Delta radiomics analysis of Magnetic Resonance guided radiotherapy imaging data can enable treatment response prediction in pancreatic cancer.

BACKGROUND: Magnetic Resonance Image guided Stereotactic body radiotherapy (MRgRT) is an emerging technology that is increasingly used in treatment of visceral cancers, such as pancreatic adenocarcinoma (PDAC). Given the variable response rates and short progression times of PDAC, there is an unmet clinical need for a method to assess early RT response that may allow better prescription personalization. We hypothesize that quantitative image feature analysis (radiomics) of the longitudinal MR scans acquired before and during MRgRT may be used to extract information related to early treatment response. METHODS: Histogram and texture radiomic features (n = 73) were extracted from the Gross Tumor Volume (GTV) in 0.35T MRgRT scans of 26 locally advanced and borderline resectable PDAC patients treated with 50 Gy RT in 5 fractions. Feature ratios between first (F1) and last (F5) fraction scan were correlated with progression free survival (PFS). Feature stability was assessed through region of interest (ROI) perturbation. RESULTS: Linear normalization of image intensity to median kidney value showed improved reproducibility of feature quantification. Histogram skewness change during treatment showed significant association with PFS (p = 0.005, HR = 2.75), offering a potential predictive biomarker of RT response. Stability analyses revealed a wide distribution of feature sensitivities to ROI delineation and was able to identify features that were robust to variability in contouring. CONCLUSIONS: This study presents a proof-of-concept for the use of quantitative image analysis in MRgRT for treatment response prediction and providing an analysis pipeline that can be utilized in future MRgRT radiomic studies.

Dose-mass inverse optimization for minimally moving thoracic lesions.

In the past decade, several different radiotherapy treatment plan evaluation and optimization schemes have been proposed as viable approaches, aiming for dose escalation or an increase of healthy tissue sparing. In particular, it has been argued that dose-mass plan evaluation and treatment plan optimization might be viable alternatives to the standard of care, which is realized through dose-volume evaluation and optimization. The purpose of this investigation is to apply dose-mass optimization to a cohort of lung cancer patients and compare the achievable healthy tissue sparing to that one achievable through dose-volume optimization. Fourteen non-small cell lung cancer (NSCLC) patient plans were studied retrospectively. The range of tumor motion was less than 0.5 cm and motion management in the treatment planning process was not considered. For each case, dose-volume (DV)-based and dose-mass (DM)-based optimization was performed. Nine-field step-and-shoot IMRT was used, with all of the optimization parameters kept the same between DV and DM optimizations. Commonly used dosimetric indices (DIs) such as dose to 1% the spinal cord volume, dose to 50% of the esophageal volume, and doses to 20 and 30% of healthy lung volumes were used for cross-comparison. Similarly, mass-based indices (MIs), such as doses to 20 and 30% of healthy lung masses, 1% of spinal cord mass, and 33% of heart mass, were also tallied. Statistical equivalence tests were performed to quantify the findings for the entire patient cohort. Both DV and DM plans for each case were normalized such that 95% of the planning target volume received the prescribed dose. DM optimization resulted in more organs at risk (OAR) sparing than DV optimization. The average sparing of cord, heart, and esophagus was 23, 4, and 6%, respectively. For the majority of the DIs, DM optimization resulted in lower lung doses. On average, the doses to 20 and 30% of healthy lung were lower by approximately 3 and 4%, whereas lung volumes receiving 2000 and 3000 cGy were lower by 3 and 2%, respectively. The behavior of MIs was very similar. The statistical analyses of the results again indicated better healthy anatomical structure sparing with DM optimization. The presented findings indicate that dose-mass-based optimization results in statistically significant OAR sparing as compared to dose-volume-based optimization for NSCLC. However, the sparing is case-dependent and it is not observed for all tallied dosimetric endpoints.

Clinical feasibility of TBI with helical tomotherapy.

Our purpose was to present the clinical feasibility of TBI with helical tomotherapy (HT) in four patients with AML. Treatment planning, delivery, dose verification and summation, toxicity and patient outcomes for each patient are presented. TBI prescription was set in such a manner that 80% of the clinical target volume received 12 Gy in six fractions, at two fractions per day. Dose reconstruction was carried out by recontouring the regions of interest in the daily pretreatment megavoltage computed tomography of each individual fraction and calculating its corresponding dose. A deformable registration model was used for dose summation of all individual fractions. Differences between planned and delivered doses were calculated. Average planned and delivered doses to the regions of interest differed by up to 2.7%. TBI toxicity was limited to radiotherapy oncology group grade 1 dermatitis in all patients and grade 1 headache in one patient. Two patients are alive with no evidence of disease and no GVHD. Two patients died of GVHD, but there was no evidence of disease at the time of death. We conclude that HT simplifies the process of TBI. Dose verification is possible with HT showing small differences between plan and delivered doses.

Altered calcium dynamics mediates P19-derived neuron-like cell responses to millimeter-wave radiation.

Intracellular calcium oscillations have long been recognized as a principal mediator of many vital cellular activities. Furthermore, Ca(2+) dynamics can be modulated by external physical cues, including electromagnetic fields. While cellular responses to low-frequency electric fields have been established, the possible non-thermal effects of millimeter-wave (MMW) radiation are still a subject of discussion and debate. We used mouse embryonic stem cell-derived neuronal cells and a custom-built 94 GHz applicator to examine in real time the altered Ca(2+) oscillations associated with MMW stimulation. MMW irradiation at 18.6 kW/m(2) nominal power density significantly increased the Ca(2+) spiking frequency in the cells exhibiting Ca(2+) activity. The N-type calcium channels, phospholipase C enzyme, and actin cytoskeleton appear to be involved in mediating increased Ca(2+) spiking. Reorganization of the actin microfilaments by a 94 GHz field seems to play a crucial role in modulating not only Ca(2+) activity but also cell biomechanics. Many but not all observed cellular responses to MMW were similar to thermally induced effects. For example, cell exposure to a 94 GHz field induced nitric oxide production in some morphologically distinct neuronal cells that could not be reproduced by thermal heating of the cells up to 42 degrees C. The highest observed average temperature rise in the MMW exposure chamber was approximately 8 degrees C above the room temperature, with possible complex non-uniform microscopic distribution of heating rates at the cell level. Our findings may be useful to establish quantitative molecular benchmarks for elucidation of nociception mechanisms and evaluation of potential adverse bioeffects associated with MMW exposure. Moreover, control of Ca(2+) dynamics by MMW stimulation may offer new tools for regulation of Ca(2+)-dependent cellular and molecular activities, for example, in tissue engineering applications.

PET imaging of heat-inducible suicide gene expression in mice bearing head and neck squamous cell carcinoma xenografts.

The ability to achieve tumor selective expression of therapeutic genes is an area that needs improvement for cancer gene therapy to be successful. One approach to address this is through the use of promoters that can be controlled by external means, such as hyperthermia. In this regard, we constructed a replication-deficient adenovirus that consists of a mutated herpes simplex virus 1 thymidine kinase (mTK) fused to enhanced green fluorescent protein (EGFP) under the control of the full-length human heat shock (HS) 70b promoter. The virus (AdHSmTK-EGFP) was evaluated both in vitro and in vivo in oral squamous cell carcinoma SCC-9 cells for expression of both mTK and EGFP. The in vitro expression of mTK-EGFP was validated using both (3)H-penciclovir and fluorescence-activated cell sorting assays. These studies show that specific expression could be achieved by heating the cells at 41 degrees C for 1 h, whereas little expression was observed using high doses of virus without hyperthermia. The vector was also evaluated in vivo by direct intratumoral injection into mice bearing SCC-9 xenografts. These studies demonstrated tumor expression of mTK-EGFP after ultrasound heating of the tumors by radioactive biodistribution assays, histology and microPET imaging. These in vivo results, which demonstrate HS-inducible transgene expression using PET imaging, provide a means for noninvasive monitoring of heat-induced gene therapy in local tumors, such as oral squamous cell carcinomas.

Modeling of carbon fiber couch attenuation properties with a commercial treatment planning system.

The purpose of this work is to evaluate the modeling of carbon fiber couch attenuation properties with a commercial treatment planning system (TPS, Pinnacle3, v8.0d). A carbon fiber couch (Brain-Lab) was incorporated into the TPS by automatic contouring of all transverse CT slices. The couch shape and dimensions were set according to the vendor specifications. The couch composition was realized by assigning appropriate densities to the delineated contours. The couch modeling by the TPS was validated by absolute dosimetric measurements. A phantom consisting of several solid water slabs was CT scanned, the CT data set was imported into the TPS, and the carbon fiber couch was auto-contoured. Open (unblocked) field plans for different gantry angles and field sizes were generated. The doses to a point at 3 cm depth, placed at the linac isocenter, were computed. The phantom was irradiated according to the dose calculation setup and doses were measured with an ion chamber. In addition, percent depth dose (PDD) curves were computed as well as measured with radiographic film. The calculated and measured doses, transmissions, and PDDs were cross-compared. Doses for several posterior fields (0 degree, 30 degrees, 50 degrees, 75 degrees, 83 degrees) were calculated for 6 and 18 MV photon beams. For model validation a nominal field size of 10 x 10 cm2 was chosen and 100 MU were delivered for each portal. The largest difference between computed and measured doses for those posterior fields was within 1.7%. A comparison between computed and measured transmissions for the aforementioned fields was performed and the results were found to agree within 1.1%. The differences between computed and measured doses for different field sizes, ranging from 5 x 5 cm2 to 25 x 25 cm2 in 5 cm increments, were within 2%. Measured and computed PDD curves with and without the couch agree from the surface up to 30 cm depth. The PDDs indicate a surface dose increase resulting from the carbon fiber couch field modification. The carbon fiber couch attenuation for individual posterior oblique fields (75 degrees) can be in excess of 8% depending on the beam energy and field size. When the couch is contoured in Pinnacle3 its attenuation properties are modeled to within 1.7% with respect to measurements. These results demonstrate that appropriate contouring together with relevant density information for the contours is sufficient for adequate modeling of carbon fiber supporting devices by modern commercial treatment planning systems.

Nonthermal effects of radiofrequency-field exposure on calcium dynamics in stem cell-derived neuronal cells: elucidation of calcium pathways.

Intracellular Ca(2+) spikes trigger cell proliferation, differentiation and cytoskeletal reorganization. In addition to Ca(2+) spiking that can be initiated by a ligand binding to its receptor, exposure to electromagnetic stimuli has also been shown to alter Ca(2+) dynamics. Using neuronal cells differentiated from a mouse embryonic stem cell line and a custom-built, frequency-tunable applicator, we examined in real time the altered Ca(2+) dynamics and observed increases in the cytosolic Ca(2+) in response to nonthermal radiofrequency (RF)-radiation exposure of cells from 700 to 1100 MHz. While about 60% of control cells (not exposed to RF radiation) were observed to exhibit about five spontaneous Ca(2+) spikes per cell in 60 min, exposure of cells to an 800 MHz, 0.5 W/kg RF radiation, for example, significantly increased the number of Ca(2+) spikes to 15.7+/-0.8 (P<0.05). The increase in the Ca(2+) spiking activities was dependent on the frequency but not on the SAR between 0.5 to 5 W/kg. Using pharmacological agents, it was found that both the N-type Ca(2+) channels and phospholipase C enzymes appear to be involved in mediating increased Ca(2+) spiking. Interestingly, microfilament disruption also prevented the Ca(2+) spikes. Regulation of Ca(2+) dynamics by external physical stimulation such as RF radiation may provide a noninvasive and useful tool for modulating the Ca(2+)-dependent cellular and molecular activities of cells seeded in a 3D environment for which only a few techniques are currently available to influence the cells.

The effects of 41 degrees C hyperthermia on the DNA repair protein, MRE11, correlate with radiosensitization in four human tumor cell lines.

PURPOSE: The goal of this study was to determine if reduced availability of the DNA repair protein, MRE11, for the repair of damaged DNA is a basis for thermal radiosensitization induced by moderate hyperthermia. To test this hypothesis, we measured the total amount of MRE11 DNA repair protein and its heat-induced alterations in four human tumor cell lines requiring different heating times at 41 degrees C to induce measurable radiosensitization. MATERIALS AND METHODS: Human colon adenocarcinoma cell lines (NSY42129, HT29 and HCT15) and HeLa cells were used as the test system. Cells were irradiated immediately after completion of hyperthermia. MRE11 levels in whole cell extract, nuclear extract and cytoplasmic extracts were measured by Western blotting. The nuclear and cytoplasmic extracts were separated by TX100 solubility. The subcellular localization of MRE11 was determined by immunofluorescence staining. RESULTS: The results show that for the human tumor cell lines studied, the larger the endogenous amount of MRE11 protein per cell, the longer the heating time at 41 degrees C required for inducing measurable radiosensitization in that cell line. Further, the residual nuclear MRE11 protein level, measured in the nuclear extract and in the cytoplasmic extract as a function of heating time, both correlated with the thermal enhancement ratio (TER). CONCLUSIONS: These observations are consistent with the possibility that delocalization of MRE11 from the nucleus is a critical step in the radiosensitization by moderate hyperthermia.

Gene expression does not change significantly in C3H 10T(1/2) cells after exposure to 847.74 CDMA or 835.62 FDMA radiofrequency radiation.

In vitro experiments with C3H 10T(1/2) mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.

Experience with a small animal hyperthermia ultrasound system (SAHUS): report on 83 tumours.

An external local ultrasound (US) system was developed to induce controlled hyperthermia of subcutaneously implanted tumours in small animals (e.g., mice and rats). It was designed to be compatible with a small animal positron emission tomography scanner (microPET) to facilitate studies of hyperthermia-induced tumour re-oxygenation using a PET radiopharmaceutical, but it is applicable for any small animal study requiring controlled heating. The system consists of an acrylic applicator bed with up to four independent 5 MHz planar disc US transducers of 1 cm in diameter, a four-channel radiofrequency (RF) generator, a multiple thermocouple thermometry unit, and a personal computer with custom monitoring and controlling software. Although the system presented here was developed to target tumours of up to 1 cm in diameter, the applicator design allows for different piezoelectric transducers to be exchanged and operated within the 3.5-6.5 MHz band to target different tumour sizes. Temperature feedback control software was developed on the basis of a proportional-integral-derivative (PID) approach when the measured temperatures were within a selectable temperature band about the target temperature. Outside this band, an on/off control action was applied. Perfused tissue-mimicking phantom experiments were performed to determine optimum controller gain constants, which were later employed successfully in animal experiments. The performance of the SAHUS (small animal hyperthermia ultrasound system) was tested using several tumour types grown in thighs of female nude (nu/nu) mice. To date, the system has successfully treated 83 tumours to target temperatures in the range of 41-43 degrees C for periods of 65 min on average.

Non-invasive estimation of hyperthermia temperatures with ultrasound.

Ultrasound is an attractive modality for temperature monitoring because it is non-ionizing, convenient, inexpensive and has relatively simple signal processing requirements. This modality may be useful for temperature estimation if a temperature-dependent ultrasonic parameter can be identified, measured and calibrated. The most prominent methods for using ultrasound as a non-invasive thermometer exploit either (1) echo shifts due to changes in tissue thermal expansion and speed of sound (SOS), (2) variation in the attenuation coefficient or (3) change in backscattered energy from tissue inhomogeneities. The use of echo shifts has received the most attention in the last decade. By tracking scattering volumes and measuring the time shift of received echoes, investigators have been able to predict the temperature from a region of interest both theoretically and experimentally in phantoms, in isolated tissue regions in vitro and preliminary in vivo studies. A limitation of this method for general temperature monitoring is that prior knowledge of both SOS and thermal-expansion coefficients is necessary. Acoustic attenuation is dependent on temperature, but with significant changes occurring only at temperatures above 50 degrees C, which may lead to its use in thermal ablation therapies. Minimal change in attenuation, however, below this temperature range reduces its attractiveness for use in clinical hyperthermia. Models and measurements of the change in backscattered energy suggest that, over the clinical hyperthermia temperature range, changes in backscattered energy are dependent on the properties of individual scatterers or scattering regions. Calibration of the backscattered energy from different tissue regions is an important goal of this approach. All methods must be able to cope with motion of the image features on which temperature estimates are based. A crucial step in identifying a viable ultrasonic approach to temperature estimation is its performance during in vivo tests.

Localized versus regional hyperthermia: comparison of xenotransplants treated with a small animal ultrasound system and waterbath limb immersion.

The response of xenotransplants were compared with waterbath immersion vs focal ultrasound (US) hyperthermia using tumour growth delay, immunhistochemistry and histopathology assays. Waterbath hyperthermia was performed by limb immersion. Precautions were taken to minimize total body heating by surrounding the mouse with plastic insulators. Thermometry was performed with clinical-grade, 20-gauge needle thermocouples and monitored with a Labthermics unit. Significant differences in cytotoxicity between ultrasound and waterbath treatment of tumors at 43 degrees C were observed as determined by TUNNEL assay. Conversely, contralateral (non-treated) tumours in animals treated with similar temperature demonstrated no significant differences between modalities. Western blot analysis revealed increased hsp70 induction at 43 degrees C in waterbath vs focal ultrasound hyperthermia. Comparison of tumour growth delay between tumours heated with waterbath vs ultrasound at 43 degrees C but not at 41 degrees C revealed significant differences. This is the first study comparing localized vs regional hyperthermia using the small animal ultrasound system (SAHUS) delivery system. Consistent ultrasound hyperthermia can be achieved throughout a xenotransplant. At equivalent temperature of 43 degrees C for 60?min, waterbath hyperthermia demonstrated greater local response vs ultrasound hyperthermia.

Transfection of human tumour cells with Mre11 siRNA and the increase in radiation sensitivity and the reduction in heat-induced radiosensitization.

Double-strand DNA breaks (DSBs) are potentially lethal DNA lesions induced by ionizing radiation. In eukaryotes, DSBs can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). DNA repair protein Mre11 participates in both the NHEJ and HR DNA repair pathways. Hyperthermia has been used clinically as a radiosensitizer. However, the mechanisms by which radiosensitization is induced by hyperthermia, especially moderate hyperthermia (41 degrees C) are not fully understood. Previous studies suggest that 41 degrees C reduces the nuclear Mre11 protein level in a manner that correlates with heat-induced changes in radiation sensitivity. Therefore, siRNA technology was used in the present study to reduce Mre11 gene expression to determine if reduced Mre11 protein levels induced radiosensitization and if such radiosensitization is similar to that induced by moderate hyperthermia. The results show that (1) the cellular level of the Mre11 protein was reduced about 60 +/- 18% by a 24-h treatment with siRNA. Results from the Mre11 protein turnover assay showed a half-life of 11.6 +/- 0.5 h for the Mre11 protein, which is consistent with reduction in protein level in 24 h after Mre11 siRNA treatment assuming a delay of 4-8 h to reduce RNA levels. After 48 h in siRNA, cellular Mre11 protein levels increased to approximately pretreatment levels. NSY cells were sensitized to ionizing radiation after 24 h of treatment with Mre11 siRNA. Two hours at 41 degrees C did not increase the radiation sensitivity of cells with a reduced Mre11 protein level following a 24-h siRNA treatment. These data support the conclusion that the DSB repair protein, Mre11, appears to be a target for radiosensitization by moderate hyperthermia.