Detection of Circulating Cancer Cells by Reverse Transcription-Polymerase Chain Reaction for Uroplakin II in Peripheral Blood of Patients with Urothelial Cancer

Patients and Blood Samples.

The first sample set consisted of peripheral blood obtained from 56 urothelial cancer patients who were hospitalized and treated at Kyoto University Hospital during the period of January 1, 1999, to December 31, 1999. All 14 patients with locally invasive cancers (pT_2–4N₀M₀) and 13 patients with metastases (5 had loco-regional lymph node metastases without distant metastases and 8 had distant metastases) treated during the above period were enrolled in this study. In addition, 29 randomly selected patients with superficial urothelial cancers (pT_a-1N₀M₀) treated endoscopically were also investigated. The histopathological diagnosis was TCC in all cases. Clinicopathological profiles of the 43 patients with localized disease are summarized in Table 1⇓ . Blood sampling was performed before any treatment for the first sample set.

The second sample set consisting of peripheral blood from 10 healthy volunteers and 10 patients with untreated renal cell carcinomas (6 patients with metastasis and 4 patients without metastasis) was prepared for the present study as a control. In addition, sequential blood sampling over the treatment course was performed to yield a third sample set for three patients with nodal or distant metastases that were treated with systemic chemotherapy.

Grading and Staging of Primary Tumors, and Estimation of Metastatic Tumor Volumes.

Grade and stage of primary tumors were classified according to the WHO criteria (16) and TNM classification (17) , respectively. The volume of metastatic tumor was estimated from an imaging study.

Tumor Specimens.

Tumor tissue samples of 23 TCCs (7 were grade G1, 8 were G2, and 8 were G3), 1 bladder squamous cell carcinoma, and 3 renal cell carcinomas were freshly frozen for the expression study. Of the 23 TCC samples, 17 were primary tumors (3 renal pelvic tumor, 4 ureteral tumors, and 10 bladder tumors) and 6 were metastatic tumors (3 lymph node, 2 liver, and 1 lung metastatic tumor). Total cellular RNA was extracted as described elsewhere and subjected to RT-PCR.

Cell Line.

HT1197, a UP-expressing TCC cell line, was purchased from American Type Culture Collection and used as a positive control for UP II gene expression in each RT-PCR assay. Cells were maintained in RPMI 1640 medium supplemented with 10% FCS.

RNA Extraction and Nested RT-PCR.

Five ml of peripheral blood were collected, and the mononuclear cell layer was separated using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) and a Leucosep tube (Greiner Labortechnik, Frickenhausen, Germany). Total RNA was extracted using TRIzol Reagent (Life Technologies, Rockville, MD), and cDNA was synthesized using the first strand DNA synthesis kit (Amersham Pharmacia Biotech). Five μl of total RNA were added to the reverse transcription reaction. Nested PCR for UP II was performed as described by Yuasa et al. (14 , 15) . The two primer pairs used were the first sense primer, 5′-TCCCCAGGGGCTGCAGACTT-3′, the first antisense primer, 5′-GGTTTGTCACCTGGTATGCACT-3′, the second sense primer, 5′-CATCTCAAGCCTCTCTCTGGTC-3′, and the second antisense primer, 5′-AGCCAGCACCACTGTCCACC-3′. The first primer pair produces a PCR product 268 bp in size, and the second primer pair produces a PCR product of 203 bp in size. These primer pairs were designed to be located on the sequence of human UP II cDNA, which is highly homologous to bovine UP II cDNA, and to be intron-spanning on the genomic sequence of the bovine UP II gene. These primer pairs are thought to be intron-spanning on the genomic sequence of the human UP II gene because PCR product on human genomic DNA is ∼400 bp in length and larger than that of RT-PCR. The 25-μl PCR mixture consisted of 1 mm MgCI₂, 200μ m deoxynucleotide triphosphates, 25 pmol of each primer, 0.2 units of AmpliTaq polymerase (Roche Molecular Systems, Branchburg, NJ), PCR buffer supplied with the polymerase, and 1μ l of template. For the template, 1 μl of cDNA was used in the first round of PCR and 1 μl of PCR product was used in the second round of PCR. In both rounds of PCR, the conditions were 95°C for 60 s, 55°C for 60 s, and 72°C for 90 s, for 35 cycles. Eight μl of PCR product of 203 bp were separated on a 2% agarose gel, stained in 0.5 μg/ml of ethidium bromide solution for 30 min, and visualized on a UV transilluminator. We have used the result of the dilution experiment described below as a positive control throughout the study.

The PCR products of several blood samples as well as HT 1197 cells that were positive for UP II expression were sequenced with ABI prism 330 (Perkin-Elmer, Norwalk, CT). All were confirmed to be identical to the UP II cDNA sequence. PCR for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed and used as an internal control. A negative control was included in each step of the RT-PCR by adding water in place of reverse transcriptase and AmpliTaq-polymerase.

The nested RT-PCR was performed twice for each total RNA sample. A third nested RT-PCR was performed only when the results were inconclusive.

Sensitivity Testing of the Nested RT-PCR Assay.

HT1197 cells (1 × 10⁶) were suspended in 1 ml of PBS and serially diluted from 10 to 10⁶ times. One ml of each diluted sample was added to 5 ml of peripheral blood from a healthy volunteer and subjected to the nested RT-PCR assay described above.

Ethical Issues.

Written informed consent was obtained from all of the patients as well as the 10 healthy volunteers who participated in this study before blood sampling.

UP II mRNA Expression in Tumor Tissues.

The 17 primary and 6 metastatic TCCs were all positive for UP II mRNA expression by both the first round of RT-PCR and the nested PCR. On the other hand, UP II-specific products were not detected even by the nested PCR in a bladder squamous cell carcinoma and in three renal cell carcinomas (Fig. 1)⇓ .

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Fig. 1.

Detection of mRNA for UP II and representative results of RT-PCR analysis. The PCR product corresponding to UP II was positive in Lanes 3, 4, 6, 7, 10, and 12. P1, P2, and P3, serially diluted positive controls (HT1197); N, negative control; Lanes 1–4, frozen tissue samples: Lane 1, renal cell carcinoma; Lane 2, squamous cell carcinoma of the bladder; Lane 3, TCC of the bladder; Lane 4, metastatic TCC of the lymph node; Lanes 5–12, peripheral blood samples from patients with TCC; Lanes 5 and 6, T_a-1N₀M₀; Lanes 7 and 8, T_2–4N₀M₀; Lanes 9 and 10, N_1–2M₀; Lanes 11 and 12, M₁.

Sensitivity of the Nested RT-PCR for UP II mRNA.

The UP-II-positive control cell line, HT 1197, was sequentially attenuated and added to peripheral blood of a healthy volunteer negative for UP II mRNA expression and examined for UP II mRNA expression. UP II mRNA could be detected even in a sample in which the cell suspension was diluted up to 10⁵ times (Fig. 2)⇓ . The limit of this assay therefore was calculated to be two cancer cells in 1 ml of blood. We defined this band intensity as a standard for positivity throughout the study.

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Fig. 2.

Dilution experiment. HT1197 cells, 10⁶ cells/ml in concentration, were serially diluted from 10 to 10⁶ times. One ml of diluent was added to 5 ml of peripheral blood from a healthy volunteer and subjected to the nested RT-PCR assay for UP II. UP II mRNA could be detected in 10⁵ times the diluted sample (two cancer cells in 1 ml of blood).

Detection of UP II mRNA in the Peripheral Blood of Patients and Controls.

Peripheral blood from 56 urothelial cancer patients with localized tumors (n = 43), loco-regional lymph node metastases (n = 5), or distant metastases (n = 8) was analyzed for the presence of UP II mRNA (Fig. 1)⇓ . In 4 (7%) of 56 urothelial cancer patients, a discrepancy in the results of duplicated experiment was obtained. Of the four patients, one with muscle invasive cancer was determined as negative, and three patients (two had muscle-invasive cancers and one had lymph node metastasis) were judged positive after a third experiment.

UP II mRNA-specific PCR product was detected in 3 (10.3%) of 29 patients with superficial cancers (pT_a-1N₀M₀), 4 (28.6%) of 14 patients with muscularly invasive cancers (pT_2–4N₀M₀), 2 (40.0%) of 5 loco-regional node-positive patients (pN_1–2M₀), and 6 (75.0%) of 8 patients with distant metastases (Tables 1⇓ and 2)⇓ . Positive rates, therefore, increased with tumor extension (P = 0.0033, Kruskal-Wallis test).

As for the second sample set, blood samples were drawn from 10 healthy volunteers and 10 renal cancers. None showed positive expression of UP II by the nested RT-PCR.

Relation between UP II mRNA Positivity and Metastatic Tumor Volumes.

The relation between positivity on UP II RT-PCR assay and tumor volume in the metastatic lesions in the 13 metastatic cases was investigated (Table 2)⇓ . No significant correlation was found. In loco-regional lymph node metastasis cases, however, both cases of microscopic involvement were negative for UP II expression.

Influence of Systemic Chemotherapy on Detection of UP-II-positive Cells in Metastatic Patients.

Multiple blood sampling was done for three patients (sample set 3) who underwent systemic combination chemotherapy consisting of cisplatin, epidoxorubicin, and methotrexate for their metastatic diseases (Table 3)⇓ . UP-II-positive cells were detected in the blood collected before chemotherapy in two patients (patients 8 and 11), whereas UP II was negative in patient 53. These three patients were treated with three to four cycles of chemotherapy. Patients 8 and 11 responded to chemotherapy well, and their UP-II-positive cells disappeared from the first cycle through to the final cycle of chemotherapy. In case 53, rather conflicting results were obtained for UP II RT-PCR. UP-II-positive cells could not be detected before chemotherapy, although the metastatic tumor burden was very large. UP-II-positive cells, however, were once detected just after the first cycle of chemotherapy, although 90% of lymph node metastases and liver metastases were eradicated by that time. This patient has thus far undergone four cycles of chemotherapy and now harbors only small metastatic foci in the liver and the lumbar vertebral bones. RT-PCR for UP II in the blood was consistently negative after two cycles of the chemotherapy.