Immune checkpoint inhibitors (ICI) dramatically improved outcomes of patients with non-small cell lung cancer (NSCLC). The gut microbiome has emerged as a biomarker of response to ICI in NSCLC and a potential therapeutic target. High fiber and Mediterranean diets were associated with prolonged response in patients with melanoma treated with ICI. The objective of this study was to assess the effect of diet on outcome and microbiome composition. Prospective collection of clinical and dietary data were assessed in 102 patients with NSCLC amenable to ICI. We employed a standardize food frequency questionnaire (FFQ) specific to the NSCLC population designed by a clinical nutritionist. FFQ answers were recorded electronically and analyzed using a calculation tool to estimate the median daily fiber (g/day), and surrogate markers of the Mediterranean diet; Monounsaturated fatty acids (MUFA) and Polyunsaturated fatty acids (PUFA) intake (g/1000kcal). Then correlated with progression-free survival (PFS) and overall survival (OS). Microbiome was profiled in a sub-group of 76 patients using shot-gun metagenomic, MetaPhlan4 pipeline was used to detect species, diversity index and LefSe were calculated. Among the 102 patients with NSCLC the median fiber intake was 16 g/day and did not correlate with outcome. However, Spearman correlation showed a significant association between increased PUFA or MUFA intake and longer OS (p<0,001 and p<0,001). Moreover, MUFA dietary intake correlated with a prolonged PFS (median PFS high MUFA intake: 20.0 months versus low MUFA intake: 9.1 months, p=0.02). Additionally, MUFA and PUFA intake correlated with the increase abundance of Eubacterium, Alistipes and Bifidobacterium. In this cohort, patients had a very low fiber diet, however a high MUFA and PUFA intake was associated with beneficial response and an overrepresentation of known beneficial immunogenic bacteria. These results raise the possibility that evidence-based dietary recommendations counseling using educational tools may represent an important strategy to employ the gut microbiome as a potential therapeutic target in improving the response to ICI.
Antibiotics (ATB) induce intestinal dysbiosis and decrease the efficacy of immune checkpoint inhibitors (ICI).1,2 DAV132 is an orally administered colon-targeted ATB adsorbent designed to prevent ATB-induced dysbiosis.3 We investigated whether DAV132 co-administered with ATB could protect gut microbiota diversity and composition. Moreover, in murine avatar tumor model, we assessed anti-PD-1 efficacy through fecal microbiota transplantation (FMT) in germ-free (GF) or antibiotic-treated specific pathogen-free (SPF) mice.
Methods
Twenty-four human healthy volunteers (HV) were randomized to receive either ceftazidime-avibactam (CZA, 2g/0.5g q8h IV for 5 days) or CZA+DAV132 (12g PO tid for 7 days). CZA plasmatic and fecal pharmacodynamic levels were measured using HPLC-MS/MS. Microbiome was profiled with 16S and shotgun metagenomics at different timepoints. FMT in GF or ATB-treated SPF mice was performed using fecal samples from 3 HV and 2 HV respectively, in each group before (D1) or after 6 days (D6) of CZA+/-DAV132; subsequently mice were inoculated with MCA-205 tumor and treated intraperitoneally with anti-PD-1, 4 times every 3 days. Immunological population of tumor infiltrating lymphocytes were analyzed by flow cytometry.
Results
DAV132 did not impact plasmatic CZA concentrations, but significantly reduced ceftazidime concentration in feces compared to HV treated with CZA alone (p<0.001). DAV132 significantly prevented the reduction in microbiota alpha-diversity at D6 (p=0.0019) and was associated with a more rapid return to baseline microbiota composition (figure 1). Significantly more bacteria associated with better response to ICI were preserved in the DAV group compared to CZA, among which Faecalibacterium praunistzii and several Alistipes spp. FMT in GF mice transplanted with feces collected at D1 exhibited a significant anti-PD-1 activity. This anti-tumor response was inhibited in mice transplanted with D6 feces from any of the 3 CZA-treated HV. Conversely, the anti-tumor response was maintained in mice transplanted with D6 feces from any of the 3 HV treated with CZA + DAV132 (figure 2). Similar results were observed upon FMT using samples from HVs into ATB-treated SPF mice. Flow cytometry on tumor T cell infiltrates demonstrated that CZA decreased CD8+T cell infiltration and CD8+/Tregulatory ratio, compared to CZA + DAV132 treated HVs (figure 3).
Conclusions
DAV132 strongly prevented CZA-induced dysbiosis in HV without influencing plasmatic concentrations. In avatar mice FMT from HV treated with CZA+DAV132 was able to preserve anti-PD-1 cancer efficacy. These results provide rationale to launch clinical trials combining DAV132 in patients on ATB amenable to ICI.
Acknowledgements
This work was funded by Da Volterra, a French biotech company, through the sharing of fecal samples and a collaboration agreement with Pr. Routy's lab.
References
Derosa L, Routy B, Desilets A, Daillère R, Terrisse S, Kroemer G, Zitvogel L. Microbiota-centered interventions: the next breakthrough in Immuno-Oncology? Cancer Discov. 2021;11(10):2396–2412. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren K, Mondragón L, Jacquelot N, Qu B, Ferrere G, Clémenson C, Mezquita L, Masip JR, Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria JC, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018;359(6371):91–97. Vehreschild MJGT, Ducher A, Louie T, Cornely OA, Feger C, Dane A, Varastet M, Vitry F, de Gunzburg J, Andremont A, Mentré F, Wilcox MH. An open randomized multicenter Phase 2 trial to assess the safety of DAV132 and its efficacy to protect gut microbiota diversity in hospitalized patients treated with fluoroquinolones. J Antimicrob Chemother. 2022;77(4):1155–1165.
Ethics Approval
All animal studies were approved by the Institutional Animal Care Committee (CIPA) and carried out in compliance with the Canadian Council on Animal Care guidelines (Ethics numbers: C18029BRs).
<div>Abstract<p>Several approaches to manipulate the gut microbiome for improving the activity of cancer immune-checkpoint inhibitors (ICI) are currently under evaluation. Here, we show that oral supplementation with the polyphenol-rich berry camu-camu (CC; <i>Myrciaria dubia</i>) in mice shifted gut microbial composition, which translated into antitumor activity and a stronger anti–PD-1 response. We identified castalagin, an ellagitannin, as the active compound in CC. Oral administration of castalagin enriched for bacteria associated with efficient immunotherapeutic responses (<i>Ruminococcaceae</i> and <i>Alistipes</i>) and improved the CD8<sup>+</sup>/FOXP3<sup>+</sup>CD4<sup>+</sup> ratio within the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase in taurine-conjugated bile acids. Oral supplementation of castalagin following fecal microbiota transplantation from ICI-refractory patients into mice supported anti–PD-1 activity. Finally, we found that castalagin binds to <i>Ruminococcus bromii</i> and promoted an anticancer response. Altogether, our results identify castalagin as a polyphenol that acts as a prebiotic to circumvent anti–PD-1 resistance.</p>Significance:<p>The polyphenol castalagin isolated from a berry has an antitumor effect through direct interactions with commensal bacteria, thus reprogramming the tumor microenvironment. In addition, in preclinical ICI-resistant models, castalagin reestablishes the efficacy of anti–PD-1. Together, these results provide a strong biological rationale to test castalagin as part of a clinical trial.</p><p><i>This article is highlighted in the In This Issue feature, p. 873</i></p></div>
