Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review

ABSTRACT Introduction: despite being extremely effective in some cases, up to 70% of patients with melanoma do not respond to anti-PD-1/PD-L1 (primary resistance) and many of the responders eventually progress (secondary resistance). Extensive efforts are being made to overcome this resistance through new strategies, especially aimed at modulating the intestinal microbiota. Objective: to assess whether fecal microbiota transplantation (FMT), associated with immunotherapy, is beneficial in the clinical course of patients with refractory melanoma. Methods: this is a scope review, based on studies collected on the MEDLINE, ScienceDirect, The Cochrane Library, Embase and BMJ Journals; using the terms: “Antibodies, Monoclonal”; “Drug Resistance, Neoplasm”; “Fecal Microbiota Transplantation”; “Host Microbial Interactions”; “Immunotherapy”; “Melanoma”; and “Microbiota”. Clinical trials, in English, with relevant data on the subject and fully available were included. A cut-off period was not determined, due to the limited amount of evidence on the topic. Results: crossing the descriptors allowed the identification of 342 publications and, after applying the eligibility criteria, allowed the selection of 4 studies. From the analyses, it was observed that a considerable part of those studied overcame resistance to immune checkpoint inhibitors after FMT, with better response to treatment, less tumor growth and increased beneficial immune response. Conclusion: it is noted that FMT favors the response of melanoma to immunotherapy, translated into significant clinical benefit. However, further studies are necessary for the complete elucidation of the bacteria and the mechanisms involved, as well as for the translation of new evidence to oncological care practice.


INTRODUCTION
C ancer is a state that involves both genetic and epigenetic interactions, culminating in a disorderly, autonomous, and uncontrollable proliferation of cells, which may eventually migrate to adjacent structures.This migratory growth characteristic belongs to invasive neoplasms, being called "malignant tumors".There are more than 200 types of cancer, each with specific clinical and morphological characteristics, with melanoma being the third most common and the most lethal among skin cancers, with an estimated increase of 50% in new cases per year by 2040, according to data from the International Agency for Research on Cancer (IARC) 1,2 .
Melanoma is a neoplasm originating from the skin formed from the transformation of melaninproducing cells (melanocytes) and may appear in skin without previous lesions (60-80%) or with pigmented lesions (20-40%), such as melanocytic nevi 3 .Although it has a lower incidence when compared with nonmelanomas, melanoma is the most important form of skin tumor, as it grows and spreads more quickly, has a higher risk of recurrence, and a worse prognosis, with a worldwide survival estimate in five years of 69%, 73% in developed countries and 56% in developing ones 4,5 .
Among the risk factors for the development of this tumor, the inheritance of sun-sensitive skin stands out (Fitzpatrick skin types I and II -light skin, hair, and metastases), adjuvant treatment, systemic therapy, and/ or radiotherapy 14 .
In cases of unresectable, metastatic cutaneous melanoma or with high rates of recurrence (stages III and IV), specific therapies should be considered, such as immunotherapy with immune checkpoint inhibitors (ICIs).Currently, the drugs most used in this treatment are the humanized monoclonal antibodies (mAbs) anti-PD-1 (pembrolizumab and nivolumab) and anti-PD-L1 (atezolizumab, avelumab, and durvalumab), which act by inhibiting the activity of the programmed cell death protein-1 (PD-1) 15,16 .These drugs bind to the PD-1 receptor, an immunological receptor present on the surface of tumor cells, preventing the receptor from interacting with specific ligands (PD-L1 and PD-L2).
Blocking the PD-1 pathway signaling cascade inhibits the negative regulation of the immune system, reversing the suppression of T cells and culminating in the anti-tumor response 17 .
Anti-PD-1/PD-L1 demonstrated superior efficacy and a more favorable toxicity profile than other mAbs, such as ipilimumab, an inhibitor of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), also widely used in the therapeutic approach of melanoma 18,19 .
However, although these blockers show extreme effectiveness in some cases, up to 70% of patients do not respond to PD-1 blockade (primary resistance) and many who do respond eventually progress (secondary resistance).Therefore, efforts are currently being made to understand in detail the mechanisms underlying resistance, aiming to elucidate biomarkers that can potentially help in identifying responders, as well as finding new strategies that can decrease anti-PD-1/PD-L1 resistance [20][21][22][23][24] .
In search of advances in medicine, researchers returned to a Chinese practice dating back to the fourth century, during the Dong Jin dynasty, known as fecal microbiota transplantation (FMT).This practice consists of transferring fecal material from a healthy donor to a sick recipient, to repopulate the latter's intestine.Despite being very old, FMT was scientifically reported for the first time in 1958, when it was successfully used in the treatment of four patients with pseudomembranous colitis [25][26][27] .Even in the face of apparent efficacy, only in the last ten years has FMT begun to be widely studied, eyes, and susceptibility to sunburn).Other factors include the presence of pigmented lesions, such as freckles, atypical nevi, or large numbers of common nevi (more than 50), intermittent sun exposure, sunburn (especially during childhood), artificial ultraviolet radiation (eg, use of tanning beds), proximity to the equator, and previous melanoma 6,7 .Positive family history for both melanoma and multiple atypical nevi is also considered a relevant factor.Mutations in the CDKN2A and CDK4 genes have been detected in some families with hereditary melanoma, conferring an approximately 60-90% increased risk for this neoplasm 8,4 .In addition, the immune status also seems to influence the development of melanoma, as this neoplasm is frequent in immunocompromised patients due to organ transplantation, human immunodeficiency virus (HIV) infection, hematologic malignancy, or use of immunosuppressants 9 .
The diagnostic suspicion, in general, is based on the identification of a new pigmented lesion (ab initio) or phenotypic alterations of a pre-existing melanocytic nevus.During the assessment, the mnemonic "ABCDE" has been shown to be useful: A -asymmetry; B -irregular edges; C -non-uniform coloring; D -diameter > 6mm; E -evolution, including rapid growth, pruritus, ulceration, and bleeding.The greater the positivity of these signs, the greater the probability of the lesion being a malignant melanoma.However, the definitive diagnosis is made through histopathological examination, which can identify the superficial disseminated, nodular, acral lentiginous, or lentigo maligna patterns 10,11 .
In terms of prognosis and treatment, staging the disease is essential, in accordance with the American Joint Commission on Cancer (AJCC) protocol, which divides patients into four distinct groups.The group is defined according to the phase the patient is in, in which stages I and II are considered clinically localized disease, stage III, locoregional disease, including individuals with metastases to regional or in-transit lymph nodes, and stage IV, comprising individuals with distant metastasis 12 .
After histopathological confirmation and staging, the treatment to be adopted is established 13 .

RESULTS
The combination of search terms resulted in the identification of 342 publications.After removal of duplicates (n=27), 315 references were submitted to screening, with analysis of titles and abstracts, of which 302 were excluded by reviewers.The 13 remaining studies were read in full by two independent examiners for the application of the eligibility criteria, culminating in the final inclusion of four trials that met these criteria.
The selected studies were published between 2019 and 2021.
After conducting the steps of identification, screening, and eligibility of the articles, it was possible to stratify and gather the main evidence related to the application of the ICI-FMT combined therapy in patients with melanoma.Studies extracted from each of the databases were recorded in a single Microsoft Excel 16.0 spreadsheet, with the aim of detecting repeated citations between platforms, forming the list of studies and assessing eligibility.
Therefore, due to the favorable pre-existing history, the present study aims to evaluate whether the ICI-FMT combined therapy positively impacts the clinical course of patients with melanoma refractory to isolated immunotherapy, representing a potential step during the therapeutic process of this neoplasm.

METHODS
This is a descriptive study of exploratory nature in literature in the scope review modality, which is a research method that makes it possible to identify and analyze a set of scientific evidence to obtain a reliable understanding about a particular theme of study.This method makes it possible to summarize the state of knowledge in a given area, as well as pointing out gaps that need to be filled with new research.Therefore, it entails support for decision-making and improvement of clinical practice 34 .
We used the Preferred Reporting Items for
The results of clinical trials that investigated the impact of FMT on the effectiveness of the immune checkpoint inhibitor are summarized in Table 1. to the anergy or "exhaustion" phenotype, represents a form of local immunocompromise that allows tumors to escape immunological surveillance 42,43 .

DISCUSSION
Blocking the PD-1/PD-L1 pathway (Figure 1) is considered one of the main targets of immunotherapy against cancer, with drugs already approved by the Food and Drug Administration (FDA), such as pembrolizumab, nivolumab, atezolizumab, and cemiplimab 24,[44][45][46][47] .Table 2 highlights each drug, with its respective indications and main adverse effects.

Barbosa
Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review
(GIT).The GIT hosts the largest number and diversity of bacterial collections that colonize the human body.It is estimated that its microbial population reaches 10 11 to 10 12 CFU/mL of intestinal contents, with about 700 different species of microorganisms, most of which are bacteria.However, although bacteria can be found throughout the GIT, the largest number resides in the colon 56,57 .
In the ileum, the bacterial population is to overcome this resistance to therapy, especially in the approach to melanoma [20][21][22][23][24] .

Intestinal Microbiota
The intestinal microbiota, previously referred to as flora, is a dynamic mixture of microorganisms, whose composition varies along the gastrointestinal tract of high-throughput sequencing technologies, the importance of the intestinal microbiota in the modulation of chemotherapy drugs was listed and the search for new influenceable drugs is now increasingly recognized 63,64 .
However, the microbiota is extremely dynamic and may be influenced by numerous factors.For example, most neoplasms reach extremes of age, when the structural ecology of the gut may be immature or disturbed by a lifetime's exposure to environmental modifiers 65 .Furthermore, pathological states or specific therapies can also create an imbalance in the microbiota composition, increasing the influence of deleterious bacteria, as well as reducing the effectiveness of antineoplastic agents and exacerbating their toxicity.
Therefore, the intestinal microbiota has been researched for the management of cancer, with the development of promising strategies, such as the modulation of the patients' intestinal microorganisms' content [28][29][30][31][32][33] .

Fecal microbiota transplantation
FMT consists of transferring a solution of fecal material from a donor to a recipient, aiming to directly change the microbial composition of the patient and confer benefits to his health.The first known report of the use of feces as a therapy was described by Ge Hong, in China, in the fourth century, for the treatment of a variety of conditions, including diarrhea 27,66,67 .However, it was only in the 1950s that the practice was scientifically described by Eiseman et al. (1958)  25 , via fecal enema, for the treatment of pseudomembranous colitis, introducing FMT in conventional medicine.
In general, the process initially involves selecting a donor with no personal or family history of metabolic, autoimmune, or malignant diseases, as well as screening for possible pathogens.The stool is then prepared by being mixed with water or normal saline, followed by a filtration step to remove any particulate matter.The mixture can be administered by nasogastric tube, nasojejunal tube, esophagogastroduodenoscopy, colonoscopy, retention enema, or lyophilized tablets.
The choice of route depends on the institution facility, the physician's experience, and the safety offered to the patient 68 .
Most of the clinical experience with FMT has been derived from the treatment of recurrent or refractory

L1 in mice
Given the evidence that the intestinal microbiota influenced the response to chemotherapy  Aiming once again to determine the differences in intestinal microbial composition between immunotherapy responders and non-responders, Gopalakrishnan et al. (2018) 72 showed that, in the R group (complete or partial response or stable disease for at least six months), there was a predominance of Faecalibacterium (belonging to the Ruminococcaceae family, order Clostridiales), while in the NR group (progressive or stable disease for less of six months), there was a predominance of Bacteroides thetaiotaomicron, Escherichia coli, and Anaerotruncus colihominis.In view of the results, the possible mechanisms by which the microbiota patterns could influence the response were described: in R patients, bacteria mainly exerted anabolic functions and biosynthesis of amino acids, in contrast to NR, in which predominantly catabolic functions were observed.Furthermore, in the R group, there was a greater infiltration of CD8+ T cells in the tumor, as well as a greater diversity of immune cells.In order to confirm this relationship, fecal material from group R was transferred to germ-free mice, followed by inoculation of neoplastic melanoma cells and administration of PD 1 inhibitor.After two weeks, the transplanted mice evolved with a better response to therapy, lower tumor growth rate, and intestinal microbiota enriched with Faecalibacterium.In addition, they had a higher amount of CD8+ T cells, consistent with the results in human models.Then human fecal material was transferred to germ-free mice, followed by inoculation of melanoma cells.They observed that the mice that received fecal material from the R group showed less tumor growth and greater infiltration of effector CD8+ T cells compared with those that received the microbiota from the NR group.The response to anti PD 1 therapy was also consistent with the response of human donors.However, there was a portion that did not have the same intestinal pattern as the donor, and there was no response to therapy.In view of this, they concluded that, although most mice mimic the donor's response, certain bacteria may have different patterns of expansion and, therefore, generate changes in the recipient's phenotype.

Influence of FMT on the response to anti-PD-1/PD-L1 in humans
Based on the recognition of the influence of  In the study carried out by Maleki et al.  3).
Despite the promising results of ICI-FMT therapy, there are still concerns about its long-term safety.In 2019, two independent clinical trials reported that two patients developed bacteremia by extendedspectrum β-lactamase-producing E. coli after receiving FMT from the same donor, leading to the death of one of them 75 .This study prompted the FDA to issue a safety bulletin warning of the risk of post-FMT infection.In addition, a recent retrospective cohort study analyzed donor stools and showed that six of the 66 individuals tested (9%) were positive for multidrug-resistant pathogens 76 .
Therefore, periodic screening of donor stools should be performed to strictly limit the spread of organisms that can lead to adverse events, which is especially relevant for immunocompromised patients.
Additional clinical investigations, which allow a better understanding of the source, the procedure, and the phenotype of both the recipient and the donor are essential for the success of ICI-FMT combined therapy 27 .
This review presented consistent evidence regarding the use of ICI-FMT therapy in patients with refractory melanoma.However, it is worth mentioning its limitations.Among them, stands out the scarcity of studies that offer relevant data on the application of fecal microbiota transplantation associated with immunotherapy in individuals affected by melanoma.
In addition, the few studies found also had limitations, mainly small sample size.Thus, we haighlight the need to conduct new randomized and controlled clinical trials, with a well-defined methodological design, prolonged follow-up time, representative sample, and low risk of bias, to produce robust results and allow a more in-depth analysis of the benefits of this alternative therapy.

CONCLUSION
FMT has a positive effect on the response of melanoma to ICIs, translated into a significant clinical benefit.However, there is still no consensus regarding the specific bacteria that are associated with a superior response, although certain species belong to phylogenetically related groups.These disagreements can be explained by the use of different genomic sequencing techniques, as well as by geographic influences and dietary variations.
In addition, due to the limited amount of literature on the subject, it is not possible to determine the causal mechanism between the intestinal microbiota and the response to anti-PD-1/PD-L1 drugs.Nonetheless, we postulate that stimulation of responses by T cells Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review against microbial antigens is involved, which in turn aids the response against the tumor.In view of this, new studies aimed at the complete elucidation of the bacteria, the mechanisms involved, and the translation of new evidence to the care practice in oncology are indispensable.

R E S U M O R E S U M O
strategy, through the following steps: (i) theme identification; (ii) formulation of the research question; (iii) collection of articles through searches on electronic platforms; (iv) eligibility of studies with application of inclusion and exclusion criteria; (v) assessment of the quality of evidence; (vi) elaboration of the collection instrument with the information to be extracted; (vii) data analysis, interpretation, synthesis, and discussion; and (viii) presentation of results.The final protocol was prospectively registered in the Open Science Framework platform.The research question was formulated based on the PICO strategy (Population, Intervention, Control, Outcome) 35 , where: Population -patients with melanoma refractory to immunotherapy with ICIs; Intervention -FMT + ICI; Control -ICIs monotherapy;Outcome -clinical benefit.Thus, we formulated the following guiding question: "Does the combined ICI-FMT therapy have a positive impact on the clinical course of patients with refractory melanoma?".To answer Barbosa Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review

PD- 1 /
PD-L1 pathway as a therapeutic target PD-1 is a protein of the CD28 superfamily, characterized by generating negative signals when binding to PD-L1 and PD-L2 proteins.Both PD-1 and its ligands are widely expressed in different cell types (B cells, CD4+ and T CD8+ cells, natural killer cells, and dendritic cells) that physiologically act by limiting the activation and proliferation of T cells, as well as promoting immunological tolerance to self-antigens, preventing auto-inflammatory reactions and autoimmunity in the healthy host 40,41 .However, after continuous exposure and recognition of tumor antigens, tumor-specific effector T cells induce increased expression of PD-1 and secrete interferon-gamma (IFN-γ), a chemical signal that induces the expression of B7 H1 (PD-L1) in neoplastic cells.The PD-1/PD L1 interaction strongly suppresses T cell activation, disrupting its anti-tumor activity.This interruption of the T cell antineoplastic response, related

represented by 10 7
to 10 8 CFU/mL, consisting of facultative anaerobes, Enterobacteria, and obligate anaerobes, such as Veilonella, Enterococcus, Clostridium, Bacteroides, and Lactobacillus.On the other hand, in the large intestine, the population reaches the 10 10 and 10 11 CFU/mL and the most frequently found genera are Bacteroides, Bifidobacterium, Escherichia coli, Bacillus, Clostridium, Eubacterium, Fusobacterium, Peptostreptococcus, and Ruminococcus 56-58 .As a rule, facultative anaerobic bacteria such as E. coli, Enterococci faecalis, and E. faecium are the first to colonize the newborn's GIT, due to the high level of oxygen that initially exists.As these microorganisms consume the gas, the medium becomes more suitable for restricted anaerobic bacteria such as Bacteriodes, Bifidobacterium, and Clostridium.The intestinal microbiota acquired in the postnatal period is composed of a vast diversity of bacteria and performs numerous functions in the human host 56 .Among its various functions, the immunological stands out.The human intestinal mucosa is the main interface between the immune system and the external environment.The intestine is considered the largest immunological organ in the human body, housing about 80% of the cells of this system, being mainly responsible for the production of immunoglobulins essential for innate and adaptive immunity.In addition, it stimulates the immune system by recruiting immune cells and activating the function of epithelial cells, thus being able to modulate the organism's multiple response pathways and tumor progression itself56,[59][60][61][62] .Moreover, the intestinal microbiota also influences the effectiveness of numerous drugs.With regard to antineoplastic agents, one of the first drugs to prove the influence of the intestinal microbiota on its response was cyclophosphamide.With the advancement However, clinical data of anti-PD-1/PD L1 therapy show considerable rates of limited response, in which a large group of patients suffered primary resistance, not responding to therapy, and part of the responders developed acquired resistance over the course of treatment43 .Extensive efforts are being made Such responses were explained by divergences at the immunological level, the JAX group exhibiting a greater density of specific T cells against the tumor and a greater intratumoral accumulation of CD8+ T cells.After receiving treatment with PD-1 pathway inhibitors, significantly greater efficacy was observed in JAX mice.Subsequently, to establish a cause-effect relationship, the fecal material of one of the JAX mice was transferred to TAC ones, generating a reduction in the tumor growth rate.In addition, some TAC mice were transplanted with JAX fecal microbiota associated with an anti-PD-L1, showing an even more effective response in tumor control.The analysis of the fecal material verified the predominance of bacteria of the genus Bifidobacterium (B.longum, B. breve, and B. adolescentis), which were found to be four hundred times more abundant in JAX mice.The presence of Bifidobacterium was strictly related to specific immune cytotoxicity against the tumor, translated by an exacerbated regulation of type-I IFN genes in antigen presenting cells (APCs) present in peripheral lymphoid organs, as well as a higher rate of maturation of dendritic cells and increased activity of effector CD8+ T cells 69 .Based on the study by Sivan et al. (2015) 69 , new research was carried out aiming to list the differences in the constitution of the intestinal microbiota between individuals responsive (R) and non-responsive (NR) to immunotherapy, such as the studies by Frankel et al. (2017) 70 and McCulloch et al. (2022) 71 .The former used a technique for sequencing the intestinal components of 39 individuals with metastatic melanoma before starting therapy with ICIs (ipilimumab, nivolumab, and pembrolizumab, either in monotherapy or combined).In general, all the R presented a microbiota enriched in Bacteroides caccae and Streptococcus parasanguinis, compared with the NR.In the second study, the analysis of the composition of the microbiota of 94 patients allowed the identification of the predominance of the following bacteria in group R: Ruminococcus (Mediterraneibacter) torques, Blautia (B.producta, B. wexlerae, and B. hansenii), Eubacterium rectale, Ruminococcus (Mediterraneibacter) gnavus, and Anaerostipes hadrus.On the other hand, in patients not responsive to anti-PD 1, a predominance of Prevotella spp., Oscillibacter spp., Alistipes spp., and Sutterellaceae spp. was observed.Subsequently, the fecal samples from these patients were taken for transcriptomic analysis, identifying a considerable increase of superoxide dismutase 2 (SOD2), pro-inflammatory cytokines (IL-1β and CXCL8), and transcription factors (NFKBIZ, NFKBIA, TNFAIP3, and LITAF) in NR.
fecal transplantation associated with immunotherapy on the course of melanoma in mice, clinical trials were carried out with promising results.The trial conducted by Davar et al. (2021) 38 analyzed the effect of FMT in 15 individuals with melanoma resistant to anti PD 1 therapy.After transplantation, six of the 15 patients (40%) showed an increase in the diversity of the intestinal microbiota (with a predominance of Ruminococcaceae, Bifidobacteriaceae, and Lachnospiraceae), a greater response to anti PD 1, an increase in CD8+ T cells activation, and a decrease in IL 8 producing myeloid cells.Therefore, it was shown that resistance to immunotherapy was overcome, as these patients, previously unresponsive to anti-PD-1 therapy, showed post-FMT clinical benefit, with tumor reduction and/or long-term disease stability.In addition, FMT has also been shown to modulate the levels of circulating chemical signals, since the R group showed a decrease in multiple cytokines associated with resistance to anti-PD-1 and an increase in biomarkers associated with a beneficial response to treatment.R patients were down regulated in circulating IL-8 as well as in tumor-producing IL-8 myeloid cells; IL-8 has been associated with low anti-PD-1 activity in several cancers, including melanoma.Similar results were listed in the studies by Youngster et al. (2019) 36 and Baruch et al. (2021) 39 , and in both there was a post-FMT increase in the infiltration of CD8+ T cells, as well as APCs (CD68+) in the intestine and in the tumor.Both examined therapyresistant subjects with metastatic melanoma and defined resistance as the inability to achieve a lasting response to anti-PD-1 therapy.In the test performed by Youngster et al. (2019) 36 , three patients (60%) overcame resistance to immunotherapy.Furthermore, in this same study, there was a post-FMT increase in bacteria from the Paraprevotellaceae family, which have been associated with responsiveness to treatment, and a significant decrease in β-proteobacteria, which have been linked to resistance to treatment.In the phase 1 clinical trial carried out by Baruch et al. (2021) 39 , patients were treated with vancomycin and neomycin, aiming to eradicate their native microbiota, before receiving the lyophilized solution of fecal material associated with immunotherapy.They observed positive responses in three individuals, two partial and one complete.It is also worth noting that the patients with the highest response rate had an abundance of Ruminococcus (R. gnavus and R. callidus) and Bifidobacterium adolescentis, considered favorable to immunotherapy, while those with a lower response rate had a microbiota enriched in bacteria from the Clostridiaceae family.The researchers also showed an increase in the beneficial immune response.

(
2020)37, two individuals who had resistant melanoma underwent FMT from two different donors.Both patients showed an improvement in immune response, with an increase in the populations of CD39+ and CD8+ T cells, in addition to a decrease in the levels of dysfunctional PD1+, CD38+, and CD8+ T cells.Patient 1 also displayed stabilization of a large skin lesion.Some clinical trials aiming to evaluate the efficacy and safety of the ICI-FMT combination are in progress (Table

Table 1 -
Selected clinical trials on the application of FMT associated with ICI in patients with refractory melanoma.

Table 3 -
74going clinical trials with the application of combined ICI-FMT therapy in cancer patients (www.clinicaltrials.gov)74.