We examined 74 surgically resected invasive gastric adenocarcinomas that were confined to the gastric wall (not invading the adjacent organs) and their normal tissues from the surgical pathology files of the first department of pathology, Kochi Medical School and its affiliated hospitals from 1994 to 2001.
The definitions used for histological classification were based on the criteria of Lauren15
and the World Health Organisation’s Histological typing of oesophageal and gastric tumours
(second edition, 1990)16
: diffuse type (24 tumours), intestinal type (43 tumours), and solid type (seven tumours). We examined one minority tumour type, carcinoma showing a solid growth pattern,16
and it will be referred to as solid-type carcinoma. Solid-type carcinoma is carcinoma showing a solid growth pattern; the neoplastic cells are closely packed and form large tumour nests, and the tumours have well defined boundaries; these neoplastic cells may be undifferentiated or poorly differentiated (containing a few glandular structures or mucus secreting cells).16
The depth of tumour invasion was classified as submucosa (29 tumours: six diffuse type, 21 intestinal type, and two solid type), muscularis propria (14 tumours confined to the muscularis propria: five diffuse type, eight intestinal type, and one solid type), and subserosa (31 tumours: 13 diffuse type, 14 intestinal type, and four solid type). In our present study, we classified the tumours confined to the submucosa as early cancers and those invading the muscularis propria and subserosa as advanced cancers.
Immunohistochemical studies were performed by the streptavidin biotin method using the Histofine SAB-PO(M) kit (Nichirei, Tokyo, Japan). Five monoclonal antibodies against ASMA, HCD, CD34, CD31, and low molecular weight cytokeratins (CK) were used. Table 2 details the monoclonal antibodies and staining procedures used. We examined immunoreactivity for HCD in all of the tumours, to distinguish ASMA positive stromal cells from smooth muscle cells, which are positive for both ASMA and HCD.8
We also examined immunoreactivity for CD31 in all of the tumour tissues, to distinguish CD34 positive stromal cells from vascular endothelial cells, which are positive for both CD34 and CD31.3
Vascular endothelial cells were used as the internal positive control for CD34 and CD31 immunostaining. We did not apply digital subtraction.2
As in our recent studies of gastric cancer, immunostaining for CK was also performed to define the tumour border in every specimen examined.2,7
Monoclonal antibodies used for immunohistochemical analysis
In the 13 diffuse-type carcinomas invading the subserosa, double immunostaining for ASMA and CD34 was performed. ASMA and CD34 were labelled with alkaline phosphatase–fast blue and 3,3`-diaminobenzidine tetrachloride (DAB), respectively.1
After immunostaining, we examined ASMA positive stromal cell distribution in the main body of gastric carcinoma tissues. The assessment was not based on the tumour growing edge and ulcerated surface. Tumours were classified into two types, namely: ASMA(+), tumours having ASMA positive stromal cells in the tumour stroma (fig 1A; note: fig 1B,C shows the expression of HCD and CK, respectively, in the same site as figs 1Aand 2A; note: fig 2B shows the expression of HCD in the same sites as fig 2A); and ASMA(−), tumours having no ASMA positive stromal cells within the tumour stroma. Statistical analysis was carried out using Fisher’s exact probability test. Values of p < 0.05 were considered to be significant. We did not perform statistical analysis in the solid-type carcinomas because of the low numbers of tumours analysed.
Figure 1 Expression of α smooth muscle actin (ASMA) and CD34 in diffuse-type advanced gastric carcinoma tissue. Stains for (A) ASMA, (B) high molecular weight caldesmon (HCD), and (C) low molecular weight cytokeratins (CK). ASMA positive stromal cells (more ...)
Figure 2 α Smooth muscle actin (ASMA) positive stromal cells in intestinal-type advanced gastric carcinoma tissue. Staining for (A) ASMA and (B) high molecular weight caldesmon (HCD). ASMA positive stromal cells—ASMA positive and HCD negative cells—are (more ...)