De Clercq Et Al. CCR, 2021. Fecal Microbiota Transplantation From

1 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

2 3 Q1xx Fecal Microbiota Transplantation from Overweight or

4 Obese Donors in Cachectic Patients with Advanced

5 Gastroesophageal Cancer: A Randomized, Double-blind,

6 Q2xx Placebo-controlled, Phase II Study 1 2 1 1 7 AUxx Nicolien C. de Clercq , Tom van den Ende , Andrei Prodan , Robert Hemke, Mark Davids , 8 Helle K. Pedersen4, Henrik B. Nielsen4, A.K. Groen1, Willem M. de Vos5,6, Hanneke W.M. van Laarhoven2, 9 and Max Nieuwdorp1

1011 13 ABSTRACT ◥ 153012 Purpose: Cachexia is a multifactorial syndrome, associated with microbiota composition (metagenomic sequencing) and metabo- 1631 poor survival in patients with cancer and is influenced by the gut lites (untargeted metabolomics). 1732 microbiota. We investigated the effects of fecal microbiota trans- Results: Allogenic FMT did not improve any of the cachexia 1833 plantation (FMT) on cachexia and treatment response in patients outcomes. Patients in the allogenic group (n 12) had a ¼ 1934 with advanced gastroesophageal cancer. higher DCR at 12 weeks (P 0.035) compared with the autol- ¼ 2035 Experimental Design: In a double-blind randomized placebo- ogous group (n 12), longer median OS of 365 versus 227 days, ¼ 2136 controlled trial performed in the Amsterdam University Medical HR 0.38 (0.14–1.05; P 0.057) and PFS of 204 versus 93 days, ¼ ¼ 2237 Center, we assigned 24 cachectic patients with metastatic HER2- HR 0.50 (0.21–1.20; P 0.092). Patients in the allogenic group ¼ ¼ 2338 negative gastroesophageal cancer to either allogenic FMT (healthy showed a significant shift in fecal microbiota composition after 2439 obese donor) or autologous FMT, prior to palliative chemotherapy FMT (P 0.010) indicating proper engraftment of the donor ¼ 2540 (capecitabine and oxaliplatin). Primary objective was to assess the microbiota. 2641 effect of allogenic FMT on satiety. Secondary outcomes were other Conclusions: FMT from a healthy obese donor prior to first-line 2742 features of cachexia, along with disease control rate (DCR), overall chemotherapy did not affect cachexia, but may have improved 2843 survival (OS), progression-free survival (PFS), and toxicity. Finally, response and survival in patients with metastatic gastroesophageal 2944 exploratory analyses were performed on the effect of FMT on gut cancer. These results provide a rational for larger FMT trials.

45 Introduction ularly affected by the intake domain due to mechanical and digestive 56 problems, leading to loss of appetite and early satiety (9). Currently, 57 46 Cachexia is associated with reduced tolerance to anticancer therapy applied multimodal treatment interventions for cachexia are based on 58 47 and decreased survival (1–4). The definition of this multifactorial nutritional support and appetite stimulation (9). However, these 59 48 syndrome includes the ongoing loss of (skeletal) muscle mass (with or interventions often lack efficacy in counteracting cachexia and have 60 49 without fat mass loss), which cannot be fully reversed by conventional no effect on survival in patients with gastroesophageal cancer. 61 50 nutritional support and leads to progressive functional impairment (5). In recent years, it has become evident that the intestinal 62 51 The pathophysiology of cancer cachexia can be divided into four major microbes, the so-called gut microbiota, play a crucial role in regulating 63 52 domains: reduced dietary intake, elevated catabolism, a reduction in different aspects of cancer cachexia, including satiety and appetite 64 53 storage capacity (fat and muscle loss), and a deterioration in perfor- regulation (10–12), host metabolism (13, 14), and systemic inflam- 65 54 mance status (6–8). Patients with gastroesophageal cancer are partic- mation (15). This is mainly through circulating bacterial components 66 and their metabolites interacting with different organ systems (Fig. 1; 67 1Department of Internal and Vascular Medicine, Amsterdam UMC, University of ref. 12). Both cancer and most anticancer treatments are able to directly 68 Q3xx Amsterdam, Amsterdam, the Netherlands. 2Department of Medical Oncology, or indirectly alter the gut barrier function. This can lead to an 69 Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, imbalance in the composition of the gut microbiota (16). In turn, 70 Q4xx Amsterdam, the Netherlands. 3Department of Radiology and Nuclear Medicine, this affects the pathways involved in the pathophysiology of cancer 71 Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. cachexia, including satiety (12), which alters eating behavior and host 72 4Clinical Microbiomics, Copenhagen, Denmark. 5Laboratory of Microbiology, 6 metabolism (Fig. 1; ref. 14). For example, in a cancer cachexia mice 73 Wageningen University, Wageningen, the Netherlands. Human Microbiome fi Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland. model, oral administration of speci c Lactobacillus spp. partly restored 74 the gut microbiota composition, reduced cachexia parameters and 75 Note: Supplementary data for this article are available at Clinical Cancer prolonged survival (17). 76 Research Online (http://clincancerres.aacrjournals.org/). Furthermore, the gut microbiota has also been implicated in 77 Q5xx Corresponding Authors: Tom van den Ende, Oncology, Amsterdam UMC, modulating the response and toxicity to several classes of anticancer 78 Amsterdam 1058BZ, the Netherlands. E-mail: agents through immunomodulation and host metabolism (Fig. 1; 79 [email protected]; and Nicolien de Clercq, Q6xx [email protected] ref. 18). Translational studies found a link between several different 80 microbial species and the response to checkpoint inhibitors (19, 20). 81 Clin Cancer Res 2021;XX:XX–XX Also, the effect of platinum agents seems to be partly driven by 82 doi: 10.1158/1078-0432.CCR-20-4918 microbiome-related attenuation of the tumor microenvironment (21). 83 Ó2021 American Association for Cancer Research. In line with these findings, manipulation of the gut microbiota could 84

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microbiota transplantation (FMT), that is, the administration of feces 94 Translational Relevance through a nasoduodenal tube from a healthy donor in the gut of a 95 In the randomized phase II TRANSIT study, we assessed the patient to treat disorders associated with gut microbiota aberrations. 96 effect of allogenic fecal microbiota transplantation (FMT) from This concept has been proven to be safe and effective for patients with 97 obese donors on cachexia in patients with HER2-negative recurrent Clostridioides difficile (formerly Clostridium difficile) infec- 98 advanced gastroesophageal cancer scheduled to receive first-line tions and has become the treatment of choice when resistance occurs to 99 chemotherapy. There was no difference between the autologous antibiotic treatment (22). Also, human studies have revealed that 100 (control arm) and allogenic FMT on any cachexia parameter. metabolic traits are transmissible via FMT, including feeding behav- 101 However, in the allogenic group, we observed better disease control ior (23, 24), glucose metabolism (25–27), and most notably body 102 rate and a numerical improvement in survival. On the basis of composition (28). 103 translational microbiome analyses, engraftment of allogenic donor To improve cachexia in patients with gastroesophageal cancer, we 104 transplant was observed. We were not able to link the microbiome conducted a randomized double-blind placebo-controlled pilot trial 105 to cachexia as our intervention did not alter cachexia. Exploratory investigating the effect of allogenic FMT from healthy obese donors 106 analyses linking the microbiome to response or survival did not versus autologous FMT. We hypothesized that an allogenic FMT from 107 reveal any difference in bacterial strains between responders and an obese donor would reduce early satiety, improve metabolism and 108 nonresponders. Our trial provides a rationale for larger FMT trials body composition. Primarily, to test this hypothesis, we assessed 109 to unravel the mechanistic biology behind chemotherapy response cachexia-related parameters. Secondarily, we evaluated the efficacy 110 and microbiome modulation. Future translational studies may of chemotherapy and survival in both groups. Exploratory mechanistic 111 include more in depth analyses of the microbiome such as analyses were performed on the basis of intestinal microbiota and 112 multi-kingdom profiling and evaluate the interaction between the plasma metabolite composition before and after FMT. 113 immune system and tumor biology.

Materials and Methods 114 Study design and participants 115 87 potentially overcome the alterations in the metabolic pathways and We performed a double-blind randomized controlled trial 116 88 subsequently offset cancer-related cachexia while at the same time with patients recruited at the Amsterdam UMC (Amsterdam, the 117 89 serve as a means for improving clinical efﬁcacy of currently used cancer Netherlands; Dutch Trial Register; NL5829). Eligible patients were 118 90 therapy. men and women older than 18 years, with histologically proven 119 91 Several interventions are now being investigated to modulate gut inoperable HER2-negative locally advanced or metastatic esophageal, 120 92 microbiota composition in humans. One of these strategies is fecal gastric, or gastroesophageal junction adenocarcinoma, who were 121

Figure 1. Cancer Treatment Role of the gut microbiota in cancer cachexia and anti-tumor response. The gut microbiota influences important physiologic functions, including host metabolism and immunity Gut microbiota through microbiota-derived metabolites. Both cancer and anticancer therapy disturb the gut microbiota composition, resulting in intestinal dysbio- sis and gut barrier dysfunction. In turn, microbiota-derived metabolites are affected, leading to dysregulation in metabolic and immunologic pathways, including appetite and satiety (gastrointestinal hormones) and sys- Plasma metabolites temic inflammation (such as CRP, IL6, TGFb,andadiponectin).Systemic inflammation is the main driver leading to four domains associated with cancer cachexia: reduced intake, elevated catabolism, reduced storage, and decreased performance. As for Reduced Elevated Reduced Decreased Metabolism Immunomodulation antitumor response, the gut micro- intake catabolism storage performance biota affects the tumor microenvironment through several mechanisms, including host metabolism and immunomodulation (IL1, IL6, TGFb, and T- Cachexia cell response). CRP, C-related protein; Tumor IL, interleukin; MIC1, macrophage x inhibitory cytokine-1; TGFb, trans- xxxxQ7 forming growth factor b. x

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2 Clin Cancer Res; 2021 CLINICAL CANCER RESEARCH Fecal Microbiota Transplantation for Gastroesophageal Cancer

124 scheduled to receive first-line chemotherapy in a 3-weekly schedule: donor and recipients delivered a fresh fecal sample (produced within 184 125 oral capecitabine 1,000 mg/m2 twice per day 1–14 and intravenous 6 hours before use). After randomization, the feces were mixed until 185 126 oxaliplatin 130 mg/m2 day 1. Patients had to meet the criteria fully homogenized. This fecal solution was then filtered to remove 186 127 for cachexia: weight loss >5% over past 6 months (in absence of food-derived debris. The filtrate was transferred to a 1,000-mL sterile 187 128 simple starvation); or BMI < 20 and any degree of weight loss >2%; or bottle and stored at room temperature (17C). Before and after fecal 188 129 (CT scan based) appendicular skeletal muscle index consistent with processing, samples were taken to study procedural effects on micro- 189 130 sarcopenia (males <7.26 kg/m2; females <5.45 kg/m2) and any degree bial composition. 190 131Q8xx of weight loss >2%.5Additional eligibility criteria included a perfor- To remove endogenous fecal contamination, patients first under- 191 132 mance status score of 0, 1, or 2 according to the guidelines of the went bowel lavage with polyethylene glycol solution (Klean-Prep, 192 133 Eastern Cooperative Oncology Group (ECOG; ref. 29). Patients with Norgine BV) through a nasoduodenal tube, followed by infusion of 193 134 an ECOG performance status higher than 2 were excluded as these the gut microbiota solution in approximately 30 minutes (Fig. 2). 194 135 patients are not eligible for treatment with chemotherapy. Finally, Remaining study procedures are described in the Supplementary 195 136 patients should be using a proton pump inhibitor (PPI) because the use Materials and Methods section. 196 137 of a PPI is common in patients with gastroesophageal cancer and can 138 have a major influence on the gut microbiome composition (30). Outcomes 197 139 Patients with noncancer-related gastrointestinal symptoms such as The primary outcome was to assess the effect of allogenic FMT on 198 140 chronic nausea, altered taste sensation, or swallowing difficulties were satiety after 4 weeks, determined by VAS questionnaires (Supplemen- 199 141 excluded because of their potential influence on the primary endpoint. tary Materials and Methods). A high VAS score (>5) indicates an 200 142 Patients with a mechanical obstruction impairing the endoscopic increased feeling of satiety. To examine additional domains of cachex- 201 143 placement of a nasoduodenal tube were also excluded. ia, secondary outcomes included validated questionnaires to deter- 202 144 To be eligible as a feces donor, subjects had to be older than 18 years mine intake (mini nutritional assessment, dysphagia using Atkinsons- 203 145 of age with a BMI > 25 kg/m2 (overweight or obese), without scale, VAS appetite). Also, in fasting plasma samples, gastrointestinal 204 146 any known underlying disease or use of medication and no signs of hormones involved in appetite regulation (ghrelin and leptin), low- 205 147 insulin resistance and/or metabolic syndrome (31), because FMT grade inflammation (C-reactive protein, IL6, TGFb activated and 206 148 using metabolic syndrome donors adversely affects metabolism in latent, adiponectin and MIC-1) were measured. In addition, REE, 207 149 humans (26), whereas healthy overweight or obese donor FMT BMI as well as muscle and fat mass measured using CT scans and 208 150 improves bodyweight in human subjects with underweight (28, 31). bioelectrical impedance analysis (for body composition) were deter- 209 151 A detailed description of patient and donor selection is available in the mined at baseline and 12 weeks and finally, performance status (ECOG 210 152 Supplementary Data. performance score). 211 153 This trial was approved by the medical ethical review committee of Secondary oncological outcomes included: disease control rate 212 154 the Amsterdam Medical Center (AMC; Amsterdam, the Netherlands) (DCR) within 3 months of enrollment by RECIST version 1.1, overall 213 155 and conducted in accordance with Good Clinical Practice guidelines survival (OS), progression-free survival (PFS), and chemotherapy 214 156 and the Declaration of Helsinki. An independent Data Safety Mon- toxicity (graded with the Common Terminology Criteria for Adverse 215 157 itoring board (DSMB) was assigned to safeguard the interests of the Events version 4.03). Responders to chemotherapy were defined as 216 158 participants, assess the safety and efficacy of the FMT during the trial, patients with stable disease (SD) or partial response (PR); Nonrespon- 217 159 and monitor the overall conduct of the study. All patients provided ders as patients with progression by the RECIST. OS was defined as 218 160 written, informed voluntary consent. Every author had access to the time from randomization to death. PFS was defined as time from 219 161 study data and reviewed and approved the final manuscript. randomization until disease progression or death from any cause, 220 whichever occurred first. The cutoff for follow-up was 1 year from 221 162 Randomization and masking randomization. The analysis for toxicity comprised all patients who 222 163 In this double-blind randomized controlled trial, patients were received the intervention (FMT). To explore potential microbial- 223 164 randomly assigned (1:1) to either receive allogenic (donor; group metabolite pathways, involved in cachectic and oncological outcomes, 224 165 A) or autologous FMT (group B) using computer-generated random- gut microbiota composition (as determined by shotgun sequencing 225 166 ization. FMT donors and recipients were matched for sex. performed by Clinical Microbiomics with Illumina Novaseq 6000) and 226 fasting plasma metabolites (Metabolon) were measured at all time- 227 167 Procedures points. Extensive description of methods is available in the Supple- 228 168 There were three study visits: the first visit (V1) was 1 week before mentary Data. 229 169xx start of chemotherapy (baseline, including FMT), week 4 (V2), and 170 week 12 (V3; Supplementary Fig. S1). At every study visit, patients Statistical analysis 230 171 provided fresh morning fecal samples and completed the visual analog We based our sample size calculation on the satiety VAS- 231 172 scale (VAS) questionnaires. Furthermore, cachexia parameters were questionnaire results of the FATLOSE1 study (healthy lean donor 232 173 measured [body mass index ((BMI), resting energy expenditure (REE), fecal infusions in metabolic syndrome patients; ref. 25). We calculated 233 174 BIA)] and fasting blood samples were drawn. Also, patients completed that with a mean 15 mm (SD 10 mm) decrease in VAS score upon an 234 175 questionnaires regarding nutritional intake 3 days prior to each study allogenic lean donor FMT versus a 5 mm increase upon autologous 235 176 visit. Response evaluation and level of sarcopenia was done by CT at FMT based on a two-sided alpha of 0.05 and 80% power, we needed 16 236 177 baseline and after three cycles of chemotherapy. Adverse events (AE; subjects in total. Subjects withdrawing for medical reasons (including 237 178 graded with Common Terminology Criteria for Adverse Events antibiotic treatment) or death during the study period were replaced by 238 179 version 4.03) and performance score (graded with ECOG) were new subjects. Our study was not powered to detect a difference in other 239 180 monitored during each study visit. cachexia or oncological outcomes. 240 181 FMT was performed as previously described by de Groot and All analyses regarding cachexia and oncological outcomes were 241 182 colleagues (Fig. 2; ref. 26). Briefly, on the day of fecal infusion, both performed in the intention-to-treat population. For the microbiome 242

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A FecaldonationB Homogenization C Filtration D LaxatingE Infusion

Onthe day of the FMT, Fecesis diluted with Fecalsolution was Toremove endogenous Infusion of the gut both donor and recipient 500 mL of sterile saline ltered to remove fecal contamination, microbiota solution delivered fresh fecal (0.9% NaCl). Next, the food-derived debris and patients rst underwent through the sample. Patients were solution is mixed until pouredin a sterile bottle. bowel lavagewith nasoduodenal tube in randomized to either fully homogenized. polyethylene glycol approximately 30 allogenic (donor) or solution (Kleanprep). minutes. autologous FMT.

Figure 2. Fecal microbiota transplantation procedure.

245 analyses, 1 patient was removed because of a failed FMT. Comparisons package (v2.5.6). Nonparametric tests were used to assess correlations 270 246 between the two intervention groups (unpaired) were performed using (Spearman rho). The Benjamini–Hochberg procedure was used to 271 247 the Mann–Whitney U or x2 test unless otherwise stated. Statistical correct P values for multiple comparisons. 272 248 comparisons between (paired) visits (V1, V2, and/or V3) were per- The XGBoost (v. 0.90) implementation of gradient boosted trees 273 249 formed using the Wilcoxon signed-rank test. The tests were performed was used in prediction models for response structured in a nested 274 250 two sided with a P < 0.05 considered statistically significant. cross-validation system to prevent overfitting and ensure robustness of 275 251 OS and PFS were calculated using the Kaplan–Meier method, HRs results (Supplementary Materials and Methods). 276 252 with the use of the Cox proportional hazards model, and testing for 253 statistical significance using the Breslow–Wilcoxon test. 254 To evaluate the effect of allogenic and autologous FMT on the Results 277 255 overall composition of the gut microbiota, multilevel principal com- Patients characteristics 278 256 ponent analysis (PCA) was performed on center log-ratio transformed Between August 2016 and January 2019, 24 patients were enrolled 279 257 species-level microbial composition using the mixOmics (v6.12.0) and randomly assigned to receive allogenic FMT (n 12) or autol- 280 ¼ 258 R package, removing between-individual variance and decomposing ogous FMT (n 12; Fig. 3). One patient did not undergo anTex FMT due 281 ¼ 259 only within-individual variance. Significance was tested using to severe constipation and seven subjects were replaced by new subjects 282 260Q9xxx MANOVA on the first 10 principal components and comparing because of antibiotic use (n 3), death (n 3), or withdrawal from 283 ¼ ¼ 261 the F statistic with 1,000 permutations where time was shuffled chemotherapy after two doses (n 1) during the 12 weeks of study. All 284 ¼ 262 within a subject and FMT allocation among subjects. randomized patients, including the aforementioned, were included in 285 263 To calculate the difference in microbiome composition between a the intention to treat analysis for response and survival (N 24). 286 ¼ 264 patient and its corresponding donor, binary Jaccard index was used Patient demographics and baseline disease characteristics are listed 287 265 (e.g., method to evaluate the resembles between donor and recipient). in Table 1. The majority of patients were male (92% in the allogenic 288 266 Plots were constructed using the ggplot2 (v3.3.0) and ggpubr (v0.3.0) group and 67% in the autologous group) with a median age of 289 267 packages. Alpha (Shannon and species richness) and beta-diversity 65 years (39–73) in the allogenic group and 62 years (51–78) in the 290 268 (Bray–Curtis) metrics were calculated in R (v4.0) using the vegan R autologous group, all patients had metastatic disease (Table 1). In the 291

4 Clin Cancer Res; 2021 CLINICAL CANCER RESEARCH Fecal Microbiota Transplantation for Gastroesophageal Cancer

Assessed for eligibility (n = 55)

Excluded (n = 31) Not meeting inclusion criteria (n = 12) Declined to participate (n = 16) Other reasons (n = 3)

Randomized (n = 24)

Allocated to allogenic FMT (n = 12) Allocated to autologous FMT (n = 12) Received allocated intervention (n = 12) Received allocated intervention (n = 11) Did not receive allocated intervention (n = 0) Did not receive allocated intervention (n = 1)

Lost to follow-up (n = 0) Lost to follow-up (n = 0)

Discontinued intervention (n = 0) Discontinued intervention (n = 0)

Analyzed (n = 12) Analyzed (n = 12) Excluded from analysis (n = 0) Excluded from analysis (n = 0)

Figure 3. Enrollment ﬂow chart.

294 allogenic group, there were 2 patients with gastric cancer (17%) versus there was also no apparent change in caloric intake between baseline 311 295 1 in the autologous group (8%), 3 patients who received previous and week 4 in both groups (Supplementary Table S2). Moreover, 312 296 gastroesophageal cancer–related surgery (25%) versus 5 (42%), 3 there was no statistically significant difference in change in any 313 297 patients with grade 2–3 dysphagia (25%) versus 8 (67%), and 8 patients other measure related to cachexia between both groups (Supple- 314 298 with two or more metastatic sites (67%) versus 5 (42%) in the mentary Table S3). Important to note, patients in the autologous 315 299 autologous group. Median time from randomization to FMT was group had a significantly higher level of dysphagia (P 0.018) but 316 ¼ 300Q10xxx 2 days (IQR,1–4) in the autologous group and 4 days (IQR, 1–8) in the not of satiety (P 0.557) at baseline compared with the allogenic 317 ¼ 301 allogenic group. group (Supplementary Table S3). 318 302 We enrolled four healthy overweight (n 1) or obese donors (n 3) 2 ¼ ¼ 303 with a median BMI of 30 kg/m (26–33). Donor baseline character- Effect of FMT on adherence to chemotherapy and toxicity 319 fi x 304 istics are depicted in Supplementary Table S1. Eighteen of 24 patients completed the rst three cycles of x 320 CAPOX without dose modifications (completion rate, 75%); there xxxQ12x 321 305 Effect of FMT on cachexia outcomes was no difference in completion rate between the autologous and 322 306 There was no significant difference in satiety levels (VAS ques- allogenic group (P 0.336). One or more doses of capecitabine and/ 323 ¼ 307 tionnaire) at week 4 between the autologous group [mean 4.25, or oxaliplatin were omitted in 6 patients because of grade ≥2 324 ¼ 308 95% confidence interval (CI) 1.63–5.96] and the allogenic group neuropathy (n 5), grade ≥3nauseaand/orvomiting(n 1). 325 ¼ ¼ ¼ 309 (mean 4.71, CI 2.03–6.47; P 0.663). In line with this finding, Three patients in the autologous group died before end of study due 326 ¼ ¼ ¼

AACRJournals.org Clin Cancer Res; 2021 5 de Clercq et al.

Q11 Table 1. Baseline characteristics (n 24). In the autologous group, 3 of 12 patients (25%) needed treatment 347 mmmm ¼ with antibiotics during the first cycle of chemotherapy, while none of 348 Allogenic Autologous the patients in the allogenic group received antibiotics. To explore the 349 Characteristic (N 12) (N 12) ¼ ¼ effect of antibiotics on oncological outcomes, we performed a sensi- 350 Age – years tivity analysis by omitting patients who received antibiotics; patients in 351 Median 65 62 the allogenic group (n 12) had a DCR of 83% versus 56% (P 0.16) 352 ¼ ¼ Range 39–73 51–78 in the autologous FMT group (n 9), median OS was 365 versus 353 ¼ Sex 158 days (HR 0.29; 95% CI 0.09–0.92; P 0.035) and median PFS 354 ¼ ¼ ¼ Male 11 (92) 8 (67) was 204 versus 89 days (HR 0.52; 95% CI 0.20–1.37; P 0.124) for 355 ¼ ¼ ¼ Female 1 (8) 4 (33) the allogenic and autologous group, respectively. 356 Subsite of tumor Esophagus 9 (75) 10 (83) Effect of FMT on gut microbiota composition 357 Gastroesophageal 1 (8) 1 (8) junction Next, we analyzed the effect of FMT on the gut microbiota com- 358 xx position in the two groups (n 23), only excluding one participant 359 Stomach 2 (17) 1 (8) ¼ Histology who did not receive autologous FMT due to constipation. Reassur- 360 Adenocarcinoma 11 (92) 10 (83) ingly, there was a significant decrease in the binary Jaccard 361 Squamous cell carcinoma 1 (8) 2 (17) index between baseline (V1) and 4 weeks (V2) in the allogenic group 362 Extent of disease (P 0.01), which was not present in the autologous FMT group 363 ¼ Metastatic 12 (100) 12 (100) (Fig. 5A). Second, there was a clear engraftment of donor species after 364 No. of metastatic sites FMT, defined as species being present in the donor, absent in recipient 365 1 4 (33) 7 (58) prior to FMT and present after FMT (Supplementary Fig. S3). Thus, 366 ≥2 8 (67) 5 (42) the microbiome composition from the allogenic recipients resembled 367 Previous surgery Yes 3 (25) 5 (42) the donor microbiome more closely after the FMT compared with 368 No 9 (75) 7 (58) baseline. To further explore the impact of FMT on the overall 369 Previous cytostatic therapy community composition of the gut microbiota, we performed a 370 Yes 7 (58) 5 (42) multilevel PCA, examining within-individual variation in microbiota 371 No 5 (42) 7 (58) composition (i.e., pre-FMT baseline microbial composition compared 372 ECOG performance-status score with microbiota composition observed at 4 and 12 weeks post-FMT). 373 0 or 1 10 (83) 9 (75) There was a clear shift after FMT in the allogenic group, while no such 374 2 2 (17) 3 (25) shift could be detected in the autologous group (Fig. 5B). To extend 375 Dysphagia (grade) our understanding of the physiologic mechanism of the improved 376 0 or 1 9 (75) 4 (33) fi 2 or 3 3 (25) 8 (67) DCR in the allogenic FMT group, we aimed to identify speci c 377 Enteral feeding bacterial species and/or bacterial communities that where enriched 378 Yes 1 (8) 1 (8) or deprived in the allogenic group compared with the autologous FMT 379 No 11 (92) 11 (92) group. However, no significant differences were found in any of the 380 alpha-diversity measures (Shannon index and species richness; Sup- 381 Note: Data shown are for the intention-to-treat population. Parenthesis indicate plementary Fig. S4) between patients who received allogenic or 382 the percentage of patients. ECOG performance-status score ranges from 0 to 4, autologous FMT at any of the three visits. Moreover, no significant 383 with 0 indicating fully active and higher scores indicating greater restrictions in relation between gut microbiota diversity (both alpha and beta diver- 384 physical activities. sity) and DCR, OS, or PFS were observed (Supplementary Fig. S5). 385 Also, no individual species or groups of functionally related species 386 were found to be associated with DCR, OS, or PFS at 4 or 12 weeks 387 329 to progression of disease. The incidence of common AEs associated following allogenic FMT (Supplementary Figs. S6 and S7). A machine 388 330 with CAPOX (nausea/vomiting, anorexia, neuropathy) was similar learning model for DCR based on the feces sample obtained in week 4 389 331 between both groups (Supplementary Table S4). also showed no predictive value (AUC: 0.52; top 15 microbes; Sup- 390 plementary Fig. S8). 391 332 Effect of FMT on response and survival 333 There were no complete responders in either group after three Effect of FMT on plasma metabolites 392 334 cycles of CAPOX. In the allogenic group, 7 patients had a PR (58%), To further elucidate potential metabolic mechanisms explaining the 393 335 3 SD (25%), and 2 had disease progression (17%). In the autologous beneficial effect of allogenic FMT, we explored the change of plasma 394 336 group, 4 patients had a PR (33%), 1 SD (8%), and 7 had disease metabolites after FMT. We found a significant effect of chemotherapy 395 337 progression (58%). Exploratory analysis of response revealed in the on the plasma metabolome, visible as a marked shift in the multilevel 396 338 allogenic group a higher DCR 83% compared with the autologous PCA plot between baseline, week 4 and week 12 (Supplementary 397 339 group 42%, P 0.035 (Fig. 4A). Median OS was 365 days and Fig. S9). However, there was no clear difference in change between the 398 ¼ 340 227 days in the allogenic and autologous group, respectively (HR two intervention groups. In line, we observed no specific plasma 399 ¼ 341 0.38; 95% CI 0.14–1.05; P 0.057; Fig. 4B). Median PFS was metabolite that was significantly different between both intervention 400 ¼ ¼ 342 204 days in the allogenic group and 93 days in the autologous arm groups and no association with DCR following allogenic donor FMT. 401 343 (HR 0.50, 95% CI 0.21–1.20; P 0.092; Fig. 4C). Per protocol The model was a poor predictor of DCR based on the plasma sample 402 ¼ ¼ ¼ 344 analyses (without failed FMT; N 23) showed comparative results drawn in week 4 (AUC: 0.49) or week 12 (AUC: 0.60; top 15 403 ¼ 345 (Supplementary Fig. S2). metabolites of both models (Supplementary Figs. S10 and S11). 404

6 Clin Cancer Res; 2021 CLINICAL CANCER RESEARCH Fecal Microbiota Transplantation for Gastroesophageal Cancer

ABC

100 Allogenic FMT 100 * Allogenic FMT Allogenic FMT 10 Autologous FMT Autologous FMT Autologous FMT 75 75

50 50 OS (%) 5 PFS (%)

No. of pts 25 25 P = 0.057 P = 0.092 0 RNR 0100200300400 0 100 200 300 400 Time (days) Time (days) No. at risk: No. at risk: Allogenic 12 12 10 8 Allogenic 12 10 8 3 Autologous 12 10 7 6 Autologous 12 5 5 2

Figure 4. DCR (A), overall survival (B), and progression-free survival (C), intention-to-treat analysis. A, DCR: allogenic versus autologous FMT (P 0.035). Kaplan–Meier ¼ estimates of overall 1-year survival (B) and PFS (C) in patients randomized for allogenic (blue) or autologous (red) FMT. R, responder (stable disease or partial response); NR, nonresponder (progression).

407 Discussion with a 5-year survival of less than 20% (32–35). The initial hypothesis 427 408 To our knowledge, this is the first randomized controlled trial of was to modulate the microbiome through FMT from a healthy 428 409 donor FMT derived from healthy obese donors prior to first-line (noninsulin resistant) obese donor in an attempt to counteract cancer 429 410 palliative chemotherapy in patients with advanced gastroesophageal cachexia and consequently improve therapeutic response. In contrast 430 411 cancer. Several conclusions can be drawn from this pilot study. First, to the hypothesis, we did not observe any statistically significant 431 412 allogenic FMT did not improve satiety or cachexia-related parameters. change in any of the cachexia-related parameters in the allogenic 432 413 However, based on exploratory efficacy analyses, we observed better donor FMT group. This could be due to several factors including: (1) 433 414 DCR in the allogenic group and higher median survival (OS and PFS). The patients suffered from refractory cachexia and therefore no robust 434 415 Second, we observed a significant and prolonged shift in gut micro- intervention could have altered their metabolic state; or (2) other 435 416 biota composition up to 12 weeks in the allogenic group after FMT factors apart from low-grade systemic inflammation and therapy- 436 417 (confirming that allogenic transplantation was sustainable, despite related side effects had a larger impact on cachexia than the 437 418 treatment with chemotherapy). We could not identify specific intes- donor FMT. Moreover, several baseline characteristics might 438 419 tinal bacterial species that were associated with oncological outcomes have affected the primary endpoint including the distribution 439 420 in the allogenic donor group. This may have been due to the limited between both groups of dysphagiaandpreviouscancer-related 440 421 sample size combined with the multidimensional effects of transfer- surgery. It is important to note that we based our hypothesis on 441 422 ring an entire microbial ecosystem on the gut microbiota composition previous studies, indicating that cachexia was associated with 442 423 and functionality. decreased PFS, OS and increased toxicity in various cancer 443 424 For advanced gastroesophageal cancer, response to first-line palli- types (1, 3). However, results from a recently published study 444 425 ative chemotherapy is heterogeneous and survival rates are still poor, addressing the relationship between survival and cachexia in 445

AB0.01 Allogenic Autologous Baseline Week 4 Week 12 0.6 20

0.5 PC2 (7%) −20 Binary Jaccard distance Jaccard Binary

−40 Baseline Week 4 Week 12 −25 0 25 −25 0 25 Time PC1 (9.4%)

Figure 5. Effect of FMT on gut microbiota composition. A, Jaccard distance in Allogenic group. The Jaccard distance measures dissimilarity between sample sets (in a binary manner: presence or absence).The larger the Jaccard distance, the more the gutmicrobiotacomposition of recipients differ from the donor. Thesmaller the distance, the more similar. There is a signiﬁcant decrease in Jaccard distance (e.g., increase in similarity) at week 4 and week 12 versus baseline. Thus, the gut microbiota of patients receiving allogenic FMT becomes more similar to the donor gut microbiota composition. B, “Multilevel” PCA: Only within-individual variance is depicted. Allogenic subjects (left) show a shift in microbiome composition after FMT; autologous subjects show no visible shift.

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448 patients with advanced gastroesophageal cancer suggested that immune system (42). In our study, we did not perform extensive 508 449 response to chemotherapy and survival in advanced gastroesoph- analyses on the tumor immune microenvironment or different 509 450 ageal cancer depends on factors beyond cachexia. (36) immune-cell subtypes in the systemic circulation. 510 451 Despite the fact that we did not find any effect of the intervention on In conclusion, this hypothesis generating study suggests that 511 452 cachectic features, the results from our exploratory analyses for healthy obese donor FMT was not able to alter cachexia in patients 512 453 response and survival favored the allogenic group. Survival in the with advanced gastroesophageal cancer through the manipulation of 513 454 allogenic group was also higher compared with historical data in the gut microbiota. On the basis of secondary efficacy analyses, 514 455 patients with advanced gastroesophageal cancer treated with CAPOX chemotherapy response and survival seemed to favor the allogenic 515 456 or doublet chemotherapy (37, 38). In this regard, the observed intervention group. However, larger studies in humans are essential 516 457 improvement in both DCR and PFS suggests that (repetitive) treat- to replicate these findings and address the link between the gut 517 458 ment with obese donor FMT could benefit patients with advanced microbiota composition and innate/adaptive immunity in relation to 518 459 gastroesophageal cancer. Importantly, the incidence of common AEs chemotherapy response. Ultimately, this could lead to the develop- 519 460 associated with CAPOX (nausea/vomiting, anorexia, neuropathy) was ment of personalized treatment modalities, such as subject-specific 520 461 similar between both groups. microbiome-based prebiotics and probiotics enhancing the efficacy of 521 462 The gut microbiota can directly and indirectly influence the phar- anticancer agents. 522 463 macological effects of chemotherapy through several mechanisms, 464 including immunomodulation and metabolism (18, 21, 39). To extend Authors’ Disclosures 523 465 our understanding of the role of the gut microbiome and its association H.B. Nielsen reports personal fees from Clinical Microbiomics during the 524 466 with the favorable oncological outcomes in the allogenic group, we conduct of the study. W. de Vos reports personal fees from Caelus Health and A- 525 467 investigated a potential link between the intervention, inflammation, Mansia Biotech outside the submitted work; in addition, W. de Vos has a patent 526 468 and plasma metabolites. However, no difference in proinflammatory for use of FMT in cancer cachexia pending. H.W.M. van Laarhoven reports 527 469 cytokines known to be related to tumor progression, nor specific personal fees from BMS and MSD; grants and personal fees from Lilly; grants, 528 personal fees, and non-financial support from Nordic Pharma and Servier; grants 529 470 metabolites that could potentially explain the difference in response and non-financial support from Bayer, Celgene, Janssen, Merck, and Roche; 530 471 between the two groups, were found. grants from Philips outside the submitted work. M. Nieuwdorp reports other from 531 472 Some limitations need to be acknowledged. First, our primary Caelus Health and Kaleido Biosciences outside the submitted work; in addition, 532 473 endpoint was satiety, which is usually altered in patients with gastro- M. Nieuwdorp has a patent for using obese donor FMT for cachectic patients with 533 advanced gastroesophageal cancer pending. No disclosures were reported by the 534 474 esophageal cancer, leading to inadequate intake and in some cases Q13 475 cachexia. However, it is a subjective outcome measure and not always other authors. xxx535 476 related to cachexia. Therefore, we assessed other cachexia-related Authors’ Contributions 477 parameters including: body composition, cytokines, and intake which 536 N.C. de Clercq: Conceptualization, data curation, formal analysis, investigation, 537 478 did not show any difference between the intervention and placebo methodology, writing–original draft, project administration. T. van den Ende: 538 479 group. Formal analysis, investigation, writing–review and editing. A. Prodan: Data curation, 539 480 Second, our study was not powered to detect a difference in software, formal analysis, methodology, writing–review and editing. R. Hemke: 540 481 response rate or survival. However, despite potentially being under- Software, formal analysis, methodology, writing–review and editing. M. Davids: 541 482 powered, a numerically higher median survival in the allogenic Data curation, software, formal analysis, methodology, writing–review and editing. 542 483 group was observed, which warrants further investigation in a larger H.K. Pedersen: Data curation, software, formal analysis, methodology, writing– 543 review and editing. H.B. Nielsen: Data curation, software, formal analysis, meth- 544 484 phase II trial. odology, writing–review and editing. A.K. Groen: Software, supervision, methodology, 545 fi 485 Third, even though microbiome analyses revealed a signi cant shift writing–review and editing. W.M. de Vos: Software, supervision, methodology, 546 486 in microbiome composition after allogenic FMT, we did not identify a writing–review and editing. H.W. van Laarhoven: Conceptualization, supervision, 547 487 specific microbe or group of microbes mediating the beneficial onco- project administration, writing–review and editing. M. Nieuwdorp: Conceptualiza- 548 xxxQ14 488 logical outcomes of the allogenic group. In this regard, it is important tion, supervision, project administration, writing–review and editing. 549 489 to stress that bacteria are not the only microorganisms present in the 490 gut, but rather coexist alongside with fungi, unicellular parasites, and Acknowledgments 550 491 phages, which were not investigated in this study. Therefore, multi- We acknowledge Ineke Heikamp-de Jong for support in the fecal sample DNA 551 fi 492 kingdom profiling (e.g., viruses, phages, parasites, etc.) is essential to isolation. Also, Harry Buller€ for his scienti c recommendations as a member of the 552 DSMB. Finally, we respectfully acknowledge our participants who selflessly helped to 553 493 exclude that other components of the gut microbiota, comprising > complete this project. 554 fi 494 60% of the feces, might explain the bene cial effects of obese donor M. Nieuwdorp is supported by a personal ZONMW-VIDI grant 2013 555 495 FMT on response and survival. (40) Moreover, the type and abundance (016.146.327) and W.M. de Vos by a personal Spinoza Award 2018 and SIAM 556 496 of proteins and metabolites produced by the gut microbiota will not Gravitation Grant 024.002.002 of the Netherlands Organization for Scientific 557 497 only depend on its composition, but also on the ecological networks Research. H.W.M. van Laarhoven has received unrestricted research grants from 558 498 formed between members of the microbial community as well as on Amgen, Bayer Schering Pharma AG, BMS, Celgene, Eli Lilly and Company, Glax- 559 oSmithKline Pharmaceuticals, MSD, Nordic Pharma Group, Philips, and Roche 560 – 499 host microbe interactions (e.g., cometabolites; ref. 41). Pharmaceuticals. 561 500 Fourth, in this study, patients in the autologous group received an 501 FMT from their own feces, though studies have shown that autologous The costs of publication of this article were defrayed in part by the payment of page 562 502 FMT can also change host metabolism (26). Future studies could charges. This article must therefore be hereby marked advertisement in accordance 563 503 alternatively subdivide the subjects in chemotherapy treatment with or with 18 U.S.C. Section 1734 solely to indicate this fact. 564 504 without FMT. 505 Finally, the beneficial oncological outcomes in the allogenic group Received December 21, 2020; revised February 25, 2021; accepted April 15, 2021; 565 506 might be caused by modulation of the host innate and adaptive published first xx xx, xxxx. 566

8 Clin Cancer Res; 2021 CLINICAL CANCER RESEARCH Fecal Microbiota Transplantation for Gastroesophageal Cancer

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