Development of a biodosimeter for radiation triage using novel blood protein biomarker panels in humans and non-human primates.

Purpose: In a significant nuclear event, hundreds of thousands of individuals will require rapid triage for absorbed radiation to ensure effective medical treatment and efficient use of medical resources. We are developing a rapid screening method to assess whether an individual received an absorbed dose of ≥2 Gy based on the analysis of a specific panel of blood proteins in a fingerstick blood sample.Materials and methods: We studied a data set of 1051 human blood samples obtained from radiotherapy patients, normal healthy individuals, and several special population groups. We compared the findings in humans with those from irradiation studies in non-human primates (NHPs).Results: We identified a panel of three protein biomarkers, salivary alpha amylase (AMY1), Flt3 ligand (FLT3L), and monocyte chemotactic protein 1 (MCP1), which are upregulated in human patients receiving fractionated doses of total body irradiation (TBI) therapy as a treatment for cancer. These proteins exhibited a similar radiation response in NHPs after single acute or fractionated doses of ionizing radiation.Conclusion: Our work provides confidence in this biomarker panel for biodosimetry triage using fingerstick blood samples and in the use of NHPs as a model for irradiated humans.

Development of a point-of-care radiation biodosimeter: studies using novel protein biomarker panels in non-human primates.

Purpose: There is a need to rapidly triage individuals for absorbed radiation dose following a significant nuclear event. Since most exposed individuals will not have physical dosimeters, we are developing a method to assess exposure dose based on the analysis of a specific panel of blood proteins that can be easily obtained from a fingerstick blood sample.Materials and methods: In three large non-human primate (NHP) studies, animals were exposed to single acute total body doses of x-ray or gamma radiation. A total of 895 blood samples were obtained at baseline and for 7 days after exposure, to evaluate the temporal progression of markers in each of 10 animals (5M/5F) in six dose groups receiving 0-10 Gy. We used tandem mass spectrometry and immunoassay techniques to identify radiation-responsive proteins in blood plasma samples.Results: A blood protein biomarker panel was developed based on analysis of blood plasma samples obtained from several irradiation studies in NHPs that aimed to simulate acute radiation injury in humans from a nuclear exposure event. Panels of several subsets of proteins were shown to accurately classify plasma samples into two exposure groups either above or below a critical dose threshold with sensitivities and specificities exceeding 90%.Conclusion: This study lays the groundwork for developing a radiation biodosimetry triage tool. Our results in NHPs must be compared with those in human patients undergoing radiotherapy to determine if the biomarker panel proteins exhibit a similar radiation response and allow adequate classification power in humans.

Prioritized selection of oligodeoxyribonucleotide probes for efficient hybridization to RNA transcripts.

Only a small fraction of short oligonucleotide probes bind efficiently to complementary segments in long RNA transcripts. Technologies such as array-based transcript profiling and antisense control of gene expression would benefit greatly from a method for predicting probes that bind well to a given target RNA. To develop an algorithm for prioritizing selection of probes, we have analyzed predicted thermodynamic parameters for the binding of several large sets of probes to complementary RNA transcripts. The binding of five of these sets of probes to their RNA targets has been reported by others. In addition, we have used a method for light-directed synthesis of oligonucleotide arrays that we developed to generate two new arrays of surface-bound probes and measured the binding of these probes to their RNA targets. We considered predicted free energies for intramolecular base pairing of the oligonucleotide and its RNA target as well as the predicted free energy of intermolecular hybridization of probe and target. We find that a reliable predictor of probes that will hybridize significantly with their targeted transcripts is the predicted free energy of hybridization minus the predicted free energy for intramolecular folding of the probe.

Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays.

We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5' exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.