ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single‐strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10‐&mgr;m paraffin‐embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5‐8 in human gliomas utilizing paraffin‐embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure >90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR‐SSCP analysis and subsequently confirmed by PCR‐based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu). A silent mutation in exon 8 was found in a cerebral hemispheric AA (codon 273, GCG > GCA: Ala > Ala). The yield of p53 mutations compares favorably with the reported incidences in AA and GB using frozen tissues (18% vs. 20‐40%). This study demonstrates the feasibility of evaluating p53 gene mutations in archived glioma specimens using PCR on paraffin sections. Application of this method should facilitate investigation of the role of p53 gene mutations in tumor biology.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;The spectrum of p53 gene mutations was investigated in thyroid carcinomas with respect to histopathological classification. In all histological subtypes of thyroid carcinoma that had previously revealed positivity in immuno‐histochemical staining for p53 protein, single‐stranded conformation polymorphism analysis and direct sequencing were performed to detect point mutations between exons 5 and 8. In well differentiated papillary and follicular carcinomas, in which we had already known that 11.1 and 14.3% of the cases, respectively, revealed p53 over‐expression as determined by immunohistochemistry, genetic aberrations were undetectable. In poorly differentiated carcinoma, in which 40.9% had revealed overexpression, two of six cases revealed point mutations at codon 244 in exon 7 and at codon 278 in exon 8. In undifferentiated carcinoma, in which 63.6% had revealed overexpression, four of six cases examined showed point mutations at codon 157 in exon 5, at codon 248 in exon 7, and at codon 273 and a two‐base insertion between codons 266 and 267 in exon 8. These results strongly suggest the crucial role of p53 gene aberration and protein overexpression in a biologically aggressive subtype, possibly as a stepwise participation in the process of tumor dedifferentiation in human thyroid carcinomas.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;We observed a potentially misinterpretable polymerase chain reaction (PCR) amplification product generated with standard primers used to detect the major breakpoint region (mbr) of chromosomal translocation t(14;18). This unexpected phenomenon was initially detected during attempts to transform follicular lymphomas in vitro with Epstein‐Barr virus (EBV). Additional studies were performed using the EBV‐producing cell line MCUV5, cell lines from EBV‐transformed normal B‐lymphocytes, and an excised lymph node from a patient with documented EBV‐associated infectious mononucleosis. These samples consistently produced a 167‐base pair product, which was indistinguishable from a t(14;18) lymphoma product when viewed on ethidium bromide‐stained gels. Through DNA sequencing and gene bank analysis, the product was identified as a portion of the EBV genome. A mbrspecific 20‐base oligonucleotide probe was able to discriminate between true translocations and the EBV‐related amplifications. These results underscore the importance of employing a specific detection system, and comprehensively screening primers when working with PCR.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;Epstein‐Barr virus (EBV) has been detected in African Burkitt's lymphoma, posttransplant lymphoproliferative disease, and a variable fraction of Hodgkin's lymphomas. To assess if EBV is associated with other lymphoid proliferations, we evaluated a wide variety of benign and malignant lymphoid lesions, using polymerase chain reaction and a sensitive in situ hybridization method. Abundant EBV+cells were seen in posttransplant lymphomas, some B cell immunoblastic lymphomas, and in tonsils from patients with infectious mononucleosis. Intermediate numbers of EBV+cells were seen in a mixed B cell lymphoma, peripheral T cell lymphomas, and in syncytial variants of Hodgkin's disease as well as a lymph node from a patient with infectious mononucleosis. Low numbers of EBV+cells were detected in normal and reactive lymph nodes, B and T cell lymphomas, and Hodgkin's lymphomas. The variable extent of EBV infection in lymphoid lesions suggests that EBV may play a variety of roles in the development of malignant and nonmalignant lymphoid lesions.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;Although undifferentiated carcinoma (UC) and squamous cell carcinoma (SCC) of the nasopharynx are regarded as two distinct histopathologic and clinical entities, it is unclear whether, like UC, SCC carries Epstein‐Barr virus (EBV) genomes. We used the polymerase chain reaction (PCR) on paraffin‐embedded biopsy specimens to test for the presence of EBV DNA in 20 cases of UC and 9 cases of SCC. Multiple copies of the viral genome were regularly detected in all UCs; however, of the nine cases of SCC, seven had no detectable EBV DNA and two contained viral genomes in a low copy number. In parallel, a marked difference in the serum levels of anti‐EBV antibodies between patients with UC and SCC was found. Our findings provide evidence for the specific association of EBV with UC in Italian patients and prove by means of a highly sensitive molecular technique that SCC is occasionally related to EBV DNA. Because of the absence of EBV DNA in most cases of SCC and the minimal viral DNA copy number in the few EBV‐associated cases of SCC, a different pathway of oncogenic transformation and growth of the nasopharyngeal epithelium is suggested for SCC and UC.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;Twenty‐five pituitary adenomas were analyzed for expression of various chromogranin/secretogranin (Cg/Sg) messenger RNA (mRNA) transcripts by in situ hybridization (ISH). An additional five adenomas were also analyzed by Northern hybridization. Immunohistochemical staining for CgA and for SgIV (with monoclonal antibody HISL‐19) was also performed. Most prolactin and adrenocorticotropin adenomas did not express CgA mRNA or protein, whereas growth hormone (GH) tumors had low to moderate amounts of CgA mRNA by Northern and in situ hybridization analyses and were focally positive for CgA protein. CgB, SgII, SgIII, and SgV mRNA transcripts were present in most adenomas, and SgIV protein was detected in all groups of tumors. A GH and a null cell adenoma cultured for 7 days also expressed CgA/Sg mRNA transcripts and protein. Paraffin sections of some adenomas that were negative for CgA protein had detectable CgA mRNA by in situ hybridization analysis. These results indicate that CgA mRNA and protein are more commonly expressed in glycoprotein hormoneproducing tumors compared with other types of pituitary adenomas and that ISH for CgA may detect the mRNA transcripts for CgA even when CgA protein is not detected by immunohistochemistry.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;We studied growth hormone (GH) and prolactin (PRL) messenger ribonucleic acids (mRNAs) in 14 cases with densely granulated somatotroph (DG) adenomas and 10 cases with sparsely granulated somatotroph (SG) adenomas using in situ hybridization with digoxigenin‐labeled probes and correlated these data with their immunohistochemical results. A good correlation between in situ hybridization results and immunohistochemical data was found in most cases examined. The DG adenomas generally had a diffuse and intense GH immunoreactivity and GH hybridization signal, whereas in SG adenomas the number of cells exhibiting a GH mRNA signal and the strength of the GH mRNA signal in these cells were relatively lower than those of DG adenomas, indicating that lower expression of the GH mRNA signal is responsible for lower GH production in SG adenomas. In addition, PRL mRNA expression differed in the two types of adenoma; in DG adenomas, seven cases (50%) expressed PRL mRNA signal with a focal or scattered distribution despite normal serum PRL levels, but two showed no PRL immunoreactivity. In contrast, in SG adenomas, only one case contained a few cells possessing a PRL mRNA signal despite having no PRL immunoreactivity. It can be concluded that DG and SG adenomas, which have been considered variants of the same tumor, display definite differences as to GH or PRL gene expression in each type of adenoma.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65‐kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogensMycobacterium tuberculosis, Mycobacterium avium‐intracellulare, andMycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, includingM. kansasiiandMycobacterium leprae, in formalin‐fixed paraffin‐embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin‐embedded tissue sections.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

&NA;Reexpression of the insulin‐like growth factor type II (IGF‐II) gene has recently been described in hepatocellular carcinoma (HCC). In this study, we used a nonisotopic in situ hybridization method to analyze the expression of IGF‐II mRNA in a series of 28 HCCs arising on cirrhotic and noncirrhotic livers. An immunohistochemical method was used to detect IGF‐II peptide. Hepatitis B virus (HBV) status and the histological differentiation degree were also evaluated. Increased expression of IGF‐II mRNA was found in 4 of 28 HCCs, and 7 of 17 cirrhotic patients showed IGF‐II mRNA in the cirrhotic nodules surrounding the HCC. A slightly higher rate of positivity for IGF‐II mRNA was found in the HBV‐negative patients than in HBV‐positive ones. Positive immunostaining for the IGF‐II peptide in the HCC and/or in surrounding cirrhotic nodules was found in 10 of 28 cases. The normal hepatocytes of the noncirrhotic patients were always negative for IGF‐II peptide and mRNA. The similarities between our results and those from experimental models in woodchucks seem to support the concept that heterogeneous phenotypic groups could exist in human HCCs.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID