Challenges & recommendations for identification of human epidermal growth factor receptor -2 (HER2)-low metastatic breast cancer in India: Expert opinion statement

Neeraj Arora; Jyoti Bajpai; Amanjit Bal; Atul Batra; Anurag Gupta; Deepak Kumar Mishra; Geetashree Mukherjee; Trupti Pai; Mayur Parihar; Geeta V. Patil Okaly; Shilpa Prabhudesai; Milap Shah; Somashekhar S.P.

doi:10.25259/IJMR_2009_2024

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Review Article

162 (

); 279-292

doi:

10.25259/IJMR_2009_2024

Challenges & recommendations for identification of human epidermal growth factor receptor -2 (HER2)-low metastatic breast cancer in India: Expert opinion statement

Neeraj Arora¹, Jyoti Bajpai², Amanjit Bal⁵, Atul Batra⁶, Anurag Gupta⁷, Deepak Kumar Mishra⁹, Geetashree Mukherjee^10,, Trupti Pai³, Mayur Parihar¹¹, Geeta V. Patil Okaly¹², Shilpa Prabhudesai⁴, Milap Shah⁸, Somashekhar S.P.¹³

1Department of Molecular Biology, & Molecular Genetics, Unipath Specialty Laboratory, Ahmedabad, Gujarat, India

2Department of Medical Oncology, Tata Memorial Centre, Mumbai, Maharashtra, India

3Department of Pathology, Tata Memorial Centre, Mumbai, Maharashtra, India

4Center of Oncopathology, Tata Trust Mahalaxmi Engineering Estate, Mumbai, Maharashtra, India

5Department of Histopathology, Post Graduate Institute of Medical Education & Research, Chandigarh, India

6Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India

7Department of Genomics, Ampath, Hyderabad, Telangana, India

8Department of Histopathology, Yashoda Hospital, Hyderabad, Telangana, India

9Department of Hematology and Molecular Pathology, Tata Medical Center, Kolkata, West Bengal, India

10Department of Histopathology, Tata Medical Center, Kolkata, West Bengal, India

11Department of Hematology and Cytogenetics, Tata Medical Center, Kolkata, West Bengal, India

12Department of Histopathology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India

13Department of Oncology, Aster CMI Hospital, Bengaluru, Karnataka, India

For correspondence: Dr Geetashree Mukherjee, Department of Histopathology, Tata Memorial Centre, Kolkata 700 160, West Bengal, India e-mail: mukherjeegeetashree54@gmail.com

Received: 2024-12-05, Accepted: 2025-06-11, Published: 2025-11-12

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Abstract

Results from a recent Phase 3 clinical trial (DESTINY-Breast04) established the efficacy of the antibody-drug conjugate, trastuzumab deruxtecan (T-DXd) in patients with metastatic breast cancer with immunohistochemistry (IHC) score 1+ or 2+ and without in situ hybridization amplification, defining a new category of metastatic breast cancer known as human epidermal growth factor receptor 2 (HER2)-low. Across studies of patients with primary or metastatic breast cancer, approximately 50 per cent have HER2-low tumours, thereby emphasizing the importance of accurately identifying these tumours. Results from the DESTINY-Breast06 trial further corroborated the DESTINY-Breast04 results showing T-DXd as a new standard of care for patients with HER2-low and HER2-ultralow (defined as IHC score 0 with membrane staining) metastatic breast cancer following one or more lines of hormone therapy. Differentiating between HER2 IHC scores 0 and 1+ shows significant interobserver variability, possibly because the low scores did not have any clinical relevance until now. To establish a standardized approach in scoring of HER2-low tumours in India, a panel of experts comprising histopathologists, molecular pathologists, and clinicians met to discuss guidelines on HER2 testing. The identification of HER2-low expression depends on multiple methodological pre-analytical and analytical variables, including sample handling, fixation, processing, antigen clones, staining methodology, and substrates. The panel also focused on the challenges in the interpretation of HER2-low status. Challenges in the pre-analytical and analytical phases could be addressed by rigorous quality control procedures and training the pathologists. In the post-analytical phase, the subjective mode of HER2 assessment and HER2 intratumoural heterogeneity in HER2-low breast cancer are factors that influence HER2-low assessment. The panel recommended robust standard operating procedures to overcome these challenges. The central point of discussion was to implement clear guidelines, careful supervision of pre-analytical and analytical issues, and specialized training for accurate HER2 testing that would help select patients eligible for novel therapies.

Keywords

Analytical factors

breast cancer

HER2

immunohistochemistry

in situ hybridization

trastuzumab deruxtecan

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Overexpression/amplification of human epidermal growth factor receptor 2 (HER2) is reported in almost 15 to 20 per cent of patients with breast cancer and is associated with an aggressive disease characterised by high recurrence rates and poor prognosis^1-3. Besides being prognostic, HER2 testing also confers a predictive value as patients with overexpressed HER2 are eligible for HER2-directed therapies. Thus, precise and reproducible HER2 testing techniques and strategies are vital for selecting patients who can benefit from novel HER2-directed targeting agents. An update from American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines 2023 reported a new subgroup, HER2-low, with immunohistochemistry (IHC) score of 1+ or 2+ without HER2 gene amplification by in situ hybridization (ISH) testing⁴.

HER2-low comprises approximately 50 per cent of patients who express HER2 protein in breast cancer⁵.The emergence of HER2-low phenotype adds a novel dimension to the biological and clinical intricacies of breast cancer. Several studies and meta-analyses have investigated the prognosis of HER2-low breast cancer, but with variable findings^6-12. Treatments with antibody-drug conjugate (ADC)-based targeted therapy have revolutionized the management landscape for patients with metastatic breast cancer (mBC) where chemotherapy was the only option¹³. Multiple translational research studies on different classes of ADCs have gained momentum globally^14-16. Trastuzumab deruxtecan (T-DXd) demonstrated a statistically significant and clinically meaningful overall and progression-free survival benefit compared with physician’s choice of chemotherapy, as well as improvement in objective response rate in patients with hormone receptor-positive or hormone receptor-negative HER2-low breast cancer in the DESTINY-Breast04 trial^13,17. Based on the findings from the DESTINY-Breast04 trial, T-DXd was approved for HER2-low unresectable or metastatic breast cancer in patients who had received at least one prior line of chemotherapy in the metastatic setting or developed disease recurrence within six months of completing adjuvant chemotherapy. Other ADCs with different payloads targeting HER2-low expression including trastuzumab duocarmazine (SYD 985, TULIP trial¹⁸) and disitamab vedotin (RC48) are being investigated in HER2-low breast cancer^19-21. The Phase 3 DESTINY-Breast06 study (NCT04494425) showed T-DXd therapy significantly improved progression-free survival versus chemotherapy in HER2-low mBC with acceptable safety profile. HER2-ultralow (defined as HER2 IHC score 0 with membrane staining: incomplete and faint staining in ≤10% of tumour cells) results were consistent with the HER2-low results²². Several clinical trials investigating the combination of novel HER2-directed ADCs and immune checkpoint inhibitors are ongoing^23,24. The emergence of treatment options for patients with HER2-low breast cancer emphasizes the requirement of more robust and reproducible HER2 testing methods for patient identification²⁵.

Currently, IHC and ISH are standard methods for determination of HER2 status employed to generate a comprehensive histopathology report to facilitate treatment decisions in patients with breast cancer²⁶. However, there are several methodological, analytical, and technical nuances along with high inter- and intra-observer variability that hinder the routine diagnostic assessment of HER2-low status. In a retrospective blinded assessment of 170 breast cancer biopsies evaluated by 18 pathologists from Yale University, there was a poor concordance (≤70%) for HER2-low identification²⁷. Accurate determination of HER2 status is critical as patients may be deprived of available HER2-directed therapy in case of inaccurate results, resulting in poor prognosis. This expert opinion focuses on challenges and recommendations for HER2-low testing in patients with breast cancer in India.

Materials & Methods

We employed a 3-step process for developing recommendations for HER2-low testing in patients with breast cancer in India. A meeting was conducted among 13 experts who discussed the key aspects of molecular testing for patients with breast cancer. The multi-disciplinary team of experts comprised histopathologists, molecular pathologists, medical and surgical oncologists, cytogeneticists, and clinicians with extensive experience in the management of patients with breast cancer. The specialists provided insights based on their clinical experience regarding the challenges in HER2 testing in India and provided strategic and implementable recommendations to overcome them. An evidence-based literature search that included a review of relevant international and regional guidelines was conducted in February 2024. This review article provides an overview of the recommendations approved by all authors, supported by the current evidence. They have been adapted to local healthcare framework.

Challenges and recommendations for HER2 testing in India

The interpretation of HER2-low expression levels is based on multiple diagnostic and analytical parameters. Despite the availability of comprehensive ASCO/CAP guidelines, the assessment of the HER2-low status can be a challenging task since multiple parameters play a significant role in pathological analysis of the sample. We discuss the challenges and recommendations during the pre-analytical, analytical, and post-analytical phases of diagnostic testing for HER2 biomarkers, especially HER2-lowin patients with breast cancer.

Pre-analytical phase

The expert panel recommended that all newly diagnosed patients with primary, recurrent, or metastatic breast cancer must undergo testing for estrogen hormone receptor, progesterone hormone receptor, HER2, and Ki67 in line with the ASCO/CAP recommendations^28-30. Vigilant management of biopsy and surgical specimens in the pre-analytical phase is required to minimise the false-negative and false-positive cases³¹. A core tissue biopsy from primary tumour is the preferred sample for testing in line with recommendations from ASCO/CAP as well as UK Breast Cancer HER2 Testing Guideline Recommendations and South Asian Association for Regional Cooperation (SAARC) consensus statement^30,32-35. Core biopsy allows for earlier, easier, and better fixation of tissue samples compared to radical mastectomy; therefore, core biopsy samples are preferred over surgical samples. In studies comparing core biopsy to modified radical mastectomy and surgical samples, core biopsy samples were associated with higher expression levels of biomarkers, including hormonal markers (estrogen and progesterone receptors) and HER2^36-38. In case of metastasis, metastatic tissue sample should also be obtained for HER2 testing wherever possible^39-41.

Any alterations to the pre-analytical variables, including fixation, antigen retrieval, antibody clones, enzymatic activity, reaction time, temperature, and substrate concentration can hamper the HER2 staining intensity and ISH sensitivity^42,43. Though most of the pre-analytical factors have been standardized in all laboratories, tissue fixation remains the foremost challenge. Improper fixation techniques, including improper fixatives, fixation time, and prolonged cold ischemia time are primary causes of improper tissue preservation and ancillary testing failure especially in developing countries where the samples may be transported from hospital to a central laboratory/referral centre for further testing. Shorter time to fixation has been associated with better HER2 staining^44,45. For optimal tissue handling, ASCO/CAP guidelines recommend the time from tissue removal to fixation must be ≤1 h and samples should to be fixed in 10 per cent neutral buffered formalin for 6-72 h²⁹. For mastectomies and lumpectomies, the ideal slicing of the samples should be 5-10 mm after appropriate inspection of margins, and tissues for HER2 testing should be fixed in a sufficient volume of 10 per cent neutral buffered formalin⁴. Refrigerated, improperly fixed frozen tissue samples are likely to undergo autolysis resulting in false-negative results^46-48. Prolonged ischemia negatively impacts the number of positive cells as well as the intensity of staining^45,49. The paraffin-embedded FFPE (formalin-fixed paraffin-embedded) sections should be ∼5 µm thick^45,50,51. Any discrepancies in obtaining tissue and tissue fixation parameters should be notified to the testing facility.

If the factors associated in the pre-analytical phase of the HER2 assay are optimized, the errors in overall assay can be minimised⁵². Considering the importance of safeguarding the biospecimen for analysis, it is critical for the healthcare practitioners to be vigilant about the effect of pre-analytical factors on the assay results. Several medical professionals play a role in specimen acquisition, handling, and transport of the samples⁵³. Thus, appropriate quality control, training, and awareness among the teams who are involved in the pre-analytics is paramount.

Analytical phase

The analytical factors that play a crucial role in accurate HER2 testing include the staining methodology, type of antibody clones and assay used, the testing platform, and tissue controls. These factors can impact the reproducibility and sensitivity of the HER2 identification tests. The challenges involved in this phase include non-validated antibody clones, non-standardized testing strategies, use of several clones without appropriate calibrations, staining issues, and lack of adequate training of laboratory personnel⁵⁴.

The two techniques approved by the US Food & Drug Administration (FDA) and recommended by ASCO/CAP as well as Royal College of Pathologists for determination of HER2 status are IHC and ISH^4,51. Both of these techniques identify different parameters related to HER2⁵⁵. IHC detects the expression and intensity of HER2 protein in the cell membranes using a scoring system (score 0: no staining or incomplete and faint/barely perceptible membrane staining in ≤10% of tumour cells; score 1+: incomplete and faint/barely perceptible membrane staining in >10% of tumour cells; score 2+: weak/moderate complete membrane staining in >10% of tumour cells or complete and intense membrane staining in ≤10% of tumour cells; and score 3+:complete and intense membrane staining in >10% of tumour cells) and ISH detects gene amplification. The equivocal results (score 2+) on IHC need to be confirmed with ISH testing as per current ASCO/CAP guidelines⁴. The guidelines at the moment do not present any alternative probes to chromosome enumeration probe 17 (CEP17) for HER2 testing in clinical practice, citing insufficient evidence regarding its clinical reliability and validity⁴.

IHC testing remains the preferred primary diagnostic technique for HER2 testing. The IHC assay involves identification of specific antigens in FFPE tissues through antibodies. The choice of antigen retrieval techniques, use of antibody clones, and assay platforms play a major role in the sensitivity of the IHC test. Several validated and standardized antibody clones are available for HER2 testing. In a comparative analysis of several HER2 assay kits, a high concordance (98.2%) was observed between the IHC kits⁵⁶. Similar results were reported in other studies comparing the standardized antibody assays with ISH testing^57,58. However, the panel recommended that appropriate in-house validation techniques are required for each assay. Also, appropriate low and high HER2 controls should be setup routinely for accurate HER2 detection^26,59. The panel recommended a caution while selecting the tissue sample for IHC testing as the samples near the edges of the tumour are prone to higher staining in the cytoplasmic region compared to other samples. Caution should be maintained while evaluating IHC samples to consider only the membrane staining and not the cytoplasmic staining. Tumour heterogeneity (intratumoural as well as between primary and metastatic samples), understaining due to antibody dilution and other pre-analytical factors should also be considered in case of negative results and the tests should be repeated as required⁶⁰. Repeating the tests in case of equivocal results can assist in eliminating the false-negative results. Additionally, appropriate training of laboratory personnel and adherence to good laboratory practices is of utmost importance for accurate IHC analysis.

Approach to HER2-low IHC interpretation

The threshold of IHC to detect minor differences in protein expression levels, particularly the discrimination between HER2-low and HER2 IHC0, which now encompasses a subset ‘HER2- ultralow’ is challenging due to inherent limitations of the technique⁶¹. A practical and helpful approach is to follow the magnification rule where faint or barely perceptible staining would be revaluated at 40×magnification⁶². Staining that is unequivocally visible only at 20×magnification is likely to represent a weak intensity score. However, a distinct intense and complete membrane staining perceived at low magnification (2.5×–5×) corresponds to strong staining (score 3+ if complete and >10% of the tumour cells), which mimics the positive control. Staining that is unequivocally visible only at 10×magnification most likely corresponds to at least moderate intensity. This rule may lead to increased reproducibility in IHC scoring⁶². Therefore, best practices for distinguishing IHC 0 vs. 1+ include using ASCO/CAP recommended scoring criteria, reviewing at 40× to detect faint or focal areas of expression, considering second reviews for cases close to the threshold and using controls with a range of protein expression⁴. In case of HER2 IHC2+ scoring, the results of IHC test are reported as equivocal, which mandate further assessment through ISH.

ISH detects gene amplification using single or dual probes (based on the HER2 CEP17 ratio and the HER2 signals/neoplastic cell, segregated into 5 ISH groups)⁶³. The HER2 status with groups 2,3, and 4 are determined as positive or negative by correlating with the IHC score⁶⁴. Several ISH testing methods are available. The methods can broadly be classified as dual probe and single probe. The identification of HER2 on ISH platform is dependent upon the type of probe used. Conventionally, fluorescence ISH (FISH) has been regarded as a standard platform for ISH HER2 testing. However, with advent of other bright-field platforms like dual-colour ISH (DISH) and chromogenic ISH (CISH) assays, ISH testing turnaround time has significantly reduced⁶⁵. Though the panel did not recommend any particular ISH technique, the experts strongly recommended that ISH test should be performed by validated standardized test and interpreted by trained and competent pathologists. The ISH techniques are described in brief below.

Fluorescence in situ hybridization

Utilizing fluorescent-labelled probes, FISH is a cytogenetic technique employed to identify specific DNA patterns in specimen samples. The FISH assay is utilised to assess the HER2 gene copy number within the nuclei of tumour cells⁶⁶. Despite the established robustness of the FISH test in evaluating HER2 gene amplification, it has certain disadvantages, including the need for fluorescent microscope, technical expertise, increased time for procedure⁶⁷, and higher reagent cost⁶⁸. In addition, FISH tends to obscure tissue architecture and it is difficult at times to distinguish invasive tumour nuclei from non-invasive tumour nuclei or non-neoplastic nuclei⁶⁹. Hence, alternative ISH methods were developed to surmount the limitations of FISH in detecting HER2 amplification, including CISH, DISH, silver-enhanced ISH (SISH), and bright-field double ISH (B-DISH). These recent techniques combine practical advantages of IHC analysis with the reproducibility of the FISH method⁷⁰.

Chromogenic in situ hybridization

The CISH technique uses peroxidase enzyme-labelled probes to visualize target genes in tissue samples. Standard bright-field microscope is utilised in CISH allowing the simultaneous assessment of tissue morphology and copy number alterations, and prompt analysis of tumour heterogeneity. It is fully automated and provides permanent signals that can be archived⁷¹. A study on 50 breast cancer cases displaying chromosome 17 polysomy reported 98 per cent concordance between FISH and CISH indicating that CISH can be a precise and practical alternative to FISH for diagnosing HER2 gene amplification in breast cancerspecimens⁷².

Dual-colour in situ hybridization

DISH is also a fully automated bright-field ISH assay for the simultaneous detection of HER2 and CEP17 signals on the same FFPE breast cancer tissue section and is recommended by the ASCO/CAP guidelines^4,73,74. DISH assay allows probe signal interpretation without destroying histologic features, thus allowing for a more accurate evaluation of the relevant nuclei. Moreover, results are available in a short time period as the assay is completely automated^73,74. Although both DISH and FISH are based on the same approach (hybridisation of labelled DNA), there are key differences in workflow and analysis which are presented in Table^69,75.

Table. Key differences in fluorescent in-situ hybridization (FISH) and dual-colour in-situ hybridization (DISH) techniques

FISH	DISH
Microscope
FISH slides require a dedicated fluorescent microscope that includes: Objectives specifically designed for fluorescence Filter sets to excite & view fluorescent dyes FISH slides must be reviewed under, low light or darkroom conditions, & at high magnification	DISH slides are viewed using a standard bright-field microscope at both high & low magnification Lower magnification allows pathologist to identify invasive carcinoma and internal controls DISH slides can be easily compared to haematoxylin & eosin stain & HER2 IHC
Slides
Light causes photo bleaching of fluorophores, so: Special conditions are necessary for storage Repeated reviewing of slides is limited as fluorescent microscopy causes photo bleaching FISH slides have limited shelf life as fluorophores fade over time	Chromogenic substrates are resistant to light, so DISH slides can be archived and reviewed with ease
Interpretation
Tissue autofluorescence can mask signals Does not allow for comprehensive analysis of tissue with morphological context More difficult to assess intratumoural genetic heterogeneity	DISH bright-field assay with counterstain enables: To review the critical morphological context to identify invasive component Identification of areas of HER2 amplification heterogeneity
Turnaround time
For most laboratories, FISH must be sent to a dedicated laboratory increasing the average time for obtaining results	DISH can be performed in-house. The fully automated protocol can be run on standardized instruments enabling same day results.

IHC, Immunohistochemistry. Source: ref 69,75

Post-analytical phase

Intratumoural HER2 heterogeneity

The prime challenges in the post-analytical phase are related to the interobserver variability. This may be due to a lack of consistent internal positive control for HER2 in the normal breast tissue^63,76. Post-analytical phase also encompasses a series of activities starting from interpretation of results to receipt of the pathology report by the physician. Several challenges summarized below may hamper the interpretation of results.

Intratumoural HER2 heterogeneity is characterized by two distinct clones of cancer cells within a lesion with difference in the HER2 status²⁵. This may either be observed in clustered pattern where cells with different amplification are seen in clusters or the cells may be interspersed in a diffuse or mosaic pattern^64,77. Intratumoural heterogeneity is one of the important causes of false positives and false negatives in HER2 report and thus can significantly impact therapeutic decisions^1,78,79. HER2 heterogeneity has been frequently reported in breast carcinomas with an equivocal (2+) HER2 score⁸⁰. The current ASCO/CAP guidelines recommend that if there is a second population of cells with increased HER2 signals/cell and this cell population is more than 10 per cent of tumour cells on the slide, a separate counting of at least 20 non-overlapping cells must also be performed within this cell population and reported⁴. It is thus important to select appropriate sample for evaluation as well as scan the entire field for heterogeneity. Additionally, ISH is preferred as the bright-field slide used for IHC can be used directly for scanning in ISH. Using core biopsy sample rather than excision sample has yielded better results⁸¹; however, in cases of heterogeneous cancers, it would be beneficial from re-testing at recurrence and/or metastases and on the excision specimens. In cases with intratumoural heterogeneity, panel recommended counting at least 60 cells to detect intermixed type or cluster-type heterogeneity, and to include the proportion of amplified and non-amplified cells in the ISH report^82,83.

CEP17 copy number gain

CEP17 copy number gain is observed in 3-46 per cent of cases⁸⁴. Though the prognostic value of CEP17 copy number gain is yet to be completely elucidated, it may impact the interpretation of HER2 status⁷⁷. The ASCO/CAP 2013 guidelines recommended repeating the ISH test with an alternative probe; however, this recommendation was subsequently removed in 2018 guidelines^28,64.

Change in HER2 status

Discordance between hormone receptor status prior to and after neoadjuvant therapy ranges widely from 8 to 33 per cent^38,85. Though the exact mechanism of this change is not understood, it may be due to selective targeting of positive or negative clones during neoadjuvant therapy and as a result of intratumoural heterogeneity. Thus, a careful evaluation of ISH sample after neoadjuvant therapy is important to rule out false-positive or negative results⁸⁶. The pathologists should be made aware of pre-operative chemotherapy/radiotherapy through appropriate sections in the pathology request form for their consideration⁵¹.

Similar ‘receptor conversion’ may occur during disease progression or metastatic stage in both HER2 as well as hormone receptors. The rate of conversion from positive to negative status ranged from 0 to 5.9 per cent and from negative to positive status ranged from 0.7 to 8.9 per cent. Similar factors as those involved with neoadjuvant therapy may be responsible for this conversion⁸⁶. Hormone receptor as well as HER2 status may differ between metastatic samples collected from the same patient at a single timepoint, and even within a single organ, different metastases may have different expression of HER2³⁸. Thus, a critical analysis of metastatic sample or reanalysis at metastasis, wherever possible, is recommended by the expert panel⁶⁴.

Other recommendations

Accurate detection of HER2-low and HER2-ultralow is critical for management of patients with breast cancer. However, interobserver variability plays an important role in the interpretation of the assay. Also, the IHC assays techniques currently in use are calibrated to detect levels of HER2 expression 100-10,000 fold above normal breast. With the advent of ADCs for treatment of patients with HER2-low, interpretation of HER2-low is challenging as the lower limit of detection for these assays may be below the calibration limits of the assay⁸⁷. Hence validation of the detection of HER2-low and HER2-ultralow is very critical and use of standardized controls will be helpful for the calibration and validation of the IHC assays.

Robust SOPs are vital not just for the diagnostic techniques but also for preparing a pathology report. Regular audits of the testing facilities can ensure that the SOPs and quality control measures are being followed. The quality of results depends heavily on the laboratory personnel. HER2 testing should be conducted by appropriately trained and certified pathologists. In a real-world study assessing the interpretation proficiency of pathologists for HER2-low assessment, pre-training (baseline) scores and a post 4-h training scores were evaluated for 74 pathologists. Irrespective of the training, the overall concordance rate was >80 per cent for HER2-low assessment⁸⁸. In another study where standardized training was implemented for pathologists, the concordance rates for IHC0 and IHC1+were 70 and 77 per cent, respectively⁸⁹. Thus, training programs for pathologists and technicians for HER2 analysis are very important for accurate diagnosis of HER2-low status.

Additionally, the HER2 tests at the laboratories can be certified using the College of American Pathologists (CAP) or National Accreditation Board for Testing and Calibration Laboratories (NABL) accreditation. Sample archives and laboratory documentation must be also effectively planned and organized to facilitate re-testing if required²⁶.

The reporting of results is an equally important step. The HER2 status report should be standardized to include all pertinent information needed for physicians to take therapeutic decisions. Hence, a comprehensive report of HER2 status is required to enable accurate treatment decisions and individualized patient management. The panel recommended that the IHC and ISH testing results should be included in the same report for ease of decision making. The pre-analytical factors that may obscure the results should be included in the report along with the details of antibody, assay, and the type of ISH technique used. The report should also include details on the intensity and percentage of positivity in tumour cells for accurate interpretation of the HER2-low and HER2-ultralow status. As recommended by ASCO/CAP guidelines, the panel agreed that the HER2-low status need not be included in the final results. The results should include the interpretation including HER2 and ISH scoring along with a footnote describing the ASCO/CAP HER2 status⁹⁰. The recommendations from international guidelines regarding the HER2-low/ultralow category can also be incorporated in the footnotes to facilitate clinical decisions⁴. Box 1 presents sample template for defining the HER2 status in the pathology report along with interpretation of results. The recommendations for pre-analytical, analytical, and post-analytical phases are summarised in figure.

Box 1.

Sample template to define HER2 status in a pathology report with interpretation

HER2 -Low

Weak to moderate complete membrane staining observed in >10% of tumour cells (IHC 2+ equivocal not amplified) or the incomplete membrane staining that is faint/barely perceptible in >10% of tumour cells (IHC 1+).

HER2 – Ultra-Low

Membrane staining that is incomplete and is faint/barely perceptible and in ≤10% of tumour cells (IHC 0).

Note:

-

Examine HER2 IHC at high power (40x) when discriminating 0 from 1+ staining.
-

Consider second pathologist review when results are close to the 0 vs. 1+ interpretive threshold
-

Use controls with a range of protein expression (including 1+) to help ensure the assay has an appropriate limit of detection.

Interpretation of the results

HER2-Low: Tumours with HER2 IHC 1+ or 2+ and ISH non-amplified. A major breakthrough in this area is the use of new anti-HER2 antibody-drug conjugates (ADCs), particularly trastuzumab deruxtecan (T-DXd). The DESTINY-Breast04 trial^1,2 results showed significant improvements in progression-free survival and response rates, leading to FDA approval of T-DXd in August 2022 for metastatic HER2-low breast cancer.

HER2-Ultra-Low: Tumours with faint, incomplete HER2 staining in ≤10% of cells and FISH-negative. Ongoing trials like DESTINY-Breast06 are exploring whether patients with ultra-low HER2 expression, previously classified as HER2-0, may still benefit from ADC therapies.

Advancements in diagnostic tools and techniques for HER2 evaluation

The evolving landscape of HER2-directed therapy has led to investigation for novel diagnostic methods and more sensitive assays to better identify HER2 expression levels. Alternative methodologies under evaluation include HERmark™ (highly sensitive and specific technique to detect HER2 proteins in FFPE tissue samples covering IHC scores ranging from 0 to 3+)⁹⁰, real-time polymerase chain reaction (PCR; gene amplification analysis by examining the mRNA extracted from FFPE samples)⁹¹, multiplex ligation-dependent probe amplification (determines multiple gene amplifications and various gene deletion portions)⁹², time resolved fluorescence resonance energy (quantifies the fluorescence signals produced from energy transfers between 2 adjacent molecules)⁹³, and reverse phase protein array technology with laser capture microdissection enrichment of tumour epithelium⁹⁴.

Recommendations for pre-analytical to post-analytical phases of HER2 testing. FFPE, formalin-fixed paraffin-embedded; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; SOP, standard operating procedure.

Machine learning and artificial intelligence in digital pathology are gaining momentum. The artificial intelligence (AI)-based techniques to optimize predicting the HER2 status with respect to speed, precision, and cost-effectiveness, thereby facilitating the HER2-directed treatment, are also being developed^95-98. A multi-institutional study reported higher accuracy for HER2 0 and 1+ tumour interpretation with AI algorithm assistance for pathologist compared to no AI algorithm assistance (0.93 vs. 0.8)⁸⁰. In a machine learning-based model, a high consensus was reported with pathologist-reported scores for HER2 using whole-slide images⁹⁹. In a cohort analysis of 363 patients with breast cancer, HER2 IHC score 1+ was refined into four categories: faint intensity in ≥20 per cent of cells regardless of the circumferential completeness; weak complete staining in ≤10 per cent; weak incomplete staining in >10 per cent; and moderate incomplete staining in ≤10 per cent. With application of artificial neural network analysis, 63 per cent of HER2-negative cases were reclassified as HER2-low along with a high interobserver agreement¹⁰⁰. Machine learning-based algorithms can thus help pathologists in HER2 assessments. However, implementation of these technologies in the real world is associated with several challenges as any minor variations in pre-analytical factors may result in critical discrepancies in the slide quality making the slides unquantifiable through the algorithms. Additionally, lack of standardization between the algorithms also poses a challenge which needs to be resolved before the techniques can be implemented in real-world HER2 assessment¹⁰¹.

To combat the issue of tumour heterogeneity, next-generation sequencing and gene protein assays combining the benefits of both IHC and ISH into a single slide for assessing HER2 status are under investigation¹⁰². Use of liquid biopsy assays using cell-free tumour DNA, circulating tumour cells, and extracellular vesicles is also under consideration for HER2 identification⁵⁸.

Discussion & Conclusion

With the advent of novel targeted therapy options for patients with HER2-low breast cancer, the management of this subgroup has undergone enormous transformation with pathology playing a very crucial role. Pathologists are now entrusted with a challenging task of accurately identifying HER2-low and in the future, even HER2-ultralow breast cancers for providing optimal therapy. Although the techniques used for assessment, IHC and ISH, are well established, precise evaluation of HER2 status could be subjective due to several factors involved in the pre-analytical, analytical, and post-analytical phases of the workflow. Robust specimen preparation with strict quality control procedures using standardized control is important to improve the sensitivity, specificity, and reproducibility. Within the post-analytical factors, standardization of report structure along with incorporation of relevant details for accurate interpretation of HER2 status is important for facilitating optimal management. Assessment for HER2-low status demands incorporating novel technologies and standardizing the testing workflows coupled with well-defined guidelines. Our expert opinion paper presents recommendations (Box 2) regarding optimal timing and best practices to ensure quality during various phases of HER2 testing among patients with breast cancer in the Indian scenario.

Box 2.

Expert panel recommendations and best practices for HER2 testing in breast cancer

All newly diagnosed patients with primary, recurrent, or metastatic breast cancer must undergo testing for oestrogen hormone receptor, progesterone hormone receptor, HER2, and Ki67.
A core tissue biopsy sample from primary/metastatic tumour is preferred.
Pre-analytical factors should be standardized for optimal sample collection and preparation for assay.
For analysis, robust procedures to be set along with appropriate positive and negative controls.
Immunohistochemistry (IHC) testing should be repeated in case of equivocal results and reflex in situ hybridization (ISH) testing should be requested.
Pathologists should carefully scrutinize entire area prior to counting; cytoplasmic staining should not be scored.
Interpretation of results should be performed considering factors that may impact the HER2 status.
Laboratories should strictly adhere to the standard operating practices and diagnostic workflows for quality assurance; regular audit is important.
Comprehensive report including both IHC and ISH results to be prepared.
Tissue storage and archival should be planned appropriately in case of further requests.
All efforts must be taken to reduce the turnaround time for optimal management of patients.
Clinicians should be made aware of the importance of pre-analytical factors in accurate and reliable diagnosis of HER2 status.
Training of pathologists involved in IHC and ISH assays is important.

The IHC test for HER2 is both prognostic and predictive, used to identify patients eligible for targeted therapies like T-DXd. However, its accuracy is limited. The current scoring system (0, 1+, 2+, 3+) may misclassify patients—those scored as 1+ may have no real HER2 expression, while some patients scored as 0 and excluded may benefit from treatment with targeted agents. Therefore, having a uniform scoring system to detect low levels of HER2 is an important step forward in identifying eligible patients. We have presented updated guidelines which can give accurate and consistent results when supported with the right training. These recommendations will optimize the management algorithm for patients with HER2 low and HER2 ultra-low breast cancer.

Acknowledgment

Authors acknowledge Fortrea Scientific Pvt. Ltd. for medical writing support that was funded by AstraZeneca Pharma India Ltd in accordance with GPP 2022 guidelines.

Financial support & sponsorship

The expert meeting was funded by AstraZeneca Pharma India limited. The manuscript writing support was funded by AstraZeneca Pharma India Ltd.

Conflicts of Interest

None

Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation

The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.

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Challenges & recommendations for identification of human epidermal growth factor receptor -2 (HER2)-low metastatic breast cancer in India: Expert opinion statement

Abstract

Keywords

Analytical factors

breast cancer

HER2

immunohistochemistry

in situ hybridization

trastuzumab deruxtecan

Materials & Methods

Challenges and recommendations for HER2 testing in India

Pre-analytical phase

Analytical phase

Approach to HER2-low IHC interpretation

Fluorescence in situ hybridization

Chromogenic in situ hybridization

Dual-colour in situ hybridization

Post-analytical phase

Intratumoural HER2 heterogeneity

CEP17 copy number gain

Change in HER2 status

Other recommendations

Advancements in diagnostic tools and techniques for HER2 evaluation

Discussion & Conclusion

Acknowledgment

Financial support & sponsorship

Conflicts of Interest

Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation

References

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