Boron Neutron Capture Therapy (BNCT) Mediated by Maleimide-Functionalized Closo-Dodecaborate Albumin Conjugates (MID:BSA) for Oral Cancer: Biodistribution Studies and In Vivo BNCT in the Hamster Cheek Pouch Oral Cancer Model.

BACKGROUND: BNCT (Boron Neutron Capture Therapy) is a tumor-selective particle radiotherapy that combines preferential boron accumulation in tumors and neutron irradiation. Although p-boronophenylalanine (BPA) has been clinically used, new boron compounds are needed for the advancement of BNCT. Based on previous studies in colon tumor-bearing mice, in this study, we evaluated MID:BSA (maleimide-functionalized closo-dodecaborate conjugated to bovine serum albumin) biodistribution and MID:BSA/BNCT therapeutic effect on tumors and associated radiotoxicity in the hamster cheek pouch oral cancer model. METHODS: Biodistribution studies were performed at 30 mg B/kg and 15 mg B/kg (12 h and 19 h post-administration). MID:BSA/BNCT (15 mg B/kg, 19 h) was performed at three different absorbed doses to precancerous tissue. RESULTS: MID:BSA 30 mg B/kg protocol induced high BSA toxicity. MID:BSA 15 mg B/kg injected at a slow rate was well-tolerated and reached therapeutically useful boron concentration values in the tumor and tumor/normal tissue ratios. The 19 h protocol exhibited significantly lower boron concentration values in blood. MID:BSA/BNCT exhibited a significant tumor response vs. the control group with no significant radiotoxicity. CONCLUSIONS: MID:BSA/BNCT would be therapeutically useful to treat oral cancer. BSA toxicity is a consideration when injecting a compound conjugated to BSA and depends on the animal model studied.

Evaluation of local, regional and abscopal effects of Boron Neutron Capture Therapy (BNCT) combined with immunotherapy in an ectopic colon cancer model.

OBJECTIVE: The aim of the present study was to evaluate the local and regional therapeutic efficacy and abscopal effect of BNCT mediated by boronophenyl-alanine, combined with Bacillus Calmette-Guerin (BCG) as an immunotherapy agent in this model. METHODS: The local effect of treatment was evaluated in terms of tumor response in the irradiated tumor-bearing right hind flank. Metastatic spread to tumor-draining lymph nodes was analyzed as an indicator of regional effect. The abscopal effect of treatment was assessed as tumor growth inhibition in the contralateral (non-irradiated) left hind flank inoculated with tumor cells 2 weeks post-irradiation. The experimental groups BNCT, BNCT + BCG, BCG, Beam only (BO), BO +BCG, SHAM (tumor-bearing, no treatment, same manipulation) were studied. RESULTS: BNCT and BNCT + BCG induced a highly significant local anti-tumor response, whereas BCG alone induced a weak local effect. BCG and BNCT + BCG induced a significant abscopal effect in the contralateral non-irradiated leg. The BNCT + BCG group showed significantly less metastatic spread to tumor-draining lymph nodes vs SHAM and vs BO. CONCLUSION: This study suggests that BNCT + BCG-immunotherapy would induce local, regional and abscopal effects in tumor-bearing animals. BNCT would be the main effector of the local anti-tumor effect whereas BCG would be the main effector of the abscopal effect. ADVANCES IN KNOWLEDGE: Although the local effect of BNCT has been widely evidenced, this is the first study to show the local, regional and abscopal effects of BNCT combined with immunotherapy, contributing to comprehensive cancer treatment with combined therapies.

Paget's disease of the jaws: Histopathological features of a series of 31 cases.

The aim of the present study was to analyze the histopathological features of Paget's disease of the jaws observed in a series comprising 31 cases. The study comprised all cases of Paget's disease of the jaws filed in the archives of the Surgical Pathology Laboratory of the Oral Pathology Department, School of Dentistry, University of Buenos Aires, between 1960 and 2018. Their microscopic features were evaluated, and available clinical data and radiographic studies were analyzed. Paget's disease of the jaws accounted for 0.05% of retrieved oral-maxillofacial pathologies. Microscopically, all cases showed lamellar bone trabeculae with the characteristic mosaic pattern. Twenty cases (64%) showed osteoblastic-osteoclastic activity, and all showed areas of necrosis. Cemento-osseous trabeculae were observed in 15 cases (48%), and cementicles were observed in 13 (42%). Osteomyelitis was seen in 11 cases (35%), all of which showed cemento-osseous trabeculae with a mosaic structure, sclerosis and necrosis, and chronic inflammation with abscess formation. Mean age was 61 years (44-85 years); 19 cases were women. Localization was the maxilla in 13 cases (42%), and the disease involved other skeletal bones in five cases. To our knowledge, this is the largest series of Paget's disease of the jaws reported to date. Paget's disease is infrequent in the jaws and has distinct histopathological features that not only differ from those observed at other skeletal sites but also require differential diagnosis from other pathologies affecting the jaws exclusively.

El objetivo del presente trabajo fue analizar las características histopatológicas de la enfermedad de Paget en los maxilares en una serie de 31 casos. El estudio comprendió todos los casos de enfermedad de Paget de los maxilares provenientes del Laboratorio de Patología Quirúrgica de la Cátedra de Anatomía Patológica de la Facultad de Odontología de la Universidad de Buenos Aires, entre 1960 y 2018. Se evaluaron las características microscópicas y se analizaron los datos clínicos y estudios radiográficos disponibles. La enfermedad de Paget en los maxilares representó el 0,05% de las patologías buco-maxilofaciales. Microscópicamente, todos los casos mostraron trabéculas óseas laminares con el característico patrón en mosaico. Veinte casos (64%) mostraron actividad osteoblástica-clástica y todos los casos mostraron necrosis focal. En 15 casos (48%) las trabéculas presentaron aspecto cemento-óseo y en 13 casos (42%) se observaron cementículos. Once casos (35%) presentaron cuadros osteomielíticos y todos ellos mostraron trabéculas cemento-óseas con estructura en mosaico, esclerosis y necrosis, e infiltrado inflamatorio crónico con formación de abscesos. La media de edad fue 61 años (44-85 años), y 19 fueron mujeres. Trece casos (42%) se localizaron en maxilar superior y 5 casos presentaron compromiso de otros huesos. A nuestro entender, esta es la serie más grande de enfermedad de Paget en los maxilares reportada hasta la fecha. La enfermedad de Paget es poco frecuente en los maxilares y presenta características histopatológicas que además de ser diferentes a las observadas en otros sitios del esqueleto plantean el diagnóstico diferencial con otras entidades que se presentan exclusivamente en los maxilares.

Paget's disease of the jaws: Histopathological features of a series of 31 cases / Enfermedad de Paget en los maxilares. Características histopatológicas de una serie de 31 casos

ABSTRACT The aim of the present study was to analyze the histopathological features of Paget's disease of the jaws observed in a series comprising 31 cases. The study comprised all cases of Paget's disease of the jaws filed in the archives of the Surgical Pathology Laboratory of the Oral Pathology Department, School of Dentistry, University of Buenos Aires, between 1960 and 2018. Their microscopic features were evaluated, and available clinical data and radiographic studies were analyzed. Paget's disease of the jaws accounted for 0.05% of retrieved oral-maxillofacial pathologies. Microscopically, all cases showed lamellar bone trabeculae with the characteristic mosaic pattern. Twenty cases (64%) showed osteoblastic-osteoclastic activity, and all showed areas of necrosis. Cemento-osseous trabeculae were observed in 15 cases (48%), and cementicles were observed in 13 (42%). Osteomyelitis was seen in 11 cases (35%), all of which showed cemento-osseous trabeculae with a mosaic structure, sclerosis and necrosis, and chronic inflammation with abscess formation. Mean age was 61 years (44-85 years); 19 cases were women. Localization was the maxilla in 13 cases (42%), and the disease involved other skeletal bones in five cases. To our knowledge, this is the largest series of Paget's disease of the jaws reported to date. Paget's disease is infrequent in the jaws and has distinct histopathological features that not only differ from those observed at other skeletal sites but also require differential diagnosis from other pathologies affecting the jaws exclusively.

RESUMEN El objetivo del presente trabajo fue analizar las características histopatológicas de la enfermedad de Paget en los maxilares en una serie de 31 casos. El estudio comprendió todos los casos de enfermedad de Paget de los maxilares provenientes del Laboratorio de Patología Quirúrgica de la Cátedra de Anatomía Patológica de la Facultad de Odontología de la Universidad de Buenos Aires, entre 1960 y 2018. Se evaluaron las características microscópicas y se analizaron los datos clínicos y estudios radiográficos disponibles. La enfermedad de Paget en los maxilares representó el 0,05% de las patologías buco-maxilofaciales. Microscópicamente, todos los casos mostraron trabéculas óseas laminares con el característico patrón en mosaico. Veinte casos (64%) mostraron actividad osteoblástica-clástica y todos los casos mostraron necrosis focal. En 15 casos (48%) las trabéculas presentaron aspecto cemento-óseo y en 13 casos (42%) se observaron cementículos. Once casos (35%) presentaron cuadros osteomielíticos y todos ellos mostraron trabéculas cemento-óseas con estructura en mosaico, esclerosis y necrosis, e infiltrado inflamatorio crónico con formación de abscesos. La media de edad fue 61 años (44- 85 años), y 19 fueron mujeres. Trece casos (42%) se localizaron en maxilar superior y 5 casos presentaron compromiso de otros huesos. A nuestro entender, esta es la serie más grande de enfermedad de Paget en los maxilares reportada hasta la fecha. La enfermedad de Paget es poco frecuente en los maxilares y presenta características histopatológicas que además de ser diferentes a las observadas en otros sitios del esqueleto plantean el diagnóstico diferencial con otras entidades que se presentan exclusivamente en los maxilares.

Optimization of the classical oral cancerization protocol in hamster to study oral cancer therapy.

OBJECTIVE(S): The hamster carcinogenesis model recapitulates oral oncogenesis. Dimethylbenz[a]anthracene (DMBA) cancerization induces early severe mucositis, affecting animal's welfare and causing tissue loss and pouch shortening. "Short" pouches cannot be everted for local irradiation for boron neutron capture therapy (BNCT). Our aim was to optimize the DMBA classical cancerization protocol to avoid severe mucositis, without affecting tumor development. We evaluated BNCT in animals cancerized with this novel protocol. MATERIALS AND METHODS: We studied: Classical cancerization protocol (24 applications) and Classical with two interruptions (completed at the end of the cancerization protocol). BNCT mediated by boronophenylalanine (BPA) was performed in both groups. RESULTS: The twice-interrupted group exhibited a significantly lower percentage of animals with severe mucositis versus the non-interrupted group (17% versus 71%) and a significantly higher incidence of long pouches (100% versus 53%). Tumor development and the histologic characteristics of tumor and precancerous tissue were not affected by the interruptions. For both groups, overall tumor response was more than 80%, with a similar incidence of BNCT-induced severe mucositis. CONCLUSION(S): The twice-interrupted protocol reduced severe mucositis during cancerization without affecting tumor development. This favored the animal's welfare and reduced the number of animals to be cancerized for our studies, without affecting BNCT response.

Nucleolar organizer regions in human oral verrucous carcinoma and adjacent lining epithelium.

The epithelium adjacent to an oral squamous cell carcinoma is at risk of undergoing precancerous changes. Even after such changes occur, however, the adjacent epithelium remains histologically similar to normal mucosa. We investigated five argyrophilic nucleolar organizer region (AgNOR)-related features in samples of oral verrucous carcinoma (VeCa) and their corresponding adjacent lining epithelium (adj. VeCa). Morphometric characteristics of AgNORs in oral adj. VeCa and oral VeCa were compared to normal mucosa epithelium, squamous cell carcinoma and oral mucosa epithelium adjacent to squamous cell carcinoma findings that we published earlier. Although adj. VeCa and normal oral mucosa were histologically similar, total AgNOR volume differentiated adj. VeCa from normal oral mucosa, but revealed no significant difference between VeCa and adj. VeCa. Total AgNOR volume/nuclear volume discriminated VeCa from adj. VeCa and normal oral mucosa. Certain AgNOR parameters provide a complementary tool for discriminating VeCa from adj. VeCa and normal oral mucosa, and also for detecting incipient malignant changes in epithelium adjacent to VeCa. Use of the AgNOR technique is cost-effective, because it can be performed on paraffin sections.

Different oral cancer scenarios to personalize targeted therapy: Boron Neutron Capture Therapy translational studies.

Boron neutron capture therapy (BNCT) is a targeted therapy, which consists of preferential accumulation of boron carriers in tumor followed by neutron irradiation. Each oral cancer patient has different risks of developing one or more carcinomas and/or oral mucositis induced after treatment. Our group proposed the hamster oral cancer model to study the efficacy of BNCT and associated mucositis. Translational studies are essential to the advancement of novel boron delivery agents and targeted strategies. Herein, we review our work in the hamster model in which we studied BNCT induced mucositis using three different cancerization protocols, mimicking three different clinical scenarios. The BNCT-induced mucositis increases with the aggressiveness of the carcinogenesis protocol employed, suggesting that the study of different oral cancer patient scenarios would help to develop personalized therapies.

Electroporation optimizes the uptake of boron-10 by tumor for boron neutron capture therapy (BNCT) mediated by GB-10: a boron biodistribution study in the hamster cheek pouch oral cancer model.

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.

Translational boron neutron capture therapy (BNCT) studies for the treatment of tumors in lung.

PURPOSE: Boron neutron capture therapy (BNCT) combines selective accumulation of 10B carriers in tumor tissue with subsequent neutron irradiation. BNCT has been proposed for the treatment of multiple, non-resectable, diffuse tumors in lung. The aim of the present study was to evaluate the therapeutic efficacy and toxicity of BNCT in an experimental model of lung metastases of colon carcinoma in BDIX rats and perform complementary survival studies. MATERIALS AND METHODS: We evaluated tumor control and toxicity in lung 2 weeks post-BNCT at 2 dose levels, including 5 experimental groups per dose level: T0 (euthanized pre-treatment), Boronophenylalanine-BNCT (BPA-BNCT), BPA + Sodium decahydrodecaborate-BNCT ((BPA + GB-10)-BNCT), Beam only (BO) and Sham (no treatment, same manipulation). Tumor response was assessed employing macroscopic and microscopic end-points. An additional experiment was performed to evaluate survival and oxygen saturation in blood. RESULTS AND CONCLUSIONS: No dose-limiting signs of short/medium-term toxicity were observed in lung. All end-points revealed statistically significant BNCT-induced tumor control vs Sham at both dose levels. The survival experiment showed a statistically significant 45% increase in post-treatment survival time in the BNCT group (48 days) versus Sham (33 days). These data consistently revealed growth suppression of lung metastases by BNCT with no manifest lung toxicity. Highlights Boron Neutron Capture Therapy suppresses growth of experimental lung metastases No BNCT-induced short/medium-term toxicity in lung is associated with tumor control Boron Neutron Capture Therapy increased post-treatment survival time by 45.

Different expression patterns of carbonic anhydrase IX in oral lichen planus and leukoplakia.

Tumor hypoxia is an important indicator of cancer prognosis. Among the different genes that are upregulated by hypoxia is carbonic anhydrase IX, which combines carbon dioxide and water to form bicarbonate and hydrogen. Although expression of this enzyme is very low in normal tissues, carbonic anhydrase IX is overexpressed in several types of cancer. The aim of the present work was to analyze carbonic anhydrase IX expression in the two most frequent potentially malignant oral disorders: oral lichen planus and oral leukoplakia. Immunohistochemical analysis of oral lichen planus and oral leukoplakia biopsies was performed using anticarbonic anhydrase IX antibody. Samples of normal mucosa served as controls. Statistical analysis was performed by Fischer's exact test. The enzyme was detected in the epithelium of both lesions. The staining was more intense in the basal layer and decreased towards the surface in oral lichen planus. Conversely, the most intense reaction was observed in the superficial layers in leukoplakia, and staining intensity decreased towards the basal membrane. No carbonic anhydrase IX expression was seen in normal mucosa samples. Carbon anhydrase IX expression in lichen and leukoplakia epithelia shows that hypoxia plays a role in the pathogenesis of both lesions. The different distribution patterns provides further evidence of the different biological behavior of these two entities, which under certain circumstances can have similar clinical and histological features.

La hipoxia tumoral es un importante indicador de pronóstico en cáncer. Entre los distintos genes que son activados por hipoxia, uno de los principales es la anhidrasa carbónica IX (CAIX), que combina CO2 con H2O para sintetizar HCO3 y H+. Aunque la expresión de esta enzima es muy baja en tejidos normales, se sobreexpresa en varios tipos de cáncer. La finalidad del presente trabajo fue analizar la expresión de CAIX en las dos lesiones orales potencialmente malignas más frecuentes: el liquen plano y la leucoplasia. Se utilizó una técnica inmuno histoquímica con un anticuerpo específico contra CAIX, en biopsias de liquen plano oral y leucoplasia oral. Se utilizaron mucosas normales como controles. Se realizaron análisis estadísticos utilizando test exacto de Fischer. La identificación de la enzima fue positiva en el epitelio de ambas lesiones. En los líquenes la reacción es más intensa en los estratos basales, disminuyendo hacia la superficie. Inversamente, las leucoplasias mostraron marcación más intensa en estratos superficiales, con disminución hacia la membrana basal. Las mucosas normales resultaron negativas. La expresión de CAIX en el epitelio de líquenes y leucoplasias indica que la hipoxia juega algún papel en la patogenia de ambas lesiones. El diferente patrón de distribución es una evidencia más del diferente comportamiento biológico de dos entidades las cuales en ciertas circunstancias pueden manifestar cuadros clínicos e histológicos semejantes.

El acceso digital a la bibliografía científica / Digital access to scientific literature

Se considera la importancia del acceso digital a la bibliografía necesaria para la formación profesional y sus principales herramientas: revistas de publicación únicamente electrónica y acceso a bases de datos como BIREME, SciELO y PubMed. Se analizan los fundamentos del movimiento hacia el acceso abierto de las publicaciones de trabajos de investigación, sus motivaciones, ventajas y consecuencias positivas y negativas (AU)

The importance of digital access to the necessary scientific literature that is a must for professional training is considered. The most useful tools are on line only journals and access to databases such as BIREME, SciELO and PubMed. The trend towards the Open Access to research publications, its motivations, advantages and positive and negative consequences are analyzed (AU)

Different expression patterns of carbonic anhydrase IX in oral lichen planus and leukoplakia

Tumor hypoxia is an important indicator of cancer prognosis. Among the different genes that are upregulated by hypoxia is carbonic anhydrase IX, which combines carbon dioxide and water to form bicarbonate and hydrogen. Although expression of this enzyme is very low in normal tissues, carbonic anhydrase IX is overexpressed in several types of cancer. The aim of the present work was to analyze carbonic anhydrase IX expression in the two most frequent potentially malignant oral disorders: oral lichen planus and oral leukoplakia. Immunohistochemical analysis of oral lichen planus and oral leukoplakia biopsies was performed using anticarbonic anhydrase IX antibody. Samples of normal mucosa served as controls. Statistical analysis was performed by Fischer's exact test. The enzyme was detected in the epithelium of both lesions. The staining was more intense in the basal layer and decreased towards the surface in oral lichen planus. Conversely, the most intense reaction was observed in the superficial layers in leukoplakia, and staining intensity decreased towards the basal membrane. No carbonic anhydrase IX expression was seen in normal mucosa samples. Carbon anhydrase IX expression in lichen and leukoplakia epithelia shows that hypoxia plays a role in the pathogenesis of both lesions. The different distribution patterns provides further evidence of the different biological behavior of these two entities, which under certain circumstances can have similar clinical and histological features (AU)

La hipoxia tumoral es un importante indicador de pronóstico en cáncer. Entre los distintos genes que son activados por hipoxia, uno de los principales es la anhidrasa carbónica IX (CAIX), que combina CO2 con H2O para sintetizar HCO3 y H+. Aunque la expresión de esta enzima es muy baja en tejidos normales, se sobreexpresa en varios tipos de cáncer. La finalidad del presente trabajo fue analizar la expresión de CAIX en las dos lesiones orales potencialmente malignas más frecuentes: el liquen plano y la leucoplasia. Se utilizó una técnica inmuno histoquímica con un anticuerpo específico contra CAIX, en biopsias de liquen plano oral y leucoplasia oral. Se utilizaron mucosas normales como controles. Se realizaron análisis estadísticos utilizando test exacto de Fischer. La identificación de la enzima fue positiva en el epitelio de ambas lesiones. En los líquenes la reacción es más intensa en los estratos basales, disminuyendo hacia la superficie. Inversamente, las leucoplasias mostraron marcación más intensa en estratos superficiales, con disminución hacia la membrana basal. Las mucosas normales resultaron negativas. La expresión de CAIX en el epitelio de líquenes y leucoplasias indica que la hipoxia juega algún papel en la patogenia de ambas lesiones. El diferente patrón de distribución es una evidencia más del diferente comportamiento biológico de dos entidades las cuales en ciertas circunstancias pueden manifestar cuadros clínicos e histológicos semejantes (AU)

Boron neutron capture therapy (BNCT) translational studies in the hamster cheek pouch model of oral cancer at the new "B2" configuration of the RA-6 nuclear reactor.

Boron neutron capture therapy (BNCT) is based on selective accumulation of B-10 carriers in tumor followed by neutron irradiation. We demonstrated, in 2001, the therapeutic effect of BNCT mediated by BPA (boronophenylalanine) in the hamster cheek pouch model of oral cancer, at the RA-6 nuclear reactor. Between 2007 and 2011, the RA-6 was upgraded, leading to an improvement in the performance of the BNCT beam (B2 configuration). Our aim was to evaluate BPA-BNCT radiotoxicity and tumor control in the hamster cheek pouch model of oral cancer at the new "B2" configuration. We also evaluated, for the first time in the oral cancer model, the radioprotective effect of histamine against mucositis in precancerous tissue as the dose-limiting tissue. Cancerized pouches were exposed to: BPA-BNCT; BPA-BNCT + histamine; BO: Beam only; BO + histamine; CONTROL: cancerized, no-treatment. BNCT induced severe mucositis, with an incidence that was slightly higher than in "B1" experiments (86 vs 67%, respectively). BO induced low/moderate mucositis. Histamine slightly reduced the incidence of severe mucositis induced by BPA-BNCT (75 vs 86%) and prevented mucositis altogether in BO animals. Tumor overall response was significantly higher in BNCT (94-96%) than in control (16%) and BO groups (9-38%), and did not differ significantly from the "B1" results (91%). Histamine did not compromise BNCT therapeutic efficacy. BNCT radiotoxicity and therapeutic effect at the B1 and B2 configurations of RA-6 were consistent. Histamine slightly reduced mucositis in precancerous tissue even in this overly aggressive oral cancer model, without compromising tumor control.

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.

Assessing advantages of sequential boron neutron capture therapy (BNCT) in an oral cancer model with normalized blood vessels.

BACKGROUND: We previously demonstrated the therapeutic success of sequential boron neutron capture therapy (Seq-BNCT) in the hamster cheek pouch oral cancer model. It consists of BPA-BNCT followed by GB-10-BNCT 24 or 48 hours later. Additionally, we proved that tumor blood vessel normalization with thalidomide prior to BPA-BNCT improves tumor control. The aim of the present study was to evaluate the therapeutic efficacy and explore potential boron microdistribution changes in Seq-BNCT preceded by tumor blood vessel normalization. MATERIAL AND METHODS: Tumor bearing animals were treated with thalidomide for tumor blood vessel normalization, followed by Seq-BNCT (Th+ Seq-BNCT) or Seq-Beam Only (Th+ Seq-BO) in the window of normalization. Boron microdistribution was assessed by neutron autoradiography. RESULTS: Th+ Seq-BNCT induced overall tumor response of 100%, with 87 (4)% complete tumor response. No cases of severe mucositis in dose-limiting precancerous tissue were observed. Differences in boron homogeneity between tumors pre-treated and not pre-treated with thalidomide were observed. CONCLUSION: Th+ Seq-BNCT achieved, for the first time, response in all treated tumors. Increased homogeneity in tumor boron microdistribution is associated to an improvement in tumor control.

Angiogenesis in potentially malignant lesions and carcinomas during experimental oral carcinogenesis: a preliminary study in the hamster cheek pouch.

AIM: To evaluate vascular morphology and density, angiogenic switch activation, vascular endothelial growth factor (VEGF) expression, and endothelial cell (EC) proliferation in the hamster cheek pouch (HCP) model of oral cancer. MATERIALS AND METHODS: Immunohistochemical detection of factor VIII, 5'-Bromo-2'-Deoxyuridine (BrdU) and VEGF was performed in pre-malignant and tumoral tissues. RESULTS: Activation of angiogenesis was detected adjacent to epithelial dysplasia. Vascularized area and perimeter (p<0.001) increased in dysplasias and tumors. Tumor blood vessels exhibited an enhanced vascular compression (p<0.001) and structural alterations. EC proliferation was similar in dysplasias and carcinomas. An increase in vascular density, EC proliferation and VEGF expression was found in potentially malignant tissues but not in carcinomas. CONCLUSION: The angiogenic switch occurs in the dysplastic stage preceding tumor development in the HCP model of oral cancer. In potentially malignant tissues, increased VEGF expression favors EC proliferation and an increase in vascular density. Conversely, in tumors, VEGF is no longer of pivotal importance.

Nitric oxide synthase 2 (NOS2) expression in histologically normal margins of oral squamous cell carcinoma

The activity of Nitric Oxide Synthase 2 (NOS2) was found in oral squamous cell carcinomas (OSCC) but not in normal mucosa. Molecular changes associated to early carcinogenesis have been found in mucosa near carcinomas, which is considered a model to study field cancerization. The aim of the present study is to analyze NOS2 expression at the histologically normal margins of OSCC. Study DESIGN: Eleven biopsy specimens of OSCC containing histologically normal margins (HNM) were analyzed. Ten biopsies of normal oral mucosa were used as controls. The activity of NOS2 was determined by immunohistochemistry. Salivary nitrate and nitrite as well as tobacco and alcohol consumption were also analyzed. The Chi-squared test was applied. RESULTS: Six out of the eleven HNM from carcinoma samples showed positive NOS2 activity whereas all the control group samples yielded negative (p = 0.005). No statistically significant association between enzyme expression and tobacco and/or alcohol consumption and salivary nitrate and nitrite was found. CONCLUSIONS: NOS2 expression would be an additional evidence of alterations that may occur in a state of field cancerization before the appearance of potentially malignant morphological changes

Nitric oxide synthase 2 (NOS2) expression in histologically normal margins of oral squamous cell carcinoma.

UNLABELLED: The activity of Nitric Oxide Synthase 2 (NOS2) was found in oral squamous cell carcinomas (OSCC) but not in normal mucosa. Molecular changes associated to early carcinogenesis have been found in mucosa near carcinomas, which is considered a model to study field cancerization. The aim of the present study is to analyze NOS2 expression at the histologically normal margins of OSCC. STUDY DESIGN: Eleven biopsy specimens of OSCC containing histologically normal margins (HNM) were analyzed. Ten biopsies of normal oral mucosa were used as controls. The activity of NOS2 was determined by immunohistochemistry. Salivary nitrate and nitrite as well as tobacco and alcohol consumption were also analyzed. The Chi-squared test was applied. RESULTS: Six out of the eleven HNM from carcinoma samples showed positive NOS2 activity whereas all the control group samples yielded negative (p=0.005). No statistically significant association between enzyme expression and tobacco and/or alcohol consumption and salivary nitrate and nitrite was found. CONCLUSION: NOS2 expression would be an additional evidence of alterations that may occur in a state of field cancerization before the appearance of potentially malignant morphological changes.

Boron neutron capture therapy (BNCT) for liver metastasis in an experimental model: dose­response at five-week follow-up based on retrospective dose assessment in individual rats.

Boron neutron capture therapy (BNCT) was proposed for untreatable colorectal liver metastases. Employing an experimental model of liver metastases in rats, we recently demonstrated that BNCT mediated by boronophenylalanine (BPA-BNCT) at 13 Gy prescribed to tumor is therapeutically useful at 3-week follow-up. The aim of the present study was to evaluate dose­response at 5-week follow-up, based on retrospective dose assessment in individual rats. BDIX rats were inoculated with syngeneic colon cancer cells DHD/K12/TRb. Tumor-bearing animals were divided into three groups: BPA-BNCT (n = 19), Beam only (n = 8) and Sham (n = 7) (matched manipulation, no treatment). For each rat, neutron flux was measured in situ and boron content was measured in a pre-irradiation blood sample for retrospective individual dose assessment. For statistical analysis (ANOVA), individual data for the BPA-BNCT group were pooled according to absorbed tumor dose, BPA-BNCT I: 4.5­8.9 Gy and BPA-BNCT II: 9.2­16 Gy. At 5 weeks post-irradiation, the tumor surface area post-treatment/pre-treatment ratio was 12.2 ± 6.6 for Sham, 7.8 ± 4.1 for Beam only, 4.4 ± 5.6 for BPA-BNCT I and 0.45 ± 0.20 for BPA-BNCT II; tumor nodule weight was 750 ± 480 mg for Sham, 960 ± 620 mg for Beam only, 380 ± 720 mg for BPA-BNCT I and 7.3 ± 5.9 mg for BPA-BNCT II. The BPA-BNCT II group exhibited statistically significant tumor control with no contributory liver toxicity. Potential threshold doses for tumor response and significant tumor control were established at 6.1 and 9.2 Gy, respectively.