#255: Detection of prostate marker mRNA in lymph nodes of prostate cancer patients U. Schmidt, A....
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#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 46 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Sample conc. Cross. pt. water standard 100 pg 17,9 standard 10 pg 21,8 standard 1 pg 25,8 standard 100 fg 30,1 Pos. 1:10 22,5 Pos. 1:100 25,9 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 5.0 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 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 pg 17,0 standard 1 pg 20,6 standard 100 fg 24,3 standard 10 fg 27,9 1 21,4 2 18,5 3 20,3 4 21,8 5 21,4 6 21,2 7 21,4 8 32,8 10,0 0,1 1,0 46 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 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 pg 19,8 standard 10 pg 23,6 standard 100 fg 27,5 1 27,9 2 25,4 3 23,8 4 21,8 5 25,1 6 21,6 7 25,7 8 35,9 10,0 0,1 1,0 46 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 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 n GAPDH- PCR+ n PSA- PCR+ PSA -TL per LN n PSM - PCR + PSM -TL per LN n PSM ‘- PCR + PSM ‘-TL per LN PCa N1 13 13/13 19/19 1,7 x 10E6 13/13 17/19 1,0 x 10E6 8/13 9/19 2,6 x 10E6 ptPCa N1 4 4/4 10/10 1,4 x 10E5 4/4 10/10 8,3 x 10E5 2/4 4/10 1,8 x 10E6 PCa N0 29 29/29 44/46 3,8 x 10E5 24/29 33/46 5,5 x 10E5 8/29 11/46 5,3 x 10E5 ptPCa N0 11 11/11 18/18 5,7 x 10E5 9/11 13/18 7,0 x 10E5 1/11 2/18 2,9 x 10E5 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´).
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Transcript of #255: Detection of prostate marker mRNA in lymph nodes of prostate cancer patients U. Schmidt, A....
#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.
ConclusionsIn 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
4619 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Sample conc. Cross. pt.
waterstandard 100 pg 17,9standard 10 pg 21,8standard 1 pg 25,8standard 100 fg 30,1Pos. 1:10 22,5Pos. 1:100 25,9
Standards for GAPDH
lo
g F
2/F
1 fl
uo
resc
ence
number of cycles
31
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.02.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9
log number of cycles
cro
ssin
g p
oin
ts Crossing points
linear regression
unknown concentration
Standard curve for GAPDH
ResultsWith 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.
waterstandard 10 pg 17,0standard 1 pg 20,6standard 100 fg 24,3standard 10 fg 27,91 21,42 18,53 20,34 21,85 21,46 21,27 21,48 32,8
10,0
0,1
1,0
4619 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
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.
waterstandard 100 pg 19,8standard 10 pg 23,6standard 100 fg 27,51 27,92 25,43 23,84 21,85 25,16 21,67 25,78 35,9
10,0
0,1
1,0
4621 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
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 n
GAPDH-
PCR+
n
PSA-
PCR+
PSA-TL per
LN
n
PSM-
PCR +
PSM-TL per
LN
n
PSM‘-
PCR +
PSM‘-TL per
LN
PCa
N1
13 13/13 19/19
1,7 x 10E6
13/13 17/19
1,0 x 10E6
8/13 9/19
2,6 x 10E6
pt PCa
N1
4 4/4 10/10
1,4 x 10E5
4/4 10/10
8,3 x 10E5
2/4 4/10
1,8 x 10E6
PCa
N0
29 29/29 44/46
3,8 x 10E5
24/29 33/46
5,5 x 10E5
8/29 11/46
5,3 x 10E5
pt PCa
N0
11 11/11 18/18
5,7 x 10E5
9/11 13/18
7,0 x 10E5
1/11 2/18
2,9 x 10E5
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´).
