Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody-interleukin 2 fusion proteins.

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Polymorphisms of the SIPA1 gene and sporadic breast cancer susceptibility

Background The novel breast cancer metastasis modulator gene signal-induced proliferation-associated 1 (Sipa1) underlies the breast cancer metastasis efficiency modifier locus Mtes 1 and has been shown to influence mammary tumour metastatic efficiency in the mouse, with an ectopically expressing Sipa1 cell line developing 1.5 to 2 fold more surface pulmonary metastases. Sipa1 encodes a mitogen-inducible GTPase activating (GAP) protein for members of the Ras-related proteins; participates in cell adhesion and modulates mitogen-induced cell cycle progression. Germline SIPA1 SNPs showed association with positive lymph node metastasis and hormonal receptor status in a Caucasian cohort. We hypothesized that SIPA1 may also be correlated to breast carcinoma incidence as well as prognosis. Therefore, this study investigated the potential relationship of SIPA1 and human breast cancer incidence by a germline SNP genotype frequency association study in a case-control Caucasian cohort in Queensland, Australia. Methods The SNPs genotyped in this study were identified in a previous study and the genotyping assays were carried out using TaqMan SNP Genotyping Assays. The data were analysed with chi-square method and the Monte Carlo style CLUMP analysis program. Results Results indicated significance with SIPA1 SNP rs3741378; the CC genotype was more frequently observed in the breast cancer group compared to the disease-free control group, indicating the variant C allele was associated with increased breast cancer incidence. Conclusion This observation indicates SNP rs3741378 as a novel potential sporadic breast cancer predisposition SNP. While it showed association with hormonal receptor status in breast cancer group in a previous pilot study, this exonic missense SNP (Ser (S) to Phe (F)) changes a hydrophilic residue (S) to a hydrophobic residue (F) and may significantly alter the protein functions of SIPA1 in breast tumourgenesis. SIPA1 SNPs rs931127 (5' near gene), and rs746429 (synonymous (Ala (A) to Ala (A)), did not show significant associations with breast cancer incidence, yet were associated with lymph node metastasis in the previous study. This suggests that SIPA1 may be involved in different stages of breast carcinogenesis and since this study replicates a previous study of the associated SNP, it implicates variants of the SIPA1 gene as playing a potential role in breast cancer.

Predictive Factors for Local Control in Primary and Metastatic Lung Tumours after Four to Five Fraction Stereotactic Ablative Body Radiotherapy: A Single Institution's Comprehensive Experience

AIMS: We report the outcomes of a large lung stereotactic ablative body radiotherapy (SABR) programme for primary non-small cell lung cancer (NSCLC) and pulmonary metastases. The primary study aim was to identify factors predictive for local control.

MATERIALS AND METHODS: In total, 311 pulmonary tumours in 254 patients were treated between 2008 and 2011 with SABR using 48-60 Gy in four to five fractions. Local, regional and distant failure data were collected prospectively, whereas other end points were collected retrospectively. Potential clinical and dosimetric predictors of local control were evaluated using univariate and multivariate analyses.

RESULTS: Of the 311 tumours, 240 were NSCLC and 71 were other histologies. The 2 year local control rate was 96% in stage I NSCLC, 76% in colorectal cancer (CRC) metastases and 91% in non-lung/non-CRC metastases. Predictors of better local control on multivariate analysis were non-CRC tumours and a larger proportion of the planning target volume (PTV) receiving ≥100% of the prescribed dose (higher PTV V100). Among the 45 CRC metastases, a higher PTV V100 and previous chemotherapy predicted for better local control.

CONCLUSIONS: Lung SABR of 48-60 Gy/four to five fractions resulted in high local control rates for all tumours except CRC metastases. Covering more of the PTV with the prescription dose (a higher PTV V100) also resulted in superior local control.

Études des rôles physiopathologiques de PTEN dans le tractus reproducteur femelle

La tumeur des cellules de la granulosa (GCT) représente 5% des cas de cancers ovariens chez la femme. Bien que considérées comme peu malignes, la mort survient dans 80% des cas suite à une recrudescence de la maladie. En dépit de ces statistiques sinistres, peu d’études ont été portées sur ce type de cancer. Le premier objectif de cette étude consistait à élucider les mécanismes moléculaires causant les GCT en démontrant l’implication de la voie de signalisation PI3K/AKT dans leur étiologie. Pour ce faire, nous avons employé la technologie Cre-Lox afin de cibler le gène Pten (antagoniste de cette voie) spécifiquement dans les cellules de la granulosa chez la souris. Ces souris (Ptenflox/flox;Amhr2cre/+) ont occasionnellement développé des GCT, soutenant notre hypothèse de l’importance de la voie PI3K/AKT dans leur étiologie. La voie WNT/CTNNB1 est une autre voie de signalisation qui a récemment été impliquée dans le développement des GCT. Dans le cadre de ce projet, nous avons également testé l’existence possible d’une synergie fonctionnelle entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la maladie. Pour ce faire, nous avons créé le modèle transgénique Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+, chez lequel les cellules de la granulosa présentant non seulement une désinhibition de la voie PI3K/AKT, mais aussi une suractivation de la voie WNT/CTNNB1. Tel que prédit, les souris Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ ont développé une forme de GCT beaucoup plus agressive que celle observée chez les femelles Ptenflox/flox;Amhr2cre/+. Spécifiquement, le développement des tumeurs se déclenchait plus tôt, leur croissance était beaucoup plus rapide, nous avons pu observer des métastases pulmonaires et la dissémination des cellules tumorales dans la cavité péritonéale, et la maladie était invariablement fatale avant l’âge de 8 semaines. Le modèle Ptenflox/flox;Ctnnb1flox (ex3)/+;Amhr2cre/+ a donc servi à démontrer l'existence d'une synergie entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la GCT. De façon inattendue, les souris Ptenflox/flox;Amhr2cre/+ ont aussi présenté un phénotype de sous-fertilité qui n’était pas d’origine ovarienne. Il a récemment été démontré que la souche Amhr2cre dirige l’expression de Cre non seulement aux cellules de la granulosa, mais aussi au stroma utérin et au myomètre. Le second objectif de ce travail était donc de démontrer si et comment le phénotype d’infertilité chez les souris Ptenflox/flox;Amhr2cre/+ pouvait découler d’un défaut utérin. Lors de l'implantation, les cellules du stroma utérin se différencient en cellules déciduelles pour former la décidua maternelle (DM), qui se régresse ensuite par apoptose afin de faciliter l’invasion des cellules trophoblastiques. De plus, la DM, en collaboration avec le tissu foetal, recrute des uNKs dont le rôle est de remodeler les artères spiralées pour augmenter l’apport sanguin maternel vers le foetus en développement. Nous avons pu démontrer que l'utérus des femelles gestantes Ptenflox/flox;Amhr2cre/+ présentait une DM anormalement résistante à l'apoptose, moins de uNKs et des artères spiralées non-remodelées. Par conséquent, l’invasion des cellules du trophoblaste était restreinte, compromettant le développement et la survie de l'embryon. Nous avons donc établi pour la première fois l’importance de Pten lors de la décidualisation et de l’invasion du trophoblaste.

Correlação da ciclooxigenase-2 com Ki-67, P53 e Caspase-3 nas neoplasias de mama de cadela

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nefroblastoma em cão de 4 meses: relato de caso

Renal nephroblastoma is a malignant tumor composed by embryonic tissue that affects mainly puppies. This works reports the occurrence of unilateral renal nephroblastoma in a puppy with four months. The diagnosis was obtained by histopathological examination of mass obtained by exploratory laparotomy, with realization of nephrectomy. The animal died at 15 days post-operatively by the presence of pulmonary metastases. The survival rate with surgical excision of the mass, with or without complementary therapies, can vary with the stage of disease.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

Expression of the CD44v2-10 isoform confers a metastatic phenotype: Importance of the heparan sulfate attachment site CD44v3

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.

Multilobular osteochondrosarcoma of the hard palate in a dog

A 14-year-old castrated male Rhodesian Ridgeback was presented with a history of sneezing and epistaxis. Diagnostic procedures included physical examination, regional and thoracic radiography, computed tomography and histological examination of an incisional biopsy. A multilobular osteochondrosarcoma of the hard palate with pulmonary metastases was diagnosed. Surgical resection of the primary tumour was achieved with clean margins and the defect was repaired using bilateral mucosal transposition flaps from the lips. Wound dehiscence and oesophageal stricture were postoperative complications, but these resolved with treatment. A long-term survival time of 14 months resulted, with good quality of life and function during this time.

Metastases and primary tumors around dental implants: A literature review and case report of peri-implant pulmonary metastasis

To perform a literature review on peri-implant metastases and primary malignoma and report a case of a pulmonary metastasis around dental implants of the anterior mandibular jaw that mimicked peri-implantitis.

Undifferentiated pulmonary adenocarcinoma of clear cells associated to hypertrophic osteopathy in a dog

Background: Most of the primary pulmonary tumors in dogs are malignant and from epithelial origin, being bronchioalveolar tumors more prevalent. Adenocarcinoma of clear cells, however, is a very rare pulmonary tumor and its origin is still unknown. It is related to several clinical abnormalities, including hypertrophic osteopathy, an unusual paraneoplastic syndrome characterized by a periosteal reaction along the shaft of long bones. Because of the unusual presentation of the pulmonary adenocarcinoma, the aim of this study was to describe the radiographic, histopathological, and immunohistochemical fi ndings of a dog affl icted with hypertrophic osteopathy secondary to an undifferentiated pulmonary adenocarcinoma of clear cells. Case: A 12-year-old, 45 kg, not castrated male Great Dane dog was presented with painful swelling of all four limbs and moderate respiratory distress. Radiographic examination and computed tomography of the limbs showed palisade-like periosteal bone proliferation involving radius, ulna, femur, patella, tibia, fi bula, tarsus, metacarpal, metatarsal and digits, suggesting hypertrophic osteopathy. Radiographic examination and computed tomography of the lungs also showed a round mass well delimited localized in the right diaphragmatic lobe. A lobectomy of the right diaphragmatic lobe and partial lobectomy of accessory lobe were performed. A poorly differentiated clear squamous cell carcinoma was diagnosed by histological examination. An immune-panel of CK5/CK6, CK7, p63 and TTF-1 was used for immunophenotyping. Immunostaining was weakly positive for CK5/CK6 and negative to all others. Therefore, the diagnosis was poorly differentiated clear cell adenocarcinoma. The dog showed improvement in clinical signs seven days after surgery. One month postoperatively, radiographic examination of the limbs showed less intense periosteal reaction and initiation of bone remodeling. Discussion: Primary pulmonary tumors are considered very infrequent in small animals, but its true incidence rate is dif- fi cult to establish in animal populations. The histological origin of the tumor in the present case, as verifi ed in the literature, is not well established by histological analysis. In these situations, the immunohistochemistry panel may be useful. The modifi cation of the diagnosis between histological analysis and by immunohistochemistry, among other factors, might be due to transdifferentiation from one phenotype to another at various stages in the neoplastic process. The clear cell appearance observed in this case may be verifi ed in all types of carcinoma due to intracellular accumulation of glycogen, most of which is dissolved during the preparation of paraffi n sections. This uncommon neoplasm apparently did not infl uence the radiographic or tomographic fi ndings of the hypertrophic osteopathy in the present case. The frequency of metastases depends on the histological type of the tumor, being common in the pulmonary adenocarcinoma and usually to tracheobronchial lymph nodes and pulmonary parenchyma. Although in this case the imaging studies did not show metastases to other pulmonary lobes, the histological exams showed metastatic lesions that may be associated to the dog’s death after the surgery.

[Pulmonary microscopic tumor embolism syndrome]

HISTORY: A 76-year-old woman and a 62-year-old man were both referred to our clinic because of an unexplained weight loss, increasing dry cough and shortness of breath. INVESTIGATIONS: Investigations revealed an adenocarcinoma of the colon with retroperitoneal, mediastinal and supraclavicular lymph node metastasis and poorly differentiated carcinoma of the prostate with extensive bone metastases. During their hospital stay both patients developed increasing shortness of breath and clinical signs of right heart failure. Echocardiography confirmed severe pulmonary hypertension and dilatation of the right ventricle in both patients. Despite the high degree of clinical suspicion CT scans of the thorax could not demonstrate pulmonary embolism. DIAGNOSIS, TREATMENT AND COURSE: During the following days the patients condition deteriorated further and both patients' died from irreversible right heart failure. Both autopsies showed extensive metastatic adenocarcinoma with marked angiosis carcinomatosa of the lungs with numerous occlusions of small arteries and arterioles and resulting cor pulmonale. Thrombotic pulmonary embolism could not be detected. CONCLUSION: In patients with malignant neoplasms, especially adenocarcinomas, dyspnea and signs of increasing pulmonary artery pressure, the possibility of a microscopic pulmonary tumor embolism should be considered after exclusion of more usual causes especially thrombotic pulmonary embolism. In selected cases a cytologic examination of blood aspirated from a wedged pulmonary artery catheter can be performed to prove angiosis is carcinomatosa.

Exophytic mass of the gingiva as the first manifestation of metastatic pulmonary adenocarcinoma

BACKGROUND: Metastasis of a malignant tumor to the oral cavity is rare, but it can be the first manifestation of a primary tumor. METHODS: The clinicopathologic features of a gingival metastasis originating from lung adenocarcinoma in a female patient are described. A 57-year-old woman showed a rapidly growing, painless, exophytic mass in the left mandibular gingiva. The whole lesion was excised, and histologic and immunohistochemical analyses were performed. RESULTS: The histopathologic sections showed a proliferation of poorly differentiated spindle and pleomorphic cells. Because the differentiation between carcinoma and sarcoma of spindle cell tumors was difficult, additional immunohistochemical evaluation was performed. The intraoral healing after tumor removal was uneventful. The discrepancy between the histopathologic results and the clinical findings led to a thorough examination by the patient's physician. Finally, a biopsy of the lungs confirmed a poorly differentiated adenocarcinoma with multiple metastases, including the oral cavity. CONCLUSIONS: An exophytic lesion on the gingiva can be the first sign of metastatic adenocarcinoma to the oral mucosa. This case emphasizes that even apparently benign-looking gingival lesions in anamnestically healthy patients need to be examined histopathologically.