Radiation Signatures Figure 6. PB metagene profiles of human radiation exposure and chemotherapy treatment are accurate and specific relative to each other. (A) The heat map on the left depicts the expression profiles of genes (rows) selected to discriminate the human samples (columns); high expression is depicted as red, and low expression is depicted as blue. A leave-one-out cross-validation assay (at right) demonstrated that the PB metagene of radiation was capable of distinguishing healthy donors (black), non-irradiated patients (gray), irradiated patients (red), pre- chemotherapy treatment patients (green), and post-chemotherapy patients (blue). A ROC curve analysis was used to define a cut-off for sensitivity and specificity of the predictive model of radiation. The dotted line represents this threshold of sensitivity and specificity. (B) The heatmap on the left depicts an expression profile of chemotherapy treatment that distinguishes chemotherapy-treated versus untreated patients. A leave-one-out cross- validation assay demonstrated that this PB metagene of chemotherapy treatment could accurately distinguish pre-chemotherapy patients (green), chemotherapy-treated patients (blue), healthy individuals (black), pre-irradiated patients (gray) and irradiated patients (red). doi:10.1371/journal.pone.0001912.g006 This study revealed that sex differences can impact the accuracy of this approach, particularly in distinguishing mice exposed to lower dose irradiation from non-irradiated controls. These results imply that aspects of the PB response to ionizing radiation are specified by sex-associated genes. Whitney et al. [12] previously showed that sex differences were associated with variation in PB autosomal gene expression in healthy individuals. Our study suggests that sex differences may contribute to characteristically distinct PB molecular responses to environmental stress (radiation) and the accuracy of PB gene expression profiling for medical screening can be affected by sex. These sex-related differences in PB response to ionizing radiation are perhaps illustrated by the fact that only 2 genes overlapped between the male and female PB signatures of 50 cGy (Ccng1 and Dda3). Interestingly, differences in genotype did not significantly impact the accuracy of the PB gene expression signatures to distinguish radiation response such that PB signatures from C57Bl6 mice displayed 100% accuracy in predicting the status of BALB/c mice and vice versa. This observation demonstrates that, while genotype differences can account for some variation in PB gene expression [12], the alterations in PB gene expression induced by 3 different levels of radiation exposure are such that PB expression profiling is highly accurate in distinguishing all irradiated mice across different genotypes. Very few genes were found in common between the 2 strains of mice at each level of radiation exposure, indicating that diverse sets of genes contribute to the PB response to radiation and that unique sets of genes can be identified which are predictive of radiation response. The time of PB collection following radiation exposure had no significant impact on the accuracy of PB signatures to predict radiation status or distinguish different levels of exposure. First, the accuracy of PB signatures to predict radiation status and distinguish different levels of radiation exposure did not decay over time. Second, when we applied a PB signature from a single time point (6 hrs) against PB samples collected from mice at other time points (24 hr and 7 days), the accuracy of the prediction remained 100% in all cases. Therefore, time as a single variable did not lessen the accuracy of this approach to distinguish irradiated from non-irradiated animals. However, the content of the genes which comprised the PB signatures changed significantly as a function of time and ,20% of the genes overlapped between the PB signatures of radiation at 6 hr, 24 hr, and 7 days. Taken together, these data indicate that PB predictors of radiation response do change over time, but PB signatures can continuously PLoS ONE | www.plosone.org 7 April 2008 | Volume 3 | Issue 4 | e1912