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We read with interest the study by Kartal et al 1 showing that the gut-microbiota-derived biomarkers for disease stratification are often shared by subjects across disease cohorts. Here, we confirmed their observations with findings from a newly diagnosed natural killer/T-cell lymphoma (NKTCL) cohort, in which the gut biomarkers were significantly overlapped with those of multiple disease cohorts and consistently enriched/depleted in subjects with those diseases. Importantly, many of the shared biomarkers were remarkably associated with patient outcomes in our cohort, implying that they may have broad prognostic values in multiple diseases.
‘Microbiota-gut-lymphoma axis’ represents a fascinating avenue of microbiota-mediated lymphomagenesis and intervention opportunity,2 but the implications of gut microbiota in NKTCL remain enigmatic. To identify gut microbiota-derived diagnostic biomarkers for NKTCL, we recruited a discovery cohort consisting of 30 treatment-naïve patients and 20 healthy controls (HCs), and a validation cohort, including 12 patients and 13 HCs, respectively (online supplemental materials and methods). We applied shotgun metagenomic sequencing to their faecal samples, profiled their gut metagenomes using mOTUs2 V.2.5,3 and trained a patient-stratification classifier with all species-level taxonomic features using the LASSO algorithm implemented in SIAMCAT.4 Our classifier achieved an accuracy of 0.868 area under the receiver operating characteristic curve (AUROC) on the discovery cohort, and 0.910 AUROC on the validation cohort (figure 1A). To increase the sample size for model training, we retrained a LASSO classifier for the NKTCL using all the samples from both cohorts, and achieved an accuracy of 0.813 AUROC in cross-validation, which strongly support the role of gut microbiota as diagnostic biomarkers for NKTCL.
To examine the specificity of the NKTCL gut-microbiota-derived signature, we applied the all-sample NKTCL classifier to 29 public gut microbiota cohorts (online supplemental table S1). We observed an overall false positive rate (FPR) of 3.1% in the HCs, but higher FPRs in patients of several cohorts (figure 1B), especially those of the pancreatic cancer (Kartal_DE_2022_PC, Kartal_ES_2022_PC, Nagata_JP_2022_PC), Crohn’s Disease (He_2017_CD, Franzosa_2018_CD, Forslund_2015_CD) and liver disease (Qin_2014_LD). These results imply significant overlaps in the biomarkers between these diseases and NKTCL, which was confirmed using LEfSe analysis5 (figure 1C). Importantly, these biomarkers were consistently enriched/depleted in most cohorts, including the enrichment of oral-derived taxa of Veillonella and Streptococcus in the patients, and known beneficial species in HCs such as Faecalibacterium prausnitzii, Eubacterium rectale and Bifidobacterium adolescentis 1 6 7 (figure 1C). These findings indicate that our classifier can accurately distinguish NKTCL patients from HCs; nevertheless, due to the shared biomarkers with other diseases, combination of selected clinical indicators with microbial biomarkers would be salutary for a distinctive diagnostic model.
Survival data were available for the NKTCL patients in the discovery cohort. Notably, many identified microbiome biomarkers, especially those shared by multiple diseases, could significantly predict the overall survival (OS) and progression-free survival (PFS) of the patients, including Streptococcus parasanguinis, Romboutsia timonensis and Veillonella atypica (online supplemental figure 1A–D). Finally, we created a Streptococcus parasanguinis–Romboutsia timonensis index (SRI) as the relative abundance ratio of the two species, and obtained the best prognostic prediction power than other individual species and combinations. Namely, NKTCL patients with higher SRI scores showed significantly worse OS and PFS than those with lower SRI scores (figure 1D–E). Furthermore, we observed remarkable correlations between high SRI score and multiple adverse prognostic factors of NKTCL, including PINK-E, stage, lymph node involvement and responses to first-line treatment (all p<0.05; figure 1F).
Overall, our results lend support for gut microbiota as a potent assistive diagnostic tool for NKTCL. Moreover, the SRI score, based on the shared biomarkers, may have extensive prognostic utility in multiple diseases and deserves further scrutiny (online supplemental discussion).
Patient consent for publication
This study was performed in accordance with the Declaration of Helsinki and rules of good clinical practice, and the study was approved by the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University, Zhenzhou, China. Participants gave informed consent to participate in the study before taking part.
We would like to thank all the clinical doctors from the Lymphoma Diagnosis and Treatment Centre of Henan Province for their kind suggestions, and we also thank all the generous participants of this study for their supports.
ZS and GH are joint first authors.
ZS and GH contributed equally.
Contributors Study concept and design: MZ, W-HC and ZL. Samples collection: ZS, LZ, XL, XW, LL and XF. Data acquisition: ZS, GH, MWL, ZS, ZL, XZ and LT. Analysis and interpretation of data: W-HC, MZ, ZL, ZS, GH and MWL. Technical and material support: LZ, XL, XW, LL, XF, ZS, ZL, XZ and LT. Drafting of the manuscript: GH and ZS. Revising of the manuscript: W-HC, ZL and MZ. All the authors approved the final version of the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (81970184; 82170183; U1904139; 82070209; 82070210).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.