<div>Abstract<p>Tumor hypoxia has been shown to predict poor patient outcomes in several cancer types, partially because it reduces radiation’s ability to kill cells. We hypothesized that some of the clinical effects of hypoxia could also be due to its impact on the tumor microbiome. Therefore, we examined the RNA sequencing data from the Oncology Research Information Exchange Network database of patients with colorectal cancer treated with radiotherapy. We identified microbial RNAs for each tumor and related them to the hypoxic gene expression scores calculated from host mRNA. Our analysis showed that the hypoxia expression score predicted poor patient outcomes and identified tumors enriched with certain microbes such as <i>Fusobacterium nucleatum</i>. The presence of other microbes, such as <i>Fusobacterium canifelinum</i>, predicted poor patient outcomes, suggesting a potential interaction between hypoxia, the microbiome, and radiation response. To experimentally investigate this concept, we implanted CT26 colorectal cancer cells into immune-competent BALB/c and immune-deficient athymic nude mice. After growth, in which tumors passively acquired microbes from the gastrointestinal tract, we harvested tumors, extracted nucleic acids, and sequenced host and microbial RNAs. We stratified tumors based on their hypoxia score and performed a metatranscriptomic analysis of microbial gene expression. In addition to hypoxia-tropic and -phobic microbial populations, analysis of microbial gene expression at the strain level showed expression differences based on the hypoxia score. Thus, hypoxia gene expression scores seem to associate with different microbial populations and elicit an adaptive transcriptional response in intratumoral microbes, potentially influencing clinical outcomes.</p>Significance:<p>Tumor hypoxia reduces radiotherapy efficacy. In this study, we explored whether some of the clinical effects of hypoxia could be due to interaction with the tumor microbiome. Hypoxic gene expression scores associated with certain microbes and elicited an adaptive transcriptional response in others that could contribute to poor clinical outcomes.</p></div>
<p>Demographic variables stratified by low and high hypoxia scores, showing no significant differences to be controlled for in the Cox proportional hazards model described in Figure 1D.</p>
<p>Cox proportional hazards model results for the interaction terms between hypoxia (binary, low or high) with microbe relative abundance in COADREAD tumors treated with radiotherapy.</p>
The rapid advancement of immunotherapies in oncology precipitates the need for clinicogenomic databases to verify published findings and to augment related discoveries. Growing bodies of real-world data (RWD) present such opportunities. We used one RWD source, Aster Insights' ORIEN Avatar® database, to develop criteria for estimating real-world progression-free survival (rwPFS) endpoints and applied these criteria to a melanoma cohort, investigating association of rwPFS with a published, validated, prognostic, IFN-γ-related gene signature developed from clinical trial patients.1
Methods
A cohort of 239 patients with melanoma and RNA-seq data were identified from the ORIEN Avatar® database (April 2023 release). This pan-cancer database is maintained by Aster Insights, which receives specimens and data submissions (comprised of whole-exome tumor/germline sequencing, RNA sequencing, and lifetime follow-up) from ORIEN members, a consortium research network of 18 U.S. cancer centers. Melanoma samples were collected from either the primary tumor site or a metastasis. rwPFS endpoints were identified from normalized clinical data. The following were considered uncensored events: 1) annotated progression/recurrence in clinical records, 2) drug therapy annotated as stopped due to progression, 3) identification of new metastases, and 4) death. Patients without an event were right-censored at last follow-up. RNA-seq analysis utilized a pipeline incorporating RSEM. Gene expression was quantified as Transcript Per Million (TPM), log2(TPM+1) transformed, and ComBat normalized to adjust for batch effects related to preservation method. A summary score was calculated for an 18 gene, IFN-γ-related signature previously reported to predict clinical response in PD-1 blockade.1
Results
112 patients in the melanoma cohort had immune-checkpoint inhibitor (ICI) therapy, RNA-seq prior to ICI therapy, and an evaluable rwPFS endpoint. Patient characteristics are shown in table 1. Median follow-up for the cohort was 4.4 years; median rwPFS was 1.5 years. Groups derived from a median cut of the IFN-γ-related signature demonstrated a marginally significant association with rwPFS (p=0.059). Higher expression of the signature associated with longer survival (figure 1). Identifying an optimal cutpoint for defining risk groups was outside the scope of this study, but stronger separation of groups was observed with lower cutpoints.
Conclusions
The observation of longer rwPFS in the high IFN-γ-related signature group corresponds with the original report of the signature, in which high expression of the signature was associated with a favorable response to ICI treatment.1 Derivation of PFS endpoints from real-world data has recently been demonstrated2; the current study provides additional confirmation of the utility of rwPFS.
References
Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, Albright A, Cheng JD, Kang SP, Shankaran V, Piha-Paul SA, Yearley J, Seiwert TY, Ribas A, McClanahan TK. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017;127(8):2930–2940. Griffith SD, Tucker M, Bowser B, Calkins G, Chang C-H. Generating Real-World Tumor Burden Endpoints from Electronic Health Record Data: Comparison of RECIST, Radiology-Anchored, and Clinician-Anchored Approaches for Abstracting Real-World Progression in Non-Small Cell Lung Cancer. Adv Ther. 2019;36:2122–2136.
Ethics Approval
For this study, ORIEN members utilized a standard protocol, Total Cancer Care (TCC®; NCT03977402), to which patients provided an IRB-approved written informed consent at their participating institutions.
