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Abstract Background Argonaute genes are essential for RNA interference, stem cell maintenance and differentiation. The Piwi-like genes, a subclass of the Argonaute genes, are expressed mainly in the germline. These genes may be re-expressed in tumors, and expression of the Piwi-like genes is associated with prognosis in several types of tumors. Methods We measured the expression of Piwi-like mRNAs (Piwi-like 2–4) in 125 soft tissue sarcoma (STS) samples by qPCRs. Statistical tests were applied to study the correlation of expression levels with tumor-specific survival for STS patients. Results In multivariate Cox’s regression analyses, we showed that low Piwi-like 2 and Piwi-like 4 mRNA expression were significantly associated with a worse prognosis (RR = 1.87; p = 0.032 and RR = 1.82; p = 0.039). Low expression of both genes was associated with a 2.58-fold increased risk of tumor-related death (p = 0.01). Piwi-like 4 and combined Piwi-like 2 and 4 mRNA levels correlated significantly with prognosis (RR = 3.53; p = 0.002 and RR = 5.23; p = 0.004) only for female but not for male patients. However, combined low Piwi-like 2 and 3 transcript levels were associated with worse survival (RR = 5.90; p = 0.02) for male patients. Conclusions In this study, we identified a significant association between the expression of Piwi-like 2 and 4 mRNAs and the tumor-specific survival of soft tissue sarcoma patients. Furthermore, a connection between sex and the impact of Piwi-like mRNA expressions on STS patients’ prognosis was shown for the first time.

Abstract Background It is well known that osteopontin (OPN) plays an important role in tumor progression and that a high OPN expression level in several tumor entities correlates with poor prognosis in cancer patients. However, little is known about the prognostic relevance of the OPN mRNA splice variants. Methods We analyzed the mRNA expression levels of different OPN splice variants in tumor tissue of 124 soft tissue sarcoma (STS) patients. Quantitative real-time PCR (qRT-PCR) was used to analyze the mRNA expression level of three OPN splice variants (OPN-a, -b and -c). Results The multivariate Cox's proportional hazard regression model revealed that high mRNA expression levels of OPN splice variants are significantly associated with poor prognosis in STS patients (n = 124). Women (n = 68) with high mRNA expression levels of OPN-a and OPN-b have an especially elevated risk of tumor-related death (OPN-a: RR = 3.0, P = 0.01, CI = 1.3-6.8; OPN-b: RR = 3.4, P = 0.01, CI = 1.4-8.2). In particular, we found that high mRNA expression levels of OPN-b and OPN-c correlated with a high risk of tumor-related death in STS patients that received radiotherapy (n = 52; OPN-b: RR = 10.3, P < 0.01, CI = 2.0-53.7; OPN-c: RR = 11.4, P < 0.01, CI = 2.2-59.3). Conclusion Our study shows that elevated mRNA expression levels of OPN splice variants are negative prognostic and predictive markers for STS patients. Further studies are needed to clarify the impact of the OPN splice variants on prognosis.

Abstract Background The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet. Methods Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified. Results A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA. Conclusion An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested.

Abstract Background Members of the urokinase-type plasminogen activator (uPA) system are up-regulated in various solid malignant tumors. High antigen levels of uPA, its inhibitor PAI-1 and its receptor uPAR have recently been shown to be associated with poor prognosis in soft-tissue sarcoma (STS) patients. However, the mRNA expression of uPA system components has not yet been comprehensively investigated in STS patients. Methods The mRNA expression level of uPA, PAI-1, uPAR and an uPAR splice variant, uPAR-del4/5, was analyzed in tumor tissue from 78 STS patients by quantitative PCR. Results Elevated mRNA expression levels of PAI-1 and uPAR-del4/5 were significantly associated with clinical parameters such as histological subtype (P = 0.037 and P < 0.001, respectively) and higher tumor grade (P = 0.017 and P = 0.003, respectively). In addition, high uPAR-del4/5 mRNA values were significantly related to higher tumor stage of STS patients (P = 0.031). On the other hand, mRNA expression of uPA system components was not significantly associated with patients' survival. However, in STS patients with complete tumor resection (R0), high PAI-1 and uPAR-del4/5 mRNA levels were associated with a distinctly increased risk of tumor-related death (RR = 6.55, P = 0.054 and RR = 6.00, P = 0.088, respectively). Strikingly, R0 patients with both high PAI-1 and uPAR-del4/5 mRNA expression levels showed a significant, 19-fold increased risk of tumor-related death (P = 0.044) compared to the low expression group. Conclusion Our results suggest that PAI-1 and uPAR-del4/5 mRNA levels may add prognostic information in STS patients with R0 status and distinguish a subgroup of R0 patients with low PAI-1 and/or low uPAR-del4/5 values who have a better outcome compared to patients with high marker levels.

Abstract Background Osteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet. Methods This study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR. Results No correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Cox's proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively. Conclusion Our data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.

Abstract Background Survivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma. Methods Tumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS). Results We detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Cox's regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus. Conclusion Survivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.

The shape of a wave carries all information about the spatial and temporal structure of its source, given that the medium and its properties are known. Most modern imaging methods seek to utilize this nature of waves originating from Huygens' principle. We discuss the retrieval of the complete kinetic energy distribution from the acoustic trace that is recorded when a short ion bunch deposits its energy in water. This novel method, which we refer to as Ion-Bunch Energy Acoustic Tracing (I-BEAT), is a refinement of the ionoacoustic approach. With its capability of completely monitoring a single, focused proton bunch with prompt readout and high repetition rate, I-BEAT is a promising approach to meet future requirements of experiments and applications in the field of laser-based ion acceleration. We demonstrate its functionality at two laser-driven ion sources for quantitative online determination of the kinetic energy distribution in the focus of single proton bunches.

Laser-accelerated proton bunches with kinetic energies up to several tens of MeV and at repetition rates in the order of Hz are nowadays achievable at several research centres housing high-power laser system. The unique features of such ultra-short bunches are also arousing interest in the field of radiological and biomedical applications. For many of these applications, accurate positioning of the biological target is crucial, raising the need for on-site imaging. One convenient option is proton radiography, which can exploit the polyenergetic spectrum of laser-accelerated proton bunches. We present a Monte Carlo (MC) feasibility study to assess the applicability and potential of laser-driven proton radiography of millimetre to centimetre sized objects. Our radiography setup consists of a thin time-of-flight spectrometer operated in transmission prior to the object and a pixelated silicon detector for imaging. Proton bunches with kinetic energies up to 20 MeV and up to 100 MeV were investigated. The water equivalent thickness (WET) of the traversed material is calculated from the energy deposition inside an imaging detector, using an online generated calibration curve that is based on a MC generated look-up table and the reconstructed proton energy distribution. With a dose of 43 mGy for a 1 mm thin object imaged with protons up to 20 MeV, the reconstructed WET of defined regions-of-interest was within 1.5% of the ground truth values. The spatial resolution, which strongly depends on the gap between object and imaging detector, was 2.5 lp mm−1 for a realistic distance of 5 mm. Due to this relatively high imaging dose, our proposed setup for laser-driven proton radiography is currently limited to objects with low radio-sensitivity, but possibilities for further dose reduction are presented and discussed.

A novel irradiation platform for preclinical proton therapy studies foresees proton imaging for accurate setup and treatment planning. Imaging at modern synchrocyclotron-based proton therapy centers with high instantaneous particle flux is possible with an integration mode setup. The aim of this work is to determine an object’s water-equivalent thickness (WET) with a commercially available large-area CMOS sensor. Image contrast is achieved by recording the proton energy deposition in detector pixels for several incoming beam energies (here, called probing energies) and applying a signal decomposition method that retrieves the water-equivalent thickness. A single planar 114 mm × 65 mm CMOS sensor (49.5 µm pixel pitch) was used for this study, aimed at small-animal imaging. In experimental campaigns, at two isochronous cyclotron-based facilities, probing energies suitable for small-animal-sized objects were produced once with built-in energy layer switching and the other time, using a custom degrader wheel. To assess water-equivalent thickness accuracy, a micro-CT calibration phantom with 10 inserts of tissue-mimicking materials was imaged at three phantom-to-detector distances: 3 mm, 13 mm, and 33 mm. For 3 mm and 13 mm phantom-to-detector distance, the average water-equivalent thickness error compared to the ground truth was about 1% and the spatial resolution was 0.16(3) mm and 0.47(2) mm, respectively. For the largest separation distance of 33 mm air gap, proton scattering had considerable impact and the water-equivalent thickness relative error increased to 30%, and the spatial resolution was larger than 1.75 mm. We conclude that a pixelated CMOS detector with dedicated post-processing methods can enable fast proton radiographic imaging in a simple and compact setup for small-animal-sized objects with high water-equivalent thickness accuracy and spatial resolution for reasonable phantom-to-detector distances.

PurposeThe Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates.MethodsA water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 μs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA).ResultsOptimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 μA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water.ConclusionOptimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.

Laser-driven acceleration of particle beams is an emerging modality under research for biomedical applications. The spatially resolved diagnostics of laser-accelerated proton bunches is crucial for their application. The RadEye detector, featuring up to 10 cm x 5 cm area of online complementary metal-oxide-semiconductor (CMOS) detector made of 48 μm pixels, is established for x-ray, proton and ion beam diagnostics. We exploit the usually undesired ‘Image lag’ phenomenon of incomplete pixel reset to generate 2D-images with a larger dynamic range than the single frame range of 12-bit. Using 532 nm laser pulses and computer simulations for single-slit diffraction, calibration factors to stack multiple readouts were successfully derived to quantitatively reconstruct spatial information about an optical beam and hence extend the dynamic range of the detector compared to a single frame. The final goal is focus quantification for a permanent magnet quadrupole system for protons and terawatt (TW-class) laser focus diagnostics.

We simulated a doublet of permanent magnet quadrupoles (PMQs) to estimate the sensitivity on positioning precision and its impact on the spectral properties of transported protons. The study guided the construction and testing of a focusing setup for laser-accelerated proton bunches with energies between 6 and 10 MeV. Our results shed light on possible applications that may arise from broad input particle spectra.

Laser-accelerated proton bunches with kinetic energies up to several tens of MeV and at repetition rates in the order of Hz are nowadays achievable at several research centres housing high-power laser system. The unique features of such ultra-short bunches are also arousing interest in the field of radiological and biomedical applications. For many of these applications, accurate positioning of the biological target is crucial, raising the need for on-site imaging. One convenient option is proton radiography, which can exploit the polyenergetic spectrum of laser-accelerated proton bunches. We present a Monte Carlo (MC) feasibility study to assess the applicability and potential of laser-driven proton radiography of millimetre to centimetre sized objects. Our radiography setup consists of a thin time-of-flight spectrometer operated in transmission prior to the object and a pixelated silicon detector for imaging. Proton bunches with kinetic energies up to 20 MeV and up to 100 MeV were investigated. The water equivalent thickness (WET) of the traversed material is calculated from the energy deposition inside an imaging detector, using an online generated calibration curve that is based on a MC generated look-up table and the reconstructed proton energy distribution. With a dose of 43 mGy for a 1 mm thin object imaged with protons up to 20 MeV, the reconstructed WET of defined regions-of-interest was within 1.5% of the ground truth values. The spatial resolution, which strongly depends on the gap between object and imaging detector, was 2.5 lp mm−1 for a realistic distance of 5 mm. Due to this relatively high imaging dose, our proposed setup for laser-driven proton radiography is currently limited to objects with low radio-sensitivity, but possibilities for further dose reduction are presented and discussed. [ABSTRACT FROM AUTHOR]