Abstract
BackgroundBiomarkers for predicting late normal tissue toxicity to radiotherapy are necessary to personalize treatments and to optimize clinical benefit. Many radiogenomic studies have been published on this topic. Conversely, proteomics approaches are not much developed, despite their advantages.MethodsWe used the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach to analyze differences in protein expression levels in ex-vivo irradiated (8 Gy) T lymphocytes from patients with grade ≥ 2 radiation-induced breast fibrosis (grade ≥ 2 bf+) and patients with grade < 2 bf + after curative intent radiotherapy. Patients were selected from two prospective clinical trials (COHORT and PHRC 2005) and were used as discovery and confirmation cohorts.ResultsAmong the 1979 quantified proteins, 23 fulfilled our stringent biological criteria. Immunoblotting analysis of four of these candidate proteins (adenylate kinase 2, AK2; annexin A1; heat shock cognate 71 kDa protein; and isocitrate dehydrogenase 2) confirmed AK2 overexpression in 8 Gy-irradiated T lymphocytes from patients with grade ≥ 2 bf + compared with patients with grade < 2 bf+. As these candidate proteins are involved in oxidative stress regulation, we also evaluated radiation-induced reactive oxygen species (ROS) production in peripheral blood mononuclear cells from patients with grade ≥ 2 bf + and grade < 2 bf+. Total ROS level, and especially superoxide anion level, increased upon ex-vivo 8 Gy-irradiation in all patients. Analysis of NADPH oxidases (NOXs), a major source of superoxide ion in the cell, showed a significant increase of NOX4 mRNA and protein levels after irradiation in both patient groups. Conversely, only NOX4 mRNA level was significantly different between groups (grade ≥ 2 bf + and grade < 2 bf+).ConclusionThese findings identify AK2 as a potential radiosensitivity candidate biomarker. Overall, our proteomic approach highlights the important role of oxidative stress in late radiation-induced toxicity, and paves the way for additional studies on NOXs and superoxide ion metabolism.
Highlights
Biomarkers for predicting late normal tissue toxicity to radiotherapy are necessary to personalize treatments and to optimize clinical benefit
To identify proteins that might predict the risk of grade ≥ 2 bf+, we compared the proteomic data of control (0 Gy) and irradiated (8 Gy) T lymphocytes from patients with (n = 2) and without (n = 2) grade ≥ 2 bf + obtained by 2D nano-LC/MS/MS analysis after isobaric tags for relative and absolute quantitation (iTRAQ) labeling
We focused on NOX4 because it was the most abundant isoform in Peripheral Blood Mononuclear Cells (PBMCs)
Summary
Biomarkers for predicting late normal tissue toxicity to radiotherapy are necessary to personalize treatments and to optimize clinical benefit. RT use for cancer treatment inevitably involves exposure of the surrounding normal tissues, and may cause late and irreversible toxicities. One of the most promising approaches for clinical use is the radiation-induced CD8 lymphocyte apoptosis (RILA) assay developed by our group several years ago [5, 6]. This assay is based on the measurement of the radiation-induced apoptosis rate in T lymphocytes isolated from a whole blood sample, 2 days after ex-vivo exposure to 8 Gy of radiation. We and others consistently reported low RILA values in all patients with radiation-induced late toxicity, and high RILA values in all patients without late toxicities [7,8,9,10]
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