HER2 Assessment Research Articles

Impact of HER2-positivity on prognosis and targeted therapeutic outcomes in advanced biliary tract cancer.

629 Background: HER2 overexpression and amplification have been identified as a promising druggable target in biliary tract cancer (BTC). This study aims to characterize the clinical and molecular outcomes associated with HER2 positivity in BTC and evaluate the prognostic implications of HER2-targeted therapies. Methods: A retrospective study was conducted involving patients with advanced BTC diagnosed at Yonsei Cancer Center (YCC) and the University of Texas MD Anderson Cancer Center (MDACC) between 2009 and 2023. Patients were classified as HER2-positive either by immunohistochemistry (IHC 3+ or 2+/ISH+) or by next-generation sequencing (NGS, ERBB2 amplification). Overall survival (OS) and palliative first-line progression-free survival (PFS) were analyzed using Kaplan-Meier and Cox regression methods. Results: A total of 389 advanced BTC patients were initially screened (310 from YCC; 79 from MDACC). Among the 310 BTC patients from YCC with available HER2 IHC results, 78 (25.2%) were HER2-positive and 232 (74.8%) were HER2-negative. HER2 positivity was significantly more common in gallbladder cancer (55.1%) compared to its prevalence in intrahepatic or extrahepatic cholangiocarcinoma (22.8%, p<0.001). Of the 201 patients with available NGS data, 186 (92.5%) were ERBB2 non-amplified; among these, 27 (14.9%) were HER2-positive by IHC. For survival analysis, 310 patients were eligible: 239 from YCC (both HER2-positive and negative) and 71 from MDACC (all HER2-positive). Kaplan-Meier survival analysis indicated a trend towards shorter OS in HER2-positive patients compared to HER2-negative patients (median OS, 13.7 vs. 17.1 months, p=0.084, HR=1.246, 95% CI=0.97–1.60) regardless of exposure to HER2-targeted therapy, while first-line PFS on chemotherapy only was significantly shorter in the HER2-positive group (5.1 vs. 7.4 months, p<0.001, HR=1.906, 95% CI=1.46-2.48). Among HER2-positive patients, 56.8% received HER2-targeted therapy in the second line setting. HER2-positive patients who did not receive HER2-targeted therapy had a significantly poorer prognosis compared to HER2-negative patients (median OS 8.1 vs. 17.1 months, HR=2.46, 95% CI=1.73-3.48), whereas those receiving HER2-targeted therapy had comparable OS to HER2-negative patients (median OS 18.2 vs. 17.1 months, HR=0.95, 95% CI=0.71-1.27). Multivariable analysis identified a history of surgical treatment, radiation therapy, HER2 positivity, and HER2-targeted therapy as independent prognostic factors for OS. Conclusions: HER2 positivity is associated with a poorer prognosis in advanced BTC. However, HER2-targeted therapies improve outcomes, suggesting their potential inclusion in standard care for HER2-positive subgroups. Discrepancies between IHC and NGS in HER2 assessment emphasize the need for optimized testing guidelines in BTC.

HER2 Status in Gastric and Gastroesophageal Carcinomas: Evaluation of Histopathological Fingings, Paired ResectionBiopsy Specimens, and the Effect of Neoadjuvant Therapy: A Single Center Study.

Accurately determining the human epidermal growth factor receptor 2 (HER2) status is crucial in identifying suitable candidates for targeted therapy in gastric cancer, considering the cost and potential side effects of anti-HER2 treatments. This study aimed to assess HER2 overexpression/amplification prevalence in gastric and gastroesophageal cancer patients, its correlation with clinicopathological characteristics, and the consistency of HER2 status between biopsy and radical specimens. We analyzed data from 667 specimens of 600 gastric/gastroesophageal cancer patients at Dokuz Eylül University Faculty of Medicine from 2012 to 2021. The correlation of HER2 expression and amplification status and clinicopathological parameters were assessed. Furthermore, we compared HER2 status concordance between paired biopsy-radical specimens when both endoscopic and radical materials were present. Additionally, we compared HER2 status before and after treatment in patients receiving neoadjuvant chemotherapy. In our study, +3 HER2 immunohistochemistry results were more common in gastroesophageal junction tumors (23%). Human epidermal growth factor receptor 2 immunohistochemistry positivity and HER2 gene amplification were found to be significantly more common in males, people over 65 years of age, and intestinal morphology. Overall concordance of HER2 status between radical and biopsy materials was 95.5%. The high HER2 concordance between the paired biopsy and radical samples holds significant importance in clinical management. This finding is particularly noteworthy since the HER2 assessment can generally be conducted on small/limited biopsy materials in routine practice. Our study is the most extensive series from Türkiye and the Balkans, which will shed light on our country's data by investigating the relationship of HER2 with clinicopathological parameters in gastric/gastroesophageal carcinomas.

Artificial Intelligence for Predicting HER2 Status of Gastric Cancer Based on Whole-Slide Histopathology Images: A Retrospective Multicenter Study.

Human epidermal growth factor receptor 2 (HER2) positive gastric cancer (GC) shows a robust response to the combined therapy based HER2-targeted therapy. The application of these therapies is highly dependent on the evaluation of tumor HER2 status. However, there are many risks and challenges in HER2 assessment in GC. Therefore, an economically viable and readily available instrument is requisite for distinguishing HER2 status among patients diagnosed with GC. The study has innovatively developed a deep learning model, HER2Net, which can predict the HER2 status by quantitatively calculating the proportion of HER2 high-expression regions. The HER2Net is trained on an internal training set derived from 531 hematoxylin & eosin (H&E) whole slide images (WSI) of 520 patients. Subsequently, the performance of HER2Net is validated on an internal test set from 115 H&E WSI of 111 patients and an external multi-center test set from 102 H&E WSI of 101 patients. The HER2Net achieves an accuracy of 0.9043 on the internal test set, and an accuracy of 0.8922 on an external test set from multiple institutes. This discovery indicates that the HER2Net can potentially offer a novel methodology for the identification of HER2-positive GC.

Exploring the heterogeneity of HER2 gene status and expression in non-positive breast cancer patients: insights from immunohistochemistry and fluorescence in situ hybridization

Breast cancer became the most prevalent malignancy among women, and HER2 expression status is critical for treatment decisions. With the emergence of ADC drugs, HER2 low-expressing patients who previously did not respond well to traditional anti-HER2 therapies may now benefit. In this study, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were applied to assess HER2 expression in 349 patients with HER2-non-positive breast cancer. Our analysis revealed that HER2-low tumors exhibited fewer grade III tumors (39.74% and 55.65%, respectively, P = 0.005) and higher positivity for estrogen receptor (ER, 88.89% vs. 61.74%, P < 0.001) and progesterone receptor (PR, 84.62% vs. 57.39%, P < 0.001) compared to HER2-ZERO tumors. Of the 349 cases, IHC was ultimately evaluated in 327, the antibodies demonstrated only 64.22% (95% CI: 58.76–69.42%) agreement between clone 4B5 and clone EP3. Pathologist 1, who had more extensive working experience, demonstrated higher consistency (94.19%) with the gold standard when using clone EP3, compared to Pathologist 2 (74.31%). FISH analysis revealed significant differences in HER2/CEP17 ratio and average HER2 copy numbers between HER2-ZERO and HER2-low tumors, but no clear cut-off value could be identified. Notably, HER2/CEP17 ratio mostly between 1 and 2, with HER2-ZERO tumors primarily ≤ 1.4, and average HER2 copy numbers were mostly ≥ 2 and < 4, with HER2-ZERO tumors primarily ≤ 2.5. Despite distinct clinicopathological features, FISH remains inadequate for distinguishing HER2-low from HER2-ZERO expression. Further studies are needed to improve HER2 assessment in this challenging subset of patients.

Automatic image generation and stage prediction of breast cancer immunobiological through a proposed IHC-GAN model.

Invasive breast cancer diagnosis and treatment planning require an accurate assessment of human epidermal growth factor receptor 2 (HER2) expression levels. While immunohistochemical techniques (IHC) are the gold standard for HER2 evaluation, their implementation can be resource-intensive and costly. To reduce these obstacles and expedite the procedure, we present an efficient deep-learning model that generates high-quality IHC-stained images directly from Hematoxylin and Eosin (H&E) stained images. We propose a new IHC-GAN that enhances the Pix2PixHD model into a dual generator module, improving its performance and simplifying its structure. Furthermore, to strengthen feature extraction for HE-stained image classification, we integrate MobileNetV3 as the backbone network. The extracted features are then merged with those generated by the generator to improve overall performance. Moreover, the decoder's performance is enhanced by providing the related features from the classified labels by incorporating the adaptive instance normalization technique. The proposed IHC-GAN was trained and validated on a comprehensive dataset comprising 4,870 registered image pairs, encompassing a spectrum of HER2 expression levels. Our findings demonstrate promising results in translating H&E images to IHC-equivalent representations, offering a potential solution to reduce the costs associated with traditional HER2 assessment methods. We extensively validate our model and the current dataset. We compare it with state-of-the-art techniques, achieving high performance using different evaluation metrics, showing 0.0927 FID, 22.87 PSNR, and 0.3735 SSIM. The proposed approach exhibits significant enhancements over current GAN models, including an 88% reduction in Frechet Inception Distance (FID), a 4% enhancement in Learned Perceptual Image Patch Similarity (LPIPS), a 10% increase in Peak Signal-to-Noise Ratio (PSNR), and a 45% reduction in Mean Squared Error (MSE). This advancement holds significant potential for enhancing efficiency, reducing manpower requirements, and facilitating timely treatment decisions in breast cancer care.

HER2-Targeted Affibody Molecule 99mTc-ABH2 for Imaging HER2 Expression in Breast Cancer.

Accuracy in in vivo assessment of human epidermal growth factor receptor type 2 (HER2) status is crucial for predicting the response to HER2-targeted therapies in breast cancer. This study assessed the safety, feasibility, and diagnostic accuracy of 99mTc-ABH2, a reengineered affibody molecule with radionuclide labeling, for HER2 expression in breast cancer using SPECT/CT imaging, compared with 18F-FDG PET/CT. Thirty-six patients suspected of primary breast cancer were enrolled in this prospective, single-center study from March to July in 2023. Each participant underwent SPECT/CT imaging with 99mTc-ABH2 SPECT/CT and 18F-FDG PET/CT. The imaging results were validated against immunohistochemistry and fluorescence in situ hybridization, employing visual scores and quantitative values for analysis. Optimal imaging contrast was observed around 2 hours postinjection of 99mTc-ABH2. Among the evaluated 27 patients with immunohistochemistry and fluorescence in situ hybridization findings, the 99mTc-ABH2 SPECT/CT visual evaluation displayed a sensitivity of 71.4%, specificity of 72.2%, and accuracy of 71.9%. Using an SUVmax cutoff of 2.32, the sensitivity, specificity, and accuracy for detecting HER2 status were 92.86%, 72.22%, and 81.25%, respectively. Notably, 99mTc-ABH2 precisely identified all immunohistochemistry (3+) tumors and showed increased uptake in bone and lymph node metastases. Meanwhile, 18F-FDG PET/CT showed no significant difference in uptake between HER2 (2+/3+) and HER2 (0/1+) tumors (P = 0.81). This study validated 99mTc-ABH2 SPECT/CT as a promising diagnostic tool for precise HER2 assessment. The robust sensitivity for immunohistochemistry (3+) tumors and the visualization of metastases highlight its diagnostic significance, potentially impacting patient outcomes positively.

Impact of Warm Ischemia Time on HER2 Expression in Breast Cancer Surgical Specimens.

Tissue sample quality control has become increasingly crucial as these samples are integral to basic research and clinical practice, particularly in immunohistochemistry and gene panel testing. Standard PREanalytical Code (SPREC) was developed to standardize pre-analytical processes, including warm ischemia time (WIT), cold ischemia time (CIT), and fixation time (FT), which can influence the surgical specimen quality. This study investigated the impact of WIT, CIT, and FT on estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression in breast cancer (BC) surgical specimens. We enrolled 277 patients with first-time BC who underwent surgery at Kanagawa Cancer Center between May 2018 and April 2019. WIT, CIT, and FT were recorded using SPREC ver. 3.0, and their effects on ER, PgR, and HER2 expression were analyzed using immunohistochemistry. Surgical specimens were compared with preoperative needle biopsy samples from the same tumors to control for WIT, CIT, and FT variability. The median WIT, CIT, and FT were 23 min, 37 min, and 43 h, respectively. Compliance with the American Society of Clinical Oncology/College of American Pathologists guidelines was 91.7% for CIT and 94.9% for FT. ER and PgR expression in surgical specimens decreased with prolonged CIT and FT, but differences were non-significant. However, HER2 expression increased significantly when WIT exceeded 30 min. WIT could significantly influence HER2 expression in BC surgical specimens, highlighting the need for meticulous WIT control during BC surgery to ensure accurate HER2 assessment, which is critical for guiding therapeutic decisions.

Abstract A053: Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers

Abstract Triple-negative breast cancer (TNBC) presents significant clinical challenges due to its aggressive phenotype, rapid progression, and limited targeted treatment options. TNBC often mimics benign lesions on ultrasound and metastatic features can be missed in routine mammography screening leading to misdiagnosis and delayed treatment. These limitations led us to investigate whether a blood-based liquid biopsy could provide a non-invasive, sensitive method for early TNBC detection, potentially overcoming the constraints of conventional imaging techniques. To discover biomarkers for TNBC, we performed targeted bisulfite sequencing on a cohort of 46 breast cancer solid tissue biopsy samples, comprising 19 TNBC and 27 non-TNBC cases. We identified 150 differentially methylated regions (mean length 703bp) separating TNBC and non-TNBC samples. Upon linking each region to its nearest gene, we observed several genes previously associated with prognosis and survival (HOXB3, PAX9, SOX9) substantiating the clinical relevance of these biomarkers. Using public TCGA data (369 breast cancer samples), we integrated expression and methylation data and observed directionally consistent changes. For example, HOXB3 showed increased methylation at its associated differential regions and decreased expression in TNBC cases. To ascertain whether these biomarkers could be useful in a liquid biopsy approach, we performed targeted bisulfite- sequencing on cell-free DNA samples derived from prospectively collected patients with breast cancer. All samples had independent tissue-based assessment of ER, PR and HER2 using immunohistochemistry or in situ hybridization. We then built a feed-forward neural network classifier with classes TNBC and non-TNBC that used transfer-learning and the identified biomarkers to learn methylation signatures in the biopsy samples and map them to cell-free DNA. To emulate clinical workflow, where subtyping follows cancer diagnosis and tissue of origin identification, we evaluated cfDNA samples correctly identified as breast cancer using two independent machine learning models trained using 2,393 prospectively collected multi- indication cancer and non-cancer samples (NCT05435066). Subsequent TNBC subtyping of breast cancer-positive cases (N=56) accurately identified 77% (10/13) of TNBC samples and 91% (39/43) of non-TNBC cases, with overall accuracy of 84%. The estimated tumor content range for correctly predicted TNBC samples was 0.5-33% with the three mis-classified samples all showing very low tumor content (0.22%, 0.28%, 0.59%). Non-TNBC cases showed a tumor content range of 0.07-36% for correct and 2.3-14% for misclassified samples. In conclusion, we identified novel methylation biomarkers that distinguish TNBC in the cell-free DNA of patients without needing invasive tissue biopsies. The high sensitivity of our assay at low tumor fractions has the potential to improve outcomes through earlier intervention and may enable earlier screening in high-risk groups, monitoring of minimal residual disease and longitudinal monitoring during treatment. Citation Format: Jocelyn Charlton, Shiva Farashahi, Kade Pettie, Elie Massaad, Josh Hubbell, Feras Hantash, Kieran I Chacko. Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A053.

Artificial Intelligence in Breast Cancer Diagnosis and Treatment: Advances in Imaging, Pathology, and Personalized Care.

Breast cancer is the most prevalent cancer worldwide, affecting both low- and middle-income countries, with a growing number of cases. In 2024, about 310,720 women in the U.S. are projected to receive an invasive breast cancer diagnosis, alongside 56,500 cases of ductal carcinoma in situ (DCIS). Breast cancer occurs in every country of the world in women at any age after puberty but with increasing rates in later life. About 65% of women with the BRCA1 and 45% with the BRCA2 gene variants develop breast cancer by age 70. While these genes account for 5% of breast cancers, their prevalence is higher in certain populations. Advances in early detection, personalised medicine, and AI-driven diagnostics are improving outcomes by enabling a more precise analysis, reducing recurrence, and minimising treatment side effects. Our paper aims to explore the vast applications of artificial intelligence within the diagnosis and treatment of breast cancer and how these advancements can contribute to elevating patient care as well as discussing the potential drawbacks of such integrations into modern medicine. We structured our paper as a non-systematic review and utilised Google Scholar and PubMed databases to review literature regarding the incorporation of AI in the diagnosis and treatment of non-palpable breast masses. AI is revolutionising breast cancer management by enhancing imaging, pathology, and personalised treatment. In imaging, AI can improve the detection of cancer in mammography, MRIs, and ultrasounds, rivalling expert radiologists in accuracy. In pathology, AI enhances biomarker detection, improving HER2 and Ki67 assessments. Personalised medicine benefits from AI's predictive power, aiding risk stratification and treatment response. AI also shows promise in triple-negative breast cancer management, offering better prognosis and subtype classification. However, challenges include data variability, ethical concerns, and real-world validation. Despite limitations, AI integration offers significant potential in improving breast cancer diagnosis, prognosis, and treatment outcomes.

Should a borderline negative HER2 result in a core biopsy of invasive carcinoma of the breast have HER2 assessment repeated in the excision specimen?

AimThe 2015 UK guidelines for HER2 assessment in breast cancer recommended repeat assessment if the core biopsy was scored as 2+ on HER2 immunohistochemistry (IHC) with borderline negative in situ...

Systematic assessment of HER2 status in ductal carcinoma in situ of the breast: a perspective on the potential clinical relevance

In many countries, hormone receptor status assessment of ductal carcinoma in situ (DCIS) is routinely performed, as hormone receptor-positive DCIS patients are eligible for adjuvant anti-hormonal treatment, aiming to reduce the ipsilateral and contralateral breast cancer risk. Although HER2 gene amplification and its associated HER2 protein overexpression constitute a major prognostic and predictive marker in invasive breast carcinoma, its use in the diagnosis and treatment of DCIS is less straightforward. HER2 immunohistochemistry is not routinely performed yet, as the role of HER2-positivity in DCIS biology is unclear. Nonetheless, recent data challenge this practice. Here, we discuss the value of routine HER2 assessment for DCIS. HER2-positivity correlates strongly with DCIS grade: around four in five HER2-positive DCIS show high grade atypia. As morphological DCIS grading is prone to interobserver variability, HER2 immunohistochemistry could render grading more robust. Several studies showed an association between HER2-positive DCIS and ipsilateral recurrence risk, albeit currently unclear whether this is for overall, in situ or invasive recurrence. HER2-positive DCIS tends to be larger, with a higher risk of involved surgical margins. HER2-positive DCIS patients benefit more from adjuvant radiotherapy: it substantially decreases the local recurrence risk after lumpectomy, without impact on overall survival. HER2-positivity in pure biopsy-diagnosed DCIS is associated with increased upstaging to invasive carcinoma after surgery. HER2 immunohistochemistry on preoperative biopsies might therefore provide useful information to surgeons, favoring wider excisions. The time seems right to consider DCIS subtype-dependent treatment, comprising appropriate local treatment for HER2-positive DCIS patients and de-escalation for hormone receptor-positive, HER2-negative DCIS patients.

Weakly-supervised deep learning models enable HER2-low prediction from H &E stained slides

BackgroundHuman epidermal growth factor receptor 2 (HER2)-low breast cancer has emerged as a new subtype of tumor, for which novel antibody–drug conjugates have shown beneficial effects. Assessment of HER2 requires several immunohistochemistry tests with an additional in situ hybridization test if a case is classified as HER2 2+. Therefore, novel cost-effective methods to speed up the HER2 assessment are highly desirable.MethodsWe used a self-supervised attention-based weakly supervised method to predict HER2-low directly from 1437 histopathological images from 1351 breast cancer patients. We built six distinct models to explore the ability of classifiers to distinguish between the HER2-negative, HER2-low, and HER2-high classes in different scenarios. The attention-based model was used to comprehend the decision-making process aimed at relevant tissue regions.ResultsOur results indicate that the effectiveness of classification models hinges on the consistency and dependability of assay-based tests for HER2, as the outcomes from these tests are utilized as the baseline truth for training our models. Through the use of explainable AI, we reveal histologic patterns associated with the HER2 subtypes.ConclusionOur findings offer a demonstration of how deep learning technologies can be applied to identify HER2 subgroup statuses, potentially enriching the toolkit available for clinical decision-making in oncology.

Development of a deep-learning model tailored for HER2 detection in breast cancer to aid pathologists in interpreting HER2-low cases.

Over 50% of breast cancer cases are "Human epidermal growth factor receptor 2 (HER2) low breast cancer (BC)", characterized by HER2 immunohistochemistry (IHC) scores of 1+ or 2+ alongside no amplification on fluorescence in situ hybridization (FISH) testing. The development of new anti-HER2 antibody-drug conjugates (ADCs) for treating HER2-low breast cancers illustrates the importance of accurately assessing HER2 status, particularly HER2-low breast cancer. In this study we evaluated the performance of a deep-learning (DL) model for the assessment of HER2, including an assessment of the causes of discordances of HER2-Null between a pathologist and the DL model. We specifically focussed on aligning the DL model rules with the ASCO/CAP guidelines, including stained cells' staining intensity and completeness of membrane staining. We trained a DL model on a multicentric cohort of breast cancer cases with HER2-IHC scores (n = 299). The model was validated on two independent multicentric validation cohorts (n = 369 and n = 92), with all cases reviewed by three senior breast pathologists. All cases underwent a thorough review by three senior breast pathologists, with the ground truth determined by a majority consensus on the final HER2 score among the pathologists. In total, 760 breast cancer cases were utilized throughout the training and validation phases of the study. The model's concordance with the ground truth (ICC = 0.77 [0.68-0.83]; Fisher P = 1.32e-10) is higher than the average agreement among the three senior pathologists (ICC = 0.45 [0.17-0.65]; Fisher P = 2e-3). In the two validation cohorts, the DL model identifies 95% [93% - 98%] and 97% [91% - 100%] of HER2-low and HER2-positive tumours, respectively. Discordant results were characterized by morphological features such as extended fibrosis, a high number of tumour-infiltrating lymphocytes, and necrosis, whilst some artefacts such as nonspecific background cytoplasmic stain in the cytoplasm of tumour cells also cause discrepancy. Deep learning can support pathologists' interpretation of difficult HER2-low cases. Morphological variables and some specific artefacts can cause discrepant HER2-scores between the pathologist and the DL model.

Population based assessment of HER-2 and PD-L1 positivity in gastric and gastroesophageal junction adenocarcinomas in a Canadian population.

e16038 Background: Current standard of care for advanced gastric and gastroesophageal cancers combines systemic chemotherapy with either human epidermal growth factor receptor 2 (HER-2) or programmed death ligand 1 (PD-L1) targeting therapy. New data is emerging that for patients whose tumours are positive for both HER-2 overexpression and have a PD-L1 combined positive score (CPS) &gt;1%, combining all three treatment modalities results in superior outcomes. There is limited international data, and no Canadian data, exploring the frequency of patients who have tumours that are both HER-2 and PD-L1 positive who will be eligible for this novel treatment approach. Objective: To determine the proportion of patients in Nova Scotia with HER-2 positive gastric and GEJ adenocarcinomas who have a PD-L1 CPS score above established clinical thresholds. Methods: A retrospective review of PD-L1 and HER-2 status for all patients diagnosed with gastric and GEJ adenocarcinomas in Nova Scotia between June 3, 2022 and August 13, 2023 was completed. PD-L1 CPS was evaluated using the 28-8 assay and was considered positive if &gt;1%. HER-2 overexpression was evaluated by immunohistochemistry and in cases with equivocal expression, status was based on fluorescence in situ hybridization. Results: Over the study period, 56 cases of gastric and 42 cases of GEJ adenocarcinoma were identified for a total of 98 cases. The median age of the study population was 73 years (range 38 - 91). 73 patients (74%) were male and 25 patients (26%) were female. HER-2 expression was evaluated in 92 cases, and PD-L1 CPS was evaluated in 95 cases. 19 cases (21%) were HER-2 positive and 86 cases (91%) were PD-L1 positive. 17 HER-2 positive cases were also PD-L1 positive (89%). Conclusions: This study provides population level data in a Canadian context on patients with gastric and GEJ adenocarcinoma with respect to HER-2 overexpression and PD-L1 status. This data will be helpful in planning allocation of clinical and financial resources as a new standard of care for these patients is established.

Prediction of response to neoadjuvant chemotherapy by MammaTyper® across breast cancer subtypes: A retrospective cross-sectional study

BackgroundNeoadjuvant chemotherapy (NACT) is widely used in the treatment of triple-negative and HER2-positive breast cancer (BC), but its use in estrogen receptor (ER) and/or progesterone receptor (PR) positive/HER2-negative BC is questioned because of the low pathologic complete response (pCR) rates. This retrospective study assessed the mRNA-based MammaTyper® assay's capability of predicting pCR with NACT, and ER, PR, Ki67, and HER2 status at immunohistochemical (IHC) through transcriptomics. MethodsDiagnostic biopsies from 76 BC patients treated at the Cremona Hospital between 2012-2018 were analyzed. Relative mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 were measured using the MammaTyper® kit and integrated into a pCR score. Predicting capability of pCR and standard IHC biomarkers could be assessed with ROC curves in 75 and 76 patients, respectively. ResultsOverall, 68.0% patients obtained a MammaTyper® high-score and 32.0% a MammaTyper® low-score. Among high-score patients, 62.7% achieved pCR, compared to 16.7% in the low-score group (p = 0.0003). The binary MammaTyper® score showed good prediction of pCR in the overall cohort (area under curve [AUC] = 0.756) and in HR+/HER2-negative cases (AUC = 0.774). In cases with residual disease, the continuous MammaTyper® score correlated moderately with residual tumor size and decrease in tumor size. MammaTyper® showed substantial agreement with IHC for ESR1/ER and ERBB2/HER2, and moderate agreement for PGR/PR and MKI67/Ki67. ConclusionOverall, MammaTyper® pCR score may serve as a standardized tool for predicting NACT response in HR+/HER2-negative BC, potentially guiding treatment strategies. Additionally, it could provide a more standardized and reproducible assessment of ER, PR, HER2, and Ki67 status.

Abstract PO5-07-11: Use of 64Cu-DOTA trastuzumab -PET to predict response to trastuzumab-deruxtecan (TDXd) in patients with metastatic disease to the brain: Study in Progress

Abstract Background: We have utilized 64Cu-DOTA trastuzumab-PET imaging in patients with advanced breast cancer. In our experience, uptake on 64Cu-DOTA trastuzumab-PET correlated with the qualitative assessment of HER2 by IHC. In women treated with the antibody-drug conjugate (ADC), ado-trastuzumab emtansine (TDM1), we identified a threshold level of 64Cu-DOTA trastuzumab-PET uptake that predicted for lack of response to TDM1. As patients are living longer with advanced breast cancer, the incidence of brain metastasis has increased. The ADC, trastuzumab-deruxtecan (T-DXd) has demonstrated activity in patients with metastatic HER2 positive breast cancer with brain metastases. The DESTINY 04 trial included patients with HER2 1+ and 2+ disease and demonstrated superior survival compared to standard of care. It is not feasible to biopsy all brain metastases to determine tumor markers. Given the increased incidence of brain metastases in this population and the efficacy of T-DXd in patients with HER2+ metastatic disease to the brain, we initiated a study of 64Cu-DOTA trastuzumab-PET/MRI of the brain prior to institution of T-Dxd. Endpoints Primary: 1. 64Cu-DOTA-trastuzumab PET-CT will identify tumor heterogeneity of trastuzumab uptake in women with metastatic breast cancers. 2. The degree of uptake of 64Cu-DOTA-trastuzumab PET-CT will predict for response to Enhertu. Secondary: 1. Uptake of 64Cu-DOTA-trastuzumab PET-CT will be predictive of response duration to Enhertu. Eligibility: Patients must have documented recurrent breast cancer that is HER2 1+, 2+ or 3+, evidence of brain metastases by CT or MRI of the brain and are candidate for treatment with trastuzumab-deruxtecan. The CNS disease must be stable and not in need of intervention. Methods: Prior to treatment staging workups includes pathologic review of HER2 status, disease staging with 18FDG-PET or CT of the chest/abdomen/pelvis and bone scan, and 64Cu-DOTA-trastuzumab PET/MRI of the brain. After initiation of T-Dxd, MRI of the brain is repeated every 6 weeks for the first 24 weeks and every 9 weeks thereafter. Progress: We have enrolled 1 patients to date, received PET and dynamic contrast-enhanced MRI. Four lesions were analyzed in this patient. The mean values of Ktrans (volumetric perfusion to tumor, s−1) in these lesions near a prior resection cavity were 0.210 s−1, indicating sufficient perfusion for ADC uptake. Corresponding to this perfusion data, the mean SUVmax of these lesions on the 64Cu-DOTA-trastuzumab PET scans was 5.38. Citation Format: Joanne Mortimer, Kofi Poku, Jessica Liu, Russell Rockne, Chen Bihong, Ryan Woodall, Vikram Adhikarla. Use of 64Cu-DOTA trastuzumab -PET to predict response to trastuzumab-deruxtecan (TDXd) in patients with metastatic disease to the brain: Study in Progress [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 PO5-07-11.

Abstract PO3-15-07: False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil

Abstract Introduction: Breast cancer (BC) is the second leading cause of cancer death in women worldwide. One of the major advances in BC management was its molecular classification, especially regarding the Human Epidermal growth factor Receptor 2 (HER2). HER2 positive tumors are at greater risk of visceral metastasis and are associated with worse survival rates. Thus, the use of specific drugs to target this pathway is essential and false-negative have a high impact on patient care. HER2 status is usually accessed using immunohistochemistry (IHC), but false-negatives results occur. When IHC results are inconclusive, in-situ hybridization (ISH) testing is necessary. Objective: to determine if there is variability between the positivity rates between laboratories that performed the ISH, according to the IHQ, and to evaluate if there are false-negative results and possible prognostic impact. Methods: retrospective, observational study in a public hospital in the city of Curitiba (Brazil), followed by cross-sectional analysis of histological samples. All cases of BC that underwent HER2 assessment by IHC and ISH between January 2008 and December 2018 were included. Participants classified as HER2 negative by IHC and patients whose medical records were not available were excluded. A new analysis of all IHC slides and of cases with negative ISH was performed. Results: We identified 205 people with an average age of 53.52 (± 11.89) years. Most cases were classified as clinical stage I and II (68.3%), and the most prevalent histological features were Invasive Ductal Carcinoma (82.4%) and luminal molecular subtype (49.8%). Initial ISH testing was performed by four laboratories. There was a significant difference in HER2 positivity rates between these laboratories, even after individual reanalysis of all IHC slides. Of the 114 cases with negative ISH, it was possible to obtain histological material in 82 of them to perform a new standardized ISH. The false-negative rate for HER2 in this 82 patients was 41.46%, with 10 cases out of 24 for positive IHC (3+) and 22 out of 58 for inconclusive IHC (2+). In this paper, false-negative results did not impact in survival, most likely to differences between groups. Conclusion: our work showed different rates of positivity for HER2 among laboratories that performed confirmatory test with ISH. The performance of a second standardized ISH proved that the difference between the laboratories was due to false-negative results. There was no difference in recurrence and cancer-specific survival in this false-negative sample. Keywords: breast cancer. Biomarkers. Health technology assessment. HER2 receptor. DIfferences between laboratories reagarding HER2 false negatives –insert figrue image– Citation Format: Bruno Batista, Sergio Ioshii, Sergio Padilha, Jacqueline Nabhen, Tayza Ostroski, Caroline Batista. False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil [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 PO3-15-07.

Abstract 1037: Clinical application of next-generation digital PCR capable of real-time analysis to HER2 measurement as a novel diagnostic in breast cancer: A multicenter retrospective study

Abstract HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Here, we developed a clinically reliable HER2 testing method enabling ultra-fast detection of HER2 gene amplification with high accuracy by using the digital real-time PCR (drPCR) system. To test the clinical validity and standardize procedures of drPCR-based HER2 status evaluation, three independent breast cancer cohorts from different institutions were enrolled, which assigned as a training (SCHU hospital, n = 103) and two validation sets (SNU hospital, n = 170; CNUH hospital, n = 45), and the drPCR assay was compared with current standard HER2 testing methods. In the training cohort, the HER2/CEP17 ratio values from FISH and drPCR tests were highly correlated (r2 = 0.81; P &amp;lt; 0.001), and the drPCR results displayed 98.1% concordance to HER2 status defined by IHC and/or FISH with 92.6% sensitivity and 100% specificity. Eight samples further verified by targeted NGS showed 100% concordance of dPCR to NGS. Consistently, two validation cohorts also showed high concordance of drPCR to IHC and/or ISH results (accuracy = 97.1% and 97.8% in SNU and CNUH cohorts, respectively). The optimal cutoff for HER2 positivity in the drPCR assay was set as a HER2/CEP17 ratio ≥ 1.9 with AUC of 0.963 based on the results from training cohort, and the same cut-off for drPCR was applicable to two independent validation cohorts, supporting the clinical validity of our drPCR-based HER2 assessment. In some discordant cases, low tumor purity (≤ 25%) was observed and microdissection partly improved the drPCR results. The discordance between drPCR and ISH results was also found in marginal HER2+ cases with HER2/CEP17 ratio 2-3, but these cases showed inter-observer variability when re-evaluating the ISH/IHC data due to intratumoral HER2 heterogeneity. Of note, in HER2 IHC3+ cases with negative drPCR results, re-evaluation of IHC using an artificial intelligence (AI)-based HER2 scoring system revised the HER2 IHC 3+ score to 2+, and ISH assessment also confirmed that these cases are indeed HER2-negative, proving the high accuracy of HER2 CN drPCR assay. In conclusion, given the advantages of drPCR-based HER2 assessment with high accuracy, sensitivity, and simplicity, the drPCR assay could be a complementary or alternative method to IHC and ISH to greatly improve current HER2 testing. Citation Format: Hayeon Kim, Soo Young Park, Sohyeon Yang, Min Ji Song, Da Sol Kim, Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Nam Hun Heo, Hee-Young Won, Ji-Hye Park, Minsik Song, Seung-Shick Shin, Do Young Lee, Si-Hyong Jang, Jeong-Yeon Lee, Han Suk Ryu. Clinical application of next-generation digital PCR capable of real-time analysis to HER2 measurement as a novel diagnostic in breast cancer: A multicenter retrospective study [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 1037.

Abstract 3705: Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies

Abstract Background. HER2 testing in tissue biopsies is extensively performed in breast cancer (BC) patients. However, tissue biopsy is invasive and repeated examination is impractical. Additionally, lack of concordance between primary tumor and metastatic sites are often observed. Liquid biopsy, on the other hand, enables less invasive and recurrent sample collection for ongoing monitoring. In this study, we developed an end-to-end Research Use Only assay for concomitant evaluation of HER2 gene amplification and protein overexpression in circulating tumor cells (CTCs). Methods. Blood was drawn from healthy volunteers into Streck Cell-Free DNA tubes, spiked with SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and processed on ANGLE's Parsortix® PC1 System, a microfluidic device that captures CTCs from metastatic breast cancer patients’ blood based on their size and deformability, without relying on specific markers. Captured cells were harvested and spun onto positively charged slides and stained with the Portrait HER2 immunofluorescence (IF) panel, consisting of a nuclear dye, epithelial and mesenchymal markers, leukocytes’ markers, and an antibody targeting HER2. Slides were then de-stained and subjected to HER2 FISH using a commercially available kit. All slides were imaged twice using BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. First, an automated scan after IF was performed to identify CTCs based on marker expression and morphology profiles. Second, an automated relocation of the CTCs based on IF results and reimaging using higher magnification to ensure precise analysis of the FISH signals in single cells. The workflow is being implemented in a cohort of BC patients. Results. Following IF, HER2 protein expression in SKBR3 cells was significantly higher (p&amp;lt;0.0001) than that of HER2-negative cells, with 100% analytical specificity and sensitivity. Following FISH, 100% of the cells were de-stained and presented sharp HER2/CEP17 foci, with 96% of the SKBR3 cells and only 4% of the Hs 578T cells displaying HER2 amplification. Additionally, in a small cohort of metastatic BC patients’, 100% of the identified CTCs are retained and evaluable from IF to FISH. Conclusions. An assay integrating HER2 identification by IF and FISH to characterize CTCs in metastatic BC patients was successfully developed. The assay enables the identification of both HER2 protein and gene copy information from each target cell with minimal cell loss, with the potential for minimally invasive patient monitoring during treatment and better classification of patients who may qualify and benefit from HER2-targeted therapies. An added value of the assay is the practicality of utilizing preserved blood samples, allowing streamline batch shipping of clinical samples. Citation Format: Mariacristina Ciccioli, Alex Young, Laila Vlietinck, Chassidy Johnson, Anne-Sophie Pailhes-Jimenez. Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies [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 3705.

Detection of HER2 expression using 99mTc-NM-02 nanobody in patients with breast cancer: a non-randomized, non-blinded clinical trial

Background99mTc radiolabeled nanobody NM-02 (99mTc-NM-02) is a novel single photon emission computed tomography (SPECT) probe with a high affinity and specificity for human epidermal growth factor receptor 2 (HER2). In this study, a clinical imaging trial was conducted to investigate the relationship between 99mTc-NM-02 uptake and HER2 expression in patients with breast cancer.MethodsThirty patients with pathologically confirmed breast cancer were recruited and imaged with both 99mTc-NM-02 SPECT/computed tomography (CT) and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT. According to the treatment conditions before recruitment, patients were divided into two groups, the newly diagnosed group (n = 24) and the treated group (n = 6). The maximal standard uptake value (SUVmax) of 18F-FDG and SUVmax and mean SUV (SUVmean) of 99mTc-NM-02 in the lesions were determined to analyze the relationship with HER2 expression.ResultsNo meaningful relationship was observed between 18F-FDG uptake and HER2 expression in 30 patients with breast cancer. 99mTc-NM-02 uptake was positively correlated with HER2 expression in the newly diagnosed group, but no correlation was observed in the treated group. 99mTc-NM-02 uptake in HER2-positive lesions was lower in those with effective HER2-targeted therapy compared with the newly diagnosed group. 99mTc-NM-02 SPECT/CT detected brain and bone metastases of breast cancer with a different imaging pattern from 18F-FDG PET/CT. 99mTc-NM-02 showed no non-specific uptake in inflamed tissues and revealed intra- and intertumoral HER2 heterogeneity by SPECT/CT imaging in 9 of the 30 patients with breast cancer.Conclusions99mTc-NM-02 SPECT/CT has the potential for visualizing whole-body HER2 overexpression in untreated patients, making it a promising method for HER2 assessment in patients with breast cancer.Trial registrationNCT04674722, Date of registration: December 19, 2020.