Melanoma amelanótico em cães: estudo retrospectivo de 35 casos (2004-2010) e caracterização imuno-histoquímica

Melanomas são tumores agressivos de melanócitos que ocorrem principalmente na cavidade oral, nas junções mucocutâneas e na pele de cães. Este tipo de neoplasma pode apresentar diversos graus de pigmentação melânica, incluindo total ausência (melanomas amelanóticos [MA]). Os arquivos de biópsia do SPV-UFRGS, que compreendem o período de 2004 a 2010, foram revisados e levantados os casos de neoplasias melanocíticas em cães. Realizou-se estudo retrospectivo de 35 casos de MA e caracterização pela imuno-histoquímica (IHQ). As principais raças acometidas foram o Poodle, Dachshund e Cocker Spaniel, mas o maior número de casos foi observado em cães sem raça definida (SRD). A idade média desses cães foi de 10,7 anos (variação de 5 a 18 anos) e não houve predileção por sexo. As principais localizações incluíram cavidade oral (57,1%) e dígitos (17,1%). Histologicamente, 40% dos MA foram classificados como epitelioides, 34,3% como mistos e 25,7% como fusiformes. Na avaliação IHQ, 86,6% dos casos foram positivos para a vimentina, 70% para a proteína S-100 e 56,6% para o melan-A. Os resultados obtidos neste trabalho possibilitam concluir que os cães com MA caracterizavam-se por serem velhos. A forma celular mais observada foi a epitelioide. Devido a pouca diferenciação desses tumores, ressalta-se a importância da realização do painel imuno-histoquímico, sobretudo da proteína S-100, que apresentou melhor marcação que o melan-A.

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Hyperthermia increases the metastatic potential of murine melanoma

Hyperthermia, either alone or combined with radio-, immuno- or chemotherapy, can control tumor growth, but its effect on metastasis is still controversial. In the present study, we investigated the influence of hyperthermia on the metastatic potential of B16-F10 murine melanoma cells. Incubation of melanoma cells at 43ºC for 30 min led to a significant decrease in cell viability. About half of the cells survived the acute exposure to heat. These thermoresistant cells displayed a longer lag phase as compared to control unheated B16-F10 melanoma cells. Other parameters of cell growth such as doubling time and saturation density were equivalent in both control and thermoresistant cells. Both control and treated cells were adherent, but thermoresistant cells failed to spread during the first 48 h after heat exposure. B16-F10 cells colonize the lungs of C57BL/6J mice when injected intravenously; the number of lung colonies is a measure of the metastatic potential of injected cells. Median values of 22, 10.5 and 31 colonies per injected mouse were observed for control cells, cells heated to 43ºC for 30 min and thermoresistant cells, respectively, with statistically significant differences between groups (Mann-Whitney test, P<0.02). Thus, despite its cytotoxic action, heat exposure induced the acquisition of a more metastatic phenotype in a subpopulation of B16-F10 cells

von Willebrand factor antigen levels in plasma of patients with malignant breast disease

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 ± 78 U/dl in patients with cancer, 148.4 ± 59 U/dl in patients with benign disease and 130.6 ± 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 ± 113 U/dl, stage IIIB = 194.0 ± 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 ± 65 U/dl, stage IIA = 146.9 ± 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression.

High-dose cyclophosphamide followed by autologous peripheral blood progenitor cell transplantation improves the salvage treatment for persistent or sensitive relapsed malignant lymphoma

Trials have demonstrated that high-dose escalation followed by autologous transplantation can promote better long-term survival as salvage treatment in malignant lymphomas. The aim of the present nonrandomized clinical trial was to demonstrate the role of high-dose cyclophosphamide (HDCY) in reducing tumor burden and also to determine the effectiveness of HDCY followed by etoposide (VP-16) and methotrexate (MTX) in Hodgkin's disease plus high-dose therapy with peripheral blood progenitor cell (PBPC) transplantation as salvage treatment. From 1998 to 2000, 33 patients with a median age of 33 years (13-65) affected by aggressive non-Hodgkin's lymphoma (NHL) (60.6%) or persistent or relapsed Hodgkin's disease (39.4%) were enrolled and treated using high dose escalation (HDCY + HDVP-16 plus HDMTX in Hodgkin's disease) followed by autologous PBPC transplantation. On an "intention to treat" basis, 33 patients with malignant lymphomas were evaluated. The overall median follow-up was 400 days (40-1233). Thirty-one patients underwent autografting and received a median of 6.19 x 10(6)/kg (1.07-29.3) CD34+ cells. Patients who were chemosensitive to HDCY (N = 22) and patients who were chemoresistant (N = 11) presented an overall survival of 96 and 15%, respectively (P<0.0001). Overall survival was 92% for chemosensitive patients and 0% for patients who were still chemoresistant before transplantation (P<0.0001). Toxicity-related mortality was 12% (four patients), related to HDCY in two cases and to transplant in the other two. HDCY + HDVP-16 plus HDMTX in only Hodgkin's disease followed by autologous PBPC proved to be effective and safe as salvage treatment for chemosensitive patients affected by aggressive NHL and Hodgkin's disease, with acceptable mortality rates related to sequential treatment.

Vertical growth phase and positive sentinel node in thin melanoma

Sentinel node (SN) status is the most important prognostic factor for localized melanoma. Usually, patients with Breslow thickness of less than 1.0 mm are not included in SN protocols. However, the literature presents a rate ranging from 3 to 7% of nodal recurrence in thin melanoma. Ulceration, regression and high mitotic rate have been considered to be indications for an SN biopsy. The metastatic potential of the vertical growth phase is uncertain. To correlate pathological features in thin melanoma with SN metastasis, we reviewed 358 patients submitted to SN biopsy. Seventy-seven patients with lesions of 1 mm or smaller were included in the study group. Histological evaluation of the primary tumor included thickness, Clark level, mitotic rate, ulceration, regression, and growth phase. Lymphoscintigraphy was performed on all patients. Lymphatic mapping and gamma probe detection were both used for SN biopsy. Histological examination of SN consisted of hematoxylin-eosin and immunohistochemical staining. Median follow-up was 37 months. Six patients had micrometastases. Statistical analysis by the Fisher test showed that ulceration (P = 0.019), high mitotic rate (P = 0.008) and vertical growth phase (P = 0.002) were positively correlated with micrometastases. If other studies confirm these results, more melanoma patients must be submitted to SN biopsy.

No evidence for mutations in exons 1, 8 and 18 of the patched gene in sporadic skin lesions of Brazilian patients

There is strong evidence that the patched (PTCH) gene is a gene for susceptibility to the nevoid basal cell carcinoma syndrome. PTCH has also been shown to mutate in both familial and sporadic basal cell carcinomas. However, mutations of the gene seem to be rare in squamous cell carcinomas. In order to characterize the role of the gene in the broader spectrum of sporadic skin malignant and pre-malignant lesions, we performed a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of genomic DNA extracted from 105 adult patients (46 females and 59 males). There were 66 patients with basal cell carcinomas, 30 with squamous cell carcinomas, 2 with malignant melanomas and 7 patients with precancerous lesions. Two tissue samples were collected from each patient, one from the central portion of the tumor and another from normal skin. Using primers that encompass the entire exon 1, exon 8 and exon 18, where most of the mutations have been detected, we were unable to demonstrate any band shift. Three samples suspected to present aberrant migrating bands were excised from the gel and sequenced directly. In addition, we sequenced 12 other cases, including tumors and corresponding normal samples. A wild-type sequence was found in all 15 cases. Although our results do not exclude the presence of clonal alterations of the PTCH gene in skin cancers or mutations in other exons that were not screened, the present data do not support the presence of frequent mutations reported for non-melanoma skin cancer of other populations.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of ß-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM ß-estradiol inhibited cell proliferation in 30% (0.70 ± 0.03 x 10(5) cells) and increased tyrosinase activity in 50% (7130.5 ± 376.5 cpm/10(5) cells), as compared to untreated cells (1.0 ± 0.05 x 10(5) cells and 4769 ± 25.5 cpm/10(5) cells, respectively). Both responses were completely (100%) blocked by 1 µM tamoxifen. Higher concentrations (up to 1.6 nM) or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-³H]-ß-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 µM, Kd = 0.14 µM, maximal displacement of 93%) or by 10 µM tamoxifen (displacement of 60%). ß-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with ß-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

The P48T germline mutation and polymorphism in the CDKN2A gene of patients with melanoma

CDKN2A has been implicated as a melanoma susceptibility gene in some kindreds with a family history of this disease. Mutations in CDKN2A may produce an imbalance between functional p16ink4a and cyclin D causing abnormal cell growth. We searched for germline mutations in this gene in 22 patients with clinical criteria of hereditary cancer (early onset, presence of multiple primary melanoma or 1 or more first- or second-degree relatives affected) by secondary structural content prediction, a mutation scanning method that relies on the propensity for single-strand DNA to take on a three-dimensional structure that is highly sequence dependent, and sequencing the samples with alterations in the electrophoretic mobility. The prevalence of CDKN2A mutation in our study was 4.5% (1/22) and there was a correlation between family history and probability of mutation detection. We found the P48T mutation in 1 patient with 2 melanoma-affected relatives. The patient descends from Italian families and this mutation has been reported previously only in Italian families in two independent studies. This leads us to suggest the presence of a mutational "hotspot" within this gene or a founder mutation. We also detected a high prevalence (59.1%) of polymorphisms, mainly alleles 500 C/G (7/31.8%) or 540 C/T (6/27.3%), in the 3' untranslated region of exon 3. This result reinforces the idea that these rare polymorphic alleles have been significantly associated with the risk of developing melanoma.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Increased sympathetic activity in normotensive offspring of malignant hypertensive parents compared to offspring of normotensive parents

Malignant hypertension seems to be the consequence of very high blood pressure. Furthermore, an increase in sympathetic and renin-angiotensin system activity is considered to be the main mechanisms producing malignant hypertension. In the present study, 10 offspring of malignant hypertensive (OMH) parents (age 28 ± 5 years, 7 males, 3 females, 2 white and 8 non-white) and 10 offspring of normotensive (ONT) parents (age 28 ± 6 years, 2 males, 8 females, 3 white and 7 non-white) were evaluated. The OMH group had significantly higher (P < 0.05) casual blood pressure (125 ± 10/81 ± 5 mmHg) compared with ONT (99 ± 13/67 ± 5 mmHg). The increase in blood pressure was greater in OMH (Δ SBP = 17 ± 2 vs Δ SBP = 9 ± 1 mmHg in ONT) during cold pressor testing, but they had a lower increase in heart rate (Δ HR = 13 ± 2 vs Δ HR = 20 ± 3 bpm in ONT) during isometric exercise (handgrip test). Sympathetic activity, measured by microneurography, was significantly higher (P < 0.05) before exercise in OMH (17 ± 6 vs 11 ± 4 burst/min in ONT) and exhibited a greater increase (Δ = 18 ± 10 vs Δ = 8 ± 3 burst/min in ONT) during isometric exercise. This study showed increased sympathetic activity in OMH before exercise and a greater response during isometric exercise, suggesting an autonomic abnormality before exercise and a greater sympathetic response to physical stress in OMH compared to ONT.

Multigenerational Brazilian family with malignant hyperthermia and a novel mutation in the RYR1 gene

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1). Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test (CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser, was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2 MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4932Ser variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its location within the protein.

Use of the caffeine-halothane contracture test for the diagnosis of malignant hyperthermia in Brazil

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine. Deaths due to MH have been reported in Brazil. The first Malignant Hyperthermia Diagnostic and Research Center in Latin America was inaugurated in 1993 at the Federal University of Rio de Janeiro, Brazil. The center followed the diagnostic protocols of the North America MH Group, in which the contractures of biopsies from the vastus lateralis muscle are analyzed after exposure to caffeine and halothane (CHCT). CHCT was performed in individuals who survived, their relatives and those with signs/symptoms somewhat related to MH susceptibility (MHS). Here, we report data from 194 patients collected over 16 years. The Southeast (N = 110) and South (N = 71) represented the majority of patients. Median age was 25 (4-70) years, with similar numbers of males (104) and females (90). MHS was found in 90 patients and 104 patients were normal. Abnormal responses to both caffeine and halothane were observed in 59 patients and to caffeine or halothane in 20 and 11 patients, respectively. The contracture of biopsies from MHS exposed to caffeine and halothane was 1.027 ± 0.075 g (N = 285) and 4.021 ± 0.255 g (N = 226), respectively. MHS was found in patients with either low or high blood creatine kinase and also, with a low score on the clinical grading scale. Thus, these parameters cannot be used with certainty to predict MHS. We conclude that the CHCT protocol described by the North America MH Group contributed to identification of MHS in suspected individuals at an MH center in Brazil with 100% sensitivity and 65.7% specificity.