因是肿瘤特异性基因,本研究检测了MAGE1、2、3、4基因mRNA在非小细胞肺癌(NSCLC)淋巴结中的表达情况,以分析MAGE基因在检测NSCLC淋巴结微转移中的意义。方法采用逆转录-聚合酶链反应(RTPCR)方法检测53例NSCLC患者的111组淋巴结中MAGE1、2、3、4mRNA的表达。结果应用常规病理检查和应用RTPCR技术检测MAGE基因mRNA表达的淋巴结转移阳性率分别为27.9%(31/111)和41.4%(46/111),差异有显著性(P<0.05)。80组常规病理检查阴性淋巴结中至少有一种MAGE基因表达的淋巴结有19组,阳性率是23.8%(19/80)。31组常规病理检查阳性淋巴结标本中至少有一种MAGE基因表达的阳性率是87.1%(27/31)。非肺癌患者淋巴结四种MAGE基因表达均为0。结论应用RTPCR法检测非小细胞肺癌淋巴结中MAGE1、2、3、4基因mRNA的表达,有助于诊断淋巴结微转移,提高肺癌TNM分期的准确性。     

Establishment and characterization of a human gastric carcinoma cell line that is highly metastatic to lymph nodes

The actual mechanisms by which carcinoma cells metastasize to lymph nodes are still unclear, and there is a need to establish in vivo experimental models suitable for the investigation of lymph node metastasis. For the purpose, we established a highly lymph node-metastasizing line, designated AZL5G, derived from a human gastric cancer cell line, AZ521, which had low capacity for lymph node metastasis. AZL5G cells transplanted orthotopically in the nude mouse stomach metastasize predominantly to the regional lymph nodes, showing little potential for hematogenous metastasis. AZL5G tumors developing in the stomach and regional lymph nodes showed poorly differentiated adenocarcinoma with medullary growth, and their histologic appearance strongly resembled that of parental AZ521. The growth activities in vitro of low-metastatic AZ521 and high-metastatic AZL5G were almost the same, but the tumorigenicity in vivo of AZL5G was significantly higher than that of AZ521. AZL5G cells also showed clearly higher abilities of cell locomotion and adhesion to type IV collagen and fibronectin in vitro as compared with AZ521 cells. Flow cytometric analysis demonstrated that the expression of integrin beta1 subfamily except for alpha6 integrin was generally increased in AZL5G cells than in AZ521 cells. Especially, the expression of alpha1 and alpha2 integrins in AZL5G cells was clearly higher than in AZ521, while alpha(v)beta3 integrin, E-cadherin, ICAM-1 and CD44H were not expressed by either cell line. The cell adhesion blocking assay showed that DGEA-containing peptide, which is composed of alpha2 integrin recognition sequence, significantly reduced the adhesiveness of AZL5G cells to type IV collagen as well as to type I collagen and laminin. Furthermore, the administration of anti-alpha2 integrin mAb or DGEA peptide in AZL5G-transplanted nude mice produced a significant reduction in the number of lymph node metastases. These data suggest that the up-regulation of alpha2 integrin expression by gastric cancer cells may play a critical role in the process of lymph node metastasis through the increased adhesiveness to type IV collagen. In conclusion, we established a gastric cancer cell line, AZL5G, with a highly metastatic potential to lymph nodes. This well-characterized line and its in vivo experimental model should be useful for investigation of the mechanisms of lymph node metastasis and for establishment of a new therapeutic approach for human gastric cancer.     

绿色荧光蛋白pAcGFP标记的人乳腺癌细胞株的建立

目的 构建绿色荧光蛋白（pAcGFP）标记的MCF-7细胞系.方法 应用Fugene HD Transfection Reagent转染MCF-7细胞.经G418筛选获得稳定表达pAcGFP的MCF-7细胞系,用倒置荧光显微镜检测pAcGFP的表达,用流式细胞仪检测pAcGFP转染率.通过生长曲线和体外侵袭实验,对转染和未转染pAcGFP细胞的生物学行为进行综合分析.结果 获得了稳定表达pAcGFP的乳腺癌细胞株MCF7-pAcGFP,转染率约为85%.生长曲线和体外侵袭力与未转染前相似（P＞0.05）.结论 MCF7-pAcGFP细胞株具备稳定表达AcGFP的能力,转染pAcGFP对MCF-7细胞的生长和转移特性并没有影响.     

Significant correlation between micrometastasis in the lymph nodes and reduced expression of E-cadherin in early gastric cancer

Background. E-cadherin has been recognized as an impor-tant factor associated with tumor metastasis. However, the relationship between micrometastasis in the lymph nodes and the expression of E-cadherin in the primary tumor in gastric cancer remains unclear. Methods. Two consecutive sections of 4522 lymph nodes from 162 patients with early gastric cancer were prepared for simultaneous hematoxylin and eosin (H&E) and cytokeratin (CK) staining. Sections of primary tumors from 135 of these patients were prepared for E-cadherin immunostaining. Results. The incidence of lymph node involvement was significantly increased, from 6.8% (11/162 patients) by H&E staining, to 27% (43/162 patients) by CK immunostaining ( P < 0.0001). Micrometastasis in the lymph node was found in 32 of 151 (21%) patients who had no lymph node metastasis evidenced by H&E staining. Micro-lymph node metastasis was frequently found in tumors with a diameter more than 1.0 cm, of those that were poorly differentiated, deeply invaded, showed lymphatic on vascular invasion, and in those that showed reduced expression of E-cadherin. Loss of expression of E-cadherin in the primary tumor was closely correlated with micro-lymph node metastasis. Patients with tumors with micro-lymph node metastasis detected by CK immunostaining had a significantly lower 5-year survival rate ( P < 0.01) than those without such metastases. Conclusion. Tumors more than 1.0 cm in diameter and those that exhibit poor differentiation, deep invasion (i.e., to the submucosa), lymphatic or vascular invasion, and reduced expression of E-cadherin are risk factors for lymph node metastasis in early gastric cancer. Thus, it is recommended that cancers confined to the mucosa (m-cancers) that are more than 1.0 cm in diameter should not be treated with limited surgery without lymphadenectomy. Received: March 27, 2001 / Accepted: May 10, 2001     

肺癌淋巴结微转移灶检测的分子生物学意义

目的 通过免疫组化的方法检测非小细胞肺癌患者术后常规病理检查阴性的淋巴结的微转移灶，研究其分子生物学意义。方法 以行肺癌根治性手术的39名患者为对象，用免疫组化角蛋白(CK)染色的方法检测术后常规病理学检查阴性的90枚淋巴结中的微转移灶，同时也用免疫组化的方法检测相应患者原发肺癌病灶中的p53、p21^ras和Ki67的表达。结果 22个患者(56．4％)的26枚淋巴结(28．89％)检出微转移移灶。原发肺癌灶中有p53、p21^ras和Ki67表达的患者的淋巴结微转移灶检出率显著高于无这些蛋白表达的患者。肿瘤直径大于3cm的患者和肿瘤直径小于3cm的患者的淋巴结微转移灶的检出率分别为55．6‰和58．3％，两者的差别无统计学意义(P=1．000)。结论 原发肺癌病灶中p53、p21^ras和Ki67的表达和淋巴结微转移灶的存在有一定的关系。     

Clinical significance of micrometastasis to lymph nodes in gastrointestinal tract cancers]

Surgery is the main therapy for malignancies of the gastrointestinal tract. Lymph node metastasis is one of the major factors in predicting patients' clinical course and choosing appropriate adjuvant therapy after surgery. The concept of micrometastasis to regional lymph nodes emerged over 10 years ago, but its significance has been controversial. To clarify the relevance of micrometastasis of gastrointestinal tract cancers, we have established RT-PCT based-diagnostic methods using multi-markers such as CEA, CK20, and Mage 3. Prospective studies have shown that not a few micrometastasis-positive patients with carcinoma of the colon, stomach, and esophagus suffered disease recurrence, even though they did not show histologically positive lymph node metastasis. They were initially diagnosed as node-negative, and thus predicted to be disease free. A retrospective study of 62 patients with stage II node-negative colorectal cancer showed that 5-year overall survival was 78.2% among micrometastasis-positive patients, against 95.3% micrometastasis-negative patients. Moreover, there was a marked difference in 5-year disease-free survival, with 61.4% versus 88.4%, respectively. These data warrant further prospective study with a large population since RT-PCR based detection systems for micrometastasis appear to have the potential to improve conventional diagnosis and therapy for colorectal cancer.     

结直肠癌淋巴结微转移对预后影响的意义

背景与目的:淋巴结微转移的预后价值尚无一致意见,本研究旨在探讨结直肠癌淋巴结微转移的预后意义。方法:收集江汉大学附属医院1988年～2001年结直肠癌根治性手术切除、有完整淋巴结检查资料的标本80例,其中直肠癌30例,结肠癌50例。经过溶脂法检查淋巴结共3869枚,平均每例48.36枚,经4μm间断连续切片、常规HE染色和免疫组化染色,确定有无淋巴结转移与微转移(微转移灶为瘤团直径<0.2mm或瘤细胞数<50个)。数据经SAS8.1统计学软件进行Cox模型回归分析及Spearman等级相关分析。结果:共检出转移淋巴结232枚(6%),微转移淋巴结39枚(1.01%)。直肠癌中淋巴结转移数与患者生存时间有关(χ2=9.94,P=0.0016);淋巴结微转移与淋巴结转移数(r=0.44,P=0.016)等病理学指标有关。结肠癌中淋巴结转移数(χ2=9.52,P=0.002)、AJCC淋巴结分期(χ2=5.73,P=0.0167)与患者生存时间有关,淋巴结微转移与Dukes’淋巴结分期(r=0.314,P=0.008)有关。结论:淋巴结微转移与结直肠癌预后相关,但不能作为独立的预后指标。     

乳腺癌ER功能状态及腋淋巴结转移的检测

目的　探讨ER功能检测及RT PCR检测乳癌淋巴结转移的临床应用。方法　应用凝胶迁移阻滞法及配体亲和组织化学法检测乳癌患者ER的功能状态。以患者腋窝淋巴结的RNA作模板 ,应用特异性引物扩增相应的Keratin19片段 ,据此判断有无腋窝淋巴结转移。结果　在 5 5例ER阳性的标本中 ,ERE结合阳性者达 71%。在HE染色淋巴结阴性的患者中 ,有 12 .8%经RT PCR检测阳性。结论　ERE结合功能有助于全面反映患者ER功能状态。RT PCR检测淋巴结Keratin19表达可作为乳腺癌转移的标志。     

微转移检测评估胃癌肝十二指肠韧带淋巴结转移及临床意义分析

目的 检测胃癌患者肝十二指肠韧带淋巴结微转移,评估No.12淋巴结转移情况及其临床意义,为合理选择淋巴结清扫范围及评估预后提供依据.方法 回顾性分析251例施行No.12淋巴结清扫的D2+胃癌根治手术患者的临床资料,连续切片并免疫组织化学方法检测210例常规病理检查No.12阴性淋巴结的微转移,分析No.12淋巴结转移与临床病理参数以及各亚组之间的关系,并进一步预后分析.结果 淋巴结微转移的检出率为5.71%(12/210),No.12淋巴结转移率从常规病理检查的16.30%提高到21.11%.No.12淋巴结转移与浸润深度、肿瘤大小、临床分期、其它组淋巴结转移有显著相关性(P＜0.01).No.12a、12b淋巴结转移率分别为17.93%、11.16%,No.12a淋巴结转移与No.12b转移存在显著相关性(P＜0.05).临床分期与No.12淋巴结转移为独立预后因素.结论 免疫组织化学方法检测细胞角蛋白敏感性高,可提高淋巴结转移的检出率.No.12淋巴结转移影响预后.进展期胃癌No.12组淋巴结清扫应作为常规,特别是No.12a与12b清扫,即肝十二指肠韧带的脉络化清扫.     

乳腺癌前哨淋巴结微转移研究中存在的问题

介绍乳腺癌前哨淋巴结微转移研究中存在的问题,包括检测方法、判断标准、临床意义等.     

Comparative T-cell oligoclonality in lung, tumor and lymph nodes in human non-small cell lung cancer

In lung cancer as in other malignancies, tumor formation induces the development of local and systemic antitumoral immune responses. The tumor itself becomes surrounded by a local stroma reaction containing inflammatory cells, a large part of which being tumor infiltrating T-lymphocytes. This study was designed to investigate the potential clonality of these T-cells in non-small cell lung cancer. Two complementary methods where used: exploration of the Vbeta TCR repertoire usage in flow cytometry and analysis of the Vgamma TCR repertoire in multiplex PCR and gradient gel electrophoresis. These techniques were applied respectively to eluted fresh lymphocytes and extracted DNA from healthy lung tissue, tumor and lymph nodes from 44 patients. There was a good correlation between the two techniques used. An oligoclonal repertoire restriction was noted in most of the cases and in the three types of tissues studied suggesting the presence of tumor-specific clones. Moreover, Vbeta14 appeared to be the most frequent specificity used whatever the tissue considered, while Vbeta13.1 appeared to be selectively used in the stroma reaction of epidermoid lung carcinomas. A restricted TCRgamma band was also present in these tumors, and two more bands of TCRgamma where selectively present in adenocarcinomas. The demonstration of both alpha-beta and gamma-delta TCR restriction suggests both the recruitment of specific T-cells and their local proliferation within the tumoral tissue. The same feature in healthy lung tissue indicates that it might already be the site of specific anti-tumoral T-cell reactivity. In conclusion, this study reports on the presence of oligoclonal T-cell responses in most cases of non-small cell lung cancer. The comparison of tumor, healthy tissue and lymph nodes showed some degree of patient-dependent similarities suggestive of tumor specificity.