Commemoration of Jack Fowler's life, work, impact and legacy.

Jack Fowler [formally Professor John Francis Fowler PhD, DSc, MD (Hon), FInstP, FRCR, FBIR, FAAPM, FASTRO, FACRO] was a remarkable scientist, known to many in the field of clinical radiation biology as at the forefront of applying linear-quadratic dose-fractionation-time modelling to help improve a wide range of cancer treatments using radiotherapy. His death on 1st December 2016 after a long career of 60 years was marked by Obituaries in six scientific journals in his field e.g.1-4 Jack is remembered for his quantification of biologically effective dose in a wide variety of radiotherapy practices and modified protocols (supported by experimental-system studies), his extensive publications, his didactic lecturing and teaching abilities, and his warm personality.

A restatement of the natural science evidence base concerning the health effects of low-level ionizing radiation.

Exposure to ionizing radiation is ubiquitous, and it is well established that moderate and high doses cause ill-health and can be lethal. The health effects of low doses or low dose-rates of ionizing radiation are not so clear. This paper describes a project which sets out to summarize, as a restatement, the natural science evidence base concerning the human health effects of exposure to low-level ionizing radiation. A novel feature, compared to other reviews, is that a series of statements are listed and categorized according to the nature and strength of the evidence that underpins them. The purpose of this restatement is to provide a concise entrée into this vibrant field, pointing the interested reader deeper into the literature when more detail is needed. It is not our purpose to reach conclusions on whether the legal limits on radiation exposures are too high, too low or just right. Our aim is to provide an introduction so that non-specialist individuals in this area (be they policy-makers, disputers of policy, health professionals or students) have a straightforward place to start. The summary restatement of the evidence and an extensively annotated bibliography are provided as appendices in the electronic supplementary material.

Mathematical models of tissue stem and transit target cell divisions and the risk of radiation- or smoking-associated cancer.

There is compelling biological data to suggest that cancer arises from a series of mutations in single target cells, resulting in defects in cell renewal and differentiation processes which lead to malignancy. Because much mutagenic damage is expressed following cell division, more-rapidly renewing tissues could be at higher risk because of the larger number of cell replications. Cairns suggested that renewing tissues may reduce cancer risk by partitioning the dividing cell populations into lineages comprising infrequently-dividing long-lived stem cells and frequently-dividing short-lived daughter transit cells. We develop generalizations of three recent cancer-induction models that account for the joint maintenance and renewal of stem and transit cells, also competing processes of partially transformed cell proliferation and differentiation/apoptosis. We are particularly interested in using these models to separately assess the probabilities of mutation and development of cancer associated with "spontaneous" processes and with those linked to a specific environmental mutagen, specifically ionizing radiation or cigarette smoking. All three models demonstrate substantial variation in cancer risks, by at least 20 orders of magnitude, depending on the assumed number of critical mutations required for cancer, and the stem-cell and transition-cell mutation rates. However, in most cases the conditional probabilities of cancer being mutagen-induced range between 7-96%. The relative risks associated with mutagen exposure compared to background rates are also stable, ranging from 1.0-16.0. Very few cancers, generally <0.5%, arise from mutations occurring solely in stem cells rather than in a combination of stem and transit cells. However, for cancers with 2 or 3 critical mutations, a substantial proportion of cancers, in some cases 100%, have at least one mutation derived from a mutated stem cell. Little difference is made to relative risks if competing processes of proliferation and differentiation in the partially transformed stem and transit cell population are allowed for, nor is any difference made if one assumes that transit cells require an extra mutation to confer malignancy from the number required by stem cells. The probability of a cancer being mutagen-induced correlates across cancer sites with the estimated cumulative number of stem cell divisions in the associated tissue (p<0.05), although in some cases there is sensitivity of findings to removal of high-leverage outliers and in some cases only modest variation in probability, but these issues do not affect the validity of the findings. There are no significant correlations (p>0.3) between lifetime cancer-site specific radiation risk and the probability of that cancer being mutagen-induced. These results do not depend on the assumed critical number of mutations leading to cancer, or on the assumed mutagen-associated mutation rate, within the generally-accepted ranges tested. However, there are borderline significant negative correlations (p = 0.08) between the smoking-associated mortality rate difference (current vs former smokers) and the probability of cancer being mutagen-induced. This is only the case where values of the critical number of mutations leading to cancer, k, is 3 or 4 and not for smaller values (1 or 2), but does not strongly depend on the assumed mutagen-associated mutation rate.

The role of gene mutations and gene products in intestinal tissue reactions from ionising radiation.

The response of the intestine to (low linear-energy-transfer) ionising radiation is reviewed regarding the cellular basis to the reactions, the regenerative processes which restore the tissue, and external agents which aid its recovery. In the steady-state, it is generally considered that the crypt cell lineages in both small and large intestine are maintained by a small number of stem cells, but there are differences for example in the composition of their niche residence and in the numbers of transit cell generations. Various cell surface markers are now available to indentify particular lineage cell types. Radiation doses up to 1Gy cause apoptotic stem-cell death in particular locations, at higher doses to >6Gy Lgr5+ stem cells are required for normal intestinal recovery, and at >8Gy some crypts are sterilised and the probability of animal death from intestinal injury increases with higher doses. Mutations in repair genes, tumour suppressor genes, and survival genes cause various degrees of stem cell and clonogenic cell radiosensitisation. Recent evidence is suggesting much plasticity in the crypt cell lineage, potentially contributing to flexibility in the hierarchical lineage, clonogen number variations and the sensitisation differences. Knockout mice for many different genes have been used to detect their role in both steady state and in irradiated conditions, expected to lead to further insight to the damage and restorative processes. Many different external agents have been used to ameliorate intestinal reactions, including prostaglandins, interleukins, angiogenic and epithelial growth factors, other cytokines, and intraluminal factors.

Human epidermal stem cells: Role in adverse skin reactions and carcinogenesis from radiation.

In human skin, keratinopoiesis is based on a functional hierarchy among keratinocytes, with rare slow-cycling stem cells responsible for the long-term maintenance of the tissue through their self-renewal potential, and more differentiated daughter progenitor cells actively cycling to permit epidermal renewal and turn-over every month. Skin is a radio-responsive tissue, developing all types of radiation damage and pathologies, including early tissue reactions such as dysplasia and denudation in epidermis, and later fibrosis in the dermis and acanthosis in epidermis, with the TGF-beta 1 pathway as a known master switch. Also there is a risk of basal cell carcinoma, which arises from epidermal keratinocytes, notably after oncogenic events in PTCH1 or TP53 genes. This review will cover the mechanisms of adverse human skin reactions and carcinogenesis after various types of exposures to ionizing radiation, with comparison with animal data when necessary, and will discuss the possible role of stem cells and their progeny in the development of these disorders. The main endpoints presented are basal cell intrinsic radiosensitivity, genomic stability, individual factors of risk, dose specific responses, major molecular pathways involved and the cellular origin of skin reactions and cancer. Although major advances have been obtained in recent years, the precise implications of epidermal stem cells and their progeny in these processes are not yet fully characterized.

Lack of Correlation between Stem-Cell Proliferation and Radiation- or Smoking-Associated Cancer Risk.

BACKGROUND: A recent paper by Tomasetti and Vogelstein (Science 2015 347 78-81) suggested that the variation in natural cancer risk was largely explained by the total number of stem-cell divisions, and that most cancers arose by chance. They proposed an extra-risk score as way of distinguishing the effects of the stochastic, replicative component of cancer risk from other causative factors, specifically those due to the external environment and inherited mutations. OBJECTIVES: We tested the hypothesis raised by Tomasetti and Vogelstein by assessing the degree of correlation of stem cell divisions and their extra-risk score with radiation- and tobacco-associated cancer risk. METHODS: We fitted a variety of linear and log-linear models to data on stem cell divisions per year and cumulative stem cell divisions over lifetime and natural cancer risk, some taken from the paper of Tomasetti and Vogelstein, augmented using current US lifetime cancer risk data, and also radiation- and tobacco-associated cancer risk. RESULTS: The data assembled by Tomasetti and Vogelstein, as augmented here, are inconsistent with the power-of-age relationship commonly observed for cancer incidence and the predictions of a multistage carcinogenesis model, if one makes the strong assumption of homogeneity of numbers of driver mutations across cancer sites. Analysis of the extra-risk score and various other measures (number of stem cell divisions per year, cumulative number of stem cell divisions over life) considered by Tomasetti and Vogelstein suggests that these are poorly predictive of currently available estimates of radiation- or smoking-associated cancer risk-for only one out of 37 measures or logarithmic transformations thereof is there a statistically significant correlation (p<0.05) with radiation- or smoking-associated risk. CONCLUSIONS: The data used by Tomasetti and Vogelstein are in conflict with predictions of a multistage model of carcinogenesis, under the assumption of homogeneity of numbers of driver mutations across most cancer sites. Their hypothesis that if the extra-risk score for a tissue type is high then one would expect that environmental factors would play a relatively more important role in that cancer's risk is in conflict with the lack of correlation between the extra-risk score and other stem-cell proliferation indices and radiation- or smoking-related cancer risk.

A modelled comparison of prostate cancer control rates after high-dose-rate brachytherapy (3145 multicentre patients) combined with, or in contrast to, external-beam radiotherapy.

BACKGROUND AND PURPOSE: To analyse biochemical relapse-free-survival results for prostate cancer patients receiving combined external beam and high-dose-rate brachytherapy, in comparison with expected results using projections based on dose/fractionation/response parameter values deduced from a previous external-beam-alone 5969-patient multicentre dataset. MATERIAL AND METHODS: Results on a total of 3145 prostate cancer patients receiving brachytherapy (BT) as part or all of their treatment were collected from 10 institutions, and subjected to linear-quadratic (LQ) modelling of dose response and fractionation parameters. RESULTS: Treatments with BT components of less than 25Gy, 3-4 BT fractions, doses per BT fraction up to 6Gy, and treatment times of 3-7weeks, all gave outcomes expected from LQ projections of the external-beam-alone data (α/ß=1.42Gy). However, BT doses higher than 30Gy, 1-2 fractions, 9 fractions (BT alone), doses per fraction of 9-15Gy, and treatment in only 1week (one example), gave local control levels lower than the expected levels by up to ∼35%. CONCLUSIONS: There are various potential causes of the lower-than-projected control levels for some schedules of brachytherapy: it seems plausible that cold spots in the brachytherapy dose distribution may be contributory, and the applicability of the LQ model at high doses per fraction remains somewhat uncertain. The results of further trials may help elucidate the true benefit of hypofractionated high-dose-rate brachytherapy.

International workshop: radiation effects on mutation in somatic and germline stem cells.

PURPOSE: New developments in knowledge of radiation effects on tissue stem cells were discussed in a Workshop held at the Radiation Effects Research Foundation (RERF) in Hiroshima, Japan, 18-19 January 2012. RESULTS: Stem cells and their niche in intestinal mucosa, haemopoietic tissue, hair follicles, and spermatogenesis were discussed variously with regard to radiosensitivity, repair, regeneration, age-dependency of effects, genetic effects, and protection aspects. These tissues all possess a common basic template, but there are structural and hierarchical differences between tissues which continue to be elucidated in terms of a stem-cell age structure and niche regulatory signals which together govern radiation responses. CONCLUSIONS: Stem cells and their niche have become much better characterized in recent years, and their radiation response can be elucidated in detail in experimental systems to help underpin both protection and therapeutic recommendations established from human epidemiological evidence. This report summarizes the presentations at the meeting, and concludes with some remaining questions which may be answered with the help of this type of research.

Dose-fractionation sensitivity of prostate cancer deduced from radiotherapy outcomes of 5,969 patients in seven international institutional datasets: α/ß = 1.4 (0.9-2.2) Gy.

PURPOSE: There are reports of a high sensitivity of prostate cancer to radiotherapy dose fractionation, and this has prompted several trials of hypofractionation schedules. It remains unclear whether hypofractionation will provide a significant therapeutic benefit in the treatment of prostate cancer, and whether there are different fractionation sensitivities for different stages of disease. In order to address this, multiple primary datasets have been collected for analysis. METHODS AND MATERIALS: Seven datasets were assembled from institutions worldwide. A total of 5969 patients were treated using external beams with or without androgen deprivation (AD). Standard fractionation (1.8-2.0 Gy per fraction) was used for 40% of the patients, and hypofractionation (2.5-6.7 Gy per fraction) for the remainder. The overall treatment time ranged from 1 to 8 weeks. Low-risk patients comprised 23% of the total, intermediate-risk 44%, and high-risk 33%. Direct analysis of the primary data for tumor control at 5 years was undertaken, using the Phoenix criterion of biochemical relapse-free survival, in order to calculate values in the linear-quadratic equation of k (natural log of the effective target cell number), α (dose-response slope using very low doses per fraction), and the ratio α/ß that characterizes dose-fractionation sensitivity. RESULTS: There was no significant difference between the α/ß value for the three risk groups, and the value of α/ß for the pooled data was 1.4 (95% CI = 0.9-2.2) Gy. Androgen deprivation improved the bNED outcome index by about 5% for all risk groups, but did not affect the α/ß value. CONCLUSIONS: The overall α/ß value was consistently low, unaffected by AD deprivation, and lower than the appropriate values for late normal-tissue morbidity. Hence the fractionation sensitivity differential (tumor/normal tissue) favors the use of hypofractionated radiotherapy schedules for all risk groups, which is also very beneficial logistically in limited-resource settings.

Human papillomavirus 16 E6 increases the radiosensitivity of p53-mutated cervical cancer cells, associated with up-regulation of aurora A.

PURPOSE: To examine the effect of the human papillomavirus (HPV) type 16-E6 (HPV 'early' gene) oncoprotein on in vitro radiosensitivity of HPV-negative/p53 mutant C33a cervical cancer cells. METHODS AND MATERIALS: The human cervical cancer cell line C33a was stably transfected with either the HPV16 E6 cDNA cloned into the vector pcDNA3.0 (C33aE6) or the empty-vector control (C33aV). Radiosensitivity, DNA damage, and cell cycle measurements were made using standard clonogenic assays, immunofluorescent assessment of nuclear histone H2AX phosphorylated on serine-139 (gamma-H2AX) foci, and flow cytometry. Western immunoblotting and fluorescence confocal microscopy were used to analyse the changes in cellular proteins. Real-time polymerase chain reaction (PCR) was used to compare levels of aurora A mRNA. RESULTS: Compared to C33aV cells, C33aE6 cells showed enhanced radiation cell killing. This was associated with a large amount of polyploidy which was followed by late cell death in C33aE6 cells. Aurora A was highly expressed in C33aE6 cells at pre- and post-irradiation times compared to C33aV cells. Silencing aurora A resulted in a reduced amount of residual gamma-H2AX foci in C33aE6 cells, and diminished the difference in radiosensitivity between the C33aE6 and C33aV cells. CONCLUSION: Our in vitro results indicate that genetic instability could be augmented in the HPV-infected cancer cells by up-regulation of aurora A, especially against a background of dysfunctional p53. Further studies are needed to examine whether aurora A could be a viable therapeutic target in HPV-related tumours.

Human exposure to high natural background radiation: what can it teach us about radiation risks?

Natural radiation is the major source of human exposure to ionising radiation, and its largest contributing component to effective dose arises from inhalation of (222)Rn and its radioactive progeny. However, despite extensive knowledge of radiation risks gained through epidemiologic investigations and mechanistic considerations, the health effects of chronic low-level radiation exposure are still poorly understood. The present paper reviews the possible contribution of studies of populations living in high natural background radiation (HNBR) areas (Guarapari, Brazil; Kerala, India; Ramsar, Iran; Yangjiang, China), including radon-prone areas, to low dose risk estimation. Much of the direct information about risk related to HNBR comes from case-control studies of radon and lung cancer, which provide convincing evidence of an association between long-term protracted radiation exposures in the general population and disease incidence. The success of these studies is mainly due to the careful organ dose reconstruction (with relatively high doses to the lung), and to the fact that large-scale collaborative studies have been conducted to maximise the statistical power and to ensure the systematic collection of information on potential confounding factors. In contrast, studies in other (non-radon) HNBR areas have provided little information, relying mainly on ecological designs and very rough effective dose categorisations. Recent steps taken in China and India to establish cohorts for follow-up and to conduct nested case-control studies may provide useful information about risks in the future, provided that careful organ dose reconstruction is possible and information is collected on potential confounding factors.

International perspectives on quality assurance and new techniques in radiation medicine: outcomes of an IAEA conference.

The International Atomic Energy Agency organized an international conference called, "Quality Assurance and New Techniques in Radiation Medicine" (QANTRM). It dealt with quality assurance (QA) in all aspects of radiation medicine (diagnostic radiology, nuclear medicine, and radiotherapy) at the international level. Participants discussed QA issues pertaining to the implementation of new technologies and the need for education and staff training. The advantage of developing a comprehensive and harmonized approach to QA covering both the technical and the managerial issues was emphasized to ensure the optimization of benefits to patient safety and effectiveness. The necessary coupling between medical radiation imaging and radiotherapy was stressed, particularly for advanced technologies. However, the need for a more systematic approach to the adoption of advanced technologies was underscored by a report on failures in intensity-modulated radiotherapy dosimetry auditing tests in the United States, which could imply inadequate implementation of QA for these new technologies. A plenary session addressed the socioeconomic impact of introducing advanced technologies in resource-limited settings. How shall the dual gaps, one in access to basic medical services and the other in access to high-quality modern technology, be addressed?