Abstract
BackgroundInter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility. Here, we characterised the intrapatient and inter-patient heterogeneity in biomarkers of DNA damage and repair and radiation-induced apoptosis.MethodsWe enrolled 85 of 172 patients with locally advanced nasopharynx cancer from a randomised controlled phase II/III trial of induction chemotherapy added to chemo-radiotherapy. G0 blood lymphocytes were harvested from these patients, and irradiated with 1, 4, and 8 Gy ex vivo. DNA damage induction (1 Gy 0.5 h) and repair (4 Gy 24 h) were assessed by duplicate γH2AX foci assays in 50–100 cells. Duplicate FLICA assays performed at 48 h post-8 Gy were employed as surrogate of radiation-induced apoptosis; %FLICA-positive cells were quantified by flow cytometry.ResultsWe observed limited intrapatient variation in γH2AX foci and %FLICA readouts; median difference of duplicate foci scores was − 0.37 (IQR = − 1.256-0.800) for 1 Gy 0.5 h and 0.09 (IQR = − 0.685-0.792) for 4 Gy 24 h; ICC of ≥0.80 was observed for duplicate %FLICA0Gy and %FLICA8Gy assays of CD4+ and CD8+ T lymphocytes. As expected, we observed wide inter-patient heterogeneity in both assays that was independent of intrapatient variation and clinical covariates, with the exception of age, which was inversely correlated with %FLICAbackground-corrected (Spearman R = − 0.406, P < 0.001 [CD4+]; R = − 0.220, P = 0.04 [CD8+]). Lastly, an exploratory case-control analysis indicates increased levels of γH2AX foci at 4 Gy 24 h in patients with severe late radiotherapy-induced xerostomia (P = 0.05).ConclusionHere, we confirmed the technical reproducibility of DNA damage response assays for clinical implementation as biomarkers of clinical radiosensitivity in nasopharynx cancer patients.
Highlights
Inter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility
We report the level of intrapatient variation between duplicates of three molecular assays of double-strand break (DSB) induction, DSB repair, and radiation-induced lymphocyte apoptosis (RILA) performed under the same laboratory conditions – the γH2AX foci assay (1 Gy 0.5 h [induction] and 4 Gy 24 h [repair]) and a FLuorescent Inhibitor of CAspase (FLICA) assay, respectively, in a cohort of locally advanced nasopharyngeal carcinoma (NPC) patients; these patients were volunteers recruited from a randomised controlled phase II/III trial of concurrent chemo-radiotherapy with and without induction chemotherapy (NCC0901)
It was apparent that FLICA assay of CD4+ and CD8+ T lymphocytes may be better suited for clinical implementation as biomarkers compared to FLICA assay of the overall lymphocyte population due to their higher reliability
Summary
Inter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility. While clinical (cigarette smoking, connective tissue disease, etc.), treatment (concurrent administration of chemotherapy, surgery, etc.), and dosimetric (total dose and dose per fraction) factors account in part for some of the inter-individual heterogeneity [2, 3, 5, 6], it has been estimated that intrinsic host genomic and epigenomic factors constitute the main determinants of tumour and normal tissue radiation sensitivity in 70% of the population [7, 8] This has supported the concept that molecular assays of cellular responses to radiation representing correlates of intrinsic radiosensitivity may predict for individual clinical responses to radiotherapy, raising the possibility of personalised dose prescription. We compared the observed levels of intrapatient differences against inter-patient heterogeneity, and examined for correlation between the cellular responses, in addition to their dependence on clinical, tumour and treatment characteristics
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