Comparative Detection of Lymph Node Micrometastases of Stage II Colorectal Cancer by Reverse Transcriptase Polymerase Chain Reaction and Immunohistochemistry

Noura, Shingo; Yamamoto, Hirofumi; Ohnishi, Tadashi; Masuda, Norikazu; Matsumoto, Takashi; Takayama, Osamu; Fukunaga, Hisanori; Miyake, Yasuhiro; Ikenaga, Masakazu; Ikeda, Masataka; Sekimoto, Mitsugu; Matsuura, Nariaki; Monden, Morito

doi:10.1200/jco.2002.10.023

Cited by 133 publications

(102 citation statements)

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“…The RNeasy minikit (Qiagen, Hilden, Germany) was used to extract a minimal amount of RNA from each tissue sample, as described previously. 26 Semiquantitative duplex. RT-PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Immunohistochemical analysis of CD31, Ang-2, and VEGF was performed using the Vectastain avidin-biotin complex peroxidase kit (Vector Laboratories, Burlingame, CA), as described previously. [25][26][27] Heat antigen retrieval was performed in 10 mM citrate buffer, pH 6.0, at 95°C for 40 minutes. Primary antibodies were applied to sections at dilutions of 1:500 for CD31, 1:50 for Ang-2, and 1:50 for VEGF.…”

Section: Methodsmentioning

confidence: 99%

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Hepatic expression of ANG2 RNA in metastatic colorectal cancer

et al. 2004

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We examined the RNA content of the gene encoding angiopoietin (Ang)-2, a modifier of angiogenesis, in hepatic metastases of colorectal cancer (CRC) to explore the role of this protein in neovascularization of metastatic foci. Metastatic CRC exhibited notable blood flow and tumor vessel formation at tumor frontiers. Reverse-transcription polymerase chain reaction assays indicated that the ANG2 RNA content was greater in metastatic CRC than in primary CRC. Investigation of metastatic foci using laser capture microdissection revealed that the RNA content of ANG2, but not ANG1, increased from the bordering liver region to the periphery of the metastatic disease, and also from the periphery to the intermediate portion of the metastatic lesion; immunohistochemical analysis confirmed that there was a corresponding gradual increase in Ang-2 protein expression. Tie-2, a receptor for angiopoietins, was preferentially expressed in the bordering liver region rather than in metastatic CRC. Vascular endothelial growth factor (VEGF) also exhibited an expression pattern similar to that of Ang-2, and there was a significant correlation between the RNA content of ANG2 and that of VEGF in dissected samples (P ‫؍‬ .002). Western blot analysis suggested that expression of Ang-1, Ang-2, Tie-2, and VEGF may be regulated at a transcriptional level. C olorectal cancer (CRC) is a common malignancy worldwide. Although 84%-92% of patients with CRC are treated with surgical resection, more than half of these patients subsequently develop disease recurrence, 1 the most common type of recurrence being CRC metastatic to the liver, which often is associated with mortality. 2,3 Metastasis of CRC to the liver is a complex multistep process, which includes adherence of metastatic cells to endothelial cells (ECs), invasion across the endothelial basement membrane, cell proliferation, and neovascularization. 4 It was reported that tumor vessels appeared in human liver metastases when metastatic foci grew to 200 m in diameter and that the density of tumor vessels increased as tumor size increased. 5 Angiogenesis is essential for tumor growth and expansion, because the resulting blood vessels supply malignant cells with sufficient oxygen and nutrition. 6,7 Therefore, interruption of this process is considered to be a strategy for preventing CRC metastases to the liver.Recent studies have focused on novel endothelial growth factors such as angiopoietins (Ang), which are ligands for the endothelium-specific tyrosine kinase receptor Tie-2. 8 -10 Ang molecules play crucial roles in normal vascular development and in embryonic angio-

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“…The RNeasy minikit (Qiagen, Hilden, Germany) was used to extract a minimal amount of RNA from each tissue sample, as described previously. 26 Semiquantitative duplex. RT-PCR.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Hepatic expression of ANG2 RNA in metastatic colorectal cancer

et al. 2004

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“…Because up to 25% of patients with node-negative colorectal cancer ultimately die as a result of disease relapse, occult cancer metastasis has been suspected as a potential marker for systemic spread of tumor cells (1). However, the prognostic value of molecular tumor cell detection in patients with node-negative colorectal cancer has remained uncertain because of lack of evidence from prospective studies (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

“…With IHC and molecular genetics methods, occult tumor cells in regional lymph nodes are detected in 25% to 50% of patients with node-negative colorectal cancer (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Because up to 25% of patients with node-negative colorectal cancer ultimately die as a result of disease relapse, occult cancer metastasis has been suspected as a potential marker for systemic spread of tumor cells (1).…”

Section: Introductionmentioning

confidence: 99%

Micrometastasis Volume in Lymph Nodes Determines Disease Recurrence Rate of Stage II Colorectal Cancer: A Prospective Multicenter Trial

Yamamoto

Murata

Fukunaga

et al. 2016

Clinical Cancer Research

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Purpose: We reported in a retrospective study that the presence of micrometastasis in lymph nodes, when assessed by carcinoembryonic antigen (CEA)-specific RT-PCR, is a significant prognostic factor in stage II colorectal cancer. The aim of this study was to clarify the clinical value of micrometastasis in a prospective multicenter trial.Experimental Design: From November 2001 to December 2005, a total of 419 colorectal cancer cases were preoperatively registered at a central data center. Of them, 315 node-negative stage II colorectal cancer cases were enrolled. After RNA quality check, 304 colorectal cancer cases were analyzed for CEA mRNA in lymph nodes by both conventional RT-PCR (a band method) and quantitative RT-PCR. Long-term prognosis of the patients was determined by each method.Results: A positive band for CEA mRNA was detected in 73 (24.0%) of 304 patients. Postoperative adjuvant chemotherapy was applied in 31 CEA band-positive cases with an oral 5-fluorouracil derivative HCFU (1-hexylcarbamoyl-5-fluorouracil) for 1 year, whereas chemotherapy was not administered to CEA band-negative group. Multivariate Cox regression analyses revealed that a high micrometastasis volume (high MMV, n ¼ 95) was an independent poor prognostic factor for 5-year disease-free survival (DFS; P ¼ 0.001) and 5-year overall survival (OS; P ¼ 0.016).Conclusions: This prospective clinical trial demonstrates that micrometastasis volume is a useful marker in identifying patients who are at high or low risk for recurrence of stage II colorectal cancer.

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“…Several molecular markers including CEA, CK, MUC2 and p53 have been used to detect micrometastases in regional lymph nodes. However, to date, these markers investigated are unreliable due to low sensitivity and/or low specificity (Sanchez-Cespedes et al, 1999;Noura et al, 2002b;Rosenberg et al, 2002;Ho et al, 2004;Lee et al, 2006). Depending on the respective marker and method used, micrometastases were detected in 20 -100% of resected histologically negative lymph nodes of CRC patients with varying results regarding their prognostic value (Futamura et al, 1998).…”

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confidence: 99%

CpG-island methylation of the ER promoter in colorectal cancer: analysis of micrometastases in lymph nodes from UICC stage I and II patients

et al. 2009

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Patients with UICC stage II colorectal cancer (CRC) have a risk of approximately 20% to develop disease recurrence after tumour resection. The presence and significance of micrometastases for locoregional recurrence in these patients lacking histopathological lymph node involvement on routine stained HE sections is undefined. Oestrogen receptor (ER) promoter methylation has earlier been identified in CRC. Therefore, we evaluated the methylation status of the ER promoter in lymph nodes from 49 patients with CRC UICC stage I and II as a molecular marker of micrometastases and predictor of local recurrence. DNA from 574 paraffinembedded lymph nodes was isolated and treated with bisulphite. For the detection of methylated ER promoter sequences, quantitative real-time methylation-specific PCR was used. Of the 49 patients tested, 15 (31%) had ER methylation-positive lymph nodes. Thirteen of those (86%) remained disease free and two (14%) developed local recurrence. In the resected lymph nodes of 34 of the 49 patients (69%), no ER promoter methylation could be detected and none of these patients experienced a local relapse. The methylation status of the ER promoter in lymph nodes of UICC stage I and II CRC patients may be a useful marker for the identification of patients at a high risk for local recurrence.

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Comparative Detection of Lymph Node Micrometastases of Stage II Colorectal Cancer by Reverse Transcriptase Polymerase Chain Reaction and Immunohistochemistry

Abstract: Micrometastases detected by RT-PCR, but not IHC, may be of clinical value in identifying patients who may be at high risk for recurrence of CRC and who are therefore likely to benefit from systemic adjuvant therapy.

Cited by 133 publications

References 35 publications

Hepatic expression of ANG2 RNA in metastatic colorectal cancer

Hepatic expression of ANG2 RNA in metastatic colorectal cancer

Micrometastasis Volume in Lymph Nodes Determines Disease Recurrence Rate of Stage II Colorectal Cancer: A Prospective Multicenter Trial

CpG-island methylation of the ER promoter in colorectal cancer: analysis of micrometastases in lymph nodes from UICC stage I and II patients

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