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Monoclonal antibody MAT-1 against human tyrosinase can detect melanogenic cells on formalin-fixed paraffin-embedded sections.

Immunohistochemical localization of tyrosinase was examined with a monoclonal antibody (MoAb MAT-1) against human tyrosinase on routine formalin-fixed paraffin-embedded sections of 3 normal skin specimens, 15 melanocytic tumors (6 pigmented nevi, 3 juvenile melanomas and 6 malignant melanomas) and 3 non-melanocytic tumors. In the melanotic melanomas, almost all tumor cells were clearly stained with the antibody. In the nevocytic nevi, the nevus cells in lower epidermis and upper dermis were positive for MoAb MAT-1, but negative in middle and lower dermis. All three juvenile melanomas, one amelanotic melanoma, and three non-melanocytic tumors were entirely negative for MoAb MAT-1. Thus, MoAb MAT-1 could recognize the cells with melanogenic activity on routine formalin-fixed paraffin-embedded sections. However, the staining quality was not adequate for normal epidermal melanocytes, indicating that small technical innovations in the immunostaining process such as formalin fixation after PBS washing are required. Nevertheless, MoAb MAT-1 can be expected to be very useful for identifying melanogenic cells on paraffin-embedded sections, because we have to date no other antibody available for it.

Antibodies, Monoclonal↗

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: an alternative method for HER-2/neu analysis.

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.

Breast Neoplasms↗

Renal chromophobe cell carcinoma: limitations of paraffin-embedded tissue.

Chromophobe cell carcinoma of the kidney is distinguished from other renal cortical tumors by the ultrastructural presence of numerous cytoplasmic vesicles of unknown composition. Failure to detect vesicles in paraffin-embedded tissue in the archival material of 10 putative chromophobe cell tumors stimulated an investigation into the effects of fixatives and dehydrating solvents on vesicle integrity. We found that although vesicles are removed by dehydrating agents during paraffin embedding, osmium tetroxide postfixation prevents vesicle loss during dehydration for electron microscopy. We conclude that paraffin-embedded tissue is useless for histochemical studies of vesicle composition and cannot be employed for the ultrastructural confirmation of a histologic diagnosis of chromophobe cell carcinoma since the intracellular vesicles are no longer present.

Adenocarcinoma↗

[A flow cytometric study of the DNA content from paraffin-embedded samples of keratoacanthoma and squamous cell carcinoma].

DNA-flow cytometry was performed on paraffin-embedded tissues from 7 cases of keratoacanthoma (KA) and squamous cell carcinoma (SCC). Proliferation index of tumor cells (% greater than 4SD) was defined as the percentage of cells that exceeded the diploid peak (G0G1) plus 4 standard deviations (SD). Proliferation index (% greater than 4SD) was lower than 20% in six out of 7 cases of KA, but higher than 25% in six out of 7 cases of SCC. One case of SCC showed a DNA aneuploidy. These results indicate that DNA-flow cytometry analysis using paraffin-embedded tissues may be a useful tool in differential diagnosis between keratoacanthoma and squamous cell carcinoma.

Aged↗

[The accelerated method of paraffin embedding in pathohistological practice].

An accelerated method of paraffin embedding of placentas in pathohistological practice is suggested allowing to get micropreparations stained with hematoxylin and eosin and other methods from 500 500 and more blocks 4-5 hours after cutting. This enables to analyze material from groups of risk among children born alive and rapid informing neonatologists and pediatricians. The method is based on rejection of formaldehyde fixation (this excludes a toxic effect on the personnel). Use of acetone combines fixation and dehydration. The reagents usable for technical reasons make the proposed method cost effective.

Acetone↗

Human papilloma virus infection and Ki-ras oncogene in paraffin-embedded squamous carcinomas of the cervix.

42 paraffin-embedded squamous cervical carcinomas were screened for the presence of human papilloma virus (HPV; 6b, 11, 16, 18) and for activation of the Ki-ras oncogene family by polymerase chain reaction. In 72% of cases we found one or more HPV types, but no mutations of the Ki-ras gene (codon 12-1 and 61-1, 61-2 and 61-3) were found. We conclude that mutations of the Ki-ras oncogene, at the positions analyzed, are not likely to be involved in the events leading to cervical carcinogenesis.

Carcinoma, Squamous Cell↗

Highly sensitive HLA-DNA typing from formalin-fixed and paraffin-embedded tissue samples.

Nucleotide sequences have been determined for more than 1700 different alleles at the core of the human leukocyte antigen (HLA) system. The highly polymorphic character of these genes affects adaptive immune response and is also useful for forensic applications. HLA typing from formalin-fixed and paraffin-embedded tissue provides abundant useful information for both clinical settings and forensic investigations. This study, which investigated the potential use of DNA from formalin-fixed and paraffin-embedded tissue samples in an HLA PCR sequence-specific primer and probe (SPP) system, showed that tissue fixed in formalin for less than 3 days and embedded in paraffin can serve as a useful source of DNA for PCR-SPP typing kits.

Autopsy↗

Detection of clonal T-cell receptor gamma gene rearrangements in paraffin-embedded tissue by polymerase chain reaction and nonradioactive single-strand conformational polymorphism analysis.

The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor gamma (TCR-gamma) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vgamma1-8, Vgamma9, Vgamma10, Vgamma11, and Jgamma1/Jgamma2 consensus primers were used for TCR-gamma gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1-5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-gamma gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue.

Base Sequence↗

[Detection of p53 gene mutation in paraffin-embedded asbestos-related lung cancer tissue].

Paraffin-embedded asbestos-related lung cancer tissue of ten cases was analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to study its genetic mutation of exon 5, 7 and 8 of anti-oncogene p53 by silver stain. Results showed that fragment of exon 7 or 8 of p53 gene in four cases was positive in silver-staining analysis of PCR-SSCP. Exon 7 or 8 fragments of p53 gene was detected positive for mutation in seven of these ten cases with autoradiographic analysis of PCR-SSCP. No exon 5 fragment was found positive for mutation in these ten cases with both methods. Agreement between results of silver staining and those of autoradiographic PCR-SSCP was 60%. Hence, autoradiographic method could be replaced by silver staining, a simpler and more sensitive one, in PCR-SSCP to study gene mutation of asbestosrelated lung cancer.

Adenocarcinoma↗

Comparison of techniques for the successful detection of BRCA1 mutations in fixed paraffin-embedded tissue.

Genomic DNA isolated from archived paraffin-embedded tissues (PETs) has important applicability in genetic epidemiological studies. To determine the accuracy of the sequence data, using DNA derived from PET among patients with known mutations characterized from blood, we conducted a blinded factorial experiment to simultaneously examine the influence of mutation type, age of the PET, PCR product type, and Taq DNA polymerase on BRCA1 gene mutation detection. The probability of detecting sequencing artifacts was also investigated. We found that: (a) gene detection was most accurate for newer PET; (b) high fidelity Taq with shorter PCR amplicon length yielded the highest mutation detection success rate and lowest artifact rate; and (c) base substitutions were more often correctly identified than frameshift mutations or wild-type sequences. We concluded that DNA derived from PET that archived for less than 18 years can be used successfully for detecting BRCA1 gene mutations if quality control is strictly maintained.

DNA Mutational Analysis↗

Immunofluorescent staining of influenza virus antigen in fixed and paraffin-embedded tissue of experimentally infected hamsters.

Sections of formalin-fixed, paraffin-embedded tissue of experimentally influenza virus-infected hamsters were treated with 0.25% trypsin and tested for virus antigen by indirect immunofluorescent staining. The results were comparable to those obtained with aceton-fixed cryo-microtome sections. As far as we know, this is the first description of influenza virus demonstration in formalin-fixed, paraffin-embedded tissue after reactivation by trypsin-treatment. This technique may be useful for influenza virus detection in human autopsy cases. It allows an etiological diagnosis even when fresh tissue for cryocut sections or virus cultivation is not available.

Animals↗

Evaluation of oligonucleotide arrays for sequencing of the p53 gene in DNA from formalin-fixed, paraffin-embedded breast cancer specimens.

BACKGROUND: Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. METHODS: DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip; Affymetrix), and the results were compared with the results obtained from direct sequencing. RESULTS: DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016). CONCLUSIONS: A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.

Breast Neoplasms↗

Fluorescent in situ hybridization (FISH) on paraffin-embedded placental tissues as an adjunct for understanding the etiology of early spontaneous abortion.

OBJECTIVES: An investigation of first-trimester spontaneous abortions (SAs) for those cases in which karyotype is not available was designed to test the efficiency of fluorescence in situ hybridization (FISH) on paraffin-embedded tissues combined with pathological examination for understanding the etiology of SAs. METHODS: Pathological examination of 202 placental tissues from SAs was performed. FISH analysis was then carried out on paraffin-embedded tissue sections from the same abortion products with probes specific for chromosomes 13, 16, 18, 21, X, Y. RESULTS: FISH could be achieved in 196 cases (97%). After pathological analysis alone, the etiology of SAs was evoked in 40 cases. The suspected diagnosis was confirmed by FISH in 26 cases (13.2%). After combined pathological and FISH analysis, the etiology of SAs was identified in 83 from the 196 cases (42.3%) with the probe set used. CONCLUSION: The present study demonstrates the value of FISH on paraffin-embedded tissues as an adjunct for understanding the etiology of SAs for those cases in which karyotype is not available. Combination of pathological and FISH analysis increases the yield of diagnosis by a factor of 3.2. The results also demonstrate that predictions of the karyotype from pathological examination should be avoided.

Abortion, Spontaneous↗

Isolation of nucleic acid from paraffin embedded tissue for PCR amplification and sequencing of TcR V beta genes.

PCR analysis of DNA and RNA from fresh and frozen tissue is now a routine practice in most laboratories. Furthermore DNA extracted from paraffin embedded tissue is routinely used for clonality studies and for HLA typing. However the use of RNA from paraffin embedded tissue is more problematical due to degradation and only short sequences are suitable for amplification. In this report we examine the suitability of extracted nucleic acid from paraffin embedded tissue for the PCR amplification of TcR V beta genes and sequencing of resultant PCR products.

Amino Acid Sequence↗

Improved immunohistochemical detection of p53 protein in paraffin--embedded tissues reveals elevated levels in most head and neck and lung carcinomas: correlation with clinicopathological parameters.

We have analyzed the expression of the p53 tumor suppressor gene in paraffin-embedded sections of normal and malignant head and neck and lung tumors by immunohistochemistry using the PAb 1801 monoclonal antibody (MAb). The PAb 1801 does not consistently detect its p53 epitope in tissue fixed in 10% buffered formaldehyde. However, the antibody is effective in AMeX-fixed specimens, thereby permitting the improved morphologic localization of p53 phosphoprotein in paraffin embedded tissue. Of 33 primary head and neck carcinomas analyzed from AMeX-fixed, paraffin-embedded sections, 21 (64%) showed heterogeneous staining with PAb 1801. All 33 normal samples of head and neck tissues were negative. Similarly, 13 out of 20 lung carcinomas (65%) showed heterogeneous staining while none of normal lung tissues were positive. The data indicate a strong positive correlation between p53 detection by PAb 1801 and carcinomas of the head and neck and of lung. However, there was no obvious correlation between p53 staining and the number of involved nodes, the stage of disease or the degree of differentiation in these carcinomas.

Biomarkers, Tumor↗

Chemical characterization of a lambda I amyloid protein isolated from formalin-fixed and paraffin-embedded tissue sections.

Amyloid protein was isolated from formalin-fixed paraffin-embedded heart tissue sections from a patient with primary (AL) amyloidosis by extraction with 6 M guanidine HCl. SDS-PAGE analysis of extracted material showed a major band at 16 kDa and a minor band at 18 kDa. Edman degradation analysis before and after pyroglutamate aminopeptidase treatment showed that the amyloid protein contained N-terminal pyroglutamic acid and was derived from an immunoglobulin lambda light chain. Analysis of tryptic peptides from the extract identified the amyloid protein as a lambda I. Of particular interest is that almost the entire amyloid protein amino acid sequence could be obtained from the cardiac sections. These results demonstrate that formalin-fixed paraffin-embedded tissue sections can be used for extensive biochemical characterization of amyloid proteins and will become a valuable source for isolation and extensive biochemical characterization of amyloid proteins as they are now a valuable source for isolation of DNA for genetic analysis.

Amino Acid Sequence↗

Identification of MEN1 mutations in sporadic enteropancreatic neuroendocrine tumors by analysis of paraffin-embedded tissue.

Gastrinomas and other gastrointestinal neuroendocrine tumors may occur sporadically or as part of the inherited syndrome multiple endocrine neoplasia type 1 (MEN1). Mutations in the recently identified MEN1 gene have been described in sporadic gastrinomas and insulinomas. This study describes techniques used to identify mutations in the MEN1 gene in DNA extracted from paraffin-preserved tissue. Two novel mutations are identified in the MEN1 gene from nine archived paraffin-embedded neuroendocrine tumors, demonstrating that retrospective genetic analysis can be used to identify mutations in the MEN1 gene from preserved tissue. Conditions are provided by which paraffin-embedded tissue can be used as a source of genetic material for sequence information of sufficient quality for mutational studies of the MEN1 gene. It should also be possible to apply this retrospective genetic analysis of paraffin-embedded tissue to other disease models.

DNA, Neoplasm↗

CD163: a specific marker of macrophages in paraffin-embedded tissue samples.

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody's usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163- with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163-. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.

Antigens, CD↗