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Expression of nerve growth factor receptor in paraffin-embedded soft tissue tumors.

Identification of growth factors and receptors in mesenchymal tumors may be crucial to understanding of growth regulation in sarcomas. During an immunohistochemical study of the expression of growth factors and receptors in human soft tissue tumors (STT), only 1 antisera capable of working in paraffin-embedded tissue was noted. A detailed study of 141 STT was undertaken to determine the frequency of expression of nerve growth factor receptor (NGF-R), its specificity and sensitivity for neural tumors, and the effect of fixation on detection. In normal mesenchymal tissue, only nerve sheath and perivascular staining was seen. No immunoreactivity was seen in many tumors including rhabdomyosarcoma, angiosarcoma, liposarcoma, Ewing's sarcoma, and alveolar soft part sarcoma. Less than 15% of tumors of smooth muscle, fibrous, or fibrohistiocytic origin showed immunoreactivity, usually focal. In contrast, a high frequency of immunoreactivity was noted in tumors of neural origin (74%). This included granular cell tumors (100%), Schwannoma/neurofibroma (91%), malignant Schwannoma (78%), neuroblastoma/neuroepithelioma (60%), and paraganglioma (57%). A high rate of reactivity was also seen in synovial sarcomas (80%), undifferentiated sarcomas (60%), and hemangiopericytomas (43%), suggesting a potential relationship to the neural phenotype. Among the neural tumors, Bouin's fixation was superior to formalin, suggesting that immunoreactivity for NGF-R is affected by fixation. This antibody may be a useful adjunct marker diagnostically.

Histological Techniques↗

Flow cytometric analysis of DNA ploidy level in paraffin-embedded tissue of non-small-cell lung cancer.

Investigations regarding the prognostic value of DNA content (ploidy) and proliferative characteristics [percentage of cells in S-phase or S-phase fraction (SPF)] have been greatly facilitated by the application of flow cytometry (FCM) using nuclei isolated from paraffin-embedded tissue. We have applied this technique to tumor sections from patients presenting with non-small-cell lung cancer (NSCLC) in 1980 and 1981. From 67 out of 115 patients material of sufficient quantity and quality was obtained to perform DNA-FCM. A multivariate analysis including stage of disease (UICC), age, tumor histology and treatment modality was performed to examine the prognostic significance of DNA-FCM in NSCLC. Aneuploidy was found in 65% of cases. In our study, the DNA content was not related to histology, stage of disease or treatment modality, nor to the length of survival (log rank test P = 0.62). Calculation of SPF was possible in 49/67 cases. The SPF was not related to histology, stage of disease or treatment modality, but a significant prognostic value was found for survival; patients with a high SPF died earlier (P = 0.04) and this was especially true for squamous cell carcinoma (P = 0.02). This study demonstrates the prognostic importance of DNA-FCM-derived information in NSCLC using a multivariate analysis; however further prospective studies in larger patient populations are needed.

Adenocarcinoma↗

Warthin's tumor with malignant lymphoma. DNA analysis of paraffin-embedded tissue.

The authors report a case of malignant lymphoma, small lymphocytic type, involving the lymphoid stroma of a Warthin's tumor of the parotid gland. This was confirmed by the presence of a monoclonal immunoglobulin heavy chain gene rearrangement, demonstrated by Southern blot hybridization of DNA extracted from paraffin-embedded tissue. Similar techniques showed only germline immunoglobulin gene bands in two control cases of Warthin's tumor.

Adenolymphoma↗

bcl-2 gene rearrangement analysis of Japanese follicular lymphomas by polymerase chain reaction in formalin-fixed, paraffin-embedded tissue specimens.

The prevalence of chromosomal translocation t(14;18) was examined in 41 cases of follicular lymphoma covering all histopathological subtypes, using the polymerase chain reaction and hybridization method with non-radioactive oligonucleotide probes. DNAs were extracted from unfixed fresh-frozen and/or formalin-fixed paraffin-embedded biopsy specimens. The t(14;18) chromosomal translocation, resulting in bcl-2-JH (immunoglobulin heavy chain gene joining segment) fusion gene, was detected in 7 (36.8%) of 19 follicular small cleaved cell lymphomas and 6 (54.5%) of 11 follicular mixed, small cleaved cell and large cell lymphomas. Meanwhile, 11 cases of follicular large cell lymphoma revealed no bcl-2-JH fusion gene. There was a statistically significant difference in the frequency of bcl-2-JH rearrangement between follicular, small cleaved cell and large cell lymphoma (two-sided Fisher exact test, P = 0.049). The difference of bcl-2 rearrangement frequency between follicular, mixed and large cell lymphoma was also statistically significant (P = 0.012).

Base Sequence↗

Identification of rhinopharyngeal plasmacytic tumours by immunofluorescence on paraffin-embedded sections.

Solitary myeloma is an uncommon lesion which may occur in many organs, mainly in the rhinopharynx. This lesion usually appears as non-secretory, and it is constituted by either typical plasma cells or large and atypical cells resembling epithelial anaplastic cells. Therefore, and immunofluorescent method on paraffin-embedded sections may be applied to reveal the presence and the monoclonality of the intracytoplasmic Ig in the pathological cells. In this paper we report a study on 22 patients suspected to bear a rhinopharyngeal tumor investigated by immunofluorescence. Moreover, in 4 cases peripheral blood lymphocytes were also studied. The results are discussed on the basis of the modern immunological trends.

Adult↗

Immunochemical demonstration of different antigens in single cells in paraffin-embedded histological sections.

A new modification of the double staining immunochemical technique is described, which is capable of demonstrating the presence of several antigens in single cells at the light microscopical level. This method was employed by the authors in order to detect the simultaneous occurrence of different types of cytoplasmic immunoglobulin chains in neoplastic plasma cell precursors in paraffin-embedded lymph-node specimens from patients with B-cell malignancies.

Animals↗

Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspiramurdochii.

Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.

Animals↗

Anti-BLA.36 monoclonal antibody shows reactivity with Hodgkin's cells and B lymphocytes in frozen and paraffin-embedded tissues.

A newly developed monoclonal antibody, anti-BLA.36, raised against a Hodgkin cell line, was shown to have reactivity with Reed-Sternberg cells and activated B lymphocytes and appears to be distinct from other antibodies which identify antigens of hematopoietic cells. Anti-BLA.36 was evaluated in B5-fixed paraffin-embedded tissue from 16 cases of Hodgkin's disease of various types and 35 cases of non-Hodgkin's lymphomas representative of the different major subtypes. The specificity of anti-BLA.36 was compared with other antibodies that have been used to mark Hodgkin's cells and B lymphocytes: namely, L26, LN-1, Leu-M1 and Ber-H2, as well as UCHL-1, a pan-T lymphocyte marker. In addition, a subset of the cases was evaluated using frozen tissue in order to validate the staining characteristics of anti-BLA.36 as observed in fixed paraffin sections. Anti-BLA.36 was found to react with Hodgkin's cells more consistently than the other antibodies used in this panel. The antibody reacted with an antigen on Reed-Sternberg cells and their variants (Hodgkin's cells) in all four subtypes of Hodgkin's disease, and with a subset of reactive and malignant B lymphocytes, but not with T lymphocytes. It may, therefore, be useful in the evaluation of non-Hodgkin's lymphomas. Finally, this is the first antibody raised to a Hodgkin's cell line which also consistently marks reactive and malignant B cells, but not T cells. The implications of this observation are discussed in relation to the cellular origin of the Reed-Sternberg cell and the overall nature of Hodgkin's disease.

Antibodies, Monoclonal↗

Different opinions on classification of DNA histograms produced from paraffin-embedded tissue.

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin-embedded adrenal adenomas or non-neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.

Adenoma↗

Detection of cytomegalovirus in formalin-fixed paraffin-embedded donor, native and allograft liver tissue using a multiplex polymerase chain reaction-liquid hybridization assay.

A multiplex polymerase chain reaction (PCR)-liquid hybridization assay detecting a minimum 10 fg of cytomegaloviral (CMV) DNA has been developed. This assay successfully detected viral nucleic acids in 25/26 (96.2%) routinely embedded allograft liver biopsies with CMV hepatitis. No CMV DNA was detected in 15 native and 12 donor liver tissues sampled at the time of liver transplantation. These results indicate that archived formalin-fixed, paraffin-embedded liver tissues obtained from patients with significant liver disease can be successfully analyzed for CMV by PCR, retrospectively. Failure to detect CMV in native and donor livers implies that any latent virus present within these tissues is very focally distributed, or is below the threshold of detection applicable to PCR.

Base Sequence↗

Epithelial markers in synovial sarcoma. An immunohistochemical study on paraffin embedded tissues.

Immunohistochemical studies on synovial sarcomas have proved the potentiality of these neoplasm for epithelial and mesenchymal differentiation and antibodies detecting epithelial cells have been found to be helpful in determining the histological types. In this study different epithelial markers directed against various cytokeratins, HMFG-2 and EMA were investigated on paraffin embedded tissues of 13 cases of synovial sarcomas, with regard to their reliability in unmasking the epithelial components demonstrable in this type of neoplasm. The results lead to three conclusions: firstly, synovial sarcomas possess the capacity for generating different epithelial cell types with uncommon compositions of intermediate filaments as well as of membrane proteins, secondly, these features may be expressed in a heterogenous pattern even within the same tumour and finally, the use of wide range anti-cytokeratin antibodies covering the spectrum of basic as well as acidic type proteins seems to be necessary for the detection of all epithelial components demonstrable in synovial sarcomas.

Antibodies↗

Flow-cytometric DNA analysis of paraffin-embedded renal cell carcinoma tissue from patients treated by parenchymal-sparing surgery.

OBJECTIVE: To determine the utility of deoxyribonucleic acid (DNA) ploidy pattern, detected by flow cytometry, in predicting the outcome of renal cell carcinoma (RCC). PATIENTS AND METHODS: DNA content was retrospectively assessed by flow cytometry in 28 primary paraffin-embedded RCCs from 24 patients (16 men and eight women, mean age 61.9 years, range 40-89) who had undergone renal parenchymal-sparing surgery. RESULTS: Tumour recurred locally in three patients, two with diploid tumours and one with aneuploid tumour. Two patients with diploid tumours had metastatic disease 11-58 months after surgery. Only one patient with aneuploid tumour developed metastatic disease. There was no difference in local recurrence or metastases with the type of renal parenchymal-sparing surgery used (enucleation or partial nephrectomy) nor any significant difference in overall survival (P = 0.11). CONCLUSION: While DNA content might be of considerable predictive value for patients with RCC, these results showed that there was no difference in mortality rate, recurrence rate, or type of renal parenchymal-sparing surgery used between aneuploid and diploid tumours.

Adult↗

Hodgkin's disease associated with chronic myeloid leukemia. Determination of bcr-abl rearrangement in paraffin-embedded tumors using the polymerase chain reaction.

A patient showing lymph node enlargement and tumors in the neck and axilla in the course of Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia (CML) was seen. There were no hematological findings suggestive of crisis. RNA was isolated from formalin-fixed and paraffin-embedded tissue specimens for the polymerase chain reaction of bcr-abl. CML-type products were detected in the peripheral blood, but no bcr-abl products were found in the lymph nodes. These findings indicate a very rare case in which Hodgkin's disease developed during the course of CML.

Base Sequence↗

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens.

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.

Adult↗

Ploidy analysis in paraffin-embedded malignant fibrous histiocytoma by DNA cytofluorometry and flourescence in situ hybridization.

To prove the relationship between chromosomal aberration and DNA ploidy in human malignant fibrous histiocytoma (MFH), fluorescence in situ hybridization (FISH) and DNA cytofluorometry were performed in this study. For FISH study, the nucleus of each tumor cell was isolated from paraffin-embedded tissue of nine MFHs. Five chromosome-specific DNA probes (1p36, 1q12, 8q21.3, 11 centromere, and 17 centromere) were hybridized on cell nuclei. Cells with more than three probe signals were regarded as chromosome polysomy. All of the tumors analyzed by FISH had extra copies. The average percentage of polysomy in all tumors was high, ranging from 10.2% to 49.2%. The DNA ploidy patterns, and the percentage of hyperdiploid cells showing a greater DNA content than diploid cells, were obtained from DNA cytofluorometry. Three of nine were diploid patterns and six were non-diploid patterns, and the percentage of hyperdiploid cells in all tumors was high, ranging from 9.1% to 61.9%. The percentage of polysomy could be correlated with the percentage of hyperdiploid cells in each cell. In this study, we found that the DNA ploidy change was closely correlated with aberrations of chromosome copy number in MFH. In addition, the alterations of specific chromosome copy number could be detected in MFH showing diploid cells. Thus, these data indicate that FISH and DNA cytofluorometry are available as a cytogenetic tool for the analysis of interphase nuclei of bone and soft tissue tumors including MFH.

Biopsy↗

An anti-CD3 epsilon serum detects T lymphocytes in paraffin-embedded pathological tissues in many animal species.

The in situ identification of T cells has been difficult and restricted to frozen tissue samples of a limited range of species. In this study, we demonstrate that an antiserum recognizing a phylogenetically conserved part of the CD3 epsilon cytoplasmic tail detects T cells of many avian and mammalian species in formalin-fixed paraffin-embedded tissue sections. This antiserum enables the morphological characterization of normal and pathological lymphoid tissues and lymphoid infiltrations in experimental work and in animal disease.

Animals↗

Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas.

Comparative genomic hybridization serves as a screening test for regions of copy number changes in tumor genomes. We have applied the technique to map DNA gains and losses in 33 cases of formalin-fixed, paraffin-embedded primary breast tumors (13 fibroadenomas and 10 diploid and 10 aneuploid carcinomas). No genomic imbalances were found in fibroadenomas. Recurrent findings in adenocarcinomas include copy number increases for chromosomes 1q (14 of 20 samples), 8q (10 of 20), 17q (5 of 20), 6p (3 of 20), 13q (3 of 20), and 16p (3 of 20), and copy number decreases for chromosomes 22 (7 of 20), 17p (6 of 20), and 20 (3 of 20). Regional high level copy number increases were observed on chromosome bands 1q32, 8p11, 8q24, 10p, 11q13, 12p, 12q15, 17q11-12, and 17q22-24. The majority of the samples were studied for gene amplification of c-myc, c-erbB2, cycD1, and int-2 by means of Southern blot analysis. The comparison with DNA ploidy measurements revealed a different distribution and a significantly higher number of chromosomal aberrations in aneuploid tumors than in diploid tumors and in fibroadenomas.

Aneuploidy↗

Expression of 20 KD homologous restriction factor of complement on myocardial cells: an immunohistochemical study using acetone-fixed and paraffin-embedded tissues.

Expression of 20 KD homologous restriction factor of complement (HRF20) (CD59) in normal human heart was immunohistochemically examined with monoclonal antibody (MAb) 1F5. HRF20 was clearly demonstrated on the cell surface membrane and intercalated discs of myocardial cells throughout the ventricular walls. Therefore, this factor may protect normal cardiomyocytes from complement deposition, which has been demonstrated on infarcted cardiomyocytes. Expression of HRF20 was also observed on endothelial cells of the endocardium and on blood vessels, including arteries, capillaries, and veins, and on the Schwann-cell sheath of peripheral nerve fibers. In the present study, we found that the epitopes of HRF20 were well preserved for immunohistochemistry even in acetone-fixed and paraffin-embedded tissues, as well as in frozen tissues that have been conventionally used for HRF20. This method using paraffin sections allows for easier staining and greater accuracy in histological examination.

Acetone↗