Oncology
in IHC 0 or 1+ cases. Aside from this, they are challenging and time-consuming techniques; FISH requires fluorescence microscopy, specialist training and high-quality equipment, making it technically demanding and relatively expensive. Interpretation can be complex, especially in borderline or heterogeneous tumours, where signal counting is subjective. CISH is more accessible than FISH, as it uses
standard brightfield microscopy and produces a permanent, stainable signal, but still requires careful technique and standardisation. CISH is also easier to integrate into routine pathology labs, but remains a manual, labour-intensive process with limited scalability for higher throughputs. Neither ISH method is well suited to distinguishing between HER2-low and -ultralow cases, and neither offers a practical solution for improving reproducibility in the low expression range on a routine basis. For these reasons, ISH techniques have not been widely applied to HER2 subgroup classification.
The promise of novel quantitative methods for accurate classification Legacy IHC methods fail to deliver sufficient reproducibility at the thresholds that now determine eligibility for treatment, meaning that patients may be misassigned and either under- or over-treated. A method that provides reproducibility at the lower end of the scoring scale is therefore essential to ensure patients receive the most appropriate care. Molecular assays – such as RT-qPCR – offer a quantitative, objective alternative that may support more confident HER2 classification. These tests are less vulnerable to pre- analytical artifacts, and mRNA levels correlate well with HER2 pathway activation and drug response. Results of quantitative molecular assays are also resistant to differences in biopsy tissue quality, making them highly reproducible across testing sites. Studies have also demonstrated that RT-qPCR-based assessment of the mRNA expression of breast cancer biomarkers showed high concordance with IHC,15 further strengthening the case for integrating RT-qPCR into the HER2 testing workflow.
Figure 1: MammaTyper provides quantitative data on mRNA expression, allowing distinction between HER2-negative, HER2-ultralow, HER2-low and HER2-positive subgroups.
MammaTyper is an RT-qPCR-based assay that quantifies the mRNA levels of key breast cancer biomarkers, including ERBB2 – the gene encoding HER2. The result allows classification into distinct categories (Figure 1). MammaTyper’s analytical robustness was demonstrated in a large multi- centre validation study by Varga et al.,15
which
showed near-perfect inter- and intra-laboratory reproducibility in the quantitative assessment of several key breast cancer biomarkers – as well as in subtype determination – across 10 pathology labs globally (Table 3). This low inter- and intra-site variance
indicates that pathology institutions can perform the assay in house, and integrate it into routine diagnostic services. The study also demonstrated that the test delivers standardised, numerical results, overcoming the variability and technical and interpretive pitfalls associated with IHC and ISH methods. This suggests that running the MammaTyper test on core needle biopsies represents a reliable, efficient and reproducible alternative for improving breast cancer classification and refining HER2-low categorisation. Another recent study from Atallah et al.4
at the clinical benefits of this approach based on a well-characterised HER2-positive cohort. It looked at the difference in the distribution of
looked
molecular subtypes between IHC-based and MammaTyper-based molecular subtyping. The cohort was divided into two groups: a group of patients who received targeted adjuvant anti-HER2 therapy, and a chemotherapy-only group where the patients were diagnosed before approval of anti-HER2 therapy. In both study groups, tumour clinicopathological parameters such as tumour grade and size, and LN metastasis were matched to ensure that anti- HER2 therapy would be the primary predictive factor for patient survival. ERBB2 mRNA levels correlated positively with HER2 gene copy numbers, and were significantly higher in IHC 3+ cases compared with IHC 2+/ISH-positive cases. This aligns with prior findings that RT-qPCR correlates better with HER2 protein levels than FISH, and may surpass FISH in identifying patients with HER2 protein overexpression. The study concluded that the MammaTyper mRNA multigene assay provides a more accurate and objective evaluation of HER2 status and molecular subtyping than traditional IHC, with ERBB2 mRNA levels more predictive of trastuzumab benefit. MammaTyper-defined HER2 subtypes showed significantly better responses to anti-HER2 therapy, and the assay demonstrated high sensitivity and specificity for HER2 detection, strongly correlating with IHC results. It also revealed false-positive HER2 classifications by IHC, particularly in borderline or heterogeneous tumours. These findings support the use of mRNA-based molecular subtyping to improve treatment guidance and patient outcomes, while highlighting the need for further validation and practical implementation. Caselli et al. (2021)16
aimed to investigate Table 3: The inter- and intra-site variance of breast cancer biomarker detection results.
discrepancies in the assessment of key biomarkers including HER2 by comparing
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