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PubMed1994

Expression of EGF, EGF-receptor, p53, v-erb B and ras p21 in colorectal neoplasms by immunostaining paraffin-embedded tissues.

Immunohistochemical studies were performed to clarify the significance of the expression or overexpression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v-erb B, ras p21 in 23 cases each of tubular adenoma and adenocarcinoma. The expression of EGF, EGFR, p53, v-erb B, and ras p21 in paraffin-embedded tissues, from 46 patients with colorectal tumors (adenoma: 23 cases; 14 mild dysplasia, six moderate dysplasia, three severe dysplasia, adenocarcinoma: 23 cases; 17 well differentiated, two moderately differentiated, three poorly differentiated, one mucinous carcinoma was analyzed immunohistochemically using anti-EGF, EGFR, p53, v-erb B and ras p21 antibodies. The EGF and ras p21 tended to express more strongly in carcinoma cases than in the adenoma cases, and in severe and moderate dysplasia than in mild dysplasia (EGF: stained positive in five adenomas [21.74%] and 17 adenocarcinomas [73.91%]; ras p21: stained positive in six adenomas [26.09%] and 14 adenocarcinomas [60.87%]. The EGFR stained positive in two adenomas (8.70%) and two adenocarcinomas (8.70%). The p53 and v-erb B showed positive staining only in the carcinoma cases (p53: stained positive in four cases [17.39%]; v-erb B: stained positive in eight cases [34.78%]). This study suggests that these factors seem to have some role in the progression of colon neoplasms. It suggests that genetic alteration is not always equal to the overexpression of protein products, but that it reflects them well, and that the staining makes some contribution to differential diagnosis in colorectal neoplasms.

Adenocarcinoma↗

PubMedDate not supplied

[Evaluation of the micro-histological technique for blood clot paraffin embedding in diagnosis by fine-needle aspiration].

Since a certain amount of blood is nearly always present in the material obtained through needle-aspiration, we have considered it helpful to take advantage of this event, in order to increase the information which is drawn from this type of investigation. We have evaluated the material obtained by means of ultrasound guided fine-needle aspiration of 287 lesions of superficial and deep sites, and we have used 1) a microhistologic method, which employs formalin fixation and paraffin embedding of blood clot (while the blood acts as a support of cells and tissue fragments), and 2) the classic smear cytologic method. The percentage of diagnostic accuracy was similar using both methods, but the microhistologic method presented a few advantages, i.e. greater diagnostic reliability, and long preservation of the residual material, which can subsequently be used for further special stains. These considerations are particularly true for hepatic lesions, which are more than a half of all cases.

Adolescent↗

PubMed2005

Detection of papillomavirus proteins and DNA in paraffin-embedded tissue sections.

The key events during the papillomavirus life cycle can be mapped in infected tissue samples by antibody detection and in situ hybridization. The ease of immuno-detection varies for different proteins and is dependent on antigen availability. Epitope exposure is sometimes necessary, because the antigen may become masked after formalin fixation and paraffin embedding of the infected tissue. Visualization of both nucleic acid and protein targets can be done simultaneously by combining in situ hybridization and immuno-detection methods.

Antigens, Viral↗

PubMed2004

Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections.

The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid malignant tumors and its expression has been correlated with disease progression and poor survival. With the advent of targeted therapies, especially IMC-C225 (Cetuximab), a monoclonal antibody (MAb) directed against the EGFR, there is an increasing interest in immunohistochemistry (IHC)-based EGFR screening methods using paraffin-embedded tumor specimens to select cancer patients eligible for treatment with Cetuximab. With the EGFRpharmDX kit, a complete assay for demonstration of EGFR is now available. Because no information about the preservation of the EGFR under various conditions of fixation is available, we performed a prospective study on a panel of commonly used fixatives to determine optimal tissue preservation protocols. The stability of the epitope on cut tissue sections stored for a period up to 24 month was also tested using material originating from patients with head and neck cancer, non-small-cell lung carcinomas, and colorectal adenocarcinomas. Depending on the fixative used and the time of storage of cut tissue sections, a variation in the determined level of EGFR expression was demonstrated compared with the most optimal fixation procedure.

Adenocarcinoma↗

PubMed1990

Detection and typing of papillomavirus DNA in formaldehyde-fixed paraffin-embedded tissue.

Clinical specimens from nine patients with papillomatosis of the vocal cords and three patients with vocal cord polyps were evaluated for the presence of human papillomavirus (HPV) DNA using two complementary molecular hybridization techniques. In one method, involving polymerase chain reaction (PCR) amplification, HPV DNA sequences were replicated in vitro from tissue DNA extracted from paraffin sections prior to hybridization. Polymerase chain reaction amplification was compared with the standard method of Southern blot hybridization. Results of the two techniques for all nine laryngeal papillomas agreed completely: five patients harbored HPV type 6 and four HPV type 11. Both PCR amplification and Southern blot hybridization found two of the three polyps to be free of HPV infection, while PCR detected HPV type 18 in one polyp specimen that was reported negative by Southern blot hybridization, suggesting a greater sensitivity of PCR. Our results demonstrate that PCR amplification is as reliable and at least as sensitive as Southern blot hybridization. Moreover the PCR technique opens the way to the undertaking of a whole variety of retrospective studies using formaldehyde-fixed paraffin-embedded tissues.

Blotting, Southern↗

PubMed1995

E-cadherin expression in bladder cancer using formalin-fixed, paraffin-embedded tissues: correlation with histopathological grade, tumour stage and survival.

To determine the potential prognostic value of epithelial cadherin (E-cadherin), a Ca(2+)-dependent cell-cell adhesion molecule, we have analysed its immunoreactivity and cellular localisation in 67 transitional cell carcinomas (TCC) using an avidin-biotin immunoperoxidase technique on formalin-fixed, paraffin-embedded tissues. These results were correlated with histopathological grade, tumour stage, presence of metastases and survival. In addition, 10 cystitis and 11 normal bladder biopsies were evaluated as controls. E-cadherin was expressed in a normal membranous pattern in all normal and 7 of 10 cystitis biopsies. Loss of normal surface E-cadherin expression was found in 3 of 15 superficial tumours and in 48 of 52 invasive cancers. Abnormal immunoreactivity was strictly related to tumour differentiation and stage. Fifteen of 20 well-differentiated (grade I) tumours showed preserved membranous E-cadherin immunoreactivity, while 46 of 47 moderate and poorly differentiated tumours (grades II and III) demonstrated abnormal staining patterns. Loss of membranous E-cadherin immunoreactivity was also associated with advanced tumour stage. There was a significantly higher 5-year survival rate for patients with preserved membranous staining compared with patients with abnormal staining.

Cadherins↗

PubMed1994

Adenovirus myocarditis: retrospective diagnosis by gene amplification from formalin-fixed, paraffin-embedded tissues.

Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viruses in clinical material. Recent advances in molecular techniques permit direct detection of viral-specific nucleic acid in tissue samples. This report describes a protocol for DNA extraction and amplification of adenovirus genome from formalin-fixed, paraffin-embedded human tissues that detects as little as 10 copies of viral genome in a background of 0.5 micrograms of human DNA. This sensitive assay permitted the examination of archived tissues to establish a retrospective diagnosis of adenoviral myocarditis in two pediatric patients.

Adenovirus Infections, Human↗

PubMed1984

Hepatic involvement in hairy cell leukemia: diagnosis by tartrate-resistant acid phosphatase enzyme histochemistry on formalin fixed and paraffin-embedded liver biopsy specimens.

The leukemic infiltrate in the liver in hairy cell leukemia may be easily overlooked in conventional histological slides and an exact classification may not always be possible. In order to facilitate the identification of tricholeukocytes , it is suggested to perform the tartrate-resistant acid phosphatase stain on routinely formalin fixed and paraffin-embedded liver biopsy specimens. Thereby it will be in most cases possible to demonstrate sufficiently enough enzyme activity to establish unequivocally the diagnosis of hairy cell leukemia.

Acid Phosphatase↗

PubMed1994

Alteration of immunoreactivity by hydrated autoclaving, microwave treatment, and simple heating of paraffin-embedded tissue sections.

The effects of treatment in a hydrated autoclave (121 degrees C, 2 atm for 20 min), microwave oven (in water), and simple heating (60 degrees C overnight in distilled water or 90 degrees C for 10 min in ZnSO4) on the stainability of 56 antigens by commercially available antibodies in formalin-fixed paraffin-embedded tissue sections were evaluated. The detectability of nuclear antigens, glycoprotein, lymphocytic surface markers, and chromogranin A was significantly and reproducibly improved by these treatments, whereas the detectability of viral antigens and peptide hormones was attenuated or unchanged. This enhancement includes not only the distinctiveness of the positive staining, but also the number of positive cells, as revealed by comparing serial sections. Among these four heating procedures, microwave heating and autoclaving were more effective than the others on p53, c-erbB-2, and CA125, whereas simple heating was best for smooth-muscle actin (HHF35 and CGA7). Generally the effects of the heating procedures for these antigens were consistent among the cases, but the effects on GFAP varied with the case. The alterations we observed could significantly influence the interpretation of immunohistochemical staining of currently popular tumor markers such as p53 in terms of their prevalence (28% vs 64% in gastric cancer; 36% vs 82% in metastatic liver cancer) and other diagnostically important markers.

Antigen-Antibody Reactions↗

PubMed1989

Supernumerary chromosome 1 in interphase nuclei of atypical germ cells in paraffin-embedded human seminiferous tubules.

The so-called atypical germ cells or cells of carcinoma in situ morphologically resemble neoplastic cells in seminoma. Since seminomas show numerical aberrations of chromosome 1 we have used a DNA probe specific for chromosome region 1q12 to determine whether such aberrations can be detected in atypical germ cell nuclei in paraffin-embedded seminiferous tubules as well. One-third of intratubular nuclei, containing atypical germ cells, consistently showed three hybridization signals in contrast to two signals regularly observed in normal intestine and in spermatogonia. We thus show that cytogenetic studies of precancerous cells can be performed directly on the tissue where these cells originate.

Chromosome Aberrations↗

PubMed1982

Demonstration of lactoperoxidase in paraffin-embedded lactating bovine breast by immunocytochemical techniques.

Immunocytochemical methods have been applied to the demonstration of lactoperoxidase (LP) in the lactating bovine breast. Trypsin digestion and a MAGIC (Wachsmuth ED: Histochemie, 37:251, 1973) procedure, using anti-LP serum and then LP solution in sequence, made the sensitive and specific demonstration of LP in formalin-fixed paraffin-embedded sections possible. Peroxidase of the breast has been shown to be inducible by estrogen stimulation and it is considered to be a possible marker of estrogen dependency in breast carcinomas. The present technique, applied to human tissues, even on fixed and embedded biopsies, might make possible the detection of LP in human breast cancers and, thereby, an evaluation of estrogen dependency of such cancers.

Animals↗

PubMed1991

Quantification of estrogen receptors on paraffin-embedded tumors by image analysis.

Emphasis on early detection of breast carcinomas has increased the number of instances in which an insufficient amount of tissue is available for biochemical estrogen receptor (ER) assay. Image analysis, used together with immunohistochemistry, introduces the possibility of quantifying molecules, such as ER, in routinely processed tissues. To explore that possibility, sections from 40 formalin-fixed, paraffin-embedded breast carcinomas with biochemically determined ER values were reacted with H222 anti-ER antibody (ER-ICA) and quantified on a CAS 200 image analysis system. A minimum of ten fields comprising at least 15,000 microns 2 of nuclear area were analyzed in each case. If the antigen distribution were not homogeneous, proportional sampling of the different tumor areas was carried out. Of the 31 ER-positive (10 to 344 fmol/mg) tumors, 27 (87%) were immunoreactive by the ER-ICA assay (greater than 5% positive nuclear area), which represents an improvement over simple microscopic evaluation (68%). Bivariate analyses showed statistically significant, albeit only modest, correlation between ER values and the percentages of positive area (r = 0.556) and positive stain (r = 0.518). The following obstacles were found to interfere with a proper correlation: (a) antigen loss during fixation and processing; (b) intratumoral antigenic heterogeneity; and directly associated with it, (c) interobserver variability. Uniform tissue handling or, alternatively, the use of internal controls to compensate for fixation-induced differences, together with thorough assessment of the tissue, should reduce these obstacles and facilitate accurate and reproducible quantification.

Antibodies, Monoclonal↗

PubMed1987

Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides.

The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.

Base Sequence↗

PubMedDate not supplied

Detection of Dirofilaria (Nochtiella) repens DNA by polymerase chain reaction in embedded paraffin tissues from two human pulmonary locations.

We here report two new cases of human pulmonary dirofilariasis in men aged 62 and 64 from Northern and Central Italy, respectively. Both were asymptomatic and the infections were discovered incidentally when chest radiographs taken for another reason revealed the presence of a coin lesion. The initial clinical diagnosis was oriented towards a lung tumour, and an excisional lung biopsy after thoracotomy was necessary for the final diagnosis. Pathologically, the lesion consisted of a roundish subpleural infarct with a central thrombotic artery containing sections of an immature nematode: Dirofilaria repens was diagnosed histologically in one case, the necrotic condition of the worm allowing only genus identification, Dirofilaria, in the other case. In both samples, PCR analysis amplified a 246 bp product, specific for the IpS insert 11 of D. repens. The authors stress the role of PCR in the diagnosis of this parasite from paraffin samples, also in cases in which identification of the species was not possible by conventional morphology due to poor conservation of the worm. These cases represent the first PCR-based diagnosis of D. repens in a human pulmonary dirofilariasis on samples embedded in paraffin.

Adult↗

PubMed2002

Intracellular distribution of intermediate filaments in vimentin-positive gastric carcinomas: confocal laser scanning microscopy using formalin-fixed paraffin-embedded specimens.

Intermediate filaments are known as cytoskeletal elements. Recently, additional vimentin expression has been reported in some carcinomas; however, the function of such expression remains unclear. We studied the intracellular distribution of low-molecular weight cytokeratin and vimentin by immunohistochemistry in 17 vimentin-positive gastric carcinomas using confocal laser scanning microscopy. All materials were formalin-fixed and paraffin-embedded. Low-molecular weight cytokeratin expression showed a membranous pattern with a prominent deposition just below the cytoplasmic membrane in both tubular and solid components of the carcinomas. This unique membranous deposition was frequently absent in diffuse components. On the other hand, vimentin expression showed a fibrillary pattern in all components and also showed a unique basal distribution in the tubular components. We also recognized an aggregate pattern of the intermediate filament expression in diffuse components. We conclude that the significance of vimentin expression in carcinoma cells cannot be explained as a simple substitution for low-molecular weight cytokeratin because the distribution of vimentin and low-molecular weight cytokeratin is different.

Adenocarcinoma↗

PubMed2005

Accurate genotyping from paraffin-embedded normal tissue adjacent to breast cancer.

Genetic polymorphism analysis for disease risk is widely used in epidemiology studies; blood or oral cavity cells are the most widely used source of DNA. However, these types of samples are not always available, particularly for studies that were conducted years ago. An alternative potential source of patient DNA exists in the form of paraffin-embedded normal tissue adjacent to tumor samples, which are collected and stored routinely for clinical use. The use of such samples can be conceptually problematic, however, due to the presence of field cancerization in the surrounding normal tissue, with the possible presence of chromosomal loss. Specifically, loss of heterozygosity (LOH) might bias the genotyping results and cause genotype misclassification. However, field cancerization and LOH might not be an issue because LOH is not easily found unless there is careful microdissection of only tumor cells (leaving stromal, inflammatory and fat cells), for example, laser-capture microdissection. In this study, we set out to determine the degree of genotype misclassification from normal tissues adjacent to tumors, if any, by comparing these results with blood genotyping. We examined samples from 106 subjects with breast cancer, analyzing five different genotypes selected from regions commonly known to have LOH in breast cancer. These genotypes were methylenetetrahydrofolate reductase (MTHFR), oxoguanosine glycosylase 1 (hOGG1), dopamine beta-hydroxylase (DBH), dopamine receptor D2 (DRD2) and NAD(P)H dehydrogenase quinone 1 (NQO1), conducted by using real-time PCR and TaqMan genotyping analyses. We found that among these five genotypes and 106 comparisons, there was a 100% concordance for genotyping from normal tissue adjacent to tumor and from blood. Our findings indicate that the use of adjacent normal tissues provides accurate genotyping results with high specificity. Although this study only used breast tumor samples, and may be applicable only to breast cancer studies, we expect the results to be applicable to other types of cancers also.

Biopsy↗

PubMed1977

[Immunofluorescence studies of experimental glomerulonephritis in formalin-fixed and paraffin-embedded material (author's transl)].

Immunofluorescence studies are an essential part of the diagnosis in glomerulonephritis. For these studies unfixed, frozen material was used only, on account of intense background staining of formalin-fixed slides. A method developed by Huang (1975) was tested on experimental glomerulonephritis. He succeeded in minimizing the background staining of formalin-fixed liver tissue with pronase-treatment. Our results show no differences in the immunofluorescence of the experimental glomerulonephritis between frozen and paraffin-embedded sections. It seems to be possible therefore to investigate a glomerulonephritis in formalin-fixed material by this method.

Animals↗

PubMed1997

Bicolor fluorescence in situ hybridization on nuclei from formalin-fixed, paraffin-embedded testicular germ cell tumors: comparison with standard metaphase analysis.

Rearrangements of chromosome 12, especially i(12p), are common in testicular germ cell tumors (TGCT). We have developed 12p and 12q chromosome arm-specific painting probes for fluorescence in situ hybridization (FISH) through the use of chromosome microdissection. We developed a method to hybridize these probes to interphase nuclei released from formalin-fixed, paraffin-embedded tumors (PET). In this study, we compared simultaneous bicolor PET FISH painting with metaphase chromosome analysis of fresh tissue from the same tumor. Bicolor PET FISH produced patterns of 12p and 12q hybridization consistent with expectations based on metaphase chromosome analysis; adjoined 12p and 12q regions appeared to detect "normal" chromosome 12s, whereas relatively large isolated regions of 12p were suggestive of i(12p), and small 12p regions probably represent cryptic rearrangements of 12p. Fourteen tumors with successful cytogenetic analyses and available archival material from the same tumor source were selected for study. In a blinded analysis, PET FISH painting assessment was in very close agreement with karyotypic findings in seven subjects, in close agreement in five, and showed less agreement in two. Differences may be due in part to clonal selection during culture for metaphase studies, or regional selection within the tumor. Although PET FISH painting should not replace standard chromosome analysis, this study shows that it can reliably predict chromosome 12 constitution in TGCT, can serve as a useful adjunct to standard cytogenetics when such analysis is unsuccessful, and can provide limited karyotyping of archival materials.

Cell Nucleus↗