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Microbiota and radiation-induced bowel toxicity: lessons from inflammatory bowel disease for the radiation oncologist Miguel R Ferreira, Ann Muls, David P Dearnaley, H Jervoise N Andreyev

New gastrointestinal symptoms are frequent after pelvic radiotherapy and can greatly affect the quality of life of cancer survivors. The effect of radiation on the intestinal microbiota, and the clinical implications of a modified microbial balance after radiotherapy are now beginning to emerge. In this Personal View, we show the importance of the microbiota for intestinal homoeostasis, and discuss the similarity between inflammatory bowel disease, which has been extensively researched, and radiation-induced gastrointestinal toxicity. By use of microbiota profiles for risk assessment and manipulation of the intestinal flora for prevention and treatment of radiation, enteropathy could become a reality and would be of substantial relevance to the increasing numbers of long-term cancer survivors.

Introduction The discovery of ionising radiation by William Roentgen in 1895 started a new era in cancer research and therapeutics.1 The first attempt to use radiotherapy in cancer management occurred in 1896,1 with damaging effects to the intestine being described 1 year later.2 Thus, gastrointestinal radiation-induced toxicity was one of the first reported challenges of radiation therapy. The intestine is within the treatment field for all intraabdominal, retroperitoneal, and pelvic tumours. Radiation enteropathy can be defined as a progressive, ischaemic, profibrotic (promotes fibrosis) process occurring after irradiation of the pelvis, abdomen, or both, driven by inadequately defined pathophysiological processes.3 In clinical practice and human studies, the presence of pronounced gastrointestinal toxicity is defined by the development of any new gastrointestinal symptoms during or after radiotherapy. This definition has major shortcomings—eg, new gastrointestinal symptoms after radiotherapy can arise because of causes other than radiation (eg, tumour recurrence or infectious gastrointestinal problems) and few studies have related changes that are observed in the bowel (eg, telangectasia, bile salt malabsorption) with bowel symptoms4,5—but at present is the only realistic definition pending the development of objective biomarkers of toxicity. Worldwide, about 300 000 patients per year receive pelvic radiation as part of their cancer treatment,6 and in the UK alone, about 17 000 patients; about 90% of these patients report a change in their bowel function, which results in a negative effect on daily activity in up to 50%.4 Intestinal toxicity is a multifactorial problem, related not only to the method and dose of radiation delivered, but also to an intrinsic process within tissues responding to cellular injury, which is independent of the radiation dose. Individual genetic background and expression pattern, premorbid conditions such as diet and smoking, as well as the cellular microenvironment1,4,7 could be important factors in the development of intestinal toxicity, although their exact contributions are unknown. www.thelancet.com/oncology Vol 15 March 2014

The human gastrointestinal tract contains 1–2 kg of bacteria8 and their role in initially driving or controlling intestinal inflammation has become increasingly evident.9 Intestinal microbiota, a term which collectively refers to the microorganisms that live in the gut, could play an important part in the development and perpetuation of intestinal radiation injury. This Personal View explores mechanisms that could be changed in the host–microbiota relationship after pelvic irradiation, and draws parallels with findings in inflammatory bowel disease.

Lancet Oncol 2014; 15: e139–47 Institute of Cancer Research, London, UK (M R Ferreira MD, Prof D P Dearnaley FRCR); Royal Marsden NHS Foundation Trust, London, UK (M R Ferreira, A Muls MSc, Prof D P Dearnaley, H J N Andreyev FRCP); and Instituto Gulbenkian Ciencia, Oeiras, Portugal (M R Ferreira) Correspondence to: Dr Miguel Reis Ferreira, Institute of Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, UK [email protected]

The gut and the microbiota: a balancing act Composition of the microbiota, orthobiosis, and dysbiosis The intestine of a healthy adult hosts up to 10¹⁴ microbes,10 with 300–500 species of bacteria, dominated by two phyla: Firmicutes and Bacteroidetes. Proteobacteria, Actinobacteria, Fusobacteria, Cyanobacteria, and Verrucomicrobia are present in much smaller numbers.11–13 Other organisms present in our gut include eukaryotes (0·5%), archaea, (0·8%), and viruses (5·8%).14 A healthy microbiota is difficult to define because every individuals’ intestinal flora is a unique mix of bacterial species and strains. However, certain bacterial families and classes are associated with gut health15—meaning that that even if species slightly differ, an overall homology in intestinal microbial groups defines a healthy gut flora. Furthermore, the microbial composition in a healthy individual is stable for 60% of the species with time in one individual.13 Three clusters of microbial populations, referred to as enterotypes, have been described in healthy individuals. Even though they are similar, they are enriched in particular microorganisims: enterotype one, enriched with Bacteroides; enterotype two, enriched with Prevotella; and enterotype three, the most frequent, enriched with Ruminococcus14—however, this classification is controversial.15 The human intestine is colonised by microbes immediately after birth. Environmental factors such as diet and genetic background of the human host then stochastically shape the microbiota into a stable e139

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community of symbionts. The resilience of the microbiota to environmental factors within an individual is shown by stability with time.16 The term orthobiosis is used for the balance and collectively beneficial effect of the indigenous microbiota on human health. By contrast, the global imbalance of bacterial populations in the gut is termed dysbiosis, which is associated with several disease processes including inflammatory bowel disease.17 The specific bacterial groups that make up orthobiotic microbiota are species-specific, as shown in a study of reciprocally transplanted microbiota between mice and zebrafish: after transplantation, a shift in the microbiota back towards the conventional profile of their respective species was seen.18

Host–microbiota symbiosis Microbes obtain nutrients from the human host, and protection from competing species by the immune system.8 However, microbiota also benefit the host through different interactions—eg, microbiota play a central part in the development of the epithelium. Germfree animal models have decreased epithelial proliferation in their crypts of Lieberkuhn compared with conventionally raised (reared normally) and A

Orthobiosis

Activation of survival pathways Gut homoeostasis Attenuation of inflammation Maintenance of mucosal barrier

B

Dysbiosis

Chemotaxis Adaptive immunity Inflammation Impairment of barrier function Secretion of antimicrobial peptides Secretion of interferons

Commensal microorganism Pathogenic microorganism Formyl ligand

M cell

Macrophage

Microbe-associated molecular pattern Pathogen-associated molecular pattern Reactive oxygen species

Enterocyte

Dendritic cell

Toll-like receptor Nucleotide oligomerisation domain Formylated peptide receptor

Figure 1: Interactions between the microbiota and the intestinal epithelium (A) Orthobiosis, through different interactions with intestinal tissues, protects them from effects of radiation. (B) Inversely, dysbiosis potentially contributes to deleterious effects provoked by radiation to the healthy gut.

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conventionalised (born germ-free and later colonised with a normal microbiota) animals;19 this is an evolutionarily conserved pattern.20 Furthermore, a molecule, competence and sporulation factor, produced by the Gram-positive Bacillus subtilis interacts with epithelial cells, activating survival pathways and enhancing antioxidant capacity.21 B subtilis seems to counteract cytotoxic effects of both radiotherapy and chemotherapeutic drugs,22 thus could be an inherent property of the microbiota that could be exploited for patient benefit during cancer therapy. The enteric flora educates or primes the immune system from early life, whereas the immune system shapes and closely interacts with the microbiota.23 Germ-free animals have a rudimentary mucosal-associated lymphoid tissue, and some defects of cell-mediated immunity.8 The immune system has to distinguish between beneficial commensal microorganisms and harmful pathogens within a heterogeneous gut microbial community.12,24 Some bacterial species directly contact epithelial cells, immune cells, or both. Antigens ingested by microfold cells in Peyer’s patches can also be sampled by dendritic cells. To sense the microbiota, the host uses pattern recognition receptors (eg, Toll-like receptors and nucleotideoligomerisation domains), which are seen in the intestinal epithelium and in immune cells. These receptors recognise bacterial components that could be microbeassociated molecular patterns, or pathogen-associated molecular patterns. Toll-like receptors (TLRs) are transmembrane proteins that are located at the cell surface, or intracellularly within the endosomal membranes. TLRs recognise different components of bacteria. Pathogenassociated molecular pattern–TLR binding leads to an immune response to eradicate potentially harmful pathogens, whereas microbe-associated molecular pattern–TLR binding tends to activate pathways involved in gut homoeostasis (figure 1).12,25 The compound CBLB502 (also known as Entolimod) has shown the potential relevance of TLRs for patients undergoing radiotherapy. CBLB502 is derived from the bacterial component flagellin and has shown activity in mouse and primate animal models.25,26 The compound has clinical potential for patients undergoing radiotherapy because in mice and primate models, CBLB502 has been shown to protect tissues at risk of toxicity—flagellin interaction leads to NFκB activation by binding to TLR5, inhibiting P53 function, and inducing factors contributing to cell protection and tissue regeneration, including reactive oxygen species scavengers, apoptosis inhibitors, and cytokines—while eliciting a tumouricidal response, making it a potential radiation response modifier for normal tissues.25,26 Formylated peptide receptors are expressed in neutrophils, macrophages, and intestinal epithelial cells. They recognise bacterial cell wall products tagged with an N-formyl group, leading to radical oxygen species www.thelancet.com/oncology Vol 15 March 2014

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formation, which, at the high concentrations elicited by pathogens, are microbicidal. They also lead to chemotaxis and upregulate proinflammatory pathways. By eliciting small increases in reactive oxygen species through formylated peptide receptor activation, commensal microorganisms in the gut enhance antioxidant capacity, cytoprotection, and epithelial restoration.27 By eliciting such responses, the commensal microbiota is counteracting the effects of radiation. However, by promoting an oxidative burst, potential pathogens might be responsible for enhancing the radiation response, leading to further deleterious effects on the normal gut. The human gut microbial genome, collectively known as the intestinal microbiome, is enriched with metabolic genes involved in the degradation and processing of dietderived substrates, producing energy sources used by both the host and the symbiont. The microbiota also produces vitamins and aminoacids.10,12 A dysbiotic milieu may change the metabolism of the host and provoke increased inflammation. Dysbiosis affects the metabolism of short-chain fatty acids, which are known to have immunomodulatory properties and play a part in radiation-induced bowel toxicity.28,29 Moreover, microbial imbalances contribute to carcinogenesis in the colon through chronic inflammation, immune evasion or suppression, and modification of the metabolism of carcinogenic substances.30

Microbiota and radiation-induced bowel toxicity Pelvic radiation disease is defined as transient or longer term problems that arise in healthy tissues as a result of radiotherapy to a tumour of pelvic origin. Its symptoms range from mild to very severe.31 Radiation enteropathy, one of the syndromes of pelvic radiation disease, affects the intestine (figure 2), and has been arbitrarily divided into acute (until 90 days after last treatment) and late enteropathy (occurring thereafter). Acute changes in the gastrointestinal tract are mediated by the cytotoxic effect of radiation to the rapidly proliferating epithelium; these changes are amplified by inflammation. Both the cytotoxic effect to the proliferating epithelium and the inflammation determine increases in free radicals, damaging the DNA, and producing functional effects.32 Eosinophilic crypt abscesses are the most characteristic change seen acutely with radiotherapy.4 Activation of the coagulation system leads to ischaemia and further necrosis.33 Increased formation of thrombin, a serine protease, is important in promoting thrombus formation and platelet activation, but also in regulating inflammation by enhancing epithelial permeability and recruiting neutrophils and monocytes.33 This mechanism can lead to ulceration, which exposes the underlying tissues to bacteria, changes the host– microbial interaction, and increases inflammation as the immune system struggles to contain bacterial translocation. The ulcer might then progress to fibrosis, a process initially driven by a TGFβ1-mediated phenotypic switch, which turns fibroblasts into collagen www.thelancet.com/oncology Vol 15 March 2014

Radiotherapy

Cytotoxic effect to the intestinal epithelium

Cell death

Direct inflammatory effects

Disruption of intestinal microenvironment

Activation of the coagulation system

Vasodilation

Cytotoxic effect to the endothelium

↑Thrombin Chemotaxis

Disruption of barrier function

Acute gastrointestinal symptoms

Inflammation

Bacterial translocation

Amplification of reaction

Loss of stem cells

Incomplete healing

Ischaemia

Intestinal fibrosis

Late gastrointestinal symptoms

Figure 2: Mechanisms of radiation-induced gastrointestinal symptoms Several mechanisms act concurrently to provoke radiotherapy-induced gastrointestinal symptoms, both in the acute and late settings.

matrix-producing myofibroblasts. However, ulceration is not necessary for a fibrotic process to occur, as microvascular lesions and inflammation are sufficient to initiate fibrosis, which progresses independently of inflammation.34 Furthermore, intestinal tissues with a delayed radiation response can become necrotic, eliciting a sustained inflammatory response.35 Radiation toxicity has been viewed as a result of the cytotoxic effects on target cells, defining tissues as either early or late reacting according to their radiosensitivity, repair capacity, proliferation rate, and tissue organisation. Although radiation kills cells by direct or indirect effects on the DNA,22 toxicity is also mediated by other mechanisms: indirect effects (eg, inflammation or bystander effects); and functional effects (eg, modification of gene expression).3 In addition to the repetitive and cumulative nature of damage to normal tissue induced by fractionated radiotherapy, a non-healing acute response might progress into a late effect. The frequency or severity of these responses, termed consequential effects, are affected by the extent of the acute changes in the same tissue, including their grade or duration.35 Dysbiosis might play a part in radiation enteropathy. The gut flora is heterogeneous, and different microorganisms have various radiosensitivities. Furthermore, commensal microorganisms are in close interaction with an environment that substantially changes during pelvic radiotherapy. Therefore, not only is dysbiosis likely to develop, but it might also contribute to toxicity.

Preclinical studies In the 1960s, irradiated germ-free mice were shown to develop fewer gastrointestinal symptoms, leading to e141

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studies with antibiotics as radiation response modifiers, which had conflicting results.36 In 1981, irradiation of rats was shown to cause a substantial increase in the number of bacteria on their intestinal villi,2 suggesting that radiation-induced changes to the intestine are associated with changes in the microbiota. In mice, lipopolysaccharide (a component of the external membrane of Gram-negative bacteria), acts as a radioprotectant in irradiated mice. Lipopolysaccharide is thought to act as a radioprotectant through a mechanism involving tumour necrosis factor (TNF) receptor 1 and APOBEC1. APOBEC1 binds and stabilises cyclooxygenase 2 RNA in subepithelial fibroblasts, leading to an increase in prostaglandin E2. TNFα is probably the relevant ligand involved in this pathway.37–39 Lipopolysaccharide also promotes the gene expression of the radioprotectant interleukin 1α in mice, an effect which is dependent on the murine P-glycoprotein.40 Germ-free mice are radioresistant to total body irradiation with 10–22 Gy followed by a bone marrow transplant (to avoid marrow failure). Animals colonised with only Bacteroides thetaiotaomicron and Escherichia coli have similar outcomes. By contrast, conventionally raised and conventionalised animals have radiosensitive phenotypes.41 Because germ-free animals are known to have an impaired epithelial turnover,19 this eliminates differences due to germ-free rearing. A protein called fasting-induced adipose factor, also known as angiopoietinlike protein 4, is involved in this difference.41 A parallel here with inflammatory bowel disease is noteworthy; colonisation with a microbiota is necessary for the development of colitis in mouse models, whereas germfree mice are resistant.8 Probiotics are live organisms that, when consumed in an adequate amount, confer a health effect on the host, whereas prebiotics are non-digestible foods that promote the growth or activity of specific microorganisms, leading to a health benefit. Synbiotics are mixtures of prebiotics and probiotics.8 The use of these three types of supplement has been explored preclinically in rodents with single fraction irradiation. Results have been encouraging,42,43 leading to reduced radiation-induced diarrhoea and histological damage, but no studies have used a probiotic specifically designed to reduce toxicity.42,43 The medical relevance of these observations, shown by a small number of studies, is nevertheless questionable. Single-fraction radiotherapy does not adequately reproduce the conditions of fractionated therapy. Moreover, radiosensitivity differs between animal models44 and the microbiota is also known to vary between species.45 Finally, these studies focus only on acute radiation-induced changes.

Clinical studies Bacterial overgrowth Small intestinal bacterial overgrowth is a manifestation of radiation enteropathy, both in the acute and late settings.4 e142

A study of 41 patients with late intestinal toxicity showed the relation between migrating motor complex patterns (aborally migrating bands of regular contractions in the intestines) and overgrowth of Gram-negative bacilli. The investigators concluded that impaired motility is a cause of gastrointestinal colonisation of Gram-negative bacilli.46 However, this associative study did not have baseline data, making it difficult to discern cause and effect. In a cohort study of 39 patients undergoing pelvic radiotherapy, glucose hydrogen breath testing, with suboptimum specificity and sensitivity, was used at baseline and at 4–5 weeks of treatment. The incidence of small intestinal bacterial overgrowth at 4–5 weeks was 26%.47

Dysbiosis and radiation A further study assessed faecal microbial populations of ten patients undergoing pelvic radiotherapy by DNA fingerprinting and sequencing of the 16S rRNA gene (unique to prokaryotes) at baseline, during, and after treatment. The clinical endpoint was the onset of diarrhoea. The six symptomatic patients showed a progressively increasing dysbiotic pattern, which was maintained after radiotherapy.48 In a subgroup analysis comparing microbial profiles, a clustering of patients with diarrhoea was reported, with an overall increase in Bacilli and Actinobacteria, and a decrease in Clostridia. However, this study had a small number of patients and only addressed acute diarrhoea, which is not the only symptom of acute radiation enteropathy. The investigators did not record if dysbiosis started before or after the onset of diarrhoea, so causality could not be determined.

Surgery and gastrointestinal toxicity Several studies have investigated the predictive value of previous abdominal surgery on the development of gastrointestinal toxicity. A high incidence of rectal bleeding after radiotherapy was associated with previous abdominal surgery (namely cholecystectomy and appendectomy49) in some, but not all studies.50 Notably, although appendectomy has been suggested as a risk factor of late radiation-induced rectal bleeding, substantial evidence shows that the surgery is protective against the development of ulcerative colitis.51 Valdagni and colleagues49 suggest that a possible mechanism for this potential increase in susceptibility in rectal toxicity after appendectomy is that appendectomy results in the removal of an important reservoir of gut-protecting bifidobacteria; they argue that this removal could lead to intestinal dysbiosis, but this finding needs further corroborative research. Despite their presence in the appendix, bifidobacteria are present in higher densities in the distal colon,52 and appendectomy has not been associated with increased toxicity in other areas of the bowel. The microbiota is known to be changed not only structurally, but also functionally after bariatric surgery, www.thelancet.com/oncology Vol 15 March 2014

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with a shift towards a Proteobacteria-dominated flora.15 Notably, Proteobacteria and other Gram-negative bacilli are known to be increased before radiation-induced late intestinal toxicity.46 Although these findings are intriguing, available studies showing the bacterial profile or function modification after surgery are scarce. Dissociation of the effect of the microbiota from changes in gut immunity and inflammation after surgery is also difficult. Moreover, patients undergoing abdominal surgery are often treated with large doses of antibiotics, which could be a cause of long-term imbalance of the microbiota and pathogen-derived colitis. Further studies in this specialty are therefore necessary to assess the effect of surgery on the structure and function of gut commensal microoganisms and its possible role in the predisposition to radiotherapyinduced gastrointestinal toxicity.

Microbiota interventions Table 1 shows clinical studies investigating probiotic, prebiotic, or synbiotic interventions.53 Although each study shows significant benefits in the prevention or treatment of radiotherapy-induced gastrointestinal symptoms, the evidence is not sufficiently robust to affect clinical practice. Few of the studies used widely accepted toxicity endpoints and none have been reproduced in large cohorts. Nevertheless, the studies show that attempts at bacteriotherapy seem safe in the context of radiation enteropathy. Type of study

Preparation

Salminen et al (1988)

Preventive, randomised controlled trial

Lactobacillus acidophilus; 2 × 10⁹ CFU per day plus lactulose

Urbancsek et al (2001)

Therapeutic, double-blind randomised controlled trial with placebo

Delia et al (2007)

Giralt et al (2008)

Similarities between radiation-induced gastrointestinal toxicity and inflammatory bowel disease Clinical comparison There are several mechanisms involving the microbiota which seem to be common between inflammatory bowel disease and radiation enteropathy. Inflammatory bowel disease involves the colon and small intestine, with the two main forms being Crohn’s disease and ulcerative colitis. Clinically, inflammatory bowel disease and radiation enteropathy are often characterised by symptoms such as diarrhoea, rectal bleeding, and malabsorption. Another common feature is pronounced infiltration of innate and adaptive immune cells into the lamina propria, accompanied by epithelial destruction and mucosal ulceration. Although ulcerative colitis tends to be confined to the mucosa, Crohn’s disease and radiation enteropathy tend to be transmural.2,54 Submucosal fibrosis, which seems to be triggered in part by oxidative stress, can feature in radiation enteropathy, ulcerative colitis, and Crohn’s disease.2,55 Furthermore, barrier function is impaired in these diseases because defects in the epithelial tight junctions lead to increased paracellular permeability.2,32,54 Studies suggest that patients with Crohn’s disease and patients with ulcerative colitis have a 29–46% increased risk of severe acute or chronic complications after radiation treatment (table 2). The risk of pronounced acute toxicity has led some oncologists to conclude that patients with Number of patients Type of randomly assigned malignancy to treatment/ analysed

Type of treatment

Effect of intervention

24/21

Gynaecological tumours

50 Gy to pelvis and 80 Gy to Reduction of diarrhoea tumour (brachytherapy boost) (p

Microbiota and radiation-induced bowel toxicity: lessons from inflammatory bowel disease for the radiation oncologist.

New gastrointestinal symptoms are frequent after pelvic radiotherapy and can greatly affect the quality of life of cancer survivors. The effect of rad...
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