Centrosome amplification in chondrosarcomas: A primary cell culture and cryopreserved tumor sample study.

The genetics background underlying the aggressiveness of chondrosarcoma (CS) is poorly understood. One possible cause of malignant transformation is chromosomal instability, which involves an error in mitotic segregation due to numerical and/or functional abnormalities of centrosomes. The present study aimed to evaluate centrosome amplification in cryopreserved samples of tumor tissue from patients with CS. An analysis was performed on 3 primary cultures of tumors from patients who underwent surgery between January 2012 and December 2012 at the Department of Orthopedics at the Barretos Cancer Hospital (Barretos, Brazil). Additionally, cryopreserved tumor specimens were analyzed from 10 patients. The data were assessed using immunocytochemistry and immunohistochemistry staining techniques with monoclonal antibody anti-γ-tubulin. A total of 4 samples of CS cultured cells were obtained from 3 patients. A recurrence of a histological grade III tumor was detected in a female patient with Ollier's syndrome. The other 2 cases were grade I and III. The incidence of centrosome amplification in the primary cultures ranged from 15-64% of the cells. Whereas control cultured fibroblasts showed baseline levels of 4% amplified cells. For the cryopreserved specimens, two independent observers analyzed each sample and counted the cells stained with γ-tubulin, verifying the percentage of affected cells to be a mean of 14%, with the number of clusters ranging between 0-6 per slide. In conclusion, centrosome amplification was found to be a consistent biological feature of CS and may underlie chromosomal instability in this tumor.

Clinicopathological correlation and prognostic significance of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common type of cancer and the fourth most frequent cause of cancer death. Literature indicates that vascular endothelial growth factor is a predominant angiogenic factor and that angiogenesis plays an important role in the progression of CRC. PATIENTS AND METHODS: The present series consisted of tissue samples obtained from 672 patients who had undergone large bowel resection between 2005 and 2010 at the Braga Hospital, Portugal. Archival paraffin-embedded CRC tissue and normal adjacent samples were used to build up tissue microarray blocks and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression was immunohistochemically assessed. RESULTS: We observed an overexpression of VEGF-C in CRC when tumour cells and normal-adjacent tissue were compared (p=0.004). In tumour samples, VEGF-C-positive cases were associated with VEGFR-3 expression (p=0.047). When assessing the correlation between VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expressions and the clinicopathological data, it was revealed that VEGF-A positive cases were associated with male gender (p=0.016) and well-differentiated tumours (p=0.001); VEGF-C with colon cancers (p=0.037), exophytic (p=0.048), moderately-differentiated (p=0.007) and T3/T4 (p=0.010) tumours; VEGFR-2 with invasive adenocarcinoma (p=0.007) and VEGFR-3 with the presence of hepatic metastasis (p=0.032). Overall survival curves for CRC were statistically significant for rectal cancer, VEGF-C expression and stage III (p=0.019) and VEGFR-3 expression and stage IV (p=0.047). CONCLUSION: Quantification of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression seems to provide valuable prognostic information in CRC and the correlation with clinicopathological data revealed an association with characteristics that contribute to progression, invasion and metastasis leading to poorer survival rates and prognosis.

Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia.

OBJECTIVE: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. METHODS: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). RESULTS: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. CONCLUSIONS: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Role of endoglin and VEGF family expression in colorectal cancer prognosis and anti-angiogenic therapies.

Colorectal cancer (CRC) is one of the cancer models and most of the carcinogenic steps are presently well understood. Therefore, successful preventive measures are currently used in medical practice. However, CRC is still an important public health problem as it is the third most common cancer and the fourth most frequent cause of cancer death worldwide. Nowadays, pathologic stage is a unique and well-recognized prognostic indicator, however, more accurate indicators of the biologic behavior of CRC are expected to improve the specificity of medical treatment. Angiogenesis plays an important role in the growth and progression of cancer but its role as a prognostic factor is still controversial. Probably the most important clinical implication of tumor angiogenesis is the development of anti-angiogenic therapy. The goal of this review is to critically evaluate the role of angiogenic markers, assessed by either endoglin-related microvessel density or expression of vascular endothelial growth factor family members in the CRC setting and discuss the role of these angiogenic markers in anti-angiogenic therapies.

Analysis of Survival Rates of Patients Diagnosed With Incipient Esophagic Cancer.

BACKGROUND: Esophagic cancer incidence is extremely variable worldwide. Also, the global survival rate has not oscillated significantly since last decade. Most of the worse prognoses are found among patients with advanced stages. Despite that, around 10% of cases occur in patients with initial stage, which strongly associate these patients with unfavorable prognosis. We sought to analyze the impact of time free of disease and global survival rates of patients with initial stage of esophagic cancer. METHODS: We studied 18 patients with initial stage of esophagic cancer (stage 0 and I), examined and treated at Hospital de Cancer de Barretos between 1990 and 2005. RESULTS: The vast majority of patients were male (83.3%) with age up to 49 yarest old (77.8%), squamous cell carcinoma (SCC) (88.9%) and stage I (83.3%). Most of them were smoker (60.0%) and etilist (62.5%). There were 38.9% of the patients with comorbities like dysphagia and epigastralgia correlated to other pathological conditions. We found free disease rates of 100% and 82.5%, respectively for 12 and 36 months. The significant prognostic evidence was the age, epigastralgia symptoms and chemotherapy. From 18 patients, 6 passed away during the period of 36 months follow up due to cancer consequences. The probabilities of global survival were 76.7% and 64.4% after 12 and 36 months, respectively, and none of the analyzed variables influenced in theses rates. CONCLUSIONS: Our data ratifies those from previous reported. The global survival rates were worse than reported by literature, maybe in consequence of the poor clinical condition of many patients which limited the option for more aggressive therapy.

MET Is highly expressed in advanced stages of colorectal cancer and indicates worse prognosis and mortality.

UNLABELLED: The aim of the present study was to evaluate by immunohistochemistry the prognostic meaning of the tumor marker MET (hepatocyte growth factor) in patients submitted to surgical resection due to primary colorectal adenocarcinoma. PATIENTS AND METHODS: A retrospective study was carried out that included 286 consecutive patients with colorectal adenocarcinoma, submitted to surgical resection at Barretos Cancer Hospital, from 1993 to 2002. The histopathological expression of the MET tumor marker was evaluated using an anti-protein monoclonal antibody against MET by the streptavidin-biotin-peroxidase technique. The expression of the tumor marker was semi-quantitative, and the slide samples were independently analyzed by three pathologists unaware of patient clinical and histopathological data. RESULTS: The tumor marker expression was positive in 236 (79%) out of a total of 286 patients. This expression was statistically significantly different between stages I and IV (p=0.004), for overall survival (p=0.009), and for cancer-related mortality rates (p=0.022). However, no association between the tumor marker and recurrence (p=0.89) or disease-free interval (p=0.91) was observed. CONCLUSION: MET has shown significant expression at advanced stages of the disease, as well as for overall survival and cancer-related mortality rates demonstrating to be a valuable marker for poor prognosis in colorectal cancer patients.

Liquid-based cytology in DNA-based molecular research: viability and potential application.

OBJECTIVE: To evaluate the effectiveness of UCM and PreservCyt liquid media in molecular analysis using exfoliated oral cells and analyze Tp53 codon 72 extracted from these cells. STUDY DESIGN: Exfoliated oral cells were placed in UCM and PreservCyt preservation media. DNA was isolated by proteinase K/phenol-chloroform protocol. Quantity and quality was measured by Nanodrop 1000. To evaluate the applicability of the isolated DNA to molecular techniques, DNA from oral cells was used to analyze Tp53 codon 72 polymorphism and GADPH genes. RESULTS: DNA was successfully isolated from all samples, with an average concentration of 56.52 +/- 34.68 ng/microL for UCM and 42.13 +/- 25.50 ng/microL for PreservCyt. Average 260/280 ratios were 1.83 +/- 0.06 for UCM and 1.74 +/- 0.11 for PreserveCyt. Analysis of Tp53 codon 72 by polymerase chain reaction-restriction fragment length polymorphism was performed for all samples, with 4% of Pro/Pro, 60% of Arg/Arg and 36% of Arg/Pro genotype. In the analysis of GADPH multiplex polymerase chain reaction, fragments varying from 100 to 400 bp were obtained from all samples. CONCLUSION: Cytologic liquid media are suitable for preserving cell samples for posterior DNA isolation and molecular analysis and could become reliable research tools.

Immunohistochemical expression and distribution of VEGFR-3 in malignant mesothelioma.

Homogeneity of mesothelial and lymphatic endothelial cells, express some markers that are presumed to be exclusive of the endothelium was recently reported. This similarity is important to improve the diagnosis and prognosis of malignant mesothelioma (MM). Additionally, some of these markers provide the rationale for specific molecular-targeted novel therapies aimed at MM, an aggressive malignant neoplasm, with an usually dismal prognosis. The goal of our study was to determine the prevalence and expression pattern of VEGF receptor-3 (VEGFR-3) immunoreactivity in MM and whether this immunoreactivity occurs in different phenotypes of this neoplasm. Formalin-fixed and paraffin-embedded samples from 29 MM cases and 5 metastatic carcinomas were immuno-stained for VEGFR-3 according to the streptavidin-biotin-peroxidase complex technique using a primary antibody (Zymed Laboratories, CA, USA) diluted at 1:200. Lymphatic vessels (LV) were outlined mainly in the peripheral area surrounding the neoplasms. Blood vessels were only rarely positive for VEGFR-3 in a pattern easily distinguishable from LV. In 25 out of 29 cases (86.2%) LV were strongly positive for VEGFR-3: 14 cases (48.2%) exhibited positive VEGFR-3 reactivity in malignant cells. Epitheliod MM showed a moderate to intense VEGFR-3 positive reaction in LV from 8 out of 19 cases. Among the other histological subtypes, a positive VEGFR-3 reaction was noted in malignant cells from two cases of transitional and one case of pleomorphic MM. Malignant cells from two out of three biphasic and one out of three sarcomatoid MM were also positive for VEGFR-3. Interestingly, one case of the multicystic subtype was negative for VEGFR-3 in malignant cells and faintly positive in an occasional LV. All cases of metastatic carcinoma were negative for VEGFR-3 in the neoplastic cells. In conclusion, VEGFR-3 was expressed in malignant cells from different subtypes of MM, reinforcing the putative role of this marker as a potential therapeutic target in this group of neoplasia.

Cyclooxygenase-2 and epidermal growth factor receptor expressions in different histological subtypes of cervical carcinomas.

This study was designed to evaluate the significance of cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) expression in a series of cervical adenocarcinoma (AC), cervical adenosquamous carcinoma (ASC), and cervical squamous cell carcinoma (SCC). One hundred thirty cases of cervical carcinoma (30 ASC, 50 AC, and 50 SCC) were analyzed for COX-2 and EGFR expressions using specific primary antibodies. Samples were scored semiquantitatively as follows: (-), 0% of immunoreactive cells; (+), <5% of immunoreactive cells; (++), 5% to 50% of immunoreactive cells; and (+++), >50% of immunoreactive cells. The COX-2 expression was more frequently positive than EGFR in all cervical cancers studied. The COX-2 expression was also more prominent in AC than in ASC (P = 0.003). Expression of either COX-2 or EGFR was significantly different when comparing SCC with AC (P < 0.001 and P = 0.04, respectively). There was no significant correlation between COX-2 and EGFR expressions and age at diagnosis, recurrence, distant metastasis, and/or positive status of regional lymph nodes, neither between COX-2 and EGFR coexpression and the clinical data analyzed. Nevertheless, our data support that there are significant differences between EGFR and COX-2 expressions in the 3 different histogenetic types of cervical cancer. Also, in terms of therapeutic strategies, our data can be valuable in the selection of patients eligible to receive specific EGFR/COX-2-targeted therapy.

Screening for cervical cancer in high-risk populations: DNA pap test or Hybrid Capture II test alone?

This study was designed to evaluate whether Hybrid Capture II (HC2) test alone refer women to colposcopy as appropriately as DNA Papanicolaou (Pap) test, in the context of a high-risk group of women using the recently validated DNACitoliq LBC system. Women with suspected cervical disease were included in this cross-sectional study at a tertiary center in São Paulo, Brazil, for further workup. All women had cervical material collected for LBC and HC2 for high-risk human papillomavirus (hrHPV)-DNA test. Irrespective of cytology and HC2 results, colposcopy, and cervical biopsy when applicable, was systematically performed. All tests were performed blindly. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of both methods were computed in relation to histology. A total of 1,080 women were included: 36.4% (393/1080) had ACUS+, 10.2% (110/1080) were high-grade squamous intraepithelial lesions (HSIL) or cancer. Mean age was 33.5 years. All women underwent colposcopy, and cervical biopsies were performed in 38.4% (415/1080): 33% (137/415) of the biopsies were negative, 14.4% (155/415) were low-grade squamous intraepithelial lesions (LSIL), 10.7% (116/415) were HSIL, and 0.6% (7/415) were cancer. HC2 sensitivity to diagnose biopsy-proven HSIL was 100%. Because all HSIL cases had a positive HC2 test, sensitivity could not be improved by adding LBC. Specificity and positive and negative predictive values of DNA Pap were not significantly different from HC2 test alone when considering LSIL+ histology as "gold standard" and HSIL+ histology. As a screening strategy for women with high-risk for cervical cancer, DNA Pap test does not seem to add substantially to HC2 alone in terms of appropriately referring to colposcopy.

Molecular strategies for identifying human papillomavirus infection in routinely processed samples: focus on paraffin sections.

OBJECTIVE: To optimize the protocols of human papillomavirus (HPV) detection in clinical samples, we used polymerase chain reaction (PCR)-based techniques in paraffin-embedded tissue sections and compared the results with those obtained with PCR and Hybrid Capture II (HC2) performed in liquid-based cytology (LBC) preservation medium. MATERIALS AND METHODS: Forty-five consecutive cervical biopsy specimens were taken from women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, São Paulo, Brazil, during 2003 and 2004. The biopsy specimens were analyzed for HPV by a modified GP5+/GP6+ PCR protocol, and the results were compared with those obtained by PGMY PCR and HC2 in samples collected in LBC preservation medium. RESULTS: beta-Globin was detected in 100% of the multiplex PCR system from LBC samples and 66.7% with PCO4+/PCO3+ PCR in biopsy specimens. Of the three methods, PGMY PCR system and HC2 were equally effective in detecting HPV; both detected 13 cases in 45 samples (28.9%). The GP5+/GP6+ PCR applied in biopsy specimens showed a 20% HPV detection rate (9/45). CONCLUSIONS: Our PCR protocols worked reasonably well and allowed us to compare the three molecular methods with histological and cytological findings. The reproducibility of the results makes the technique applicable in archival materials.

Infection with Mycobacterium ulcerans induces persistent inflammatory responses in mice.

Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections with Mycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouse M. ulcerans induces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not from M. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.

Is agNOR and DNA ploidy analysis useful for evaluating thyroid neoplasms?

OBJECTIVE: To correlate the subjective AgNOR counting method and DNA content with histologic diagnoses of thyroid cancer and invasion. STUDY DESIGN: Eighty-one consecutive cases of thyroid carcinoma were selected for DNA and AgNOR analysis. The diagnoses were: papillary carcinoma (n = 40), follicular carcinoma (n = 31), Hürthle cell adenocarcinoma (n = 4), and undifferentiated carcinoma (n = 6). Seven normal thyroids were used as controls. DNA quantitative measurement was performed with Vidas 2.0 software (Kontron Bildanalyse, Munich, Germany) connected to an MPM 210 photometer microscope (Carl Zeiss, Oberkochen, Germany). The DNA index was obtained using histograms. Counting the NORs was performed by subjectively counting the NORs in 200 malignant cells. RESULTS: DNA ploidy analysis showed all Hürthle cell adenocarcinomas, 21 (67%)follicular tumors, 23 (57%) papillary tumors and 4 (67%) undifferentiated carcinomas to be aneuploid. DNA analysis correlated with histologic type of the tumor (p = 0.032). There was no statistical significance to the AgNOR counting variables studied. Statistical analysis showed correlation between ploidy and histologic diagnosis, but not AgNOR counting, to have prognostic value. CONCLUSION: DNA ploidy is more useful than subjective counting of NORs as an adjunct method for thyroid lesion analysis.

Desmoplastic small round cell tumour: Cytological and immunocytochemical features.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive neoplasm. The cytological diagnosis of these tumors can be difficult because they show morphological features quite similar to other small round blue cells tumors. We described four cases of DSRCT with cytological sampling: one obtained by fine needle aspiration biopsy (FNAB) and three from serous effusions. The corresponding immunocytochemical panel was also reviewed. METHODS: Papanicolaou stained samples from FNAB and effusions were morphologically described. Immunoreaction with WT1 antibody was performed in all cytological samples. An immunohistochemical panel including the following antibodies was performed in the corresponding biopsies: 34BE12, AE1/AE3, Chromogranin A, CK20, CK7, CK8, Desmin, EMA, NSE, Vimentin and WT1. RESULTS: The smears showed high cellularity with minor size alteration. Nuclei were round to oval, some of them with inconspicuous nucleoli. Tumor cells are clustered, showing rosette-like feature. Tumor cells in effusions and FNA were positive to WT1 in 3 of 4 cytology specimens (2 out 3 effusions and one FNA). Immunohistochemical reactions for vimentin, NSE, AE1/AE3 and WT1 were positive in all cases in tissue sections. CONCLUSION: The use of an adjunct immunocytochemical panel coupled with the cytomorphological characteristics allows the diagnosis of DSRCT in cytological specimens.

High-risk HPV detection by Hybrid Capture and ploidy determination by a computer-assisted system in cervical biopsies.

OBJECTIVE: To correlate high-risk HPV (hrHPV) detection by Hybrid Capture II (HC2) (Digene, Gaithersburg, Maryland, U.S.A.) with DNA content (ploidy) of cervical biopsies analyzed by a computer-assisted system. STUDY DESIGN: Cervical biopsies from 54 women examined at Leonor Mendes de Barros Hospital, São Paulo, as part of the Latin American Screening study during 2002--2003, were tested for hrHPV with HC2. All patients had been referred for colposcopic examination due to an abnormal cervical cytology. The final diagnosis included 30 cervicitis, 14 cervical intraepithelial neoplasia (CIN) 1, 5 CIN 2, 4 CIN 3 and 1 squamous cell carcinoma (SCC). Five-micrometer sections of each biopsy were stained with Feulgen-tionine and evaluated with the CAS 200 System (Becton Dickinson, U.S.A.), using the 3.0 software (version 8.1) of the DNA Quantitative Measurement Program (Becton Dickinson). Ploidy was evaluated from histograms obtained by analyzing atypical nuclei. RESULTS: Of the 30 cervicitis cases, 28 (93.3%) were diploid, and hrHPV was detected in 8 (28.5%) of the cases. Two tetraploid cervicitis lesions were observed, 1 positive and 1 negative for hrHPV. Among the CIN 1 lesions, 8 (57.1%) were diploid and 6 (42.8%) aneuploid. Of the latter, 4 (66.6%) were negative and 2 (33.3%) positive for hrHPV. Of the 5 CIN 2 lesions, 2 were diploid, 2 aneuploid and 1 tetraploid; all were positive for hrHPV. All CIN 3 lesions and the SCC proved to be aneuploid and positive for hrHPV. CONCLUSION: The data suggest that the majority of cervicitis and CIN 1 lesions are diploid and negativef or hrHPV. This is in sharp contrast to high grade CIN 2-3 lesions, all of which were positive for hrHPV in this study and also aneuploid, consistent with their progressive potential.