7 Clin Pathol 1997;50:741-748 741

Gastric mucous neck cell and intestinal phenotypes in gastric 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 , 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 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 . 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 ) or tissue was either fixed for three to four hours in Brunner's 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 , adeno- 13 May 1997 ( Clin Pathol 1997;50:741-748) carcinomas of the (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 7HGM-1D7-B; in mucins and then with . 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 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 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 (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 , 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 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. to the mucous vacuole of the goblet cells of the To obtain maximum staining of antigens in small and . It was demonstrated paraffin sections of the routinely fixed and in all 136 samples of normal colon and 32 of processed tissue, sections were taken down to small bowel, in the goblet cells of intestinal water, placed in 0.05 M citrate buffer, pH 6, metaplasia in six stomachs and in 61 of which had been brought to the boil in a micro- 101 primary colorectal .7 " wave oven, again heated to boiling point in the 17NM was not present in the normal mucosa Cell phenotypes in gastric carcinoma 743

of any of the 32 stomachs examined in the cur- macroscopically normal mucosa were available rent study, but was present in the goblet cells of for study (table 1). all examples of intestinal metaplasia seen in Antibody 7HGM-1D7-B stained the cyto- J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from eight of the 15 stomachs with a diffuse-type plasm of the epithelial cells of stomach, cancer and seven of 13 stomachs with an intestine, breast, ducts, and the transitional intestinal-type cancer for which "Swiss rolls" of epithelium of the kidney calyces, as well as http://jcp.bmj.com/ on September 27, 2021 by guest. Protected copyright.

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Figure 1 Immunohistochemical staining ofsections ofhuman stomach for antigens DlO, 17NM, and PCNA. (A) Corpus mucosa showing cell nuclei stainedfor PCNA and mucous neck cellsfor antigen D10. PCNA positive nuclei (black) are mainly present in the lower pit and isthmus regions and above the mucous neck cells (staining brown for Dl 0). (B) A typical diffuse-type gastric carcinoma (specimen 6 in table 1) showing most tumour cells staining stronglyfor D10. (C-G) Serial sections of a Dl 0 positive, diffuse-type gastric carcinoma (specimen 8). (C) Shows that most of the cancer cells stain for D10 and are widely disseminated throughout the stomach wall. Two areas (arrows) near the surface of the tumour stain for acid mucins with the Alcian blue counter stain and contain relativelyfew cells stainingfor D10. (D) Shows that cells in the two Alcian blue positive areas marked in C, together with scattered, solitary cells in the adjacent tissue, stain stronglyfor antigen 1 7NM (therefore, this Dl 0 positive tumour includes foci ofdiffuse tumour cells of the intestinal goblet cell phenotype). (E) A higher power view ofC to show part of the deeper edge ofone of the Alcian blue stained areas and the presence of numerous large, round, Alcian blue positive cancer cells which, unlike the surrounding smaller tumour cells, do not stain for antigen D10. (F) A higher power view ofD corresponding to part of the section area seen in E to show that the majority of the Alcian blue positive, D10 negative cells seen in E stain stronglyfor 1 7NM. (G) An area of a section stained with the AB-dPAS stain corresponding to part of that seen in E; this shows the large Akian blue positive cells seen in E stainingpurplefor acid mucins, and the smaller, adjacent cancer cells in the right halfof the photomicrograph (corresponding to the DJ0 positive ceUs seen in E) staining redfor neutral mucins. (H) An intestinal-type cancer (specimen 13) showing thefocal cell stainingfor D10 (arrows) typical ofintestinal-type cancers that express D10. (Original magnifications:A, B, E, lj G, H x250; C, D x12.) 744 Hughes, Bhathal

Table 1 Antigen expression and synthesis by 15 diffuse, 16 intestinal, and one widely disseminated throughout the tissue as indeterminate type gastric carcinomas single cells, or in loose, irregular groups. Three J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from Antigen Other of the diffuse cancers (specimens 5, 10, and 12 Specimen histological Intestinal in table 1) showed occasional tubules in some numbers* D10 17NM Mucin type featurest metaplasiat areas, which in each case constituted less than - 10% of the sectioned part of the tumour. Diffuse type 6 +++§ M, Al + These tubules lacked the more ordered colum- 9 +++ N + nar cell arrangement of those seen in the 10 +++ N Tubular + 14 +++ N intestinal-type cancers. 18 +++ N All but one of the 16 tumours classified as 23 +++ - M intestinal-type showed well formed glands/ 7 ++ N 32 ++ - M + tubules or a papillary pattern of growth 12 +++ + M Tubular + throughout the greater part ofthe tumour. The 8 +++ ++ N, A mucinous cancer (specimen 3) consisted pre- 17 +++ ++ M,A + 2 + +++ A + dominantly of lakes of extracellular 5 + +++ A Tubular enclosing groups of cells, but with some more 16 +++ M + superficial parts of the tumour consisting of 24 +++ A Intestinal type tubules, the appearance suggesting an origin in 25 ++ N, A Tubular + an intestinal-type rather than a diffuse-type 31 ++ - N Tubular, pd cancer. I 13 + A Papillary 11 + + N, A Tubular + One tumour (specimen 4) was an 28 + ++ N, M Tubular, pd - indeterminate-type carcinoma consisting pre- 3 - +++ M Mucinous dominantly of solid cords of cells with one area 1 - ++ M Tubular NA 15 - ++ M,A Tubular of papillary and tubular carcinoma, as well as 20 - ++ M, A Tubular, pd NA one intramucosal and another submucosal area 21 - ++ M Tubular + of a diffuse growth pattern that together repre- 27 - ++ M,A Tubular + 19 - -- Papillary + sented less than - 10% of the tumour tissue in 22 - - N Papillary + the sections examined. 26 - - Tubular, solid NA 29 - - N Tubular, solid + 30 - - M Tubular, solid ANTIGEN EXPRESSION IN CANCERS Indeterminate type Antigen DI 0 was present in gastric cancer cells 4 +++ + M, A Solid, diffuse as cytoplasmic granules. Granular and diffuse *The specimens are not in order of their accession, but for both diffuse and intestinal types have staining for D10 was also seen in the stroma of been arranged in order of their expression of antigen, first for D10 and then for 17NM. many areas containing large numbers of DI0 tRecords the presence oftubules in some areas ofthe otherwise diffuse-type cancers, and the gen- eral histological pattern(s) of tumour growth seen in sections of the intestinal-type cancers. pd, positive cancer cells in diffuse-type cancers, but poorly differentiated; solid, growing mainly as solid cords of tumour cells. pools of mucin were not present in these tIntestinal metaplasia in the non-neoplastic mucosa as demonstrated by staining of goblet cells tumours. Antigen 17NM was present in the with antibody 17NM-20-20. +, metaplasia present; -, metaplasia not found; NA, insufficient mucosa available for evaluation. mucigen granules or mucous vacuoles of http://jcp.bmj.com/ §A subjective estimate of the proportion of cancer cells staining for antigen made by comparison cancer cells, in extracellular mucus surround- with the numbers ofcancer cells seen in an adjacent section stained with antibody 7HGM-1D7-B. ing tumour cells, and in pools of secreted -, no cells stained; +, an occasional cell stained; ++, numerous cells stained; +++, most cells stained. mucus. ¶The predominant histochemical staining of intra- and extracellular mucins by the AB-dPAS A subjective estimate of the proportions of stain, seen generally or in different areas of the cancers. -, no staining; A, acid mucin; N, neutral cancer cells staining for D 10 or 1 7NM in mucin; M, a mixture of acid and neutral mucins. representative areas of sections of the 32 NA, "Swiss rolls" of mucosa not available. tumours was made using adjacent sections on September 27, 2021 by guest. Protected copyright. hepatocytes, and the neuroendocrine cells in stained with antibody 7HGM-1D7-B as a . It did not stain the stratified measure of total numbers of cancer cells epithelium of skin or oesophagus. Therefore, present. Results are shown in table 1. the tissue distribution of the antigen identified resembles that of cytokeratins 8 and 18.12 The Diffuse-type gastric cancers antibody stained all cancer cells in all of the Eleven of the 15 diffuse-type cancers showed gastric adenocarcinomas tested, and was par- numerous to most cancer cells staining strongly ticularly useful in evaluating sections of the for antigen D10 (fig 1 B). When not completely diffuse-type carcinomas. obscured by D10 positive cytoplasmic gran- ules, these tumour cells generally showed some TUMOUR CLASSIFICATION degree of cytoplasmic staining with the Alcian Fifteen ofthe cancers were classified as diffuse- blue counter stain and slight to strong staining type (table 1). These were from 10 men (aver- for predominantly neutral mucins with the age age 54 years, median 52) and five women AB-dPAS stain. Sections from three ofthese 1 1 (average age 54 years, median 55), with cancer diffuse-type tumours (specimens 12, 8, and 17) sites in cardia (one), corpus (four), and antrum also showed some areas with occasional to (10). Of the remaining 17 tumours, 16 were of numerous, large signet ring cells with granular the intestinal type and one was indeterminate; that did not stain for D10 but the they were from 12 men (average age 61 years, majority of which stained strongly for antigen median 60) and five women (average age 73 17NM. These large signet ring cells stained years, median 74), with cancer sites in the car- predominantly for acid mucins with the dia (six), corpus (three), and antrum (seven), AB-dPAS stain, in contrast with the neutral and one unspecified site. mucins of the D10 positive tumour cells seen All 15 tumours of diffuse type consisted pre- throughout the bulk of these three cancers (figs dominantly of small, pleomorphic cancer cells 1C-G). Foci of cancer cells expressing the Cell phenotypes in gastric carcinoma 745

intestinal goblet cell phenotype were, therefore, not stain for 17NM did not stain for mucins present in these three otherwise uniformly D10 with the AB-dPAS stain. positive, advanced diffuse-type cancers. J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from The remaining four diffuse-type gastric can- Discussion cers were signet ring cell carcinomas from one Antigen D10 is characteristic of certain cells of male and three female patients (specimens 2, 5, organs derived from the foregut that typically 16, and 24). Sections of these four tumours synthesise neutral mucins.6 It is invariably were characterised by discrete areas showing expressed in normal gastric mucosa and, intense staining for antigen 17NM (fig 2A); therefore, could be expected to occur in some these areas corresponded to pools of extracel- mucus secreting gastric tumours. On the other lular mucin staining predominantly for acid hand, antigen 17NM is specific to intestinal mucins with the AB-dPAS stain. 1 7NM in goblet cells and only appears in the stomach in these areas was also mostly extracellular. The intestinal metaplasia. Therefore, these two stroma lying outside ofthe pools ofmucin con- antigens represent universally expressed, mu- tained few tumour cells in two of these tually exclusive and distinctive tissue specific tumours; one contained numerous cancer cells cell markers of potential use in tracing the cell (including signet ring cells) many of which did lineages of gastric and intestinal adenocarcino- not stain for 1 7NM (as shown in figs 2A and B mas. In the following discussion, we will refer for specimen 2); and the fourth tumour showed to the D10 phenotype as that of a mucous neck innumerable, small, irregularly shaped and dif- cell, although the similarity in cytology of fusely scattered cells all of which appeared to mucous neck cells to the mucous cells of the stain for 17NM in their cytoplasm. Tumour cardiac, pyloric and Brunner's glands is well cells in these areas also stained predominantly documented'3 '4 and further supported by the for acid mucins with the AB-dPAS stain (figs presence of D10 in each of these tissues.6 Cells 2A-F). Two of the four tumours (specimens 2 staining for 17NM will be referred to as of an and 5) showed occasional small groups of cells intestinal goblet cell phenotype. (< 40 cells) staining for D10 in areas outside This study has shown that the majority the pools of mucin and in which tumour cells (84%) of gastric carcinomas display one or did not stain for 1 7NM. Therefore, foci of can- both of the cell phenotypes investigated, and cer cells expressing the DI0 phenotype were that there is some correlation between the pre- present in these two tumours which otherwise dominant phenotype displayed and the histo- uniformly expressed the intestinal goblet cell logical classification of the tumours. As both phenotype. D10 and 17NM are normally associated with mucin producing cells, the generally lower Intestinal-type gastric cancers prevalence of antigen positive cells in the Staining for antigens D10 and 17NM was not intestinal-type cancers was probably related to as consistent in the tumour cells of the their generally poorer mucin content as dem-

intestinal-type cancers as in the diffuse-type onstrated by histochemical staining for mucins. http://jcp.bmj.com/ and, when present, was generally only focal and The four diffuse cancers that showed extensive seen in occasional, scattered cells (figs 1H, 2G pools of extracellular mucin, as well as the one and H). Ofthe 16 intestinal-type cancers, three intestinal-type mucinous cancer, all displayed stained exclusively for D10, two stained for predominantly the intestinal goblet cell pheno- both D10 and 17NM, and another six stained type, and this was in keeping with the active exclusively for 1 7NM. D1 0 positive cells secretion of acid mucins normally exhibited by stained predominantly for neutral mucins, and this cell phenotype. In contrast, the diffuse on September 27, 2021 by guest. Protected copyright. 1 7NM positive cells for acid mucins. The cancers with cells exclusively or predominantly remaining five cancers did not stain for either expressing D 10 did not show pools of extracel- DI0 or 1 7NM, and showed little or no mucin lular mucin except in those cancers containing with the AB-dPAS stain. When present in the segregated cells of the intestinal goblet cell same tumour, the two antigens were generally phenotype (as illustrated in fig 1D). The two segregated in different regions of the tumours. phenotypes studied revealed that Lauren's diffuse-type cancers are a heterogeneous group Indeterminate-type gastric cancers that can be subdivided into cancers that exclu- In specimen 4, the solid cords of cancer cells sively express either the mucous neck cell or and the glandular areas showed the great intestinal goblet cell (signet ring cell) pheno- majority of cells staining for D10, but there type, or both phenotypes in segregated areas. were also rare, scattered cells staining for As either antigen can be expressed in a diffuse 17NM. The small areas of diffuse-type cancer or intestinal-type cancer, the phenotypes do present in this specimen (see above) did not not strictly characterise histological tumour stain for either 17NM or D10 antigen, but type. However, if the apparent differences in stained for acid mucins with the AB-dPAS prevalence ofthe two phenotypes in diffuse and stain. intestinal-type cancers are real, the differences in behaviour of two such disparate cancer cell Colonic signet ring cell carcinomas and phenotypes could help to explain the generally adenocarcinomas accepted biological relevance of Lauren's None of the four signet ring cell cancers of the tumour classification. colon or the five adenocarcinomas of the colon All of the diffuse-type gastric carcinomas stained for D1 0, but all of these tumours displayed predominantly one or other of the except for two of the adenocarcinomas stained two cell phenotypes. Generally, the two antigen for 17NM; the two adenocarcinomas that did markers and associated mucin profiles dis- 746 Hughes, Bhathal

played by the malignant cells matched those of are rather more biochemically mature than normal cell types (the mucous neck cell their lack of cell cohesion would indicate,' and

the two J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from and the intestinal goblet cell), suggesting that the continued expression of D10 by the major- many tumour cells in the diffuse-type cancers ity of these cancers supports such an were fully differentiated, at least with respect to interpretation. However, there were many can- these tissue specific components. Because of cer cells that did not stain for D 10 or 1 7NM in their continued synthesis of mucins, Lauren sections of most of the diffuse-type cancers, suggested that the cells of diffuse-type cancers and it is these unstained cells that may be the

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Figure 2 Immunohistochemical staining ofsections ofhuman stomachfor antigens D10 and 1 7NM. (A-F) Serial sections of a 1 7NM positive diffuse-type gastric carcinoma (specimen 2 in table 1). (A) Low power view showing areas containing cancer cells and pools ofextracellular mucin (lower left and upper right) staining strongly for antigen 1 7NM. In an adjacent section (not shown), stained with AB-dPAS, these 1 7NM positive areas stained strongly for acid mucins. Scattered groups ofcancer ceUs showing cytoplasmic stainingfor 1 7NM (arrow) are also present in the generally unstained stroma between the pools of mucin. (B) The area corresponding to that outlined in A in a section stained with antibody 7HGM-ID7-B to show the presence of numerous cancer cells in the stroma, many ofwhich did not stainfor 1 7NM in section A. (C) A higher power view of the area marked by the arrow in section A to show a group of the 1 7NM positive cancer cells. (D) The area corresponding to that in C in a section stainedfor antigen D10 to show large signet ring cells similar in size to the 1 7NM positive cells in C stainingfor acid mucins with the Alcian blue counterstain but notfor Dl0. (E) The area corresponding to that in C and D in a section stained with the 7HGM-1D7-B antibody showing that there are many other smaller cancer cells present besides the large, Alcian blue positive signet ring cells. (F) The area corresponding to that in C, D, and E in a section stained with AB-dPAS showing that the large signet ring cells contain predominantly acid mucins. (G and H) Serial sections ofan intestinal-type gastric cancer (located in the right halfof the mucosa and ) and adjacent mucosa that contains normal pyloric glands and an area of intestinal metaplasia (arrow). (G) Shows the pyloric glands stainingfor D10 while the area of intestinal metaplasia (arrow) and the carcinoma are unstained. (H) Shows the reverse, wvith the normal pyloric glands unstainedfor 1 7NM, but goblet cells in the intestinal metaplasia stainingfor 1 7NM andfocal staining of the carcinoma. (Original magnifications: A x25; B x60; C, D, E, F x250; G, H xl5.) Cell phenotypes in gastric carcinoma 747

most active in proliferation. In the case of the goblet cell phenotype and secreting acid four diffuse-type cancers with tumour cells mucins histologically were classic signet ring predominantly or exclusively of a phenotype cell carcinomas.9 The cell phenotype displayed J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from foreign to the stomach, one could argue that by these four tumours suggests a relation with, the cells have arisen from the same cell lineage and perhaps origin in, intestinal metaplasia, as that giving rise to intestinal metaplasia in the this being the only non-neoplastic tissue in the stomach. Intestinal metaplasia has been re- stomach to express the 1 7NM antigen. Intesti- ported to be more frequent and widespread in nal metaplasia is reported to arise from within stomachs with intestinal-type cancers than in the isthmus of the glands of intact gastric those with the diffuse-type,' 4 although the mucosa,22 or during the reparative process of relation remains uncertain.'5 We found intesti- erosions. 23 In the early stages of the develop- nal metaplasia in almost 50% of the stomachs ment of intestinal metaplasia in intact mucosa, bearing each type of cancer as demonstrated by the proliferative zone may remain in the staining for the 17NM antigen in "Swiss rolls" isthmus with the surface epithelium and pits of gastric mucosa from this small series of 32 showing metaplastic cells in continuity with the stomachs. These proportions need to be deeper normal .22 Transformed confirmed in a larger series of cancers. cells developing at such a site could give rise to The consistent expression of D10 by the 17NM positive diffuse-type carcinomas in an majority of the diffuse-type cancers suggests analogous fashion to the D10 positive diffuse that this group of tumours could have arisen cancers that may develop from pre-mucous from the cell lineage that normally differenti- neck cells, as suggested above. Since there was ates into mucous neck cells and the glands of evidence of tubule formation in three of the 15 cardia and pylorus. Previous investigators have diffuse-type carcinomas, it is also possible that identified dysplasia in the proliferative zone of some of the tumours studied were originally non-metaplastic gastric glands and proposed intestinal-type cancers that were then over- this as the site of origin of diffuse-type grown by a diffuse-type tumour, although there cancers.'6 Also, histological and histochemical was no histological evidence to support this. evidence that the mucous neck cell may give Both the D10 and 17NM cell phenotypes rise to diffuse-type carcinoma has been pre- were co-expressed in individual tumours of a sented by others,'7 and similarity in lectin quarter of all the gastric cancers examined. staining and in histochemical and ultrastruc- Since no more than 10% of subjects with gas- tural features between normal mucous neck tric carcinoma are reported to show macro- cells and the neoplastic cells demonstrated.'8 scopically discrete synchronous cancers,24 colli- Our previous observations on cell proliferation sion tumours are an unlikely explanation of the in normal human gastric mucosa suggest that mixed cell phenotypes found in some of our mucous neck cells are mature cells that rarely specimens. A stratification of tumour cells divide, although pre-mucous neck cells in the which parodies normal gastric cell types has

isthmus that show some cytoplasmic granules been observed in the early stages of develop- http://jcp.bmj.com/ staining for D10 can do so.'9 ment of gastric carcinoma by other It should be noted that DI 0 is also investigators,25 and segregation of the two cell characteristic of pyloric gland metaplasia phenotypes was noted in the mucosa ofsome of found in inflammatory conditions of the stom- the diffuse-type cancers studied here. There- ach, small bowel, biliary tree, and gall bladder.6 fore, it appears that some transformed gastric This form of metaplasia is said to be derived cells are sufficiently plastic and primitive to be

from an ulcer associated cell lineage (UACL) capable of divergent differentiation into either on September 27, 2021 by guest. Protected copyright. distributed throughout the gastrointestinal the DI 0 or 1 7NM phenotype. Clonal selection tract, which, in response to mucosal ulceration, could account for the predominance with time buds from the base of glands and then prolifer- of one or other phenotype or, when there is no ates to form the metaplastic glands.20 Antigen selective advantage, the growth of both cell D10 has been demonstrated in the UACL in phenotypes would result in the regional differ- the .6 The UACL has been ences in phenotype expression that we ob- described as having the differentiation pro- served within some individual tumours. gramme of Brunner's glands but acquiring the Since normal Brunner's glands form by proliferative organisation of the gastric gland.2' budding off from the crypts of the duodenal If the UACL is distinct from the gastric stem mucosa,26 the stem cells in the crypts of the cell lineage normally giving rise to mucous must be capable of divergent differ- neck cells, it is possible that DIO positive, entiation into either mucosa containing intesti- diffuse-type cancers may also arise indirectly, nal goblet cells (displaying antigen 17NM) or that is by transformation of cells of the UACL Brunner's glands (displaying antigen Dl 0). It as they proliferate in response to the chronic appears that in the course of development of gastritis which frequently precedes gastric car- diffuse-type cancers the transformed gastric cinoma. The UACL does not stain for 17NM stem cells consistently regain such plasticity and is not known to be directly associated with and go on to express one or both of the two intestinal metaplasia or intestinal goblet cells, phenotypes studied. Why the mucous neck cell and is unlikely therefore to give rise to gastric phenotype rather than that of the intestinal cancers other than those displaying antigen goblet cell is then more frequently expressed by D1O. such cancers may be explained on the basis that The small group of four diffuse-type cancers this would be the more normal direction in dif- that were distinguished by predominantly or ferentiation for gastric cells (differentiation exclusively displaying the 1 7NM intestinal into mucous neck cells or mucous cells of the 748 Hughes, Bhathal

cardiac and pyloric glands). The co-existence 8 Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, of both phenotypes found in some individual Bifulco C, et al. Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 1993;171:83- J Clin Pathol: first published as 10.1136/jcp.50.9.741 on 1 September 1997. Downloaded from cancers is also in keeping with a capacity for 98. divergent 9 "Watanabe H, Jass JR, Sobin LH, in collaboration with differentiation by the neoplastic cells, pathologists in eight countries. Histological typing of and helps to explain the confusing histochemi- oesophageal and gastric tumours. In: WHO international cal histological classification of tumours. Berlin: Springer-Verlag, heterogeneity of these tumours. 1990:1-43. This study has shown that gastric adenocar- 10 Cook HC. . In: Bancroft JD, Stevens A, eds. cinomas are characterised by two cell pheno- The theory and practice of histological techniques. Edinburgh: Churchill Livingstone, 1982:180-216. types, one of which is foreign to the normal 11 Hughes NR. Gland heterogeneity and colorectal neoplasia. stomach mucosa and both of which are University of Sydney: PhD thesis, 1988. 12 Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The normally present in the duodenum. Reversion catalog of human cytokeratins: patterns of expression in of the transformed gastric stem cell to a more normal epithelia, tumors and cultured cells. Cell 1982;31: primitive gut stem cell lineage possessing the 11-24. 13 Bloom W, Fawcett DW. A textbook ofhistology. Philadelphia: same capacity for divergent differentiation as WB Saunders Company, 1975:639-57. the normal duodenal stem cell is the most eco- 14 Katsuyama T, Spicer SS. Histochemical differentiation of complex carbohydrates with variants of the concanavalin nomical explanation of the consistent associ- A-horseradish peroxidase method. Jf Histochem Cytochem ation of the two phenotypes with the non- 1978;26:233-50. 15 Dixon MF. Progress in gastric cancer. In: Kirkham N, Hall cohesive diffuse-type carcinoma. These PA, eds. Progress in pathology. Vol 1. Edinburgh: Churchill cancers typically show one of the two antigens Livingstone, 1995:13-29. 16 Ghandur-Mnaymneh L, Paz J, Roldan E, Cassady J. predominating, usually DI0, and being Dysplasia ofnonmetaplastic gastric mucosa. A proposal for strongly expressed by the majority ofthe cancer its classification and its possible relationship to diffuse-type cells. In contrast, gastric carcinoma. Am J Surg Pathol 1988;12:96-114. intestinal-type cancers with 17 Yamashina M. A variant of early gastric carcinoma. their more ordered tubular structure more fre- Histologic and histochemical studies of early signet ring quently display the intestinal goblet cell cell carcinomas discovered beneath preserved surface epithelium. Cancer 1986;58: 1333-9. phenotype and typically express the antigens in 18 Tatematsu M, Furihata C, Katsuyama T, Miki K, Honda H, relatively few tumour cells. Konishi Y, et al. Gastric and intestinal phenotypic expressions of human signet ring cell carcinomas revealed by their biochemistry, mucin histochemistry, and ul- The work was supported by a grant from the National Health trastructure. Cancer Res 1986;46: 4866-72. and Medical Research Council ofAustralia. This work was pre- 19 Hughes NR, Bhathal PS. Do gastric mucous neck cells sented in part at the Australian Gastroenterology Week 1996, divide? [abstract]. IntJ' Surg Pathol 1996;3:217. the Annual Scientific Meeting of the Gastroenterological Soci- 20 Wright NA, Pike C, Elia G. Induction of a novel epidermal ety of Australia, held in Adelaide, September 1996, and growth factor-secreting cell lineage by mucosal ulceration published in abstract form in J Gastroenterol Hepatol 1996;11 in human gastrointestinal stem cells. 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