Immunotherapy in Colorectal Cancer: Potential of Fecal Transplant and Microbiota-augmented Clinical Trials

Abstract

Purpose of review.

This review summarizes the role of the microbiome in colorectal cancer (CRC) in the setting of immunotherapy and emphasizes the potential of microbiota-influencing strategies with a focus on the use of fecal microbiota transplant (FMT).

Recent findings.

Observations from preclinical and clinical studies suggest that the human gut microbiome is implicated in the CRC carcinogenesis and is integral in determining the clinical response and toxicity to immunotherapy. Among the therapeutic methods devised to exploit the microbiome, FMT is the most direct method and is backed by the highest level of evidence of efficacy in nonneoplastic disease settings. Furthermore, a favorable microbiome has the potential to overcome immunotherapy resistance and ameliorate immune-related adverse events (irAEs). To this end, clinical trials are underway to evaluate the potential of FMT and microbiota-augmented methods in the setting of immunotherapy in CRC.

Summary.

Evidence from animal studies, retrospective studies, and smaller-scale prospective human studies have led to initiation of a number of microbiota-augmented clinical trials in CRC. Given the intimate relationship between the gut microbiota and the immune system as well as antitumor immune responses, potentiating immunotherapy and managing its toxicity are major areas of research in microbiota-augmented therapies in cancer. Therefore, evaluation of the patient microbiome as a routine part of clinical outcome analysis is warranted in future clinical trials.

Introduction

The Role of Immunotherapy in Colorectal Cancer

Colorectal cancer (CRC) is 3rd in global incidence and 2nd in global mortality according to 2018 data [1]. Furthermore, 25% present with stage 4 disease, 25–50% with early-stage disease then develop advanced disease; in addition, metastatic CRC (mCRC) has a 5-year survival of 12.5% [2–5]. In 2017, immunotherapy gained FDA approval as 2^nd line therapy in mCRC in tumors positive for deficient mismatch repair/microsatellite-high (dMMR/MSI-H) based on the excellent tumor response, progression-free survival, and overall survival data of NCT01876511 for pembrolizumab and CheckMate 142 for nivolumab [6–8]. However, dMMR/MSI-H is seen in only 15% of CRC [9], and their proficient MMR/MSI- low (pMMR/MSI-L) counterparts are poor responders. Lack of tumor mutational burden (TMB), tumor-infiltrating lymphocytes (TIL), low PD-L1, and IFNγ expression have been cited as mechanisms of resistance in pMMR/MSI-L CRC [10, 11]. Moreover, pMMR/MSI-L CRC has poorer prognosis in early-stage compared to dMMR/MSI-H counterparts [12] and make up the overwhelming majority of mCRC (96–98%) [13]. Therefore, a majority of CRC patients are resistant to immunotherapy and cannot derive benefit from immunotherapy alone.

In addition, immunotherapy is associated with frequent and often unpredictable immune-related adverse events (irAE). Most common irAEs are dermatological, gastrointestinal, hepatic, and endocrine, but can involve any organ system including pulmonary, renal, neurological, and hematologic. iRAEs are usually manageable with corticosteroids as the cornerstone of treatment but occasionally can be refractory to multiple lines of immunosuppressive agents and at times fatal [14]. Furthermore, combination immunotherapy is emerging as a viable treatment option in advanced cancer and is associated with a significantly higher incidence of iRAEs. For example, rash and pruritis occur in 15–20% of patients treated with anti-PD-1/PD-L1 monotherapy compared to 40% in patients treated with anti-PD-1 + anti-CTLA-4 therapy; and diarrhea and colitis occur in 10–20% of anti-PD-1/PD-L1 versus 44% in anti-PD-1 + anti-CTLA-4 [15]. Therefore, along with treatment resistance, toxicity is an important impediment to the use of immunotherapy.

Role of the Microbiota in Cancer

Human microbiota is the collective aggregate of symbiotic microorganisms residing within or on human tissue primarily along the gastrointestinal tract and respiratory tract as well as the skin whereas the microbiome refers to the collective genomes of such microorganisms. The human microbiota is emerging as an important aspect of health and disease along with environment and genetics/epigenetics and is implicated in cardiovascular, neurologic, metabolic, and gastrointestinal disorders [16–20]. Interestingly, the microbiome in certain cases may be superior to the genome in predicting disease phenotype [21]; and specific bacterial taxa and signatures may predict mortality [22]. Moreover, evidence is emerging for the role of microbiota in cancer. For example, studies have found general shifts in resident microbiota and reduction in microbial diversity to be associated with carcinogenesis [23, 24]. Furthermore, a well-noted association between certain bacterial species and several cancer types have been recognized, including Salmonella typhi and Helicobacter spp. with biliary tract cancer [25, 26]; Helicobacter pylori with gastric cancer [27, 28]; and Streptococcus bovis with CRC [29]. Other studies have even implicated the gut microbiota in non-GI cancer as well [30, 31]. Microbiota within the tumor microenvironment (TME) are also associated with clinical outcomes in GI cancer [32, 33]. Consensus on microbiota as an etiopathogenetic factor in cancer is not yet established, but its importance alongside environment and genetic/epigenetics in carcinogenesis cannot be ignored [34].

The leading hypothesis behind the contributing mechanism of gut dysbiosis in the pathogenesis of cancer is the propagation of chronic inflammation by genotoxins [35–38] and alteration of signaling pathways involved in carcinogenesis [39, 40]. Other mechanisms may include release of metabolites with context-dependent tumorigenic effects; for example, the production of secondary bile acids may alter immune function and influence tumor growth in the context of hepatobiliary cancer [41, 42]; or levels of circulating estrogens may be altered in the context of breast cancer [43]. Beyond the resident gut microbiota, the existence of microbiota within the TME has been observed in tumors proximal and distant from mucosal surfaces with resident microbiota. The explanation for the presence of microbiota in tumors distant from microbiota is unclear, although some studies suggest the role of altered mucosal integrity of microbiota-resident systems such as the GI tract and preferential homing of bacteria for the TME due to the rich vasculature and nutrients [44–46]. To this end, metastases can carry bacteria from the microbiome of primary tumors to distal tissues [47]. Thus, tumor microbiota may have an important role in the context of cancer progression, treatment, including resistance to chemotherapy [48] and altered clinical outcomes [32].

Gut Microbiota in CRC

The cancer-microbiota association has emerged in large part from studies evaluating CRC. Evidence mostly comes from preclinical and clinical metagenomic studies evaluating the entirety of the microbiome as well as preclinical studies using gnotobiotic hosts such as germ-free and antibiotic-treated mice and fecal microbiota transfer (FMT). In a recent metagenomic/metabolomic analysis of biopsy specimens from colonoscopies in 616 patients, a dose-dependent increase in Fusobacterium nucleatum spp. continuously from intramucosal carcinoma to more advanced stages of carcinoma was seen. Branched-chain amino acids, phenylalanine, and bile acids were also elevated in intramucosal carcinomas [49]. Furthermore, a meta-analysis of metagenomic analyses, including geographically diverse studies with 767 colorectal cancer specimens, identified a group of bacterial species enriched in CRC. This CRC signature maintained its accuracy in each single study [50]. Translational studies have evaluated certain microbiota signatures to aid in CRC screening either as standalone tests or alongside existing stool-based testing methods such as FIT-C. As standalone or in combination with established stool-based tests, panels of microbiome evaluated using various sequencing methods yielded high test accuracies (AUC 0.73–0.98) across different ethnic populations [51–56]. Indeed, studies using metagenomic methods and gnotobiotic mice have provided evidence implicating the gut microbiota in colorectal carcinogenesis and have suggested the potential of exploiting the gut microbiota in the screening, diagnosis and treatment of CRC.

Microbiota in Cancer Treatment Response and Toxicity

Chemotherapy/Radiation and Microbiota

Several studies have found an association between the gut microbiota and the anticancer activity of multiple chemotherapeutic agents, including 5-fluorouracil (5-FU), cyclophosphamide, gemcitabine, and oxaliplatin [57–61]. Putative mechanisms of altered efficacy may be different for each chemotherapeutic agent. Altered gut integrity and bacterial translocation has been cited as the mechanism of increased cyclophosphamide activity [58]. On the other hand, irinotecan is metabolized twice into an inactive compound, which in turn is metabolized back into its active metabolite by gut microbiota. Inhibition of this metabolic process reduces irinotecan-induced toxicity in mice [62]. Furthermore, F. nucleatum is associated with resistance to oxaliplatin and 5-FU via yet other mechanisms [48].

Less is known for the association between gut microbiota and radiation therapy. In mice and humans, radiation induces significant alterations in the composition of gut flora [63, 64]. Reduction of Firmicutes and increase in Proteobacteria may be associated with an increase in radiation-induced colitis susceptibility [65, 66]. Further studies are necessary to determine the effects of gut dysbiosis a on chemotherapy as well as radiation therapy response and toxicity.

Immunotherapy and Microbiota

Gut microbiota may influence antitumor immunity via several different mechanisms. Microbial components or pathogen-associated molecular patterns (PAMPs) may enhance the action of antigen presenting cells (APCs) via pattern recognition receptors (PRRs) such as toll-like receptors (TLRs); may enhance the cytokine production of immune cells, and may have distant effects via microbe-derived metabolites. Furthermore, tumor microbiota may alter the expression of clinically significant immune checkpoints [67–69]. Accordingly, preclinical and clinical evidence suggest that the microbiome is essential to immunotherapy response and is important in influencing irAE severity. Moreover, experiments in which fecal specimens from immunotherapy responder (R) and non-responder (NR) patients are transferred to gnotobiotic mice via FMT have demonstrated that the responder phenotype can be transferred and recapitulated [70–72]. Many studies have also uncovered a correlation between specific microbiota taxa and response to immunotherapy [73, 74, 70–72]. For example, in NSCLC and RCC patients treated with anti-PD-1 agents, A. municiphila was enriched in R versus NR (n= 60) (n=40), respectively [70]. On the other hand, in metastatic melanoma patients treated with anti-PD-1 agents (n=43), Clostridiales and Ruminococcaceae were enriched in R patients whereas Bacteroides thetaiotaomicron, E. coli, and Anaerotruncus colihominis were enriched in NR patients [72]. Another study in metastatic melanoma patients treated with anti-PD-1 agents (n=39) showed enrichment of Bacteroides caccae and Streptococcus parasanguinis in R versus NR [74].

As expected, the microbiota is also strongly associated with toxicity associated with immunotherapy, or immune-related adverse events (irAE). In metastatic melanoma patients treated with anti-CTLA-4 agent (n=34), enrichment of a number of species within the Bacteroidetes phylum was associated with protection against colitis, whereas decreased activation of metabolic pathways involved in vitamin B synthesis and polyamine transport in the gut were associated with susceptibility [75]. In another study of melanoma patients (n=26) treated with anti-CTLA-4 agent, there was a dynamic reduction in microbiome diversity with a specific reduction in the Firmicutes phylum that occurred concomitantly with colitis onset whereas enrichment of Bacteroidetes was associated with protection [73]. Interestingly, however, certain species may be associated with both favorable response as well as toxicity. For example, Firmicutes may be associated with favorable response as well as toxicity [73]. It is unclear whether the response and toxicity can be uncoupled as numerous studies support the evidence that irAEs are associated with a favorable response in multiple cancer types.

Microbiota in CRC and Immunotherapy

In CRC, specific bacterial species have been associated with favorable response to immunotherapy, including A. muciniphila, B. fragilis, Bifidobacterium spp., E limosum, Faecalibacterium spp. and A shahii [70, 71, 73, 61, 76–78]. Furthermore, FMT of A. shahii in antibiotic-treated colon tumor mice model restored therapeutic response [61], and a pooled metagenome study showed correlation of A. muciniphila and R. champanellensis with response to anti-PD-1 agent [79]. Another study identified a microbiota signature associated with treatment response in mouse colon cancer model [80]. In mice bearing MC38 tumors, oral administration of B. fragilis, B. thetalotaomicron, or B. cepacian restored the response to anti-CTLA-4 agent following antibiotic treatment; B. fragilis also restored therapeutic response in germ-free mice bearing MC38 tumors. Moreover, enrichment in Bacteroides spp. was associated with a response following the FMT of human specimens to mice [76]. Efficacy of microbiota-augmented methods to enhance immunotherapy or to treat irAEs, specifically in CRC, has not yet been demonstrated.

Role of Fecal Microbiota Transplant

Several methods have been devised to alter and shape the microbiome into a favorable composition conducive to desired clinical outcomes in the clinical setting. For example, the simplest method is the use of certain antibiotics to deplete unfavorable bacteria while leaving favorable species intact. Dietary interventions or probiotics are yet other methods of shaping the microbiota. Although simple in execution, there is difficulty in precisely targeting specific bacteria with existing antibiotics. Another analogous but more precise way may be to exploit bacteriophages for depletion of target bacteria, as is done in the field of nutrition. However, the most direct way of shaping the microbiome with the most clinical evidence for efficacy is the FMT.

FMT as Therapy for CRC

FMT has been used to treat nonneoplastic diseases with success and tolerable safety, most notably in C. difficile associated disease (CDAD) refractory to conventional antibiotic therapy. FMT may be delivered via several different routes, including via colonoscopy, enema, nasogastric tube, or oral capsules. Whether one route is more effective than the other remains a topic of controversy [81, 82]. Regardless of the route of delivery, however, safety profile seems to be excellent even in immunocompromised patients [83, 84].

Evidence for the use of FMT in the setting of cancer is limited [85, 86], although more evidence exists for their use in hematologic malignancies, specifically in the setting of acute graft-versus-host disease following hematopoietic stem cell transplant [87, 88]. However, more clinical trials in solid cancer are starting to emerge, including melanoma [NCT03341143, NCT03353402], prostate cancer [NCT04116775], and GI cancers (Table 1).

Table 1:

Ongoing clinical trials evaluating the role of microbiome in cancer immunotherapy and other select cancer treatments.

ID	Phase	Indication	Design	Main Outcome Measure	Direct Microbiome Intervention	Status
NCT04264975	Phase I	Solid Cancer	Evaluation of FMT in modulating efficacy of immune therapy	ORR*	FMT***	Recruiting
NCT04130763	Phase 1	Gastrointestinal cancer	Evaluation of FMT in improving the efficacy of anti-PD-1 therapy	ORR	FMT	Recruiting
NCT04163289	Phase 1	Renal Cell Carcinoma	Evaluation of FMT in prevention of immune-related colitis	Immune-related colitis incidence and severity	FMT	Recruiting
NCT04116775	Phase 2	Prostate cancer	Evaluation of FMT in improving the efficacy of anti-PD-1 therapy	ORR	FMT	Recruiting
NCT03819296	Phase 1	Melanoma GU cancer	Evaluation of FMT in treatment of immune-related colitis	Immune-related colitis incidence and severity	FMT	Not yet recruiting
NCT03353402	Phase 1	Melanoma	Evaluation of FMT in treatment potentiation in patients who failed anti-PD-1 therapy	ORR	FMT	Recruiting
NCT03341143	Phase 2	Melanoma	Evaluation of FMT in improving the efficacy of anti-PD-1 therapy	ORR	FMT	Recruiting
NCT03688347	Observational	Lung cancer	Evaluation of the correlation between microbiome and efficacy and safety of immune therapy and other cancer treatments	ORR, adverse event incidence	None	Active, not recruiting
NCT03586297	Observational	Breast cancer	Evaluation of the correlation between microbiome diversity and pathological complete response after neoadjuvant chemotherapy	pCR**	None	Recruiting
NCT03569137	Observational	Hematologic malignancy	Evaluation of the correlation between microbiome biomarkers and ibrutinib-associated colitis	Colitis incidence and severity	None	Not yet recruiting

^*ORR: overall response rate

^**pCR: pathological complete response

^***FMT: fecal microbiota transplant

FMT to Manage Treatment-related Adverse Events

Options are limited in the management of irAEs beyond corticosteroids, and toxic immunosuppressants, which are suboptimal therapies in frail advanced cancer patients and so FMT represents an attractive therapeutic option in this patient population. Recent case reports highlight the potential of manipulating the microbiota for the treatment of irAEs that are refractory to corticosteroids and other immunosuppressants [89]. Large scale studies evaluating irAEs are necessary to establish FMT as part of the therapeutic armamentarium.

FMT limitations

Several limitations should be noted in the use of FMT. First, the composition of a ‘favorable’ microbiome in any context has not been established and will be elusive to determine given the heterogeneity based on geographical, ethnic, and technical diversity [50]. Second, the risk of adverse events is not trivial and includes potentially fatal systemic inflammatory response syndromes, inadvertent transfer of pathogenic organisms, including highly resistant organisms. However, the improved resolution of the microbial composition, as well as improved screening methods, will help to minimize this risk. Third, there is also the potential of conferring risk of chronic diseases given that microbiome is implicated in various chronic disease processes, including obesity, atherosclerosis, and other inflammatory disorders. In mice, FMT of obese mouse fecal specimens can lead to the adoption of the same phenotype. These limitations should be taken into account in the risk vs. benefit debate as well as in the decision to use FMT.

Conclusion

The gut microbiome is emerging as an important component in colorectal carcinogenesis. Enhancing IO therapy efficacy and minimizing irAEs are important hurdles for maximizing the benefit of IO therapy in CRC. In this regard, the gut microbiome may potentiate the efficacy of immunotherapy and aid in overcoming the resistance or prevent or aid in treating irAEs. Several methods of manipulating the microbiome have been devised and are being evaluated. Among them, FMT represents the most direct and arguably most effective method of altering the microbiome. However, limitations exist in such clinical application of microbiome in CRC treatment, including the unclear definition of favorable microbiome composition and risk of adverse events, including the transfer of pathogenic microorganisms or disease traits. Therefore, further research is necessary for elucidating the science of the microbiome and its translation to the clinical setting. To this end, future clinical trials, especially of IO therapies in CRC, should analyze the individual patient microbiomes to facilitate this process.