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. 2020 Oct;31(10):1366-1375.
doi: 10.1016/j.annonc.2020.06.003. Epub 2020 Jun 20.

Fusobacterium nucleatum persistence and risk of recurrence after preoperative treatment in locally advanced rectal cancer

G Serna  1 , F Ruiz-Pace  2 , J Hernando  3 , L Alonso  1 , R Fasani  1 , S Landolfi  4 , R Comas  2 , J Jimenez  1 , E Elez  3 , S Bullman  5 , J Tabernero  6 , J Capdevila  3 , R Dienstmann  2 , P Nuciforo  7
Affiliations

Fusobacterium nucleatum persistence and risk of recurrence after preoperative treatment in locally advanced rectal cancer

G Serna et al. Ann Oncol. 2020 Oct.

Abstract

Background: Accumulating evidence has identified Fusobacterium as an important pathogenic gut bacterium associated with colorectal cancer. Nevertheless, only limited data exist about the role of this bacterium in locally advanced rectal cancer (LARC). In this study, we quantified Fusobacterium nucleatum in untreated and post-neoadjuvant chemoradiotherapy (nCRT) samples from LARC patients and investigated its association with therapy response and survival.

Patients and methods: A total of 254 samples from 143 patients with rectal adenocarcinomas were analyzed for the presence and abundance of F. nucleatum using RNA in situ hybridization and digital image analysis. Assay accuracy was determined using infected cell lines and tumor samples with available quantitative PCR data. We studied the impact of F. nucleatum load on pathologic complete response and relapse-free survival. Treatment-induced changes were evaluated in paired pre- and post-nCRT samples (n = 71). Finally, tumor microenvironment changes during nCRT were assessed in paired samples (n = 45) by immune contexture analysis.

Results: F. nucleatum tissue levels by RNA in situ hybridization strongly correlated with quantitative PCR (r = 0.804, P < 0.001). F. nucleatum abundance was higher in untreated [median, 7.4; 95% confidence interval (3.7-16.2)] compared with treated [median, 1.6; 95% confidence interval (1.3-2.4)] tumors (P <0.001) with 58% (73/126) and 26% (22/85) positive tumors, respectively (P < 0.001). Baseline F. nucleatum levels were not associated with pathologic complete response. F. nucleatum positivity after nCRT, but not baseline status, significantly increased risk of relapse [hazard ratio = 7.5, 95% confidence interval (3.0-19.0); P < 0.001]. Tumors that turned F. nucleatum-negative after nCRT had a strong increase in CD8+ T cells post-nCRT (P < 0.001), while those that persisted F. nucleatum-positive after nCRT lacked CD8+ T cells induction in post-nCRT samples compared with baseline (P = 0.69).

Conclusion: F. nucleatum persistence post-nCRT is associated with high relapse rates in LARC, potentially linked to suppression of immune cytotoxicity.

Keywords: Fusobacterium nucleatum; locally advanced rectal cancer; microbiome; preoperative chemoradiotherapy.

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Conflict of interest statement

Disclosure JT reports personal financial interest in the form of scientific consultancy role for Array Biopharma, AstraZeneca, Bayer, BeiGene, Boehringer Ingelheim, Chugai, Genentech, Inc., Genmab A/S, Halozyme, Imugene Limited, Inflection Biosciences Limited, Ipsen, Kura Oncology, Lilly, Merck Sharp & Dohme, Menarini, Merck Serono, Merrimack, Merus, Molecular Partners, Novartis, Peptomyc, Pfizer, Pharmacyclics, ProteoDesign SL, Rafael Pharmaceuticals, F. Hoffmann-La Roche Ltd, Sanofi, SeaGen, Seattle Genetics, Servier, Symphogen, Taiho, VCN Biosciences, Biocartis, Foundation Medicine, HalioDX SAS, and Roche Diagnostics. JC reports scientific consultancy role (speaker and advisory roles) from Novartis, Pfizer, Ipsen, Exelixis, Bayer, Eisai, Advanced Accelerator Applications, Amgen, Sanofi, ITM, Sirlex and Merck Serono. Research grants from Novartis, Pfizer, AstraZeneca, Advanced Accelerator Applications, Eisai, and Bayer. RD has an advisory role at Roche and Boehringer-Ingelheim, has received a speaker’s fee from Roche, Ipsen, Amgen, Sanofi, Servier, Merck Sharp & Dohme and further received direct research funding from Merck and Pierre Fabre. PN has consulted for Bayer, Novartis, Merck Sharp & Dohme, and Targos, and received compensation. The other authors have declared no conflicts of interest.

Figures

Figure 1
Figure 1
RNA-ISH assay for intratumoral Fusobacterium nucleatum visualization and quantification. (A) Representative RNA-ISH images of HCT116 cell lines infected with increasing dose of Fusobacterium nucleatum (multiplicity of infection: 0, 1, 10, 100 from left to right; digital magnification, 30×). Upper: RNA-ISH; bottom: RNA-ISH after digital image analysis (DIA). (B) Boxplot of F. nucleatum densities quantification by DIA in infected cell lines (multiplicity of infection: 0, 1, 10, 100). (C) Boxplot of log10-transformed Fusobacterium densities by DIA by qPCR categories (high: ct value ≤30; intermediate: ct value 31–35; low: ct value 36–39 and negative: ct value >40). (D–F) Representative RNA-ISH images showing patterns of F. nucleatum infection in rectal cancer tissue samples (digital magnification, upper:10×, bottom: 30×). (D) Non-adhesive F. nucleatum; (E) adhesive F. nucleatum in correspondence of ulceration; (F) invasive F. nucleatum. ISH, in situ hybridization; qPCR, quantitative PCR.
Figure 2
Figure 2
Kaplan–Meier curves for relapse-free survival (RFS). (A) Control cohort by Fusobacterium nucleatum baseline status. (B) Treated cohort by F. nucleatum status in pre-neoadjuvant chemoradiotherapy (nCRT) tumor samples. (C) Treated cohort by F. nucleatum status in post-nCRT tumor samples. (D) Paired treated cohort grouped according to the shift in F. nucleatum status between pre-nCRT and post-nCRT paired samples. N-N: patients who maintained negative F. nucleatum status before and after treatment. P-N: patients in whom F. nucleatum was negative after treatment. P-P: patients with a positive F. nucleatum status in both samples. HR, hazard ratio.
Figure 3
Figure 3
Tumor immune microenvironment modulation according to treatment-induced microbiotype status change. (A) Multiple parallel coordinate plot and boxplot of the change of CD8+ cells density values in three groups of patients defined based on the change of Fusobacterium nucleatumstatus from pre-nCRT samples to post-nCRT samples (N-N, negative-negative; P-N, positive-negative; P-P positive-positive). Each line joins both samples belonging to the same patient. The blue lines highlight the increase in CD8+ while the pink lines indicate a decrease in these values. (B) Representative immunohistochemistry images of CD8 immune cells staining in pre-nCRT and post-nCRT paired samples (digital magnification, 10×). nCRT, neoadjuvant chemoradiotherapy.
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