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PubMed2004

[Diagnostic significance and clinical application of specific chimeric genes in soft tissue sarcomas by RT-PCR using paraffin-embedded tissues: a study of 103 specimens].

OBJECTIVE: To investigate the expression of the chimeric genes resulting from the specific chromosomal translocations in soft tissue sarcomas (STS) and its diagnostic significance for STS. METHODS: The variety of fusion transcripts were detected in 103 cases of STS, including 30 cases of synovial sarcoma (SS), 15 cases of rhabdomyosarcoma (RMS), 25 cases of Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/pPNET), 12 cases of dermatofibrosarcoma protuberans (DFSP), 14 cases of aveolar soft part sarcoma (ASPS), 3 cases of leiomyosarcoma (LMS), 2 cases of malignant fibrous histocytoma (MFH), and 2 cases of fibrosarcoma (FS); and 20 cases of control tumors by reverse transcription-polymerase chain reaction (RT-PCR) using formalin fixed, paraffin embedded specimens. RESULTS: Of the 34 cases of SS 28 (93.3%) expressed SSX-SYT chimeric transcripts (14 were positive for SYT-SSX1, 9 for SYT-SSX2). Four of the six cases of alveolar RMS had a PAX3/PAX7-FKHR fusion transcript. None of the 9 cases of embryonic and polymorphic RMS expressed PAX3/PAX7-FKHR. Of the 25 cases of ES/pPNET, 19 were positive for EWS-FLI1 fusion transcript and 1 for EWS-ERG fusion transcript. COL1A1-PDGFB fusion transcript was expressed in 8 of the 12 cases (66.7%) of DFSP. Of the fourteen cases of ASPS, ten expressed ASPL-TFE3 fusion transcript. None of the 3 cases of LMS, 2 cases of MFH, 2 cases of FS, and 20 control cases contained any of the fusion transcript. CONCLUSION: Chimeric gene transcript resulting from specific chromosomal translocations is a reliable index for the molecular diagnosis of STS and RT-PCR assay for detection of specific fusion gene provides a useful tool for confirmation of the diagnosis of STS in diagnostically difficult cases and in retrospective studies.

Artificial Gene Fusion↗

PubMed1997

Lymphoid infiltrates in B cell non Hodgkin's lymphoma: comparing nuclear characteristics between lymph node and bone marrow; and evaluating diagnostic features of bone marrow infiltrates in paraffin embedded tissues.

Distinguishing non Hodgkin's lymphoma from benign lymphoid aggregates in bone marrow is well recognised to be difficult. Our objective was to evaluate nuclear morphology, and to perform morphometry on benign and neoplastic lymphoid infiltrates, to establish if objective criteria were of value in the diagnosis of neoplasia. By comparing neoplastic infiltrates in bone marrow with infiltrates in lymph nodes, the validity of grading non Hodgkin's lymphoma on the basis of bone marrow histology alone was assessed. 82 cases of B cell non Hodgkin's lymphoma (44 low grade and 38 high grade), known to have both lymph node and bone marrow involvement at the time of presentation, were compared with bone marrow trephines containing reactive lymphoid infiltrates. The results suggest that in paraffin embedded tissue from bone marrow trephines, nuclear morphology, nuclear area and the nuclear contour index cannot, in most cases, be used to distinguish reactive from neoplastic lymphoid infiltrates although mean nuclear area supports a diagnosis of a neoplastic infiltrate if it can be shown that the nuclei are larger than would be expected in reactive infiltrates. Such differences are subtle and often not appreciable without the use of quantitative techniques. The frequent occurrence of discordant infiltrates in high grade lymphomas means grading of lymphoma on the basis of bone marrow appearances is often unrealistic.

B-Lymphocytes↗

PubMed1993

Methodology of immunohistological detection of oestrogen receptor in human breast carcinoma in formalin-fixed, paraffin-embedded tissue: a comparison with frozen section methodology.

We describe a method of immunohistochemically assessing estrogen receptor status on routinely processed formalin-fixed tissue, using a commercially available monoclonal antibody (Abbott H222), with pronase predigestion of tissue sections and overnight antibody incubation. The staining was assessed using the H score system. A series of 94 cases of breast cancer were analysed and the results were compared with assessment by oestrogen receptor immunocytochemical assay performed on frozen section. Direct comparison of the paired sets of H scores obtained with frozen tissue and formalin-fixed tissue showed a highly significant correlation of 0.8 (P < 0.001) between the two methods of oestrogen receptor assessment. Chi-squared analysis using H score cut off points of 50 and 100 also showed a similar significant association (P < 0.001). We conclude that this oestrogen receptor method, applicable to formalin-fixed, paraffin-embedded tissue, gives accurate results on routinely fixed tissue and could be used as an alternative to other methods.

Adenocarcinoma↗

PubMed1989

Primary invasive and in situ vaginal carcinoma. Flow cytometric analysis of DNA aneuploidy and cell proliferation from archival paraffin-embedded tissue.

Eleven invasive and five non-invasive primary vaginal carcinomas were studied by DNA flow cytometry using archival paraffin-embedded tissue as starting material. Overall frequency of DNA aneuploidy in the invasive carcinomas was 8/11 (73%). DNA aneuploidy occurred in all four advanced stage (III-IV) and in 4/7 (57%) of the early stage (I-II) carcinomas. Among the squamous cell carcinomas aneuploid DNA content was also associated with non-keratinizing tumor type. Invasive vaginal carcinomas showed a high median S-phase fraction (SPF) (18.4%, range 6.9-31.8%). High SPF values were associated with advanced stage and non-keratinizing tumors. Corrected 5-year survival rate in invasive vaginal cancer was 44%, with no significant relation to DNA ploidy or SPF. In situ carcinomas were almost as often DNA-aneuploid (3/5, 60%) as the invasive carcinomas and had comparable median SPF value (13.4%, range 5.5-24.6). One in situ carcinoma with a high DNA-index and SPF relapsed, but overall 5-year survival rate was 100%. In conclusion, both invasive and in situ vaginal carcinomas frequently contain DNA-aneuploid stemlines and show a high SPF. Although DNA aneuploidy and high SPF correlate with advanced stage and non-keratinizing tumor type, they do not have much prognostic relevance in vaginal neoplasia.

Aneuploidy↗

PubMed1996

Comparative genomic hybridization analysis of archival formalin-fixed paraffin-embedded uveal melanomas.

Comparative genomic hybridization (CGH) was used to analyze seven autologous uveal melanomas with both formalin-fixed, paraffin-embedded and fresh-frozen specimens. In addition, DNA from two archival formalin-fixed tumors more than 45 years old were also analyzed. The most frequent genetic changes were loss of chromosome 3; increase in copy number of 6p and loss of 6q; and increase in copy number of 8q. A comparison of CGH data from the fresh-frozen tumors and their autologous formalin-fixed tumors revealed a correlation coefficient of 0.83. Comparative genomic hybridization (CGH) analysis of 45-year-old specimens identified genetic changes similar to those found in more recent tumors including loss of chromosome 3 and increase in copy numbers of 6p and 8q. The results indicate that there is a good agreement between data obtained from formalin-fixed and fresh-frozen specimens using CGH. Furthermore, the results demonstrate the applicability of this technique in analyzing archival formalin-fixed tumors that were previously not accessible to cytogenetic analysis.

Analysis of Variance↗

PubMed1990

Malignant pleural mesothelioma and epidermal growth factor receptor (EGF-R). Relationship of EGF-R with histology and survival using fixed paraffin embedded tissue and the F4, monoclonal antibody.

The expression of epidermal growth factor receptor (EGF-R) in 34 formalin fixed paraffin embedded specimens of malignant mesothelioma was examined using the F4 antibody. Eight samples of reactive pleura showed homogenous cytoplasmic staining with the antibody. EGF-R positive cells (greater than or equal to 5%) were found in 68% of the mesotheliomas examined. EGF-R positivity was more commonly seen in the epithelial histological subtype than in the sarcomatous or mixed subtypes. Patients with less than 5% of mesothelioma cells staining positive for EGF-R had a significantly shorter survival (median 299 days) compared with patients whose tumours had a greater number of cells positive for EGF-R (median 446 days) (P = 0.04). However, when the histological subgroup was also taken into consideration (epithelial type had a significantly longer survival than the sarcomatous or mixed) the survival difference in relation to EGF-R positivity was no longer significant (P = 0.08). EGF-R could not be used to distinguish between malignant and benign mesothelial tissue and was not an independent prognostic factor for survival.

Adult↗

PubMed2003

The accuracy of telediagnosis of frozen sections is inferior to that of conventional diagnosis of frozen sections and paraffin-embedded sections.

The accuracy of telepathology diagnosis and conventional diagnosis of frozen sections was compared, using the diagnosis established on paraffin-embedded tissue as a reference. Out of a total of 270 cases, remote frozen-section diagnosis was correct in 227 cases (84.1%) and incorrect in 23 cases (8.5%). The latter comprised 12 false positive diagnoses of malignancy (4.4%) and 11 false negative diagnoses (4.1%). A diagnosis was not possible in 20 cases (7.4%). In contrast, the conventional frozen-section diagnosis was correct in 269 cases (99.6%) and incorrect in 1 case (0.4%), the latter being a false negative diagnosis. The average time needed to make a remote diagnosis was 14.2 min (SD 9). Manual examination was not found to be essential for remote frozen-section diagnosis. Overall slide quality was rated as 'satisfactory' to 'fair' by the six pathologists concerned. An improvement in the quality of slides is necessary to guarantee an acceptable level of accuracy of remote frozen-section diagnosis; a shortening of the time needed for diagnosis is a further requirement for the successful implementation of a routine telepathology frozen-section service.

False Positive Reactions↗

PubMed1989

Monoclonal antibody to a proximal nephrogenic renal antigen: immunohistochemical analysis of formalin-fixed, paraffin-embedded human renal cell carcinomas.

An IgG1 murine monoclonal antibody (PN-15) to an epitope preserved in formalin-fixed, paraffin-embedded tissue sections of renal cell carcinomas was developed following immunization of mice with the microsomal fraction of human renal cortical tissue homogenates. An immunohistochemical screening procedure was utilized against fixed, embedded tissue sections of renal cell carcinomas. This antibody identified a Mr 200,000 determinant on immuno-Western blot analysis, and the recognized epitope was shown to reside in the carbohydrate domain of a Mr 200,000 glycoprotein (gp200). Analysis of its tissue distribution was performed by indirect immunoperoxidase staining of fixed, embedded tissue sections of the following: 36 different types of normal tissues; 45 primary and 38 metastatic renal cell carcinomas; and 124 cases of 39 different types of neoplasms. In the normal kidney, gp200 was localized along the brush border of the proximal tubule and the luminal surface of Bowman's capsule adjoining the outgoing proximal tubule. Shared tissue localization was also noted in breast lobules and ducts, parathyroid gland, and epididymis. The gp200 was expressed by 93% of primary and 84% of metastatic renal cell carcinomas, primarily on the cell surface membrane. Shared antigenic expression was noted in relatively few other tumors including: mammary carcinomas; teratocarcinomas; and parathyroid adenomas. The PN-15 monoclonal antibody holds promise as a useful reagent in retrospective investigations of fixed, embedded cases of renal cell carcinomas. The antibody may also be of practical utility in diagnostic immunohistochemistry by helping to retrospectively define the primary site of metastatic clear cell tumors, especially when fresh frozen tissue from such tumors is not available.

Antibodies, Monoclonal↗

PubMed1988

Nuclear DNA content in prostatic carcinoma measured by flow cytometry: a retrospective study on paraffin-embedded tissue.

The nuclear DNA content in 67 prostatic carcinomas was measured by flow cytometry of nuclear suspensions prepared from paraffin-embedded tissue blocks. A method was developed that provided nuclear suspensions of good quality whether tissue blocks from transurethral resections or transvesical prostatectomies were used. Thirty (43.7%) of the tumors were found to be aneuploid. No significant correlation between clinical stage or histological grade and aneuploidy was demonstrated.

Aged↗

PubMed1993

Caveats in the estimation of DNA-ploidy in paraffin embedded specimens of primary prostate cancer and lymph node metastases by flow and image cytometry.

DNA ploidy assessments of 72 archival paraffin embedded specimens were made utilizing flow cytometry (FCM) and image cytometry (ICM). Forty four specimens were obtained from primary prostatic tumours and 28 from metastatic lymph nodes. Eighteen of 30 FCM diploid range tumours were aneuploid by ICM. Nineteen of 22 FCM tetraploid range tumours were ICM aneuploid. All FCM non-diploid range specimens were non-diploid range by ICM, and all ICM diploid range specimens were diploid range by FCM. ICM more easily identifies small numbers of aneuploid cells than FCM. Discrepancy between FCM and ICM measurements was particularly high in lymph node specimens. FCM diploid range distributions mostly occurred in lymph nodes with a small tumour fraction whereas the number of aneuploidy specimens increased with larger tumour volume. Lymphocytes are different from solid tumour cells since they are not fixed in tissue and will probably be more easily dissolved in cell suspension when sections are disintegrated. This may influence FCM measurements and has to be considered when DNA ploidy measurements of lymph node metastases are made.

Adenocarcinoma↗

PubMed2004

HOPE technique enables Western blot analysis from paraffin-embedded tissues.

In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

Blotting, Western↗

PubMed2004

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Acetic Acid↗

PubMed1993

[Variation of the diploid pattern in tissues used as normal controls of solid tumors: advantages and disadvantages of flow cytometric analysis of paraffin-embedded tissues].

The aim of the present work was to study the factors that influence the variation of diploid patterns in 75 areas of histologically normal gastric tissue, located in surgically resected stomachs due to early gastric cancer. Each sample was used as a diploid reference of its respective tumoral area. Fifty five samples were embedded in formalin and 20 corresponded to fresh tissue. It was found that in the older tissues, the cellular debris increased, the localization channel of the peaks shifted to the left (lower than channel 60) and the peak shape was deformed, with an increase in the variation coefficient (around 6) and an elevation and lengthening of phase S. When fresh tissue was used, the first peak was localized around channel 60-70, with a significant reduction of cellular debris, a lower variation coefficient amplitude (2-5) and a phase S adhered to the basal line. Aiming to observe the influence of paraffin embedding over time, 21 tissue samples originally assayed as fresh tissues, were reanalyzed one year later. The same alterations initially observed in old tissues were again noticed. According to these results, it is necessary to select adequate controls for flow cytometric analysis to obtain an adequate histogram interpretation, and specially to calculate the ADN index.

Adult↗

PubMed1994

Detection of canine parvovirus DNA in paraffin-embedded tissues by polymerase chain reaction.

Canine Parvovirus (CPV) is seemingly a 'new' virus which suddenly appeared during the mid-1970's in an epizootic of disease in dogs. The virus is very similar to the feline panleukopenia virus (FPV), and recent studies have underlined the possible emergence of CPV as a variant of a virus from some other carnivore--possibly from FPV (Parrish, 1990). Several conserved amino-acid changes between CPV and FPV isolates have been defined by cloning and sequencing the capsid-protein gene. An alternative to cloning and sequencing the entire capsid-protein gene would be to use PCR amplification of short regions of the gene containing the appropriate variable amino-acid codons. In addition, use of PCR would also facilitate the study of virus samples which cannot be recovered as infectious agents, e.g. after having undergone formalaldehyde fixation and paraffin-embedding procedures. This study reports on the amplification of CPV DNA from 15-year-old tissue sections which have been prepared by formaldehyde or paraformaldehyde-lysine-periodate-glutaraldehyde fixation, using PCR with various primer pairs within the capsid-protein gene of CPV.

Animals↗

PubMed2001

Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

DNA↗

PubMed2001

Cellular localization of BM88 mRNA in paraffin-embedded rat brain sections by combined immunohistochemistry and non-radioactive in situ hybridization.

With the advent of gene cloning and sequencing, it has become increasingly common to identify novel genes for which no antibody is available. The best approach to study the expression and the distribution of these new genes is by in situ hybridization. One of the challenges with this method is to define the exact cellular subtype where the gene of interest is expressed. Conventional isotopic in situ hybridization methods lack precision for cellular identification because radioactive probes often result in a scattered signal. To identify the exact cellular subtype expressing BM88, we established a rapid colocalization method using non-isotopic in situ hybridization followed by chromogenic immunohistochemistry on the same tissue section. We demonstrated that BM88, which was identified from subtractive hybridization experiments between normal and ischemic tolerant brain tissue, was expressed exclusively in neurons in normal adult rat brain. Paraffin-embedded tissue was used as it resulted in better preservation of tissue and cellular morphology, thus allowing for more accurate histological localization of gene expression. It also allowed for retrospective studies on a number of archived tissue samples.

Animals↗

PubMed1993

Evaluation of sensitivity, specificity, and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T-cell receptor genes in formalin-fixed, paraffin-embedded sections.

Following our recent reports of detecting clonal immunoglobulin and T-cell receptor gene rearrangements by the polymerase chain reaction, we have improved and simplified the technique for use in diagnostic histopathology laboratories and determined, on coded samples, the sensitivity, specificity, and reproducibility of the modified methodology in distinguishing malignant lymphoma from reactive lymphoid hyperplasia and nonlymphoid tumors. Using only three primer pairs for the immunoglobulin heavy chain and T-cell receptor beta and gamma chain genes on well-characterized lesions of widely varying morphology and immunophenotype, clonal rearrangements were detected in 65% of B-cell lymphomas, and 77-82% of T-cell tumors. Specificity and observer consistency ranged from 93-97%. The method requires very careful control, particularly to avoid misinterpretation of results because of contamination and nonspecific amplification, but in its present form is relatively simple and inexpensive, and gives results on single paraffin-embedded sections within 24 h.

Antibody Specificity↗

PubMed1998

New monoclonal antibodies to the T cell antigens CD4 and CD8. Production and characterization in formalin-fixed paraffin-embedded tissue.

We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli. Purified fusion protein was used to immunize mice. The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue. In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed. In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.

Antibodies, Monoclonal↗