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Role of P53 and MDM2 in Treatment Response of Human Germ Cell Tumors

From the Department of Pathology/Laboratory for Experimental Patho-Oncology, University Hospital Rotterdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotterdam, Rotterdam, the Netherlands, and Department of Hematology/Oncology, University of Tübingen, Tübingen, Germany.

Address reprint requests to Department of Pathology/Laboratory for Experimental Patho-Oncology, University Hospital Rotterdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotterdam, Bldg Be 431, Rm 430b, PO Box 1738, 3000 DR Rotterdam, the Netherlands; email: Looijenga{at}leph.azr.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Testicular germ cell tumors (TGCTs) of adolescents and adults are very sensitive to systemic treatment. The exquisite chemosensitivity of these cancers has been attributed to a high level of wild-type P53.

MATERIALS AND METHODS: To clarify the role of P53 in treatment sensitivity and resistance of TGCTs, we performed immunohistochemistry and Western blotting analysis on a series of 39 fresh-frozen primary TGCTs before therapy (unselected series). In a series of formalin-fixed paraffin-embedded TGCTs of patients with fully documented clinical course, including treatment-sensitive (n = 17) and -resistant (n = 18) tumors, P53 status was assessed by immunohistochemistry and mutation analysis. In addition, the involvement of MDM2, a P53 antagonist, was investigated by immunohistochemistry, reverse transcriptase polymerase chain reaction, and in situ hybridization.

RESULTS: Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%, and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the positive TGCTs, most tumors, ie, 49%, 41%, and 33%, showed 1% to 10% positive nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis revealed only one silent P53 mutation in one of the responding patients. All embryonal carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA, while a heterogeneous pattern was found for the other histologic components. Amplification of MDM2 was detected in one out of 12 embryonal carcinomas.

CONCLUSION: Although our results are in line with previous findings of the presence of wild-type P53 in TGCTs, they show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and inactivation of P53 is not a common event in the development of cisplatin resistance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
TESTICULAR GERM CELL tumors (TGCTs) of adolescents and adults, ie, seminomas and nonseminomas, are of particular interest from a clinical point of view because of their exquisite sensitivity to treatment. While seminomas and nonseminomas can be effectively treated with cisplatin-based chemotherapy, seminomas are also highly sensitive to irradiation (reviewed in^1-3). Approximately 80% of patients with metastatic disease can be cured by systemic treatment.⁴ In addition, TGCTs are also biologically interesting, since they mimic embryonal development to a certain extent (reviewed in ^5-8). Seminomas show characteristics of early (primordial) germ cells, whereas nonseminomas can be composed of embryonal carcinoma, being the stem-cell component, which may differentiate to either yolk sac tumor and choriocarcinoma (the extra-embryonic lineages) or teratoma (the somatic lineage).⁹ Another intriguing clinical finding is that fully differentiated ("mature") teratoma components are resistant to chemotherapy.^10-12 Surgery is needed to remove these lesions because of their potential for secondary malignant transformation, which can lead to non–germ cell malignancies.¹³

Various investigations have focused on the P53 pathway to explain the chemosensitivity of TGCTs. Most studies reported a high level of wild-type P53 protein based on immunohistochemistry. An overview of the different studies is given in Table 1,^14-31 indicating that although a high percentage of positive tumors are reported, the majority of these cases show less than 30% of positive tumor nuclei. In fact, a significant number of cases are scored as containing between 1% and 10% positive nuclei, and some of the tumors showed no staining at all. In contrast to many other solid cancers, P53 mutations have hardly been identified in unselected TGCTs (see Table 2 for publications and results^{20,21,29,31-40}). Out of the 281 sequence-verified tumors, 19 (6.7%) were demonstrated to have a mutation. Comparison of two independent human TGCT-derived cell lines, one with functional and one with a mutant P53, showed that the former was more sensitive to cisplatin than the latter.³³ These studies led to the conclusion that high levels of wild-type P53 account for the exquisite chemosensitivity of TGCTs. However, we found no differences in treatment sensitivity between a well-characterized TGCT-derived cell line with functional P53 (NTera2) and a line without (NCCIT).⁴¹ Inactivation of P53 by the HPV16-E6 protein in the cisplatin-sensitive NTera2 cell line did not result in resistance. In addition, the only resistant TGCT-derived cell line (2102Ep) had functional P53.

The objective of the present study was to clarify the role of P53 and MDM2 in sensitivity and resistance of TGCTs to cisplatin-based chemotherapy. To exclude interobserver variability, we applied both Western blotting and immunohistochemistry to assess P53 protein level. By assuming a crucial role of a high-level wild-type P53 in chemosensitivity of TGCTs and an association between P53 mutations and chemotherapy resistance, high levels of P53 protein and no or only small numbers of mutations were expected in a group of unselected cases and in chemosensitive cases. In contrast, resistant cancers would show either a low level of wild-type P53 and/or an increased frequency of P53 mutations or inactivation by overrepresentation of MDM2 encoded by the full-length mRNA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Material
Fresh-frozen and formalin-fixed paraffin-embedded tissue blocks from 39 unselected patients (23 nonseminomas and 16 seminomas) were collected between 1991 and 2000 in close collaboration with urologists and pathologists in the southwestern part of the Netherlands. They were retrieved from the archive of the Laboratory for Experimental Patho-Oncology, Department of Pathology, University Hospital Rotterdam/Daniel. For these patients, no data on the clinical course were available.

From 17 patients treated at the University Hospital Rotterdam/Daniel between 1991 and 1994 who remained continuously disease-free after initial treatment, formalin-fixed paraffin-embedded material of the primary TGCT was collected. The series consisted of nine seminomas and eight nonseminomas.

Formalin-fixed, paraffin-embedded samples from 18 chemotherapy-refractory patients diagnosed between 1991 and 1998, treated within various trials led by Tübingen University, Germany, were investigated. Patients were considered refractory when progression or relapse occurred despite adequate initial and salvage treatment, including high-dose chemotherapy with autologous stem-cell transplantation. The material of nine patients was obtained at initial diagnosis; in eight cases, the material was sampled after exposure to chemotherapy, and in one case, material from both the primary tumor and a metastatic tumor in relapse was available. The group of refractory tumors consisted of 16 nonseminomas, one seminoma, and one secondary non–germ cell malignancy. Table 3 summarizes the characteristics of the responding and refractory patients. All cases were reviewed and diagnosed by J.W.O. according to the World Health Organization classification, and the fully documented clinical course was available for these patients.

Immunohistochemical Detection of P53 and MDM2
Paraffin sections of 3-µm thickness were mounted on aminopropylethoxysilane-coated slides, deparaffinized in xylene, and rehydrated. Pressure cooking in citrate buffer 0.01 mol/L, pH 6.0 (1.2 bar), was used for antigen retrieval. All antibodies were diluted in phosphate-buffered saline (PBS) with 1% bovine serum albumin. Primary antibodies (mouse monoclonal anti-P53, Do-7, 1/50 [Dako, Glostrup, Denmark]; mouse monoclonal anti-MDM2, Ab1, clone smp14, 1/50 [Neomarkers, Fremont, CA]) were incubated at room temperature for 1 hour (P53) or overnight (MDM-2). Biotin-labeled rabbit-antimouse immunoglobulins and a biotinylated AP-streptavidin complex (both Dako) were subsequently applied for 30 minutes each. A solution of new fuchsin, naphthol, and levamisole in a Tris HCL buffer 0.2 mol/L (pH 8.0) was used as chromogen; slides were counterstained with Mayer’s hematoxylin stain. Only red nuclear staining was considered positive. For a negative control, the primary antibody was omitted on serial slides. Appropriate positive control sections were stained simultaneously (colon cancer for P53, breast cancer for MDM2). Two investigators (A.-M.F.K. and F.M.) independently assessed samples. In case of discordance, slides were re-evaluated. For evaluation of P53, 300 cells were scored in three randomly selected high-power fields. Furthermore, four randomly selected slides were stained and evaluated independently in the pathology department of the University of Tromso (Norway), as described before.⁵⁰

Immunoprecipitation and Western Blotting
Cultured cells and fresh-frozen tissue samples were used for immunoprecipitation and Western blotting. Samples containing only a limited nontumor component were selected. For immunoprecipitation studies, cells were washed twice in ice-cold PBS and harvested in lysis buffer (Tris HCl 50 mmol/L [pH 8.0], EDTA 5 mmol/L, NaCl 150 mmol/L, 0.75% NP40, Pefabloc SC 1 mmol/L [Roche, Mannheim, Germany], sodium fluoride 50 mmol/L, sodium o-vanadate 10 mmol/L, and one tablet of Complete mini [Roche] per 10 mL of lysis buffer). Protein was subjected to immunoprecipitation by incubating 1 µg of monoclonal antibody against MDM2 (IF-2; Oncogene Science, Cambridge, MA). Specific complexes were collected after incubation with Protein A agarose (Roche). After the precipitates were washed in lysis and wash buffer (Tris HCl 10 mmol/L [pH 8.0], EDTA 1 mmol/L, and sodium o-vanadate 1 mmol/L [Sigma-Aldrich]), they were electrophoresed in 8% sodium dodecyl sulfate (SDS)–polyacrylamide gels, transferred to PVDF membrane (Amersham/Pharmacia, Buckinghamshire, United Kingdom), and probed for MDM2 as described below.

For Western blot analyses, cells were washed with ice-cold PBS, scraped into lysis buffer, and cooled on ice. After centrifugation and denaturation of the proteins at 95°C, the protein lysate (50 µg/lane) was electrophoresed on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. The filters were blocked with PBS containing 3% nonfat dried milk and 0.1% Tween-20 and probed with the respective antibodies. MDM2 was detected using IF-2 antibody 5.0 µg/mL (Oncogene Science), and P53 was detected using Do-7 antibody 0.08 µg/mL (Dako). The input for Western blotting was standardized using the nuclear replication protein A, a nuclear protein present at relatively constant levels in human cells.^51,52 After washing and subsequent incubation with horseradish peroxidase–conjugated rabbit-antimouse antibody (Dako), specific complexes were detected using a chemiluminescent technique according to the manufacturer’s recommendations (ECL kit; Amersham/Pharmacia).

Amplification Analysis for MDM2 Using Fluorescence In Situ Hybridization
Frozen tissue sections of 5 µm were mounted on aminopropylethoxysilane-coated slides, air dried, submerged in methanol/acetone (1:1) at -20°C for 20 minutes, and air dried again. Sections were digested with 0.0005% pepsin (Sigma-Aldrich) in HCl 0.01 mol/L for 1 minute at 37°C, rinsed in demi-water, and dehydrated. YAC-MDM2 probe 75Ia4 (chromosome 12 band q14, obtained from CEPH, Paris, France) was used in combination with a chromosome 12 centromeric probe (p12H8).^53,54 DNA of the YAC-MDM2 was isolated and human sequences were amplified by inter-ALU polymerase chain reaction (PCR), using the primers Alu-1 and Alu-2.⁵⁵ The centromeric probe was labeled with digoxigenin-11-deoxyuridine triphosphate and the YAC-MDM2 probe was labeled with biotin-16-deoxyuridine triphosphate (Roche) using a nick-translation kit (Gibco). After denaturation (73°C for 5 minutes in Hybmix), the probes were preannealed with an excess of Cot-1 DNA (Life Technologies/Gibco). The denatured probe mix was added to denatured slides (3 minutes in 70% formamide/two times standard saline citrate (2xSSC) [pH 7.0] and 5 minutes in 70% ethanol at -20°C and then dehydrated) and hybridized for 48 hours. Slides were washed in 50% formamide/2xSSC. The hybrids were visualized with fluorescein isothiocyanate–conjugated sheep-antidigoxigenin antibody (Roche) and Cy3-conjugated avidin antibody (1/50; Jackson ImmunoResearch, West Grove, PA). The MDM2 gene was considered amplified when at least twice as many hybridization signals were found for MDM2 than for the centromeric probe.

DNA Isolation
Of formalin-fixed paraffin-embedded tissue blocks considered suitable for DNA isolation without further processing, four sections of 25 µm were used. In case of significant nontumor components, tumor areas were microdissected from four to eight 10-µm-thick unstained sections. The material was deparaffinized with xylene, washed in 100% ethanol, and air dried. The tissue was incubated in lysis buffer (Tris 50 mmol/L [pH 8], EDTA 100 mmol/L, NaCl 100 mmol/L, and 1% SDS) containing 25 µL of proteinase K (10 mg/mL) overnight at 55°C followed by incubation with RNase at 37°C for 30 minutes. After phenol/chloroform extraction, DNA was precipitated with isopropanol, washed with 70% ethanol, air dried, and resuspended in a suitable volume of TE buffer. Fresh-frozen material was processed in an identical manner, apart from omission of deparaffinization and rehydration.

Single-Strand Conformation Polymorphism and Sequencing of P53 Exons 5 to 8
Isolated DNA was used as a template for radioactive PCR using alpha-³²P as label. PCR settings and primer sequences are given in Table 4. The PCR products were run on a nondenaturing gel (8% acrylamide/bisacrylamide 49:1, 10% glycerol) for 16 hours at 8 watts.

Reverse Transcription PCR for the MDM2 Splice Variants
RNA was isolated from 14 snap-frozen TGCTs (10 seminomas and four embryonal carcinomas) with Trizol reagent (Life Technologies/Gibco) according to the manufacturer’s instructions. For reference, the histologic composition of the tumor under investigation was checked by microscopic analysis of a parallel section stained with hematoxylin and eosin. RNA was isolated in the same way from four cell lines (Tera1, NTera2, 2102EP, and NCCIT). All RNA samples were pretreated with RNase-free DNase I according to the standard method.⁵⁶ The RNA pellets were dissolved in DEPC-treated water. First-strand cDNA was synthesized from 1 µg of oligodeoxythymidine and random hexamer–primed DNase I–treated RNA in a total volume of 40 µL according to standard procedures. The cDNA quality was checked by PCR with the primers HPRT 244 and HPRT 246, which amplify a specific 587-base pair fragment from mRNA encoding the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT).⁵⁷

Presence of MDM2 splice variants was determined by PCR amplification on the generated cDNA, using gene-specific primer combinations spanning the complete coding region. The following primers were used: forward, 5'-GGCCCGGAGAGTGGAATG-3'; reverse, 5'-ATAAATTTCAGGTTGTCTAAATTC-3' (annealing at 58°C). The expected size of the full-length transcript is 1,685 base pairs.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immunohistochemistry for P53 and MDM2
Unselected tumor samples. To study the presence of P53 and MDM2 protein in a randomly collected series of TGCTs, immunohistochemical analysis was performed on paraffin-embedded tissue sections of a series of 39 TGCTs (16 seminomas and 23 nonseminomas). In this series, the clinical outcome of which is not known, but frozen tissue is available for Western blotting (see below), 36% of the tumors showed no staining for P53 at all, including seven seminomas and 10 nonseminomas (see Table 5). In 49% of the cases, 1% to 10% of the tumor nuclei were positive. In 15% of the cases, there was 10% to 30% positive staining. None of the tumors showed more than 30% positive tumor nuclei. Positive cells were found in all different histologic elements except choriocarcinoma, which was completely negative. Representative examples are shown in Fig 1A and 1B. In total, 1% or more positive tumor nuclei for P53 were identified in 25 (64%) of the 39 TGCTs. To confirm our results, parallel sections of a selected number of TGCTs (n = 4) were stained and evaluated independently; the results were the same.

View larger version (136K): [in this window] [in a new window]   Fig 1. Representative examples of the immunohistochemical stainings for P53 and MDM2. (A) Nonseminoma with strong nuclear P53 staining; (B) seminoma with single P53-positive cells; (C) seminoma without positive MDM2 staining; (D) embryonal carcinoma with strongly positive nuclear MDM2 staining (all x200).

Chemosensitive and refractory tumor samples. To shed light on the actual role of P53 and MDM2 in chemosensitivity and resistance, we investigated a series of patients with TGCTs responding (n = 17) or not responding (n = 18) to chemotherapy by using immunohistochemistry for P53 and MDM2. The clinical data of the patients and histology of the tumors are listed in Table 3. The results of immunohistochemistry are also summarized in Table 5, indicating that the findings for P53 in the responding TGCTs are comparable to the results in the unselected series; 41% of the responding TGCTs (six seminomas and one nonseminoma) showed no staining at all; 41% had 1% to 10% positive nuclei, and 12% of the TGCTs had 10% to 30% positive nuclei. Only one tumor showed more than 30% positive nuclei. In the series of refractory cases, a trend toward a lower number of completely negative cases (17%, three nonseminomas) and a higher number of positive cases (33% for 10% to 30% positive nuclei, and 17% for > 30% positive nuclei) was observed, compared with the unselected and responding series. No differences were identified between cases obtained before (n = 11) and after (n = 7) exposure to chemotherapy. MDM2 also showed a higher number of positive cases in the nonresponding series (15 of 18) compared with the unselected (28 of 39) and responding (11 of 17) series. This is explained by the higher number of nonseminomas in the first series. Again, no differences between chemotherapy-naive and exposed tumors were observed.

Western Blotting and Immunoprecipitation of P53 and MDM2
As immunohistochemistry can be influenced by differences in fixation, pretreatment protocols, the method of staining and scoring, and particularly by selection of regions in case of tumor heterogeneity, we performed Western blotting and immunoprecipitation for P53 and MDM2. Fourteen snap-frozen TGCTs (seven seminomas and seven nonseminomas, all with a limited nontumor component), one normal testicular parenchyma sample, and three well-characterized TGCT-derived cell lines (NTera2, NCCIT, and 2102Ep) were investigated. All TGCTs, the normal testis, and the NTera2 and NCCIT cell lines showed a low level of P53. This was comparable to the level found in the breast carcinoma–derived cell line MCF-7, known to have a wild-type and low-level P53. The level was lower than that found in the breast carcinoma cell lines SKBR-3 and T47D and a colon cancer cell line, all of which are known to have mutant P53 and thus a higher protein level (see Fig 2). The only resistant cell line (2102Ep) showed the highest level of P53 in our series, in accordance with our previous data.⁴¹ Immunoprecipitation of MDM2 performed on one seminoma and two nonseminomas demonstrated, in concordance with the immunohistochemical results, a high level in embryonal carcinomas and a low level in the other histologic components. Rehybridization of the blots with the P53-specific antibody showed that most of P53 was bound to MDM2.

View larger version (33K): [in this window] [in a new window]   Fig 2. Western blotting for P53. All nonseminomas and seminomas showed low levels of P53 compared with SKBR-3 and colon tumor (high expression) and MCF-7 and normal testis (low expression). Replication protein A (RPA) was used as a loading control. Percentage of positive nuclei by immunohistochemistry is given in parentheses.

View larger version (106K): [in this window] [in a new window]   Fig 3. Representative examples of fluorescence in situ hybridization with centromere 12 (red) and MDM2 (green) probes on frozen sections of (A) an embryonal carcinoma and (B) a seminoma. Note the increased MDM2 copies compared with the centromere in the embryonal carcinoma. (C) Examples of reverse transcription PCR for expression and alternative transcripts of MDM2. Only the full-length mRNA (1,685 base pairs) is present in the embryonal carcinomas (ECs) and nonseminoma-derived cell lines but not in seminoma (SE).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
TGCTs, ie, seminomas and nonseminomas, account for 1% to 3% of all neoplasms in men. They are the most common cancers in white males between 15 and 45 years of age.⁵⁹ These tumors are unique in their responsiveness to cisplatin-based chemotherapy and are considered a model for curative disease.¹ This phenomenon has been explained by high levels of wild-type P53, as demonstrated by immunohistochemistry in a number of studies (see Tables 1 and 2). On average, 70% of the tumors were considered positive, although in most cases, only a minority of tumor cells showed a P53 signal and even completely negative tumors were identified. In analyses of these data by the fraction of positive cells, approximately 32% of the tumors contained no P53-positive cells, approximately 45% showed positivity in more than 5% of tumor cells, and approximately 23% had a P53 signal in 30% or more tumor cells. These numbers are in the same range as our results (see Table 5). Regarding the putative role of P53 in the favorable response of TGCTs to therapeutic interventions, it is important to realize that in both the unselected series and the series of responsive tumors, more than one third of the cases showed no P53 staining.

To assess the actual level of P53 protein in TGCTs, we performed Western blotting on seminomas, nonseminomas, and well-characterized nonseminoma-derived cell lines. These experiments demonstrated convincingly that the protein level of P53 is in the range of control samples with a low level of P53, ie, normal testis and the breast carcinoma cell line MCF-7.⁶⁰ The protein level was far lower than that found in a colon cancer–derived and two breast carcinoma–derived cell lines, all known to have a high level of P53. The discrepancy between our results and the finding of a high level of P53 in mouse teratocarcinomas⁶¹ may indicate that mouse teratocarcinomas are not a proper model for human TGCTs. This assumption is supported by further discrepancies such as the absence of seminoma components, a prepubertal clinical presentation, and a diploid chromosomal constitution of mouse tumors (reviewed in^62-64).

To study the role of P53 in chemotherapy resistance in vivo, we investigated the presence of P53 by immunohistochemistry on a clinically well-defined group of patients with nonresponding/refractory TGCTs. All patients had failed adequate first-line treatment. In the course of the disease, most of them had been considered suitable candidates for salvage high-dose chemotherapy but had progressed or relapsed afterward. The treatment-resistant tumors did not show reduced levels of P53 compared with the responsive and the unselected groups. In contrast, the resistant group contained fewer cases without P53 signal in any fraction of cells (17%) and more cases with a high percentage of positive tumor nuclei (both between 10% and 30% and > 30%). This finding cannot be explained by a prior exposure of tumor cells to chemotherapy in the resistant group, as no difference was seen between tumors sampled before and after chemotherapy. Furthermore, mature teratomas, known to be intrinsically resistant to cisplatin-based chemotherapy,^10-12 also showed positive staining for P53 in all three investigated groups. Therefore, a loss or lack of high levels of P53 protein does not account for a resistant phenotype in our series.

None of the tumors of chemosensitive and of the nonresponding patients contained a mutation within exons 5 to 8 of the P53 gene, supporting the previously described low frequency of P53 mutations in TGCTs. At the same time, the findings indicate that P53 mutations are not a common means by which these tumors develop chemotherapy resistance. This is in line with data from TGCT-derived cell lines, where inactivation of P53 in a cisplatin-sensitive cell line did not alter the response to cisplatin. Furthermore, a cell line with inactive P53 was still highly sensitive to chemotherapy, and another cell line with a high level of functional P53 has been demonstrated to be resistant.^41,65

In contrast, one study described P53 mutations in specimens from four out of 23 patients sampled at relapse. All of these patients died of their disease. Histologically, three patients were diagnosed as having pure mature teratoma and one patient had both a mature teratoma and a rhabdomyosarcoma component, the latter probably representing a secondary non–germ cell malignancy.³³ The authors connected the resistant phenotype to the presence of a P53 mutation. However, since mature teratomas are intrinsically resistant to chemotherapy, it remains difficult to judge the impact of the P53 mutation in the development of cisplatin resistance. Moreover, P53 mutations are more frequently detected in rhabdomyosarcoma than in TGCTs,⁶⁶ which might explain the presence of the P53 mutation in the mixed tumor. An additional explanation for the different results between the studies may be a lower number of mature teratomas and a lower number of specimens sampled at relapse in our series. In any case, the majority of resistant cases both in the former and in the present study cannot be explained by inactivating P53 mutations. This is in agreement with our conclusion that the sensitivity to treatment in a significant number of TGCTs cannot be explained by a high level of wild-type P53.

In the absence of mutations, P53 can be inactivated by alternative mechanisms, including overrepresentation of MDM2. A high level of MDM2 has been described in TGCTs in two investigations, with no correlation of the findings to treatment response.^20,49 Both studies showed more positive staining in nonseminomas compared with seminomas. We found high levels of MDM2, specifically in embryonal carcinoma components regardless of the treatment outcome, associated with amplification of the MDM2 gene in only one case. The MDM2 protein was encoded by the full-length mRNA, indicating the absence of the formerly reported alternative splice products.^67,68

In summary, our data show that not all TGCT are characterized by a high level of P53. Therefore, the level of wild-type P53 in TGCTs does not explain their overall sensitivity to cisplatin-based chemotherapy. This does not exclude the possibility that functional P53 is necessary for a favorable response of TGCTs to chemotherapy. Loss of high levels of P53 protein or inactivation of P53 by mutation might contribute to the resistant phenotype in a minority of cases at most. It is the strength of the current investigation that immunohistochemical and molecular studies were performed not only on cell lines and unselected samples but specifically on clinically clearly defined samples from patient cohorts with documented treatment-sensitive and cisplatin-refractory disease.

The following hypothesis might explain the P53 findings so far. First, positive staining for P53 in TGCT cells might reflect a physiologic reaction of these cells to apoptotic stimuli rather than being an indication of an overall high level of wild-type P53 in these tumor cells. This is supported by the spatial relation between positive staining for P53 and the presence of apoptotic bodies in most of the tumors studied (see Fig 1A). Second, exposure to DNA-damaging agents, particularly cisplatin-based chemotherapy, induces apoptosis in the TGCTs by a P53-independent mechanism with a lower threshold than the P53-dependent mechanism. In refractory tumors, inactivation of P53 might become secondarily relevant, resulting in positive selection of tumor cells with inactive P53. Again, inactivation of P53 by MDM2 does not seem to interfere with chemoresponsiveness in these tumors. Thus, future experiments need to seek alternative explanations for the unique chemosensitivity of TGCTs.

	ACKNOWLEDGMENTS

 
Supported by the Dutch Cancer Society (L.H.J.L. and J.W.O.), including KWF-DDHK grant no. 99 1876 (A.-M.F.K., P.C.vanW., and M.M.), and the European Society for Medical Oncology (F.M.).

We thank P. Busch for staining of P53 on a randomly selected series of germ cell tumors, Elisabetta Citterio for providing the replication protein A antibody, and W. Dinjes for providing the P53 SSCP primers.

	NOTES

 
The first two authors contributed equally to the work.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
1. Einhorn LH: Do all germ cell tumor patients with residual masses in multiple sites require postchemotherapy resections? J Clin Oncol 15: 409-410, 1997[Free Full Text]

2. International Germ Cell Consensus Classification: A prognostic factor-based staging system for metastatic germ cell cancers—International Germ Cell Cancer Collaborative Group. J Clin Oncol 15: 594-603, 1997[Abstract/Free Full Text]

3. Gospodarowicz MK, Sturgeon JFG, Jewett MAS: Early stage and advanced seminoma: Role of radiation therapy, surgery, and chemotherapy. Semin Oncol 25: 160-173, 1998[Medline]

4. Bosl GJ, Motzer RJ: Testicular germ-cell cancer. N Engl J Med 337: 242-253, 1997[Free Full Text]

5. Van Gurp RJLM, Oosterhuis JW, Kalscheuer V, et al: Human testicular germ cell tumors show biallelic expression of the H19 and IGF2 gene. J Natl Cancer Inst 86: 1070-1075, 1994[Abstract/Free Full Text]

6. Verkerk AJMH, Ariel I, Dekker MC, et al: Unique expression patterns of H19 in human testicular cancers of different etiology. Oncogene 14: 95-107, 1997[CrossRef][Medline]

7. Looijenga LHJ, Gillis AJM, Van Gurp RJHLM, et al: X inactivation in human testicular tumors: XIST expression and androgen receptor methylation status. Am J Pathol 151: 581-590, 1997[Abstract]

8. Looijenga LHJ, Oosterhuis JW: Pathogenesis of testicular germ cell tumours. Rev Reprod 4: 1-10, 1999[Abstract]

9. Ulbright TM: Germ cell neoplasms of the testis. Am J Surg Pathol 17: 1075-1091, 1993[CrossRef][Medline]

10. Oosterhuis JW: The metastasis of human teratomas, in Damjanov I, Knowles B, Solter D (eds): The Human Teratomas (ed 1). Clifton, NJ, Humana Press, 1983, pp 137-171

11. Oosterhuis JW, Suurmeyer AJH, Sleyfer DT, et al: Effects of multiple-drug chemotherapy (cis-diammine-dichloroplatinum, bleomycin, and vinblastine) on the maturation of retroperitoneal lymph node metastases of nonseminomatous germ cell tumors of the testis. Cancer 51: 408-416, 1983[CrossRef][Medline]

12. Oosterhuis JW, Andrews PW, De Jong B: Mechanisms of therapy-related differentiation in testicular germ cell tumours, in McBrien D (ed): Proceedings of the Fourth Symposium of the International Agency of Cancer Research. Oxford, United Kingdom, IRC Press, 1986, pp 65-90

13. Ulbright TM: Testis risk and prognostic factors: The pathologist’s perspective. Urol Clin North Am 26: 611-626, 1999[CrossRef][Medline]

14. Baltaci S, Orhan D, Turkolmez K, et al: P53, bcl-2 and bax immunoreactivity as predictors of response and outcome after chemotherapy for metastatic germ cell testicular tumours. BJU Int 87: 661-666, 2001[CrossRef][Medline]

15. Heidenreich A, Sesterhenn IA, Mostofi FK, et al: Prognostic risk factors that identify patients with clinical stage I nonseminomatous germ cell tumors at low risk and high risk for metastasis. Cancer 83: 1002-1011, 1998[CrossRef][Medline]

16. Heidenreich A, Schenkman NS, Sesterhenn IA, et al: Immunohistochemical and mutational analysis of the p53 tumour suppressor gene and the bcl-2 oncogene in primary testicular germ cell tumours. APMIS 106:90-99; discussion 99-100, 1998

17. Eid H, Geczi L, Magori A, et al: Drug resistance and sensitivity of germ cell testicular tumors: Evaluation of clinical relevance of MDR1/Pgp, p53, and metallothionein (MT) proteins. Anticancer Res 18: 3059-3064, 1998[Medline]

18. Eid H, Van der Looij M, Institoris E, et al: Is p53 expression, detected by immunohistochemistry, an important parameter of response to treatment in testis cancer? Anticancer Res 17: 2663-2669, 1997[Medline]

19. Chou PM, Barquin N, Guinan P, et al: Differential expression of p53, c-kit, and CD34 in prepubertal and postpubertal testicular germ cell tumors. Cancer 79: 2430-2434, 1997[CrossRef][Medline]

20. Guillou L, Estreicher A, Chaubert P, et al: Germ cell tumors of the testis overexpress wild-type p53. Am J Pathol 149: 1221-1228, 1996[Abstract]

21. Lothe RA, Peltomaki P, Tommerup N, et al: Molecular genetic changes in human male germ cell tumors. Lab Invest 73: 606-614, 1995[Medline]

22. Albers P, Orazi A, Ulbright TM, et al: Prognostic significance of immunohistochemical proliferation markers (Ki-67/MIB-1 and proliferation-associated nuclear antigen), p53 protein accumulation, and neovascularization in clinical stage A nonseminomatous testicular germ cell tumors. Mod Pathol 8: 492-497, 1995[Medline]

23. Riou G, Barrois M, Prost S, et al: The p53 and mdm-2 genes in human testicular germ-cell tumors. Mol Carcinog 12: 124-131, 1995[Medline]

24. Lewis DJ, Sesterhenn IA, McCarthy WF, et al: Immunohistochemical expression of P53 tumor suppressor gene protein in adult germ cell testis tumors: Clinical correlation in stage I disease. J Urol 152: 418-423, 1994[Medline]

25. Ulbright T, Orazi A, DeRiese W, et al: The relationship of p53, PCNA, and S-phase in non-seminomatous germ cell tumors of the testis. Lab Invest 68: 71A, 1993

26. Ulbright TM, Orazi A, de Riese W, et al: The correlation of P53 protein expression with proliferative activity and occult metastases in clinical stage I non-seminomatous germ cell tumors of the testis. Mod Pathol 7: 64-68, 1994[Medline]

27. Bartkova J, Bartek J, Lukas J, et al: p53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia. Int J Cancer 49: 196-202, 1991[Medline]

28. DeRiese WTW, Orazi A, Foster RS, et al: The clinical relevance of p53 expression in early stage nonseminomatous germ cell tumor. J Urol 149: 311A, 1993

29. Heimdal K, Lothe RA, Lystad S, et al: No germline TP53 mutations detected in familial and bilateral testicular cancer. Genes Chromosom Cancer 6: 92-97, 1993[Medline]

30. Przygodzki RM, Moran CA, Suster S, et al: Primary mediastinal and testicular seminomas: A comparison of K-ras-2 gene sequence and p53 immunoperoxidase analysis of 26 cases. Hum Pathol 27: 975-979, 1996[CrossRef][Medline]

31. Bokemeyer C, Kuczyk MA, Serth J, et al: Treatment of clinical stage I testicular cancer and a possible role for new biological prognostic parameters. J Cancer Res Clin Oncol 122: 575-584, 1996[CrossRef][Medline]

32. Kim SK, Cho BK, Paek SH, et al: The detection of p53 gene mutation using a microdissection technique in primary intracranial germ cell tumors. Int J Oncol 18: 111-116, 2001[Medline]

33. Houldsworth J, Xiao H, Murty VV, et al: Human male germ cell tumor resistance to cisplatin is linked to TP53 gene mutation. Oncogene 16: 2345-2349, 1998[CrossRef][Medline]

34. Kuczyk MA, Serth J, Bokemeyer C, et al: Alterations of the p53 tumor suppressor gene in carcinoma in situ of the testis. Cancer 78: 1958-1966, 1996[CrossRef][Medline]

35. Schenkman NS, Sesterhenn IA, Washington L, et al: Increased p53 protein does not correlate to p53 gene mutations in microdissected human testicular germ cell tumors. J Urol 154: 617-621, 1995[CrossRef][Medline]

36. Fleischhacker M, Strohmeyer T, Imai Y, et al: Mutations of the p53 gene are not detectable in human testicular tumors. Mod Pathol 7: 435-439, 1994[Medline]

37. Peng HQ, Hogg D, Malkin D, et al: Mutations of the p53 gene do not occur in testis cancer. Cancer Res 53: 3574-3578, 1993[Abstract/Free Full Text]

38. Serth J, Kuczyk MA, Derendorf L, et al: Identification of frequent p53 gene alterations in testicular cancer by in situ molecular genetic and immunohistochemical analysis. J Urol 151: 408A, 1994

39. Wei YD, Zheng J, Qian SX, et al: p53 gene mutations in Chinese human testicular seminoma. J Urol 150: 884-886, 1993[Medline]

40. Strohmeyer TG, Fleischhacker M, Imai Y, et al: Status of the p53 tumor suppressor gene and the MDM-2 gene in human testicular tumors. J Urol 149: 311A, 1993

41. Burger H, Nooter K, Boersma AW, et al: Distinct p53-independent apoptotic cell death signalling pathways in testicular germ cell tumour cell lines. Int J Cancer 81: 620-628, 1999[CrossRef][Medline]

42. Kubbutat MH, Jones SN, Vousden KH: Regulation of p53 stability by Mdm2. Nature 387: 299-303, 1997[CrossRef][Medline]

43. Haupt Y, Maya R, Kazaz A, et al: Mdm2 promotes the rapid degradation of p53. Nature 387: 296-299, 1997[CrossRef][Medline]

44. Reifenberger G, Liu L, Ichimura K, et al: Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res 53: 2736-2739, 1993[Abstract/Free Full Text]

45. Jiang M, Shao ZM, Wu J, et al: p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. Int J Cancer 74: 529-534, 1997[CrossRef][Medline]

46. Moller MB, Nielsen O, Pedersen NT: Oncoprotein MDM2 overexpression is associated with poor prognosis in distinct non-Hodgkin’s lymphoma entities. Mod Pathol 12: 1010-1016, 1999[Medline]

47. Endo K, Ueda T, Ohta T, et al: Protein expression of MDM2 and its clinicopathological relationships in human hepatocellular carcinoma. Liver 20: 209-215, 2000[CrossRef][Medline]

48. Bartel F, Meye A, Wurl P, et al: Amplification of the MDM2 gene, but not expression of splice variants of MDM2 MRNA, is associated with prognosis in soft tissue sarcoma. Int J Cancer 95: 168-175, 2001[CrossRef][Medline]

49. Eid H, Institoris E, Geczi L, et al: mdm-2 expression in human testicular germ-cell tumors and its clinical value. Anticancer Res 19: 3485-3490, 1999[Medline]

50. Wester K, Wahlund E, Sundstrom C, et al: Paraffin section storage and immunohistochemistry: Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen. Appl Immunohistochem Mol Morphol 8: 61-70, 2000[CrossRef][Medline]

51. Fairman MP, Stillman B: Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J 7: 1211-1218, 1988[Medline]

52. Wood RD, Robins P, Lindahl T: Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell 53: 97-106, 1988[CrossRef][Medline]

53. Looijenga LHJ, Smit VTHBM, Wessels JW, et al: Localization and polymorphism of a chromosome 12-specific alpha satellite DNA sequence. Cytogenet Cell Genet 53: 216-218, 1990[Medline]

54. Looijenga LHJ, Oosterhuis JW, Smit VTHBM, et al: Alpha satellite DNA on chromosomes 10 and 12 are both members of the dimeric suprachromosomal subfamily, but display little identity at the nucleotide sequence level. Genomics 13: 1125-1132, 1992[CrossRef][Medline]

55. Liu P, Siciliano J, Seong D, et al: Dual Alu polymerase chain reaction primers and conditions for isolation of human chromosome painting probes from hybrid cells. Cancer Genet Cytogenet 65: 93-99, 1993[CrossRef][Medline]

56. van Roozendaal CE, Gillis AJ, Klijn JG, et al: Loss of imprinting of IGF2 and not H19 in breast cancer, adjacent normal tissue and derived fibroblast cultures. FEBS Lett 437: 107-111, 1998[CrossRef][Medline]

57. Gibbs RA, Nguyen PN, McBride LJ, et al: Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc Natl Acad Sci U S A 86: 1919-1923, 1989[Abstract/Free Full Text]

58. Carbone D, Chiba I, Mitsudomi T: Polymorphism at codon 213 within the p53 gene. Oncogene 6: 1691-1692, 1991[Medline]

59. Bergström R, Adami H-O, Mohner M, et al: Increase in testicular cancer incidence in six European countries: A birth cohort phenomenon. J Natl Cancer Inst 88: 727-733, 1996[Abstract/Free Full Text]

60. Elledge RM, Lock-Lim S, Allred DC, et al: p53 mutation and tamoxifen resistance in breast cancer. Clin Cancer Res 1: 1203-1208, 1995[Abstract]

61. Lutzker SG, Levine AJ: A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat Med 2: 804-810, 1996[CrossRef][Medline]

62. Van Berlo RJ, Oosterhuis JW, Schrijnemakers E, et al: Yolk-sac carcinoma develops spontaneously as a late occurrence in slow-growing teratoid tumors produced from transplanted 7-day mouse embryos. Int J Cancer 45: 153-155, 1990[Medline]

63. Van Berlo RJ, De Jong B, Oosterhuis JW, et al: Cytogenetic analysis of murine embryo-derived tumors. Cancer Res 50: 3416-3421, 1990[Abstract/Free Full Text]

64. Walt H, Oosterhuis JW, Stevens LC: Experimental testicular germ cell tumorigenesis in mouse strains with and without spontaneous tumours differs from development of germ cell tumours of the adult human testis. Int J Androl 16: 267-271, 1993[Medline]

65. Burger H, Nooter K, Kortland CJ, et al: Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines. Br J Cancer 77: 1562-1567, 1998[Medline]

66. Taylor AC, Shu L, Danks MK, et al: P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines. Med Pediatr Oncol 35: 96-103, 2000[CrossRef][Medline]

67. Sigalas I, Calvert AH, Anderson JJ, et al: Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: Transforming ability and frequent detection in human cancer. Nat Med 2: 912-917, 1996[CrossRef][Medline]

68. Lukas J, Gao DQ, Keshmeshian M, et al: Alternative and aberrant messenger RNA splicing of the mdm2 oncogene in invasive breast cancer. Cancer Res 61: 3212-3219, 2001[Abstract/Free Full Text]

Submitted July 20, 2001; accepted December 3, 2001.

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