Metagenomic Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma Patients

Arthur E Frankel, Laura A Coughlin, Jiwoong Kim, Thomas W Froehlich, Yang Xie, Eugene P Frenkel, Andrew Y Koh, Arthur E Frankel, Laura A Coughlin, Jiwoong Kim, Thomas W Froehlich, Yang Xie, Eugene P Frenkel, Andrew Y Koh

Abstract

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT-particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P). IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
Study schema.
Figure 2
Figure 2
MSS identifies specific bacterial species that are enriched in the gut microbiomes of melanoma patients who are responding to ICT therapy. Relative abundance of gut bacterial taxa as determined by MetaPhlAn analysis of MSS data generated from fecal specimens collected from melanoma patients prior to receiving ipilimumab/nivolumab, pembrolizumab, ipilimumab alone, or nivolumab alone. Differential taxonomic abundance was analyzed by linear discriminate analysis coupled with effect size measurements (LEfSe) projected as a histogram (A, C and E) or cladrogram (B, D and F). All listed bacterial groups were significantly (P < .05, Kruskal-Wallis test) enriched for their respective groups (responder versus progressive).
Figure 3
Figure 3
Unbiased metabolomics analysis of stool metabolites from adult melanoma patients prior to treatment with ICT. UPLC-MS–based global profiling of metabolites in feces of adult melanoma patients receiving immune checkpoint inhibitor therapy (n = 39). Data were log transformed and mean centered. Venn diagrams of metabolites (A) significantly increased or (B) decreased when comparing the ICT responder group versus the progressive group for all ICTs, IN only, and P only. The heat maps show the normalized relative abundances of stool metabolites comparing responders to those with progressive disease for (C) all ICTs, (D) IN only, and (E) P only (q < 0.05, unpaired t test with Welch's correction followed by false discovery rate correction). Orange colors indicate relative abundance increase, and blue indicates relative abundances decrease (responders:progressive, log2 transformed).

References

    1. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23–34.
    1. Horvat TZ, Adel NG, Dang TO, Momtaz P, Postow MA, Callahan MK, Carvajal RD, Dickson MA, D'Angelo SP, Woo KM. Immune-related adverse events, need for systemic immunosuppression, and effects on survival and time to treatment failure in patients with melanoma treated with ipilimumab at Memorial Sloan Kettering Cancer Center. J Clin Oncol Off J Am Soc Clin Oncol. 2015;33:3193–3198.
    1. Frankel AE, Frankel EP. Melanoma metamorphoses: advances in biology and therapy. J Cancer Sci Ther. 2017;9:325–335.
    1. Gibney GT, Weiner LM, Atkins MB. Predictive biomarkers for checkpoint inhibitor-based immunotherapy. Lancet Oncol. 2016;17:e542–e551.
    1. Roberts EW, Broz ML, Binnewies M, Headley MB, Nelson AE, Wolf DM, Kaisho T, Bogunovic D, Bhardwaj N, Krummel MF. Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma. Cancer Cell. 2016;30:324–336.
    1. Salerno EP, Bedognetti D, Mauldin IS, Deacon DH, Shea SM, Pinczewski J, Obeid JM, Coukos G, Wang E, Gajewski TF. Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk. Oncoimmunology. 2016;5:e1240857.
    1. Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science. 2012;336:1268–1273.
    1. Iida N, Dzutsev A, Stewart CA, Smith L, Bouladoux N, Weingarten RA, Molina DA, Salcedo R, Back T, Cramer S. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment. Science. 2013;342:967–970.
    1. Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, Benyamin FW, Lei YM, Jabri B, Alegre ML. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy. Science. 2015;350:1084–1089.
    1. Vetizou M, Pitt JM, Daillere R, Lepage P, Waldschmitt N, Flament C, Rusakiewicz S, Routy B, Roberti MP, Duong CP. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science. 2015;350:1079–1084.
    1. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1) Eur J Cancer. 2009;45:228–247.
    1. Fan D, Coughlin LA, Neubauer MM, Kim J, Kim MS, Zhan X, Simms-Waldrip TR, Xie Y, Hooper LV, Koh AY. Activation of HIF-1alpha and LL-37 by commensal bacteria inhibits Candida albicans colonization. Nat Med. 2015;21:808–814.
    1. Simms-Waldrip TR, Sunkersett G, Coughlin LA, Savani MR, Arana C, Kim J, Kim M, Zhan X, Greenberg DE, Xie Y. Antibiotic-induced depletion of anti-inflammatory Clostridia is associated with the development of graft-versus-host disease in pediatric stem cell transplantation patients. Biol Blood Marrow Transplant. 2017;23:820–829.
    1. Piper HG, Fan D, Coughlin LA, Ho EX, McDaniel MM, Channabasappa N, Kim J, Kim M, Zhan X, Xie Y. Severe gut microbiota dysbiosis is associated with poor growth in patients with short bowel syndrome. JPEN J Parenter Enteral Nutr. 2016
    1. Segata N, Waldron L, Ballarini A, Narasimhan V, Jousson O, Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Methods. 2012;9:811–814.
    1. Abubucker S, Segata N, Goll J, Schubert AM, Izard J, Cantarel BL, Rodriguez-Mueller B, Zucker J, Thiagarajan M, Henrissat B. Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput Biol. 2012;8:e1002358.
    1. Kim J, Kim MS, Koh AY, Xie Y, Zhan X. FMAP: functional mapping and analysis pipeline for metagenomics and metatranscriptomics studies. BMC Bioinformatics. 2016;17:420.
    1. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–336.
    1. Sinha R, Ahn J, Sampson JN, Shi J, Yu G, Xiong X, Hayes RB, Goedert JJ. Fecal microbiota, fecal metabolome, and colorectal cancer interrelations. PLoS One. 2016;11:e0152126.
    1. Dehaven CD, Evans AM, Dai H, Lawton KA. Organization of GC/MS and LC/MS metabolomics data into chemical libraries. J Chem. 2010;2:9.
    1. Hodi FS, Chesney J, Pavlick AC, Robert C, Grossmann KF, McDermott DF, Linette GP, Meyer N, Giguere JK, Agarwala SS. Combined nivolumab and ipilimumab versus ipilimumab alone in patients with advanced melanoma: 2-year overall survival outcomes in a multicentre, randomised, controlled, phase 2 trial. Lancet Oncol. 2016;17:1558–1568.
    1. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122–133.
    1. Fouhy F, Clooney AG, Stanton C, Claesson MJ, Cotter PD. 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform. BMC Microbiol. 2016;16:123.
    1. Kennedy K, Hall MW, Lynch MD, Moreno-Hagelsieb G, Neufeld JD. Evaluating bias of illumina-based bacterial 16S rRNA gene profiles. Appl Environ Microbiol. 2014;80:5717–5722.
    1. Tremblay J, Singh K, Fern A, Kirton ES, He S, Woyke T, Lee J, Chen F, Dangl JL, Tringe SG. Primer and platform effects on 16S rRNA tag sequencing. Front Microbiol. 2015;6:771.
    1. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, Gobourne A, No D, Liu H, Kinnebrew M, Viale A. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature. 2015;517:205–208.
    1. Looft T, Allen HK. Collateral effects of antibiotics on mammalian gut microbiomes. Gut Microbes. 2012;3:463–467.
    1. Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly YM, Glickman JN, Garrett WS. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013;341:569–573.
    1. Vucenik I, Shamsuddin AM. Protection against cancer by dietary IP6 and inositol. Nutr Cancer. 2006;55:109–125.
    1. Liu X., Villalta P.W., Sturla S.J. Simultaneous determination of inositol and inositol phosphates in complex biological matrices: Quantitative ion-exchange chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2009;23:705–712.
    1. Morais S.M., Silva K.A., Araujo H., Vieira I.G., Alves D.R., Fontenelle R.O., Silva A.M. Anacardic acid constituents from cashew nut shell liquid: NMR characterization and the effect of unsaturation on its biological activities. Pharmaceuticals. 2017;10(1):31.
    1. Gnanaprakasam J.N., Estrada-Muniz E., Vega L. The anacardic 6-pentadecyl salicylic acid induces macrophage activation via the phosphorylation of ERK1/2, JNK, P38 kinases and NF-kappaB. Int Immunopharmacol. 2015;29:808–817.
    1. Hollands A, Corriden R, Gysler G, Dahesh S, Olson J, Raza Ali S, Kunkel MT, Lin AE, Forli S, Newton AC. Natural product anacardic acid from cashew nut shells stimulates neutrophil extracellular trap production and bactericidal activity. J Biol Chem. 2016;291:13964–13973.
    1. Peled JU, Devlin SM, Staffas A, Lumish M, Khanin R, Littmann ER, Ling L, Kosuri S, Maloy M, Slingerland JB. Intestinal microbiota and relapse after hematopoietic-cell transplantation. J Clin Oncol. 2017;35(15):1650–1659.
    1. Gopalakrishnan VS, Christine, Reuben Alexandre, Karpinets Tatiana, Hutchinson Diane, Prieto Peter A, Tetzlaff Michael T, Lazar Alexander, Davies Michael A. Association of diversity and composition of the gut microbiome with differential responses to PD-1 based therapy in patients with metastatic melanoma. 2017 ASCO-SITC Clinical Immuno-Oncology Symposium; Orlando, FL, USA. J Clin Oncol. 2017;35
    1. Chaput N., Lepage P., Coutzac C., Soularue E., Le Roux K., Monot C., Boselli L., Routier E., Cassard L., Collins M. Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab. Ann Oncol. 2017;28(6):1368–1379.
    1. Dethlefsen L., Relman D.A. Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci U S A. 2011;108(Suppl. 1):4554–4561.
    1. David L.A., Maurice C.F., Carmody R.N., Gootenberg D.B., Button J.E., Wolfe B.E., Ling A.V., Devlin A.S., Varma Y., Fischbach M.A. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505:559–563.
    1. Hickey CA, Kuhn KA, Donermeyer DL, Porter NT, Jin C, Cameron EA, Jung H, Kaiko GE, Wegorzewska M, Malvin NP. Colitogenic Bacteroides thetaiotaomicron antigens access host immune cells in a sulfatase-dependent manner via outer membrane vesicles. Cell Host Microbe. 2015;17:672–680.
    1. Berezow AB, Ernst RK, Coats SR, Braham PH, Karimi-Naser LM, Darveau RP. The structurally similar, penta-acylated lipopolysaccharides of Porphyromonas gingivalis and Bacteroides elicit strikingly different innate immune responses. Microb Pathog. 2009;47:68–77.
    1. Selby MJ, Engelhardt JJ, Johnston RJ, Lu LS, Han M, Thudium K, Yao D, Quigley M, Valle J, Wang C. Preclinical development of ipilimumab and nivolumab combination immunotherapy: mouse tumor models, in vitro functional studies, and cynomolgus macaque toxicology. PLoS One. 2016;11:e0161779.
    1. Wang H, Hu S, Chen X, Shi H, Chen C, Sun L, Chen ZJ. cGAS is essential for the antitumor effect of immune checkpoint blockade. Proc Natl Acad Sci U S A. 2017;114:1637–1642.
    1. Derrien M, van Hylckama Vlieg JE. Fate, activity, and impact of ingested bacteria within the human gut microbiota. Trends Microbiol. 2015;23:354–366.
    1. McNulty NP, Yatsunenko T, Hsiao A, Faith JJ, Muegge BD, Goodman AL, Henrissat B, Oozeer R, Cools-Portier S, Gobert G. The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins. Sci Transl Med. 2011;3:106ra106.
    1. Lou HR, Mondal S. Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils. EMBO Reports. 2015;16(2):149–163.
    1. Hemshekhar M. M Sebastin Santhosh, K Kemparaju and KS Girish, Emerging roles of anacardic acid and its derivatives: a pharmacological overview. Basic Clin Pharmacol Toxicol. 2012;110:122–132.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner