The Multigaussian method: a new approach to mitigating spatial heterogeneities with multichannel radiochromic film dosimetry.

The main objective of multichannel radiochromic film dosimetry methods is to correct, or at least mitigate, spatial heterogeneities in the film-scanner response, especially variations in the active layer thickness. To this end, films can also be scanned prior to irradiation. In this study, the abilities of various single channel and multichannel methods to reduce spatial heterogeneities, with and without scanning before irradiation, were tested. Red, green and blue single channel models, two additive channel independent perturbation (CHIP) models and two multiplicative CHIP models were compared with the Multigaussian method. The Multigaussian method is a new approach to multichannel dosimetry, based on experimental findings. It assumes that the probability density function of the response vector formed by the pixel values of the different color channels, including irradiated and non-irradiated scans, follows a multivariate Gaussian distribution. The Multigaussian method provided more accurate doses than the other models under comparison, especially when incorporating the information of the film prior to irradiation. The relative dose differences between reference doses measured with MatriXX and film doses were examined. After applying inter-scan and lateral corrections, the lowest mean absolute errors were 0.8% and 1.0% for the Multigaussian method with and without the information of the scan before irradiation, respectively. Followed by the uniform multiplicative CHIP and red single channel models, using pixel values and net optical density, respectively, both with 1.1%.

Grid patterns, spatial inter-scan variations and scanning reading repeatability in radiochromic film dosimetry.

PURPOSE: When comparing different scans of the same radiochromic film, several patterns can be observed. These patterns are caused by different sources of uncertainty, which affect the repeatability of the scanner. The purpose of this work was to study these uncertainties. METHODS: The variance of the scanner noise, as a function of the pixel position, was studied for different resolutions. The inter-scan variability of the scanner response was analyzed taking into account spatial discrepancies. Finally, the distance between the position of the same point in different scans was examined. RESULTS: The variance of noise follows periodical patterns in both axes, causing the grid patterns. These patterns were identified for resolutions of 50, 72 and 96dpi, but not for 150dpi. Specially recognizable is the sinusoidal shape with a period of 8.5mm that is produced with 72dpi. Inter-scan variations of the response caused systematic relative dose deviations larger than 1% in 5% of the red channel images, 9% of the green and 51% of the blue. No systematic deviation larger than 1% was found after applying response corrections. The initial positioning and the speed of the scanner lamp vary between scans. CONCLUSIONS: Three new sources of uncertainty, which influence radiochromic film dosimetry with flatbed scanners, have been identified and analyzed in this work: grid patterns, spatial inter-scan variations and scanning reading repeatability. A novel correction method is proposed, which mitigates spatial inter-scan variations caused by deviations in the autocalibration of the individual Charge Coupled Device detectors.

On multichannel film dosimetry with channel-independent perturbations.

PURPOSE: Different multichannel methods for film dosimetry have been proposed in the literature. Two of them are the weighted mean method and the method put forth by Micke et al. ["Multichannel film dosimetry with nonuniformity correction," Med. Phys. 38, 2523-2534 (2011)] and Mayer et al. ["Enhanced dosimetry procedures and assessment for EBT2 radiochromic film," Med. Phys. 39, 2147-2155 (2012)]. The purpose of this work was to compare their results and to develop a generalized channel-independent perturbations framework in which both methods enter as special cases. METHODS: Four models of channel-independent perturbations were compared: weighted mean, Micke-Mayer method, uniform distribution, and truncated normal distribution. A closed-form formula to calculate film doses and the associated type B uncertainty for all four models was deduced. To evaluate the models, film dose distributions were compared with planned and measured dose distributions. At the same time, several elements of the dosimetry process were compared: film type EBT2 versus EBT3, different waiting-time windows, reflection mode versus transmission mode scanning, and planned versus measured dose distribution for film calibration and for γ-index analysis. The methods and the models described in this study are publicly accessible through IRISEU. Alpha 1.1 (http://www.iriseu.com). IRISEU. is a cloud computing web application for calibration and dosimetry of radiochromic films. RESULTS: The truncated normal distribution model provided the best agreement between film and reference doses, both for calibration and γ-index verification, and proved itself superior to both the weighted mean model, which neglects correlations between the channels, and the Micke-Mayer model, whose accuracy depends on the properties of the sensitometric curves. With respect to the selection of dosimetry protocol, no significant differences were found between transmission and reflection mode scanning, between 75 ± 5 min and 20 ± 1 h waiting-time windows or between employing EBT2 or EBT3 films. Significantly better results were obtained when a measured dose distribution was used instead of a planned one as reference for the calibration, and when a planned dose distribution was used instead of a measured one as evaluation for the γ-analysis. CONCLUSIONS: The truncated normal distribution model of channel-independent perturbations was found superior to the other three models under comparison and the authors propose its use for multichannel dosimetry.

In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated ß1 integrin and induction of FAK/PI3K/Akt motility signaling.

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active ß1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active ß1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of ß1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of ß1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with ß1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented ß1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, ß1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through ß1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a ß1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Gafchromic EBT2 film dosimetry in reflection mode with a novel plan-based calibration method.

PURPOSE: A dosimetric system formed by Gafchromic EBT2 radiochromic film and Epson Expression 10000XL flatbed scanner was commissioned for dosimetry. In this paper, several open questions concerning the commissioning of radiochromic films for dosimetry were addressed: (a) is it possible to employ this dosimetric system in reflection mode; (b) if so, can the methods used in transmission mode also be used in reflection mode; (c) is it possible to obtain accurate absolute dose measurements with Gafchromic EBT2 films; (d) which calibration method should be followed; (e) which calibration models should be used; and (f) does three-color channel dosimetry offer a significant improvement over single channel dosimetry. The purpose of this paper is to help clarify these questions. METHODS: In this study, films were scanned in reflection mode, the effect of surrounding film was evaluated and the feasibility of EBT2 film dosimetry in reflection mode was studied. EBT2's response homogeneity has been reported to lead to excessive dose uncertainties. To overcome this problem, a new plan-based calibration method was implemented. Plan-based calibration can use every pixel and each of the three color channels of the scanned film to obtain the parameters of the calibration model. A model selection analysis was conducted to select lateral correction and sensitometric curve models. The commonly used calibration with fragments was compared with red-channel plan-based calibration and with three-channel plan-based calibration. RESULTS: No effect of surrounding film was found in this study. The film response inhomogeneity in EBT2 films was found to be important not only due to differences in the fog but also due to differences in sensitivity. The best results for lateral corrections were obtained using absolute corrections independent of the dose. With respect to the sensitometric curves, an empirical polynomial fit of order 4 was found to obtain results equivalent to a gamma-distributed single hit model based on physical assumptions. Three-channel dosimetry was found to be substantially superior to red-channel dosimetry. CONCLUSIONS: Reflection mode with Gafchromic EBT2 radiochromic film was found to be a viable alternative to transmission mode. The same methods that are used in transmission mode can be followed in reflection mode. A novel plan-based method was developed for calibration and multichannel dosimetry. This novel method offers increased robustness against film response inhomogeneities and reduces considerably the time required for calibration.

Blocking of CDCP1 cleavage in vivo prevents Akt-dependent survival and inhibits metastatic colonization through PARP1-mediated apoptosis of cancer cells.

The CUB domain-containing protein-1 (CDCP1) is a transmembrane molecule that has recently been implicated in cancer progression. In this study we have established a novel mechanism for initiation of CDCP1-mediated signaling in vivo and demonstrated that specific 135→70-kDa processing of cell-surface CDCP1 by extracellular serine proteases is a prerequisite for CDCP1-dependent survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a survival program involving recruitment of Src and PKCδ, Src-mediated phosphorylation of cell-surface-retained 70-kDa CDCP1, activation of Akt and suppression of PARP1-induced apoptosis. We demonstrate in vivo that phosphorylated Src, PKCδ and Akt all constitute activated elements of a CDCP1-signaling axis during tissue colonization of tumor cells. Preventing in vivo cleavage of CDCP1 with unique anti-CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling associated with the 70-kDa CDCP1, and induced PARP1 cleavage and PARP1-mediated apoptosis, ultimately resulting in substantial inhibition of tissue colonization by tumor cells. The lack of CDCP1 cleavage in the lung tissue of plasminogen-knockout mice along with a coordinated reduction in tumor cell survival in a lung retention model, and importantly rescue of both by in vivo supplied plasmin, indicated that plasmin is the crucial serine protease executing in vivo cleavage of cell-surface CDCP1 during early stages of lung colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream from CDCP1-induced pro-survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.