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.