The biomarker triage, and what is left when it finds nothing
Three positive markers now organise a category defined by absence, and most of the work of this chapter is with the patients in whom none of the three is found.
1 · Biomarker-driven treatment selection
This chapter is about how to approach the patient. Risk, disease burden, symptom load, biomarker triage and what the line of treatment is for. The trial record itself, arranged by line, is in Metastatic therapy from first line onwards. Where a decision here rests on a result, the evidence for it is tabulated there and is pointed to rather than restated.
The division matters because the two questions come apart in clinic. Which regimen has the better hazard ratio is answered by Metastatic therapy from first line onwards. Whether this patient should start a regimen at all, and what to measure to find out if it worked, is answered here.
Triple-negative is defined by what is not there. Three receptor assays report below threshold and the tumour is assigned to a group on that basis. The category is therefore an artefact of an assay panel, and the biological diversity inside it is real1. The classification argument is developed in Triple-negative and other biologically diverse phenotypes.
What has changed is that management no longer runs on the absence. Three positive markers now determine the first decision, and they are checked in a fixed order.
PD-L1 determines whether first-line treatment includes a checkpoint inhibitor First-line chemoimmunotherapy and the PD-L1 threshold. It is an assay output with an antibody clone, a scoring algorithm and a cut-point attached, not a property of the tumour.
Germline BRCA1 and BRCA2 status, extended to PALB2 and to somatic BRCA alterations, determines eligibility for PARP inhibition PARP inhibition for germline carriers. It also changes family counselling, which is a second reason to test early.
HER2 expression level determines whether trastuzumab deruxtecan is available, because a proportion of tumours called triple-negative score immunohistochemistry 1+ or 2+ without amplification. The eligibility argument is in Expression and alteration-defined treatment across subtypes.
Trop-2 expression is measured in trials and is not a selection criterion. In the biomarker analysis of ASCENT, sacituzumab govitecan outperformed chemotherapy across high, medium and low Trop-2 expression, and the low-expression group was too small for a firm conclusion2. Tumour mutational burden is high in only a small minority of breast cancers and is not a routine selection tool here3.
In practice
Order all three at the first metastatic presentation, not sequentially as options run out.
PD-L1 on the most recent available tissue, with the assay and cut-point named in the report.
Germline BRCA1 and BRCA2 if never tested, with PALB2 included.
HER2 immunohistochemistry on recent tissue, read for the 0 against 1+ boundary specifically.
The order matters because the first-line decision depends on PD-L1 and cannot wait for the others. The others determine the second and third decisions and there is time to get them right.
2 · Androgen receptor and other subtype-directed targets
Advanced disease · TNBC · any
Finding something to aim at in a disease defined by absence
Is the androgen receptor a target in a subtype defined by the absence of two other receptors?
Rationale
A minority of triple-negative tumours express the androgen receptor and carry a luminal transcriptional programme. If that receptor drives growth in the way the oestrogen receptor does elsewhere, an antagonist already licensed in another disease would be available immediately.
Experiment
A single-arm study of enzalutamide in advanced triple-negative disease with any nuclear androgen receptor staining. Clinical benefit was reported at a fixed time point, in all enrolled patients and in a subgroup defined by a higher staining threshold.
Finding
A minority of patients had clinical benefit, and the proportion was higher in the subgroup defined by the higher threshold.
Limitation
There is no control arm, so nothing separates drug effect from the natural history of a slower-growing luminal subset. The threshold that defined the better-performing subgroup was applied to the same data that suggested it, which is the general problem Expression and alteration-defined treatment across subtypes sets out.
Next question
Does the androgen receptor mark a targetable dependency, or simply identify the patients in this category whose disease was never going to behave like the rest of it?
MDV3100-11
Transcriptional subtyping divides triple-negative disease into groups with different biology, including a luminal androgen receptor group1. The clinical question is whether any of those groups has a therapy attached. Mostly they do not.
Heavily pretreated women with HER2-mutated metastatic breast cancer that was HER2-negative on local testing, enrolled as one cohort of an open-label basket study of HER2-altered solid tumours; those with hormone receptor-positive disease also received fulvestrant.
Single arm of tucatinib plus trastuzumab, with fulvestrant added for hormone receptor-positive disease.
Overall response rate.
Overall response rate was 41.9% (90% CI 26.9 to 58.2) in the 31 patients. The secondary endpoints of duration of response and progression-free survival were 12.6 months (90% CI 4.7 to not estimable) and 9.5 months (90% CI 5.4 to 13.8) respectively.
Patients with metastatic androgen receptor-positive breast cancer, defined as at least 10% expression, with the phase Ib part open to oestrogen receptor-positive as well as triple-negative disease and the randomised phase II part restricted to triple-negative disease.
Phase Ib gave enzalutamide 160 mg with taselisib to find dose-limiting toxicities and the maximum tolerated dose; phase II randomised patients to enzalutamide alone or enzalutamide plus taselisib 4 mg until disease progression.
Clinical benefit rate at 16 weeks.
The clinical benefit rate among evaluable patients receiving the combination was 35.7% and median progression-free survival was 3.4 months. The abstract gives no clinical benefit rate for the enzalutamide-alone arm, so the record carries no comparison.
Locally advanced or metastatic triple-negative breast cancer with any nuclear androgen receptor staining above 0% on an immunohistochemistry assay optimised for breast cancer.
Single arm of enzalutamide 160 mg once daily until disease progression, with no control arm.
Clinical benefit rate at 16 weeks, in all enrolled patients and in the evaluable subgroup whose tumours expressed androgen receptor in at least 10% of nuclei.
Of 118 patients enrolled, 78 were evaluable. Clinical benefit rate at 16 weeks was 25% (95% CI 17% to 33%) in the intention-to-treat population and 33% (95% CI 23% to 45%) in the evaluable subgroup. Median progression-free survival was 2.9 months (95% CI 1.9 to 3.7) and 3.3 months (95% CI 1.9 to 4.1) respectively.
Patients with oestrogen and progesterone receptor-negative advanced breast cancer whose primary tumour or a metastatic site was androgen receptor-positive on central immunohistochemistry, defined as more than 10% nuclear staining.
Single arm of bicalutamide 150 mg daily.
Clinical benefit rate, defined as complete response, partial response or stable disease beyond six months.
The six-month clinical benefit rate was 19% (95% CI 7% to 39%) and median progression-free survival was 12 weeks (95% CI 11 to 22 weeks). There was no comparator arm.
Three of those four rows tested androgen receptor blockade, and they agree on the shape of the answer. A minority of selected patients avoid progression for a few months. The measure is a clinical benefit rate at a fixed timepoint. No trial carried a chemotherapy control arm. Activity is real and it is not a treatment, and no androgen receptor directed agent is approved in this setting.
What separates the three is the assay rather than the drug. TBCRC 011 required more than 10% nuclear staining on central immunohistochemistry7. MDV3100-11 admitted any nuclear staining above zero, then reported an evaluable subgroup defined at 10% or more6. TBCRC 032 set its threshold at 10% and randomised between enzalutamide and enzalutamide with taselisib5. The clocks differ as well. Clinical benefit was scored at six months in the first trial and at sixteen weeks in the other two. Three studies of the same target with three eligibility definitions and two scoring windows cannot be ranked against each other.
The measurement problem sits underneath all of it. In MDV3100-11 benefit was higher in the receptor-defined subgroup than in the whole enrolled population, which is what an imperfect selection assay produces. TBCRC 032's investigators concluded that androgen receptor protein expression alone does not identify androgen receptor-dependent tumours, and that the luminal androgen receptor subtype and receptor splice variants may5. A threshold that has not been reproduced between laboratories cannot support an indication however the responses distribute around it.
The assay also discards most patients before it selects any. TBCRC 011 screened 424 patients with oestrogen and progesterone receptor-negative disease, and 12% scored androgen receptor-positive7. A single-arm study inside a selected fraction that small has to show a large effect to earn a randomised test. None of these did. The one randomisation in the table, inside TBCRC 032, compared two experimental arms, closed early, and left no clinical benefit rate on the record for the enzalutamide-alone arm.
SGNTUC-019 is in this table for a different reason, and its population column is the part to read. It enrolled heavily pretreated patients selected by an activating HER2 mutation in disease that local testing called HER2-negative, across receptor status rather than within triple-negative disease4. It is one cohort of a single-arm basket study. Its response rate belongs to a genotype and not to this subtype. The alteration-defined options that do cross this subtype are collected in Antigen-defined and alteration-defined options.
The general lesson recurs across this chapter. A transcriptional subtype is a research classification. Converting it into an eligibility criterion requires an assay that ordinary laboratories can run reproducibly, and the gap between those two things is the same gap described for HER2 scoring in HER2-low and ultralow reframed as a heterogeneity problem. The trial design consequences are set out in Clinical trials and evidence interpretation.
This subtype produces the fastest clinical deterioration in breast cancer, and the interval between a decision and its consequences is short.
Two practical features follow. The first is that a line of therapy must be assessed early. Waiting twelve weeks for a scan in a patient deteriorating over three is a decision to spend the remaining time on a failing regimen. The second is that performance status falls faster than in other subtypes, so eligibility for anything, including trials, is lost quickly.
The judgment that is hardest and most important is when not to treat. The evidence for it is indirect and consistent. Response rates on single-agent chemotherapy in the refractory setting are in single figures, as the control arm of ASCENT shows Conjugates from refractory disease to the first line. A patient with declining performance status has a low probability of benefit and a high probability of toxicity, and the toxicity arrives first.
In practice
Set a reassessment interval that matches the tempo of the disease, not the convention of the department. In rapidly progressive disease that is often six weeks.
Decide in advance what would make you stop, and say it out loud before starting. A named stopping rule is easier to act on than a judgment made in the room.
Involve palliative care in parallel with active treatment rather than after it8. The referral trigger that works is disease status, not exhaustion of options Palliative and end-of-life care.
Performance status is the variable that closes doors. Record it at every visit, because the trial that is open today will not be open at the next decline.
4 · The unmet need after conjugate failure
The population that defines this disease has moved. The unmet need used to sit after chemotherapy. It is now after conjugates, and the evidence base behind that position is retrospective.
The relevant data are the sequential conjugate series, and they were not assembled inside this subtype. Among 85 patients with HER2-low metastatic breast cancer treated with both trastuzumab deruxtecan and sacituzumab govitecan, progression-free survival on the second conjugate was shorter than on the first in 75.2%. Fourteen of the 85 were still on treatment at the data cutoff9. Longer progression-free survival on the first conjugate predicted longer progression-free survival on the second, which is the signature of partial rather than complete cross-resistance. Hormone receptor status was not associated with that second interval, which is what allows the series to be read here at all. A second conjugate is a reasonable option with a smaller expected magnitude.
What makes the position difficult is that three of the payloads now in routine use are topoisomerase I inhibitors. Irinotecan works through the same mechanism, and its active metabolite is the payload of sacituzumab govitecan. A patient who has received sacituzumab govitecan, datopotamab deruxtecan and trastuzumab deruxtecan has had three exposures to the same payload class delivered by three different antibodies. That is not three lines of non-cross-resistant therapy, whatever the line count says.
The mechanisms that are not antigen loss remain unmeasurable in clinic. PTEN loss was associated with de novo resistance to trastuzumab deruxtecan and not to sacituzumab govitecan, despite the shared payload class9. That association is retrospective and it points at handling rather than at target. The full taxonomy is in Conjugate resistance, antigen loss, payload efflux, and repair capacity.
The only prospective study addressing this position.
Trial
Population
Experimental vs control
Primary endpoint
TRADE-DXd2 · 357 · ongoing
HER2-low or HER2 immunohistochemistry 0 disease progressing on a previous antibody-drug conjugate, hormone receptor positive and negative cohorts
datopotamab deruxtecan or trastuzumab deruxtecan after previous conjugate exposure, non-comparative cohorts
objective response rate in the first and second conjugate cohorts
TRADE-DXd will give the first prospective estimate of how much activity remains after a conjugate fails. It enters patients on the state of their disease after a previous conjugate, which is the population every retrospective series has had to assemble after the fact. It enrols by HER2 result rather than by subtype, with separate hormone receptor-positive and negative cohorts, so only part of it will speak to this chapter. Its cohorts are non-comparative, so it will not rank sequences. What it will and will not settle is set out in Unresolved questions and what reads out next.
Lehmann BD, Bauer JA, Chen X, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011 121:2750-2767. PMID 21633166
Bardia A, Tolaney SM, Punie K, et al. Biomarker analyses in the phase III ASCENT study of sacituzumab govitecan versus chemotherapy in patients with metastatic triple-negative breast cancer. Ann Oncol 2021 32:1148-1156. PMID 34116144
Barroso-Sousa R, Jain E, Cohen O, et al. Prevalence and mutational determinants of high tumor mutation burden in breast cancer. Ann Oncol 2020 31:387-394. PMID 32067680
Okines AFC, Curigliano G, Mizuno N, et al. Tucatinib and trastuzumab in HER2-mutated metastatic breast cancer: a phase 2 basket trial. Nat Med 2025 31:909-916. PMID 39825152
Lehmann BD, Abramson VG, Sanders ME, et al. TBCRC 032 IB/II Multicenter Study: Molecular Insights to AR Antagonist and PI3K Inhibitor Efficacy in Patients with ARMetastatic Triple-Negative Breast Cancer. Clin Cancer Res 2019 26:2111-2123. PMID 31822498
Traina TA, Miller K, Yardley DA, et al. Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer. J Clin Oncol 2018 36:884-890. PMID 29373071
Gucalp A, Tolaney S, Isakoff SJ, et al. Phase II trial of bicalutamide in patients with androgen receptor-positive, estrogen receptor-negative metastatic breast cancer. Clin Cancer Res 2013 19:5505-5512. PMID 23965901
Temel JS, Greer JA, Muzikansky A, et al. Early palliative care for patients with metastatic non-small-cell lung cancer. N Engl J Med 2010 363:733-742. PMID 20818875
Mai N, Klar Lieberman MM, Ferraro E, et al. Sequential antibody-drug conjugate therapy in patients with metastatic breast cancer treated with sacituzumab govitecan and trastuzumab deruxtecan. JCO Precis Oncol 2025 9:e2400898. PMID 40440568