Analysis of breast cancer by small angle X-ray scattering (SAXS)

Small angle X-ray scattering (SAXS) images of normal breast tissue and benign and malignant breast tumour tissues, fixed in formalin, were measured at the momentum transfer range of 0.063 nm(-1) <= q (=4 pi sin(theta/2)/lambda) <= 2.720 nm(-1). Four intrinsic parameters were extracted from the scattering profiles (1D SAXS image reduced) and, from the combination of these parameters, another three parameters were also created. All parameters, intrinsic and derived, were subject to discriminant analysis, and it was verified that parameters such as the area of diffuse scatter at the momentum transfer range 0.50 <= q <= 0.56 nm(-1), the ratio between areas of fifth-order axial and third-order lateral peaks and third-order axial spacing provide the most significant information for diagnosis (p < 0.001). Thus, in this work it was verified that by combining these three parameters it was possible to classify human breast tissues as normal, benign lesion or malignant lesion with a sensitivity of 83% and a specificity of 100%.

Identification of Neoplasias of Breast Tissues Using a Powder Diffractometer

An investigation was carried out to study the potential use of the angular distribution of scattered photons by human breast samples for a rapid identification of neoplasias of breast tissues. This technique has possible applications as diagnostic aid for breast cancer. In this work, a commercial powder diffractometer was used to obtain the scattering profiles from breast tissues histopathologically classified as normal breast tissues, fibroadenomas (benign breast diseases) and carcinomas (malignant breast diseases), in the interval 0.02 angstrom(-1) < x < 0.62 angstrom(-1). The experimental methods and data corrections are discussed in detail, and they included background subtraction, polarization, self-attenuation and geometric effects. The validation of the experimental procedure was achieved through an analysis of water sample. The results showed that the scattering profile is a unique impression of each type of tissue, being correlated with their microscopic morphological features. Multivariate analysis was applied to these profiles in order to verify if the information carried by these scattering profiles allow the differentiation between normal, benign and malignant breast tissues. The statistical analysis results showed that a correct identification of 75% of the analyzed samples is accomplished. The values of sensibility and specificity of this method in correctly differentiating between normal and neoplastic samples were 95.6% and 82.3%, respectively, while the values for differentiation between benign and malignant neoplasias were 78.6% and 62.5%. These initial results indicate the feasible use of commercial powder diffractometer to provide a rapid diagnostic with a high sensitivity.

A perivascular epithelioid cell tumor of the stomach: An unsuspected diagnosis

Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasia and currently well recognized as a distinct entity with characteristic morphological, immunohistochemical and molecular findings. We report a case of PEComa arising in the antrum of a 71-year-old female with melena. The tumor, located predominantly in the submucosa as a well delimited nodule, measured 3.0 cm in diameter and was completely resected, with no evidence of the disease elsewhere. Histologically, it was composed predominantly of eosinophilic epithelioid cells arranged in small nests commonly related to variably sized vessels, with abundant extracellular material, moderate nuclear variation and discrete mitotic activity. No necrosis, angiolymphatic invasion or perineural infiltration was seen. Tumor cells were uniformly positive for vimentin, smooth muscle actin, desmin and melan A. Although unusual, PEComa should be considered in the differential diagnosis of gastric neoplasia with characteristic epithelioid and oncocytic features and prominent vasculature: (C) 2010 Baishideng. All rights reserved.

The Ultrastructural Study of Tumorigenic Cells Using Nanobiomarkers

Despite recent advances, patients with malignant brain tumors still have a poor prognosis. Glioblastoma (WHO grade 4 astrocytoma), the most malignant brain tumor, represents 50% of all astrocytomas, with a median survival rate of <1 year. It is, therefore, extremely important to search for new diagnostic and therapeutic approaches for patients with glioblastoma. This study describes the application of superparamagnetic nano-particles of iron oxide, as well as monoclonal antibodies, of immunophenotypic significance, conjoined to quantum dots for the ultrastructural assessment of glioblastoma cells. For this proposal, an immunophenotypic study by flow cytometry was carried out, followed by transmission electron microscopy analysis. The process of tumor cell labeling using nanoparticles can successfully contribute to the identification of tumorigenic cells and consequently for better understanding of glioblastoma genesis and recurrence. In addition, this method may help further studies in tumor imaging, diagnosis, and prognostic markers detection.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Vandetanib Plus Docetaxel/Prednisolone in Patients with Hormone-Refractory Prostate Cancer

Vandetanib (ZACTIMA(TM)) is a once-daily oral anticancer drug that selectively inhibits vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection signaling. This randomized (1: 1), double-blind study evaluated vandetanib (100mg/day) or placebo in combination with docetaxel (D; 75mg/m(2) every 3 weeks) and prednisolone (P; 2 x 5 mg/day) in 86 patients with metastatic hormone-refractory prostate cancer (mHRPC). The primary assessment was prostate-specific antigen (PSA) response (confirmed reduction of >= 50% from baseline) and a greater number of patients showed a PSA response with placebo + DP (67%) versus vandetanib + DP (40%); hazard ratio = 2.23 (one-sided 80% confidence limit = 2.90; one-sided p = 0.99). More patients experienced progression events (disease progression or death from any cause) with vandetanib + DP (65%) versus placebo + DP (60%); hazard ratio = 1.13 (one-sided 80% confidence limit = 1.44; one-sided p = 0.67). The overall incidence of adverse events was similar in both groups, although more patients experienced adverse events, leading to permanent discontinuation with vandetanib + DP (28%) versus placebo + DP (12%). However, the safety and tolerability profile for vandetanib was similar to that previously reported; adverse events that occurred more frequently in the vandetanib + DP arm were hypertension (14% vs. 2%), erythematous rash (14% vs. 2%), and exfoliative rash (12% vs. 2%). In this study of patients with mHRPC, vandetanib + DP did not demonstrate any efficacy benefit, compared with placebo + DP.

Mucin pattern reflects the origin of the adenocarcinoma in Barrett's esophagus: a retrospective clinical and laboratorial study

Background: Mucin immunoexpression in adenocarcinoma arising in Barrett's esophagus (BE) may indicate the carcinogenesis pathway. The aim of this study was to evaluate resected specimens of adenocarcinoma in BE for the pattern of mucins and to correlate to the histologic classification. Methods: Specimens were retrospectively collected from thirteen patients who underwent esophageal resection due to adenocarcinoma in BE. Sections were scored for the grade of intestinal metaplasia. The tissues were examined by immunohistochemistry for MUC2 and MUC5AC antibodies. Results: Eleven patients were men. The mean age was 61 years old (varied from 40 to 75 years old). The tumor size had a mean of 4.7 +/- 2.3 cm, and the extension of BE had a mean of 7.7 +/- 1.5 cm. Specialized epithelium with intestinal metaplasia was present in all adjacent mucosas. Immunohistochemistry for MUC2 showed immunoreactivity in goblet cells, while MUC5AC was extensively expressed in the columnar gastric cells, localizing to the surface epithelium and extending to a variable degree into the glandular structures in BE. Tumors were classified according to the mucins in gastric type in 7/13 (MUC5AC positive) and intestinal type in 4/13 (MUC2 positive). Two tumors did not express MUC2 or MUC5AC proteins. The pattern of mucin predominantly expressed in the adjacent epithelium was associated to the mucin expression profile in the tumors, p = 0.047. Conclusion: Barrett's esophagus adenocarcinoma shows either gastric or intestinal type pattern of mucin expression. The two types of tumors developed in Barrett's esophagus may reflect the original cell type involved in the malignant transformation.

Intrahepatic Glissonian Approach for Pure Laparoscopic Left Hemihepatectomy

Background: Recent advances in laparoscopic devices and experience with advanced techniques have increased the indications for laparoscopic liver. Aim: The aim of this work was to present a video with technical aspects of a pure laparoscopic left hemi-hepatectomy (segments 2, 3, and 4) by using the intrahepatic Glissonian approach and control of venous outflow without hilar dissection or the Pringle maneuver. Patient and Method: A 63-year-old woman with a 5-cm solitary liver metastasis was referred for treatment. Four trocars were used. The left lobe was pulled upward and the lesser omentum was divided, exposing Arantius' ligament. This ligament is a useful landmark for the identification of the main left Glissonian pedicle. A small anterior incision was made in front of the hilum, and a large clamp was introduced behind the Arantius' ligament toward the anterior incision, allowing control of the left main sheath. Ischemic discoloration of the left liver was achieved and marked with cautery. The vascular clamp was replaced by a stapler. If ischemic delineation was coincident with a previously marked area, the stapler was fired. The left hepatic vein was dissected and encircled. Parenchymal transection and vascular control of the hepatic veins were accomplished with a Harmonic scalpel and an endoscopic stapling device, as appropriate. All these steps were performed without the Pringle maneuver and without hand assistance. Results: Operative time was 220 minutes with minimum blood loss. Hospital stay was 4 days. Pathology showed free surgical margins. The patient is alive with no signs of recurrence 18 months after the operation. Conclusion: Totally laparoscopic left hemihepatectomy is safe and feasible in selected patients and should be considered for patients with benign or malignant liver neoplasms. The described technique, with the use of the intrahepatic Glissonian approach and control of venous outflow, may facilitate laparoscopic left hemihepatectomy by reducing the technical difficulties in pedicle control and may decrease bleeding during liver transection.

Activation of Neural and Pluripotent Stem Cell Signatures Correlates with Increased Malignancy in Human Glioma

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal-and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies.

Expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in medullary thyroid carcinoma: Prognostic implications

Background: Metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) participate in the degeneration of the extracellular matrix and are associated with carcinogenesis. MMP-2 is one of the main metalloproteinases active in neoplasia and is a marker of the malignant phenotype. Since the biological behavior of medullary thyroid carcinoma (MTC) varies widely, the present study was undertaken to determine if there is a correlation between the clinical evolution of MTC and the immunohistochemically detected expression of these enzymes in thyroid surgical specimens containing MTC. If so, their expression would be a novel indicator of the prognosis of MTC. Methods: Thirty-seven patients with MTC who had undergone thyroid surgery were followed for an average of 73 months. Immunohistochemical staining for metalloproteinase-related enzymes was performed in surgical paraffin blocks. The clinical status of the patients after surgery and at the end of the study period was characterized to determine correlations between these and the immunohistochemical markers. A value of p < 0.05 was considered statistically significant. Results: At the end of the study period, 15 patients (40.5%) were alive and without evidence of MTC, 17 (45.9%) had persistent MTC, and 5 (13.5%) had a relapse of their neoplasia. Four patients (10.8%) died during the course of the study. There was a significant correlation (p = 0.0005) between the immunohistochemical staining for MMP-2 and the clinical condition of the patients at the end of the study period, and a correlation between the state of apparent cure compared to persistence of MTC after thyroid surgery (p = 0.0207). No significant correlations were observed between either TIMP-2 expression or immune marking of metastatic lymph nodes and the clinical variables studied. Conclusion: Immunohistochemical expression of MMP-2 in thyroid surgical specimens from patients with MTC is a novel indicator of the prognosis of this cancer.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas

Background: Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas. Methods: Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting. Results: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue. Conclusion: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of HDAC mRNA in glioblastomas.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Hemangiossarcoma primário intrauterino em um macaco aranha de cara vermelha (Ateles paniscus)

Hemangiosarcoma is a common neoplasm in dogs and less frequently seen in cats. In nonhuman primates, this tumor is rarely reported. A 17 year-old female spider monkey (Ateles paniscus) was submitted an ultrasound exam due to gestation suspicion, which was seen a circular mass intra-uterine measuring 1.3 cm. New exams shown increase of the mass to 4.4 x 3.0 cm associated with a viable fetus. Was realized cesarean with ovariohysterectomy and excision of the mass; however the animal died in less than 24 hours after the surgery. In the necropsy, severe hemoabdomen was evidenced, although the surgical stumps were properly ligated and the complete sutures. Macroscopically, the uterine mass was soft, dark heterogeneous and measuring 5.0 cm in diameter. Histologically was visualized proliferation of spindle cells that form vascular channels replete of erythrocytes and some with thrombus, marked pleomorphism, nucleolus evident, binucleated cells and mitotic figures were rare. The immunohistochemistry (IHC) was performed, using streptavidin-biotin peroxidase technique (Dako Cytomation, USA) with the use of antibodies CD31 (clone JC/70A), CD34 (clone QB-END/10). The IHC showed a specific antigen-antibody reaction for CD31. According to localization, morphology and IHC, the present study reports a primary uterine hemangiosarcoma in a spider monkey that caused hemostatic abnormalities and consequent death of the animal.