Biomarker testing in positive, randomized phase III trials in the post-CDK4/6i setting in advanced, HR+/HER2– breast cancer.
| Trial | Biomarker | Specimen source and timing for biomarker testing | Biomarker assessment site | Stratified according to biomarker | Assay | Included alterations | Rule for assignment of biomarker-indeterminate patients | Patient distribution according to biomarker status (% of ITT) |
|---|---|---|---|---|---|---|---|---|
| VIKTORIA-1 [86] | PIK3CA-WT | Archival or fresh biopsy. If neither available: ctDNA from blood samples | Central | NA1 | Therascreen PIK3CA Rotor-Gene Q PCR test (QIAGEN) | 10 PIK3CA mutations: C420R, E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, H1047L, H1047R, H1047Y | Identified PIK3CA status was an inclusion criterion | Identified PIK3CA status was an inclusion criterion |
| CAPItello-291 [82] | PIK3CA, AKT1, PTEN | Tumor tissue from the most recently collected tumor sample (primary or recurrent) | Central | No | China:OncoScreen Plus (Burning Rock Biotech)All other countries:FoundationOne CDx (Foundation Medicine) | Activating mutations in PIK3CA and AKT1 and inactivating alterations in PTEN genes | Patients without a qualifying alteration or with an unknown test result were included in the AKT pathway-non-altered population | 40.8% with confirmed AKT pathway alterations59.2% without confirmed AKT pathway alterations (of which 25.3% had an unknown AKT pathway alteration status) |
| VERITAC-2 [52] | ESR1 | ctDNA from pretreatment blood samples (prior to randomization) | Central (for some patients: local laboratory results were used) | Yes | China:NGS by OrigmedAll other countries:NGS by Foundation Medicine | Not reported | Patients with non-informative ESR1 test (result “unknown”) were stratified as patients without ESR1 mutation | 43.3% with ESR1 mutation56.7% without ESR1 mutation (percentage of patients with a possibly unknown result not reported) |
| EMERALD [41] | ESR1 | ctDNA in blood samples at screening | Central | Yes | Guardant360 CDx (Guardant Health) | ESR1 missense mutations in codons 310–547 | Patients without detectable ctDNA in the blood sample/where ESR1 status could not be determined are included in the group of patients without ESR1 mutation | 47.8% with ESR1 mutation52.2% without ESR1 mutation (percentage of patients without detectable ctDNA not reported) |
| EMBER-3 [46] (Imlunestrant vs. Fulvestrant) | ESR1 | ctDNA in blood samples before treatment administration (after randomization) | Central | No | China:OncoCompass Target (Burning Rock Biotech)All other countries:Guardant360 CDx (Guardant Health) | 34 ESR1 variants annotated as oncogenic or likely oncogenic by the OncoKB database | Patients without detectable ctDNA or with an unknown ESR1 mutation status (analytical failure, missing sample) are included in the group of patients without ESR1 mutation | 38.7% with ESR1 mutation58.2% without ESR1 mutation (of which 5% have an unknown ESR1 mutation status) |
| PADA-1 [57, 58] | ESR1 | ctDNA in blood samples at inclusion and repeatedly (during surveillance2 period: every 2 cycles) | Central | NA1 | Multiplex ddPCR (QX200 system; Bio-Rad Laboratories, Marnes-la-Coquette, France) | ESR1 mutations in hotspot codons 380, 536, 537, and 538 | Rising ESR1 mutation was an inclusion criterion | Rising ESR1 mutation was an inclusion criterionAfter randomization3: 46.6% of patients had no ESR1 mutation detected in pretreatment sample |
| SERENA-6 [45] | ESR1 | ctDNA in blood samples at inclusion and repeatedly (during surveillance2 period: every 2–3 months) | Central | NA1 | Guardant360 CDx (Guardant Health) | 11 ESR1 mutations E380Q, V422del, S463P, L536H, L536P, L536R, Y537C, Y537D, Y537N, Y537S, D538G | ESR1 mutation was an inclusion criterion | ESR1 mutation was an inclusion criterion |
| DESTINY-Breast06 [97] | HER2-low HER2-ultralow | Tissue sample at time of metastatic disease or later (most recent pre-randomization tumor sample) | Central | Yes (HER2-low vs. ultralow) | VENTANA HER2/neu (4B5) assay | HER2-low: IHC 1+ or IHC 2+ and ISH−HER2-ultralow: IHC 0 with membrane staining; also known as IHC > 0 and < 1+ | HER2-low or HER2-ultralow status was an inclusion criterion | 81.8% HER2-low17.6% HER2-ultralow |
Biomarker testing strategies and ascertainment rules in randomized phase III trials in the post-CDK4/6i setting. Trials are presented in which a primary endpoint was evaluated in a prospectively defined biomarker-selected population. Only trials reported as full publications were included. Data presented in this table were derived exclusively from the original or follow-up study report or protocol, no additional sources were consulted. 1 Only biomarker-defined patients were included in the study; 2 during the surveillance period, patients were monitored for ESR1. In case of rising (PADA-1) or detection (SERENA-6) of ESR1 mutation, patients were randomized to the respective treatments; 3 after randomization, ESR1 testing was repeated before treatment initiation, referred to as the pretreatment sample. CDK4/6i: CDK4/6 inhibitor; ctDNA: circulating tumor DNA; ddPCR: droplet digital polymerase chain reaction; IHC: immunohistochemistry; ISH: in situ hybridization; ITT: intention-to-treat; NA: not applicable; NGS: next-generation sequencing; WT: wild-type.
During the preparation of this work, the authors used Claude (Anthropic, Opus 4.8) and ChatGPT (OpenAI, GPT-5.3) for language refinement, improvement of readability, and translation of selected expressions. After using the tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.
AMH: Conceptualization, Investigation, Writing—original draft, Writing—review & editing. RB: Conceptualization, Investigation, Supervision, Visualization, Writing—original draft, Writing—review & editing. Both authors read and approved the submitted version.
AMH received travel support from Janssen and Amgen. RB received honoraria from H+O Health Communications, Medtis, Novartis, AstraZeneca, Impulze, Janssen, Silamed, Daiichi-Sankyo, Gilead, Stemline Menarini, MSD, OncoPulse; research funding from Gilead; travel support from Daiichi-Sankyo, Astra Zeneca, Servier and Roche; is a mentee in the ENDEAVOUR-Breast program; his wife is employed by CSL Behring AG; and he is listed on a patent application (provisional application no. 63/503,528) submitted to Massachusetts General Hospital.
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This study received Institutional Funding from the Department of Medical Oncology and the Medical Faculty of the University of Bern. The funders had no role in the conception and conduct of the review, decision to publish, or preparation of the manuscript.
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