anti-HER2 Antibody-drug Conjugate Research Articles

Abstract PO2-13-03: Spatial HER2 heterogeneity on HER2-PET in HER2-low and negative disease: potential biomarker for antibody-drug conjugate effect

Abstract Background: Anti-HER2 antibody drug conjugates (ADCs) can assert an effect in part of patients with HER2-low breast cancer. In light of patient burden and financial sustainability, biomarkers to select responding patients are urgently needed, but so far, none have been identified. In the IMPACT trial (NCT01957332), patients with newly diagnosed and non-rapidly progressive metastatic breast cancer (MBC) of all subtypes (including HER2-negative, -low, and –positive disease) were included. We assessed the performance of Zirconium-89 (89Zr)-trastuzumab positron emission tomography (HER2-PET) in HER2-low and negative MBC. Methods: All patients received extensive workup, including a metastasis biopsy and HER2-PET at baseline. HER2 status was determined by immunohistochemistry (IHC) and in situ hybridization. ⁸⁹Zr-trastuzumab uptake was quantified as maximum and mean standardized uptake values (SUVmax, SUVmean) in metastases and healthy background tissue. Quantitative ⁸⁹Zr-trastuzumab uptake of all metastases and corresponding biopsied metastasis, was related to HER2 IHC status. Results: In 200 patients, ⁸⁹Zr-trastuzumab uptake was quantified in 5,163 metastases. With increasing HER2 IHC status, uptake was higher (geometric mean SUVmax 7.0, 7.6, 7.3, and 17.4 with HER2 IHC 0, 1, 2, or 3+, in respectively 71, 71, 20, and 24 biopsied metastases (P  0.001 between IHC 0 and 3+). SUVmax in lesions was heterogeneous within and between patients for all HER2 IHC groups. All HER2 IHC groups, also HER2-negative and -low, had uptake exceeding that of the healthy background (geometric mean tumor-to-background ratio SUVmax >1 in all groups, increasing with higher HER2 IHC). Of patients with a HER2 IHC 0 biopsy, 27.8% had at least one metastasis elsewhere in the body, with uptake exceeding the overall 90th SUVmax percentile of 14.6; in HER2 low IHC1+ and 2+, this was 31.9% and 30%. Conclusions: In this largest series with HER2-PET in patients with MBC, we show that ±30% of patients with HER2-low or -negative IHC in the biopsy, in fact, have metastases with high HER2 uptake elsewhere in the body. This spatial heterogeneity provides novel insights into HER2-negative and -low disease compared to standard HER2 IHC of a single biopsy. As HER2-PET uptake is related to ADC response in HER2 positive MBC1, HER2-PET is a potential biomarker for anti-HER2 ADC effect in HER2-low and -negative MBC as well. Reference: 1. Gebhart G, et al. Ann Oncol 2016;27(4): 619-624. Supported by the Dutch Cancer Society grant 2012-5565 Citation Format: Carolien Schroder, Jasper van Geel, Bertha Eisses, Adrienne Brouwers, Sjoerd Elias, Frederike Bensch, Evelien Kuip, Agnes Jager, Andor Glaudemans, Bert van der Vegt, Willemien Menke-van der Houven van Oordt, Elisabeth de Vries. Spatial HER2 heterogeneity on HER2-PET in HER2-low and negative disease: potential biomarker for antibody-drug conjugate effect [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 PO2-13-03.

Abstract PO1-27-07: Is HER2-low a biologically distinct breast cancer (BC) subtype? Prognosis and pathological complete response rate after neoadjuvant treatment (NAT) in early breast cancer (BC) HER2 negative

Abstract Background: Overexpression of HER2 is a significant prognostic and predictive factor in early breast cancer. HER2 positive tumors are those scored by immunohistochemistry (IHC) 3+ or 2+ with amplification by in situ hybridization (ISH). These tumors may benefit from HER2-targeted treatment and additionally, in early HER2-positive BC, achieving a complete pathological response after neoadjuvant treatment improves patient survival. In recent years, new anti-HER2 antibody-drug conjugates have proven effective for HER2 low BC (defined as IHC score 1+ or 2+ not amplified) in the metastatic disease setting; however, in HER2-low early BC, studies testing novel anti-HER2 antibody-drug conjugates as neoadjuvant therapy (NAT) are ongoing. Currently, we don´t know exactly whether HER2 expression can influence the pathological response rate after NAT or disease-free survival (DFS) in these patients compared to HER2-zero (IHC 0). The aim of our study is to determine the impact on response rate to NAT and survival outcomes in early HER2-negative BC. Methods: We conducted a retrospective study in two hospitals in Andalucia, Spain. Patients with early HER2 negative BC treated with NAT from January 2015 to June 2022 were included. Patients were divided into HER2 low patients (IHC 1+ or 2+ not amplified) and HER2 0 (IHC 0). We collected clinical and pathological characteristics, pathological response rate using the Residual Cancer Burden (RCB) system where a complete pathological response (pCR) was defined as ypT0/ypTis and ypN0, and survival outcomes. The primary objective of the study was to analyze the pCR rate after NAT according to HER2 score, and secondary objectives were to assess disease-free survival and overall survival (DFS and OS rates). Results: 574 patients were included (100% women). 397 patients had hormone receptors (luminal BC) on these tumors, and 177 did not have them, i.e., triple-negative BC (TNBC). 225 patients were HER2-zero, and 312 patients were HER2-low (253/312 patients had luminal BC and 59/312 patients were TNBC). The pCR in HER2-low patients was 17.3% and 23.1% in HER2-zero patients (p=0.047). For luminal BC patients, the pCR was 16.7% in HER2-low tumors and 13.7% in HER2-zero tumors (p=0.29). Similarly, there was no difference in pCR rates in TNBC patients (45.8% in HER2-low and 53.8% in HER2-zero tumors, respectively, (p=0.958). The overall survival (OS) at 96 months was 88.7% (95% CI 85.6%-92%). Regarding survival outcomes, neither HER2 expression level was associated with higher 8-year DFS: 81.1% in HER2-low patients (95% CI 75.7%-86.8%) and 74.9% in HER2-zero patients (95% CI 62.3%-90.1%) p=0.64. There was no difference between histological subtypes at 8-years regarding DFS rate: 80.5% (95% CI 74.3%-87.2%) in luminal HER2-low patients and 76.3% (95% CI 60.8%-95.7%) in luminal HER2-zero patients, p=0.22; 83.8% in TNBC HER2-low (95% CI 74.5%-94.2%) and 77.5% in TNBC HER2-zero patients (95% CI 69.4%-86.6%) p=0.26. We also didn´t find significant differences in terms of OS. For HER2-low patients, the 8-year OS was 90.9% (95% CI 87%-94.9%) and for HER2-zero patients, it was 86.4% (95% CI 80.8%- 92.5%) p=0.46. There were also no differences in OS for different histological subgroups with an 8-year OS in luminal HER2-low breast cancer patients of 92.5% (95% CI 88%-96.7%) and 87% in HER2-zero (95% CI 78.9%-96.1%) p=0.91. In patients with triple-negative breast cancer phenotype, the OS was 84.2% for HER2-low (95% CI 74.5%-95.2%) and 85.3% for HER2-zero (95% CI 77.9%-93.4%) p=1. Conclusions: We found no difference in survival outcomes and response rate to conventional NAT between HER2-low and HER2-zero expression levels. Therefore, our data do not support the hypothesis of HER2-low as a biologically specific breast cancer subtype. Further research on this approach with HER2 antibody-drug conjugate schemes as an alternative to traditional NAT schemes is warranted. Citation Format: Ana Gil-Torralvo, Yeray Rodriguez, Alejandro Falcon, David Morales, Mónica Cejuela, Isabel Miras, Marta Amérigo, Manuel Ruíz - Borrego, Juan Bayo, Francisco Javier Salvador Bofill. Is HER2-low a biologically distinct breast cancer (BC) subtype? Prognosis and pathological complete response rate after neoadjuvant treatment (NAT) in early breast cancer (BC) HER2 negative [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 PO1-27-07.

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.

Abstract PO5-24-07: HER2 EXPRESSION HETEROGENEITY PATTERN IN INVASIVE BREAST CARCINOMAS: FREQUENCY, DISTRIBUTION AND RELATION TO MORPHOLOGICAL VARIABLES

Abstract Human epidermal growth factor receptor 2 (HER2), a critical contributor to breast cancer development, is found to be amplified and overexpressed in approximately 15-20% of cases, making it a significant factor in carcinogenesis. This scenario was targeted by anti-HER2 drugs approved by several regulatory agencies in the past 15 years. Recently, novel anti-HER2 antibody-drug conjugates (ADCs) were approved to metastatic breast cancer with some HER2 expression albeit not due to amplification of the gene. Despite being classified as HER2 negative, most of these tumors exhibit detectable amounts of HER2 protein, reinforcing the challenging task of HER2 expression evaluation. In this scenario, it is very important to evaluate the spectrum of HER2 expression within a tumor due to its heterogeneity. 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. Pathological variables (histological subtype, histological grading, and hormonal receptors expression) were collected in pathology reports. Only patients with available slides and blocks were enrolled. HER2 slides were evaluated by three observers in consensus to access ASCO-CAP 2018 guidelines HER2 expression; presence or absence of HER2 expression heterogeneity; pattern of heterogeneity; frequency of the primary and secondary HER2 score in heterogeneous cases; and frequency of HER2 score in cases with homogeneous expression. Statistical analyses were performed with Chi-Square and Mann–Whitney U test using SSPS (26.0) program. 353 cases were included in this study. 104 (29,5%) were under 50 years and 249 (70,5%) over 50 years. 54,1% cases were left sided and 45,9% right sided. IBC of no special type comprehended 324 cases (91,8%) and invasive lobular carcinomas (ILC) were represented by 26 cases (7,4%); less than 1% of the cases were rare subtypes. Overall, HER2 results was negative in 296 (83,9%) cases and positive in 42 (11,9%) cases; HER2 2+ accounted by 15 cases (4,2%). Out of the negative cases, 235 (79.4%) were classified as 0+ and 61 (20.6%) were classified as 1+. Homogenous HER2 expression was observed in 287 cases (81,3%) with predominance of 0+ expression (80,8% - 232 cases), followed by 1+ (9,4% - 27 cases). ITH was present in 66 cases (18,7%), in which the subtyping was represented by 39 scattered (59,1%), 25 clustered (37,9%) and 2 mosaic (3%). The most prevalent score in this group was 1+ (51,5% - 34 cases), followed by 3+ (25,8% - 17 cases); the secondary score was predominantly categorized as 0+ (58,3%), followed by 1+ (20%) and 2+ (20%); only one case showed as 3+ expression. In the ILC group, 24 cases (92,3%) exhibited homogeneous HER2 expression, with predominant 0+ score (75% - 18 cases), while 2 cases (7,7%) showed heterogeneous expression. Out of the cases with heterogeneous HER2 expression, one exhibited a clustered heterogeneity pattern, with a primary HER2 score of 1+ and a secondary score of 0+. The other case displayed a scattered heterogeneity pattern, with a primary HER2 score of 3+ and a secondary score of 2+. There was a statistically significant association between HER2 expression heterogeneity and histologic grade (p = 0.005), and a marginal association with estrogen (p = 0.060) and progesterone (p = 0.060) expression. Tumor size wasn’t associated with heterogeneity (p = 0,071). We conclude that heterogeneity is prevalent in HER2 expression, especially in IBC, and should be addressed in pathology reports, especially in the ADCs scenario. Citation Format: Angela Flavia Waitzberg, Lucas de Figueiredo Barbosa, Adilson Monteiro dos Santos Filho, Ana Luiza da Cruz, Lisandra Gonzalez Porta Nova, Karla Calaça Kabbach Prigenzi. HER2 EXPRESSION HETEROGENEITY PATTERN IN INVASIVE BREAST CARCINOMAS: FREQUENCY, DISTRIBUTION AND RELATION TO MORPHOLOGICAL VARIABLES [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-07.

Abstract PO5-06-14: Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma

Abstract Background: Triple-negative (TN) invasive lobular carcinoma (ILC) of the breast is a rare entity, comprising roughly 2% of primary ILC. Clinicopathologic studies have shown that TN ILC is associated with more pleomorphism, higher nuclear grade, older patient age, and worse disease-related outcomes compared to estrogen receptor-positive (ER+) ILC. Intrinsic subtyping indicates that TN ILC less frequently expresses basal markers than TN invasive ductal carcinoma (IDC). This prompted investigation of the genomic landscape and clinical management of TN ILC to provide insight into potential therapeutic approaches. Methods: Twenty patients with TN ILC were included in this study. Three patients were treated at a single academic center and underwent comprehensive genomic profiling (CGP) of breast or metastatic tissue; clinicopathologic details were obtained from their electronic health records. Seventeen patients with TN ILC who underwent CGP of breast or metastatic biopsies were included from the Foundation Medicine, Inc. database. PD-L1 testing for all patients was determined by immunohistochemistry (IHC) using the VENTANA SP142 or Dako 22C3 commercial assays. HER2 expression was determined by IHC. Negative HER2 expression was defined as an IHC score of 0, or IHC 1+ or 2+ with non-amplified fluorescence in situ hybridization (FISH). Results: In the Foundation Medicine database of patients with TN ILC, the most frequent genomic alterations were in CDH1 (15/17 patients [88.2%]), TP53 (10/17 patients [58.8%]), and PIK3CA (10/17 patients [58.8%]). Short variant mutations in ERBB2 were found in 3/17 patients (17.6%). Fourteen patients were HER2-low (HER2 IHC 1+ or 2+ with non-amplified FISH). No cases were MSI high or expressed PD-L1 (8 of 17 cases underwent PD-L1 assessment by SP142). Median tumor mutational burden (TMB) was 5 muts/Mb. Among the three patients with TN ILC treated at a single center, the most common somatic mutations were in CDH1 and TP53, and no cases were MSI high or expressed PD-L1 (by SP142 or 22C3). All three patients had pleomorphic ILC, histologic grade 2 or 3, and HER2 IHC 0. One patient with mpT1cN1a(sn) ER+/HER2- ILC of the right breast in 2011 received adjuvant TAC (docetaxel, doxorubicin, cyclophosphamide), breast/axillary radiation, and ten years of adjuvant endocrine therapy. She was diagnosed with TN ILC of the left supraclavicular nodes in 2022; she did not exhibit a response to docetaxel and carboplatin with pembrolizumab, and she subsequently received palliative radiation followed by capecitabine. Another patient with locally advanced TN ILC progressed on the KEYNOTE-522 regimen, immediately developed metastases, and is currently receiving palliative sacituzumab govitecan. The third patient with cT3N1 TN ILC progressed on neoadjuvant therapy (through the I-SPY 2 trial) with oral paclitaxel, encequidar, and dostarlimab, followed by dostarlimab with dose-dense doxorubicin and cyclophosphamide. Her surgical pathology demonstrated ypT3N3a disease with 10 × 7 cm of tumor, and she developed skin metastases on the chest wall within weeks post-operatively. She received comprehensive chest wall and nodal radiation and is currently receiving palliative capecitabine with neratinib given a high variant allele frequency driver somatic ERBB2 mutation. Conclusions: The most common somatic mutations among TN ILC patients included in this study were in CDH1, TP53, and PIK3CA. Currently the management of TN ILC is extrapolated from that of TN IDC, however our patients did not respond to chemoimmunotherapy. The presence of HER2-low status or somatic ERBB2 mutations may provide opportunities for treatment with an anti-HER2 antibody-drug conjugate or tyrosine kinase inhibitor. Studies with larger sample sizes of TN ILC are needed for molecular profiling and assessment of outcomes with specific therapeutic approaches. Citation Format: Hemali Batra-Sharma, Smruthy Sivakumar, Prashanth Ashok Kumar, Ethan Sokol, Rebecca Shatsky, Jeffrey Ross. Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-14.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-04.

Abstract PO3-20-06: TRUDI: A phase II study of neoadjuvant TRastuzumab derUxtecan and Durvalumab for stage III HER2-expressing Inflammatory breast cancer

Abstract Background: Despite being a rare presentation of breast cancer, inflammatory breast cancer (IBC) is responsible for up to 10% of all breast cancer-related deaths. Most IBCs express HER2, with up to 40% of all IBCs being HER2-positive and 40% being HER2-low. Additionally, IBC more often exhibits PD-L1 expression compared to non-IBC, suggesting a preeminent role for immune evasion in the development and progression of IBC. No systemic treatment approaches have been yet developed specifically for IBC, and standard treatments commonly lead to poor outcomes. Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate currently approved for patients with pretreated HER2-positive and HER2-low metastatic breast cancer. Early-phase data highlighted the safety and potential synergy of combining T-DXd with the immune checkpoint inhibitor durvalumab. The aim of TRUDI is to evaluate the clinical activity and safety of neoadjuvant T-DXd with durvalumab among patients with HER2-expressing IBC. Trial Design: TRUDI is an ongoing, open label, multicenter, two cohort investigator-initiated phase II neoadjuvant trial for patients with stage III HER2-expressing IBC. The trial was activated in May 2023, with enrollment ongoing. Eligible participants are women and men with previously untreated, stage III (T4d, any N) breast cancer, clinically determined to be IBC. Patients will be included in two cohorts, depending on the locally determined HER2 status (with any hormone receptor [HR] status): Cohort 1 (n=36) for patients with HER2-positive disease (HER2 IHC 3+ or 2+/ISH amplified); Cohort 2 (n=27) for patients with HER2-low disease (HER2 IHC 1+ or 2+/ISH not amplified). Patients will receive eight cycles of T-DXd (5.4 mg/kg IV every 21 days) combined with durvalumab (1125 mg IV every 21 days), followed by modified radical mastectomy and post-mastectomy radiation. Post-surgical systemic treatment will follow local standards. Tumor tissue will be collected at baseline, cycle 1 day 8, and at surgery; blood will be collected at baseline, cycle 4 day 1, cycle 7 day 1 and at surgery; stool will be collected at baseline, after 3-6 weeks of treatment and surgery. The primary endpoint is pathologic complete response (pCR; ypT0/Tis ypN0). Patients in each cohort will be enrolled based upon Simon two-stage designs: in Cohort 1, if ≥8/20 patients with HER2-positive IBC experience pCR then a total of 36 patients will be enrolled. In Cohort 2, if ≥1/18 patients with HER2-low IBC experience pCR then a total of 27 patients will be enrolled. Secondary endpoints include the residual cancer burden at surgery, event free survival, distant progression or distant disease-free survival, as well as safety of the regimen. Exploratory objectives will investigate biomarker analyses on tissue, blood, and microbiome to explore the association of HER2 expression, immune variables, and stool composition on the efficacy of T-DXd with durvalumab among patient with HER2-expressing IBC. To our knowledge, this is the first and only ongoing study testing the combination of an anti-HER2 antibody-drug conjugate with immunotherapy for the neoadjuvant treatment of patients with IBC. Clinical trial information: NCT05795101. Citation Format: Paolo Tarantino, Samuel Niman, Antonio Giordano, Faina Nakhlis, Jennifer Bellon, Wendy Woodward, Azadeh Nasrazadani, Sadia Saleem, Anthony Lucci, Michelle DeMeo, Naoto Ueno, Sara Tolaney, Vicente Valero, Meredith Regan, Rachel Layman, Filipa Lynce. TRUDI: A phase II study of neoadjuvant TRastuzumab derUxtecan and Durvalumab for stage III HER2-expressing Inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-20-06.

Abstract PO2-04-03: BL-M07D1, an antibody-drug conjugate directed to HER2 in patients with locally advanced or metastatic Breast Cancer with HER2-positive/low-expression and other solid tumors: Results from a first-in-human phase 1 study

Abstract Background: BL-M07D1 is an anti-HER2 antibody-drug conjugate (ADC) comprised of a humanized anti-HER2 antibody, a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor (Ed-04). Methods: This study included subjects with locally advanced or metastatic HER2 expressing (positive/low) breast cancer (BC) and other solid tumors. BL-M07D1 would be administered at doses of 1.0mg/kg Day 1 & Day 8 every 3 weeks (D1D8 Q3W) or 2.6, 3.2, 3.8, 4.4, 5.0, 5.6, 6.2, 6.8 and 7.4 mg/kg Day 1 every 3 weeks (D1Q3W) during dose escalation (D-ESC). A subset of patients (pts) will be enrolled in the dose-expansion phase (D-EXP) at D1 Q3W regimens. Results: As of June 25, a total of 75 pts have been treated with at least one dose of BL-M07D1, with 22 pts in the D-ESC phase and 53 in the D-EXP phase. Among the 75 pts, 1 received 1.0 mg/kg D1D8Q3W and 74 were treated on the D1Q3W schedule. Dose-limiting toxicity (DLT) was observed at 6.2mg/kg (febrile neutropenia and G3 thrombocytopenia lasting >7 days in one patient). The maximum tolerated dose (MTD) has not been reached yet. D-EXP dose levels included 3.8, 4.4, 5.0 and 5.6mg/kg D1 Q3W. This study enrolled 62 patients with BC, 6 with gastric cancer, 4 with colorectal cancer and 3 with non-small cell lung cancer. The most common TEAEs ( >10%, all grade/≥G3) were leukopenia (79%/24%), neutropenia (69%/39%), anemia (63%/11%), nausea (47%/0%), thrombocytopenia (37%/9%), vomiting (37%/1%), decreased appetite (27%/0%), lymphopenia (24%/8%), alopecia (23%/0%), asthenia (19%/0), gamma-glutamyl transferase increased (17%/0%), aspartate aminotransferase increased (17%/0%), constipation (13%/0%), diarrhea (12%/0%), hypertriglyceridemia (12%/0%), urinary tract infection (11%/1%), COVID-19 (11%/0%), occult blood positive (11%/0%), pyrexia (11%/0%). No ILD was observed. Forty-five pts with BC were evaluated for efficacy (i.e., had at least one post-baseline tumor assessment). Updated efficacy and pharmacokinetic (PK) results will be presented at the meeting. Conclusions: BL-M07D1 demonstrated encouraging efficacy in heavily pretreated HER2 expressing cancers, especially in HER2+ BC. The safety profile showed adequate safety and tolerability. Clinical trial identification: NCT05461768. Efficacy in Patients with Breast Cancer a. 8/9 pts received prior HER2-ADC with microtubule inhibitor. 1/9 pts received prior HER2-ADC with TOPI inhibitor. b. 6 pts with tumor shrinkage are still on-going. c. All pts with tumor shrinkage are still on-going. Citation Format: Erwei Song, Herui Yao, Meili Sun, Hong Zong, Rongbo Lin, Wen Zou, Muran Ding, Jing Yu, Sa Xiao, Hongwei Wang, Hai Zhu, Martin Olivo, Yi Zhu. BL-M07D1, an antibody-drug conjugate directed to HER2 in patients with locally advanced or metastatic Breast Cancer with HER2-positive/low-expression and other solid tumors: Results from a first-in-human phase 1 study [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 PO2-04-03.

Abstract PO1-04-01: Phase 1 dose escalation study of ARX788, a next-generation anti-HER2 antibody drug conjugate, in heavily pretreated breast cancer patients

Abstract Introduction: ARX788 is a next-generation anti-HER2 antibody-drug conjugate (ADC) conjugated to amberstatin269 (AS269), a potent cytotoxic tubulin inhibitor. ARX788 is highly stable with nearly identical PK profiles for the total ADC and the total antibody due to proprietary site-specific oxime conjugation chemistry. The stability of ARX788 results in limited systemic toxicity and increased targeted delivery of payload to tumor cells. Clinical benefit of ARX788 has been observed in patients (pts) with HER2-positive (HER2-pos) breast cancer (BC) in multiple clinical trials and recently in a randomized, controlled, phase 3 registrational trial conducted in China. Methods: ARX788-1711 (NCT03255070)was a phase 1 dose-escalation study of ARX788 monotherapy in pts with advanced solid tumors with HER2 expression. There was no limit to the number of prior therapies. An objective of the trial was investigating the safety and tolerability of ARX788 in pts with BC. Results: Between August 2020 and June 2023, 42 pts with HER2-pos and HER2-low BC with a median of 6 prior lines of therapy received intravenous ARX788 at either 1.5, 1.6, or 1.7 mg/kg every 21 days (Q3W) or every 28 days (Q4W). Treatment regimens were combined for these analyses. ARX788 was generally well tolerated. Most adverse events (AEs) were grade (Gr) 1 or 2, the most common being ocular, alopecia, nausea, and fatigue. Gr3 treatment-related AEs (TRAEs) occurred in 23.8% of pts and included nausea, ocular, AST and ALP increase (4.8% each); one pt (2.4%) had Gr3 pneumonitis/interstitial lung disease. A Gr3 ocular SAE occurred 48 days after the pt’s last study dose and was resolving 9 days after onset. There were no Gr4 or Gr5 events. Treatment discontinuation due to drug-related AEs occurred in 7.1% of pts. Overall response rate (ORR) per RECIST v1.1 was analyzed in groups by HER2-low or HER2-pos tumor status. In pts with HER2-pos BC (n=11) the ORR was 54.5% (4 confirmed, 2 unconfirmed); in pts with HER2-low BC (n=30) the ORR was 23.3% (5 confirmed, 2 unconfirmed). The disease control rate (confirmed complete or partial response + stable disease) was 81.8% and 76.7%, for HER2-pos and HER2-low BC, respectively. Six of 9 pts with confirmed responses had a duration of response greater than 5 months. Six of eight pts with prior trastuzumab deruxtecan (T-DXd) exposure also had prior trastuzumab emtansine (T-DM1). Two pts with prior T-DM1 and T-DXd had significant target lesion reductions of 55% and 32%; these were a HER2-low pt with 6 prior cancer therapy regimens and a HER2-pos pt with 9 prior cancer therapy regimens, respectively. Both pts came off treatment before response confirmation. As of the data cut-off (19 Jun 2023), 4 of 9 confirmed responders remain on treatment and continue to experience clinical benefit including one pt with prior T-DM1 exposure with a durable response of 15.8 months. Importantly, significant target lesion reductions were observed in heavily pretreated pts who also received prior T-DXd and T-DM1. Discussion: Based on the promising safety and antitumor activity of ARX788 in this heavily pretreated pt population, a phase 2 study is open (NCT04829604) and enrolling pts with HER2-pos BC who have been previously treated with T-DXd. Citation Format: Sophia Frentzas, Haesong Park, George Budd, Vinod Ganju, Catherine Shannon, Sara Hurvitz, Katharine Cuff, Peter Lau, Richard Eek, Joyce O'Shaughnessy, Fran Boyle, Sandra Aung, Colin Hessel, Janice Lu. Phase 1 dose escalation study of ARX788, a next-generation anti-HER2 antibody drug conjugate, in heavily pretreated breast cancer patients [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 PO1-04-01.

Abstract PO3-02-07: HER2-low status in inflammatory breast cancer (IBC) is associated with hormone receptors positivity but not with pathological response to neoadjuvant chemotherapy or overall survival

Abstract Background: Half of HER2-negative breast cancers (BC) show HER2-low expression. The strong efficacy of recent anti-HER2 antibody-drug conjugates (ADC) in HER2-low tumors has risen the interest of HER2-low as a proper BC subtype. However, HER2-low non-inflammatory breast cancer (non-IBC) appears to have only marginal clinicopathological differences compared to HER2-0. Little is known about HER2-low clinical significance in IBC. Methods: Data from patients diagnosed with HER2-negative IBC between 1990 and 2021 were collected from the Institut Paoli-Calmettes European Comprehensive Cancer Center database. Two tumor groups were defined (HER2-low and HER2-0). HER2-low tumors were defined by HER2 IHC score of 1+ or 2+ with negative FISH, and HER2-0 by IHC score of 0+. Hormone Receptor (HR) status was determined according to the French guidelines (oestrogen and/or progesterone receptors by IHC with a 10% threshold for HR-positivity). Clinicopathological characteristics, pCR (defined as [ypT0/ypTis] and [pN0sn or ypN0]), and OS rates were compared between the two tumor groups. Results: The individual data of 256 IBC patients were analyzed. Thirty-seven percent of tumors were HER2-low and 63% were HER2-0. Among patients with stage III (n = 226) and stage IV (n = 30) IBC, 35% and 53% had HER2-low tumors, respectively. HR positivity, age over 50 years, SBR grade, and menopausal status were associated with HER2-low in univariate analysis, but only HR positivity remained significant in a binary logistic regression (OR: 1.95 [1.06-3.58]; p=3.15E-02). In stage III patients, the pCR rate after neoadjuvant chemotherapy was similar in HER2-low (24%) and HER2-0 (29%) patients (p=0.550). No difference was observed by analyzing pCR by HR status. In univariate analysis, overall survival was affected by the clinical axillary lymph node invasion the presence of metastasis, the pathological type and menopausal status, but not by HER2 status. Conclusion: In IBC, HER2-low is associated with HR positivity but has no effect on pathological response to chemotherapy and overall survival in our retrospective cohort, consistently with data available in non-IBC patients. Of 256 IBC patients, 37% were HER2-low. These results may be useful to design future clinical trials testing anti-HER2 ADCs in IBC. Citation Format: Alexandre de Nonneville, Pascal FINETTI, Laurys Boudin, Lucas Usclade, Patrice Viens, Frederic VIRET, Emilie Mamessier, Anthony Gonçalves, François BERTUCCI. HER2-low status in inflammatory breast cancer (IBC) is associated with hormone receptors positivity but not with pathological response to neoadjuvant chemotherapy or overall survival [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-02-07.

Abstract PO2-07-05: Deep learning model for automated quantification of HER2 expression in invasive breast cancers from immunohistochemical whole slide images

Abstract Introduction Human epidermal growth factor receptor 2 (HER2) protein overexpression and/or HER2 gene amplification is found in about 20% of invasive breast cancers. Considering the results of the DESTINY-Breast trials confirming the remarkable efficacy of anti-HER2 antibody drug-conjugate (T-Dxd) in both HER2-overexpressed and HER2-low tumors, it is necessary to identify not only HER2 (immunohistochemistry (IHC) score 3+) overexpressing tumors, but also HER2-low tumors. The latter category, defined as IHC1+ or IHC2+ but non-amplified, has proven to be challenging even for experienced pathologists, with high inter-observer variability. Here, we validate the performance of a deep learning (DL) model at: 1) predicting the IHC score from IHC histological features and 2) identifying HER2-low tumors with high sensitivity. Methods 675 HER2 stained IHC slides from primary breast cancer patients were selected based on pathology reports across three different cohorts (KCL_GSTT_1 n=369; Cypath_Breast n=214; KCL_GSTT_2 n=92). The slides were digitized and reviewed by expert breast pathologists. Specifically, Cypath_Breast and KCL_GSTT_2 were each annotated by one expert pathologist, while KCL_GSTT_1 was annotated by 5 expert pathologists with the ground truth defined as a majority vote between the 5 annotators. Cypath_Breast was assigned as the “discovery” set; while KCL_GSTT_1 and KCL_GSTT_2 were used as two independent validation cohorts. The model was specifically trained to extract features from IHC tiles to ensure they would capture both the staining intensity and the staining location on the cells (membrane, cytoplasmic or nuclei). The one-vs-one (OVO) and one-vs-rest (OVR) AUC as well as sensitivity and specificity to HER2-low and positive tumors were used as metrics to assess the performance of our model. An expert pathologist reviewed the most predictive regions of HER2 expression. Results After review by expert pathologists, 59 slides from KCL_GSTT_1 were removed either because of folded tissue, blurriness or because there was too little tissue. The IHC scores (0/1+/2+/3+) for the 3 cohorts were distributed as follows; Cypath_Breast: 42/120/36/16; KCL_GSTT_1: 120/90/52/48; KCL_GSTT_2: 54/22/15/1. For KCL_GSTT_1, the average agreement between the 5 expert pathologists amounted to a Cohen’s Kappa score of 0.63. Following training on Cypath_Breast, the performance of the model are presented in An ablation study proved that a feature extractor trained on IHC tiles outperformed an in-house extractor solely trained on H&E tiles (OVO AUC 0.95 vs 0.93 on KCL_GSTT_1; DeLong p< 0.001). Specifically, the model performed best at distinguishing HER2-negative (HER2 IHC 0) from HER2-low and HER2-positive tumors with a sensitivity (0 vs 1+/2+/3+) of 0.95 [0.93 - 0.98] and a specificity (0 vs 1+/2+/3+) of 0.78 [0.72 - 0.84] on KCL_GSTT_1. We observed the same tendency on KCL_GSTT_2 with a sensitivity (0 vs 1+/2+/3+) of 0.97 [0.91 - 1.00] and a specificity of 0.91 [0.83 - 0.98]. More importantly, the DL model reached a Cohen’s Kappa score of 0.72 [0.67 - 0.77] with the ground truth on KCL_GSTT_1, surpassing the average agreement Kappa score between expert pathologists (0.63). The most predictive regions showed cytoplasmic staining in tumor regions. Conclusion Our model provides a path towards a fully automated workflow for identifying up to 95% of breast cancer patients who could potentially benefit from anti-HER2 targeted therapies. Additionally, we show the utility of AI-based tools to minimize discrepancies among pathologists and assist in the diagnostic patient pathway. Table 1: Results of external validation on KCL_GSTT_1 and KCL_GSTT_2 <grpahic> Cohorts OVO AUC OVR AUC IHC 0 OVR AUC IHC 1+ OVR AUC IHC 2+ OVR AUC IHC 3+ KCL_GSTT_1 0.95 0.95 0.80 0.91 0.99 [0.93 - 0.96] [0.94 - 0.95] [0.79 - 0.82] [0.90 - 0.92] [0.99 - 1.00] KCL_GSTT_2 0.92 0.97 0.78 0.84 N/A [0.90 - 0.93] [0.96 - 0.98] [0.76 - 0.81] [0.80 - 0.88] Citation Format: Pierre-Antoine Bannier, Loïc Herpin, Rémy Dubois, Lydwine Van Praet, Charles Maussion, Ellen Amonoo, Anca Mera, Jasmine Timbres, Cheryl Gillett, Elinor Sawyer, Patrycja Gazinska, Piotr Ziolkowski, Roberto Salgado, Sheeba Irshad. Deep learning model for automated quantification of HER2 expression in invasive breast cancers from immunohistochemical whole slide images [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 PO2-07-05.

Abstract RF02-05: Report on Cohort Expansion of Phase I Study to Investigate the Safety, Tolerability, Pharmacokinetics and Antitumor Activity of BB-1701 in Patients with Locally Advanced/Metastatic HER2 Low Breast Cancer

Abstract Background: BB-1701 is an antibody-drug conjugate (ADC) consisting of a humanized IgG1κ monoclonal anti-HER2 antibody with the same sequence as trastuzumab and eribulin linked together by a cathepsin-cleavable valine-citrulline linker. Eribulin is an inhibitor of microtubule approved for the treatment of metastatic breast cancer and advanced liposarcoma, following prior therapies. When BB-1701 targets HER2 expressing cancer cells and it is internalized, free eribulin is cleaved from the ADC by cathepsin b and causes cytotoxicity to cancer cells. The neighboring cells were affected with bystander effects, like cytotoxicity on tumor cells and non-cytotoxic effects on microenvironment, by free eribulin released from dying cancer cells. Here, we report preliminary results from the ongoing cohort expansion of a phase I study of BB-1701 in patients with HER2 low breast cancer (ClinicalTrials.gov Identifier: NCT 04257110). Methods: Patients ≥ 18 years of age with confirmed locally advanced/metastatic HER2 low breast cancer (IHC 2+ and FISH negative or IHC 1+), that had progressed on at least two lines of prior standard therapies, with ECOG PS ≤ 2 and measurable disease (RECIST 1.1) were enrolled. HER2 expressions was determined by IHC and FISH before enrollment. Total of four dose levels of BB-1701 (1.0 mg/kg Q3W, 1.2 mg/kg Q3W, 1.4 mg/kg Q3W and 1.6 mg/kg Q3W) were administered during the cohort expansion. The safety, tolerability and preliminary anti-tumor activity of BB-1701 were assessed during study. Radiographic assessment was conducted every 6 weeks. Results: As of 30 June 2023, 40 patients with HER2 low breast cancer were enrolled in the four dose levels, 5 patients at 1.0 mg/kg Q3W, 20 patients at 1.2 mg/kg Q3W, 5 patients at 1.4 mg/kg Q3W and 10 patients at 1.6 mg/kg Q3W. Median age was 55 years (range 30-74), 97.5% were female, and 30.0%/70.0% patients were ECOG PS 0/1. The median prior lines of systemic therapy were 3 (range 2 – 9). All subjects experienced at least one treatment emergent adverse events and treatment-related adverse events (TRAEs) occurred in 30 subjects. The most common all grade TRAEs (≥20%) were peripheral neuropathy, aspartate aminotransferase increased, alanine aminotransferase increased, anemia, white blood cell count decreased. Grade 3 TRAEs were peripheral neuropathy (3 subjects), peripheral sensory neuropathy (2 subjects), neutrophil count decreased (2 subjects), platelet count decreased (1 subject), white blood cell count decreased (1 subject). There were no grade 4 or grade 5 events. The treatment related serious adverse events (SAEs) were peripheral sensory neuropathy (1 subject) and infusion related reaction (1 subject). There was no interstitial lung disease reported. No patients discontinued the study treatment due to AE. Of the 40 patients enrolled, 38 patients were evaluable for antitumor activity. At 1.0 mg/kg dose level, of 5 patients treated, 2 achieved partial response (PR), 2 had stable disease (SD), best overall response rate (BOR) was 40.0% (2/5) and disease control rate (DCR) was 80.0% (4/5). At 1.2 mg/kg dose level, of 18 patients treated, 5 achieved PR, 11 had SD with BOR of 27.8% (5/18) and DCR of 88.9% (16/18). At 1.4 mg/kg dose level, of 5 patients treated, 2 achieved PR, 2 had SD with BOR of 40.0% (2/5), and DCR was 80.0% (4/5). One patient who achieved PR was previously progressed on sacituzumab govitecan. At 1.6 mg/kg dose level, of 10 patients treated, 1 achieved CR, 3 achieved PR, 2 had SD with BOR of 40.0% (4/10), and DCR of 60.0% (6/10). Among these patients, the patient who achieved CR was previously treated by disitamab vedotin, and one patient who achieved PR was previously treated by trastuzumab deruxtecan. Conclusions: BB-1701 has demonstrated promising preliminary antitumor activity in HER2 low breast cancer including patients who received prior anti-HER2 ADCs, and a manageable safety profile. Citation Format: Fei Ma, Pamela Munster, Xiaoxiang Guan, Jingfen Wang, Herui Yao, Erika Hamilton, Xian Wang, Yehui Shi, Xinjun Liang, Nong Xu, Huoling Tang, Yuhong Zhou, Ziping Wei. Report on Cohort Expansion of Phase I Study to Investigate the Safety, Tolerability, Pharmacokinetics and Antitumor Activity of BB-1701 in Patients with Locally Advanced/Metastatic HER2 Low Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-05.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Abstract 2111: Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy

Abstract Background: T-DXd is an FDA-approved anti-HER2 Antibody Drug Conjugate (ADC), which has shown clinical efficacy in breast cancers with either overexpression or low-level expression of HER2. The benefit of T-DXd across the spectrum of HER2 expression has called into question the relevance of HER2 expression level as relevant parameter for T-DXd sensitivity. We sought to experimentally address the relevance of HER2 expression level upon the degree of T-DXd response. Methods: A series of isogenic models was generated that expressed different levels of surface HER2 using a doxycycline-inducible promoter. These were analyzed for impact of differential expression level upon T-DXd internalization and drug response. Human breast cancer samples were analyzed for HER2 expression levels and association with clinical response to T-DXd under an IRB approved protocol. Results: Induction of different levels of HER2 at specified doxycycline concentrations was verified in multiple models using immunoblotting and FACS. These different levels appeared to correspond to clinical breast cancer immunohistochemistry (IHC) scoring levels to 0, 1+, 2+, and 3+. The models were analyzed for T-DXd response and showed increasing levels of HER2 led to increase lysosomal uptake of T-DXd (p<0.0001). Moreover, elevation of HER2 levels led to lower concentrations of T-DXd needed to suppress tumor growth. Analyses of the effects of increasing levels of HER2 upon xenograft response to T-DXd are ongoing. Finally, retrospective analyses of a cohort of 50 breast cancer patients treated with T-DXd revealed that higher levels of HER2 corresponded to longer time on treatment (time to progression). Conclusion: We observe a consistent relationship between elevated expression of HER2 and breast cancer response to T-DXd. The findings highlight the relevance of HER2 expression level as a biomarker of T-DXd response and support the potential value of therapeutic strategies that can induce HER2 surface expression levels to enhance response. Citation Format: Wanyi Chen, Joshua Drago, Avantika Gupta, Bo Liu, Sarat Chandarlapaty. Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2111.

Abstract 6788: Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models

Abstract Robust techniques are needed to screen the anti-tumor activity of antibody-drug conjugates (ADCs) in vitro. Traditional monolayer models provide a cost-effective and scalable option but lack many features of the tumor microenvironment, such as complex cell-cell and cell-extracellular matrix interactions. Tumor spheroids afford a more translational model for quantification of ADCs. Here, spheroids are used to compare the mechanism of action (MoA) of anti-HER2 ADCs. Single spheroids were formed from mono- or co-cultures of HER2 over-expressing cancer cells and HER2 negative control cells. Incucyte® Nuclight Green Lentivirus was used to label HER2 positive BT474 cells, whilst HER2 negative MDA-MB-231 cells were left unlabeled. Antibodies were added after 72 hours of spheroid formation in 96-well ultra-low attachment plates. Brightfield and fluorescence images were captured every 3 hours using the Incucyte® Live-Cell Analysis System and quantified, for metrics such as spheroid size and fluorescence intensity, to give an indication of ADC cytotoxicity. Co-culture spheroids were dissociated at assay endpoint and the remaining proportion of green and unlabeled cells was measured using the iQue® Advanced Flow Cytometry Platform. The area of the BT474 mono-culture spheroids decreased in a concentration-dependent manner in the presence of both ADCs tested (trastuzumab emtansine and trastuzumab deruxtecan), indicating increased cytotoxicity. In contrast, only the highest concentration of trastuzumab, which is the monoclonal antibody backbone on which they are based, resulted in a reduction in spheroid size. This displays the increased anti-tumor activity due to the addition of the ADC payloads. In the co-culture model, only the trastuzumab deruxtecan induced a reduction in spheroid area, with the size of the spheroids in the presence of trastuzumab emtansine and trastuzumab remaining comparable to control levels. Analysis of the spheroid composition using flow cytometry showed that for the BT474 cells, again, percentage cell death compared to the IgG control was greater for the two ADCs, at 96.4% and 97.2%, respectively. This death was greater than with trastuzumab, which induced 46.4% cell death. Unlike in the mono-culture assays, a high level of death of the HER2 low expressing cells was also seen in the co-culture in the presence of trastuzumab deruxtecan (94.1% death compared to control). This displays the unique bystander activity of trastuzumab deruxtecan, which has led to its approval as a treatment for both HER2 high and HER2 low breast cancers. These data display how the Incucyte® Live-Cell Analysis System combined with iQue® Advanced Flow Cytometry can be leveraged to provide comprehensive assessment of the in vitro function of ADCs and how it can enable differences in their MoAs to be distinguished in 3D spheroid models. Citation Format: Kirsty McBain, Kalpana Barnes, Nicola Bevan. Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6788.

HER2-low Breast Cancer: Now and in the Future.

Breast cancer is a heterogeneous disease, and its subtypes are characterized by hormone receptor and human epidermal growth factor receptor 2 (HER2) expression status. "HER2-low" tumors, which exhibit a low level of HER2 expression (immunohistochemistry 1+ or 2+ without gene amplification), were conventionally considered not amenable to anti-HER2 targeting agents based on the results of a phase III trial of trastuzumab. However, this perspective is being challenged by the emergence of novel anti-HER2 antibody-drug conjugates, such as trastuzumab-deruxtecan. These innovative therapies have demonstrated remarkable efficacy against HER2-low breast cancer, shedding new light on a previously overlooked category of breast cancer. Such promising results highlight the need for in-depth investigations of the biology and prognostic implications of HER2-low tumors. In this review, we comprehensively summarize the current evidence surrounding this topic and highlight areas that warrant further exploration and research in the future.

Systemic Therapies for HER2-Positive Advanced Breast Cancer.

Despite recent advances, HER2-positive advanced breast cancer (ABC) remains a largely incurable disease, with resistance to conventional anti-HER2 drugs ultimately unavoidable for all but a small minority of patients who achieve an enduring remission and possibly cure. Over the past two decades, significant advances in our understanding of the underlying molecular mechanisms of HER2-driven oncogenesis have translated into pharmaceutical advances, with the developing of increasingly sophisticated therapies directed against HER2. These include novel, more potent selective HER2 tyrosine kinase inhibitors (TKIs); new anti-HER2 antibody-drug conjugates; and dual epitope targeting antibodies, with more advanced pharmacological properties and higher affinity. With the introduction of adjuvant T-DM1 for incomplete responders to neoadjuvant therapy, fewer patients are relapsing, but for those who do relapse, disease that may be resistant to standard first- and second-line therapies requires new approaches. Furthermore, the risk of CNS relapse has not been abrogated by current (neo)adjuvant strategies; therefore, current research efforts are being directed towards this challenging site of metastatic disease. In this article, we review the currently available clinical data informing the effective management of HER2-positive breast cancer beyond standard first-line therapy with pertuzumab, trastuzumab, and taxanes, and the management of relapse in patients who have already been exposed to both these agents and T-DM1 for early breast cancer (EBC). We additionally discuss novel anti-HER2 targeted agents and combinations in clinical trials, which may be integrated into standard treatment paradigms in the future.

Prevalence and Therapeutic Targeting of High-Level ERBB2 Amplification in NSCLC

IntroductionERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody–drug conjugate (ADC) therapeutic strategy. MethodsOur institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. ResultsHigh-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. ConclusionsHigh-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.

Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.