This research process mainly referred to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) framework [6]. Scientific documents including peer-reviewed journals, academic conference reports, and conference proceedings were searched using combinations of keywords with Boolean operators ("gastrointestinal microbiota" OR "gut microbiome" OR "intestinal microflora" OR "intestinal bacterial" OR "microbiota composition" AND "radiation enteritis" OR "radiation enteropathy" OR "radiation injury" OR "radiation-induced toxicities" OR "pelvic radiotherapy") in PubMed, MEDLINE, Web of Science and Google Scholar to ascertain relevant literature. An online search was conducted between May 15 and June 1, 2022. Retrieved results were not date-limited. A second search of the references lists from the literature was conducted to reduce the possibility of missing qualified articles.
Studies from the searched database were screened via title and abstract, and confirmed by two authors. Then, full texts for the identified articles were retrieved and reviewed, while ambiguous studies were discussed by two reviewers. After that, studies were assessed and data was extracted. Finally, the studies and data were synthesized and analyzed.
The causes and symptoms of radiation enteritis
According to Global Cancer Statistics 2018, the new incidence of cancers occurred at prostate, colon, stomach, rectum, cervix uteri, bladder, corpus uteri, anus, vagina and penis account for 27% among all cancers.[7] Radiotherapy is a common therapeutic modality for varieties of cancers and plays a dominant role in abdominopelvic malignancy.However, treatment is often accompanied by toxic and side effects. In 1895, X-rays was discovered by Wilhelm Roentgen. Two years later, the radiation-induced damage to the gut was first reported.[8, 9]
Radiation enteritis (RE) is thought to be mainly caused by radiation-induced endothelial system damage, leading to a systemic inflammatory response mediated by leukocyte recruitment[10]. Under irradiation, tissues rich in radiation sensitive vascular layers, such as endothelial cells and epithelial cells, produces high levels of proinflammatory mediators and reactive oxygen species, leading microvascular leakage, high levels of adhesion molecules and leukocyte infiltration[11].
RE is generally divided into acute and chronic forms. The acute form often occurs within the first three months of radiotherapy and usually subsides and recovers within three months. Common symptoms of acute radiation enteritis include abdominal pain, diarrhea, urgency, bloating, fecal incontinence, flatulence, rectal bleeding and weight loss. The chronic type develops months to years after radiotherapy, and has been recognized to cause chronic diarrhea, hemafecia, anemia, perforation, ileus, and nutritional deficiency.[12] The Radiation Therapy Oncology Group (RTOG) - European Organization for Research on Treatment of Cancer (EORTC) acute and late effects scale[13] and the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Effects (CTCAE) version 5.0[14] were in common use for reporting toxicity.
Pathophysiological stages and clinical development of mucositis
At present, it is generally accepted that the formation of radiation enteropathy is accompanied by persistent overexpression of inflammatory and fibrotic cytokines. First, mucosal barrier destruction and inflammation, and then gradually develop into progressive arteriosclerosis and intestinal wall fibrosis.[15, 16]
In 2004, a study revealed five pathological stages of mucositis. The initiation phase is the generation of reactive oxygen species (ROS), activation of the innate immune response. The next phase is upregulation and message generation such as cytokines, producing tissue injury and apoptosis. The third one is the amplification phase. Inflammation is aggravated and apoptosis occurs beneath the mucosal surface. Next, the ulcerative phase is characterized by the discontinuity of the mucosa, allowing bacterial colonization and cell wall products releasing. The final phase is the ulcer healing. Extracellular matrix released new messenger molecules to promote cell proliferation, thus fibrosis is facilitated.[17] In effect, such artificial division is unlikely to fully reflect the actual processes. Rather, the process is dynamic as well as unique individually.
When exposed to radiation, DNA strand breaks can cause cell death or damage[18]. Non-DNA damage is initiated by a variety of mechanisms, some of which are mediated by the production of reactive oxygen species[19]. The damage of endothelial cell wall was observed by electron micrograph within 1 week after acute exposure to radiation and at least 5 days before epithelial cell wall rupture[20]. Damage signals at the cellular and tissue levels occur within seconds of radiation exposure. Fractionated planning of radiation dosing lead to sustained and overlapping damage signals and tissue change. For gastrointestinal mucosa, symptoms (burning and modest pain) related to atrophic changes usually begin at patients have received radiation doses 10Gy. Ulceration always occurs between the second and third week of radiation (cumulative radiation doses of 20-40Gy). The patient may suffer from contiguous and extremely pain and dysfunction. Lesions lasted up to 6 weeks after the radiotherapy. [21]
The gut microbiota and its impact the on the host
The gut microbiome is a community composed of bacteria, fungi, archaea, protozoa and viruses. It is considered to regulate a variety of immune pathways, thus affecting host health and disease.[22] The diversity of the gut microbiome is defined as the number and relative abundance distribution of these different types of gastrointestinal tract microorganisms.[23]
Since the preliminary study and visualization of cells via microscopy in 1660s, humans began to investigated microscopic organisms. Some studies investigated with denaturing gradient gel electrophoresis (DGGE). Microbiome identification was limited to hundreds of culturable species, but with the progress of whole genome sequencing, the research of microorganisms has undergone great innovation. The microbial 16S ribosomal RNA (16S rRNA) gene sequencing, a high-throughput sequencing technology, has been used to investigate microbiota composition of many mammalian species, including humans.[24] This deep sequencing technology is aiming at the hyper-variable regions V1-V9 of the bacterial 16S rRNA genes. At present, the regions used for deep sequencing of 16S rRNA gene mainly include V4 region, V3-V4 region, and V4-V5 region. Compared to culture-dependent or -independent methods, it provides a new method for analyzing microbial communities and has been successfully applied to the characterization of intestinal microbial diversity.[25, 26]
An overall homology of intestinal microbial groups means a healthy intestinal microbiota. There are up to 100 trillion microorganisms (including bacteria, fungi and viruses) in the intestines of a healthy adult. Among them, there are 300-500 kinds of bacteria, which are mainly composed of two phyla: Firmicutes (30%-50%) and Bacteroidetes(20%-40%).[27] The intestinal microbiota of healthy human is comprised of 109 species, 59 genera, 38 orders, 23 classes, 18 families, and 8 phyla. Firmicutes, Actinobacteria, and Bacteroidetes make up a majority of the bacterial species. These three kinds of bacteria accounted for 40%, 30%, and 20%, respectively. In Firmicutes, the Clostridia class is the most abundant class species, accounting for 20.3%, followed by Bacteroidia, Bifidobacteriales, Enterobacterales, and Lactobacillales, accouting for 18.5%, 16.6%, 14%, and 14%, respectively.[28]
Several studies contrasted alpha diversity of gastrointestinal microbiome between cancer patients and healthy volunteers before radiotherapy. The consistent result of these studies was that the alpha diversity of cancer patients is significantly lower than that of healthy controls. [29-32]There is no conclusive evidence that intestinal microbial flora plays an active role in the development of radiation induced mucositis. However, it is known that microbiota can stimulate the production of pro-inflammatory cytokines. [33] Through producing metabolites and antigens, gut microbiota interacts with host immunity and metabolisms, thus promotes intestinal inflammation and plays important roles in cancer risk. In addition, some products may make a difference to the progression of colorectal cancer by stimulating colonic inflammation and tumor induced toxicities.[34, 35]
Another study showed that there were significant differences (P < 0.005) in microbial composition between cancer patients and healthy individuals. The researchers investigated the relative abundance of bacterial taxon at each phylum-level in fecal samples from nine patients with gynecological cancer from this study and six healthy women previously studied by the authors. Cancer patients were consistent with healthy individuals in nine bacterial phyla, and while their relative abundance of the dominant bacterial phylum is different.[29]
In following studies, gut microbiota composition was shown to affect the response of melanoma patients, and those suffering from rectal cancer or pancreatic cancer, to anti-tumor therapy, as well as tumor control[36-38]. In July, 2020, Jang et al. published their research results, that is, there was significant statistical difference of microbiota between complete response (CR) and non-CR patients as well Bacteroidales was more abundant in the latter. Duodenibacillus massiliensis was related to the increase of CR rate[39].
The changes of gut microbiota during radiation injury
Crawford and Gordon revealed that sterile mice were significantly resistant to lethal radiation-induced intestinal injury and had less radiation-induced epithelial cell damage than conventionally raised mice with symbiotic gut microflora. The microbiota increases the sensitivity of mesenchymal endothelial and immune cells to total body irradiation induced apoptosis. It was also found that the fasting induced fat factor (FIAF), a key mediator of microbiota promoting host energy storage capacity, might be involved in the radiation resistance of germ-free mice. [40] However, this animal experiment did not find specific types of bacteria related to radiation-induced intestinal injury, nor could it clarify which microbiota regulated host genes dominated the resistance to radiation-induced intestinal endothelial cell apoptosis.
In another animal experiment, Lavelle A and Sokol H investigated that the mice which recovered from high-dose radiation and live normal life spans, harbored distinct gut microbiota after radiation. This kind of gut microbiota could protect recipients against radiation-induced injury. After radiation, with hematopoietic recovery and gastrointestinal repair, the abundances of the bacterial taxa Enterococcaceae and Lachnospiraceae were elevated. These two kinds of bacteria are also more abundant in leukemia patients with mild gastrointestinal dysfunction after radiotherapy. Metabonomics showed that the concentrations of microbial derived propionic acid and tryptophan metabolites increased in the feces of mice surviving after radiotherapy. These metabolites might play a long-term role in radiation protection, and could alleviate hematopoietic disorders, reduce gastrointestinal syndrome, and reduce pro-inflammatory response[41].
It was presented from two studies that lower diversity was associated with radiation-related enteritis.[32, 42] Ferreira, M.R. reported that prostate cancer patients with radiation enteropathy (RE) have higher counts of Faecalibacterium, Roseburia,and Clostridium IV (p < 0.05). Meanwhile, they found that intestinal mucosa cytokines were related to RE and negatively correlated with counts of Propionibacterium and Roseburia. [42] Two studies conducted in China showed that diarrhoea induced by radiotherapy was associated with a higher ratio of Firmicutes to Bacteroides. [32, 43] E Husebye et.al. quantitatively analyzed gram-negative bacilli (GNB) by the glucose gas test in the culture of gastric and duodenal samples collected from 41 consecutive female patients with symptoms of late radiation enteropathy. Based on the research results, they conjectured that proximal intestinal dyskinesia and gram-negative bacterial colonization might be secondary to the injury of distal intestine after pelvic radiotherapy. [44] Another study was reported that gut anaerobic counts and aerobic microbiota (Enterobacteriaceae and Lactobacillus) might be generally reduce two hours after exposed to radiation. At the same time, it also had an impact on imbalance of the gut bacterial community structure, recruitment of leucocytes, and pathogenic effects on the epithelial mucosa ensues. However, these phenomena compensated in duration. After 24 h, the differences were not significant between irradiated and sham animals. [45]
Yuan et al. conducted a one-arm clinical study in 11 patients undergoing pelvic cancer radiotherapy, and through pyrosequencing analysis of 16S rRNA gene, it was found that the microbial disorders before treatment and the microbial disorders caused by radiotherapy during treatment were related to pelvic radiation disease.[32]Zhongqiu Wang et.al. investigated the alterations in gut microbial profiles and their relevance with enteritis in 18 cervical cancer patients undergoing pelvic radiotherapy. They revealed that microbial diversity and richness were significantly changed in patients who developed diarrhea, which was one of the symptoms of acute radiation enteritis. During that time, the α‐diversity reduced but β‐diversity increased.[43] Another study involved 10 patients undergoing 5week pelvic radiotherapy and 5 controls. The fecal microbial populations were assessed using DNA fingerprinting and sequencing of the 16S rRNA gene, before, during, and after treatment. Six patients suffered from diarrhea, and showed significant alterations in their microbial profiles during and 7 weeks after radiotherapy. The study also showed that whether patients developed diarrhea during radiotherapy was related to the initial microbial composition. A subgroup analysis of patients who reported diarrhoea was performed to compare their bacterial profiles. The results showed that the Bacilli class and Actinobacteria phylum increased while the Clostridia decreased [31]. Unfortunately, only a small number of patients was enrolled in this study, bias might arised. In addition, acute diarrhoea was not the only symptom of acute RE. The extent of RE did not be fully characterized. Furthermore, it did not be investigated if dysbiosis started before or after the begining of diarrhoea, so causal relationship could not be determined.
It was observed that the island Hispanic Puerto Ricans (HPR) patients had the similar within-sample alpha diversity with the mainland non-Hispanic whites (NHW) patients during chemoradiotherapy (CRT) while several taxa were significantly different in abundances in both groups at the end of CRT. Turicibacter and Eubacteriaceae were over-represented taxa in NHW, whereas some taxas (Muribaculaceae, Prevotella 2 and 7, Gemella, Bacillales Family XI, Catenibacterium, Sutterella, Pasteurellales, and Pasteurellaceae genera) were more enriched in the gut of HPR. It was predicted that there were significant differences in the microbiota related to phenylacetic acid and phenylethylamine degradation pathways and L-methionine synthesis pathways in HPR. Thus, HPR patients receiving CRT might had greater potential for inflammatory reaction[46].
Gastrointestinal toxicity monitoring is closely related to the feeling and description of patients. The method of data collection is clinician based. Adverse events in clinical trials are assessed and recorded by clinicians, yet clinician accuracy in assessing symptoms has been questioned.[47] Fromme EK. et.al. compared clinician records of chemotherapy related toxicities with patient-reported symptoms using the Quality-of-Life Questionnaire C30. They found that the uncorrected agreement was 59.4%.[48] Trotti A. et.al. conducted an exploratory study which suggests patient-reported outcome instruments may could be used in patients with high degree of compliance and engagement, thereby improving the quality and comprehensiveness of collected data on treatment related adverse reactions. However, this modality may also lead to increased cost and administrative burden when conducting trials.[49] These factors other than treatment hinder the research of radiation enteropathy.
Effect of radiotherapy on intestinal flora
The number of unique sequences showed a decreasing trend between initial samples and follow-up samples and a significant reduction of estimated operation classification units (OTUs) through the intraperitoneal radiotherapy. This downward trend was also identified in the Shannon diversity index (H). This prompted that radiotherapy might affect the abundance of the gut microbial community in cancer patients.[29]
A prospective observational study, included in 9 patients receiving pelvic radiotherapy, was performed by Young-Do Nam et al. to identify the impact of radiation on gut microbiota. It was revealed that the numbers of species-level taxa were obviously reduced after radiotherapy in the fecal samples. In addition, the abundance of each community largely changed. After radiation therapy, the Fusobacterium was increased by 3% while the phyla Firmicutes was decreased by 10%. The whole gut microbial composition was gradually rebuilt after the pelvic radiotherapy.[29]It was observed that Lactobacillaceae at the family level and Lactobacillus at the genus level were more abundant in the stool samples collected from the treated (either chemotherapy and/or radiotherapy) gastrointestinal tract neoplasm patients than non-treated patients[50]. Another study was to prospectively investigate changes in the composition and diversity of the gut microbies during and after pelvic CRT. The 16Sv4 rRNA gene sequencing was used to analyzed the rectal swabs collected from 58 pelvic cancer patients from two institutions before, during, and 12weeks after CRT. They found out that the reduction of gut microbiome diversity and richness levels were sustained throughout CRT. Since then, the microbial diversity of gut tended to return to baseline levels or near baseline levels at the 3months follow-up period after CRT. Gut microbiome structure and composition remained significantly changed. During CRT, the Proteobacteria increased while the Clostridiales decreased. After CRT, the Bacteroides species increased.[51]
In animal experiments, the change of intestinal flora affected by radiotherapy was also observed. Tissue samples from the irradiated small intestine of male C57Bl/6J mice were processed through a culture-dependent method. Then it was observed that compared to the tissues from sham animals, the tissue samples from the irradiated small intestine showed more pronounced changes in the bacterial composition. At 16 hours after high dose irradiation, there was a prompt and obvious decrease in the Lactobacillus group, Enterobacteriaceae group, and the total aerobic counts. However, the differences between sham and irradiated mice were not significant 24 hours after irradiation.[45] Kim YS et al. investigated the effects of radiotherapy on the alterations of the intestinal microbiota of mice using high-throughput 16S rRNA gene sequencing. They found that at the genus level, bacterial compositions of the intestines altered significantly, which might be induced by irradiation treatment. In the large intestine, the level of the genus Alistipes increased, while in the small intestine, the level of the genus Corynebacterium increased after irradiation treatment. In addition, the number of operational taxonomic units increased in the small intestine but was not observed in the large intestine. When compared to the corresponding control group, the level of the genera Prevotella in the irradiated large intestine and the levels of genera Alistipes in the irradiated small intestine were lower[52].
Studies on the relationship between enteritis and intestinal microbiome in patients underwent radiotherapy/ chemoradiotherapy (RT/CRT) were showed in the Table 1.
The gut flora modulation related therapeutic approaches for radiation enteritis
Probiotics, one kind of viable microbial dietary supplement, are live microbial organisms that could reduce the translocation of pathogens, enhance antipathogenic activity, and promote intestinal immune barrier function, thus playing beneficial roles in cancer prevention and treatment.[53] In 2002, Pietro Delia reported that the use of VSL#3, a novel and highly efficient preparation of probiotic lactobacilli, could reduce the incidence of diarrhea in patients receiving radiotherapy.[54] Then, they expanded the sample size and completed a double-blind, placebo-controlled trial. In this trail, 239 patients were enrolled in the control group and 243 patients in the trial group. Less VSL#3 patients were ultimately found to have radiation-induced diarrhea than placebo patients (31.6% vs. 51.8%, P < 0.001). Daily bowel movements were more frequent and the mean time of taking loperamide was longer in placebo patients than the VSL#3 recipients. Additionally, it is reassuring that no case of bacteremia, sepsis, or septic shock, which were attributed to the probiotic lactobacilli of VSL#3, was observed.[55] Encouraging findings have been achieved in some researches, but the analysis might be limited by the presence of significant statistical heterogeneity among clinical trials. Not all probiotics exert favorable effects due to the variability of patient characteristics and probiotics. Further high‐quality trials are needed to address the ideal bacterial strain type and dosage. In addition, cancer patients are often at risk of treatment‐related immunosuppression. Microbial agents may have adverse effects on them, thus safety concerns regarding the use of probiotics in the clinic should also be carefully investigated.[56]
Accumulating evidence suggests that gut microbiota is closely associated with radiation enteritis. To ascertain the relationship between the intestinal bacterial pattern and radiotherapy susceptibility, Ming Cui et al had conducted a series of experiments in a mouse model. It showed that gastrointestinal bacterial community composition was associated with radiotherapy susceptibility to radiation toxicity. Fecal microbiota transplantation (FMT) preserved the intestinal bacterial composition, retained the gene expression profile of the small intestine, promoted angiogenesis and increased the intestinal epithelial integrity in irradiated animals. After fecal flora transplantation, the intestinal function and survival rate of mice receiving radiotherapy were significantly improved.[57] Some experiments were designed to evaluate the efficacy and safety of FMT in patients with chronic radiation enteritis (CRE). Fresh fecal samples were processed to prepare suspension, which was infused into the patient’s intestines once through nasojejunal transendoscopic enteral tubing. Three out of five patients underwent the trail responded to FMT. They presented improvement in diarrhea, abdominal/rectal pain, rectal hemorrhage, fecal incontinence and a decrease in Karnofsky Performance Status (KPS) score. But the efficacy was not long-lasting. A mild and transient FMT-related adverse event (transient nausea) was observed in one patient. The study suggests that FMT might be effective and safe in ameliorating intestinal injury in patients with CRE for some time.[58]
Rosli D. et.al. reported that supplementation of partially hydrolyzed guar gum (PHGG), a prebiotic and natural water-soluble fiber, had the potential to increase the bifidobacterial count and relief the diarrhea after pelvic radiation treatment[59]. Other intervention approaches, including prebiotics, synbiotics, antibiotics and so on, have also been found to reduce the incidence of tumorigenesis by modulating the gut microbiome.[60-62]