Supplement: Long-term Follow-up of Bladder Cancer Patients with Disseminated
Tumour Cells in Bone Marrow
M. Retza,*, J. Roteringb, R. Nawrotha, A. Buchnerc, M. Stöckleb, J.E. Gschwenda, J. Lehmannd
a
Department of Urology, Technische Universität München, Rechts der Isar Medical Centre,
Munich, Germany
b
Department of Urology, Saarland University, Homburg/Saar, Germany
c
Department of Urology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich,
Germany
d
Urology Practice Prüner Gang, Kiel, Germany
* Corresponding author. Department of Urology, Technische Universität München, Rechts der
Isar Medical Centre, Ismaninger Str. 22, 81675 Munich, Germany. Tel.: + 49 89 4140 2522;
Fax: +49 89 4140 4843.
E-mail address: margitta.retz@lrz.tum.de (M. Retz).
1. Patients and methods
1.1. Bone marrow sampling from bladder cancer patients
Preoperative bone marrow samples were collected from all 51 cystectomised patients in a
standardised procedure. To exclude contamination of bone marrow samples with cutaneous
epithelial cytokeratin-20 (CK20)–positive Merkel cells, a small skin incision was applied
above the spina iliaca anterior, and a Jamshidi needle (MD TECH, Gainesville, FL, USA) was
passed through the incision into the bone marrow compartment from both anterior iliac crests.
Bone marrow samples were aspirated into heparin tubes (10 ml) prior to radical cystectomy.
In some cases, bone marrow samples could only be collected from one side of the anterior
iliac crest because of age-related punctio sicca. Bone marrow samples were immediately
processed by Ficoll-isopaque density gradient centrifugation to isolate mononuclear blood
cells, allowing the separation of red blood cells and granulocytes from mononuclear blood
cells. After repeated washings with phosphate-buffered saline (Life Technologies, Eggenstein,
Germany), cells were pelleted and subsequently lysed with RNAzol (WAK-Chemie Medical,
Bad Homburg, Germany) [1].
1.2. Isolation of total RNA
Total RNA was isolated from mononuclear cell fractions in bone marrow based on the
guanidinium thiocyanate-phenol-chloroform single-step isolation method [2]. Total RNA
from mononuclear cell fractions was extracted using 1 ml of RNAzol, as recommended by the
suppliers. To optimise the yield of RNA, 5 µg of glycogen (Boehringer, Mannheim, Germany)
were added to the aqueous phase before precipitation with isopropanol. The RNA preparations
were dissolved in 40 µl RNase-free water. RNA recovery and purity were controlled by
absorption measurement at 260 nm and 280 nm (Gene Quant II calculator, Pharmacia Biotech,
Freiburg, Germany). Samples of 2 µg of the extracted total RNA were separated in 1%
agarose gel (Small DNA agarose; Biozym, Hessisch Oldendorf, Germany). The integrity of
ribosomal RNA was determined and samples with evidence of ribosomal RNA degradations
were discarded.
1.3. Cytokeratin-20 reverse-transcriptase polymerase chain reaction
Two micrograms of total RNA from each sample were dissolved in a volume of 10 µl of
RNase-free water, denatured for 10 min at 70°C, and then quickly chilled on ice. The
complementary DNA (cDNA) was synthesised in a total volume of 20 µl containing 4 µl of
5× first-strand buffer, 2 mmol/l of dithiothreitol, 200 U of SuperScript II reverse transcriptase
(Life Technologies), 20 units of RNase inhibitor, 5 µmol/l of random hexamers, and 1 mmol/l
of deoxynucleotide triphosphate (dNTP) mix (Perkin-Elmer Applied Biosystems, Weiterstadt,
Germany). The reaction mix was incubated for 10 min at 24°C and 60 min at 42°C. Reverse
transcriptase was then inactivated for 3 min at 94°C. The CK20-specific polymerase chain
reaction (PCR) was performed as a nested protocol using the same primer pairs for both PCR
reactions. Primers were synthesised by MWG-Biotech (Ebersberg, Germany): CK20-A sense,
5′-CGGCGGGGACCTGTTTGT and CK20-B antisense, 5′CAGTGTTGCCCAGATGCTTGTG. For the first PCR, 20 µl of the cDNA synthesis mix
were used in a final volume of 50 µl. The reaction mix contained 3 µl of 10× tricine buffer III
[3], 250 µmol/l of dNTP mixture, 1.5 µmol/l of CK20-A sense, 1.5 µmol/l of CK20-B
antisense primer, and 5 U of Taq DNA polymerase (Life Technologies).
The cycling protocol for the initial CK20 PCR is listed in Table 1, starting with a
modified touchdown protocol [4] (Table 1a) followed by 40 PCR cycles (Table 2b). The
second CK20 PCR was performed in a similar manner to the first PCR. One microliter of the
first PCR reaction mix was formulated with 1.5 µmol/l of the CK20-A sense and CK20-B
antisense primers in a final volume of 50 µl. The cycling protocol for the second PCR again
started with a touchdown protocol (Table 1a) followed by 21 PCR cycles (Table 1c). The
CK20 PCR resulted in a 485-bp product. To monitor the cDNA synthesis, a PCR for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed from the cDNA
preparation using GAPDH sense primer 5′-CCAGCCGAGCCACATCGCTC and GAPDH
anti-sense primer 5′-ATGAGCCCCAGCCTTCTCCAT. The cycling protocol for the GAPDH
PCR was performed as for CK20 but only included Table 1a and 1c. The resulting PCR
product migrated as a 359-bp fragment in 2% agarose-gel electrophoresis. We used 2 µg of
total RNA from the colon carcinoma–derived cell line WIDR as a CK20-positive control.
The absence of contamination was routinely checked by reverse-transcriptase PCR
(RT-PCR) assay of control samples in which RNA was replaced by sterile water. A second
negative control was performed by omitting RT from a positive control sample. All RT-PCR
products were separated by electrophoresis in a 2% agarose gel (Small DNA agarose) in Trisacetate-EDTA buffer and visualised by ethidium-bromide staining. The molecular weights
were determined using a DNA molecular weight marker (100-bp DNA ladder, Life
Technologies). Bone marrow samples were analysed by two independent CK20 PCRs.
1.4. Sequencing of the reverse-transcriptase polymerase chain reaction products
Representative amplification products of the specific CK20 RT-PCR were extracted from
agarose gels using the QIAEX II Gel Extraction Kit (QIAGEN, Hilden, Germany) and
sequenced by an automatic sequencer ALFexpress (Pharmacia). The results obtained were
verified using the BLAST programme (http://blast.ncbi.nlm.nih.gov).
Author contributions: M. Retz had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Retz, Lehmann.
Acquisition of data: Rotering.
Analysis and interpretation of data: Retz, Lehmann, Nawroth.
Drafting of the manuscript: Retz, Buchner, Nawroth.
Critical revision of the manuscript for important intellectual content: Stöckle, Buchner,
Gschwend.
Statistical analysis: Lehman.
Obtaining funding: Stöckle, Gschwend.
Administrative, technical, or material support: Rotering, Lehmann.
Supervision: Retz.
Other (specify): None.
Financial disclosures: I certify that all conflicts of interest, including specific financial
interests and relationships and affiliations relevant to the subject matter or materials discussed
in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria,
stock ownership or options, expert testimony, royalties, or patents filed, received, or pending),
are the following: None.
Funding/Support and role of the sponsor: The Hensel Foundation of Kiel, Germany, and the
Gruber Foundation of Munich, Germany sponsored the design and conduct of this study.
References
[1] Soeth E, Vogel I, Röder C, et al. Comparative analysis of bone marrow and venous blood
isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells
using reverse transcription PCR. Cancer Res 1997;57:3106–10.
[2] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid-guanidiniumthiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9.
[3] Ponce MR, Micol JL. PCR amplification of long DNA fragments. Nucleic Acids Res
1992;20:623.
[4] Charnock-Jones DS. Amplification and direct sequencing of DNA fragments using
fluorescently labelled dye-terminators. Int Biotechn Lab 1993;11:12.
Fig. 1 – Bone marrow samples were collected from both the anterior iliac crest left (l)
and iliac crest right (r). In some cases, bone marrow samples could be collected from
only one side of the anterior iliac crest because of punctio sicca. After RNA extraction
and cDNA synthesis, we performed a polymerase chain reaction (PCR) for cytokeratin20 (CK20) and glyceraldehyde-3-phosphate dehydrogenase (as a control), as described.
The figure shows one out of two independent experiments in bone marrow from eight
patients. As a positive control for the CK20 PCR, we used a cDNA from the colon
carcinoma–derived cell line, WIDR.
CK20 = cytokeratin-20; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
Table 1 – Cycling protocol for the cytokeratin-20 reverse-transcriptase polymerase chain
reaction (PCR). The procedure was performed as a double PCR starting with a nine-cycle
touchdown protocol (a), followed by 40 cycles (b) in the first and 21 cycles (c) in the second
PCR.
(a) Touchdown cycles
Step
Denaturation time and
Annealing and extension time and temperature
temperature
No. of
cycles
1
60 s/94°C
60 s/70°C
1
2
20 s/94°C
55 s/69°C
1
3
20 s/94°C
50 s/68°C
1
4
20 s/94°C
45 s/67°C
1
5
20 s/94°C
40 s/66°C
1
6
20 s/94°C
35 s/65°C
1
7
20 s/94°C
30 s/64°C
1
8
20 s/94°C
25 s/63°C
1
9
20 s/94°C
20 s/62°C
1
(b) First PCR
Step
Denaturation time and
Annealing time and
Extension time and
No. of
temperature
temperature
temperature
cycles
10–49
20 s/94°C
30 s/61°C
20 s/71°C
40
50
–
–
5 min /72°C
–
(c) Second PCR
Step
Denaturation time and
Annealing time and
Extension time and
No. of
temperature
temperature
temperature
cycles
10–30
20 s/94°C
30 s/61°C
20 s/71°C
21
31
–
–
5 min/72°C
–
Table 2 – Analysis of bone marrow samples from bladder cancer patients with the cytokeratin20 reverse-transcriptase polymerase chain reaction according to tumour stage
Patient
Gender
Age, yr pT
pN
Metastasis Grade RT-PCR
no.
1
CK20
status
M
78
4a
0
0
3
Left (−) right
Neg
(−)
2
M
77
2
0
0
3
Left (−) right
Neg
(−)
3
M
70
2
0
0
2
Left (−) right
Pos
(+)
4
F
59
3
0
0
2
Left (−) right
Neg
(−)
5
M
35
1+
0
0
3
Left (+) right
Pos
(−)
CIS
6
M
68
2
0
0
3
Right (−)
Neg
7
M
68
2
0
0
3
Right (−)
Neg
8
M
73
2
0
0
3
Left (−) right
Neg
(−)
9
M
69
1+
0
0
3
Right (−)
Neg
CIS
10
M
61
2
0
0
3
Left (−)
Neg
11
M
71
2
0
0
3
Right (+)
Pos
12
M
81
3
0
0
3
Left (+) right
Pos
(+)
13
M
86
3
1
0
3
Left (+) right
Pos
(+)
14
F
80
2
0
0
3
Left (−) right
Neg
(−)
15
M
66
1+
0
0
3
Left (−)
Neg
CIS
16
M
73
3
2
0
2
Left (−)
Neg
17
M
73
3
0
0
2
Left (−) right
Neg
(−)
18
M
73
3
2
0
3
Left (−)
Neg
19
M
69
3
1
0
2
Left (−)
Neg
20
M
77
3
0
0
2
Right (+)
Pos
21
M
73
3
0
0
3
Left (−) right
Neg
(−)
22
M
62
2
0
0
3
Left (−) right
Neg
(−)
23
M
76
3
0
0
3
Left (+) right
Pos
(−)
24
M
56
2
0
0
3
Left (−) right
Pos
(+)
25
M
62
2
0
0
3
Left (−) right
Pos
(+)
26
F
69
3
0
0
2
Left (−) right
(−)
Neg
27
M
75
2
0
0
3
Left (−) right
Neg
(−)
28
M
61
2
0
0
3
Left (−)
Neg
29
M
58
2
2
0
3
Right (+)
Pos
30
M
61
3
2
0
3
Right (−)
Neg
31
M
64
2
0
0
3
Left (−) right
Neg
(−)
32
M
80
3
0
0
3
Right (−)
Neg
33
M
68
4a
1
0
3
Left (+)
Pos
34
F
74
4a
0
0
3
Left (−) right
Neg
(−)
35
F
75
3
0
0
3
Right (−)
Neg
36
F
78
3
0
0
3
Left (+) right
Pos
(+)
37
M
76
3
1
0
3
Left (−) right
Neg
(−)
38
M
65
3
2
0
3
Left (−) right
Neg
(−)
39
M
68
4a
0
0
3
Left (+) right
Pos
(+)
40
M
79
3
2
0
3
Left (−) right
Neg
(−)
41
M
76
1+
CIS
0
0
3
Left (−) right
(−)
Neg
42
F
69
1
0
0
3
Right (−)
Neg
43
M
63
1+
0
0
3
Right (−)
Neg
0
0
3
Left (−) right
Neg
CIS
44
M
55
1
(−)
45
M
55
1+
0
0
3
M
74
1
Neg
(−)
CIS
46
Left (−) right
0
0
3
Left (−) right
Pos
(+)
47
F
76
1
0
0
3
Left (+)
Pos
48
F
77
3
0
0
3
Left (−)
Neg
49
M
61
1
0
0
3
Left (−) right
Pos
(+)
50
M
64
2
0
0
3
Left (−) right
Neg
(−)
51
M
59
2
0
0
3
Left (−) right
Neg
(−)
RT-PCR = reverse-transcriptase polymerase chain reaction; CK20 = cytokeratin-20; CIS =
carcinoma in situ.
Left (−): Bone marrow sample from the left iliac crest is CK20 negative.
Left (+): Bone marrow sample from the left iliac crest is CK20 positive.
Right (−): Bone marrow sample from the right iliac crest is CK20 negative.
Right (+): Bone marrow sample from the right iliac crest is CK20 positive.
Analysis: If one bone marrow sample from the left or right side of the iliac crest shows a
positive CK20 PCR, the patient is considered CK20 positive. PCR results have to be
confirmed by a second PCR.