Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms.

Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone-induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration-dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration-dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT-mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT-mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress.