REFINING HER2 IHC ASSESSMENT GUIDED BY CAP REPORTING TEMPLATE: ENABLING ACCURATE CLASSIFICATION ACROSS SOLID TUMORS AND ADDRESSING INTRATUMORAL HETEROGENEITY – Medical News Asia

Introduction

Immunohistochemistry (IHC) assessment of human epidermal growth factor receptor 2 (HER2) status in oncology has undergone a change in paradigm, from a dichotomous ‘positive’ or ‘negative’ to a spectrum of HER2 expression. This is driven by the recent development of newer HER2-directed agents and studies of these compounds in an increasing variety of tumor types, which have demonstrated the importance of a more graduated approach to identify patients who are likely to respond to treatment.^1-3

The binary classification of HER2 status (‘positive’ or ‘negative’) was based on the responses of breast cancer patients in studies of earlier classes of HER2-directed agents. HER2-positive tumors were previously defined as IHC 3+ or IHC 2+ with gene amplification by reflex in situ hybridization (ISH) per ASCO/CAP (American Society of Clinical Oncology/College of American Pathologists) guidelines.³ Patients meeting these criteria were eligible for HER2-directed agents, while those who failed to meet these criteria were classified as HER2-negative and not eligible for HER2-directed agents.³

However, studies have demonstrated that a new generation of HER2-directed therapies exert clinically meaningful responses even in those patients with lower levels of HER2 expression previously classified as ‘negative’ (e.g., IHC 0+, IHC 1+, or IHC 2+ with negative ISH).¹ This has led to an evolving understanding of HER2-positivity, where all patients with varying levels of HER2 expression may be eligible for HER2-directed agents (covered in a previous article).¹ Furthermore, with the expansion of HER2-directed therapies beyond breast and gastric cancer to pan-tumor indications,⁴ there remains a need for IHC scoring guidelines in different tumor types (also covered in a previous article).

This article aims to discuss the clinical impact of these recent developments, including changes to the CAP reporting template for HER2 status in breast cancer, and the adaptation of HER2 scoring criteria for pan-tumor indications. Guidance on the assessment of HER2 in specimens with intratumoral heterogeneity (ITH), as well as HER2 assay validation, are also discussed, and these topics aim to enhance confidence in HER2 status assessment.

The updated CAP template for HER2 reporting in breast cancer

Studies of newer agents in breast cancer patients with lower levels of HER2 expression have demonstrated marked responses.¹ This has led to updates in the most recent version of the CAP reporting template for biomarker testing of breast cancer specimens, to include a description distinguishing IHC 0 and IHC 0+ specimens (Table 1).^5,6 This is an important revision to previous templates, where IHC 0+ specimens were classified as IHC 0.^5,6 This updated reporting template also includes standardized comments that describe how these categories relate to the ‘HER2-low’ and ‘HER2-ultralow’ expression which were defined in clinical trials of the newer HER2-directed therapies, and notes that patients with HER2 IHC 0+, IHC 1+ or IHC 2+/ISH negative tumors may still be eligible for such therapies in the metastatic setting.⁶

Table 1. Reporting results of HER2 testing of breast cancers by IHC (CAP guidelines version 1.6.1.0)

Reporting of HER2 scoring in non-breast cancers

For non-breast cancers, such as the gastric/gastroesophageal junction (GEJ), colorectal, and gynecological cancers, CAP biomarker reporting templates include specific IHC scoring recommendations adapted from clinical trials of HER2-directed agents (Table 2).^8-13 Notably, staining patterns and definitions of IHC scores differ among tumor types. For example, while IHC 3+ in breast cancer is defined by intense circumferential or membranous staining,³ in gastric, colorectal, or gynecological tumor specimens, IHC 3+ is defined by intense basolateral or lateral staining (Tables 1 and 2 and Figure 1), a pattern that is consistently observed in gastric cancer specimens. The CAP guidance for gynecological and colorectal cancers added that specimens could be evaluated with gastric cancer HER2 scoring criteria in addition to specific HER2 scoring systems from clinical trials.^9,14 Notable clinical trials whose HER2 scoring systems are included in CAP guidance are the HERACLES trial for colorectal cancer and study NCT01367002 for gynecologic cancers.^9,14 A comparison of IHC staining patterns in breast and gastric tumor specimens is provided in Figure 1.^3,8,15 Application of these scoring criteria should be guided with reference to the intended treatment strategy and relevant clinical indications.

Table 2. Summary of HER2 scoring criteria in non-breast solid tumors

Figure 1. Examples of HER2 IHC 3+ specimens of breast and gastric tumor per ASCO/CAP guidance

The challenges of intratumoral heterogeneity for interpreting HER2 status

Besides differences in IHC staining characteristics and scoring criteria between various tumor types, there may also be differences in HER2 distribution patterns within the primary tumor and/or between the primary tumor and its metastases.¹⁶ Coexistence of subpopulations or subclones of cells with different genotypes or phenotypes within a primary tumor, or between a primary tumor and its metastases is known as ITH.¹⁶ Patterns of HER2 ITH may be broadly classified as genetic or non-genetic ITH.¹⁶ Genetic ITH patterns are categorized as (1) clustered type, with two distinct areas with different HER2 status, (2) mosaic type, characterized by diffuse intermingling of cells with different HER2 statuses, or (3) scattered type, caused by isolated HER2 overexpressing tumor cells in a predominantly HER2 non-amplified tumor (Figure 2).¹⁶ Non-genetic ITH is defined by the presence of tumor cells with HER2 gene amplification without HER2 protein expression intermixed with tumor cells with concordant HER2 amplification and expression.¹⁶

Figure 2. Examples of HER2 genetic intratumoral heterogeneity in breast cancer

HER2 ITH is reported in up to 40% of breast cancers.¹⁶ Although it can be found in tumors with any level of IHC positive staining, ITH is more commonly associated with equivocal cases and is rare in IHC 3+ cases.¹⁶ In gastric/GEJ cancers, HER2 ITH has also been reported in 14–79% and 23–54% of cases by IHC and fluorescence in situ hybridization (FISH), respectively.¹⁶ Less is known about HER2 ITH in colorectal and gynecological cancers. However, limited evidence suggests that mosaic and clustered HER2 expression patterns occur in approximately 12–40% of these tumors. This indicates that a significant proportion of colorectal and gynecological tumors harbor clinically relevant HER2 ITH.^17,18

Impact of intratumoral heterogeneity on HER2-directed therapy

HER2 ITH impacts the efficacy of HER2-directed therapies in multiple ways. It may lead to inappropriate allocation of patients to HER2-directed therapies,¹⁶ and tumors with HER2 ITH may bind or take up HER2-directed agents with a lower efficiency than those with more consistent expression.¹⁶ In breast cancer studies, patients with HER2 ITH treated with HER2-directed therapies had shorter disease-free survival, decreased overall survival and poorer responses to therapy.^19-22 For example, in a study of 164 patients, pathologic complete response rate was 0% in patients with HER2 ITH versus 55% in those without HER2 ITH.²¹ Similarly, results from gastric cancer studies also suggest worse outcomes with HER2-directed therapies in patients with HER2 ITH compared to non-ITH patients.²³

A study comparing the HER2 status in metastatic colorectal cancers (CRCs), showed a discrepancy of almost 19.5% between primary tumors and their paired metastases.²⁴ This discrepancy concerned the IHC 0, IHC 1+, and IHC 2+ non-amplified categories, and it raises the question of retesting of different tumor sites for HER2 status.²⁴

Recommendations for HER2 intratumoral heterogeneity evaluation

Guidance from ASCO-CAP and other expert groups offer several approaches to address the challenges posed by ITH for HER2 status evaluation.^7,25 For breast cancer, recommendations include examining HER2 IHC specimens at higher magnification (40x), and having a second pathologist review the results when discrimination between IHC 0 and IHC 1+ for samples is close to the interpretive threshold.^7,25 If only a small biopsy sample was tested negative for IHC, repeat testing on a larger subsequent specimen should be considered.^6,25

The CAP reporting template for breast cancer includes specific items to report and comment on the characteristics of HER2 ITH samples.⁶ Reporting of HER2 clustered heterogeneity is required only if clustered heterogeneity is present as discrete, separate populations, one of which has IHC 3+ staining.⁶ Clustered heterogeneity is reported as⁶:

Not applicable
Not identified (IHC 3+ staining is homogeneous throughout sample)
Present (distinct IHC 3+ as well as non-3+ staining populations)

When clustered heterogeneity with IHC 3+ staining is present in >10% of the cells, the case is reported as 3+. The percentage of tumor cells with overexpression must be reported, as well as the IHC score of the non-IHC 3+ areas.⁶ If IHC 3+ staining is present in ≤10% of cells, the result should be classified as HER2 equivocal (IHC 2+), with clustered pattern noted and additional sampling considered.⁶ Uncommon staining patterns should be described in the “Other (specify)” or comments section.⁶

Clustered HER2 amplification detected by ISH generally corresponds to IHC 3+ staining.⁶ In cases with heterogeneity, the proportion of HER2-amplified cells identified by ISH should be reported, along with their corresponding IHC scores.⁶ Additionally, ISH analysis should be performed separately in IHC 3+ and non–IHC 3+ regions, with a clear description of the observed heterogeneity.⁶

Retesting of multiple biopsy specimens is also recommended for gastric and gynecological cancers.^8,9 In patients who develop metastatic disease, testing of specimen from a newly acquired biopsy from the metastatic site should also be considered.^7,25 Where repeated testing of HER2 on additional and resection specimens have been performed, the results from all specimens should be considered when finalizing HER2 status and reporting data to colleagues.²⁶

Guidance on laboratory validation of HER2 IHC assays

In addition to the ASCO-CAP recommendations for routine clinical HER2 IHC evaluation using validated tests, the CAP has also provided a guideline offering recommendations on the analytic validation of laboratory-developed IHC assays and verification of FDA-cleared IHC assays for diagnostic purposes.²⁷ This includes guidance on extending the use of a previously validated assay to a new tumor type.²⁷ For example, if a laboratory intends to utilize a HER2 IHC assay and scoring criteria combination previously validated in non-colorectal tumors for CRC, the laboratory director has the discretion to extend the initial non-CRC validation to CRC by assessing a representative sample of colorectal tumors.²⁷

If a laboratory is validating a new HER2 assay and intends to use the same scoring criteria in breast and colon cancers, then both cancer types should be included in the set of 20 positive and 20 negative tissues constituting the validation.²⁷ However, this guidance should not be interpreted as a recommendation that every assay, scoring system, and tumor type combination is to be subjected to the requirement of 20 positive and 20 negative cases for each validation.²⁷ It is at the laboratory director’s discretion whether these different combinations require separate validations.

Moreover, the same CAP guideline provides recommendations related to changes in antibody clone or assay components. When changes to assays or reagents have been made, e.g., a new fixative type, antigen retrieval method, tissue processing equipment, automated testing platform or changes to environmental conditions of testing, the laboratory should test a sufficient number of tissues to ensure that the expected results are achieved consistently.²⁷ The number of specimens tested is at the discretion of the laboratory director.²⁷ If an antibody clone is changed for an existing validated assay, a full revalidation (equivalent to initial analytic validation) is suggested.²⁷ This is an important consideration as limited data suggests there is variation in performance between commonly used anti-HER2 antibody clones, such as Pathway^® 4B5 and Herceptest™ DG44, when evaluating HER2-low breast cancer specimens.²⁸

Conclusions and future directions

Currently, the most appropriate approach to HER2 IHC evaluation is to use a well-validated IHC assay and scoring system specific to the tumor type, in alignment with guidance from expert groups such as ASCO/CAP.^3,8,29 The use of reporting templates can further assist by ensuring assay results are reported in a clear and consistent manner. Future development of HER2 IHC assessment may include using artificial intelligence-based image analysis for the evaluation of specimens. This may provide a more quantitative and objective approach and reduce intra-observer variability.²⁵ Prior to clinical deployment, these newer tools will need to be thoroughly validated in large cohorts, including various levels of HER2 positivity.²⁵

References

Venetis K, et al. Front Mol Biosci 2022;9:834651.

Ko HC, et al. Front Oncol 2025;15:1565872.

Wolff AC, et al. J Clin Oncol 2018;36:2105–2122.

US Food and Drug Administration. FDA grants accelerated approval to fam-trastuzumab deruxtecan-nxki for unresectable or metastatic HER2-positive solid tumors. Available at: https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-fam-trastuzumab-deruxtecan-nxki-unresectable-or-metastatic-her2. Accessed 7 May 2026.

Titus K. Taking on low, ultralow HER2 breast cancer. Available at: https://www.captodayonline.com/taking-on-low-ultralow-her2-breast-cancer/. Accessed 7 May 2026.

College of American Pathologists. Template for Reporting Results of Biomarker Testing of Specimens from Patients with Carcinoma of the Breast (Version 1.6.1.0, June 2025). Available at: https://documents.cap.org/documents/New-Cancer-Protocols-June-2025/Breast.Bmk_1.6.1.0.-REL_CAPCP.pdf. Accessed 7 May 2026.

Wolff AC, et al. J Clin Oncol 2023;41:3867–3872.

Bartley AN, et al. J Clin Oncol 2017;35:446–464.

College of American Pathologists. Template for Reporting Results of Biomarker Testing of Specimens From Patients With Carcinoma of Gynecologic Origin (version 1.3.0.0, September 2025). Available at: https://documents.cap.org/protocols/Gynecologic.Bmk_1.0.0.0.REL_CAPCP.pdf. Accessed 7 May 2026.

College of American Pathologists. Template for Reporting Results of HER2 (ERBB2) Biomarker Testing of Specimens from Patients with Adenocarcinoma of the Stomach or Gastroesophageal Junction. Available at: https://documents.cap.org/protocols/cp-gastric-HER2biomarker17-1001.pdf. Accessed 7 May 2026.

Fader AN, et al. J Clin Oncol 2018;36:2044–2051.

Valtorta E, et al. Mod Pathol 2015;28:1481–1491.

National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines^®) Gastric Cancer Version 2.2026. Available at: https://www.nccn.org/professionals/physician_gls/pdf/gastric.pdf. Accessed 7 May 2026.

College of American Pathologists. Template for Reporting Results of Biomarker Testing of Specimens From Patients With Carcinoma of the Colon and Rectum. Available at: https://documents.cap.org/protocols/ColoRectal.Bmk_1.3.0.0.REL_CAPCP.pdf. Accessed 7 May 2026.

Abrahao-Machado LF, Scapulatempo-Neto C. World J Gastroenterol 2016;22:4619–4625.

Hou Y, et al. Cancers (Basel) 2023;15(10):2664.

Hashimoto T, et al. Br J Cancer 2023;129:1176–1183.

Shen S, et al. Mod Pathol 2023;36:100299.

Rye IH, et al. Mol Oncol 2018;12:1838–1855.

Dai W, et al. Mod Pathol 2025;38:100785.

Filho OM, et al. Cancer Discov 2021;11:2474–2487.

Lee HJ, et al. Am J Clin Pathol 2015;144:570–578.

Yagi S, et al. Gastric Cancer 2019;22:518–525.

D’Angelo F, et al. Diagn Pathol 2024;19:88.

Rakha EA, et al. Mod Pathol 2025;39:100925.

Muller KE, et al. Am J Clin Pathol 2019;152:7–16.

Goldsmith JD, et al. Arch Pathol Lab Med 2024;148:e111–e153.

Kain ZE, et al. Virchows Arch Published online September 16, 2025.

College of American Pathologists. Principles of Analytic Validation of Immunohistochemical Assays. Available at: https://www.cap.org/protocols-and-guidelines/cap-guidelines/current-cap-guidelines/principles-of-analytic-validation-of-immunohistochemical-assays. Accessed 7 May 2026.

The article is sponsored by Daiichi Sankyo and AstraZeneca.

MED-HK-TRAS-00133 2026/06

HK-13134 06/2026

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).

© Copyright 2026 MediPaper Medical Communications Ltd. – Refining HER2 IHC Assessment Guided by Cap Reporting Template: Enabling Accurate Classification Across Solid Tumors and Addressing Intratumoral Heterogeneity.