Abstract PD6-01: Novel approach to HER2 quantification: Digital pathology coupled with AI-based image and data analysis delivers objective and quantitative HER2 expression analysis for enrichment of responders to trastuzumab deruxtecan (T-DXd; DS-8201), specifically in HER2-low patients

Introduction: Enfortumab Vedotin (EV), an antibody-drug conjugate (ADC) targeting Nectin-4, has emerged as a new standard-of-care for patients with metastatic urothelial cancer (UC). Promising results have also been observed in bladder cancer (BLCA) patients with ADCs targeting HER2. To identify predictive biomarkers of ADC sensitivity, we characterized a large and expanding biobank of patient-derived organoid (PDO) and xenograft (PDX) models for Nectin-4 and HER2 expression, and Nectin-4 and HER2-targeted ADC sensitivity. Methods: We generated 19 PDOs and 34 PDXs that reflect the genomic and biologic heterogeneity of human UC. A subset of these models was further characterized using a multiplatform approach, including bulk and single-cell DNA and RNA sequencing and immunoblot for Nectin-4 and HER2 expression. We leveraged these models to study the biologic determinants of sensitivity to EV and the HER2-targeted ADC trastuzumab deruxtecan (T-DXd). PDX models were treated with EV (5 mg/kg IV on day 1, 3, 6) or T-DXd (10 mg/kg IV on day 1, 21, 42) or vehicle only as control. We also studied sensitivity to T-DXd and EV, as well as to their cytotoxic payloads (exatecan and vedotin) in vitro using paired PDO models. Results: We observed high degree of variability of HER2 and Nectin-4 expression across UC PDOs and PDXs. In PDO models, sensitivity to EV correlated strongly with Nectin-4 expression, whereas HER2 expression was not strongly correlated with sensitivity to T-DXd. We also noted a poor correlation between Nectin-4 expression in PDOs and PDXs derived from the same patient. A subset of models, for example, SMBO-104 (ERBB2 wildtype with low HER2 expression), exhibited significantly greater sensitivity to EV than to T-DXd. Conversely, T-DXd was significantly more active than EV in SMBO-170, a model with ERBB2 amplification (~40 copies) and HER2 3+ expression. Sensitivity to exatecan, an analogue of the T-DXd payload deruxtecan, as measured by inhibition of cell proliferation and induction of apoptosis, was more predictive of T-DXd sensitivity than HER2 expression level. Conclusion: In this study, we generated and biologically characterized UC patient-derived models with the goal of identifying predictive biomarkers of sensitivity to Nectin-4 and HER2-targeted ADCs. We observed significant heterogeneity of HER2 and Nectin-4 expression across models. Models were identified in which T-DXd was superior to EV, and vice versa. Sensitivity to the T-DXd cytotoxic payload was more predictive of PDX response than HER2 expression levels, with significant tumor regression observed with T-DXd in BLCA models with low HER2 expression. Our data suggest that clinical trials of HER2-targeted ADCs such as T-DXd should not be restricted to patients with HER2 overexpression and that novel biomarkers will be needed to identify those UC patients most likely to benefit from FDA-approved and investigational ADCs. Citation Format: Xinran Tang, Ziyu Chen, Jasmine Thomas, Karan Nagar, John Christin, Naryan Rustgi, Sizhi Gao, Elisa de Stanchina, Jonathan Coleman, Michael Shen, Hikmat Al-Ahmadie, Gopa Iyer, Kwanghee Kim, David Solit. Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived 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 6006.

Silver Jubilee of HER2 targeting: a clinical success in breast cancer.

Introduction/BackgroundLow grade serous carcinoma (LGSC) is a subtype of ovarian cancer characterized by chemoresistance and a slow growth rate. Advances in HER2-targeted antibody drug conjugates (ADCs) have demonstrated efficacy in...

Background: Invasive Lobular Carcinoma (ILC) is a molecular subtype of breast cancer that is usually hormone receptor (HR) positive and E-cadherin negative. Ninety percent of ILC carry CDH1 mutations. Although ILC patients have a seemingly favorable outlook at diagnosis, they suffer from late recurrences and hence the long-term outcomes are not as favorable suggesting resistance to conventional therapies. In vitro and in vivo studies have demonstrated cross talk between EGFR/HER2 with HR in inducing endocrine resistance1. Increase in HER2 expression at the time of recurrence has been reported, while HER2 activating mutations occur in approximately 15.4% of ILC’s2. Importantly, HER2 mutations are associated with significantly worse prognosis in patients with CDH1 mutated ILC3. We believe that HER2 is a viable target in ILC, and endocrine resistant IDC when combined with endocrine therapy. HER2 targeted therapy, such as Trastuzumab is not currently used to treat patients with normal HER2 expression as a recent clinical trial NSAPB B-47 found that Trastuzumab has very little effect on these patients. We hypothesize that ONT-380 (Tucatinib), a tyrosine kinase inhibitor (TKI) that is currently in clinical trials for HER2 overexpressing metastatic breast cancer, will be effective in treating normal HER2 expressing ILC and HR positive IDC that are relatively endocrine resistant. Combined treatment with TKI and anti-estrogen could improve responses to endocrine therapy. Methods: We have studied effects of ONT-380 in combination with Tamoxifen on cell proliferation using in vitro cell culture models. We used ERα positive cell lines with varying levels of HER2 expression BT474 (HER2+), and cell lines expressing basal level of HER2, ZR-75-1 (IDC), T47D (IDC), SUM44PE (ILC), and MDA-MB-134-VI (ILC) to understand the effects of simultaneous targeting of HER2 and ERα. We have used MTT assay (Promega) to compare cell proliferation in drug treated and untreated cells. Western blot analysis was used to measure the levels of pHER2Y877, pHER2Y1221/1222,pAKTS473, total HER2 and total AKT. Basal level and drug induced alteration in phosphorylation status of downstream targets of HER2 and ERα signaling will be assessed using Human RTK (Receptor Tyrosine Kinase) phosphorylation antibody array (Abcam). Results: Western blot analysis of whole cell extracts showed that the designated HER2 positive cells (BT474) have higher level of HER2 protein, while the other cells (ZR-75-1, T47D, SUM44PE, MDA-MB-134) have significantly lower level of HER2 protein. HER2 was not detectable in MCF7 cells. Similarly, pAKT, a downstream effector of EGFR/ HER2 signaling was higher in the HER2 positive cell lines. Treatment of ILC cell line MDA-MB-134-VI with ONT-380 (100nM) or Tamoxifen (10 μM) alone or in combination showed additive inhibition of cell proliferation when the two drugs were combined as opposed to single drug treatment. We have observed similar effect of ONT-380 and Tamoxifen on SUM44PE, BT474 and ZR-75-1 cells. Furthermore, treatment of ZR-75-1 cells with ONT-380 and Tamoxifen markedly reduced pAKT level, while the effect of treatment with individual drugs was barely detectable. Similar studies are underway for endocrine resistant ILC and additional IDC cell lines. Conclusion: Targeting HER2 kinase activity in normal HER2 expressing ILC and endocrine resistant IDC with small molecule inhibitors of tyrosine kinase could be a viable option for treating these cancers. Further in vitro and in vivo studies are underway to study the efficacy of this drug combination. 1. Schafer et.al. 2002 (PMID: 12650703) 2. Deniziaut et.al. 2016 (PMID: 27602491) 3. Ping et.al. 2016 (PMID: 27811364) Citation Format: Nikhil Pramod, Sivanesan Dhandayuthapani, Sarmila Majumder, Bhuvaneswari Ramaswamy. Unlocking Her2 as a target in invasive lobular cancer and endocrine resistant invasive ductal carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-22.

AKT Inhibition Relieves Feedback Suppression of Receptor Tyrosine Kinase Expression and Activity

Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² > 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [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 PO3-13-11.

Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin's lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery, and is non-competitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ∼15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors. Citation Format: Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2015-LB-231

Human epidermal growth factor receptor 2 (HER2) is an established therapeutic target in multiple solid tumors, particularly breast and gastric cancers. Significant advancements have been made in the development of HER2-targeted therapies, including monoclonal antibodies, tyrosine kinase inhibitors, antibody-drug conjugates (ADC), and novel bispecific antibodies. These agents have revolutionized the treatment landscape for HER2-positive metastatic cancers, resulting in improved progression-free and overall survival, and quality of life for patients. Beyond breast and gastric cancers, HER2 expression/amplification has been observed in other solid tumors, such as colorectal, lung, bladder, ovarian, and biliary tract cancers, offering new opportunities for personalized therapy in a larger patient population. In this review, we highlight the current state of HER2-targeted treatment strategies across HER2-expressing solid tumors, discussing the clinical efficacy, adverse event profiles, and challenges of existing therapies. We explore emerging treatment approaches, including novel agents such as HER2-targeting ADCs, combination therapies, and strategies to overcome resistance mechanisms. Additionally, we examine the role of HER2 expression heterogeneity, biomarker-driven patient selection, and diagnostic tools for patient selection and optimization of treatment outcomes. This review also investigates ongoing challenges in expanding HER2-targeted therapies, including addressing intrinsic and acquired resistance and tailoring strategies to low HER2-expressing or HER2-mutant tumors. Lastly, we provide insights into future directions, emphasizing the importance of precision oncology to broaden the therapeutic opportunities of HER2-targeted therapies across diverse HER2-driven malignancies.

Background: Expression of p95HER2, a truncated form of HER2 lacking the extracellular domain, has been associated with resistance to trastuzumab (T)-based therapy in metastatic breast cancer patients. Conversely, high levels of HER2 have been correlated with increased clinical benefit from anti-HER2 therapy in the neoadjuvant setting. In this work we correlated the expression of p95HER2 and HER2 with pathological complete response (pCR) following T, lapatinib (L) or the combination of both agents (T+L) with paclitaxel. Methods: p95HER2 and HER2 were quantified by VeraTag® and HERmark® (Monogram Biosciences), respectively, in primary tumors of 455 patients enrolled in the phase III neoadjuvant study NeoALTTO (Baselga J. et al. Lancet, 2012). The relationship of pCR status to p95HER2 and HER2 was studied using logistic regression models, which accounted for stratification factors and treatment. Unless specified, p95HER2 and HER2 were included as log terms. Results: p95HER2 was measurable in 283 cases (62%) and HER2 in 327 cases (72%). A positive correlation between p95HER2 and HER2 levels was found in the 274 cases (60%) where quantification of both markers was available. Increased HER2 was strongly associated with increased pCR rate in patients treated with the combination of T+L (OR 5.09, 95%CI 2.27-11.44; p<0.01), with a trend observed also in the L-only arm (OR 1.88, 95% CI 0.96-3.70; p = 0.067). Overall, patients with tumors that were HER2-positive (>17.8 RF/mm2) by HERmark had a higher pCR rate than those that were HER2-negative by HERmark (39% vs. 11%, respectively; p<0.001). Increasing p95HER2 levels did not predict for pCR in the L or T+L arms and showed weak evidence (p = 0.073) of an increase in pCR in the T arm. In an unplanned analysis, we examined the odds of achieving response to anti-HER2 therapy in patients with HER2 levels above and below the median (100 RF/mm2, HER2 entered as a binary covariate). HER2 levels above the median predicted a higher response rate to T (OR 3.6, 95% CI 1.2-11; p<0.05) and, more significantly, to T+L (OR 6.01, 95% CI 2.51-14.4; p<0.001). In particular, patients treated with T+L had a higher probability to achieve pCR compared to T alone when HER2 was above the median (T+L = 73% pCR vs. T = 43% pCR, p<0.01; OR 3.74, 95% CI 1.57-8.90), but not when HER2 was below the median (T+L = 29% pCR vs. T = 19% pCR, p>0.2; OR 1.84, 95% CI 0.74-4.55). When tumors were divided in hormone receptor (HR)-positive and HR-negative groups, total levels of HER2 still predicted response to T or T+L. Conclusions: Increasing HER2 protein expression correlated with increased benefit of adding L to T compared to T alone. In tumors above the median of HER2 expression, the levels of HER2 predicted for response to both T and T+L. Our interpretation is that, in the neoadjuvant setting, the association between p95HER2 expression and response to anti-HER2 therapy is likely a consequence of the correlation between p95HER2 and total HER2 levels. HER2 expression seems to be a stronger predictor of pCR than p95HER2 for response to T, L and especially, T+L. Future studies to understand the impact of p95HER2 and HER2 expression on disease-free and overall survival following anti-HER2 therapy are warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-42.

The clinical significance of human epidermal growth factor receptor 2 (HER2) low and HER2(0) expression in hormone receptor-positive (HR+) breast cancer patients remains uncertain. This study aimed to explore the clinical and pathological characteristics, prognosis, and endocrine therapy (ET) sensitivity among HR+ breast cancer patients with HER2 low and HER2(0) expression. We conducted a retrospective analysis of 390 HR+, HER2-negative breast cancer patients who underwent radical surgery at The First Affiliated Hospital of Bengbu Medical University between December 2014 and December 2017. HER2 status was classified per ASCO/CAP 2018 guidelines: tumors with an immunohistochemistry (IHC) score of 0 were defined as HER2(0), and those with a score of 1+ or 2+ with negative FISH for ERBB2 gene amplification were categorized as HER2 low. Clinical and pathological characteristics, ET sensitivity, and survival outcomes were analyzed. Primary endpoints included disease-free survival (DFS) and overall survival (OS), with ROC curve analysis employed to determine prognostic thresholds. Of the 390 HR+ breast cancer patients, 32.6% had HER2(0) expression [HER2(0) cohort] and 67.4% had HER2 low expression (HER2-low cohort). Most baseline characteristics were balanced between the two cohorts, and the HER2(0) cohort had significantly worse DFS and OS than the HER2-low cohort (both P<0.05). Within the same pN stage (pN0-2), patients with HER2 low expression had a better prognosis (all P<0.05). But at pN3, patients had worse survival outcomes (P=0.003). Patients with the IHC score >4 had significantly better OS (P<0.001) and DFS (P=0.003). No significant difference in OS and DFS was observed in the IHC score ≤4 group between cohorts (both P>0.05), whereas in the IHC score >4 group, the HER2-low cohort had better OS (P<0.001) and DFS (P=0.01). Patients with HER2 low expression have distinct clinical characteristics and a better prognosis compared to those with HER2(0) expression. Our study provides further real-world evidence for personalized treatment of breast cancer patients.

HER2-positive breast cancer is characterized by overexpression of the HER2 receptor, driving aggressive tumor growth. Antibody-drug conjugates (ADCs), like trastuzumab emtansine (Kadcyla, T-DM1) and trastuzumab deruxtecan (Enhertu, T-DXd), are effective in targeting HER2-positive tumors, though their efficacy across varying HER2 levels remains under study. This study compares Kadcyla and Enhertu regarding cell proliferation, ADC internalization, and bystander cytotoxicity in cancer cell lines with high (SK-BR-3/SKOV-3), moderate (JIMT-1), and low (MDA-MB-231/MDA-MB-435) HER2 expression. It also examines off-site ADC toxicity in primary non-tumor cells caused by off-target payload delivery. The xCELLigence system, which works via label-free impedance measurements on living cells, was employed to monitor real-time cell proliferation and viability. Both, Kadcyla and Enhertu, eliminate tumor cells, effectively at nanomolar concentrations, correlating with HER2 expression levels. Interestingly, Kadcyla's effect appears at least 24 hours earlier than Enhertu, whereas the unconjugated antibody Trastuzumab itself has little to no effect on tumor cell growth. Low HER2-expressing cells were killed at 1 µM, the highest dose tested. ADC internalization was analyzed using pH-sensitive, fluorescently labeled Trastuzumab in flow cytometry and microscopy, assessing uptake by mean fluorescence intensity (MFI) in viable cells. This analysis allowed the comparison of the rate and extent of Trastuzumab uptake across the tested cell lines. A co-culture system evaluated bystander effects, with high HER2-expressing SKOV-3/SK-BR-3 cells co-cultured with luciferase-labeled HER2-negative LN-229 cells. After treatment with Kadcyla or Enhertu, luciferase assays were conducted to assess whether the cytotoxic payload released by the ADCs in HER2-positive cells could affect the viability of neighboring HER2-negative cells. Enhertu demonstrated a bystander effect, whereas Kadcyla, on the other hand, showed a more limited activity, reflecting the non-cleavable linker and the membrane-impermeable nature of its payload DM1. Toxicity analyses of Kadcyla and Enhertu on non-tumor cells, and especially on bone marrow, complement on-target investigations by revealing critical results about their safety profiles. While these ADCs are designed to selectively target tumor cells, off-site toxicity can affect healthy tissues and impair immune system function. Studies on hematopoietic stem cells are essential to assess potential adverse effects on hematopoietic regeneration and differentiation, aiming to reduce unintended immunosuppression. These findings support the optimization of ADC design, to enhance both selectivity and therapeutic windows. Citation Format: Veronica Bergo, Philipp Metzger, Valerie Oberhardt, Jan Eric Ehlert, Holger Weber. In vitro comparison of Kadcyla and Enhertu in breast cancer with varying HER2 expression: proliferation, internalization, bystander effects and toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2891.

109 Background: Peptide vaccines comprised of HLA class II epitopes, which elicit CD4+ T cell responses, play a critical role in potentiating immune responses. We are conducting a randomized phase II trial of AE37, a hybrid peptide created by the addition of the Ii-Key moiety (LRMK) to the HER2 helper epitope, AE36 (HER2 aa776-790). Here, we present efficacy data focusing on outcomes in patients with low HER2 (IHC 1+ or 2+) expression and triple negative breast cancer (TNBC). Methods: The trial is enrolling node positive or high risk node negative breast cancer patients with any degree of HER2 expression (IHC 1+, 2+ or 3+ or FISH &gt; 1.2) rendered disease-free following standard of care therapy. Patients are randomized to receive either AE37+GM-CSF or GM-CSF alone in 6 monthly intradermal inoculations followed by booster inoculations administered every 6 months. Results: The trial has enrolled 254 patients; 105 in the vaccine group (VG) and 149 in the control group (CG). After a median follow-up of 22.3 months, the disease-free survival (DFS) rate in the VG is 90.3% vs 81.1% in the CG (p=.46), a 49% risk reduction. Evaluating patients with low HER2 expression (IHC 1+ or 2+), there are 53 VG patients and 77 CG patients. The groups are well-matched with respect to the percentage of patients with high grade tumors, tumors &gt; 2cm, the rate of node positivity and ER/PR status (all p&gt;.5). The DFS rate in the VG of low HER2 expressers is 89.8% vs 68.2% in the CG (p=.12), a 68% risk reduction. When limiting analyses to patients with TNBC (ER/PR negative, HER2 1+ or 2+), there are 13 VG patients and 23 CG patients. The groups are again well-matched with the exception of control patients having a larger percentage of tumors &gt; 2 cm (70% vs 31%; p=.02). The DFS rate in the VG of TNBC patients is 83.3% vs 47.6% in the CG (p=.23), a 68% risk reduction. Conclusions: Early analyses suggest clinical benefit to vaccination with AE37, particularly in patients with low HER2-expressing tumors. Importantly, the benefit appears to persist in TNBC patients. Patients will continue to be followed per protocol for 5 years; however, these data suggest that a subsequent phase III trial should evaluate the vaccine in patients with low HER2-expressing disease to include TNBC.

To investigate inhibitory activities of a homogenous anti-human epidermal growth factor receptor 2 (HER2)-antibody drug conjugate (ADC) on the proliferation of nine tumor cell lines with different levels of HER2 expressions, and its activities on the tumor growth of five xenograft mouse models. The HER2 expression levels of BT-474, Calu-3, MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, SK-OV-3, HCC1954, NCI-N87 tumor cell lines were measured using QIFI KIT. For the in vitro anti-proliferation assay, serial diluted anti-HER2-ADC, ado-trastuzumab emtansine, AS269, pAF-AS269 and paclitaxel were added to the seeded cells, and after 72 or 96 hours of incubation, the cell proliferation was analyzed. For the in vivo activity, 5-6 weeks old mice were inoculated with four HER2 positive tumor cell lines HCC1954, BT-474, SK-OV-3, NCI-N87 or one HER2 negative tumor cell line MDA-MB-468. Different amounts of anti-HER2-ADC, ado-trastuzumab emtansine, trastuzumab, paclitaxel and phosphate buffered saline control were injected after the tumor volume reached a certain size, then the tumor growth inhibition was analyzed. The expression levels of the six high HER2-expression cell lines SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 were between 430 000 to 800 000 receptors per cell, which were 50 times higher than those of the other three low HER2 expression tumor cell lines MDA-MB-231, MCF-7, MDA-MB-468. Anti-HER2-ADC had inhibition effects on cell lines with high level of HER2 expression in the in vitro anti-proliferation assay. The half maximal inhibitory concentrations of anti-HER2-ADC on SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 tumor cell lines were 46 pmol/L, 17 pmol/L, 17 pmol/L, 161 pmol/L, 125 pmol/L, 50 pmol/L, respectively. Anti-HER2-ADC had a dose dependent antitumor activity in vivo in all the HER2 positive xenograft mouse models. In NCI-N87 xenograft tumor model, the same dose of anti-HER2-ADC showed better anti-tumor activity compared with trastuzumab and ado-trastuzumab emtansine, and its relative tumor proliferation rates were about 1/30 to 1/20 of the two. In HCC1954 xenograft tumor model, the complete regression of the tumor was observed. As expected, anti-HER2-ADC had no tumor inhibitory effects on MDA-MB-468 xenograft models with low HER2 expression. The antitumor activities of anti-HER2-ADC in HER2 positive xenograft tumor models were the same as or better than the activities of ado-trastuzumab emtansine. The homogenous site-specific anti-HER2-ADC obtained using unnatural amino acid technology can inhibit the growth of high HER2-expression tumor cells with high potency both in vivo and in vitro.

Background: 15-20% of metastatic breast cancers are characterized by overexpression or amplification of human epidermal growth factor receptor 2 (HER2). First-line treatment for HER2-positive metastatic breast cancer includes the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab in combination with chemotherapy. Second-line therapy includes T-DM1, an antibody-drug conjugate (ADC) consisting of trastuzumab conjugated to maytansinoid (DM1). Until recently, patients who progress on T-DM1 were left with few treatment options. Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) consisting of an anti-HER2 (human epidermal growth factor receptor 2) antibody linked to a novel topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker and was recently approved for unresectable or T-DM1-refractory HER2+ breast cancer. In addition, DS-8201a is currently being evaluated as a treatment in breast cancers with low and medium expression of HER2. To better understand the potential for DS-8201a as a treatment in these cancer types, we evaluated this therapy in a panel of ER+/- XPDX models with differential HER2 expression. In addition, we compared DS-8201a activity to T-DM1 and benchmarked efficacy of the topoisomerase I inhibitor irinotecan in all tested models. Methods: One hundred (50ER+/50ER-) breast XPDX models were evaluated in this study. Models were established and characterized for estrogen receptor and HER2 expression by IHC and profiled using WES and RNAseq. For in vivo studies, DS-8201a and T-DM1 were administered IV at 3 mg/kg and irinotecan IP at 100 mg/kg on a weeklyx3 schedule. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Models were grouped by ER positivity (+/-) and model HER2 score and designated as null (0), low (1+), medium (2+) or high (3+); models established from clinically HER2+ patients were also noted. In the ER+ group, HER2 low and medium expressing models accounted for over 80% of the total, while 15% were categorized as high and less than 5% as null. All models with high and 30% with medium HER2 staining were from clinically HER2+ patients, no low or null models were established from breast cancer assigned HER2+ clinically. In the ER- group, HER2 low and medium expressing models accounted for 75% of the total, while 12% were categorized as high and 13% as null. All models with high and 13% with medium HER2 staining were from clinically HER2+ patients; 5% of low or null models were established from breast cancer assigned HER2+ clinically. In vivo, 65% of the ER+ group reported sensitivity to DS-8201a versus 25% to T-DM1; 10% of DS-8201a, and 40% of T-DM1 sensitive models were high expression models and the remaining low and medium with no null models reporting sensitivity to either agent. 35% of tested ER+ models were sensitive to both HER2 therapies and 50% of models sensitive to DS-8201a were also sensitive to irinotecan. In the ER- group, 70% reported sensitivity to DS-8201a versus 25% to T-DM1; 20% of DS-8201a and 25% of T-DM1 sensitive models were high expression models and the remaining low and medium with one null model (ST069) sensitive to both agents. 25% of tested ER- models were sensitive to both HER2 therapies and 40% of models sensitive to DS-8201a were also sensitive to irinotecan. Conclusion: We have compared activity of DS-8201a, T-DM1 and irinotecan in a panel of ER+/- XPDX models and correlated activity based on HER2 expression. This data and panel can be utilized as a valuable tool in better understanding the potential for DS-8201a and other HER2-targeting therapies as a treatment in cancers driven by HER2 expression. Citation Format: Alyssa Simonson, Peter Forofontov, Johnnie R Flores, Kimberly Hernandez, April Cabang, Amy Lang, Gladys Rodriguez, Kyriakos Papadopoulos, Murali Beeram, Arthur Rosenthal, Brittany DeBerry, Lon Smith, Ronald Drengler, Amita Patnaik, Drew Rasco, Luis Rodriguez, Steven Abbate, Scott Ulmer, Michael Wick. Correlation of HER2 receptor expression and in vivo activity of the HER2-targeting therapies trastuzumab deruxtecan (DS-8201a) and T-DM1 activity in a panel of breast XPDX models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-04.

Discerning subsets of breast cancer with very low and absent HER2 protein expression