Cytochrome P450 CYP3A4 /5: A new prognostic factor for osteosarcoma.

Abstract

Osteosarcoma is a primary malignancy of bone with a tendeney for early pulmonary metastasis. There is a need to identify patients who may benefit from more aggressive pre-operative therapy or perhaps require less aggressive chemotherapy. A reliable biomarker predicting clinical outcome at initiation of treatment has not yet been identified. We hypothesized that one or more drug-metabolizing enzymes, mainly cytochromes P450 are related to drug resistance and/or poor clinical outcome in osteosarcoma. To test the hypothesis, tissue microarray blocks containing 18 osteogenic and 109 soft tissue sarcoma primary tumors were used to identify the expression of P450s 1A1/2, 1B1, 2B6, 2D6, and 3A4/5 as well as metallothionein, MDR1, UGT2B7 and mdm-2 by enzyme-linked avidin-biotin-complexed immunocytochemistry. CYP1A1/2, CYP1B1, CYP3A4/5, MT, UGT2B7, MDR-1 and mdm-2 were detected in 59%, 64%, 76%, 87%, 63%, 90% and 94% of soft tissue tumors, and in 83%, 67%, 83%, 72%, 78%, 72% and 11% of osteosarcomas, respectively. CYP2B6 and CYP2D6 immunoreactivities were seen in 3% and 20% of soft tissue tumors, respectively, and were not detected in osteosarcomas. In addition, osteosarcomas were heterogenous with respect to the expression CYP3A4/5, an enzyme involved in the metabolic activation and detoxification of ifosfamide, etoposide, and doxorubicin, which are used in the treatment of osteosarcoma. This observation was extended by developing a quantitative immunofluorescence technique to assess the levels of CYP3A4/5 in 18 archival primary osteosarcoma sections. Expression of CYP3A4/5 was found to be significantly higher in primary biopsies of patients that developed distant metastatic disease compared to biopsies from metastatic disease-free patients (p ≤ 0.0004). Results from clonogenic assays using two human osteosarcoma cell lines showed that cells expressing CYP3A4 are more resistant to etoposide compared to non-expressing cells. No difference in resistance was evident among the cell lines when exposed to ifosfamide or doxorubicin or etoposide in the presence of ketoconazole, a potent inhibitor of CYP3A4. This thesis provides evidence that selective P450 isoenzymes are expressed in sarcomas. Moreover, increased levels of CYP3A4/5 expression appears to predict poor clinical outcome in osteosarcomas, and may confer protection through mechanisms of resistance other than regular detoxification pathways.Ph.D.Health and Environmental SciencesToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131733/2/3057937.pd