HISTOPATHOLOGY
HER2 classification HER2-positive
IHC score 3+
2+ HER2-low
HER2-ultralow (emerging)
HER2-negative 1+ or 2+ 0 (with faint
staining <10 %) 0
ISH status Not required Amplified (ISH+)
Non-amplified (ISH−) Not applicable
Not applicable Treatment eligibility
Trastuzumab, pertuzumab, T-DM1
Trastuzumab, pertuzumab, T-DM1
T-DXd Not currently eligible
(may be included in future) Not eligible for HER2-
targeted therapies Table 1. HER2 classification categories with associated IHC/ISH results and treatment options. to ADCs,9 making consistent and more
accurate classification across the entire HER2 expression spectrum of growing importance.
Diagnostic challenges using HER2 classification With the increasing recognition of HER2- low and -ultralow groups, HER2 testing has become more complicated. The diagnostic tools to determine HER2 status – namely IHC and ISH techniques – were not designed to detect slight variances at the lower end of the expression scale. Consequently, these methods struggle to provide the precision necessary to guide treatment; accurate classification is vital for determining HER2 status and directing the use of specific therapies. Misclassifying HER2-low or -ultralow tumours as HER2 0 can deny patients access to ADCs like T-DXd, while incorrectly assigning HER2-low status may lead to unnecessary exposure to costly and potentially toxic therapies. It is therefore vital that diagnostic methods identify subtle differences to ensure patients receive the most appropriate therapy.
Challenges of IHC Immunohistochemistry is still the most widely used method for breast cancer diagnosis and has historically provided vital information for treatment planning. However, its results are semi-quantitative and subjective, giving rise to two
primary sources of variability: technical inconsistency and human interpretation. The results from IHC can vary depending on a range of factors – including fixation quality, staining procedure, antibody clone, detection system and scoring threshold – as well as there being no globally standardised protocol for HER2 IHC. These issues are more evident at the lower end of the expression range, where distinguishing IHC 0 from 1+ or 2+ is especially problematic. This issue has been reported in a number of studies around the world. For example, Zaakouk et al. in the UK and Republic of Ireland looked at classification by 16 expert pathologists. Each pathologist scored 50 digitally scanned HER2 IHC slides, showing absolute agreement in only 6% of cases, all of which were HER2 3+. Poor agreement was found in 10% of cases, largely owing to the heterogeneous nature of HER2 expression, cytoplasmic staining and low expression spanning the 10% cut-off value.10
Inter-observer
agreement was only fair to moderate in HER2-low cases, highlighting the challenge of classifying low-range HER2 scores, even for experienced pathologists.10 Another study by Baez-Navarro et al.
across multiple centres in Europe found similar results. Pathologists scored 105 HER2-negative breast cancer cases across two rounds. Complete inter-observer agreement was very limited in the first round – 4.7% of cases; and only slightly
Incorrectly assigning HER2-low status may lead to unnecessary exposure to costly and potentially toxic therapies. It is therefore vital that diagnostic methods identify subtle
differences to ensure patients receive the most appropriate therapy
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WWW.PATHOLOGYINPRACTICE.COM February 2026
improved by clustering IHC categories in the second round, with Fleiss’ kappa remaining in the fair-to-moderate range.11 Similarly, US-based data support
these findings. A study by Robbins et al. that included 18 specialist pathologists from 15 institutions showed significant discordance within the intermediate categories: <1% agreement for 1+ and 3.6% agreement for 2+.12
Substantial
discordance was also observed within IHC 0 cases, with an overall agreement of only 25% and weak inter-rater reliability metrics.12 Finally, Fernandez et al. reported
similar conclusions in a study that analysed datasets from both Yale University and the College of American Pathologists, finding poor agreement between pathologists, especially in 0 and 1+ cases.13
Evidence from these studies
show that traditional IHC methods do not provide the necessary reproducibility at the thresholds that now determine access to treatment.
Limitations of ISH In situ hybridisation methods provide a more objective assessment of patients by detecting HER2 gene amplification, but they also are quite limited. Their main drawback is the fact that their routine use is restricted to being used as confirmatory tests for IHC 2+ cases; not to clarify HER2 status in IHC 0 or 1+ cases. In addition, they are time-consuming and challenging techniques, requiring specialist training and high-quality equipment, which makes them technically demanding and relatively expensive. FISH requires fluorescence microscopy, with the interpretation of results often quite complex, especially in borderline or heterogeneous tumours, where signal counting is subjective. CISH uses standard brightfield microscopy to produce a permanent, stainable signal – which makes it more accessible than FISH – and it is easier to integrate into routine pathology laboratories. However, it is still a manual, labour-intensive process with
Notes Clear eligibility; strong, complete membrane staining Confirmatory ISH testing required
Supported by DESTINY-Breast04 and -Breast06 trials8,9 Subject of ongoing research; potential for ADC response
in DESTINY-Breast06 trial9 No detectable membrane staining
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