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Fresh Tissue (fresh + tissue)
Selected AbstractsQuantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissueTHE PROSTATE, Issue 8 2009Thomas J. Walton Abstract BACKGROUND Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ER,) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. METHODS Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ER,), estrogen receptor alpha (ER,), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann,Whitney U -test. Correlation coefficients were analyzed using Spearman's test. RESULTS Significant positive correlations were seen when AR and AR-dependent PSA, and ER, and ER,-dependent PGR were compared, indicating a representative population of RNA transcripts. ER, gene expression was significantly over-expressed in the cancer group compared with benign controls (P,<,0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P,<,0.05). There were no significant differences in AR, ER, or PSA expression between the groups. This study represents the first to show an upregulation of ER, gene expression in laser microdissected prostate cancer specimens. CONCLUSIONS In concert with recent studies the findings suggest differential production of ER, splice variants, which may play important roles in the genesis of prostate cancer. Prostate 69: 810,819, 2009. © 2009 Wiley-Liss, Inc. [source] ,-Methylacyl-CoA racemase (AMACR) in fine-needle aspiration specimens of prostate lesionsDIAGNOSTIC CYTOPATHOLOGY, Issue 11 2009Gordana Kai, M.D. Abstract The elevated expression of P504S gene and its product ,-methylacyl-CoA racemase (AMACR) can serve as a molecular marker for prostate cancer. The goal of this study is to investigate P504S/AMACR expression in fine-needle aspiration smears and correlate it with cytological diagnosis. Immunocytochemistry was performed in 35 patients with morphological diagnosis of prostate carcinoma (n = 16), atypia (n = 15), and benign hyperplasia (n = 4). Among 16 malignant cases there were two low-grade, eight intermediate, and six high-grade prostate carcinomas. Cytoplasmic positivity is analyzed qualitatively as predominantly diffuse or focal and quantitatively as <5%, 5,50%, and >50% of cells. Benign cases showed no P504S/AMACR expression. Positive staining was recorded in 75% of malignant cases, but in the majority of them it was weak and focal or diffuse and in a small amount of cells. The most intensive staining was seen in low-grade carcinomas and some atypical cases. This observation indicates a correlation between P504S/AMACR expression and differentiation of cells. P504S/AMACR staining might be of great value in cytodiagnosis of prostate lesions as well as an example of the characterization of cells at the molecular level using fresh tissue obtained by fine-needle aspiration. Diagn. Cytopathol. 2009. © 2009 Wiley-Liss, Inc. [source] Fine needle aspiration biopsy of epithelioid hemangioendothelioma of the oral cavity: Report of one case and review of literatureDIAGNOSTIC CYTOPATHOLOGY, Issue 3 2006Guo-Xia Tong M.D., Ph.D. Abstract Epithelioid hemangioendothelioma (EHE) is an uncommon vascular tumor with biological behavior intermediate between hemangioma and angiosarcoma. It rarely occurs in the oral cavity. We report a case of an 81-yr-old woman with a 2-mo history of a 2 × 2 cm2 submucosal buccal mass. Fine needle aspiration (FNA) smears were paucicellular and showed mainly single atypical large epithelioid cells in a bloody background. The atypical cells had abundant dense cytoplasm, some with fine vacuoles. Occasionally, cells with large cytoplasmic lumina were seen. Cytology preparations from fresh tissue received for frozen section revealed numerous neoplastic cells with large intracytoplasmic lumina, some of which contained red blood cells. In addition, cells with distinct intranuclear inclusions were present. Histologic sections and immunohistochemical stains confirmed the diagnosis of EHE. Although the histologic features of EHE are well recognized, reports of FNA cytology findings are sparsely existent in the literature as several case reports. The characteristic cytological features of EHE are reviewed in this report. We believe that the diagnosis of this rare tumor can be suggested when an adequate FNA specimen is obtained. Diagn. Cytopathol. 2006;34:218,223. © 2006 Wiley-Liss, Inc. [source] Human T-lymphotropic virus type-1 related adult T-cell leukemia/lymphoma presenting as a parotid mass diagnosed by fine-needle aspiration biopsyDIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004Guo-Xia Tong M.D., Ph.D. Abstract A 48-yr-old black woman with a history of blood transfusions for menorrhagia secondary to uterine fibroids but no known Caribbean association presented with a 6-wk history of a rapidly enlarging right parotid mass. At the time of presentation, she could not close her right eye. An aspiration biopsy showed small, medium, and large lymphoma cells with angulated nuclei, red macronucleoli, and basophilic cytoplasm with fine vacuoles. Flow cytometry indicated a (CD25+/CD7,) T-cell lineage, suggesting an human T-lymphotropic virus (HTLV) 1-related T-cell leukemia/lymphoma, which was confirmed by polymerase chain reaction (PCR)-based amplification on DNA extracted from fresh tissue with specific oligonucleotide primers for HTLV-1 DNA sequence. Histology showed interstitial infiltration and destruction of the parotid parenchyma by lymphoma cells without involvement of adjacent lymph nodes. Total body CT scan and magnetic resonance imaging (MRI) studies were negative for lymphadenopathy but showed liver metastasis. To our knowledge, this is the first reported case of HTLV-1-related primary parotid lymphoma as the initial presentation of adult T-cell leukemia/lymphoma. Diagn. Cytopathol. 2004;31:333,337. © 2004 Wiley-Liss, Inc. [source] Localization of insulin-like growth factor binding protein-2 in chondrocytes of bovine articular cartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2003Teresa I. Morales Purpose: Previous work indicated that transforming growth factor (TGF-,) treatment of bovine articular cartilage resulted in an accumulation of insulin-like growth factor binding protein-2 (IGF-BP-2). The purpose of the work presented in this paper was to define the localization of the IGF-BP-2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF-,. Method: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF-,1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti-IGF-BP-2 performed. Results: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF-BP-2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF-, treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue. Conclusions: It is concluded that IGF-BP-2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF-, increases the number of cells with positive immunostaining. These data help to support the postulate that TGF-, exerts at least some of its actions in articular cartilage via cross-talk mechanisms involving the IGF-BP-2 system. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justifiedTHE JOURNAL OF PATHOLOGY, Issue 4 2005Robyn Lynne Ward Abstract This study prospectively examines the accuracy of immunohistochemical staining in the identification of mismatch repair defective (MMRD) colorectal cancer in routine clinical practice. The potential impact of this information on decisions regarding adjuvant treatment and germline testing were quantified. A consecutive series of fresh tissue (836 cancers) was obtained from 786 individuals undergoing curative surgery for colorectal cancer at one institution. As part of normal practice, each tumour was screened for the expression of MLH1 and MSH2 by immunohistochemical staining (IHC) and relevant clinicopathological details were documented. Microsatellite instability (MSI) was assessed using standard markers. Overall, 108 (13%) tumours showed loss of staining for either MLH1 (92 tumours) or MSH2 (16 tumours). The positive predictive value of mismatch repair IHC when used alone in the detection of MSI tumours was 88%, and the negative predictive value was 97%. Specificity and positive predictive value were improved by correlation with microsatellite status. Tumour stage (HR 3.5, 95% CI 2.0,6.0), vascular space invasion (HR 1.9, 95% CI 1.2,3.0) and mismatch repair deficiency (HR 0.2, 95% CI 0.05,0.87) were independent prognostic factors in stages II and III disease. Screening by mismatch repair IHC could reasonably have been expected to prevent ineffective treatment in 3.6% of stage II and 7.6% of stage III patients. The frequency of germline mismatch repair mutations was 0.8%, representing six unsuspected hereditary non-polyposis colorectal cancer (HNPCC) cases. Routine screening of colorectal cancers by mismatch repair IHC identifies individuals at low risk of relapse, and can prevent unnecessary adjuvant treatments in a significant number of individuals. Abnormal immunohistochemistry should be confirmed by microsatellite testing to ensure that false-positive results do not adversely impact on treatment decisions. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Salinity-induced changes in essential oil, pigments and salts accumulation in sweet basil (Ocimum basilicum) in relation to alterations of morphological developmentANNALS OF APPLIED BIOLOGY, Issue 2 2010N. Bernstein The objective of the project was to study salinity-induced effects on essential oil, pigments and salts accumulation in sweet basil (Ocimum basilicum, the cultivar Perrie) in relation to the alteration of plant morphological development and yield production. Hydroponically grown plants were exposed to one of six NaCl concentrations (1, 25, 50, 75, 100 and 130 mM NaCl). Inhibitory effects of salinity on biomass production of the shoot and the root, and area of individual leaves were apparent already under cultivation with 25 mM NaCl. Elevation of salinity from 1 to 100 mM NaCl induced 63% and 61% reductions in fresh and dry herb biomass production, respectively. The stress-induced reduction of foliage biomass sourced mainly from inhibition of leaf area development rather than reduction of internode and leaf number. Cl and Na concentrations in the leaves, stems and roots increased with elevation of NaCl concentration in the cultivation solution. While the extent of Cl accumulation was leaves>stems>roots, Na was largely excluded from the leaves and was preferentially accumulated in roots and the stems, potentially accounting for the moderate sensitivity of the leaf tissue to salinity. Salt stress increased the contents of essential oil and carotenoids in the leaves that may further account for the moderate sensitivity of sweet basil to salinity and suggest a potential for agro-industrial production. A twofold increase in both carotenoid concentration and the percent of essential oil in the fresh tissue was observed by elevation of the salinity from 1 to 130 mM NaCl. Overall, the stress induced increase of the percent of essential oil in the tissue in the salinity range 1,75 mM NaCl was about 50%, and thereby compensated for the similar reduction of biomass production in this salinity range, so that oil production on per plant basis was not reduced by salinity. [source] Change in proteoglycan metabolism is a characteristic of human patellar tendinopathyARTHRITIS & RHEUMATISM, Issue 10 2010John Parkinson Objective To determine differences in the metabolism of proteoglycans and the gene expression of proteinases and their inhibitors between patellar tendons exhibiting chronic overuse tendinopathy and normal patellar tendons in humans. Methods Rates of loss and synthesis of proteoglycans were determined. Radiolabeled and total proteoglycans retained in and lost from the tissue were analyzed by fluorography and Western blotting. Levels of messenger RNA for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-1, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4 were determined in fresh tissue. Results The rate of loss of 35S-labeled proteoglycans was greater in abnormal tendons, as was the rate of synthesis of proteoglycans. Fluorography and Western blotting revealed the presence of greater amounts of large proteoglycans (aggrecan and versican) in abnormal tendons, and these proteoglycans were rapidly lost from the matrix of abnormal tendons. There was no significant difference in the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, TIMP-2, TIMP-3, or TIMP-4. There was a significant increase in the expression of MMP-9 and TIMP-1 in abnormal tendons. Conclusion Our findings suggest that a change in the proteoglycan content of the extracellular matrix in abnormal tendons results from the altered metabolism of the cells, reflected in the enhanced synthesis of the large proteoglycans aggrecan and versican, and does not appear to result from changes at the level of gene expression. [source] Absence of c- kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patientsTHE JOURNAL OF PATHOLOGY, Issue 1 2004Kazuo Kinoshita Abstract Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c- kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c- kit gene mutations and that sporadic GISTs from non-NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c- kit gene mutations and ten sporadic GISTs from ten non-NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c- kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax-embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c- kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non-NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c- kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c- kit gene mutations and none of the ten GISTs derived from non-NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non-NF1 patients. Copyright © 2004 John Wiley & Sons, Ltd. [source] Comparison of 11 endogenous control genes for normalization of mRNA obtained from paraffin-embedded tissuesAPMIS, Issue 12 2009REKHA PAI Real-time reverse transcriptase PCR (RT-PCR) based assays are being increasingly used in characterization of gene expression. Good quality mRNA is an essential prerequisite for such assays. While fresh tissues provide quality mRNA, the same may not be true of tissues which are formalin-fixed and paraffin-embedded (FFPE). This emphasizes the need to identify a good endogenous control gene to normalize for differences in quality and RNA recovery. We attempted to characterize gene expression patterns of 11 commonly used endogenous control genes among 20 FFPE tissues (both neoplastic and normal). Pearson's coefficient of correlation was determined by comparing the expression of each gene against the mean expression of all other genes. ,2 microglobulin (,2M) and ,-actin (,A) (r = 0.95 and 0.94, respectively) were found to be stably expressed across all tissues. However, ,A had greater accuracy (2 × SD) than ,2M and therefore may be a better choice of an endogenous control for experiments that require normalization while using FFPE tissues. [source] PlGF expression in pre-invasive and invasive lesions of uterine cervix is associated with angiogenesis and lymphangiogenesisAPMIS, Issue 11 2009SHOUHUA YANG Most vascular endothelial growth factors (VEGF) have been shown to be associated with lymphangiogenesis and angiogenesis in various cancers. However, whether placental growth factor (PlGF), a rarely mentioned VEGF member, is involved in the pathogenesis of uterine cervical lesions remains unclear. To address this issue, we examined the relationship between PlGF expression and clinicopathologic variables in patients with pre-invasive and invasive lesions of uterine cervix. Sixty-two cervical specimens were immunostained with PlGF polyclonal antibody to define PlGF expression, and monoclonal antibodies D2-40 and CD34 to evaluate the lymphatic vessel density (LVD) and blood vessel density (BVD) of the lesions. PlGF mRNA level was detected by RT-PCR in all lesions from fresh tissues. We found that the levels of PlGF protein and mRNA expression were related to clinical stages (p < 0.05), but not to other clinicopathologic variables. No significant difference in PlGF expression was observed between squamous carcinoma and adenocarcinoma. Increased LVD and BVD were all associated with advanced stages (p < 0.001). Although LVD was strongly correlated with BVD, only high LVD was associated with pelvic lymphatic metastasis. Moreover, the level of PlGF expression was associated with both BVD(r = 0.715, p < 0.001) and LVD(r = 0.321, p < 0.05). Together, our study suggests that PlGF may participate in both tumor-associated angiogenesis and lymphangiogenesis of cervical carcinogenesis. [source] Prostate carcinoma tissue proteomics for biomarker discoveryCANCER, Issue 12 2003Yaxin Zheng M.D. Abstract BACKGROUND The advent of the prostate-specific antigen (PSA) test has had a profound impact on the diagnosis and treatment of prostate carcinoma. However, the use of PSA levels alone for screening for prostate carcinoma was compromised by the variations in the amount of PSA produced by the benign prostatic tissue specimens. Proteins were involved in various pathways that determine the behavior of a cell. Therefore, information regarding proteins may reveal drug targets and/or markers for early detection. METHODS The authors used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to determine the protein profiles from fresh tissues of the prostate. Laser capture microdissection was performed to isolate pure populations of cells. RESULTS The authors identified a protein with an average m/Z of 24,782.56 ± 107.27 that was correlated with the presence of prostate carcinoma. Furthermore, using laser capture microdissection, they demonstrated that the origin of this protein, which the authors designated PCa-24, was derived from the epithelial cells of the prostate. PCa-24 expression was detected in 16 of 17 (94%) prostate carcinoma specimens but not in paired normal cells. In addition, this protein was not expressed in any of the 12 benign prostatic hyperplasia specimens that were assayed. CONCLUSIONS PCa-24 may be useful a marker for prostate carcinoma. Cancer 2003;98:2576,82. © 2003 American Cancer Society. [source] | |||||||||||||