HER2 Copy Number Research Articles

Mouse Models of HER2+ Human Breast Cancer: A Literature Review

Introduction: HER2+ breast cancer (BC) makes up 15-20% of BC cases, where HER2 overexpression drives BC progression via proliferative signals. Targeted therapies inhibit HER2's activity but face challenges like resistance and metastasis. Thus, HER2+ BC is a developing research area where many preclinical studies are being conducted, and mice are often used due to their genetic and physiological similarities to humans. This study aims to review and compare existing mouse models to determine the best model of HER2+ BC. Methods: A literature search on PubMed in February 2024 using search terms including HER2, neu, neuNT, MMTV, and mammary tumors identified 16 primary research articles that used Neu-overexpressing mice. The papers were categorized under five models: MMTV-Neu, MMTV-NeuNT, FloxNeo-NeuNT, MMTV-NIC, and MMTV-HER2. Results: Among examined studies, Andrechek et al.'s FloxNeo-NeuNT model had the longest average tumor onset at 447 days, while Kuang et al.'s MMTV-NIC had the shortest at 126 days. Ursini-Segal et al.'s MMTV-NIC mice showed 100% mammary tumor incidence. Most models resulted in mammary adenocarcinomas, with lung metastases commonly observed, especially in papers that used MMTV-NIC. Discussion: Human HER2+ BC is commonly adenocarcinoma and often metastasizes to lungs, bones, liver, and brain. The mouse models were also found to be adenocarcinomas, but they primarily showed lung metastasis, possibly due to insufficient tumor detection methods for brain and bone metastasis. Neu copy numbers in Andrechek et al.’s FloxNeo-NeuNT model also align with findings on HER2 copy number amplification in human HER2+ BC. Conclusion: We have found that MMTV-NIC is the optimal mouse model for HER2+ BC research due to its short latency, 100% tumor incidence, and high rate of lung metastasis.

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...

HER2 amplification subtype intrahepatic cholangiocarcinoma exhibits high mutation burden and T cell exhaustion microenvironment

ObjectiveThis study aimed to establish a uniform standard for the interpretation of HER2 gene and protein statuses in intrahepatic cholangiocarcinoma (ICC). We also intended to explore the clinical pathological characteristics, molecular features, RNA expression and immune microenvironment of HER2-positive ICC.MethodsWe analyzed a cohort of 304 ICCs using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to identify HER2 status. Comprehensive analyses of the clinicopathological, molecular genetic, and RNA expression characterizations of ICCs with varying HER2 statuses were performed using next-generation sequencing. We further investigated the tumor microenvironment of ICCs with different HER2 statuses using IHC and multiplex immunofluorescence staining.ResultsHER2/CEP17 ratio of ≥ 2.0 and HER2 copy number ≥ 4.0; or HER2 copy number ≥ 6.0 were setup as FISH positive criteria. Based on this criterion, 13 (4.27%, 13/304) samples were classified as having HER2 amplification. The agreement between FISH and IHC results in ICC was poor. HER2-amplified cases demonstrated a higher tumor mutational burden compared to non-amplified cases. No significant differences were observed in immune markers between the two groups. However, an increased density of CD8 + CTLA4 + and CD8 + FOXP3 + cells was identified in HER2 gene-amplified cases.ConclusionFISH proves to be more appropriate as the gold standard for HER2 evaluation in ICC. HER2 gene-amplified ICCs exhibit poorer prognosis, higher mutational burden, and T cell exhaustion and immune suppressed microenvironment.

Analysis of Factors Associated With Pathological Complete Response in Patients With HER2-Positive Breast Cancer Receiving Neoadjuvant Chemotherapy

PurposeThis study aimed to examine the impact of the level of HER2 overexpression on pathologic and clinical outcomes in HER2-positive breast cancer (BC) patients treated with neoadjuvant therapy (NAT). MethodsWomen with Stage II or III HER2-positive BC who received anthracycline-taxane-trastuzumab NAT regimens followed by curative-intent surgery were included. Patients were classified according to tumor HER2 expression into HER2-high (immunohistochemistry (IHC) 3+ or fluorescence in situ hybridization (FISH) HER2/CEP17 ratio ≥5 or HER2 copy number ≥10) and HER2-intermediate (IHC 2+ with HER2/CEP17 ratio ≥2 to <5 or copy number ≥4 to <10). Univariate and multivariate logistic regression analyses were performed using HER2 expression as a categorical variable. The primary outcome was pathological complete response (pCR). Estimated 3-year disease-free survival (DFS) and Overall Survival (OS) were secondary outcomes. ResultsAmong 161 patients with HER2-positive BC, 139 (86%) and 22 (14%) were classified as HER2-high and HER2-intermediate, respectively; 105 (65.2%) had hormone receptor (HR)-positive tumors; 72 (45%) achieved a pCR. In the overall population, pCR rates of 18% and 49% were achieved in HER2-intermediate and HER2-high cases, respectively (odds ratio [OR] = 0.23 95% CI 0.07-0.72; P = .007). No pCRs were observed among HR-positive, HER2-intermediate cases. Estimated 3-year DFS was 97.1% versus 89.3% for patients achieving a pCR versus those with residual disease, respectively (P = .0011). ConclusionWe found that patients with HER2-high disease were more likely to achieve pCR after NAT compared to patients with HER2-intermediate BC, a subgroup of patients that may benefit from more personalized NAT strategies.

Image analysis for bright-field HER2 in situ hybridization: validation for clinical use.

The aim of the present study was to develop and validate a quantitative image analysis (IA) algorithm to aid pathologists in assessing bright-field HER2 in situ hybridization (ISH) tests in solid cancers. A cohort of 80 sequential cases (40 HER2-negative and 40 HER2-positive) were evaluated for HER2 gene amplification with bright-field ISH. We developed an IA algorithm using the ISH Module from HALO software to automatically quantify HER2 and CEP17 copy numbers per cell as well as the HER2/CEP17 ratio. We observed a high correlation of HER2/CEP17 ratio, an average of HER2 and CEP17 copy number per cell between visual and IA quantification (Pearson's correlation coefficient of 0.842, 0.916, and 0.765, respectively). IA was able to count from 124 cells to 47,044 cells (median of 5565 cells). The margin of error for the visual quantification of the HER2/CEP17 ratio and of the average of HER2 copy number per cell decreased from a median of 0.23 to 0.02 and from a median of 0.49 to 0.04, respectively, in IA. Curve estimation regression models showed that a minimum of 469 or 953 invasive cancer cells per case is needed to reach an average margin of error below 0.1 for the HER2/CEP17 ratio or for the average of HER2 copy number per cell, respectively. Lastly, on average, a case took 212.1s to execute the IA, which means that it evaluates about 130 cells/s and requires 6.7s/mm2. The concordance of the IA software with the visual scoring was 95%, with a sensitivity of 90% and a specificity of 100%. All four discordant cases were able to achieve concordant results after the region of interest adjustment. In conclusion, this validation study underscores the usefulness of IA in HER2 ISH testing, displaying excellent concordance with visual scoring and significantly reducing margins of error.

Trastuzumab Deruxtecan in Advanced Solid Tumors With Human Epidermal Growth Factor Receptor 2 Amplification Identified by Plasma Cell-Free DNA Testing: A Multicenter, Single-Arm, Phase II Basket Trial.

HERALD/EPOC1806 was conducted as a multicenter phase II trial assessing trastuzumab deruxtecan (T-DXd) therapy for patients with human epidermal growth factor receptor 2 (HER2)-amplified progressive stage solid tumors detected by cell-free DNA (cfDNA) testing. Patients exhibited advanced solid tumors with HER2 amplification that was identified via next-generation sequencing of cfDNA testing, without the requirement for immunohistochemical HER2 testing. The studied group was administered T-DXd at 5.4 mg/kg once every 3 weeks until onset of disease progression or intolerable toxicity. Overall, 4,734 patients underwent cfDNA testing from December 2019 to January 2022, and 252 demonstrated HER2 amplification. Finally, the study included 62 patients with 16 cancer types with a median baseline plasma HER2 copy number (CN) of 8.55 (range, 2.4-73.9). Confirmed overall response rate (ORR) by investigator assessment was 56.5% (95% CI, 43.3 to 69.0), thus showing a value beyond the 5% threshold. Responses were evaluated for 13 cancer types, including KRAS-mutant colorectal (1/3), PIK3CA-mutant endometrial (5/6), and tissue HER2-negative gastric (1/2) cancers. Plasma HER2 CN above versus below the baseline median value did not differ for impact response; however, clearance of HER2 amplification in cfDNA on cycle 2 day 1 had higher response values compared with persistence. Median progression-free survival and response duration were 7.0 (95% CI, 4.9 to 9.7) and 8.8 (95% CI, 5.8 to 11.2) months, respectively, with the majority of complications being mild to moderate. Interstitial lung diseases were identified in 16 (26%) patients, including 14 patients with grade 1 disease, one patient with grade 2 disease, and one patient with grade 3 disease. T-DXd treatment demonstrated high ORR with durable response in patients with advanced HER2-amplified solid tumors determined with cfDNA testing.

Abstract PO4-26-05: Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution

Abstract Background: HER-2 critically guides therapies and is prognostic in breast cancer (BC). Current ASCO/CAP guidelines for immunohistochemistry (IHC) define 3+ as positive, 0 and 1+ as negative, and 2+ as equivocal, with additional testing recommended. Current guidelines for in situ hybridization (ISH) define a HER-2/CEP17 ratio of ≥2.0, with an average HER-2 copy number of ≥4.0 signals/cell as ISH positive. With &amp;lt; 4.0 copies HER-2/cell, and for various other ISH groups that are borderline, further specific additional testing is recommended. Data suggest that HER-2 copy number does have clinical relevance in HER-2+ BC, however most clinical trials evaluating HER-2 targeted therapeutics have defined HER-2+ as specifically a HER-2/CEP17 ratio ≥2.0. Limited studies have explored HER-2 copy number and its clinical implication in the HER-2 1+, and 2+ ISH negative population, recently described as HER-2 low. Further, the guidelines for HER-2 testing have changed twice over the past decade, and little is known about the impact of these changes. Design: From 2001- March 2023, all new invasive BC cases tested at University of Vermont (UVM) for HER-2 with both IHC and ISH were analyzed. All testing was performed on formalin-fixed paraffin-embedded tissue, using the HercepTest kit (Dako Corp, Carpinteria, California) (2001-2012) 4B5 Rabbit Monoclonal Antibody (Ventana) (2013-2023) for IHC and the INFORM dual ISH DNA probe (Ventana) for CISH. Interpretation was performed using published criteria at the time of original testing, and all cases with both IHC and CISH testing were independently analyzed by two experienced reviewers. Results: 2734 cases had dual testing with IHC and CISH. 2407 patients (88%) had a HER-2/CEP17 ratio &amp;lt; 2.0, of these, 6.4% had a HER-2 copy number ≥4 and &amp;lt; 6. Only 12% of cases overall had a ratio ≥ 2.0. In IHC 0 and 1+ cases with HER-2/CEP17 ratio of &amp;lt; 2, 0.4% and 4.3% have excess HER-2 copies/cell (defined as ≥ 4), respectively, compared to 13.8% for IHC 2+. For IHC 2+ cases, only 13.6% overall demonstrate amplification, with however, again, an additional 13.8% having excess copy number. Interestingly, 9.6% of the IHC 3+ cases had a ratio of &amp;lt; 2.0, however 50% of those demonstrate HER-2 amplification. Overall, 6.4% of HER-2 non-amplified cases with a ratio &amp;lt; 2.0 have an excess HER-2 copy number. In our full institutional pathology database, 11.2% of cases are HER2+, by either IHC or ISH. Lastly, 43% of cases overall, at UVM, from 2018 – March 2023 would fall into the current classification as HER-2 low (IHC 1+, or IHC 2+ and ISH negative), as well. Additionally, we have evaluated the change in IHC and ISH results that occurred with the changes in the ASCO/CAP guidelines in both 2013 and 2018, further data will be available for presentation. Conclusion: In this large single institutional analysis of dual HER-2 testing, only 12% of cases demonstrate HER-2 amplification, and overall in our institutional pathology database, from 2001 – March 2023, 11.2% of cases are HER-2+ by either IHC or ISH. In our dual tested cohort, only 13.6% of IHC 2+ cases demonstrate amplification, and 13.8% are non-amplified, but have excess HER-2 copy number. We demonstrate a numerical increase in the rate of amplification, and copy number in non-amplified cases, associated with IHC score. In all cases tested with IHC at UVM from 2018 – March 2023, we find 43% of cases meet the current criteria for HER-2 low. Finally, the ASCO/CAP guideline changes from 2013 to 2018 had modest impact on the overall percentage of HER-2+ cases in our series, further data on this impact will be available for presentation. Table. Simultaneously tested IHC and CISH data for new invasive breast cancer cases Citation Format: Xiaocao Xu, Abiy Ambaye, Kara Landry, Peter Kaufman. Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution [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 PO4-26-05.

Abstract PO4-20-11: Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay

Abstract A 72-year-old female presented to the emergency department with sudden onset of imbalance and cognitive decline. Two years ago, she was diagnosed with stage III estrogen receptor (ER) positive, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative (immunohistochemistry score: 0) invasive breast cancer. She subsequently underwent lumpectomy, adjuvant radiation and chemotherapy. She had been taking adjuvant letrozole at the time of presentation. Magnetic resonance imaging (MRI) of the brain showed obstructive hydrocephalus without any parenchymal lesion or leptomeningeal enhancement. MRI of spine, computerized tomography of chest/abdomen/pelvis and bone scan were unrevealing except a left adrenal mass which was equivocal for metastasis. Cerebrospinal fluid (CSF) analysis showed rare malignant cells. Based on the morphology of malignant cells and the patient’s prior history of breast cancer, the diagnosis of leptomeningeal disease with breast primary was made. There were insufficient cells for additional studies. CNSide, a microfluidic platform that uses antibody capture method to detect CSF tumor cells, showed 4678 tumor cells per milliliter (ml) of CSF. Immunocytochemistry of these cells showed that the cells were ER negative (criterion: &amp;lt; 2.6% ER positive cells) and programmed cell death ligand 1 (PD-L1) negative (criterion: &amp;lt; 3.4% PD-L1 positive cells). Fluorescence in situ hybridization (FISH) testing showed 22% of tumor cells with HER2 amplification (either HER2 to CEP17 ratio &amp;gt;/= 2.0 or HER2 copy number &amp;gt;/= 6). She underwent ventriculoperitoneal (VP) shunt placement with complete recovery of her neurological symptoms. Five weeks later, she was started on trastuzumab deruxtecan (T-DXd) mainly because of the HER2 FISH results from CNSide. She did not receive central nervous system(CNS) irradiation due to complete resolution of neurological symptoms with VP shunt placement. After two cycles of T-DXd, MRI brain showed mild leptomeningeal enhancement along bilateral superior cerebellar sulci. Positron emission tomography/computed tomography(PET/CT) scan showed a new sclerotic rib focus, thought to represent treatment response. It also showed decrease in size of the adrenal mass. The adrenal mass was hypermetabolic on PET, consistent with malignancy. CSF cytology was negative, while CSF tumor cell count significantly reduced to 6.72 cells/ml using CNSide. There were no new neurological symptoms. Despite MRI brain findings, she was continued on T-DXd given overall favorable clinical, radiological and CSF data. After 7th cycle of T-DXd, repeat CSF cytology continued to remain negative, CNSide CSF tumor cell count decreased further to 6.27 cells/ml, MRI brain showed interval resolution of prior leptomeningeal enhancement, CT scan showed further decrease in size of the adrenal mass (baseline 5.4cm to 3cm), stable rib sclerotic lesion and no additional metastatic lesion. To date, she has undergone eight cycles of T-DXd which has effectively controlled her disease for a period of seven months. She has not needed VP shunt revision and continues to do well. To our knowledge, this is the first case demonstrating the activity of T-DXd against CSF tumor cells where a small subset exhibited HER2 amplification. This case also highlights the benefits of using advanced and highly sensitive CSF assays to diagnose and surveil leptomeningeal disease and to assess HER2 signaling. This case is well-timed especially in the era of next-generation, CNS penetrating, potent HER2-directed antibody-drug conjugate like T-DXd which can mount antitumor activity even with minimal HER2 expression in tumors. Citation Format: Sarah Stanford, Zeni Kharel, Lauryn Hemminger, Tyler Schmidt, Sara Hardy, Jason Zittel, Nimish Mohile, Ajay Dhakal. Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay [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 PO4-20-11.

NSABP FB-10: a phase Ib/II trial evaluating ado-trastuzumab emtansine (T-DM1) with neratinib in women with metastatic HER2-positive breast cancer

BackgroundWe previously reported our phase Ib trial, testing the safety, tolerability, and efficacy of T-DM1 + neratinib in HER2-positive metastatic breast cancer patients. Patients with ERBB2 amplification in ctDNA had deeper and more durable responses. This study extends these observations with in-depth analysis of molecular markers and mechanisms of resistance in additional patients.MethodsForty-nine HER2-positive patients (determined locally) who progressed on-treatment with trastuzumab + pertuzumab were enrolled in this phase Ib/II study. Mutations and HER2 amplifications were assessed in ctDNA before (C1D1) and on-treatment (C2D1) with the Guardant360 assay. Archived tissue (TP0) and study entry biopsies (TP1) were assayed for whole transcriptome, HER2 copy number, and mutations, with Ampli-Seq, and centrally for HER2 with CLIA assays. Patient responses were assessed with RECIST v1.1, and Molecular Response with the Guardant360 Response algorithm.ResultsThe ORR in phase II was 7/22 (32%), which included all patients who had at least one dose of study therapy. In phase I, the ORR was 12/19 (63%), which included only patients who were considered evaluable, having received their first scan at 6 weeks. Central confirmation of HER2-positivity was found in 83% (30/36) of the TP0 samples. HER2-amplified ctDNA was found at C1D1 in 48% (20/42) of samples. Patients with ctHER2-amp versus non-amplified HER2 ctDNA determined in C1D1 ctDNA had a longer median progression-free survival (PFS): 480 days versus 60 days (P = 0.015). Molecular Response scores were significantly associated with both PFS (HR 0.28, 95% CI 0.09–0.90, P = 0.033) and best response (P = 0.037). All five of the patients with ctHER2-amp at C1D1 who had undetectable ctDNA after study therapy had an objective response. Patients whose ctHER2-amp decreased on-treatment had better outcomes than patients whose ctHER2-amp remained unchanged. HER2 RNA levels show a correlation to HER2 CLIA IHC status and were significantly higher in patients with clinically documented responses compared to patients with progressive disease (P = 0.03).ConclusionsThe following biomarkers were associated with better outcomes for patients treated with T-DM1 + neratinib: (1) ctHER2-amp (C1D1) or in TP1; (2) Molecular Response scores; (3) loss of detectable ctDNA; (4) RNA levels of HER2; and (5) on-treatment loss of detectable ctHER2-amp. HER2 transcriptional and IHC/FISH status identify HER2-low cases (IHC 1+ or IHC 2+ and FISH negative) in these heavily anti-HER2 treated patients. Due to the small number of patients and samples in this study, the associations we have shown are for hypothesis generation only and remain to be validated in future studies.Clinical Trials registration NCT02236000

Abstract 6439: Next-generation sequencing based detection of HER2 copy number predicts pathological response to neoadjuvant therapy in HER2 positive breast cancer

Abstract Purpose: Achieving pathologic complete response (pCR) following neoadjuvant chemotherapy is associated with better prognosis for HER2 positive breast cancer. However, clinical and biological heterogeneity of HER2 positive disease significantly affect prognosis and treatment response. Early prediction of treatment benefit could enable tailored anti-HER2 therapy. Next-generation sequencing (NGS) based genomic tests including copy number (CN) detection are widely used in breast cancer patients. We aim to evaluate the association of HER2 copy number detected by NGS with pCR rate of HER2 positive breast cancer after neoadjuvant chemotherapy. Methods: HER2 positive breast cancer patients underwent neoajuvant chemotherapy in Henan Cancer Hospital From January 2022 to March 2023 were included in the study. Pretreatment tumour specimens were subjected to pan-cancer 1021-gene panel genomic sequencing. Copy number of HER2 was obtained from the panel. Copy number of &amp;gt;=4 was classified as HER2 amplified and HER2 copy number &amp;lt;4 was classified as HER2 non-amplified. All patients underwnt definitive surgery after receieveing neoadjuvant chemotherapy containing taxanes, trastuzumab and pertuzumab. pCR was defined as pathological stage ypT0/Tis ypN0. Results: In total, 299 HER2 positive breast cancer patients were eligible for the analysis. 87 were classified as HER2 non-amplified (29.1%) by copy number and 212 patients were classified as amplified group (70.9%). The HER2 non-amplified group tends to have a higher proportion of hormone receptor positive (HR+) patients (71.3% vs. 50.5%, p=0.002). Other clinicopathological parameters were well distributed between the groups. 91.3% of patients in HER2 amplified group had strong positive HER2 expression (IHC 3+), while only 10.5% of patients in HER2 non-amplified group had HER2 expression of IHC 3+. Patients with HER2 IHC 3+ had a PCR rate of 75.8% and Patients with HER2 IHC 2+ had a pCR rate of 20.0% (20.0% vs. 75.8% p&amp;lt;0.001). Patients in the amplified group had a pCR rate of 76.9% and pCR rate of the non-amplified group was12.6% (12.6% vs. 76.9% p&amp;lt;0.001). In a multivariable logistic regression with HER2 copy number by NGS, HR status and HER2 IHC, the HER2 copy number predicted pCR rate independently of HER2 IHC and HR status. Conclusions: NGS based detection of HER2 copy number could predict response to neoadjuvant therapy in HER2 positive patients. The discordance between IHC/FISH and NGS based detection of HER2 amplification may reflect HER2 heterogeneity. HER2 heterogeneity promotes resistance to anti-HER2 therapy. Noval antibody-drug conjugates (ADC) with bystander effect, such as Trastuzumab deruxtecan, have shown anti-tumor effect even in HER2 low expression tumors. Thus, prospective study is needed to optimize anti-HER2 therapy for patients with HER2 heterogeneity. Citation Format: Hao Dai, Dechuang Jiao, Xuhui Guo, Jianghua Qiao, anfang Li, Zhenduo Lu, Xiuchun Chen, Chengzheng Wang, Min Yan, Zhenzhen Liu. Next-generation sequencing based detection of HER2 copy number predicts pathological response to neoadjuvant therapy in HER2 positive breast cancer [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 6439.

All-In-One OsciDrop Digital PCR System for Automated and Highly Multiplexed Molecular Diagnostics.

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all-in-one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation-induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette-based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning-driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high-multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost-effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics.

Response to anti-HER2 neoadjuvant chemotherapy in HER2-positive invasive breast cancers with different HER2 FISH patterns

AimsHuman epidermal growth factor receptor 2 (HER2)-positive patients with breast cancer may have different HER2/CEP17 ratios and HER2 copy numbers, with inconsistent responses to anti-HER2 neoadjuvant chemotherapy (NACT). Our study...

Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer

BackgroundOverexpression of human epidermal growth factor receptor 2 (HER2) caused by HER2 gene amplification is a driver in breast cancer tumorigenesis. We aimed to investigate the prognostic significance of manual scoring and digital image analysis (DIA) algorithm assessment of HER2 copy numbers and HER2/CEP17 ratios, along with ERBB2 mRNA levels among early-stage HER2-positive breast cancer patients treated with trastuzumab.MethodsThis retrospective study comprised 371 early HER2-positive breast cancer patients treated with adjuvant trastuzumab, with HER2 re-testing performed on whole tumor sections. Digitized tumor tissue slides were manually scored and assessed with uPath HER2 Dual ISH image analysis, breast algorithm. Targeted ERBB2 mRNA levels were assessed by the Xpert® Breast Cancer STRAT4 Assay. HER2 copy number and HER2/CEP17 ratio from in situ hybridization assessment, along with ERBB2 mRNA levels, were explored in relation to recurrence-free survival (RFS).ResultsThe analysis showed that patients with tumors with the highest and lowest manually counted HER2 copy number levels had worse RFS than those with intermediate levels (HR = 2.7, CI 1.4–5.3, p = 0.003 and HR = 2.1, CI 1.1–3.9, p = 0.03, respectively). A similar trend was observed for HER2/CEP17 ratio, and the DIA algorithm confirmed the results. Moreover, patients with tumors with the highest and the lowest values of ERBB2 mRNA had a significantly worse prognosis (HR = 2.7, CI 1.4–5.1, p = 0.003 and HR = 2.8, CI 1.4–5.5, p = 0.004, respectively) compared to those with intermediate levels.ConclusionsOur findings suggest that the association between any of the three HER2 biomarkers and RFS was nonlinear. Patients with tumors with the highest levels of HER2 gene amplification or ERBB2 mRNA were associated with a worse prognosis than those with intermediate levels, which is of importance to investigate in future clinical trials studying HER2-targeted therapy.

Predictive markers for pathological complete response (pCR) after neo-adjuvant chemotherapy in HER2-positive breast carcinoma.

Patients with HER2-positive invasive breast cancer that is node-positive and/or larger than 3 cm are generally treated with neoadjuvant chemotherapy (NAC). We aimed to identify predictive markers for pathological complete response (pCR) after NAC in HER2-positive breast carcinoma. Hematoxylin/eosin-stained slides of 43 HER2-positive breast carcinoma biopsies were histopathologically reviewed. Immunohistochemistry (IHC) was performed on pre-NAC biopsies, comprising HER2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, epidermal growth factor receptor (EGFR), mucin-4 (MUC4), p53 and p63. Dual-probe HER2 in situ hybridization (ISH) was performed to study the mean HER2 and CEP17 copy numbers. ISH and IHC data were retrospectively collected for a validation cohort, comprising 33 patients. Younger age at diagnosis, 3+ HER2 IHC scores, high mean HER2 copy numbers and high mean HER2/CEP17 ratios were significantly associated with an increased chance of achieving a pCR, and the latter two associations were confirmed in the validation cohort. No other immunohistochemical or histopathological markers were associated with pCR. This retrospective study of two community-based NAC-treated HER2-positive breast cancer patient cohorts identified high mean HER2 copy numbers as a strong predictor for pCR. Further studies on larger cohorts are required to determine a precise cut-point for this predictive marker.

Abstract A005: Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study

Abstract The HER2 diagnostics is necessary for selection of patients harboring HER2 gene amplification or protein overexpression who will benefit from anti-HER2 therapies in breast cancer. However, HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Thus, a new method is needed to accurately quantify HER2 levels. 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, a potential new diagnostic platform with improved performance by integrating both real-time and digital PCR technologies. For drPCR-based HER2 copy number (CN) measurement, primer-probe sets specific to HER2 gene and a genomic region adjacent to chromosome 17 centromere (CEP17) were designed, and the optimal drPCR condition was determined in clinical breast tumor specimens. 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: Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Soo Young Park, Ji-Hye Park, Hee-Young Won, Min Ji Song, Da Sol Kim, Hayeon Kim, Sohyeon Yang, Nam Hun Heo, Minsik Song, Seung-Shick Shin, Do Young Lee, Han Suk Ryu, Si-Hyong Jang, Jeong-Yeon Lee. Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A005.

CCQM-K176 breast cancer biomarker HER2 copy number variation (CNV) measurement

Main textBreast cancer is the most common malignant tumor in women all over the world. Breast cancers with amplification of the human epidermal growth factor receptor 2 (HER2) gene (referred to as HER2-positive breast cancer) accounts for about 20% ~ 30% of invasive breast cancer, and this type of tumor is characterized by high invasiveness, high risk of recurrence, rapid progression and poor prognosis. The determination of HER2 gene expression or copy number variant (CNV) (HER2 copy number normalized to cell number or a reference gene) in tumor cells of breast cancer patients is beneficial to the choice of treatment, through targeted therapies such as trastuzumab, and prognosis. Testing of the core competencies of laboratories to deliver services of CNV measurements for different genetic analytes has not been covered previously. Agreement was received in the October 2019 meeting of the NAWG in Torino to conduct the HER2 CNV measurement as a key comparison. Evidence of successful participation in formal, relevant international comparisons is needed to support calibration and measurement capability claims (CMCs) made by national metrology institutes (NMIs) and designated institutes (DIs).Ten National Metrology Institutions participated in the Track A Key Comparison: CCQM-K176 "Breast cancer biomarker HER2 copy number variation (CNV) measurement". Participants were requested to evaluate the copy number concentration (expressed in [μL-1]) and copy number ratio of defined genomic sequences in eukaryotic intact genomic DNA at an indicative concentration of 90~130 ng/μL. Participation in K176 required assay selection/design and quantitative detection of the analyte in the buffer. The objectives of the key comparison were to measure 1) HER2 copy number concentration; 2) the copy number concentration of the single copy reference gene ribonuclease P RNA component H1 (RPPH1); 3) the HER2/ RPPH1 copy number ratio, using two defined sequences representative of the two genes (HER2 and RPPH1) in mixed genomic DNA samples, and 4) provide evidence for CMC claims by participating laboratories when measuring target sequence content in purified genomic DNA. All participants performed measurements by digital PCR (dPCR), with alternative assays and pre-treatment of samples (for example, restriction enzyme digestion). Interlaboratory reproducibility (%CV) was less than 13% and 5% for copy number concentration and HER2/RPPH1 copy number ratio, respectively, demonstrating very high interlaboratory reproducibility in CNV measurements.Successful participation in CCQM-K176 demonstrates the following measurement capabilities in determining copy number ratio (of a defined target sequence expressed relative to a reference gene sequence) in the range from 1.0 to 40.0 at a background of relatively high concentration of intact (non-fragmented) genomic DNA. The study will also support participants' claim for measurement of defined DNA sequence/gene copy number concentration in the range ~104-106 μL-1 (taking into account both reported reference gene and HER2 copy number concentration values).To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

HER2/ERBB2 copy number analysis by targeted next-generation sequencing in breast cancer.

A combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) is the current standard of care for HER2 evaluation in breast cancer. Here, we investigate the potential clinical utility of next-generation sequencing (NGS)-derived HER2/ERBB2 copy number (CN) data for predicting HER2 status as defined by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. In total, 294 locally recurrent and metastatic breast cancers previously tested by targeted hybrid capture-based NGS and by HER2 IHC/FISH were included. Analyses focused on the ERBB2 median log2 ratios and start-end genomic coordinates from NGS, average HER2 CN and HER2/CEP17 ratios from FISH, and the HER2 IHC scores. We also determined a more stringent log2 ratio cutoff to predict HER2-positive status with 100% specificity. Sixty-four (22%) cases were HER2 positive and 230 (78%) were HER2 negative by ASCO/CAP guidelines. The ERBB2 median log2 ratios from NGS strongly correlated with HER2 status by IHC/FISH (area under receiver operator characteristic curve = 0.951). ERBB2 log2 ratio more than 1.7 was 100% specific for HER2-positive results by IHC/FISH. Start and end genomic coordinates for regions of gain near ERBB2 by NGS also predicted HER2 status. Copy number data from our NGS panel strongly correlate with HER2 status. Using a stringent cutoff, ERBB2 log2 ratio accurately predicts HER2 positivity with high specificity. The NGS CN assessment may have utility in determining HER2 status in certain clinical settings.

HER2 amplification level by in situ hybridization predicts survival outcome in advanced HER2-positive breast cancer treated with pertuzumab, trastuzumab, and docetaxel regardless of HER2 IHC results

BackgroundThe role of HER2 amplification level in predicting the effectiveness of HER2-directed therapies has been established. However, its association with survival outcomes in advanced HER2-positive breast cancer treated with dual HER2-blockade remains unexplored.MethodsThis is a single-center retrospective study of patients with advanced HER2-positive breast cancer treated with first-line pertuzumab, trastuzumab, and docetaxel. The primary objective was to ascertain the relationship between treatment outcomes and the level of HER2 amplification by in situ hybridization (ISH).ResultsA total of 152 patients were included with a median follow-up duration of 50.0 months. Among the 78 patients who received ISH, a higher HER2/CEP17 ratio correlated significantly with longer PFS (HR 0.50, p = 0.022) and OS (HR 0.28, p = 0.014) when dichotomized by the median. A higher HER2 copy number also correlated significantly with better PFS (HR 0.35, p < 0.001) and OS (HR 0.27, p = 0.009). In multivariate analysis, the HER2/CEP17 ratio was an independent predictive factor for PFS (HR 0.66, p = 0.004) and potentially for OS (HR 0.64, p = 0.054), along with HER2 copy number (PFS HR 0.85, p = 0.004; OS HR 0.84, p = 0.049). Furthermore, the correlation between HER2 amplification level by ISH with PFS and OS was consistent across the HER2 IHC 1+/2+ and 3+ categories.ConclusionsThis is the first study to report that a higher level of HER2 amplification by ISH is associated with improved PFS and OS in advanced HER2-positive breast cancer treated with dual HER2-blockade. Notably, HER2 amplification level had a predictive role regardless of IHC results. Even in patients with HER2 protein expression of 3+, treatment outcome to HER2-directed therapy was dependent on the level of HER2 gene amplification.

HER2 amplification in cases with low HER2 expression by immunohistochemistry (IHC): A Single Institutional Experience

Abstract Introduction/Objective HER2 testing in invasive breast carcinoma (IBC) is the standard of care to determine patient eligibility for HER2 directed therapy with trasuzumab. Initial testing of tissue by HER2 immunohistochemistry (IHC) can be positive, negative, or equivocal. In equivocal cases as well as negative cases in our institution HER2 FISH is also performed. According to ASCO/CAP guidelines a subset of equivocal and negative IHC cases are considered HER2 positive if they meet criteria for increased HER2/CEP17 ratio and HER2 copy number by FISH, allowing more patients to be treated with HER2 directed therapies. HER2 grouping per ASCO is as follows; Group 1: HER2 copy number ≥4 signals/cell, Group 2: &amp;lt;4.0 signals/cell, Group 3: ≥6 signals/cell, Group 4: HER2 copy number ≥4 and &amp;lt;6 signals/cell, Group 5: HER2 copy number &amp;lt;4 signals/cell Methods/Case Report All cases of IBC resection with HER2 IHC and FISH results from Jan 2018-Dec 2021 were included in this study. All cases positive for HER2 by IHC alone (3+ staining) were excluded. The ASCO/CAP 2018 guidelines for HER2 testing in breast cancer were used to evaluate HER2 positivity by FISH and the relation to IHC scores Results (if a Case Study enter NA) A total of 2630 IBC cases with both HER2 IHC and FISH testing were included. Of these, 131 (5.0%) were in FISH group 1, 17 (0.7%) in group 2, 11 (0.4%) in group 3, 161 (6.1%) in group 4, and 2310 (87.8%) in group 5. A total of 142 (5.4%) cases that were HER2 equivocal or negative by IHC were HER2 positive by FISH. Specifically, 0.3% of cases with an IHC score of 0, 1.4% with a score of 1+, and 14.6% of cases with a score of 2+ were positive by FISH. Of the 14.6% IHC 2+ cases that were FISH positive 13.3% were in FISH group 1 (115 cases) and 1.3% (11 cases) in FISH group 3. Of FISH positive cases 1.4% had an IHC score of 0, 9.9% had an IHC score of 1 and 88.7% had a score of 2 Conclusion At our institution 14.6% of IBC with equivocal HER2 staining on IHC, 1.4% of cases with negative (1+) staining and 0.3% of cases with negative (0) were HER2 FISH positive. Since HER2 FISH is not reflexively performed on cases HER2 negative by IHC, this additional 1.7% will qualify for HER2 targeted therapy Interpretation of low HER2 expression by IHC by traditional IHC assay may miss a small percentage of cases eligible for HER2 directed therapy. Accurate interpretation of HER2 low by IHC is critical as recent clinical trials have shown that low HER2 expression with IHC scores of 1+ or 2+ without gene amplification by FISH could benefit from Trastuzumab Deruxtecan

Decreased HER2 expression in endometrial cancer following anti-HER2 therapy.

Trastuzumab has demonstrated clinical efficacy in the treatment of HER2-positive serous endometrial cancer (EC), which led to its incorporation into standard-of-care management of this aggressive disease. Acquired resistance remains an important challenge, however, and its underlying mechanisms in EC are unknown. To define the molecular changes that occur in response to anti-HER2 therapy in EC, targeted next-generation sequencing (NGS), HER2 immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) were performed on pre- and post-treatment tumour samples from 14 patients with EC treated with trastuzumab or trastuzumab emtansine. Recurrent tumours after anti-HER2 therapy acquired additional genetic alterations compared with matched pre-treatment ECs and frequently showed decreased HER2 protein expression by IHC (7/14, 50%). Complete/near-complete absence of HER2 protein expression (score 0/1+) observed post-treatment (4/14, 29%) was associated with retained HER2 gene amplification (n = 3) or copy number neutral status (n = 1). Whole-exome sequencing performed on primary and recurrent tumours from the latter case, which exhibited genetic heterogeneity of HER2 amplification in the primary tumour, revealed selection of an early HER2-non-amplified clone following therapy. Our findings demonstrate that loss of target expression, by selection of HER2-non-amplified clones or, more commonly, by downregulation of expression, may constitute a mechanism of resistance to anti-HER2 therapy in HER2-positive EC. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.