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Keywords:

  • kidney;
  • cell adhesion molecule;
  • metastasis;
  • prognosis;
  • polymerase chain reaction

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

To evaluate the significance of the presence of circulating renal cell carcinoma (RCC) cells in the development of metastases, the authors extended a previous study to quantify cadherin-6 mRNA levels in association with the pattern of metastasis.

METHODS

Cadherin-6 mRNA levels were measured in peripheral blood samples from 66 patients with RCC, including 55 patients who had newly diagnosed RCC (43 without metastases and 12 with metastases) and 11 patients who had recurrent RCC. For quantitative polymerase chain reaction analysis, a cutoff value was determined in blood samples from 25 healthy volunteers and was verified in samples from 5 healthy controls and from 10 patients who had other malignancies. The correlation between the site of metastases and the cadherin-6 mRNA level was analyzed, and a follow-up study (median, 39 months) to track subsequent metastases was performed after patients underwent nephrectomy.

RESULTS

Cadherin-6 was found in 69.9% of patients with metastases and in 34.9% of patients without apparent metastases (P = 0.0099). In the group of patients with recurrent RCC, patients who had only pulmonary metastases had a significantly lower positivity rate (25.0%) compared with patients who had distant metastases (85.7%; P = 0.044). Among 43 patients with newly diagnosed RCC, 5 of 15 patients who were positive for cadherin-6 had metastases after nephrectomy, whereas only 2 of the 28 patients with negative cadherin-6 status had recurrent disease (P = 0.0398). In addition, the recurrence-free survival of patients who were positive for cadherin-6 was poorer compared with the survival of patients who were negative for cadherin-6 (P = 0.062).

CONCLUSIONS

The quantification of cadherin-6 mRNA in peripheral blood may be a significant predictive marker for current and future metastases. However, subsequent metastases did not always correlate with levels of cadherin-6 mRNA. This may have been due either to the small numbers of circulating tumor cells or to the low levels cadherin-6 mRNA in circulating tumor cells. Cancer 2004. © 2004 American Cancer Society.

Renal cell carcinoma (RCC) has an unpredictable clinical course and a tendency to recur and metastasize, even several years after surgery. Approximately 30% of patients with RCC already have metastatic disease at the time of diagnosis.1 Although several new therapeutic modalities (e.g., cytotherapy, immunotherapy, and gene therapy) have been developed recently for the treatment of patients with metastatic RCCs, the prognosis for these patients remains poor. It is possible, however, that survival rates may be improved if these treatment modalities were carried out at an earlier stage in metastatic disease, i.e., during micrometastasis. From this point of view, a number of investigations have attempted to evaluate the malignant potential of RCC cells by detecting genetic alterations and trying to identify a molecular marker that is predictive of metastases.

Giroldi et al. showed that cell-cell adhesion molecules, the so-called cadherins, play an essential role in the preservation of normal tissue integrity and that their disruption correlates with the most initial phase of the metastatic cascade in many carcinomas, including urologic malignancies.2 Cadherin-6, which is a Type 2 cadherin that probably is associated with tumor origin, is expressed frequently in RCC instead of E-cadherin.3 We previously reported that cadherin-6 had prognostic value, especially in patients with E-cadherin-absent RCC.4 Among patients with RCC who have abnormal cadherin-6 levels, survival rates are poorer statistically compared with the rates among patients with RCC who have normal cahderin-6 levels.4 In addition, we have noticed that mRNA expression of cadherin-6 often was increased in patients who had RCC with abnormal cadherin-6 levels5 and that this molecule also was expressed at the metastatic site (date not shown).

Recent investigations have revealed that micrometastasis can be predicted with polymerase chain reaction (PCR)–based molecular techniques using circulating blood.6–9 Although specific molecular markers for the detection of RCC cells in the peripheral blood have not been established, several basic investigations have been performed.5, 10–12 In the current study, we extended our previous work,5 in which we attempted to detect circulating RCC cells by using a quantitative PCR method for the detection of cadherin-6 mRNA as a new molecular marker, and evaluated its clinical value for predicting micrometastasis and evaluating the metastasis-related biologic potential of RCC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients

Blood samples were collected from patients with RCC, including 55 patients with newly diagnosed RCC and 11 patients with recurrent RCC, after informed consent was obtained from each patient. Patients with recurrent RCC had undergone previous nephrectomy and had metastatic lesions. Among the 66 patients, the male:female ratio was 46:20. Among the 55 patients with newly diagnosed RCC, 43 patients had localized RCC without evidence of metastasis, and 12 patients had advanced disease with metastases (Table 1). Peripheral blood samples were collected from all patients with newly diagnosed RCC (55 patients) before nephrectomy and from 26 patients during the period 1–4 weeks after nephrectomy. In addition, peripheral blood samples were collected from patients at the time of diagnosis of recurrence with metastatic disease. The median follow-up after nephrectomy was 39 months (range, 30–60 months). Peripheral blood samples from 25 healthy volunteers were used as a control to determine the cutoff value of this assay. To verify the validity of the cutoff value, additional samples from 5 healthy controls and from 10 patients with other urologic malignancies (6 urothelial carcinomas, 3 prostate carcinomas, and 1 testicular carcinoma) also were analyzed.

Table 1. Summary of Patients with Renal Cell Carcinoma and Quantification of Cadherin-6 mRNA
CharacteristicNo. of patientsM/FTumor classificationTumor gradeCadherin-6 mRNA (relative ratio)
T1T2T3T4Tx123XMedian (range)Average (SD)
  1. M/F: male/female ratio; SD: standard deviation; RCC: renal cell carcinoma; meta: metastasis; Nx: nephrectomy.

Newly diagnosed RCC             
 Without metastases (localized)4330/1324711011719700.15 (0.003–1.25)0.316 (0.404)
 With metastasesA128/40260402640.32 (0.135–3.31)0.60 (0.869)
Recurrent RCC             
 With metastases (post-Nx)B118/3045110.345 (0.01–4.985)0.817 (1.427)
Any RCC metastases (A + B)2316/70.325 (0.01–4.985)0.704 (1.147)

RNA Extraction from Blood Samples and cDNA Synthesis

Blood samples were diluted with the same volume of phosphate buffered saline (PBS) and then layered onto 20 mL of Ficoll-Hypaque (Pharmacia). After centrifugation at 1500 revolutions per minute for 10 minutes, the buffy coat was washed with PBS, and the cell pellet was frozen in liquid N2. Total RNA was isolated from the cell pellet by the standard TRIzol (Gibco BRL) method according to the manufacturer's instructions. Complementary DNA was generated from 1 μg total RNA via randomly primed reverse transcriptase PCR.

Quantitative PCR for Cadherin-6

The quantitative PCR assay was performed using equation image of the volume of cDNA generated by 1 μg total RNA in an ABI PRISM 7700 real-time thermal cycler. Upstream and downstream PCR primers and the TagMan probe for cadherin-6 were 5′-GCA CTC ACT ACG CAC A-3′, 5′-ACC CTT TGG TGA TCA G-3′, and 5′-TGC CAT CAG CAT GAG AAC TTA CCG CTA CTT-3′, respectively. TagMan Ribosomal RNA control reagent (Perkin Elmer) was used according to the manufacturer's instructions as an internal control for correction of the mRNA quantity of each sample. A standard curve for the assay was created by quantitative measurement of cadherin-6 mRNA expression in serial dilutions of SKRC-33 total RNA. Quantitative analyses were performed twice for each sample to confirm the reproducibility of the assay.

Evaluation of Cadherin-6 Expression and Correlation between Metastatic Status and Quantity of Cadherin-6 mRNA

The cutoff value of cadherin-6 mRNA expression for this assay was determined by calculating the average value + 2 × the standard deviation (SD) in the healthy control group. The quantity of cadherin-6 mRNA from each blood sample was estimated by comparison with the standard curve of cadherin-6 expression using the SKRC-33 cell line. Comparative analyses of the associations between cadherin-6 levels and the presence and site of metastatic disease were performed. In newly diagnosed patients who had localized or locally advanced RCC, the correlation between the detection of cadherin-6 mRNA and metastases following nephrectomy also was analyzed. In those patients, a plot of recurrence-free survival with metastasis also was constructed using the Kaplan–Meier method, and differences between the two groups were estimated with the log-rank test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cutoff Value and Cadherin-6 mRNA Levels in the Samples

Compared with the cutoff values in 25 healthy volunteers, cadherin-6 mRNA levels in 5 additional healthy volunteers were observed at the lower cutoff value. In all patients with malignancies other than RCC, cadherin-6 mRNA levels in peripheral blood samples were within the lower cutoff range (Fig. 1).

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Figure 1. Illustration of the cutoff value for cadherin-6 mRNA levels and the validation of this cutoff level using peripheral blood samples from a healthy control group and from a group of patients with urologic malignancies other than renal cell carcinoma. The cutoff value for the assay was 0.24 (the relative score of cadherin-6 mRNA in SKRC-33 cells) and was determined by calculating the mean plus 2 standard deviations in the healthy control group (n = 25). Additional samples from the control group and from the group of patients with other malignancies showed the lower level of cutoff value in cadherin-6 mRNA. The y-axis shows the relative score compared with the cadherin-6 mRNA level in the SKRC-33 cell line, and circles in the Standard column indicate serially diluted cadherin-6 mRNA levels in SKRC-33 cells. Y-axis: relative ratio compared to expression level of cadherin-6 in SKRC-33 cells.

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Patient Profiles and Metastatic Status during Follow-Up

Of 55 patients with newly diagnosed RCC, 43 patients had no metastatic lesions, and 12 patients had metastases in lung, bone, liver, or other organs. Among the 43 patients who were without metastases, 7 had recurrences with metastatic disease after nephrectomy. Conversely, 11 patients who had recurrences after nephrectomy also had metastases in lung, bone, liver, brain, or other organs.

Quantitative PCR for Cadherin-6 According to the Presence of Metastases

Among the patients who had metastatic disease (including patients with newly diagnosed disease and patients with recurrent disease), 16 of 23 patients (69.9%) were positive for cadherin-6 mRNA, whereas positive expression of cadherin-6 was detected in 15 of 43 patients (34.9%) who were without metastases (newly diagnosed patients). The difference between positive cadherin-6 mRNA rates with and without metastases was statistically significant (chi-square statistic, 7.24; P = 0.0099) (Table 1, Fig. 2).

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Figure 2. Cadherin-6 mRNA levels in peripheral blood from patients with renal cell carcinoma (n = 66) according to metastasis (meta) status show that the rate of positive cadherin-6 results among patients who had metastatic disease was statistically greater than the rate among patients without metastatic disease (P = 0.0099). Y-axis: relative ratio compared to expression level of cadherin-6 in SKRC-33 cells.

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Quantitative PCR for Cadherin-6 According to the Site of Metastases

Detection rates in patients who had metastases after undergoing nephrectomy were 1 in 4 among patients with pulmonary metastases alone and 6 in 7 among patients with metastases to other organs (chi-square statistic, 4.06; P = 0.044). Conversely, among the 12 new patients who had metastatic disease, positive cadherin-6 mRNA status before nephrectomy was noted in 9 (Fig. 3). In new patients without metastatic disease, although only 1of 10 patients with T1a tumors had a positive result, the positive rate was not associated with either T classification or tumor grade.

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Figure 3. Cadherin-6 mRNA levels in patients with metastatic renal cell carcinoma according to the site of metastasis show that a lower positivity rate was observed in patients who had pulmonary metastases (meta) alone. Nx: nephrectomy. Y-axis: relative ratio compared to expression level of cadherin-6 in SKRC-33 cells.

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Correlation between the Quantitative Assay for Cadherin-6 and Later Metastases

Among 43 patients with newly diagnosed RCC, a higher rate of recurrence with metastases was observed in patients who had detectable cadherin-6 mRNA levels in peripheral blood compared with patients who had negative cadherin-6 results (5 of 15 patients [33.3%] vs. 2 of 28 patients [7.1%], respectively; P = 0.0398) (Fig. 4). The correlation between the detection of cadherin-6 mRNA and later disease recurrence in 26 patients who had both prenephrectomy and postnephrectomy blood samples available is shown in Table 2. Of the six patients who continuously had positive results for cadherin-6, three had metastases. At the time of recurrence, cadherin-6 mRNA remained detectable in five of the seven patients who had positive cadherin-6 status before nephrectomy; in contrast, of the two patients who had negative cadherin-6 status before nephrectomy, only one remained negative for cadherin-6 after this procedure. Recurrence-free survival after nephrectomy in the RCC patient who was positive for cadherin-6 mRNA seemed to be poorer compared with the survival of patients who were negative for cadherin-6. However, the difference between the two groups was not statistically significant (P = 0.062; log-rank test) (Fig. 5).

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Figure 4. Recurrence (black dots) was subsequently documented in 5 of 15 patients who were positive for cadherin-6 mRNA before nephrectomy (Nx), compared with 2 of 28 patients who were negative for cadherin-6. Y-axis: relative ratio compared to expression level of cadherin-6 in SKRC-33 cells.

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Table 2. Correlation between Detection of Cadherin-6 and Subsequent Metastases
Detection of cadherin-6Subsequent status (no. of patients)
Pre-NxPost-NxMetastasisNo metastasis
  1. Nx: nephrectomy.

NegativeNegative213
PositiveNegative14
NegativePositive01
PositivePositive32
TotalNegative or positive620
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Figure 5. Recurrence-free survival curves in patients with renal cell carcinoma after nephrectomy show that the patients who were positive for cadherin-6 mRNA (+; n = 15) measured in their peripheral blood seemed to have a higher rate of recurrence compared with patients who were negative for cadherin-6 (−; n = 28). However, the difference between the two groups was not significant (P = 0.062). Y-axis: Recurrence-free survival rate.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A definitive tumor marker that is expressed in RCC has not been identified to date. Recent studies have demonstrated that RCC cells can be detected in the circulating blood, i.e., from renal and peripheral veins, by means of molecular biologic techniques. McKiernan et al. reported that MN/CA9, which is a relatively specific membrane oncoprotein, is a useful tool for detecting clear cell RCC cells in the peripheral blood independent of tumor stage using reverse transcriptase-PCR (RT-PCR) technology.11 Uemura et al.10 showed that MN/CA9 was expressed in > 90% of clear cell-type RCCs and was useful for the detection of RCC cells in the peripheral blood.13 However, those authors reported that the detection rate of MN/CA9 mRNA in the peripheral blood from healthy volunteers also was relatively high (32.3%), suggesting that positive expression of MN/CA9 is present in normal blood cells or in cells that have been contaminated by the blood sampling procedure.10 Therefore, this indicator remains controversial among investigators.10, 11

Conversely, Ashida et al.12 demonstrated that the von Hippel–Lindau (VHL) gene mutation, which is specific for clear cell-type RCC, is useful for the detection of tumor cells. VHL gene mutations were detected in peripheral blood before surgery in 11.8% of patients with sporadic clear cell RCC. This method is considered specific for clear cell RCC, but it is necessary to identify the presence and type of mutation of the VHL gene prior to the analysis of blood samples. It is worth noting that the detection rate of RCC cells by VHL mutation was remarkably high (77.8%) in renal venous blood during nephrectomy and in peripheral blood in the 24 hours after nephrectomy, indicating that RCC cells may be spread into the circulation system by surgical manipulation. In addition, de la Taille et al.14 showed that a combined, blood-based RT-PCR analysis of MN/CA9 and prostate-specific membrane antigen is a useful clinical tool for the detection of vascular invasion of RCC, indicating that a PCR-based blood assay using two or three molecules associated with RCC may help to identify micrometastases.

Thus, as shown in our current and previous work, RCC cells frequently appear in the circulating blood system before and during nephrectomy, independent of the presence of apparent metastatic disease. According to the previous literature, in patients with localized RCC, it is emphasized that the positive rate of RCC cells is between 32% and 46%11, 14 in peripheral blood but > 70%12 in renal venous blood during nephrectomy. Because the dissemination of tumor cells into blood circulation is essential in the early stages of metastasis, patients who have localized RCC with positive levels of cadherin-6 mRNA are considered a group that is at high risk for later metastases. Conversely, it also has been speculated that certain molecules in the metastatic cascade, e.g., those of invasion, proliferation, and angiogenesis, are indispensable in the establishment of metastases.

Several factors influence the probability of detection. Tumor volume may be associated with the positive rate of this assay. In the group with metastases, the detection rate was higher in patients who had tumor-bearing kidney compared the rate in patients after nephrectomy. Furthermore, a low detection rate (1 of 10 patients) was observed in patients who had localized T1a tumors that measured < 4.0 cm in greatest dimension. Even in patients with metastatic disease, no definitive reason was identified for the low detection rate in those who had only pulmonary metastases. Our previous study showed that a concentration > 10 cells/mL was required for detection by nested PCR,5 indicating that at least 1 RCC cell in 105 mononuclear cells was required for detection using a PCR system, because the mononuclear cell concentration typically ranged between 4000 and 9000 cells/μL. Therefore, it is possible that the numbers of circulating tumor cells were not great enough to detect using the PCR system. Alternatively, the level of cadherin-6 mRNA expression of circulating tumor cells may have been too low for detection. The reason for this may be that normal lung epithelial cells express cadherin-6, and RCC cells with cahderin-6 may attach to pulmonary tissue by means of homophilic cadherin adhesion, suggesting that RCC cells that pass through the pulmonary circulation may lack mRNA expression of cadherin-6.

The current quantitative detection system may possess prognostic value, as positive cadherin-6 mRNA status in the blood appeared to be correlated with later recurrence. However, some exceptions were observed in the correlation between the assay results and the occurrence of metastases. This finding may indicate that the risk of metastasis is associated not only with the presence of tumor cells in the blood circulation but also with the metastatic potential of other tumor cells, e.g., angiogenesis, proliferation, and invasion, as discussed above. Thus, further investigation with longer follow-up will be is needed to confirm the predictive value of the quantitative assay for cadherin-6 mRNA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Risa Kawamoto for her technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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