Cutaneous schistosomiasis and leishmaniasis coinfection: a case report.

BACKGROUND: The polymorphic clinical presentations of schistosomiasis and leishmaniasis allow their inclusion in the differential diagnoses of several conditions. Although an overlap in distribution of these diseases has been reported in endemic areas, coinfection with cutaneous schistosomiasis and cutaneous leishmaniasis in the same patient is rare. OBJECTIVES: We report an unusual case of concomitant cutaneous schistosomiasis and cutaneous leishmaniasis. Actions for the management and diagnosis were proposed. METHODS: A patient presented with cutaneous lesions on the abdomen and left elbow. The presence of degenerated ova of Schistosoma mansoni in the skin biopsy led to perform a complementary investigation with immunohistochemical techniques, rectal biopsy and abdominal ultrasonography. After the left elbow lesions had failed to improve after several weeks of standard treatment, a new biopsy was performed and led to diagnosis of another infection. RESULTS: The patient lived in an endemic area for two infectious diseases (schistosomiasis and leishmaniasis). Biopsies revealed chronic granulomatous dermatitis. Degenerated S. mansoni eggs were found in the abdominal lesion and in a rectal biopsy specimen. Ultrasonography revealed hepatic involvement. Despite combination treatment with oxamniquine and praziquantel, a cutaneous lesion persisted on the left elbow; a new biopsy revealed amastigote forms of Leishmania. The patient was successfully treated with intramuscular and intralesional meglumine antimoniate. CONCLUSIONS: The presence of a similar granulomatous infiltrate in lesions caused by the two different infectious agents led to a delay in the diagnosis of cutaneous leishmaniasis. This report serves as a warning of the unusual possibility of cutaneous schistosomiasis and leishmaniasis coinfection in an endemic area.

M2 macrophages coexist with a Th1-driven profile in periapical cysts.

AIM: To evaluate the participation of both Th1 and Th2 responses in periapical cysts by assessing the presence of M2 macrophages, as well as acute IL-1 ß, TNF-α and IL-6 cytokines. METHODOLOGY: Twenty-four cases of periapical cysts were selected. Immuno-expressions of IL-1 ß, IL-6, TNF-α and CD163 were analysed in the cystic capsules in both superficial and deeper regions. Data were analysed with paired Wilcoxon test and Spearman correlation coefficient (P ≤ 0.05). RESULTS: There was a higher expression of IL-1ß, IL-6, TNF-α and M2 macrophages in the superficial region (P < 0.001) of cystic capsules. All acute cytokines had significant positive correlations amongst them regardless of the cystic capsule region. Regarding CD163, positive correlations occurred only with TNF-α (P = 0.007; r = 0.537) and IL-6 (P = 0.018; r = 0.478) in the superficial regions of the cystic capsule. CONCLUSIONS: M2 macrophages participated actively in the inflammatory response of periapical cysts and correlated with the expression of certain acute Th1-related cytokines. This illustrates the coexistence of an acute and chronic Th2-driven immune response in these lesions. Although M2 macrophages favour the healing process, their presence is not sufficient for periapical cyst regression, once an acute active response has occurred due to an infectious stimuli.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Abstract Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Resumo Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 μg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer.

OBJECTIVES: Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS: Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS: Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS: Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 µg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Prognostic Value of ADAMTS Proteases and Their Substrates in Epithelial Ovarian Cancer

Background: ADAMTS are metalloproteases with disintegrin and thrombospondin motifs. They are secreted proteases playing a role in biological processes such as inflammation, angiogenesis, and urogenital development. ADAMTS have specific substrates, such as the proteoglycans (PG) versican, aggrecan, and brevican. Despite data indicating a role of ADAMTS in tumor invasion and metastases, effects played by these molecules in cancer progression are still controversial. In ovarian cancer, the importance of ADAMTS gene mutations was recently described and related to chemotherapy outcome. Objective: To analyze protein levels of ADAMTS-1, -4, and -5, and TIMP-3 in human ovarian cancer classified as benign, borderline, or malignant. We also assessed the expression of the ADAMTS substrates aggrecan, brevican, and versican in these neoplasms. Correlations between overall survival and protein expression were performed. Methods: Tumors were classified according to the WHO Classification of Tumors of Female Reproductive Organs. Protein and PG expression was studied by immunohistochemistry. Differences in labeling were analyzed by percent measurements of stained areas. Results: ADAMTS-1, ADAMTS-5, and its tissue inhibitor TIMP-3 are increased in borderline and malignant tumors compared to benign neoplasms. Aggrecan and versican levels were increased in malignant subtypes compared to benign ovarian cancer. Higher ADAMTS-1, TIMP-3, and versican expression was associated with a shorter overall survival. Conclusions: Comparison of protease, TIMP-3, and substrate expression showed that in malignant tumors all ADAMTS and TIMP-3 expression levels were significantly raised compared to the substrates studied.

Are podoplanin and ezrin involved in the invasion process of the ameloblastomas?

The association between podoplanin and ezrin in the process of odontogenic tumors invasion has been suggested, but was not studied yet. Our purpose was to investigate the relationship between podoplanin and ezrin expressions in the odontogenic epithelium of ameloblastomas. Forty-seven ameloblastomas were analyzed by immunohistochemistry using anti-podoplanin and anti-ezrin antibodies. The expressions of both proteins were evaluated using a score method and the comparison and association between these proteins were verified, respectively, by Wilcoxon Signed-Rank test and by Spearman's rank correlation coefficient, using a statistical significance level of 0.05. The majority of tumors (87.2%) exhibited strong membranous expression of podoplanin in the peripheral cells. Cytoplasmic expression of ezrin in the peripheral cells of ameloblastomas was stronger than its membranous expression. No statistically significant correlation was observed between podoplanin and ezrin. However, there was statistically significant difference between membranous podoplanin and membranous ezrin expressions, between cytoplasmic podoplanin and membranous ezrin expressions, and between cytoplasmic podoplanin and cytoplasmic ezrin expressions. There was no statistical difference between membranous podoplanin and cytoplasmic ezrin expressions. These results suggest a synergistic role of both proteins in the process of invasion of ameloblastomas.

B-1 cells and concomitant immunity in Ehrlich tumour progression.

Concomitant immunity is a phenomenon in which a tumour-bearing host is resistant to the growth of an implanted secondary tumour. Metastases are considered to be secondary tumours that develop spontaneously during primary tumour growth, suggesting the involvement of concomitant immunity in controlling the rise of metastases. It has been demonstrated that B-1 cells, a subset of B-lymphocytes found predominantly in pleural and peritoneal cavities, not only increase the metastatic development of murine melanoma B16F10, but also are capable of differentiating into mononuclear phagocytes, modulating inflammatory responses in wound healing, in oral tolerance and in Paracoccidiose brasiliensis infections. Here, we studied B-1 cells' participation in concomitant immunity during Ehrlich tumour progression. Our results show that B-1 cells obtained from BALB/c mice previously injected with Ehrlich tumour in the footpad were able to protect BALB/c and BALB/Xid mice against Ehrlich tumour challenge. In addition, it was demonstrated that BALB/Xid show faster tumour growth and have lost concomitant immunity, and that this state can be partially restored by reconstituting these animals with B-1 cells. However, further researches are required to establish the mechanism involving B-1 cells in Ehrlich tumour growth.

Immunohistochemical evidence for myofibroblast-like cells associated with liver injury induced by aflatoxin B1 in rainbow trout (Oncorhynchus mykiss).

In mammalian species, profibrogenic cells are activated to become myofibroblasts in response to liver damage. Few studies have examined hepatic myofibroblasts and their role in liver damage in teleosts. The aim of the present study was to investigate the involvement of myofibroblast-like cells in rainbow trout (Oncorhynchus mykiss) with hepatic damage induced by aflatoxin B1 (AFB1). Histopathological and immunohistochemical analyses characterized alterations in the liver stroma during the carcinogenic process. Anti-human α-smooth muscle actin (SMA) and anti-human desmin primary antibodies were used in immunohistochemistry. Only the anti-SMA reagent labelled cells in trout liver. In the livers of control fish, only smooth muscle in blood vessels and around bile ducts was labelled. In the livers from AFB1-treated fish, SMA-positive cells were present in the stroma surrounding neoplastic lesions and in areas of desmoplastic reaction. These observations indicate that in teleosts, as in mammals, the myofibroblast-like cell is involved in fibrosis associated with liver injury. Chronic liver injury induced in trout by aflatoxin may provide a useful model system for study of the evolution of such mechanisms.

Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients.

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. M. leprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

p16 (INK4a) has clinicopathological and prognostic impact on oropharynx and larynx squamous cell carcinoma.

CDKN2A encodes proteins such as p16 (INK4a), which negatively regulate the cell-cycle. Molecular genetic studies have revealed that deletions in CDKN2A occur frequently in cancer. Although p16 (INK4a) may be involved in tumor progression, the clinical impact and prognostic implications in head and neck squamous cell carcinoma (HNSCC) are controversial. The objective of this study was to evaluate the frequency of the immunohistochemical expression of p16 (INK4a) in 40 oropharynx and 35 larynx from HNSCC patients treated in a single institution and followed-up at least for 10 years in order to explore potential associations with clinicopathological outcomes and prognostic implications. Forty cases (53.3%) were positive for p16 (INK4a) and this expression was more intense in non-smoking patients (P = 0.050), whose tumors showed negative vascular embolization (P = 0.018), negative lymphatic permeation (P = 0.002), and clear surgical margins (P = 0.050). Importantly, on the basis of negative p16 (INK4a) expression, it was possible to predict a probability of lower survival (P = 0.055) as well as tumors presenting lymph node metastasis (P = 0.050) and capsular rupture (P = 0.0010). Furthermore, increased risk of recurrence was observed in tumors presenting capsular rupture (P = 0.0083). Taken together, the alteration in p16 (INK4a) appears to be a common event in patients with oropharynx and larynx squamous cell carcinoma and the negative expression of this protein correlated with poor prognosis.

Prognostic significance of CD24 and claudin-7 immunoexpression in ductal invasive breast cancer.

This study aimed to identify the CD24 and CD44 immunophenotypes within invasive ductal breast carcinoma (IDC) subgroups defined by immunohistochesmistry markers and to determine its influence on prognosis as well as its association with the expression of Ki-67, cytokeratins (CK5 and CK18) and claudin-7. Immunohistochemical expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with subgroups defined as luminal A and B; HER2 rich and triple negative, or with the other markers and prognosis was analyzed. CD44+/CD24- and CD44-/CD24+ were respectively present in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD44+/CD24+ was observed in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44+/CD24- phenotype was more common in the basal subgroups but absent in HER2 tumors, whereas luminal tumors are enriched in CD44-/CD24+ and CD44+/CD24+ cells. The frequency of CD44+/CD24- or CD44-/CD24+ was not associated with clinical characteristics or biological markers. There was also no significant association of these phenotypes with the event free (DFS) and overall survival (OS). Single CD44+ was evident in 57.9% of the tumors and was marginally associated to grading and not to any other tumor characteristics as well as OS and DFS. CD24+ was positive in 74.7% of the tumors, showing a significant association with estrogen receptor, progesterone receptor and Ki-67 and a marginal association with CK18 and claudin-7. Expression of claudin-7 and Ki-67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found (P=0.03). CK5, CK18 and Ki-67 expression had no influence in OS or DFS. Single CD24+ (P=0.07) and claudin-7 positivity (P=0.05) were associated with reduced time of recurrence, suggesting a contribution of these markers to aggressiveness of breast cancer.

B-1 cells modulate the kinetics of wound-healing process in mice.

Wound healing is a complex phenomenon whose mechanisms are not fully understood. Although inflammatory cells are recruited to the site of the lesion there are no reports concerning the participation of B lymphocytes in tissue repair. As demonstrated in our laboratory, B-1 cells migrate to a non-specific inflammatory focus and differentiate into a phagocyte. It has been reported that BALB/Xid mice are deficient in B-1 cells. Using this model, here we report that BALB/Xid mice have an increased inflammatory response, a delayed wound-healing process, a prominent neutrophilic infiltration of the lesion, and an increased neovascularization of the lesion as compared with BALB/c and BALB/Xid reconstituted with B-1 cells. The infiltration of B-1 cells into the wound was demonstrated. As B-1 cells secret and use interleukin 10 (IL-10) as an autocrine growth factor, the possible participation of this interleukin in the kinetics of wound healing was investigated. Results show that C57/BL6 IL-10 KO mice had an increased inflammatory response when compared with C57/BL6 and C57/BL6 IL-10 KO mice reconstituted with B-1 cells, thus suggesting that the observed effects of B-1 cells in the healing process is mediated by this interleukin. However, the mechanisms by which IL-10 influence these phenomena remain to be uncovered.

Continuous and high-level in vivo delivery of endostatin from recombinant cells encapsulated in TheraCyte immunoisolation devices.

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 microg/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 microg/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma.

AIMS: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. METHODS AND RESULTS: Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. CONCLUSIONS: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Expression of vascular endothelial growth factor-C does not predict occult lymph-node metastasis in early oral squamous cell carcinoma.

Strong vascular endothelial growth factor-C (VEGF-C) expression has been correlated to occurrence of lymph-node metastases in patients with oral squamous cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a decisive factor in the prognosis of patients with early OSCC. The aim of this study was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult lymph-node metastases were analyzed for VEGF-C expression by malignant cells. Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection. No statistically significant difference was found between OSCC with and without occult lymph-node metastasis in regard to VEGF-C immunoexpression by malignant cells and clinicopathologic features. Independently of VEGF-C expression, lymph-node metastasis (pN+) was the most significant prognostic factor for overall survival of patients with OSCC (p=0.030). These findings indicate that isolated VEGF-C expression by malignant cells is not of predictive value for occult lymph-node metastasis in the early stages of OSCC.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8 percent) of the tumors were HPV DNA positive while 29.1 percent of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia.

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Cáncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P=0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P=0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Morphometric analysis of the tumor associated tissue eosinophilia in the oral squamous cell carcinoma using different staining techniques.

In a previous study, we found tumor-associated tissue eosinophilia (TATE) to be a favourable prognostic indicator for oral squamous cell carcinomas. Special techniques such as autofluorescence or immunohistochemistry are reported to be sometimes necessary to detect the presence of intact and degranulating eosinophils within the tumors. The aim of this study was to compare the number of eosinophils identified routinely with hematoxylin and eosin stain and by immunohistochemistry in oral squamous cell carcinomas with TATE. Thirty specimens of oral squamous cell carcinoma of the tongue, floor of the mouth, retromolar area and inferior gingiva with TNM stages II and III were used for histopathological analysis. Three-micrometer sections were stained with hematoxylin and eosin and immunohistochemically with monoclonal anti-human granulocyte-associated antigen using a standard streptavidin-biotin-peroxidase complex technique. The number of eosinophils/mm2 in the invasive front of the tumors was automatically quantified in a x400 field using an image computer analyser. Univariate statistical analysis was carried out using Student's t test. The computer-assisted morphometric results showed that there was no statistically significant difference (p>0.05) in the number of eosinophils/mm2 identified by hematoxylin and eosin or immunostaining technique in oral squamous cell carcinomas with TATE. This result suggests that hematoxilyn and eosin routine stain is a useful technique for measuring eosinophils in squamous cell carcinoma with eosinophilic tumor infiltration.