Orchestrated Biosciences

Tumor mutational burden (TMB) is our best predictor of immune checkpoint blockade (ICB) response, yet it's only modestly accurate (AUC ~0.6) and reads DNA alone. I asked whether a tumor's RNA adds independent signal. I built a cohort-scale pipeline for non-reference RNA features (alternative splicing, intron retention, RNA editing, transposable-element activation, fusion transcripts) across five melanoma ICB cohorts, and ran the Evo2 genomic foundation model on novel splice junctions. Using strict leave-one-cohort-out testing, nothing in the RNA layer beat plain TMB — and I found why: every RNA signal was a proxy for how inflamed the tumor already was, and that proxy flips sign between cohorts, which may explain why so many RNA biomarkers fail to replicate. Reframing from response to survival surfaced the real result: subclonal mutation burden — a measure of how disordered a tumor's evolution is — predicted worse overall survival across three cohorts (HR 1.49, p=0.0004), separable from both TMB and immune state. The takeaway: how many mutations a tumor carries shapes whether it responds; how disordered its evolution is shapes how long the patient lives. A rigorously reported negative plus a novel, cross-cohort survival signal — and an honest account of where the signal actually lives.