Surveying the Challenges to Improve Linear Accelerator-based Radiation Therapy in Africa: a Unique Collaborative Platform of All 28 African Countries Offering Such Treatment.

Radiation therapy is a critical component for curative and palliative treatment of cancer and is used in more than half of all patients with cancer. Yet there is a global shortage of access to this treatment, especially in Sub-Saharan Africa, where there is a shortage of technical staff as well as equipment. Linear accelerators (LINACs) offer state-of-the-art treatment, but this technology is expensive to acquire, operate and service, especially for low- and middle-income countries (LMICs), and often their harsh environment negatively affects the performance of LINACs, causing downtime. A global initiative was launched in 2016 to address the technology and system barriers to providing radiation therapy in LMICs through the development of a novel LINAC-based radiation therapy system designed for their challenging environments. As the LINAC prototype design phase progressed, it was recognised that additional information was needed from LMICs on the performance of LINAC components, on variables that may influence machine performance and their association, if any, with equipment downtime. Thus, a survey was developed to collect these data from all countries in Africa that have LINAC-based radiation therapy facilities. In order to understand the extent to which these performance factors are the same or different in high-income countries, facilities in Canada, Switzerland, the UK and the USA were invited to participate in the survey, as was Jordan, a middle-income country. Throughout this process, LMIC representatives have provided input on technology challenges in their respective countries. This report presents the method used to conduct this multilevel study of the macro- and microenvironments, the organisation of departments, the technology, the training and the service models that will provide input into the design of a LINAC prototype for a LINAC-based radiation therapy system that will improve access to radiation therapy and thus improve cancer treatment outcomes. It is important to note that new technology should be introduced in a contextual manner so as not to disrupt existing health systems inadvertently, especially with regards to existing staffing, infrastructure and socioeconomic issues. A detailed analysis of data is underway and will be presented in a follow-up report. Selected preliminary results of the study are the observation that LINAC-based facilities in LMICs experience downtime associated with failures in multileaf collimators and vacuum pumps, as well as power instability. Also, that there is a strong association of gross national product per capita with the number of LINACs per population.

The survival of fetal and bone marrow monocyte-derived alveolar macrophages is promoted by CD44 and its interaction with hyaluronan.

Alveolar macrophages maintain lung homeostasis by performing important roles in immunosurveillance and lung surfactant catabolism. They express high levels of CD44 and are one of the few macrophage populations that constitutively bind hyaluronan, a ligand for CD44 and component of pericellular and extracellular matrices. Using adoptive transfer experiments and a mouse model of inflammation, we found that alveolar macrophages are initially depleted after an inflammatory insult then rapidly self-renew and return to original numbers after the resolution phase. Monocytes recruited to an inflamed lung differentiate and contribute to the alveolar macrophage pool, but this occurs over a much slower time frame than alveolar macrophage self-renewal. CD44 expression on both fetal and bone marrow-derived alveolar macrophages promoted their survival and provided a competitive advantage over CD44-deficient alveolar macrophages at homeostasis and after inflammation. CD44-mediated hyaluronan binding was induced by the alveolar environment, and this interaction promoted alveolar macrophage survival both ex vivo and in vivo. Without CD44, alveolar macrophages lacked a hyaluronan coat, were more susceptible to death, and were present at lower numbers in the alveolar space. This demonstrates a new role for CD44 and hyaluronan in promoting alveolar macrophage survival.

CD45 regulates GM-CSF, retinoic acid and T-cell homing in intestinal inflammation.

CD45 is a leukocyte-specific tyrosine phosphatase important for T-cell development, and as a result, CD45-/- mice have substantially reduced numbers of T cells. Here we show that, upon dextran sodium sulfate (DSS)-induced colitis, CD45-/- mice have equivalent intestinal pathology and T-cell numbers in their colon as C57BL/6 mice and show enhanced weight loss. CD45-/- mice have a greater percentage of α4ß7+ T cells prior to and after colitis and an increased percentage of T cells producing inflammatory cytokines in the inflamed colon, suggesting that CD45-/- effector T cells preferentially home to the intestine. In DSS-induced colitis in CD45RAG-/- mice lacking an adaptive immune system, CD45 was required for optimal granulocyte-macrophage colony-stimulating factor (GM-CSF) and retinoic acid (RA) production by innate immune cells. Addition of CD45+/+ T cells led to greater weight loss in the RAG-/- mice compared with CD45RAG-/- mice that correlated with reduced α4ß7+ T cells and lower recruitment to the colon of CD45RAG-/- mice in DSS-induced colitis. Addition of exogenous GM-CSF to CD45RAG-/- mice rescued RA production, increased colonic T-cell numbers, and increased weight loss. This demonstrates opposing effects of CD45 in innate and adaptive immune cells in proinflammatory responses and the expression of the gut-homing molecule, α4ß7.

ENLIGHT and LEIR biomedical facility.

Particle therapy (including protons and carbon ions) allows a highly conformal treatment of deep-seated tumours with good accuracy and minimal dose to surrounding tissues, compared to conventional radiotherapy using X-rays. Following impressive results from early phase trials, over the last decades particle therapy in Europe has made considerable progress in terms of new institutes dedicated to charged particle therapy in several countries. Particle therapy is a multidisciplinary subject that involves physicists, biologists, radio-oncologists, engineers and computer scientists. The European Network for Light Ion Hadron Therapy (ENLIGHT) was created in response to the growing needs of the European community to coordinate such efforts. A number of treatment centres are already operational and treating patients across Europe, including two dual ion (protons and carbon ions) centres in Heidelberg (the pioneer in Europe) and Pavia. However, much more research needs to be carried out and beamtime is limited. Hence there is a strong interest from the biomedical research community to have a facility with greater access to relevant beamtime. Such a facility would facilitate research in radiobiology and the development of more accurate techniques of dosimetry and imaging. The Low Energy Ion Ring (LEIR) accelerator at CERN presents such an opportunity, and relies partly on CERN's existing infrastructure. The ENLIGHT network, European Commission projects under the ENLIGHT umbrella and the future biomedical facility are discussed.

A Monte Carlo-based treatment-planning tool for ion beam therapy.

Ion beam therapy, as an emerging radiation therapy modality, requires continuous efforts to develop and improve tools for patient treatment planning (TP) and research applications. Dose and fluence computation algorithms using the Monte Carlo (MC) technique have served for decades as reference tools for accurate dose computations for radiotherapy. In this work, a novel MC-based treatment-planning (MCTP) tool for ion beam therapy using the pencil beam scanning technique is presented. It allows single-field and simultaneous multiple-fields optimization for realistic patient treatment conditions and for dosimetric quality assurance for irradiation conditions at state-of-the-art ion beam therapy facilities. It employs iterative procedures that allow for the optimization of absorbed dose and relative biological effectiveness (RBE)-weighted dose using radiobiological input tables generated by external RBE models. Using a re-implementation of the local effect model (LEM), the MCTP tool is able to perform TP studies using ions with atomic numbers Z ≤ 8. Example treatment plans created with the MCTP tool are presented for carbon ions in comparison with a certified analytical treatment-planning system. Furthermore, the usage of the tool to compute and optimize mixed-ion treatment plans, i.e. plans including pencil beams of ions with different atomic numbers, is demonstrated. The tool is aimed for future use in research applications and to support treatment planning at ion beam facilities.

A possible biomedical facility at the European Organization for Nuclear Research (CERN).

A well-attended meeting, called "Brainstorming discussion for a possible biomedical facility at CERN", was held by the European Organization for Nuclear Research (CERN) at the European Laboratory for Particle Physics on 25 June 2012. This was concerned with adapting an existing, but little used, 78-m circumference CERN synchrotron to deliver a wide range of ion species, preferably from protons to at least neon ions, with beam specifications that match existing clinical facilities. The potential extensive research portfolio discussed included beam ballistics in humanoid phantoms, advanced dosimetry, remote imaging techniques and technical developments in beam delivery, including gantry design. In addition, a modern laboratory for biomedical characterisation of these beams would allow important radiobiological studies, such as relative biological effectiveness, in a dedicated facility with standardisation of experimental conditions and biological end points. A control photon and electron beam would be required nearby for relative biological effectiveness comparisons. Research beam time availability would far exceed that at other facilities throughout the world. This would allow more rapid progress in several biomedical areas, such as in charged hadron therapy of cancer, radioisotope production and radioprotection. The ethos of CERN, in terms of open access, peer-reviewed projects and governance has been so successful for High Energy Physics that application of the same to biomedicine would attract high-quality research, with possible contributions from Europe and beyond, along with potential new funding streams.

Investigating the robustness of ion beam therapy treatment plans to uncertainties in biological treatment parameters.

Uncertainties in determining clinically used relative biological effectiveness (RBE) values for ion beam therapy carry the risk of absolute and relative misestimations of RBE-weighted doses for clinical scenarios. This study assesses the consequences of hypothetical misestimations of input parameters to the RBE modelling for carbon ion treatment plans by a variational approach. The impact of the variations on resulting cell survival and RBE values is evaluated as a function of the remaining ion range. In addition, the sensitivity to misestimations in RBE modelling is compared for single fields and two opposed fields using differing optimization criteria. It is demonstrated for single treatment fields that moderate variations (up to ±50%) of representative nominal input parameters for four tumours result mainly in a misestimation of the RBE-weighted dose in the planning target volume (PTV) by a constant factor and only smaller RBE-weighted dose gradients. Ensuring a more uniform radiation quality in the PTV eases the clinical importance of uncertainties in the radiobiological treatment parameters, as for such a condition uncertainties tend to result only in a systematic misestimation of RBE-weighted dose in the PTV by a constant factor. Two opposed carbon ion fields with a constant RBE in the PTV are found to result in rather robust conditions. Treatments using two ion species may be used to achieve a constant RBE in the PTV irrespective of the size and depth of the spread-out Bragg peak.

FLUKA simulations of the response of tissue-equivalent proportional counters to ion beams for applications in hadron therapy and space.

For both cancer therapy with protons and ions (hadron therapy) and space radiation environments, the spatial energy deposition patterns of the radiation fields are of importance for quantifying the resulting radiation damage in biological structures. Tissue-equivalent proportional counters (TEPC) are the principal instruments for measuring imparted energy on a microscopic scale and for characterizing energy deposition patterns of radiation. Moreover, the distribution of imparted energy can serve as a complementary quantity to particle fluences of the primary beam and secondary fragments for characterizing a radiation field on a physical basis for radiobiological models. In this work, the Monte Carlo particle transport code FLUKA is used for simulating energy depositions in TEPC by ion beams. The capability of FLUKA in predicting imparted energy and derived quantities, such as lineal energy, for microscopic volumes is evaluated by comparing it with a large set of TEPC measurements for different ion beams with atomic numbers ranging from 1 to 26 and energies from 80 up to 1000 MeV/n. The influence of different physics configurations in the simulation is also discussed. It is demonstrated that FLUKA can simulate energy deposition patterns of ions in TEPC cavities accurately and that it provides an adequate description of the main features of the spectra.

Benchmarking nuclear models of FLUKA and GEANT4 for carbon ion therapy.

As carbon ions, at therapeutic energies, penetrate tissue, they undergo inelastic nuclear reactions and give rise to significant yields of secondary fragment fluences. Therefore, an accurate prediction of these fluences resulting from the primary carbon interactions is necessary in the patient's body in order to precisely simulate the spatial dose distribution and the resulting biological effect. In this paper, the performance of nuclear fragmentation models of the Monte Carlo transport codes, FLUKA and GEANT4, in tissue-like media and for an energy regime relevant for therapeutic carbon ions is investigated. The ability of these Monte Carlo codes to reproduce experimental data of charge-changing cross sections and integral and differential yields of secondary charged fragments is evaluated. For the fragment yields, the main focus is on the consideration of experimental approximations and uncertainties such as the energy measurement by time-of-flight. For GEANT4, the hadronic models G4BinaryLightIonReaction and G4QMD are benchmarked together with some recently enhanced de-excitation models. For non-differential quantities, discrepancies of some tens of percent are found for both codes. For differential quantities, even larger deviations are found. Implications of these findings for the therapeutic use of carbon ions are discussed.

ENLIGHT and other EU-funded projects in hadron therapy.

Following impressive results from early phase trials in Japan and Germany, there is a current expansion in European hadron therapy. This article summarises present European Union-funded projects for research and co-ordination of hadron therapy across Europe. Our primary focus will be on the research questions associated with carbon ion treatment of cancer, but these considerations are also applicable to treatments using proton beams and other light ions. The challenges inherent in this new form of radiotherapy require maximum interdisciplinary co-ordination. On the basis of its successful track record in particle and accelerator physics, the internationally funded CERN laboratories (otherwise known as the European Organisation for Nuclear Research) have been instrumental in promoting collaborations for research purposes in this area of radiation oncology. There will soon be increased opportunities for referral of patients across Europe for hadron therapy. Oncologists should be aware of these developments, which confer enhanced prospects for better cancer cure rates as well as improved quality of life in many cancer patients.

Modulation of proto-oncogene expression by polychlorinated biphenyls in 3T3-L1 cell line.

The effects of two substituted polychlorinated biphenyls, the 3,4,5,3',4,5' (PCB-169) and the 2,3,4,2',4',5' (PCB-138) forms, were examined on the expression of c-myc, c-jun, c-ras, and jun-b in 3T3-L1 cells. Northern blot analysis demonstrated that the two PCBs, which exhibit a coplanar and di-ortho-substituted configuration, activated these oncogenes differently. PCB-138 markedly induced overexpression of ras, jun, and myc, whereas PCB-169 led to the overexpression of jun-b. High-performance liquid chromatography analysis of the cell samples treated in medium without serum revealed a higher intracellular concentration of the 2,3,4,2',4',5'-hexachlorobiphenyl (hexaCB), whereas the 3,4,5,3',4'5'-hexaCB reached the same concentration in the sonicated samples of cells with or without serum. These results indicated that there was a relationship between PCB structure, bioavailability, and the capacity to stimulate oncogene expression.

Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes.

The yeast and human RAD51 genes encode strand-transfer proteins that are thought to be involved in both recombinational repair of DNA damage and meiotic recombination. In yeast, the Rad51 family of related proteins also includes Rad55, Rad57 and Dmc1. In mammalian cells, five genes in this family have been identified (HsRAD51, XRCC2, XRCC3, RAD51B/hREC2 and HsDMC1), and here we report the isolation of the sixth member, RAD51C. RAD51C was originally identified by a computer screen of the EST database. A full-length approximately 1.3 kb cDNA clone has been isolated that encodes a protein of 376 aa, having a 18-26% aa identity with other human Rad51 family members. RAD51C includes a previously mapped sequenced-tagged site location near the end of chromosome 17q. The RAD51C transcript is expressed in various human tissues, with highest level of expression in testis, followed by heart muscle, spleen and prostate. Yeast two-hybrid experiments indicate that the Rad51C protein binds to two other members of the Rad51 protein family (Xrcc3 and Rad51B) but not to itself. These findings suggest that Rad51C may function similarly to the yeast Rad55 or Rad57 proteins, rather than as a Rad51 functional homolog.

Suppression of apoptosis by overexpression of Bcl-2 or Bcl-xL promotes survival and mutagenesis after oxidative damage.

Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, and its close relative Bcl-xL, are both effective inhibitors of apoptosis induced by a wide variety of stimuli in many different cell types. In a previous study, we reported that suppression of apoptosis by Bcl-2 or Bcl-xL, markedly elevates the levels of radiation-induced mutations at the specific locus thymidine kinase. We investigated the effect of the Bcl-2 or Bcl-xL overproduction on hydrogen peroxide-induced mutagenesis. Oxidative DNA damage has been implicated in biological processes such as mutagenesis, carcinogenesis and aging. Overexpression of either Bcl-2 or Bcl-xL enhances oxidative stress mutagenesis in cells with wild type p53 as well as with mutated p53 protein. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.

Synthesis and properties of oligonucleotides containing the mutagenic base O4-benzylthymidine.

The preparation of synthetic oligodeoxynucleotides containing O4-benzylthymidine (Tbn) is described. The use of standard and t-butylphenoxyacetyl amino protecting groups is compared. The thermal stabilities of duplexes containing Tbn paired with adenine and guanine have been measured.

Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate.

A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry. Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G. st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis. The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.

1,N6-ethenoadenine is preferred over 3-methyladenine as substrate by a cloned human N-methylpurine-DNA glycosylase (3-methyladenine-DNA glycosylase).

A lethal DNA adduct induced by methylating agents, 3-methyladenine (m3A), is removed by both the constitutive (Tag) and inducible (AlkA) bacterial m3A-DNA glycosylases. The human 3-methyladenine-DNA glycosylase also releases m3A as well as other methylated bases. The rate of release of m3A from alkylated DNA by the purified or recombinant human m3A glycosylase is much higher than that of the other methylated bases. We now find that a partially purified recombinant human m3A-DNA glycosylase, expressed in Escherichia coli, releases at least 10-fold more 1,N6-ethenoadenine (epsilon A) than m3A from DNA. epsilon A is completely unrelated to m3A since it is a heterocyclic adduct produced by the carcinogen vinyl chloride. The rates of release of epsilon A and m3A were both dependent on protein concentration and time. The differential release of epsilon A and m3A occurs regardless of whether DNA containing each adduct is assayed separately or is assayed in a mixed substrate containing both DNAs. This result raises the question of what structural features are involved in recognition and excision by the human m3A-DNA glycosylase and what may be its primary substrate.

All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.