High efficacy of particle beam therapies against tumors under hypoxia and prediction of the early stage treatment effect using 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography.

OBJECTIVE: Compared with radiation therapy using photon beams, particle therapies, especially those using carbons, show a high relative biological effectiveness and low oxygen enhancement ratio. Using cells cultured under normoxic conditions, our group reported a greater suppressive effect on cell growth by carbon beams than X-rays, and the subsequent therapeutic effect can be predicted by the cell uptake amount of 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT) the day after treatment. On the other hand, a hypoxic environment forms locally around solid tumors, influencing the therapeutic effect of radiotherapy. In this study, the influence of tumor hypoxia on particle therapies and the ability to predict the therapeutic effect using 18F-FLT were evaluated. METHODS: Using a murine colon carcinoma cell line (colon 26) cultured under hypoxic conditions (1.0% O2 and 5.0% CO2), the suppressive effect on cell growth by X-ray, proton, and carbon irradiation was evaluated. In addition, the correlation between decreased 18F-FLT uptake after irradiation and subsequent suppression of cell proliferation was investigated. RESULTS: Tumor cell growth was suppressed most efficiently by carbon-beam irradiation. 18F-FLT uptake temporarily increased the day after irradiation, especially in the low-dose irradiation groups, but then decreased from 50 h after irradiation, which is well correlated with the subsequent suppression on tumor cell growth. CONCLUSIONS: Carbon beam treatment shows a strong therapeutic effect against cells under hypoxia. Unlike normoxic tumors, it is desirable to perform 18F-FLT positron emission tomography 2-3 days after irradiation for early prediction of the treatment effect.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation.

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs' production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

Exposure of the cytoplasm to low-dose X-rays modifies ataxia telangiectasia mutated-mediated DNA damage responses.

We recently showed that when a low X-ray dose is used, cell death is enhanced in nucleus-irradiated compared with whole-cell-irradiated cells; however, the role of the cytoplasm remains unclear. Here, we show changes in the DNA damage responses with or without X-ray microbeam irradiation of the cytoplasm. Phosphorylated histone H2AX foci, a surrogate marker for DNA double-strand breaks, in V79 and WI-38 cells are not observed in nucleus irradiations at ≤ 2 Gy, whereas they are observed in whole-cell irradiations. Addition of an ataxia telangiectasia mutated (ATM) kinase inhibitor to whole-cell irradiations suppresses foci formation at ≤ 2 Gy. ABL1 and p73 expression is upregulated following nucleus irradiation, suggesting the induction of p73-dependent cell death. Furthermore, CDKN1A (p21) is upregulated following whole-cell irradiation, indicating the induction of cell cycle arrest. These data reveal that cytoplasmic radioresponses modify ATM-mediated DNA damage responses and determine the fate of cells irradiated at low doses.

Stability of daily rectal movement and effectiveness of replanning protocols for sparing rectal doses based on the daily CT images during proton treatment for prostate cancer.

PURPOSE: To evaluate the optimal period of replanning to spare the rectal dose by investigating daily rectal movements during computed tomography (CT) image-guided proton therapy for prostate cancer. MATERIALS AND METHODS: To evaluate the optimum reference period for replanning, we analyzed 1483 sets of daily CT (dCT) images acquired from 40 prostate cancer patients and measured the daily rectal movement along the anterior-posterior direction based on the simulator CT (sCT) images and dCT images. We calculated daily dose distributions based on initial plans on the sCT images and replans on the dCT images for 13 representative patients, and evaluated daily dose volume histograms (DVHs) for the prostate, seminal vesicles, and rectum. RESULTS: The rectal anterior side on the dCT images around the seminal vesicles largely deviated toward the anterior side relative to the position on the reference sCT images, but the deviation decreased by referring to the dCT images and became nearly zero when we referred to the dCT images after 10-day treatment. The daily DVH values for the prostate showed good dose coverage. For six patients showing rectal movement toward the anterior side, the daily rectal DVH (V77% ) showed a 3.0 ± 1.7 cc excess from the initial plan and this excess was correlated with 9.9 ± 6.8 mm rectal movement. To identify the patients (37.5% in total) for whom the replanning on the 10th-day and 20th-day CT images reduced the V77% excess to 0.4 ± 1.5 cc and -0.2 ± 1.3 cc, respectively, we evaluated the accumulated mean doses with a 1.2 cc criterion. CONCLUSION: Our data demonstrate that the daily movement of the rectal anterior side tends to move toward the anterior side, which results in a rectal overdose, and the mean of the movement gradually decreases with the passage of days. In such cases, replanning with the reference CT after 10 days is effective to spare the rectal dose.

Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation.

BACKGROUND: Radiation therapy is an important alternative treatment for advanced cancer. The aim of the current study was to disclose distinct alterations of the biological characteristics of gene expression features in pancreatic cancer cells, MIAPaCa-2, following proton and X-ray irradiation. MATERIALS AND METHODS: Using cDNA microarray, we examined the gene expression alterations of MIAPaCa-2 cells following proton or X-ray irradiation. We also isolated the surviving MIAPaCa-2 cells after irradiation and analyzed their gene expression profiles. RESULTS: Although the cytocidal effects of both types of irradiation were similar at sufficient doses in vitro and in vivo, the affected gene expression profile alterations of MIAPaCa-2 cells irradiated with protons were distinct from those irradiated with X-ray. Interestingly, clustering analysis of gene expression of the surviving MIAPaCa-2 cells was also completely discernible between the two types of irradiation. However, a similar cytocidal effect was still observed in the proton- and X-ray-irradiated surviving cells after re-irradiation, commonly showing biological effects related to apoptosis and cell cycle processes. CONCLUSIONS: Proton irradiation treatment for pancreatic cancer provides the distinct biological effect of steady gene expression alterations compared to X-ray irradiation; however, surviving cells from both types of irradiation were still susceptible to the cytocidal effects induced by proton re-irradiation treatment.

Positioning accuracy and daily dose assessment for prostate cancer treatment using in-room CT image guidance at a proton therapy facility.

PURPOSE: To evaluate the effectiveness of CT image-guided proton radiotherapy for prostate cancer by analyzing the positioning uncertainty and assessing daily dose change due to anatomical variations. MATERIALS AND METHODS: Patients with prostate cancer were treated by opposed lateral proton beams based on a passive scattering method using an in-room CT image-guided system. The system employs a single couch for both CT scanning and beam delivery. The patient was positioned by matching the boundary between the prostate and the rectum's anterior region identified in the CT images to the corresponding boundary in the simulator images after bone matching. We acquired orthogonal kV x-ray images after couch movement and confirmed the body position by referring to the bony structure prior to treatment. In offline analyses, we contoured the targeted anatomical structures on 375 sets of daily in-room CT images for 10 patients. The uncertainty of the image-matching procedure was evaluated using the prostate contours and actual couch corrections. We also performed dose calculations using the same set of CT images, and evaluated daily change of dose-volume histograms (DVHs) to compare the effectiveness of the treatment using prostate matching to the bone-matching procedure. RESULTS: The isocenter shifts by prostate matching after bone matching were 0.5 ± 1.8 and -0.8 ± 2.6 mm along the superior-inferior (SI) and anterior-posterior (AP) directions, respectively. The body movement errors (σ) after couch movement were 0.7, 0.5, and 0.3 mm along the lateral, SI and AP direction, respectively, for 30 patients. The estimated errors (σ) in the prostate matching were 1.0 and 1.3 mm, and, in conjunction with the movement errors, the total positioning uncertainty was estimated to be 1.0 and 1.4 mm along the SI and AP directions, respectively. Daily DVH analyses showed that in the prostate matching, 98.7% and 86.1% of the total 375 irradiations maintained a dose condition of V95%  > 95% for the prostate and a dose constraint of V77%  < 18% for the rectum, whereas 90.4% and 66.1% of the total irradiations did so when bone matching was used. The dose constraint of the rectum and dose coverage of the prostate were better maintained by prostate matching than bone matching (P < 0.001). The daily variation in the dose to the seminal vesicles (SVs) was large, and only 40% of the total irradiations maintained the initial planned values of V95% for high-risk treatment. Nevertheless, the deviations from the original value were -4 ± 7% and -5 ± 11% in the prostate and bone matching, respectively, and a better dose coverage of the SV was achieved by the prostate matching. CONCLUSION: The correction of repositioning along the AP and SI direction from conventional bone matching in CT image-guided proton therapy was found to be effective to maintain the dose constraint of the rectum and the dose coverage of the prostate. This work indicated that prostate cancer treatment by prostate matching using CT image guidance may be effective to reduce the rectal complications and achieve better tumor control of the prostate. However, an adaptive approach is desirable to maintain better dose coverage of the SVs.

Effects of organ motion on proton prostate treatments, as determined from analysis of daily CT imaging for patient positioning.

PURPOSE: We quantified interfractional movements of the prostate, seminal vesicles (SVs), and rectum during computed tomography (CT) image-guided proton therapy for prostate cancer and studied the range variation in opposed lateral proton beams. MATERIALS/METHODS: We analyzed 375 sets of daily CT images acquired throughout the proton therapy treatment of ten patients. We analyzed daily movements of the prostate, SVs, and rectum by simulating three image-matching strategies: bone matching, prostate center (PC) matching, and prostate-rectum boundary (PRB) matching. In the PC matching, translational movements of the prostate center were corrected after bone matching. In the PRB matching, we performed PC matching and correction along the anterior-posterior direction to match the boundary between the prostate and the rectum's anterior region. In each strategy, we evaluated systematic errors (Σ) and random errors (σ) by measuring the daily movements of certain points on each anatomic structure. The average positional deviations in millimeter of each point were determined by the Van Herk formula of 2.5Σ + 0.7σ. Using these positional deviations, we created planning target volumes of the prostate and SVs and analyzed the daily variation in the water equivalent length (WEL) from the skin surface to the target along the lateral beam directions using the density converted from the daily CT number. Based on this analysis, we designed prostate cancer treatment planning and evaluated the dose volume histograms (DVHs) for these strategies. RESULTS: The SVs' daily movements showed large variations over the superior-inferior direction, as did the rectum's anterior region. The average positional deviations of the prostate in the anterior, posterior, superior, inferior, and lateral sides (mm) in bone matching, PC matching, and PRB matching were (8.9, 9.8, 7.5, 3.6, 1.6), (5.6, 6.1, 3.5, 4.5, 1.9), and (8.6, 3.2, 3.5, 4.5, 1.9) (mm), respectively. Moreover, the ones of the SV tip were similarly (22.5, 15.5, 11.0, 7.6, 6.0), (11.8, 8.4, 7.8, 5.2, 6.3), and (9.9, 7.5, 7.8, 5.2, 6.3). PRB matching showed the smallest positional deviations at all portions except for the anterior portion of the prostate and was able to markedly reduce the positional deviations at the posterior portion. The averaged WEL variations at the distal and proximal sides of planning target volumes were estimated 7-9 mm and 4-6 mm, respectively, and showed the increasing of a few millimeters in PC and PRB matching compared to bone matching. In the treatment planning simulation, the DVH values of the rectum in PRB matching were reduced compared to those obtained with other matching strategies. CONCLUSION: The positional deviations for the prostate on the posterior side and the SVs were smaller by PRB matching than the other strategies and effectively reduced the rectal dose. 3D dose calculations indicate that PRB matching with CT image guidance may do a better job relative to other positioning methods to effectively reduce the rectal complications. The WEL variation was quite large, and the appropriate margin (approx. 10 mm) must be adapted to the proton range in an initial planning to maintain the coverage of target volumes throughout entire treatment.

Development of the breast immobilization system in prone setup: The effect of bra in prone position to improve the breast setup error.

PURPOSE/OBJECTIVE(S): Accurate and reproducible positioning of the breast is difficult due to its deformability and softness; thus, targeting a breast tumor or tumor bed with fractionated radiotherapy using external beam radiation is difficult. The aim of this study was to develop a novel bra to aid in breast immobilization in the prone position. MATERIALS & METHODS: To assess the accuracy of prone position fixation of breast tumors, 33 breast cancer patients with 34 lesions were recruited. The bra used in this verification was customized from a commercially available bra. Duplicate MRI were acquired in the prone position, alternating with and without the bra, and for each series, patients were asked to step off the MRI table and re-set up in the prone position. Patients were also asked to remove and re-fit the bra for the second MRI. Each pair of images were superimposed to match the shape of the skin surface, and the maximum difference in tumor geometric center in three axes was measured. The required set up margin was calculated as: required margin = mean difference in geometric center + 2.5 standard deviation. The volumetric overlap of the tumor, as well as contouring uncertainties, was evaluated using contour analysis software. RESULTS: The median breast size was 498 cc. The required margins for the lateral, vertical, and longitudinal directions were estimated to be 4.1, 4.1, and 5.0 mm, respectively, with the bra, and 5.1, 6.9, and 6.7 mm, respectively, without the bra. These margins covered the dislocation of more than 33 lesions in total. With the bra, 33 lesions had achieved an objective overlap of 95% and 99% with 2 and 4 mm margins, respectively, whereas 4 and 8 mm, respectively, were needed without the bra. CONCLUSION: The use of an immobilizing bra reduced the setup margin for prone position fixation of breast tumors.

Reprint of localized dose delivering by ion beam irradiation for experimental trial of establishing brain necrosis model.

Localized dose delivery techniques to establish a brain radiation necrosis model are described. An irradiation field was designed by using accelerated protons or helium ions with a spread-out Bragg peak. Measurement of the designed field confirmed that a high dose can be confined to a local volume of an animal brain. The irradiation techniques described here are very useful for establishing a necrosis model without existence of extraneous complications.

Localized dose delivering by ion beam irradiation for experimental trial of establishing brain necrosis model.

Localized dose delivery techniques to establish a brain radiation necrosis model are described. An irradiation field was designed by using accelerated protons or helium ions with a spread-out Bragg peak. Measurement of the designed field confirmed that a high dose can be confined to a local volume of an animal brain. The irradiation techniques described here are very useful for establishing a necrosis model without existence of extraneous complications.

Localized radiation necrosis model in mouse brain using proton ion beams.

Brain radiation necrosis is the most serious late adverse event that occurs after 6 months following radiation therapy. Effective treatment for this irreversible brain necrosis has not been established yet. This study tries to establish brain radiation necrosis mouse model using proton or helium beam. The right cerebral hemispheres of C57BL/6J mouse brains were irradiated at doses of 40, 50, 60 Gy with charged particles. In 60 Gy group, brain necrosis that recapitulates human disease was detected after 8 months.

Predictive Value of Early-Stage Uptake of 3'-Deoxy-3'-18F-Fluorothymidine in Cancer Cells Treated with Charged Particle Irradiation.

UNLABELLED: The aim of this study was to investigate whether 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) can monitor the early response of tumor cell proliferation to charged particle irradiation in vitro and in vivo. METHODS: In vitro, after 0.1, 0.5, 1, 5, and 10 Gy of proton or carbon ion irradiation, (18)F-FLT cell uptake was examined at 24 h and cell proliferation ability was measured from days 1 to 4. In vivo, after 0.5, 1, and 5 Gy of proton or carbon ion irradiation, (18)F-FLT PET imaging was performed on tumor-bearing BALB/c nu/nu mice at 24 h and tumor growth was measured from days 1 to 7. Tumor-to-background ratios of standardized uptake values were calculated to assess the (18)F-FLT accumulation in tumors. Both cells and mice also received x-irradiation as a control. RESULTS: In vitro, (18)F-FLT cell uptake was significantly lower after 1 Gy of proton irradiation (P < 0.05) and carbon ion irradiation (P < 0.05) and after 5 Gy of x-irradiation (P < 0.01), but cell proliferation ability at these doses did not show significant differences until day 3. In vivo, (18)F-FLT tumor uptake was significantly lower after 1 Gy of proton (P < 0.001) and carbon ion irradiation (P < 0.01) and after 5 Gy of x-irradiation (P < 0.001), but tumor growth did not significantly differ at these doses until day 4 after proton irradiation, day 3 after carbon ion irradiation, and day 5 after x-irradiation. CONCLUSION: The reduction in (18)F-FLT uptake after charged particle irradiation was more rapid than the change in tumor growth in vivo or the change in cell proliferation ability in vitro. Therefore, (18)F-FLT is a promising tracer for monitoring the early response of cancer to charged particle irradiation.

Relative biological effectiveness of therapeutic proton beams for HSG cells at Japanese proton therapy facilities.

We investigated the relative biological effectiveness (RBE) of therapeutic proton beams at six proton facilities in Japan with respect to cell lethality of HSG cells. The RBE of treatments could be determined from experimental data. For this purpose, we used a cell survival assay to compare the cell-killing efficiency of proton beams. Among the five linear accelerator (LINAC) X-ray machines at 4 or 6 MeV that were used as reference beams, there was only a small variation (coefficient of variation CV = 3.1% at D10) in biological effectiveness. The averaged value of D10 for the proton beams at the middle position of the spread-out Bragg peak (SOBP) was 4.98. These values showed good agreement, with a CV of 4.3% among the facilities. Thus, the average RBE10 (RBE at the D10 level) at the middle position of the SOBP beam for six facilities in Japan was 1.05 with a CV of 2.8%.

Development of a therapeutically important radiation induced promoter.

Radio-genetic therapy is a combination of radiation therapy and gene therapy that may solve some of the problems associated with conventional radiotherapy. A promoter responsive to radiation was obtained from a promoter library composed of DNA fragments created by linking the TATA box signal to randomly combined binding sequences of transcription factors that are reactive to radiation. Each promoter connected to the luciferase gene, was evaluated by luciferase expression enhancement in transfected cells after X-ray irradiation. The reactivity of the best promoter was improved by the random introduction of point mutations and the resultant promoter showed more than a 20-fold enhancement of the luciferase expression after X-ray irradiation at 10 Gy. The expression of downstream genes was also enhanced in stably transfected cells not only by X-rays but also by proton beam irradiation; and either enhancement was attenuated when an anti-oxidant was added, thus suggesting the involvement of oxidative stress in the promoter activation. Constructed promoters were also activated in tumors grown in mice. In addition, cell killing with the fcy::fur gene (a suicide gene converting 5-fluorocytosin to highly toxic 5-fluorouracil) increased dose-dependently with 5-fluorocytosin only after X-ray irradiation in vitro. These results suggest that promoters obtained through this method could be used for possible clinical applications.

Regulation of gene expression in retrovirus vectors by X-ray and proton beam radiation with artificially constructed promoters.

BACKGROUND: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated. METHODS: Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8. RESULTS: Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin. CONCLUSIONS: Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.