In this competing renewal application, the applicant will expand on his observations which show mutations in TP53, widely variable p53 protein expression, bcl-2 protein expression, and defective apoptosis. All of these suggest that drug resistance in germ cell tumors (GCTs) is partly related to defects in the cell death pathway. GCTs display unusual sensitivity to chemotherapy. Only 20% to 30% of tumors are resistant to cisplatin-based chemotherapy. GCTs also display somatic differentiation (teratoma, usually histologically benign). Thus, teratomas are also """"""""drug resistant"""""""", but the presence of teratoma after chemotherapy rarely leads to a patient's death. Therefore, two forms of drug resistance are present. Thus, the applicant will study the genetic events associated with drug resistance in drug-sensitive (patient cured) undifferentiated GCTs, drug-resistant (patient relapsed or dead of disease) undifferentiated GCTs, and drug-resistant differentiated GCTs (teratomas).
In Specific Aim 1, the frequency of TP53 mutations in GCTs will be estimated. His preliminary data show that TP53 mutations exist in some drug-resistant GCTs.
In Specific Aim 2, the apoptotic response and the expression of p53-dependent apoptosis or cell cycle regulatory proteins (e.g., bax, bcl-2, p21) will be assessed. His preliminary data in two GCT cell lines (one drug-sensitive (TP53 wild-type) and one drug-resistant (TP53 mutant)) show failure to upregulate p21, no change in apoptotic rate in the TP53 mutant line compared to the wild-type when exposed to cisplatin, and marked differences in p53, bax, and bcl-2 protein expression in sensitive and resistant GCTs.
In Specific Aim 3, comparative genomic hybridization (CGH) will be used to evaluate GCTs for regions of genomic excess or loss limited to drug-resistant tumors. His preliminary data have identified six amplified regions on five different chromosome arms. In each of these specific aims, he will further determine whether an association exists in univariate analysis between genetic events (markers) and clinical endpoints (e.g., cured vs. dead), initially using the odds-ratio as the measure of association, followed by multivariable analysis to establish better methods of treatment planning. On each tumor, SSCP and sequencing will be performed for TP53 mutations; immunohistochemistry performed for bax, bcl-2, and p21 expression and TUNEL for apoptotic rate; and CGH performed seeking regions of genomic imbalance. From these data, the applicant will begin to dissect the resistance pathway(s), possibly identify new genes associated with GCT drug resistance, and determine whether they have independent clinical predictor value.
