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Analysis of K-ras gene mutations in periampullary cancers, gallbladder cancers and cholangiocarcinomas from paraffin-embedded tissue sections.

Point mutations of the K-ras gene were analyzed in 25 periampullary cancers (21 ampulla vater cancers, two common bile duct cancers and two duodenal cancers), two gallbladder cancers and six cholangiocarcinomas. DNA extracted from the paraffin-embedded tissues was amplified with the polymerase chain reaction and subsequently analyzed by direct cycle sequencing at codons 12, 13 and 18 of the K-ras gene. Codon 61 was first screened with single strand conformation polymorphism and then sequenced by direct cycle sequencing. No point mutation was found in any of the 25 periampullary cancers or the two gallbladder cancers. These results are similar to previous reports. Mutation of the K-ras gene seems not to play an important role in tumorigenesis of periampullary cancer. In two of six (33%) cholangiocarcinoma patients, point mutations were found. Both mutations were transitions, GGT to GAT at codon 12. The incidence of mutation was greater than that in Thailand (about 8%) but less than that in Japan (about 60%). Mutation of the K-ras gene may play varied roles in the tumorigenesis of cholangiocarcinoma, depending on geographic area.

Adult↗

Effects of trypsinization and microwave treatment on lectin labelling of microglial cells in paraffin-embedded sections from pre- and postnatal bovine brains.

The effects of microwave heat treatment on lectin histochemical staining of microglial cells with Griffonia simplicifolia B4 isolectin (GSA I-B4) and Ricinus communis agglutinin-I (RCA-I) in paraffin-embedded pre- and postnatal bovine brain tissue fixed in two different fixatives (Bouin's fluid and 4% neutral buffered formaldehyde) were examined, and the results compared with lectin labelling obtained in untreated and trypsinized serial sections. The results indicate that lectin labelling of bovine microglial cells depends on the kind of lectin applied, the fixative used for tissue preservation, the isotype of microglia to be labelled, and the pretreatment of tissue sections. In brain tissue fixed in Bouin's fluid, GSA I-B4 staining of both microglial isotypes, i.e., amoeboid and ramified microglial cells, was achieved without trypsinization. Staining of sections with RCA-I, however, yielded negative results both on untreated and on trypsinized sections. These findings suggest that species-specific differences in the density of binding sites accessible to GSA I-B4 and RCA-I, respectively, may exist. Pretreatment of sections by microwave irradiation had different effects depending on the lectin and fixative used and on the microglial isotype to be stained. Microwave heat treatment of sections prior to incubation with RCA-I enabled the labelling of amoeboid and ramified microglial cells. The latter cell type, however, was exclusively stained in brain tissue fixed in Bouin's fluid. With GSA I-B4, exclusively the labelling of ramified microglial cells in sections fixed in Bouin's fluid was improved. It is assumed that by microwave pretreatment of sections from bovine brain the access of both lectins to their receptors, i.e., D-galactose residues on microglial cells, may be facilitated.

Animals↗

Detection of hepatitis C virus RNA using ligation-dependent polymerase chain reaction in formalin-fixed, paraffin-embedded liver tissues.

Reverse transcription polymerase chain reaction (RT-PCR) has been used to detect hepatitis C virus (HCV) sequences in liver tissue. However, RT-PCR has a variable detection sensitivity, especially on routinely processed formalin-fixed, paraffin-embedded (FFPE) specimens. RNA-RNA and RNA-protein cross-links formed during formalin fixation is the major limiting factor preventing reverse trans criptase from extending the primers. To overcome this problem, we applied the ligation-dependent PCR (LD-PCR) for the detection of HCV RNA in FFPE liver tissue. This method uses two capture probes for RNA isolation and two hemiprobes for the subsequent PCR. Despite cross-links, the capture probes and the hemiprobes are able to form hybrids with HCV RNAs released from the FFPE tissue. The hybrids are isolated through binding of the capture probes to paramagnetic beads. The hemiprobes are then ligated by a T4 DNA ligase to form a full probe that serves as a template for the Taq DNA polymerase. A total of 22 FFPE liver specimens, 21 with hepatocellular carcinoma (HCC) and 1 with biliary cirrhosis secondary to bile duct atresia were selected for this study, of which 13 patients were HCV seropositive and 9 seronegative. HCV RNA was detectable by ID-PCR from all 13 HCV-seropositive HCCs and from 5 of 8 HCV-seronegative HCCs but not from the HCV-seronegative liver with biliary atresia. By contrast, RT-PCR detected HCV sequences in only 5 of the HCV-sero-positive and in 1 of the HCV-seronegative HCCs. To resolve the discordance between the LD-PCR and RT-PCR results, RT-PCR was performed on frozen liver tissue of the discrepant specimens, which confirmed the LD-PCR positive results. In conclusion, LD-PCR is a more sensitive method than RT-PCR for the detection of HCV sequences in routinely processed liver tissues. A high rate of HCV infection (86%) is found in HCC specimens, indicating a previously underestimated role of HCV in HCC pathogenesis.

Base Sequence↗

Three immunostaining techniques for the localization of leukocyte common antigen in formalin-fixed, paraffin-embedded dermatological biopsy specimens.

Although leukocyte common antigen is a valuable discriminant between lymphoreticular proliferations and other histologically similar neoplasms, several publications have described difficulties in adequately labeling this determinant in formalin-fixed, paraffin-embedded tissues. We studied 662 paraffinized cutaneous and noncutaneous neoplasms for the presence of leukocyte common antigen, using an extended avidin-biotin-peroxidase complex (ABC) method, a combined ABC-peroxidase-antiperoxidase technique, and a biotin-strept-avidin procedure. Each of the three immunohistochemical techniques produced effective results in the detection of leukocyte common antigen in all malignant lymphomas, but none of them produced false-positive staining in nonhematopoietic lesions. These results suggest that antibodies to leukocyte common antigen are highly specific and that the methods employed in this study allow for excellent diagnostic sensitivity in the recognition of cutaneous lymphomas.

Biopsy↗

Antigen retrieval for bcl-2 protein in formalin-fixed, paraffin-embedded sections.

A commercial monoclonal antibody to bcl-2 protein (bcl-2, 124) is useful in immunohistochemical detection of this 25 kDa oncogene product in frozen sections. In order to overcome the difficulty in identifying bcl-2 protein in formalin-fixed, paraffin-embedded sections, its antigenicity was retrieved by a variety of pretreatments. The heating pretreatment by hydrated autoclaving was the best method for retrieving the antigenicity, and deionized water was the most adequate solution for soaking sections. This antigen retrieval sequence was helpful to examine the localization of bcl-2 protein in archival sections of normal lymphoid and nonlymphoid tissues and follicular lymphomas.

Antibodies, Monoclonal↗

Monoclonal antibodies to human germ cell tumors from "routine" paraffin-embedded pathological specimens.

We have established a method for monoclonal antibody (MoAb) preparation from routine paraffin-embedded tissue of human seminoma as an immunogen. Three 40-microns thick sections were deparaffinized and rehydrated. An eight-week-old BALB/c mouse was immunized intraperitoneally with this extract, which showed no detectable protein bands on sodium laurylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Five monoclonal antibodies (MoAbs) with different characteristics were obtained; one reacted with the nucleus, two with the cytoplasm, and two with the cytoplasmic membrane. One of the MoAbs 5G9 reacted with spermatogonia in normal human tissues and with seminoma, embryonal carcinoma and choriocarcinoma in the testicular tumors.

Animals↗

Improved method for release of cell nuclei from paraffin-embedded cell material of squamous cell carcinomas.

An improved method of releasing cell nuclei from paraffin-embedded highly keratinized squamous cell carcinomas by pretreatment with 85% formic acid-0.3% H2O2 followed by enzymatic treatment with subtilisin Carlsberg is described. After DAPI staining the resulting suspensions of cell nuclei were analyzed by DNA flow cytometry, in addition to microscopy. The total yield of released cell nuclei was improved and the proportion of tumor cell nuclei in the suspensions increased from 12% to 53% using this method compared to the conventional preparation technique. Cytoplasmic residue disappeared nearly completely. Thus, the finding of false aneuploid cell populations representing diploid cells with autofluorescent cytoplasm could be avoided. In addition, even small true aneuploid cell populations could be detected due to the increased proportion of released tumor cell nuclei and the lower proportion of background in the > 2C region.

Carcinoma, Squamous Cell↗

Detection of cytomegalovirus in paraffin-embedded postmortem coronary artery specimens of heart transplant recipients by the polymerase chain reaction: implications of cytomegalovirus association with graft atherosclerosis.

To assess the presence of cytomegalovirus in various tissues and its relevance to the development of graft atherosclerosis, 54 postmortem paraffin-embedded tissue samples from 15 heart transplant recipients who survived more than 100 days were analyzed by the polymerase chain reaction for cytomegalovirus. Eight patients had known previous cytomegalovirus exposure; 7 patients did not. Of the eight with known previous exposure, three patients (38%) died of graft atherosclerosis versus four (57%) of the seven patients without previous exposure. Of the 54 specimens, 49 were positive for beta-globin (inclusive positive control), including 38 coronary artery, five lung, five gastrointestinal, and one kidney. Only two coronary artery specimens were cytomegalovirus positive in a single patient with known cytomegalovirus exposure who did not have evidence of graft atherosclerosis at autopsy. Other tissues tested showed positive lung and stomach specimens in the patient with cytomegalovirus-positive coronary artery specimens and positive kidney, lung, and gastrointestinal specimens in a second patient. No specimens were cytomegalovirus positive in the remaining patients, despite the presence of graft atherosclerosis or previous cytomegalovirus exposure. Our data do not support the hypothesis that graft atherosclerosis is associated with latent cytomegalovirus infection of the coronary arteries. The role of cytomegalovirus in the pathogenesis of graft atherosclerosis is unknown, but possibly it represents a modulation of the immune system by remote infection.

Adolescent↗

Demonstration of gene-amplification by PCR in archival paraffin-embedded breast cancer tissue.

The polymerase chain reaction (PCR) is a powerful tool to examine genetic alterations in tumor samples. We describe a simple, rapid, nonisotopic PCR method to semi-quantitatively determine the number of gene copies in human formalin-fixed paraffin-embedded tissue. The procedure is exemplified by analysis of 15 years old breast cancer samples to determine the presence of amplification of c-erb-B2. The samples were obtained from routine specimens kept in pathological archives. Patients with amplified samples showed a poor prognosis, both for recurrences and death in breast cancer.

Archives↗

Comparison of fresh, ethanol-preserved, and paraffin-embedded samples in DNA flow cytometry.

Fresh, ethanol-preserved, and formalin-fixed and paraffin-embedded samples taken from the same part of 15 human tumors, and from one normal spleen and one pancreas were analyzed for nuclear DNA content by flow cytometry. The coefficient of variation (CV) values of the G1 peaks were smaller in the fresh than in the other samples (P less than 0.001). The DNA ploidy of the tumors was the same in all types of samples. The DNA indices (DIs) measured from either ethanol-preserved or formalin-fixed tissue correlated strongly with those obtained from fresh tissue (P less than 0.001), although they tended to be somewhat smaller in the fresh samples. The S-phase fractions measured from all types of samples were of the same order of magnitude in most cases (P less than 0.001). Uninterpretable histograms were most often obtained from fresh samples. Identical DI values and rather constant S-phase fractions were obtained from ethanol-preserved samples stored at 4 degrees C for up to 5 months. It is concluded that all three types of samples are suitable for the determination of DNA ploidy, DI, and S-phase fraction and that 50% ethanol is suitable for long-term preservation of flow cytometric samples.

DNA, Neoplasm↗

Thick-section fluorescence in situ hybridization on formalin-fixed, paraffin-embedded archival tissue provides a histogenetic profile.

Fluorescence in situ hybridization has become a major tool for analysis of gene and chromosome copy number in normal and malignant tissue. The technique has been applied widely to fresh tissue and dispersed formalin-fixed, paraffin-embedded archival tissue, but its use on sections of archival tissue has largely been limited to sections < 6 mu thick. This does not provide intact, uncut nuclei for accurate analysis of gene or chromosome copy number. We report here a method of hybridization to sections > 20 microns thick that overcomes these difficulties. Key developments were the use of DNA probes directly labeled with fluorochromes and optical sectioning using laser-scanning confocal microscopy.

Chromosomes, Human, Pair 1↗

Cyclin D1 protein in multiple myeloma and plasmacytoma: an immunohistochemical study using fixed, paraffin-embedded tissue sections.

In this study, we analyzed 69 plasma cell neoplasms, including 54 multiple myelomas (MMs), 3 cases of plasma cell leukemia, and 12 plasmacytomas, for expression of cyclin D1 protein using an immuno-histochemical method applied to routinely fixed, paraffin-embedded tissue sections. Cyclin D1 was expressed in 18 (26%) of 69 plasma cell neoplasms, including 16 (30%) of 54 MMs and 2 (17%) of 12 plasmacytomas. Three cases of plasma cell leukemia were negative. Cyclin D1 expression correlated with the cytologic differentiation and histologic stage of MM. Twelve (75%) of 16 MMs had intermediate or immature cytologic features and were histologic Stage III. With use of a polymerase chain reaction assay, we also analyzed six cases (three cyclin D1 positive, three cyclin D1 negative) for the t(11;14); one MM carried the t(11;14) and expressed cyclin D1 protein. We conclude that cyclin D1 expression occurs in approximately one-quarter of plasma cell neoplasms and correlates with the degree of cytologic differentiation and histologic stage. The relatively high frequency of cyclin D1 expression, compared with the less than 5% incidence of the t(11;14) detected by conventional cytogenetics reported in the literature, suggests that upregulation of cyclin D1 protein might be the result of mechanisms other than the t(11;14).

Adult↗

Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.

Animals↗

Monoclonal antibody selectively reactive with myelin sheaths of the peripheral nervous system in paraffin-embedded material.

We have produced a monoclonal antibody (PM43) selectively reactive with formalin-fixed paraffin-embedded peripheral nervous system (PNS) myelin. The hybridomas were generated by fusion of mouse myeloma cell line Sp2/0 with spleen cells of BALB/c mice immunized with cultured human melanocytes. Hybridomas were screened by the indirect immunoperoxidase assay. Ouchterlony analysis showed the immunoclass of PM43 to be IgM. By the immunoaffinity chromatography technique, among others a 43-kDa protein was isolated from PNS myelin. The antigenic determinant of PM43 in the mouse is expressed with a similar tissue distribution as observed in man. Expression of the antigenic determinant does not become visible until after birth in mice. PM43 opens further possibilities for the use of anti-myelin antibodies in the study of myelination and demyelination processes in the PNS and remyelination processes in the central nervous system.

Animals↗

Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue.

The influence of eight decalcifying agents on the immunoreactivity of formalin-fixed, paraffin-embedded tissue for immunoglobulins, lysozyme, factor VIII-related antigen and keratin was studied using the unlabelled antibody peroxidase-antiperoxidase (PAP) method. Limited studies were also performed on tissues fixed in acid-formalin mixtures. All tissues were stained using an indirect immunoperoxidase method with mouse monoclonal antibodies to IgM, kappa and lambda light chains. The results suggest that, with controlled trypsinization of sections, it was possible to obtain optimal immunostaining for all tested antigens, with adequate preservation of histological structure, after decalcification in neutral EDTA or 10% aqueous acetic or formic acid. Tissue treated with agents containing mineral acids exhibited variable immunoreactivity and impaired counterstaining.

Animals↗

Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.

Animals↗

A correlative chemical and histochemical study of the O-acetylated sialic acids of human colonic epithelial glycoproteins in formalin fixed paraffin embedded tissues.

Procedures are described for the isolation and identification of epithelial glycoproteins from formalin fixed, paraffin embedded specimens of human large intestine. The side chain O-acetylation patterns of the sialic acids of these glycoproteins were surprisingly similar to those of purified glycoproteins prepared from epithelial cells obtained from the same tissue before fixation. These results were consistent with those obtained by histochemical procedures performed on representative sections taken from the same tissue blocks. The methodology described permits a direct correlation of chemical and histochemical results obtained from the study of colonic epithelial glycoproteins of both normal and diseased tissues. It eliminates some of the difficulties associated with interpretation of the results by either discipline and may provide new information which would be unavailable by either chemistry or histochemistry alone.

Acetylation↗

DNA ploidy, tumour site, and prognosis in colorectal cancer. A flow cytometric study of paraffin-embedded tissue.

DNA ploidy patterns were studied by flow cytometry in nuclear suspensions from 149 paraffin-embedded colorectal adenocarcinomas. The DNA ploidy of rectal tumours was not significantly different from that of colonic tumours. Patients with DNA diploid tumours had a significant survival advantage compared with patients with non-diploid tumours, but DNA ploidy did not confer any significant additional prognostic information when tumour site, Dukes's stage, the invasiveness of the tumour, and the number of lymph node metastases were adjusted for in a proportional hazards regression analysis (Cox). It is concluded that DNA ploidy does not contribute significantly to the explanation of why patients with rectal cancer have a poorer prognosis than those with colonic cancer.

Adenocarcinoma↗