HER2 Assay Research Articles

The efficacy and potential mechanisms of pyrotinib in targeting EGFR and HER2 in advanced oral squamous cell carcinoma

BackgroundHuman epidermal growth factor receptor 2 (HER2) plays an important role in the progression of multiple solid tumors and induces resistance to epidermal growth factor receptor (EGFR) target treatment. However, the expression status and the clinical significance of HER2 in oral squamous cell carcinoma (OSCC) is still controversial. Pyrotinib (PYR) is a promising novel EGFR/HER2 dual inhibitor, whose efficacy in OSCC has not been determined.Methods57 locally advanced de novo OSCC patients were included in this study to investigate the relationship between the HER2 expression levels and the prognosis by the tissue microarray analysis (TMA). In vitro and in vivo experiments were performed to retrieve the efficacy of PYR in OSCC. The main downstream of HER2 was evaluated by western blotting in OSCC cell lines and xenograft tumors to explore the potential mechanism of PYR.ResultsThis study revealed the primary tumor of OSCC had higher HER2 expression levels. Patients with HER2 overexpression had poor overall survival (P < 0.014) and poor disease free survival (P < 0.042). In vitro, PYR suppressed the proliferation, colony formation and migration of OSCC cells. It also promoted apoptosis of OSCC cells and induced cell cycle arrest. Furthermore, PYR was able to inhibit the occurrence and development of OSCC effectively in vivo. Western blotting revealed that PYR suppressed OSCC by inhibiting the phosphorylation of HER2, AKT and ERK.ConclusionsThis study exhibited the anti-OSCC effects of PYR in vitro and in vivo, and demonstrated PYR inhibited OSCC cells by inducing apoptosis via the HER2/ AKT and ERK pathway. The result of this study also indicated locally advanced OSCC patients might benefit from HER2 assay and EGFR/HER2 dual inhibit treatment.

Development and Validation of a HER2-Low Focused Immunohistochemical Scoring System With High-Interobserver Concordance: The Australian HER2-Low Breast Cancer Concordance Study

The DESTINY Breast-04 trial revealed survival advantages of trastuzumab deruxtecan for women with metastatic HER2-low breast cancer (1+ or 2+ immunohistochemistry [IHC], without amplification). Although this trial applied the 2018 Americal Society of Clinial Oncology (ASCO)/College of American Pathologists (CAP) HER2 IHC scoring criteria, the subjectivity and imprecision in IHC scoring have raised concerns that patients’ treatment may be misaligned. Our group of 9 experienced breast pathologists collated a deidentified set of 60 breast cancer core biopsies from 3 laboratories, evaluated with the Ventana 4B5 HER2 assay and mostly scored locally as HER2 0 or 1+. Based on ASCO/CAP 2018 criteria and our extensive experience of reporting HER2 IHC, we specified scoring conventions for cancers with low levels of HER2 protein expression, articulating specific scoring pitfalls. Each pathologist then reviewed digitized whole slide images of the IHC slides and scored the HER2 expression for each case. At a subsequent consensus workshop, we reviewed the cases jointly to establish consensus scores for each case and determine the percentage of HER2 expressing tumor cells. Consensus was reached on all cases, with 40 classified as 1+ and 3 as 2+ (not amplified), totaling 43 (71.7%) HER2-low cancers. The remaining cases were HER2 0. In 93.3% of cases (56/60), the consensus score matched with the majority opinion of pathologists’ independent scores. Seven (41.2%) of the 17 cases reported locally as HER2 0 were classified as HER2 low. Conversely, among 32 cases with local scores of 1+, 7 (21.8%) were reclassified as ultralow or null. Individual pathologists’ accuracy in matching the consensus scores ranged from 73.3% to 91.67% (mean, 80.74%). Among HER2-low cancers those in which <20% of the tumor cells expressed HER2 had the lowest concordance levels. Observers Cohen’s κ coefficients for concordance were excellent for 4, good in 1, and moderate in the 4 observers. This reference set of cases with expert consensus HER2 scores will be invaluable for peer training and development of our national external quality assurance program for HER2-low cancers. For assessing breast cancers at the low end of HER2 protein expression, our targeted scoring criteria and explicit instruction on pitfalls improved pathologists’ accuracy and concordance.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² &amp;gt; 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

Abstract PO4-25-12: Improving HER2 Low Scoring Consistency and Accuracy: Insights from the Australian HER2 Low Concordance Study for Invasive Breast Cancer

Abstract Introduction: The Destiny Breast-04 Trial (DB-04) demonstrated the survival benefits of Trastuzumab Deruxtecan (T-DXd) for women with metastatic HER2 Low breast cancer, characterised by 1+ or 2+ IHC staining without amplification. While the DB-04 study applied the standard 2018 ASCO CAP IHC scoring criteria, in clinical practice, distinguishing HER2 0 from 1+ cancers is challenging as i) HER2 Low is not a biologically distinct subset of breast cancer, ii) there are no reference standards for HER2 Low cancers, iii) second-tier test, like ISH, are not applicable, and iv) there are no known controls for cases that have 0 or 1+ HER2 scores. For two decades this distinction was clinically immaterial, but now differentiating between HER2 0 and 1+ has now become crucial for determining patient eligibility for T-DXd therapy. Concerns regarding the subjectivity, imprecision and poor concordance between pathologists in scoring IHC in HER2 Low cancers raise the potential for misalignments in patient treatment. Ensuring pathologists have access to focused training for interpreting IHC scores at the low end of the HER2 expression spectrum, quality assurance procedures and reference sets are essential steps to help pathologists assess HER2 Low breast cancers more accurately and consistently. Design: In this study, a group of 9 experienced breast pathologists compiled a deidentified set of 60 breast cancer core biopsies from 3 laboratories. The Ventana 4B5 HER2 assay had been used for evaluation and the local laboratories had scored the samples as HER2 0 or 1+. We teased out the ASCO CAP 2018 criteria and used our collective expertise of reporting HER2 IHC for many years to specify HER2 Low-focused scoring conventions, including some potential pitfalls. Subsequently, using these conventions, each pathologist reviewed digitized whole slide images of the IHC slides and scored HER2 expression for each case. At a consensus workshop, the cases were jointly reviewed to establish consensus scores and determine the percentage of HER2-expressing tumor cells in each case. We then evaluated the concordance between individual pathologists' HER2 scores and the consensus opinion and ascertained reasons for discordance. Results: Among the cases discussed during the consensus conference, 43 out of 60 (71.7%) were classified as HER2 Low, with 40 cases designated as 1+ and three as 2+ (known to be not amplified). The consensus score matched the majority opinion of the pathologists' independent scores in 93.3% (56 out of 60) of the cases. Utilizing the HER2 Low-focused IHC scoring conventions, 7 out of 17 (41.2%) cases locally reported as HER2 0 were reclassified as HER2 Low. Conversely, among the 32 cases with local scores of 1+, 7 (21.8%) were reclassified as ultralow or null. When compared to the consensus score, individual pathologists' scores demonstrated concordance levels ranging from 71.7% to 91.7%, with a mean concordance rate of 81.3%. Cases with less than 20% of tumor cells expressing HER2 had lower inter observer concordance. This reference set of cases with expert consensus HER2 scores obtained through our study will be invaluable for peer training and the development of external quality assurance programs for HER2 Low cancers, including the quality assurance program of the Royal College of Pathologists of Australasia. Conclusion: This study revealed that when breast pathologists were provided explicit instructions on scoring pitfalls and HER2 Low-focused scoring conventions, their HER2 scores were concordant with expert consensus scores in 71.7% to 91.7% of cases. Discordant cases primarily involved cases with less than 20% of tumor cells expressing HER2. Utilising such an approach, peer training and quality assurance procedures will improve the accuracy and consistency of HER2 IHC assessment for better patient care. Reassessing older cases using HER2 Low focused scoring conventions may result in revisions of HER2 scores from HER2 Low to zero, and vice versa. Table. Individual pathologists' concordance with the consensus HER2 IHC score Applying our HER2 Low-focused IHC scoring conventions in a set of 60 core biopsies of invasive breast cancer with low or 0 HER2 protein expression. Citation Format: Gelareh Farshid, Beena Kumar, Nirmala Pathmanathan, Hema Mahajan, Ben Dessauvagie, Jane Armes, Cameron Snell, Amardeep Gilhotra. Improving HER2 Low Scoring Consistency and Accuracy: Insights from the Australian HER2 Low Concordance Study for Invasive Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-12.

Abstract 3758: Evaluation of breast cancer-derived extracellular vesicles as diagnostic indicator for HER2 status

Abstract HER2-low is a new distinct type of breast cancer that is characterized by low levels of the HER2 protein. As treatment paradigms for HER2-low breast cancer are rapidly evolving with the approval of antibody-drug conjugates, identification of HER2-low breast cancer with more sensitive and reproducible HER2 assays is needed. We aimed to investigate the utility of liquid biopsy in isolating tumor-derived extracellular vesicles (TDEs) for diagnosis of HER2 status. We adopted an affinity-based approach to isolate TDEs and performed CNV analysis of the ERBB2/neu genes using the tumor DNA present in TDEs (etDNA). HER2 status of etDNA had been determined by droplet digital PCR using HER2/EIF2C ratio, a highly accurate method to quantify DNA copy number. In our study, we utilized a variety of breast cancer cell lines categorized as HER2-low (MDA-MB-231, MCF7, ZR-75-1) and HER2-high (MDA-MB-453, BT-474, SK-BR-3) to confirm HER2 expression in extracellular vesicles (EVs) by flow cytometry. This analysis successfully demonstrated that EVs effectively reflect the HER2 status of the tumor cells, evidenced by a strong Pearson correlation coefficient (r=0.987, p&amp;lt;0.001). Further clinical validation was conducted with plasma samples from 22 breast cancer patients. In the clinical study, we observed a significant correlation between the CNV of tumor DNA present in TDEs and the HER2 status of primary tumors (p&amp;lt;0.01). Additionally, the Receiver Operating Characteristic (ROC) analysis yielded an impressive area under the curve (AUC) value of 0.838 (p&amp;lt;0.01), demonstrating a high level of accuracy in differentiating between high and low HER2 groups, with a sensitivity of 55.6% and a specificity of 100%. In summary, we show potential diagnostic tools for real-time, less invasive determination of HER2 status in primary tumors, providing continuous monitoring methods for HER2 status. Although further validation in larger cohorts and prospective studies is needed, our study indicates that TDE analysis could contribute to the assays for identification of the HER2 status in breast cancer patients. Citation Format: Sol Moon, Min Woo Kim, Young Kim, Suji Lee, Hyojung Lee, Joon Ye Kim, Seung Il Kim, Jee Ye Kim. Evaluation of breast cancer-derived extracellular vesicles as diagnostic indicator for HER2 status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3758.

Impact of HER2 assay sensitivity on the ability to detect tumors with low to ultra-low HER2 expression: A TMA study on more than 10,000 tumors from more than 100 tumor entities

Abstract Introduction/Objective Detection of low level HER2 expression is important for therapy with new HER2 inhibitors in breast cancer. HER2 assays with high sensitivity are therefore needed. These may identify low level HER2 expression in various types other than breast cancer. Methods/Case Report A tissue microarray containing &amp;gt;10,000 tissue samples from more than 100 different tumor types and subtypes was analyzed for immunohistochemical expression of HER2 using different immunohistochemistry protocols and anti-HER2 antibodies, including the HercepTest and laboratory developed tests designed for high sensitivity. HER2 IHC evaluation included the recording of HER2 low (1+), ultralow (any staining, less than 1+), and 0 (negative). Results (if a Case Study enter NA) Irrespective of the assay sensitivity all assays showed the expected/tolerable association with HER2 FISH data in 308 breast cancers. In non-breast cancers, the use of different assays yielded variable rates of low or ultra-low HER2 expression in different tumor entities. For example, low (1+) and ultra-low (+) expression was observed in 7-24% (1+) and 11-28% (+) of serous ovarian cancers, 6.2-24% (1+) and 3.4-19% (+) of endometrioid endometrial cancers, 5.5-14% (1+) and 7.7-13% (+) of intestinal gastric cancers, 1.8-9.2% (1+) and 4.5- 11% (+) of ductal adenocarcinomas of the pancreas, 19-33% (1+) and 5.5-11% (+) of muscle invasive urinary bladder cancers, 2.9-16% (1+) and 4.3-12% (+) of adenocarcinomas of the colon. Conclusion Low level HER2 expression occurs commonly in many cancer types. The rate of identifiable tumors with HER2 low or HER2 ultra-low expression varies depending on assay parameters that can easily be modified. Irrespective of assay sensitivity, our data identify various tumor entities with significant fractions of “HER2-low” cases that might benefit from therapy with new antibody drug conjugates.

110P Detection of HER2 overexpression in biliary tract cancers: Comparison of AmoyDx® HER-2 (29D8) assay with ventana PATHWAY anti-HER-2/neu (4B5) assay

110P Detection of HER2 overexpression in biliary tract cancers: Comparison of AmoyDx® HER-2 (29D8) assay with ventana PATHWAY anti-HER-2/neu (4B5) assay

Synthesis of novel pyrimido[4,5-b]quinolines as potential anticancer agents and HER2 inhibitors.

A series of N-arylpyrimido[4,5-b]quinolines 3a-e and 2-aryl-2,3-dihydropyrimido[4,5-b]quinoline-4(1H)-ones 5a-e was designed and synthesized as potential anticancer agents against breast cancer. Compounds 3e, 5a, 5b, 5d, and 5e showed promising activity against the MCF-7 cell line. Among them, compound 5b was the most active with IC50 of 1.67 μM. Compound 5b promoted apoptosis and induced cell cycle arrest at S phase. 5b increased the level of pro-apoptotic proteins p53, Bax, and caspase-7 and inhibited the anti-apoptotic protein Bcl-2. Furthermore, all the synthesized compounds were docked into the crystal structure of HER2 (PBD: 3 pp0). Compounds 3e, 5a, 5b, 5d, and 5e showed good energy scores and binding modes. Finally, Compound 5b was evaluated on the HER2 assay and revealed good inhibition with IC50 of 0.073 μM.

Comprehensive Analysis of Human Epidermal Growth Factor Receptor 2 Through DNA, mRNA, and Protein in Diverse Malignancies.

Human epidermal growth factor receptor 2 (HER2) expression (protein immunohistochemistry [IHC] or gene amplification [copy-number variation, CNV]) predicts anti-HER2 therapy responsiveness, although recently it was shown that even low HER2-expressing breast cancers benefit from trastuzumab-deruxtecan. Little is known about HER2 transcriptomic (mRNA) expression, and comparisons between genomic, mRNA, and protein HER2 assays. HER2 status was evaluated using clinical-grade IHC (protein), quantitative reverse transcription polymerase chain reaction (mRNA), and next-generation sequencing (NGS; amplifications). Multi-institutional HER2 testing was performed on 5,305 diverse cancers including non-small-cell lung (n = 1,175), breast (n = 1,040), and colon cancers (n = 566; N = 3,926 tested for CNV; N = 1,848, mRNA; N = 2,533, IHC). Overall, 4.1% (161/3,926) had NGS HER2 amplification; 33.3% (615/1,848) had mRNA overexpression; and 9.3% (236/2,533) were IHC-positive. In 723 patients with all three tests (CNV/mRNA/IHC), various amplification/expression patterns occurred: 7.5% (54/723) had all three HER2 tests positive; 62.8% (454/723) had all three tests negative. Discrepant patterns between amplification and overexpression emerged. For instance, 20% (144/723) of patients had mRNA overexpression alone with negative CNV and IHC. A range in values for only mRNA+ cases occurred in different tumor types (eg, 16.9%, breast; 5%, hepatobiliary). There were 53 patients with various tumors from our institution who had all three assays attempted; 22 tested positive for HER2, and seven received anti-HER2 therapy: two patients achieved response: one (esophageal cancer), complete response (≥42 months); one (cholangiocarcinoma), who only had HER2 mRNA positivity (tissue was inadequate for IHC and CNV assessment), partial response (≥24 months) on HER2-based regimens. We demonstrate variability of HER2 (protein and mRNA) expression and amplification using comprehensive assays (CNV, mRNA, and IHC) among diverse cancers. As HER2-targeted therapy indications expand, the relative importance of these modalities merits further evaluation.

Impact of HER2 assay sensitivity on the ability to detect tumors with low to ultra-low HER2 expression: A TMA study on more than 10,000 tumors from more than 100 tumor entities.

3137 Background: Detection of low level HER2 expression (HER2 low) is a prerequisite for therapy with new HER2 inhibitors in advanced breast cancer. Currently, tumors with immunohistochemical 1+ expression, and possibly also those with lower expression, are considered HER2 low. Therefore, the question of optimal sensitivity of HER2 assays is of increasing importance for clinical HER2 testing. In addition, there is hope that tumor types other than those with typical 3+ positivity may also benefit from these therapies. The aim of this study was to systematically investigate the influence of HER2 immunohistochemistry assay parameters on the detection rate of tumors with HER2 low expression in different tumor types. Methods: A tissue microarray containing &gt;10,000 tissue samples from more than 100 different tumor types and subtypes was analyzed for immunohistochemical expression of HER2 using different immunohistochemistry protocols and anti-HER2 antibodies, including the HercepTest and laboratory developed tests designed for high sensitivity. HER2 IHC evaluation included the recording of HER2 low (1+), ultralow (any staining, less than 1+), and 0 (completely negative). Results: Irrespective of the assay sensitivity all assays showed the expected/tolerable association with HER2 fluorescence in situ data in a cohort of 356 breast cancers. For the HercepTest, HER2 amplification was seen in 93% of 28 3+ cases, 55% of 11 2+ cases and in 1.5% of 274 0 and 1+ cases. For the most sensitive assay, amplification was seen in 90% of 30 3+ cases, 19% of 37 2+ cases and in 0.8% of 249 0 and 1+ cases. In non-breast cancers, the use of different assays resulted in highly variable results regarding the range of tumors with low or ultra-low HER2 expression in different tumor entities. For example, low (1+) and ultra-low (+) expression was observed in 7-24% (1+) and 11-28% (+) of serous ovarian cancers, 6.2-24% (1+) and 3.4-19% (+) of endometrioid endometrial cancers, 5.5-14% (1+) and 7.7-13% (+) of intestinal gastric cancers, 1.8-9.2% (1+) and 4.5-11% (+) of ductal adenocarcinomas of the pancreas, 19-33% (1+) and 5.5-11% (+) of muscle invasive urinary bladder cancers, 2.9-16% (1+) and 4.3-12% (+) of adenocarcinomas of the colon, 0-7.7% (1+) and 2.4-4.6% (+) of clear cell renal cell carcinomas, 4.8-6.5% (1+) and 6.0-8.6% (+) squamous cell carcinomas (SQCC) of the larynx, 6.3-7.1% (1+) and 2.4-6.3% (+) SQCC of the esophagus, 0.8-26% (1+) and 2.4-12% (+) SQCC of the cervix, as well as 1.5-5.1% (1+) and 1.5-2.2% (+) SQCC of the vulva. Conclusions: Low level HER2 expression occurs commonly in many cancer types. The rate of identifiable tumors with HER2 low or HER2 ultra-low expression greatly varies depending on assay parameters that can easily be modified. Clinical trials are needed to identify the HER2 expression thresholds to identify patients who may benefit from anti-HER2-low therapies.

Abstract P6-04-16: ART: Automated Region segmentation of Tumor on HER2-stained breast cancer tissue

Abstract Background Computational pathology-based methods, eg, Quantitative Continuous Scoring (QCS) [Gustavson, SABCS 2020], are built to provide objective and quantitative methods to assess HER2 expression in breast cancer (BC). For accurate HER2 quantification, it is important to exclude non-invasive epithelium from analysis since HER2 overexpression could be more frequent in ductal carcinoma in situ (DCIS) and pleomorphic lobular carcinoma in situ (PLCIS) than in invasive BC [Lari, J Cancer 2011]. Generally, computational pathology-based approaches require experts to delineate the invasive BC regions of interest for analysis and exclude all benign/non-invasive epithelium. Developing a tool that delineates the invasive BC regions automatically without human intervention to avoid subjectivity of manual annotation by pathologists is ideal. We developed a novel, deep-learning–based system, called DualScaleNet, to perform Automated Region segmentation of Tumor (ART) by automatically identifying the invasive BC regions and excluding benign/non-invasive epithelium on HER2-stained digitized images. Identification and diagnosis of these regions, especially the in situ tumors are a challenge as they can mimic benign and invasive lesions causing wrong HER2 evaluation. Additional stains (eg, p63 for myoepithelial cells or laminin for basement membrane) are often required for diagnosis [Pinder, Mod Pathol 2010] but were not available for this study. Methods DualScaleNet works simultaneously on HER2-stained immunohistochemistry (IHC) image patches at 2 different resolutions. The target branch uses a higher resolution RGB image (0.5 μm/pixel) to learn accurate local details; the context branch uses a lower resolution image (4.0 μm/pixel) to incorporate more context in visual learning. The algorithm generates 4 output image layers representing probabilities of 4 classes: invasive tumor, ductal/lobular carcinoma in situ, benign epithelium, and other tissue. The final segmentation result is generated by assigning each image pixel the class with the largest probability value. The algorithm was trained using ground truth (GT) annotations generated by 5 pathologists using 6157 square field of views (FOVs), 200-500 μm in size. These FOVs were collected from 850 whole slide images (WSI), spanning 9 commercial BC sample cohorts stained with different HER2 assays and scanned by several versions of the Aperio AT2 scanner. The samples included a mixture of biopsies and resections and covered different BC histologies and HER2 staining intensities. To evaluate the reproducibility of tumor area detection by human pathologists, an interpathologist comparison in detection of invasive tumor regions was performed using 225 FOVs annotated by multiple pathologists. Results Analysis generated an average Dice/F1 Score of 81.6% among different pathologists for invasive cancer. On the same sample set (independent of the ART training set), the invasive cancer detection by the ART algorithm was on par with human pathologists, achieving a similar average Dice/F1 score of 80.7%. Conclusions Novel deep learning-based ART algorithm provides accurate segmentation of invasive cancer on HER2-stained IHC images. The performance was verified against the GT annotations provided by multiple pathologists. Since the algorithm is trained using annotations from multiple pathologists, it is not possible to generate higher accuracy with computational pathology than is achievable between independent pathologists. Importantly, the same WSI read by the ART will consistently output the exact same tumor region identification result thus removing the inherent human subjectivity and variability, while improving the turnaround time for analysis. This development serves as the necessary foundation upon which a computational pathology-based diagnostic can be built. Citation Format: Ansh Kapil, Anatoliy Shumilov, Philipp Wortmann, Sihem Khelifa, Jessica Chan, Michel Vandenberghe, Craig Barker, Mark Gustavson, Danielle Carroll, Hadassah Sade, Günter Schmidt. ART: Automated Region segmentation of Tumor on HER2-stained breast cancer tissue [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-16.

Abstract HER2-17: HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer

Abstract Background: The HER2 antibody drug conjugate (ADC) fam-trastuzumab deruxtecan-nxki (T-DXd) significantly improves outcomes over standard chemotherapy in pts with HER2-LOW (IHC 1+ and IHC 2+ FISH-) metastatic breast cancer (MBC). Data from the DAISY (NCT04132960) trial in pts with HER2 IHC 0 or “ultra low” MBC revealed median progression-free survival (PFS) of 4.2 mos vs 6.7 mos in HER2-LOW and 11.1 mos in HER2 3+ pts (Diéras V, et al. Cancer Res. 2022;82(4_Suppl):PD8-02). There is an urgent need to develop methods to accurately discern HER2 LOW expression versus HER2 IHC 0/ultra-low expression so appropriate pts may receive T-DXd. We have developed a quantitative HER2 assay using reverse phase protein array (RPPA) technology with laser capture microdissection (LCM) enrichment of tumor epithelium that measures HER2 expression over a 65-fold dynamic range in HER2 IHC 0 to 3+ breast cancers. In I-SPY 1 and 2 trials we found that RPPA-assessed quantitative HER2 and activated/phosphorylated HER2 expression predicted for pathologic complete response in pts with HER2 IHC 0, HER2-LOW and HER2 3+ disease with various HER2 and other targeted agents. We then developed and validated the first CLIA/CAP-accredited quantitative HER2 protein expression and activation assay, and now explore quantitative HER2 expression in HER2 IHC 0/ultra low disease. Given the recent approval of sacituzumab govitecan-hziy in TNBC and the results of the TROPiCS-02 (NCT03901339), we also evaluated quantitative RPPA-based TROP-2 expression levels in HER2 IHC 0 breast cancer. Methods: LCM-enriched tumor epithelium was obtained from freshly cut FFPE core needle and resected breast cancer samples from 175 pts with pathology-determined HER2 IHC 0 status (N=68 ER+ and N=107 ER-). Quantitative HER2 output is generated as a relative intensity unit (RU) that is interpolated to an internal calibrator curve and a population referent comprised of known HER2+ (IHC 3+ and 2+/FISH+), HER2-LOW and HER2 IHC 0/ultra-low tumors to generate population-based cut-points as a referent. RPPA-based quantitative HER2 expression are reported as one of four levels of expression across the HER2 dynamic range observed: “non/extremely low”, “modest“, “moderate”, and “high”. Quantitative TROP2 protein expression levels were also measured for each case by the RPPA assay on the same lysate using the same adjectival determinants of relative expression as HER2. Results: For the HER2 IHC 0 ER+ cohort, we observed HER2 protein expression over a 25-fold dynamic range and that 57% (N=39) had HER2 non/extremely low, 37% (N=25) modest, and 4 (6%) moderate/high HER2 expression by RPPA. For the HER2 IHC 0 ER- cohort, we observed HER2 protein expression over a 15-fold dynamic range and that 71% (N=76) had non/extremely low, and 31% (N=29%) modest HER2 expression. We observed TROP-2 protein expression over a 35-fold dynamic range across all tumors measured, and it was found to be at least modestly expressed in 95% (37/39) ER+ and 95% (72/76) ER- of the IHC 0/ultra-low tumors that were also found to be HER2 non/extremely low by RPPA. Conclusions: LCM-RPPA quantitative assessment of HER2 expression showed that nearly 40% of ER+ IHC 0 and 30% of ER- IHC 0 “ultra-low” breast cancers actually had modest to moderate HER2 expression. Interestingly, this frequency approximates the response rate of 30% seen with T-DXd in HER2 IHC 0 pts in the DAISY trial. Quantitatively measured TROP2 is at least modestly expressed in the vast majority of RPPA-assessed HER2 IHC 0 cancers regardless of ER status, making a TROP2-directed ADC an attractive therapeutic option for these pts. If validated in larger studies, LCM-RPPA-based HER2 expression could provide a better understanding of the potential for therapeutic efficacy with T-DXd in patients with HER2 IHC “ultra-low” disease, and may better define true ultra-low HER2 expression in HER2 IHC 0 tumor biopsies at baseline and refine the lower limit of the HER2-LOW designation. Citation Format: Emanuel F. Petricoin, Brian A. Corgiat, Joyce O’Shaughnessy, Patricia LoRusso, Kris Weinberg, Justin Davis, Chelsea Gawryletz. HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-17.

Abstract HER2-13: HER2-13 Proficiency assessment of HER2-low breast cancer scoring with the Ventana PATHWAY 4B5 and Dako HercepTest HER2 assays and the impact of pathologist training

Abstract Background: Based on the results of the DESTINY-Breast04 study, the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan (T-DXd) and the Ventana PATHWAY 4B5 companion diagnostic were recently approved by the US Food and Drug Administrated for the treatment and identification of patients with HER2-low (IHC2+/ISH- or IHC1+) (Modi et al. N Engl J Med 2022). The efficacy of T-DXd in HER2-low mBC highlights the need to distinguish lower ranges of HER2 IHC expression, which has been reported to be more challenging than scoring high HER2 expression (Fernandez et al. JAMA Oncol 2022). Here we report on current real-world HER2-low interpretation proficiency and the impact of training for participating pathologists in HER2-low scoring. Methods: Pathologists from laboratories across the US, EU, Japan, Australia, and Brazil were invited to use a digital pathology platform (Pathotrainer) to interpret HER2 digital images using ASCO/CAP 2018 scoring criteria. Two whole-slide imaging sample sets of representative study cases were compiled for Ventana PATHWAY 4B5 or Dako HercepTest (HcT) stained tumors. Another sample set (n = 25) was developed for a 4 hour virtual training session based on the ASCO/CAP 2018 guideline with some practical considerations. A steering committee (SC) of 8 pathology experts was formed to guide the study. Pathologists’ score was compared with a reference score as determined by independent review of 3 experts in HER2 pathology. Paired study cases (n = 14) considered challenging due to difficult-to-interpret staining patterns were reevaluated by the SC members. Concordance and efficacy of training were measured by Cohen’s weighted kappa (κ) coefficient, overall rater agreement (ORA), and receiver operating characteristic curve statistics. The primary endpoint was real-world concordance and ORA. The secondary endpoints were post-training concordance and ORA and correct identification of HER2 IHC 0 and HER2-low. Results: Pre-training baseline or real-world scores were taken by 77 pathologists in 14 countries (n = 49 for 4B5, n = 28 for HcT) and 74 pathologists completed post-training scores (n = 48 for 4B5, n = 26 for HcT). HER2 scoring proficiency of pathologists was high for both assays when assessed on ASCO/CAP binary HER2-negative and -positive status, irrespective of training (4B5: κ = 0.96, ORA =98.9.%; HcT: κ = 0.84, ORA = 94.3%) Concordance per ORA for the new 3-tier classification (HER2 IHC 0 vs HER2-low [IHC 2+/ISH- or IHC 1+] vs HER2-positive [IHC 3+ or IHC 2+/ISH+]) was greater than 80% for both assays at baseline and after training (see Table). In a subgroup analysis assessing training effect for 4B5 assay, concordance rates for HER2 IHC 0 were 74.6% at baseline and 89.2% after training (P &amp;lt; 0.001), and for HER2-low, NPA was 80.6% before and 91.1% after training (P &amp;lt; 0.001); there were no statistically significant changes after training for HcT (data not shown). Conclusions: Results from this real-world global study demonstrate that overall score concordance with a new category of HER2-low was above the 80% ORA benchmark for both 4B5 and HcT and is higher than previously reported (Fernandez et al. JAMA Oncol 2022). These data demonstrate pathologists’ ability to achieve an acceptable level of accuracy for identifying HER2 IHC 0 and HER2-low patients even after short-term training; however, additional training techniques and experience are needed to further improve accuracy. Acknowledgment Judy Yu, a former AstraZeneca employee, provided expertise and technical insights to support the study. Editorial Acknowledgment Under guidance of the authors, assistance in medical writing and editorial support was provided by Toinette Labuschagné, MSc, of ApotheCom, and was funded by Daiichi Sankyo. Funding This study was funded by Daiichi Sankyo and AstraZeneca. Table. Summary of Pathologist Concordance and Interobserver Variability Citation Format: Josef Rüschoff, Alexander Penner, Ian O. Ellis, M. Elizabeth H. Hammond, Annette Lebeau, Robert Y. Osamura, Frédérique Penault-Llorca, Federico Rojo, Neil Atkey, Andreas H. Scheel, Corrado D´Arrigo, Hans-Ulrich Schildhaus, Akira Moh, Chirag Desai, Giuseppe Viale. HER2-13 Proficiency assessment of HER2-low breast cancer scoring with the Ventana PATHWAY 4B5 and Dako HercepTest HER2 assays and the impact of pathologist training [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-13.

Abstract P5-06-03: Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms

Abstract Background Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. In addition, the development of quantitative, reproducible, and more sensitive HER2 assays is expected to enable the identification of patients with HER2-low MBC that may benefit from novel HER2-targeted therapies. Here we report a comprehensive liquid biopsy platform including immunofluorescent HER2 and ER quantitative protein expression in CTCs (ctcIF) coupled with the determination of ERBB2 amplification and the number of Large-scale State Transitions (LST) by single-cell CTC genomics (ctcDNA), and ctDNA alterations in plasma. Methods Blood samples were collected for cell-based and cell-free analysis from 62 patients with documented MBC and from 24 blood donors (HD) with no known cancer history. After isolation, nucleated cells were plated, and slides and plasma were bio-banked. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms, and ctcIF enables quantitation of HER2 and ER protein expression. ctcDNA was analyzed by low-pass whole-genome sequencing (WGS), allowing detection of ERBB2 amplification (ERBB2amp) and quantification of large-scale state transitions (LST+) in individual CTCs. cfDNA from plasma was analyzed using a validated NGS panel (56 genes of interest) to detect ctDNA alterations. Results Within this cohort of 62 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 87%, 70%, and 59%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (11%, and 2%, respectively). A variant allele frequency (VAF) of &amp;gt; 40%, which is required for detecting a two-fold ERBB2 amplification by ctDNA, was not present among 86% and 0% of MBC detected by the ctcDNA and ctDNA platforms, respectively, suggesting that the ctcDNA platform can identify ERBB2amp among patients with a low ctDNA fraction. HER2+ or ER+ expression by ctcIF were detected in 37% and 58% of MBC patients, respectively. At the cellular level, across 62 patients, CTC with detectable ERBB2amp by ctcDNA had a higher median expression of HER2 protein by ctcIF compared to CTC with ERBB2nonamp (1772 MFI vs 122.5 MFI, respectively; p&amp;lt; 0.001). Similarly, at the patient level, among patients with circulating ERBB2amp, HER2 protein was detected by ctcIF in 100% of MBC patients (p&amp;lt; 0.001), suggesting a very high positive correlation between the presence of ctcDNA genomic ERBB2amp and ctcIF HER2 protein expression. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that among MBC, 11% were ERBB2amp, 26% were HER2 expressing (HER2+ and ERBB2nonamp), and 60% were HER2neg (HER2- and ERBB2nonamp). Combination models of the three individual platforms (ctcIF, ctcDNA, and ctDNA) were able to provide potentially clinically actionable biomarker data (LST+ CTC, ERBB2amp CTC, HER2+ CTC, ER+ CTC and 1A+ SNVs) to 79% of MBC patients while retaining 100% specificity. Conclusions Here we reported a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in late MBC patients. The comprehensive liquid biopsy platform’s combined utility can aid biomarker profiling of MBC among often biologically heterogeneous tumor sites of metastatic disease and those inaccessible by conventional tissue biopsy. Citation Format: Giuseppe Di Caro, Ernest T. Lam, Megan M. Slade, Anna Lundberg, Martin Blankfard, Nilesh Dharajiya, Alisa Tubbs, Rick Wenstrup, Lee Schwartzberg. Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-03.

HER2 testing in colorectal cancer: Concordance analysis between breast and gastric scoring algorithms from the MOUNTAINEER trial.

198 Background: HER2 overexpression/amplification (HER2+) occurs in 3%-5% of patients (pts) w/ metastatic colorectal cancer (mCRC). Rates of HER2+ can increase to ~10% in pts w/ RAS/BRAF wild-type mCRC tumors. The MOUNTAINEER trial (NCT03043313) evaluated the efficacy and safety of the investigational combination of tucatinib with trastuzumab in pts with HER2+ and RAS wild-type mCRC. Established regional guidelines for mCRC recommend HER2 testing and HER2-directed treatment options; however, there is currently no established best practice for HER2 testing and interpretation in mCRC. Here, we present data from a concordance analysis comparing breast and gastric HER2 testing algorithms in the mCRC setting. Methods: The MOUNTAINEER trial enrolled pts w/ HER2+ mCRC identified using ≥1 method: tissue-based local immunohistochemistry (IHC), in situ hybridization (ISH), and/or next-generation sequencing (NGS) testing. Archival or fresh tumor tissue was submitted to a sponsor-designated central laboratory for confirmatory HER2 testing w/ IHC/FISH per the package insert of the FDA approved assay and scored by both the breast and gastric algorithms for HER2 IHC. A positive result per the breast scoring criteria for IHC requires circumferential membrane staining for HER2, while the gastric criteria allows for circumferential, basolateral, or lateral staining patterns. Results: A total of 114 pts were enrolled with HER2+ tumors per ≥1 local testing methods; 69 pts were HER2+ by NGS, 46 by IHC 3+, and 36 by ISH. Of 105 pts who had tissue available for central HER2 testing w/IHC/FISH, 98 had valid HER2 results; 82/98 (83.7%) of pts had tumors centrally confirmed as HER2+ using both the breast and gastric algorithms. Tissue samples from pts in the MOUNTAINEER trial had 100% concordance between breast and gastric algorithms in HER2 status and 99% concordance in HER2 IHC score. Conclusions: Central pathology testing using both the breast and gastric criteria showed high concordance between these two commonly used algorithms. A high central confirmation rate of local HER2+ results was also observed. These data support the use of either the breast or gastric algorithms to identify HER2+ mCRC tumors until an FDA-approved HER2 assay is available for mCRC. Clinical trial information: NCT03043313 . [Table: see text]

Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer

AbstractThe efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Retrospective study to estimate the prevalence of HER2-low breast cancer (BC) and describe its clinicopathological characteristics.

1087 Background: Approximately 50% of BCs traditionally categorized as HER2 negative (HER2-neg) express low levels of HER2 (IHC 1+ or IHC 2+/ISH-; Miglietta, NPJ Breast Cancer 2021). HER2-targeted therapies for HER2-low metastatic BC (mBC) are under investigation (eg, T-DXd in the phase 3 DESTINY-Breast04 study; NCT03734029), but HER2 assays currently used to select patients (pts) for approved anti-HER2 therapies are optimized for high HER2 expression and are not validated for HER2-low detection. A recent study found relatively poor agreement (&lt;70% interrater agreement) in evaluation of IHC scores of 0 and 1+ using current HER2 assays (Fernandez, JAMA Oncol 2022). Our objectives were to assess the prevalence of HER2-low among HER2-neg based on rescored HER2 IHC slides after training on low-end expression scoring and to describe pt characteristics of HER2-low vs HER2 IHC 0 mBC. Preliminary results are reported for 233 of 1000 planned pts. Methods: This multicenter, retrospective study (NCT04807595) included pts with confirmed HER2-neg unresectable/mBC diagnosed between 2015 and 2017. Local laboratories, blinded to historical HER2 scores, rescored HER2 IHC-stained slides. HER2 was assessed using Ventana 4B5 and other assays. BCs were categorized as HER2-low or HER2 IHC 0. The prevalence of HER2-low BC among pts originally scored as HER2-neg was measured. Demographics (eg, age, country, race) and clinicopathological characteristics were examined via medical charts/electronic health records. Concordance between historical HER2 scores and rescores was assessed. Results: HER2 rescores were obtained for 233 pts (mean age, 54 y). HER2-low prevalence was 63.2% overall and numerically greater in hormone receptor (HR)–positive vs HR-negative subgroups (66.1% vs 54.8%; Table). No notable differences in prevalence were seen among different HER2 assays or in demographic/baseline disease characteristics between the HER2-low and HER2 IHC 0 groups. Concordance rate between historical and rescored slides for HER2-status classification was 82.3%. The presentation will include an expanded data set (≈400 pts) with additional results. Conclusions: Data on HER2-low prevalence in BC is limited. Preliminary data from this study of mBC samples suggest a somewhat higher prevalence estimate (≈63%) than a previous study of primary BC samples (≈50%). Concordance was 82%; ongoing analyses with updated data will clarify the concordance between rescored and historical HER2 slides. These data can support development of best practices for identifying pts with HER2-low expression who may benefit from HER2-targeted therapies. Clinical trial information: NCT04807595. [Table: see text]

IS ADVANCED ALPHA-FETOPROTEIN PRODUCING GASTRIC CANCER TREATABLE?

We illustrated a patient with advanced alpha-fetoprotein producing gastric cancer, who was treated with chemotherapy combined with trastuzumab, resulting in complete response in four months after therapy. It has been 5 years after his initial therapy, he is now healthy and shows no evidence of tumor recurrence. The efcacy of the trastuzumab therapy in breast cancer patients prompted studies to evaluate potential clinical benet of the drug in patients with HER2 positive gastric cancer. The largest study, ToGA trial, has published a promising result leading FDA to approve the use of trastuzumab for HER2 positive gastric and gastroesophageal cancer. We discuss the need for the standardized assay method for HER2 status. We also bring attention to the existence of intratumoral heterogeneity in gastric cancer tissue which may cause false negative test for HER2 assay

Abstract P1-02-02: Examination of low Her2 expression in breast cancer

Abstract Introduction: Antibody-drug conjugates (ADC) are designed to effectively deliver cytotoxic agents directly to malignant cells. Trastuzumab deruxtecan (Tdx), an ADC of trastuzumab, an enzyme-cleavable linker, and a cytotoxic topoisomerase I inhibitor, has been shown to have antitumor activity in patients with breast cancer with low levels of HER2. However, current companion diagnostic tests for HER2-targetting therapies, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), were optimized for high (gene amplified) levels of HER2. We hypothesize that the current common assays used in clinic do not efficiently differentiate between patients whose cancers have “0” HER2 expression and “1+” HER2 expression and thus could miss patients who would benefit from treatment with this drug. Methods: Here, we evaluated two years of HER2 surveys from the College of American Pathologists’ (CAP) Proficiency Testing Surveys for HER2 expression in breast cancer from 2019 and 2020. Participating laboratories received two tissue microarrays (TMAs) of 10 breast cancer cores, each Laboratories stained for HER2 using the standard IHC assay used in their labs. The scores were returned to the CAP as part of their quality assessment program. Each survey dataset covers the scores from around 1400 labs of 20 cores each as well as supplemental questions regarding the methodology used. We summarized the relative frequency and distribution of each score given to every core. A second independent analytic dataset was selected from the archives of the Department of Pathology at Yale, from breast biopsies in 2018. The set, enriched in HER2 2+ and 3+ cases, was read by eighteen board-certified pathologists, most with over 5 years’ experience, participating in an interobserver variability study. Hematoxylin and eosin (H&amp;E) and HER2 IHC digitally scanned images of 170 independent cases were provided. Pathologists scored the cases as 0,1, 2, or 3+. Fisher’s exact test was used to compare the 0/1+ concordant cases to 2+/3+ cases. All tests were two-sided at a significant level 0.05. Statistical analysis was performed using Graphpad Prism Version 9.0.1 and the dplyr package in R Version 1.0.143. This study was approved by Yale Human Investigation IRB protocol ID 9505008219. Results: We found that 65% of the 80 cores evaluated in the CAP survey (52/80) had a concordance rate ≥90%. This high concordance was limited to scores of 0 and 3+. The lowest concordance was found between 0 versus 1+. Of the 80 cores, 56 were considered negative (HER2 score of 0 or 1). In 25% of those cores there was &amp;lt; 70% concordance (n=15; 6 in 2019 and 9 in 2020). Analytic concordance was assessed in the independent, Yale cohort where we found that of the 170 cases, 92 were read as 0 by at least one pathologist. Of these 92, 24 were concordant (26%), defined as a ≥90% agreement. In comparison, 45/170 were read as 3+ by at least one pathologist. Again,. using a 90%definition of concordance, 26 of 45 cases (58%) were concordant. Comparison of 0/1+ concordant cases versus 2+/3+ concordant cases showed a significant difference (χ2 = 12.07, p&amp;lt;0.0005). Conclusions: Assessment of laboratory performance of around 1400 CAP labs using common current HER2 assays on CAP survey specimens, there is significant discordance in the evaluation of 0 vs. 1+ cases. In a separate selected breast biopsy cohort examined by 18 breast pathologists, we showed that discordance between scores of 0 vs 1+ is significantly larger than that between 2+ and 3+. Given the efficacy of T-DXd, we believe patients may be mis-assigned for treatment or no treatment if the decision depends on performance of the standard current HER2 assays. Citation Format: Aileen I Fernandez, Matthew Liu, Andrew Bellizzi, Jane Brock, Oluwole Fadare, Krisztina Hanley, Malini Harigopal, Julie M. Jorns, M. Gabriela Kuba, Amy Ly, Mirna Podoll, Kimmie Rabe, Mary Ann Sanders, Kamaljeet Singh, Olivia L Snir, Rinda Soong, Shi Wei, Hannah Wen, Serena Wong, Esther Yoon, Lajos Pusztai, Emily Reisenbichler, David L. Rimm. Examination of low Her2 expression in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-02.

HER-2 Status and Heterogeneity in Gastric Cancer: An Experience at a Community Hospital

Background: The efficacy of the trastuzumab therapy in breast cancer patients prompted studies to evaluate potential clinical benefit of the drug in patients with HER2 positive gastric cancer. Unlike in breast cancer, the studies, in gastric cancer to date have yielded inconsistent findings regarding the frequency of HER-2 status. Furthermore, few studies have shown that intratumoral heterogeneity can lead to false negative results. All studies were done at the large medical centers. At the community hospital, we wish to assay HER-2 status intratumoral heterogeneity in patients with gastric cancer, and compare the results with that of large medical centers. Materials and Methods: We investigated 185 cases of gastric cancer specimens using immunohistochemistry (IHC) for HER2 status. The matched biopsy and resection specimen of 30 patients were also investigated for their correlation and the presence of intratumoral heterogeneity. Results: The positive HRE2 status was 29.7% (n=55), falling between the published data .One of two neuroendocrine tumors was HER2 positive, which was never reported before. We found the concordance rate of 83% in 30 matched biopsy and gastrectomy specimens; 57% of the gastrectomy specimens showed intratumoral heterogeneity. Conclusion: Anti-HER2 targeted therapy has been approved by FDA, Japan, and European Medicine Agency, as a standard initial treatment in HER2 positive gastric cancer. Overall, both biopsy and resection specimens are appropriate for HER-2 testing. The wide range of HER-2 frequency giving in the literature indicated that some problems exist. We are hoping that future study will clarify those confusing problems and standardized HER-2 assay method will become available. It is crucial for accurate selection of patients who could really benefit from a targeted therapy.