Biomarker & Molecular Testing in Breast Cancer: An Overview

Why molecular testing matters here

Breast cancer is not one disease but a family of biologically distinct entities that happen to arise in the same organ. Two tumors that look similar under the microscope can behave very differently and respond to entirely different drug classes. Molecular and biomarker testing is what resolves that ambiguity — it defines the tumor's lineage and hormonal dependence, estimates the risk of recurrence, and identifies vulnerabilities that specific therapies can exploit. In modern practice, a handful of biomarker results assembled at diagnosis effectively route a patient into one of several major treatment pathways. This page is an orientation to those tests; each named biomarker links to a fuller detail article. Throughout, biomarker results are connected to therapy classes and eligibility concepts, not to individualized treatment recommendations.

It is useful to group the tests by what they are for. Some establish diagnosis and lineage and simultaneously carry prognostic and predictive weight. Others are purely prognostic — they estimate risk. A third group is predictive: they gate access to a particular drug class.

What gets tested, grouped by purpose

Lineage-defining biomarkers that are also predictive and prognostic

Three receptor assays form the backbone of every invasive breast cancer workup and are performed by immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue.

Estrogen receptor (ER) and progesterone receptor (PR/PgR) are scored as the percentage of tumor nuclei staining. Under the ASCO/CAP framework, ER is reported positive at ≥1%, with a distinct ER-low positive (1–10%) category flagged as a biologically separate group, and clearly positive tumors at ≥10% [1]. PR is positive at ≥1% [1]. Together these define hormone-receptor status. ER positivity gates endocrine therapy classes — selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs) — and, in combination with other factors, informs use of CDK4/6 inhibitors. An ER-positive/PR-negative profile predicts a somewhat less favorable endocrine response [1].

HER2 (ERBB2) is assessed by IHC (scored 0, 1+, 2+, 3+ per membrane-staining criteria) with equivocal 2+ results reflexed to in-situ hybridization (ISH), interpreted through the HER2/CEP17 ratio and copy-number algorithm [2]. A HER2-positive result (IHC 3+ or ISH-amplified) gates the anti-HER2 antibody and antibody-drug-conjugate classes (trastuzumab, pertuzumab, T-DM1, tucatinib-based regimens) [2,3]. The evidence has also carved out a "HER2-low" concept (IHC 1+ or 2+/ISH-negative) that gates trastuzumab deruxtecan in the metastatic setting per DESTINY-Breast04 [4]. Importantly, ASCO/CAP has not endorsed "HER2-low" as a formal interpretive category, and this remains a fast-moving, contested area of assay interpretation [3,4].

Prognostic biomarkers (risk estimation)

Ki-67, an IHC proliferation index reported as the percentage of positive tumor nuclei, contributes to risk stratification. Assay standardization remains a recognized limitation, and the International Ki-67 Working Group has issued guidance to constrain that variability [5]. It functions as one input among several rather than a standalone gate, though it has featured in some adjuvant decision frameworks.

Two validated multigene assays refine prognosis in HR-positive/HER2-negative early disease. The 21-gene recurrence score (Oncotype DX), an RT-PCR assay on FFPE, yields a continuous score interpreted alongside age and nodal status; it informs whether adjuvant chemotherapy should be added to endocrine therapy, validated prospectively in TAILORx (node-negative) and RxPONDER (1–3 nodes) [6,7]. The 70-gene signature (MammaPrint) delivers a binary low- versus high-genomic-risk classification and, in MINDACT, identified clinically high-risk patients who could safely forgo chemotherapy [8]. These assays are among the clearest examples of testing that de-escalates rather than escalates therapy.

Predictive biomarkers that gate specific drug classes

Germline BRCA1/BRCA2 testing is performed on blood or saliva. A pathogenic or likely pathogenic variant gates the PARP-inhibitor class — olaparib in the adjuvant setting (OlympiA) and olaparib or talazoparib in metastatic disease (OlympiAD, EMBRACA) — and also informs risk-reducing surgical discussions [9,10,11].

PIK3CA mutation, detectable by PCR or NGS on tumor FFPE or plasma ctDNA, gates the PI3K-inhibitor class (alpelisib) combined with fulvestrant in HR-positive/HER2-negative advanced disease, per SOLAR-1 [12].

PD-L1, scored by IHC using assay-specific cutoffs — the Combined Positive Score on the 22C3 assay at CPS ≥10 for triple-negative breast cancer (TNBC) — gates the immune-checkpoint-inhibitor class (pembrolizumab) in PD-L1-positive metastatic TNBC per KEYNOTE-355 [13]. Antibody- and platform-specific scoring makes assay harmonization a persistent challenge.

Mismatch repair/MSI (dMMR/MSI-H) and tumor mutational burden (TMB) are relevant to the tumor-agnostic checkpoint-inhibitor indication. MMR protein loss by IHC, MSI-high status, or TMB ≥10 mut/Mb can support pembrolizumab use in rare qualifying breast cancers [14].

NTRK gene fusion is screened by pan-TRK IHC and confirmed by NGS or FISH; a fusion gates the TRK-inhibitor class (larotrectinib, entrectinib) and is enriched in secretory carcinoma via ETV6::NTRK3 [15].

ESR1 mutation (acquired) is an emergent resistance biomarker detected in plasma ctDNA. An activating ligand-binding-domain mutation gates the oral SERD elacestrant in ER-positive/HER2-negative advanced disease after prior endocrine therapy, per EMERALD [16].

How results steer treatment

The logic is modular. HR status opens or closes the endocrine and CDK4/6 pathway. HER2 status opens the anti-HER2 pathway, with HER2-low extending antibody-drug-conjugate eligibility. In HR-positive/HER2-negative early disease, multigene assays adjudicate the chemotherapy question. In advanced disease, a sequence of predictive markers — gBRCA (PARP inhibitors), PIK3CA (PI3K inhibitors), ESR1 (elacestrant), and in TNBC PD-L1 (checkpoint inhibitors) — layer additional class-level options as the disease evolves. Tumor-agnostic markers (MSI-H/dMMR, TMB-high, NTRK fusion) provide options that cut across histology in rare cases. In every instance the biomarker establishes eligibility for a class, not a directive.

Specimen and testing realities

ER, PR, and HER2 are exquisitely preanalytics-sensitive: cold ischemia under one hour and fixation of 6–72 hours in 10% neutral buffered formalin are specified precisely because deviations degrade staining and can misclassify a tumor [1,2]. HER2 IHC 2+ automatically reflexes to ISH — a built-in second test rather than a repeat order. Germline BRCA testing uniquely requires a blood or saliva sample, not tumor. A major shift is the rise of liquid biopsy (plasma ctDNA): PIK3CA can be interrogated in tissue or plasma, and ESR1 resistance mutations are best captured longitudinally in ctDNA as they emerge under endocrine pressure [12,16]. Tissue remains the reference standard for most markers, but plasma enables serial monitoring without repeat biopsy.

What's emerging

Several areas are moving quickly and should be read as provisional. The HER2-low (and now "HER2-ultralow") construct is reshaping how the lower end of the HER2 spectrum is interpreted, ahead of formal guideline endorsement of the category [3,4]. ctDNA-based detection of acquired resistance — ESR1 being the prototype — is expanding the concept of testing at progression rather than only at diagnosis [16]. Assay standardization for Ki-67 and PD-L1 remains unresolved and limits cross-study comparability [5,13]. Readers should treat cutoffs, categories, and companion-diagnostic pairings as subject to revision and verify current assay versions and primary readouts at the time of use.

References

1. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and Progesterone Receptor Testing in Breast Cancer: ASCO/CAP Guideline Update. J Clin Oncol / Arch Pathol Lab Med. 2020. doi:10.1200/JCO.19.02309.
2. Wolff AC, Hammond MEH, Allison KH, et al. HER2 Testing in Breast Cancer: ASCO/CAP Clinical Practice Guideline Focused Update. J Clin Oncol. 2018. doi:10.1200/JCO.2018.77.8738.
3. Wolff AC, Somerfield MR, Dowsett M, et al. HER2 Testing in Breast Cancer: ASCO–CAP Guideline Update. J Clin Oncol. 2023. doi:10.1200/JCO.22.02864.
4. Modi S, Jacot W, Yamashita T, et al. Trastuzumab Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer. N Engl J Med. 2022. doi:10.1056/NEJMoa2203690.
5. Nielsen TO, Leung SCY, Rimm DL, et al. Assessment of Ki67 in Breast Cancer: Updated Recommendations From the International Ki67 in Breast Cancer Working Group. J Natl Cancer Inst. 2021. doi:10.1093/jnci/djaa201.
6. Sparano JA, Gray RJ, Makower DF, et al. Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer (TAILORx). N Engl J Med. 2018. doi:10.1056/NEJMoa1804710.
7. Kalinsky K, Barlow WE, Gralow JR, et al. 21-Gene Assay to Inform Chemotherapy Benefit in Node-Positive Breast Cancer (RxPONDER). N Engl J Med. 2021. doi:10.1056/NEJMoa2108873.
8. Cardoso F, van't Veer LJ, Bogaerts J, et al. 70-Gene Signature as an Aid to Treatment Decisions in Early-Stage Breast Cancer (MINDACT). N Engl J Med. 2016. doi:10.1056/NEJMoa1602253.
9. Tutt ANJ, Garber JE, Kaufman B, et al. Adjuvant Olaparib for Patients With BRCA1- or BRCA2-Mutated Breast Cancer (OlympiA). N Engl J Med. 2021. doi:10.1056/NEJMoa2105215.
10. Robson M, Im SA, Senkus E, et al. Olaparib for Metastatic Breast Cancer in Patients With a Germline BRCA Mutation (OlympiAD). N Engl J Med. 2017. doi:10.1056/NEJMoa1706450.
11. Litton JK, Rugo HS, Ettl J, et al. Talazoparib in Patients With Advanced Breast Cancer and a Germline BRCA Mutation (EMBRACA). N Engl J Med. 2018. doi:10.1056/NEJMoa1802905.
12. André F, Ciruelos E, Rubovszky G, et al. Alpelisib for PIK3CA-Mutated, Hormone Receptor–Positive Advanced Breast Cancer (SOLAR-1). N Engl J Med. 2019. doi:10.1056/NEJMoa1813904.
13. Cortes J, Cescon DW, Rugo HS, et al. Pembrolizumab plus Chemotherapy in Advanced Triple-Negative Breast Cancer (KEYNOTE-355). N Engl J Med / Lancet. 2020–2022.
14. Marabelle A, et al. Pembrolizumab in MSI-H / TMB-high Tumors (tumor-agnostic) (KEYNOTE-158). Lancet Oncol / J Clin Oncol. 2020.
15. Drilon A, et al.; Doebele RC, et al. Larotrectinib / Entrectinib in TRK Fusion–Positive Cancers. N Engl J Med / Lancet Oncol. 2018–2020.
16. Bidard FC, Kaklamani VG, Neven P, et al. Elacestrant versus Standard Endocrine Therapy in ER+/HER2- Advanced Breast Cancer (EMERALD). J Clin Oncol. 2022. doi:10.1200/JCO.22.00338.

Reference details (author lists, primary readout citations, and current CAP protocol version) should be verified against the source at the time of use.