Presentation on theme: "#255: Detection of prostate marker mRNA in lymph nodes of prostate cancer patients U. Schmidt, A. Meye, R. Kranz, S. Füssel, U. Fiedler, M.P. Wirth Department."— Presentation transcript:
#255: Detection of prostate marker mRNA in lymph nodes of prostate cancer patients U. Schmidt, A. Meye, R. Kranz, S. Füssel, U. Fiedler, M.P. Wirth Department of Urology, Technical University of Dresden Material & Methods Lymph nodes (2-6) were obtained from 57 patients mostly undergoing radical prostatectomy. One half of each node was examined by a pathologist, the other half was snap-frozen before the extraction of RNA. Contaminating DNA was removed by DNase I digestion. When available, 4 µg of RNA were reverse-transcribed into cDNA which served (2µl per reaction) as starting material for the quantitative PCR analyses (LightCycler). The quality of the cDNA was checked with a specific PCR for the house keeping gene GAPDH. The results of this PCR were also used for normalization. External standards (plasmids with cloned PCR products) were used for the quantification. The quantitative PCRs employed hybridisation probes for the detection of the PCR products. The amount of specific transcripts was normalized per amount of GAPDH per µg totalRNA. Conclusions In contrast to qualitatitative RT-PCR where only a yes/no answer is possible, quantitative RT-PCR assays allow a sensitive detection and a precise masurements of transcript levels of target genes. Follow- up screenings represent a tool to determine cut-off levels for specific transcripts involved in metastatic processes. 10,0 0,1 1,0 46192021222324252627282930313233343536373839404142434445 Sample conc. Cross. pt. water standard 100 pg17,9 standard 10 pg21,8 standard 1 pg25,8 standard 100 fg30,1 Pos. 1:1022,5 Pos. 1:10025,9 Standards for GAPDH log F2/F1 fluorescence number of cycles 31 17 18 19 20 21 22 23 24 25 26 27 28 29 30 5.02.02.12.22.214.171.124.126.96.36.199.03.13.23.33.188.8.131.52.83.94.04.14.184.108.40.206.220.127.116.11 log number of cycles crossing points Crossing points linear regression unknown concentration Standard curve for GAPDH Results With the quantitative LightCycler assay we detected PSA transcripts in all analysed lymph nodes. We detected PSM transcripts in all positive lymph nodes and the lymph nodes of 35/40 N0 patients. PSM´ specific transcripts were detected in 10/17 N+ and in 9/40 N0 patients. Lymph nodes verified as positive by histopathology were RT-PCR positive for all three tested prostate specific markers. Considering the GAPDH ratios, lymph nodes of N+ patients contained nearly 8 times more PSA, 2.5 times more PSM and 8 times more PSM´transcripts in comparison to those of N0 patients. N+ patients with a hormone pre-treatment showed an obvious decrease in the average amount of PSA transcripts. In contrast to that, PSM and PSM´ levels were only slightly influenced by hormonal pre- treatment. Lymph nodes of patients with a Gleason Score of 3 or less did not contain PSM´ transcripts. Overview of the quantitative PCR‘s of LN RNA Overview of the qualitative PCR´s of LN RNA Analyses for PSA sample conc. cross pt. water standard 10 pg17,0 standard 1 pg20,6 standard 100 fg24,3 standard 10 fg27,9 121,4 218,5 320,3 421,8 521,4 621,2 721,4 832,8 10,0 0,1 1,0 46192021222324252627282930313233343536373839404142434445 1 2 3 4 5 6 7 8 M Comparison of the quantitative (LightCycler) and the qualitative (agarose gel) PCR analyses for PSA as a marker. log F2/F1 number of cycles Analyses for PSM 1 2 3 4 5 6 7 8 M Sample conc. cross. pt. water standard 100 pg19,8 standard 10 pg23,6 standard 100 fg27,5 127,9 225,4 323,8 421,8 525,1 621,6 725,7 835,9 10,0 0,1 1,0 4621222324252627282930313233343536373839404142434445 Comparison of the quantitative (LightCycler) and the qualitative (agarose gel) PCR analyses for PSM as a marker. log F2/F1 number of cycles The transcript levels were referred to 1µg of total RNA and a mean GAPDH level. The presented means were deduced from all values including those that were zero. [LN: lymph node, TL: transcript levels, pt: hormone pre-treatment] Measurements of transcript levels in lymph nodes Introduction and Objectives The technique of reverse transcriptase polymerase chain reaction (RT-PCR) allows the specific and sensitive quantification of the expression levels of single genes. In prostate carcinoma, regional lymph nodes are the primary site of colonization of metastasizing tumour cells. So far, metastases in the regional lymph nodes can be elucidated only by histopathology. The aim of this study was to establish a sensitive RT-PCR method for the detection of prostate specific markers in lymph nodes including the determination of specific cut-off levels. We used the prostate specific antigen (PSA) and two splice variants of the prostate specific membrane antigen (PSM and PSM´).