ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The breakpoints of the translocation t(2;5)(p23;q35) associated with Ki-1-positive anaplastic large-cell lymphoma (Ki-1 ALCL) involve a novel tyrosine kinase gene,ALK, at 2p23 and the nucleophosmin gene,NPM, at 5q35. Reverse transcriptase-polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 ALCL cases with the translocation, resulting from genomic breakpoints within the same respective introns ofNPMandALK. To examine the feasibility of long-range DNA PCR with the same exonicNPMandALKprimers for the detection of the genomicNPM-ALKrearrangement, we examined 20 cases of Ki-1 ALCL previously characterized byNPM-ALKRT-PCR. Ten cases were positive for theNPM-ALKfusion RNA and 10 were negative. We first confirmed that both theNPMandALKnormal introns are relatively short, approximately 1 and 2 kb, respectively, suggesting that the largest possible size for the chimericNPM-ALKintron would be about 3 kb. All 10 cases positive by RT-PCR were also positive by long-range DNA PCR. The DNA PCR products ranged, as expected, from the sizes of the normal introns, between 0.5 and 2.5 kb. All 10 RT-PCR-negative cases were also negative by long-range DNA PCR, and control templates for RT-PCR and long-range DNA PCR were successfully amplified. Thus, we have shown that the introns involved by theNPM-ALKrearrangement seen in some Ki-1 lymphomas are relatively short, making the genomic rearrangement amenable to reliable detection by longrange DNA PCR. Furthermore, the variability observed in the sizes of chimeric introns is evidence against clustering of the genomic breakpoints within these introns.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The detection of clonality in B-cell lymphomas has been facilitated by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy-chain gene (IgH) complementarity determining region 3 (CDR3) and size fractionation by polyacrylamide gel electrophoresis (PAGE). However, the detection of minor clonal populations and biallelic rearrangements and the isolation of monoclonal products from gels are sometimes problematic. This study evaluated whether denaturing gradient gel electrophoresis (DGGE), a technique that separates DNA based on nucleotide sequence rather than length, could alleviate these problems. A total of 32 selected cases was studied with a diagnosis of monoclonal (n= 10). polyclonal (n= 9). and indeterminate (n= 13) IgH gene rearrangements, which were determined by analysis of seminested IgH CDR3 PCR products in 8% PAGE. These cases were evaluated using DGGE of seminested IgH CDR3 PCR products that included a 40-bp GC clamp on the Jh primer. DGGE allowed the discrimination of monoclonal populations in 9 of 13 cases where 8% PAGE results were indeterminate. In addition, DGGE demonstrated biallelic IgH rearrangements in three cases where 8% PAGE revealed only one predominant product. DGGE facilitated the purification and isolation of clonal IgH CDR3 products for sequencing without prior cloning. As an adaptation of current IgH PCR protocols. DGGE can enhance the construction of tumor-specific CDR3 primers/probes for investigations of minimal residual disease.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

A supportive or causal role for human herpesvirus 6 (HHV-6) in lymphoproliferative disorders is still controversial. Different results were obtained in both tissue-based and serological investigations. We investigated 243 lymph node and salivary gland tissue biopsies for the presence of viral DNA by using a newly developed, highly sensitive nested polymerase chain reaction method. HHV-6 was detected in 39% of the non-Hodgkin's lymphomas, in 52% of Hodgkin's diseases, 64% of non-neoplastic lymph nodes. 23% of tumor metastases, and 50% of salivary gland biopsies. When correlating the patients' ages with the occurrence of HHV-6, we found a significantly higher percentage of positive samples in patients younger than 60 years of age (54%) than in older patients (35%). This age-related-difference was found in all the lymphoproliferative disorders studied as well as in salivary gland biopsies. Taking patient's ages into account, we found no significant difference between the various groups of disorders concerning the percentage of HHV-6-positive samples.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to identify breast carcinoma cells in bone marrow aspirates with high sensitivity and specificity. This assay relies on the detection of cytokeratin 19 (K19) RNA by nested primer PCR followed by annealing to a (32P)-labeled internal sequence probe and autoradiography. In reconstitution experiments, this assay is capable of detecting 10 fg of admixed mammary tumor RNA in 1 μg of normal marrow RNA (a dilution of 1:107). Thirty of 30 primary breast tumor specimens, 19 of 19 cytologically positive bone marrow aspirate specimens, and three of 11 aspirate negative/biopsy positive specimens showed detectable K19 transcript. This assay shows high specificity, with 50 of 52 negative control aspirates showing no detectable amplification product. False-positive amplification was noted in two of 18 aspirates obtained from patients with active chronic myelogenoirs leukemia. Of stage II and III postsurgical breast carcinoma patients with histologically negative bone marrows and no radiographic bone disease. 14 of 30 were K19 positive by PCR. RT-PCR analysis of K19 transcript is a highly sensitive and specific method of detecting and monitoring low-level metastatic disease in patients with primary carcinoma of the breast. The presence of K19 RNA in histologically negative bone marrows suggests that this assay may prove a powerful monitor for patients undergoing curative therapy as well as a novel prognostic indicator.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We examined 232 breast carcinomas for c-erhB-2 amplification by Southern analysis using two different cDNA probes. Using these same probes, 95 of these tumors were also examined for mRNA expression by Northern analysis. Amplification was detected in 20 and 17% of the tumors with the probes pHER 2 and pCER 204, respectively, but only 10% showed amplification with both probes. A significantly higher incidence (p < 0.01) of mRNA overexpression was detected with the pHER 2 probe (34%) compared with the pCER 204 probe (16%), with only 11% of tumors demonstrating overexpression with both probes. A total of 10 tumors (11%) exhibited amplification as well as overexpression with pHER 2, whereas significantly fewer (3%) manifested both abnormalities with the larger pCER 204 probe (p < 0.05). Amplification of c-erbB-2, as detected with the pHER 2 probe but not with the pCER 204 probe, was significantly associated with the absence of both estrogen and progesterone receptors (p < 0.05 and p < 0.01. respectively). No relationship was found with other clinical prognostic indicators, such as nodal involvement and metastases. As determined by either probe, overexpression was not associated with prognostic indicators. There was no significant difference in the c-erbB-2 status of tumors from different racial groups.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6–50% cells positive) or focal (<5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2–11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6–100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6–50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in <5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01: and PAb1801. p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immuno-histology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

This pilot project analyzed the tumor suppressor genes p53 and Rb in 13 cases of aggressive fibromatoses and six cases of palmar fibromatoses (Dupuytren contracture). Immunohistochemistry, reverse transcription polymerase chain reaction, polymerase chain reaction followed by single-strand confirmation polymorphism analysis, and Southern blot to detect gene rearrangements were used. No abnormalities were detected in p53. The aggressive fibromatoses demonstrated a lack of Rb immunohistochemical staining and decreased mRNA for Rb. No structural mutation in the coding sequence of the Rb gene was detected. The decreased level of Rb gene expression, despite a normal coding sequence, may indicate increased proliferation and may suggest potential treatment schemes.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Using polymerase chain reaction (PCR)-SSCP (single-strand conformation polymorphism) and PCR-DNA sequencing analysis, we screened 61 cases of laryngeal squamous cell carcinoma for mutations in exons 5–8 of the p53 gene. Mutations were found in 31.3% (19 of 61) of the laryngeal cancers. Seventeen of 19 (84.2%) cases showing p53 gene mutations were stage III and IV. which suggests that p53 gene mutation is a rather late event in tumor development and is involved in the progression of laryngeal squamous cell carcinoma. A high frequency of G:C to T:A transversion (50%, seven of 14), especially G to T (35.7%, five of 14), was noted in laryngeal carcinoma samples in our study. This finding may point toward an environmental carcinogen (such as tobacco smoke) as an important agent in the genesis of laryngeal squamous cell carcinoma. Most of the p53 gene mutations (18 of 19) found in our studies could change the protein and thus may cause an inactivation of the p53 tumor suppressor gene, strongly suggesting that p53 gene mutation plays a crucial role in the progression of laryngeal carcinoma.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with laborintensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5 + / GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slotblot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

ISSN:1052-9551    

出版商:OVID    

年代:1996

数据来源: OVID