Biomedical subjects

Tony E Godfrey

Publications and source records attributed to Tony E Godfrey.

22 records · Page 2Linked to original sources

PubMed2002

Rapid, quantitative reverse transcriptase-polymerase chain reaction: application to intraoperative molecular detection of occult metastases in esophageal cancer.

OBJECTIVE: Our earlier data showed that quantitative reverse transcriptase-polymerase chain reaction can discriminate patients with node-negative cancer who are at high risk for recurrence. The objective of this study was to determine whether a new, more rapid quantitative reverse transcriptase-polymerase chain reaction assay could provide this information in a time frame suitable for intraoperative decision making. METHODS: We studied formalin-fixed, archived lymph nodes from 30 patients with histologically determined node-negative esophageal cancer with rapid quantitative reverse transcriptase-polymerase chain reaction to measure expression of carcinoembryonic antigen messenger RNA. We also performed rapid quantitative reverse transcriptase-polymerase chain reaction on 37 snap-frozen lymph nodes from 23 patients. Eleven of the 23 patients had benign esophageal disorders (negative control group). The other 12 had esophageal cancer, 6 with histologically determined positive lymph nodes and 6 with histologically determined negative lymph nodes. RESULTS: In the retrospective analysis of archival tissue from 30 patients with esophageal cancer with histologically determined negative lymph nodes, rapid quantitative reverse transcriptase-polymerase chain reaction predicted disease recurrence with a sensitivity and a specificity of 90% and 80%, respectively, and was comparable to conventional quantitative reverse transcriptase-polymerase chain reaction. In the frozen-tissue analysis rapid quantitative reverse transcriptase-polymerase chain reaction detected significantly higher levels of carcinoembryonic antigen expression in all 12 of the histologically determined positive lymph nodes than in the benign nodes. For 2 of these 12 nodes the intraoperative frozen-section analysis had negative histologic results, and N1 status was determined only on final pathologic examination. Rapid (intraoperative) quantitative reverse transcriptase-polymerase chain reaction discriminated both nodes as positive. Among the 14 histologically determined negative nodes, 1 of 3 nodes from 1 patient showed increased carcinoembryonic antigen according to rapid quantitative reverse transcriptase-polymerase chain reaction, and this patient had a clinical recurrence. CONCLUSIONS: In our study we were able to rapidly discriminate patients with node negative-esophageal cancer who had a high risk of recurrence. In frozen tissues rapid quantitative reverse transcriptase-polymerase chain reaction correlated with final pathologic report for 11 of 12 patients. In the 1 discordant case, the quantitative reverse transcriptase-polymerase chain reaction result was positive and may have detected microscopically occult metastasis, because this patient did have disease recurrence. Rapid quantitative reverse transcriptase-polymerase chain reaction was more sensitive than intraoperative frozen sections for detecting metastatic disease. These data suggest that rapid quantitative reverse transcriptase-polymerase chain reaction may have a prognostic role and could guide intraoperative decisions.

Aged↗

PubMed2002

Real-time quantitative polymerase chain reaction: a potential tool for genetic analysis in neuropathology.

Since its introduction in the early- to mid-1980s, the polymerase chain reaction (PCR) has been modified and optimized for an increasing number of applications. Early on, the focus was on the amplification of a specific nucleic acid template into quantities amenable to identification and experimental manipulation. While this remains an important application, recent technology has allowed the use of PCR to accurately quantitate the amount of a specific nucleic acid template present in a complex sample. Rather than simply analyzing the final product amount following the course of sequential cycles of amplification, quantitative PCR allows one to measure the accumulation of PCR product during the course of the reaction ("real-time PCR"). Under the appropriate conditions the number of PCR cycles required for the accumulation of a specific amount of product (during the exponential phase of the reaction) is a reflection of the relative amount of nucleic acid template present in the sample under analysis. Real-time quantitative PCR allows one to analyze a relatively large number of samples in a short period of time, potentially allowing multiple markers to be applied on a sample within a time frame consistent with clinical settings. In this overview, we will highlight the uses of real-time quantitative PCR as a potential diagnostic tool in neuropathology, focusing on the analysis of CNS tumors.

Brain Neoplasms↗

PubMed2002

Temperature-controlled primer limit for multiplexing of rapid, quantitative reverse transcription-PCR assays: application to intraoperative cancer diagnostics.

BACKGROUND: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate. METHODS: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples. RESULTS: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1-2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples. CONCLUSION: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.

Base Sequence↗

PubMedDate not supplied

Workshop on cancer biometrics: identifying biomarkers and surrogates of cancer in patients: a meeting held at the Masur Auditorium, National Institutes of Health.

The current excitement about molecular targeted therapies has driven much of the recent dialog in cancer diagnosis and treatment. Particularly in the biologic therapy of cancer, identifiable antigenic T-cell targets restricted by MHC molecules and the related novel stress molecules such as MICA/B and Letal allow a degree of precision previously unknown in cancer therapy. We have previously held workshops on immunologic monitoring and angiogenesis monitoring. This workshop was designed to discuss the state of the art in identification of biomarkers and surrogates of tumor in patients with cancer, with particular emphasis on assays within the blood and tumor. We distinguish this from immunologic monitoring in the sense that it is primarily a measure of the tumor burden as opposed to the immune response to it. Recommendations for intensive investigation and targeted funding to enable such strategies were developed in seven areas: genomic analysis; detection of molecular markers in peripheral blood and lymph node by tumor capture and RT-PCR; serum, plasma, and tumor proteomics; immune polymorphisms; high content screening using flow and imaging cytometry; immunohistochemistry and tissue microarrays; and assessment of immune infiltrate and necrosis in tumors. Concrete recommendations for current application and enabling further development in cancer biometrics are summarized. This will allow a more informed, rapid, and accurate assessment of novel cancer therapies.

Alternative Splicing↗