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Gut microbiome influences efficacy of PD-1–based immunotherapy against epithelial tumors

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Science  05 Jan 2018:
Vol. 359, Issue 6371, pp. 91-97
DOI: 10.1126/science.aan3706
  • Fig. 1 Antibiotics compromise the efficacy of PD-1 blockade in mouse tumor models and cancer patients.

    (A) Tumor growth kinetics of RET melanoma (left) and MCA-205 sarcoma in mice (right) treated with four injections of PD-1 (clone RMP1-14) mAb (αPD-1) or isotype control mAb (Iso Ctrl) in the presence or absence of broad-spectrum antibiotics (ATB). Data are means ± SEM of tumor sizes for 10 to 12 mice per group. (B) Survival curves of RET-bearing mice (left) and MCA-205–bearing mice (right) treated with PD-1 mAb combined with CTLA-4 mAbs. Each line represents one survival curve for each group of five mice from two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 [analysis of variance (ANOVA) and log-rank (Mantel-Cox) analysis]; ns, not significant. (C to E) Kaplan-Meier estimates for progression-free survival (PFS) or overall survival (OS) of cancer patients. (C) All cancer patients (n = 249); (D) patients with advanced non–small cell lung cancer (NSCLC, n = 140, fig. S1A; fig. S1D for the validation cohort); (E) renal cell carcinoma (RCC, n = 67, fig. S1B). [See fig. S1C and tables S1 to S6 for data on patients with urothelial carcinoma (n = 42) treated with PD-1/PD-L1 mAb who did or did not receive ATB.] The points represent data censored at the last time the patient was known to be alive and without progression. P values are shown [log-rank (Mantel-Cox) analysis].

  • Fig. 2 Metagenomic analysis of fecal samples predicts response at 3 months of PD-1 mAb treatment in cancer patients.

    (A) Shotgun sequencing of fecal samples at diagnosis with representation of gene and MGS counts for all cancer patients according to clinical outcome (PFS at 6 months). Data are means ± SEM of counts for patients experiencing PFS shorter or longer than 6 months. Gene or MGS richness did not predict PFS at 3 months. (B and C) Shotgun sequencing of fecal samples at diagnosis with representation of the relative abundance of each MGS in responders (R) (partial response or stable disease) over nonresponders (NR) (progression or death) defined using the best clinical response according to RECIST1.1 criteria (B) or PFS at 3 months (C) and the corresponding P value on the entire cohort of n = 100 (60 NSCLC and 40 RCC) patients (B) and excluding those who took ATB (C) (fig. S5B), n = 78 (42 R, 36 NR); see also fig. S5A for all patients. T0 samples were analyzed; when not available, T1 specimens were used, as there was no statistical difference between T0 and T1 (fig. S3A). (D) Frequency of patients with detectable A. muciniphila in their feces according to PR (partial response), SD (stable disease), or PD (progressive disease) clinical status, as assessed by metagenomics and analyzed by Cochran-Armitage test. (E and F) Circulating memory T cell immune responses directed against commensals detected during PD-1 blockade and evaluation of the time to progression. (E) Heat map of the P values for each cytokine and each commensal, segregating NSCLC+RCC patients’ PFS according to the median value of cytokine production of the whole cohort. Significant P values (<0.05, Student t test) are indicated with an asterisk for the relevant commensals. (F) Univariate analysis and Kaplan-Meier curves showing PFS for peripheral blood memory Th1 and Tc1 immune responses directed against A. muciniphila and E. hirae 13144, respectively. See fig. S9 for PFS curves for nonspecific T cell receptor–driven cytokine release. (G) Culturomics-based analyses of fecal samples in 16 R and 16 NR NSCLC patients (defined as the best clinical outcome) before therapy, each commensal colony having been identified by mass spectrometry. Colored bars show relative frequencies of each commensal in all fecal cultures in R over NR patients, with P values of the difference shown at the right.

  • Fig. 3 Fecal microbiota transplantation (FMT) of stool samples from NSCLC and RCC patients into mice dictates outcome after PD-1 blockade.

    (A) Experimental setting: FMT was performed in germ-free recipients or after 3 days of ATB in SPF mice. Two weeks later, MCA-205 sarcoma cells were inoculated and PD-1 mAb was injected intraperitoneally every 3 days starting on D+5. (B) Representative MCA-205 tumor growth curves after FMT from NSCLC patients into germ-free (left) or ATB-treated mice (right) during therapy with PD-1 mAb. (C) Means ± SEM of tumor sizes after Iso Ctrl mAb (left) or after PD1 mAb (center and right) in MCA-205–bearing, SPF-reared mice treated with ATB and then receiving FMT from eight NSCLC patients (n = 4 NR, n = 4 R) before PD1 mAb. Pooled data from all patients (left and center) or individual patients (right) are shown; each fecal sample was transferred into five mice per group. (D and E) Flow cytometry analysis of CXCR3 (D) or PD-L1 (E) expression on tumor-infiltrating lymphocytes (TILs) (D) or CD3+CD4+ splenocytes (E) in 38 and 80 animals, respectively, analyzed at killing times corresponding to those in (C). (F) Monitoring of RENCA progression via bioluminescence imaging of luciferase activity in ATB-treated mice (n = 133) after FMT with feces from three R and four NR RCC patients and treated with a combination of PD-1 and CTLA-4 mAbs. All experiments included five to seven mice per group and were performed at least twice in similar conditions yielding similar results. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA and Student t test). Dx, last IVIS (bioluminescent imaging) measurement; D0, day of randomization.

  • Fig. 4 Biological importance of A. muciniphila during anticancer PD-1 blockade treatment.

    (A and B) ATB-induced dysbiosis was restored by oral administration of A. muciniphila (Akkermansia) alone or combined with Enterococcus hirae 13144 (E. hirae) to recipient mice receiving PD-1 mAb. Commensals and PD-1 mAb regimens were administered five times every 3 days against RET melanomas (A) and four times in luciferase-expressing orthotopic LLC non–small cell lung cancers treated, or not, with radiotherapy plus PD-1 mAb (B). (C) Means ± SEM of tumor sizes at time of killing in mice exhibiting NR FMT-induced dysbiosis and compensated with A. muciniphila alone or combined with E. hirae (also refer to fig. S12) or control bacteria (fig. S13) during PD-1 mAb–based therapy. Each color represents one NR donor of feces transferred into five mice per group. (D and E) Flow cytometry analysis of CCR9 and CXCR3 expression in mLN-residing CD4+ TCM at 48 hours (D) and at D+17 among the tumor-infiltrating lymphocytes (TILs) (E) after the first injection of PD-1 mAb in ATB-treated animals compensated with a mixture of A. muciniphila and E. hirae. Data are pooled from two independent experiments composed of five to seven mice per group; each symbol represents one mouse. (F and G) Immunohistochemical determination of CD4 and FoxP3 infiltrates in D+10-treated tumor beds [representative micrograph in (F)] calculated by image analyzer for the experimental setting described in Fig. 3C and calculation of the ratios between these two values on the whole tumor sample [(G), left]; Spearman correlation between CD4/FoxP3 ratios and tumor size at the time of killing [(G), right]. (H) Effects of neutralizing IL-12p40 mAb on the anticancer efficacy of PD-1 inhibition alone (left) or combined with Akkermansia (right). Data are means ± SEM of tumor sizes at killing; each symbol represents one tumor and each group comprises five mice. One representative experiment out of four is shown. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA and Student t test). Dx, last IVIS measurement; D0, day of randomization.

Supplementary Materials

  • Gut microbiome influences efficacy of PD-1–based immunotherapy against epithelial tumors

    Bertrand Routy, Emmanuelle Le Chatelier, Lisa Derosa, Connie P. M. Duong, Maryam Tidjani Alou, Romain Daillère, Aurélie Fluckiger, Meriem Messaoudene, Conrad Rauber, Maria P. Roberti, Marine Fidelle, Caroline Flament, Vichnou Poirier-Colame, Paule Opolon, Christophe Klein, Kristina Iribarren, Laura Mondragón, Nicolas Jacquelot, Bo Qu, Gladys Ferrere, Céline Clémenson, Laura Mezquita, Jordi Remon Masip, Charles Naltet, Solenn Brosseau, Coureche Kaderbhai, Corentin Richard, Hira Rizvi, Florence Levenez, Nathalie Galleron, Benoit Quinquis, Nicolas Pons, Bernhard Ryffel, Véronique Minard-Colin, Patrick Gonin, Jean-Charles Soria, Eric Deutsch, Yohann Loriot, François Ghiringhelli, Gérard Zalcman, François Goldwasser, Bernard Escudier, Matthew D. Hellmann, Alexander Eggermont, Didier Raoult, Laurence Albiges, Guido Kroemer, Laurence Zitvogel

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S16
    • Tables S1 to S18
    • References

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