In vivo differentiation and genomic evolution in adult male germ cell tumors.

Germ cell tumors (GCTs) are the most common solid malignancy in young adult men, but the genes and genomic regions involved in their etiology are not fully defined. We report here an investigation of DNA copy number changes in GCTs using 1 Mb BAC arrays. As expected, 12p gain was the defining genomic alteration, occurring in 72/74 GCTs. Parallel expression profiling of these tumors identified potential oncogenes from gained regions (LYN and RAB25) and potential tumor suppressor genes in regions of loss (SYNPO2, TTC12, IGSF4, and EPB41L3). Notably, we observed specific genomic alterations associated with histology, including gain of 17p11.2-q21.32 and loss of 2p25.3 in embryonal carcinoma, gain of 8p23.3-12 and loss of 5p15.33-35.3, 11q23.1-25, and 13q12.11-34 in seminoma, and gain of 1q31.3-42.3, 3p, 14q11.2-32.33, and 20q and loss of 8q11.1-23.1 in yolk sac tumors (YST). Many significant genes that mapped to these regions had previously been associated with specific histologies, such as EOMES (chr3) and BMP2 (chr20) in YST and SPRY2 (chr13) and SOX17 (chr8) in seminomas. Additionally, our results suggest a model in which histologic differentiation of GCTs may drive genomic evolution.

Predicting outcome to chemotherapy in patients with germ cell tumors: the value of the rate of decline of human chorionic gonadotrophin and alpha-fetoprotein during therapy.

PURPOSE: The prognostic significance of the rate of decline of the serum tumor marker alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) during the first two cycles of chemotherapy in germ cell tumor (GCT) patients was initially reported by us, but its value has been debated. We re-examined this issue in the context of the International Germ Cell Cancer Collaborative Group (IGCCCG) risk classification system and investigated the role of including in the analysis patients whose markers normalized early. PATIENTS AND METHODS: One hundred eighty-nine GCT patients with elevated AFP/HCG marker values treated with platinum-based chemotherapy between 1986 and 1998 were included in this analysis. Patients were classified as good, intermediate, or poor risk by the IGCCCG criteria and as having satisfactory or unsatisfactory marker decline. Risk and marker decline were correlated with response, event-free survival, and overall survival. RESULTS: Satisfactory marker decline predicted improved complete response (CR) proportion and event-free and overall survival (P <.0001). The CR proportion, 2-year event-free, and 2-year overall survival rates for patients with a satisfactory and unsatisfactory marker decline were 92% versus 62%, 91% versus 69%, and 95% versus 72%, respectively. Marker decline remained a significant variable for all three end points when adjusted for risk (P <.01) with the outcome differences most pronounced in the poor-risk group. CONCLUSION: The rate of marker decline during chemotherapy has prognostic value independent of risk and may play a significant role in the management of poor-risk patients. It is appropriate to include patients whose markers normalized early.

Low-volume nodal metastases detected at retroperitoneal lymphadenectomy for testicular cancer: pattern and prognostic factors for relapse.

PURPOSE: To determine the incidence, pattern, and predictive factors for relapse in patients with low-volume nodal metastases (stage pN1) at retroperitoneal lymphadenectomy (RPLND) and identify who may benefit from chemotherapy in the adjuvant or primary setting. PATIENTS AND METHODS: Fifty-four patients with testicular nonseminomatous germ cell tumor had low-volume retroperitoneal metastases (pathologic stage pN1, 1997 tumor-node-metastasis classification) resected at RPLND, 50 of whom were managed expectantly without adjuvant chemotherapy. The dissection was bilateral in 12 and was a modified template in 38 patients. Retroperitoneal metastases were limited to microscopic nodal involvement in 14 patients. Follow-up ranged from 1 to 106 months (median, 31.4 months). RESULTS: Eleven patients (22%) suffered a relapse at a median follow-up of 1.8 months (range, 0.6 to 28 months). The most frequent form of recurrence was marker elevation in nine (18%) patients. Persistent marker elevation after orchiectomy and before retroperitoneal lymphadenectomy was a significant independent predictor of relapse (relative risk, 8.0; 95% confidence interval, 2.3 to 27.8; P =.001). Four of five (80%) patients with elevated markers (alpha-fetoprotein alone in three, alpha-fetoprotein and beta human chorionic gonadotropin in one) suffered a relapse, compared with seven of 45 (15.6%) patients with normal markers. CONCLUSION: Clinical stage I and IIA patients with normal markers who have low-volume nodal metastases have a low incidence of relapse and can be managed by observation only if compliance can be assured. In contrast, patients with elevated markers before retroperitoneal lymphadenectomy have a high rate of relapse and should be considered for primary chemotherapy.

ID gene expression varies with lineage during differentiation of pluripotential male germ cell tumor cell lines.

Human male germ cell tumors (GCTs) comprise an excellent model system for understanding the molecular events controlling cellular differentiation and lineage decision. Pluripotential embryonal carcinoma cell lines derived from GCTs can be induced to undergo terminal differentiation along specific lineages dependent upon the differentiating agent. We report here that one such cell line, NTera2/clone D1 (NT2/D1), previously shown to undergo differentiation along a neuronal lineage by all-trans-retinoic acid (RA), can be induced along a distinct non-neuronal lineage by the mammalian morphogens, bone morphogenetic proteins-2 and -4 (BMP-2 and -4). Very little is known regarding the molecular events that govern such human lineage decisions. In this study, the role of the ID (inhibitor of differentiation and DNA-binding) family of genes that act as inhibitors of the function of helix-loop-helix (HLH) transcriptional activators involved in lineage commitment was investigated using two pluripotential GCT cell lines as a model system. In the differetiation programs studied, Id1 was noted to decline, an event often associated with the decrease in proliferative rate occurring during differentiation. However, differences in the expression of ID2 and ID3 family members were detected between the programs. Notably, an increase in Id3 during RA-induced differentiation of NT2/D1 cells was observed, while Id2 levels increased during BMP-2 and -4 treatment of NT2/D1 cells and during the induction of an endodermal-like differentiation program in the cell line, 27X-1. The pluripotential male GCT cell lines comprise a unique system in which the roles of specific genes such as the ID family of genes in human cell differentiation and lineage decision can be studied.

Role of postchemotherapy adjunctive surgery in the management of patients with nonseminoma arising from the mediastinum.

PURPOSE: To evaluate the role of postchemotherapy surgery in patients with nonseminomatous germ cell tumors arising from the anterior mediastinum. PATIENTS AND METHODS: Thirty-two patients with nonseminoma arising from a mediastinal primary site were treated on a clinical trial at our center, and they underwent postchemotherapy surgery. The results of postchemotherapy surgical resection, frequency of viable tumor found during postchemotherapy surgery, and prognostic factors for survival were assessed. RESULTS: Complete resection of all gross residual disease was achieved in 27 patients (84%). Histologic analysis of resected residua postchemotherapy revealed viable tumor in 66%, teratoma in 22%, and necrosis in 12% of the specimens. Viable tumor included embryonal carcinoma, choriocarcinoma, yolk sac carcinoma, seminoma, and teratoma with malignant transformation to nongerm cell histology (eg, sarcoma). Clinical characteristics associated with a shorter survival after surgery included the presence of viable tumor in a resected specimen (P =.003) and more than one site resected during surgery (P =.06). There were no statistically significant differences in survival for patients who underwent surgical resection with normal markers compared with patients with elevated serum tumor markers (P =.33). A trend toward shorter survival was found in patients with increasing tumor markers before surgery compared with patients with normal and declining serum tumor markers (P =.09). CONCLUSION: Surgical resection of residual mass after chemotherapy plays an integral role in the management of patients with primary mediastinal nonseminoma. Teratoma and viable tumor were found in the majority of resected residua after chemotherapy. Because patients who undergo conventional salvage chemotherapy programs rarely achieve long-term disease-free status, selected patients with elevated markers after chemotherapy are considered candidates for surgical resection.

Expression of ID genes in differentiated elements of human male germ cell tumors.

The ID genes are members of a family of genes that encode helix-loop-helix (HLH)-containing proteins. The Id proteins, unlike other HLH proteins, lack an adjacent DNA binding domain and hence act as dominant negative regulators of HLH transcription factors that have been implicated in control of cellular differentiation. Although the role of Id genes in murine development has been documented, their roles in human embryogenesis remain unknown. In this study, human male germ cell tumors (GCTs) were used as a model for examining the expression of the ID genes in various histologies that are reflective of different temporal phases of human development. In seminomas, little or no expression of IDI, ID2, and ID3 was detected, consistent with the uncommitted germ cell-like phenotype of this tumor histology. Likewise, GCTs with histologies reflective of extraembryonic and embryonic patterns of differentiation exhibited patterns of expression of the three ID genes often similar to those noted during murine development. It was also evident, as revealed by ID expression patterns, that despite the overall aberrant spatial differentiation patterns displayed by these tumors, some tissue-tissue interactions reminiscent of those observed during normal embryogenesis are retained. Thus, adult male GCTs offer a unique system in which the role of genes such as the IDs can be studied in human embryogenesis.

Phase II trial of pyrazoloacridine in patients with cisplatin-refractory germ cell tumors.

Thirteen patients with cisplatin-refractory germ cell tumors were treated on a Phase II trial with pyrazoloacridine. Pyrazoloacridine was given intravenously at 600 mg/m2 every three weeks. The median nadir leucocyte count was 2.5 cells/mm3, hemoglobin was 10.8 g/dl, and platelet count was 126,000 cells/m3. None of the thirteen evaluable patients achieved a major response. Pyrazoloacridine is not efficacious in the treatment of cisplatin-refractory germ cell tumors.

Paclitaxel, ifosfamide, and cisplatin second-line therapy for patients with relapsed testicular germ cell cancer.

PURPOSE: To evaluate the dose, toxicity, and efficacy of paclitaxel in combination with ifosfamide and cisplatin as salvage therapy for patients with relapsed testicular germ cell tumors (GCTs). PATIENTS AND METHODS: Thirty patients with previously treated GCTs were treated with paclitaxel and ifosfamide plus cisplatin (TIP) as second-line therapy. All had favorable prognostic features for response (testis primary tumor site and prior complete response to first-line chemotherapy program). Four cycles of paclitaxel, ifosfamide 5 g/m(2), and cisplatin 100 mg/m(2) were given 21 days apart with granulocyte colony-stimulating factor support, followed by resection of radiographic residua. The dose of paclitaxel was increased among cohorts with dose levels of 175, 215, and 250 mg/m(2); the largest dose was selected for the phase II part of the trial. RESULTS: Twenty-three (77%) of 30 patients achieved a complete response to chemotherapy alone, and one patient achieved a durable partial response with normal tumor markers. Therefore, 24 (80%) achieved a favorable response. Eleven patients with normalized markers after chemotherapy underwent resection of residual tissue, with only necrosis found in 10 and mature teratoma in one. Two patients relapsed, and 22 (73%) of the favorable responses remain durable at a median follow-up duration of 33 months. Myelosuppression was the major toxicity, and two patients had grade 3 neurotoxicity. CONCLUSION: Four cycles of TIP was associated with a high proportion of patients who achieved a complete response, a lack of relapse, and relative tolerability as an ifosfamide-containing salvage regimen for testicular GCTs. The high durable complete response proportion emphasizes the importance of patient selection according to prognostic factors for a favorable outcome to conventional-dose salvage therapy.

Sequential dose-intensive paclitaxel, ifosfamide, carboplatin, and etoposide salvage therapy for germ cell tumor patients.

PURPOSE: To evaluate the efficacy and toxicity of sequential, dose-intensified chemotherapy with paclitaxel/ifosfamide and carboplatin/etoposide administered plus peripheral blood-derived stem-cell (PBSC) support for patients with germ cell tumors (GCT) who have unfavorable prognostic features in response to conventional-dose salvage programs. Carboplatin was dose escalated by target area under the curve (AUC; in [milligrams per milliliter] x minutes) among patient cohorts, and pharmacokinetic studies were performed for comparison. PATIENTS AND METHODS: Thirty-seven previously treated patients who had cisplatin-resistant GCT and unfavorable prognostic features for response to conventional-dose salvage therapy were treated. Two cycles of paclitaxel 200 mg/m(2) plus ifosfamide 6 g/m(2) were given 2 weeks apart with leukapheresis, followed by three cycles of carboplatin plus etoposide given 14 to 21 days apart with reinfusion of PBSCs. The dose of etoposide was 1, 200 mg/m(2), and the carboplatin target AUC ranged among cohorts from 12 to 32 (mg/mL) x min. Pharmacokinetic studies of carboplatin were performed for comparison of target to measured AUC. RESULTS: Twenty-one patients (57%) achieved a complete response and an additional two patients (5%) achieved a partial response with normal tumor markers; therefore, 23 (62%) achieved a favorable response. Eight patients relapsed, and 15 (41%) of the favorable responses remained durable at a median follow-up of 30 months. Myelosuppression was the major toxicity; 58% of carboplatin/etoposide cycles were associated with hospitalization for nadir fever. The AUC of carboplatin measured in serum was lower than the target AUC; this may be related to underestimation of the glomerular filtration rate used in the dosing formula. CONCLUSION: Dose-intense therapy with sequential, accelerated chemotherapy of paclitaxel/ifosfamide and carboplatin/etoposide administered with PBSC support was relatively well tolerated. The durable complete response proportion was substantial in patients with unfavorable prognostic features for achieving durable complete response to conventional-dose salvage programs. Optimal dosing of carboplatin in the high-dose setting warrants further investigation.

Recognizing abnormal marker results that do not reflect disease in patients with germ cell tumors.

PURPOSE: The judicious use of serum alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG) and lactate dehydrogenase is key to appropriate management of patients with germ cell tumors. Elevated AFP and/or HCG generally indicate active disease. We describe patients with germ cell tumors who had elevated serum AFP and/or HCG but no active disease, despite careful repeat evaluation. MATERIALS AND METHODS: Histories of 6 cases of germ cell tumors that remained in remission despite abnormal serum AFP and/or HCG were reviewed. RESULTS: Markers were only modestly elevated, remained constant or spontaneously normalized during repeat measurements, and there was no other clinical or radiographic evidence of disease. Patients were treated conservatively with physical examination, radiological tests and repeat marker assays, with no relapse to date. CONCLUSIONS: Stable, low increases in serum AFP and HCG may not represent active disease. Careful repeat evaluation will determine whether the markers increase. If no change is noted after appropriate studies have been reviewed by an experienced practitioner to exclude active disease from diagnosis, then consideration should be given to managing such cases with close surveillance to avoid unnecessary chemotherapy.

High-dose chemotherapy as primary treatment for poor-risk germ-cell tumors: the Memorial Sloan-Kettering experience (1988-1999).

Although the majority of patients with poor-risk germ-cell tumors (GCTs) will achieve a durable complete remission (CR) with standard first-line therapy, 20% to 30% of them will either relapse or fail to achieve an initial CR and eventually die. For this reason, the strategy of using high-dose (HD) chemotherapy with autologous stem-cell support has been investigated to improve the chances of cure attainable in the salvage setting, but at a cost of significant morbidity and mortality. Treatment using HD therapy in the first-line setting offers the promise of reducing morbidity and mortality while increasing efficacy. At Memorial Sloan-Kettering Cancer Center (MSKCC), trials were conducted to test this hypothesis. Patients at high risk of relapse following conventional therapy were identified, based on post-treatment serum marker concentrations that failed to appropriately decline by predicted half-life after several cycles of standard treatment. These patients received first-line HD treatment. Patients received a 2-drug HD regimen in one trial and an intensified 3-drug regimen in another, each with autologous bone marrow transplantation. These patients had improved overall and event-free survival rates (p = 0.001 and 0.003, respectively) compared with historical controls who underwent standard first-line treatment, with a lower incidence of treatment-related mortality than patients who received HD therapy in the salvage setting. Randomized trials are under way to prospectively verify these results.

Germ cell tumor clinical trials in North America.

The management of germ cell tumors (GCT) in men has been the focus of intense clinical investigation over the past 25 years. Following clinical trials which demonstrated that GCTs are curable, considerable effort has been directed at refinement of the multidisciplinary treatment plan. During the past decade, there has been a major emphasis on identifying those clinical prognostic features most closely associated with a high likelihood of a complete response (good risk) and a low likelihood of complete response (poor risk). Randomized good- and poor-risk clinical trials have been conducted which clarify the role of bleomycin and the number of cycles of therapy in good-risk patients and the role of high-dose cisplatin (200 mg/m(2)/cycle) and ifosfamide in poor-risk patients. The efficacy of high-dose chemotherapy with stem cell rescue has led to a randomized trial of its use following standard induction therapy as compared to standard induction therapy in previously untreated poor-risk patients. Salvage chemotherapy for patients in first or second relapse, or those who have not achieved an initial complete response, initially tested the role of ifosfamide; efforts are now focused on the role of paclitaxel and high-dose therapy in specific patient subgroups. High-dose chemotherapy with stem cell rescue is the treatment of choice for patients in second relapse with gonadal primary tumors who have responsive disease and factors which predict a reasonable likelihood of achieving a disease-free status. A general understanding of tumor biology and specific knowledge regarding GCTs will be very important in the next decade of discovery as the pathways of malignant transformation, progression, and drug resistance become better known and serve as targets for new therapy.

Telomerase activity in germ cell cancers and mature teratomas.

BACKGROUND: An inverse relationship has been reported between the presence of telomerase enzymatic activity and the induction of differentiation in human tumor cell lines. Male germ cell tumors represent an attractive clinical model to assess this relationship further because high telomerase activity is present in normal germ cell progenitors and in embryonal carcinomas that can differentiate into mature teratomas. To investigate how telomerase activity and the differentiation state of germ cell tumors are related, telomerase activities and telomere lengths were measured in benign testicular tissues, germ cell cancers, and mature or immature teratomas. METHODS: By use of a modified telomeric repeat amplification protocol (TRAP) assay, telomerase activity was measured in four specimens of benign testicular tissue, in 27 germ cell cancers, in seven mature teratomas, and in one immature teratoma. Telomere lengths were measured in all specimens by restriction digestion of genomic DNA and Southern blot hybridization analysis. Associations between telomerase activity and tissue histopathology were assessed with two-sided Fisher's exact tests. RESULTS: Telomerase activity was detected in all examined germ cell cancers and in the benign testicular tissue specimens. In marked contrast, telomerase activity was not detected in any mature teratoma (P<.0001). Very long telomeres were detected in some mature teratomas, consistent with telomerase repression as a late event in teratoma formation. The immature teratoma, with malignant transformation, had high telomerase activity. CONCLUSION: Telomerase is active in germ cell cancers and repressed in mature teratomas. The absence of telomerase activity may contribute to the limited proliferative capacity of mature teratomas. These findings support the existence of an inverse relationship between telomerase activity and the differentiation state of clinical germ cell tumors.

A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors.

Cytogenetic and molecular genetic analyses have shown that the 12q22 region is recurrently deleted in male germ cell tumors (GCTs), suggesting that this site may harbor a tumor suppressor gene (TSG). Previous loss of heterozygosity (LOH) analyses identified a consensus minimal deleted region between the markers D12S377 and D12S296, and a YAC clone contig covering the region was generated. Here, we describe a high-resolution sequence-ready physical map of this contig covering a 3-Mb region. The map comprised of 52 cosmids, 49 PACs, and 168 BACs that were anchored to the previous YAC contig; 99 polymorphic, nonpolymorphic, EST, and gene-based markers are now placed on this map in a unique order. Of these, 61 markers were isolated in the present study, including one that was polymorphic. In addition, we have narrowed the minimal deletion to approximately 830 kb between D12S1716 (proximal) and P382A8-AG (distal) by LOH analysis of 108 normal-tumor DNAs from GCT patients using 21 polymorphic STSs. These physical and deletion maps should prove useful for identification of the candidate TSG in GCTs, provide framework to generate complete DNA sequence, and ultimately generate a gene map of this segment of the chromosome 12. [The sequence data described in this paper have been submitted to the Genome Survey Sequence under accession nos. AQ254896-AQ254955 and AQ269251-AQ269266. Online supplementary material is available at http://www.genome.org]

Chromosomal amplification is associated with cisplatin resistance of human male germ cell tumors.

Chemotherapy resistance of tumors is an important biological and clinical problem. Studies from many tumor types have indicated that resistance may be based on multiple genetic pathways. Human male germa cell tumors (GCTs) are an especially good model system to study the genetic basis of tumor sensitivity and resistance to chemotherapy. GCTs are exquisitely sensitive to treatment with DNA-damaging drugs such as cisplatin, rarely exhibit TP53 gene mutations, express normal p53 protein, and undergo p53-mediated apoptosis upon drug treatment. A small proportion of tumors (20-30% of metastatic lesions) escape the apoptotic response and result in treatment resistance. We have recently shown (J. Houldsworth, et al., Oncogene, 16: 2345-2359, 1998) that in a subset of such tumors, resistance is linked to TP53 gene mutations. In a further search for genetic mechanisms underlying resistance, we subjected a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemotherapy-resistant tumors to comparative genomic hybridization analysis to identify possible amplified regions (implying amplified/overexpressed genes) associated with resistance. With the exception of 12p11.2-12, high level amplification was not detected in any of the sensitive tumors. We have identified eight amplified regions (1q31-32, 2p23-24, 7q21, 7q31, 9q22, 9q32-34, 15q23-24, and 20q11.2-12) in five resistant tumors, which suggests that chromosomal and, hence, gene amplification may comprise a pathway to drug resistance. Identification of amplified/overexpressed genes at these sites may elucidate new genetic pathways of chemotherapy resistance in GCTs and possibly also in other tumors.

FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas.

A subset of male germ cell cancers presenting with advanced stage abundantly express the fibroblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undifferentiated embryonal carcinomas (ECs). During induced differentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or differentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or differentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated differentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RA-treatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably differ between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were significantly more likely to express FGF4 mRNA (P < or = 0.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the antitumorigenic actions of RA, and is selectively expressed in specific histopathologic subsets of germ cell tumors. Taken together, these findings indicate how differentiation and anti-tumorigenic retinoic acid signals can be dissociated in germ cell cancer.

Human male germ cell tumor resistance to cisplatin is linked to TP53 gene mutation.

Male germ cell tumors (GCTs) are uniquely sensitive to cisplatin-based chemotherapy, with more than 90% of newly diagnosed cases cured. The underlying cause for resistance to treatment in 20-30% of metastatic lesions remains to be identified. Unlike other solid tumors, no mutations in the TP53 gene have been identified to date in random panels of GCT specimens, which could account for the exquisite sensitivity of these tumors to genotoxic insult. However, in a panel of resistant GCTs that did either not respond to cisplatin-based chemotherapy or subsequently relapsed and resulted in the death of the patient, we have now identified a subset of tumors to contain TP53 mutations within exons 6-9. A cell line derived from one of these tumors (228A) displayed the same TP53 mutation as the tumor specimen, expressed only mutant TP53 mRNA, and exhibited a relative resistance to cisplatin in vitro in comparison to a cell line (218A) derived from a responsive tumor with wild-type TP53. The resistant cell line displayed a much reduced apoptotic cell death and did not exhibit an induction of transcription of the p53-responsive genes WAF1 and MDM2 following cisplatin treatment, compared to that observed in the sensitive cell line. The levels of bax, an agonist of apoptosis, were found to be reduced in the resistant cell line. The simplest explanation for the resistance of this subset of GCTs that are resistant to cisplatin-based chemotherapy, is the inability of the cells to mount an apoptotic response following exposure due to a functionally inactivating mutation in the TP53 gene.