HER2 Expression In Tumors Research Articles

64Cu]Cu-NOTA-Trastuzumab and [89Zr]Zr-DFO-Trastuzumab in Xenografts with Varied HER2 Expression.

Positron emission tomography (PET) has potential as a complementary technique to biomarker analysis, especially for human epidermal growth factor receptor 2 (HER2)-expressing tumors characterized by high heterogeneity. In this study, zirconium-89 (89Zr) and copper-64 (64Cu) labeled trastuzumab were employed to monitor varying levels of tumoral HER2 expression. Additionally, we studied the use of the cholesterol-depleting lovastatin as a pharmacological approach to enhance cell-surface HER2 expression in tumors with moderate to low HER2 levels, aiming to increase antibody accumulation in these tumor types. Both 89Zr- and 64Cu-labeled trastuzumab effectively monitor HER2 expression levels in xenografts exhibiting varying HER2 expression. No significant difference in tumor uptake was observed between 89Zr- or 64Cu-labeled trastuzumab, and tumor uptake for both radioimmunoconjugates positively correlated with HER2 protein levels. These findings underscore the potential of PET to monitor HER2 protein levels across heterogeneous tumors. Furthermore, our results suggest that further optimization of statin dosing and timing could offer a promising strategy to enhance trastuzumab accumulation in HER2-high, HER2-moderate, and HER2-low tumors.

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.

Imaging and Monitoring HER2 Expression in Tumors during HER2 Antibody-Drug Conjugate Therapy Utilizing a Radiolabeled Site-Specific Single-Domain Antibody Probe: 68Ga-NODAGA-SNA004-GSC.

The overexpression of HER2 is pivotal in the initiation and progression of breast cancer. Developing HER2-targeted radiotracers is crucial for noninvasive assessment of HER2 expression, patient selection for HER2-targeted therapy, monitoring treatment response, and identifying resistance. Here, we reported a nonsite-specific coupled radiotracer, 68Ga-NOTA-SNA004-His6, and a site-specific coupled radiotracer, 68Ga-NODAGA-SNA004-GSC, based on a novel HER2 nanobody, SNA004. Both radiotracers exhibited high affinity, specific targeting, and rapid clearance in vitro and in vivo. Additionally, these tracers and trastuzumab showed noncompetitive binding to HER2. Compared to 68Ga-NOTA-SNA004-His6, 68Ga-NODAGA-SNA004-GSC demonstrated significantly reduced renal and liver uptake. PET/CT imaging with 68Ga-NODAGA-SNA004-GSC sensitively detected the responsiveness of various tumor models to trastuzumab and its antibody-drug conjugates (ADCs). Overall, the site-specific coupled radiotracer 68Ga-NODAGA-SNA004-GSC offered significant advantages in biodistribution and signal-to-noise ratio, making it a valuable tool for monitoring HER2 expression levels before, during, and after trastuzumab and ADC treatment.

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.

Abstract P6-04-05: A fully automatic artificial intelligence system for accurate and reproducible HER2 IHC scoring in breast cancer

Abstract Objective: Tumor HER2 expression is a key prognostic and treatment influencing factor in breast cancer. As with all immunohistochemistry (IHC) staining, visual interpretation of HER2 expression is subjective, which leads to intra- and inter-pathologist variability. Recent findings on the efficacy of HER2-targeted therapy on HER2-low patients raise the need for accurate and reproducible scoring. We developed a fully automated, artificial intelligence (AI) -based algorithm for HER2 scoring. The algorithm was based on ASCO/CAP 2018 guidelines and validated against rigorous ground truth (GT) established by multiple blinded expert pathologists. Methods: Algorithm development: We developed a solution that employs two steps: The first step consists of an ensemble of Deep Learning networks that process tissue regions and classify them as various tissue classes: Invasive cancer, Ductal Carcinoma In Situ (DCIS) and other morphologies. These networks were trained on slides that were automatically labeled by a separate AI system that analyzed the corresponding H&amp;E slides and projected its findings to the HER2 IHC slides using a registration algorithm. To further enrich the training set, especially with rare and difficult cases, a team of 8 expert pathologists manually marked tissue areas and assigned them to one of the tissue classes. In total, the training set consisted of 6,400 manual annotations and 1,300 automatically-annotated slides, both collected from 9 laboratories and scanned using 3 different scanners. The second step is an ensemble of Object Detection networks that process only the regions classified as invasive cancer, detect the tumor cells within them, and classify their staining pattern (e.g., Not stained, Moderate incomplete, etc.). Finally, the detected cells are counted, and the ASCO/CAP guidelines are applied to derive the slide-level HER2 score. Validation: The validation set was comprised of 453 HER2 slides stained using the VENTANA anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody as per manufacturer’s instructions. HER2 slides included biopsies and excisions with different breast cancer diagnoses (e.g., Infiltrating Ductal Carcinoma (IDC), Infiltrating Lobular Carcinoma (ILC), rare invasive subtypes, with and without DCIS) from 3 different laboratories. Ground truth was established by the consensus scores of a panel of 3 pathologists, who scored HER2 according to the guidelines without additional clinical considerations, such as scoring borderline 1+/2+ cases as 2+ to have additional tests performed. Results: The algorithm showed very high performance for detecting invasive cancer in HER2 tissue sections, with AUC of 0.967 (measured on 4-fold Cross-Validation classifying invasive vs. other regional classes). The algorithm demonstrated an overall accuracy of 80.3% for the HER2 scores when compared to the GT. When using different cutoffs for binary classification the resulting performance was: for 0 vs 1+/2+/3+ Kappa was 0.800; 0/1+ vs 2+/3+ Kappa was 0.728; for 0/1+/2+ vs 3+ Kappa was 0.954. The Quadratic Kappa between the AI score and the GT was 0.898, which is considered almost perfect. The performance of the AI was similar across the different laboratories and diagnoses(e.g. IDC, ILC). Conclusion: This study reports the successful development and independent validation of a fully automatic AI-based solution for accurate HER2 scoring in breast cancer. AI solutions, such as the one reported here, could be used as decision-support tools for pathologists in routine clinical practice, enhancing the reproducibility and consistency of HER2 scoring, thus enabling optimal treatment pathways and better patient outcomes. Accurate and automatic IHC scoring solutions can also contribute to the development of new prognostic, predictive and companion diagnostic tools. Citation Format: Yuval Globerson, Lilach Bien, Jonathan Harel, Giuseppe Mallel, Geraldine Sebag, Michel Vandenberghe, Craig Barker, Tsuyoshi Matsuo, Charo Garrido, Judith Sandbank, Chaim Linhart. A fully automatic artificial intelligence system for accurate and reproducible HER2 IHC scoring in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-05.

Phase 2 clinical study evaluating the efficacy and safety of disitamab vedotin with or without pembrolizumab in patients with HER2-expressing urothelial carcinoma (RC48G001).

TPS594 Background: Urothelial carcinoma (UC) is the 10th most diagnosed cancer worldwide, and nearly 50% of patients cannot tolerate the current first-line (1L) standard of care. There is a need to improve outcomes in 1L and later lines of therapy. HER2 is overexpressed in UC and may be associated with poor outcomes. Despite this, no HER2-directed therapies are currently approved for UC. Disitamab vedotin (DV; RC48-ADC) is an investigational antibody-drug conjugate comprising a novel HER2-directed monoclonal antibody, disitamab, conjugated to monomethyl auristatin E (MMAE) via a protease-cleavable mc-vc linker. DV elicits antitumor activity through multimodal mechanisms of action including MMAE-mediated direct cytotoxicity, bystander effect, and immunogenic cell death. Methods: RC48G001 (NCT04879329) is a phase 2, multi-cohort, open-label, multicenter trial to evaluate DV in patients with HER2-expressing locally advanced/metastatic UC (LA/mUC). Patients are enrolled in 3 cohorts based on prior treatment and tumor HER2 expression by immunohistochemistry (IHC) and gene amplification (via in situ hybridization [ISH]), assessed centrally. Cohorts A and B are enrolling patients who have received prior systemic therapy (1 or 2 lines, including platinum-containing chemotherapy) and whose tumors are HER2-positive (IHC 3+, or 2+ and ISH-positive) or HER2-low (IHC 2+ and ISH-negative, or IHC 1+), respectively. Cohort C is enrolling 1L treatment-naive patients in the LA/mUC setting, whose tumors have HER2-positive or HER2-low expression. Cohorts A and B will evaluate DV as monotherapy (intravenous [IV] administration, once every 2 weeks [Q2W]). Cohort C will evaluate DV (IV, Q2W) +/– pembrolizumab (IV, Day 1 of each 6-week cycle). Patients in all cohorts must have measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 and an Eastern Cooperative Oncology Group Performance Status of 0–1 in Cohorts A and B, and 0–2 in Cohort C. The primary endpoint is confirmed objective response rate (cORR) per blinded independent central review (BICR). Secondary endpoints include cORR by investigator assessment, overall survival, duration of response, progression-free survival, disease control rate per RECIST v1.1 by BICR and investigator, safety, and pharmacokinetic parameters. Enrollment for all cohorts is ongoing in North America and Europe, and planned in Latin America, Asia-Pacific, and Israel. Clinical trial information: NCT04879329 .

Molecular imaging of HER2 expression in breast cancer patients using a novel peptide-based tracer 99mTc-HP-Ark2: a pilot study

BackgroundDue to the temporal and spatial heterogeneity of human epidermal growth factor receptor 2 (HER2) expression in breast tumors, immunohistochemistry (IHC) cannot accurately reflect the HER2 status in real time, which may cause misguided treatment decisions. HER2-specific imaging can noninvasively determine HER2 status in primary and metastatic tumors. In this study, HER2 expression in breast cancer patients was determined in vivo by SPECT/CT of 99mTc-HP-Ark2, comparing with PET/CT of 18F-FDG lesion by lesion.MethodsA novel HER2-targeted peptide probe 99mTc-HP-Ark2 was constructed. Biodistribution and nanoScan SPECT/CT imaging were performed in mice models. The correlation between the quantified tumor uptake and HER2 expression in tumor cells was analyzed. In the pilot clinical study, a total of 34 breast cancer patients (mean age ± SD: 49 ± 10 y) suspected of having breast cancer according to mammography or ultrasonography were recruited at Peking Union Medical College Hospital, and 99mTc-HP-Ark2 SPECT/CT and 18F-FDG PET/CT were carried out with IHC and fluorescence in situ hybridization as validation.ResultsSmall animal SPECT/CT of 99mTc-HP-Ark2 clearly identified tumors with different HER2 expression. The quantified tumor uptake and tumor HER2 expression showed a significant linear correlation (r = 0.932, P < 0.01). Among the 36 primary lesions in the 34 patients, when IHC (2 +) or IHC (3 +) was used as the positive evaluation criterion, 99mTc-HP-Ark2 SPECT/CT imaging with a tumor-to-background ratio of 1.44 as the cutoff value reflected the HER2 status with sensitivity of 89.5% (17/19), specificity of 88.2% (15/17) and accuracy of 88.9% (32/36), while the 18F-FDG PET/CT showed sensitivity of 78.9% (15/19), specificity of 70.6% (12/17) and accuracy of 75.0% (27/36). In particular, 100% of IHC (3 +) tumors were all identified by 99mTc-HP-Ark2 SPECT/CT imaging.Conclusion99mTc-HP-Ark2 SPECT/CT can provide a specific, noninvasive evaluation of HER2 expression in breast cancer, showing great potential to guide HER2-targeted therapies in clinical practice.ClinicalTrials.gov Trial registration: NCT04267900. Registered 11th February 2020. Retrospectively registered, https://www.clinicaltrials.gov/ct2/results?pg=1&load=cart&id=NCT04267900.

Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts: Efficacy and Selection of Companion Imaging Counterpart.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPTNeg-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as 99mTc(CO)3-ADAPT6 or the affibody molecule 99mTc-ZHER2:41071, are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1–based therapy.

HER2-targeted dual radiotracer approach with clinical potential for noninvasive imaging of trastuzumab-resistance caused by epitope masking.

Rationale: The decreased HER2-accessibility by epitope masking is a primary trastuzumab-resistance mechanism. In this study, we developed a HER2-targeted dual radiotracer approach to predict the HER2-trastuzumab engagement noninvasively.Methods: Two novel HER2-specific VHHs, MIRC208 and MIRC213, were acquired by immunizing alpaca with human HER2 protein, and were site-specifically labeled with 99mTc. Biodistribution and SPECT/CT imaging studies were performed in mice bearing HER2-positive and HER2-negative tumors. The HER2 binding sites of 99mTc-MIRC208 and 99mTc-MIRC213 were investigated by cell binding and SPECT/CT imaging studies. We evaluated the therapeutic predictive ability of our dual-radiotracer imaging approach for trastuzumab treatment in mice bearing MUC4-positive tumors (trastuzumab-resistant JIMT-1 and 87MUC4) and MUC4-negative tumors (trastuzumab-sensitive 7HER2 and NCI-N87). The preliminary clinical studies of 99mTc-MIRC208 were performed in two patients with HER2-positive breast tumors.Results: 99mTc-MIRC208 and 99mTc-MIRC213 clearly visualized HER2-positive tumors, but not HER2-negative tumors. 99mTc-MIRC208 competes with trastuzumab for HER2-binding while 99mTc-MIRC213 recognizes HER2 on an epitope that is not masked by MUC4. The SPECT/CT studies with 99mTc-MIRC208 and 99mTc-MIRC213 clearly showed that the MUC4-negative and trastuzumab-sensitive 7HER2 and NCI-N87 tumors had very similar tumor uptake with the SUV208/SUV213 (2 h) ratios of 1.11 ± 0.17 in 7HER2 and 1.25 ± 0.22 in NCI-N87. However, the MUC4-positive JIMT-1 tumors showed the decreased SUV208/SUV213 (2 h) ratio (0.63 ± 0.07), which correlated well with the low response rate to trastuzumab therapy. The SUV208/SUV213 (2 h) ratio was reduced to 0.72 ± 0.02 in MUC4-expressing NCI-N87 cells, and resulting in the decreased trastuzumab sensitivity, further supporting the correlation between the SUV208/SUV213 (2 h) ratio and trastuzumab-sensitivity. The primary and metastatic HER2-positive lesions of patients were clearly visualized by 99mTc-MIRC208 SPECT at 2 h post injection.Conclusion: Overall, we demonstrated that the dual radiotracer imaging strategy is a valid noninvasive approach for the cancer patient selection before trastuzumab therapy. 99mTc-MIRC213 SPECT is utilized to quantify the tumor HER2 expression and screen HER2-positive cancer patients, while 99mTc-MIRC208 SPECT is used to determine the HER2-accessibility of trastuzumab. The SUV208/SUV213 (2 h) ratio is an important biomarker to determine the responsiveness of trastuzumab therapy.

Haploinsufficiency Interactions between RALBP1 and p53 in ERBB2 and PyVT Models of Mouse Mammary Carcinogenesis.

Simple SummaryRlip knockout has been reported to prevent cancer in highly cancer-susceptible mice lacking p53, and Rlip knockdown kills many types of cancer cells. In humans, breast cancer shows diverse characteristics, including HER2-driven subtypes and viral-driven subtypes. HER2 can be targeted; however, escape of the cancer from targeted therapies remains a problem. In this work we evaluated the capacity of Rlip knockout to prevent breast cancer in genetically engineered mouse models of HER2-driven breast cancer (Erbb2 model) and polyomavirus-driven breast cancer (PyVT model). We found that in Erbb2 mice, Rlip knockout significantly delayed oncogenesis and reduced the expression of genes associated with poor prognosis in patients. In PyVT mice, Rlip knockout did not delay oncogenesis or tumor growth, but Rlip knockdown reduced tumor metastasis to the lung. We conclude that Rlip inhibitors may significantly improve survival in HER2-positive patients, but are unlikely to offer benefits to patients with polyomavirus-associated tumors.We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Machine learning models to quantify HER2 for real-time tissue image analysis in prospective clinical trials.

3061 Background: Patient eligibility for HER2-targeting treatments is commonly informed by testing tumor HER2 expression using immunohistochemistry. As HER2 expression is visually assessed by pathologists, inter- and intra-rater variability might affect treatment decisions. Here, we report the development of an automated machine learning (ML)-based algorithm to quantify HER2 cell membrane expression across a diversity of breast cancer phenotypes as a clinical tool for monitoring HER2 testing quality. Methods: A total of 689 breast cancer tissue samples were either procured (Avaden Biosciences) or were anonymized samples from the AstraZeneca biobank comprising tissues from primary and metastatic tumors, core needle biopsies and surgical resections, lobular and ductal carcinomas, across tumor grades and HER2 expression levels. Samples were stained for HER2 detection (Ventana HER2 (4B5) Assay) and digitized (Leica Biosystems) across 5 laboratories in the US. Whole-slide images (WSIs) were stratified into training (n = 407), validation (n = 110), and test sets (n = 172). Multiple convolutional neural network based ML models (PathAI, Boston, MA) were trained using 190,000 manual annotations provided by 30 board-certified pathologists to identify artifacts, invasive tumor, identify individual cancer cells and measure tumor cell membrane HER2 expression as partial or complete, and negative, weak-or-moderate, or intense. Cell-level scores were validated against a consensus of manual cell counts from 5 independent pathologists in 320 representative regions of test set WSIs. HER2 scores were generated by automatically applying rules derived from 2018 ASCO/CAP guidelines and then compared in the test set with consensus scores from 3 independent pathologists. Results: Cell counts provided by the ML model were strongly consistent with cell counts obtained by pathologist consensus in all cell-types except for faintly positive HER2 cells where ML-based quantification identified more cells on average. Automatically generated ML-ASCO/CAP HER2 scores using WSI showed substantial consistency across IHC categories with the consensus of pathologists (ICC 0.88, 95%CI 0.82-0.92) in the test set and improved further when ML models were trained to agree with pathologists by adjusting cut offs (ICC 0.91, 95%CI 0.89-0.94). The ML-based model was deployed through the PathAI cloud platform to calculate HER2 testing quality control metrics in real-time in multicentric clinical trials. Conclusions: Automated image analysis of HER2-stained breast cancer tissues using ML-based models is consistent with pathologist consensus across breast cancer tissue types. The results support evidence that ML-based algorithms can help pathologists assess HER2 testing reproducibility in clinical trials.

Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex.

Molecular imaging of tumor HER2 expression may allow patient selection for HER2-targeted therapies. Our aim was to introduce hexahistidine (His6) peptides into pertuzumab Fab to enable labeling with the [99mTc(CO)3(H2O)3]+ complex and study these radioimmunoconjugates for microSPECT/CT imaging of HER2-positive tumor xenografts in mice. Fab were produced by papain digestion of pertuzumab and reacted with sulfo-SMCC for conjugation to His6-containing peptides (CGYGGHHHHHH). His6-peptide conjugation was measured by a radiometric assay. His6-pertuzumab Fab were labeled at 0.4-1.0MBq/μg with [99mTc(CO)3(H2O)3]+ for 1h at 37°C. HER2 immunoreactivity was assessed in a direct (saturation) binding assay using HER2-overexpressing SK-BR-3 human breast cancer (BC) cells. MicroSPECT/CT and biodistribution studies were performed in NOD/SCID mice with HER2-positive s.c. SK-OV-3 human ovarian cancer, or MDA-MB-361 or MDA-MB-231 human BC xenografts at 4 or 24h post i.v. injection of [99mTc]His6-pertuzumab Fab (29-49MBq, 70μg). The specificity of tumor uptake was assessed by comparison to irrelevant [99mTc]Fab 3913 in SK-OV-3 tumor-bearing mice. SDS-PAGE analysis demonstrated cleavage of pertuzumab to produce Fab, which eluted as a single peak with a retention time of 13.8min on SE-HPLC. Fab were conjugated to 2.1 ± 0.5 His6 peptides and labeled with [99mTc(CO)3(H2O)3]+ to a radiochemical purity of 92-97 % at 0.4-0.8MBq/μg. [99mTc]His6-pertuzumab Fab exhibited saturable and specific binding to SK-BR-3 cells with a KD = 51.3 ± 5.2 × 10-9M and Bmax = 3.5 ± 0.1 × 106 receptors/cell. SK-OV-3 tumors were imaged at 4 and 24h p.i [99mTc]His6-pertuzumab Fab. Tumor uptake at 24h p.i. was 4.1 ± 0.6 %ID/g, which was 13-fold significantly greater than [99mTc]Fab 3913 (0.3 ± 0.0 %ID/g; P < 0.01). MicroSPECT/CT imaged HER2-overexpressing MDA-MB-361 tumors but not MDA-MB-231 tumors with low HER2 expression. Tumor uptake was 5.2-fold significantly greater at 24h p.i. in MDA-MB-361 than MDA-MB-231 tumors (3.2 ± 0.1 %ID/g vs. 0.8 ± 0.1 %ID/g; P < 0.05). MicroSPECT/CT with [99mTc]His6-pertuzumab Fab imaged tumors in NOD/SCID mice that exhibited intermediate or high HER2 expression, but not tumors with low HER2. [99mTc]His6-pertuzumab Fab is promising for SPECT imaging of tumor HER2 expression.

Targeting and imaging of HER2 overexpression tumor with a new peptide-based 68Ga-PET radiotracer

Human epidermal growth factor receptor 2 (HER2) overexpression, as a predictive biomarker, is associated with more tumor aggressiveness and worse clinical outcomes in cancer, whereas it’s accurate identification has led to the choice of effective treatments in many patients. In this study, a peptide-based PET probe (68Ga-DOTA-(Ser)3-LTVSPWY) was developed for imaging HER2 expression in tumors. The DOTA-(Ser)3-LTVSPWY was labeled with 68Ga and then was evaluated in vitro with HER2-positive SKOV-3 cell line; moreover, the in vivo biodistribution and PET/CT imaging were performed in xenografted tumor-bearing nude mice. The 68Ga-DOTA-(Ser)3-LTVSPWY displayed the high radiochemical purity greater than 95% and good stability in normal saline and human serum. The cellular binding experiments showed that the cell uptake in HER2-positive ovarian cancer cells could be effectively blocked by non-labeled peptide. The Kd and Bmax values for radiolabeled peptide were obtained at 2.5 ± 0.6 nM and (3.4 ± 0.2) × 105 sites per cell, respectively. Biodistribution study demonstrated that tumor-to-blood and tumor-to-muscle ratios were about 1.73 ± 0.36 and 3.78 ± 0.17 at 120 min after the injection of the radiolabeled peptide, respectively. Tumor imaging by PET/CT exhibited high contrast tumor image at 60 min after injection in animal models. Consequently, the results were indicative of the specific accumulation of 68Ga-DOTA-(Ser)3-LTVSPWY peptide in HER2-positive tumors and the suitability of its application as a PET probe for the diagnosis of HER2-overexpression tumor.

Radionuclide therapy using ABD-fused ADAPT scaffold protein: Proof of Principle

Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.

Imaging and monitoring HER2 expression in breast cancer during trastuzumab therapy with a peptide probe 99mTc-HYNIC-H10F.

The novel molecular imaging probe 99mTc-HYNIC-H10F was developed for patient screening and efficacy monitoring of trastuzumab therapy by SPECT imaging of HER2 expression in breast cancer. 99mTc-HYNIC-H10F was developed by labeling H10F peptide with 99mTc following an optimized protocol. Biodistribution and SPECT/CT were performed in mouse models bearing HER2-positive SK-BR3 and HER2-negative MDA-MB-231 human breast cancer xenografts, respectively. The treatment response to trastuzumab was monitored and quantified by SPECT/CT in two HER2-positive breast cancer models (SK-BR3 and MDA-MB-361). The preliminary clinical study was performed in two patients with breast cancer. SPECT/CT with 99mTc-HYNIC-H10F showed that the SK-BR3 tumors were clearly visualized, while the signals from MDA-MB-231 tumors were much lower. The tumor uptake of 99mTc-HYNIC-H10F could be blocked by excess unlabeled H10F peptide but not by excess trastuzumab. The growth of two HER2-positive tumors was prominently suppressed at day 11 post-treatment. However, SPECT/CT reflected much earlier therapy response at day 4 post-treatment. The HER2 expression in tumors of breast cancer patients could be detected by 99mTc-HYNIC-H10F SPECT/CT imaging. 99mTc-HYNIC-H10F specifically accumulates in HER2-positive tumors. Compared with trastuzumab, 99mTc-HYNIC-H10F binds to a different domain of HER2 antigen, providing new opportunities to monitor HER2 expression levels before/during/after trastuzumab treatment for more effective personalized treatment.

Abstract P3-01-08: HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup

Abstract Introduction: CTCs can overexpress HER2 discordant from tumor HER2 expression. We aimed to describe characteristics of a CTC-defined group of pts with metastatic breast cancer (MBC) that is tumor HER2- and CTC HER2+ (HER2 tumor- CTC+). Methods: We retrospectively analyzed data from pts treated at Northwestern University who had serial evaluation of CTCs and circulating tumor DNA (ctDNA). We included pts with pathologically confirmed HER2- MBC and HER2+ CTCs. CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was determined using the CellSearch CXC Kit in 7.5 cc whole blood, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Results: Among 98 pts with HER2- MBC and CTC analysis, 46 (47%) had at least 1 HER2+ CTC. In this cohort the median age was 53. At initial BC diagnosis, 80% had early stage or locally advanced BC and 20% had de-novo metastatic disease. Baseline histology was 65% ductal, 20% lobular, 2% mixed ductal and lobular, and 13% unknown. Pathology of metastatic tumor was hormone receptor positive (HR+)/HER2- in 78% and triple negative in 22%. Detailed HER2 immunohistochemistry (IHC) and FISH results from metastases were available from 63% of pts of whom 72% had an IHC score of 0 or 1 and 28% had an IHC score of 2 with negative FISH testing. The median time from the most recent pathologic metastatic tumor assessment to the detection of a HER2+ CTC was 6.5 mo. Twenty-two pts had simultaneous (within 8 weeks) HER2- tumor confirmation and HER2+ CTC detection. The median lines of endocrine therapy (ET) for MBC prior to detection of HER2+ CTCs was 1 (range 0-5, 41% no ET, 17% 1 line, 41% &amp;gt;2 lines). Pts received a median of 2 (range 0-10) prior systemic therapies for MBC prior to detection of HER2+ CTCs, (20% 0 lines, 41% 1-3 lines, and 39% &amp;gt;4 lines). Among these 46 pts, CTCs were analyzed longitudinally in 104 samples, with HER2+ CTCs detected in 77 samples. Number of HER2+ CTCs at initial detection ranged from &amp;lt;5 in 24%, 5-50 in 43%, and &amp;gt;50 in 33%, with a median of 11.5 HER2+ CTCs. CTC clusters were noted in 37% of pts. At initial detection the proportion of CTCs that were HER2+ was 0-25% in 13% of pts, 26-50% in 46% of pts, and 51-100% in 41% of pts. Seven pts had ERBB2 aberrations in ctDNA. Of 12 pts with tumor sequencing, 2 had ERBB2 mutations, 1 had ERBB3 amplification, and 1 had overexpression of ERBB3 RNA. After detection of HER2+ CTCs, 18 pts received HER2 directed therapy (with chemotherapy in 13 pts, with endocrine therapy in 4 pts, and as monotherapy in 1 pt). Imaging demonstrated a partial response or stable disease in 9 pts (clinical benefit rate 50%), including in 1 pt with trastuzumab monotherapy, progressive disease in 8 pts, and not evaluated in 1 pt. Conclusions: HER2+ CTCs are frequently detected simultaneously or soon after HER2- tumor assessment in MBC. Within this newly defined subgroup, the several responses seen with HER2 targeted therapy serve as a proof of concept that HER2 tumor- CTC+ patients can benefit from HER2 targeted therapy. Future studies are needed to determine a clinically relevant threshold for HER2+ CTCs to guide further study of HER2 therapy combinations in HER2 tumor- CTC+ pts. Citation Format: Shah AN, Gerratana L, Zhang Q, Davis AA, Zhang Y, Flaum L, Behdad A, Platanias L, Gradishar WJ, Cristofanilli M. HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-08.

SPECT/CT Imaging of the Novel HER2-Targeted Peptide Probe 99mTc-HYNIC-H6F in Breast Cancer Mouse Models.

Overexpression of human epidermal growth factor receptor 2 (HER2) plays important roles in tumorigenesis and tumor progression in breast cancer. Nuclear imaging of HER2 expression in tumors might detect all HER2-positive tumors throughout the body and guide HER2-targeted therapies for patients. We therefore aimed to develop a HER2-targeted peptide probe for breast cancer imaging. A novel SPECT imaging probe, 99mTc-HYNIC-H6F, was prepared and then evaluated in breast cancer animal models. Methods: The HER2-targeted peptide H6F (YLFFVFER) was conjugated with the bifunctional chelator hydrazinonicotinamide (HYNIC). 99mTc-HYNIC-H6F was prepared, and the in vivo characteristics of 99mTc-HYNIC-H6F were investigated in MDA-MB-453 (HER2-positive) and MDA-MB-231 (HER2-negative) models using small-animal SPECT/CT. Moreover, to investigate the specificity of the H6F peptide toward HER2 and the potential applications in monitoring therapies involving trastuzumab, unlabeled H6F and trastuzumab were used as blocking agents in cell competition studies and SPECT imaging. Results: A standard tricine/trisodium triphenylphosphine-3,3',3″-trisulfonate labeling procedure demonstrated that the radiochemical purity was greater than 95%. 99mTc-HYNIC-H6F displayed excellent HER2-binding specificity both in vitro and in vivo. SPECT/CT imaging revealed that the MDA-MB-453 tumors were clearly visualized (percentage injected dose per gram, 3.58 ± 0.01 at 30 min after injection), whereas the signals in HER2-negative MDA-MB-231 tumors were much lower (0.73 ± 0.22 at 30 min after injection). Tumor uptake of MDA-MB-453 was blocked by the coinjection of excess H6F but not by excess trastuzumab. Conclusion: The 99mTc-HYNIC-H6F peptide probe specifically accumulates in HER2-positive tumors and is therefore promising for the diagnosis of HER2-positive cancers. Because 99mTc-HYNIC-H6F and trastuzumab target different regions of the HER2 receptor, this radiotracer also has great potential for monitoring the therapeutic efficacy of trastuzumab by rechecking the expression level of HER2 without blocking effect during therapy.

Occurrence and outcome of de novo metastatic breast cancer by subtype in a large, diverse population.

To examine the occurrence and outcomes of de novo metastatic (Stage IV) breast cancer, particularly with respect to tumor HER2 expression. We studied all 6,268 de novo metastatic breast cancer cases diagnosed from 1 January 2005 to 31 December 2011 and reported to the California Cancer Registry. Molecular subtypes were classified according to HER2 and hormone receptor (HR, including estrogen and/or progesterone receptor) expression. Multivariable logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of Stage IV versus Stage I-III breast cancer; Cox proportional hazards regression was used to assess relative hazard (RH) of mortality. Five percent of invasive breast cancer was metastatic at diagnosis. Compared to patients with earlier stage disease, patients with de novo metastatic disease were significantly more likely to have HER2+ tumors (HR+/HER2+: OR 1.29, 95% CI 1.17-1.42; HR-/HER2+: OR 1.40, 95%CI 1.25-1.57, vs. HR+/HER2-). Median survival improved over time, but varied substantially across race/ethnicity (Asians: 34months; African Americans: 6months), neighborhood socioeconomic status (SES) (highest: 34months, lowest: 20months), and molecular subtype (HR+/HER2+: 45months; triple negative: 12months). In a multivariable model, triple negative (RH 2.85, 95% CI 2.50-3.24) and HR-/HER2+ (RH 1.60, 95% CI 1.37-1.87) had worse, while HR+/HER2+ had similar, risk of all-cause death compared to HR+/HER2- breast cancer. De novo metastatic breast cancer was more likely to be HER2+. Among metastatic tumors, those that were HER2+ had better survival than other subtypes.

Monitoring therapeutic response of human ovarian cancer to trastuzumab by SPECT imaging with 99mTc-peptide-ZHER2:342

IntroductionPatients with human epidermal growth factor receptor 2 (HER2)-positive cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. How to systemically assess tumor HER2 expression and identifying appropriate use of anti-HER2 therapies by noninvasive imaging in vivo is an urgent issue. The purpose of this study was to evaluate SPECT imaging of 99mTc-Gly-(D)Ala-Gly-Gly-ZHER2:342 (99mTc-peptide-ZHER2:342) for monitoring therapeutic response to trastuzumab in nude mice bearing HER2-positive SKOV-3 xenografts. MethodsNude mice bearing HER2-positive SKOV-3 xenografts were treated with trastuzumab (treatment group) or saline (control) with ten mice in each group. Mice in trastuzumab-treated group were given trastuzumab intraperiotoneally 4mg/kg on day 1 and 2mg/kg on day 8; Mice in control group were given physiological saline on day 1 and 8. Mice body weights and tumour volume were monitored every three days during treatment. In vivo SPECT imaging was performed in mice of the two groups using 99mTc-peptide-ZHER2:342 before treatment, on day 8 and 15 after treatment. Radiolabeled probe uptake in tumours was measured as the ratio of radioactive counts in the tumour to that in the contralateral equivalent region (T/NT). After SPECT imaging on day 15, all the mice were euthanized, biodistribution studies of the SKOV-3 xenografts were carried out to validate the imaging results and HER2 expression of the transplanted tumours was analyzed by immunohistochemistry (IHC). Correlation analysis was performed between T/NT ratios acquired by in vivo SPECT imaging on day 15 and the HER2 level of tumours. In vitro cell binding capacity of 99mTc-ZHER2:342 with SKOV-3 cells in the absence and presence of varying amount of trastuzumab were also conducted in the study. ResultsTwenty mice body weight in the two groups gradually increased during treatment, but there was no statistical difference (p>0.05). Though volumes of SKOV-3 xenografts gradually increased in each group during the treatment, the transplanted tumours in trastuzumab-treated group had a slower growth than those in control group (p<0.05). Compared with the baseline, the results of in vivo imaging showed that radionuclide accumulation in transplanted tumours reduced significantly in trastuzumab-treated group after treatment (p<0.05), whereas the tumour accumulation in control group increased after treatment. Biodistribution studies demonstrated that the results corresponded well with in vivo imaging data. Immunohistochemical staining confirmed the significant reduction in tumor HER2 level upon trastuzumab treatment, and there was an obviously positive correlation between T/NT ratios and HER2 level of tumours with correlation coefficient rs=0.919, p<0.05. There was no significant significance in cell binding ratios between varying amount of trastuzumab and the absence of trastuzumab (p>0.05). ConclusionsThe early response to trastuzumab in mice bearing SKOV-3 xenografts was successfully monitored by SPECT imaging using 99mTc-peptide-ZHER2:342. This approach may be valuable in monitoring the therapeutic response in HER 2-positive tumours under HER2-targeted therapy.

Functional Imaging of Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast Cancer Using 64Cu-DOTA-Trastuzumab PET

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG. (64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.