R Berbeco

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

Residual motion of lung tumours in gated radiotherapy with external respiratory surrogates

Due to respiration, many tumours in the thorax and abdomen may move as much as 3 cm peak-to-peak during radiation treatment. To mitigate motion-induced irradiation of normal lung tissue, clinics have employed external markers to gate the treatment beam. This technique assumes that the correlation between the external surface and the internal tumour position remains constant inter-fractionally and intra-fractionally. In this work, a study has been performed to assess the validity of this correlation assumption for external surface based gated radiotherapy, by measuring the residual tumour motion within a gating window. Eight lung patients with implanted fiducial markers were studied at the NTT Hospital in Sapporo, Japan. Synchronized internal marker positions and external abdominal surface positions were measured during the entire course of treatment. Stereoscopic imaging was used to find the internal markers in four dimensions. The data were used retrospectively to assess conventional external surrogate respiratory-gated treatment. Both amplitude- and phase-based gating methods were investigated. For each method, three gating windows were investigated, each giving 40%, 30% and 20% duty cycle, respectively. The residual motion of the internal marker within these six gating windows was calculated. The beam-to-beam variation and day-to-day variation in the residual motion were calculated for both gating modalities. We found that the residual motion (95th percentile) was between 0.7 and 5.8 mm, 0.8 and 6.0 mm, and 0.9 and 6.2 mm for 20%, 30% and 40% duty cycle windows, respectively. Five of the eight patients showed less residual motion with amplitude-based gating than with phase-based gating. Large fluctuations (>300%) were seen in the residual motion between some beams. Overall, the mean beam-to-beam variation was 37% and 42% from the previous treatment beam for amplitude- and phase-based gating, respectively. The day-to-day variation was 29% and 34% from the previous day for amplitude- and phase-based gating, respectively. Although gating reduced the total tumour motion, the residual motion behaved unpredictably. Residual motion during treatment could exceed that which might have been considered in the treatment plan. Treatment margins that account for motion should be individualized and daily imaging should be performed to ensure that the residual motion is not exceeding the planned motion on a given day.

Accuracy of tumor motion compensation algorithm from a robotic respiratory tracking system: a simulation study.

The Synchrony Respiratory Tracking System (RTS) is a treatment option of the CyberKnife robotic treatment device to irradiate extra-cranial tumors that move due to respiration. Advantages of RTS are that patients can breath normally and that there is no loss of linac duty cycle such as with gated therapy. Tracking is based on a measured correspondence model (linear or polynomial) between internal tumor motion and external (chest/abdominal) marker motion. The radiation beam follows the tumor movement via the continuously measured external marker motion. To establish the correspondence model at the start of treatment, the 3D internal tumor position is determined at 15 discrete time points by automatic detection of implanted gold fiducials in two orthogonal x-ray images; simultaneously, the positions of the external markers are measured. During the treatment, the relationship between internal and external marker positions is continuously accounted for and is regularly checked and updated. Here we use computer simulations based on continuously and simultaneously recorded internal and external marker positions to investigate the effectiveness of tumor tracking by the RTS. The Cyberknife does not allow continuous acquisition of x-ray images to follow the moving internal markers (typical imaging frequency is once per minute). Therefore, for the simulations, we have used data for eight lung cancer patients treated with respiratory gating. All of these patients had simultaneous and continuous recordings of both internal tumor motion and external abdominal motion. The available continuous relationship between internal and external markers for these patients allowed investigation of the consequences of the lower acquisition frequency of the RTS. With the use of the RTS, simulated treatment errors due to breathing motion were reduced largely and consistently over treatment time for all studied patients. A considerable part of the maximum reduction in treatment error could already be reached with a simple linear model. In case of hysteresis, a polynomial model added some extra reduction. More frequent updating of the correspondence model resulted in slightly smaller errors only for the few recordings with a time trend that was fast, relative to the current x-ray update frequency. In general, the simulations suggest that the applied combined use of internal and external markers allow the robot to accurately follow tumor motion even in the case of irregularities in breathing patterns.

Internal-external correlation investigations of respiratory induced motion of lung tumors

In gated radiation therapy procedures, the lung tumor position is used directly (by implanted radiopaque markers) or indirectly (by external surrogate methods) to decrease the volume of irradiated healthy tissue. Due to a risk of pneumothorax, many clinics do not implant fiducials, and the gated treatment is primarily based on a respiratory induced external signal. The external surrogate method relies upon the assumption that the internal tumor motion is well correlated with the external respiratory induced motion, and that this correlation is constant in time. Using a set of data that contains synchronous internal and external motion traces, we have developed a dynamic data analysis technique to study the internal-external correlation, and to quantitatively estimate its underlying time behavior. The work presented here quantifies the time dependent behavior of the correlation between external respiratory signals and lung implanted fiducial motion. The corresponding amplitude mismatch is also reported for the lung patients studied. The information obtained can be used to improve the accuracy of tumor tracking. For the ten patients in this study, the SI internal-external motion is well correlated, with small time shifts and corresponding amplitude mismatches. Although the AP internal-external motion reveals larger time shifts than along the SI direction, the corresponding amplitude mismatches are below 5 mm.

Synchronized moving aperture radiation therapy (SMART): improvement of breathing pattern reproducibility using respiratory coaching

Recently, at Massachusetts General Hospital (MGH) we proposed a new treatment technique called synchronized moving aperture radiation therapy (SMART) to account for tumour motion during radiotherapy. The basic idea of SMART is to synchronize the moving radiation beam aperture formed by a dynamic multileaf collimator with the tumour motion induced by respiration. The two key requirements for being able to successfully use SMART in clinical practice are the precise and fast detection of tumour position during the simulation/treatment and the good reproducibility of the tumour motion pattern. To fulfil the first requirement, an integrated radiotherapy imaging system is currently being developed at MGH. The results of a previous study show that breath coaching techniques are required to make SMART an efficient technique in general. In this study, we investigate volunteer and patient respiratory coaching using a commercial respiratory gating system as a respiration coaching tool. Five healthy volunteers, observed during six sessions, and 33 lung cancer patients, observed during one session when undergoing 4D CT scans, were investigated with audio and visual promptings, with free breathing as a control. For all five volunteers, breath coaching was well tolerated and the intra- and inter-session reproducibility of the breathing pattern was greatly improved. Out of 33 patients, six exhibited a regular breathing pattern and needed no coaching, four could not be coached at all due to the patients medical condition or had difficulty following the instructions, 13 could only be coached with audio instructions and 10 could follow the instructions of and benefit from audio-video coaching. We found that, for all volunteers and for those patients who could be properly coached, breath coaching improves the duty cycle of SMART treatment. However, about half of the patients could not follow both audio and video instructions simultaneously, suggesting that the current coaching technique requires improvements.

Evaluation of the combined effects of target size, respiratory motion and background activity on 3D and 4D PET/CT images

Gated (4D) PET/CT has the potential to greatly improve the accuracy of radiotherapy at treatment sites where internal organ motion is significant. However, the best methodology for applying 4D-PET/CT to target definition is not currently well established. With the goal of better understanding how to best apply 4D information to radiotherapy, initial studies were performed to investigate the effect of target size, respiratory motion and target-to-background activity concentration ratio (TBR) on 3D (ungated) and 4D PET images. Using a PET/CT scanner with 4D or gating capability, a full 3D-PET scan corrected with a 3D attenuation map from 3D-CT scan and a respiratory gated (4D) PET scan corrected with corresponding attenuation maps from 4D-CT were performed by imaging spherical targets (0.5-26.5 mL) filled with (18)F-FDG in a dynamic thorax phantom and NEMA IEC body phantom at different TBRs (infinite, 8 and 4). To simulate respiratory motion, the phantoms were driven sinusoidally in the superior-inferior direction with amplitudes of 0, 1 and 2 cm and a period of 4.5 s. Recovery coefficients were determined on PET images. In addition, gating methods using different numbers of gating bins (1-20 bins) were evaluated with image noise and temporal resolution. For evaluation, volume recovery coefficient, signal-to-noise ratio and contrast-to-noise ratio were calculated as a function of the number of gating bins. Moreover, the optimum thresholds which give accurate moving target volumes were obtained for 3D and 4D images. The partial volume effect and signal loss in the 3D-PET images due to the limited PET resolution and the respiratory motion, respectively were measured. The results show that signal loss depends on both the amplitude and pattern of respiratory motion. However, the 4D-PET successfully recovers most of the loss induced by the respiratory motion. The 5-bin gating method gives the best temporal resolution with acceptable image noise. The results based on the 4D scan protocols can be used to improve the accuracy of determining the gross tumor volume for tumors in the lung and abdomen.

Localized Dose Enhancement to Tumor Blood Vessel Endothelial Cells via Megavoltage X-rays and Targeted Gold Nanoparticles: New Potential for External Beam Radiotherapy

PURPOSE Tumor endothelial cell damage during radiation therapy may contribute significantly to tumor eradication and treatment efficacy. Gold nanoparticles (AuNPs) delivered preferentially to the walls of tumor blood vessels produce low-energy, short-range photoelectrons during external beam radiotherapy, boosting dose to the tumor microvasculature. In this study dosimetry at the single-cell level is used to estimate the anticipated AuNP-mediated dose enhancement to tumor endothelial cells during 6-MV X-ray irradiation. METHODS AND MATERIALS Endothelial cells are modeled as thin slabs with 100-nm-diameter AuNPs attached within the blood vessel. The number of photoelectrons emitted per AuNP per gray of X-rays is computed at multiple points along the external beam central axis by use of a Monte Carlo-generated energy fluence spectrum. The energy deposited from AuNP emissions to the endothelium is calculated based on an analytic method incorporating the energy-loss formula of Cole. The endothelial dose enhancement factor (EDEF) is the ratio of the overall (externally plus internally generated) dose to endothelial cells in the presence of AuNPs to the dose without AuNPs (from the external beam only). RESULTS At 20-cm depth, the EDEF is 1.7 (70% dose increase) for an intravascular AuNP concentration of 30 mg/g. Most of this dose enhancement arises from the low-energy (approximately 100 keV) portion of the linear accelerator X-ray spectrum. Furthermore, for AuNP concentrations ranging from 7 to 140 mg/g, EDEF values of 1.2 to 4.4 (20-340% dose increase) are calculated. CONCLUSIONS In contrast to calculations assuming that AuNPs distributed homogeneously throughout the target volume (macrodosimetry), our cellular microdosimetry calculations predict a major dose enhancement to tumor microvasculature from conventional linear accelerator X-rays. This effect may enable the delivery of ablative therapeutic doses to these sensitive microstructures while maintaining established dose constraints for the organs at risk.

Clinical Utility of 4D FDG-PET/CT Scans in Radiation Treatment Planning

PURPOSE The potential role of four-dimensional (4D) positron emission tomography (PET)/computed tomography (CT) in radiation treatment planning, relative to standard three-dimensional (3D) PET/CT, was examined. METHODS AND MATERIALS Ten patients with non-small-cell lung cancer had sequential 3D and 4D [(18)F]fluorodeoxyglucose PET/CT scans in the treatment position prior to radiation therapy. The gross tumor volume and involved lymph nodes were contoured on the PET scan by use of three different techniques: manual contouring by an experienced radiation oncologist using a predetermined protocol; a technique with a constant threshold of standardized uptake value (SUV) greater than 2.5; and an automatic segmentation technique. For each technique, the tumor volume was defined on the 3D scan (VOL3D) and on the 4D scan (VOL4D) by combining the volume defined on each of the five breathing phases individually. The range of tumor motion and the location of each lesion were also recorded, and their influence on the differences observed between VOL3D and VOL4D was investigated. RESULTS We identified and analyzed 22 distinct lesions, including 9 primary tumors and 13 mediastinal lymph nodes. Mean VOL4D was larger than mean VOL3D with all three techniques, and the difference was statistically significant (p < 0.01). The range of tumor motion and the location of the tumor affected the magnitude of the difference. For one case, all three tumor definition techniques identified volume of moderate uptake of approximately 1 mL in the hilar region on the 4D scan (SUV maximum, 3.3) but not on the 3D scan (SUV maximum, 2.3). CONCLUSIONS In comparison to 3D PET, 4D PET may better define the full physiologic extent of moving tumors and improve radiation treatment planning for lung tumors. In addition, reduction of blurring from free-breathing images may reveal additional information regarding regional disease.

A technique for respiratory-gated radiotherapy treatment verification with an EPID in cine mode

Respiratory gating based on external surrogates is performed in many clinics. We have developed a new technique for treatment verification using an electronic portal imaging device (EPID) in cine mode for gated 3D conformal therapy. Implanted radiopaque fiducial markers inside or near the target are required for this technique. The markers are contoured on the planning CT set, enabling us to create digitally reconstructed radiographs (DRRs) for each treatment beam. During the treatment, a sequence of EPID images can be acquired without disrupting the treatment. Implanted markers are visualized in the images and their positions in the beams eye view are calculated off-line and compared to the reference position by matching the field apertures in corresponding EPID and DRR images. The precision of the patient set-up, the placement of the beam-gating window, as well as the residual tumour motion can be assessed for each treatment fraction. This technique has been demonstrated with a case study patient, who had three markers implanted in his liver. For this patient, the intra-fractional variation of all marker positions in the gating window had a 95% range of 4.8 mm in the SI direction (the primary axis of motion). This was about the same (5 mm) as the residual motion considered in the planning process. The inter-fractional variation of the daily mean positions of the markers, which indicates the uncertainty in the set-up procedure, was within +8.3 mm/−4.5 mm (95% range) in the SI direction for this case.

The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy

A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.