The hamster cheek pouch model for field cancerization studies

External carcinogens, such as tobacco and alcohol, induce molecular changes in large areas of oral mucosa, which increase the risk of malignant transformation. This condition, known as ‘field cancerization’, can be detected in biopsy specimens using histochemical techniques, even before histological alterations are identified. The efficacy of these histochemical techniques as biomarkers of early cancerization must be demonstrated in appropriate models. The hamster cheek pouch oral cancer model, universally employed in biological studies and in studies for the prevention and treatment of oral cancer, is also an excellent model of field cancerization. The carcinogen is applied in solution to the surface of the mucosa and induces alterations that recapitulate the stages of cancerization in human oral mucosa. We have demonstrated that the following can be used for the early detection of cancerized tissue: silver staining of nucleolar organizer regions; the Feulgen reaction to stain DNA followed by ploidy analysis; immunohistochemical analysis of fibroblast growth factor‐2, immunohistochemical labeling of proliferating cells to demonstrate an increase of epithelial cell proliferation in the absence of inflammation; and changes in markers of angiogenesis (i.e. those indicating vascular endothelial growth factor activity, endothelial cell proliferation and vascular density). The hamster cheek pouch model of oral cancer was also proposed and validated by our group for boron neutron capture therapy studies for the treatment of oral cancer. Clinical trials of this novel treatment modality have been performed and are underway for certain tumor types and localizations. Having demonstrated the efficacy of boron neutron capture therapy to control tumors in the hamster cheek pouch oral cancer model, we adapted the model for the long‐term study of field cancerized tissue. We demonstrated the inhibitory effect of boron neutron capture therapy on tumor development in field cancerized tissue with acceptable levels of mucositis, a dose‐limiting side‐effect.     

Therapeutic effect of boron neutron capture therapy (BNCT) on field cancerized tissue: inhibition of DNA synthesis and lag in the development of second primary tumors in precancerous tissue around treated tumors in DMBA-induced carcinogenesis in the hamster cheek pouch oral cancer model

OBJECTIVE: We previously reported the therapeutic success of different BNCT protocols in the treatment of oral cancer, employing the hamster cheek pouch model. The aim of the present study was to evaluate the effect of these BNCT protocols on DNA synthesis in precancerous and normal tissue in this model and assess the potential lag in the development of second primary tumors in precancerous tissue. The data are relevant to potential control of field cancerized tissue and tolerance of normal tissue. MATERIALS AND METHODS: We evaluated DNA synthesis in precancerous and normal pouch tissue 1-30 days post-BNCT mediated by boronophenylalanine (BPA), GB-10 (Na(2)(10)B(10)H(10)) or (BPA+GB-10) employing incorporation of 5-bromo-2'-deoxyuridine as an end-point. The BNCT-induced potential lag in the development of second primary tumors from precancerous tissue was monitored. RESULTS: A drastic, statistically significant reduction in DNA synthesis occurred in precancerous tissue as early as 1 day post-BNCT and was sustained at virtually all time-points until 30 days post-BNCT for all the protocols. The histological categories evaluated individually within precancerous tissue (dysplasia, hyperplasia and NUMF [no unusual microscopic features]) responded similarly. DNA synthesis in normal tissue treated with BNCT oscillated around the very low pre-treatment values. A BNCT-induced lag in the development of second primary tumors was observed. CONCLUSIONS: BNCT induced a drastic fall in DNA synthesis in precancerous tissue that would be associated to the observed lag in the development of second primary tumors. The minimum variations in DNA synthesis in BNCT-treated normal tissue would correlate with the absence of normal tissue radiotoxicity. The present data would support the control of field-cancerized areas by BNCT.     

P27、P16和pRb 蛋白在金黄地鼠颊囊癌变过程中的表达及意义

目的：检测pRb、p27和p16在地鼠颊囊黏膜癌前病变和鳞癌中的表达。方法：用60只金黄地鼠建立了颊囊粘膜癌前病变的动物模型，用免疫组化SABC法研究了p27、pRb和p16的表达。结果：p27、pRb和p16在口腔癌前病变和鳞癌中均有表达的异常。结论：p27、pRb和p16参与了黏膜癌前病变和鳞癌的发生与发展，它们的异常表达有助于口腔粘膜癌前病变和鳞癌的早期诊断，为研究癌前病变和鳞癌的基因治疗也提供了一定的理论依据。     

Detection of apoptotic cells in whole saliva of patients with oral premalignant and malignant lesions: a preliminary study

OBJECTIVE: The purpose of this study was to identify and measure apoptotic cells in whole saliva of patients with oral premalignant and malignant lesions and explore its utility as a prognostic indicator. STUDY DESIGN: A fluorescent TUNEL technique (APO-BrdU TUNEL) modified by our lab was applied to unstimulated whole saliva from 8 healthy volunteers, 16 patients with oral leukoplakia and/or lichen planus, 10 untreated and 5 treated cases with oral malignant lesion. RESULTS: The apoptotic cells in whole saliva were detected in 4 groups of study subjects. The apoptotic cells demonstrated morphology similar to normal exfoliated epithelial cells of oral mucosa. The fraction of apoptotic cell in treated malignant patients (18.18+/-12.65) was significantly higher than that in healthy volunteers (6.99+/-6.52), premalignant patients (4.43+/-5.52), and untreated malignant patients (3.40+/-5.14) (P<.05). CONCLUSION: Detection of apoptotic epithelial cells in whole saliva appears to have some clinical potential in monitoring reaction to chemoradiotherapy and may reveal some insight into the mechanism of oral carcinogenesis.     

Papillomaviruses: the current status in relation to oral disease

Human papillomaviruses of different types are associated with a variety of benign oral lesions and may be associated with some premalignant and malignant oral lesions. However, since it is now clear that a variant of human papillomavirus 16 is harbored by normal oral mucosa, as well as by premalignant and malignant lesions, such associations may not necessarily always be causal. The rapid progress of recent research in this field is reviewed, with particular reference to oral disease, and the current status is discussed.     

Histochemical studies on Concanavalin A-binding in experimental carcinoma of the mouse submandibular gland

The lectin, Concanavalin A(Con A) has been used to localize specific sugar residues (D-glucose, D-mannose and D-fructose) in premalignant lesions and squamous-cell carcinomas induced following cryosurgery of the mouse submandibular gland. The original Con A-horseradish peroxidase (HRP) technique as well as its combination with periodate oxidation and subsequent reduction by borohydrate were used to compare the epithelial elements during submandibular gland carcinogenesis. Granules in the granular convoluted tubule cells which were weakly reactive to the Con A-HRP method were not present in the premalignant duct like structures. The epithelium of premalignant lesions, duct-like structures, multicystic lesions, and squamous-cell carcinomas were positive for the cell-surface and intercellular substances; and basement membranes and stromal fibers were also positive. The results indicated that throughout malignant transformation of the ductal segments, premalignant epithelia lost Con A-HRP-staining granules and that Con A-binding patterns in induced squamous-cell carcinomas were similar to those found in squamous-cell epithelium.     

An experimental model to demonstrate the carcinogenic action of oral chronic traumatic ulcer

BACKGROUND: Oral chronic traumatic ulcer (CTU) is caused by constant irritation by dental edges or restorations and could behave as a pre-malignant lesion in a field initiated by carcinogens such as tobacco or alcohol. METHODS: We developed an experimental model in the hamster cheek pouch, combining the chemical carcinogen 7,12-dimethyl-benzanthracene (DMBA) with CTU. RESULTS: The successive or simultaneous action of both agents induced a significantly larger number of endophytic carcinomas than larger doses of DMBA alone. CTU alone failed to induce tumor development. Ploidy analysis revealed significantly higher malignancy indices in endophytic than in exophytic carcinomas. 5-Bromo-2-deoxyuridine labeling evidenced greater proliferation around the ulcers in chemically cancerized epithelium than around ulcers in healthy epithelium. CONCLUSIONS: The results show that CTU acts as a tumor promoter in this model. This finding is clinically relevant in that CTU may increase the risk of malignant transformation in patients with subclinical tumor initiation.     

Argyrophilic nucleolar organizer regions in inflammatory, premalignant, and malignant oral lesions: a quantitative and qualitative assessment.

BACKGROUND AND OBJECTIVE: Argyrophilic nucleolar organizer regions (AgNORs) have found widespread application in the past, especially in tumor histopathology. This study was undertaken to evaluate the significance of various AgNOR parameters and to assess their role in differentiating hyperplastic, premalignant, and malignant lesions. MATERIALS AND METHODS: The study sample consisted of archival biopsy specimens of ten squamous cell carcinomas, ten premalignant lesions, and five inflammatory lesions. Two biopsies from normal mucosa acted as control. AgNORs were assessed both quantitatively and qualitatively. The data were analyzed using Student's independent t-test, one-way analysis of variance (ANOVA), and multiple range test (Tukey-HSD). RESULTS: Quantitatively significant difference existed in the number of AgNORs between the normal mucosa, inflammatory lesions, and carcinomas, but the premalignant lesions failed to differ significantly from the normal mucosa. The number of AgNORs was found to be related to epithelial proliferation. Qualitatively, in terms of size, shape, and pattern of distribution, the normal mucosa and inflammatory lesion were alike, but the premalignant and malignant lesions differed significantly from the normal, with a marked degree of AgNOR pleomorphism being observed in carcinomas. CONCLUSIONS: AgNOR quantity is strictly proportional to the proliferative activity of the cell and does not necessarily indicate malignancy. It is the qualitative characteristics of AgNOR that help to differentiate hyperplastic, premalignant, and malignant lesions.     

Ki-67 expression in non-tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumours

Objective:  The aim of this study was to determine whether the differential assessment of epithelial proliferation is useful to diagnose premalignant fields and assess the risk of multiple tumours.
Material and methods:  We analysed 83 oral carcinomas with associated non-tumour epithelium classified as distant or close according to its distance (> or <1 cm) from the invasion point, and as squamous hyperplasia, mild, moderate, severe dysplasia or carcinoma in situ . Twenty-five healthy oral mucosa samples were used as controls. An immunohistochemical technique was applied using Mib-1. Ki-67 in premalignant epithelium was assessed in basal layer, parabasal layer, medium and upper third.
Results:  Parabasal expression was significantly higher or showed a tendency to be higher in close and distant epithelia with any histological grade than in the controls. Parabasal Ki-67 significantly differed between distant epithelia associated with multiple vs single tumours ( P  < 0.001) and between distant epithelia associated with multiple tumours vs controls ( P  < 0.001). This difference was not observed between distant epithelia associated with single tumours and controls ( P  = 0.175). The cut-off point that differentiated epithelia associated with multiple tumours was >50% of Ki-67 + parabasal cells in distant epithelia, which yielded 0.88 sensitivity and 0.79 specificity.
Conclusions:  The concept of a precancerous field may be linked to an increase in the proliferative activity of parabasal cells.     

Increase in placental glutathione S-transferase in human oral epithelial dysplastic lesions and squamous cell carcinomas

This study investigated the immunohistochemical expression of human placental glutathione S-transferase (GST-φ) in the epithelium of oral premalignant and malignant lesions. Epithelial lining of normal oral mucosa, hyperplastic lesions and oral epithelium exhibiting mild dysplasia showed weak to moderate GST-φ staining. Moderate epithelial dysplasia revealed an increased antibody content while severe dysplasia. carcinoma- in-situ (CIS) and squamous cell carcinoma (SCC) demonstrated markedly increased antibody binding. The GST-φ staining was evident mainly in the cytoplasm. Severe dysplasia. CIS and SCC were also characterized by areas of cells with intensive nuclear GST-φ staining. These findings support the hypothesis that GST-φ plays a role in human oral carcinogenesis and may be used as a tumor marker for human oral premalignant and malignant lesions.     

Sanguinaria-associated oral leukoplakia: comparison with other benign and dysplastic leukoplakic lesions

OBJECTIVE: This study was undertaken to compare and contrast biomarkers and ploidy data from maxillary gingiva leukoplakias associated with dentifrices and mouthrinses containing the herbal compound sanguinaria with other forms of oral benign and premalignant mucosal keratosis. STUDY DESIGN: Representative archived specimens of benign keratosis, sanguinaria-associated keratosis, and keratosis with dysplasia were used for computerized image analysis and biomarker immunohistochemical assays to assess ploidy, DNA content, and p53 and proliferating cell nuclear antigen immunoreactivity of nuclei. RESULTS: DNA content was significantly higher and higher numbers of cell populations with hyperploid nuclei were encountered in the dysplastic group than in the other two groups (P <.001). Sanguinaria-associated keratosis did not harbor significant numbers of p53-expressing nuclei, yet it showed a significant elevation in proliferating cell nuclear antigen-labeled nuclei in total, in the basal layer, and in the spinous layer in comparison with benign keratoses (P <.001). In addition, 1.5% of the sanguinaria-associated leukoplakia epithelial cell population was characterized by nuclei with a greater than 4-fold increase in DNA content. CONCLUSIONS: Sanguinaria-associated keratoses show some marker and image analysis profiles similar to those of non-sanguinaria dysplastic lesions of the lip and mucosa. Preparations containing sanguinaria should be avoided until the risk for malignant transformation is determined.     

Cell proliferation and tumour suppressor gene expression in iodine unstained area surrounding oral squamous cell carcinoma

The purpose of this study was to investigate the relationship between epithelial dysplasia unstained with iodine and the expression of proliferating cell nuclear antigen (PCNA) and/or tumour suppressor gene (p53) and the existence of glycogen. Thirty cases of squamous cell carcinomas arising from the buccal mucosa and floor of the mouth were examined. Iodine unstained areas were diagnosed histopathologically as mild, moderate or severe epithelial dysplasia. Normal oral mucosa stained with iodine was used as a control group. There was no histochemical difference in the distribution or ratio of PAS-positive cells between the control and the mild epithelial dysplasia groups, however PAS stained areas of the moderate and the severe dysplasia groups were significantly decreased. Ultrastructurally, glycogen granules were not recognized in the moderate or severe dysplastic epithelia. Immunoreactive ratios of PCNA and p53 in the moderate and severe dysplastic groups were significantly higher than those of the control and the mild dysplasia groups. The positive ratio of PCNA was higher than that of p53, although the immunostaining patterns of PCNA- and p53-positive cells were quite similar. These results suggest that mild dysplastic epithelia that are stained with iodine may be in the category of normal epithelia, whereas both moderate and severe dysplasia that are un-stained with iodine may be suspected of malignant lesions.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.     

Progress in determining the malignant potential of oral lesions

The dilemma in managing patients with potentially malignant oral lesions and field change is of deciding which mucosal lesions or areas will progress to carcinoma. Although dysplasia may be predictive, this is not invariable, and there can be considerable inter- and intraexaminer variation in that diagnosis. Recent data on molecular and DNA changes in potentially malignant lesions suggest that it is now feasible to identify those lesions that are truly potentially malignant.     

Comparison of BrdU and cyclin A as markers of the S-phase in oral precancerous lesions

A study comparing bromodeoxyuridine (BrdU) and cyclin A as markers of cells in the S-phase in oral precancerous lesions was performed. These were also compared with the growth fraction (GF) as assessed by Ki-67. Biopsies of 15 lesions were obtained, presenting clinically as leukoplakia or erythroplakia of the lateral tongue or floor of mouth. Half of each biopsy was incubated in BrdU and routinely fixed and processed. Sequential sections from each block were cut and stained immunohistochemically with antibodies against the following proteins: BrdU, Ki-67 and cyclin A. Stained sections were quantified and the labelling indices (LI) expressed per 100 of the total nucleated cell population (%) and per millimetre basement length (/mm). The mean LI% for BrdU was 11.24% (SD 2.83), for cyclin A it was 12.76% (SD 3.88) and the GF% was 29.25% (SD 11.88). The mean LI/mm for BrdU was 40.93/mm (SD 11.88), for cyclin A it was 47.59/mm (SD 18.28) and the GF/mm was 110.72/mm (SD 49.30). The BrdU and cyclin A indices were significantly correlated with each other. In the more dysplastic cases, the cyclin A LI was quantitatively much larger than that for BrdU, suggesting that the protein was being overexpressed. It was concluded that as a tool to study the kinetic aspects of the cell cycle in dysplastic lesions cyclin A was limited by the fact that it is overexpressed. In minimally dysplastic lesions and normal epithelia, however, cyclin A may be a viable alternative to BrdU for the study of the S-phase.     

Histological and histochemical studies of oral cancer.

Increasing attempts are now being made to measure and to assess the relative importance of histologic features in oral premalignant and malignant conditions in order to improve the accuracy of diagnosis and prognosis. Though certain individual features, such as DNA content of cell nuclei, or stereological parameters like nuclear : cytoplasmic ration or desmosome density can be very accurately quantified, no single feature can completely characterize the neoplastic process. Multifactorial studies are thus of great importance, even though it is not practicable to measure all the parameters included with the sam accuracy; in such studies retrospective computation of the relative importance of individual parameters is of greater value than arbitarily assigned weights. So far as early prediction of malignant transformation in oral premalignant lesions is concerned, great interest is now being shown in the reactivity of glucose-6-phosphate dehydrogenase in epithelial cells. For predicting the prognosis of patients with established squamous cell carcinoma of the mouth, it is now realized that thenature and intensity of the host immune inflammatory response seen in the connective tissue within the surrounding the tumour and in affected regional lymph nodes, is at least as important as are the features of the tumour cells themselves.     

Papillomaviruses: their possible role in oral disease

Papillomaviruses are ubiquitous DNA viruses that are epitheliotropic and produce a range of epithelial neoplasms, both benign and malignant, in animals and man. Human papillomaviruses are associated with a variety of rare and uncommon oral lesions, and there has been increasing suspicion that they may be implicated also in some premalignant and malignant oral lesions.     

Evaluation of nuclear morphometry and DNA ploidy status for detection of malignant and premalignant oral lesions: quantitative cytologic assessment and review of methods for cytomorphometric measurements.

PURPOSE: Detection of a precancerous or cancerous lesion when small is one of the most important factors to improve 5-year survival rates of oral cancer. Although surgical biopsy is the most definitive method for diagnosing oral lesions, it is impractical to routinely subject large numbers of patients to biopsy. Recently, cytomorphometric assessments improved by advanced computer-assisted image analysis systems have gained importance. This study was established to evaluate the efficacy of nuclear cytomorphometric analysis and DNA ploidy status for the detection of oral malignancies. Methods used for cytomorphometric analysis were also reviewed. PATIENTS AND METHODS: Oral mucosal smears (n = 44) were obtained from patients (n = 22) presenting with various oral lesions using a cytobrush immediately before biopsy. Cytomorphometric measurements and nuclear Feulgen DNA content analysis were carried out after the Feulgen staining procedure. Smears from the lesion site constituted the study group whereas contralateral healthy mucosal sites served as control. RESULTS: DNA ploidy analysis revealed 20 diploid (90.9%) and 2 aneuploid DNA patterns (9.1%) sampled from the lateral margin of the tongue and floor of the mouth. When only malignant lesions were considered, aneuploidy rate was 16.7% whereas a diploid pattern was indicated for 83.3% of the sample. With cytomorphometric measurements, a statistically significant difference was shown for nuclear perimeter, area, diameter equivalent to circle, minimum and maximum Feret, intensity, DNA content (c) and DNA index values. CONCLUSIONS: Cytomorphometric analysis via oral brush biopsy is a valuable adjunct to biopsy for identification of premalignant and early stage cancerous oral lesions as a rapid and minimally invasive procedure with high specificity and sensitivity rates, requiring no topical or local anesthetic.     

Alterations in the volume of the intercellular space between epithelial cells of the hamster cheek-pouch: quantitative studies of normal and carcinogen-treated tissues

The present report investigated the extent of the epithelial dysplastic feature known as "loss of cellular adherence" at the ultrastructural level by quantifying the volume of the intercellular space during hamster cheek-pouch carcinogenesis. Following topical application of DMI3A to cheek-pouches, lesions were classified as hyper-plasia, dysplasia and carcinoma, with untreated pouches serving as a control. Stereological point counting procedures were used to determine the volume density of intercellular space in defined basal, spinous and granular layers for each group. In general, progressive increases in volume density were detected within each stratum during carcinogenesis. These results indicate that increasing separation of epithelial cells occurs during carcinogenesis, although it is not yet known whether this results from loss of cohesion between specialised (i.e., desmosomal) or non-specialised membrane areas. In addition, a simple indicator of pathological alteration, the Pathological Alteration Ratio (PAR), is described and was used to evaluate existing published data for intercellular spaces in various oral mucosal conditions. Values of the PAR were found to be substantially higher in carcinogen-treated epithelia than in reports describing changes in wound healing, lichen planus and leukoplakia simplex. These objective techniques are of value for investigating the pathogenesis of diseased epithelium and may find applications in the diagnosis of oral premalignant lesions.