Lymphatic invasion using D2-40 monoclonal antibody and its relationship to lymph node micrometastasis in pN0 gastric cancer

Abstract

The monoclonal antibody D2-40 is a specific lymphatic endothelial markers and D2-40 staining have been applicable to evaluate lymphatic invasion in various malignant neoplasms. In the present study, we investigated lymph node micrometastasis determined by immunohistochemistry (IHC) and reverse transcription -polymerase chain reaction (RT -PCR) in all dissected lymph nodes obtained from 80 patients with node-negative gastric cancer, and analysed the relationship between micrometastasis and clinicopathological findings including lymphatic invasion of the resected primary tumour using D2-40 immunohistochemical staining. The incidence of micrometastasis determined by IHC and RT -PCR was 11.3% (nine out of 80) and 31.3% (25 out of 80), respectively. Although haematoxylin -eosin (HE) staining revealed lymphatic invasion in 11.3% (nine out of 80) of patients, D2-40 staining uncovered new invasion in 23.8% (19 out of 80) of patients. In the diagnosis of HE and D2-40 staining, the incidence of micrometastasis was significantly higher in patients with lymphatic invasion than in those without lymphatic invasion (P ¼ 0.0150 and Po0.0001, respectively). Micrometastasis correlated more closely with D2-40 than with HE staining. We demonstrated a high incidence of micrometastasis and lymphatic invasion and a correlation between them even in pN0 gastric cancer. When planning less invasive treatment, the presence of such occult cancer cells should be considered.

B7‐H3 expression in gastric cancer: A novel molecular blood marker for detecting circulating tumor cells

“…The gastric cancer cell lines, MKN-7, MKN-45, MKN-74, and NUGC-4, were cultured in RPMI 1640 (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal calf serum (Mitsubishi Kasei, Tokyo, Japan), 100 units ⁄ mL penicillin and 100 units ⁄ mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO 2 , as previously described (27,28) and then examined by immunocytochemistry and RT-PCR.…”

“…Total RNA was isolated and purified using phenol-chloroform as previously described. (27,28) The concentration and purity of the total RNA were determined using a GeneQuant pro UV ⁄ Vis spectrophotometer (Amersham Pharmacia Biotech, Cambridge, UK).…”

“…All total RNA samples were reverse transcribed using the Advantage RT-for-PCR kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) as previously described. (27,28) Samples were analyzed by quantitative RT-PCR (qRT-PCR) using the LightCycler System (Roche Diagnostics, Mannheim, Germany). Reaction mixtures contained cDNA from 250 ng of RNA, each primer, probe, MgCl 2 and LightCycler FastStart DNA Master hybridization probes (Roche).…”

“…The gastric cancer cell lines, MKN-7, MKN-45, MKN-74, and NUGC-4, were cultured in RPMI 1640 (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal calf serum (Mitsubishi Kasei, Tokyo, Japan), 100 units ⁄ mL penicillin and 100 units ⁄ mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO 2 , as previously described (27,28) and then examined by immunocytochemistry and RT-PCR.Patients. Blood specimens were collected preoperatively from 95 patients (64 men and 31 women; age range, 35-87 years; average, 68 years) with gastric cancer who underwent curative gastrectomy with lymphadenectomy at Kagoshima University Hospital (Kagoshima, Japan) between 2003 and 2005.…”

Clinical significance of circulating tumor cells in blood from patients with gastric cancer

“…The greatest advantage of the RT‐PCR assay is its high sensitivity for the molecular detection of CTC. We previously investigated its sensitivity using an in vitro model system with serially diluted gastric tumor cells mixed with peripheral blood cells from healthy donors 14. The findings of this cell spiking study showed that the RT‐PCR assay detected 10 tumor cells/107 donor‐derived peripheral blood cells.…”