HER2-low and HER2-ultralow breast cancer in the antibody-drug conjugate era: diagnostic challenges and reporting workflow

Abstract

Background

Human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugates (ADCs) have expanded the clinical relevance of HER2 expression beyond the traditional HER2-positive versus HER2-negative framework. HER2-low breast cancer, commonly defined as immunohistochemistry (IHC) 1 + or IHC 2 + with negative in situ hybridization (ISH), is now treatment-relevant in selected advanced-disease settings. HER2-ultralow disease, characterized as IHC 0 with membrane staining below the IHC 1 + threshold, further challenges routine pathological interpretation.

Main body

This pathology-centered review summarizes the evolution of HER2 classification from binary status to a clinically relevant expression continuum and clarifies the definitions of true HER2-zero, HER2-ultralow, HER2-low, and HER2-positive disease. Particular emphasis is placed on the zero/ultralow/1 + boundaries, reflex ISH or fluorescence in situ hybridization (FISH) testing for IHC 2 + cases, assay and fixation variables, nonspecific staining, intratumoral heterogeneity, specimen discordance, and interobserver variability. DESTINY-Breast06 and subsequent regional approvals have made HER2-ultralow actionable in defined hormone receptor-positive advanced-disease settings. A practical workflow is proposed for assessment, quality control, consensus review, and reporting.

Conclusions

HER2-low and HER2-ultralow should be interpreted as clinically relevant extensions of established HER2 testing rather than as simple new labels. Responsible implementation requires standardized IHC scoring, careful morphological correlation, validated assays, transparent reporting language, and multidisciplinary communication. A reproducible pathology-centered workflow is essential for translating ADC-era advances into precise and equitable breast cancer care.

Background

Breast cancer remains a major global health burden, and biomarker-based classification is central to modern treatment selection [1–3]. HER2 biology and early trastuzumab trials established HER2 as a clinically actionable target [4, 5]. The original clinical function of HER2 assessment was to identify tumors with HER2 protein overexpression or ERBB2 gene amplification that were eligible for conventional HER2-targeted treatment, a framework standardized through ASCO/CAP guidance [6–9] and regional testing recommendations [10]. In this framework, tumors with IHC 3 + staining or IHC 2 + staining with positive in situ hybridization were classified as HER2-positive, whereas IHC 0, IHC 1+, and IHC 2+/ISH-negative tumors were grouped as HER2-negative. This binary model was clinically effective in the trastuzumab and subsequent anti-HER2 therapy era [5, 11, 12].

Accordingly, this review keeps the conventional IHC score and ISH result at the center of interpretation while using HER2-low and HER2-ultralow as clinically useful descriptors. This distinction is essential: the pathology report should provide granular and reproducible biomarker information, whereas treatment eligibility depends on disease setting, prior therapy, regulatory indication and multidisciplinary judgment.

The therapeutic landscape has changed with HER2-directed ADCs, particularly trastuzumab deruxtecan (T-DXd). ADCs combine target recognition with cytotoxic payload delivery and may exert bystander activity in heterogeneous tumors [13, 14]. Trials in HER2-expressing breast cancer established the activity of T-DXd [15–19], and subsequent regulatory approvals made HER2-ultralow actionable in defined hormone receptor-positive advanced-disease settings [20–22].

This shift exposes a diagnostic problem: conventional HER2 IHC was optimized to identify overexpression or amplification, not to reproducibly separate true zero, ultralow, and 1 + staining. Although established guidelines remain the basis of scoring [6–10], low-level interpretation is sensitive to assay performance and observer thresholds [23–30]. The pathology report should therefore preserve the conventional IHC score and ISH result while communicating clinically relevant low-expression patterns without implying treatment eligibility.

This review focuses on diagnostic implementation. We searched PubMed/MEDLINE, Web of Science, Embase, society guidelines, regulatory documents, and relevant pathology and oncology literature using terms related to HER2-low, HER2-ultralow, HER2 scoring, interobserver variability, ADCs, T-DXd, and digital pathology. Priority was given to guidelines, pivotal trials, regulatory sources, pathology-focused studies, and reproducibility data.

The review follows a diagnostic sequence from classification and operational definitions to sources of diagnostic error, clinical implications, reporting workflow, and future standardization. Trial eligibility categories are not automatically equivalent to routine diagnostic categories: trials often use central testing and protocol-specific thresholds, whereas routine practice involves varied specimens, platforms, and historical reports. The practical question is whether low-expression labels can be defined and reported reproducibly without false precision.

Evolution of HER2 classification in breast cancer

From binary status to low-expression categories

The traditional HER2 classification system was shaped by the success of HER2-targeted therapy in amplified and overexpressing tumors. Trastuzumab established HER2 as a predictive therapeutic target in metastatic and early breast cancer [5, 11, 12], and later HER2-directed regimens further consolidated the HER2-positive treatment paradigm [31–33]. In parallel, HER2 testing guidelines standardized the interpretation of IHC and ISH results [6–9], with Chinese guidance supporting application in local diagnostic practice [10]. In that historical setting, the clinically important task was to identify HER2 positivity, not to define subtle differences among negative or weakly stained cases.

Low-expression categories were previously underemphasized because treatment selection prioritized specificity for HER2-positive disease. The ADC era adds a second requirement: sensitivity to weak expression must improve without encouraging overcalling.

HER2 IHC has therefore not become a new test; the clinical question asked of an established test has changed. The historical HER2-negative group contains tumors ranging from no membrane staining to IHC 2+/ISH-negative disease, and ADC activity at lower expression levels made this heterogeneity clinically relevant [13, 14, 18, 19].

HER2-low as an ADC-era category

HER2-low breast cancer is generally defined as IHC 1 + or IHC 2+/ISH-negative disease [18, 34, 35]. It is not HER2-positive disease and does not imply ERBB2 amplification. Instead, it describes a subset of tumors historically classified as HER2-negative that retain low but detectable HER2 protein expression. DESTINY-Breast04 established the treatment relevance of this category in the metastatic setting, making the low end of the HER2 IHC spectrum clinically consequential [18].

The emergence of HER2-low changed the relationship between pathology and oncology. A score that previously carried little consequence beyond being HER2-negative could now guide consideration of HER2-directed ADC therapy in appropriate advanced disease settings. This does not mean that HER2-low is a fully established intrinsic biological subtype. Molecular and neoadjuvant analyses suggest associations with hormone receptor status and luminal biology [36, 37], whereas early breast cancer cohorts have reported variable clinicopathological or prognostic findings [38–41]. Additional studies emphasize molecular, endocrine, or treatment-response heterogeneity [42–44]. HER2-low is therefore best understood as a treatment-relevant expression category whose biological independence remains under investigation.

HER2-ultralow as an actionable category in defined advanced-disease settings

HER2-ultralow refers to IHC 0 tumors with detectable but extremely faint incomplete membrane staining below the IHC 1 + threshold. It distinguishes IHC 0 with membrane staining from true HER2-zero while remaining within the conventional IHC 0 score [19, 20].

DESTINY-Breast06 showed longer progression-free survival with T-DXd than with physician’s-choice chemotherapy in hormone receptor-positive HER2-low or HER2-ultralow metastatic breast cancer after endocrine-based therapy; the primary analysis was conducted in HER2-low disease, with consistent results in the exploratory HER2-ultralow cohort [19]. Approvals in the United States, European Union, and Japan subsequently made HER2-ultralow treatment-relevant in defined hormone receptor-positive advanced breast cancer settings [20–22]. This clinical actionability does not remove the diagnostic challenge: pathologists must still distinguish true zero from minimal membrane staining near the detection limit and document the conventional IHC score.

HER2 expression as a practical continuum

HER2 expression can be considered a practical continuum from true HER2-zero to HER2-positive disease, but these categories do not represent equivalent biological subtypes or a single therapeutic gradient. Their meaning depends on the clinical setting and applicable indication [13, 14, 18–22].

For diagnostic pathology, the practical response is not to abandon established categories but to report them more precisely. The IHC score and ISH result remain the diagnostic foundation, while low-expression descriptors add clinical context. Figure 1 summarizes the shift from binary treatment selection to contextual interpretation of a low-expression continuum.

Fig. 1.

Evolution from the traditional binary HER2 model to an ADC-era HER2 expression continuum. The figure summarizes the transition from a conventional HER2-positive/HER2-negative framework to a continuum-based interpretation in the antibody-drug conjugate era. The traditional model grouped IHC 0, IHC 1+, and IHC 2+/ISH-negative tumors as HER2-negative, whereas current interpretation increasingly distinguishes true HER2-zero, HER2-ultralow, HER2-low IHC 1+, HER2-low IHC 2+/ISH-negative, and HER2-positive disease. This refined classification supports ADC-era clinical decision-making while preserving conventional IHC scoring and ISH/FISH testing as the diagnostic foundation

Pathological definitions of HER2-low and HER2-ultralow breast cancer

Conventional HER2 IHC scoring

HER2-low and HER2-ultralow categories are derived from the conventional HER2 IHC scoring system; they are not independent testing methods. Routine HER2 scoring is based on the intensity, completeness, and percentage of membranous staining in invasive tumor cells, as defined by ASCO/CAP guidelines [6–9] and regional testing recommendations [10]. IHC 0 indicates no membranous staining or incomplete faint/barely perceptible staining in no more than 10% of invasive tumor cells. IHC 1 + indicates faint or barely perceptible incomplete membrane staining in more than 10% of invasive tumor cells. IHC 2 + indicates weak to moderate complete membrane staining in more than 10% of invasive tumor cells and requires reflex ISH testing. IHC 3 + indicates strong complete circumferential membrane staining in more than 10% of invasive tumor cells and is interpreted as HER2-positive disease.

Because this scoring system was originally designed to identify HER2-positive tumors, its performance at the lower end of the expression spectrum requires careful attention. Small technical or interpretive differences may shift a case across adjacent low-expression categories. The following sections define these categories before their operational distinctions are summarized in Table 1.

Table 1.

HER2 expression categories in breast cancer

HER2 category	Operational definition	ADC-era interpretation	Practical note
True HER2-zero	IHC 0 with no discernible membranous staining in invasive tumor cells	Not considered HER2-low by routine IHC	Confirm technical adequacy; do not score benign/in situ or artifact staining
HER2-ultralow	IHC 0 with very faint incomplete membrane staining below the IHC 1 + threshold	Treatment-relevant in defined hormone receptor-positive advanced-disease settings; diagnostic reproducibility remains limited	Preserve IHC 0; add an ultralow descriptor only when clinically and regionally applicable
HER2-low (IHC 1+)	Faint/incomplete membrane staining in > 10% invasive tumor cells	ADC-relevant category in selected advanced settings	Distinguish from ultralow and from IHC 2+
HER2-low (IHC 2+/ISH-negative)	Weak to moderate complete staining in > 10% invasive tumor cells; ISH/FISH-negative	ADC-relevant category with confirmed non-amplification	Reflex ISH/FISH is required for all IHC 2 + cases
HER2-positive	IHC 3 + or IHC 2 + with HER2 amplification	Established HER2-driven, anti-HER2-targetable disease	Report separately from HER2-low categories

Abbreviations: ADC antibody-drug conjugate, FISH fluorescence in situ hybridization, HER2 human epidermal growth factor receptor 2, IHC immunohistochemistry, ISH in situ hybridization

Two aspects of the scoring system are particularly relevant for low-expression interpretation. The first is the recognition of incomplete membrane staining, which requires careful separation of true membrane signal from cytoplasmic or background staining. The second is the estimation of tumor-cell percentage, which becomes difficult when weak staining is focal or heterogeneous. Both issues should be explicitly considered when historical IHC 0 cases are rescored or when borderline cases are reviewed.

HER2-low

HER2-low is defined as IHC 1 + or IHC 2+/ISH-negative invasive breast cancer [18, 34, 35]. IHC 1 + tumors show faint incomplete membrane staining in more than 10% of invasive tumor cells. IHC 2 + tumors require ISH or FISH testing; only those without ERBB2 amplification are included in the HER2-low category. IHC 2+/ISH-positive tumors are HER2-positive and should not be grouped with HER2-low disease [6, 8–10].

This distinction is important for both clinical care and reporting. A report should preserve the exact IHC score and ISH result rather than simply stating HER2-negative. In multidisciplinary discussion, the term HER2-low may be useful when the clinical context involves ADC therapy or trial eligibility, but it should not replace standardized HER2 reporting [9, 45].

In routine reports, exact wording matters. A concise statement such as “HER2 IHC 1+; HER2-low in relevant clinical contexts” is more informative than “HER2-negative” alone. For IHC 2 + cases, the ISH/FISH result should be directly linked to the final interpretation, because an equivocal IHC result without amplification has different implications from amplified HER2-positive disease.

HER2-ultralow

HER2-ultralow is a treatment-relevant descriptor within the conventional IHC 0 range. It denotes IHC 0 with detectable membrane staining that remains below the IHC 1 + threshold, whereas true HER2-zero indicates no discernible membrane staining [19, 20].

Recognition remains technically demanding because the signal is close to the detection limit and may be confounded by background or cytoplasmic staining, edge artifact, benign epithelium, ductal carcinoma in situ, fixation quality, or archival tissue [23–26]. Borderline cases should be assessed on invasive carcinoma with appropriate controls and, when clinically consequential, second review.

Reporting should preserve the conventional score and add the descriptor only when clinically relevant under institutional and regional practice. A suitable formulation is: “HER2 IHC 0 with membrane staining (HER2-ultralow in applicable clinical and regulatory contexts).” This wording communicates the staining pattern without converting the pathology report into a treatment recommendation.

Treatment eligibility remains a multidisciplinary decision based on hormone receptor status, disease stage, prior therapy, and the applicable regulatory indication [20–22].

Distinguishing adjacent categories

A pathology-centered framework should distinguish true HER2-zero, HER2-ultralow, HER2-low IHC 1+, HER2-low IHC 2+/ISH-negative, and HER2-positive disease. These adjacent categories differ in diagnostic reproducibility and clinical evidence; they should be treated as expression categories rather than assumed biological subtypes [34, 35].

The most difficult boundaries are true zero versus ultralow, ultralow versus IHC 1+, and IHC 1 + versus IHC 2+. Table 1 consolidates the operational definitions and reporting implications; detailed sources of variability are addressed in Sect.  4.

Diagnostic challenges in low-level HER2 expression

Low-level HER2 interpretation extends routine IHC into the range where pre-analytical, analytical, biological, and observer variation have the greatest effect [6–10, 23–26]. The main challenge is therefore reproducible recognition and reporting of weak membrane staining, not awareness of the terminology.

The true zero/ultralow boundary

The most challenging boundary is true HER2-zero versus HER2-ultralow. The distinction rests on the presence of minimal membrane staining near the visual detection limit, and historical IHC 0 reports were not generated with this separation in mind [19, 20, 46].

Under-recognition may occur when weak membrane staining is dismissed, whereas over-recognition may result from cytoplasmic staining, background, edge artifact, benign epithelium, or ductal carcinoma in situ [23–26]. Both errors can alter treatment screening or real-world classification.

The ultralow/IHC 1 + boundary

The ultralow/IHC 1 + boundary depends on both staining intensity and the proportion of invasive tumor cells. IHC 1 + requires faint incomplete membrane staining in more than 10% of invasive tumor cells [6–10], whereas HER2-ultralow remains within IHC 0 under trial and regulatory definitions [19, 20]. Estimating weak patchy staining around this threshold is inherently subjective.

Retrospective studies should therefore report specimen type, assay, scoring criteria, number of reviewers, consensus procedures, and agreement statistics; otherwise, apparent biological differences may reflect classification variability.

The IHC 1+/2 + boundary and reflex testing

The IHC 1+/2 + boundary remains important because IHC 2 + cases require reflex ISH or FISH testing. IHC 1 + represents faint incomplete membrane staining in more than 10% of invasive tumor cells, while IHC 2 + represents weak to moderate complete membrane staining in more than 10% of invasive tumor cells [6–9]. Regional guidance similarly emphasizes standardized interpretation and reflex testing [10]. Misclassifying a true 2 + case as 1 + may omit necessary ISH/FISH testing, while overcalling 1 + as 2 + may lead to unnecessary testing. Because IHC 2+/ISH-positive tumors are HER2-positive and IHC 2+/ISH-negative tumors are HER2-low, this boundary determines both diagnostic classification and downstream clinical interpretation.

Pre-analytical and analytical variables

Low-level HER2 interpretation is particularly sensitive to tissue handling and assay performance. Cold ischemia time, fixation duration, section thickness, antigen retrieval, antibody clone, staining platform, detection chemistry, and tissue age can all alter weak membranous staining [6, 8–10]. Studies of low ERBB2 protein expression and laboratory reproducibility further show why low-expression interpretation is vulnerable to method and observer effects [23–25, 47]. A staining protocol that reliably identifies IHC 3 + tumors may still be insufficiently reproducible for distinguishing true zero, ultralow, and 1 + cases. This is why low-expression interpretation should be embedded within validated laboratory workflows and external quality assurance rather than treated as a purely visual exercise.

Analytical variability is also relevant when institutions compare old and new results. A patient may appear to convert from HER2-zero to HER2-low because of biological change, but the same pattern may also result from a different antibody clone, platform sensitivity, fixation interval, or scoring threshold. Reports and research studies should avoid interpreting all discordance as biological unless technical factors have been considered.

Archival tissue deserves special caution. Older formalin-fixed paraffin-embedded blocks may show antigen degradation, and previously stained slides may fade. When historical IHC 0 cases are reassessed for HER2-ultralow expression, freshly cut sections from representative blocks are preferable. If staining quality is compromised by fixation artifact, tissue exhaustion, or excessive background, repeat staining may be more appropriate than forcing a low-expression label.

Morphological correlation and false staining

HER2 scoring must be performed on invasive carcinoma. Ductal carcinoma in situ, benign breast epithelium, stromal elements, inflammatory cells, necrotic tissue, crushed tissue, and edge-artifact regions should not determine the HER2 score of invasive disease. This is particularly important for HER2-ultralow, because the expected membrane signal is extremely faint and can be confused with non-specific staining.

Cytoplasmic staining is a common pitfall. HER2 scoring is based on membranous staining, not isolated cytoplasmic or granular background signal. Pathologists should confirm that staining outlines the tumor cell membrane, even if incomplete, before assigning ultralow or low-level expression. Corresponding hematoxylin and eosin slides should be reviewed to confirm invasive tumor areas and avoid scoring benign or in situ components.

Heterogeneity and specimen discordance

HER2 expression may be heterogeneous and may differ across biopsy, resection, residual disease, recurrence, and metastasis. Intratumoral heterogeneity, including coexistence of tumor-cell populations with different HER2 protein expression or amplification, can complicate biomarker interpretation and treatment selection [48]. Low-expression studies and receptor-conversion cohorts document discordance across specimens and disease sites [37, 49–55]. Because small foci of weak staining can alter classification, limited biopsies may not represent the full tumor.

When recurrent or metastatic tissue is available and the result could influence treatment or trial eligibility, reassessment should be considered. Discordance should not automatically be interpreted as biological conversion, because platform, fixation, sampling, and scoring differences may contribute [51–58].

Interobserver variability

Interobserver variability is greatest at the IHC 0/ultralow/1 + and 1+/2 + boundaries [23–26]. Differences arise from recognition of faint membrane staining, percentage estimation, heterogeneity, artifacts, and laboratory performance.

Second-review triggers should be predefined for clinically consequential ultralow cases, 0/1 + borderline cases, and heterogeneous staining. Research cohorts should use independent double reading, consensus adjudication, and agreement statistics; interpretive consensus cannot compensate for technically inadequate slides.

Implications for clinical trials and real-world evidence

Diagnostic variability can bias clinical trials and retrospective cohorts. Historical IHC 0 reports often lack a description of faint membrane staining, so reclassification requires available tissue, standardized staining, predefined rescoring, and documented tissue quality [46].

The problem is particularly relevant to hormone receptor-positive early breast cancer, where prognostic associations remain inconsistent and many studies did not separate true zero from ultralow [36–44]. Table 2 links the major diagnostic risks to practical mitigation strategies.

Table 2.

Diagnostic pitfalls and pathology strategies

Diagnostic issue	Why it matters	Practical strategy
True HER2-zero vs. HER2-ultralow	May affect treatment consideration, trial screening, or retrospective reclassification	Score invasive tumor only; exclude background/artifact; use second review for borderline cases
HER2-ultralow vs. IHC 1+	Threshold-sensitive and less reproducible	Apply strict IHC 1 + criteria; estimate tumor-cell percentage; consensus review if borderline
IHC 1 + vs. IHC 2+	Determines need for reflex ISH/FISH	Assess membrane completeness and intensity; perform ISH/FISH for all IHC 2 + cases
Pre-analytical/analytical variation	Can produce false-negative or falsely weak staining	Standardize fixation, platform, controls, and repeat testing when quality is inadequate
Tumor heterogeneity	May cause sampling bias across blocks or specimens	Review representative tissue; document heterogeneity and specimen limitations
Primary-metastatic discordance	Primary tumor may not reflect current disease	Retest recurrent/metastatic tissue when clinically meaningful

Abbreviations: FISH fluorescence in situ hybridization, HER2 human epidermal growth factor receptor 2, IHC immunohistochemistry, ISH in situ hybridization

Clinical implications of HER2-low and HER2-ultralow breast cancer

Low-level HER2 expression is no longer merely a negative result. DESTINY-Breast04 established HER2-low as treatment-relevant after prior chemotherapy, whereas DESTINY-Breast06 and subsequent regional approvals extended actionability to HER2-ultralow in defined hormone receptor-positive advanced-disease settings [18–22, 59]. The pathology consequence is the need for precise, qualified reporting rather than therapeutic decision-making by the pathologist.

HER2-low as a treatment-relevant category

DESTINY-Breast04 established HER2-low as a treatment-relevant category in unresectable or metastatic breast cancer [18]. This changed the meaning of IHC 1 + and IHC 2+/ISH-negative results. A score that once simply belonged to the HER2-negative group may now influence therapeutic options in the appropriate setting. This is why pathology reports should retain the exact IHC score and ISH result.

HER2-low should nevertheless not be equated with HER2-positive disease. HER2-low tumors are not defined by overexpression or amplification and should not be interpreted as HER2-driven cancers in the classical sense [6, 9, 10]. Their treatment relevance derives from ADC drug delivery and, potentially, bystander killing rather than from classical HER2 oncogene addiction [13, 14, 18]. This distinction is important in reports and multidisciplinary discussion because patients and clinicians may misinterpret HER2-low as a conventional HER2-positive result.

HER2-ultralow as a treatment-relevant category

DESTINY-Breast06 enrolled patients with hormone receptor-positive HER2-low or HER2-ultralow metastatic breast cancer after endocrine-based therapy and no prior chemotherapy for metastatic disease. T-DXd improved progression-free survival versus physician’s-choice chemotherapy; the primary endpoint was assessed in HER2-low disease, and results were consistent in the exploratory HER2-ultralow cohort [19].

Regulatory approvals in the United States, European Union, and Japan subsequently recognized HER2-ultralow in defined hormone receptor-positive advanced breast cancer indications [20–22]. Accordingly, HER2-ultralow is actionable in these settings, but it is not interchangeable with HER2-low or HER2-positive disease, and its diagnostic reproducibility remains less established. Reports should retain IHC 0 and specify membrane staining when the ultralow descriptor is applied.

HR-positive breast cancer and early disease

Hormone receptor-positive disease is central to the current ultralow indication and dominates many HER2-low cohorts [18, 19, 36–41, 59]. In early breast cancer, however, low-expression categories remain primarily research variables: studies report inconsistent clinicopathological and outcome associations and often do not separate true zero from ultralow [38–44].

The unresolved question is whether HER2 fine stratification adds prognostic information beyond established luminal factors. This requires standardized rescoring and adjustment for hormone receptor expression, Ki-67, grade, nodal status, systemic therapy, and follow-up [60–68].

Predictive versus prognostic value

HER2-low and HER2-ultralow have predictive relevance for T-DXd in defined advanced-disease settings [18–22, 59], but this does not establish independent prognostic value in early breast cancer. Historical IHC 0 groups may contain both true zero and ultralow tumors, further confounding retrospective comparisons.

Early-disease studies should therefore analyze low-expression categories alongside established clinicopathological variables and avoid using HER2-low as a stand-alone risk marker until supported by robust evidence [38–44, 60–68].

Trial eligibility, reassessment, and communication

Eligibility increasingly depends on the exact IHC score and ISH result. Reassessment of recurrent or metastatic tissue is reasonable when clinically meaningful and technically feasible, because receptor discordance is well documented [51–58]. Retesting should be targeted rather than indiscriminate.

Patients and clinicians may misinterpret HER2-low as HER2-positive or assume that any detectable staining guarantees access to T-DXd. Reports should therefore preserve the conventional score, use explanatory comments when needed, and defer treatment eligibility to the multidisciplinary team. Table 3 summarizes the evidence that underpins these reporting implications.

Table 3.

Key evidence for HER2 low expression

Evidence/source	Study context	Key finding	Pathology relevance
DESTINY-Breast04 [18]	Unresectable/metastatic HER2-low breast cancer	T-DXd improved PFS and OS versus chemotherapy	Established HER2-low as a treatment-relevant category
DESTINY-Breast06 and regional approvals [19–22]	HR-positive HER2-low or HER2-ultralow advanced breast cancer; DESTINY-Breast06 enrolled metastatic disease after endocrine-based therapy	T-DXd prolonged PFS; results were consistent in the exploratory HER2-ultralow cohort	Established HER2-ultralow as actionable in defined regional indications; preserve IHC 0 with membrane staining
DAISY trial [14]	Metastatic breast cancer with variable HER2 expression	Showed activity across a HER2 expression spectrum	Supports continuum-based interpretation and quantitative interest
Miglietta primary-recurrent study [49]	Matched primary and recurrent breast cancer samples	HER2-low expression may be unstable over time	Supports reassessment in recurrence or metastasis
Miglietta residual-disease study [50]	Matched baseline and residual disease after neoadjuvant therapy	HER2-low status may change after treatment	Supports careful interpretation of residual disease cohorts

Abbreviations: HER2 human epidermal growth factor receptor 2, HR hormone receptor, IHC immunohistochemistry, OS overall survival, PFS progression-free survival, T-DXd trastuzumab deruxtecan

Reporting workflow for HER2-low and HER2-ultralow assessment

The reporting workflow operationalizes established HER2 guidance for low-expression assessment by linking specimen adequacy, conventional scoring, reflex ISH, review of borderline cases, and documentation of limitations.

Confirm the correct tissue and tumor component

HER2 scoring should begin with confirmation of specimen type and invasive carcinoma on the corresponding H&E slide. Benign epithelium, ductal carcinoma in situ, stromal cells, inflammatory cells, necrosis, and artifact-prone areas should not determine the HER2 score of invasive disease. The report or study protocol should specify whether assessment was performed on core biopsy, surgical resection, residual disease after neoadjuvant therapy, recurrence, or metastasis. This is particularly important because low-level expression may be heterogeneous across treatment and disease states [49, 50] and may vary between primary and metastatic lesions [51–53]. Additional receptor-conversion studies further support careful interpretation across disease sites [54, 55].

Verify assay quality before low-expression interpretation

Before assigning low-expression categories, the HER2 IHC run should be technically adequate. Internal controls, fixation quality, tissue preservation, section quality, background staining, and tumor cellularity should be assessed. If interpretation may influence treatment or trial screening and the slide is technically compromised, repeat staining on a freshly cut section or another representative block should be considered. Laboratories should avoid assigning HER2-ultralow on slides with poor preservation, excessive background, or edge artifact.

Score conventionally before applying ADC-era terminology

The first interpretive step should always be conventional HER2 scoring: 0, 1+, 2+, or 3+ [6–9]. HER2-low and HER2-ultralow categories should be derived from, not substituted for, the conventional score. Regional recommendations can support implementation in local practice [10]. IHC 3 + is HER2-positive. IHC 2 + requires reflex ISH/FISH. IHC 1 + is included in HER2-low. IHC 0 should be evaluated for the presence or absence of extremely faint incomplete membrane staining only when ultralow classification is clinically or scientifically relevant.

Use reflex ISH/FISH correctly

All IHC 2 + cases require ISH or FISH testing. This is a non-negotiable step because IHC 2 + alone cannot distinguish HER2-positive from HER2-low disease. IHC 2+/ISH-positive tumors are HER2-positive; IHC 2+/ISH-negative tumors are HER2-low according to established testing frameworks [6, 8–10] and ADC-era eligibility criteria [18]. Reports should include both the IHC score and the ISH/FISH result, because both are required for clinical interpretation.

Reassess IHC 0 cases cautiously

For IHC 0 cases, ultralow assessment should focus on invasive tumor membrane staining. Cytoplasmic or background staining, benign ducts, ductal carcinoma in situ, stromal staining, and edge artifact must be excluded. Detectable membrane staining below the IHC 1 + threshold may support an ultralow descriptor in an applicable clinical context; absence of membrane staining remains true HER2-zero [19, 20].

Institutional policy should specify when the descriptor is reported, which assay and specimen requirements apply, and when second review is required.

A practical formulation is: “HER2 IHC 0 with membrane staining (HER2-ultralow in applicable clinical and regulatory contexts).” This preserves the conventional score, communicates the clinically relevant pattern, and avoids implying treatment eligibility.

Review borderline cases

Borderline cases at the true zero/ultralow, ultralow/1+, and 1+/2 + boundaries should be eligible for second review by a breast pathologist. If reviewers disagree, consensus adjudication should be used. In research cohorts, independent dual scoring and kappa statistics should be reported. Borderline review is especially important when historical IHC 0 cases are rescored for ultralow status or when trial eligibility depends on the result [23, 24, 26, 46].

Report clinically useful information

A practical report should include the HER2 IHC score, ISH/FISH result when applicable, final interpretation, specimen type, tumor component assessed, and relevant limitations [6, 9, 45]. For IHC 1 + or IHC 2+/ISH-negative tumors, the report may add HER2-low in relevant clinical contexts. For IHC 0 with membrane staining, the ultralow formulation above may be used when applicable.

The report should identify the tested specimen and document limitations such as low tumor cellularity, post-treatment change, poor fixation, or heterogeneity. These details help determine whether additional review or repeat testing is warranted.

A short internal checklist can reduce variability: confirm invasive tumor and assay adequacy; assign the conventional IHC score; complete ISH/FISH for IHC 2+; assess clinically relevant IHC 0 cases for membrane staining; review borderline cases; document heterogeneity and limitations; and verify final wording.

Reassess recurrent or metastatic disease when clinically relevant

Reassessment of recurrent or metastatic tissue may be useful when the result could alter treatment or trial eligibility [51–55]. It should use validated assays and account for specimen, treatment, and laboratory differences; the goal is clinically rational updating, not automatic retesting.

Workflow summary

Figure 2 summarizes the workflow: confirm invasive carcinoma and assay quality, assign the conventional HER2 score, complete ISH/FISH for IHC 2+, identify clinically relevant IHC 0 with membrane staining, review borderline cases, and report the specimen and limitations. The workflow adds contextual information without replacing established HER2 testing principles.

Fig. 2.

Pathology-centered workflow for HER2 low-expression assessment and reporting in breast cancer. The workflow begins with specimen selection and adequacy assessment, confirmation of invasive carcinoma, and technically adequate HER2 IHC. Conventional HER2 IHC scoring remains the basis for classification. IHC 3 + is interpreted as HER2-positive, IHC 2 + requires reflex ISH/FISH testing, IHC 1 + is categorized as HER2-low, and IHC 0 cases may be reassessed for very faint incomplete membrane staining when ultralow classification is clinically relevant. Borderline cases, including 0 versus ultralow, ultralow versus 1+, 1 + versus 2+, and heterogeneous staining, should undergo second pathologist review or consensus review. Final reports should include IHC score, ISH/FISH result when applicable, HER2 category, specimen type, and clinically relevant interpretive comments

Future directions

The next phase of HER2 low-expression assessment should focus less on terminology expansion and more on reproducibility. HER2-low and HER2-ultralow will be clinically useful only if pathologists can identify and report them consistently across laboratories, specimen types and disease settings.

Future work should therefore be evaluated by measurable diagnostic endpoints, including improved interobserver agreement, fewer discordant borderline classifications, better concordance between local and central scoring, transparent reporting of limitations and clearer integration of HER2 results into treatment discussions. Without such endpoints, additional terminology will not necessarily improve patient care.

Training and external quality assurance

Training and external quality assessment should include true zero, ultralow, 1+, 2+/ISH-negative, and positive cases, with emphasis on borderline staining, artifacts, heterogeneous tumors, and small biopsies [6–10, 23–26].

Digital slide libraries can expose pathologists to identical difficult cases, support consensus discussion, and provide more realistic low-expression training than textbook examples alone.

Digital pathology and artificial intelligence

Digital pathology may support training, remote consultation, image archiving and quantitative analysis. QuPath and other digital pathology platforms provide practical tools for image analysis [69–71], while AI pathology reviews describe both the feasibility and limitations of computational interpretation [72, 73]. Validation studies and deep-learning applications demonstrate technical potential but also highlight the need for careful generalization [74–76]. Separating true zero, ultralow and 1 + staining remains difficult near the limits of IHC detection; AI models will require multicenter validation, consensus-annotated ground truth and platform-independent testing before clinical implementation. At present, AI should be considered an adjunct, not a replacement for pathologist interpretation.

Dynamic HER2 assessment

Future studies should distinguish biological change from technical variation when comparing biopsy, resection, residual disease, recurrence, and metastasis [43, 49–58].

HR-positive early breast cancer

A major unresolved question is whether true zero, ultralow, 1+, and 2+/ISH-negative categories add prognostic information in hormone receptor-positive early breast cancer. Studies should use current rescoring criteria, ISH/FISH confirmation for IHC 2+, dual review, agreement analysis, adequate event numbers, and long-term outcomes [38–44, 65–68].

Predefined endpoints, transparent exclusions, adjustment for established luminal risk factors, and sensitivity analyses are essential to avoid false subgroup signals.

Harmonizing clinical and pathological definitions

Clinical trial terminology should be harmonized with routine pathology reporting. Future guidance should define when low/ultralow descriptors are appropriate, how borderline cases are handled, and how conventional scores and limitations are communicated [64, 77, 78].

Recommendations should distinguish ideal research standards from minimum safe clinical standards for laboratories with limited access to repeat staining, central review, or digital consultation.

Limitations and scope of this review

This review has several limitations that define the scope of its conclusions. First, it is a pathology-centered narrative review based on a targeted literature search rather than a formal systematic review or meta-analysis; accordingly, completeness of evidence capture cannot be guaranteed and no formal risk-of-bias assessment was performed. Second, the evidence and regulatory landscape for HER2-low and particularly HER2-ultralow breast cancer is evolving rapidly, and approved indications, companion-diagnostic requirements, and reporting practices differ across regions and may change over time.

Third, the strongest therapeutic evidence for HER2-ultralow disease currently derives from hormone receptor-positive advanced breast cancer, whereas its prognostic significance and clinical utility in early-stage disease remain less well established. Fourth, low-level HER2 assessment still depends on semi-quantitative immunohistochemical interpretation and is affected by interobserver variability, assay platform, tissue quality, and intratumoral heterogeneity, which may limit transferability of reporting approaches across laboratories. Finally, the workflow proposed here is an evidence-informed pathology framework rather than a prospectively validated diagnostic algorithm. These limitations reinforce the need for guideline harmonization, interlaboratory validation, and prospective studies, while also underscoring the practical value of transparent and standardized low-expression HER2 reporting.

Conclusions

The ADC era has changed the clinical meaning of low-level HER2 expression. HER2-low is treatment-relevant in selected advanced-disease settings, and DESTINY-Breast06 together with subsequent regional approvals has made HER2-ultralow clinically actionable in defined hormone receptor-positive advanced breast cancer settings [18–22, 59].

For diagnostic pathology, the central challenge remains reproducibility at the true zero/ultralow/1 + boundaries. Responsible implementation requires conventional IHC scoring, reflex ISH/FISH when indicated, morphological correlation, assay quality control, and consensus review of clinically consequential borderline cases [6, 9, 23–26, 45].

HER2-low and HER2-ultralow are expression categories rather than established intrinsic biological subtypes. Their predictive role in defined advanced-disease indications should not be conflated with prognostic significance in early breast cancer, which remains uncertain [38–44].

The goal is not to multiply labels, but to report reproducible, clinically contextual information. Preserving the conventional HER2 result while adding an appropriate low-expression descriptor provides a practical bridge between pathology and precision oncology.

Authors' contributions

LK and JZ contributed equally to this work. LK conceived the review, designed the manuscript framework, performed the literature search, drafted the manuscript, and prepared the tables and figures. JZ performed literature screening, summarized the pathological and clinical evidence, drafted and revised the manuscript, and contributed to table and figure preparation. LH contributed to literature collection, reference verification, and revision of the clinical evidence sections. YZ contributed to interpretation of pathology-related content and revision of the diagnostic workflow and reporting sections. HH contributed to manuscript review, reference checking, and editing of tables and figures. LX conceptualized and supervised the work, provided critical intellectual revision, secured funding, and approved the final manuscript. All authors read and approved the final manuscript.

Funding

Supported by Sanming Project of Medicine in Shenzhen (No. SZSM202411003).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.