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Detection of Germ-Cell Tumor Cells in Peripheral Blood Progenitor Cell Harvests: Impact on Clinical Outcome¹

Departments of Internal Medicine [M. H.] and Hematology and Oncology [O. R., A. S., W. S., D. H., J. B.], Charité Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, 13353 Berlin, Germany

	ABSTRACT

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Our study was conducted to evaluate the impact of tumor cell contamination in peripheral blood progenitor cell (PBPC) harvests on the clinical outcome of patients with germ-cell tumors undergoing high-dose chemotherapy (HDCT) and autologous PBPC reinfusion.

Samples of mononuclear cells from progenitor cell harvests of 57 patients with advanced or recurrent germ-cell tumors were retrospectively screened for contaminating tumor cells using immunocytochemical staining for cytokeratin filaments and reverse transcription-PCR (RT-PCR) testing for germ-cell alkaline phosphatase mRNA. The results were correlated to clinical prognostic variables as well as to the overall and event-free survival of these patients.

Tumor cell contamination was detected in PBPC harvests of 16 of 57 enrolled patients (28%), and, among these, in 14 of 51 (27%) who underwent HDCT. The presence of contaminating tumor cells as detected by either immunocytochemical staining, RT-PCR, or both was strongly associated with a reduced overall survival (43% versus 71%, P = 0.0037) and event-free survival (0% versus 52%, P = 0.0005) after 1 year. In multivariate analysis, the demonstration of contaminating tumor cells had a higher predictive value for a poor event-free survival than other known prognostic variables.

The presence of contaminating tumor cells in PBPC harvests of patients with germ-cell tumors seems to predict a poor overall and event-free survival in patients undergoing HDCT and autologous PBPC reinfusion.

	Introduction

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Current treatment modalities of metastatic germ-cell tumors result in CRs³ and lasting remissions in the majority of patients (1) . Even those who do not respond to conventional-dose treatment may still benefit from HDCT, followed by reinfusion of autologous hematopoietic progenitor cells harvested from the bone marrow or the peripheral blood (2 , 3) . A potential limitation of HDCT is the risk of tumor cell contamination of autologous progenitor cell products (4) , which is increasingly being regarded as a clinically relevant adverse prognostic factor (5) .

We have recently established a sensitive technique for the detection of germ-cell tumor cells in autologous PBPC products of patients with advanced germ-cell tumors (6) . In the present analysis, we correlate these results to the overall and event-free survival of germ-cell tumor patients after HDCT. In addition, we investigate the interaction of tumor cell contamination as a prognostic variable with clinical indicators for treatment outcome after HDCT that have been defined previously (7) .

	Patients and Methods

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Patients.
The patient characteristics are shown in Table 1 . As described previously, patients with germ-cell tumors who progressed during or who relapsed after cisplatin-based chemotherapy (n = 57) were enrolled in consecutive clinical trials that included conventional-dose salvage treatment, followed by HDCT (8) . The clinical trials and the study on tumor cell contamination presented here were approved by the local ethics committee, and all patients gave their written and informed consent before participation.

RNA Preparation.
Samples of peripheral blood MNCs were separated on a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden), washed twice with PBS containing 1% FCS, counted, and tested for viability by means of trypan blue exclusion. Cells (2–3 x 10⁷) were lysed in TRIzol reagent (Life Technologies, Inc., Grand Island, NY) and sheared to homogeneity, following a one-step guanidinium isothiocyanate/phenol RNA preparation technique (9) . Chloroform was added, and the RNA contained in the aqueous upper phase was precipitated in isopropanol at -80°C, washed in 80% ethanol, and resuspended in RNase-free water. The integrity of RNA was examined by RT-PCR analysis for ß₂ microglobulin mRNA (10) .

cDNA Synthesis and PCR.
RNA (10 µg ) was incubated with 50 ng of a ₁₅(dT) primer at 60°C for 10 min. After the addition of a deoxynucleotide mix (10 mM), DTT (10 mM), and first-strand reaction buffer, 100 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) were added to a final volume of 50 µl. The cDNA synthesis reaction was carried out at 37°C for 40 min. After heat inactivation at 95°C for 5 min and subsequent maintenance at 4°C, 5 µl of each reaction product were subjected to PCR analysis.

PCR analysis was performed as described previously (6) . In brief, PCR was carried out at 94°C (40 s), 65°C (1 min), and 72°C (1 min) for 40 cycles, followed by a final 5 min at 72°C. All reagents used for PCR analysis were purchased from InViTek (Berlin, Germany). Twenty microliters of the final PCR products were separated on a 1% agarose gel (Oncor Appligene, Illkirch, France). For reamplification, 5 µl of the PCR product were again subjected to 40 cycles of PCR using a nested sense primer. For ß2 microglobulin and GCAP mRNA amplification, the primers used were the same as described previously (6) .

Immunocytochemical Staining.
For immunocytochemical staining, 10 µg of a commercially available murine F(ab)₂ fragment directed against cytokeratin filaments (EpiMet Tumor Cell Detection Kit; Baxter Biotech, Unterschleißheim, Germany) were used. Cell suspensions were attached to slides by cytocentrifugation, air dried, and stored at -20°C. After thawing, the slides were again allowed to dry and fixed in acetone. The germ-cell tumor cell line Tera-2 (American Type Culture Collection, Manassas, VA) was used as a positive control for each immunostaining experiment. Cytospin preparations of peripheral blood MNCs from healthy donors served as a negative control. A standard protocol, as outlined in the kit, was followed. In brief, the slides were permeabilized in methanol with 4% formaldehyde. Because it was linked to alkaline phosphatase, the anticytokeratin F(ab)₂ fragment allowed immediate staining with a chromogenic substrate.

Tumor cells were considered immunocytochemically positive when staining was observed on >70% of the cytoplasm. Routinely, an overall number of 1–2 x 10⁶ MNCs, with 5 x 10⁵ cells per slide, was analyzed and tumor cells were quantified.

Definitions.
A clinical CR was present if there was disappearance of all radiological manifestations and normalization of tumor markers by chemotherapy alone. Patients with normalization of tumor markers and complete resection of necrosis/mature teratoma or undifferentiated viable tumor were considered having pathological CR or surgical CR, respectively. Patients with normalization of tumor markers, but radiological evidence of disease, were considered PRm-. Patients with a reduction of radiological manifestations of 50% or more or with a decline of tumor markers of 90% or more were considered PRm+. All other patients were classified as having stable disease or PD.

Sensitivity to cisplatin was assessed as reported previously (11) . Any disease was considered sensitive to cisplatin when more than stable disease was achieved for >4 weeks. Any disease was considered refractory to cisplatin when stable disease or better was achieved, but when there was evidence of tumor progression within 4 weeks of the last cisplatin-based treatment. Any disease was considered absolutely refractory to cisplatin when not even stable disease was achieved following cisplatin-based chemotherapy.

The progenitor cell harvest of a patient was defined as positive for contaminating tumor cells if the RT-PCR for GCAP mRNA was positive in two separate analyses of an RNA sample or if cytokeratin-positive cells were detected on at least two cytospin preparations.

Statistical Analysis.
Two categories of patients with and without tumor cell contamination of PBPC products were formed, and clinical variables of patient and tumor characteristics were compared between these categories using the Mann-Whitney U test for continuous variables and the ² test for discontinuous variables.

The median follow-up time for patients still alive was 16 months, with a range of 3–63 months. Overall and event-free survival rates were calculated according to the Kaplan-Meier method (12) . Univariate comparisons of the event-free and overall survival rates were performed using the log-rank test. The Cox proportional hazards regression model was used for the multivariate survival analysis (13) . All calculations were performed with SPSS 8.0 computer software.

	Results

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Tumor Cell Detection.
PBPC samples from 57 patients were processed and analyzed by immunocytochemical staining for cytokeratin (n = 42), by RT-PCR for GCAP mRNA (n = 48), or both (n = 33). For technical reasons, not all samples could be tested by both methods. Immunocytochemical staining identified positive cells in PBPC samples of 7 of 42 (17%) patients. In the RT-PCR analysis, five of these samples were positive for GCAP mRNA and two samples were negative. RT-PCR gave positive results in PBPC samples of 14 of 48 (29%) patients. Seven samples tested positive by RT-PCR were negative by immunocytochemical staining. In two of the samples that were positive by RT-PCR, immunocytochemical staining analyses were not performed. Altogether, PBPC samples of 16 of 57 (28%) patients tested positive in either immunocytochemical staining, RT-PCR, or both. The median number of tumor cells detected by immunocytochemical staining was 2 in 10⁶ mononuclear cells (range, 1–12 in 10⁶ MNCs). The numbers of tumor cells detected were similar in the five samples that were positive by RT-PCR and the two samples that were negative.

Of the 57 patients included, only 51 underwent HDCT. Among these, 13 of 42 harvests tested were positive by RT-PCR (29%), and 4 of 37 harvests tested by immunocytochemical staining were positive (11%). In the remaining patients, either RT-PCR (n = 9) or immunocytochemical staining (n = 14) could not be performed.

All patients were divided into two groups depending on whether or not contaminating tumor cells were detected in their PBPC products. Patient and tumor characteristics at study entry (n = 57; Table 1 ) and immediately before HDCT (n = 51; Table 2 ) were compared between these groups. None of the clinical parameters correlated with the presence of contaminating tumor cells in PBPC products. Subsequently, all patients were assigned to risk categories according to a clinical model that was previously shown to predict treatment outcome after HDCT (7) . Again, there was no difference in the distribution of patients with PBPC products positive for contaminating tumor cells between these clinically defined prognostic groups (Table 2) .

Survival Analysis.
The overall and event-free survival rates of all 57 patients 1 year after study entry were 64% and 38%, respectively. Among those 51 patients who actually received HDCT, the maximum response to HDCT was CR in 22, PRm- in 10, PRm+ in 8, and stable disease/PD in 10 patients. In one patient, no follow-up data were available. The overall survival at 1 year was 43% and the event-free survival at 1 year was 0% in 16 patients in whom PBPC products were positive for contaminating tumor cells detected by either immunocytochemical staining, RT-PCR, or both. These figures compare with an overall survival at 1 year of 71% and an event-free survival at 1 year of 52% in 41 patients in whom PBPC products were negative for contaminating tumor cells (P = 0.0037 for overall survival and P = 0.0005 for event-free survival; Fig. 1 B).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Kaplan-Meier plots for the event-free survival in all patients included (A), in patients with and without tumor cell contamination (B), in patients with at least PRm- after HDCT with or without tumor cell contamination (C), and in good-prognosis patients with or without tumor cell contamination (n = 35; D).

When analyzed separately, both RT-PCR and immunocytochemical staining were able to identify patients with a reduced event-free survival following HDCT (RT-PCR: 0% versus 51% after 6 months, P = 0.012; immunocytochemical staining: 0% versus 50% after 6 months, P = 0.008).

The maximum response to HDCT was compared with the presence of tumor cell contamination in the harvests. Thirty-two of 51 patients (63%) who actually underwent HDCT achieved at least PRm-. Among these, 14 patients (44%) relapsed. Of those who relapsed, 5 of 14 (36%) patients had PBPC harvests positive for tumor cells. In contrast, only 2 of 18 (11%) patients who did not relapse during the observation period had positive harvests (P = 0.035). A survival analysis among those 32 patients who had achieved at least PRm- showed that patients with harvests positive for tumor cell contamination had a poorer overall and event-free survival (overall survival 1 year after HDCT: 0% versus 78%, P = 0.001; event-free survival 6 months after HDCT: 0% versus 80%, P < 0.001; Fig. 1 C).

Correlation with Risk Categories.
In previous studies, prognostic variables for response to HDCT could be identified, resulting in a clinical model for the prediction of treatment response after HDCT (7) . In univariate and multivariate analyses, these factors were evaluated in all 57 patients included in the present analysis, with the information about tumor cell contamination in PBPC products added as a new variable. The results are shown in Table 3 . In univariate analysis, all parameters were shown to predict a poor overall and event-free survival, with the exception of a mediastinal primary tumor. In multivariate analysis, tumor cell contamination of PBPC products showed the highest predictive value for a poor event-free survival at 1 year (Table 3) .

	Discussion

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Tumor cell contamination in PBPC products that are reinfused after potentially curative HDCT has become an important issue in a number of solid tumors. Particularly in germ-cell tumors, contamination of PBPC products with malignant cells could be of major importance because HDCT has become a common treatment modality and hematogeneous dissemination occurs in the majority of patients with advanced disease. However, sensitive techniques for the detection of tumor cells in PBPC harvests of patients with germ-cell tumors have only recently become available (6 , 14) .

We have previously demonstrated the high sensitivity and specificity of RT-PCR analysis for GCAP mRNA for the detection of cells from germ-cell tumor cell lines Tera-1 and Tera-2 (6) . To date, no false positive RT-PCR results were obtained in peripheral blood samples (n = 40) and progenitor cell harvests (n = 20) of healthy donors or patients with hematological malignancies. The comparison with immunocytochemical staining for cytokeratin-positive cells (one tumor cell in 10⁵ MNCs detected) had demonstrated that the sensitivity of the RT-PCR assay for GCAP mRNA was superior in one cell line (Tera-1; one tumor cell in at least 10⁶ MNCs detected), but inferior to immunocytochemical staining in another (Tera-2; one tumor cell in 10⁴ MNCs detected), apparently depending on the degree of mRNA expression. We concluded that, whenever feasible, both RT-PCR for GCAP mRNA and immunocytochemical staining for cytokeratin-positive cells should be performed.

In the present analysis, PBPC harvests of 57 patients were retrospectively analyzed using both RT-PCR for GCAP mRNA and immunocytochemical staining for cytokeratin-positive cells. At the time of analysis for tumor cell contamination, all laboratory investigators were blinded for any clinical data. RT-PCR analysis yielded positive results in a higher number of harvests than immunocytochemical staining for cytokeratin. With one exception, the number of tumor cells detected by immunocytochemical staining was low and did not differ significantly between those tested positive and those tested negative by RT-PCR for GCAP mRNA. Nonetheless, two of seven harvests positive for cytokeratin staining were negative by RT-PCR. As deduced from the sensitivity of tumor cell detection observed in our previous study, the amount of GCAP mRNA may vary in different germ-cell tumor cell lines in vitro and, possibly, also in patients with germ-cell tumors (6) . Schär et al. (15) showed that the differential expression of alkaline phosphatase isoenzymes in germ-cell tumors depends on the respective degree of tumor differentiation. It should be noted that both RT-PCR analysis for GCAP mRNA and immunocytochemical staining for cytokeratin filaments were independently able to identify patients with a reduced event-free survival. Therefore, the analysis for contaminating tumor cells by immunocytochemical staining may add information, confirming the RT-PCR results and identifying tumor cells in PBPC harvests that were possibly missed by RT-PCR analysis. However, whenever immunocytochemical staining is not feasible (e.g., when no fresh specimens are available for analysis), RT-PCR analysis for GCAP mRNA can be used alone.

Overall, 16 of 57 samples (28%) tested positive with either RT-PCR or immunocytochemistry. We then correlated the information about tumor cell contamination of PBPC products with clinical data of all 57 patients at study entry (Table 1) and of those 51 patients who subsequently received HDCT (Table 2) . None of the clinical variables could be used to predict tumor cell contamination of PBPC products. In contrast, the presence of tumor cell contamination strongly correlated with a reduced overall and event-free survival following HDCT (Fig. 1 C).

Does the presence of tumor cell contamination add prognostic information with respect to treatment outcome following HDCT? Three observations support this assumption. First, 32 of 51 patients who underwent HDCT achieved at least a PRm- or better. Even in this subgroup of patients with a favorable response to HDCT, tumor cell contamination was clearly associated with a significantly shorter overall and event-free survival (Fig. 1 C). Second, based on clinical prognostic variables, 35 patients were assigned to a good risk group (7) . Also, among this subgroup of patients with no apparent clinical risk factors for treatment failure after HDCT, both overall and event-free survival were significantly worse in patients in whom tumor cell contamination of PBPC products could be demonstrated as compared with patients in whom no contaminating tumor cells were identified. Finally, the negative impact of tumor cell contamination was supported by a multivariate analysis that identified tumor cell contamination of PBPC products as the strongest independent adverse prognostic factor among all variables tested.

The results of the present analysis may be relevant for the treatment of patients with germ-cell tumors with HDCT, and the prognostic impact of tumor cell contamination of PBPC products needs to be further studied. Several important questions should be further explored. Do contaminating tumor cells in PBPC products contribute to the relapse of patients after HDCT, beyond their apparent role of heralding an unfavorable course of the disease? Will attempts to reduce tumor cell contamination of PBPC products result in an improved survival after HDCT? And, more importantly, how should we presently treat patients with PBPC products that are positive for contaminating tumor cells?

From the present analysis, we must conclude that the demonstration of tumor cells in PBPC products of patients with germ-cell tumors as assessed by RT-PCR for GCAP or by immunocytochemical staining for cytokeratin-positive cells predicts a poor overall and event-free survival despite the use of HDCT and that tumor cell contamination is an independent adverse prognostic factor in these patients.

	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 Supported by a grant from the Wilhelm Sander-Stiftung (Munich, Germany).

² To whom requests for reprints should be addressed, at Medizinische Klinik und Poliklinik, Charité Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Phone: 49-30-45053539; Fax: 49-30-45053900; E-mail: hildebra{at}charite.de

³ The abbreviations used are: CR, complete remission; HDCT, high-dose chemotherapy; PBPC, peripheral blood progenitor cell; RT-PCR, reverse transcription-PCR; GCAP, germ-cell alkaline phosphatase; PD, progressive disease; MNC, mononuclear cell; PRm, partial remission, marker.

Received 6/20/00; revised 10/ 2/00; accepted 10/13/00.

	REFERENCES

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