Aims—To investigate the role of CD34 positive stromal cells, namely dendritic interstitial cells, in gastric carcinomas, the distribution of CD34 positive stromal cells in gastric adenocarcinomas (GCs), with special reference to two histological types (diffuse (D-type) and intestinal (I-type)), was examined.
Methods—In total, 55 surgically resected GCs (15 D-type and 40 I-type) and their normal tissues were examined. To distinguish CD34 positive stromal cells from vascular endothelial cells and to recognise the tumour border, immunostaining for CD34, CD31, and low molecular weight cytokeratins was performed.
Results—In the 15 D-type GCs, eight of the nine D-type GCs invading the muscularis propria and subserosa had a large number of CD34 positive stromal cells in the tumour stroma, whereas all six D-type GCs confined to the submucosa had no CD34 positive stromal cells in the tumour stroma. All of the 40 I-type GCs had no CD34 positive stromal cells, regardless of tumour depth.
Conclusions—These results suggest that CD34 expression in stromal cells is associated with progression of D-type GCs, and that absence of expression is also seen in I-type GCs that are progressing.
- stromal cell
- diffuse type
- intestinal type
- gastric adenocarcinoma
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The CD34 molecule is a 110 kDa transmembrane cell surface glycoprotein,1 which was originally described as a marker for human haematopoietic stem cells.2 CD34 has no significant sequence homology to any known proteins.2 CD34 positive stromal cells, namely dendritic interstitial cells,3 are distributed throughout the human body including the gastrointestinal tract.3–12 In the gastrointestinal tract, CD34 positive stromal cells are distinct from interstitial cells of Cajal.10,11 An immunohistochemical study using confocal laser microscopy revealed that interstitial cells of Cajal are positive for c-kit and negative for CD34, whereas CD34 positive stromal cells are negative for c-kit.10,11 It has been suggested that CD34 positive stromal cells throughout the human body play a supportive role, not only in the maturation or proliferation of adjacent mesenchymal and epithelial stem cells, but also in immune mediated responses.4–6
Recently, we reported that a lack of CD34 expression in colorectal tumour stromal cells is associated with desmoplastic stroma formation in well and moderately differentiated adenocarcinomas.12 However, the relation between CD34 positive stromal cell distribution and histological types of gastric cancer has not been studied.
In our present study, to elucidate the importance of CD34 positive stromal cells in tumour stroma formation and progression of gastric adenocarcinomas with special reference to histological types, we examined the distribution of CD34 positive stromal cells in diffuse-type (D-type) and intestinal-type (I-type) gastric adenocarcinomas and their normal tissues.
Materials and methods
We examined 55 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 1999. The definitions used for histological classification were based on the criteria of Lauren13; 15 D-type and 40 I-type tumours were identified. Depth of tumour invasion was classified as submucosa (27 tumours; six D-type, 21 I-type), muscularis propria (13 tumours confined to the muscularis propria; five D-type, eight I-type), and subserosa (15 tumours; four D-type, 11 I-type). 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). Three monoclonal antibodies against CD34, CD31, and low molecular weight cytokeratins (LMW-CKs; cytokeratins 8 and 18) were used. Table 1 lists the monoclonal antibodies and staining procedures. We examined immunoreactivity for CD31 in all of the tumours and their normal tissues, to distinguish CD34 positive stromal cells from vascular endothelial cells, which are positive for both CD34 and CD31.14 Vascular endothelial cells were used as the internal positive control of immunostaining for CD34 and CD31. (We did not apply digital subtraction.) Similar to our recent study regarding gastric cancer, immunostaining for LMW-CKs was also performed, to recognise the tumour border in every specimen examined.15
After immunostaining, we examined CD34 positive stromal cell distribution in gastric adenocarcinomas and normal tissues of the 55 cases. Regarding the tumours, the number of CD34 positive stromal cells was classified into two groups, namely: (+), tumours having CD34 positive stromal cells in the tumour stroma (fig 1A; note: fig 1B, C shows the expression of CD31 and LMW-CKs, respectively, at the same site as fig 1A); and (−), tumours having no CD34 positive stromal cells in the tumour stroma (fig 2). Statistical analysis was carried out using Fisher's exact probability test and p values < 0.05 were considered to be significant.
Table 2 summarises the results.
In all of the 55 cases examined, CD34 positive stromal cells were mainly in the perivascular area of the normal submucosa, muscularis propria, and subserosa. CD34 positive stromal cells were not detected in the lamina propria but they were distributed in the areas adjacent to the muscularis mucosa. Results concerning the normal gastric wall were the same as the recent report by Kim et al,16 who found that CD34 was expressed in stromal cells around vessels and muscle bundles in the gastric wall.16
In the 15 D-type gastric adenocarcinomas examined, eight tumours had CD34 positive stromal cells in the tumour stroma, whereas the remaining seven tumours had no CD34 positive stromal cells in the tumour stroma (table 2). All of the eight tumours were advanced cancers, and had a large number of CD34 positive stromal cells (fig 1A; note: fig 1B, C shows the expression of CD31 and LMW-CKs, respectively, at the same site as fig 1A). Eight of the nine advanced cancers had CD34 positive stromal cells in the tumour stroma classified as (+), whereas all of the six early cancers had no CD34 positive stromal cells and were classified as (−) (p = 0.0014).
In contrast, all 40 I-type gastric adenocarcinomas examined had no CD34 positive stromal cells classified as (−), regardless of tumour depth (fig 2).
Regarding the 28 advanced cancers, eight of the nine D-type adenocarcinomas were classified as (+), whereas all of the 19 I-type adenocarcinomas were classified as (−) (p = 0.0000029).
CD34 positive stromal cells were absent in the tumour stroma of well and moderately differentiated adenocarcinomas of the colorectum.12 However, in our present study, we detected CD34 positive stromal cells in the D-type advanced gastric adenocarcinomas. To our knowledge, this is the first report to detect large numbers of CD34 positive stromal cells in malignant epithelial tumour stroma.
CD34 positive stromal cells, namely dendritic interstitial cells, are distributed in the connective tissue surrounding mammary acini,4 salivary gland acini,5 thyroid follicles,6 hair follicles,7 sweat glands,8 and endocervical and deep endometrial glands of the uterus,9 whereas no CD34 positive stromal cells are detected in the lamina propria of the colorectum12 and stomach. In the colorectum12 and stomach, CD34 positive stromal cells are found only in the submucosa, subserosa, muscularis propria, and muscularis mucosa. These results suggest that CD34 positive stromal cells are essential for the maintenance of gastrointestinal mesenchymal elements including smooth muscles and vessels, but not for normal gastrointestinal epithelial cell maturation and proliferation.
During haematopoietic differentiation, the expression of CD34 decreases and terminally differentiated cells do not express CD34.17 Accordingly, in view of the distribution of CD34 positive stromal cells, the stroma of D-type gastric adenocarcinoma invading the muscularis propria and subserosa may be immature, whereas that of I-type gastric adenocarcinoma may be mature. Further studies including molecular and cell biological analyses are needed to confirm the biological meaning of CD34 expression.
D-type gastric carcinoma-like carcinomas are also reported in the colorectum, although at a very low frequency.18 Further examination of CD34 expression in tumour stromal cells should be performed in malignant neoplasms of other organs showing a D-type gastric carcinoma-like growth.
In conclusion, D-type advanced gastric adenocarcinomas had large numbers of CD34 positive stromal cells in the tumour stroma, whereas D-type early gastric adenocarcinomas had no CD34 positive stromal cells in the tumour stroma. In contrast, regardless of tumour depth, I-type gastric adenocarcinomas had no CD34 positive stromal cells in the tumour stroma. These results suggest that CD34 expression is associated with progression of diffuse carcinomas, and that absence of this expression is also seen in intestinal carcinomas that are progressing. To elucidate the clinical relevance of these immunohistochemical findings, further clinicopathological investigations are needed.
The authors thank Ms M Mitani, Ms H Yamasaki, Ms N Nakamura, and Mr T Tokaji, first department of pathology, Kochi Medical School and Ms Y Nabeshima, department of pathology, Chikamori Hospital for their excellent technical assistance. This work was supported by Foundation for Promotion of Cancer Research in Japan, and Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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