What information can we gain from performing adaptive radiotherapy of head and neck cancer patients from the past 10 years?

The aim of the review was to present the current literature status about replanning regarding anatomical and dosimetric changes in the target and OARs in the head and neck region during radiotherapy, to discuss and to analyze factors influencing the decision for adaptive radiotherapy of head and neck cancer patients. Significant progress has been made in head and neck patients' evaluation and qualification for adapted radiotherapy over the past ten years. Many factors leading to anatomical and dosimetric changes during treatment have been identified. Based on the literature, the most common factors triggering re-plan are weight loss, tumor and nodal changes, and parotid glands shrinkage. The fluctuations in dose distribution in the clinical area are significant predictive factors for patients' quality of life and the possibility of recovery. It has been shown that re-planning influence clinical outcomes: local control, disease free survival and overall survival. Regarding literature studies, it seems that adaptive radiotherapy would be the most beneficial for tumors of immense volume or those in the nearest proximity of the OARs. All researchers agree that the timing of re-planning is a crucial challenge, and there are still no clear consensus guidelines for time or criteria of re-planning. Nowadays, thanks to significant technological progress, the decision is mostly made based on observation and supported with IGRT verification. Although further research is still needed, adaptive strategies are evolving and now became the state of the art of modern radiotherapy.

Impact of COVID-19 on the performance of a radiation oncology department at a major comprehensive cancer centre in Poland during the first ten weeks of the epidemic.

The outbreak of SARS-CoV-2 coronavirus rapidly altered radiotherapy service delivery around the world. AIM: The main objective of this study was to assess the impact of precautionary measures implemented in response to the COVID-19 pandemic on the performance of a radiation oncology departments and on mitigation the risk of COVID-19 contagion between and among patients and staff. METHODS: The study period was from March 15 until May 22, 2020. We evaluated total number of patients irradiated and those who initiated treatments, taking into account tumours localisations. We assessed the relationship of potential risk of contagion with patients' domiciles locations in regions with high number of COVID19 case. RESULTS AND CONCLUSIONS: The number of patients treated with radiotherapy during the study period decreased due to precautionary measures. After five weeks, the number of radiotherapy treatments began to increase. Just over half of the radiotherapy patients (53.5%) treated at the GPCC reside in the city of Poznan or in one of the ten surrounding counties where COVID19 incidence was low and reached at the end of the study period cumulative number of cases n = 204. The precautionary measures were effective qRT-PCR tests were performed in 1545 individuals (patients and hospital staff) revealing four staff members and none patient with a positive PCR result. Immunoglobulin testing was performed in 1132 individuals (patients and hospital staff). A total of 63 individuals were positive for antibodies.

Low dose out-of-field radiotherapy, part 2: Calculating the mean photon energy values for the out-of-field photon energy spectrum from scattered radiation using Monte Carlo methods.

PURPOSE: During radiotherapy, leakage from the machine head and collimator expose patients to out-of-field irradiation doses, which may cause secondary cancers. To quantify the risks of secondary cancers due to out-of-field doses, it is first necessary to measure these doses. Since most dosimeters are energy-dependent, it is essential to first determine the type of photon energy spectrum in the out-of-field area. The aim of this study was to determine the mean photon energy values for the out-of-field photon energy spectrum for a 6 MV photon beam using the GEANT 4-Monte Carlo method. MATERIAL AND METHODS: A specially-designed large water phantom was simulated with a static field at gantry 0°. The source-to-surface distance was 92cm for an open field size of 10×10cm2. The photon energy spectra were calculated at five unique positions (at depths of 0.5, 1.6, 4, 6, 8, and 10cm) along the central beam axis and at six different off-axis distances. RESULTS: Monte Carlo simulations showed that mean radiation energy levels drop rapidly beyond the edge of the 6 MV photon beam field: at a distance of 10cm, the mean energy level is close to 0.3MeV versus 1.5MeV at the central beam axis. In some cases, the energy level actually increased even as the distance from the field edge increased: at a depth of 1.6cm and 15cm off-axis, the mean energy level was 0.205MeV versus 0.252MeV at 20cm off-axis. CONCLUSION: The out-of-field energy spectra and dose distribution data obtained in this study with Monte Carlo methods can be used to calibrate dosimeters to measure out-of-field radiation from 6MV photons.

Low dose out-of-field radiotherapy, part 1: Measurement of scattered doses.

PURPOSE: To measure out-of-field doses in a phantom model to better quantify this radiation. MATERIAL AND METHODS: The individual contribution of photons and neutrons to the total out-of-field dose for 6 MV and 20 MV photons at open beam were measured in a purpose-designed water phantom. Radiation doses were measured at seven separate points (P1-P7) in the phantom with thermoluminescent detectors (TLD 100, 600, and 700) and GAFchromic™ EBT films. RESULTS: At a prescribed dose of 75Gy to the isocentre, the photon dose level in the close-to-field area (P2) ranged from 2.0-2.5Gy for 6 MV and 1.5-2.0Gy for 20 MV; the total out-of-field doses at P2 and P7, respectively, were estimated to be as follows: for 6 MV: TLD 100 (<3.23% and<0.14%); radiochromic film (<2.52% and <0.03%); and for 20 MV: TLD 100 (<2.94% and <0.78%); TLD 700 (<2.02% and <0.14%); and radiochromic film (<1.73% and <0.01%). Although the dose decreased rapidly as the distance from the central beam axis increased, even distant doses could be as high as several centigrays. The neutron dose for 20 MV photons at a distance of 25cm from the isocentre was 4.0mSv/Gy. CONCLUSION: Our results show that in the close-to-field area, the dose level could be as high as 1.5Gy assuming a prescribed dose of 75Gy to the isocentre. By contrast, the doses delivered to more distant areas from the planning target volume were much lower (centigrays). These findings show that both 6 MV and 20 MV photons could produce dosimetrically important dose levels outside of the field. The data reported here may be of value to study the potential impact of even very low doses of radiation on human tissues.

Low dose out-of-field radiotherapy, part 3: Qualitative and quantitative impact of scattered out-of-field radiation on MDA-MB-231 cell lines.

PURPOSE: Patients who undergo external beam radiotherapy are at risk of developing second tumours due to scattered radiation outside the path of the primary beam. The aim of this study was to experimentally determine the in vitro radiobiological effects of scattered radiation in cells located outside the primary photon beam and to compare this to the effects that occur in cells inside the primary beam. The comparison was performed by assessing cell viability, DNA damage, and apoptosis. MATERIAL AND METHODS: Cells from the human breast cancer line MDA-MB-231 were inserted in a water phantom and irradiated at varying doses (1.5, 2.0, 2.5, and 3.0Gy). The cells were placed at two geometrical points: in the central beam axis and at 10cm out-of-field. The dose was constant in both geometrical points. Survival fraction, number of DNA double strand-breaks, and cleaved poly-(ADP-ribose) polymerase (PARP) levels were determined by clonogenic assay and flow cytometry. RESULTS: A slight, non-significant decrease of 3 to 5% in cell survival fraction was observed in cells irradiated outside the primary field. The number of PARP-positive cells and DNA double strand-breaks both increased after out-of-field irradiation. CONCLUSION: Scattered irradiation appears to induce an in vitro biological response on out-of-field cells that is stronger than the effect of primary radiation on in-field cells, independent of the bystander effect. These findings suggest that the biological response of healthy tissues outside the primary beam might be higher than previously believed.

Analysis of the retina in the zebrafish model.

The vertebrate retina is remarkably conserved in evolution. Its relative simplicity and well-defined architecture make it particularly suitable for developmental and functional analysis of neuronal networks in the vertebrate central nervous system. The zebrafish model is at the forefront of these studies. It makes it possible to apply a wide variety of parallel embryological, genetic, and imaging tools to study the eye. Here we discuss experimental approaches that range from cell lineage analysis to the imaging of synaptic calcium currents and atomic force microscopy. These methods are currently used in zebrafish to model morphogenetic events during early development of the eye primordium, cell fate decisions during retinal neurogenesis, and the differentiation and function of the many fine structural features that underlie the detection and processing of light stimuli in the eye.

Analysis of cilia structure and function in zebrafish.

Cilia are microtubule-based protrusions on the surface of most eukaryotic cells. They are found in most, if not all, vertebrate organs. Prominent cilia form in sensory structures, the eye, the ear, and the nose, where they are crucial for the detection of environmental stimuli, such as light and odors. Cilia are also involved in developmental processes, including left-right asymmetry formation, limb morphogenesis, and the patterning of neurons in the neural tube. Some cilia, such as those found in nephric ducts, are thought to have mechanosensory roles. Zebrafish proved very useful in genetic analysis and imaging of cilia-related processes, and in the modeling of mechanisms behind human cilia abnormalities, known as ciliopathies. A number of zebrafish defects resemble those seen in human ciliopathies. Forward and reverse genetic strategies generated a wide range of cilia mutants in zebrafish, which can be studied using sophisticated genetic and imaging approaches. In this chapter, we provide a set of protocols to examine cilia morphology, motility, and cilia-related defects in a variety of organs, focusing on the embryo and early postembryonic development.

Impact of the Intra- and Inter-observer Variability in the Delineation of Parotid Glands on the Dose Calculation During Head and Neck Helical Tomotherapy.

The intra- and inter-observer variability in delineation of the parotids on the kilo-voltage computed tomography (kVCT) and mega-voltage computed tomography (MVCT) were examined to establish their impact on the dose calculation during adaptive head and neck helical tomotherapy (HT). Three observers delineated left and right parotids for ten randomly selected patients with oropharynx cancer treated on HT. The pre-treatment kVCT and the MVCT from the first fraction of irradiation were selected to delineation. The delineation procedure was repeated three times by each observer. The parotids were delineated according to the institutional protocol. The analyses included intra-observer reproducibility and inter-structure, -observer and -modality variability of the volume and dose. The differences between the left and right parotid outlines were not statistically significant (p > 0.3). The reproducibility of the delineation was confirmed for each observer on the kVCT (p > 0.2) and on the MVCT (p > 0.1). The inter-observer variability of the outlines was significant (p < 0.001) as well as the inter-modality variability (p < 0.006). The parotids delineated on the MVCT were 10% smaller than on the kVCT. The inter-observer variability of the parotids delineation did not affect the average dose (p = 0.096 on the kVCT and p = 0.176 on the MVCT). The dose calculated on the MVCT was higher by 3.3% than dose from the kVCT (p = 0.009). Usage of the institutional protocols for the parotids delineation reduces intra-observer variability and increases reproducibility of the outlines. These protocols do not eliminate delineation differences between the observers, but these differences are not clinically significant and do not affect average doses in the parotids. The volumes of the parotids delineated on the MVCT are smaller than on the kVCT, which affects the differences in the calculated doses.

Impact of the Intra- and Inter-observer Variability in the Delineation of Parotid Glands on the Dose Calculation During Head and Neck Helical Tomotherapy.

The intra- and inter-observer variability in delineation of the parotids on the kilo-voltage computed tomography (kVCT) and mega-voltage computed tomography (MVCT) were examined to establish their impact on the dose calculation during adaptive head and neck helical tomotherapy (HT). Three observers delineated left and right parotids for ten randomly selected patients with oropharynx cancer treated on HT. The pre-treatment kVCT and the MVCT from the first fraction of irradiation were selected to delineation. The delineation procedure was repeated three times by each observer. The parotids were delineated according to the institutional protocol. The analyses included intra-observer reproducibility and inter-structure, -observer and -modality variability of the volume and dose. The differences between the left and right parotid outlines were not statistically significant (p > 0.3). The reproducibility of the delineation was confirmed for each observer on the kVCT (p > 0.2) and on the MVCT (p > 0.1). The inter-observer variability of the outlines was significant (p < 0.001) as well as the inter-modality variability (p < 0.006). The parotids delineated on the MVCT were 10% smaller than on the kVCT. The inter-observer variability of the parotids delineation did not affect the average dose (p = 0.096 on the kVCT and p = 0.176 on the MVCT). The dose calculated on the MVCT was higher by 3.3% than dose from the kVCT (p = 0.009). Usage of the institutional protocols for the parotids delineation reduces intra-observer variability and increases reproducibility of the outlines. These protocols do not eliminate delineation differences between the observers, but these differences are not clinically significant and do not affect average doses in the parotids. The volumes of the parotids delineated on the MVCT are smaller than on the kVCT, which affects the differences in the calculated doses.

Gene-modified tumor vaccine secreting a designer cytokine Hyper-Interleukin-6 is an effective therapy in mice bearing orthotopic renal cell cancer.

Although renal cell cancer (RCC) is known to be immunogenic, clinical efficacy of various immunotherapeutic approaches remains unsatisfactory. Novel targeted therapies showing cytostatic rather than cytotoxic activity are unable to cure RCC patients. In our studies, we evaluated the therapeutic efficacy of whole-cell vaccine based on irradiated murine RENCA cells genetically modified to secrete designer cytokine--Hyper-IL6 (H6)--comprising IL-6 and soluble IL-6 receptor. An orthotopic RCC model based on a subcapsular implantation of RENCA cells into kidneys of Balb/C mice was employed. The efficacy of RENCA-H6 vaccine was compared with control vaccine (RENCA-wt) in relation to naive (non-immunized) animals. Three sets of vaccination experiments were carried out in a (i) protective, (ii) palliative and (iii) adjuvant (following nephrectomy) setting. The influence of vaccination on survival of RCC-bearing animals was analyzed. Specificity of vaccine-induced immune response was studied using model antigen-GFP. RCC-bearing animals immunized with RENCA-H6 vaccine showed prolonged survival compared with other groups. In palliative and adjuvant settings the survival RENCA-H6-immunized animals exceeded 75%. Administration of RENCA-H6 inhibited formation and recruitment of Treg cells (CD4+CD25+Foxp3+) and increased maturation of DCs. RENCA tumors in RENCA-H6- vaccinated animals contained large populations of NK cells and activated CD4+, CD8+ T cells. In addition, in mice vaccinated with RENCA-H6 cells large population of CD4+ and CD8+ memory cells (CD62Llow) were detected. In the orthotopic RCC model, RENCA-H6 vaccine showed high therapeutic potential, which resulted from modulation of numerous immunological mechanisms.

The efficacy and reliability of lung protection during total body irradiation of patients with disseminated malignancies.

The main problem in total body irradiation (TBI) is obtaining a homogenous dose distribution inside the whole irradiated body and ensuring appropriate dose reduction in the lungs. The process of irradiation should be comfortable for the patients and repeatable despite the size and age diversity among patients. The aim of this paper was 1) to check accuracy of the applied dose algorithm and reliability of the measurement technique used in the lung region during TBI taken alternatively on a Cobalt-60 unit and on 15 MV linear accelerators, and 2) to check if the described methodology made it possible to obtain reproducibly of the lowered level of the dose to the lungs for a diverse group of patients. TBI was performed as a preparatory regiment in children and adults with disseminated malignancies undergoing bone marrow transplantation (a dose of 12.6 Gy in the midline/central beam axis). Two consecutive groups of patients were retrospectively included in the study: 15 irradiated with Cobalt-60 and 15 with 15 MV photons. The doses were evaluated for three sections passing through the middle of the lungs and at their upper and lower sides. Two types of detectors: semiconductor and thermoluminescent ones were used simultaneously. The measured doses were evaluated statistically to reveal agreement between readings of the two types of detectors and agreement between the measured doses and those previously calculated. The results of measurements exhibited a not Gaussian-type distribution (dissymmetry). The Wilcoxon-type test revealed compliance between the doses measured with thermoluminescent (TL) and semiconductor (SEM) detectors for all sections passing through the lungs (p>0.05), excluding the lung exit (middle and lower sides) with the Cobalt therapy. The t-Student test used to compare the measured doses with those previously calculated revealed agreement (p>0.05) between the measured doses and those calculated for all lung sections for the 15 MV photon therapy, while for Cobalt therapy such an agreement was at some points doubtful. The calculation algorithm and measurement techniques have proved to be correct, which was revealed by agreement between the doses measured and those calculated. The shielding of the lungs during both types of fields was effective and reproducible as indicated by agreement between the doses measured with the two types of detectors. Better agreement between the measured and calculated doses was found for 15 MV photons than for the Cobalt unit.

How the implementation of an in-vivo dosimetry protocol improved the dose delivery accuracy in head and neck radiotherapy.

Doses were measured in-vivo at the entrance using semiconductor detectors for patients with head and neck tumors. Regular measurements started on January 1st and continued till June 30th, 2001. Then the evaluation of the discrepancies between the measured and calculated doses was made, and it resulted in the changes in the protocol of in-vivo dosimetry made effective by February 1st, 2002. The collection of the measurements was censored by January 30th 2003. The number of patients in the two groups was 285 (1st) and 407 (2nd), respectively. The results exhibited the not-Gaussian distribution of the measured doses in both groups. The average number of dose checks per patient increased from 4.9 to 6.0 (1st vs. 2nd group). The mean relative difference between the measured and calculated doses was: -1.5% vs. -0.5%, whereas the standard deviation (1 SD) decreased from 6.1% to 5.6%. The Mann-Whitney U test detected a significant difference between the 1st and the 2nd group (p=0.00000), which justified the conclusion that the changes implemented in the protocol improved dose delivery accuracy and reproducibility of irradiation.

Thermoluminescent dosimetry in rotary-dual technique of the total skin electron irradiation.

The aim of the study was to discuss the results of thermoluminescent dosimetry (TLD) in rotary-dual technique of the total skin electron irradiation (TSEI RD), to confirm beam calibration and monitor unit calculations and to provide data for making clinical decisions. Between May 2001 and April 2002, in 3 cases of mycosis fungoides, 736 dosimetric checks were performed in 34 points at the skin. CaF2:MnTLD-400 cubes (1/8"x1/8"x0.015") were used for in vivo dosimetry. Doses were computed and analyzed for all locations. Percent of described dose and SD for the following localizations from 34 points were: anterior abdomen (reference point) 100+/-6%, upper back 100+/-8%, right calf 98+/-10%, left foot (mid dorsum) 97+/-8%, posterior neck 93+/-6%, right hand (mid dorsum) 78+/-10%, hand fingers 57+/-10%, top of right shoulder 56+/-14%, left groin 35+/-20%, perineum 22+/-17%. The correlations between patient's height and measured doses were sufficient for the following localizations: scalp (top rear), occiput, elbows, hand fingers and hands (mid dorsum). The correlations between obesity index and measured doses were sufficient for the following localizations: shoulders and lateral neck, groins, and perineum. Dosimetric checks at the reference point confirm that our beam calibration technique and monitor unit calculation are accurate. TLD shows that for some parts of the skin such as shoulder, hands and perineum boost fields were required. The correlations with obesity index and height for several sites suggest that boost fields must be customized for each patient.

Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio).

Although the zebrafish has become an important model for genetic analysis of the vertebrate auditory system, a comprehensive description of the zebrafish ear has been provided for embryonic and larval development only (Haddon and Lewis [1996] J. Comp. Neurol. 365:113). Here we describe the development of sensory maculae in juvenile fish and the morphology of the adult zebrafish ear. This description was obtained via three-dimensional reconstruction of serial sections and confocal microscopy of immunolabeled preparations and includes the Weberian ossicles and fluid spaces. Phalloidin staining, which labels actin filaments of stereocilia, was used to delineate the sensory epithelia, to visualize the distribution of hair cells, to estimate their density in different areas of the maculae, and to perform hair cell counts. Morphology of ciliary bundles in different regions of the lagena, saccule, utricle, macula neglecta, and cristae was characterized with an anti-acetylated tubulin antibody and by phalloidin staining. We have identified two antibodies characterized by region-specific staining patterns in the inner ear epithelia. Zn-1 antibody staining largely correlates with the presence of short-bundle hair cells in the peripheral regions of sensory epithelia. Zn-4 antibody, on the other hand, labels a zone of epithelial cells surrounding the sensory maculae. These analyses extend previous observations of cell-type heterogeneity in both sensory and nonsensory epithelia of the fish ear.

A mutation of early photoreceptor development, mikre oko, reveals cell-cell interactions involved in the survival and differentiation of zebrafish photoreceptors.

To gain insight into mechanisms involved in photoreceptor development, we characterized a zebrafish mutation in the mikre oko locus that produces early loss of photoreceptor cells. mikre oko photoreceptors lose their elongated morphology at the time of wild-type outer segment formation and undergo cell death within a few days. To investigate whether this phenotype involves cell-cell interaction defects, we performed analysis of genetically mosaic animals. Interactions of mikre oko photoreceptors with wild-type cells rescue several aspects of the mutant phenotype. When placed in a wild-type environment, mikre oko photoreceptor cells retain elongated morphology and survive longer. Moreover, although mutant mikre oko photoreceptor outer segments develop only infrequently and are usually disorganized, mikre oko cone and rod cells in mosaic retinas develop robust outer segments that closely resemble the wild type. In contrast to the outer segments, the proximal regions of mikre oko photoreceptor cells, including their inner segments, the nuclear regions, and the synaptic termini, retain the mutant appearance. mikre oko outer segment rescue is not mediated by interactions with the retinal pigment epithelium. These studies demonstrate that the differentiation of outer segments is surprisingly independent from the more proximal photoreceptor cell features and that outer segment development includes retinal pigment epithelium-independent cell-cell interactions.

Mutation of the zebrafish glass onion locus causes early cell-nonautonomous loss of neuroepithelial integrity followed by severe neuronal patterning defects in the retina.

Mutation of the glass onion locus causes drastic neuronal patterning defects in the zebrafish retina and brain. The precise stratified appearance of the wild-type retina is absent in the mutants. The glass onion phenotype is first visible shortly after the formation of optic primordia and is characterized by the rounding of cells and disruption of the ventricular surface in the eye and brain neuroepithelia. With exception of the dorsal- and ventral-most regions of the brain, neuroepithelial cells lose their integrity and begin to distribute ectopically. At later stages, the laminar patterning of retinal neurons is severely disrupted. Despite the lack of lamination, individual retinal cell classes differentiate in the glass onion retina. Mosaic analysis reveals that the glass onion mutation acts cell nonautonomously within the retina and brain, as neuroepithelial cell morphology and polarity in these tissues are normal when mutant cells develop in wild-type hosts. We conclude that the glass onion mutation affects cell-cell signaling event(s) involved in the maintenance of the neuroepithelial cell layer shortly after its formation. The disruption of neuroepithelial integrity may be the cause of the neuronal patterning defects following neurogenesis. In addition, the expression of the glass onion phenotype in a subset of neuroepithelial cells as well as its onset following the initial formation of the neuroepithelial sheets indicate the presence of genetically distinct temporal and spatial subdivisions in the development of this histologically uniform tissue.

Improvement of counting helminth eggs with internal standard.

The sanitary regulations introduce a requirement for parasitological control of sewage water used for irrigation and fertilisation of crops. The existing methods of estimation of helminth eggs, based on the coproscopic method using sedimentation, desorption, centrifugation and flotation of the material, allow for only 30-75% efficiency. The aim of this paper is to present an improved quantitative estimation method. The proposed method is based on an internal standard. A predetermined number of eggs of Ascaris lumbricoides suis L is coloured with crystalline violet. These marked eggs are added to sample being examined. Counting the marked eggs in the sample and comparing them with their introduced number allows to estimate the efficiency of the procedure. This results in an increase in the efficiency of the method to 100%. It is possible to calculate the real number of helminth eggs in the sample on the basis of the estimated efficiency of the procedure. The sensitivity of the proposed method is at the level of about 0.4 ppm. The necessary number of replications in the method is about 10 times lower than that in the existing methods. The number of natural (not coloured) eggs does not influence the efficiency of the method with an internal standard.