HISTOPATHOLOGY ≤34.0 34.5 35.0 35.5 36.0 36.5 37.0 HER2 0 / HER2-ultralow IHC 0 37.5 38.0 38.5 39.0 39.5 HER2-low 38.3 IHC 1+ / IHC 2+ (FISH neg) 40.0 40.5 41.0 41.5 HER2-positive 40.4 IHC 2+ (FISH pos) / IHC 3+
Fig 1. Molecular assays provide quantitative data on mRNA expression. For example, MammaTyper allows the distinction between HER2-negative, HER2-ultralow, HER2-low and HER2-positive subgroups.
limited scalability for laboratories with large sample numbers, as well requiring careful technique and standardisation. Neither approach effectively distinguishes HER2-low from HER2-ultralow cases, and neither provides a practical route to improving reproducibility in the low- expression range on a routine basis. As a result, ISH techniques have seen limited application in tackling the key diagnostic challenges related to HER2.
Evolving role of quantitative methods It is clear that a method that provides reproducibility at the lower end of the scoring scale is required, as treatment decisions are increasingly centred around subtle distinctions in HER2 expression. In this context, molecular assays – such as those based on RT-qPCR – provide quantitative, objective and reproducible methods that may support more confident HER2 classification. For example, MammaTyper is an RT-qPCR- based assay that measures mRNA levels of key breast cancer biomarkers, including ERBB2, which is the gene encoding HER2. This allows classification into distinct categories, while delivering standardised, numerical results to avoid many of the technical and interpretive pitfalls of IHC (Fig 1).
The robustness of this technique
was clearly demonstrated in a large multi-centre validation study by Varga et al, finding near-perfect inter- and
RT-qPCR can support IHC and help to bridge the HER2 diagnostic gap, offering an
additional technique where IHC alone may not provide sufficient clarity
intra-laboratory reproducibility across 10 pathology laboratories in Europe.14 Similarly, Atallah et al. investigated the clinical benefits of this approach based on a well-characterised HER2-positive cohort. The research team concluded that ERBB2 mRNA levels were more predictive of treatment benefit from trastuzumab than IHC scores alone, and revealed cases that were falsely classified as HER2- positive by IHC, particularly in borderline or heterogeneous tumours.15 Collectively, this evidence highlights the value of RT- qPCR, and supports the integration of this method into HER2 testing workflows. Caselli et al. also investigated
inconsistencies in the assessment of various key biomarkers for breast cancer, including oestrogen receptor,
progesterone receptor, HER2 and the Ki67 protein.16
Researchers compared IHC and
FISH data with the results of manual and semi-automated re-evaluation of the original IHC slides, and with RNA expression data from the same tissue block using MammaTyper. They found that these key biomarkers at the mRNA level showed a high degree of correlation with IHC results overall. However, the improved sensitivity of the RNA-based method for detecting weak biological signals was highlighted in discordant cases, often with low protein expression and mRNA levels near the cut-off. mRNA quantification with RT-qPCR was able to clarify borderline HER2 classifications, including potential false positives missed by FISH. These findings demonstrate that mRNA assays can offer more precise and standardised evaluation of key breast cancer biomarkers and resolve equivocal HER2 cases. Researchers concluded that RT-qPCR could serve as a complementary method to be used in all cases of unclear results or close to the cut-off values. As Caselli et al. concluded, this
molecular assay is intended to complement IHC, and by no means replace it. Compared to techniques like FISH, it is faster, less labour intensive and more scalable across laboratories. MammaTyper can be seamlessly integrated into existing workflows, since it runs on standard RT-qPCR instruments and does not need specialised or proprietary equipment (Fig 2). The process requires minimal
≥42.0
February 2026
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