Acquired Loss of Function of CYP2C19 in Cancer Patients: Studies to Determine the Prevalence and the Possible Molecular Mechanisms of This Genotype- Phenotype Discordance

Abstract

Variability in the way patients metabolise antineoplastic drugs can have substantial impact of the effectiveness and safety of these agents. Approximately 3% of Caucasians are homozygous for null function alleles in the gene encoding the drug metabolising enzyme CYP2C19 and are consequently poor metabolisers of certain drugs, including several chemotherapeutic agents. Two previous studies have reported that up to 37% of cancer patients have a poor metaboliser status but are not homozygous for null variants of the gene. The aim of this thesis was to define the prevalence of this genotype-phenotype discordance, and to investigate possible molecular mechanisms involved in the regulation of CYP2C19. An initial clinical study was undertaken in patients with multiple myeloma (n = 25) in which 28% of the subjects were found to be poor metabolisers of the CYP2C19 probe drug proguanil, a significantly (p < 0.0001) higher prevalence than that predicted from CYP2C19 genotype. This was the first report of compromised CYP2C19 activity in patients with haematological malignancy. A second clinical study to investigate the role of disease burden in discordant CYP2C19 activity was then undertaken in two cohorts of patients with gastro-intestinal cancer: A) resected patients with no currently evaluable disease (n = 25) and B) patients with stage III/IV disease (n = 25). CYP2C19 activity was probed on three separate occasions in each patient. At test 1 the proportion of discordant patients was 17% in the no evaluable disease cohort and 20% in the stage III/IV disease cohort. Dynamic and reversible changes in phenotype were observed in 15 individuals. Studies to elucidate factors which regulate transcription of the CYP2C19 gene were also undertaken. Culture of human cells (HCT116 and HepG2) with the DNA methyltransferase inhibitor 5azaDC increased CYP2C19 transcription ≥18-fold. This acquired change in transcription was not directly due to changes in the methylation status CpG islands identified in CYP2C19. However the treatment also increased mRNA expression (p < 0.05) for a number of transcription factor genes known to regulate CYP2C19 mRNA expression: ESR1, GATA4, NR1I2, and NR1I3. The relationship between CYP2C19 and these transcription factors was then examined in human liver tissue ex vivo (n = 31). Strong correlations (RS > 0.70, p < 0.001) were observed between CYP2C19 gene expression and ESR1, NR1I3, NR3C1 mRNA. Differential expression (p < 0.002) was observed in 31 circulating microRNAs between discordant (n = 5) and concordant (n = 6) patients in a microarray analysis. Future studies to assess the role of post-transcriptional regulation of CYP2C19 may help to identify the factors which lead to an acquired loss of function in some cancer patients. The phenomenon of acquired loss of CYP2C19 activity appears to be relatively common in cancer patients. Further work is required to understand the mechanism(s) that result in downregulation of this drug metabolising enzyme. However, measurement of CYP2C19 activity rather than CYP2C19 genotype may have more clinical relevance for determination of adverse chemotherapeutic outcomes.