Heterogeneity Of HER2 Expression Research Articles

Clinical significance of HER2 expression between primary tumors and metastatic lymph nodes in patients with extramammary Paget's disease.

e21568 Background: HER2 has been reported to be a potential oncogene and therapeutic target in extramammary Paget's disease (EMPD). However, the expression of HER2 in EMPD, especially in matched metastatic lymph nodes (LNs), is limited. Here, we investigated the heterogeneity of HER2 expression between primary tumors and positive nodes and its clinical significance in advanced EMPD patients and detected the efficacy of HER2-targeted antibody drug conjugates (ADCs) in EMPD. Methods: A total of 170 patients with pathologically confirmed EMPD of the scrotum and/or penis treated at SYSUCC from 2003 to 2023 were included. HER2 IHC staining (anti-HER2/neu (4B5), Ventana) and scoring were performed according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2023 guidelines. Outcome relationships were explored using Cohen's kappa coefficient, multivariate Cox regression and log-rank analysis. Results: A total of 102 primary tumor and 32 metastatic specimens were ultimately subjected to HER2 IHC staining. HER2 high-expression (IHC: 2/3+) was detected in 71.6% (0: 9.8%, 1+: 18.6%, 2+: 40.2% and 3+: 31.4%) and 68.8% (0: 21.9%, 1+: 9.4%, 2+: 28.1% and 3+: 40.6%) of the primary tumors and metastatic LNs, respectively. HER2-high EMPD patients experienced poorer outcomes, with a 5-year OS of 56.5%, than did HER2-low patients (P = 0.032). A multivariate Cox regression model showed that a positive lymph node (HR 15.39, 95% CI 3.79~62.43; P < 0.001) and HER2 high-expression in primary tumors (HR 3.68, 95% CI 1.49~9.09; P = 0.005) were independent poor prognostic factors. Notably, in 31 paired samples, 35.5% (n = 11) of patients had inconsistent HER2 expression between primary tumors and corresponding lymph node metastases. Six (19.4%) patients exhibited a shift from high expression of HER2 in primary tumors to low expression of HER2 in the lymph nodes, and 5 (16.1%) patients exhibited the reverse change. Advanced EMPD patients with HER2 high-expression were treated with Disitamab Vedotin (RC48), for which the ORR was 80% and the DCR was 100% (n = 5). No adverse events (AEs) above grade 3-4 were observed. Conclusions: HER2 was highly expressed in both primary and metastatic LNs in EMPD patients. However, there was some heterogeneity in HER2 expression between paired primary and corresponding metastatic LNs. The expression status of HER2 should be evaluated in both primary tumors and corresponding lymph node metastases. A positive lymph node and HER2 high-expression are poor prognostic factors in patients with EMPD. RC48 demonstrated significant efficacy in treating patients with HER2-high advanced EMPD and has promising potential that requires further validation.

Trastuzumab deruxtecan in HER2-positive advanced gastric cancer: exploratory biomarker analysis of the randomized, phase 2 DESTINY-Gastric01 trial

Trastuzumab deruxtecan (T-DXd) showed statistically significant clinical improvement in patients with human epidermal growth factor receptor 2-positive (HER2+) gastric cancer in the DESTINY-Gastric01 trial. Exploratory results from DESTINY-Gastric01 suggested a potential benefit in patients with HER2-low gastric cancer. Spatial and temporal heterogeneity in HER2 expression or gene alteration, an inherent characteristic of gastric cancer tumors, presents a challenge in identifying patients who may respond to T-DXd. Specific biomarkers related to therapeutic response have not been explored extensively. Exploratory analyses were conducted to assess baseline HER2-associated biomarkers in circulating tumor DNA and tissue samples, and to investigate mechanisms of resistance to T-DXd. Baseline HER2-associated biomarkers were correlated with objective response rate (ORR) in the primary cohort of patients with HER2+ gastric cancer. The primary cohort had 64% concordance between HER2 positivity and HER2 (ERBB2) plasma gene amplification. Other key driver gene amplifications, specifically MET, EGFR and FGFR2, in circulating tumor DNA were associated with numerically lower ORR. Among 12 patients with HER2 gain-of-function mutations, ORR was 58.3% (7 of 12). ORR was consistent regardless of timing of immunohistochemistry sample collection. Further investigations are required in larger studies.

Abstract PO4-15-11: HER2 STATUS AND TUMOR HETEROGENEITY IN INVASIVE BREAST CARCINOMAS: CLINICAL PATHOLOGICAL IMPACT

Abstract Human epidermal growth factor receptor 2 (HER2) is a major player in breast carcinogenesis with a range of 15-20% of cases harboring gene amplification and overexpression turning the tumor eligible to over eight anti-HER2 drugs in the past 15 years. Recently, novel anti-HER2 antibody-drug conjugates (ADCs) treatment was recently approved to metastatic breast cancer bearing some HER2 expression albeit not due to gene amplification. Immunohistochemically classified, by ASCO-CAP Guidelines (2018), as 1+ cases were enrolled in clinical trials and benefited by the addition of ADCs to chemotherapy. Despite being classified as HER2 negative, most tumors exhibit detectable levels of HER2 protein, reinforcing the importance of HER2 expression evaluation. HER-2 intratumoral heterogeneity (ITH) is reported in up to 40% of breast cancers and is defined as the coexistence of subpopulations of tumor cells with different HER2 gene or protein expression. In this scenario, it becomes very important to evaluate the spectrum of HER-2 expression in correlation with pathological factors as ITH is associated with poor prognosis and anti-HER2 resistance. We designed a retrospective linear study to reanalyze all HER2 immunohistochemical core biopsy slides of patients with primary invasive breast carcinomas (IBC) diagnosed and treated at São Paulo Federal University Hospital between 2019 and 2023. Clinical variables (age, laterality, tumor size by ultrasound imaging, BI-RADS) were collected from clinical files. HER2 slides were reviewed by three observers and reported regarding status by ASCO-CAP 2018 guidelines, HER2 heterogeneity and patterns. Statistical analyses were performed with Chi-Square test of independence, Fisher's exact test and Mann–Whitney U test using IBM® SPSS® Statistics (26.0) software. 353 cases were included; the mean age of the patients at the time of diagnosis was 58 years old. 191 cases (54,1%) were left sided and 162 cases (45,9%) were right sided. Regarding BI-RADS report, the major category was 4 (140 cases), followed by 5 (119 cases). Out of the 164 patients who underwent clinical follow-up at our institute, 14 deaths were reported. Concerning pathological features, IBC of no special type was the prevalent histological subtype with 304 cases (86,1%), followed by invasive lobular carcinoma, with 26 cases (7,4%). 296 cases (83,9%) were HER2 negative and 42 (11,9%) were HER2 positive; 15 cases were HER2 2+ (4,2%). Amongst negative cases, 235 were HER2 0+ and 61 were HER2 1+. Considering ITH, 287 cases (81,3%) were HER2 homogenous with 0+ score as the most prevalent, presenting with 232 cases (80%). 66 cases (18,7%) had ITH, with 1+ as the most prevalent primary score, with 34 cases (51,5%), followed by 3+, with 17 cases (25,8%). Not only HER2 positive carcinomas, but also the presence of heterogeneity in HER2 expression was associated with death outcome (p=0,003 and p=0,001, respectively). Also, HER2 positive IBC had larger tumor size compared to HER2 negative carcinomas (mean size of 25,6 mm x 31,3 mm, respectively; p=0,012); HER2 heterogeneity showed no statistical significance when associated with tumor size (p=0,165). We conclude that ITH is prevalent in HER2 expression and should be addressed in pathology reports since it may play an additional role in tumor progression and drug response, especially in the ADCs scenario. Citation Format: Karla Calaça Kabbach Prigenzi, Lisandra Gonzalez Porta Nova, Adilson Monteiro dos Santos Filho, Ana Luiza da Cruz, Lucas de Figueiredo Barbosa, Angela Flavia Waitzberg. HER2 STATUS AND TUMOR HETEROGENEITY IN INVASIVE BREAST CARCINOMAS: CLINICAL PATHOLOGICAL IMPACT [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 PO4-15-11.

Establishment and characterization of multiple patient-derived organoids from a case of advanced endometrial cancer.

Patient-derived organoids (PDOs) retain the original tumor's characteristics to a large degree and allow direct evaluation of the drug sensitivity, thereby emerging as a valuable resource for both basic and preclinical researches. Whereas most past studies stereotypically adopted a single PDO as an avatar of the patient, it remains to be investigated whether this assumption can be justified even for the tumor with spatial diversity. To address this issue, we established and characterized multiple PDOs originating from various sites of a patient with advanced uterine carcinosarcoma (UCS). Specifically, cancer cells were separately sampled from three sites; resected UCS tumor tissue, the peritoneal lavage fluid, and an intra-uterine brushing of the tumor. The three derived PDOs were morphologically undistinguishable, displaying typical carcinoma organoids-like appearance, but two of them proliferated at a faster rate. The primary tumor harbored mutations in TP53 and STK11 along with amplifications in CCNE1, ERBB2, and KRAS. These two mutations and the CCNE1 amplification were detected in all PDOs, while either KRAS or ERBB2 amplification was selectively observed in each PDO in a mutually exclusive manner. Observed intra-tumor heterogeneity in HER2 expression was differentially reproduced in the PDOs, which mirrored each PDO's sensitivity to HER2 inhibitors. Inter-PDO heterogeneity was also evident in sensitivity to standard cytotoxic agents. Lastly, a drug screening identified four candidate reagents commonly effective to all PDOs. Collectively, we showed that multiple PDOs could help reproduce the spatial diversity of a tumor and serve as a valuable resource in UCS research in many respects.

Molecular imaging predicts lack of T-DM1 response in advanced HER2-positive breast cancer (final results of ZEPHIR trial)

Efficacy of the human epidermal growth factor receptor (HER)2-targeting trastuzumab emtansine (T-DM1) in breast cancer (BC) relies on HER2 status determined by immunohistochemistry or fluorescence in-situ hybridization. Heterogeneity in HER2 expression, however, generates interest in “whole-body” assessment of HER2 status using molecular imaging. We evaluated the role of HER2-targeted molecular imaging in detecting HER2-positive BC lesions and patients unlikely to respond to T-DM1. Patients underwent zirconium-89 (89Zr) trastuzumab (HER2) PET/CT and [18F]-2-fluoro-2-deoxy-D-glucose (FDG) PET/CT before T-DM1 initiation. Based on 89Zr-trastuzumab uptake, lesions were visually classified as HER2-positive (visible/high uptake) or HER2-negative (background/close to background activity). According to proportion of FDG-avid tumor load showing 89Zr-trastuzumab uptake (entire/dominant part or minor/no part), patients were classified as HER2-positive and HER2-negative, respectively. Out of 265 measurable lesions, 93 (35%) were HER2-negative, distributed among 42 of the 90 included patients. Of these, 18 (19%) lesions belonging to 11 patients responded anatomically (>30% decrease in axial diameter from baseline) after three T-DM1 cycles, resulting in an 81% negative predictive value (NPV) of the HER2 PET/CT. In combination with early metabolic response assessment on FDG PET/CT performed before the second T-DM1 cycle, NPVs of 91% and 100% were reached in predicting lesion-based and patient-based (RECIST1.1) response, respectively. Therefore, HER2 PET/CT, alone or in combination with early FDG PET/CT, can successfully identify BC lesions and patients with a low probability of clinical benefit from T-DM1.

Assessment of the expression pattern of HER2 and its correlation with HER2-targeting antibody-drug conjugate therapy in urothelial cancer

BackgroundHuman epidermal growth factor receptor 2 (HER2) overexpression is related to anti-HER2 therapy in many tumors. RC48- antibody-drug conjugate (ADC) has shown promising efficacy in patients with HER2-positive locally advanced or metastatic urothelial carcinoma (UC). The characteristic expression and scoring systems of HER2 are nonexistent in UC. We aimed to explore HER2 status and its correlation with the efficacy of HER2-targeting ADC therapy in UC. MethodsA total of 137 and 43 patients were enrolled in cohort 1 and cohort 2, respectively, from March 2009 to December 2018. The patients in cohort 2 were enrolled in a phase II study of RC48-ADC. UC samples were tested for HER2 status using immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). The 2018 ASCO/CAP HER2 scoring system was adopted and modified to score HER2 expression in UC. ResultsThe HER2-positive (IHC 2+ or 3+) rate was 24.1% (33/137). In HER2 IHC 2+ or 3+ patients, the HER2 gene amplification rate was 31% (13/42). The objective response rates (ORRs) in RC48-ADC-treated patients with IHC 3+, IHC 2+ and FISH+, IHC 2+ and FISH- were 58.8%, 66.7% and 40%, respectively. The ORR showed a trend toward a better benefit for RC48-ADC therapy in patients with HER2 amplification than in those without amplification (61.5% vs. 44.8%, P = 0.059). The heterogeneity of HER2 expression in the primary tumor was 55.5% (15/27), and the ORR was not significantly different between patients with tumor heterogeneity and homogeneity. ConclusionsIHC testing should be performed to assess the HER2 status before the initiation of HER2-ADC therapy. There was a trend toward a better benefit for patients with HER2 amplification, and tumor heterogeneity did not influence the drug efficacy.

Assessment of HER2 in Gastric-Type Endocervical Adenocarcinoma and its Prognostic Significance

As the most common type of human papillomavirus-independent endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs. Although anti-HER2 therapy has been proven effective in many cancers, it has not been used in ECAs, including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in the tumor type selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using immunohistochemistry and fluorescent in situ hybridization and investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (10/58[17.2%]) and amplification (9/58 [15.5%]), as well as high immunohistochemistry-fluorescent in situ hybridization concordance rate (9/10 [90%]) was found in GEAs. A lateral/basolateral staining pattern (“U-shaped”) was observed, at least focally, in most of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (P = .047 and P = .032, respectively). Programmed death-ligand 1 expression was associated with worse progression-free survival (P = .032), whereas mutant-type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER2 testing guideline for GEAs.

Unlocking the Resistance to Anti-HER2 Treatments in Breast Cancer: The Issue of HER2 Spatial Distribution

Approximately 15% of breast cancers are classified as HER2-positive, with an amplification of the ERBB2 gene and/or an overexpression of the HER2 protein. Up to 30% of HER2-positive breast cancers shows heterogeneity in HER2 expression and different patterns of spatial distribution, i.e., the variability in the distribution and expression of the HER2 protein within a single tumour. Spatial heterogeneity may potentially affect treatment, response, assessment of HER2 status and consequently, may impact on the best treatment strategy. Understanding this feature can help clinicians to predict response to HER2-targeted therapies and patient outcomes, and to fine tune treatment decisions. This review summarizes the available evidence on HER2 heterogeneity and spatial distribution and how this may affect current available treatment choices, exploring possible opportunities for overcoming this issue, such as novel pharmacological agents, belonging to the group of antibody-drug conjugates.

Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas

AbstractHER2 is an established therapeutic biomarker in advanced or recurrent endometrial serous carcinoma. Current clinical guidelines recommend HER2 testing exclusively in this endometrial carcinoma (EC) subtype; however, the full spectrum of ECs harboring HER2 amplification remains ill-defined. The present study characterizes the clinicopathologic and molecular features of HER2-amplified ECs across all histologic subtypes. Retrospective analysis of our institutional cohort of 2,042 ECs subjected to targeted clinical massively parallel sequencing identified 77 (3.8%) cases with HER2 amplification, a group comprised of serous (n = 29), endometrioid (low-grade, n = 2, high-grade, n = 1) and clear cell (n = 4) carcinomas, carcinosarcomas (n = 18) and high-grade ECs with ambiguous features (HGEC, n = 23). A co-existing TP53 mutation was identified in 94% (72/77) of HER2-amplified ECs. Other recurrent genetic alterations included amplification of CCNE1 (22%) and ERBB3 (10%), FBXW7 mutations or deletions (13%), and mutations in PIK3CA (40%) and PPP2R1A (13%). The HER2 immunohistochemistry score was 2+ or 3+ for all evaluable cases (n = 61). Apart from carcinosarcomas, which often showed lower HER2 expression, particularly in the sarcomatous component, HER2 immunohistochemical staining pattern and intensity were similar across EC subtypes. Intratumor heterogeneity in HER2 expression was common and correlated with genetic heterogeneity as detected by fluorescence in-situ hybridization. These results demonstrate the frequent co-occurrence of HER2 amplification with TP53 mutation and high-grade histology, rather than being specific to serous carcinoma, per se. Overall, these findings suggest that HER2 targeted therapy may be more broadly applicable to all high-grade EC histotypes and consideration should be given to expanding therapeutic eligibility.

Heterogeneity of HER2 Expression in Circulating Tumor Cells of Patients with Breast Cancer Brain Metastases and Impact on Brain Disease Control.

Simple SummaryResults from a previous study suggested that the number of circulating tumor cells (CTC) might have a role as a biomarker of early distant brain failure in patients with breast cancer brain metastases (BCBM). However, it remains largely underexplored whether heterogeneous HER2 expression in CTC may have a prognostic implication. We evaluated the status of HER2 expression in CTC before and after radiotherapy/radiosurgery for BCBM and observed that the presence of HER2 expression in any moment was associated with longer distant brain failure-free survival, irrespective of the primary immunophenotype of the breast tumor. This finding suggests that the status of HER2 expression in CTC has the potential to improve the treatment selection for patients with BCBM.HER2 expression switching in circulating tumor cells (CTC) in breast cancer is dynamic and may have prognostic and predictive clinical implications. In this study, we evaluated the association between the expression of HER2 in the CTC of patients with breast cancer brain metastases (BCBM) and brain disease control. An exploratory analysis of a prospective assessment of CTC before (CTC1) and after (CTC2) stereotactic radiotherapy/radiosurgery (SRT) for BCBM in 39 women was performed. Distant brain failure-free survival (DBFFS), the primary endpoint, and overall survival (OS) were estimated. After a median follow-up of 16.6 months, there were 15 patients with distant brain failure and 16 deaths. The median DBFFS and OS were 15.3 and 19.5 months, respectively. The median DBFFS was 10 months in patients without HER2 expressed in CTC and was not reached in patients with HER2 in CTC (p = 0.012). The median OS was 17 months in patients without HER2 in CTC and was not reached in patients with HER2 in CTC (p = 0.104). On the multivariate analysis, DBFFS was superior in patients who were primary immunophenotype (PIP) HER2-positive (HR 0.128, 95% CI 0.025–0.534; p = 0.013). The expression of HER2 in CTC was associated with a longer DBFFS, and the switching of HER2 expression between the PIP and CTC may have an impact on prognosis and treatment selection for BCBM.

Temporal heterogeneity of HER2 expression in metastatic gastric cancer: a case report

Gastric cancer is a disease with high heterogeneity, and this heterogeneity may result in an uneven distribution of subclones with varied genetic properties at disease locations (spatial heterogeneity) or temporal changes in subclonal composition (temporal heterogeneity). We present the case of a 69-year-old woman with metastatic gastric cancer who presented for axillary lymph node enlargement and underwent axillary lymphadenectomy. Pathological evidence showed human epidermal growth factor receptor 2 (HER2)(3+). Abdominal computed tomography revealed a mass in the gastric body, gastroscopic biopsy showed HER2(3+). After tumor shrinkage by preoperative translational chemotherapy (oxaliplatin, calcium folate, fluorouracil) and targeted therapy (trastuzumab), she had laparoscopic-assisted total gastrectomy. However, HER2 immunohistochemistry was found to be diffusely negative in the surgically removed tissue, and there was no evidence of HER2 amplification in the whole exon sequencing either. After 10 months of trastuzumab treatment, her disease progressed. Although trastuzumab treatment was initially beneficial, the residual HER2-negative subclones may cause tumor recurrence and metastasis due to temporal heterogeneity, as shown in this case.

Therapeutic Advances and Challenges in the Management of HER2-Positive Gastroesophageal Cancers.

Gastroesophageal cancer is one of the most common cancers in the world, with a high rate of mortality. While there has been significant progress over the past decade, particularly with the addition of anti-HER2 therapies to platinum-based chemotherapy agents in the advanced setting, the prognosis remains poor and the treatment options for this disease entity remain limited. In this review, we discuss the current therapeutic landscape for HER2-positive gastroesphageal cancer and the seminal clinical trials that have shaped our approach to this disease entity. In addition, we highlight some of the challenges to the understanding and management of this disease, specifically discussing the breadth of molecular diversity and intratumoral heterogeneity of HER2 expression that impact the clinical efficacy and prognosis. Furthermore, we discuss the potential role of next-generation sequencing (NGS) and circulating-tumor DNA (ctDNA) as complementary tools to immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) to guiding clinical decision making. Finally, we highlight promising clinical trials of new treatment regimens that will likely reshape the therapeutic approach to this disease entity.

Abstract 214: The Breast Single-Cell Atlas: Single-cell transcriptomics for personalised medicine

Abstract Breast Cancer (BC) patient stratification is driven by receptor status and histological grading and subtyping, with about 20% of patients for which absence of any actionable biomarkers results in no clear therapeutic intervention. Here, we evaluated the potentiality of single cell RNA-sequencing (scRNA-seq) for automated diagnosis and drug treatment of BC. We transcriptionally profiled 35,276 individual cells from 33 BC cell-lines covering all BC subtypes plus primary cancer cells from three patients to obtain a BC cell atlas. scRNA-seq successfully measured the expression of clinically relevant receptors and was used to automatically group cancer cell lines according to their tumor subtype. In most cell lines, as well as patients' cells, we observed a high degree of heterogeneity in the expression of BC receptors. We thus asked whether such heterogeneity impacts a cell line overall drug sensitivity. By correlating the percentage of cells expressing a given drug target (e.g. HER2, etc.) to the known IC50 of the relevant drug across the 33 cell lines, we observed a significant negative correlation (the higher the % of cells, the lower the IC50). This means that even within a genomically stable isogenic cell line, cells with differential drug sensitivity can co-exist. We then focused on the MDAMB361 cell-line of the luminal B subtype with a gain in genomic copy number of the locus containing the ERRB2 gene coding for HER2. Despite HER2 amplification, scRNA-seq showed that only 64% of cells express its mRNA. We used fluorescence-activating cell sorting to isolate HER2 expressing cells (HER2+) from non-expressing cells (HER2-). After a week, both subpopulations re-established the original heterogeneity, thus showing that heterogeneity in HER2 expression in these cells is dynamic. As expected, HER2 targeting drugs such as Afatinib and Erlotinib, have a higher IC50 in this cell line as compared to cell lines uniformly expressing HER2. We developed a bioinformatics approach named DEEP (Drug Estimation from Expression Profiles) to automatically predict responses to more than 450 anticancer agents starting from scRNA-seq and confirmed the validity of the approach using published large-scale studies on drug sensitivity. We then applied DEEP to the MDAMB361 cell-line to identify drugs able to selectively inhibit growth of the HER2- subpopulation. Etoposide was predicted to selectively inhibit growth of the HER2- cells but not HER2+ cells. We experimentally confirmed this prediction by performing cell viability assays at different dosages. Finally, we used DEEP to primary cancer cells of three patients end confirmed its validity experimentally. We found that scRNA-seq can be used to improve cancer diagnosis and predict drug sensitivity and transcriptional heterogeneity is common, dynamic and plays a relevant role in determining drug sensitivity. Our BC cell atlas and DEEP approach are a unique resource for the BC research community. Citation Format: Gaetano Viscido, Gennaro Gambardella, Diego di Bernardo. The Breast Single-Cell Atlas: Single-cell transcriptomics for personalised medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 214.

Heterogeneity of HER2 expression in circulating tumor cells of patients with breast cancer brain metastases and impact on brain disease control.

2029 Background: The HER2 expression switching in circulating tumor cells (CTC) of breast cancer is dynamic and may have prognostic and predictive clinical implications. This study aims to analyse the association between expression of HER2 in CTC of patients with breast cancer brain metastases (BCBM) and brain disease control. Methods: Exploratory analysis of a prospective assessment (NCT02941536) of CTC before (CTC1) and 4–5 weeks after (CTC2) stereotactic radiotherapy/radiosurgery (SRT). CTC were isolated and quantified by a method of isolation by size of tumors and analyzed by immunocytochemistry to evaluate the expression of HER2. Distant brain failure-free survival (DBFFS), the primary endpoint, and overall survival (OS) were estimated by Kaplan-Meier estimator. Log-rank tests were applied in order to compare the survival curves. For multivariate analysis of prognostic factors that affected DBFFS and OS, the Cox proportional model was adjusted. Results: The median age at SRT was 54 (34-70), the diagnosis-specific graded prognostic assessment (DS-GPA) was 1–2 in 17.5% and 2.5–4 in 82.5% and the primary immunophenotype (PIP) was HER2-enriched in 51%, luminal B (LB) in 31% and triple negative (TN) in 18% of the total of 39 patients. CTC were detected in all 39 patients before SRT and the median CTC1 was 2 CTC/mL. After SRT, CTC were detected in 34 of 35 patients (4 deaths between CTC1 and CTC2) and the median CTC2 was 2.33 CTC/mL. HER2 was expressed in CTC1 and/or CTC2 in 9 patients, of which only 2 patients had PIP HER2-enriched. After a median follow-up of 16.6 months, there were 15 patients with distant brain failure and 16 deaths. The median DBFFS and OS were 15.3 and 19.5 months, respectively. Median DBFFS was 7 months in patients with PIP TN and was not reached in PIP LB and HER2-enriched (p = 0.036); 14 months in patients with DS-GPA 1-2 and 7 months with DS-GPA 2.5-4 (p = 0.017); 10 months in patients without HER2 expressed in CTC and not reached in patients with HER2 expressed in CTC (p = 0.012). Median OS was 4.8 months in patients with PIP TN and was not reached in PIP LB and HER2-enriched (p = 0.0026); 19.54 months in patients with DS-GPA 1-2 and 7.6 months with DS-GPA 2.5-4 (p = 0.00088); 17 months in patients without HER2 expressed in CTC and not reached in patients with HER2 expressed in CTC (p = 0.104). On multivariate analysis, DBFFS was superior in patients with PIP HER2-enriched (HR 0.128, 95% CI 0.025–0.534; p = 0.013) and OS was superior in patients with PIP HER2-enriched (HR 0.073, 95% CI 0.018-0.288; p < 0.0001) and LB (HR 0.224, 95% CI 0.062–0.816; p = 0.023). The status of expression of HER2 in CTC was not included in Cox model for DBFFS due to lack of events in patients with HER2 expressed in CTC. Conclusions: The expression of HER2 in CTC was associated with a longer DBFFS and the switching of HER2 expression between PIP and CTC may have impact on prognosis and treatment selection of BCBM.

HER2 Expression, Test Deviations, and Their Impact on Survival in Metastatic Gastric Cancer: Results From the Prospective Multicenter VARIANZ Study.

PURPOSETrastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2–positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC.METHODSPatients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR.RESULTSFive hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2− mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab.CONCLUSIONSignificant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.

Abstract PS3-03: [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models

Abstract Introduction: Paclitaxel (PTX) is one of the most commonly used first-line chemotherapies in the treatment of breast cancer. However, it is reported that overall PTX response rate is between 30-60% for the treatment of metastatic breast cancer. Thus, novel discoveries of molecular mechanisms that account for ineffective response is critical in breast cancer treatment. [89Zr]-Pertuzumab (antibody that targets HER2 receptors) and 2-deoxy-2- [fluorine-18] fluoro-D-glucose ([18F]-FDG) positron emission tomography/computed tomography (PET/CT) imaging allows us to noninvasively measure the amount and heterogeneity of HER2 expression and tumor glucose metabolism. These methods are clinical translatable and can provide insight into tumor biology changes during the course of therapy. This study evaluates whether HER2 expression level is correlated with PTX treatment efficacy in controlled pre-clinical mouse models of HER2+ breast cancer. Experimental Design: BT474 (1×107), MDA-MB-361 (6×106), or MDA-MB-231 (2×106) cells were subcutaneously injected into athymic nude mice (N = 7 per cell line). When the tumor volume reached approximately 200 mm3, mice were enrolled in the study. Tumor size was measured with calipers weekly until enrolled and every 3 days after experiment started. In vivo HER2 expression level was determined by [89Zr]-Pertuzumab PET/CT imaging at one week prior to initiation of treatment and confirmed with immunohistochemistry staining. PTX (15 mg/kg) was administered via i.v. on days 0 and 3. In vivo tumor metabolism was quantified by [18F]-FDG PET/CT imaging on day 0, 3 and 6. Mean, standard deviation, sum, and frequency histogram of standard uptake value (SUV) were quantified. Tumors were harvested at day 6 for histological analysis. Hematoxylin and eosin (H&amp;E) and cleaved caspase 3 immunohistochemistry (IHC) staining were used to determine tumor apoptosis. Pearson’s correlation and ANOVA were used as statistical analysis. Results: [89Zr]-Pertuzumab SUVmean of BT474 (HER2+) tumors were 4.9±1.5, MDA-MB-361 (HER2+) tumors were 1.4±0.2 (p&amp;lt;0.0001, compared with BT474), and MDA-MB-231 (HER2-) tumors were 1.1±0.4 (p&amp;lt;0.0001, compared with BT474). [18F]-FDG SUV sum was positively correlated with tumor volume (R2=0.1669, p=0.0250). Tumor volumes showed no significant changes during the treatment. However, normalized [18F]-FDG SUV concentration changes from day 0 to day 3 was negatively correlated with baseline [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show paclitaxel treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. [89Zr]-Pertuzumab PET/CT imaging quantitively measured HER2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. This discovery will help identify chemotherapy responders and potentially enhance clinical decision making. Acknowledgments: We thank funding sources American Cancer Society RSG-18-006-01-CCE and NIH NCI R01CA240589. Citation Format: Yun Lu, Adriana Massicano, Patrick Song, Meng Li, Suzanne Lapi, Anna Sorace. [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-03.

Details of human epidermal growth factor receptor 2 status in 454 cases of biliary tract cancer

Human epidermal growth factor receptor 2 (HER2)-targeted therapy has improved clinical outcomes in patients with HER2-positive breast and gastric cancers, although ineffective or recurrent cases are present. One reason for this is the heterogeneity of HER2 expression in cancer cells. The aim of this study was to investigate the clinicopathological characteristics and HER2 status of patients with biliary tract cancers (BTCs). We examined HER2 protein expression by immunohistochemistry, HER2 gene amplification by fluorescence in situ hybridization, and both HER2 protein and gene levels simultaneously by gene-protein assay. Samples were collected from 454 patients who underwent surgical resection for BTCs (110 intrahepatic cholangiocarcinomas [ICC], 67 perihilar extrahepatic cholangiocarcinomas [ECC-Bp], 119 distal extrahepatic cholangiocarcinomas [ECC-Bd], 80 gallbladder carcinomas [GBC], and 79 ampullary carcinomas [AVC]). HER2 status was assessed according to the guidelines for HER2 testing in gastroesophageal adenocarcinoma. HER2-positive status was detected in 14.5% of BTCs (3.7% of ICC, 3.0% of ECC-Bp, 18.5% of ECC-Bd, 31.3% of GBC, and 16.4% of AVC). Furthermore, HER2-positivity tended to correlate with low histological grade, tumor histology, and macroscopic features in certain tumors. HER2 heterogeneity was common and highly frequent (83%) in BTC cases. Reduced HER2 protein expression in the deeper invasive areas with simultaneous dedifferentiation was frequently observed in HER2-positive cancer cells. The findings of this study suggest that a large subgroup of HER2-positive BTC cases can be considered for HER2-targeted therapy. Moreover, the HER2 status in BTCs should be determined carefully using a sensitive approach toward larger cancer tissues.

Abstract 2775: Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model

Abstract Introduction: The overall goal of this study is to quantify amount and heterogeneity of Her2 expression and determine if its expression affects its response to chemotherapy in breast cancer. Paclitaxel (PTX) is one of the most commonly used first-line drugs in the treatment of breast cancer. However, it is reported that overall PTX response rate is 30%-60%, as first-line treatment of metastatic breast cancer, and the main treatment failure is PTX resistance. Thus, novel discoveries of molecular mechanisms that account for ineffective response or resistance to chemotherapy is critical in breast cancer treatment. This study evaluates whether Her2 expression level is correlated with PTX treatment efficacy in a controlled pre-clinical mouse model. Experimental Design: BT474 (1 × 107) or MDA-MB-231 (2 × 106) cells were subcutaneously injected into athymic nude mice (n = 10). Mice were enrolled in the study once they achieved tumor volume of 200 mm3. Tumor size were measured with caliper weekly or every 3 days after experiment started. In vivo Her2 expression level was determined by [89Zr]-Pertuzumab (Her2 antibody) positron emission tomography/computed tomography (PET/CT) imaging at one week prior to initiation of treatment. PTX (15 mg/kg) was administrated via i.v. at day 0 and day 3. In vivo tumor metabolism was quantified by 2-deoxy-2-[fluorine-18]fluoro- D-glucose ([18F]-FDG) PET/CT imaging at day 0, day 3 and day 6. Mean, standard deviation, and frequency histogram of standard uptake value (SUV) was quantified. Tumors were harvested at day 6 for histological analysis. Pearson's correlation test was used as statistical analysis. Results: BT474 (Her2+) tumors had significantly increased [89Zr]-Pertuzumab uptake compared to MDA-MB-231 (Her2-) tumors, which indicated the specificity and accuracy of [89Zr]-Pertuzumab PET/CT imaging. [89Zr]-Pertuzumab SUV varies within BT474 tumors, and tumor growth rate was positively correlated with mean Her2 expression (R2=0.9011, p=0.001). During PTX treatment, tumor volumes did not reveal significant changes. However, [18F]-FDG SUV concentration, normalized to percent changes from day 0 to day 3, was negatively correlated with initial [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show Her2 expression level is positively correlated with chemotherapy response in mouse models of human breast cancer. This discovery will help studies of PTX resistance in breast cancer and potentially guide clinical administration of combined therapies. [89Zr]-Pertuzumab PET/CT imaging quantitively measured Her2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. Citation Format: Yun Lu, Adriana I. Massicano, Suzanne E. Lapi, Meng Li, Patrick N. Song, Anna G. Sorace. Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2775.

Impact of 68Ga-NOTA-MAL-MZHER2 PET imaging in advanced gastric cancer patients and therapeutic response monitoring

Clinical PET imaging of human epidermal growth factor receptor 2 (HER2) can noninvasively detect HER2 overexpression in lesions. A novel 68Ga-NOTA-MAL-MZHER2 (68Ga-HER2) affibody was developed for clinical PET/CT, and its safety, tissue dosimetry, ability to detect HER2-positive lesions, and utility for HER2-targeted therapy in patients with advanced gastric cancer (AGC) were evaluated. Thirty-four patients with AGC (23 with HER2-positive and 11 with HER2-negative primary lesions) were included and underwent PET/CT after an injection of approximately 3.7MBq/kg body weight 68Ga-HER2 affibody. Thirteen patients (8 HER2-positive and 5 HER2-negative patients) were scanned at 1, 2, and 3h post-injection to determine the best imaging timepoint, and the remaining patients were scanned at the optimized timepoint. All patients underwent standard 18F-FDG PET/CT within 7d to identify viable lesions. The SUVmax of lesions larger than 1.0cm were analyzed. Five lesion maxima were analyzed for each organ. (1) The 68Ga-HER2 affibody was safe and effective, and optimal image contrast was observed 2h post-injection; the average effective absorbed dose was 0.0215mSv/MBq. (2) The HER2-positive group had significantly higher 68Ga-HER2 affibody uptake than the HER2-negative group (SUVmax 10.7 ± 12.5 vs 3.8 ± 1.7, p = 0.005). The specificity and sensitivity were 100 and 55.4%, respectively, with a SUVmax cutoff value of 6.6. The SUVmax of the lesions ranged from 1.6 to 73.0, suggesting heterogeneity in HER2 expression. (3) 68Ga-HER2 affibody uptake showed an organ-dependent difference in patients with HER2-positive expression. Bone metastases had the highest uptake (SUVmax 40.5 ± 24.9), followed by liver metastases (SUVmax 11.9 ± 3.9) and lymph node metastases (SUVmax 5.6 ± 3.7), while the uptake in other lesions, including in the primary lesion, was relatively lower (SUVmax 7.3 ± 3.7). (4) Patients receiving therapy had a non-significantly lower lesion SUVmax than patients not receiving therapy (SUVmax 8.8 ± 4.9 vs 11.8 ± 15.2) (p = 0.253). Additionally, the 68Ga-HER2 affibody detected positive lesions in 1/11 patients with HER2-negative primary gastric cancer, which was confirmed by second generation gene sequencing. (5) Moreover, ten patients underwent baseline PET/CT followed by targeted anti-HER2 therapy. Patients with lesions showing high avidity to the 68Ga-HER2 affibody showed longer progression-free survival (PFS) than those with lesions showing low avidity (4-9m vs 2-3m). 68Ga-HER2 affibody PET/CT is a feasible method to noninvasively detect the HER2 status in AGC patients and enable early detection with a low dose. Ongoing anti-HER2 therapy did not influence 68Ga-HER2 affibody imaging, which allowed repeated evaluations to monitor the HER2 status after anti-HER2 therapy. This method provides an in vivo understanding of AGC biology that will ultimately help oncologists improve individualized therapy plans.

Abstract 2615: Heterogeneity of HER2 expression in gastric cancer (GC) leads to high deviation rates between local and central testing and hampers efficacy of anti-HER2 therapy: Survival results from the VARIANZ study

Abstract Background: HER2 is a membrane-bound receptor tyrosine kinase that belongs to the epidermal growth factor receptor family. The anti-HER2 directed monoclonal antibody trastuzumab is approved for 1st-line treatment of stage IV HER2+ GC, while other drugs addressing HER2 (pertuzumab, TDM1, lapatinib) failed to improved survival outcomes. Until today, resistance against HER2-directed treatment of GC is poorly understood. The VARIANZ study aims to assess the biologic background of resistance to anti-HER2 therapy in GC. Methods: This academic network study funded by the German Federal Ministry of Education and Research (BMBF 01ZX1610E) recruited patients (pts) who received medical treatment for stage IV GC in 34 sites. HER2 expression was verified centrally by two dedicated GI pathologists using immunohistochemistry (DCS, HI608C0I) and chromogenic in situ hybridization (Zytomed Systems, C-3022-40). Treatment and survival outcomes were reported by investigators. Results: 502 pts were enrolled from May 2014 to Oct 2017. At present, 442 samples were fully characterized for HER2. According to criteria from the ToGA study, 73 of 442 samples were found HER2+ in central testing. In 57 samples with a HER2+ status diagnosed by local pathologists, HER2 positivity could not be confirmed centrally. Deviation rate between local and central testing was 23%. Centrally confirmed HER2-positive GC displayed a higher percentage of tumor cells staining positive for HER2 (49.72 ± 28.58% [SD] vs. 11.35 ± 17.74% [SD]; p&amp;lt;0.001) and a higher HER2/CEP17 ratio (5.78 ± 2.55 [SD] vs. 1.43 ± 0.41 [SD]; p&amp;lt;0.001). Survival outcomes indicate that only pts with centrally confirmed HER2+ status benefit from trastuzumab with an overall survival of 30.7 months (95%-CI 10.7 - 50.8; n=28) versus 7.9 months (95%-CI 2.8 - 12.9, n=33); HR for death was 3.9 in pts with unconfirmed vs. confirmed HER2+ GC receiving trastuzumab plus chemotherapy; p&amp;lt;0.0001. Conclusions: Variability between local and central HER2 assessment in GC is significant. Pts with centrally unconfirmed HER2+ status have no benefit from anti-HER2 therapy. HER2 status should be assessed in highly qualified laboratories and cut-offs for defining HER2+ should be reconsidered. Alternative treatment options–other than trastuzumab–should be investigated in pts with unconfirmed or borderline HER2 expression. Citation Format: Florian Lordick, Ivonne Haffner, Birgit Luber, Dieter Maier, Elba Raimundez, Jan Hasenauer, Albrecht Kretzschmar, Ludwig Fischer von Weikersthal, Miriam Ahlborn, Jorge Riera Knorrenschild, Gabriele Siegler, Beate Rau, Stefan Fuxius, Thomas Decker, Katrin Schierle, Christian Wittekind. Heterogeneity of HER2 expression in gastric cancer (GC) leads to high deviation rates between local and central testing and hampers efficacy of anti-HER2 therapy: Survival results from the VARIANZ study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2615.