HER2 Unraveled—The Vital Distinction Between Overexpression and Gene Amplification in Cancer – Medical News Asia

Significance of HER2 overexpression and HER2 amplification

Human epidermal growth factor receptor 2 (HER2) is a crucial biomarker in contemporary cancer management.¹ Alterations in the HER2 protein or in the protein-coding gene, erythroblastic leukemia viral oncogene homolog 2 (ERBB2; hereafter referred to as HER2 in line with common practice), have significant clinical implications.¹ Overexpression of the HER2 protein, gene amplification or activating mutations can overactivate or enhance downstream HER2 signaling pathways.^2,3 Increased HER2 signaling drives key processes in tumorigenesis, including cellular transformation, survival, proliferation, and invasion.^2,3

Among these alterations, overexpression and gene amplification are closely related.¹ Amplification refers to an increase in HER2 gene copy number, which often leads to overexpression, defined as an increased abundance of HER2 receptors on the cell surface (Figure 1).^1,2,4-8 However, gene amplification does not always correlate with protein overexpression, which can also occur through other mechanisms such as transcriptional regulation and post-transcriptional events.^9,10 Biological differences between HER2-overexpressing and HER2-amplified tumors remain poorly understood due to limited data.¹¹

In gastric and breast cancers, HER2 amplification is associated with poor prognosis and aggressive disease features, including shorter time to relapse and a higher propensity for brain metastases.^12,13 An analysis of a phase III study suggests that the HER2-directed antibody trastuzumab may be less effective in HER2-amplified breast cancers compared with HER2-overexpressing tumors, likely due to the higher receptor abundance in the latter.¹¹

An accurate assessment of HER2 status is important in the diagnostic evaluation of patients with solid tumors.¹ For those whose tumors harbor HER2 alterations, multiple guideline-recommended HER2-targeted therapies are available, including monoclonal antibodies, antibody–drug conjugates (ADCs), and tyrosine kinase inhibitors (TKIs), with additional treatments in development.¹ Therapy selection should be guided by the approved treatment indication, taking into account tumor type, specific alteration, and clinical context.^14-17

Currently, immunohistochemistry (IHC) and in situ hybridization (ISH) are the most commonly used methods to assess HER2 overexpression and gene amplification, respectively.¹⁸ In breast and gastric cancers, standardized IHC and ISH scoring criteria determine HER2 positivity, with IHC as the initial test and in situ hybridization (ISH) used for equivocal cases (IHC 2+).^14-16 For other cancers, such as non-small cell lung cancer (NSCLC), molecular testing is recommended to detect activating HER2 mutations.¹⁹ Recently, HER2-targeted therapy has become available for patients with advanced or metastatic solid tumors meeting the IHC 3+ criteria, and, in these patients, confirming the presence of HER2 amplification is not necessary.¹⁷ Because the type of the HER2 alteration present in the tumor cells may impact a patient’s eligibility for targeted therapy,^14-17selecting the appropriate assessment method is essential to guide patients toward optimal therapeutic options.

Figure 1. Definition and testing methods of HER2 gene amplification and HER2 overexpression.

Prevalence of HER2 overexpression and HER2 amplification in solid tumors

The prevalence of HER2 overexpression and HER2 amplification varies widely across tumor types (Figure 2).² Amplification of the HER2 gene and HER2 overexpression as defined by the IHC 3+ criteria are found in 20% and 10% of breast cancers, 40% and 15–37% of salivary gland cancers and 2% and 3% of lung cancers, respectively.² This variability between amplification and overexpression in different solid tumors underscores the importance of tumor-specific testing strategies to guide appropriate HER2-directed therapy.

Figure 2: Prevalence of HER2 amplification and HER2 overexpression by tumor types.

Current standards and interpretation of HER2 testing guidelines for overexpression and amplification

Different diagnostic tests are required to identify different types of HER2 alterations, a topic covered in detail in article 5 of this series. IHC uses an anti-HER2 antibody as a probe to visualize HER2 protein abundance in a biopsy or surgical specimen and thereby detecting overexpression.⁵ Amplification of the HER2 gene is detected by ISH using a DNA probe that can detect increases in gene copy number.⁵ The DNA probes used in ISH may be coupled to a fluorescent (FISH), chromogenic (CISH) or silver (SISH) detection system.⁵ Single-probe ISH can measure HER2 copies per nucleus, and dual-probe ISH using an additional probe chromosome 17 centromere protein (CEP17) can determine the HER2:CEP17 ratio.⁵ The HER2:CEP17 ratio is considered to be a more accurate measure of amplification status as it is less affected by the mitotic index of the specimen, section thickness, and other potential sources of error.⁵ Recommendations for HER2 testing are available from the National Comprehensive Cancer Network (NCCN), the American Society of Clinical Oncology (ASCO)-College of American Pathologists (CAP), and other expert groups (Table 1).^{4,14,19,21-45}

Specific and detailed scoring criteria for the interpretation of both ISH and IHC assays are well established for breast and gastric cancer.^14,15 For other solid tumors, a general HER2 IHC scoring template, which adopts the gastric HER2 IHC scoring, provided by the CAP is available to guide assessment.²⁷ Additionally, specific IHC scoring criteria have been developed and utilized as enrollment criteria in clinical trials.^28,43 The appropriate testing modality varies by tumor type and may be impacted by tissue availability.

Table 1. Summary of HER2 testing recommendations in guidelines.^{4,14,19,21-45}

HER2 ISH scoring criteria in breast and gastric cancers

For patients with breast or gastric cancer, HER2 status needs to be accurately evaluated to identify patients who may be eligible for targeted therapies. This may require the use of both IHC and ISH. Detailed IHC scoring guidelines for breast and gastric cancers are available from ASCO-CAP,^14,15 and these have been reviewed in detail previously. These guidelines also make recommendations for ISH scoring for these two tumor types, with dual-probe ISH as the preferred approach for breast cancer and the only approach recommended for gastric cancer (Figure 3).^{14,15,17,21,36,37,45} For patients with breast cancer, ISH scores are categorized into five groups based on HER2:CEP17 ratio and average number of signals per cell.¹⁵ Groups 1 and 5 (HER2 positive and negative, respectively) account for ~95% of tumors tested.¹⁵ Specimens in Groups 2–4 need additional data from IHC testing to confirm their HER2 status.¹⁵ For those with gastric cancer, single probe ISH is not recommended.³⁶ HER2 positive is defined as HER2:CEP17 ratio ≥2.0 or an average HER2 copy number of ≥6 signals per cell.³⁶ In summary, assessment of HER2 status with both ISH and IHC is vital to ensure that all breast and gastric cancer patients eligible for HER2-directed therapies are identified.

Figure 3. Testing and interpretation of HER2 ISH in breast and gastric cancer

Concordance between IHC and ISH evaluation of HER2

Despite the mechanistic relationship between gene amplification and protein overexpression, the concordance between IHC and ISH evaluation of HER2 is not absolute. The reported concordance between IHC 3+ and ISH testing is 91% for both breast and gastric cancers.^46,47 Polysomy 17 is the likely cause of this inconsistency.⁹ Conversely, some breast cancers test positive for HER2 gene amplification but do not over-express HER2; these patients are less likely to benefit from HER2-directed therapies.⁹

HER2 Amplification Assessment via NGS — Opportunities and Limitations

Although ISH is the guideline-recommended standard for detecting HER2 gene amplifications in breast, gastric, and other cancers,^14,15 the use of next-generation sequencing (NGS) to detect mutations and amplifications is recommended in guidelines for some tumor types (e.g., NSCLC and colorectal cancer),^19,34,41 and there is increasing interest in this approach. NGS allows the assessment of large numbers of genes and can make more efficient use of limited biopsy specimens or permit the use of liquid biopsies. However, challenges exist in the concordance between NGS estimates of gene amplification and ISH. A study comparing tissue-based NGS and dual-probe ISH for measuring HER2 amplifications for breast cancer determined NGS cutoff values (copy number >4.09 and <2.91) for HER2 amplification status that had 95% sensitivity and 99% specificity with reference to ISH.⁴⁸ However, the same cutoff had lower sensitivity (65%) with gastric cancer specimens, possibly due to tumor heterogeneity.⁴⁸ Similarly, a study by Shah et al. that included multiple tumor types found varying concordance between copy number gain by NGS and standard IHC/ISH definitions of positivity by tumor and specimen type.⁴⁹ Tissue NGS and circulating tumor DNA (ctDNA) NGS positive predictive value (PPV) for HER2 positivity by IHC/ISH was highest for breast cancer, 97% and 93% respectively, but PPV was lower for non-breast cancer tumors (76% tissue, 44% ctDNA).⁴⁹ Considerable discordance may exist between NGS and tissue IHC/ISH. For example, in two studies of biliary tract cancer, both using the same ctDNA NGS assay, positive percent agreement between ctDNA NGS and tissue IHC/ISH varied between 36.4% and 75.9%.^50,51 Further research should be conducted to determine appropriate diagnostic cutoff values for different NGS panels in specific tumor types.

Precision in HER2 testing allows targeted treatment

Due to the discordance of HER2 status assessed by IHC, ISH, or NGS,⁴⁹ HER2 gene amplification and HER2 overexpression should not be considered as clinically equivalent.⁹ NGS has the potential to serve as a tumor-agnostic approach for assessing HER2 amplification, but currently, concordance with established methods is highly variable.^48,49 HER2 overexpression assessed by IHC remains the only HER2 alteration with a tumor-agnostic indication for HER2-targeted therapy.¹⁷ At present, HER2 testing strategies should be aligned with current guidelines, treatment indication and individualized clinical objectives.

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The article is sponsored by Daiichi Sankyo and AstraZeneca.

MED-HK-TRAS-00121 HK-12585 12/2025

Disclaimer

This article is not medical advice. Patients should seek personal assessment by a licenced specialist. Physicians are recommended to read the full publication(s) as cited in the article before making medical decisions. This article does not supersede nor replace the published article(s).