Background Immune checkpoint blocker (ICB) therapy has changed the treatment landscape of solid cancers, with remarkable upfront responses; however, most patients develop secondary resistance and relapse within a few months. Currently, biomarkers to predict secondary resistance to ICB are unidentified. We hypothesized that gut microbiome and microbial metabolome could predict and modulate secondary resistance to ICB therapy across solid cancers.

Methods We conducted a prospective study to evaluate the differential composition of the gut microbiome and microbial metabolome in patients with durable responses-DR (defined as progression-free survival, PFS >1 year) versus those with progressive disease(PD) (PFS <6 months) and enrolled patients with metastatic renal cell cancer (RCC), melanoma, and head and neck cancer (HNSCC) receiving first-line ICB.

We collected paired blood and stool samples at treatment initiation, at one year if ongoing response, or at disease progression. Additional samples were collected from a subset of durable responders who subsequently lost their response. Stool metagenomics and fecal and plasma metabolomics was evaluated, with focus on microbial metabolites.

Complementarily, we conducted preclinical studies using mice models of HNSCC and RCC to elucidate the role of microbial metabolome in secondary ICB resistance. 30 mice were injected with MOC1 cells. When tumor volume reached 100mm³, they were treated with bi-weekly anti-PD1 for eight doses, with tumor measurement. A subset of ten mice with the slowest tumor growth and classified as durable responder, were randomized into two groups: one continued bi-weekly anti-PD1, while the other group also received daily Indole 3 Acetic Acid (I3A). Tumor growth and PFS were monitored for additional two weeks.

Results 220 patients and their samples were eligible, with median follow-up of 26 months. Significant differences in microbial composition and metabolite abundance was noted in DR vs PD samples. Higher abundance of Ruminococcus Gnavus(Odds ration [OR] -22.4, p-0.01, Bacteroides uniformis (OR- 7.8,p-0.005) and significantly lower abundance of Immunosuppressive microbial metabolite- I3A and indole carboxylic acid (ICA) was associated with durable response(DR) samples.

In preclinical models, administration of I3A accelerated tumor growth, with mean changes in tumor volume of 120% versus 25% (p=0.01).

Conclusions This is one of the first prospective study to demonstrate the role of microbial metabolome in predicting and mediating secondary ICB resistance. Information gained can help predict which patients are more likely to develop secondary resistance and inform future trials aimed to modulate microbial metabolome to mitigate secondary ICB resistance.