Gastric and intestinal phenotypes and histogenesis of advanced glandular stomach cancers in carcinogen‐treated, Helicobacter pylori‐infected Mongolian gerbils

Abstract

The Helicobacter pylori‐infected Mongolian gerbil (MG) has been established as an appropriate animal model for studies of stomach cancer development. However, there have hitherto been no data on the phenotypic classification of glandular stomach cancers in H. pylori‐infected and non‐infected MG. We therefore examined the phenotypes of 50 and six advanced glandular stomach cancers in H. pylori‐infected and non‐infected MG, respectively, as well as adjacent non‐neoplastic mucosa, using several gastrointestinal epithelial phenotypic markers. The lesions were divided phenotypically into 21 gastric, 24 gastric‐and‐intestinal mixed, four intestinal and one null types, with 90.0% of the lesions harboring gastric elements and 56.0% demonstrating intestinal phenotypic expression in H. pylori‐infected MG. All six lesions were classified as gastric type in non‐infected MG. There was no clear correlation with the presence of intestinal metaplasia in surrounding mucosa. In conclusion, our data suggest that most advanced adenocarcinomas retain a gastric cellular phenotype in the glandular MG stomach. Thus, it might be proposed that intestinal metaplasia is a paracancerous phenomenon rather than a premalignant condition. H. pylori infection may trigger intestinalization of both stomach cancers and non‐neoplastic mucosa. (Cancer Sci 2006; 97: 38 – 44)

Histologically, human gastric cancers present as two major groups, the ‘intestinal’ and ‘diffuse’ types of Lauren,^{( 1 )} which correspond approximately to the ‘differentiated’ and ‘undifferentiated’ types, respectively, of Nakamura et al. ^{( 2 )} and Sugano et al. ^{( 3 )} It has also been suggested that intestinal type carcinomas arise in intestinalized mucosa, whereas the diffuse type develops from the gastric mucosa proper.^{( 1} , ² , ⁴ , ^{5 )} This hypothesis is based on morphological similarities between cancers and intestinal metaplasia (IM), and on the results of comparisons of carcinomas and surrounding mucosa.^{( 6 )} However, previous studies on phenotypic expression and microsatellite instability (MSI) of each IM or stomach cancer cells have pointed to several contradictions to this hypothesis.^{( 6} , ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ^{17 )} The phenotypic expression of stomach cancer cells of each histologic type can be classified clearly into gastric and intestinal epithelial cell types by immunohistochemistry and mucin histochemistry using gastrointestinal epithelial cell phenotypic markers such as human gastric mucin (HGM), intestinal type alkaline phosphatase (I‐ALP), paradoxical concanavalin A staining (PCS), and Alcian blue–periodic acid Schiff staining (AB‐PAS).^{( 18} , ^{19 )}

The Helicobacter pylori‐infected Mongolian gerbil (MG) has been established as an appropriate animal model for the study of stomach cancer development, with induction of adenocarcinomas by N‐methyl‐N‐nitrosourea (MNU) and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) as the carcinogens.^{( 20} , ²¹ , ²² , ²³ , ^{24 )} We previously demonstrated promoting effects on tumor development due to H. pylori infection in the MG model,^{( 22} , ²³ , ²⁴ , ^{25 )} and several studies based on detailed histopathological assessment have shown no carcinomas in animals treated only with H. pylori infection, suggesting that H. pylori is a strong promoter of gastric carcinogenesis rather than an initiator.^{( 21} , ²² , ²³ , ^{26 )} Eradication of infection results in the curtailment of enhancing effects, particularly in the early stages of associated inflammation.^{( 21} , ²⁷ , ^{28 )} However, there have hitherto been no data on the phenotypic classification of glandular stomach cancers arising in H. pylori‐infected and non‐infected MG. As evaluation of the phenotype is very important with reference to histogenesis, the present study was conducted.

We here examine the phenotypes of advanced glandular stomach cancers, as well as adjacent non‐neoplastic mucosa, using several gastrointestinal epithelial phenotypic markers in 50 H. pylori‐infected and six non‐infected MG treated with the carcinogen MNU. An especial focus was on the comparison of small tubular lesions less than 10 mm in diameter and the surrounding mucosa in H. pylori‐infected and non‐infected MG.

Materials and Methods

Samples and tissue collection

Advanced carcinomas of the glandular stomach in 50 H. pylori‐infected and six non‐infected MG treated with MNU^{( 21} , ^{29 )} were classified histopathologically according to the Japanese Classification of Gastric Carcinomas.^{( 30 )} Tumor tissues and adjacent non‐neoplastic mucosa were fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) (pH 7.2), Bouin's solution, 10% buffered formalin, or 95% ethanol containing 1% acetic acid,^{( 25} , ²⁸ , ^{31 )} sectioned at 5 µm, and stained with hematoxylin and eosin for histologic examination. All of the cancers analyzed demonstrated invasion into the muscularis propria, the subserosa, or the serosa and the peritoneal cavity, sometimes with involvement of adjacent organs.

Immunohistochemistry and mucin histochemistry

Immunohistochemical staining was carried out with antibodies against the following antigens: HGM (Novocastra Laboratories, Newcastle‐upon‐Tyne, UK); small intestinal mucinous antigen (SIMA) (Novocastra); I‐ALP (kindly provided by Dr Kazuyuki Hirano, Department of Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan); and CD10 (Novocastra) (Table 1). The precise procedures for immunohistochemical analysis were as described previously.^{( 15} , ¹⁸ , ¹⁹ , ³² , ³³ , ^{34 )} With regard to gastric phenotypic markers, we used normal gastric mucosa and normal ileum as positive and negative controls, respectively, or vise versa for intestinal phenotypic controls. Briefly, 4 µm‐thick consecutive sections were deparaffinized and hydrated through a graded series of alcohols. After inhibition of endogenous peroxidase activity by immersion in 3% H₂O₂/methanol solution, antigen retrieval was achieved by heating in 10 mM citrate buffer (pH 6.0) in a microwave oven for 10 min at 98°C. Sections were then incubated with primary antibodies. After thorough washing in PBS, they were next incubated with biotinylated secondary antibodies, and then with an avidin–biotin horseradish peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Finally, immune complexes were visualized by incubation with 0.01% H₂O₂ and 0.05% 3,3′‐diaminobenzidine tetrachloride. Nuclear counterstaining was accomplished using Mayer's hematoxylin.

Table 1.

Phenotypic markers for gastrointestinal epithelial cells

Tissue type	Cell type	Marker
Gastric	Foveolar	HGM, PAS (mucin stained red)
	Pyloric	PCS
Intestinal	Goblet	AB (mucin stained blue), SIMA
	Absorptive	I‐ALP, CD10

AB, Alcian blue staining; HGM, human gastric mucin; I‐ALP, intestinal type alkaline phosphatase; PAS, periodic acid–Schiff staining; PCS, paradoxical concanavalin A staining; SIMA, small intestinal mucinous antigen.

For mucin histochemistry, we used PCS for identifying class III mucins in mucous neck and pyloric gland cells.^{( 35} , ^{36 )} We also carried out AB–PAS for identifying gastric surface mucous cells, with mucin staining red and goblet cells staining blue (Table 1).^{( 26} , ^{33 )}

Two independent pathologists (TM and TT) judged the histology and the immunohistochemical and mucin histochemical stainings of the phenotypic markers. With regard to the phenotypic markers, the results of immunohistochemical and mucin histochemical stainings were evaluated in terms of the percentage of positively stained cancer cells, with 10% and above considered positive, as described previously.^{( 15} , ¹⁸ , ¹⁹ , ³² , ^{34 )}

Classification of cancers.

Tumors were classified phenotypically with reference to the expression patterns of the phenotypic markers (Table 1).^{( 15} , ¹⁸ , ¹⁹ , ³² , ^{34 )} Glandular stomach cancers in which more than 10% of the section area consisted of at least one gastric or intestinal epithelial cell phenotype were classified as gastric phenotype (G type) or intestinal phenotype (I type) cancers, respectively. Those that showed both gastric and intestinal phenotypes were classified as gastric‐and‐intestinal mixed phenotype (GI type) cancers, whereas those showing neither gastric nor intestinal phenotype expression were grouped as null (N type).

Comparison of phenotypic expression between adenocarcinomas and non‐neoplastic surrounding mucosa

Non‐neoplastic glandular ducts were divided histologically and phenotypically into three types: (i) gastric phenotypic glandular (G type gland), consisting of at least one gastric epithelial cell phenotype; (ii) gastric‐and‐intestinal mixed phenotypic glandular (GI type gland), showing both gastric and intestinal phenotypes; and (iii) intestinal phenotypic glandular (I type gland), harboring at least one intestinal counterpart.^{( 15} , ¹⁸ , ^{33 )} Particular attention was paid to comparisons of adjacent normal glands with tubular adenocarcinomas less than 10 m in diameter in 13 H. pylori‐infected and five non‐infected MG.

Results

Phenotypic expression of non‐neoplastic glandular ducts in the MG glandular stomach

Typical findings for immunohistochemical and mucin histochemical staining in the non‐cancerous mucosa of glandular stomach are illustrated in Fig. 1. HGM was detected in the cytoplasm of foveolar epithelial cells of the pyloric (Fig. 1A,F) and fundic glandular ducts. It was also found in GI‐IM. PCS was observed in the cytoplasm of normal pyloric glands (Fig. 1A,G) and mucous neck cells. In GI‐IM, the cytoplasm of columnar and goblet cells was stained red and blue, respectively, with AB–PAS (Fig. 1B,H). Regarding I‐IM, the cytoplasm of goblet cells was stained blue with AB, while that of columnar cells was not stained red with PAS (Fig. 1C,I). I‐ALP and CD10 were apparent at the luminal surfaces of absorptive cells in the duodenum (Fig. 1D,E,J). SIMA was evident in the cytoplasm of goblet cells in the duodenum (Fig. 1D,K).

Figure 1.

Expression of gastrointestinal phenotypic markers in non‐neoplastic mucosa. (A,F,G) gastric (G type) glands; (B,H) gastric‐and‐intestinal mixed (GI type) glands; (C,I) intestinal (I type) glands; and (D,E,J,K) normal duodenal glandular ducts. (A) H&E staining of pyloric glandular ducts. (B) H&E staining of GI‐IM. (C) H&E staining of I‐IM. (D) H&E staining of duodenal glandular ducts. (E) Higher magnification of the red square in part D. Note the presence of CD10 at the luminal surface of absorptive cells in the duodenum. (F) Human gastric mucin is evident in the cytoplasm of normal foveolar epithelial cells in the pyloric mucosa. (G) Paradoxical concanavalin A staining is present in the cytoplasm of normal pyloric gland cells. (H) The cytoplasm of columnar and goblet cells in the IM is stained red and blue, respectively, with AB–PAS. (I) The cytoplasm of goblet cells in IM is stained blue with AB, while that of columnar cells is not stained red with PAS. (J) Intestinal type alkaline phosphatase is present at the luminal surface of absorptive cells in duodenum. (K) Higher magnification of the blue square in part D. Small intestinal mucinous antigen is evident in the cytoplasm of goblet cells in the duodenum. Original magnification: A, F and G, ×100; B, D, H and J, ×160; C and I, ×200; E and K, ×640. AB–PAS, Alcian blue–periodic acid Schiff staining; GI‐IM, gastric‐and‐intestinal mixed phenotype IM; IM, intestinal metaplasia; I‐IM, solely intestinal phenotype IM.

Phenotypic classification of advanced glandular stomach cancers in H. pylori‐infected and non‐infected MG treated with MNU

Typical findings for mucin and brush border staining in glandular stomach cancers are shown in2, 3. We evaluated 44 differentiated and six undifferentiated glandular stomach cancers of H. pylori‐infected MG phenotypically using several epithelial phenotypic markers. The lesions were divided phenotypically into 21 G, 24 GI, four I and one N type (Table 2). Of the 44 differentiated type cancers, 39 (88.6%) had gastric phenotypic expression, whereas 22 (50%) harbored intestinal elements. All six undifferentiated type cancers were judged as GI type. We also evaluated five differentiated and one undifferentiated glandular stomach cancers developing in non‐infected MG, all six lesions being of G type (Table 2). The lesions of H. pylori‐infected groups had more intestinal phenotypic expression compared with those of non‐infected groups (P < 0.05). Tumor histology was mostly homogeneous in the gerbil stomach adenocarcinomas in the current study.

Figure 2.

Histology and phenotype of well‐differentiated adenocarcinomas: (A–C) gastric (G type) and (D–G) intestinal (I type). (A) H&E staining of the G type tumor. (B) Human gastric mucin is present in the cytoplasm of tumor cells. (C) Paradoxical concanavalin A is present in the cytoplasm of cancer cells. (D) H&E staining of the I type tumor. (E) Higher magnification of the red square in part D. The cytoplasm of cancer cells is stained blue by Alcian blue–periodic acid Schiff staining. (F) Small intestinal mucinous antigen is evident in the cytoplasm of cancer cells. (G) CD10 is present at the luminal surface of cancer cells. Original magnification: A, ×40; D, ×100; B and C, ×200; E and F, ×320; G, ×640.

Figure 3.

Histology and phenotype of poorly differentiated adenocarcinomas: (A–C) gastric (G type) and (D–G) intestinal (I type). (A) H&E staining of the G type tumor. (B) Human gastric mucin is present in the cytoplasm of malignant cells. (C) Paradoxical concanavalin A is present in the cytoplasm of tumor cells. (D) The cytoplasm of tumor cells was stained blue by Alcian blue–periodic acid Schiff staining. (E) Small intestinal mucinous antigen is evident in the cytoplasm of cancer cells. (F) I‐ALP is positive at the luminal surface of cancer cells. (G) CD10 is present at the luminal surface of cancer cells. Original magnification: A–D and F, ×200; E and G, ×640.

Table 2.

Histologic and phenotypic classification in advanced carcinomas of glandular stomach in 50 Helicobacter pylori‐infected and six non‐infected Mongolian gerbils treated with N‐methyl‐N‐nitrosourea

H. pylori	Histologic classification †	Phenotypic classification ‡
		G	GI	I	N	Total
Infected	Differentiated	21	18	4	1	44
	Undifferentiated	0	6	0	0	6
	Subtotal	21	24	4	1	50
Non‐infected	Differentiated	5	0	0	0	5
	Undifferentiated	1	0	0	0	1
	Subtotal	6	0	0	0	6

^† Classification based on the structure of elements. ‘Differentiated’ includes tubular types, whereas ‘undifferentiated’ consists of signet‐ring cell and poorly differentiated types.

^‡ The numbers of adenocarcinomas possessing intestinal phenotypic expression are 24 gastric‐and‐intestinal mixed (GI) and four intestinal (I) types in H. pylori‐infected gerbils (57% among gastric [G], GI and I types), while corresponding cases do not exist in non‐infected animals, the former was significantly higher (Fisher's exact test, P < 0.05). Null (N) type is excluded from this statistical analysis.

Relationships between carcinomas of tubular structure and non‐neoplastic surrounding mucosa in the glandular stomach of H. pylori‐infected and non‐infected MG from the viewpoint of phenotypic expression

The relationship between adenocarcinomas of tubular structure, which measured less than 10.0 mm in largest dimension, and adjacent non‐neoplastic surrounding mucosa is shown in Table 3. Of 13 lesions, 12 (92.3%) had non‐neoplastic surrounding mucosa with G type glands. One G type lesion (case 7) had adjacent non‐cancerous mucosa harboring GI and I type glands, and no G type glands. The non‐neoplastic surrounding mucosa had only G type glands in seven lesions of G type (cases 1, 2, 3, 4, 5, 6 and 8) (Table 3). In the remaining six cases (cases 7, 9, 10, 11, 12 and 13), the phenotypes of the adenocarcinomas of tubular structure did not coincide with those of non‐neoplastic surrounding mucosa.

Table 3.

Relationships between carcinomas and adjacent non‐neoplastic mucosa in the glandular stomach of Helicobacter pylori‐infected Mongolian gerbils treated with N‐methyl‐N‐nitrosourea

Number	Clinicopathological findings of tumors			Phenotype of adjacent non‐neoplastic mucosa
	Histologic type	Depth	Phenotype	G type glands	GI type glands	I type glands
Case 1	well	mp	G	+	–	–
Case 2	well	mp	G	+	–	–
Case 3	well	mp	G	+	–	–
Case 4	mod	mp	G	+	–	–
Case 5	well	ss	G	+	–	–
Case 6	well	ss	G	+	–	–
Case 7	well	ss	G	–	+	+
Case 8	mod	ss	G	+	–	–
Case 9	mod	ss	G	+	+	–
Case 10	well	ss	GI	+	–	–
Case 11	well	ss	GI	+	–	–
Case 12	well	mp	I	+	–	–
Case 13	well	ss	I	+	+	–

mod, moderately differentiated adenocarcinoma; por, poorly differentiated adenocarcinoma; sig, signet‐ring cell carcinoma; well, well differentiated adenocarcinoma. Tumor types are gastric (G), intestinal (I) and gastric‐and‐intestinal mixed (GI).

Regarding the glandular stomach cancers in the non‐infected MG, the non‐neoplastic surrounding mucosa had only G type glands in all of the five G type tubular lesions.

Discussion

Our present data provide clear evidence that most advanced adenocarcinomas of the glandular stomach in H. pylori‐infected MG treated with MNU are characterized by gastric phenotypic expression. All of the six lesions were classified phenotypically as G type in non‐infected MG treated with MNU. We have shown previously that experimentally induced adenocarcinomas in the rat glandular stomach consist mainly of gastric epithelial phenotypic cancer cells, with intestinal epithelial phenotypic cancer cells appearing later in larger tumors.^{( 7} , ⁸ , ³⁷ , ^{38 )} Similarly, in mice, experimentally induced adenocarcinomas in the glandular stomach^{( 39} , ^{40 )} consist mainly of gastric epithelial phenotypic tumor cells.^{( 41 )} We and others have also reported that the phenotypic shift from gastric to intestinal phenotypic expression occurs in accordance with increasing depth of invasion in human signet ring cell carcinomas and with progression in human differentiated gastric cancers.^{( 42} , ⁴³ , ^{44 )} Early stage papillary (papillary dominant) stomach cancers show significantly higher and more widespread high‐frequency microsatellite instability (MSI‐H) than other morphological types, and inactivation of human MutL homologue 1 (hMLH1) expression by promoter hypermethylation may be an early event in carcinogenesis of this type of stomach cancer.^{( 45} , ^{46 )} MSI‐positive differentiated gastric cancers with gastric foveolar phenotypic expression in the early stages sometimes demonstrate intestinal phenotypic expression in advanced stages.^{( 47 )} Taking into account the previous reports and our present data, we consider that the same phenotypic shift occurs in both rodents and humans, and H. pylori infection may trigger intestinalization of stomach cancers and non‐neoplastic mucosa.

It has been suggested that differentiated gastric carcinomas arise from mucosa with IM, but that undifferentiated gastric cancers originate from mucosa without IM in view of morphological similarities between each cancer and the surrounding mucosa.^{( 1} , ² , ⁴ , ⁵ However, this has not been supported by previous studies on phenotypic expression and MSI of individual intestinal metaplastic or stomach cancer cells.^{( 6} , ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ^{17 )} We here found seven G type tubular lesions with non‐neoplastic surrounding mucosa solely of G type, but in the remaining six cases the tumor phenotype did not match that of the immediately adjacent normal glands in H. pylori‐infected MG (Table 3). Regarding the glandular stomach cancers in non‐infected MG, the non‐neoplastic surrounding mucosa had only G type glands in all five G type lesions. Heterotopic proliferative glands (HPG) frequently develop with H. pylori infection in the glandular stomach of infected MG, with slight dysplastic change of constituent cells.^{( 26 )} They often resemble differentiated or mucinous adenocarcinomas, but do not appear to be malignant, disappearing with little evidence of persistence after eradication of bacteria. However, HPG also show a phenotypic shift from G type to GI or I type with the appearance of Paneth cells during the overall course of H. pylori infection.^{( 26 )} Intestinalization progresses from G through GI to I type in non‐cancerous and cancerous tissue independently in humans and MG.^{( 18} , ^{48 )} Thus, it has been proposed that IM is important not as a precancerous lesion but as a paracancerous phenomenon.^{( 11} , ^{12 )} Therefore, many questions remain regarding its pathogenesis as well as the actual relationship to gastric cancers.^{( 18} , ^{48 )} In the present study, of 13 lesions, 12 (92.3%) had non‐neoplastic surrounding mucosa with G type glands in H. pylori‐infected MG treated with the carcinogens. In the rat, Tatematsu et al. have proposed pepsinogen 1‐altered pyloric glands, which are low in pepsinogen 1 but appear normal in the pyloric mucosa after MNNG treatment, as putative preneoplastic lesions in the glandular stomach.^{( 49} , ⁵⁰ , ^{51 )} Thus, a kind of G type gland may be precancerous. We therefore consider that the origin of stomach cancers might be clarified by analysis of the genetic alteration in stomach cancers and non‐neoplastic G type glands in H. pylori‐infected MG treated with the carcinogens.

Stomach cancers frequently show variable morphology within individual tumors in humans and this may reflect different genetic alterations. A strong relationship has been observed between early stage papillary stomach cancer and MSI‐H, and promoter hypermethylation of hMLH1 may be important for the development of this type of stomach cancer.^{( 45 )} Mismatch repair deficiency in MSI‐positive tumors causes multiple gene inactivations through frameshift mutations in short repetitive sequences in a heterogenous way within a histologically heterogenous tumor.^{( 52 )} Song et al.^{( 46 )} also suggested that a modest centromere numerical abnormality might be another characteristic of stomach cancer characterized by a papillary structure. Further studies of genetic alterations in stomach cancers of MG may be needed to clarify the different carcinogenetic pathways for various types of human stomach cancers.

In conclusion, our data suggest that most advanced adenocarcinomas retain a gastric cellular phenotype in the glandular MG stomach. Thus, it might be proposed that IM is a paracancerous phenomenon rather than a premalignant condition. H. pylori infection may trigger intestinalization of both stomach cancers and non‐neoplastic mucosa.