Identification of intratumor bacteria-associated prognostic risk score in adrenocortical carcinoma

Abstract

A landmark study by Poore et al. showed intratumor bacteria (ITBs) playing a critical role in most cancers by reproduction of The Cancer Genome Atlas (TCGA) transcriptome data. A recent study by Salzberg et al. argued that ITBs, being overstated as a methodology by Poore et al., were problematic. We previously reported that ITBs were prognostic in adrenocortical carcinoma (ACC), a highly aggressive rare disease using data by Poore et al., and here, we aimed to answer whether ITBs truly existed and were prognostic in ACC. ACC samples from our institutes underwent 16S rRNA sequencing [adrenocortical carcinoma blocks from Huashan Hospital and China Medical University (HS) cohort]. The ITB profile was compared to TCGA data processed by Poore et al. (TCGA-P) and TCGA data processed by Salzberg et al. (TCGA-S), respectively. The primary outcome was overall survival (OS). A total of 26 ACC cases (HS cohort) and 10 paraffin controls were sequenced. The TCGA cohort encompassed 77 cases. Two and four amid the top 10 abundant genera in HS cohort were not detected in TCGA-P and TCGA-S, respectively. Neither was alpha or beta diversity associated with survival nor could ACC be subtyped by ITB signature in the HS cohort. Notably, a five-genera ITB risk score (Corynebacterium, Mycoplasma, Achromobacter, Anaerococcus, and Streptococcus) for OS trained in the HS cohort was validated in both TCGA-P and TCGA-S cohorts and was independently prognostic. Whereas ITB signature on the whole may not be associated with ACC subtypes, certain ITB features are associated with prognosis, and a risk score could be generated and validated externally.

Importance: In this report, we looked at the role of ITBs in ACC in patients with different race and sequencing platforms. We found a five-genera ITB risk score consistently predicted overall survival in all cohorts. We conclude that certain ITB features are universally pathogenic to ACC.

Keywords: adrenocortical carcinoma; intratumor bacteria; prognosis.

Fig 1

Intratumor bacteria (ITB) exist in adrenocortical carcinoma (ACC). Sequenced by 16S rRNA are (A) the drop of read counts before and after the decontamination process; (B) the drop of blacklist contaminant after decontamination controlled by FFPE samples; and the composition of top abundant ITBs at (C) phylum (top 5) and genus (top 10) levels of 26 cases in the HS cohort. In the HS cohort, shown are the (D) overall survival and the progression-free survival, (E) three indices indicative of alpha diversity between gender; and (F) correlation between patient age and Shannon index. F, female; M, male.

Fig 2

Adrenocortical carcinoma (ACC) cannot be subtyped by intratumor bacteria (ITB) signature. Shown are (A) the association between Shannon index and overall survival/progression-ree survival; (B) the association between dichotomized survival time by median survival time and beta diversity, and (C) the subtyping of ACC using unsupervised partition around medoids (PAM) clustering of all ITB in the HS cohort; (D) demonstration of relative abundance of top 10 abundant ITB in the HS cohort composition in two other TGCA cohorts with ITB classified as “other” removed.

Fig 3

Risk score based on intratumor bacteria (ITB) features is independently prognostic. Constructed using 5 ITB features in Fig. S2, the LASSO model shows (A) Kaplan-Meier curves of ACC cases with high and low risk score compared by log-rank test, trained in HS cohort and validated in TCGA-P/TCGA-S cohorts and (B) corresponding receiver operating characteristic curve (ROC) and area under the curve (AUC) profiles; (C) forest plot showing independence of the risk score in TCGA-P/TCGA-S cohorts. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Fig 4

Genomic and genetic alterations in ACC grouped by risk score of OS. (A) Waterfall plot showing the distribution of driver genomic events including gene mutation and copy number variation in the subgroup categorized by overall survival risk score in the TCGA-S cohort; violin plot showing the difference of (B) TMB and (C) fraction genome altered between high- and low-risk score groups in the TCGA-S cohort; (D) Bubble plot showing the top 10 pathways enriched by differentially expressed genes between high- and low-risk score groups using the Kyoto Encyclopedia of Genes and Genomes database in the TCGA-P and TCGA-S cohorts. PPAR, peroxisome proliferator-activated receptor; cAMP, cyclic AMP.

Fig 5

Survival-associated ITB is metabolic in ACC in the TCGA-S cohort. (A) The metabolic pathways were differentially enriched between high- and low-risk score groups by gene set enrichment analysis. (B) Boxplot showing the difference in the immune cell infiltration scores between high- and low-risk score groups. ssGSEA, single-sample gene set enrichment analysis.