Introduction
The human gut is colonized by a large number of commensal microorganisms which play important roles in maintenance of good health. Conversely, gut microbiota have also been implicated in the pathogenesis and pathophysiology of certain diseases [
1‐
3]. Despite being a foreign entity to the host and under the constant surveillance of the immune system, gut microbiota are able to coexist synergistically with our immune system [
4]. However, perturbations in either gut microbiota or the immune system can potentially affect this mutualistic relationship, and subsequently affect the overall health of an individual [
4]. For example, gut microbiota dysbiosis, which is associated with immune activation and inflammation of the intestinal mucosa, is a known key player in the pathogenesis of inflammatory bowel disease [
5,
6].
Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer [
7]. Advances in treatment strategies have lead to high cure rates [
8], but survivors of childhood cancer are at risk of developing many therapy-related late effects, such as metabolic syndrome, cardiovascular disease and musculoskeletal disorders, later in their life [
9‐
11]. In a recent cross-sectional study of young adult survivors of childhood ALL (median age = 26 years old) with a median period of 18.5 years off-chemotherapy, we reported reduced gut microbiota diversity and distinct gut microbiota profile as compared to controls who had no history of cancer. These survivors also exhibited increased markers of immune activation [
12] and higher prevalence of metabolic syndrome [
13]. However, it is unclear if the microbiota diversity observed in these young adult survivors of ALL is a consequence of chemotherapy exposure during their childhood and has in fact, persisted over time. Understanding this is particularly important in the context of late effects in childhood cancer survivors, which include gastrointestinal complications, chronic inflammation, metabolic syndrome, and cardiovascular disease [
9,
10,
13,
14]; conditions, which have all been, associated with gut dysbiosis in the general population [
6,
15,
16].
Several recent studies have explored gut microbiota dysbiosis in children diagnosed with ALL [
17,
18]. We provide a literature summary of the study designs and findings documented in these studies to give context to our work (Additional file 1: Table
S1). Thus far, three studies have highlighted the differences in microbiota profiles between healthy children and those with ALL at diagnosis [
17‐
19]. Children diagnosed with ALL had lower bacteria diversity in their fecal and oral microbiota [
17‐
19]. Fecal microbiota among these children were enriched with certain bacteria including
Bacteroidetes,
Enterococcaceae and
Porphyromonadaceae, while depleted with
Firmicutes,
Lachnospiraceae and
Clostridia [
17,
18]. Furthermore, changes in microbiota composition observed during chemotherapy were found to be associated with adverse clinical outcomes [
20]. Hakim et al. described that participants with higher baseline relative abundance of
Proteobacteria,
Enterococcaceae and
Streptococcaceae had a greater risk of febrile neutropenia and diarrhea during treatment phase [
20]. To date, it is still poorly understood if the host gut microbiota fully recovers in children following remission from ALL.
Gut microbiota dysbiosis has been reported in children diagnosed with ALL but no longitudinal study has thus far tracked the microbiota changes during and after cessation of chemotherapy. Here, we conducted a longitudinal observational study to examine the temporal changes in gut microbiota profile in paediatric patients diagnosed with ALL who underwent chemotherapy and compared these with age- and ethnic-matched controls. Data from this study allowed us to observe the changes in gut microbiota from time of initial cancer diagnosis and the longitudinal impact of chemotherapy on gut microbiota in children with leukemia.
Discussion
While previous research by other groups have reported gut microbiota dysbiosis in children with ALL before the initiation of chemotherapy [
17‐
19] and during the first year of chemotherapy [
18,
20] compared to healthy controls, our study is the first to examine the microbiota changes longitudinally from pre-chemotherapy up to 9 months post completion of chemotherapy. When compared to healthy controls, we observed a larger inter-individual variability and a different bacterial composition among the patients with ALL especially at time of diagnosis (or pre-chemo), consistent with previous studies both in ALL and other types of cancers [
17,
18,
30]. Although the microbiota community among the patients with ALL developed greater similarities to the controls (measured with alpha diversity and phyla distribution) post-chemotherapy, there were still differences detected in microbiota composition (measured with Bray Curtis distance) and in abundance of some bacteria OTUs between the groups. Gut microbiota perturbation in our patients during ALL treatment (possibly by chemotherapy and antibiotics) may lead to long-term microbiota dysbiosis. This is not unexpected because previous studies have shown that perturbations in gut microbiota by antibiotics often lead to incomplete microbiota restoration despite cessation of antibiotics [
31,
32].
Microbiota at pre-chemo had a lower trend of median alpha diversity than that of the healthy controls. Even though the differences were not significant, the lower trend of alpha diversity in our cohort was in concordance with previous studies measuring both oral and gut microbiota compositions in children with newly-diagnosed ALL [
17‐
19]. We also observed significantly lower bacteria evenness during chemotherapy than in the controls. The same observation has been reported in patients who received conditioning chemotherapy prior to bone marrow transplantation [
33,
34] and in mice with chemotherapy-induced mucositis [
35]. It is also interesting to note that a previous study showed that gut microbiota diversity in children with ALL decreased during intensive chemotherapy but rebounded during the reduced intensity phase [
20]. In our study, majority of the during-chemo microbiota samples were collected during the maintenance (less intensive) phase and thus, we could not verify the above observations [
20].
We observed a higher relative abundance of
Bacteroidetes and lower
Firmicutes in the patients with ALL at diagnosis compared to healthy children, in concordance with other studies [
17,
18]. In particularly, gut bacteria in our patients with ALL were predominantly belonging to
Bacteroidetes phylum and
Bacteroides genus. Enrichment of bacteria belonging to the
Bacteroidetes may be a signature dysbiosis in childhood ALL as this observation is not only found in our study, but has also been reported in three previous studies of children diagnosed with ALL at different study sites [
17‐
19]. A species of
Bacteroidetes, namely enterotoxigenic
Bacteroides fragilis has been linked with the pathogenesis of colorectal cancer [
36] but its role has never been explored in patient with ALL by previous studies.
B. fragilis toxin has been shown to be able to induce expression of c-Myc, an oncoprotein, and promote human colonic epithelial cell line proliferation in vitro [
37]. Interestingly, we also observed two OTUs affiliated to
B. fragilis were high in abundance among our patients with ALL at diagnosis. However, we are not able to postulate the role of this bacterium in the leukemogenesis with our study design. Alternatively, the changes observed in pre-chemo samples relative to controls could be associated with antibiotic exposure, as all participants had received a course within a month of sampling. A follow-up study comparing microbiota changes in ALL patients with and without prior antibiotic exposure is needed to confirm the potential influence of bacteria on leukemogenesis.
Our study further extended the sampling timeline to investigate the microbiota composition after cessation of chemotherapy i.e. with the disease in remission. This is important to understand as we previously observed reduced bacteria diversity and microbiota dysbiosis in long-term childhood ALL survivors years after chemotherapy exposure [
12]. We found that the median alpha diversity of microbiota in patients with ALL measured five to nine months after completion of chemotherapy was not significantly different from that of the control group. However, while there was no difference in alpha diversity, we detected six OTUs that were differently abundant between the post-chemo patients and controls. Significant differences in both the Bray Curtis dissimilarity distance and OTUs abundances between post-chemo and control samples suggested that the perturbed microbiota in children with ALL did not fully restore to the microbial pattern of the healthy controls despite the potential microbiota modifying factors (including but not limited to chemotherapeutic drugs and antibiotics) have been removed. In this pilot study, we did not find the same differential bacteria that were perturbed in the long-term survivors reported in our previous study [
12]. This is not unexpected as subjects in the present study are children while subjects in our earlier study were adults who had ceased chemotherapy more than a decade ago. Additionally, the lifestyle behavior and eating habits of the adult survivors have likely changed considerably. Nevertheless, we do not exclude the possibility that after a longer period of time, the microbiota community in our current cohort of ALL survivors may evolve to acquire a similar dysbiosis pattern as was observed in the long-term survivors.
There are several limitations in our study. Our preliminary findings are based on a small number of subjects and hence may not sensitively identify the bacteria with lower degree of changes. Observations in this study do not suggest causal relationship between microbiota dysbiosis post-chemotherapy with the risk of future health conditions, which would require a longer follow-up study and confirmation study with a bigger group of subjects. Despite previous studies which have shown similar microbiota composition obtained from anal swab and fecal samples [
38,
39], we are aware that other studies have on the other hand, demonstrated variation in the gut microbiota composition analyzed by different sampling methods [
39,
40]. We performed anal swabs to collect fecal bacteria as opposed to collecting stool samples due to the practicality in the clinic setting and to maintain consistency with our previous study. Moreover, it is challenging to get on-demand fecal samples from young children. In this study, we used 16 s rRNA gene targeted sequencing for microbiota profiling because it is one of the most widely used and robust method to identify and quantify different bacteria taxa within microbial community comprises of large variety of species, but this technique does not allow us to measure the functional genomics of the microbiota [
41].
Antibiotics are known to cause alterations in gut microbiota composition [
42]. However, studies by Bai et al and Rajagopala et al detected gut microbiota dysbiosis in patients at the diagnosis of ALL regardless of prior exposure to antibiotic treatment [
17,
18]. Findings in these studies shown that the lower bacteria diversity in patients with ALL could not be solely explained by the use of antibiotics close to the time of fecal microbiota measurement [
17,
18]. Rather, the authors suggested that microbiota dysbiosis in these patients could be influenced not only by antibiotic usage, but also by immune system derangement. We are unable to elucidate the role of malignancy-related altered immunity in causing differences in gut bacterial composition due to the small number of our subjects and the fact that all of them received antibiotics prior to baseline (pre-chemo) sampling. Ideally, we would have enrolled a group of ALL patients not treated with antibiotics. However, it is challenging to enrol children with ALL without prior antibiotic exposure because an audit identified that > 80% of the children diagnosed in our medical centre received empirical antibiotics prescribed at the referring hospitals or by the patient’s primary care physician for fever and presumed infections before the diagnosis of ALL was made. An alternative could have been an additional control group with healthy children (without ALL) treated with antibiotics for fever or other minor illness, which could be included in future studies.
Our main objective was to discover whether gut microbiota pattern in children treated for ALL restored towards a healthy state, as represented by the healthy antibiotic-free children controls, after immune restitution following attainment of disease remission and cessation of chemotherapy. Thus, this study was not designed nor adequately powered to identify the actual causes of gut microbiota perturbation in these children. In addition to antibiotics, gut microbiota dysbiosis in children with ALL can also be affected by other factors such as ethnicity, severity of ALL, treatment intensity, episodes of opportunistic infections, and environment factors, which should be taken into account in future studies.
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