Interobserver consistency and diagnostic challenges in HER2-ultralow breast cancer: a multicenter study
In recent years, trastuzumab deruxtecan (T-DXd) has demonstrated significant clinical benefits for patients with unresectable and/or metastatic human epidermal growth factor receptor 2 (HER2)-low [immunohistochemistry (IHC) 1+ or IHC 2+/in situ hybridization-negative] breast cancer (BC), following the success of the DESTINY-Breast (DB) series of clinical trials. , , This marks a shift from prior anti-HER2 therapies, such as trastuzumab and ado-trastuzumab emtansine, which required HER2 overexpression to be effective. Preliminary results from the DB-06 clinical trial, announced in June 2024, further demonstrated that T-DXd offers clinical benefits for patients with advanced BC exhibiting HER2-ultralow (defined as HER2 IHC >0 and <1+). Compared with physician-selected single-agent chemotherapy (capecitabine, nab-paclitaxel, or paclitaxel), T-DXd significantly improved progression-free survival (PFS). The PFS results for HER2-ultralow patients [median PFS 13.2 months, hazard ratio 0.78, 95% confidence interval (CI) 0.50-1.21] aligned with those for HER2-low patients (median PFS 13.2 months, hazard ratio 0.62, 95% CI 0.51-0.74). Therefore, precise diagnosis of HER2-null and HER2-ultralow by pathologists is essential to support individualized treatment strategies for BC patients. However, there is inherent variability in HER2 IHC interpretation, both between observers and within the same observer, especially when evaluating low levels of HER2 expression. The 2023 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines acknowledge this issue and offer clinical practice recommendations for distinguishing HER2 IHC 0 from IHC 1+. Nonetheless, the assessment of HER2-low cases remains challenging. Zaakouk et al. analyzed the consistency among 16 pathologists evaluating 50 HER2 IHC cases and found moderate overall agreement (Fleiss κ = 0.433), with a high Fleiss κ of 0.803 for HER2 IHC 3+ cases. However, the consistency for HER2 IHC 0 and IHC 1+ cases was only 0.437 and 0.292, respectively. Similarly, a consistency analysis involving 18 pathologists evaluating 170 BC biopsy samples yielded comparable results, with Fleiss κ values of 0.49 and 0.35 for HER2 IHC 0 and IHC 1+ cases, respectively. Few studies have specifically examined pathologists’ ability to distinguish between HER2-null and HER2-ultralow within HER2 IHC 0 cases. Additionally, there is limited direct evidence on pathologists’ capacity to differentiate HER2-null from HER2-non-null, particularly in light of the clinical validation of T-DXd’s efficacy in HER2-ultralow patients. This study aimed to evaluate interobserver consistency among pathologists in distinguishing HER2-null from HER2-ultralow, to analyze the concordance between reassessment scores and historical scores after the expansion of the eligible patient population for T-DXd, and to explore the challenges and promising solutions for accurate diagnosis of HER2-null and HER2-ultralow by pathologists.   Sample collection This study retrospectively collected surgical specimens from 50 consecutive patients diagnosed with invasive BC at the Fourth Hospital of Hebei Medical University between February and March 2023, whose pathological reports indicated HER2 IHC 0. The ethics committee of the Fourth Hospital of Hebei Medical University approved the study protocol (approval No. 2021KY124). The study was carried out in accordance with the ethics standards of the participating institutions and the tenets of the Declaration of Helsinki. As this study did not involve interactions with human subjects or the use of personally identifiable information, informed consent was not required for utilizing existing pathology materials, and no identifiable patient information was revealed. Formalin-fixed, paraffin-embedded tissue sections of 4 μm were subjected to HER2 IHC staining using the VENTANA anti-HER2/neu (4B5) rabbit monoclonal primary antibody from Roche (Basel, Switzerland), carried out on the BenchMarK XT automated IHC platform. HER2 IHC interpretation process Thirty-six pathologists from four centers (Xingtai People’s Hospital, Cangzhou People’s Hospital, Hebei University Affiliated Hospital, and the Fourth Hospital of Hebei Medical University), all experienced in evaluating HER2 IHC slides in routine clinical practice, visually assessed the 50 HER2 IHC slides under a microscope. The participating pathologists were blinded to the original HER2 IHC pathological report results and the study’s purpose. All pathologists underwent training based on the 2023 ASCO/CAP guidelines, following their practice recommendations to differentiate between HER2 IHC 0 and 1+ using high-power magnification (×40) for HER2 IHC examination. In addition to providing HER2 IHC scores (HER2 IHC 0/1+/2+/3+), the pathologists were also required to report the percentage of HER2 membrane staining to distinguish HER2-null (no staining observed) from HER2-ultralow (incomplete and faint/barely perceptible membrane staining in ≤10% of tumor cells) within HER2 0 cases. Statistical analysis Statistical analyses were carried out using IBM SPSS Statistics (version 26.0; IBM Corporation, Armonk, NY) and GraphPad Prism (8.01; GraphPad Software, LLC, San Diego, CA). Based on the HER2 IHC interpretation results from the pathologists, three scenarios were analyzed: three categories (HER2 IHC 0/1+/2+), four categories (HER2 IHC null/ultralow/1+/2+), and binary classification (HER2 IHC null/non-null). Fleiss κ was used to assess interobserver consistency in HER2 IHC interpretation. The κ values were interpreted as follows: 0.01-0.20: slight agreement, 0.21-0.40: fair agreement, 0.41-0.60: moderate agreement, 0.61-0.80: substantial agreement, 0.81-1.00: almost perfect agreement. The study defined the degree of case interpretation consistency as follows: high consistency: ≥90% of pathologists (31-34 evaluators) agreed on the HER2 IHC result, moderate consistency: ≥75%-<90% of pathologists (26-30 evaluators) agreed, low consistency: >50%-<75% of pathologists (18-25 evaluators) agreed, and challenging cases: ≤50% of pathologists (≤17 evaluators) agreed. In this study, pathologists’ HER2 IHC assessment results were compared with the consensus scores obtained from the majority of 36 pathologists to analyze their interpretation trends. For each pathologist, the proportion of cases assessed below the consensus score ( P below ), the proportion assessed above the consensus score ( P above ), and the overall agreement rate with the consensus score ( P agreement ) were calculated. The interpretation trends were then classified based on the following criteria: (i) Stable: pathologists whose difference between the proportions of cases assessed below and above the consensus score was <0.1, and whose agreement rate with the consensus score was ≥75%; (ii) Fluctuating: pathologists whose difference between the proportions of cases assessed below and above the consensus score was <0.1, but with an agreement rate <75%; (iii) Conservative: pathologists who assessed a higher proportion of cases below the consensus score by at least 0.1; (iv) Aggressive: pathologists who assessed a higher proportion of cases above the consensus score by at least 0.1. The formulas were as follows: Stable: ∣ P b e l o w − P a b o v e ∣ < 0.1 a n d P agreement ≥ 75 % Fluctuating: ∣ P b e l o w − P a b o v e ∣ < 0.1 a n d P agreement < 75 % Conservative: P b e l o w − P a b o v e ≥ 0.1 Aggressive: P a b o v e − P b e l o w ≥ 0.1   This study retrospectively collected surgical specimens from 50 consecutive patients diagnosed with invasive BC at the Fourth Hospital of Hebei Medical University between February and March 2023, whose pathological reports indicated HER2 IHC 0. The ethics committee of the Fourth Hospital of Hebei Medical University approved the study protocol (approval No. 2021KY124). The study was carried out in accordance with the ethics standards of the participating institutions and the tenets of the Declaration of Helsinki. As this study did not involve interactions with human subjects or the use of personally identifiable information, informed consent was not required for utilizing existing pathology materials, and no identifiable patient information was revealed. Formalin-fixed, paraffin-embedded tissue sections of 4 μm were subjected to HER2 IHC staining using the VENTANA anti-HER2/neu (4B5) rabbit monoclonal primary antibody from Roche (Basel, Switzerland), carried out on the BenchMarK XT automated IHC platform.   Thirty-six pathologists from four centers (Xingtai People’s Hospital, Cangzhou People’s Hospital, Hebei University Affiliated Hospital, and the Fourth Hospital of Hebei Medical University), all experienced in evaluating HER2 IHC slides in routine clinical practice, visually assessed the 50 HER2 IHC slides under a microscope. The participating pathologists were blinded to the original HER2 IHC pathological report results and the study’s purpose. All pathologists underwent training based on the 2023 ASCO/CAP guidelines, following their practice recommendations to differentiate between HER2 IHC 0 and 1+ using high-power magnification (×40) for HER2 IHC examination. In addition to providing HER2 IHC scores (HER2 IHC 0/1+/2+/3+), the pathologists were also required to report the percentage of HER2 membrane staining to distinguish HER2-null (no staining observed) from HER2-ultralow (incomplete and faint/barely perceptible membrane staining in ≤10% of tumor cells) within HER2 0 cases.   Statistical analyses were carried out using IBM SPSS Statistics (version 26.0; IBM Corporation, Armonk, NY) and GraphPad Prism (8.01; GraphPad Software, LLC, San Diego, CA). Based on the HER2 IHC interpretation results from the pathologists, three scenarios were analyzed: three categories (HER2 IHC 0/1+/2+), four categories (HER2 IHC null/ultralow/1+/2+), and binary classification (HER2 IHC null/non-null). Fleiss κ was used to assess interobserver consistency in HER2 IHC interpretation. The κ values were interpreted as follows: 0.01-0.20: slight agreement, 0.21-0.40: fair agreement, 0.41-0.60: moderate agreement, 0.61-0.80: substantial agreement, 0.81-1.00: almost perfect agreement. The study defined the degree of case interpretation consistency as follows: high consistency: ≥90% of pathologists (31-34 evaluators) agreed on the HER2 IHC result, moderate consistency: ≥75%-<90% of pathologists (26-30 evaluators) agreed, low consistency: >50%-<75% of pathologists (18-25 evaluators) agreed, and challenging cases: ≤50% of pathologists (≤17 evaluators) agreed. In this study, pathologists’ HER2 IHC assessment results were compared with the consensus scores obtained from the majority of 36 pathologists to analyze their interpretation trends. For each pathologist, the proportion of cases assessed below the consensus score ( P below ), the proportion assessed above the consensus score ( P above ), and the overall agreement rate with the consensus score ( P agreement ) were calculated. The interpretation trends were then classified based on the following criteria: (i) Stable: pathologists whose difference between the proportions of cases assessed below and above the consensus score was <0.1, and whose agreement rate with the consensus score was ≥75%; (ii) Fluctuating: pathologists whose difference between the proportions of cases assessed below and above the consensus score was <0.1, but with an agreement rate <75%; (iii) Conservative: pathologists who assessed a higher proportion of cases below the consensus score by at least 0.1; (iv) Aggressive: pathologists who assessed a higher proportion of cases above the consensus score by at least 0.1. The formulas were as follows: Stable: ∣ P b e l o w − P a b o v e ∣ < 0.1 a n d P agreement ≥ 75 % Fluctuating: ∣ P b e l o w − P a b o v e ∣ < 0.1 a n d P agreement < 75 % Conservative: P b e l o w − P a b o v e ≥ 0.1 Aggressive: P a b o v e − P b e l o w ≥ 0.1   Patient characteristics All 50 patients included in the study were female, with a median age of 54 years and an average tumor size of 2.0 cm. Among these cases, 82% (41/50) were diagnosed as having an infiltrating duct carcinoma, not otherwise specified. Estrogen receptor positivity was observed in 76% (38/50) of the cases, and progesterone receptor positivity was seen in 74% (37/50). The Ki67 level was ≤30% in 60% (30/50) of the cases. Tumor-infiltrating lymphocytes showed low infiltration (1%-9%) in 46% (23/50) of cases and moderate infiltration (10%-39%) in 40% (20/50) of cases. Detailed baseline characteristics are presented in , available at https://doi.org/10.1016/j.esmoop.2024.104127 . Interobserver consistency among pathologists Thirty-six pathologists from four centers nationwide conducted microscopic visual evaluations on 50 HER2 IHC slides. The HER2 IHC assessment results from these pathologists are depicted in A. When categorized into three groups (HER2 IHC 0/1+/2+), the overall interobserver consistency among the pathologists was moderate, with a Fleiss κ of 0.344. Consistency within individual centers varied, with Fleiss κ ranging from 0.264 to 0.446 ( B). Further classifying HER2 IHC 0 cases into HER2-null and HER2-ultralow reduced the overall interobserver consistency by approximately 33% under the four-category classification (HER2 IHC null/ultralow/1+/2+), yielding a Fleiss κ of 0.230 (95% CI 0.229-0.230, C). Consistency within individual centers also decreased, with a reduction ranging from 16% to 37%. Giving the positive outcomes of the DB-06 clinical trial, which demonstrated clinical benefits for HER2-ultralow patients, we adjusted the classification to HER2-null versus HER2-non-null. This adjustment improved the overall interobserver consistency among the 36 pathologists compared with the four-category classification (HER2 IHC null/ultralow/1+/2+), with a Fleiss κ of 0.292 (95% CI 0.292-0.293), though the consistency remained suboptimal ( D). Notably, most centers showed improvements in consistency, with center B achieving an approximate 92% increase. Consistency of interpretation for each case As shown in , based on the HER2 scores provided by the 36 pathologists, cases were initially classified into three categories: HER2 IHC 0, IHC 1+, and IHC 2+. Of these cases, 32% (16/50) demonstrated high consistency, 36% (18/50) showed moderate consistency, 30% (15/50) had low consistency, and 2% (1/50) were classified as challenging. When the classification was expanded to four categories (HER2 IHC null/ultralow/1+/2+), the proportion of cases with low consistency increased to 48% (24/50), and the percentage of challenging cases rose significantly from 2% to 28%. Meanwhile, the number of cases with high and moderate consistency decreased by 14 and 8 cases, respectively. Simplifying the classification to two categories (HER2 IHC null/non-null) resulted in 48% (24/50) of the cases remaining in the low-consistency category, while 48% (24/50) achieved high and moderate consistency, and the number of challenging cases decreased. Analysis of trends in pathologists’ interpretation results The evaluation of HER2 IHC scores by different pathologists showed significant variability in the proportion of cases assessed in each category, particularly for HER2-null (median 43%, range 2%-74%) and HER2-ultralow (median 28%, range 0%-78%), followed by HER2 1+ (median 27%, range 8%-46%) ( A). This study also compared each pathologist’s HER2 IHC assessment results with the consensus scores to analyze their interpretation trends, which were categorized into four types: stable, fluctuating, conservative, and aggressive. As shown in B, 42% (15/36) of the pathologists were categorized as aggressive, 25% (9/36) were conservative, 22% (8/36) exhibited fluctuating assessment habits, and only 11% (4/36) were stable in their evaluations. Consistency between reassessment consensus scores and historical scores The consensus scores from the reassessment by the 36 pathologists showed a 72% (36/50) agreement with the historical scores ( , available at https://doi.org/10.1016/j.esmoop.2024.104127 ). When HER2 IHC 0 cases were subdivided into HER2-null and HER2-ultralow categories, the consistency decreased to 54% (27/50). Among these cases, 38% (19/50) were reassessed with scores higher than the historical scores, while 8% (4/50) were reassessed with scores lower than the historical scores ( , available at https://doi.org/10.1016/j.esmoop.2024.104127 ). Retrospective analysis of controversial cases Among the 50 cases analyzed using the four-category interpretation, 14 were identified as challenging . Most of these cases were invasive ductal carcinoma (86%, 12/14), histological grade II (64%, 9/14), and hormone receptor positive (79%, 11/14). Of the controversial cases, six were consensus-scored as HER2-null, five as HER2-ultralow, and three as HER2 IHC 1+. The controversies arose from several factors. HER2 expression exhibited significant heterogeneity, making it challenging for pathologists to consistently interpret the 10% cut-off between HER2-ultralow and HER2 1+. Additionally, some cases showed weak and scattered membrane staining, which pathologists may overlook during microscopic examination. The presence of cytoplasmic staining in tumor cells could also lead to misinterpretation. Furthermore, the degree to which pathologists recognized the clinical significance of differentiating HER2 IHC 0 into HER2-null and HER2-ultralow may have influenced their thoroughness in distinguishing these categories. Finally, there was some disagreement regarding the integrity of weakly stained membranes. These factors collectively contributed to the variability and difficulty in reaching a consensus .   All 50 patients included in the study were female, with a median age of 54 years and an average tumor size of 2.0 cm. Among these cases, 82% (41/50) were diagnosed as having an infiltrating duct carcinoma, not otherwise specified. Estrogen receptor positivity was observed in 76% (38/50) of the cases, and progesterone receptor positivity was seen in 74% (37/50). The Ki67 level was ≤30% in 60% (30/50) of the cases. Tumor-infiltrating lymphocytes showed low infiltration (1%-9%) in 46% (23/50) of cases and moderate infiltration (10%-39%) in 40% (20/50) of cases. Detailed baseline characteristics are presented in , available at https://doi.org/10.1016/j.esmoop.2024.104127 .   Thirty-six pathologists from four centers nationwide conducted microscopic visual evaluations on 50 HER2 IHC slides. The HER2 IHC assessment results from these pathologists are depicted in A. When categorized into three groups (HER2 IHC 0/1+/2+), the overall interobserver consistency among the pathologists was moderate, with a Fleiss κ of 0.344. Consistency within individual centers varied, with Fleiss κ ranging from 0.264 to 0.446 ( B). Further classifying HER2 IHC 0 cases into HER2-null and HER2-ultralow reduced the overall interobserver consistency by approximately 33% under the four-category classification (HER2 IHC null/ultralow/1+/2+), yielding a Fleiss κ of 0.230 (95% CI 0.229-0.230, C). Consistency within individual centers also decreased, with a reduction ranging from 16% to 37%. Giving the positive outcomes of the DB-06 clinical trial, which demonstrated clinical benefits for HER2-ultralow patients, we adjusted the classification to HER2-null versus HER2-non-null. This adjustment improved the overall interobserver consistency among the 36 pathologists compared with the four-category classification (HER2 IHC null/ultralow/1+/2+), with a Fleiss κ of 0.292 (95% CI 0.292-0.293), though the consistency remained suboptimal ( D). Notably, most centers showed improvements in consistency, with center B achieving an approximate 92% increase.   As shown in , based on the HER2 scores provided by the 36 pathologists, cases were initially classified into three categories: HER2 IHC 0, IHC 1+, and IHC 2+. Of these cases, 32% (16/50) demonstrated high consistency, 36% (18/50) showed moderate consistency, 30% (15/50) had low consistency, and 2% (1/50) were classified as challenging. When the classification was expanded to four categories (HER2 IHC null/ultralow/1+/2+), the proportion of cases with low consistency increased to 48% (24/50), and the percentage of challenging cases rose significantly from 2% to 28%. Meanwhile, the number of cases with high and moderate consistency decreased by 14 and 8 cases, respectively. Simplifying the classification to two categories (HER2 IHC null/non-null) resulted in 48% (24/50) of the cases remaining in the low-consistency category, while 48% (24/50) achieved high and moderate consistency, and the number of challenging cases decreased.   The evaluation of HER2 IHC scores by different pathologists showed significant variability in the proportion of cases assessed in each category, particularly for HER2-null (median 43%, range 2%-74%) and HER2-ultralow (median 28%, range 0%-78%), followed by HER2 1+ (median 27%, range 8%-46%) ( A). This study also compared each pathologist’s HER2 IHC assessment results with the consensus scores to analyze their interpretation trends, which were categorized into four types: stable, fluctuating, conservative, and aggressive. As shown in B, 42% (15/36) of the pathologists were categorized as aggressive, 25% (9/36) were conservative, 22% (8/36) exhibited fluctuating assessment habits, and only 11% (4/36) were stable in their evaluations.   The consensus scores from the reassessment by the 36 pathologists showed a 72% (36/50) agreement with the historical scores ( , available at https://doi.org/10.1016/j.esmoop.2024.104127 ). When HER2 IHC 0 cases were subdivided into HER2-null and HER2-ultralow categories, the consistency decreased to 54% (27/50). Among these cases, 38% (19/50) were reassessed with scores higher than the historical scores, while 8% (4/50) were reassessed with scores lower than the historical scores ( , available at https://doi.org/10.1016/j.esmoop.2024.104127 ).   Among the 50 cases analyzed using the four-category interpretation, 14 were identified as challenging . Most of these cases were invasive ductal carcinoma (86%, 12/14), histological grade II (64%, 9/14), and hormone receptor positive (79%, 11/14). Of the controversial cases, six were consensus-scored as HER2-null, five as HER2-ultralow, and three as HER2 IHC 1+. The controversies arose from several factors. HER2 expression exhibited significant heterogeneity, making it challenging for pathologists to consistently interpret the 10% cut-off between HER2-ultralow and HER2 1+. Additionally, some cases showed weak and scattered membrane staining, which pathologists may overlook during microscopic examination. The presence of cytoplasmic staining in tumor cells could also lead to misinterpretation. Furthermore, the degree to which pathologists recognized the clinical significance of differentiating HER2 IHC 0 into HER2-null and HER2-ultralow may have influenced their thoroughness in distinguishing these categories. Finally, there was some disagreement regarding the integrity of weakly stained membranes. These factors collectively contributed to the variability and difficulty in reaching a consensus .   The preliminary results of the DB-06 clinical trial confirmed that T-DXd offered clinical benefits to patients with metastatic HER2-ultralow BC, similar to those with HER2-low BC. This finding underscores the importance of pathologists accurately distinguishing between HER2-null and HER2-ultralow within HER2 IHC 0 cases. Our study evaluated the diagnostic consistency of 36 pathologists who reassessed 50 BC cases previously reported as HER2 IHC 0. The results revealed poor consistency in distinguishing between HER2-null and HER2-ultralow or HER2-null and HER2-non-null. This is the first study to directly highlight the significant challenges pathologists encounter when interpreting low HER2 expression levels. Our study demonstrated that when distinguishing between HER2 IHC 0 and 1+, 34% of the cases achieved high consistency, similar to the 26% reported by Fernandez et al. when distinguishing between HER2 IHC 0 and 1+. The reassessed consensus scores showed a 72% agreement with the historical scores, which closely aligns with the 69.9% reported by Viale et al. However, when pathologists further differentiated between HER2-null, -ultralow, and 1+, only 4% of the cases achieved high consistency. Although the proportion of cases with high consistency increased to 18% when distinguishing between HER2-null and HER2-non-null, it remained lower than the results for distinguishing only HER2 IHC 0 and 1+. This indicated that diagnosing HER2-ultralow was more challenging for pathologists. The primary reasons for this challenge could be attributed to two main factors. Firstly, distinguishing between HER2-null and HER2-ultralow depends on the presence or absence of HER2 staining. Due to the principle of microscopy magnification, faint staining was only clearly observable at high magnification (×40). Pathologists may not have accurately selected the appropriate fields of views for further confirmation at high magnification during the initial low magnification scanning, potentially leading to an underestimation of HER2-ultralow cases. Additionally, individual differences in color perception among pathologists, along with potential cytoplasmic staining and non-specific staining, contributed to significant variability in distinguishing HER2-null from HER2-ultralow cases. This variability was evident in our findings, with the greatest discrepancies observed in the assessments of HER2-null cases (range 2%-74%) and HER2-ultralow cases (range 0%-78%). Only 11% of pathologists reported stable HER2 status, with 42% tending to give higher HER2 diagnostic results and 25% tending to give lower results. Secondly, the distinction between HER2-ultralow and HER2 1+ was based on a 10% cut-off value. The semi-quantitative IHC assessment made precise quantification challenging, particularly given the heterogeneity of HER2 expression. Pathologists struggled to provide reproducible evaluations of complex HER2 membrane staining patterns, especially in cases with low-level expression of HER2. Artificial intelligence (AI), with its robust learning capabilities and advantages in quantitative image assessment, holds promise for enhancing HER2 IHC evaluations. Jung et al. found that AI-assisted pathologist assessments of HER2 IHC consistency improved from 49.3% to 74.1%, with a notable increase in the consistency of HER2 IHC 1+ cases from 26.5% to 70.7%. Developing AI algorithms tailored for low HER2 expression levels could help address the challenges pathologists face in distinguishing HER2-null, -ultralow, and 1+ cases. Common AI algorithms used for HER2 IHC evaluation included the HER2-CONNECT algorithm, convolutional neural network algorithm, HER2 4B5 algorithm, and multi-class logistic regression algorithm. , , , , , , These can be employed for automated whole-slide image (WSI) assessment of HER2 status or for AI-assisted evaluation of multiple representative regions of interest selected by pathologists. Given the challenges of accurately identifying and quantifying HER2-ultralow, WSI-based automated assessment might be more suitable for evaluating low HER2 expression cases. Currently, HER2-ultralow detection in clinical practice primarily relies on IHC. However, existing HER2 IHC testing was designed to distinguish HER2 IHC 3+ from other scores and lacks the precision needed to differentiate between HER2-null and HER2-ultralow. Several factors before and during IHC testing can affect the detection of low-level HER2 protein expression. For instance, the type of specimen and the quantity of the sample can both impact HER2 status diagnosis. Na et al. analyzed the consistency of HER2 status between core needle biopsy and surgical specimens in 1387 invasive BC patients, finding a κ value of 0.587 for HER2 classification (HER2 0/low/positive). Bar et al. found that with each additional biopsy, about one-third of patients previously classified as HER2 IHC 0 could be reclassified as HER2-low, with the likelihood of detecting HER2-low increasing with the number of biopsies. Additionally, the selection of antibodies can affect the diagnosis of HER2 status. Rüschoff et al. showed that the HercepTest antibody identified more cases of HER2-low than the VENTANA 4B5 antibody. These findings indicated that several modifiable testing parameters in IHC can influence the detection of HER2-ultralow/low. Therefore, there is a need for more stable, quantifiable, precise, and cost-effective HER2 detection methods. Some studies suggested that RNAScope might be a promising new technique to optimize the detection of HER2-ultralow and HER2-low, although further clinical data were required for validation. Although the DB-06 trial confirmed the clinical benefit of T-DXd for patients with metastatic HER2-ultralow BC, the exact lower limit of T-DXd’s efficacy, particularly the threshold for defining HER2-ultralow, remains unclear. Jung et al. explored the critical threshold for differentiating HER2-null from HER2-ultralow using an AI-based WSI analysis tool. Their results suggested that a 6.0% membrane-specific HER2 score was the optimal threshold to distinguish between HER2-null cells and cells with incomplete and faint/barely perceptible staining. However, this threshold was derived from a model-based analysis and lacks clinical validation. The ongoing DB-15 clinical trial aims to evaluate the efficacy and safety of T-DXd in patients with unresectable and/or metastatic HER2 IHC 0 BC. It will also include patients with HER2 IHC 0 (no staining) to potentially refine the patient population benefiting from T-DXd and help establish a more precise definition of HER2-ultralow. This study evaluated the ability of pathologists from multiple centers to differentiate between HER2-null and HER2-ultralow by reassessing HER2 IHC slides of BC specimens previously diagnosed as HER2 IHC 0. A key strength of this study was the inclusion of a large cohort of pathologists from various centers, which enhanced the reliability of the findings. Additionally, by exclusively focusing on cases previously diagnosed as HER2 IHC 0, the study eliminated potential confounding from other HER2 categories. Besides, in our study, all pathologists reviewed the same HER2 IHC slides, minimizing the influence of pre-analytical and analytical variables and further emphasizing the considerable variability in interpretation. However, the study had some limitations, including the relatively small number of cases reviewed. Nonetheless, the results clearly highlighted the poor interobserver consistency in distinguishing HER2-null from HER2-ultralow. Furthermore, the recent publication of the DB-06 trial results may have influenced the emphasis on accurately differentiating HER2-ultralow from HER2-null, potentially affecting the pathologists’ assessment. However, we did not survey pathologists on this matter, and the increased recognition of the clinical significance of HER2-ultralow might significantly alter the study results. It is also important to note that while the DB06 trial demonstrated the benefit of T-DXd in advanced hormone receptor-positive, HER2-ultralow BC patients, its efficacy in hormone receptor-negative patients remains unclear. This study focused on evaluating pathologists’ ability to identify HER2-ultralow cases without considering hormone receptor status. In conclusion, this study highlighted significant interobserver variability in the precise differentiation between HER2-null, -ultralow, and 1+ categories. It underscored the need for more clinical data to refine the definition of HER2-ultralow. Future efforts should focus on incorporating AI-based algorithms specifically designed for HER2-ultralow assessment, as well as more advanced detection methods, to assist pathologists in improving the accuracy and consistency of HER2 evaluations.   