For a patient with hormone‑receptor‑positive metastatic breast cancer, when should next‑generation sequencing be ordered and which specimen—metastatic site formalin‑fixed, paraffin‑embedded (FFPE) core biopsy, archival primary‑tumor block, or circulating tumor DNA (ctDNA) blood sample—should be sent for testing?

When to Order NGS in HR-Positive Metastatic Breast Cancer

Tumor next-generation sequencing should be performed as standard of care after resistance to endocrine therapy develops in all patients with hormone receptor-positive, HER2-negative metastatic breast cancer. 1

Optimal Timing for NGS Testing

• Order NGS after endocrine therapy resistance to maximize the likelihood of detecting ESR1 mutations, which are uncommon in treatment-naïve metastatic disease but emerge during aromatase inhibitor exposure 1
• Perform testing early in patients with good performance status and preferably when genomic mutation-driven therapeutic trials are available 2
• Do not delay testing until multiple lines of therapy have failed, as actionable mutations are identified in approximately 95% of metastatic breast cancer patients and can guide treatment decisions earlier in the disease course 2, 3

Which Specimen to Send

First-Line Approach: Circulating Tumor DNA (ctDNA)

• Start with plasma ctDNA testing as the initial approach, particularly when recent metastatic tissue is unavailable 1
• Plasma testing offers convenience and avoids invasive biopsy procedures 1
• Critical limitation: plasma testing misses approximately 44% of PIK3CA mutations that are detectable in tissue specimens 1, 4

Reflex to Tissue Testing

• If plasma ctDNA is negative, reflex to tumor tissue testing is mandatory to avoid false-negative results 1, 4
• Test the most recent tumor sample available, preferably from a metastatic site, as mutations can be acquired during metastatic treatment and receptor status may change from primary to metastatic disease 1
• Metastatic site FFPE core biopsy is preferred over archival primary tumor when feasible, as concordance between primary and metastatic samples for key driver genes is only 75% and metastatic lesions may harbor additional actionable alterations 3, 5

Practical Algorithm

1. If recent metastatic biopsy tissue is available: Send FFPE tissue for NGS 1
2. If no recent tissue is available: Start with plasma ctDNA testing 1
3. If plasma ctDNA is negative: Obtain tissue biopsy (if clinically feasible) and send for NGS 1, 4
4. Avoid bone biopsies when possible, as processing may affect receptor results 1

Key Actionable Alterations to Detect

Level IA Alterations (Highest Priority)

• ESR1 mutations (9% prevalence): Predict benefit from elacestrant (HR 0.55 for PFS) 1
• PIK3CA mutations (39% prevalence): Guide alpelisib plus fulvestrant therapy after CDK4/6 inhibitor failure 1, 6
• BRCA1/2 mutations (somatic 4-6% prevalence): Enable PARP inhibitor therapy (olaparib or talazoparib) 1

Level IIB Alterations

• AKT1/PTEN alterations: FDA-approved indication for capivasertib plus fulvestrant, though benefit predominantly arises from PIK3CA mutations 1
• Somatic BRCA1/2 mutations: PARP inhibitors show 50% objective response rate 1
• Germline PALB2 variants: PARP inhibitors show 82% objective response rate 1

Critical Caveats

Germline BRCA1/2 Testing

• Tumor NGS misses approximately 7% of germline BRCA1/2 mutations 1
• Patients with high likelihood of harboring germline BRCA1/2 mutations must undergo dedicated germline testing even if tumor NGS is negative 1
• High-risk features include: Young age at diagnosis, triple-negative subtype, family history of breast/ovarian cancer, Ashkenazi Jewish ancestry 1

ESR1 Mutation Testing Limitations

• ESR1 mutations are uncommon in treatment-naïve metastatic disease and in patients without prior aromatase inhibitor exposure 1
• Testing before endocrine therapy resistance develops has lower yield and may not justify the cost 1
• Current ASCO guidelines state insufficient data exist to recommend routine ESR1 testing, though the 2024 ESMO guidelines now classify ESR1 as level IA based on EMERALD trial results 1

Panel Selection Requirements

• Ensure the NGS panel includes comprehensive fusion detection capability, as actionable fusions (though rare in breast cancer) are increasingly targetable 7
• Minimum gene coverage should include: PIK3CA, ESR1, BRCA1/2, AKT1, PTEN, ERBB2, FGFR1, TP53 3, 6
• Panels targeting 89-546 genes have been successfully used in clinical practice 6

Real-World Implementation Challenges

• Only 4.7-7.4% of patients with actionable mutations receive matched therapy in real-world settings due to lack of available drugs, clinical trials, poor performance status, or financial burden 6
• Approximately 70% of patients with actionable mutations identified by NGS can be referred for clinical trials or have treatment changes when comprehensive genomic tumor boards and trial access are available 2, 3
• Higher mutational burden correlates with worse overall survival, providing additional prognostic information beyond treatment selection 3