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PubMed2003

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

DNA Primers↗

PubMed2005

Combined application of RT-PCR and immunohistochemistry on paraffin embedded sentinel lymph nodes of prostate cancer patients.

The detection of tumor cells in the sentinel lymph node (SLN) is of great importance for the prognosis of cancer patients. At present, immunohistochemistry and RT-PCR for tumor marker expression are the most sensitive techniques available for this analysis. However, so far, most RT-PCR-based analyses of SLNs have been performed on fresh material, excluding a direct comparison with the (immuno)histologic results. In our view, this does not entirely aid routine diagnosis. We established an efficient method for RNA extraction and RT-PCR from paraffin sections of SLNs from prostate cancer patients and compared the results with the (immuno)histologic data of adjacent sections. Amplifiable RNA was obtained from 133 SLNs of 68 prostate cancer patients. Correlation of PSA-specific RT-PCR with (immuno)histologic findings showed a positive and negative predictive value of 83% and 100%, respectively, for the prostate cancer patients investigated. Four of 12 patients with biochemical relapse, but without (immuno)histologically detectable tumor cells were RT-PCR-positive for PSA. We found that single sections of paraffin-embedded SLNs are suitable for routinely performed RT-PCR. Combined with (immuno)histology, PSA-specific RT-PCR is a revealing supplementary technique for the detection of tumor cells in SLNs of prostate cancer patients.

Adenocarcinoma↗

PubMed2006

Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Animals↗

PubMed2000

H-ras-1 point mutation in malignant peripheral nerve sheath tumors: polymerase chain reaction restriction fragment length polymorphism analysis and direct sequencing from paraffin-embedded tissues.

It has been shown that the NF1 (neurofibromatosis type 1) gene encodes a tumor suppressor which inactivates ras proteins. Among malignant mesenchymal tumors, H-ras-1 mutations have been found in malignant fibrous histiocytoma, leiomyosarcoma and embryonal rhabdomyosarcoma. However, studies on H-ras-1 mutation of many cases of malignant peripheral nerve sheath tumors (MPNST) have not been documented. Therefore, we investigated H-ras-1 mutations of MPNST. In 45 cases of MPNSTs of our files, DNA was extracted from the formalin-fixed paraffin-embedded tissue, and the mutations of the H-ras-1 gene were detected by using PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphisms) method and direct sequencing. We found two cases with H-ras-1 point mutation in MPNST for the first time. Both cases showed the same mutation in codon 13.1 [GGT(Gly) to AGT(Ser) transition]. Interestingly, both cases were associated with NF1. It is possibile that the mutation of the H-ras-1 gene occurred after the mutation of the NF1 gene in the MPNST.

Adolescent↗

PubMed1998

Immunohistochemical detection of the human major vault protein LRP with two monoclonal antibodies in formalin-fixed, paraffin-embedded tissues.

Multidrug resistant cancer cells frequently overexpress the 110-kd lung resistance-related protein (LRP) as detected with the monoclonal antibody (MAb) LRP-56. Recently, we identified LRP as the major vault protein (MVP), which is the major constituent of vaults, multisubunit cellular organelles. Clinically, LRP/MVP expression in cancer at time of diagnosis provided a strong and independent prognostic factor for response to chemotherapy and outcome in different tumor types, notably acute myeloid leukemia and ovarian cancer. To facilitate additional immunohistopathological studies, we have optimized LRP/MVP detection in paraffin-embedded tissues using two monoclonal antibodies, LRP-56 and LMR-5. Blocking experiments showed that LRP-56 and LMR-5 MAbs detect different epitopes of LRP/MVP. Immunohistochemical studies with both MAbs in a panel of human multidrug resistant tumor cell lines, normal tissues, and colorectal tumors showed that LRP/MVP expression can be reliably detected after formalin-fixation and paraffm-embedding using overnight incubation at 4 degrees C with the primary MAbs at 5- to 10-fold higher concentrations (ie, 1 to 10 microg/ml) as currently used with frozen sections. Both streptavidin-biotin complex and alkaline phosphatase-anti-alkaline phosphatase techniques could be successfully used for signal-amplification. Staining quality did not benefit from antigen-retrieval pretreatments. The optimized staining methodology facilitates studies in archival material on the putative role of LRP/MVP in clinical drug resistance.

Antibodies, Monoclonal↗

PubMed2002

Genetic analysis of HER-2/neu gene amplification in paraffin embedded tumour tissue in women with breast cancer.

In recent years several novel prognostic determinants of breast cancer have been identified, including HER-2/neu. The oncogene called HER-2/neu or c-erbB-2 located at 17q21 encodes a 185-kD transmembrane glycoprotein, p185. The HER-2/neu gene is frequently overexpressed in human breast cancers as a result of gene amplification and/or elevated transcription. In this study we investigated the association between the presence of HER-2/neu gene ampliflcation and appearance and/or progression of breast cancer. Paraffin embedded tumour tissue was obtained from 82 women with breast cancer. Blood samples from age matched healthy women served as control (n = 50). The amplification of HER-2/neu gene was determined by PCR amplification using appropriate primers. The amplification of HER-2/neu gene was significantly higher in women with breast cancer as compared with control (P < 0.05). Additionally, there were differences in the amplification status between node-positive and node-negative breast cancer patients. The results suggest that the presence of amplification of HER-2/neu gene may be linked with the appearance and/or progression of breast cancer.

Adult↗

PubMed1996

TP53 mutation analyses on breast carcinomas: a study of paraffin-embedded archival material.

The aim of this investigation was to examine the possibility of analysing TP53 mutations in archival paraffin-embedded material with the constant denaturant gel electrophoresis (CDGE) method. We extracted DNA from 193 archival primary breast carcinoma samples, diagnosed in 1981-83; further analysis was possible for 186 of these. TP53 mutations in exons 5-8 were detected with CDGE in 30 samples (16.1%) and 17 of these mutations were confirmed by sequencing. Immunohistochemistry demonstrated TP53 nuclear accumulation in 58 tumours (31%). A strong association between the presence of TP53 mutations and TP53 immunostaining was observed (P < 0.001). Our mutation and immunohistochemistry results are in agreement with other findings based on fresh tumour tissue. TP53 abnormalities were significantly related to high S-phase fraction, low oestrogen receptor (ER) content and high tumour grade. Survival of patients with TP53 abnormalities, in the group as a whole, did not differ from patients with normal TP53. Our study did, however, show that patients with abnormal TP53 had a significantly shorter post-recurrence survival (P = 0.005) than patients with normal TP53.

Amino Acid Sequence↗

PubMedDate not supplied

Flow-cytometric analysis of the DNA content in paraffin-embedded tissue and in fresh tumour samples obtained from renal cell carcinoma: comparison of DNA ploidy using ethidium bromide and mithramycin.

With the help of the deparaffination technique described by Heldly, it has become possible to conduct large-scale flow-cytometric (FCM) studies, including retrospective DNA analysis of renal cell carcinoma (RCC). The quality of these histograms, however, has to be comparable to the quality of the analyses from fresh tumour tissue. The present study compares the application of FCM on fresh tumour tissue from RCC with paraffin-embedded archive material, thus for the first time, the same combination of stains (ethidium bromide/mithramycin) was used for both materials. From a total of 44 tumours, 125 histograms were obtained from fresh tumour tissue. Aneuploidy was found in 78 (62.4%) of the DNA analyses (coefficient of variation, CV = 5.5%). These 78 aneuploid histograms were compared with 228 sections from the archive material which had been deparaffined and stained. This involved, a deparaffinising period of twelve hours as well the application of pepsin and protease, which was necessary to obtain high-quality histograms (CV = 6.0%). Retrospectively, a total of 58 histograms from 36 cases were available for comparison. Of the archive material, 23/36 tumours were classified as aneuploid; thus new stem cell lines could be detected retrospectively in 63.8% of the cases. The comparison of DNA indices (DI) in the two series produced no significant differences, and the comparison of the mean values for the CV-5.5% for native material histograms and 6.0% for archive material histograms-was not statistically significant. The detritus part in the archive histograms revealed a mean value of 6.3% for native histograms the mean value was 13.4%. These differences were also not significant. These results show that, using the staining combination ethidium bromide/mithramycin, the quality of the histograms obtained with archive material is equal to the quality of the histograms obtained from fresh tumour material. Furthermore, when taking into consideration the clonal heterogeneity of RCC, it is possible to use retrospective analysis to detect aneuploidy with a high degree of certainty and accuracy.

Carcinoma, Renal Cell↗

PubMed1996

In situ hybridization detection of varicella zoster virus in paraffin-embedded skin biopsy samples.

BACKGROUND: When virologic and molecular diagnostic techniques are unavailable, the diagnosis of varicella zoster virus (VZV) infection depends on clinical criteria and histologic evaluation of skin biopsy specimens or Tzank preparations. These methods can misdiagnose chickenpox and zoster, particularly when the clinical manifestations are atypical. OBJECTIVE: To improve diagnosis in these settings, we developed an in situ hybridization technique for the detection of VZV utilizing a fluorescein-labeled oligonucleotide probe visualized with anti-fluorescein alkaline phosphatase-conjugated antibody. STUDY DESIGN: We retrospectively examined 26 paraffin-embedded skin biopsy specimens with histologic features consistent with VZV or herpes simplex virus (HSV) infection and 11 control cases by in situ hybridization. In situ hybridization for VZV and HSV-1 was compared with polymerase chain reaction (PCR) for VZV and HSV-1 and clinical and histologic examination. RESULTS: Thirteen of the 26 study cases and two of the 11 control cases were positive for VZV by in situ hybridization. When compared with PCR, in situ hybridization was 92% sensitive and 88% specific. When compared with clinical diagnosis, in situ hybridization was 86% sensitive and 87% specific. All cases of chickenpox had VZV-positive inflammatory cells in the dermis but this finding was less frequent among the cases of zoster. CONCLUSIONS: This in situ hybridization technique is a sensitive and specific method for the diagnosis of VZV in skin lesions that is applicable to most histopathology laboratory settings. In addition, in situ hybridization reveals individual infected cells and may provide insight into the pathogenesis of VZV skin infection.

Chickenpox↗

PubMed1992

Demonstration of Epstein-Barr viral DNA in paraffin-embedded specimens of lymphoproliferative syndrome. Evidence for a productive infection comparable to lymphoblastoid cell lines.

B-cell lymphoproliferative syndromes (LPS) occurring in immunodeficient subjects are frequently associated with EBV infections. Histology as well as EBV-related serology are not diagnostic, but demonstration of EBV DNA in LPS suspected lesions might be useful for diagnosis. We studied four autopsied cases of LPS that developed in the setting of bone marrow transplantation, with proliferations ranging from poly- to monoclonal. Our protocol of DNA extraction allowed detection of EBV DNA in formalin-fixed, paraffin-embedded tissue specimens of all four cases. In dot blot hybridization the sensitivity in these specimens was 10% as compared to fresh frozen material, but still sufficient for a biotinylated probe. Southern blotting with the former DNA was not successful due to extensive degradation. In situ hybridization resulted in positive signals in all cases, using either 35S or 3H labeled probes. The labeling pattern suggested virus replication in B cells of LPS. By this, LPS resembles productively infected lymphoblastoid cell lines rather than latently EBV-infected Burkitt's lymphoma (BL). These findings strengthen the concept of LPS as a distinct clinicopathologic entity, differing from monoclonal, latently EBV-infected BL, as well as from polyclonal infectious mononucleosis, and the more common EBV-negative Non-Hodgkin lymphomas of the immunocompetent host.

Adult↗

PubMed1983

Comparative evaluation of immunhistochemistry and enzyme histochemistry for granulocyte visualization in formalin-fixed and paraffin-embedded liver and lung biopsies.

Two methods for the specific visualization of granulocytes (PMNs) in formalin-fixed and paraffin-embedded tissue samples were compared: (1) the well-established enzyme histochemical method using naphthol-AS-D-chloracetate as a substrate; (2) an immunoperoxidase reaction using anti-PMN serum. Both methods are highly selective for visualizing PMNs. The advantages offered by the immunohistochemical method, i.e. better visualization of the remaining tissue structures and potential combination with other staining techniques, are stressed. Problems of PMN visualization in lung tissue and effects of tissue preparation are discussed. For histomorphometric studies the immunohistochemical method is preferable.

Animals↗

PubMed1987

Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization.

A novel method for covalently binding formalin fixed paraffin embedded (FFPE) tissue sections to glass microscope slides is validated suitable for in situ hybridization (ISH). Using the organosilane methodology of Maples (1985), 100% tissue adhesion is reported with no nonspecific probe binding, staining, or autoradiographic artefacts. JC viral nucleic acid sequences are successfully detected in FFPE progressive multifocal leukoencephalopathy brain tissue and the Tm of the hybridized product is estimated. From the Tm the most stringent washing condition resulting in an optimal signal to noise ratio is determined. A comparison is made between currently used methods of tissue adhesion and the proposed organosilane methodology. This methodology greatly facilitates studies of conditions for ISH and elucidation of mechanisms of viral infections requiring consecutive FFPE sections. It is also applicable to studies using cryosections and cultured cells.

Animals↗

PubMed1984

Effects of fixation and enzymatic digestion on the immunohistochemical demonstration of laminin and fibronectin in paraffin embedded tissue.

The effects of different fixatives and enzymatic digestion procedures on the immunohistochemical demonstration of fibronectin and laminin in paraffin embedded tissues have been compared. None of the fixatives tested enabled staining of these proteins without enzymatic digestion. No intracytoplasmic laminin was found either in fixed or in fresh frozen tissue. Fixation in formol acetic acid was unsatisfactory for demonstration of fibronectin; prolonged fixation in formol sublimate was unsatisfactory for demonstration of laminin. Optimal results were achieved after fixation in routine 10% formol saline. Trypsin was completely ineffective for unmasking laminin antigens except after fixation in ethanol acetic acid; it was only partially effective for showing fibronectin antigens. The best results were obtained with protease digestion, but pepsin was an adequate, although slightly less reliable, alternative. These enzymes may be used at lower concentrations than usually recommended.

Acetates↗

PubMed1989

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique.

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.

Animals↗

PubMed1979

Immunoperoxidase localization of treponema pallidum: its use in formaldehyde-fixed and paraffin-embedded tissue sections.

The spirochete, Treponema pallidum, the causative agent of syphilis, has been successfully localized in formaldehydefixed and paraffin-embedded tissue sections using rabbit anti-T pallidum antiserum with two immunoperoxidase techniques. These techniques, the indirect peroxidase-labeled antibody method and the peroxidase-antiperoxidase (PAP) method are compared for sensitivity and degree of nonspecific staining. Both offer substantial advantages over conventional silver-impregnation techniques, but the indirect peroxidase-labeled antibody method seems better, based on the intensity of staining and the simplicity of procedure.

Antibodies, Bacterial↗

PubMed2004

Detection of (NAD(P)H:Quinone oxidoreductase-1, EC 1.6.99.2) 609C-->T and 465C-->T polymorphisms in formalin-fixed, paraffin-embedded human tumour tissue using PCR-RFLP.

NQO1 is a cytosolic flavoprotein that plays a dual role in the detoxification of potentially carcinogenic compounds and the bioreductive activation of quinone based anticancer drugs. Two polymorphic variants of NQO1 exist (NQO1*2 and NQO1*3) which cause significant phenotypic reductions in NQO1 protein content and activity. Current methods for detecting NQO1 polymorphisms commonly use PCR-RFLP techniques and have exclusively used DNA isolated from fresh tissues. This study describes a method that is suitable for analysing NQO1 polymorphisms in genomic DNA isolated from formalin-fixed paraffin-embedded tissue. The method utilises two rounds of PCR amplification using a nested primer strategy that generates specific PCR products followed by RFLP analysis using either Hinf1 (for NQO1*2) or Msp1 (for NQO1*3). Whilst existing methods proved unsatisfactory (low product yield and poor specificity), the nested primer strategy produced good quality PCR products suitable for RFLP analysis and genotyping of NQO1*2 and NQO1*3 in archival tissue samples. The ability to utilise the vast archives of human tissue held by pathology laboratories would be of considerable benefit as retrospective studies comparing NQO1 genotype status, patient history and treatment outcomes could be conducted.

Carcinoma, Transitional Cell↗

PubMed2006

Human Papillomavirus DNA and P16INK4A are not detected in renal tumors with immunohistochemistry and signal-amplified in situ hybridization in paraffin-embedded tissue.

The association between human papillomavirus (HPV) infection and carcinogenesis has long been established in literature, with the strongest evidence for its role in cervical carcinoma. The role of HPV in urological tumors has been investigated and sporadic reports have linked HPV infection to bladder, prostate, renal, penile, and testicular cancer. Although less rigorously studied, there are a few conflicting results about the role of HPV in the development of malignant renal tumors. Moreover, no data are available for association of HPV DNA and expression of P16 in benign renal tumors. Formalin-fixed, paraffin-embedded tissues from 62 renal tumors (40 clear cell, 9 papillary, and 3 chromophobe renal cell carcinomas, 1 collecting duct carcinoma, 2 urothelial carcinoma of renal pelvis and 7 oncocytomas) were immunostained with low-risk and high-risk HPV DNA (6, 11, 16, 18, 31, 33, 42, 51, 52, 56, 58). Tissue microarray sections of 62 tumors were stained with P16 by immunohistochemistry. Signal amplified colorimetric in situ hybridization was performed on microarray sections using biotinylated probes for HPV subtypes 6, 11, 16, 18. A nuclear dot-like signal was considered positive for low-risk and high-risk HPV by immunohistochemistry and in situ hybridization and nuclear or cytoplasmic staining is considered positive for P16. No staining for HPV DNA and P16 was found in any type of renal tumors. Our results support that HPV does not seem to play a role in the development of benign and malignant renal tumors.

Adenoma, Oxyphilic↗

PubMed2004

Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation.

In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.

Animals↗