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[Cancer Research 60, 3170-3174, June 15, 2000]
© 2000 American Association for Cancer Research

Clinical Investigations

Detection of Germ-Cell Tumor Cells in the Peripheral Blood by Nested Reverse Transcription-Polymerase Chain Reaction for {alpha}-Fetoprotein-Messenger RNA and ß Human Chorionic Gonadotropin-Messenger RNA1

Andreas L. A. Hautkappe2, Mengji Lu, Hendrik Mueller, Axel Bex, Andreas Harstrick, Michael Roggendorf and Herbert Ruebben

Department of Urology [A. L. A. H., H. M., A. B., H. R.], Institute of Virology [M. L., M. R.], and Department of Medicine, Tumor Research [A. H.] University of Essen, D-45122 Essen, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By establishing sensitive nested reverse transcription-PCRs for the detection of mRNA of {alpha}-fetoprotein (AFP) and ß human chorionic gonadotropin (ßhCG), we investigated the presence of circulating tumor cells in the peripheral blood of 119 patients with germ-cell tumor. A total of 336 blood samples obtained before and during therapy were examined with regard to clinical applicability. The overall ratio of positive PCR results was 26.5% and was independent of the serum concentration of AFP and hCG/ßhCG. No correlation of the positivity for AFP-mRNA to serum AFP level was found. In contrast, positive results in ßhCG-PCR were twice as frequent in patients with elevated serum hCG/ßhCG levels as in those with normal serum hCG/ßhCG levels (P = 0.012). To develop a valid correlation to tumor stage, tumor histology, and serum level of tumor markers, a subgroup of 36 patients was evaluated before definite therapy. The subgroup revealed an overall ratio of 33.3% positive PCR results. The serum level of both of the markers did not correlate with the detection of corresponding mRNA in peripheral blood samples. However, positive ßhCG-PCR results were found exclusively in patients with elevated serum hCG/ßhCG (6 of 18 versus 0 of 18; P = 0.019). Patients with stage IIC/III germ-cell tumor demonstrated nearly twice the frequency of positive PCR results as patients with stage I tumor [7 (41.2%) of 17 versus 4 (23.5%) of 17] in this subgroup. With regard to histology, positive PCR results were found mostly in embryonal carcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular cancer is the most common malignancy in young men between 20 and 40 years of age. Germ-cell tumors are potentially curable because of effective therapeutic developments in the last two decades. Nevertheless, the mortality is about 200 per year in Germany. Clinical research on testicular cancer is focused on further improvement of treatment such as risk-adapted reduction of invasive and cytostatic therapy as well as optimizing diagnostics for early detection of recurrence and posttreatment monitoring.

AFP3 and hCG/ßhCG are important tumor markers for the diagnosis and monitoring of germ-cell tumor patients (1) . The serum levels of AFP and hCG/ßhCG are dependent on the volume of the primary tumor and metastases as well as tumor cell activity. Elevation of these markers in the serum of patients reflects tumor activity, although a normal serum level of these markers does not imply the absence of tumor. A successful therapy results in a marker decrease corresponding to their half-life. Persistant elevation or increasing serum markers indicate either ineffective therapy or tumor progression. An AFP elevation in serum has been found in 50–70% of patients with NSGT, whereas seminomas do not produce AFP. ßhCG is elevated in all patients with choriocarcinoma. Forty to 60% of patients with NSGT reveal an elevated serum hCG/ßhCG as compared with 5–10% of those with seminomas. NSGT secrete both, (holo)hCG which consists of the {alpha}- and ß-subunit and the isolated ß-subunit. This leads to a correlated elevation of both these markers (2) . In contrast, the secretion of (holo)hCG and ßhCG is not well correlated in seminomas. About 30% of hCG-positive seminomas reveal an isolated elevation of ßhCG, whereas an isolated (holo)-hCG elevation can be verified in 20% of the cases. About 90% of patients with stage I-III NSGTs have an elevation of both markers AFP and hCG/ßhCG. The incidence is lower in patients with clinical stage I (3, 4, 5) . It is not clear, whether hCG/ßhCG and AFP are prognostic markers. Most authors do not see any correlation with the risk of progression (6 , 7) .

Recently, it became possible to detect specific gene transcripts by sensitive RT-PCR. This technique provides opportunities to trace tumor marker expression in tissues, even when protein products cannot be measured conventionally in the serum. In prostate cancer, the presence of circulating PSA-producing tumor cells in the peripheral blood could be verified. The correlation to either advanced or locally defined tumor is discussed controversially (8, 9, 10, 11) . Funaki et al. (12) and Komeda et al. (13) could identify AFP-mRNA indicating circulating tumor cells in the peripheral blood of patients with hepatocellular carcinoma. The presence of ßhCG-mRNA in the peripheral blood reflecting circulating tumor cells has been investigated in metastatic breast cancer (14) .

Presently, it is not known, whether detection of AFP- and ßhCG-mRNA in the peripheral blood of germ cell tumor patients can add information for diagnosis and tumor monitoring. The expression of AFP- and ßhCG-mRNA in normal human testicular tissue and germ cell tumor tissues has been examined. Berger et al. (15) detected ßhCG in cytosolic extracts of normal human testes and demonstrated ßhCG gene expression in normal testes by RT-PCR. Madersbacher et al. (16) analyzed hydrocele fluids of patients with testicular cancer for hCG and its free subunits {alpha}hCG and ßhCG by monoclonal antibody-based two-site immunoassays. Increased hydrocele levels of hCG, free {alpha}hCG, and free ßhCG were observed in 77, 54, and 92%, respectively, of patients with testicular cancer compared with those with nonmalignant hydroceles. In two patients with pure seminoma who were negative for the serum markers in the periphery but elevated for free ßhCG in hydrocele fluid, the production of this marker in the tissue could be proven by specific RT-PCR (16) .

Recently Yuasa et al. (17) demonstrated AFP-mRNA expression in 4 of 4 NSGT tissues and 6 of 10 histologically classified seminoma tissues. All of the of these NSGT tissues were positive for immunohistochemical staining of AFP, whereas AFP was not detected in any of the 10 seminomas. All of the NSGT patients of this study had elevated serum AFP levels. However, the correlation of the serum AFP level and the presence of AFP-mRNA expression in the primary tumor tissue was not analyzed. Our own unpublished data showed the presence of AFP-mRNA in 11 of 14 NSGT and ßhCG-mRNA in 10 of 14 NSGT tissues analyzed by respective RT-PCR.4 Nine of 11 seminomas tested positive for AFP-mRNA as well as ßhCG-mRNA expression. This raises the question about the basic definition of the absence of AFP-production in seminomas. In this analysis, we could not find any correlation between serum marker levels and respective mRNA expression in the tissues. These analyzed tissues were not derived from the patients in this study. There are no more data available about AFP-mRNA and ßhCG-mRNA expression in larger sample sizes of NSGT tissues.

Here we report on the detection of AFP-mRNA and ßhCG-mRNA in terms of circulating tumor cells in the peripheral blood of 119 patients with germ-cell tumor of the testis. To assess clinical feasibility we examined 336 peripheral EDTA-blood samples with sensitive RT-PCR for both markers. We correlated the PCR results with the corresponding serum marker levels, histological type, and tumor stage, to assess the clinical significance of the detection of circulating tumor cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
We established RT-PCR for AFP- and ßhCG-mRNA using a ßhCGproducing cell line JEG-3 and an AFP-producing cell line HepG2. Defined numbers of these marker-producing cells were suspended in human EDTA-blood, and the sensitivity of the specific RT-PCRs was tested in serial dilutions. Positive PCR results were obtained with samples containing 101 JEG-3 cells/ml and 103 HepG2 cells/ml.

Patient Samples.
To evaluate the feasibility in clinical samples, we investigated 336 samples from 119 patients with germ-cell tumor of different tumor stage and at various time intervals during chemotherapy. The study design has been approved by the clinical committee of the University of Essen, Medical School. Before the collection of blood samples, written informed consent was obtained from the patients to be included in the trial.

Patients were recruited from the Department of Urology, University of Essen, and from the Department of Medicine, Tumor Research, University of Essen. The mean age of patients was 34 years (range, 17–56 years; median, 33.5 years). From some patients, more than one sample was collected during therapy. From patients undergoing chemotherapy, the samples were taken only before the application of cytotoxic drugs.

The control group consisted of 23 healthy young men, ages 19–38 (mean , 28.7 years; median, 25 years) with normal serum AFP and serum hCG/ßhCG.

The serum levels of AFP and hCG/ßhCG were determined routinely with patient samples by using immunoenzymetric tests (Hybritech Inc., Cologne, Germany). The peripheral blood samples (10 ml EDTA-blood) were prepared for further RNA-extraction within 2–4 h. The specific RT-PCRs for detection of AFP- and ßhCG-mRNA were performed immediately, or were delayed until after the cell-fraction was lysed with specific lysis buffer, and were stored at -70°C. The PCR results were correlated with serum tumor marker level, tumor stage, and tumor histology. Serum levels of AFP of <8 ng/ml and hCG/ßhCG of <2 mU/ml were regarded as normal values according to the manufacturer‘s instructions.

Cell Fraction.
A standard protocol using dextrane was used to remove excessive erythrocytes from the peripheral blood. Six ml of peripheral EDTA-blood were mixed with 2 ml of dextrane T 500 (6%, Pharmacia, Freiburg, Germany) and incubated at 37°C for 30 min (18) . The supernatant containing leukocytes (peripheral blood lymphocytes) and potential tumor cells was recovered and subsequently pelleted by centrifugation at 1000 rpm for 10 min. The remaining erythrocytes were removed by further incubation in 3 ml of hemolysis buffer. Cells recovered after centrifugation were immediately lysed with lysis buffer of RNeasy Mini Kit (Qiagen, Hilden, Germany) for RNA preparation.

RNA Extraction.
Total RNA from peripheral blood mononuclear cells was prepared by using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was dissolved in 50 µl of distilled water and stored at -70°C until used. The integrity of isolated RNA was assessed by agarose gel electrophoresis.

RT-PCR for AFP- and ßhCG-mRNA.
Primers used for RT-PCR are listed in Table 1Citation . The primers for ßhCG-mRNA are specific and do not amplify luteinizing hormone ß (15 , 21) . Approximately 5 µg of total RNA were incubated with 5 mM antisense primer in a total volume of 10 µl at 65°C for 10 min. The mix was then immediately transferred to an ice box at 0°C and used for RT. The RT reaction was performed in a total volume of 20 µl with 200 units Moloney-murine leukemia virus reverse transcriptase (Life Technologies) at 37°C for 60 min. cDNAs generated in RT reactions were used as template for PCRs. The PCR mix consisted of 5 µl of template, 20 pmol of each primer, 75 nmol of MgCl2 and 2.5 units Taq polymerase in a total volume of 50 µl. The first PCR reaction was carried out over 30 cycles of 1 min at 94°C after 1.5 min at 45°C annealing temperature and 2 min at 72°C. The nested PCR was performed under the same conditions except the change of the annealing temperature to 57°C. The PCR products were visualized by agarose gel electrophoresis and stained with ethidium bromide. The PCR fragments of AFP and ßhCG-cDNA were 282 bp and 314 bp, respectively. DNA-sequencing of 10 randomly chosen samples was performed to confirm the specificity of the PCR.


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  		Table 1 RT-PCR primer for AFP-mRNA and ßhCG-mRNA

Primers for AFP-mRNA were modified according to Matsumura et al. (19) . The numbering of 5' base for ßhCG is according to the number assignment of Boorstein et al. (20) .

 
Statistical Analysis.
We ran Fisher‘s exact test to compare clinical characteristics (level of serum tumor marker, tumor stage) to the RT-PCR results of ßhCG and AFP for all of the 336 samples as well as for the subgroup of the 36 pretherapy patients and the 17 stage-I patients.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Total Samples (n = 336).
For clinical evaluation of AFP-mRNA and/or ßhCG-mRNA, 336 peripheral blood samples from 119 patients with germ-cell tumor were examined with specific RT-PCRs. The detection of these mRNAs in peripheral blood cells is supposed to reflect circulating tumor cells. Forty-nine blood samples were collected from 25 patients with seminoma. Another 287 samples were obtained from 94 NSGT-patients.

Eighty-nine samples (26.5%) were positive in at least one of the specific PCRs. The relationship between the PCR results and the corresponding serum tumor markers is shown in Table 2Citation . With regard to each marker separately, patients with a normal serum hCG/ßhCG level revealed positive PCRs in 19 (7.1%) of 267 samples. Conversely, patients with an elevated serum hCG/ßhCG disclosed positive PCRs in 12 (17.3%) of 69 samples. The correlation of elevated serum hCG/ßhCG and positive PCR results was statistically significant (P = 0.012).


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  		Table 2 Evaluation of patients with germ-cell tumor for AFP- and ßhCG-mRNA in the peripheral blood (samples, n = 336): Comparison to serological marker data

 
In contrast, no difference was found comparing AFP serum levels and corresponding AFP-PCR results. Patients with an elevated serum AFP level and those with a normal serum level had positive PCR results in 17.6% and 19.4% of samples, respectively. Considering both of the serum markers, we found positive PCR results (AFP and/or ßhCG) in 33.5% if at least one serum marker was elevated, compared with 20.4% of patients whose serum markers were both normal. This difference reached statistical significance as well (P = 0.009).

Control Group.
The serum levels of AFP and hCG/ßhCG were measured, and the corresponding RT-PCRs were performed. All of the controls had normal serum levels of AFP and hCG/ßhCG and negative results for both of the RT-PCRs.

Detection of AFP- and ßhCG-mRNA before Therapy (n = 36).
Thirty-six of 119 patients could be considered as "patients before therapy." Patients with tumor stages I and IIA were recruited before orchiectomy. Patients after orchiectomy but with advanced tumor stages (IIB-III) and large retroperitoneal tumor masses or/and distant metastases were recruited before the first cycle of chemotherapy. Twelve (33.3%) of 36 revealed a positive PCR result for at least one of the markers independent of tumor stage and/or serum marker levels. We compared RT-PCR results with serum marker levels, tumor stage, and tumor histology.

Detection of AFP- and ßhCG-mRNA in Relation to Serum Tumor Marker Level (n = 36).
The PCR results were assessed considering the absolute elevation of the serum tumor markers. The ranges for this assessment for AFP were: <=8ng/ml; >8 to <=1,000 ng/ml; >1,000 to <=10,000 ng/ml; and >10,000 ng/ml. The following values were chosen for hCG/ßhCG: <=2mU/ml; >2 to <=5,000 mU/ml; >5,000 to <= 50,000 mU/ml, and >50,000 mU/ml (Table 3)Citation . The ranges for these two markers were chosen according to the definition of different prognostic groups (good risk, intermediate risk, poor risk) for patients with NSGT by the International Germ Cell Cancer Collaborative Group, IGCCCG (22) . The detection of AFP- and ßhCG-mRNA did not depend on the tumor marker level. However, positive ßhCG-PCRs were found in 6 of 18 patients with corresponding elevated serum markers, whereas all of the patients with normal serum hCG/ßhCG were ßhCG-PCR-negative (P = 0.019).


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  		Table 3 Evaluation of pretherapy patientsa (all tumor stages): Comparison of AFP- and ßhCG-mRNA to the level of the corresponding serum marker (n = 36)

 
Detection of AFP- and ßhCG-mRNA Depending on Tumor Stage (n = 36).
The 36 patients were found to be at different tumor stages (according the Lugano classification): stage I, n = 17; stage IIA, n = 2; stage IIB, n = 0; stage IIC, n = 2; and stage III, n = 15. The corresponding PCR results with regard to tumor stage are summarized in Table 4Citation . Positive PCR results for at least one marker were found for 23.5% (4 of 17) stage I patients and 41.2% (7 of 17) stage IIC/III patients. However, the statistical significance of this difference between stage I and stage IIc/III patients could not be proven (P = 0.46).


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  		Table 4 Evaluation of pretherapy patients (n = 36): Comparison of serum AFP and hCG/ßhCG to corresponding PCR in different tumor stages

 
Patients with Clinical Stage I Tumor (n = 17).
The evaluation of this subgroup of clinical stage-I patients (10 with seminoma, 7 with NSGT) demonstrated two positive AFP-PCRs with corresponding normal and elevated serum AFP levels. In contrast to these findings, the 3 positive ßhCG-PCRs were found exclusively in patients with elevated serum hCG/ßhCG, i.e., 3 of 6 versus 0 of 11 (Table 5)Citation . This reflects the results for all of the tumor stages in the pretherapy group and revealed a statistical significance (P = 0.029) despite the small numbers.


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  		Table 5 Evaluation of patients (n = 17) with stage I germ-cell tumor: Comparison of AFP- and ßhCG-mRNA to the level of the corresponding serum marker

 
Detection of AFP- and ßhCG-mRNA Depending on Histology (n = 36).
Seven teratocarcinomas, 7 embryonal carcinomas, 11 mixed tumors, and 11 seminomas were found in the subgroup of 36 pretherapy patients. Three of 7 patients with teratocarcinomas were positive for PCR. One patient revealed a positive AFP-PCR, 1 a positive ßhCG-PCR, and 1 was positive for both PCRs. Four of seven patients with embryonal carcinoma revealed positive AFP-PCRs with an additional positive ßhCG-PCR in one patient. In 11 patients with mixed tumor, 2 were positive for AFP-PCR and 1 was positive for ßhCG-PCR. As expected, none of the seminoma patients showed a positive PCR for AFP, but 2 of 11 were positive for ßhCG (Table 6)Citation . No definite correlation of PCR results with histological cell type could be found.


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  		Table 6 Evaluation of pretherapy patients: Comparison of serum AFP and hCG/ßhCG to corresponding PCR results in the peripheral blood of patients (n = 36) with different tumor histologies

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Molecular biological aspects are increasingly playing a role in diagnostics, therapy and monitoring of malignancies. The question of circulating tumor cells in the peripheral blood of patients with malignancies and their clinical value has been investigated for different tumors (8, 9, 10, 11, 12, 13, 14 , 23) . For prostate cancer the investigation in detection of circulating tumor cells by RT-PCR for PSA-mRNA has been extensively done in the last few years. However, potential correlation of RT-PCR detection of PSA-mRNA to tumor stage or serum PSA level is undergoing controversial discussion. An improved preoperative staging with a potential change of therapeutical decisions could not be concluded from available results thus far. Concerning the markers AFP and hCG/ßhCG, first experiences in the detection of circulating tumor cells were reported for hepatocellular carcinoma and breast cancer (12, 13, 14 , 24) , but have hitherto not been investigated in germ-cell tumors.

A positive PCR for at least one marker was found in 26.5% of all of the 336 samples independent of tumor stage or serological marker level. We found 33.5% PCR-positive results if at least one serum marker was elevated. AFP-mRNA detection in the peripheral blood seemed to be independent of the corresponding serum marker in this nonstratified cohort. In contrast, more than twice as many positive ßhCG-PCRs were found in patients with elevated serum hCG/ßhCG as compared with those with normal serum levels (17.3% versus 7.1%). This is consistent with the results of Fan et al. (25) , who found positive ßhCG-PCR results in 8 (17%) of 47 apheresis products before bone marrow transplantation in 28 patients with ßhCG-secreting germ cell tumors. In contrast to our findings, they noted no positive PCR results in patients with normal serum ßhCG level.

A subgroup of 36 patients before definitive therapy was stratified. This allowed an evaluation concerning tumor stage, marker elevation, and histology. A correlation between the level of serum AFP and corresponding PCR result was not found in this subgroup.

Even in patients with distinct AFP elevation (AFP, >1000 ng/ml), we could not demonstrate more positive PCR results. These results correspond with the findings in the AFP-producing hepatocellular carcinoma. In independent studies, no correlation of PCR result and level of serum marker elevation, not even in excessive AFP elevation, could be demonstrated (12 , 26) .

Positive results for the ßhCG-PCR were found exclusively in those patients in our study who had elevated serum-hCG/ßhCG. Six (33%) of 18 patients with pathological serum hCG/ßhCG revealed a corresponding positive PCR, but none of those with normal serum marker did so. Elevated serum ßhCG/hCG was noted in 100% (6 of 6) of PCR-positive patients compared with 40% (12 of 30) of PCR-negative patients. This showed statistical significance. These results are in concordance with the experiences of Fan et al. (25) . They noted positive PCR results for ßhCG in the apheresis products in 7 of 20 of their germ-cell tumor patients. All of these patients (100%) had an elevated serum hCG in comparison with 46.2% when PCR was negative. One of two of the patients in our study with excessive serological hCG/ßhCG showed a positive PCR. However, additional studies in a large cohort need to be performed to evaluate a potential correlation between marker levels and PCR results.

Seven (41.2%) of 17 patients with clinical stage IIC/III had a positive PCR for one of the markers, whereas only 4 (23.5%) of 17 patients with clinical stage I had positive PCRs. Thus, the detection of circulating tumor cells seems to be more likely with increasing tumor volume. The statistical significance of this finding has to be confirmed with a larger number of patients. Our findings are consistent with published data for prostate cancer and hepatocellular carcinoma. The dependence of circulating tumor cells on tumor volume and extension of metastases could be demonstrated for PSA-mRNA in prostate cancer and for AFP-mRNA in hepatocellular carcinoma (13 , 24) .

Regarding histology of the primary tumor 4 (57%) of 7 patients with embryonal carcinoma showed relatively more positive PCR results in the peripheral blood than patients with teratocarcinoma (43%), mixed tumor (27.3%), and seminoma (18%), with exclusive detection of ßhCG-mRNA in the latter. Nevertheless, we could not demonstrate a definite correlation with different tumor histology. The higher rate of positive PCR results in patients with embryonal carcinoma may reflect the hypothesis of a potential prognostic factor, because the histological presence of embryonal carcinoma in the primary tumor is regarded as a prognostic factor in lower stages of germ-cell tumors (27 , 28) .

It cannot be assessed, thus far, whether the detection of AFP- and/or ßhCG-mRNA in the peripheral blood has a prognostic relevance. Positive RT-PCR results indicate the presence of circulating tumor cells in the peripheral blood but do not necessarily imply that metastases will occur or has occurred. Animal studies showed that <1% of malignant cells that are discharged by the primary tumor and circulating in the peripheral blood meet all of the necessary criteria for a successful metastasizing. A tumor is able to discharge up to 104 cells/g tissue per 24 h. However, the incidence of clinically verified metastases is much lower. Different factors may cause the different results for mRNA expression of both markers: AFP and ßhCG expression may be heterogeneous, and tumor cells may enter the circulation intermittently. Furthermore, the potential differences of stability and integrity of both specific-mRNAs are not known. Nevertheless, based on the experiences in other tumor entities, the value of circulating tumor cells as a prognostic factor is worth being discussed (22) . Further clinical follow-up will be mandatory. In patients with clinical stage I, our data (2 of 7 NSGTs) may correspond to the known tumor recurrence rate in patients who undergo a watch-and-wait-strategy in this tumor stage.

Our findings of AFP- and/or ßhCG-mRNA detection in the peripheral blood of germ-cell tumor patients strongly suggest the presence of circulating tumor cells. These findings are dependent on tumor stage and seem to be associated with tumor histology and serological data for hCG/ßhCG. The detection of circulating germ-cell tumor cells may have other implications in tumor management. Whether these tumor cells have the capacity to contribute to recurrence and metastasis remains to be determined. The significance of PCR for the identification of patients with risk of recurrence deserves further study, especially concerning the follow-up during chemotherapy, the watch-and-wait strategy in patients with stage I tumor, and the correlation with histological cell type.


   ACKNOWLEDGMENTS
 
We thank Bianca Bulgaru for excellent technical assistance and Jean Bolte for editorial comments regarding the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by the internal IFORES-grant (Interdisziplinäre Forschungsförderung der Universität Essen) of the Medical School, University of Essen. Back

2 To whom requests for reprints should be addressed, at Department of Urology, University of Essen Medical School, Hufelandstrasse 55, D-45122 Essen, Germany. Back

3 The abbreviations used are: AFP, {alpha}-fetoprotein; hCG, human chorionic gonadotropin; NSGT, nonseminomatous germ-cell tumor; RT, reverse transcription; PSA, prostate-specific antigen. Back

4 Unpublished data. Back

Received 9/16/99. Accepted 4/17/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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