Quantitation of G0 and G1 phase cells in primary carcinomas. Antibody to M1 subunit of ribonucleotide reductase shows G1 phase restriction point block.

Human cancers have an apparent low growth fraction, the bulk of cells presumed to being out of cycle in a G0 quiescent state due to the inability in the past to distinguish G0 from G1 cells. The allosteric M1 subunit of ribonucleotide reductase (M1-RR) is constitutively expressed by cycling cells (i.e., G1, S, G2-M). It is acquired during transition from G0 to G1, lost during exit to G0 and thus distinguishes G0 from G1 cells. To estimate the proportion of G0 and G1 cells in primary human breast (n = 5) and colorectal (n = 12) adenocarcinomas, we used both analytical DNA flow cytometry (ADFC) and immunoperoxidase staining of sections with the monoclonal antibody to M1-RR (MAb M1-RR). ADFC of fresh tumors revealed a low percentage of cells in the S phase (4.0 +/- 3.4%) but immunoperoxidase staining for M1-RR revealed an unexpectedly high proportion of positive cells (52.4 +/- 12.7%) in the G1, S, G2-M phases indicating a high G1 content of primary human tumors. Thus, human cancers are blocked in transition in G1 and are not predominantly in a G0 or quiescent differentiated state. This block was interpreted to mean that human cancers are responding to putative regulatory events at a restriction point in the G1 phase, such as relative growth factor deficiency, density inhibition, antiproliferative cytokines, or gene products. Using flow cytometry for both DNA and M1-RR content we found that human colon cancer cell lines arrest in the G1 but not G0 phase upon serum deprivation or density inhibition. Similarly, human breast cancer cell lines are arrested in G1 but not G0 phase by medroxyprogesterone acetate (MPA) or tamoxifen exposure. These findings match our in situ observations, and support the concept of a restriction point block in primary human tumors.

Identification of a TLR2-regulated gene signature associated with tumor cell growth in gastric cancer.

Toll-like receptors (TLRs) are key regulators of innate immune responses, and their dysregulation is observed in numerous inflammation-associated malignancies, including gastric cancer (GC). However, the identity of specific TLRs and their molecular targets which promote the pathogenesis of human GC is ill-defined. Here, we sought to determine the clinical utility of TLR2 in human GC. TLR2 mRNA and protein expression levels were elevated in >50% of GC patient tumors across multiple ethnicities. TLR2 was also widely expressed among human GC cell lines, and DNA microarray-based expression profiling demonstrated that the TLR2-induced growth responsiveness of human GC cells corresponded with the up-regulation of six anti-apoptotic (BCL2A1, BCL2, BIRC3, CFLAR, IER3, TNFAIP3) and down-regulation of two tumor suppressor (PDCD4, TP53INP1) genes. The TLR2-mediated regulation of these anti-apoptotic and tumor suppressor genes was also supported by their increased and reduced expression, respectively, in two independent genetic GC mouse models (gp130F/F and Gan) characterized by high tumor TLR2 expression. Notably, enrichment of this TLR2-regulated gene signature also positively correlated with augmented TLR2 expression in human GC tumors, and served as an indicator of poor patient survival. Furthermore, treatment of gp130F/F and cell line-derived xenograft (MKN1) GC mouse models with a humanized anti-TLR2 antibody suppressed gastric tumor growth, which was coincident with alterations to the TLR2-driven gene signature. Collectively, our study demonstrates that in the majority of GC patients, elevated TLR2 expression is associated with a growth-potentiating gene signature which predicts poor patient outcomes, thus supporting TLR2 as a promising therapeutic target in GC.

Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes.

AIM: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. METHODS: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin-wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. RESULTS: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. CONCLUSIONS: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia-carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients.

Improved survival in esophageal cancer in the period 1978 to 1983.

The outcome of therapy of adenocarcinomas and squamous-cell carcinomas of the esophagus is so poor that the results of new approaches to therapy, such as the addition of radiotherapy or chemotherapy, are often compared with those achieved in historical controls. To determine the validity of this approach in cancers with a poor outcome, the results of therapy were analyzed at our institution from Jan 11, 1978 to Aug 9, 1981 (77 patients) and compared with the results achieved in the period from Aug 14, 1981 to Feb 19, 1984 (77 patients). The patients were evenly matched for prognostic factors. It was found that the median survival of the first group of patients (4 months) was significantly less (P less than .01) than that of the recently treated group (10 months). This was due to the better median survival of patients treated surgically from 4 months in the early group to 29 months at present (P less than .01). There was no change in the survival of the other patients. The major improvement in the outcome of surgery was due to the reduction of the perioperative mortality rate to less than 5%. There was no detectable change in patient selection for surgery. The results indicate that even in tumors with a very poor outcome, such as esophageal cancer, large changes in results can occur without a specific change in therapy, and that historical controls may be misleading.

The so-called bile duct adenoma is a peribiliary gland hamartoma.

The cell phenotype of so-called bile duct adenoma (BDA) was investigated immunohistochemically using monoclonal antibodies to two recently identified antigens (designated D10 and 1F6) extracted from human liver and cultured biliary epithelium. The acini and tubules of BDA consisted of serous and mucous cells that expressed D10 and 1F6. The intrahepatic peribiliary glands of normal liver, comprising intramural mucous glands and extramural tubuloalveolar seromucinous glands, similarly expressed D10 and 1F6 antigens. Antigen 1F6 was present in the cells forming the canals of Hering and normal bile ductules but not in interlobular and larger bile ducts. Proliferating bile ductules associated with large bile duct obstruction and alcoholic cirrhosis or the epithelia of the von Meyenberg complex and polycystic liver did not exhibit this combined profile of D10 and 1F6 expression and mucous cells. These findings suggest an origin of BDA from peribiliary glands rather than from bile ductules or ducts. Consistent with this view was our finding that 18 of the 30 BDA were spatially related to a large-calibre bile duct. Therefore, BDA, well known for its benign behavior is a small mass of disorganized but mature peribiliary gland acini and tubules within a variable amount of stroma and should properly be called a peribiliary gland hamartoma.

Liver failure complicating non-alcoholic steatohepatitis following allogeneic bone marrow transplantation for Shwachman-Diamond syndrome.

Bone marrow transplantation is potentially curative therapy for the hematologic complications associated with Shwachman-Diamond syndrome (SDS). This syndrome is, however, also associated with significant pancreatic and hepatic dysfunction, which may complicate BMT. We report a case of liver failure due to non-alcoholic steatohepatitis (NASH) following BMT for SDS. This case illustrates the need for assessing liver dysfunction pre-BMT in these patients, in addition to highlighting the potential risk posed by pre-existing steatosis for the development of rapidly progressive hepatic failure following transplantation.

Gastric mucous neck cell and intestinal goblet cell phenotypes in gastric adenocarcinoma.

AIM: To investigate the phenotype of cells comprising diffuse and intestinal-type gastric cancers using monoclonal antibodies to two antigens. One antigen (designated D10) is characteristic of gastric mucous neck cells, cardiac glands, pyloric glands, and Brunner's glands. The second antigen (designated 17NM) is specific to the mucous vacuole of intestinal goblet cells. METHODS: Thirty two gastrectomy specimens with adenocarcinoma were studied. Serial paraffin sections were stained immunohistochemically for D10 and 17NM and histochemically for acid and neutral mucins. The cancers were classified histologically as of either diffuse or intestinal type according to Lauren. RESULTS: Of 15 diffuse-type gastric carcinomas, 11 showed the majority of cancer cells staining for D10 while four were typical signet ring cell cancers staining predominantly for 17NM; five tumours displayed both phenotypes with the two phenotypes segregated in different areas of the tumours. In contrast, of 16 intestinal-type cancers, six expressed 17NM, three D10, five neither antigen, and two expressed both antigens. One indeterminate-type cancer expressed both antigens. The staining of individual cells for D10 and 17NM was mutually exclusive in both diffuse and intestinal types. In contrast to the diffuse cancers, intestinal-type cancers typically expressed either antigen only in occasional small groups of cells and individual cells. CONCLUSIONS: In disease, the gastric stem cell can assume the capacity of the duodenal stem cell for divergent differentiation into either intestinal goblet cells (for example, as in intestinal metaplasia) or Brunner's gland cells (for example, as in pyloric gland/Brunner's gland metaplasia). With neoplastic transformation, this potential for divergent differentiation is maintained and gives rise to diffuse-type cancers that display either the D10 phenotype, the 17NM phenotype, or the clonal expression of both phenotypes. In the more cell cohesive (intestinal-type) tumours, differentiation for antigen expression is poorly developed and more frequently directed towards the intestinal goblet cell phenotype.

Phenotypic identity of gastric mucous neck cells and mucous cells of cardiac, pyloric, and Brunner's glands.

AIM: To investigate the tissue specificity of a novel monoclonal antibody raised to a tissue fraction of normal human liver and which identified certain cells of gastric and duodenal mucosa. METHODS: A total of 155 samples of various tissues obtained from 100 surgical specimens were fixed in cold ethanol-paraformaldehyde, embedded in paraffin wax, and 3 microns sections were studied by immunohistochemical and lectin staining procedures. RESULTS: Immunohistochemical staining showed a major tissue specific component which was strongly expressed by mucous neck cells of the body of the stomach, glands of the cardia and pyloric antrum, and by Brunner's glands. Staining for antigen in the periductal glands of normal major biliary and pancreatic ducts was variable and relatively weaker. It was not detected elsewhere in normal intestine or in the other normal tissues tested. Barrett's mucosa of gastric cardia type, and pyloric gland metaplasia in the gall bladder and small bowel affected with Crohn's disease stained for the antigen. The tissue distribution of the antigen was identical with that of a glycoprotein, demonstrated by an induced affinity for concanavalin A following treatment of tissue sections with periodic acid. The antigen was not sensitive to sialidase. CONCLUSIONS: The tissue component identified (designated here as antigen D10) seems to be characteristic of certain differentiated epithelial cells derived from that part of foregut giving rise to stomach, duodenum, and biliary and pancreatic ducts. The antibody will be of use in investigating pathological processes involving tissue differentiation at these sites, and in the oesophagus and intestines.

Collagenous colitis: jejunal and colorectal pathology.

AIMS: To determine: (1) whether there is an association between collagenous colitis and coeliac disease or lymphocytic colitis; (2) the distribution of lymphocyte subsets and macrophages in the lamina propria and surface epithelial layer in collagenous colitis; and (3) the colorectal distribution of the disease and whether a mucosal biopsy specimen, using a flexible sigmoidoscope, is sufficient to diagnose it. METHODS: The clinical data and colorectal biopsy specimens from 38 patients with collagenous colitis were studied. In 10, small bowel biopsy specimens were also available for review. Immunostaining of the mucosal lymphoid infiltrate with a panel of relevant antibodies was carried out on formalin fixed tissue in seven cases; in three the phenotyping was performed on fresh biopsy specimens separately frozen or fixed in B5 solution. RESULTS: Coeliac disease was found in four out of the 10 patients with collagenous colitis who had had a small bowel biopsy, in contrast to the prevalence of the disease in Australia of 1 in 3000. Collagenous colitis did not respond to gluten withdrawal. Five of 29 (17%) of the patients had a mixed pattern of lymphocytic and collagenous colitis. Immunostaining of the lymphoid infiltrate showed that the striking increase in intraepithelial lymphocytes in collagenous colitis was due to an influx of CD8 positive cells. The occurrence and severity of collagenous colitis along the large bowel were independent of the anatomical site, and in more than 90% of cases biopsy specimens from the sigmoid colon or rectum were diagnostic. CONCLUSIONS: There is a very high incidence of coeliac disease among patients with collagenous colitis so that jejunal biopsy should be an essential part of their investigations, especially if symptoms persist. However, only a minority showed a mixed pattern of lymphocytic and collagenous colitis. The intraepithelial lymphocytes in collagenous colitis are CD8 positive cells. Collagenous colitis can be diagnosed from rectal or sigmoid colon biopsy specimens in more than 90% of cases.

Compound heterozygosity for haemochromatosis gene mutations and hepatic iron overload in allogeneic bone marrow transplant recipients.

Iron overload has been proposed as a cause of liver dysfunction after BMT Factors which could be relevant to iron overload include the number of red cell transfusions and mutations within the haemochromatosis gene (HFE). Two point mutations, Cys282Tyr and His63Asp, have been described within HFE. Cys282Tyr homozygosity is associated with haemochromatosis; the effect of compound heterozygosity, Cys282Tyr/His63Asp, on iron status is variable. We analysed HFE status in 52 allograft patients surviving more than 6 months. Compound heterozygosity was identified in three patients (Cases 1-3). Iron status and liver function were evaluated and, in Cases 1 and 2, liver histology and iron content as well. Case 3 who received 12 units of red cells had a normal ferritin and liver function. Cases 1 and 2 received 29 and 59 units, respectively, and had high serum ferritins and transferrin saturations, abnormal liver function and significant hepatic iron overload on biopsy. Iron overload in Case 1 patient progressed in the context of GVHD and in the absence of further transfusion, suggesting that liver GVHD may increase hepatic iron accumulation. These cases demonstrate the variable phenotypic expression of HFE compound heterozygosity in BMT recipients, which may be only partly explained by transfusional iron loading. Venesection or chelation therapy should be considered in patients with coexistent hepatic GVHD and iron overload.

Liver plasma membrane enzyme activities following glutaraldehyde fixation.

The Wachstein-Meisel ATPase histochemical method has been previously used to demonstrate the ultrastructural localization of this enzyme in both whole liver and isolated plasma membranes following fixation in glutaraldehyde. In the present study biochemical assay, of liver plasma membrane enzymes following fixation in cold 2.5% glutaraldehyde showed that approximately 40% of Mg2+-ATPase, but only 4% of (Na+-K+)-ATPase activity remained in membranes from either control or ANIT-treated rats. In addition, 5'-nucleotidase activity was almost abolished by fixation. The present results indicate that the Wachstein-Meisel method, when applied to biliary canaliculi, can reliably be used to demonstrate the ultrastructural, histochemical localization of Mg2+-ATPase but not that of (NA+-K+)-ATPase. Furthermore, the method permits a valid comparison to be made of the relative Mg2+-ATPase activity in normal and chemically damaged biliary canaliculi.

Alpha-naphthyl-isothiocyanate-induced cholestasis in the rat: studies of liver plasma membrane enzymes.

Oral administration of a single dose of alpha-naphthyl-isothiocyanate (ANIT) to rats produced a conjugated hyperbilirubinaemia, maximal at 2 days and which subsided by 7. The activities of 3 liver plasma membrane enzymes, Mg-2+-ATPase, (Na-+-K-+)-ATPase and 5-nucleotidase, and serum bilirubin levels were studied for up to 7 days after treatment. Activities of the 3 enzymes were significantly decreased at 2 days after treatment and returned to normal by 7, thus varying inversely with the degree of hyperbilirubinaemia. Enzyme histochemistry used to demonstrate canalicular localization of Mg-2+-ATPase in sections of whole liver and of isolated plasma membrane pellets showed that the reduction in activity was not a uniform partial loss, but represented a range of reductions in most canaliculi with a few retaining normal staining intensity. The results suggest that after ANIT intoxication there is a membrane lesion which may be responsible for the observed hyperbilirubinaemia due to the failure of secretion of biliary constituents into the canaliculus. However, more direct studies are necessary to determine whether any one of these enzymes is directly involved in the transport of biliary constituents across the bile canalicular membrane.

Incidence of perilobular hemangioma in the female breast.

The perilobular hemangioma is generally considered uncommon. We found a high incidence (11%) of this little-recognized benign mammary lesion during the course of a recently completed autopsy study designed to assess bilateral, four-quadrant, and subareolar disease of the breast in 210 consecutive forensic post-mortem examinations on female subjects between 15 and 97 years of age. Despite the lesion's name, its distribution was found to involve any of the stromal components of the breast. The unexpected frequency of the perilobular hemangioma within breasts in this study and the reported rarity of mammary angiosarcomas indicate that malignant transformation, if it occurs, must be rare.

Pneumatosis cystoides gastrica associated with adenocarcinoma of the stomach.

This paper reports the finding of benign foreign-body-type multinucleated giant cells and clumps of adenocarcinoma cells in gastric brushing cytology specimens of a case confirmed by radiology and histology as pneumatosis cystoides gastrica associated with an ulcerated carcinoma. Pneumatosis cystoides gastrica should be included in the differential diagnosis of benign multinucleated giant cells found in gastric cytology smears.

Application of the isolated perfused rat liver preparation to pharmacological studies of the intrahepatic portal vascular bed.

A method is described for the perfusion of the isolated rat liver preparation which is particularly suited to a selective pharmacological study of the portal vascular bed of the liver. A synthetic medium free of vasoactive substances and composed of 2.5% bovine serum albumin in Krebs-Henseleit buffer is pumped into the portal vein at a controlled rate while the perfusion pressure is recorded electronically. The advantages of this technique are that while the viability of the isolated organ is preserved, the preparation remains haemodynamically stable and the response to vasoactive agents can be accurately and reproducibly determined. The constant perfusate flow also allows cumulative dose-response curves to be constructed, even for powerful vasoconstrictor agents. The present perfusion method has also been validated by comparing the responses obtained from a variety of vasoactive substances with those reported in other in vivo studies.

Differential role of MyD88 and Mal/TIRAP in TLR2-mediated gastric tumourigenesis.

Signalling by the toll-like receptor (TLR) family of pathogen recognition receptors has emerged as a key molecular component in the pathogenesis of an increasing number of inflammatory-related cancers, among which gastric cancer rates as the second most lethal cancer world-wide. The myeloid differentiation factor 88 (MyD88) adapter molecule has a critical role in mediating innate immune signalling by members of the TLR and interleukin (IL)-1 families, and has been associated with either pro- or antitumourigenic responses in various cancer models. However, little is known about the in vivo role of MyD88 adapter-like (Mal)/TIR-domain containing adapter protein (TIRAP), which is restricted to facilitating TLR4 and TLR2 signalling. To interrogate the role of these innate immune signalling components in gastric tumourigenesis, here we have employed the spontaneous gastric cancer gp130(F/F) mouse model, in which TLR2 promotes the growth of gastric tumours. Genetic ablation of Myd88 in gp130(F/F) mice suppressed tumourigenesis and was associated with increased apoptosis and reduced proliferation in the gastric tumour epithelium, comparable to that observed previously upon deletion of Tlr2 in gp130(F/F) mice. By contrast, the tumour burden in gp130(F/F):Mal(-/-) mice was equivalent to their gp130(F/F) littermates. At the molecular level, suppressed tumourigenesis in gp130(F/F):Myd88(-/-) mice correlated with reduced expression and activation of TLR2-regulated protumourigenic genes and signalling pathways, respectively. Consistent with the previously defined non-essential role for TLR2 in gastric tumour inflammation, the extent of inflammatory cell infiltrates in gastric tumours from gp130(F/F):Mal(-/-) and gp130(F/F):Myd88(-/-) mice remained unaltered compared with gp130(F/F) mice. Collectively, our data reveal a differential, but inflammation-independent, requirement for Mal and MyD88 during TLR2-promoted gastric tumourigenesis.