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PubMed1995

Localization of Epstein-Barr Virus-Encoded Small RNA-1 by in situ Reverse Transcription: Demonstration of cDNA Generation in Formalin-Fixed Paraffin-Embedded Tissue Sections.

Reverse transcription (RT) followed by polymerase chain reaction (RT-PCR) has been commonly used to detect viral and cellular transcripts in whole cell extracts. Application of this technique to tissue sections requires the in situ generation of cDNA. In this study, we selected an abundant transcript, Epstein-Barr virus (EBV)-encoded small RNA (EBER-1), as a model template to demonstrate cDNA generation in tissue sections. Using both digoxigenin-dUTP and primers which are complementary to EBER-1, we demonstrated specific EBER-1 cDNA generation both in vitro, and in tissue sections taken from formalin-fixed paraffin-embedded cell blocks of an EBV-infected cell line, B95-8. Furthermore, we utilized in situ RT in sections of EBV-associated nasopharyngeal carcinomas, and identified EBER-1 cDNA specifically in neoplastic cells, but not in the surrounding nonneoplastic stroma. EBER-1 cDNA was localized to the nucleus of these cells, with relative sparing of the nucleolus and the cytoplasm. No specific signal was evident if the reverse transcriptase was omitted, if 'sense' primers were used, or if RT was preceded by RNase digestion. The specificity of EBER-1 cDNA was further confirmed by in situ hybridization using the sense riboprobe, which has the same polarity as the EBER-1 transcript. Our results provide a successful example of using nonradioactive nucleotide analogue for cDNA generation in formalin-fixed, paraffin-embedded tissue sections. This approach would provide a visible assay to monitor RT in tissue sections, and allow further optimization of conditions for cDNA generation in tissue sections. Therefore, it potentially can be helpful for the future development of RT-PCR in tissue sections. Copyright 1995 S. Karger AG, Basel

Journal Article↗
PubMed1997

Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues.

The differential diagnosis of pleural mesotheliomas and lung adenocarcinomas presents a continued challenge in the practice of surgical pathology. Paraffin immunohistochemistry (IHC) using different panels of antibodies can be helpful in some cases, but, as yet, no antigen is expressed specifically in mesotheliomas nor in adenocarcinomas. Using well characterized monoclonal antibodies (MAb) that recognized distinct mesenchymal and epithelial adhesion proteins, N-cadherin (13A9 MAb) and E-cadherin (E9 MAb), respectively, we found previously that in frozen-section IHC mesotheliomas and adenocarcinomas had distinct cadherin phenotypes: mesotheliomas were positive for N-cadherin, and lung adenocarcinomas were positive for E-cadherin. Using antigen-retrieval methods, we successfully extended our study to formalin-fixed, paraffin-embedded tissue sections. Tumors from 28 patients (14 originally diagnosed as mesotheliomas, and 14 diagnosed as adenocarcinomas) were stained with 13A9 MAb and E9 MAb. Review of hematoxylin-eosin sections excluded from analysis one case previously diagnosed as mesothelioma, which represented a hemangiopericytoma. Of the remaining 27 cases, 12 of 13 mesotheliomas were positive for N-cadherin and negative for E-cadherin. The exception was a multifocal microscopic papillary tumor of apparent mesothelial origin, which was negative for both N-cadherin and E-cadherin. Conversely, 13 of 14 adenocarcinomas were E-cadherin positive and N-cadherin negative except for one adenocarcinoma with focal N-cadherin expression. One case of a poorly differentiated adenocarcinoma invading skeletal muscle was negative for both 13A9 and E9. These studies confirmed the utility of the cadherin antibodies in distinguishing pleural mesotheliomas from lung adenocarcinomas. The reactivity of the cadherin-specific antibodies with antigens in paraffin sections make them powerful and reliable markers in the practice of diagnostic surgical pathology.

Adenocarcinoma↗
PubMed1993

The polymerase chain reaction for detecting adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem.

The polymerase chain reaction (PCR) was used to detect adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem. Adenovirus DNA was successfully amplified from specimens of lung and liver from two patients with disseminated adenovirus infection confirmed by virus isolation, electron microscopy and/or immunohistochemistry. Negative results were obtained for specimens of lung from two patients with cytomegalovirus pneumonia. The specificity of the adenovirus PCR was confirmed by means of a digoxigenin-labelled probe generated in a separate PCR. Detection of viral nucleic acid by PCR in tissues obtained post mortem has considerable diagnostic potential.

Adenovirus Infections, Human↗
PubMed1996

[Detection of point mutation of p53 gene by silver staining PCR-SSCP in paraffin-embedded malignant fibrous histiocytoma].

Silver staining PCR-SSCP method was used to detect point mutation of p53 gene in paraffin-embedded malignant fibrous histiocytoma (MFH) tissues. The abnormal shifting of the single-stranded DNA (ssDNA) was identified in 9 out of 16 cases. The positive figure of SSCP was 1,4,4, 3 in exon 5, 6, 7, 8, respectively. The mutant p53 protein was detected by microwave oven treatment and ABC immunohistochemistry. Positive nuclear staining was observed in 10 cases. The positive coincidence rate was 90.0% between SSCP and p53 protein expression. The mutation of p53 gene was not correlated with the subtypes of MFH. Our results indicate that detection of point mutations with silver staining PCR-SSCP is convenient, rapid and reliable in the screening of point mutation of genes.

Genes, p53↗
PubMed2006

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues.

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

Acetates↗
PubMed2003

[Detection of COL1A1/PDGFB fusion transcripts in dermatofibroscoma protuberans by revers transcriptase-polymerase chain reaction using paraffin-embedded tissues].

OBJECTIVE: To detect the COL1A1/PDGFB fusion transcripts and discuss its clinicopathological significance in dermatofibroscoma protuberans. METHODS: Formalin fixed, paraffin-embedded tumor specimens from 12 patients with DFSP were reviewed by light microscope and the expression of COL1A1/PDGFB mRNA resulting from the reciprocal translocation t(17;22) (q22;q13.1) was detected by one-step revers transcriptase-polymerase chain reaction. The following tumor specimens were included as controls: 2 fibrosarcoma, 2 malignant fibrous histocytoma, 3 leiomyosarcoma, 1 dermarofibroma and 1 nerve shealth tumor. RESULTS: The COL1A1/PDGFB fusion transcripts were detected in 8 (67%) of 12 samples from patients with DFSP. Nucleotide sequence analysis using the PCR products confirmed that different regions of the COL1A1 gene, respectively, were fused with of PDGFB gene. No COL1A1/PDGFB fusion transcripts were detected in the control tumors. CONCLUSION: Detection of specific COL1A1/PDGFB fusion transcripts in DFSP will help to diagnose the nature of DFSP and research the mechanism of its molecular histogenesis.

Adolescent↗
PubMed2006

Methacarn fixation--effects of tissue processing and storage conditions on detection of mRNAs and proteins in paraffin-embedded tissues.

In this study, we examined suitable conditions for tissue fixation with methacarn and ethanol dehydration and storage of paraffin-embedded tissues (PETs) on gene expression analysis. With fixation and dehydration of rat liver tissues for up to 16 h (overnight) and 1 week, respectively, at 4 degrees C, integrity of extracted total RNAs and polypeptides did not vary, the former integrity being constantly lower than that with unfixed frozen tissue, while protein yield was slightly reduced with increasing dehydration. Retained expression levels of mRNAs and proteins were mostly unaffected by the period of fixation but slightly fluctuated with the length of dehydration. When PETs were stored for up to 12 months, integrity of both total RNAs and polypeptides was retained at 4 degrees C but reduced at room temperature. Reduced expression levels of mRNAs and proteins were also noted by storage at room temperature after 12 and 3 months, respectively. However, neither tissue processing nor storage affected variability in either mRNA or protein levels among samples. Thus, the results suggest that, for gene expression analysis, tissues can be fixed with methacarn and dehydrated for at least 1 day and 1 week, respectively, and PETs can be stored for at least 12 months, but a temperature of 4 degrees C is preferable.

Acetic Acid↗
PubMed1992

Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation.

Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.

Base Sequence↗
PubMed1995

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Base Sequence↗
PubMed1990

Establishment of specific monoclonal antibodies against recombinant human granulocyte colony-stimulating factor (hG-CSF) and their application for immunoperoxidase staining of paraffin-embedded sections.

Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for proliferation and differentiation of granulocytes, has been performed up to the present by use of the granulocyte colony-formation assay, because of the lack of a specific anti-G-CSF antibody. This has prevented the advancement of biological investigations of cell dynamics linked to G-CSF, e.g., cell localization of G-CSF and its pathophysiological changes. In the present work, two monoclonal antibodies (MAb), 1E7 and 4A6, against recombinant human G-CSF (rhG-CSF) were developed by cell hybridization between NS-1 myeloma cells and splenocytes from a mouse immunized with rhG-CSF. 1E7 and 4A6 were shown to be reactive with hG-CSF but not with other CSF (hGM-CSF, hIL-3, and mouse GM-CSF) by Western blot analysis. An immunoperoxidase staining method using these MAb was then established. This method was applicable to frozen sections, paraffin-embedded sections, and cells fixed with 4% paraformaldehyde. Positive staining for G-CSF was observed in tumor cells secreting G-CSF and also in Chinese hamster ovary (CHO) cells transfected with hG-CSF cDNA. However, no staining was seen in tumor cells secreting no G-CSF, untransfected CHO cells, lung fibroblasts, or bone marrow stromal cells after short periods of culture. These results confirmed the immunospecificity of MAb 1E7 and 4A6 and the validity of their application to immunohistochemistry using paraffin-embedded sections.

Animals↗
PubMed1986

Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections.

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.

Animals↗
PubMed1997

A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue.

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection.

Antibodies, Monoclonal↗
PubMed1994

[Detection of point mutation of p53 gene by non-isotopic PCR-SSCP in paraffin-embedded breast cancer tissue].

Non-isotopic PCR-SSCP method was used to detect point mutation of p53 gene in paraffin-embedded breast cancers. The abnormal shifting of the single-stranded DNA (ssDNA) was identified in 14 out of 60 cases. The point mutations of four cases were found in exons 5-6, three in exon 7 and seven in exon 8. The mutant p53 protein was detected by immunohistochemistry. The positive nuclear staining was observed in 13 cases, among which 11 cases showed abnormal shifting of ssDNA in SSCP analysis. The point mutations of the other two cases might occur in the regions which were not examined in this study. The point mutations of p53 were found in 5 cases in the absence of detectable protein. This probably resulted from the reduction or loss of antigenicity of p53 in some of the routinely processed tissues. Our results indicate that detection of point mutations of p53 gene by PCR-SSCP method is more accurate and reliable than that by immunohistochemistry and non-isotopic PCR-SSCP is especially useful in the screening of point mutations during handling a large number of samples.

Breast Neoplasms↗
PubMed1980

Detection of immune deposits in glomeruli: a comparative study of paraffin-embedded, enzyme-treated sections and cryostat sections as substrates in immunofluorescence.

Paraffin-embedded tissue can be used as substrate for immunohistochemistry after enzymatic treatment with proteases. The sensitivity of immunofluorescence on enzyme-treated paraffin sections and on unfixed cryostat sections is compared in this study. Pepsin was more efficient by weight than trypsin in restoring the antigenicity of immune deposits. Increased fluorescence intensity was obtained up to a pepsin concentration of 0.4%. Intensity was further increased when pepsin treated sections were treated with trypsin. Immune deposits were detected in enzyme treated, paraffin sections of kidneys of mice injected with anti-GBM diluted 1/200 or less and in cryostat sections of mice injected with anti-GBM diluted 1/400 or less. This small decrease in sensitivity is considered trivial compared with the advantage gained by the excellent preservation of the tissue.

Animals↗
PubMed1994

Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.

The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. All MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cystine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TGC-->TCC; Cys-->Ser) in one, in codon 635 (TGC-->CGC; Cys--Arg) in three, and in codon 635 (TGC-->TAC; Cys-->Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheochromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable methods to detect single base substitutions in DNA extracted from archival material.

Adolescent↗
PubMed2006

Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue.

PURPOSE: Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy. PATIENTS AND METHODS: Patients with newly diagnosed stage II or III breast cancer were treated with sequential doxorubicin 75 mg/M2 q2 wks x 3 and docetaxel 40 mg/M2 weekly x 6; treatment order was randomly assigned. Pretreatment core biopsy samples were interrogated for genes that might correlate with pathologic complete response (pCR). In addition to the individual genes, the correlation of the Oncotype DX Recurrence Score with pCR was examined. RESULTS: Of 70 patients enrolled in the parent trial, core biopsies samples with sufficient RNA for gene analyses were available from 45 patients; 9 (20%) had inflammatory breast cancer (IBC). Six (14%) patients achieved a pCR. Twenty-two of the 274 candidate genes assessed correlated with pCR (p < 0.05). Genes correlating with pCR could be grouped into three large clusters: angiogenesis-related genes, proliferation related genes, and invasion-related genes. Expression of estrogen receptor (ER)-related genes and Recurrence Score did not correlate with pCR. In an exploratory analysis we compared gene expression in IBC to non-inflammatory breast cancer; twenty-four (9%) of the genes were differentially expressed (p < 0.05), 5 were upregulated and 19 were downregulated in IBC. CONCLUSION: Gene expression analysis on core biopsy samples is feasible and identifies candidate genes that correlate with pCR to primary chemotherapy. Gene expression in IBC differs significantly from noninflammatory breast cancer.

Adult↗
PubMed1996

Technical approach for the study of the genetic evolution of breast cancer from paraffin-embedded tissue sections.

We have optimized a technique that allows the study of numerous chromosomal loci (n = 20-50) from single paraffin-embedded tissue sections by microsatellite length polymorphism analysis. DNA samples from normal and breast cancerous tissue can be obtained from the same section by means of microdissection. This technique was further improved by subjecting DNA to several cycles of amplification with a degenerate (universal) primer and then with specific microsatellite primers. This amplified DNA was also used to screen for mutations in the p53 gene by means of PCR-SSCP. In addition adjacent tissue sections were used to assess specific chromosome copy number by interphase cytogenetic analyses (chromosome in situ hybridization) and to analyze expression of specific genes such as p53 and ERBB2. As an example of the use of our approach we performed a detailed chromosome 17 allelotypic analysis in 22 breast tumors (5 ductal carcinomas in situ, 13 invasive ductal carcinomas, and 4 invasive lobular carcinomas). We detected mutations in the p53 gene by PCR-SSCP in 36% of the samples. Samples with significant levels of p53 protein accumulation detected by immunohistochemistry were also positive for mobility shifts in the SSCP analysis. We observed that chromosome 17 allelic losses and imbalance occurred at as early a stage as ductal carcinoma in situ (DCIS). Although in some cases we observed allelic losses or imbalance affecting the 17p13 region, close to the p53 locus, several of the tumors showed dissociation between such loss or imbalance and p53 mutation. Lobular carcinomas were predominantly disomic for chromosome 17 in contrast with ductal tumors, which often showed polysomy for chromosome 17. This comprehensive approach correlating the tumor subtype, its allelotype, with specific chromosome copy number and specific gene mutations and expression in preinvasive or early invasive breast cancer lesions will potentially provide information of relevance for a better understanding of the multistep mechanisms of breast carcinogenesis.

Alleles↗