ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Southern blotting using radiolabeled probes is a well established technique for the detection of the Philadelphia translocation in the diagnosis of chronic myelogenous leukemia (CML). However, the use of radioisotopes in the clinical setting is often problematic. Because of this we investigated the use of a digoxigenin-labeled probe and chemiluminography in the detection of the Philadelphia translocation. In this study DNA was extracted from 19 bone marrow or blood samples from patients with CML or other malignancies and subjected to Southern blotting with a probe specific for the Philadelphia translocation, Phl/bcr3. The probe was labeled with either, 32P or digoxigenin to determine the relative sensitivity and specificity of autoradiography and chemiluminography in the molecular diagnosis of the BCR/abl fusion gene. All 19 samples were tested by both methods. All blots were performed and interpreted by individuals blind to the initial patient diagnosis. In addition, 12 samples (6 positive for CML, 6 negative for CML as determined by Southern blotting with 32P-labeled probe) were subjected to triplicate Southern-blot analyses, with three separate lots of digoxigenin-labeled probe to assay batch to batch variability in the efficacy of the probe. Radiolabeled and digoxigenin-labeled probes resulted in identical diagnoses in all cases. All results obtained by molecular analysis correlated perfectly with the clinical diagnoses of the patients from whom the samples had been obtained. Preanalysis of patient samples with different batches of digoxigenin-labeled probe gave highly reproducible results. With digoxigenin-labeled probe, diagnostic results were obtained after exposure times of less than 1 h at room temperature. Comparable results with radiolabeled probe required the use of enhancing screens and a 3–4-day exposure at −70°C. Overall, we have found the use of the digoxigenin-labeled probe Phl/bcr3 and chemilumines-cence using the substrate CSPD to be at least as good and in some respects superior to the use of radiolabeled probe and autoradiography in the molecular diagnosis of CML.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Structural alterations ofp53and overexpression of the p53 protein are found in a large proportion of human cancers. In this study, we examined the frequency ofp53mutations and p53 overexpression in squamous cell carcinomas (SQCC) of head and neck. Expression of p53 was detected by immunohistochemistry (IHC) with monoclonal antibodies defining three distinct epitopes: PAb421 (species cross-reactive epitope on normal and mutated p53), PAbl801 (epitope on normal and mutated human p53), and PAb240 (conformational epitope of mutated p53 and denatured normal p53). Genetic alterations ofp53were identified by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing in selected cases. IHC assays revealed nuclear p53 immu-nostaining in 53% of cases (32 of 60) with PAbl801. 38% (23 of 60) with PAb421, and 32% (19 of 60) with PAb240. Cases positive with PAb421 or PAb240 were also positive with PAbl801, whereas PAb421 and PAb240 identified overlapping but distinct tumor subsets. Areas of carcinoma in situ present in the tumor specimens showed nuclear p53 immunostaining in 11 of 26 cases. SSCP analysis for exons 5–9. the most common sites ofp53abnormalities, revealed mutations in 26% (15 of 58) of the evaluable cases. Comparison of the SSCP results with the IHC results for PAbl801 identified 11 cases that were positive by both methods, 4 cases withp53mutations that were negative by IHC, 20 cases positive by IHC but without detectablep53mutations, and 23 cases negative by both methods. IHC with PAb240, which is thought to be specific for mutated p53, was positive in 9 cases with demon- strablep53mutations and in 9 cases with no detectable mutations. DNA sequence analysis of nine tumors identified point mutations, nonsense mutations, and frame-shift mutations. In conclusion, our study shows that p53 overexpression in SQCC of head and neck as detected by IHC is a frequent finding, and that overexpression is associated with common types ofp53mutations in some cases. However, IHC identifies p53 overexpression in some tumors with apparently normal SSCP patterns and, conversely, tumors with nonsense or frameshift mutations may not express any p53 detectable by IHC.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

In lymph node diagnosis, difficulties are frequently encountered with the differential diagnosis of reactive and neoplastic T-cell proliferations. Immunohistochemistry is of limited use, and fresh frozen material for DNA studies is not available in many cases. We have established a polymerase chain reaction (PCR) technique to amplify rearranged T-cell receptor (TCR)-γ sequences from paraffin-embedded material. Our method differs from other techniques previously described in that it uses four sets of family-specific variable (V)- γ primers. Clonality of the investigated T cells is reflected not only by the varying lengths of amplified products but also by differences in the relative amount of rearranged Vγ families. Preliminary studies indicate that this approach can provide information about the presence of predominant T-cell clones within the sample and thus help to classify the lymph node lesion. With this technique we were able to confirm clonality in seven of 12 T-cell lymphomas in paraffin-embedded tissues.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Metastasis to neck lymph nodes is often the presenting symptom of occult head and neck tumors, including undifferentiated nasopharyngeal carcinoma (UNPC). The diagnosis of the primary site of the tumor by conventional cytological analysis of tissue obtained by fine-needle aspiration (FNA) may be difficult. As Epstein-Barr virus (EBV) infection is consistently associated with UNPC, we evaluated the diagnostic significance of EBV detection using a nonradioisotopic in situ hybridization assay. The data obtained by FN A from metastatic head and neck tumors was correlated with the histology of the corresponding surgical specimens. In a series of 25 FN A specimens of cervical lymph node metastases of tumors of unknown origin, EBV was found expressed in all seven metastases of UNPC but in none of 18 metastases of tumors of different types. Therefore, detection of EBV in cervical metastatic adenopathy may be successfully used to identify the presence of occult UNPC

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

An 80-year-old woman with slowly progressive dementia of at least 1 year's duration died with an abrupt change in the rate and character of mental deterioration. Pathologic examination of the brain showed Alzheimer s disease as well as progressive multifocal leukoencephalopathy (PML) in the pariental and cerebellar areas. JC virus (JCV) was detected by polymerase chain reaction (PCR) amplification of paraffin sections from both PML and non-PML areas of the cerebrum. Analysis by in situ hybridization and reverse transcriptase PCR in situ localized JC viral DNA and RNA to the nuclei of oligodendrocytes only in the areas of demyelination. This case is noteworthy in that PML occurred in the setting of Alzheimer's disease by presumed activation of latent JCV in the absence of usual causes of immunocompromise

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Fine needle aspiration (FNA) cytology represents a reliable diagnostic procedure for preoperative identification of thyroid carcinoma. For improving its diagnostic accuracy, a technique that allows the analysis of cancer-related gene abnormalities on thyroid FNA smears has been developed. Cells were collected by scraping from thyroid smears, DNA-extracted andrasproto-oncogene sequences amplified by the polymerase chain reaction (PCR). Preliminary destaining of cytologic smears was essential for efficient PCR amplification. Twelve thyroid FNA cytologic smears, characterized by the indeterminate pattern of follicular neoplasia, were analysed for the presence ofrasmutations known to confer an oncogenic potential. The same nucleotide substitution at codon 12 of theH-rasproto-oncogene was detected in two different thyroid nodules among six cases that, at final histology, were identified as follicular carcinomas. Norasmutations were observed in the remaining six cases that were diagnosed as follicular adenoma at histologic examination. Molecular analysis of FNA smears may provide additional information on the nature of the lesion underlying thyroid neoplasia, thus improving diagnostic accuracy of conventional FNA cytology.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Nuclear retinoic acid receptors (RARs) function as ligand-activated trans-acting transcription factors and mediate the effects of retinoids on gene expression, cell growth, and differentiation. Determination of the receptors expression in premalignant and malignant lesions may provide prognostic value and direct the selection of receptor-specific retinoids in cancer prevention or treatment. We describe a sensitive and practical in situ hybridization method for the analysis of RARs in tissue sections of fixed and embedded surgical specimens. Digoxi-genin-labeled antisense and sense RNA probes were prepared for nuclear RAR-a, RAR-f3, and RAR-7. The specificity of the probes for their respective receptor mRNAs was demonstrated by Northern blot hybridization to total RNA extracted from murine and human cells. Optimal conditions for in situ localization of the RAR mRNA were established using cultured tumor cells, and these conditions were then used for the detection of RAR mRNA in formalin-fixed, paraffin-embedded sections of surgical specimens from human tumors. The hybridization stain was detected in the cytoplasm (where it was expected to be localized) and not seen in the cell nucleus. This method provides a rapid detection procedure with good resolution that allows one to clearly distinguish strongly and weakly stained cells. A comparison of receptor expression in head and neck squamous carcinoma specimens and in adjacent normal tissues revealed a significant decrease in the level of RAR-β mRNA in the tumor cells.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The analysis of the tumor suppressor gene, p53, is of fundamental importance in prognosis and staging in many cancers; however, the molecular techniques required to analyze this gene have been expensive, time consuming, and unrelatable to the histological appearance of the samples. This research explored one model of clinically testing for specific mutations in the p53 gene by scraping selected areas of stained histological slides and analyzing for “hot-spot” p53 mutations. Selectively removing samples from the stained histological slide will be of special value in examining suspicious regions in adenomas, potential metastatic regions, and the margins of resected area. A polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis approach in which naturally occurring or primer-mediated mutagenesis-induced restriction enzyme sites were utilized to test seven hot-spot mutations. These assays were able to detect one mutated sequence in 100, and therefore, were sufficiently sensitive to be used with very heterogeneous tumors. Several of the assays could be multiplexed to reduce the number of PCRs necessary to screen for the seven mutational hot spots. Furthermore, an exact determination of the base change could be obtained by direct sequencing of the PCR products. Although this form of analysis may be applicable only to certain types of cancers (e.g., bladder, brain, colon, esophageal, gastric, thyroid, and ovarian tumors), this approach can obtain detailed mutational information from specific regions of a histological slide in a cost-effective and timely manner.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID