Roswitha Runge

Fully automated interpretation of ionizing radiation-induced γH2AX foci by the novel pattern recognition system AKLIDES®

Purpose: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. Materials and methods: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to 188Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES® for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. Results: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R2 = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. Conclusion: The interpretation system AKLIDES® demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci.

Systemic radionuclide therapy in pain palliation

Several radiopharmaceuticals were investigated to determine their efficacy and toxicity in the palliation of painful bone metastases. Data on the influence of rhenium-188 hydroxyethylidene diphosphonate ( 188 Re-HEDP), rhenium-186 hydroxyethylidene diphosphonate ( 186 Re-HEDP), and strontium-89 ( 89 Sr) on pain symptoms, quality of life, and bone-marrow function were obtained in 64 patients with breast and prostate cancer. Thirty-one patients were treated with 188 Re-HEDP (3194 ± 387 MBq), 15 patients with 186 Re-HEDP (1358 ± 158 MBq), and 18 patients with 89 Sr (152 ± 19 MBq). The 188 Re-HEDP group included six breast cancer patients and 25 prostate cancer patients; the 186 Re-HEDP group included three breast cancer patients and 12 prostate cancer patients; and the 89 Sr group included three breast cancer patients and 15 prostate cancer patients. All subjects participated in an interview using a standardized sets of questions before and after the 12-week term of therapy. Blood counts were taken weekly for six weeks and after 12 weeks. Results showed that 77 percent of patients reported pain relief after treatment with 188 Re-HEDP, 67 percent after treatment with 186 Re-HEDP, and 72 percent after treatment with 89 Sr. Sixteen percent of patients treated with 188 Re-HEDP, 13 percent treated with 186 Re-HEDP, and 17 percent treated with 89 Sr were able to discontinue their analgesics and were pain-free. Patients described an improvement on Karnofsky performance status (KPS) from 73 ± 7 percent to 85 ± 8 percent 12 weeks after 188 Re-HEDP (p < 0.05), from 72 ± 13 percent to 79 ± 12 percent after 186 Re-HEDP (p = 0.251), and from 62 ± 14 percent to 69 ± 16 percent after 89 Sr (p = 0.415). Only three patients undergoing 188 Re-HEDP therapy, one undergoing 186 Re-HEDP therapy, and three undergoing 89 Sr therapy had thrombocytopenia (platelet count below 100 × 10(3)/µl) following treatment. The maximum nadir of platelet and leukocyte counts was observed between the second and fifth week after treatment for all radionuclides and was reversible within 12 weeks. The nadir was earlier for 188 Re-HEDP with a shorter physical half-life compared with 89 Sr. There were no significant differences in bone marrow toxicity (p = 0.123-0.421). Results of this study indicate that all evaluated radiopharmaceuticals were effective in pain palliation without induction of severe side effects. The increase in KPS after 188 Re-HEDP was the only statistically significant finding (p = 0.001).

Potential of a Cetuximab-based radioimmunotherapy combined with external irradiation manifests in a 3-D cell assay.

Targeting epidermal growth factor receptor (EGFR)‐overexpressing tumors with radiolabeled anti‐EGFR antibodies is a promising strategy for combination with external radiotherapy. In this study, we evaluated the potential of external plus internal irradiation by [90Y]Y‐CHX‐A″‐DTPA‐C225 (Y‐90‐C225) in a 3‐D environment using FaDu and SAS head and neck squamous cell carcinoma (HNSCC) spheroid models and clinically relevant endpoints such as spheroid control probability (SCP) and spheroid control dose 50% (SCD50, external irradiation dose inducing 50% loss of spheroid regrowth). Spheroids were cultured using a standardized platform. Therapy response after treatment with C225, CHX‐A″‐DTPA‐C225 (DTPA‐C225), [90Y]Y‐CHX‐A″‐DTPA (Y‐90‐DTPA) and Y‐90‐C225 alone or in combination with X‐ray was evaluated by long‐term monitoring (60 days) of spheroid integrity and volume growth. Penetration kinetics into spheroids and EGFR binding capacities on spheroid cells were identical for unconjugated C225 and Y‐90‐C225. Spheroid‐associated radioactivity upon exposure to the antibody‐free control conjugate Y‐90‐DTPA was negligible. Determination of the SCD50 demonstrated higher intrinsic radiosensitivity of FaDu as compared with SAS spheroids. Treatment with unconjugated C225 alone did not affect spheroid growth and cell viability. Also, C225 treatment after external irradiation showed no additive effect. However, the combination of external irradiation with Y‐90‐C225 (1 µg/ml, 24 hr) resulted in a considerable benefit as reflected by a pronounced reduction of the SCD50 from 16 Gy to 9 Gy for SAS spheroids and a complete loss of regrowth for FaDu spheroids due to the pronounced accumulation of internal dose caused by the continuous exposure to cell‐bound radionuclide upon Y‐90‐C225‐EGFR interaction.

99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4 −). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4 − (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4 − treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4 – treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4 − induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.

Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of 99mTc versus 188Re

Abstract Purpose: Cellular radionuclide uptake increases the heterogeneity of absorbed dose to biological structures. Dose increase depends on uptake yield and emission characteristics of radioisotopes. We used an in vitro model to compare the impact of cellular uptake of 188Re-perrhenate and 99mTc-pertechnetate on cellular survival. Materials and methods: Rat thyroid PC Cl3 cells in culture were incubated with 188Re or 99mTc in the presence or absence of perchlorate for 1 hour. Clonogenic cell survival was measured by colony formation. In addition, intracellular radionuclide uptake was quantified. Results: Dose effect curves were established for 188Re and 99mTc for various extra- and intracellular distributions of the radioactivity. In the presence of perchlorate, no uptake of radionuclides was detected and 188Re reduced cell survival more efficiently than 99mTc. A37, the activity that is necessary to yield 37% cell survival was 14 MBq/ml for 188Re and 480 MBq/ml for 99mTc. In the absence of perchlorate, both radionuclides showed similar uptakes; however, A37 was reduced by 30% for the beta-emitter and by 95% for 99mTc. The dose D37 that yields 37% cell survival was between 2.3 and 2.8 Gy for both radionuclides. Conclusions: Uptake of 188Re and 99mTc decreased cell survival. Intracellular 99mTc yielded a dose increase that was higher compared to 188Re due to emitted Auger and internal conversion-electrons. Up to 5 Gy there was no difference in radiotoxicity of 188Re and 99mTc. At doses higher than 5 Gy intracellular 99mTc became less radiotoxic than 188Re, probably due to a non-uniform lognormal radionuclide uptake.

[99mTc reduces clonogenic survival after intracellular uptake in NIS-positive cells in vitro more than 131I].

AIM In addition to gamma radiation of 140 keV 99mTc emits during the transition to 99Tc electrons of low energy and tiny path-lengths. These Auger electrons cannot be utilized in diagnostic procedures. However, they were discussed frequently for therapeutic application. Hitherto proof of effect of the Auger electrons from 99mTc is missing which is supplied now in an in vitro-system in comparison to beta-emitter 131I. METHODS The thyroid cell line PCCl3 (sodium iodide symporter (NIS)-positive) was incubated with 131I-sodium iodide (131I) or 99mTc-pertechnetate (99mTc) in presence or absence of perchlorate. For comparison the amount of radioactivity was adjusted to obtain the same dose from extracellular irradiation for both radionuclides. The colony forming assay detects the clonogenic cell survival as surviving fraction. In addition, intracellular radionuclide uptake was quantified. RESULTS Dose effect curves were established for 131I and 99mTc for variable extra- and intracellular distribution of the radioactivity. In presence of perchlorate no cellular uptake of radioactivity was detectable. Survival curves were largely comparable confirming the dosimetric calculations. In absence of perchlorate cellular radiotracer uptake varied from 1.39% (131I) to 1.90% 99mTc). Effects on survival were twice for the beta-emitter and ten-fold higher for 99mTc. CONCLUSIONS Intracellular uptake of 131I and 99mTc increases DNA-damage compared to strict extracellular radiotracer distribution which was demonstrated by means of colony forming assay. Increasing radiotoxicity from intracellular 99mTc is explained most likely by increased dose deposition in cellular structures due to Auger- and conversion-electrons of low range and high local energy deposition.

Cellular damage in vitro

AIM The cellular damage of ionising radiation depends on dose, physical radiation quality (e. g. LET) and intracellular radionuclide uptake. The influence of two beta emitters (188Re and 131I) on the thyroid cell line PCCl3 was studied. Furthermore, we analysed the effect of intracellular accumulation. METHODS The thyroid cell line PCCl3 was irradiated with 188Re-perrhenate or 131I-sodium iodide in presence or absence of perchlorate. The initial DNA-damage was measured in the comet assay as olive tail moment (OTM). The colony forming assay detects the clonogenic cell survival as surviving fraction. Additional the intracellular radionuclide uptake was quantified. RESULTS Dose response curves were established for irradiation with 188Re-perrhenate or 131I-iodine under various extra- and intracellular activity distribution conditions. In the presence of perchlorate DNA-damage and clonogenic cell survival for both radionuclides were comparable. In the absence of perchlorate radionuclide uptake of 1.39% (131I) and 4.14% (188Re) were measured causing twofold higher radiotoxicity. Although 131I uptake was lower than 188Re uptake the OTM values were higher und surviving fractions were lower. CONCLUSIONS 131I, compared to 188Re, has lower mean beta energy and a higher LET, and therefore, it induced a higher DNA-damage even at lower intracellular uptake. An additional explanation for the higher radiotoxicity of 131I could be the higher dose exposition caused by cross-fire through neighborhood cells.

Autoradiographic studies of rhenium-188-hydroxyethylidine diphosphonate in normal skeleton and osteoblastic bone metastases in a rat model of metastatic prostate cancer.

AimThe quantitative distribution of bone-seeking radiopharmaceuticals in trabecular bone, cortical bone and in skeletal metastases is required for calculation of radiation-absorbed dose in radionuclide therapy. An animal model of intraosseous tumor cell administration was developed to simulate osteoblastic metastases for autoradiographic study of radionuclide localization. MethodsIn 45 Copenhagen rats R3327-MATLyLu syngeneic prostate cancer cells were given intraosseously in both the femori. Rhenium-188-hydroxyethylidine diphosphonate (HEDP) was administered intravenously 17±1 days after cells instillation and these animals were euthanized at 4, 24 and 48 h after injection of the radiopharmaceutical. The uptake of radiopharmaceutical was estimated in normal skeleton and the bone metastases by means of region of interest analysis using autoradiography. The tumor to nontumor ratio and the fractional uptake in cortical bone and trabecular bone were quantified. ResultsThe uptake of rhenium-188-HEDP in cortical bone was 33.5% and in trabecular bones was 66.5% after 4 h, 34.6 and 65.4% after 24 h, and 35.9 and 64.1% after 48 h, respectively. Assuming a theoretic cortical-trabecular distribution of 50–50%, (MIRDOSE) calculation, radiation-absorbed dose to bone marrow was underestimated by 26%. In bone metastases, an inhomogeneous distribution with a minimal and maximal tumor to nontumor ratio of 3 : 1 and 14 : 1 after 4 h, 5 : 1 and 14 : 1 after 24 h, and 5 : 1 and 16 : 1 after 48 h was observed. ConclusionThe MIRDOSE model underestimates the radiation-absorbed dose to the bone marrow because of demonstrable differences in the uptake of rhenium-188-HEDP in cortical and trabecular bone and inhomogeneous uptake in skeletal metastases.

Comparison of the radiotoxicity of the 99mTc-labeled compounds 99mTc-pertechnetate, 99mTc-HMPAO and 99mTc-MIBI

Abstract Purpose: In addition to gamma radiation, 99mTc emits low-energy Auger electrons with path-lengths of nanometers to micrometers that cannot be utilized for diagnostic procedures; however, they have frequently been discussed for therapeutic applications. We compared radiotoxicity of three 99mTc-labeled radiopharmaceuticals with differences in the subcellular distribution. Materials and methods: The intracellular radionuclide uptake and subcellular distribution of [99mTc]-pertechnetate (99mTc-pertechnetate), [99mTc]Tc-hexamethyl-propylene-aminoxime (99mTc-HMPAO) and [99mTc]Tc-hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) were quantified in rat thyroid FRTL-5 cells. Radiotoxicity was compared using late phosphorylated histone H2AX (γH2AX) foci as a marker for unrepaired DNA double-strand breaks (DNA-DSB) and clonogenic cell survival. Results: 99mTc-HMPAO showed a substantially higher uptake into the nucleus and the membrane/organelles than 99mTc-pertechnetate or 99mTc-MIBI. The colony-forming assay showed that 99mTc-pertechnetate and 99mTc-HMPAO caused a similar reduction in cell survival. 99mTc-MIBI is less radiotoxic in terms of the estimated nucleus dose and induced the fewest number of γH2AX foci compared with the other 99mTc-tracers, and 99mTc-HMPAO induced a fewer number of γH2AX foci than 99mTc-pertechnetate. Conclusions: Our findings reveal that clonogenic cellular survival is not solely determined by the DNA-DSB response. This finding may suggest the involvement of extra-nuclear radiosensitive targets in cell inactivation. For example, the mitochondria or the cell membrane could be affected by 99mTc-HMPAO.

Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

UNLABELLED Technetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, (99m)Tc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much (99m)Tc can be present in exposed cells and which radiobiological effects could be estimated in (99m)Tc-overloaded cells. METHODS Sodium iodine symporter (NIS)-positive thyroid cells were used. (99m)Tc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyrophosphate kit solution or as a standard (99m)Tc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. RESULTS Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of (99m)Tc and is followed by further pertechnetate influx to an unexpectedly high (99m)Tc level. The uptake of (99m)Tc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3-5% to >80%. The maximum possible cellular uptake of (99m)Tc was 90Bq/cell. Compared with nearly pure extracellular irradiation from routine (99m)Tc complexes, cell survival was reduced by 3-4 orders of magnitude after preincubation with stannous pyrophosphate. CONCLUSIONS Intracellular (99m)Tc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.