Abstract
Modern advances in genomic and molecular technologies have sparked substantial research on the human intestinal microbiome over the past decade. A deeper understanding of the microbiome has illuminated that dysbiosis, or a disruption in the microbiome, is associated with inflammatory disease states and carcinogenesis. Novel therapies that target the microbiome and restore healthy flora may have value in dampening the immunopathologic state induced by dysbiosis. A narrative review of the literature on the use of microbiota-centered interventions (MCIs) was conducted. Several randomized clinical trials show that MCIs can augment response to immune checkpoint inhibitor (ICI) therapy in patients with metastatic cancer. Clinical trials have also demonstrated that modulation of the intestinal microbiome can enhance recovery and reduce infectious complications in the surgical management of colorectal adenocarcinoma. Overall, these major discoveries suggest future clinical applications of MCIs for a wide range of immune-mediated conditions. These results may also translate to improved patient outcomes in systemic immunotherapy for urothelial carcinoma as well as in patients recovering from radical cystectomy (RC), which is complicated by high infection rates. Further research is needed to evaluate the optimal bacterial composition of microbiota-centered therapies and the specific cellular changes that lead to improved tumor antigen recognition after microbiota-centered therapies.
Keywords
Introduction
The human intestinal microbiome is a complex community of microorganisms constituted by the collection of all gastrointestinal bacteria, viruses, protozoa, and fungi and their genetic material. In recent years, the taxonomic composition and functional characteristics of the microbiome have been extensively studied across the globe.1,2 Although numerous factors affect the distinct “enterotype” of individuals, developments in genome sequencing technologies and bioinformatics have illuminated remarkable associations between the architecture of the gut microbiome and human health.3–5 Several studies have linked persistent imbalances or deviations in the microbiome, also known as dysbiosis, to disease states such as inflammatory bowel disease, diabetes, and cancer.4,6 Research has demonstrated a significant link between dysbiotic gut microbiota and a higher abundance of opportunistic pathogens, including hydrogen sulfide- and endotoxin-producing pathogens. 7 These opportunistic pathogens increase the antigen load and sustain a higher baseline level of inflammation in the body. 8 Thus, a dysbiotic microbiome predisposes patients to a greater risk of immunopathology, which may exacerbate existing diseases or provoke underlying diseases such as cancer. Dysbiosis can occur outside of the gastrointestinal tract as well; recent research reported significant differences in relative concentrations of urinary tract microbes when comparing patients with genitourinary malignancies to controls, corroborating the role of the urinary tract microbiome in the pathogenesis of urologic cancers. 9
Dysbiosis has been shown to drive carcinogenesis through both metabolite production and immunomodulatory mechanisms. While research has historically focused on the ability of individual pathogens like Helicobacter pylori, Schistosoma haematobium, and Human Papillomavirus to initiate and perpetuate carcinogenesis, newer research has utilized a holistic approach, which deepens our understanding of the dysbiotic changes that lead to cancer by characterizing the protective processes of entire commensal microbial communities. For example, beneficial gut bacteria that produce short-chain fatty acids (SCFAs) such as butyrate and acetate through the fermentation of dietary fiber have been shown to suppress carcinogenesis.10,11 SCFAs support intestinal barrier integrity, regulate redox reactions, and promote immune function—all of which have a preventative effect on cancer development. 10 One study has shown that butyrate can even induce apoptosis in colorectal cancer cells by inhibiting the expression of histone deacetylase-regulating genes. 12 Beneficial gut bacteria also modulate anti-inflammatory interferons and interleukins in the cell microenvironment. For example, Bifidobacterium breve produces metabolites that favor the local differentiation and mitochondrial fitness of IL-10-producing regulatory T cells (Tregs), thereby supporting regulatory B cells and suppressing proangiogenic Th17 cells in the microenvironment. 13 This is in contrast to dysbiotic microbiomes, where altered signals at the colonic mucosa cause immunologic activation and dysregulation of growth pathways involving Wnt, Notch, transforming growth factor beta (TGF-β), IL-23, and nuclear factor kappa-light-chain-enhancer ofactivated B cells (NF-κB).10,14
The identification of the carcinogenic mechanisms linked to gut dysbiosis has valuable clinical implications for the treatment of cancer. Although certain microbial compositions can promote tumorigenesis, novel therapies that restore a commensal microbiome may play a unique role in combating cancer through the regulation of the local immune system and the production of anti-inflammatory SCFAs. Treatments that target the microbiome may also augment the efficacy of existing therapies by strengthening the host's ability to perform cancer immunosurveillance (Figure 1). 15 Thus, several investigators are studying microbiota-centered interventions (MCIs), such as live bacterial product capsules and fecal microbiota transplants (FMTs), as therapeutic strategies which can strengthen the efficacy of the current standard of care. The objective of this article is to review the current randomized controlled trial evidence on MCI in medical and surgical oncology. New developments and challenges associated with MCI will be discussed in the context of urologic oncology, with the aim of demonstrating applications for MCI in urothelial carcinoma management.
Mechanisms of action of microbiota-centered interventions (MCIs).
MCIs and Response to Immunotherapy
Food and Drug Administration (FDA) approval of immune checkpoint inhibitors (ICIs) for the treatment of several cancer types has revolutionized the management approach to cancer therapy over the past decade. Dysbiosis in the microbiome has been associated with resistance to immunotherapy.16–18 This is supported by the fact that most cancers responsive to programmed cell death protein 1 (PD-1)/programmed death ligand-1 (PD-L1) blockade arise at physiologic intersections where a complex microbial ecosystem exists. Furthermore, treatment with antibiotics, many of which markedly disrupt the gut microbiome, has been linked to a significant reduction in both progression-free survival and overall survival in cancers such as renal cell carcinoma. 19 Treatment with antibiotics prior to ICI therapy was shown to be more harmful than antibiotic use after ICI therapy. 20 Derosa et al. 11 inspected the landscape of bacteria associated with clinical responses to PD-1/PD-L1 blockade across stool genetic fingerprints in several independent studies, identifying the Bacteroidales order, the Verrucomicrobiaceae and Lachnospiracea families, the Eubacterium and Bifidobacterium genera, and the Ruminococcus and Collinsella species as beneficial microbiota. Conversely, they identified that gut microbiome signatures dominated by the Actinobacteria and Proteobacteria phyla (containing Escherichia coli, Shigella, and Klebsiella), the Fusobacteria genera, and the Porphyromonadaceae family are associated with nonresponders to ICI therapy. Although conclusions cannot be drawn about whether poor response to ICI therapy is consequential to the absence of beneficial bacteria or the presence of detrimental bacteria, patients who responded to immunotherapy tended to possess high relative concentrations of SCFA-producing bacteria. 11
In light of the potential therapeutic value of SCFA-producing bacteria, Dizman et al. 21 performed the first prospective randomized controlled trial that added a live bacterial product to standard-of-care ICI therapy (Table 1). The authors examined the biological effect of CMB588, a strain of Clostridium butyricum that produces SCFAs and promotes the growth of other commensal gut bacteria, in combination with nivolumab and ipilimumab for metastatic renal cell carcinoma (mRCC). Patients with previously untreated intermediate or poor-risk clear cell or sarcomatoid mRCC were randomized to receive ICI + CBM-588 80 mg bid or ICI + placebo. Metagenomic sequencing in 29 evaluable patients showed an 8-fold increase from baseline to week 12 in Bifidobacterium bifidum and a 6-fold increase in Bifidobacterium adolescentis among those patients receiving CBM-588 in addition to nivolumab and ipilimumab. C butyricum was detected only among those receiving CBM-588. Furthermore, pathogenic species such as E coli and Klebsiella spp. were less prevalent in patients who received CBM-588. Overall response rate (ORR) was significantly higher among patients receiving ICI therapy plus CBM-588 versus ICI therapy alone (58% vs 20%; P = .024). Median progression-free survival (mPFS) was also prolonged when CBM-588 was added to ICI therapy (NR vs 11 weeks; P < .001). 21
Microbiota-Centered Interventions (MCIs) in Medical Oncology.
Abbreviations: FMT, fecal microbiota trasnplant; mPFS, median progression-free survival; ORR: overall response rate; PD-1, programmed cell death protein 1.
Baruch et al. 22 also reported favorable alterations in clinical response to ICI therapy when combined with modulation of the gut microbiome in their phase I trial of 10 patients with anti-PD-1-refractory metastatic melanoma. Study participants underwent a native microbiota depletion phase with vancomycin and neomycin, followed by a FMT via oral capsules and colonoscopy from 2 donors. The donors had achieved at least 1 year of complete response to anti-PD-1 monotherapy for metastatic melanoma. Anti-PD-1 treatment with nivolumab was resumed for 6 more treatment cycles, with maintenance oral FMT every 14 days until day 90. Three participants, all of whom received FMT from donor 1, demonstrated objective responses to treatment and crossed the progression-free survival milestone of 6 months; one participant achieved a complete response while the other 2 achieved partial responses. 16S rRNA gene sequencing analysis showed that the posttreatment gut microbiomes were significantly altered from their baseline (P = .02), with a higher relative abundance of the Veillonellaceae family and a lower relative abundance of B bifidum. 22 Interestingly, patients who showed improved outcomes in Dizman et al. had an 8-fold increase in B bifidum concentrations, implying that the value of a certain species may depend upon the context and interactions of a myriad of physiologic factors.
This is supported by the finding that although 4 taxa differed significantly between the 3 responders and the 2 nonresponders from the same donor, when the concentrations of these specific microbes were examined in all 10 participants, there were some nonresponders whose microbiota compositions were similar to the responders. 22 Thus, the authors were unable to establish a clear relationship between those microbes and clinical response to ICI therapy. This echoes the concept that while research has illuminated themes in stool genetic fingerprints which are correlated with treatment response, the ideal composition of microbiota remains elusive and is likely patient dependent. The external validity of Baruch et al.'s study is limited by small sample size, but their work demonstrates that MCIs like FMT are overall safe and feasible.
MCIs and Response to Intraabdominal Surgery
Modulation of the gut microbiome may also have clinical utility in expediting recovery from surgery and reducing infectious perioperative complications. Evidence suggests that most postoperative infections are secondary to pathogens located in the gastrointestinal tract.23,24 Although preoperative antibiotics targeting these organisms have significantly reduced infection rates, 25 this approach also eradicates many of the healthy microbiota that guides recovery.26,27 The variability of the gut microbiome and its sensitivity to antibiotics varies on an individual level, 28 and one of the factors most strongly associated with the composition of individuals’ gut microbiome is diet. 29 The majority of surgical patients in the United States consume a highly processed, low-fiber Western diet (WD), which results in obese body habitus and a decrease in SCFAs, both of which are linked to delayed recovery from injury. 30
Keskey et al. 31 studied the effects of a WD on gut microbiome profiles in the context of surgical outcomes. These investigators subjected mice to either 6 weeks of a WD or 6 weeks of a standard diet (SD). WD mice either maintained their WD before surgery or received 3 versus 7 days of dietary pre-habilitation prior to surgery (3DP vs 7DP). WD mice demonstrated markedly decreased survival compared to SD mice, and longer courses of dietary pre-habilitating improved survival (29% vs 38% vs 79% vs 100%: WD vs 3DP vs 7DP vs SD, P < .001). 16S rRNA gene sequencing of stool samples showed that WD and SD mice had distinct stool microbiota signatures. Dietary pre-habilitation led to a significant proliferation of Bacteroidetes alongside a decrease in Firmicutes and Proteobacteria (containing clinically aggressive E coli) as early as 24 h from the change in diet. Functional enzyme analysis showed that after 7 days of diet pre-habilitation, there was a restoration of SCFA-producing bacteria in WD mice. This observed shift in stool microbiota profile and function is consistent with the beneficial gut microbiome signatures identified by Derosa et al. However, neither microbiota composition alterations nor SCFA concentrations were independently satisfactory in predicting survival. Rather, the functional output of the entire microbiome relative to the bacterial biomass provided a more accurate index. Furthermore, single time point measurements did not reflect the dynamic changes that occur with dietary interventions. 31 Keskey et al.'s work implies that alterations in the gut microbiome can establish a readiness to withstand the process of surgery, and may be predictive of enhanced survival.
Keskey et al.'s work in mice has been validated by studies that examined the effect of preoperative synbiotics on surgical outcomes in humans (Table 2). Synbiotic compounds modulate the intestinal microbiota through “competitive exclusion”. In competitive exclusion, microorganisms such as Bifidobacterium and Lactobacillus reinforce the natural defense mechanisms of the body by producing SCFAs, which inhibit the growth of disadvantageous microbes. 32 It is important to mention the difference between synbiotics, prebiotics, and probiotics, as evidence for the routine use of these compounds in the perioperative setting remains heterogeneous.33,34 A probiotic is a formulation containing viable, specific microorganisms that alter the microbiota of the host with the intent to provide beneficial health effects. A prebiotic is a nondigestible food ingredient that selectively encourages the growth and/or activity of certain beneficial microorganisms. 35 A synbiotic is a product that contains both probiotics and prebiotics.
Microbiota-Centered Interventions (MCIs) in Surgical Oncology.
In a prospective randomized, double-blind clinical trial of 91 patients with colorectal adenocarcinoma, Flesch et al. 36 examined postoperative infection rates in patients receiving either synbiotics or placebo administered twice daily for 5 days before and 14 days after surgery. Surgical site infection occurred in only 1 patient (2%) in the synbiotics group compared to 9 patients (21.4%) in the control group (P = .002). Additionally, the authors reported 7 nonsurgical site infections in the control group compared to zero nonsurgical site infections in the synbiotic group (P = .001). There were no statistically significant differences in mean hospitalization time, rehospitalization rates, or mortality. 36
In another prospective randomized, double-blind clinical trial, Polakowski et al. 37 found that the use of preoperative synbiotics in 73 patients with colorectal cancer not only reduced infectious complications and attenuated several inflammatory markers but also decreased length of hospital stay and antibiotic use. In this trial, the participants ingested either placebo or the same synbiotic used in Flesch et al. twice daily for 7 days prior to colorectal surgery. Postoperative infectious complications occurred in only 1 patient (2.8%) in the synbiotic group compared to 7 patients (18.9%) in the control group (P = .02). The synbiotic consumption group had significantly reduced IL-6 (P < .001) and CRP (P < .001), while the placebo group had no significant differences in IL-6 or CRP levels. Mean days of antibiotic usage was significantly lower in the synbiotic group compared to the control group (P < .001). Mean days of hospital stay was also significantly decreased for the synbiotic group compared to the control group (P < .001). There were no deaths in the synbiotic group, but 3 (8.1%) deaths were reported in the control group. 37
The results of these 2 studies are corroborated by the findings of a meta-analysis published by Kinross et al., which examined synbiotic use in surgery for 962 patients across 13 randomized controlled trials. 38 The authors found that the incidence of postoperative sepsis was reduced in the synbiotic group versus the control group (pooled OR = 0.25; 95% CI = 0.1-0.6; P = .002), and that synbiotics reduced the length of postoperative antibiotic use (weighted mean difference = −1.71; 95% CI −3.2 to −0.21; P = .03). Almost all of the studies used synbiotics that incorporated Lactobacillus, and 6 of the 13 studies used synbiotics that incorporated a Bifidobacteria species, which has demonstrated anti-inflammatory properties and is known to inhibit pathogenic organisms. The thirteen studies lacked consistency in route of administration, dosing, and type of synbiotic used, which, in addition to variations in surgical procedure and technique, serves as a significant confounder for these results. Although conclusions cannot be drawn about which specific synbiotic confers the greatest benefit in terms of reducing postoperative infectious complications, these studies demonstrate that MCIs can improve surgical outcomes by modulating opportunistic pathogens in the gut microbiome, which decreases baseline levels of inflammation and lowers the risk for sepsis. This is similar to the way that MCI performs as an immunomodulator in cancer patients on anti-PD-1/PD-L1 therapy.
Applications to Urothelial Carcinoma
The triumphs of MCIs in bolstering the response to systemic ICI therapy in cancer patients have implications for the management of bladder cancer—one of the only diseases for which the U.S. FDA has approved a live biotherapeutic product as treatment. 39 The gold-standard adjuvant therapy for patients with intermediate- and high-risk non-muscle-invasive bladder cancer (NIMBC) is intravesical Bacillus Calmette–Guerin (BCG) instillation after transurethral resection of bladder tumor (TURBT). 40 BCG works by inducing immunologic changes that trigger increased expression of major histocompatibility complexes and elevated release of urinary cytokines; this immune challenge enables functional reprogramming of the patient's own immune system, allowing previously unrecognized bladder cancer cells to be targeted by macrophages and lymphocytes for destruction. 41 However, up to 40% of patients with NMIBC will fail to respond to intravesical BCG therapy. 42 The urinary microbiome may play a role in predicting responsiveness to BCG therapy for NMIBC, especially since numerous studies have identified changes in the diversity and abundance of bacteria in the bladder among patients with urothelial carcinoma compared to healthy controls. 43 Several of these studies also demonstrated that certain lactic acid-producing bacteria (such as Lactobacillus rhamnosus and Lactobacillus casei) have anti-proliferative effects on bladder cancer cell lines in vitro and in vivo mouse models.44,45 Additionally, in a trial of 31 patients treated for NMIBC with intravesical BCG immunotherapy, Sweis et al. reported higher relative concentrations of Proteobacteria in patients with tumor recurrence (P = .035), whereas Lactobacillales were more abundant in patients without tumor recurrence (P = .049). 46 Thus, given that poor therapeutic alternatives currently exist for patients with intravesical BCG failure, 47 MCI such as synbiotic capsules to augment the efficacy of BCG therapy represents a promising source of investigation. Decreasing resistance and improving responsiveness to BCG therapy is particularly critical to decreasing morbidity and mortality from bladder cancer due to the current shortage of BCG, which has led many clinicians to strategize on how to conserve doses. 48 Nevertheless, the limited reserve of BCG has generated an opportunity to accelerate research and clinical trials exploring the relationship between the urinary tract microbiome and urothelial carcinoma. For example, the SILENTEMPIRE trial plans to investigate signatures in urinary and gut microbial profiles of NMIBC patients as predictors for BCG therapy response. 49 Moreover, with the approval of PD-1/PD-L1 inhibitors for urothelial cancer, several trials have begun to examine BCG as an MCI that can serve to enhance the efficacy of these agents in NMIBC (Table 3). The introduction of BCG to the bladder microbiome has been shown to upregulate PD-L1 expression in urothelial cancer cells, 50 and therefore these patients are likely to benefit from the combination of systemic immunotherapy and intravesical BCG. The promising therapeutic benefit of PD-L1 inhibitors in combination with a live bacterial product such as BCG is underscored by the successes of MCI and ICI therapy combinations reported by Dizman et al. and Baruch et al.
Ongoing Clinical Trials, Microbiota-Centered Interventions (MCIs) for Urothelial Carcinoma.
Abbreviations: BCG, Bacillus Calmette–Guerin; NMIBC, non-muscle-invasive bladder control; TURBT, transurethral resection of bladder tumor.
Furthermore, promising data on MCI's role in decreasing infectious complications after visceral surgery may have future implications in recovery from radical cystectomy (RC). The gold standard in the management of muscle-invasive bladder cancer is neoadjuvant chemotherapy followed by RC and the creation of a new urinary reservoir, 53 which is typically reconstructed from the terminal ileum. RC is also an option for BCG-unresponsive high-grade NMIBC patients. 54 RC is a morbid procedure plagued by disappointingly high complication rates; greater than 1/third of patients experience complications within 30 days and greater than ½ of patients experience complications within 90 days. 55 The incidence of urinary tract infection after RC is approximately 30%, and infectious complications are the strongest predictor of readmission within 90 days after RC.56,57 Given that there is a unique microbiome in the gut used to reconstruct the urinary reservoir of patients after RC, it is possible that intestinal and urinary tract dysbiosis contributes to infectious complications. Moreover, a recent study of twenty RC patients found that the urinary diversion microbiome after RC differs from the microbiome of the native bladder and ileum; this study also observed that high fungal loads were correlated with patients having any complication. 58 A deeper understanding of the urinary diversion microbial environment, combined with novel therapies targeting the human microbiome, may ultimately lead to a decline in perioperative complications after RC. Future research should include prospective randomized trials to evaluate the impact of microbiome therapies on reducing negative outcomes after RC. Future research should also examine whether patients with infectious perioperative complications have differences in baseline urine and stool microbial diversity compared to patients without infectious complications.
Conclusions
MCIs, which work by altering the host's microbiome, have demonstrated a clinically meaningful ability to strengthen the efficacy of cancer therapies through their immuno-modulatory and anti-proliferative properties. There is no doubt that MCI will complement current and yet-to-be-developed immunotherapeutic agents in the future. Moving forward, challenges still exist to define “the healthy microbiome” in terms of the optimal composition of immunogenic and suppressive bacteria, as well as their local and distant group interactions. The preferred shift in geo-distribution, circulating metabolites, and cellular immune responses to tumor antigens after MCI represents another frontier requiring further investigation. Lastly, molecular strategies to examine the pharmacokinetics of recolonization and engineering tools to develop distributable MCI formulations remain arduous and difficult to establish given the highly personalized nature of MCI therapy. Nevertheless, technologies for cancer treatment have seen accelerated growth in recent years, and these advances may help to guide the next steps associated with the promising preliminary results of MCIs for cancer therapeutics, particularly with respect to the medical and surgical treatment of urothelial carcinoma.
Footnotes
Abbreviations
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
