Chemical Repair of Radical Damage to the GC Base Pair by DNA-Bound Bisbenzimidazoles.

The migration of an electron-loss center (hole) in calf thymus DNA to bisbenzimidazole ligands bound in the minor groove is followed by pulse radiolysis combined with time-resolved spectrophotometry. The initially observed absorption spectrum upon oxidation of DNA by the selenite radical is consistent with spin on cytosine (C), as the GC• pair neutral radical, followed by the spectra of oxidized ligands. The rate of oxidation of bound ligands increased with an increase in the ratio (r) ligands per base pair from 0.005 to 0.04. Both the rate of ligand oxidation and the estimated range of hole transfer (up to 30 DNA base pairs) decrease with the decrease in one-electron reduction potential between the GC• pair neutral radical of ca. 1.54 V and that of the ligand radicals (E0', 0.90-0.99 V). Linear plots of log of the rate of hole transfer versus r give a common intercept at r = 0 and a free energy change of 12.2 ± 0.3 kcal mol-1, ascribed to the GC• pair neutral radical undergoing a structural change, which is in competition to the observed hole transfer along DNA. The rate of hole transfer to the ligands at distance, R, from the GC• pair radical, k2, is described by the relationship k2 = k0 exp(constant/R), where k0 includes the rate constant for surmounting a small barrier.

An approach to assessing the contribution of the high LET effect in strategies for Auger endoradiotherapy.

Purpose: The interest in exploiting Auger emitters in cancer therapy stems from their high linear energy transfer (LET)-type radiation damage to DNA. However, the design of Auger-emitter labeled vehicles that target the Auger cascade specifically to the DNA of tumour cells is challenging. Here we suggest a possible approach to evaluate tumour-targeting Auger-labeled conjugates by assessing the impact of a radioprotector known to be effective in protecting from low LET radiation, but not high LET radiation. Given some similarity between the energy spectrum of Auger electrons and that of secondary electrons from soft X-rays, we report the results of radioprotection experiments with 25 kVp X-rays. Materials and methods: Clonogenic survival curves for cultured human keratinocytes were established for three different irradiation conditions: 137Cs γ-rays, 25 kVp X-rays and 320 kVp X-rays, and the effect of including a new radioprotector, denoted "2PH", was investigated.Results: The extent of radioprotection by 2PH was comparable for all radiation conditions, although RBE was higher (about 1.7) for soft X-rays. Conclusions: Radioprotectors like 2PH will help to evaluate Auger endoradiotherapy strategies, by determining the relative contributions of the high-LET effects (not protected), compared to other components, such as Auger electrons not effectively targeted to DNA.

Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status.

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.

Monitoring DNA Damage and Repair in Peripheral Blood Mononuclear Cells of Lung Cancer Radiotherapy Patients.

Thoracic radiotherapy (RT) is required for the curative management of inoperable lung cancer, however, treatment delivery is limited by normal tissue toxicity. Prior studies suggest that using radiation-induced DNA damage response (DDR) in peripheral blood mononuclear cells (PBMC) has potential to predict RT-associated toxicities. We collected PBMC from 38 patients enrolled on a prospective clinical trial who received definitive fractionated RT for non-small cell lung cancer. DDR was measured by automated counting of nuclear γ-H2AX foci in immunofluorescence images. Analysis of samples collected before, during and after RT demonstrated the induction of DNA damage in PBMC collected shortly after RT commenced, however, this damage repaired later. Radiation dose to the tumour and lung contributed to the in vivo induction of γ-H2AX foci. Aliquots of PBMC collected before treatment were also irradiated ex vivo, and γ-H2AX kinetics were analyzed. A trend for increasing of fraction of irreparable DNA damage in patients with higher toxicity grades was revealed. Slow DNA repair in three patients was associated with a combined dysphagia/cough toxicity and was confirmed by elevated in vivo RT-generated irreparable DNA damage. These results warrant inclusion of an assessment of DDR in PBMC in a panel of predictive biomarkers that would identify patients at a higher risk of toxicity.

Abscopal Gene Expression in Response to Synchrotron Radiation Indicates a Role for Immunological and DNA Damage Response Genes.

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.

DNA lesions correlate with lymphocyte function after selective internal radiotherapy.

In patients with non-resectable hepatic malignancies selective internal radiotherapy (SIRT) with yttrium-90 is an effective therapy. However, previous data indicate that SIRT leads to impaired immune function. The aim of the current study was to determine the extent of DNA lesions in peripheral blood mononuclear cells of SIRT patients and to correlate these lesions with cellular immune responses. In ten patients γH2AX and 53BP1 foci were determined. These foci are markers of DNA double-strand breaks (DSBs) and occur consecutively. In parallel, lymphocyte proliferation was assessed after stimulation with the T cell mitogen phytohemagglutinin. Analyses of vital cells were performed prior to and 1 h and 1 week after SIRT. 1 h and 1 week after SIRT numbers of γH2AX and of 53BP1 foci were more than threefold larger than before (p < 0.01). Already at baseline, foci were more abundant than published in healthy controls. Lymphocyte proliferation at baseline was below the normal range and further decreased after SIRT. Prior to therapy, there was an inverse correlation between lymphocyte proliferation and the quotient 53BP1/γH2AX; which could be considered as a measure of the course of DNA DSB repair (r = - 0.94, p < 0.0001). Proliferative responses were inversely correlated with 53BP1 foci prior to therapy and γH2AX and 53BP1 foci 1 h after therapy (r < - 0.65, p < 0.05). In conclusion, DNA foci in SIRT patients were correlated with impaired in vitro immune function. Unrepaired DNA DSBs or cell cycle arrest due to repair may cause this impairment.

Radiation Therapy Modulates DNA Repair Efficiency in Peripheral Blood Mononuclear Cells of Patients With Non-Small Cell Lung Cancer.

PURPOSE: There is growing interest in developing individually tailored cancer radiation therapy (RT), wherein patients with high intrinsic radiosensitivity are identified before commencing treatment, to minimize severe adverse reactions. In a previous retrospective study of severely radiosensitive RT patients, we established a functional assay with a high predictive capability. The assay involves ex vivo irradiation of peripheral blood mononuclear cells and analysis of DNA repair using the γ-H2AX assay. It is unknown whether RS is a fixed phenomenon or is modulated under different conditions. We now report the impact of RT on the apparent radiosensitivity, as reflected by the assay. METHODS AND MATERIALS: Peripheral blood mononuclear cells of 11 patients with non-small cell lung cancer were collected before, during, and after RT. Quantitative parameters derived from the nonlinear regression analysis of γ-H2AX foci were applied to examine the cellular radiation response. RESULTS: Although the repair rate and foci yield remained constant during and after RT, the "unrepairable" component of γ-H2AX foci decreased over the course of treatment in 7 patients, signifying a generally enhanced DNA repair capacity. Interestingly, enhanced repair capacity tended to be associated with a poorer response to RT. CONCLUSIONS: Although generalization of these results into normal and tumor tissues warrants further investigation, the findings of this study have important implications in future strategies for identifying radiosensitive individuals before exposure to RT. We can anticipate that the threshold values that will discriminate radiosensitive patients in a future prospective trial will differ from those established in the retrospective study.

A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect.

PURPOSE: Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. METHODS AND MATERIALS: Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. RESULTS: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C-induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. CONCLUSIONS: In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype.

A Functional Immune System Is Required for the Systemic Genotoxic Effects of Localized Irradiation.

PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.

A Bayesian Approach for Prediction of Patient Radiosensitivity.

PURPOSE: A priori identification of the small proportion of radiation therapy patients who prove to be severely radiosensitive is a long-held goal in radiation oncology. A number of published studies indicate that analysis of the DNA damage response after ex vivo irradiation of peripheral blood lymphocytes, using the γ-H2AX assay to detect DNA damage, provides a basis for a functional assay for identification of the small proportion of severely radiosensitive cancer patients undergoing radiotherapy. METHODS AND MATERIALS: We introduce a new, more rigorous, integrated approach to analysis of radiation-induced γ-H2AX response, using Bayesian statistics. RESULTS: This approach shows excellent discrimination between radiosensitive and non-radiosensitive patient groups described in a previously reported data set. CONCLUSIONS: Bayesian statistical analysis provides a more appropriate and reliable methodology for future prospective studies.

Localized Synchrotron Irradiation of Mouse Skin Induces Persistent Systemic Genotoxic and Immune Responses.

The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFß1, and TGFß2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR.

Compromized DNA repair as a basis for identification of cancer radiotherapy patients with extreme radiosensitivity.

A small percentage of cancer radiotherapy patients develop abnormally severe side effects as a consequence of intrinsic radiosensitivity. We analysed the γ-H2AX response to ex-vivo irradiation of peripheral blood lymphocytes (PBL) and plucked eyebrow hair follicles from 16 patients who developed severe late radiation toxicity following radiotherapy, and 12 matched control patients. Longer retention of the γ-H2AX signal and lower colocalization efficiency of repair factors in over-responding patients confirmed that DNA repair in these individuals was compromised. Five of the radiosensitive patients harboured LoF mutations in DNA repair genes. An extensive range of quantitative parameters of the γ-H2AX response were studied with the objective to establish a predictor for radiosensitivity status. The most powerful predictor was the combination of the fraction of the unrepairable component of γ-H2AX foci and repair rate in PBL, both derived from non-linear regression analysis of foci repair kinetics. We introduce a visual representation of radiosensitivity status that allocates a position for each patient on a two-dimensional "radiosensitivity map". This analytical approach provides the basis for larger prospective studies to further refine the algorithm, ultimately to triage capability.

Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes.

PURPOSE: Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories. MATERIALS AND METHODS: Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA. RESULTS: Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration. CONCLUSIONS: We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher's bias and has a potential for usage in clinics and molecular epidemiology.

Radiotherapy for Non-Small Cell Lung Cancer Induces DNA Damage Response in Both Irradiated and Out-of-field Normal Tissues.

PURPOSE: To study the response of irradiated and out-of-field normal tissues during localized curative intent radiotherapy. EXPERIMENTAL DESIGN: Sixteen patients with non-small cell lung carcinoma (NSCLC) received 60 Gy in 30 fractions of definitive thoracic radiotherapy with or without concurrent chemotherapy. Peripheral blood lymphocytes (PBL) and eyebrow hairs were sampled prior, during, and after radiotherapy. Clinical variables of radiotherapy dose/volume, patient age, and use of chemoradiotherapy were tested for association with γ-H2AX foci, a biomarker of DNA damage that underlies cellular response to irradiation. RESULTS: Radiotherapy induced an elevation of γ-H2AX foci in PBL, representing normal tissues in the irradiated volume, 1 hour after fraction one. The changes correlated directly with mean lung dose and inversely with age. γ-H2AX foci numbers returned to near baseline values in 24 hours and were not significantly different from controls at 4 weeks during radiotherapy or 12 weeks after treatment completion. In contrast, unirradiated hair follicles, a surrogate model for out-of-field normal tissues, exhibited delayed "abscopal" DNA damage response. γ-H2AX foci significantly increased at 24 hours post-fraction one and remained elevated during treatment, in a dose-independent manner. This observed abscopal effect was associated with changes in plasma levels of MDC/CCL22 and MIP-1α/CCL3 cytokines. No concordant changes in size and concentration of circulating plasma exosomes were observed. CONCLUSIONS: Both localized thoracic radiotherapy and chemoradiotherapy induce pronounced systemic DNA damage in normal tissues. Individual assessment of biologic response to dose delivered during radiotherapy may allow for therapeutic personalization for patients with NSCLC. Clin Cancer Res; 22(19); 4817-26. ©2016 AACRSee related commentary by Verma and Lin, p. 4763.

Triple targeting of Auger emitters using octreotate conjugated to a DNA-binding ligand and a nuclear localizing signal.

PURPOSE: We investigated the effect of incorporation of a nuclear localization signal (NLS) into a conjugate comprising the DNA binding ligand para-iodoHoechst (PIH) and octreotate on its DNA binding and affinity to the somatostatin receptor (SSTR). Confirmation of these properties would support development of similar conjugates labelled with Auger emitters for their potential in Auger endoradiotherapy. MATERIALS AND METHODS: We synthesized conjugates of PIH and octreotate (PO) or PIH and NLS (PN) and a conjugate comprising PIH, NLS and octreotate (PNO). DNA-binding characteristics of PIH and conjugates were assessed using synthetic DNA oligonucleotides employing spectrophotometric titration of ligand solutions with DNA. We used membranes from the type 2 SSTR (SSTR2) overexpressing human non-small cell lung cancer cell line A427-7 to investigate the binding affinity of PNO. RESULTS: We demonstrated PN and PNO retain specific high affinity DNA-binding properties observed for PIH, and acquire an additional non-specific binding capacity. No DNA binding was observed for PO. PNO retains its binding affinity for SSTR. CONCLUSIONS: The DNA-binding properties of PNO and its affinity for SSTR suggests that it could potentially be used for tumour-specific delivery of PIH labelled with an Auger emitter in SSTR expressing tumours.

Strand breakage by decay of DNA-bound 124I provides a basis for combined PET imaging and Auger endoradiotherapy.

Purpose DNA ligands labelled with 125I induce cytotoxic DNA double-strand breaks (DSB), suggesting a potential for Auger endoradiotherapy. Since the 60-day half-life of 125I is suboptimal for therapy, we have investigated another Auger-emitter 124I, with shorter half-life (4.18 days), and the additional feature of positron-emission, enabling positron emission tomography (PET) imaging. The purpose of this study was to compare the two radionuclides on the basis of DNA DSB per decay. Materials and methods Using a 124I- (or 125I)-labelled minor groove binding DNA ligand, we investigated DNA breakage using the plasmid DNA assay. Biodistribution of the conjugate of the labelled ligand with transferrin was investigated in nude mice bearing a K562 human lymphoma xenograft. Results The probability of DSB per decay was 0.58 and 0.85 for 124I and 125I, respectively, confirming the therapeutic potential of the former. The crystal structure of the ligand DNA complex shows the iodine atom deep within the minor groove, consistent with the high efficiency of induced damage. Biodistribution studies, including PET imaging, showed distinctive results for the conjugate, compared to the free ligand and transferrin, consistent with receptor-mediated delivery of the ligand. Conclusions Conjugation of 124I-labelled DNA ligands to tumor targeting peptides provides a feasible strategy for Auger endoradiotherapy, with the advantage of monitoring tumor targeting by PET imaging.

Assessment and Implications of Scattered Microbeam and Broadbeam Synchrotron Radiation for Bystander Effect Studies.

Synchrotron radiation is an excellent tool for investigating bystander effects in cell and animal models because of the well-defined and controllable configuration of the beam. Although synchrotron radiation has many advantages for such studies compared to conventional radiation, the contribution of dose exposure from scattered radiation nevertheless remains a source of concern. Therefore, the influence of scattered radiation on the detection of bystander effects induced by synchrotron radiation in biological in vitro models was evaluated. Radiochromic XRQA2 film-based dosimetry was employed to measure the absorbed dose of scattered radiation in cultured cells at various distances from a field exposed to microbeam radiotherapy and broadbeam X-ray radiation. The level of scattered radiation was dependent on the distance, dose in the target zone and beam mode. The number of γ-H2AX foci in cells positioned at the same target distances was measured and used as a biodosimeter to evaluate the absorbed dose. A correlation of absorbed dose values measured by the physical and biological methods was identified. The γ-H2AX assay successfully quantitated the scattered radiation in the range starting from 10 mGy and its contribution to the observed radiation-induced bystander effect.

Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity.

Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects.

Analysis of 177Lu-DOTA-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors.

UNLABELLED: Ionizing radiation-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability, and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external-beam ionizing radiation-induced DSBs in peripheral blood lymphocytes (PBLs). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal-tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate (LuTate) peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. METHODS: We analyzed the kinetics of γ-H2AX foci in PBLs of 11 patients undergoing PRRT. The number of γ-H2AX foci was determined before and up to 72 h after treatment. These values were compared with the estimated absorbed dose to blood, spleen, bone marrow, and tumor and with subsequent PBL reduction. RESULTS: The decrease in (177)Lu activity in blood with time followed a biexponential kinetic pattern, with approximately 90% of circulating activity in blood cleared within 2 h. Absorbed dose to blood, but not to spleen or bone marrow, correlated with the administered (177)Lu activity. PRRT increased γ-H2AX foci in lymphocytes in all patients, relative to pretherapy values. The response varied significantly between patients, but the average number of foci indicated a general trend toward an increase at 0.5-4 h with a subsequent decrease by 24-72 h after treatment. The peak number of foci correlated with the absorbed dose to tumor and bone marrow and the extent of PBL reduction. CONCLUSION: γ-H2AX can be exploited in the LuTate PRRT as a biomarker of PBL cytotoxicity. Long-term follow-up studies investigating whether elevated residual γ-H2AX values are associated with acute myelotoxicity and secondary blood malignancy may be worthwhile.