Impact of a 1.5 T magnetic field on DNA damage in MRI-guided HDR brachytherapy

PurposeSome studies have suggested that the presence of a static magnetic field (SMF) during irradiation alters biological damage. Since MRI-guided radiotherapy is becoming increasingly common, we constructed a DNA-based detector to assess the effect of a 1.5 T SMF on DNA damage during high dose rate (HDR) brachytherapy irradiation.MethodsBlock phantoms containing a small cavity for the placement of plasmid DNA (pBR322) samples were 3-D printed with biocompatible tissue equivalent material. The phantom was CT scanned and an HDR brachytherapy treatment plan was designed to deliver 20 Gy and 30 Gy doses to the DNA samples in the presence and absence of a 1.5 T SMF. Relative yields of single- and double-strand breaks (SSBs and DSBs, respectively) were computed from gel electrophoresis images of the DNA band intensities and averaged over sample sizes ranging from 12 to 30. Radiation dose was also measured in the presence and absence of the 1.5 T SMF using GafChromic™ EBT3 film placed in the coronal, sagittal, and axial planes.ResultsThe average yield of DNA with SSBs and DSBs in the presence and absence of the SMF showed no statistically significant differences (all p ≥ 0.17). Differences in the net optical densities of the EBT3 films for each plane were within experimental uncertainty, suggesting no dose difference in the presence and absence of the SMF.ConclusionsHDR irradiation in the presence of the 1.5 T SMF did not alter dose deposition to the DNA cavity nor change SSB and DSB DNA damage.     

Radiosensitivity and Biological Effects of Gamma and X-Rays on Germination and Seedling Vigour of Three Coffea arabica Varieties

Effects of gamma and X-ray treatments were studied on three varieties of Coffea arabica (Kent, Mundo Novo and Geisha) to determine their radiosensitivity and relative biological effects. The coffee varieties seeds were subjected to 0, 50, 100, 150, 200 and 400 Gy of gamma and X-rays from Cobalt 60 (⁶⁰Co) source irradiation. The irradiated seeds were pre-germinated in Petri dishes placed in a germination chamber, whilst some were sown in the greenhouse for germination studies. Data were collected on germination date and rate, root and hypocotyl length to determine the relative biological effectiveness of treatments and the optimum dose. The results showed varieties responding differently to the irradiations and doses. There was a decrease in germination with increasing doses of the irradiation. The X-ray-treated seeds had less germination percentage and seedling vigour measured at 28 days after treatment compared to the gamma-irradiated seeds. The irradiation effects on germination suggest that lower doses of X-rays give the same Relative Biological Effects as higher gamma doses for both growth chamber and greenhouse germination for Geisha at LD₅₀, where the effects were similar for the two irradiations. Whereas 50–100 Gy stimulated germination and seedling vigour, 150 Gy adversely affected germination and no germination occurred at 200–400 Gy. The study concluded that all the coffee varieties evaluated are sensitive to gamma and X-ray irradiation in terms of germination, seedling vigour and biological effects with an optimum dose of 50–100 Gy. Therefore, both gamma and X-rays could be utilized in a future mutational breeding programme for coffee seedlings.

     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Ascorbic acid 2-glucocide reduces micronucleus induction in distant splenic T lymphocytes following head irradiation

PurposeEvidence from in vivo studies suggests there are enhanced radiation effects in abscopal regions after local head gamma ray irradiation. Splenocyte apoptosis and T lymphocyte micronuclei were induced at higher rates than what would be estimated given the dose at a shielded, distant position. In addition, we evaluated the radio-protective effects of ascorbic acid, acting as a radical scavenger on enhanced radiation effects in the shielded spleen following local head irradiation.Methods and materialsThe heads of C3H mice were exposed to γ-rays (10–20 Gy), while the other parts of the body were shielded with a 5 cm-thick lead block. The effective dose for the spleen was calculated at 1.0–2.0 Gy. Splenocytes were isolated 24 h after cranial irradiation and their apoptosis was measured with an Elisa kit (Roche). The induction of T lymphocyte micronuclei was studied using the cytokinesis-block micronucleus assay. The ascorbic acid glucoside, 2-O-alpha-d-glucopyranosyl-l-ascorbic acid (AA-2G), was orally administered to mice 1 h before whole body irradiation. The radio protective effects of AA-2G were estimated by comparing the induction of splenocyte damage (by apoptosis) and micronucleus induction.ResultsThe splenocyte damage, as measured by the above two methods, was more excessive than what would be expected given exposure to 1.0–2.0 Gy of radiation. Our results suggest that the effects were enhanced in a distant, non-irradiated organ after localized irradiation. Plasma ascorbic acid concentrations were increased 8–10× over control. Treatment with ascorbic acid slightly protected mouse splenocytes from the induction of apoptosis by the enhanced effects of radiation in the abscopal region. However, ascorbic acid significantly inhibited micronucleus induction in splenic T lymphocytes following local head irradiation.ConclusionsOur results suggest that ascorbic acid effectively scavenged radiation-induced radicals and protected against the enhanced effects of radiation in an abscopal region after local head gamma ray irradiation.     

Adjuvant hypofractionated radiotherapy with simultaneous integrated boost after breast-conserving surgery: A systematic literature review

PurposesSeveral studies have shown that simultaneous integrated boost provides better dose homogeneity, improves the biologically effective dose-volume histogram and reduces treatment time compared to sequential boost in breast cancer.Patients and methodsWe conducted a systematic review of published trials evaluating simultaneous integrated boost in hypofractionated radiotherapy to analyze the results in terms of overall survival, local control, early and late side effects, and radiotherapy techniques used.ResultsUpon 9 articles, the prescribed dose to the whole breast varied from 40 to 46.8 Gy. The number of fractions varies from 15 to 20 fractions. The prescribed dose per fraction to the boost varied from 2.4 Gy per fraction to 3.4 Gy per fraction for a total boost dose from 48 to 52.8 Gy.ConclusionsSimultaneous integrated boost seems effective and safe when given hypofractionated whole-breast irradiation but needs to be validated in prospective trials.     

Increased carcinoembryonic antigen expression on the surface of lung cancer cells using gold nanoparticles during radiotherapy

PurposeTumor-associated antigens are a promising target of immunotherapy approaches for cancer treatments but rely on sufficient expression of the target antigen. This study investigates the expression of the carcinoembryonic antigen (CEA) on the surface of irradiated lung cancer cells in vitro using gold nanoparticles as radio-enhancer.MethodsHuman lung carcinoma cells A549 were irradiated and expression of CEA on the cell surface measured by flow cytometry 3 h, 24 h, and 72 h after irradiation to doses of 2 Gy, 6 Gy, 10 Gy, and 20 Gy in the presence or absence of 0.1 mg/ml or 0.5 mg/ml gold nanoparticles. CEA expression was measured as median fluorescent intensity and percentage of CEA-positive cells.ResultsAn increase in CEA expression was observed with both increasing radiation dose and time. There was doubling in median fluorescent intensity 24 h after 20 Gy irradiation and 72 h after 6 Gy irradiation. Use of gold nanoparticles resulted in additional significant increase in CEA expression. Change in cell morphology included swelling of cells and increased internal complexity in accordance with change in CEA expression.ConclusionsThis study showed an increase in CEA expression on human lung carcinoma cells following irradiation. Increase in expression was observed with increasing radiation dose and in a time dependent manner up to 72 h post irradiation. The results further showed that gold nanoparticles can significantly increase CEA expression following radiotherapy.     

Biological effects induced by doses of mammographic screening

PurposeMammography is the diagnostic imaging practice used in screening to detect early lesions suspected of malignancy. It uses a low energy X-ray beam in which a low dose in the order of 2–3 mGy is delivered to patient breast cells. However, it has been speculated that it could lead to significant cell damage, when compared to conventional X-ray. We investigated the biological effects of low doses, with mean glandular doses (MGDs) of 2.5 mGy and 2.5 + 2.5 mGy, on mammary cells in vitro.MethodsWe used the non-tumorigenic cell line (MCF-10A) and two tumor cells lines (MCF-7 and MDA-MB-231). Colony formation, apoptosis, and double-strand DNA breaks (DSBs) were quantified.ResultsThe selected MGD regimens did not alter the formation of colonies by any of the cell lines. MCF-7 cells exhibited a markedly increase in apoptosis, 24 h after the single-dose protocol; MCF-10A cells underwent apoptosis only after 72 h, with both irradiation regimens, while MDA-MB-231 cells (highly invasive and metastatic) were not susceptible to apoptosis. The detection of γH2AX histone in the nuclei of irradiated cells showed that the double-dose resulted in increase of DSBs, especially in tumor cell lines.ConclusionsAlthough the health benefits of early breast screening remain indisputable, our future perspective is to better understand the biological basis for the effects of low dose radiation on breast cells and to investigate if and under what conditions there would be a risky situation in repeated mammography screening, in both asymptomatic and symptomatic women.     

Effect of X-ray irradiation on olive shoot culture evaluated by morphological measurements, nuclear DNA content and SSR and AFLP markers

Key message

This article presents the first investigation of X-ray irradiation treatments of olive shoot culture followed by detailed genetic analysis of induced mutation events. This was achieved by comparing morphological measurements, studies of nuclear DNA content and molecular marker (SSR and AFLP) analysis.

Abstract

Traditional olive cultivars often need minor phenotypic corrections, which can be assessed using mutation breeding. We investigated the effects of X-ray irradiation at 0, 10, 30 and 60 Gy delivered to in vitro grown shoots of the olive variety ‘Canino’. Morphological measurements showed that growth parameters did not decrease at a 10-Gy dose, that rooting ability was diminished at a 30-Gy dose and that shoot growth was inhibited at a 60-Gy dose. Measurements of acclimatized plants showed that nuclear DNA content decreased with irradiation dose from 2.972 to 2.963 and 2.935 pg/2C nucleus of control, for 10 and 30 Gy treated plants, respectively. SSR profiling using 6 microsatellite loci revealed no polymorphism. AFLP analysis generated in total 630 scorable fragments within 13 primer combinations, of which 179 (28.4 %) were polymorphic. Although no major morphological changes were observed at a 10-Gy dose, a high number of absent/novel fragments were detected by AFLP analysis, indicating that mutation events can also be detected for doses at which growth parameters are not affected.     

DNA synthesis in nuclei and nuclear matrices of regenerating rat liver: Effect of whole-body gamma irradiation

Summary Partial hepatectomy (PH) of rats (Wistar strain) resulted in acceleration of DNA synthesis in liver which reached a maximum at 36 h after PH. Whole-body radiation exposure (10 Gy) of the rats at 12 h after PH completely arrested this stimulation in DNA synthesis. The elevation of DNA synthetic rate in response to PH and complete obliteration of this stimulation by whole-body radiation exposure were found to be the reflection of levels of DNA polymerase-alpha in nuclei and nuclear matrices isolated from the rat livers. Studies based on assays of DNA polymerase in nuclei and nuclear matrices, with and without exogenous DNA template (activated calf thymus DNA), revealed that whole-body irradiation blocked induction of DNA polymerase-alpha and, in turn, assembling of DNA polymerizing apparatus. Irradiation of nuclei (suspended in buffer) in vitro at doses as high as 500 Gy did not have any inhibitory effect on DNA polymerase-alpha activitiy.     

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes — a Ca²⁺/Mg²⁺-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca²⁺/Mg²⁺-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca²⁺/Mg²⁺-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg²⁺-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca²⁺/Mg²⁺-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Potential interest of developing an integrated boost dose escalation for stereotactic irradiation of primary prostate cancer

IntroductionThe stereotactic irradiation is a new approach for low-risk prostate cancer. The aim of the present study was to evaluate a schema of stereotactic irradiation of the prostate with an integrated-boost into the tumor.Material and methodsThe prostate and the tumor were delineated by a radiologist on CT/MRI fusion. A 9-coplanar fields IMRT plan was optimized with three different dose levels: 1) 5 × 6.5 Gy to the PTV1 (plan 1), 2) 5 × 8 Gy to the PTV1 (plan 2) and 3) 5 × 6.5 Gy on the PTV1 with 5 × 8 Gy on the PTV2 (plan 3). The maximum dose (MaxD), mean dose (MD) and doses received by 2% (D2), 5% (D5), 10% (D10) and 25% (D25) of the rectum and bladder walls were used to compare the 3 IMRT plans.ResultsA dose escalation to entire prostate from 6.5 Gy to 8 Gy increased the rectum MD, MaxD, D2, D5, D10 and D25 by 3.75 Gy, 8.42 Gy, 7.88 Gy, 7.36 Gy, 6.67 Gy and 5.54 Gy. Similar results were observed for the bladder with 1.72 Gy, 8.28 Gy, 7.01 Gy, 5.69 Gy, 4.36 Gy and 2.42 Gy for the same dosimetric parameters. An integrated SBRT boost only to PTV2 reduced by about 50% the dose difference for rectum and bladder compared to a homogenous prostate dose escalation. Thereby, the MD, D2, D5, D10 and D25 for rectum were increased by 1.51 Gy, 4.24 Gy, 3.08 Gy, 2.84 Gy and 2.37 Gy in plan 3 compared to plan 1.ConclusionsThe present planning study of an integrated SBRT boost limits the doses received by the rectum and bladder if compared to a whole prostate dose escalation for SBRT approach.     

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA

We present an analysis of X-ray-induced damage in ΦX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L> = 1,400 nm for a dose of D = 250 Gy to < L> = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB_b> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB_b> increases almost linearly from a value of <DSB_b> = 1.3 after irradiation with D = 250 Gy to <DSB_b> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.

Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay

The comet assay was performed to elucidate the linearity of calibration curves and detection limits for DNA damage in multiple organs of whole body X-irradiated mice, and rates of reduction in DNA damage by DNA repair during the irradiation period were estimated in the respective organs by comparing the rates of increase in DNA damage at different absorbed dose rates of X-rays. Of the assay parameters, tail length and the percentage DNA in the tail showed a higher sensitivity to DNA damage in most organs than Olive tail moment. Data at the higher absorbed dose rates (2.22 or 1.44 Gy/min) showed good correlations between absorbed doses and these two parameters, with correlation coefficients of more than 0.7 in many organs. However, this assay had difficulty detecting DNA damage at the lower absorption dose rate (0.72 Gy/min). The estimated rates of increase in DNA damage and those of DNA repair during the irradiation period in the respective organs suggested differences in the radiosensitivity of nuclear DNA and DNA repair capacity among organs. Our results indicated that absorbed dose rates of 1.0–1.3 Gy/min or greater were needed to induce detectable DNA damages by the comet assay in many organs.     

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11–13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.     

Segmented photon beams technique for irradiation of postmastectomy patients

AimTo present the segmented photon beams technique (SPBT) for irradiation of postmastectomy patients.BackgroundIn majority of techniques for irradiation of posmastectomy patients, a few adjacent photon or electron beams were usually implemented in order to encompass different parts of the target. In the presented SPBT technique, the radiotherapy plan consists of 6 isocentric photon beams and the area CTV includes both the chest wall and the supraclavicular area. This makes it possible to provide a uniform dose to the CTV with no hot and cold points and enables the determination of doses for the entire volume of critical organs.Methods and materialThe treatment forward-IMRT plan comprises six isocentric 4 and 15 MV photon beams. Modulation of the dose distribution for each field was obtained by applying three segments on average. The total dose of 45 Gy was administered in 20 fractions. Dose distributions in target volume and organs at risk were evaluated for 70 randomly chosen patients.ResultsOn average, 94.8% of the CTV volume received doses within 95–107% of the prescribed dose. The average volume of the heart receiving a dose of 30 Gy and lager was 2% for patients with left breast cancer. The average dose to the lung on the irradiation side was always lower than 15.5 Gy and the average V20 Gy was below 35.5%.ConclusionsThe SPBT complies with requirements for high dose homogeneity within the target volume and satisfactory level of sparing of organs at risk.     

Complication probability model for subcutaneous fibrosis based on published data of partial and whole breast irradiation

PurposeTo extend the application of current radiation therapy (RT) based normal tissue complication probability (NTCP) models of radiation-induced fibrosis (RIF) of the breast to include the effects of fractionation, inhomogeneous dose, incomplete recovery, and time after the end of radiotherapy in partial breast irradiation (PBI).Materials and methodsAn NTCP Lyman model with biologically effective uniform dose (BEUD) with and without a correction for the effect of incomplete repair was used. The time to occurrence of RIF was also taken into account. The radiobiological parameters were determined by fitting incidences of moderate/severe RIF in published randomized studies on RT of the breast. The NTCP model was used to calculate the risk of toxicity in 35 patients treated with intensity modulated, non-accelerated PBI and the result was compared with observed incidence of RIF.ResultsWith α/β fixed at 3Gy the parameters of the model without correction for incomplete repair extracted from fitting were: 50% complication probability biologically effective dose BEUD₅₀ = 107.2 Gy (95%CI = 95.9–118.6 Gy), volume parameter n = 0.06 (95%CI = 0–0.23), and slope of dose response m = 0.22, (95%CI = 0.20–0.23). After including the correction for incomplete repair with repair halftime for subcutaneous tissue of τ = 4.4 h we obtained BEUD₅₀ = 105.8 Gy (95%CI = 96.9–114.6Gy), n = 0.15 (95%CI = 0–0.33), m = 0.22 (95%CI = 0.20–0.23). Average NTCP predicted by these models, 4.3% and 2.0% respectively, offered a good agreement with RIF incidence in our patients, 5.7%, after an average follow-up of 12 months.ConclusionThe NTCP models of RIF, incorporating the effects of fractionation, volume effect, and latency of toxicity look promising to model PBI. Clinical validation from a prospective PBI treatment study is under development and will help test this preliminary result.