7 Clin Pathol 1997;50:741-748 741 Gastric mucous neck cell and intestinal goblet cell phenotypes in gastric adenocarcinoma J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from N R Hughes, P S Bhathal Abstract Keywords: gastric carcinoma; mucous neck cells; intes- Aim-To investigate the phenotype of cells tinal goblet cells comprising diffuse and intestinal-type cancers using monoclonal anti- gastric The histological classification of gastric adeno- bodies to two antigens. One antigen carcinomas into Lauren's diffuse or intestinal (designated D10) is characteristic of gas- type' has some biological significance as the tric mucous neck cells, cardiac glands, intestinal-type cancer shows variation in geo- pyloric glands, and Brunner's glands. The graphical incidence and has a somewhat differ- second antigen (designated 17NM) is spe- ent clinical behaviour to the diffuse type.2 3 cific to the mucous vacuole of intestinal Although intestinal metaplasia is common in goblet cells. stomachs bearing intestinal-type cancers,4 the Methods-Thirty two gastrectomy speci- cell lineages of the two classes of tumour and mens with adenocarcinoma were studied. the reason for the frequent histological and Serial paraffin sections were stained im- histochemical heterogeneity seen in individual munohistochemically for D10 and 17NM specimens of gastric carcinoma remain un- and histochemically for acid and neutral clear. We report on the use of two phenotypic mucins. The cancers were classified histo- markers that identify two distinct neoplastic logically as of either diffuse or intestinal cell populations in gastric carcinoma and throw type according to Lauren. some light on the histogenesis of these Results-Of 15 diffuse-type gastric carci- tumours. nomas, 11 showed the majority of cancer cells staining for Dl0 while four were typi- cal signet ring cell cancers staining pre- Methods dominantly for 17NM; five tumours Tissues were collected over a four year period displayed both phenotypes with the two from gastrectomy specimens (as approved by phenotypes segregated in different areas the Ethics Committee of The Royal Mel- http://jcp.bmj.com/ of the tumours. In contrast, of 16 bourne Hospital) from 22 men (28-80 years intestinal-type cancers, six expressed old) and 10 women (45-82 years old) admitted 17NM, three Dl0, five neither antigen, and to this hospital for surgery for gastric adenocar- two expressed both antigens. One cinoma. The specimens were consecutive and indeterminate-type cancer expressed both unselected except that the fresh specimen antigens. The staining of individual cells needed to be available immediately after resec- for D1O and 17NM was mutually exclusive tion, and the duty pathologist was prepared to on September 27, 2021 by guest. Protected copyright. and intestinal types. In release tissue for the research after diagnostic in both diffuse had been met. Blocks of tumour and contrast to the diffuse cancers, intestinal- needs either adjacent mucosa and, when possible, long type cancers typically expressed strips of non-involved mucosa were taken from antigen only in occasional small groups of the fresh specimens for this study. In addition, cells and individual cells. sections ofthe cancers were taken subsequently Conclusions-In disease, the gastric stem from the routinely processed paraffin blocks cell can assume the capacity ofthe duode- used in producing the surgical pathology nal stem cell for divergent differentiation report. into either intestinal goblet cells (for For optimal antigen preservation, the fresh example, as in intestinal metaplasia) or tissue was either fixed for three to four hours in Brunner's gland cells (for example, as in cold fixative containing 2% paraformaldehyde, pyloric gland/Brunner's gland metapla- 75% ethanol, and 0.4% cetylpyridinium chlo- sia). With neoplastic transformation, this ride (EPF), or in Zamboni's fixative5 for one potential for divergent differentiation is hour followed by a further three hours in EPF. Department of maintained and gives rise to diffuse-type Tissues were dehydrated in isopropanol, Anatomical Pathology, cancers that display either the D1O pheno- cleared in chloroform, and vacuum embedded The Royal Melbourne type, the 17NM phenotype, or the clonal in four changes of paraffin wax over a period of Hospital, Parkville, both In the of mucosa were Victoria 3050, expression of phenotypes. one hour. The long strips fixed, Australia more cell cohesive (intestinal-type) tu- dissected free from the muscularis propria, mours, differentiation for antigen expres- fashioned into "Swiss rolls", and processed for Correspondence to: sion is poorly developed and more sectioning as described above. Professor Bhathal. frequently directed towards the intestinal Paraffin sections of four signet ring cell can- Accepted for publication goblet cell phenotype. cers of the caecum and sigmoid colon, adeno- 13 May 1997 ( Clin Pathol 1997;50:741-748) carcinomas of the ascending colon (one case) 742 Hughes, Bhathal and sigmoid colon (four cases) obtained from oven over one minute, and allowed to cool for routinely processed tissue were also studied. 20-30 minutes before staining.8 Following Two micron thick serial paraffin sections immunohistochemical staining, sections were J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from were stained immunohistochemically for anti- counter stained for five minutes with 1 % gens D 10, 1 7NM, and the antigen detected by Alcian blue in 3% acetic acid (pH 2.5) for acid the monoclonal antibody 7HGM-1D7-B; in mucins and then with haematoxylin. addition, the sections were stained histochemi- Double staining for Dl 0 and proliferating cally for acid and neutral mucins (AB-dPAS cell nuclear antigen (PCNA) was done by stain). sequentially reacting sections with a 1/500 dilution of antibody to PCNA (Cat. No. ABT PRODUCTION OF MONOCLONAL ANTIBODIES 152; American Biotech, Florida, USA), rabbit Preparation of hybridoma 5HL-5D 11 -D10, antibodies to mouse immunoglobulin (Z259; which recognises antigen DI 0, has been Dako), and gold labelled goat antibodies to described elsewhere.6 The hybridoma was rabbit immunoglobulins (Cat. No. RPN 470; grown intraperitoneally in BALB/c mice and Amersham International, Amersham, UK), the same batch of ascites fluid used for the pre- and then enhancing the gold label with the vious study was used in the present study. IntenSE M kit (Amersham International). Hybridoma 1 7NM-20-20 recognises antigen Sections were rinsed in 0.1 M glycine HCI 1 7NM and was produced by immunising mice buffer, pH 2.5, for two minutes, in Tris buffer, with isolated colonic glands from subjects with pH 7.2, for five minutes, and stained for D10 colonic carcinoma; the procedures for prepar- as before. ing cell hybrids, screening, and cloning this hybridoma have been described elsewhere.7 HISTOLOGICAL CLASSIFICATION OF GASTRIC The same batch of ascites fluid used in the pre- CANCERS vious study was used here. The tumours were classified according to A third monclonal antibody (7HGM- Lauren' and the WHO International Histologi- 1D7-B) used in this study was produced by cal Classification of Tumours,9 either as being immunising BALB/c mice with epithelium iso- of a diffuse or intestinal-type, or of an indeter- lated from surgically resected human gall blad- minate category, based on the predominant ders. Cell hybrids were prepared from the histological growth pattern ofthe tumours seen spleens of these mice and screened against a in the sections stained with antibody 7HGM- mosaic of tissues as described for antibody 1D7-B. The indeterminate category refers to 5HL-5D1 1-D 10.6 Hybridoma 7HGM-1D7-B tumours with about equal proportions of the was selected, cloned by limiting dilution, diffuse and intestinal growth patterns.9 Other grown intraperitoneally in BALB/c mice, and histological features of the tumours were the batch of the resulting ascites fluid used recorded as solid, tubular, papillary, or here. mucinous,9 and the general degree of differen- tiation of the tumours was noted. http://jcp.bmj.com/ IMMUNOHISTOCHEMISTRY A four stage immunohistochemical staining HISTOCHEMICAL STAINING procedure was used as previously described.6 Sections were digested with salivary amylase Briefly, sections were incubated sequentially in for one hour at 37°C, washed in distilled water, Tris buffer (140 mM NaCl, 50 mM Tris, stained with Alcian blue, pH 2.5, and then by 2.7 mM KC1, 0.01% merthiolate, pH 7.2) the periodic acid Schiff (PAS) reaction'" for containing 10% fetal calf serum (FCS), mouse carbohydrate (AB-dPAS stain). on September 27, 2021 by guest. Protected copyright. monoclonal antibody, rabbit immunoglobulins to mouse immunoglobulins (Z259; Dako, Results Botany, Australia), 1.5% H202 in Tris buffer, ANTIGEN EXPRESSION IN NORMAL TISSUE swine immunoglobulins to rabbit immu- As described elsewhere, the normal tissue dis- noglobulins (Z 196; Dako) and rabbit horserad- tribution of D10 is limited to gastric mucous ish peroxidase antiperoxidase (Zi 13; Dako). neck cells, the glands ofthe cardia and pylorus, The substrate 3-3'-diaminobenzidine (DAB) Brunner's glands, peribiliary glands, and the was used to demonstrate the sites of bound periductal glands of the pancreas; staining for enzyme. All immunological reagents were Dl 0 was present as cytoplasmic granules in all diluted in Tris buffer containing 10% FCS and samples of the tissues tested.6 The distribution 0.1% NaN3 (0.04% NaN, was used with the of D1 0 in the 32 specimens of stomach studied peroxidase antiperoxidase solution) and the here was similar to that reported previously. sections were washed between changes for 10 Figure 1A illustrates the spatial relation of minutes in Tris buffer. The three monoclonal mucous neck cells staining for D10 with cells of antibodies were all used at a dilution of 1/400. the proliferative zone ofgastric corpus mucosa, A 1/400 dilution of ascites fluid produced by a as revealed by staining for PCNA. non-reactive mouse hybridoma or the Tris The normal tissue distribution of antigen buffer containing 10% FCS were used as nega- 1 7NM has previously been shown to be limited tive controls.