THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.

Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity

Immunohistochemical analysis of E-cadherin has changed the way lobular neoplasia is perceived. It has helped to classify difficult cases of carcinoma in situ with indeterminate features and led to the identification of new variants of lobular carcinoma. Pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS), recently described variants of invasive and in situ classic lobular carcinoma, are reported to be associated with more aggressive clinical behaviour. Although PLC/PLCIS show morphological features of classic lobular neoplasia and lack E-cadherin expression, it is still unclear whether these lesions evolve through the same genetic pathway as lobular carcinomas or are high-grade ductal neoplasms that have lost E-cadherin. Here we have analysed a case of extensive PLCIS and invasive PLC associated with areas of E-cadherin-negative carcinoma in situ with indeterminate features, using immunohistochemistry, chromogenic in situ hybridization, high-resolution comparative genomic hybridization (CGH) and array-based CGH. We observed that all lesions lacked E-cadherin and beta-catenin and showed gain of 1q and loss of 16q, features that are typical of lobular carcinomas but are not seen in high-grade ductal lesions. In addition, amplifications of c-myc and HER2 were detected in the pleomorphic components, which may account for the high-grade features in this case and the reported aggressive clinical behaviour of these lesions. Taken together, these data suggest that at least some PLCs may evolve from the same precursor or through the same genetic pathway as classic lobular carcinomas. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Análise da imunoexpressão da podoplanina e da triptase em carcinoma epidermóide de língua e sua relação com parâmetros clinicopatológicos

Oral tongue squamous cell carcinoma (OTSCC) has an aggressive biological behavior, with a high propensity for the development of lymph node metastases. In this context, lymphangiogenesis is considered an important phenomenon for the spread of tumor cells and may be influenced by microenvironmental stimuli. Mast cells have been implicated in tumor progression, although their influence in the formation of lymphatic vessels is not well established. The aim of this study was to analyze, in a case series of OTSCC (n=50), possible correlations between lymphatic vessel density (LVD), mast cell count and clinicopathological features, including tumor-node-metastasis (TNM) stage, histological grade of malignancy (Bryne, 1998), and nodal metastasis. LVD was established as the mean number of lymphatic vessels immunostained by anti-podoplanin (D2-40) antibody, identified in five microscopic fields (200x). For the analysis of mast cells, tryptase-immunoreactive cells were quantified in five fields (400x). Both immunostainings were analyzed in the tumor center and invasion front. Intratumoral lymphatic density (ILD) was higher in cases in advanced clinical stages (III-IV), compared to those in initial stages (I-II), as well as in metastatic cases in respect of non-metastatic (p<0,05). There were no statistically significant differences between low-grade and high-grade malignancy cases with respect to ILD (p>0,05). Peritumoral lymphatic density (PLD) and mast cell counts showed no significant relations with any of the clinicopathological parameters evaluated (p>0,05). Also there were no significant correlations between LVD and mast cell counts, whether in intratumoral (r = -0,004; p=0,977) or peritumoral region (r = -0,154; p=0,285). The results of the present study suggest that intratumoral lymphatic vessels may contribute in part to the progression of OTSCC, although PLD may be insufficient to justify differences in biological behavior. This supports the hypothesis of involvement of other mechanisms in metastatic spread of malignant cells, which could complement the effects of lymphangiogenesis. Although mast cells perform several pro- and antitumoral functions, they do not appear to directly influence aggressiveness of OTSCC. In addition, the quantity of these cells may not be essential for lymphatic vessel formation.

Expressão imuno-histoquímica do ativador de plasminogênio do tipo uroquinase e seu receptor em carcinoma epidermoide de língua oral e sua relação com parâmetros clínico-patológicos

Squamous cell carcinoma (SCC ) is the most common malignancy of the oral cavity (OSCC), with a high mortality rate. Due to this, the discovery of biomarkers that facilitate the understanding of the biological behavior of the tumor and improve treatment is necessary. Urokinase type plasminogen activator (uPA) and its receptor, uPAR, are responsible for the proteolysis of structures of the basement membrana and extracellular matrix, facilitating tumor invasion. This study aims to assess the immuno expression of these proteins in 46 cases of squamous cell carcinoma of the oral tongue (OTSCC). These results were related to the presence of metastasis, clinical TNM staging, locoregional recurrence, outcome of the lesion and histological grading. Immunostaining of each case was evaluated semiquantitatively, in the front of invasion and center of the tumor, in which scores were assigned: 0 (0% of positive cells), 1 (1-10% of positive cells), 2 (11 -50% positive cells) and 3 (more than 50% positive cells). The expression of uPA was observed in 93.5% (n=43) of the cases in the front of invasion, with predominance of score 2 (n=16; 34.8%) and in 67.9% (n=31) of the cases in the center of the tumor, with predominance of score 1 (n=15; 32.6%). Overall, the immunoexpression of uPA was not associated with clinical parameters. Regarding the malignant histological grading, a higher expression of uPA was observed in cases of high-grade malignancy comp ared to low-grade malignancy (p=0.05). Regarding the morphological parameters, increased expression of uPA was observed in the worst mode of invasion (p=0.03 ). The expression of uPAR was observed in 73.9% of cases in the front of invasion, with a predominance of score 1 (n=21; 45.6 %), and in 47.5% (n=21) of the cases in the center of the tumor, with a predominance of score 0 (n=25; 54.4%). Although no statistical differences were observed in relation to lymph node metastasis, clinical TNM staging, outcome, and histological grading, there was a higher expression of uPAR in cases with locoregional recurrence (p=0.04). Regarding the tumor intra -localization, it was observed an increased expression of uPA and uPAR at the front of invasion in relation to the center of the tumor (p<0.001). Regarding the correlation between uPA and uPAR, there was no statistical sign ificance. Based on these results, it is suggested that uPA and uPAR are involved in the progression of CELO, mainly in the deeper region of the tumor.

Expressão imuno-histoquímica do ativador de plasminogênio do tipo uroquinase e seu receptor em carcinoma epidermoide de língua oral e sua relação com parâmetros clínico-patológicos

Squamous cell carcinoma (SCC ) is the most common malignancy of the oral cavity (OSCC), with a high mortality rate. Due to this, the discovery of biomarkers that facilitate the understanding of the biological behavior of the tumor and improve treatment is necessary. Urokinase type plasminogen activator (uPA) and its receptor, uPAR, are responsible for the proteolysis of structures of the basement membrana and extracellular matrix, facilitating tumor invasion. This study aims to assess the immuno expression of these proteins in 46 cases of squamous cell carcinoma of the oral tongue (OTSCC). These results were related to the presence of metastasis, clinical TNM staging, locoregional recurrence, outcome of the lesion and histological grading. Immunostaining of each case was evaluated semiquantitatively, in the front of invasion and center of the tumor, in which scores were assigned: 0 (0% of positive cells), 1 (1-10% of positive cells), 2 (11 -50% positive cells) and 3 (more than 50% positive cells). The expression of uPA was observed in 93.5% (n=43) of the cases in the front of invasion, with predominance of score 2 (n=16; 34.8%) and in 67.9% (n=31) of the cases in the center of the tumor, with predominance of score 1 (n=15; 32.6%). Overall, the immunoexpression of uPA was not associated with clinical parameters. Regarding the malignant histological grading, a higher expression of uPA was observed in cases of high-grade malignancy comp ared to low-grade malignancy (p=0.05). Regarding the morphological parameters, increased expression of uPA was observed in the worst mode of invasion (p=0.03 ). The expression of uPAR was observed in 73.9% of cases in the front of invasion, with a predominance of score 1 (n=21; 45.6 %), and in 47.5% (n=21) of the cases in the center of the tumor, with a predominance of score 0 (n=25; 54.4%). Although no statistical differences were observed in relation to lymph node metastasis, clinical TNM staging, outcome, and histological grading, there was a higher expression of uPAR in cases with locoregional recurrence (p=0.04). Regarding the tumor intra -localization, it was observed an increased expression of uPA and uPAR at the front of invasion in relation to the center of the tumor (p<0.001). Regarding the correlation between uPA and uPAR, there was no statistical sign ificance. Based on these results, it is suggested that uPA and uPAR are involved in the progression of CELO, mainly in the deeper region of the tumor.

Association of the XPD and XRCC3 gene polymorphisms with oral squamous cell carcinoma in a Northeastern Brazilian population: A pilot study

Objective to evaluate the association between XPD and XRCC3 polymorphisms and oral squamous cell carcinoma (OSCC). Design the sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). Genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results XPD-Lys/Gln was more common in IFH (n = 28; 70%) than in OSCC (n = 24; 44.4%) (OR: 0.3; p < 0.05). XPD-Gln was more frequent in high-grade lesions (0.48) than in low-grade lesions (0.21) (OR: 3.4; p < 0.05). The Gln/Gln genotype was associated with III and IV clinical stages (OR: 0.07; p < 0.05). XRCC3-Met was more frequent in OSCC (0.49) than in IFH (0.35) (OR: 2.6; p < 0.05). The Met/Met genotype was associated with the presence of metastases (OR: 8.1; p < 0.05) and with III and IV clinical stages (OR: 0.07; p < 0.05). Conclusions in this sample, the frequency of XPD-Gln in IFH suggests that this variant may protect against OSCC. The presence of the XRCC3-Met allele seems to contribute to the development of OSCC, metastases and more advanced stages in these lesions.

Association of the XPD and XRCC3 gene polymorphisms with oral squamous cell carcinoma in a Northeastern Brazilian population: A pilot study

Objective to evaluate the association between XPD and XRCC3 polymorphisms and oral squamous cell carcinoma (OSCC). Design the sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). Genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results XPD-Lys/Gln was more common in IFH (n = 28; 70%) than in OSCC (n = 24; 44.4%) (OR: 0.3; p < 0.05). XPD-Gln was more frequent in high-grade lesions (0.48) than in low-grade lesions (0.21) (OR: 3.4; p < 0.05). The Gln/Gln genotype was associated with III and IV clinical stages (OR: 0.07; p < 0.05). XRCC3-Met was more frequent in OSCC (0.49) than in IFH (0.35) (OR: 2.6; p < 0.05). The Met/Met genotype was associated with the presence of metastases (OR: 8.1; p < 0.05) and with III and IV clinical stages (OR: 0.07; p < 0.05). Conclusions in this sample, the frequency of XPD-Gln in IFH suggests that this variant may protect against OSCC. The presence of the XRCC3-Met allele seems to contribute to the development of OSCC, metastases and more advanced stages in these lesions.

Breast Carcinoma with Choriocarcinomatous Differentiation; a Rare Case

Objectives: Breast carcinoma with choriocarcinomatous differentiation is a rare entity, generally presenting with high-grade disease and an aggressive clinical course with overall survival of less than a year Case: A 69-year-old woman with a diagnosis of pT1N0M0 invasive ductal carcinoma with choriocarcinomatous differentiation received six cycles of adjuvant chemotherapy and is still disease free on the 23rd month after diagnosis, showing a better prognosis than most other cases reported in the literature. Conclusion: The reason for the poor prognosis for this type of breast carcinoma remains unclear. Standard chemotherapeutic agents administered for breast carcinoma may be used for choriocarcinomatous differentiation.

Contribution to the study of prognostic factors in canine oral squamous cell carcinoma

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Clínica

Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer

Paraffin sections (n = 168, 27 benign, 16 low malignant potential [LMP] and 125 malignant tumours) from epithelial ovarian tumours were evaluated immunohistochemically for expression of retinoblastoma gene product (pRB) and p53 protein, and the relationship among pRB, p53 and cyclin-dependent kinase inhibitor 2 (CDKN2) gene product p16INK4A (p16) was analysed, following our previous study of p16. Forty-one percent of the benign, 50% of the LMP and most (71%) of the malignant tumours showed high pRB expression. High expression of pRB (>50% pRB-positive cells) significantly correlated with non-mucinous histological subtypes. Reduced pRB expression, substage and residual disease were significant predictors for poor prognosis in stage I patients. All the benign and most of the LMP (81%) tumours were in either the p53-negative or low p53-positive category, but nearly half of the malignant tumours had high p53 expression. High p53 accumulation was found in non-mucinous, high grade and late stage tumours. For well-differentiated carcinomas, high p53 expression was a predictor of poor prognosis. However, even though high p53 expression was not associated with histological subtype, stage or the presence of residual disease, high p53 expression was not an independent predictor when all clinical parameters were combined. For all ovarian cancers, a close correlation was found between high p53 and high p16 expression. The relationship between the expression of pRB and p16 depended on tumour stage. In stage I tumours, high pRB was associated with low p16 reactivity. On the other hand, most advanced tumours showed both high pRB and high p16 reactivity. Int. J. Cancer 74:407–415, 1997. © 1997 Wiley-Liss, Inc.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice

Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

The role of goal importance in predicting university students’ high academic performance

We examined goal importance, focusing on high, but not exclusive priority goals, in the theory of planned behaviour (TPB) to predict students’ academic performance. At the beginning of semester, students in a psychology subject (N = 197) completed TPB and goal importance items for achieving a high grade. Regression analyses revealed partial support for the TPB. Perceived behavioural control, but not attitude or subjective norm, significantly predicted intention, with intention predicting final grade. Goal importance significantly predicted intention, but not final grade, indicating that perceiving a performance goal as highly, but not necessarily exclusively, important impacts on students’ achievement intentions.

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1−/−/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1−/−/PyMT mice, compared with Adamts1+/+/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1−/−/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1−/− tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45+ leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1−/− tumors, compared with Adamts1+/+ tumors. This finding is supported by significantly elevated IL-12+ cell numbers in Adamts1−/− tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.

Runs of homozygosity and a cluster of vulvar cancer in young Australian Aboriginal women

Objective A cluster of vulvar cancer exists in young Aboriginal women living in remote communities in Arnhem Land, Australia. A genetic case–control study was undertaken involving 30 cases of invasive vulvar cancer and its precursor lesion, high-grade vulvar intraepithelial neoplasia (VIN), and 61 controls, matched for age and community of residence. It was hypothesized that this small, isolated population may exhibit increased autozygosity, implicating recessive effects as a possible mechanism for increased susceptibility to vulvar cancer. Methods Genotyping data from saliva samples were used to identify runs of homozygosity (ROH) in order to calculate estimates of genome-wide homozygosity. Results No evidence of an effect of genome-wide homozygosity on vulvar cancer and VIN in East Arnhem women was found, nor was any individual ROH found to be significantly associated with case status. This study found further evidence supporting an association between previous diagnosis of CIN and diagnosis of vulvar cancer or VIN, but found no association with any other medical history variable. Conclusions These findings do not eliminate the possibility of genetic risk factors being involved in this cancer cluster, but rather suggest that alternative analytical strategies and genetic models should be explored.

High P-T granulite relicts from the Imjingang belt, South Korea:Tectonic significance

This study presents a detailed description on crustal metamorphic signatures of garnet-clinopyroxene-quartz-rutile-bearing high P-T granulites, Samgot unit, Imajingang belt, northwestern Korean Peninsula that formed during Permo-Triassic regional metamorphism related to the amalgamation of East Asian continental fragments. Lenses and blocks of high P-T granulites and garnet-bearing leucosomes occur within mafic metamorphic rocks (mainly amphibolites). The mafic blocks comprise relicts of granoblastic garnet and clinopyroxene with medium-grained quartz and rutile. These relict mineral assemblages are confined to local micro-domains and constitute remnants of peak metamorphism. Plagioclase and amphibole form only as retrograde phases in medium ton coarse-grained moats that rim grain boundaries between relict peak mineral assemblages. This microstructure represents the reaction between garnet, clinopyroxene, quartz and rutile in the presence of melt to form amphibole, plagioclase and titanite with minor biotite. The leucosome domains consist of euhedral garnets within the quartz-K feldspar-plagioclase (granitic) matrix, probably representing peritectic garnet growth along with melting. The rare earth element (REE) composition of minerals also support the peritectic garnet growth with a positive Eu/Eu* (positive Eu anomaly), while the relict garnet shows a slight negative anomaly typical for high-grade granulites. The peak-metamorphic conditions calculated from thermodynamic modeling and compositional isopleths indicate a temperature around c. 900 degrees C at a pressure around c. 20 kbar. The present P-T path indicates a clear multi-stage decompression history with initial decompression and cooling followed by a stage of decompression during hydration possibly during Late Triassic exhumation. The results from this study together with the presence of eclogites from the Hongsung area suggest that the Imjingang area and the western Gyeonggi massif likely resided at crustal levels deeper than those of the eastern and southern part of the Gyeonggi massif. (C) 2009 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.