Histamine reduces boron neutron capture therapy-induced mucositis in an oral precancer model.

OBJECTIVES: Searching for more effective and selective therapies for head and neck cancer, we demonstrated the therapeutic effect of boron neutron capture therapy (BNCT) to treat oral cancer and inhibit long-term tumor development from field-cancerized tissue in the hamster cheek pouch model. However, BNCT-induced mucositis in field-cancerized tissue was dose limiting. In a clinical scenario, oral mucositis affects patients' treatment and quality of life. Our aim was to evaluate different radioprotectors, seeking to reduce the incidence of BNCT-induced severe mucositis in field-cancerized tissue. MATERIALS AND METHODS: Cancerized pouches treated with BNCT mediated by boronophenylalanine at 5 Gy were treated as follows: control: saline solution; Hishigh : histamine 5 mg kg(-1) ; Hislow : histamine 1 mg kg(-1) ; and JNJ7777120: 10 mg kg(-1). RESULTS: Hislow reduced the incidence of severe mucositis in field-cancerized tissue to 17% vs CONTROL: 55%; Hishigh : 67%; JNJ7777120: 57%. Hislow was non-toxic and did not compromise the long-term therapeutic effect of BNCT or alter gross boron concentration. CONCLUSION: Histamine reduces BNCT-induced mucositis in experimental oral precancer without jeopardizing therapeutic efficacy. The fact that both histamine and boronophenylalanine are approved for use in humans bridges the gap between experimental work and potential clinical application to reduce BNCT-induced radiotoxicity in patients with head and neck cancer.

Biodistribution of the boron carriers boronophenylalanine (BPA) and/or decahydrodecaborate (GB-10) for Boron Neutron Capture Therapy (BNCT) in an experimental model of lung metastases.

BNCT was proposed for the treatment of diffuse, non-resectable tumors in the lung. We performed boron biodistribution studies with 5 administration protocols employing the boron carriers BPA and/or GB-10 in an experimental model of disseminated lung metastases in rats. All 5 protocols were non-toxic and showed preferential tumor boron uptake versus lung. Absolute tumor boron concentration values were therapeutically useful (25-76ppm) for 3 protocols. Dosimetric calculations indicate that BNCT at RA-3 would be potentially therapeutic without exceeding radiotolerance in the lung.

Boron neutron capture therapy for oral precancer: proof of principle in an experimental animal model.

OBJECTIVES: Field-cancerized tissue can give rise to second primary tumours, causing therapeutic failure. Boron neutron capture therapy (BNCT) is based on biological targeting and would serve to treat undetectable foci of malignant transformation. The aim of this study was to optimize BNCT for the integral treatment for oral cancer, with particular emphasis on the inhibitory effect on tumour development originating in precancerous conditions, and radiotoxicity of different BNCT protocols in a hamster cheek pouch oral precancer model. MATERIALS AND METHODS: Groups of cancerized hamsters were locally exposed to single or double (2 or 4 weeks apart) applications of BNCT at different dose levels, mediated by the boron compounds boronophenylalanine (BPA) or BPA and decahydrodecaborate (GB-10) administered jointly. Cancerized, sham-irradiated hamsters served as controls. Clinical status, tumour development from field-cancerized tissue and mucositis were followed for 8 months. RESULTS: A double application (4 weeks apart) of BNCT mediated by GB-10+ BPA at a total dose of 10 Gy in two 5-Gy doses rendered the best therapeutic advantage (63-100% inhibition of tumour development from field-cancerized tissue), minimizing dose-limiting mucositis. CONCLUSION: BNCT can be optimized for the integral treatment for head and neck cancer, considering the implications for field-cancerized tissue.

Boron neutron capture therapy (BNCT) inhibits tumor development from precancerous tissue: an experimental study that supports a potential new application of BNCT.

We previously demonstrated the efficacy of boron neutron capture therapy (BNCT) mediated by boronophenylalanine (BPA), GB-10 (Na(2)(10)B(10)H(10)) and (GB-10+BPA) to control tumors, with no normal tissue radiotoxicity, in the hamster cheek pouch oral cancer model. Herein we developed a novel experimental model of field-cancerization and precancerous lesions (globally termed herein precancerous tissue) in the hamster cheek pouch to explore the long-term potential inhibitory effect of the same BNCT protocols on the development of second primary tumors from precancerous tissue. Clinically, second primary tumor recurrences occur in field-cancerized tissue, causing therapeutic failure. We performed boron biodistribution studies followed by in vivo BNCT studies, with 8 months follow-up. All 3 BNCT protocols induced a statistically significant reduction in tumor development from precancerous tissue, reaching a maximum inhibition of 77-100%. The inhibitory effect of BPA-BNCT and (GB-10+BPA)-BNCT persisted at 51% at the end of follow-up (8 months), whereas for GB-10-BNCT it faded after 2 months. Likewise, beam-only elicited a significant but transient reduction in tumor development. No normal tissue radiotoxicity was observed. At 8 months post-treatment with BPA-BNCT or (GB-10+BPA)-BNCT, the precancerous pouches that did not develop tumors had regained the macroscopic and histological appearance of normal (non-cancerized) pouches. A potential new clinical application of BNCT would lie in its capacity to inhibit local regional recurrences.

Dosimetry and radiobiology at the new RA-3 reactor boron neutron capture therapy (BNCT) facility: application to the treatment of experimental oral cancer.

The National Atomic Energy Commission of Argentina (CNEA) constructed a novel thermal neutron source for use in boron neutron capture therapy (BNCT) applications at the RA-3 research reactor facility located in Buenos Aires. The aim of the present study was to perform a dosimetric characterization of the facility and undertake radiobiological studies of BNCT in an experimental model of oral cancer in the hamster cheek pouch. The free-field thermal flux was 7.1 x 10(9) n cm(-2)s(-1) and the fast neutron flux was 2.5 x 10(6) n cm(-2)s(-1), indicating a very well-thermalized neutron field with negligible fast neutron dose. For radiobiological studies it was necessary to shield the body of the hamster from the neutron flux while exposing the everted cheek pouch bearing the tumors. To that end we developed a lithium (enriched to 95% in (6)Li) carbonate enclosure. Groups of tumor-bearing hamsters were submitted to BPA-BNCT, GB-10-BNCT, (GB-10+BPA)-BNCT or beam only treatments. Normal (non-cancerized) hamsters were treated similarly to evaluate normal tissue radiotoxicity. The total physical dose delivered to tumor with the BNCT treatments ranged from 6 to 8.5 Gy. Tumor control at 30 days ranged from 73% to 85%, with no normal tissue radiotoxicity. Significant but reversible mucositis in precancerous tissue surrounding tumors was associated to BPA-BNCT. The therapeutic success of different BNCT protocols in treating experimental oral cancer at this novel facility was unequivocally demonstrated.

Modification of Luna's technique for staining eosinophils in the hamster cheek pouch.

The cheek pouch is an anatomical peculiarity of hamsters, widely used as an experimental model for oral cancer, and characterization of its normal cell populations and the changes they undergo in pathological conditions is of great interest. Our studies of epithelium-connective tissue interactions have revealed that hamster eosinophils are not easily recognizable because they are small and exhibit a larger nucleus:cytoplasm ratio than those in human and other animal tissues. Luna's technique is the most popular specific staining technique for eosinophils. Owing to the morphology of hamster eosinophils, however, it was necessary to modify Luna's technique to stain these cells selectively against a more contrasting background that would enable their identification and quantitation in the hamster cheek pouch. The modification involved staining the sections with a solution of 0.5% Biebrich scarlet in lithium carbonate followed by counterstaining with 1% metanil yellow in water. The eosinophils were stained selectively red against a yellow background. Our technique avoided nuclear staining and enhanced observation of selectively stained granules in a scarce cytoplasm with a contrasting background, which permits fast, reproducible studies and automated image analysis.

Fibroblast growth factor-2 expression during experimental oral carcinogenesis. Its possible role in the induction of pre-malignant fibrosis.

BACKGROUND: The fibroblastic growth factor (FGF)-2 has been shown to induce angiogenesis in several tumor types. To date, the activity of FGF during the development of oral pre-cancerous lesions has not been analyzed. We herein evaluated the role of FGF-2 in the pre-cancerous and cancerous lesions in the hamster cheek pouch oral cancer model. METHODS: Expression of FGF-2 and its receptors FGFR-2 and FGFR-3 was assessed by immunohistochemistry at different stages of the carcinogenesis protocol. Activity of FGF-2 isoforms was analyzed by Western blots. RESULTS: Increase and abnormal localization of FGF-2 expression was evident in cancerized epithelium before it was possible to detect morphologic alterations. The changes in FGF-2 are concomitant with the evolution of subepithelial fibrosis. Immunolabeling of carcinomas was faint or completely negative. Increases of FGF-2 activity are mainly due to the increase in the 18 kDa isoform. Receptors 2 and 3 of FGF are present in epithelium, fibroblasts, and vascular endothelia of control samples and in all stages of malignant transformation. CONCLUSIONS: Our results would suggest a role for FGF-2 in the epithelium-connective interactions and a deregulation of its expression in the early stages of oral cancerization. In pre-cancerous tissue FGF-2 would play a central role in the development of fibrosis and a more collateral role in the induction of angiogenesis. The data would indicate its involvement in the process via the 18 kDa isoform.

Ploidy analysis of field cancerization and cancer development in the hamster cheek pouch carcinogenesis model.

BACKGROUND: The hamster cheek-pouch carcinogenesis model is a well-known animal system that closely mimics the development of premalignant and malignant lesions in human oral cancer. Our aim was to numerically characterize the premalignant and malignant lesions and expressions of field cancerization in this model using ploidy as the end-point. METHODS: To study the DNA content and proliferation status of the cells in this model we assessed the Feulgen reaction and the immunohistochemical reaction for 5-bromo-2-deoxiuridine (BrdU) in different histological areas of serial tissue sections of the cheek pouches of animals injected with BrdU. RESULTS: Ploidy values were higher in cancerized epithelia with no unusual microscopic features (NUMF), in preneoplastic and tumor areas than in control epithelia. The aneuploidy index was higher in NUMF areas than in control and differed significantly from control in preneoplastic areas and carcinoma. CONCLUSIONS: The unexpected alteration in DNA content observed in NUMF epithelia is of great relevance as a biomarker of field cancerized areas.

A new technique for staining mast cells using ferroin.

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.

Use of correction procedures in ploidy analysis of oral carcinomas.

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

In vivo response to the different LET regions of a 55 MeV helium ion beam.

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.

Use of correction procedures in ploidy analysis of oral carcinomas

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Use of correction procedures in ploidy analysis of oral carcinomas.

Ploidy analysis is an aid in the diagnosis and evaluation of prognosis of tumors. Image analysis is a relatively simple technique to assess ploidy that can be carried out with accessible equipment. However, it requires the use of accurate technical procedures to avoid methodological errors that may bias the measurements. We previously developed two procedures that are simple to apply in routine work and serve to correct the errors derived from the measurement of large nuclei that are not fully contained in the thickness of the section and those derived from non-specific background readings. In the present study we applied these corrections to the retrospective ploidy analysis of a series of 67 patients with oral carcinoma with a follow-up time of 18 months. Thirty-four patients were alive at the end of the study, 33 were deceased. The ploidy values and the malignancy indices corresponding to the deceased and live patients with TNM stage III and IV carcinomas at the time of biopsy were significantly different. There were no significant differences in ploidy values between live and deceased patients with TNM stage I and II at the time of biopsy. The corrections improved the sensitivity of the method and thus the statistical significance of the data. These data suggest that the method proposed may be of use to estimate lesion evolution, in particular in patients with advanced oral squamous cell carcinomas.

Boron neutron capture therapy for the treatment of oral cancer in the hamster cheek pouch model.

We have proposed and validated the hamster cheek pouch model of oral cancer for boron neutron capture therapy (BNCT) studies and shown that boronophenylalanine delivers potentially therapeutic 36.9 +/- 17.5 ppm boron to tumor tissue with tumor:normal tissue and tumor:blood ratios of 2.4:1 and 3.2:1, respectively. Here we report the first evidence of the usefulness of BNCT for the treatment of oral cancer in an experimental model. We assessed the response of hamster cheek pouch tumors, precancerous tissue, and normal oral tissue to boronophenylalanine-mediated BNCT using the thermalized epithermal beam of the RA-6 Reactor at the Bariloche Atomic Center. BNCT leads to complete remission by 15 days posttreatment in 78% of tumors and partial remission in an additional 13% of tumors with virtually no damage to normal tissue.

The hamster cheek pouch as a model of oral cancer for boron neutron capture therapy studies: selective delivery of boron by boronophenylalanine.

Herein we propose and validate the hamster cheek pouch model of oral cancer for boron neutron capture therapy (BNCT) studies. This model serves to explore new applications of the technique, study the biology and radiobiology of BNCT, and assess the uptake of boron compounds and response of tumor, precancerous tissue, and clinically relevant normal tissues. These issues are central to evaluating and improving the therapeutic gain of BNCT. The success of BNCT is dependent on the absolute amount of boron in the tumor, and the tumor:blood and tumor:normal tissue boron concentration ratios. Within this context, biodistribution studies are pivotal. Tumors were induced in the hamsters with a carcinogenesis protocol that uses dimethyl-1,2-benzanthracene and mimics spontaneous tumor development in human oral mucosa. The animals were then used for biodistribution and pharmacokinetic studies of boronophenylalanine (BPA). Blood, tumor, precancerous pouch tissue surrounding tumor, normal pouch tissue, tongue, skin, cheek mucosa, palate mucosa, liver, and spleen, were sampled at 0-12 h after administration of 300 mg BPA/kg. The data reveal selective uptake of BPA by tumor tissue and, to a lesser degree, by precancerous tissue. Mean tumor boron concentration was 36.9 +/- 17.5 ppm at 3.5 h and the mean boron ratios were 2.4:1 for tumor:normal pouch tissue and 3.2:1 for tumor:blood. Higher doses of BPA (600 and 1200 mg BPA/kg) increased tumor uptake. Potentially therapeutic absolute boron concentrations, and tumor:normal tissue and tumor:blood ratios can be achieved in the hamster oral cancer model using BPA as the delivery agent.

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis.

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent.

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent.

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.

AgNOR are sensitive markers of radiation lesions in squamous epithelia.

The possibility of detection of incipient cellular alterations is central to early diagnosis and to clinician's capacity to discriminate between samples that appear similar on routine preparations. We examined the value of silver-stained nucleolar organizer regions (AgNOR) in detecting radio-induced alterations in a model of squamous epithelium biologically similar to oral mucosa. Morphometry of AgNOR has been proven to be of value in the detection of incipient cellular alterations. This method allows for the quantitative evaluation of lesions induced by high doses of radiation long before they become apparent in routine preparations. We herein examine the capacity of AgNOR to reveal the response to low doses of radiation, closer to the therapeutic or accidental dose to which the epithelium of oral mucosa may be exposed.