Detection of micrometastasis of gastric carcinoma in peripheral blood circulation

Abstract

Aim: To detect the micrometastasis of gastric carcinoma in peripheral blood circulation using immunomagnetic beads sorting technique and RT-PCR technique, and to discuss its significance and the difference between the two methods.

Methods: Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood, immunomagnetic beads sorting technique and RT-PCR technique were used to detect the disseminated carcinoma cells. HE, immunocytochemical and immunofluorescence staining were also used to identify the characteristics of the cells separated with immunomagnetic beads sorting technique.

Results: Cells expressing cytokeratin were separated and enriched from the peripheral blood specimens of patients suffering from gastric carcinoma or chronic gastritis. After HE staining, two kinds of cells with little cytoplasm were found. Majority of these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal. Immunohistochemical staining indicated that there were CD34 and CD45 expression on the cell membrane of this kind of cells and these cells also showed expressed human telomerase reverse transcriptase by immunofluorescence staining, but the expression of carcinoembryonic antigen was absent. So, these cells might hematopoiesis precursors. Another kind of cells had larger and abnormal nuclei with thicker nuclear membranes. Massed chromatins and poly-nucleoli were found in the nuclei. These cells expressed human telomerase reverse transcriptase and carcinoembryonic antigen, but CD34 and CD45 were not found on the cell membrane. So, these cells were considered as gastric carcinoma cells escaping from the original focuses and existing in the peripheral blood circulation. Carcinoma cells were found in 25 of 60(41.7%) specimens of peripheral blood from patients with gastric carcinoma, while there were no such cells separated from the blood specimens of chronic gastritis patients. The difference of positive rates of disseminated carcinoma cells between two groups was markedly significant (P<0.005). The expressions of CK20 mRNA in peripheral blood specimens were examinated with RT-PCR. CK20 mRNA was detected from 32 of 60(53.3%) peripheral blood specimens in the group of gastric carcinoma patients, while none of the specimens from patients suffering from chronic gastritis had CK20 mRNA. Significant difference was also found between two groups (P<0.005). Statistic analyses also showed that there was a significant difference between the positive rates of two methods in detecting the disseminated carcinoma cells from the peripheral blood circulation of gastric carcinoma patients (P<0.05).

Conclusion: The results demonstrated that there were disseminated carcinoma cells in the peripheral blood circulation of some patients with gastric carcinoma. Disseminated carcinoma cells can be detected from the peripheral blood samples with immunomagnetic beads sorting technique and RT-PCR technique. The positive rate of RT-PCR technique is higher than that of immunomagnetic beads sorting technique in detecting micrometastasis.

Figure 1

Immunohistochemical staining showed that there was CD34 expression on the membranes of the cells, these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal (amplification ×1000).

Figure 2

Immunohistochemical staining showed that there was CD45 expression on the membranes of the cells, these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal (amplification ×1000).

Figure 3

Immunofluorescence staining the cells, which had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal (amplification ×200), had posi-tive expressions of hTERT.

Figure 4

The cells, which had small and round nuclei, even chromatins and the thickness of nuclear membrane was nor-mal (HE staining, amplification×1000), might be hematopoie-sis precursors.

Figure 5

The cells, which had larger nuclei, abnormal forms and thicker membrane, and massed chromatins and more than one nuclei could also be found in this kind of cells, had posi-tive expression of CEA (Immunofluorescence staining, ampli-fication ×200).

Figure 6

The cells, which had larger nuclei, abnormal forms and thicker membrane, and massed chromatins and more than one nuclei could also be found in this kind of cells, had posi-tive expressions of CEA and hTERT, and negative expressions of CD34 and CD45, were considered as the disseminated car-cinoma cells escaped from the primary neoplasm focus (HE staining, amplification ×1000).

Figure 7

RT-PCR for CK20 and β-actin. M: Marker; Lanes1-4 blood from gastric cancer; Lanes5-6 blood from chronic gastritis.