Profiling the gut and oral microbiota of hormone receptor-positive, HER2-negative metastatic breast cancer patients receiving pembrolizumab and eribulin

Abstract

Aim: Changes in host-associated microbial communities (i.e., the microbiota) may modulate responses to checkpoint blockade immunotherapy. In the KELLY phase II study (NCT03222856), we previously demonstrated that pembrolizumab [anti-programmed cell death protein 1 (PD-1)] combined with eribulin (plus microtubule-targeting chemotherapy) showed encouraging antitumor activity in patients with hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (mBC) who had received prior treatments.

Methods: A total of 58 fecal and 67 saliva samples were prospectively collected from a subset of 28 patients at baseline (BL), after three treatment cycles, and end of treatment. Shotgun metagenomics, 16S rRNA gene amplicon sequencing, and bioinformatics and statistical approaches were used to characterize fecal and oral microbiota profiles.

Results: Treatment caused no substantial perturbations in gut or oral microbiota, suggesting minimal drug-related microbial toxicity. Bacteroides and Faecalibacterium were the dominant gut microbiota genera, while Prevotella and Streptococcus were present in both oral and gut samples, highlighting potential gut-oral microbial interactions. Additionally, clinical benefit (CB) appeared to be associated with gut-associated Bacteroides fragilis (B. fragilis) and a BL oral abundance of Streptococcus ≥ 30%. Notably, B. fragilis NCTC 9343 supernatant induced dose-dependent lactate dehydrogenase (LDH) release from the MCF-7 (HR-positive/HER2-negative) BC cell line.

Conclusion: These findings suggest that specific gut and oral microbiota may modulate the effectiveness of combinatory anti-BC therapies, potentially through the action of microbial metabolites.

Keywords

Microbiota / pembrolizumab / eribulin / immunotherapy / breast cancer / metastatic breast cancer / shotgun metagenomic sequencing / 16S rRNA gene amplicon sequencing

References

[1] NIH. Cancer stat facts: female breast cancer subtypes. Available from: https://seer.cancer.gov/statfacts/html/breast-subtypes.html. [Last accessed on 26 Oct 2024]

[2] Rugo HS,Im SA.Safety and antitumor activity of pembrolizumab in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer.Clin Cancer Res2018;24:2804-11

[3] Nanda R,Yau C.Effect of pembrolizumab plus neoadjuvant chemotherapy on pathologic complete response in women with early-stage breast cancer: an analysis of the ongoing phase 2 adaptively randomized I-SPY2 trial.JAMA Oncol2020;6:676-84 PMCID:PMC7058271

[4] Pérez-García JM,G Cortés M.Pembrolizumab plus eribulin in hormone-receptor-positive, HER2-negative, locally recurrent or metastatic breast cancer (KELLY): an open-label, multicentre, single-arm, phase II trial.Eur J Cancer2021;148:382-94

[5] Cortes J, O’Shaughnessy J, Loesch D, et al; EMBRACE (Eisai Metastatic Breast Cancer Study Assessing Physician’s Choice Versus E7389) investigators. Eribulin monotherapy versus treatment of physician’s choice in patients with metastatic breast cancer (EMBRACE): a phase 3 open-label randomised study. Lancet 2011;377:914-23.

[6] Lee SH,Yoon Y.Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.Nat Microbiol2021;6:277-88

[7] Sivan A,Hubert N.Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy.Science2015;350:1084-9 PMCID:PMC4873287

[8] Tanoue T,Plichta DR.A defined commensal consortium elicits CD8 T cells and anti-cancer immunity.Nature2019;565:600-5

[9] Viaud S,Mignot G.The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide.Science2013;342:971-6 PMCID:PMC4048947

[10] Iida N,Stewart CA.Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment.Science2013;342:967-70 PMCID:PMC6709532

[11] Gopalakrishnan V,Nezi L.Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.Science2018;359:97-103 PMCID:PMC5827966

[12] Davar D,McCulloch JA.Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients.Science2021;371:595-602 PMCID:PMC8097968

[13] Routy B,Derosa L.Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.Science2018;359:91-7

[14] Vétizou M,Daillère R.Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota.Science2015;350:1079-84 PMCID:PMC4721659

[15] Routy B,Daillère R,Wargo JA.The gut microbiota influences anticancer immunosurveillance and general health.Nat Rev Clin Oncol2018;15:382-96

[16] Burugu S,Nielsen TO.Immune infiltrates in the breast cancer microenvironment: detection, characterization and clinical implication.Breast Cancer2017;24:3-15

[17] Kitamoto S,Hein R,Kamada N.The bacterial connection between the oral cavity and the gut diseases.J Dent Res2020;99:1021-9 PMCID:PMC7375741

[18] Dore J,Levenez F. IHMS_SOP 03 V1: standard operating procedure for fecal samples self-collection, laboratory analysis handled within 4 to 24 hours (4 hours ˂ x ≤ 24 hours). Available from: https://brd.nci.nih.gov/brd/sop/show/2102. [Last accessed on 26 Oct 2024].

[19] Caporaso JG,Walters WA.Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.ISME J2012;6:1621-4 PMCID:PMC3400413

[20] Kiu R,Bedwell H.Genomic analysis on broiler-associated Clostridium perfringens strains and exploratory caecal microbiome investigation reveals key factors linked to poultry necrotic enteritis.Anim Microbiome2019;1:12 PMCID:PMC7000242

[21] Huson DH,Flade I.MEGAN community edition - interactive exploration and analysis of large-scale microbiome sequencing data.PLoS Comput Biol2016;12:e1004957 PMCID:PMC4915700

[22] The comprehensive R archive network. https://cran.r-project.org/. [Last accessed on 26 Oct 2024]

[23] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550 PMCID:PMC4302049

[24] Chen S,Chen Y.fastp: an ultra-fast all-in-one FASTQ preprocessor.Bioinformatics2018;34:i884-90 PMCID:PMC6129281

[25] Beghini F,Blanco-Míguez A.Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3.Elife2021;10:e65088 PMCID:PMC8096432

[26] Li D,Luo R,Lam TW.MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.Bioinformatics2015;31:1674-6

[27] Uritskiy GV,Taylor J.MetaWRAP-a flexible pipeline for genome-resolved metagenomic data analysis.Microbiome2018;6:158 PMCID:PMC6138922

[28] Wood DE,Langmead B.Improved metagenomic analysis with Kraken 2.Genome Biol2019;20:257 PMCID:PMC6883579

[29] Lu J,Thielen P.Bracken: estimating species abundance in metagenomics data.PeerJ Computer Science2017;3:e104

[30] Parks DH,Skennerton CT,Tyson GW.CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes.Genome Res2015;25:1043-55 PMCID:PMC4484387

[31] Finn RD,Eddy SR.HMMER web server: interactive sequence similarity searching.Nucleic Acids Res2011;39:W29-37 PMCID:PMC3125773

[32] Hyatt D,Hauser LJ.Gene and translation initiation site prediction in metagenomic sequences.Bioinformatics2012;28:2223-30

[33] Wu YW,Singer SW.MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets.Bioinformatics2016;32:605-7

[34] Kang DD,Kirton E.MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies.PeerJ2019;7:e7359 PMCID:PMC6662567

[35] Chaumeil PA,Hugenholtz P.GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database.Bioinformatics2019;36:1925-7 PMCID:PMC7703759

[36] vegan: an R package for community ecologists. Available from: https://github.com/vegandevs/vegan/. [Last accessed on 26 Oct 2024]

[37] Segata N,Waldron L.Metagenomic biomarker discovery and explanation.Genome Biol2011;12:R60 PMCID:PMC3218848

[38] Zhou Z,Breister AM.METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks.Microbiome2022;10:33 PMCID:PMC8851854

[39] Florensa AF,Clausen PTLC,Aarestrup FM.ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes.Microb Genom2022;8:000748 PMCID:PMC8914360

[40] Zankari E,Cosentino S.Identification of acquired antimicrobial resistance genes.J Antimicrob Chemother2012;67:2640-4 PMCID:PMC3468078

[41] Franco AA,Trucksis M,Kaper JB.Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.Infect Immun1997;65:1007-13 PMCID:PMC175082

[42] Seemann T. ABRicate. 1.0.1 ed. Available from: https://github.com/tseemann/abricate. [Last accessed on 26 Oct 2024]

[43] Dybdal-Hargreaves NF,Mooberry SL.Eribulin mesylate: mechanism of action of a unique microtubule-targeting agent.Clin Cancer Res2015;21:2445-52 PMCID:PMC4812567

[44] Swami U,Ghalib MH.Eribulin - a review of preclinical and clinical studies.Crit Rev Oncol Hematol2012;81:163-84 PMCID:PMC3954568

[45] Zhang ZY,Pelletier RD.Delineation of the interactions between the chemotherapeutic agent eribulin mesylate (E7389) and human CYP3A4.Cancer Chemother Pharmacol2008;62:707-16

[46] Mazmanian SK,Tzianabos AO.An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.Cell2005;122:107-18

[47] Dasgupta S,Ochoa-Reparaz J,Kasper DL.Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms.Cell Host Microbe2014;15:413-23 PMCID:PMC4020153

[48] Barton LL,Fauque GD.Sulfur cycling and the intestinal microbiome.Dig Dis Sci2017;62:2241-57

[49] Krinos CM,Weinacht KG,Kasper DL.Extensive surface diversity of a commensal microorganism by multiple DNA inversions.Nature2001;414:555-8

[50] Baruch EN,Ben-Betzalel G.Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.Science2021;371:602-9

[51] Dizman N,Bergerot P.Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.Nat Med2022;28:704-12 PMCID:PMC9018425

[52] Iida N,Goldszmid R,Trinchieri G.Gut commensal bacteria promote anti-tumor innate immune responses in distant tumors after immunotherapy and chemotherapy (162.5).J Immunol2012;188:162.5

[53] Li Y,Elmén L.Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5^-/- mice.Nat Commun2019;10:1492 PMCID:PMC6445090

[54] Mager LF,Pett N.Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.Science2020;369:1481-9

[55] McKee AM,Madgwick M.Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.iScience2021;24:103012 PMCID:PMC8426205

[56] Roy S.Microbiota: a key orchestrator of cancer therapy.Nat Rev Cancer2017;17:271-85

[57] Teng NMY,McKee AM,Robinson SD.Exploring the impact of gut microbiota and diet on breast cancer risk and progression.Int J Cancer2021;149:494-504 PMCID:PMC8650995

[58] Latorre-Pérez A,Iglesias JR.The Spanish gut microbiome reveals links between microorganisms and Mediterranean diet.Sci Rep2021;11:21602 PMCID:PMC8580991

[59] Han YW.Fusobacterium nucleatum: a commensal-turned pathogen.Curr Opin Microbiol2015;23:141-7 PMCID:PMC4323942

[60] Castellarin M,Freeman JD.Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.Genome Res2012;22:299-306 PMCID:PMC3266037

[61] Kostic AD,Pedamallu CS.Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.Genome Res2012;22:292-8 PMCID:PMC3266036

[62] Parhi L,Sol A.Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression.Nat Commun2020;11:3259 PMCID:PMC7320135

[63] Sulyanto RM,Beall CJ,Griffen AL.The predominant oral microbiota is acquired early in an organized pattern.Sci Rep2019;9:10550 PMCID:PMC6646312

[64] Kawasaki M,Ikeda E.Oral infectious bacteria in dental plaque and saliva as risk factors in patients with esophageal cancer.Cancer2021;127:512-9

[65] Mukherjee PK,Retuerto M.Bacteriome and mycobiome associations in oral tongue cancer.Oncotarget2017;8:97273-89 PMCID:PMC5722561

[66] Zhou CB,Jin P.Fecal signatures of Streptococcus anginosus and Streptococcus constellatus for noninvasive screening and early warning of gastric cancer.Gastroenterology2022;162:1933-47.e18

[67] Perdigon G,Alvarez S,Pesce de Ruiz Holgado AA.Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus.J Dairy Sci1987;70:919-26

[68] Kaci G,Dennin V.Anti-inflammatory properties of Streptococcus salivarius, a commensal bacterium of the oral cavity and digestive tract.Appl Environ Microbiol2014;80:928-34 PMCID:PMC3911234

[69] Nearing JT,Hayes MG.Microbiome differential abundance methods produce different results across 38 datasets.Nat Commun2022;13:342 PMCID:PMC8763921

[70] Chen F,Zhou CC.Integrated analysis of the faecal metagenome and serum metabolome reveals the role of gut microbiome-associated metabolites in the detection of colorectal cancer and adenoma.Gut2022;71:1315-25 PMCID:PMC9185821

[71] Hezaveh K,Klötgen A.Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.Immunity2022;55:324-40.e8 PMCID:PMC8888129

[72] Neff CP,Arnolds KL.Diverse intestinal bacteria contain putative zwitterionic capsular polysaccharides with anti-inflammatory properties.Cell Host Microbe2016;20:535-47 PMCID:PMC5113727

[73] Gul L,Fonseca S.Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease.J Extracell Vesicles2022;11:e12189 PMCID:PMC8783345

[74] Zhang M,Dorfman RG.Butyrate inhibits interleukin-17 and generates tregs to ameliorate colorectal colitis in rats.BMC Gastroenterol2016;16:84 PMCID:PMC4967301

[75] Kovács T,Vida A.Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors.Sci Rep2019;9:1300 PMCID:PMC6361949

[76] Mikó E,Kovács T.Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness.Biochim Biophys Acta Bioenerg2018;1859:958-74

[77] Corander J,Pensar J.Causal discovery for the microbiome.Lancet Microbe2022;3:e881-7 PMCID:PMC9680137

[78] Raza MH,Arshad A.Microbiota in cancer development and treatment.J Cancer Res Clin Oncol2019;145:49-63