Human epidermal growth factor receptor 2 (HER2) has long been a key marker for screening patients suitable for targeted therapy against HER2. This strategy is primarily applicable to breast cancer but also to several other solid tumors. Immunohistochemistry (IHC), as the core detection method for evaluating HER2 expression level, has important clinical value in guiding targeted therapy against HER2. However, with the significant clinical efficacy of novel antibody - drug conjugates (ADCs such as T - DXd) in patients with HER2 - low and HER2 - ultralow breast cancer, higher demands are being placed on the accurate interpretation of HER2 expression levels.

The IHC detection technology faces many challenges in the interpretation of HER2 low/ultralow. This is mainly due to the wide dynamic range of HER2 protein expression and intra - tumor heterogeneity, a variety of pre - analysis variables (such as antibody selection and differences in the detection process), and the inherent subjectivity of IHC score ^[1]. Together, these factors limit the accurate identification of low - level HER2 expression and highlight the urgent need to establish standardized operating procedures and develop novel detection methods that are specific to low - level HER2 expression and may have higher sensitivity. Currently, a number of emerging methods to overcome the limitations of existing testing techniques and scoring systems are in the development phase.

01. Quantitative immunofluorescence (QIF)

The principle of QIF detection is similar to that of concentration measurement in liquid samples. The objective measurement of HER2 expression level is achieved through the quantitative analysis of HER2 expression in the tumor cell region defined by pancytokeratin (CK), which overcomes the limitation of IHC detection. Breast cancer tissues are stained to quantify HER2 signals within the molecular compartments of pancytokeratin. HER2 quantification of cell lines with different levels of HER2 expression was carried out using liquid chromatography - tandem mass spectrometry (LC - MS/MS) technology to generate a cell line microarray (CMA) standard. This standard was detected on the same tray as the patient sample using an automated stainer. Cell segmentation was performed using digital images of the CMA standard, and the proteomics measurements were converted to amol/cell area. The HER2 signal was converted to amol/mm² using a standard curve for each dye lot (Figure 1)^[2]. This method provides an objective and quantitative assessment of HER2 expression, enables the detection of low levels of HER2 expression, and helps identify patients who may benefit from HER2 - targeted ADC therapy.

Figure 1: Quantitative immunofluorescence (QIF) detection principle

A study involving 51 patients with metastatic breast cancer (32 HER2+ and 19 HER2 - ) validated the ability of QIF to detect HER2 expression levels. In all patients, HER2 expression levels were positively correlated with TTNT (time from enrollment (or start of current treatment) to start of next - line treatment) and OS (overall survival). In addition, there was a significant positive correlation between TTNT and HER2 expression when divided by the quartile, and the median TTNT showed an upward trend with the increase in the quartile of HER2 expression level. Consistent results were also observed in patients with HER2+ or HER2 - metastatic breast cancer (Figure 2)^[3].

▲ Figure 2: HS - HER2 and HER2 states of TTNT with T - DXd

In addition to breast cancer, the QIF assay was also validated in 741 patients with NSCLC, in which HER2 was detected in 63% of patients and was above the limit of quantitation in 17% (Figure 3)[4]. This finding suggests that the QIF assay is expected to identify more patients who are suitable for receiving drug conjugates of HER2 - targeted antibodies, and not just those with HER2 amplification or mutation.

▲ Fig. 3: Data distribution diagram of 741 cases of non - small cell lung cancer

02. Reverse transcription polymerase chain reaction (RT - PCR)

The RT - PCR - based method is the most direct means of assessing and quantifying HER2 mRNA and is expected to assist in assessing low levels of HER2 expression.

A retrospective study of the Oncotype DX test showed that the mean risk score for relapse (RS) was 17.802 in the HER2 IHC 0 group and 18.503 in the HER2 IHC 1+ group; when RS > 25, the proportion in the HER2 IHC 0 group was about 12.4% (117 of 944 cases) and that in the HER2 IHC 1+ group was about 17.0% (230 of 1351 cases). The HER2 score by RT - PCR was 8.912 in the HER2 IHC 0 group and 9.337 in the HER2 IHC 1+ group (Figure 4)[5]. This indicates a significant difference in HER2 mRNA between patients with HER2 IHC scores of 0 and 1+, and a correlation between HER2 protein and mRNA levels in the range of low HER2 expression. However, the Oncotype DX test has not been evaluated using continuous protein measurements.

▲ Figure 4: Oncotype DX Detection Recurrence Score (RS) vs. HER2 Status

In the Xpert Breast Cancer STRAT4 test, the δCt value is used to distinguish the positive and negative of HER2 expression, that is, the Ct value of the internal reference gene (CYFIP1) minus the Ct value of the target gene (four, including HER2) to determine the level of HER2 mRNA. But compared with the QIF test, it lacks the ability to analyze spatial heterogeneity ^[6]. Currently, the Xpert Breast Cancer STRAT4 test is a CE - IVD product that has not yet been approved by the FDA.

03. RNA in situ hybridization (RNAscope)

RNAscope, an in situ hybridization (ISH) - based mRNA assay, has also been used to investigate the expression of ERBB2 in HER2 low - expression breast cancer by providing spatial information via multi - probe hybridization. RNAscope has been shown to differentiate IHC 3+ from 2+ (AUC = 0.89), but is less effective at differentiating 0 from 1+ (AUC = 0.62). In addition, its score depends on the subjective point count, lacks a standardized quantitative method, and its correlation with QIF protein level in the low expression range is poor ^[7].

04. HER2DX genomic testing

The HER2DX assay is the first assay that integrates clinical data with genomic data to capture tumor features, immune features, and pathological features simultaneously. HER2DX covers 27 genetic tests, 14 of which are associated with immunologic features (i.e., CD27, CD79A, HLA - C, IGJ, IGKC, IGL, IGLV3 - 25, IL2RG, CXCL8, LAX1, NTN3, PIM2, POU2AF1, and TNFRSF17). Four are associated with tumor cell proliferation features (i.e., EXO1, ASPM, NEK2, and KIF23), five with luminal differentiation features (i.e., BCL2, DNAJC12, AGR3, AFF3, and ESR1), and four with HER2 amplification features (i.e., ERBB2, GRB7, STARD3, and TCAP). This assay can predict two different clinical endpoints: long - term survival outcome (i.e., HER2DX risk score) and the possibility of achieving complete pathological response (pCR) (i.e., HER2DX pCR score), providing new guidance for optimizing treatment regimen (Figs. 5–6). The HER2DX test has been extensively studied and there is evidence that the HER2DX risk score may help identify a significant proportion of early - stage HER2 - positive breast cancer patients who do not require additional therapy such as Pertuzumab, Trastuzumab Deruxtecan, or Neratinib. Further studies are needed to further verify the clinical value of the HER2DX test score ^[8].

▲ Fig. 5: Clinical studies involving HER2 DX detection

▲ Figure 6: Summary of variables included in HER2 DX test and their correlation with each clinical endpoint

05. Summary

Immunohistochemistry remains the gold standard in almost all clinical pathology laboratories. However, with the increasing digitalization of pathology and the increasing need for more accurate information, new technologies seem likely to replace the subjective assessment of immunohistochemistry as a new concomitant diagnostic test. In particular, there is a requirement for accurate detection of HER2 at a low level and a more accurate quantitative method. It is believed that with the development of technology, more accurate detection techniques will be applied to detect HER2 expression and more accurately guide treatment.

References

[1] Nature reviews Drug discovery,2023,22(2):101-126.

[2] Laboratory Investigation, 2022, 102(10): 1101-1108.

[3] Annals of Oncology,2024,35:S384-S385.

[4] Modern Pathology,2024,37(9):100556.

[5] Breast Cancer Research,2024,26(1):154.

[6] NPJ Breast Cancer,2019,5(1):28.

[7] Modern Pathology,2024,37(2):100408.

[8] EBioMedicine,2022,75.

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