Distant Mucosa Research Articles

Helicobacter pylori and colorectal cancer

Recent researches of the association between helicobacter pylori (Hp) and colorectal adenocarcinoma have found that Hp infection rate is higher in the patients with colorectal cancer and other related colorectal adenoma,which suggest that Hp probably influence on local and distant intestinal mucosa epithelial cells achieve carcinogenesis by its own virulence and the role of elevated serum gastrin levels.Hp infection may be a potential risk factor of colorectal cancer. Key words: Helicobacter pylori; Colorectal neoplasms

BAP31 is frequently overexpressed in patients with primary colorectal cancer and correlates with better prognosis

We previously showed that B cell receptor associated protein 31(BAP31) was significantly upregulated in colorectal cancer compared with normal mucosa epithelia. However, its expression pattern and pathological role in colorectal cancer are not clearly understood. In this study, we investigated whether the expression of BAP31 was associated with the clinicopathological parameters of colorectal cancer. The expression pattern of BAP31 was detected by immunohistochemistry on a tissue microarray in both primary tumor and paired distant normal mucosa samples from 120 consecutive colorectal cancer patients. Furthermore, BAP31 protein expression was also determined in twenty colorectal adenomas and eight liver metastasis samples. There was positive expression of BAP31 in 64.17% of primary tumors and 6.67% in distant normal mucosa (P=0.000). Negative expression of BAP31 was correlated with distant metastasis (P=0.036) and lower tumor differentiation grade (P=0.001). Patients with BAP31-negative expression showed significantly lower overall survival rate (P=0.003) compared to patients with BAP31-positive expression. Our results demonstrate that BAP31 may serve as a candidate prognostic marker in colorectal cancer and negative BAP31 expression may lead to more aggressive invasion of colorectal cancer.

Topical and mucosal liposomes for vaccine delivery

Mucosal (and in minor extent transcutanous) stimulation can induce local or distant mucosa secretory IgA. Liposomes and other vesicles as mucosal and transcutaneous adjuvants are attractive alternatives to parenteral vaccination. Liposomes can be massively produced under good manufacturing practices and stored for long periods, at high antigen/vesicle mass ratios. However, their uptake by antigen-presenting cells (APC) at the inductive sites remains as a major challenge. As neurotoxicity is a major concern in intranasal delivery, complexes between archaeosomes and calcium as well as cationic liposomes complexed with plasmids encoding for antigenic proteins could safely elicit secretory and systemic antigen-specific immune responses. Oral bilosomes generate intense immune responses that remain to be tested against challenge, but the admixing with toxins or derivatives is mandatory to reduce the amount of antigen. Most of the current experimental designs, however, underestimate the mucus blanket 100- to 1000-fold thicker than a 100-nm diameter liposome, which has first to be penetrated to access the underlying M cells. Overall, designing mucoadhesive chemoenzymatic resistant liposomes, or selectively targeted to M cells, has produced less relevant results than tailoring the liposomes to make them mucus penetrating. Opposing, the nearly 10 µm thickness stratum corneum interposed between liposomes and underlying APC can be surpassed by ultradeformable liposomes (UDL), with lipid matrices that penetrate up to the limit with the viable epidermis. UDL made of phospholipids and detergents, proved to be better transfection agents than conventional liposomes and niosomes, without the toxicity of ethosomes, in the absence of classical immunomodulators.

Ki-67 from Clinically and Histologically “Normal” Distant Mucosa as Prognostic Marker in Early-Stage (T1-T2N0) Oral Squamous Cell Carcinoma: A Prospective Study

The aggressive behavior and long-term prognosis of oral squamous cell carcinoma (OSCC) have recently been related to the mucosa surrounding the primary mass, consisting of genetically altered cells that might be responsible for cancer progression. Early-stage T1-T2N0 OSCCs have been associated with a good prognosis; however, a certain percentage of them can be complicated by locoregional metastases. The purpose of our study was to determine whether an abnormal proliferative status can be found in clinically and histologically "normal" mucosa situated in areas distant from the primary tumor. We also sought to determine whether this is associated with a poor prognosis in terms of local recurrence or lymph node metastasis. The prospective study included 42 consecutive patients with T1N0M0 (n = 19) and T2N0M0 (n = 23) OSCC. Disease-free survival endpoints were defined as the duration between surgical resection and the diagnosis of recurrence, lymph node metastasis, or last follow-up visit. Proliferative status in distant areas (opposite cheek) was evaluated by Ki-67 expression. The mean Ki-67 value (17.6% ± 8.2%) in the distant mucosa was significantly greater (F = 13.87; P < .01) than that found in the controls (9.8 ± 3.1). "Abnormally high" Ki-67 values were detected in 13 patients with OSCC (30%). Four patients developed locoregional recurrence during follow-up. Kaplan-Meier analysis showed that Ki-67 in the distant mucosa was a significant independent prognostic factor for disease-free survival. A certain percentage of patients surgically treated for early T1-T2 OSCC will have an abnormal proliferative status in areas very distant from the primary tumor that seems to be related to a poor prognosis.

Fluorescence in situ hybridization biomarkers of gastric cancer risk in intestinalized Helicobacter pylori pangastritis.

17 Background: H. pylori (HP) intestinalized pangastritis predisposes to gastric adenocarcinoma. Biomarkers to identify the less than 10% subset of chronic gastritis patients who will develop gastric cancer are greatly needed to focus surveillance efforts onto those patients most likely to benefit. Investigations in ulcerative colitis and Barrett's esophagus reveal that chromosomal alterations in far distant and nondysplastic mucosa are powerful cancer biomarkers. We hypothesized a similar situation in gastric tumorigenesis. Methods: The full morphologic spectrum of gastric neoplasia was examined using flow-sorted gastric epithelial cells from 10 gastric cancer resections versus 20 normal gastric control patients. Then we addressed whether we could distinguish the subset of HP intestinalized pangastritis patients who develop cancer (progressors), from those who remain neoplasia-free (nonprogressors). A single nondysplastic intestinalized gastric antral biopsy collected using chromoendoscopy on each of 41 Korean progressors with gastric cancer and 30 nonprogressors without dysplasia. Interphase FISH on whole epithelial cell preparations using 4q32-q33, 10q22.1, 18q21.2 and 19p11-q11 chromosomal arm probes was performed. Results: Increasing chromosomal alterations occurred throughout the morphologic spectrum of gastric neoplasia. Significantly increased FISH alterations for chromosomes 4q32-q33 (p = 0.016) and 10q22.1 (p = 0.028) occurred as early in the neoplastic spectrum as nondysplastic distant intestinal metaplasia. Subsequent nondysplastic antral biopsy testing of progressors versus nonprogressors revealed parallel FISH results for 4q32-q33 (p = 0.039) and 10q22.1 (p = 0.023), and 19p11-q11 (p = 0.019). Receiver operator characteristic analysis for these 3 combined probes demonstrated a sensitivity of 0.53 and specificity of 0.92 with optimal choice of thresholds. Conclusions: Interphase FISH for 4q32-q33, 10q22.1, and 19p11-q11 chromosomal probes on nondysplastic intestinalized antral mucosa shows promise as a biomarker of gastric cancer in multifocal HP intestinalized pangastritis. No significant financial relationships to disclose.

Abstract 194: Loss of imprinting and abnormal expression of insulin-like growth factor II in gastric cancer

Abstract Introduction: Gastric carcinoma (GC) is a leading cause of cancer mortality worldwide, and is highly prevalent in China. Loss of imprinting (LOI) of insulin-like growth factor II (IGF-2) gene is an important epigenetic phenomenon related to carcinogenesis and tumor progression in many human cancers. To date, however, few studies have evaluated the IGF-2 LOI in GC. Aims: This study aimed: (1) to examine the prevalence of LOI of IGF-2 in tumor, matched normal gastric mucosa, and peripheral blood lymphocyte (PBL) from a well characterized series of patients with GC, and in PBL from a control group, (2) To examine the association of LOI of IGF-2 with circulating IGF-2 and tissue IGF-2 expression. Methods: IGF-2 genomic imprinting status was analyzed by PCR and RT-PCR followed by restrictive endonuclease (Apa I) digestion. Prevalence of LOI in PBL from 33 Apa I informative GC patients (21 males, age: 59.91±13.87) was compared with that from 21 informative controls (13 males, age: 56.81±13.28). Findings were also associated with the clinicopathologic parameters in patients with GC. IGF-2 level in peripheral blood (ng/ml) and tumor were determined with ELISA and immunohistochemical staining, respectively. Results: LOI of IGF-2 was positive in 48.48% (16/33) GC tumor, 21.21% (7/33) adjacent mucosa (AM), 12.12% (4/33) distant mucosa (DM), and 15.15% (5/33) PBL. The prevalence of LOI in PBL was not significantly different between GC patients (5/33, 15.15%) and controls (2/21, 9.52%) (p=0.693). Patients with LOI-positive tumor were more likely to be advanced stage (93.75% vs. 58.82%, p=0.039) or have positive lymph nodes (87.50% vs. 52.94%, p=0.057). Significantly increased blood IGF-2 level was seen in GC patients with LOI-positive tumor (808.07±255.25) when compared with those with LOI-negative tumor (396.21±198.94) (p&amp;lt;0.01). The positive rate of IGF-2 staining was significantly higher in tumor (10 of 16, 62.50%), AM (7 of 7, 100.00%), and DM (3 of 4, 75.00%) with LOI than that in tissues without LOI (tumor: 2 of 17, 11.76%; AM: 2 of 26, 7.69%; DM: 2 of 29, 6.90%) (p&amp;lt;0.01). Conclusions: The results of this study suggest that IGF-2 is a critical factor in carcinogenesis and progression of GC, with LOI of IGF-2 modulating tissue and circulating expression of IGF-2. IGF-2 LOI may represent a potential epigenetic marker for gastric cancer risk, prognosis and treatment stratification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 194.

M Cell–Targeted Ocular Immunization: Effect on Immunoglobulins in Tears, Feces, and Serum

This study investigates whether antigen-sampling M cells, present in the follicle-associated epithelium (FAE) above organized conjunctiva-associated lymphoid tissue in rabbits, bind and retro-transport secretory IgA (sIgA) from the tear film. The hypothesis that IgA-mediated uptake of antigens promotes local and systemic production of immunoglobulins was tested. sIgA binding and retro-translocation by M cells was characterized by immunocytochemistry. Immunoglobulin concentrations in tears, feces and serum were measured using enzyme-linked immunoassays (ELISA) after topical and systemic immunization with either goat IgG anti-rabbit IgA or nonspecific goat IgG. Endogenous sIgA was found associated with the apical membrane of conjunctival M cells. Exogenous anti-IgA immunoglobulins were translocated across M cells. Significant levels of sIgA against goat IgG were present in tears of pre-immune animals. Topical application of either goat IgG specific for rabbit IgA or nonspecific goat IgG led to similar increases in antigen-specific IgA in tear, feces, and serum. The antigen-specific IgG response in tears mirrored the serum response for both immunogens consistent with transudation of this immunoglobulin. The IgM response in tears and serum was weak for both immunogens. Systemic immunization did not sustain or enhance the local mucosal IgA responses. Conjunctival M cells bind and translocate sIgA from the tear film. Topical conjunctival immunization leads to generation of antigen-specific immunoglobulins from both local and distant mucosae and in serum. Natural antibodies, present in the tear film before immunization, may have contributed to similar immune responses to goat anti-rabbit IgA and nonspecific goat IgG.

CIS is a surrogate marker of genetic instability and field carcinogenesis in the urothelial mucosa

Objective To investigate whether carcinoma in situ (CIS) lesion could be considered a surrogate marker of urothelium genetic instability and field carcinogenesis or not, we evaluated DNA content, p53 overexpression, and proliferative index (Ki-67 expression) in primary tumor, in tumor-adjacent mucosa, and distant urothelial mucosa with and without presence of CIS. Patients and methods A retrospective study in radical cystectomy specimens from 49 patients was carried out. All the lesions present in each cystectomy specimen were studied, including the tumor area and the adjacent mucosa (AM). Whenever possible, the distant mucosa (DM) was also studied. When CIS was detected, this lesion and the surrounding normal mucosa were also studied. The 49 tumor areas included high grade papillary urothelial carcinoma (HGP) in 19 cases (38.8%) and invasive urothelial cell carcinomas in 30 cases (61.2%). The nuclear DNA content of cancer cells was evaluated using image cytometry allowing the determination of the DNA ploidy and 5cER parameters. The p53 and Ki-67 immunoexpression was evaluated by immunohistochemistry. Results CIS lesions were observed in the AM and DM of both tumor groups: 15.8% and 15.4% in AM and DM, for each one of them, in HGP group and 26.7% and 22.2% in AM and DM, for each one of them, in invasive tumors group. In CIS lesion aneuploid DNA content, p53 overexpression and high proliferative labeling index were observed. The so-called normal mucosa (AM and DM) with and without focus of CIS lesions were compared for genetic instability and molecular alterations profile. Statistical differences were observed between the normal mucosa with and without CIS: the so-called normal mucosa areas with focus of CIS revealed significantly higher frequencies of DNA content alterations, p53 overexpression, and higher proliferative index. These differences were significantly different in the invasive UCC group, but this profile it is also present in HPG group. Conclusion This study points out that CIS is a marker of genetic instability of the urothelium mucosa. The CIS surrounding morphologically normal urothelium showed a high frequency of abnormal DNA content, with high percentage of clear aneuploid cells (high 5cER), p53 mutated protein expression, and a proliferative status underlying a field carcinogenesis. These alterations in normal mucosa were not found when CIS was not present.

Prognostic value of Ki67 from clinically and histologically ‘normal’ distant mucosa in patients surgically treated for oral squamous cell carcinoma: a prospective study

The frequent spread of oral squamous cell carcinoma (OSCC) has been explained by the persistence of genetically altered mucosa after surgery. This study examined whether clinically and histologically 'normal' mucosa distant from the primary tumour (from the opposite cheek) has an abnormal proliferative status, and whether this is associated with poor prognosis in terms of local recurrence or lymph node metastasis. The prospective study included 47 consecutive patients with OSCC. Disease-free survival endpoints were defined as the duration between surgical resection and the diagnosis of recurrence, lymph node metastasis, death or last follow-up. Proliferative status was evaluated by Ki67 expression. The mean Ki67 value (16.5+/-8.9) in the distant mucosa was statistically higher than that in controls (8.8+/-2.9). Abnormally high Ki67 values (>20%) were detected in 11 OSCC patients (23%). Multivariate analysis showed that Ki67 value in distant mucosa was a powerful independent prognostic factor, greater than tumour differentiation or clinical stage for the disease-free survival rate; it was statistically negative regarding local recurrence. Some patients surgically treated for OSCC have an abnormal proliferative status in areas distant from the primary tumour. The Ki67 value in these areas is a promising prognostic factor.

PRb2/p130 protein in relation to clinicopathological and biological variables in rectal cancers with a clinical trial of preoperative radiotherapy

pRb2/p130 plays a key role in cell proliferation and is a considerable progress about expression patterns of pRb2/p130 in number of malignancies. However, pRb2/p130 expression and its significance in rectal cancer remain unknown. The purpose of the present study was to investigate pRb2/p130 protein patterns and their correlations with clinicopathological and biological factors in rectal cancer patients with or without preoperative radiotherapy (RT). pRb2/p130 protein was examined by immunohistochemistry in 130 primary tumors, along with the corresponding 61 distant normal mucosa specimens, 85 adjacent normal mucosa specimens, 34 lymph node metastases, and 93 primary tumor biopsies from rectal cancer patients who participated in a Swedish clinical trial of preoperative RT. The pRb2/p130 protein was mainly localized in the cytoplasm of tumor cells. In nonradiated cases, the lack of pRb2/p130 was related to advanced tumor-node-metastases stage, poorer differentiation, weak fibrosis, less inflammatory infiltration, higher Ki-67, and positive Cox-2 expression (p < 0.05). In radiated cases, the lack of pRb2/p130 was related to nonstaining of Cox-2 and survivin (p < 0.05). pRb2/p130 protein in primary tumors tended to be increased after RT (27% vs 16%, p = 0.07). pRb2/p130 was mainly localized in the cytoplasm rather than in the nucleus in rectal cancer. After RT, pRb2/p130 protein seems to be increased in primary tumors, and further the relationship of the pRb2/p130 with the clinicopathological and biological variables changed compared to the nonradiated cases. However, we did not find that the pRb2/p130 was directly related to RT, tumor recurrence, and patients' survival.

Equid Herpesvirus 2-Associated Oral and Esophageal Ulceration in a Foal

A case of a 1-month-old Thoroughbred foal with dysphagia, salivation, pyrexia, oral mucosal pustules, and esophageal ulceration is reported. Swabs from the ulcerated lesions yielded Equid herpesvirus 2 (EHV-2) in virus isolation assays, and histopathology of a biopsy from the esophageal lesion identified nuclear inclusions suggestive of herpesviruses. Immunohistochemical staining with antibodies specific for EHV-2 was positive for epithelial cells in the vicinity of the ulcer but not in more distant mucosa. Electron microscopic evaluation of the biopsy showed herpesviral particles in epithelial cells. The foal recovered over 5 days of supportive and gastroprotective therapy, and the esophageal ulcers healed. Serology and immunohistochemistry indicated that this foal likely had lesions associated with EHV-2 and not EHV-1, -4, or -5.

Smad3 knockout mice exhibit impaired intestinal mucosal healing

Altered transforming growth factor-β (TGFβ) expression may contribute to inflammatory bowel disease and modulate epithelial cell restitution. Interference with TGFβ-mediated signaling inhibits excisional skin wound healing, but accelerates healing of incisional cutaneous wounds and wounds in some other tissues. Therefore, we sought to clarify the potential role of Smad3-dependent TGFβ signaling in intestinal mucosal healing in Smad3 null mice. Jejunal serosal application of filter disks saturated with 75% acetic acid yielded a circumscribed reproducible ischemic mucosal ulcer 1 day later. We compared ulcer area at 3 and 5 days to day 1 in Smad3 knockout mice and syngeneic wild-type mice, and evaluated mucosal immunoreactivity at the ulcer edge for TGFβ, phosphorylated (activated) focal adhesion kinase (pFAK), phosphorylated extracellular signal-related kinase (pERK), proliferating cell nuclear antigen and apoptosis by TUNEL. Ulcer healing in Smad3 null mice was 17% less at day 3 (n=14, P=0.022) and 15% less at day 5 (n=14, P=0.004) than in wild-type littermates. In wild-type mice, pFAK, pERK and TGFβ immunoreactivity were elevated in epithelium immediately adjacent to the ulcer compared with more distant mucosa. However, this pattern of immunoreactivity for pFAK, pERK and TGFβ was not observed in Smad3 null mice. Smad3 null mice exhibited increased epithelial proliferation and no differences in apoptotic cell death compared with wild types, suggesting that ulcer healing may reflect differences in restitutive cell migration. Thus, Smad3-dependent disruption of the TGFβ signaling pathway impairs the healing of murine intestinal mucosal ulcers and alters patterns of activated FAK and ERK immunoreactivity important for cell migration at the ulcer edge. These studies suggest a significant role for Smad3-dependent TGFβ signaling in intestinal mucosal healing.

O.524 Prognostic Ki67 from normal distant mucosa in oral cancer

O.524 Prognostic Ki67 from normal distant mucosa in oral cancer

Antigen levels of urokinase-type plasminogen activator receptor and its gene polymorphism related to microvessel density in colorectal cancer.

We determined the distribution of genotypes and frequencies of alleles of the (CA)(n) repeat polymorphism in intron 3 of the urokinase plasminogen activator receptor (uPAR) gene, uPAR antigen levels and microvessel density (MVD) in tumour and distant mucosa samples from 52 patients with colorectal cancer. The uPAR level was higher for patients with high MVD comparing to patients with lower MVD which may suggest that uPAR can be correlated with progression of colorectal cancer. The significant relationship between the high MVD and uPAR antigen level appeared to be independent of the (CA)(n) repeat polymorphism because no differences in the level of uPAR antigen between carriers of alleles were found. The received results, indicate that uPAR might be considered as a target in colorectal cancer patients' therapy.

Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy

To investigate p73 expression in normal mucosa, primary tumor, and metastasis in relation to radiotherapy (RT) response and clinicopathologic/biologic variables in rectal cancers. p73 was immunohistochemically examined on biopsies (unirradiated, n=102), distant (from the large bowel, n=82), and adjacent (adjacent to primary tumor, n=89) normal mucosa samples, primary tumors (n=131), and lymph node metastasis (n=32) from rectal cancer patients participating in a clinical trial of preoperative RT. Seventy-four patients received surgery alone and 57 received additional RT. Cytoplasmic p73 was increased in the primary tumor compared with the distant or adjacent mucosa (p<or=0.0001). Nuclear (p=0.02) and cytoplasmic (p=0.003) p73 was higher in irradiated distant mucosa samples than in unirradiated ones, and nuclear p73 tended to be increased in irradiated primary tumors compared with unirradiated ones (p=0.06). p73 was positively related to cyclooxygenase-2 expression in irradiated tumors (p=0.03). p73-negative tumors tended to have a lower local recurrence after RT compared with unirradiated cases (p=0.06). Normal epithelial cells seem more sensitive to RT than tumor cells regarding p73 expression. Patients with p73-negative rectal tumors may have a lower risk of local recurrence after RT.

Telomere Length in the Colon Declines with Age: a Relation to Colorectal Cancer?

Telomeres shorten with age, which may be linked to genomic instability and an increased risk of cancer. To explore this association, we analyzed telomere length in normal colorectal tissue of individuals at different ages using quantitative-fluorescence in situ hybridization (Q-FISH) and quantitative-PCR (Q-PCR). Using Q-FISH, we also examined the histologically normal epithelium adjacent to, or distant from, colon adenomas and cancers, in addition to the neoplasms. Q-FISH and Q-PCR showed that telomere length was inversely associated with age until approximately ages 60 to 70; surprisingly, beyond this age, telomere length was positively associated with age. This association was found exclusively in epithelial, and not in stromal, cells. Peripheral blood lymphocytes showed an inverse association between telomere length and age, but without any apparent increase in telomere length in the oldest individuals. Telomere length in larger adenoma lesions (>2 cm) was significantly shorter than in normal adjacent (P = 0.004) or normal distant (P = 0.05) tissue from the same individuals. However, telomere length in histologically normal epithelium adjacent to cancers or in adenomas <2 cm was not statistically different from that of the normal distant mucosa or from normal controls, evidence that a telomere-shortening field effect was not present. We suggest that the positive association between telomere length and age in the oldest patients is a consequence of selective survival of elderly patients with long colonocyte telomeres.

Multifocal pathomorphological study of bladder cancer with the use of luminescence analysis.

Operation material from patients with various forms of urinary bladder cancer was examined. Systemic involvement resulting from multistage and multifocal tumor growth due to previous multicentric changes was demonstrated. Fluorescent study showed that in urinary bladder cancer tumor transformation involves not only adjacent, but also distant mucosa.

Plasminogen activator inhibitor 1 (PAI-1) 1334G/A genetic polymorphism in colorectal cancer.

Plasminogen activator inhibitor 1 (PAI-1) content in colorectal cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumours predict poor prognosis. The gene encoding PAI-1 is highly polymorphic and PAI-1 gene variability could contribute to the level of PAI-1 biosynthesis. In the present work the distribution of genotypes and frequency of alleles of the 1334G/A polymorphism in 92 subjects with colorectal cancer in samples of cancer tissue and distant mucosa samples as well as in blood were investigated. Blood samples age matched healthy individuals (n = 110) served as control. The 1334G/A polymorphism was determined by PCR amplification using allele specific primers. No differences in the genotype distributions and allele frequencies between blood, distant mucosa samples and cancer tissue were detected. However, the distribution of the genotypes of the 1334G/A polymorphism in patients differed significantly (P

Crypt organization in the progression to colorectal neoplasia: Computerized three dimensional (3D) crypt reconstruction and quantitative analysis of bcl-2, bas, mitosis and apoptosis within the crypt

AIMS: Alterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. Deregulation of bcl-2 and bax are important factors in the control of cell turnover. The aim of this study was to analyze patterns of bcl-2 and bax expression and in mitosis and apoptosis in the progression to colorectal neoplasia, and to reconstruct colorectal crypts to visually demonstrate this deregulation. METHODS: 12 patients with a sigmoid adenomatous polyp and 12 low risk controls were studied. Mucosal biopsies were taken from the polyp, tissue adjacent to and distant from the polyp and from the sigmoid colon of controls. Apoptotic and mitotic figures, and cells expressing bcl-2 or bax were counted, and labeling indices calculated for each within each crypt. Results are expressed as total labeling index (TLI) and labeling index (L1) for each of the five compartments (cpts) in which colonic crypts were divided for mitotic index (MI), apoptotic index (AI), and bd-2 and bax expression. Sample individual crypts that were complete within serially sections were idemified. The appropriate cells were marked, the images captured and stored, and then whole crypts were reconstructed in 3D using Analyze3.1 to assess the intra-erypt relationships of each variable. RESULTS: There was no significant difference between TLI and LI for bcl-2 and bax for controls vs. distant mucosa. The TL1 and L1 were significantly higher for polyp vs adjacent vs. distant mucosa for both bcl-2 (cpts 1-3, p<O.02) and bax (cpts 2-5, p<0.01). There was a significam increase in MI in polyp vs. distant mucosa (cpts 1-3, p<0.05). No difference in AI was seen between distant and adjacent mucosa, but there was a trend to a reduction in AI in polyp mucosa vs. all other sites. A total of 56 crypts were available for reconstruction. In reconstructed crypts, deregulation of bcl-2 and bax, increased mitosis, and the presence of apoptotic bodies in the basal cpts of polyps where bd-2 expression was highest, was easily visualized in still images and video sequences. CONCLUSIONS: Mitosis, expression ofbcl-2 and bax, and apoptosis maybecome deregulated within the colorectal crypt in the progression to colorectal neoplasia. In addition to quantitative analysis, computerized reconstruction with still images or video sequences of whole crypts can be useful in advancing our understanding of this process.

Antigen levels of the urokinase-type plasminogen activator and its gene polymorphisms in colorectal cancer

We analysed the distribution of genotypes of two polymorphisms in the urokinase-type plasminogen activator (uPA) gene: C→T substitution in exon 6 and T→C substitution in intron 7 in 52 subjects with colorectal cancer. Genotypes were determined in tumour tissue and distant mucosa samples by allele-specific polymerase chain reaction. The antigen levels of uPA in cancer tissue were higher than in distant mucosa as measured by enzyme-linked immunosorbent assay. The level of uPA antigens in cancer samples with the C/C genotype of C→T polymorphism in exon 6 was higher than in samples with C/T and T/T genotypes. No differences in the level of uPA antigens between the alleles of the intron 7 T→C polymorphism were found. As uPA can be involved in cancer invasion and metastasis, C/C genotype in exon 6 of uPA gene can be further considered as being related to colorectal cancer progression.