ESR1 and PIK3CA circulating tumor DNA (ctDNA) mutation status as predictive biomarkers beyond variant allele fraction (VAF) in metastatic breast cancer.

Circulating tumor DNA (ctDNA) analysis is part of the current clinical practice for the management of hormone receptor (HR)-positive/HER2-negative metastatic breast cancer, particularly for detecting actionable alterations such as ESR1 and PIK3CA. Molecular testing reports commonly include variant allele fraction (VAF) as a contextual technical parameter, whose correct interpretation is increasingly important. Plasma VAF is influenced by multiple factors, including tumor fraction, metastatic site shedding, clonal architecture of the neoplasm, copy-number state, assay sensitivity, timing of sampling, and treatment pressure. In metastatic breast cancer, ESR1 and PIK3CA alterations represent different evolutionary events: PIK3CA mutations are early, clonal, and stable; ESR1 mutations are acquired under endocrine therapy and are often subclonal and dynamic. Consequently, VAF values across these biomarkers should be interpreted within their different biological contexts rather than as directly comparable indicators of therapeutic priority. Importantly, pivotal trials (EMERALD, PADA-1, SERENA-6, SOLAR-1, BYLieve, INAVO120, CAPItello-291) support mutation detection as the key evidence-based criterion for therapy selection. In this work, we discuss why, in metastatic breast cancer, the clinically relevant criterion for selecting ESR1-or PIK3CA-directed therapies remains the presence or absence of mutations, regardless of VAF.