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Contenu archivé le 2024-04-30

Measurement of haematogenous micrometastases associated with prostate cancer by RT/PCR and DNA probe detection of prostate specific antigen(PSA) mRNA

Objectif



The aim of this research and development project is to establish a reliable, reproducible, semi-quantitative, precise and sensitive RT/PCR PSA assay, in conjunction with a state-of-the-art detection method of amplified target mRNA. PSA mRNA is exclusively transcribed by prostatic epithelial cells. The presence of such cells in the blood and/or in tissues other than the prostate gland is abnormal and can only be attributed to the spreading of prostate cancer. Hematogenous metastasis are suspected to be the carrier of prostate tumor cells to bone metastasis. The RT/PCR PSA assay will be used as a reference method to study metastasis in human prostate cancer, by evaluating the presence of circulating prostate cancer cells in peripheral blood.
After RNA isolation from the specimen, the assay utilizes a reverse transcription (RT) reaction to produce PSA cDNA which will be subsequently amplified by a polymerase chain reaction (PCR). The amplified target DNA will then be captured and its presence visualized by means of an immunoassay-like detection procedure featuring homogeneous hybridization, magnetic beads separation and a chromogenic enzymatic reaction. An innovative and commercially viable RT/PCR PSA assay that can be used for 'in vitro' diagnostic tests will be developed. The final product will be a DNA-probe kit which includes the critical reagents required to perform the assay, as has been accomplished with other widely used immunodiagnostic kits. The procedure will be optimized in terms of sensitivity, specificity, practicability, cost effectiveness, total assay time, reagent presentation, negative and positive controls. The detection procedure of the amplified target DNA will offer researchers and clinicians major advantages over the currently used methods in molecular pathology laboratories. It will be fast -requiring less than two hours after amplification, easy to use -highly skilled personnel will not be needed, cost effective -simple instruments which are commonly available in routine clinical laboratories will be utilized, and safe -no harmful, toxic or radioactive materials will be used. Cancer of the prostate is the second leading cause of cancer and cancer deaths in the European community. For patients with clinically organ confined prostate cancer, radical prostatectomy is considered to be the gold standard of therapy, and it is thought to be curative. However, up to 50 of patients thought to have organ confined disease are found to have been clinically understaged at the time of surgery. A more sensitive staging modality that could improve the detection of extraprostatic disease in patients prior to local therapy would greatly assist clinicians and have important therapeutic implications. The aim of the clinical validation will be to establish a predictive assay which can identify those patients who have circulating prostate cancer cells prior to radical prostatectomy or radiotherapy. Furture benefit will derive from the information gathered about iatrogenic factors, such as digital rectal examination (DRE), transrectal prostate biopsy, transurethral resection of the prostate (TURP) for prostate cancer, and radical prostatectomy which may produce circulating prostate cancer cells, and from determining if these cells eventually lead to metastases. An international consortium composed of highly qualified specialists will participate in validation of the assay and in clinical trials to evaluate the potential role of this innovative and commercially viable in vitro diag,ostic DNA probe kit.

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Coordinateur

Raggio-Italgene SpA
Contribution de l’UE
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Adresse
Via delle Antille 29
00040 Pomezia Roma
Italie

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