Preclinical Evaluation of Cetuximab and Benzoporphyrin Derivative-Mediated Intraperitoneal Photodynamic Therapy in a Canine Model.

Peritoneal carcinomatosis (PC) can occur as an advanced consequence of multiple primary malignancies. Surgical resection, radiation or systemic interventions alone have proven inadequate for this aggressive cancer presentation, since PC still has a poor survival profile. Photodynamic therapy (PDT), in which photosensitive drugs are exposed to light to generate cytotoxic reactive oxygen species, may be an ideal treatment for PC because of its ability to deliver treatment to a depth appropriate for peritoneal surface tumors. Additionally, epidermal growth factor receptor (EGFR) signaling plays a variety of roles in cancer progression and survival as well as PDT-mediated cytotoxicity, so EGFR inhibitors may be valuable in enhancing the therapeutic index of intraperitoneal PDT. This study examines escalating doses of benzoporphyrin derivative (BPD)-mediated intraperitoneal PDT combined with the EGFR-inhibitor cetuximab in a canine model. In the presence or absence of small bowel resection (SBR) and cetuximab, we observed a tolerable safety and toxicity profile related to the light dose received. Additionally, our findings that BPD levels are higher in the small bowel compared with other anatomical regions, and that the risk of anastomotic failure decreases at lower light doses will help to inform the design of similar PC treatments in humans.

Five-year Long-term Outcomes of Stereotactic Body Radiation Therapy for Operable Versus Medically Inoperable Stage I Non-small-cell Lung Cancer: Analysis by Operability, Fractionation Regimen, Tumor Size, and Tumor Location.

BACKGROUND: Stereotactic body radiation therapy (SBRT) is standard for medically inoperable stage I non-small-cell lung cancer (NSCLC) and is emerging as a surgical alternative in operable patients. However, limited long-term outcomes data exist, particularly according to operability. We hypothesized long-term local control (LC) and cancer-specific survival (CSS) would not differ by fractionation schedule, tumor size or location, or operability status, but overall survival (OS) would be higher for operable patients. PATIENTS AND METHODS: All consecutive patients with stage I (cT1-2aN0M0) NSCLC treated with SBRT from June 2009 to July 2013 were assessed. Thoracic surgeon evaluation determined operability. Local failure was defined as growth following initial tumor shrinkage or progression on consecutive scans. LC, CSS, and OS were calculated using Cox proportional hazards regression. RESULTS: A total of 186 patients (204 lesions) were analyzed. Most patients were inoperable (82%) with Eastern Cooperative Oncology Group performance status of 1 (59%) or 2 (26%). All lesions received biological effective doses ≥ 100 Gy most commonly (94%) in 3 to 5 fractions. The median follow-up was 4.0 years. LC at 2 and 5 years were 95.6% (95% confidence interval, 92%-99%) and 93.7% (95% confidence interval, 90%-98%), respectively. Compared with operable patients, inoperable patients did not have significant differences in 5-year LC (93.1% vs. 96.7%; P = .49), nodal failure (31.4% vs. 11.0%; P = .12), distant failure (12.2% vs. 10.4%; P = .98), or CSS (80.6% vs. 91.0%; P = .45) but trended towards worse OS (34.2% vs. 45.3%; P = .068). Tumor size, location, and fractionation did not significantly influence outcomes. CONCLUSIONS: SBRT has excellent, durable LC and CSS rates for early-stage NSCLC, although inoperable patients had somewhat lower OS than operable patients, likely owing to greater comorbidities.

Theranostic gold nanoparticles modified for durable systemic circulation effectively and safely enhance the radiation therapy of human sarcoma cells and tumors.

Radiation therapy (RT) is an integral component of the treatment of many sarcomas and relies on accurate targeting of tumor tissue. Despite conventional treatment planning and RT, local failure rates of 10% to 28% at 5 years have been reported for locally advanced, unresectable sarcomas, due in part to limitations in the cumulative RT dose that may be safely delivered. We describe studies of the potential usefulness of gold nanoparticles modified for durable systemic circulation (through polyethylene glycosylation; hereinafter "P-GNPs") as adjuvants for RT of sarcomas. In studies of two human sarcoma-derived cell lines, P-GNP in conjunction with RT caused increased unrepaired DNA damage, reflected by approximately 1.61-fold increase in γ-H2AX (histone phosphorylated on Ser(139)) foci density compared with RT alone. The combined RT and P-GNP also led to significantly reduced clonogenic survival of tumor cells, compared to RT alone, with dose-enhancement ratios of 1.08 to 1.16. In mice engrafted with human sarcoma tumor cells, the P-GNP selectively accumulated in the tumor and enabled durable imaging, potentially aiding radiosensitization as well as treatment planning. Mice pretreated with P-GNP before targeted RT of their tumors exhibited significantly improved tumor regression and overall survival, with long-term survival in one third of mice in this treatment group compared to none with RT only. Interestingly, prior RT of sarcoma tumors increased subsequent extravasation and in-tumor deposition of P-GNP. These results together suggest P-GNP may be integrated into the RT of sarcomas, potentially improving target imaging and radiosensitization of tumor while minimizing dose to normal tissues.

In-vivo light dosimetry for HPPH-mediated pleural PDT.

This study examines the light fluence (rate) delivered to patients undergoing pleural PDT as a function of treatment time, treatment volume and surface area. The accuracy of treatment delivery is analyzed as a function of the calibration accuracies of each isotropic detector and the calibration integrating sphere. The patients studied here are enrolled in a Phase I clinical trial of HPPH-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. Patients are administered 4mg per kg body weight HPPH 24-48 hours before the surgery. Patients undergoing photodynamic therapy (PDT) are treated with light therapy with a fluence of 15-60 J/cm2 at 661nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) is monitored at 7 different sites during the entire light treatment delivery. Isotropic detectors are used for in-vivo light dosimetry. The anisotropy of each isotropic detector was found to be within 15%. The mean fluence rate delivery and treatment time are recorded. A correlation between the treatment time and the treatment volume is established. The result can be used as a clinical guideline for future pleural PDT treatment.

In-vivo Light dosimetry for pleural PDT.

In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is one of the critical dosimetry quantities for predicting PDT outcome. This study examines the relationship between the PDT treatment time and thoracic treatment volume and surface area for patients undergoing pleural PDT. In addition, the mean light fluence (rate) and its accuracy were quantified. The patients studied here were enrolled in Phase II clinical trial of Photofrin-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. The ages of the patients studied varied from 34 to 69 years old. All patients were administered 2mg per kg body weight Photoprin 24 hours before the surgery. Patients undergoing photodynamic therapy (PDT) are treated with laser light with a light fluence of 60 J/cm2 at 630nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) was monitored at 7 different sites during the entire light treatment delivery. Isotropic detectors were used for in-vivo light dosimetry. The anisotropy of each isotropic detector was found to be within 30%. The mean fluence rate deliver varied from 37.84 to 94.05 mW/cm2 and treatment time varied from 1762 to 5232s. We found a linear correlation between the total treatment time and the treatment area: t (sec) = 4.80 A (cm2). A similar correlation exists between the treatment time and the treatment volume: t (sec) = 2.33 V (cm3). The results can be explained using an integrating sphere theory and the measured tissue optical properties assuming that the saline liquid has a mean absorption coefficient of 0.05 cm-1. Our long term accuracy studies confirmed light fluence rate measurement accuracy of ±10%. The results can be used as a clinical guideline for future pleural PDT treatment.

A heterogeneous algorithm for PDT dose optimization for prostate.

The object of this study is to develop optimization procedures that account for both the optical heterogeneity as well as photosensitizer (PS) drug distribution of the patient prostate and thereby enable delivery of uniform photodynamic dose to that gland. We use the heterogeneous optical properties measured for a patient prostate to calculate a light fluence kernel (table). PS distribution is then multiplied with the light fluence kernel to form the PDT dose kernel. The Cimmino feasibility algorithm, which is fast, linear, and always converges reliably, is applied as a search tool to choose the weights of the light sources to optimize PDT dose. Maximum and minimum PDT dose limits chosen for sample points in the prostate constrain the solution for the source strengths of the cylindrical diffuser fibers (CDF). We tested the Cimmino optimization procedures using the light fluence kernel generated for heterogeneous optical properties, and compared the optimized treatment plans with those obtained using homogeneous optical properties. To study how different photosensitizer distributions in the prostate affect optimization, comparisons of light fluence rate and PDT dose distributions were made with three distributions of photosensitizer: uniform, linear spatial distribution, and the measured PS distribution. The study shows that optimization of individual light source positions and intensities are feasible for the heterogeneous prostate during PDT.

Diffuse reflectance spectra measured in vivo in human tissues during Photofrin-mediated pleural photodynamic therapy.

Optimal delivery of light in photodynamic therapy (PDT) requires not only optimal placement and power of light sources, but knowledge of the dynamics of light propagation in the tissue being treated and in the surrounding normal tissue, and of their respective accumulations of sensitizer. In an effort to quantify both tissue optical properties and sensitizer distribution, we have measured fluorescence emission and diffuse reflectance spectra at the surface of a variety of tissue types in the thoracic cavities of human patients. The patients studied here were enrolled in Phase II clinical trials of Photofrin-mediated PDT for the treatment of non-small cell lung cancer and cancers with pleural effusion. Patients were given Photofrin at dose of 2 mg per kg body weight 24 hours prior to treatment. Each patient received surgical resection of the affected lung and pleura. Patients received intracavity PDT at 630nm to a dose of 30 J/cm2, as determined by isotropic detectors sutured to the cavity walls. We measured the diffuse reflectance spectra before and after PDT in various positions within the cavity, including tumor, diaphragm, pericardium, skin, and chest wall muscle in 5 patients. The measurements we acquired using a specially designed fiber optic-based probe consisting of one fluorescence excitation fiber, one white light delivery fiber, and 9 detection fibers spaced at distances from 0.36 to 7.8 mm from the source, all of which are imaged via a spectrograph onto a CCD, allowing measurement of radially-resolved diffuse reflectance and fluorescence spectra. The light sources for these two measurements (a 403-nm diode laser and a halogen lamp, respectively) were blocked by computer-controlled shutters, allowing sequential fluorescence, reflectance, and background acquisition. The diffuse reflectance was analyzed to determine the absorption and scattering spectra of the tissue and from these, the concentration and oxygenation of hemoglobin and the local drug uptake. The total hemoglobin concentration in normal tissues varied from 50 to 300 µM, and the oxygen saturation was generally above 60%. One tumor measured exhibited higher hemoglobin concentration and lower saturation.

In-vivo light dosimetry of interstitial PDT of human prostate.

We report results of in-vivo light dosimetry of light fluence (rate) in human prostate during photodynamic therapy (PDT). Measurements were made in-vivo at the treatment wavelength (732nm) in 15 patients in three to four quadrants using isotropic detectors placed inside catheters inserted into the prostate. The catheter positions are determined using a transrectal ultrasound (TRUS) unit attached to a rigid template with 0.5-cm resolution. Cylindrical diffusing fibers with various lengths are introduced into the catheters to cover the entire prostate gland. For the last four patients, distributions of light fluence rate along catheters were also measured using a computer controlled step motor system to move multiple detectors to different distances (with 0.1 mm resolution). To predict the light fluence rate distribution, a kernel-based model was used to calculate light fluence rate using either (a) the mean optical properties (assuming homogeneous optical properties) for all patients or (b) using distributions of optical properties measured for latter patients. Standard deviations observed between the calculations and measurements were 56% and 34% for (a) and (b), respectively. The study shows that due to heterogeneity of optical properties significant variations of light fluence rate were observed both intra and inter prostates. However, if one assume a mean optical properties (µa = 0.3 cm-1, µs' = 14 cm-1), one can predict the light fluence rate to within a maximum error 200% for 80% of the cases and a mean error of 105%. To improve the prediction of light fluence rate further would require determination of distribution of optical properties.

Detector Calibration factor for interstitial in-vivo light dosimetry using isotropic detectors with scattering tip.

Isotropic detectors with spherical scattering tips are commonly used for in-vivo dosimetry of light fluence rate during photodynamic therapy (PDT). These detectors are typically calibrated in-air. It has been well established that the response of an isotropic detector is a function of the refractive index (n) of the surrounding medium when it is surrounded by an infinite medium of uniform n. However, there are few, if any, studies of the isotropic detector response when the detector is placed in a secondary medium, such as air, before it is placed inside the infinite uniform medium. This condition often arises when one places the isotropic detector inside an air-filled catheter which is then inserted into a turbid medium, such as tissue. We have performed theoretical and experimental studies to determine the correction factors in water (n = 1.33), which has a refractive index similar to that of tissue (n = 1.4). We found that the resulting correction factor is almost the same (within 20%) as the correction factor for the outermost medium (the water) rather than the immediate medium surrounding the isotropic detector (air). The detector correction factor is also a function of the index of refraction of the probe material. For a 1-mm diameter probe from CardioFocus, the detector correction factor varied from 1 (in air) to 1.09 (at air-water interface) to 1.49 (completely submerged in water). At the air-water interface the spherical bulb of the isotropic detector is placed half in air and half in water. For a 0.5-mm diameter probe from the same company, it varied from 1 (in air) to 1.32 (at air-water interface) to 1.87 (in water). For a 0.3-mm diameter probe from the same company, it varied from 1 (in air) to 1.32 (at air-water interface) to 1.71 (in water). We have also found that the detector response changes by less than 10% when the detector position is varied from touching the catheter wall closest to the light source, to not touching, to touching the catheter wall farthest from the light source. The calibration factors between individual isotropic detectors of the same type varied within 5% for all detector types. Thus mean correction factor can be used for each individual isotropic detector of the same type.

Optimization of light sources for prostate photodynamic therapy.

To deliver uniform photodynamic dose to the prostate gland, it is necessary to develop algorithms that optimize the location and strength (emitted power × illumination time) of each light source. Since tissue optical properties may change with time, rapid (almost real-time) optimization is desirable. We use the Cimmino algorithm because it is fast, linear, and always converges reliably. A phase I motexafin lutetium (MLu)-mediated photodynamic therapy (PDT) protocol is on-going at the University of Pennsylvania. The standard plan for the protocol uses equal source strength and equal spaced loading (1-cm). PDT for the prostate is performed with cylindrical diffusing fibers (CDF) of various lengths inserted to longitudinal coverage within the matrix of parallel catheters perpendicular to a base plate. We developed several search procedures to aid the user in choosing the positions, lengths, and intensities of the CDFs. The Cimmino algorithm is used in these procedures to optimize the strengths of the light catheters at each step of the iterative selection process. Maximum and minimum bounds on allowed doses to points in four volumes (prostate, urethra, rectum, and background) constrain the solutions for the strengths of the linear light sources. Uniform optical properties are assumed. To study how different opacities of the prostate would affect optimization, optical kernels of different light penetration were used. Another goal is to see whether the urethra and rectum can be spared, with minimal effect on PTV treatment delivery, by manipulating light illumination times of the sources. Importance weights are chosen beforehand for organ volumes, and normalized. Compared with the standard plan, our algorithm is shown to produce a plan that better spares the urethra and rectum and is very fast. Thus the combined selection of positions, lengths, and strengths of interstitial light sources improves outcome.

In vivo measurement of fluorescence emission in the human prostate during photodynamic therapy.

Among the challenges to the clinical implementation of photodynamic therapy (PDT) is the delivery of a uniform photodynamic dose to induce uniform damage to the target tissue. As the photodynamic dose depends on both the local sensitizer concentration and the local fluence rate of treatment light, knowledge of both of these factors is essential to the delivery of uniform dose. In this paper, we investigate the distribution and kinetics of the photosensitizer motexafin lutetium (MLu, Lutrin®) as revealed by its fluorescence emission. Our current prostate treatment protocol involves interstitial illumination of the organ via cylindrical diffusing fibers (CDF's) inserted into the prostate though clear catheters. For planning and treatment purposes, the prostate is divided into 4 quadrants. We use one catheter in each quadrant to place an optical fiber-based fluorescence probe into the prostate. This fiber is terminated in a beveled tip, allowing it to deliver and collect light perpendicular to the fiber axis. Excitation light is provided by a 465 nm light emitting diode (LED) source coupled to a dichroic beamsplitter, which passes the collected fluorescence emission to a CCD spectrograph. Spectra are obtained before and after PDT treatment in each quadrant of the prostate and are analyzed via a linear fitting algorithm to separate the MLu fluorescence from the background fluorescence originating in the plastic catheter. A computer-controlled step motor allows the excitation/detection fiber to be moved along the catheter, building up a linear profile of the fluorescence emission spectrum of the tissue as a function of position. We have analyzed spectral fluorescence profiles obtained in 4 patients before and after MLu-mediated PDT. We find significant variation both within individual prostates and among patients. Within a single quadrant, we have observed the fluorescence signal to change by as much as a factor of 3 over a distance of 2 cm. Comparisons of pre- and post-PDT spectra allow a quantification treatment-induced photobleaching. Like the drug distribution, the extent of photobleaching varies widely among patients. In two cases, we observed bleaching of approximately 50% of the drug, while others exhibited negligible photobleaching.

Light Dosimetry at Tissue Surfaces for Oblique Incident Circular Fields.

Oblique incident light fields are sometimes unavoidable for photodynamic therapy of skin cancers, e.g., for large fields on uneven surface. We have performed Monte-Carlo simulation for circular fields (R = 0.25, 0.35, 0.5, 1, 2, 3, and 8 cm) for reduced scattering coefficient µs' = 10 cm-1 and attenuation coefficient µa = 0.1 - 1.0 cm-1. We used anisotropy g = 0.9 and the index of refraction n = 1.4 for all Monte-Carlo simulations. Compared to a broad beam of normal incidence, the peak fluence rate along the central-axis for a slanted beam is increased for otherwise the same geometrical conditions and optical properties. The effective attenuation coefficient is slightly decreased for a slanted beam compared to a normal incident beam. The beam profile for a slanted beam at a fixed depth is no longer symmetrical but is higher towards the lateral side of beam incidence. Since the broad beam with finite radius R can be considered as a convolution of a pencil beam, solution for a slanted pencil beam can be used to determine the light fluence distribution for circular beams with oblique beam incidence. An analytical solution can be obtained for the pencil beam obliquely incident on a semi-infinite medium. The solution can be approximated using the diffusion or P3 theory with one point source or two point sources located at appropriate depths with appropriate weights along the beam pathlength inside the phantom, with corresponding image sources to fulfill the extended boundary condition. The analytical solution agrees well with Monte-Carlo Simulation at depths z > 2cosθ t /µ' t , θt is the incident angle after refraction at the interface. Measurements using an isotropic detector were made in a liquid phantom composed of intralipid and ink to verify the Monte-Carlo simulation results.

Light Dosimetry at Tissue Surfaces for Small Circular Fields.

Small circular light fields (≤ 2 cm diameter) are sometimes used for photodynamic therapy of skin and recurrent breast cancers on the chest wall. These fields have lateral dimensions comparable to the effective mean free path of photons in the turbid medium, which causes reduced light fluence rate compared to that of a broad beam of uniform incident irradiance. We have compared Monte-Carlo simulation with in-vivo dosimetry for circular fields (R = 0.25, 0.35, 0.5, 0.75, 1, 2, 3, and 8 cm) in a liquid phantom composed of intralipid and ink (µs' = 4 - 20 cm-1 and µa = 0.1 cm-1) for wavelengths between 532 and 730 nm. We used anisotropy g = 0.9 and the index of refraction n = 1.4 for all Monte-Carlo simulations. The measured light fluence rate agrees with Monte-Carlo simulation to within 10%, with the measured value lower than that of the Monte-Carlo simulation on tissue surface. The ratio of the peak fluence rates between a circular beam and a broad beam under tissue is 0.58 - 0.96 or 0.84 - 1.00 for R between 0.5 - 2 cm and µeff = 1.1 or 2.0 cm-1, respectively. The ratio of peak fluence rate and incident irradiance for the broad beam is 5.9 and 6.4 for µeff = 1.1 and 2.0 cm-1, respectively. The optical penetration depth δ varies from 0.34 - 0.48 cm for R between 0.5 and 2 cm, with the corresponding δ = 0.51 cm for a broad beam. The ratio of fluence rate and incident irradiance above tissue surface is 1.4 - 1.8 or 1.9 - 2.2 for R between 0.5 - 2 cm and µeff = 1.1 or 2.0 cm-1, respectively. At depth of 0.2 cm inside tissue, Off-axis ratio OAR, defined as the ratio of fluence rate at off-axis distance r to that on the central axis, varies between 0.91 - 0.54 or 0.93 - 0.52 for off-axis distances r between 0.6 and 1.0 cm and µeff = 1.1 or 2.1 cm-1, respectively. In conclusion, in-vivo light dosimetry agrees with Monte-Carlo simulation for small field dosimetry provided the isotropic detector is corrected for the blind spot. The light fluence rates for small circular fields are substantially lower than that of the broad beam of the same incident irradiance.

The ratio of the spherical and flat Detectors at tissue surfaces during pleural photodynamic therapy.

An isotropic detector-based system was compared with a flat photodiode-based system in patients undergoing pleural photodynamic therapy. Isotropic and flat detectors were placed side by side in the chest cavity, for simultaneous in vivo dosimetry at surface locations for twelve patients. The treatment used 630nm laser to a total light irradiance of 30 J/cm2 (measured with the flat photodiodes) with photofrin® IV as the photosensitizer. Since the flat detectors were calibrated at 532nm, wavelength correction factors (WCF) were used to convert the calibration to 630nm (WCF between 0.542 and 0.703). The mean ratio between isotropic and flat detectors for all sites was linear to the accumulated fluence and was 3.4±0.6 or 2.1±0.4, with or without the wavelength correction for the flat detectors, respectively. The µeff of the tissues was estimated to vary between 0.5 to 4.3 cm-1 for four sites (Apex, Posterior Sulcus, Anterior Chest Wall, and Posterior Mediastinum) assuming µs' = 7 cm-1. Insufficient information was available to estimate µeff directly for three other sites (Anterior Sulcus, Posterior Chest Wall, and Pericardium) primarily due to limited sample size, although one may assume the optical penetration in all sites to vary in the same range (0.5 to 4.3 cm-1).