Hypoxia-inducible factor-1alpha and -2alpha are expressed in most rectal cancers but only hypoxia-inducible factor-1alpha is associated with prognosis.

The hypoxia-mediated response of tumours is a major determining factor in growth and metastasis. Understanding tumour biology under hypoxic conditions is crucial for the development of antiangiogenic therapy. Using one of the largest cohorts of rectal adenocarcinomas to date, this study investigated hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression in relation to rectal cancer recurrence and cancer-specific survival. Patients (n=90) who had undergone surgery for rectal adenocarcinoma, with no prior neoadjuvant therapy or metastatic disease, and for whom adequate follow-up data were available were selected. Microvessel density (MVD), HIF-1alpha and HIF-2alpha expressions were assessed immunohistologically with the CD34 antibody for vessel identification and the NB100-131B and NB100-132D3 antibodies for HIF-1alpha and HIF-2alpha, respectively. In a multifactorial analysis, results were correlated with tumour stage, recurrence rate and long-term survival. Microvessel density was higher across T and N stages (P<0.001) and associated with poor survival (hazard ratio (HR)=8.7, P<0.005) and decreased disease-free survival (HR=4.7, P<0.005). hypoxia-inducible factor-1alpha and -2alpha were expressed in >50% of rectal cancers (HIF-1alpha, 54%, 48/90; HIF-2alpha, 64%, 58/90). HIF-1alpha positivity was associated with both TNM stage (P<0.05) and vascular invasion (P<0.005). In contrast, no associations were demonstrated [corrected] between HIF-2alpha [corrected] and any pathological features or [corrected] outcome. The study showed an independent association between HIF-1alpha expression and advanced TNM stage with poor outcome. Our results indicate that HIF-1alpha, but not HIF-2alpha, might be used as a marker of prognosis, in addition to methods currently used, to enhance patient management.

Polyclonal origin of colonic adenomas in an XO/XY patient with FAP.

It is widely accepted that tumors are monoclonal in origin, arising from a mutation or series of mutations in a single cell and its descendants. The clonal origin of colonic adenomas and uninvolved intestinal mucosa from an XO/XY mosaic individual with familial adenomatous polyposis (FAP) was examined directly by in situ hybridization with Y chromosome probes. In this patient, the crypts of the small and large intestine were clonal, but at least 76 percent of the microadenomas were polyclonal in origin.

Assessment of microvessel density and carbonic anhydrase-9 (CA-9) expression in rectal cancer.

AIM: The mechanism by which neoplasias respond to hypoxia determines their biological behavior and prognosis. Understanding the biology of tumors under hypoxic conditions is crucial for the development of anti-angiogenic therapy. Using the largest cohort of rectal adenocarcinomas to date, this study aimed to assess microvessel density (MVD) and carbonic anhydrase-9 (CA-9) expression and to correlate the results with recurrence and cancer-specific survival. MATERIALS AND METHODS: Patients (n=101) who underwent surgery for rectal adenocarcinoma without previous neoadjuvant therapy or metastatic disease were selected. MVD and CA-9 expression were assessed immunohistologically by using the CD34 antibody and the MN/CA9 M75 antibody, respectively. In a multifactorial analysis, the results were correlated with tumor stage, recurrence rate, and long-term survival. RESULTS: MVD was higher with increased T- and N-stages (p<0.01) and associated positively with poor survival (hazard ratio (HR) 1.3 per 10 vessel increase, p<0.01). CA-9 was expressed in 73% of cancers. Negative lymph node status correlated with CA-9 positivity (p<0.05), reflected in a higher rate of CA-9 positivity in earlier Dukes' stages (p<0.05). CA-9 positivity across tumor node metastasis (TNM) stages approached significance (Stage I/II: 80% CA-9 positive vs. 20% CA-9 negative; Stage III: 63% CA-9 positive vs. 37% negative, p=0.051). A trend was seen towards better cancer-specific survival in patients with CA-9 positive carcinomas (HR 0.51, p=0.07) on univariate analysis. DISCUSSION: MVD was higher in more advanced T- and N-stages and may be used as a determinant of survival in patients with rectal adenocarcinomas. CA-9 expression was seen more often in earlier Dukes' stages, possibly representing an early tumor hypoxic response. CA-9 expression by adenocarcinoma cells may confer long-term survival advantage in surgically treated rectal cancer.

Can depth of tumour invasion predict lymph node positivity in patients undergoing resection for early rectal cancer? A comparative study between T1 and T2 cancers.

OBJECTIVE: The present study investigated the risk of lymph node metastasis according to the depth of tumour invasion in patients undergoing resection for rectal cancer. METHOD: The histology of patients undergoing oncological resection with regional lymphadenectomy for rectal cancer at St Marks Hospital from 1971 to 1996 was reviewed. Of the total number of 1549 patients, 303 patients with T(1) or T(2) rectal cancers were selected. The tumour type, grade, evidence of vascular invasion, depth of submucosal invasion (classed into 'sm1-3') were evaluated as potential predictors of lymph node positivity using univariate and multi-level logistic regression analysis. RESULTS: Tumour stage was classified as T(1) in 55 (18.2%) and T(2) in 248 (81.2%) patients. The incidence of lymph node metastasis in the T(1) group was 12.7% (7/55), compared to 19% (47/247) in the T(2) group. The node positive and negative groups were similar with regard to patient demographics, although the former contained a significantly higher number of poorly differentiated (P = 0.001) and extramural vascular invasion tumours (P = 0.002). There was no significant difference in the number of patients with sm1-3, or T(2) tumour depths within the lymph node positive and negative groups. On multivariate analysis the presence of extramural vascular invasion (odds ratio = 10.0) and tumour grade (odds ratio for poorly vs well-differentiated = 11.7) were independent predictors of lymph node metastasis. CONCLUSION: Whilst the degree of vascular invasion and poor differentiation of rectal tumours were significant risk factors for lymph node metastasis, depth of submucosal invasion was not. This has important implications for patients with superficial early rectal cancers in whom local excision is being considered.

Analysis of somatic molecular changes, clinicopathological features, family history, and germline mutations in colorectal cancer families: evidence for efficient diagnosis of HNPCC and for the existence of distinct groups of non-HNPCC families.

OBJECTIVE: To analyse somatic molecular changes, clinicopathological features, family history, and germline mutations in families with colorectal cancer (CRC). METHODS: Molecular changes (K-ras and beta-catenin mutations, chromosome 18q allele loss (LOH), APC LOH, microsatellite instability (MSI), and expression of beta-catenin and p53) were examined in four series of CRC patients with proven or probable hereditary disease: hereditary non-polyposis colon cancer (HNPCC); MYH associated polyposis (MAP); multiple (>5) colorectal adenomas without familial adenomatous polyposis (FAP); and other families/cases referred to family cancer clinics (FCC series). HNPCC was diagnosed using a combination of germline mutation screening and tumour studies. A series of unselected CRC patients was also studied. RESULTS: There was overlap between genetic pathways followed by each type of CRC, but significant differences included: increased frequency of K-ras mutation and reduced frequency of APC LOH in cancers from MAP, but not from multiple adenoma patients; reduced frequency of LOH in HNPCC CRCs; and increased MSI in CRCs from HNPCC, but not from FCC or multiple adenoma patients. HNPCC was apparently detected efficiently by combined germline and somatic analysis. Cancers from the FCC, unselected, and multiple adenoma series shared similar molecular characteristics. In the FCC and multiple adenoma series, hierarchical cluster analysis using the molecular features of the cancers consistently identified two distinct groups, distinguished by presence or absence of K-ras mutation. CONCLUSIONS: While K-ras mutation status is known to differentiate hereditary bowel cancer syndromes such as MAP and FAP, it may also distinguish groups of non-HNPCC, FCC patients whose disease has different, as yet unknown, genetic origins.

Loss of fragile histidine triad expression in colorectal carcinomas and premalignant lesions.

Abnormal expression of the fragile histidine triad (FHIT) candidate tumor suppressor gene has been observed in a variety of human tumors, but little is known about its expression during colorectal tumorigenesis. Sections of 70 aberrant crypt foci (ACF), 55 adenomas, 84 primary colorectal carcinomas, and 13 metastatic lesions were evaluated immunohistochemically for Fhit expression. All normal colonic epithelium showed a strong expression of Fhit; 44% of carcinomas showed a marked loss or absence of Fhit expression. The proportion of carcinomas with reduced expression showed an increasing trend (a) with decreasing differentiation and (b) in tumors with metastases (62%) compared with tumors without metastases (38%). The proportion of metastatic lesions (12 of 13) with reduced expression of Fhit was even greater. Although only a small proportion of ACF and adenomas showed a reduction of Fhit expression, the reduced expression of Fhit was strongly associated with the degree of dysplasia in both ACF (P = 0.0002) and adenomas (P = 0.0085). The findings of reduced expression of Fhit in a small proportion of colonic precancerous lesions and in increased proportions of primary and metastatic colorectal cancers suggest that Fhit plays a role in the development and progression of some colon carcinomas.

Identification of a human ribosomal protein mRNA with increased expression in colorectal tumours.

A human ribosomal protein cDNA was selected from a normal colon cDNA library on the basis of overexpression in familial adenomatous polyposis. Nucleotide sequence analysis was used to identify this cDNA as corresponding to the human equivalent of the rat ribosomal protein L31 (HL31). We have quantified the expression of HL31 mRNA in colorectal tumours and found overexpression in 23 out of 23 cases. Our results indicate that HL31 is associated with a malfunction of normal growth regulatory mechanisms in these tumours, and suggest a role for HL31 in proliferation and neoplasia.

Indeterminate colitis: a pathologist's view.

Indeterminate colitis is only a partial diagnosis, which is provisional until sufficient information becomes available for a more precise diagnosis of either ulcerative colitis or Crohn's disease. As originally defined, i.e. based only on the pathological features in the colon resected for fulminant colitis, a few patients eventually developed Crohn's disease. If the term is used only after all available clinical and investigative evidence is considered, 'indeterminate colitis' behaves clinically like ulcerative colitis. This has important implications for pouch surgery.

CD44 is associated with proliferation in normal and neoplastic human colorectal epithelial cells.

Flash-frozen biopsies obtained from surgical specimens of three adenomatous polyps and 22 colorectal adenocarcinomas (19 primary and three metastatic) were tested by immunohistochemistry for CD44 expression using F10-44-2 monoclonal antibody. CD44 positivity was correlated with proliferative status defined by Ki-67 monoclonal antibody reactivity. In normal colonic mucosa, CD44 was expressed in the proliferative zone of crypts. In tumours, CD44 expression was associated with proliferative areas irrespective of tumour stage or differentiation. Non-proliferating areas of the carcinomatous epithelium did not express CD44 although non-proliferating stromal lymphoid tissue did. There was no apparent association with tumour progression. F10-44-2-defined CD44 is consistently expressed during proliferation by normal colorectal epithelial cells and by both benign and malignant colorectal tumour cells.

Primary lymphoma of the small intestine. A clinicopathological study of 119 cases.

Small bowel lymphomas account for 20 to 40% of primary gut lymphomas in Western populations and are among the most common malignant tumours of the small bowel. We studied 119 cases of primary small bowel lymphoma presenting over 4 decades. Two thirds of the patients were men with a peak age incidence in the 7th decade. Common presenting features included abdominal pain, weight loss, small bowel obstruction, and acute abdomen. Tumours were classified using the Kiel European Association for Haematopathology Geneva Workshop scheme and phenotyped on paraffin sections; 66% were B cells, and 34% were T cell. In all cases, the antibodies L26 and polyclonal CD3 reliably distinguished between B- and T-cell tumours. Of the B-cell lymphomas, 62% were diffuse high grade, 20% were low-grade lymphomas of mucosa-associated lymphoid tissue, 11% had both low- and high-grade components, and 7% were other low-grade types. Of the T-cell lymphomas, 83% were high grade, and 49% were enteropathy associated. Most T-cell lymphomas were ulcerated plaques or strictures in the proximal small bowel; B-cell lymphomas tended to be annular or polypoid masses in the distal and terminal ileum. Survival data showed that low-grade B-cell lymphomas had the best outcome and T-cell lymphomas the worst. Adverse prognostic features included perforation, high-grade histology, multiple tumours and advanced stage.

Four-week open-label trial of metronidazole and ciprofloxacin for the treatment of recurrent or refractory pouchitis.

BACKGROUND: Preliminary data suggest that short-term antibiotic therapy with a single drug is effective for the treatment of patients with pouchitis. However, some patients are resistant to treatment. AIM: To evaluate the therapeutic efficacy of a prolonged course of a combination of two antibiotics in patients with refractory or recurrent pouchitis, as well as its impact on their quality of life. METHODS: Patients with active refractory or recurrent pouchitis were recruited. This was defined as both: (i) a history of pouchitis at least twice in the last 12 months or persistent pouchitis requiring continual intake of antibiotics; and (ii) a Pouchitis Disease Activity Index score 3 7 (best to worst pouchitis=0-18) at the beginning of therapy. Treatment consisted of a combination of metronidazole, 400 or 500 mg twice daily, and ciprofloxacin, 500 mg twice daily, for 28 days. Symptomatic, endoscopic and histological evaluations were undertaken before and after antibiotic therapy using the Pouchitis Disease Activity Index score. Remission was defined as a combination of a Pouchitis Disease Activity Index clinical score of

Gut mucosal response to food antigens in Crohn's disease.

BACKGROUND: Food antigens may contribute to gut inflammation in Crohn's disease. AIM: To assess in vivo sensitization to food antigens, ascertain whether sensitivity is gut specific, assess food sensitization in vitro, and correlate in vivo changes with histological and blood changes. METHODS: Skin testing and rectal exposure to six food antigens (cereal, cabbage, citrus, milk, yeast and peanut) and control saline were assessed double-blind by immediate and 3.5-h laser Doppler blood flowmetry, and rectal biopsies were taken. Peripheral blood lymphocyte proliferation was measured in response to the same antigens. RESULTS: Ten patients with Crohn's disease and 10 healthy controls were studied. Blood flow increased in 24 of 60 antigen sites in Crohn's disease patients and six of 60 antigen sites in controls (P < 0.0001) after 3.5 h. The Crohn's disease group demonstrated higher rectal blood flow than controls in response to all food antigens, and this was significantly different for the responses to yeast (P = 0.036) and citrus fruits (P = 0.038). Lymphocyte proliferation occurred in 32 of 60 tests in Crohn's disease patients and eight of 60 tests in controls (P < 0.0001). There were no skin responses. Submucosal oedema corresponded to increased mucosal flow. CONCLUSIONS: Crohn's disease patients demonstrate in vivo and in vitro sensitization to food antigens, which is gut specific. Mucosal flowmetry allows the identification of sensitization to gut antigens.

Molecular and cellular biology of pre-malignancy in the gastrointestinal tract.

Important pathogenic alterations within established cancers are acquired during the pre-malignant stage. These genetic alterations can be grouped into specific neoplastic pathways that differ within and between anatomical sites. By understanding the mechanisms that determine the initiation and progression of each pathway, it will be possible to develop novel approaches to the diagnosis, prevention and treatment of cancer. This chapter outlines the principles underlying the molecular characterization of pre-malignant lesions, taking colorectal neoplasia as the main model.

Pulmonary megakaryocytes: "missing link" between cardiovascular and respiratory disease?

Pulmonary megakaryocytes were quantitated in a series of 30 consecutive hospital necropsies using a two stage immunoperoxidase stain for factor VIII related antigen. In all 30 cases they were found with a mean density of 14.65 megakaryocytes/cm2 in lung sections of 5 micron in thickness. The maximum concentration of intrapulmonary megakaryocytes was consistently found to be in the central zone of the right upper lobe. Less than 22% of the observed cells possessed abundant cytoplasm, the rest appearing as effete, naked, and seminaked nuclei. The mean megakaryocyte count was found to be increased in association with both respiratory pathology (positive smoking history and impaired lung function) and cardiovascular disease states--shock; thromboembolism; myocardial infarction; and severe atheroma in the abdominal aorta, the coronary circulation, and the circle of Willis. Pulmonary megakaryocytes probably embolise from bone marrow. This may reflect stimulated thrombopoiesis, caused by increased platelet consumption in association with atherosclerotic disease, but it cannot be taken to confirm that the lung is the principal site of platelet production.

Liver disease in infancy: histological features and relationship to alpha-antitrypsin phenotype.

Sixty-nine specimens of liver tissue from 53 infants with neonatal hepatitis or its sequelae were examined without knowledge of the alpha1-antitrypsin phenotype. Distinctive, diastase-resistant, PAS-positive, pure magenta-coloured, sharply defined globules, 2-20 microns in diameter were found in periportal and paraseptal hepatocytes in all liver biopsies from eight alpha1-antitrypsin deficient (PiZZ) infants biopsied after the age of 12 weeks. Such globules were not seen in biopsies from PiZZ individuals aged less than 12 weeks nor in individuals of normal alpha1-antitrypsin phenotype (PiMM). No other specific histological abnormality was found in PiZZ individuals, but in them giant-cell transformation was infrequent and liver damage was more severe, three of 14 cases developing cirrhosis in contrast to four of 27 PiMM subjects. The pathogenesis of liver disease in PiZZ individuals is discussed.

Brush border enzymes in coeliac disease: histochemical evaluation.

Two hundred and ninety four duodenal and jejunal mucosal biopsy specimens from patients with coeliac disease, treated and untreated, and other conditions were examined histologically and by histochemical staining for five peptidase and three disaccharidase enzymes to determine profiles of activity. Suppression of activity paralleled the histology with the following enzymes: lactase, trehalase, brush border endopeptidase, dipeptidyl peptidase II and isomaltase. Lactase, trehalase, and brush border endopeptidase were specifically suppressed in untreated coeliac disease and were diagnostically useful. Examination of a combination of enzymes is recommended.

Expression of nitric oxide synthase in inflammatory bowel disease is not affected by corticosteroid treatment.

AIM: To examine the effect of corticosteroid treatment on the expression of inducible nitric oxide synthase (iNOS) in the colon of patients with inflammatory bowel disease. METHODS: Four groups of patients were studied: (1) ulcerative colitis treated with high dose corticosteroids (six patients, 10 blocks); (2) ulcerative colitis patients who had never received corticosteroids (10 patients, 16 blocks); (3) Crohn's disease treated with high dose corticosteroids (12 patients, 24 blocks); (4) Non-inflammatory, non-neoplastic controls (four patients, six blocks). Full thickness paraffin sections of colons removed at surgery were immunostained with an antibody raised against the C terminal end of iNOS. Sections were assessed semiquantitatively for the presence and degree of inflammation and immunoreactivity for nitric oxide synthase. RESULTS: Cases of ulcerative colitis and Crohn's disease with active inflammation showed strong staining for nitric oxide synthase. The staining was diffuse in ulcerative colitis and patchy in Crohn's disease, in accordance with the distribution of active inflammation. Staining was seen in epithelial cells and was most intense near areas of inflammation such as crypt abscesses. Non-inflamed epithelium showed no immunoreactivity. Treatment with corticosteroids made no difference to the amount of nitric oxide synthase. CONCLUSIONS: Expression of nitric oxide synthase is increased in both ulcerative colitis and Crohn's disease and appears to be unaffected by treatment with corticosteroids. Disease severity necessitated surgery in all the cases included in this study, regardless of whether or not the patients had received long term corticosteroid treatment. It seems therefore that a high level of iNOS expression and, presumably, production of nitric oxide characterise cases which are refractory to clinical treatment; this suggests that specific inhibition of the enzyme may be a useful therapeutic adjunct.

Imbalance between proliferation and apoptosis in the development of colorectal carcinoma.

To evaluate the relationship between cell proliferation and apoptosis in sporadic colorectal carcinogenesis, immunohistochemistry for proliferation-associated antigen Ki-67 and in situ end labelling for identifying apoptotic bodies were performed on paraffin sections from 59 adenomas and 22 carcinomas. These results were correlated with the expression of the proliferation and apoptosis modulators Bcl-2 and p53. Carcinomas showed increased proliferation and apoptosis compared with adenomas (P<0.0001, P<0.001, respectively). There were positive linear correlations between proliferation and apoptosis in adenomas and carcinomas (P<0.02, P<0.05, respectively). The proliferative rate increased significantly from mild to moderate, and from moderate to severe dysplasia (P<0.002, P<0.001, respectively). Apoptotic rate also increased in this sequence, but the increases did not reach statistical significance (both P>0.05). Expression of Bcl-2 was associated with lower apoptotic rate in adenomas (P<0.025) but not in carcinomas (P>0.25), whereas p53 expression was correlated with higher proliferative rate in both adenomas and carcinomas (P<0.01, P<0.05, respectively). An inverse relationship between Bcl-2 and p53 expression was seen in both adenomas and carcinomas (P<0.05, P<0.005, respectively). These data suggest that the normal balance between proliferation and apoptosis is disturbed in colorectal carcinogenesis, both being increased, but proliferation occurs in excess. Bcl-2 and p53 may each play a role in modulating cell apoptosis or proliferation during the development of colorectal carcinoma.

Granulomatous mastitis: a report of seven cases.

The clinical history and histological features of seven cases of granulomatous mastitis are presented. The lesion occurs in young parous women as a tender extra-areolar breast lump. Histologically, non-caseating discrete granulomas are present, confined to breast lobules with, in three cases, coalescence of the granulomas and microabscess formation. Pathogenesis of the changes is discussed. It is thought that granulomatous mastitis is an entity morphologically distinct from duct ectasia/plasma cell mastitis and the commoner forms of granulomatous breast diseases.

Follicular thyroid tumours: a study of laminin and type IV collagen in basement membrane and endothelium.

Immunocytochemical stains for laminin and type IV collagen can be used as markers for basement membrane and vascular endothelium. Thirty four follicular thyroid lesions were examined using these techniques to investigate two aspects: firstly, the relation between the extent of invasion and the integrity of basement membrane; secondly, whether the techniques could enhance the detection of tumour vascular invasion. The results showed that although basement membrane was lost in widely invasive tumours, preservation was seen in most but not all encapsulated tumours. The potential for improved recognition of vascular invasion was also found.