T. D. Murtha1, T. C. Brown1, F. Haglund2, C. C. Juhlin2, C. Larsson2, J. C. Rubinstein1, R. Korah1, T. J. Carling1 1Yale University School Of Medicine,Deparment Of Surgery,New Haven, CT, USA 2Karolinska Institutet,Department Of Oncology-Pathology,Stockholm, , Sweden
Introduction: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy that portends a poor prognosis. Adjuvant therapies, including mitotane, have demonstrated efficacy, though frequently fail due to drug resistance. Complete R0 resection offers the only hope for a durable cure. In other cancers, cytochrome P450 (CYP) mediated metabolism of anti-cancer agents has been shown to contribute to chemotherapy resistance. Recent comprehensive genetic analyses of ACCs by our group and others have shown recurrent arm-level copy number gains of chromosome 19 that carry a group of CYP genes. The role of these genes in adrenocortical carcinogenesis has yet to be clarified. Demonstrated functions of CYP enzymes in xenobiotic metabolism and biotransformation of carcinogenic compounds prompted us to investigate the potential influence of Chr 19q13 CYP genes in adrenocortical malignancy.
Methods: Using TaqMan real-time qPCR techniques, we investigated the expression patterns of seven CYP genes (CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2F1, CYP2S1, & CYP4F2) affected by predicted chromosome 19 copy gains in histologically confirmed ACC (n = 29) and normal adrenal cortex (n = 11). We used TaqMan copy number assays to verify the gene copy numbers in a subset of samples to assess the contribution of gene copy gains in promoting gene expression. Correlations between gene expression patterns and clinical parameters including patient age, gender, tumor size, pathological stage, metastatic status, and hormonal profile were also evaluated. Continuous variables and variables with > 2 dependent values were analyzed with the Mann-Whitney U and Kruskal-Wallis tests, respectively. The Spearman correlation assessed matched continuous variables while categorical variables were analyzed with the Fisher’s exact test.
Results: Two chromosome 19q13 CYP genes (CYP2A6 & CYP2A7) showed increased overall expression levels compared to normal adrenal tissue (p < .05). Increased expression of CYP2A6 and CYP2A7 was found in 98% of samples with average expression levels 25 (0.7-115.7) and 38 (1.8-368.1) fold greater than normal adrenal tissue. The remaining five CYP genes evaluated (CYP2A13, CYP2B6, CYP2F1, CYP2S1, & CYP4F2) had unaltered or decreased expression levels compared to normal adrenal cortex. Copy number analysis of selected ACC samples showed copy gains of CYP2A6 in only 27% of specimens and no copy gains in CYP2A7, suggesting alternative mechanisms underlying CYP overexpression. Analysis of patient and tumor characteristics revealed a statistically significant relationship between >10 fold overexpression of CYP2A6 and non-metastatic status (p < .05).
Conclusion: Global increase in expression of CYP2A6 and CYP2A7, possibly independent of gene copy gains, was observed in 98% of ACC samples tested. Whether the selective amplification of these CYP2 enzymes plays a role in the metastatic spread and/or chemoresistance of ACC warrants further investigation.