摘要:

This study evaluates the utility of fluorescence-based polymerase chain reaction (PCR) and PCR-SSCP methodologies to monitor the clonal relatedness of cells with bcl-2 major break point region (mbr)/JR fusion sequences in sequential samples from patients with follicular lymphoma (FL). Fluorescence-tagged PCR products from 2-A sequential samples from seven FL patients were resolved in acrylamide gels and analyzed on an Applied Biosystems' automated DNA sequencer equipped with Genescan software. The amplicons were sequenced directly using automated DNA sequencing to obtain the precise amplicon size and base sequence. Fluorescence-based PCR-single-strand conformation polymorphism (SSCP) analysis performed to distinguish amplicons of similar size but of different base sequence. Amplification products differing by as few as 5 bp resolved clearly under fluorescent PCR assay conditions making possible by visual inspection alone the distinction of two products that otherwise appeared to be of similar size by conventional gel electrophoretic methods. The size of the amplicons as determined by Genescan software correlated exactly with the sizes generated by sequence analysis confirming the precision and accuracy of the fluorescent PCR assay. Under nondenaturing conditions, the mobility profiles of the amplicons from sequential samples with identical base sequence remained indistinguishable, whereas amplicons of similar size but of dissimilar base sequence from different patients exhibited distinct migration patterns. Thus, this study demonstrates that a combination of fluorescent PCR and PCR-SSCP assays for the detection of the t( 14:18) provides an accurate measure of clonal relationship based on molecular size and sequence similarities without involving radiolabeling and sequencing strategies. Furthermore, the demonstrated preservation of junctional sequences across sequential biopsy specimens validates the use of PCR in the monitoring of minimal residual disease and eliminates concern about the detection of secondary, non-tumor-related translocations.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Hepatic resection is the treatment of choice for selected patients with liver metastases from colorectal cancer (CRC). Although the 5-year survival rate among patients after liver resection is 25–45%, 55–75% of patients die from progressive disease. The purpose of this study was to characterize molecular genetic alterations, including micro-satellite instability and allelic imbalance, in patients with potentially curative resected liver metastases from CRC and to correlate these molecular features with clinical and pathologic characteristics. We examined DNA from formalin-fixed, paraffin-embedded archival tumor specimens from 141 surgically resected hepatic metastases from CRC. We used microsatellite markers localized to chromosome arms 5q, 8p, 10q, 15q, 17p, 18p, and 18q in a polymerase chain reaction-based assay. Allelic imbalance at each locus and the presence of tumor microsatellite instability were correlated with clinicopathologic features of the tumor and clinical course of the patient. Microsatellite instability at multiple loci was seen in only 2.5% of resected liver metastases, a frequency significantly lower than that previously detected for primary CRC. Additionally, these findings had no significant correlation with disease-free survival or overall survival. Allelic imbalance at one or more loci was seen in 87% of informative tumors. Allelic imbalance on chromosome 17p was seen in 84% of informative tumors, and its presence was associated with a significantly poor disease-free survival (p = 0.015) and overall survival (p = 0.05). These data suggest that allelic imbalance on chromosome 17p is an independent prognostic parameter in patients with potentially curative resected liver metastases from CRC. Such alterations could provide a useful stratification criterion for adjuvant therapy for patients who have undergone curative resection of liver metastases from CRC.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

A first-degree relative with pancreatic cancer is found in 5% to 10% of patients with pancreatic carcinomas, suggesting an inherited predisposition for this neoplasm. The recently identified DPC4 tumor suppressor gene is a strong candidate for the gene responsible for the familial form of pancreatic carcinoma. DPC4 was identified in a consensus area of homozygous deletion in pancreatic carcinomas, and it is biallelically inactivated in approximately 50% of sporadic pancreatic carcinomas. The coding sequence of this gene is 1660 nucleotides in length, covering 11 exons. We describe optimized primers and conditions used in polymerase chain reaction and cycle sequencing of the entire DPC4 coding sequence of 25 individuals (eight with pancreatic carcinoma) from 11 kindreds with a familial aggregation of pancreatic carcinoma. No mutations in the coding sequences of the DPC4 gene were found; hence, it appears that germline mutations in DPC4 cannot account for many of the familial aggregations of pancreatic carcinoma.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Alveolar rhabdomyosarcoma is a pediatric soft-tissue tumor that is often difficult to distinguish from other small round-cell tumors. The PAX3-FKHR and PAX7-FKHR gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival material, we used reverse transcriptase-polymerase chain reaction and oligonucleotide hybridization methodology to develop an assay capable of identifying PAX3-FKHR and PAX7-FKHR fusion transcripts in formalin-fixed, paraffin-embedded tissue. Use of a control assay for wild-type FKHR mRNA indicated that RNA was successfully isolated, reverse-transcribed, and amplified in 15 of 16 archival cases. Comparison of assay results for the PAX3-FKHR and PAX7-FKHR fusions with standard molecular assays of paired frozen material revealed that all eight cases of known fusion-positive rhabdomyosarcoma were correctly identified and distinguished as PAX3-FKHR or PAX7-FKHR. The seven cases of known fusion-negative rhabdomyosarcoma showed no evidence of either product. These results indicate that we have developed a molecular assay that accurately identifies the fusion transcripts characteristic of alveolar rhabdomyosarcoma in archival samples. This assay will be useful for diagnosis and for retrospective clinicopathologic correlative studies.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Aggressive fibromatosis (also called deep fibromatosis or desmoid tumor) is a proliferation of cytologically benign-appearing fibrocytes, often resulting in significant functional loss. The nature of the lesion is controversial: some evidence suggests that it is a reactive process, whereas other evidence supports a neoplastic etiology. The pattern of X chromosome inactivation, using a technique based on polymerase chain reaction (PCR) amplification of a hyper-variable CAG repeat region flanking Hhal restriction sites of the human androgen receptor gene, was determined in four cases in which cryopreserved tumor and adjacent normal tissue were available. All four tumors demonstrated a monoclonal pattern, while the adjacent normal tissues demonstrated a polyclonal pattern. This demonstrates that aggressive fibromatosis is proliferation of cells derived from a single clone with a growth advantage, and thus is likely a neoplastic process.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The telomere repeat amplification protocol (TRAP) was recently developed to detect telomerase activity in cellular protein extracts. Teleomerase synthesizes a specific repeating nucleotide sequence onto the ends of telomeres, which stabilize eukaryotic chromosomes. Telomeric repeats are identified in the TRAP method by polymerase chain reaction amplification and incorporation of radionucleotides detected by autoradiography. Several drawbacks to this method have been recognized, including the time required to complete the assay, the resolution of the results, and the hazards of radioactive material. We have developed a new fluorescent method of detecting telomerase to alleviate these problems. Telomeric repeats are identified in the fluorescent TRAP (F-TRAP) assay by incorporation of fluorescein-labeled primers during amplification and subsequent detection with an automated DNA sequencer. This new method appears to be as sensitive as the standard TRAP assay and offers advantages in speed, resolution, cost, and safety.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Specimen identification is a carefully controlled factor in clinical laboratory testing. However, on occasion, despite surmountable efforts to prevent misidentification, a specimen is either mislabeled or an identifier is lost. Recently, we experienced a case of questionable mix-up of surgical specimens where the surgeon and patient questioned the biopsy site and size of specimen as indicated in the anatomic pathology report. Despite extensive tracking mechanisms, the perception of specimen mix-up warranted further means of identification. We utilized the PM+DQA1 amplification and typing system to confirm that typing results of a questionable biopsy were identical to typing results on a previous biopsy on the same patient and to the patient's blood. We demonstrate that this system is ideal for rapid DNA typing and identification of clinical specimens and that it can be performed on DNA isolated from paraffin-embedded tissues.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Loss of p16 expression can occur via homozygous deletion, point mutation, or hypermethylation of exon 1. Astrocytomas representing all World Health Organization (WHO) grades of malignancy were analyzed in a correlative study using multiplex polymerase chain reaction (PCR) analysis to detect deletions of the p16 gene together with immunohistochemistry to detect loss of the protein in archival specimens of the same tumors. Homozygous deletions of p16 were detected in 29% (15 of 52) of WHO grade 3 and 4 tumors. Immunostaining for pl6 protein was present in 26 tumors retaining the p16 gene and absent in 11 tumors with deletions of the p16 gene. A close correlation was found between the two detection methods, with all tumors lacking immunostaining showing homozygous loss of the p16 gene. Astrocytomas exhibiting inactivation of the p16 gene most often contained p53 gene mutations or amplified epidermal growth factor receptor genes, genetic characteristics associated with both the progressive and de novo tumor variants. Immunohistochemical evaluation may be a useful, rapid method to screen astrocytomas for loss of p16 gene expression, regardless of the underlying mechanism leading to p16 gene inactivation.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The histopathologic alterations of hepatitis C virus (HCV) infection of the liver overlap with those of other diseases, making interpretation of liver biopsy specimens in some cases insufficient to render a diagnosis. Although HCV infection can be confirmed by detection of circulating anti-HCV antibodies, immunocompromised liver transplant recipients are often unable to mount an immunologic response to the virus, resulting in false-negative serologic testing. We describe the comparison of reverse transcription-polymerase chain reaction (RT-PCR) with histopathology, serology, and immunohistochemistry for the diagnosis of HCV. Sixty-three formalin-fixed, paraffin-embedded tissue samples (40 needle biopsy specimens and 23 native liver resection specimens) from 35 transplant patients were analyzed by use of a novel method of RNA extraction followed by nested PCR for HCV as well as albumin mRNA as an internal control. HCV was detected by RT-PCR in 50 of 51 (98%) paraffin sections of liver from transplant patients with circulating anti-HCV antibodies, 15 of which lacked characteristic histologic features of HCV infection. Overall, there were no false-negative results in 36 needle biopsy specimens from patients with hepatitis C infection, but three negative results were seen in end-stage cirrhotic native livers resected from HCV-infected patients. No false-positive test results were seen among 21 negative controls (10 liver samples from immunocompetent patients with abnormalities unrelated to hepatitis C and 11 liver biopsies from immunocompetent patients without histologic evidence of liver disease). In comparison, immunohistochemistry using antibody TORDJI-22 was positive for HCV in only 15 of 32 (47%) needle biopsies positive by RT-PCR. Our results indicate that RT-PCR is a more sensitive and specific method of detecting hepatitis C in routinely processed paraffin sections of formalin-fixed liver biopsy specimens than histopathologic examination or immunohistochemistry.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID