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PubMedDate not supplied

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Animals↗
PubMed1988

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections.

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.

Animals↗
PubMed2003

Improvement in the detection rate of t(14;18) translocation on paraffin-embedded tissue: a combination approach using PCR and FISH.

AIMS: PCR has been the primary method used for the detection of t(14;18) translocation in formalin-fixed, paraffin-embedded tissues. This technique mainly targets the well-characterised breakpoint regions in chromosomes 14 and 18. FISH is now applicable on paraffin tissue sections and has been suggested to be capable of detecting essentially 100% of t(14;18) translocated cases. In this study, we described the application of both PCR and FISH for the detection of t(14;18) translocation. METHODS: Fifty follicular lymphoma cases were retrieved from the files of the Department of Pathology, University of Malaya Medical Centre (UMMC). Nested PCR amplification of MBR/JH and mcr/JH was performed in these cases, and those cases that did not demonstrate the translocation were subjected to FISH analysis. RESULTS: Thirty cases (60%) had t(14;18) translocation detected by PCR, 25 (50%) had breakpoint with MBR and five (10%) involved mcr. Twenty cases without detectable t(14;18) translocation by PCR were analysed by FISH. Eleven cases were successfully probed, and four of them showed positive translocation signal. CONCLUSIONS: The combination of PCR and FISH analysis on paraffin tissue sections for the detection of t(14;18) translocation increases the sensitivity of detection from 60 to 68%. Problems encountered in our FISH analysis on tissue sections impose certain limitations in using this technique for retrospective screening of large number of samples. Therefore, we suggested the application of PCR as the first screening tool on retrospective archival materials, followed by FISH on those PCR-negative cases.

Chromosome Fragile Sites↗
PubMed1995

Immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies.

Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location.

Animals↗
PubMed1997

[Detecting pathogens of fetal defects in paraffin embedded tissues by polymerase chain reaction].

OBJECTIVE: To examine 3 types of pathogens in dead fetus with congenital defects for exploring the pathogenesis. METHODS: The paraffin embeded brain and liver tissues from 39 fetal autopsies with congenital defects were examined for toxoplasma, cytomegalovirus, and herpes simplex II by polymerase chain reaction. Among them 16 placentae were examined as well. RESULTS: Pathogens were detected in 25 of 39 cases, a positive rate of 64%. Nine out of the 13 fetuses with urogenital defects were positive for the shove pathogens, while 8 from the 12 with neural tube defects were positive as well. CONCLUSION: Intrauterine infection was one of the important causes of birth defects. The characteristics of placenta morphologic changes were described, and the significance of placenta investigation in congenital malformation was emphasized.

Animals↗
PubMed1998

Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases.

We report the development of a reverse-transcriptase polymerase chain reaction assay that detects (in paraffin-embedded, formalin-fixed tissue) the SYT-SSXchimeric RNA transcript resulting from the t(X;18) of synovial sarcoma. The primers chosen detect both of the SSX1 and SSX2 partners, and the target sequence is small enough (87 base pairs) to be reliably detected in archival and variably processed consultation material. To demonstrate its usefulness, we applied it to 14 problematic cases, including spindle cell tumors of the thoracic region, of the neck, and of subcutaneous tissue. For instance, we show that, depending on the location, synovial sarcoma can mimic malignant solitary fibrous tumor, the spindle epithelial tumor with thymus-like differentiation, or skin adnexal tumors. Molecular detection of the SYT-SSX chimeric RNA should allow the reclassification of difficult cases in which the morphologic features overlap different entities or in which tumor nosology is still evolving.

Adult↗
PubMedDate not supplied

Diagnosis of bronchial carcinoma on sections of paraffin-embedded sputum. Sensitivity and specificity of an alternative to routine cytology.

The diagnostic accuracy of sputum cytology for the diagnosis of bronchial carcinoma using paraffin-embedded, serially sectioned and hematoxylin and eosin-stained specimens was tested in 4,297 sputum samples from 1,889 patients, 219 of whom had bronchial carcinoma. The diagnostic sensitivity depended mainly on the number of investigated samples and was 85.4% with three sufficient sputa. The sensitivity was not influenced by the histologic types, location or TNM stage of the tumor. The specificity of the method was 99.5%. In three cases localization of sputum cytologically diagnosed bronchial carcinomas was not possible immediately (occult carcinomas, pTx); in two of these cases the bronchial carcinomas were located during follow-up. The third patient died without verification of the cytologic diagnosis. According to our results, sputum cytology on serial sections is a valuable instrument for mass screening of high-risk groups for the early detection of bronchial carcinoma. Lower sensitivities of sputum cytology in mass screening programs for the early diagnosis of lung cancer are discussed critically.

Carcinoma, Bronchogenic↗
PubMed2001

Losses of chromosomal arms 1p and 19q in the diagnosis of oligodendroglioma. A study of paraffin-embedded sections.

Comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), polymerase chain reaction-based microsatellite analysis, and p53 sequencing were performed in paraffin-embedded material from 18 oligodendrogliomas and histologically similar astrocytomas. The study was undertaken because of evidence that concurrent loss of both the 1p and 19q chromosome arms is a specific marker for oligodendrogliomas. Of the six lesions with a review diagnosis of oligodendroglioma, all had the predicted loss of 1p and 19q seen by CGH, FISH, and polymerase chain reaction. Other lesions, including some considered oligodendroglioma or mixed glioma by the submitting institution, did not. There were no p53 mutations in any of the six oligodendrogliomas, whereas 5 of the 10 remaining, successfully studied cases did have p53 mutations. The results suggest that CGH and FISH performed on current or archival tissue can aid in classification of infiltrating gliomas such as oligodendrogliomas and astrocytomas. The results of the p53 studies are consistent with findings of previous investigations that such mutations are less common in oligodendrogliomas than they are in astrocytomas.

Adult↗
PubMed1994

Detection of immunophenotypic abnormalities in paraffin-embedded B-lineage non-Hodgkin's lymphomas.

The authors analyzed the frequency of immunophenotypic abnormalities in 1,474 cases of routinely fixed, paraffin-embedded B-lineage non-Hodgkin's lymphomas. B-lineage was determined by immunoreactivity for CD20 (L26, 92%); CD45RA (4KB5, an additional 3%) or immunoglobulin (Ig) light chain restriction (remaining 5%). CD45RA was found to be especially helpful on Bouin's-fixed or decalcified tissue and Ig staining was most helpful in plasmacytoid lesions. Coexpression of the T-cell marker CD43 (Leu-22) was the most common immunophenotypic abnormality, seen in 60% of mantle cell lymphomas (MCL), 39% of CLL/small lymphocytic lymphomas, 16% of diffuse large cell lymphomas (DLCL), but only 5% of follicular lymphomas (FL). Antibodies to CD45RO (A6 and UCHL1) and CD3 (polyclonal) were useful in distinguishing infiltrating T cells from B cells coexpressing CD43. Ig light chain restriction was the next commonest immunophenotypic abnormality, which was identified in 67% of plasmacytoid diffuse small cell lymphomas, 43% of MCLs, 35% of monocytoid B-cell lymphomas and 28% of FLs. Overexpression of bcl-2 oncogenic protein was observed in 71% of FLs (n = 96), but not in a control group of reactive follicular hyperplasias (n = 34). Combining two criteria increased the sensitivity of immunodiagnosis in certain circumstances.

Antigens, CD↗
PubMed1993

Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.

Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4 degrees C for 24-48 h before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

Animals↗
PubMed1987

DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study.

Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease. In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974. Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch. Thirty-micron sections were cut from each plug for dewaxing and disintegration. The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry. In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern. No apparent correlation was found between the histopathological grade and the DNA ploidy. Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy. The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.

Acid Phosphatase↗
PubMed1998

Immunocytochemical detection of Candida albicans in formalin fixed, paraffin embedded material.

AIM: To assess the ability of the commercially available monoclonal antibody 1B12 (BioGenex, San Ramon, USA) to identify C albicans in formalin fixed, paraffin wax embedded material (FFPE). METHODS: Broth cultures of 20 strains of seven Candida species were resuspended in 4% agarose blocks, fixed in formalin for 24 hours, and embedded in paraffin wax. In addition, 16 blocks of FFPE tissue known to contain periodic acid-Schiff positive fungal hyphae were examined. Antigen retrieval involved microwave treatment of specimens in citrate buffer (0.01 M; pH 6.5) before addition of 1B12 antibody for 24 hours. Bound antibody was subsequently detected using a biotinylated link antibody and a peroxidase conjugated streptavidin. RESULTS: Only C albicans strains were 1B12 positive in the agarose blocks. All FFPE tissue blocks were found to contain 1B12 positive hyphal structures, indicating the presence of C albicans. CONCLUSIONS: The ability to identify candida organisms penetrating the lesional tissue in cases of chronic hyperplastic candidosis will help to clarify the role of individual Candida spp in this important form of oral candidosis.

Antibodies, Monoclonal↗
PubMed1994

[Microwave oven in immunohistochemistry. An important tool for determination of antigens in formalin-fixed and paraffin-embedded tissue].

Immunocytochemical methods are important tools for identifying antigens in tissue sections and cell smears. Some antigens were retrieved in formalin-fixed and paraffin-embedded tissues by means of a microwave technique. This method also increased the sensitivity of the immunohistochemical detection of several other antigens. Altogether, microwave boiling of the tissue sections in citrate buffer clearly improved the immunoreactivity for cytokeratin 18, oestrogen receptor, Ki-67 protein, PCNA, p53 protein, retinoblastoma gene protein and c-erbB-2 protein. This new technique, which can be applied in every pathology laboratory, will facilitate the application of immunohistochemical methods for research and diagnostic work.

Epitopes↗
PubMed1998

Numerical abnormalities of chromosomes 1 and 10 in endometrial adenocarcinoma: fluorescence in situ hybridization analysis of 23 archival paraffin-embedded samples.

Retrospective analysis of chromosomal changes in endometrial carcinoma was performed by fluorescence in situ hybridization on free nuclei isolated from formalin-fixed paraffin-embedded tissue. We examined 23 archival samples for numerical aberrations of chromosomes 1 and 10 with the use of specific DNA probes for the pericentromeric and centromeric regions of these two chromosomes. Numerical aberrations of chromosomes 1 and 10 were detected in 39% of the case analyzed, and the frequency of trisomy 10 tended to increase as the histological grade worsened. Our findings confirm the association of cytogenetic anomalies involving chromosomes 1 and 10 with endometrial carcinoma, as reported by other studies, and suggest that changes in centromere 10 copy number may correlate with the degree of tumor differentiation.

Adult↗
PubMed1992

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

Animals↗
PubMed1984

DNA-Feulgen-cytophotometric analysis of single cells isolated from paraffin embedded tissue.

This study describes a method for the isolation of cells from paraffin embedded tissues for DNA-Feulgen-cytophotometric measurements. The relevance of the method used is demonstrated by the analysis of the DNA-distribution pattern of cells isolated from an alveolar sarcoma of the soft tissues of a 10-year old girl. The comparison between freshly prepared imprint preparations and specimens after pepsin--extraction clearly shows, that the preparation mode used did not influence the relative DNA-content. The mean values of DNA/nuclei did not show a decrease even after prolonged pepsin treatment (90 minutes). Therefore this method permits access to stored histopathological material for retrospective DNA-Feulgen-Cytophotometric investigations.

Child↗
PubMed2006

Immunohistochemical measurement of endothelial cell apoptosis and proliferation in formalin-fixed, paraffin-embedded human cancer tissue.

There is a need for simple, reproducible methodology for measurement of endothelial cell (EC) apoptosis and proliferation in formalin-fixed, paraffin-embedded (FFPE) human tissue obtained in clinical trials of potential anti-angiogenic agents. Therefore, we developed colorimetric, dual-label immunohistochemical techniques for use on FFPE tissue, based on the use of single-stranded (ss) DNA and Ki-67 as markers of EC apoptosis and proliferation, respectively. Digital image analysis was used to obtain the total tumour microvessel density (MVD), from which the EC apoptosis index (AI) and proliferation index (PI) were derived manually as the number of positive ECs per vessel. Immunohistochemical measurement of EC apoptosis and proliferation was validated on human colorectal cancer liver metastases from a randomized, placebo-controlled trial of the selective cyclooxygenase-2 inhibitor rofecoxib. Proliferating and apoptotic ECs clustered in discrete areas of the tumour vasculature. ECAI (median [inter-quartile range, IQR] 0.0018 [0.0003-0.0064]) and ECPI (median [IQR] 0.0043 [0.002-0.014]) values were low and highly variable in tumours from both placebo- and rofecoxib-treated patients. Our novel dual-label immunohistochemical methods will be generally applicable in FFPE human cancer tissue and should prove invaluable for measuring the anti-angiogenic activity of experimental therapies in clinical trials. The low absolute level of and variability in EC apoptosis and proliferation detectable in human colorectal cancer liver metastases indicates that similar intervention studies using these end-points will need to ensure adequate size in order to be able to detect anti-angiogenic activity by immunohistochemical methods.

Apoptosis↗