Assessment of DNA damage by 53PB1 and pKu70 detection in peripheral blood lymphocytes by immunofluorescence and high-resolution transmission electron microscopy.

PURPOSE: 53BP1 foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA double-strand break (DSB) marker. In addition, high-resolution transmission electron microscopy (TEM) with immunogold labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA repair process. To establish this TEM method in the PBLs of patients with cancer, we analyzed and characterized whether different modes of irradiation influence the formation of DSBs, and whether accompanying chemotherapy influences DSB formation. METHODS: We obtained 86 blood samples before and 0.1, 0.5, and 24 h after irradiation from patients (n = 9) with head and neck or rectal cancers receiving radiotherapy (RT; n = 4) or radiochemotherapy (RCT; n = 5). 53BP1 foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70 dimers and 53BP1 clusters within euchromatin and heterochromatin of PBLs. RESULTS: IFM analyses showed that during radiation therapy, persistent 53BP1 foci in PBLs accumulated with increasing numbers of administered RT fractions. This 53BP1 foci accumulation was not influenced by the irradiation technique applied (3D conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1 foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT patients than in PBLs of RT only patients. Moreover, not every residual 53BP1 focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1 clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion or, possibly, defective repair. CONCLUSION: IFM 53BP1 foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1 focus but not every 53BP1 focus represents a DSB.

Clustered DNA damage concentrated in particle trajectories causes persistent large-scale rearrangements in chromatin architecture.

BACKGROUND AND PURPOSE: High linear-energy-transfer (LET) irradiation (IR) is characterized by unique depth-dose distribution and advantageous biologic effectiveness compared to low-LET-IR, offering promising alternatives for radio-resistant tumors in clinical oncology. While low-LET-IR induces single DNA lesions such as double-strand breaks (DSBs), localized energy deposition along high-LET particle trajectories induces clustered DNA lesions that are more challenging to repair. During DNA damage response (DDR) 53BP1 and ATM are required for Kap1-dependent chromatin relaxation, thereby sustaining heterochromatic DSB repair. Here, spatiotemporal dynamics of chromatin restructuring were visualized during DDR after high-LET and low-LET-IR. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon/calcium ions or low-LET photons. At 0.1 h, 0.5 h, 5 h and 24 h post-IR fluorophore- and gold-labeled repair factors (53BP1, pATM, pKAP-1, pKu70) were visualized by immunofluorescence and transmission electron microscopy, to monitor formation and repair of DNA damage in chromatin ultrastructure. To track chromatin remodeling at damage sites, decondensed regions (DCR) were delineated based on local chromatin concentration densities. RESULTS: Low-LET-IR induced single DNA lesions throughout the nucleus, but nearly all DSBs were efficiently rejoined without visible chromatin decompaction. High-LET-IR induced clustered DNA damage and triggered profound changes in chromatin structure along particle trajectories. In DCR multiple heterochromatic DSBs exhibited delayed repair despite cooperative activity of 53BP1, pATM, pKap-1. These closely localized DSBs may disturb efficient repair and subsequent chromatin restoration, thereby affecting large-scale genome organization. CONCLUSION: Clustered damage concentrated in particle trajectories causes persistent rearrangements in chromatin architecture, which may affect structural and functional organization of cell nuclei.

Analysis of relation between hypoxia PET imaging and tissue-based biomarkers during head and neck radiochemotherapy.

BACKGROUND: Tumor hypoxia is associated with poor prognosis and outcome and can be visualized using 18F-MISO-positron emission tomography (PET) imaging. The goal of this study was to evaluate the correlation between biological markers and biological imaging in a group of patients in whom a correlation between biological imaging and outcome has previously been demonstrated. MATERIAL AND METHODS: In a prospective pilot project, 16 patients with locally advanced cancer of the head and neck underwent 18F-MISO-PET scans before and during primary radiochemotherapy in addition to 18F-FDG-PET and computed tomography (CT). Tumor biopsies were stained for three tissue-based markers (Ku80, CAIX, CD44); in addition, human papillomavirus (HPV) status was assessed. H-scores of marker expression were generated and the results were correlated with the biological imaging and clinical outcome. RESULTS: No statistically significant correlation was established between the H-scores for Ku80, CD44 and CAIX or between any of the H-scores and the imaging variables (tumor volume on 18F-FDG-PET in ml, hypoxic subvolume as assessed by 18F-MISO-PET in ml, and SUVmax tumor/SUVmean muscle during the 18F-MISO-PET). A statistically significant negative correlation was found between CD44 H-score and HPV status (p = .004). Cox regression analysis for overall survival and recurrence-free survival showed one significant result for CAIX being associated with improved overall survival [hazard ratio 0.96 (0.93-1.00), p = .047]. CONCLUSION: Expression of Ku80, CAIX and CD44 as assessed by immunohistochemistry of tumor biopsies were not correlated to one another or the biological imaging data. However, there was a significant influence of CAIX on overall survival and between CD44 and HPV.

Expression and significance of Ku80 and PDGFR-α in nasal NK/T-cell lymphoma.

BACKGROUND: To investigate the clinical and prognostic significance of Ku80 and PDGFR-α in nasal type NK/T cell lymphoma (NKTCL). METHODS: Immunohistochemistry for Ku80 and PDGFR-α was performed on tissue sections from 35 patients diagnosed with NKTCL. We analyzed the relationship between Ku80 and PDGFR-α expression and the clinicopathological features of NKTCL. We further performed multivariate analyses to identify prognostic factors for progression free survival (PFS) of patients. RESULTS: Ku80 expression rate in NKTCL was 94.3% compared with that in reactive lymphoid hyperplasia of nasopharynx (P=0.003). The positive expression rate of PDGFR-α in the group of NKTCL was 91.4% compared with that in control group (P=0.004). The positive correlation between Ku80 and PDGFR-α was also found (r=0.496, P=0.002). Ku80 and PDGFR-α expressions were not correlated with patient's gender, age, B symptoms, LDH, Ann Arbor stage, IPI score and treatment (P>0.05). High expression of Ku80 and PDGFR-α was shown to be correlated with worse PFS (P=0.003 and 0.034, respectively). Multivariate analysis with a Cox proportional hazards model further suggested that Ku80 high expression rate (HR, 11.495; P=0.009), PDGFR-α high expression rate (HR, 4.836; P=0.031) and International Prognostic Index (IPI) score of 3-4 (HR, 7.308; P=0.001) were statistically independent prognostic factors for patients' PFS. Our results suggest that Ku80 and PDGFR-α may be valuable indicators for predicting the survival of NKTCL patients. CONCLUSION: Ku80 and PDGFR-α might be effective predictive indicators for the prognosis of NKTCL. A large prospective study is required to confirm the prognostic significance of Ku80 and PDGFR-α in NKTCL.