HER2 Blockade Research Articles

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive 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 PO1-25-08.

Abstract Db1-01: Anthracyclines …to give or not to give? “For” argument

Abstract Anthracyclines have been one of the mainstays of early breast cancer treatment for decades. In a recent patient-level meta-analysis including data from over 18,000 patients, anthracycline-containing regimens were associated with improvements in disease-free survival and overall survival, regardless of estrogen receptor or nodal status. Importantly, trials specifically designed and powered to assess the safety of anthracyclines omission failed to demonstrate the non-inferiority of taxane-based anthracycline-free regimens and confirmed the benefit of these agents, particularly in patients with node-positive and hormone receptor-negative breast cancer. Several therapies incorporated into the management of early breast cancer were studied in populations largely treated with anthracycline-based chemotherapy backbones in both HER2-negative (i.e., perioperative pembrolizumab, post neoadjuvant olaparib and capecitabine) and HER2-positive disease (i.e., dual HER2 blockade, adjuvant T-DM1). It is uncertain whether the benefits demonstrated by these strategies would be maintained if anthracyclines were omitted. While anthracyclines are associated with some long-term toxicities, particularly with an increased risk of lymphoproliferative malignancies and dose-dependent cardiotoxicity, these adverse events are not associated with an increased risk of death (including deaths from cardiovascular causes) when compared to anthracycline-free regimens. The identification of risk factors for cardiotoxicity, combined with improvements in monitoring strategies, allows improvements in patient selection, early detection of myocardial damage and, possibly, implementation of cardioprotective interventions, leading to improved cardiovascular outcomes. Until high-quality evidence demonstrates the safety of omitting anthracyclines in high-risk populations, these agents should continue to be used in carefully selected patients after individual risk versus benefit assessment to be shared with the patient. Citation Format: Martine Piccart, Guilherme Nader-Marta. Anthracyclines …to give or not to give? “For” argument [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 Db1-01.

Abstract PO2-01-07: Real-world efficacy of dual HER2-blockade in combination with anthracycline-containing and anthracycline-free neoadjuvant treatment in early breast cancer (BC)

Abstract BACKGROUND: Neoadjuvant therapy with antiHER-2 blockade is the standard of care for HER-2 positive early BC. The objective of this treatment is tumor downstaging to increase resectability and pathological complete response (pCR), a reliable surrogate endpoint for survival. Adding Trastuzumab (T) to chemotherapy resulted in an improvement in pCR, however, resistance to this agent is common and the incorporation of Pertuzumab (P) to the neoadjuvant scene demonstrated an increase in pCR in the Peony (39.3% vs 21.8%) and Neosphere trials (45.8% vs 29%). Across all studies, pCR is even higher in patients (pts) with negative hormonal receptor (HR) status (50%-63%) (3, 4). Tryphaena and Berenice found low rates of cardiotoxicity with T-P combination. Recently, TRAIN-2 data showed that anthracyclines and platinum have a similar pCR (67% vs 68%), but febrile neutropenia and cardiotoxicity is higher in the anthracycline group, concluding that omitting this therapy might be a preferred approach in the presence of dual HER2 blockade. Our study investigated the pCR benefit of dual HER2 blockade with anthracyclines and platinum in HER2 positive early BC. METHODS: We conducted a retrospective, unicenter, observational study of pts, treated with neoadjuvant T-P plus chemotherapy, in HER-2 positive early BC, between April 2015 and December 2022 at University Hospital A Coruña (Spain). The primary endpoint was total pCR in breast and axila (tpCR: ypT0/is ypN0). Secondary endpoints were: association between clinicopatological characteristics and pCR; and between cardiotoxicity and treatment pattern. Statistical analyses were conducted with Statistical Package for the Social Science (SPSS). RESULTS: During the study period, from April 2015 to May 2022, 154 pts were enrrolled. The median age was 51,3 years [range 26 – 799 years], all patients were female. Infiltrating ductal carcinoma was present in 92.2% pts, and 5.8% were inflammatory BC. At diagnosis, 11.7%, 61.7% and 26.6% had stages I, II and III, respectively, and 51.3% revealed axillary node involvement. Among observed cases, 94 (61%) were positive HR; 68 (45%) had histological grade 2 and 83 (53.9%) histological grade 3 tumors; and 151 pts (99.3%) presented a ki-67 level >20%. HER2 by immunohistochemistry (IHC) resulted in 3+ for 133 pts (86.9%) and in 2+ for 20 pts (13.1%), all of them with positive result by in situ hybridation (ISH). P-T were mostly combined with anthracyclines (64.9%). tpCR rate was 50.6%, and it was not significantly different according chemotherapy approach. tpCR was significantly higher among HR negative tumours (73.3%) compared with HR positive (36.2%) (p= 0.001), and in HER2 positive 3+ (55.6%) compared with HER2 positive 2+ (15%) tumours (p= 0.001). Achievement of tpCR was not significantly associated with tumor size, stage, histological grade and ki-67 level. Breast pCR was higher than tpCR (54.2%). 13 pts (8.4%) presented recurrence disease, in 76.9% of cases was a metastatic relapse. Cardiotoxicity was low during all stages of treatment and was not significantly associated with chemotherapy pattern. CONCLUSIONS: Neoadjuvant T-P plus chemotherapy with either anthracyclines or platinum therapy, demonstrated same efficacy and cardiac safety in our real-world study. pCR rates are lower in positive HR and HER2 positive 2+, needing more investigation with combination strategies including hormonotherapy, Our results are similar to data from randomized trials and other real-world efficacy studies, but we need more follow-up to identify late cardiotoxicity by anthracyclines and recurrences in positive HR tumors. Citation Format: Beatriz Alonso de Castro, Cristina Reboredo, Lourdes Calvo, Maria-Eva Perez-Lopez, Martin Igor Gomez Randulfe Rodriguez, Sofia silva diaz, Iria Parajo Vazquez, Silvia Antolin Novoa. Real-world efficacy of dual HER2-blockade in combination with anthracycline-containing and anthracycline-free neoadjuvant treatment in early breast cancer (BC) [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 PO2-01-07.

Abstract PO1-27-05: De-escalated neoadjuvant weekly nab-paclitaxel with trastuzumab and pertuzumab in HER2-positive early breast cancer (HELEN-006):a randomized,phase 3 trial

Abstract Background: Chemotherapy de-escalation is currently under investigation as a treatment approach for HER2-positive early-stage breast cancer(EBC). We conducted a phase 3 clinical trial to evaluate the effectiveness of a weekly regimen of nab-paclitaxel monotherapy in combination with dual HER2 blockade for HER2-positive EBC. Methods: We conducted a multicenter, randomized, open-label Phase 3 trial at six hospitals in China. Eligible participants were women aged 18-70 with locally confirmed, HER2-positive, stage II-III, invasive, operable breast cancer and an Eastern Cooperative Oncology Group performance status of 0 or 1. Participants were randomly assigned (1:1) using central block randomization, with block sizes of four, stratified by hormone receptor status, tumor stage, and node status. They were assigned to receive either intravenous nab-paclitaxel (125mg/m2 on days 1, 8, and 15) for six 3-week cycles (group A), or intravenous docetaxel (75mg/m² on day 1) and carboplatin (area under the concentration-time curve 6mg/mL per min on day 1) for six 3-week cycles (group B). Both groups received concurrent trastuzumab (8mg/m2 loading dose, 6mg/m2 maintenance dose on day 1) and pertuzumab (intravenous 840mg loading dose, 420mg maintenance dose on day 1). The primary endpoint was the proportion of patients achieving a pathological complete response(pCR) in the breast and axilla (ypT0/is ypN0) as determined by a local pathologist after surgery, assessed using a modified intention-to-treat analysis. To test for non-inferiority, we used a closed test procedure, considering the nab-paclitaxel group non-inferior to the docetaxel plus carboplatin group if the lower 95% confidence interval(CI) for the odds ratio (OR) was above 0.802, which accounted for a 5.5% non-inferiority margin (10% of the 55% pCR rate for docetaxel plus carboplatin and dual HER2 blockade regimen). We planned to test for superiority only if the non-inferiority test was positive, using an alpha value of 0.05. Safety was assessed in all patients who received the study drug. The trial is registered with ClinicalTrials.gov, with the registration number NCT04547907. Findings: Between September 10, 2020, and March 1, 2023, we randomly assigned 689 women, of whom 669 started treatment (332 with nab-paclitaxel and 337 with docetaxel plus carboplatin). The nab-paclitaxel group had a higher proportion of patients achieving a pCR (220 [66.3%, 95% CI 61.2-71.4]) compared to the docetaxel plus carboplatin group (194 [57.6%, 52.3-62.9]). The OR for pCR was 1.448 (95% CI 1.058-1.981), indicating a statistically significant difference (unadjusted p=0.021). Overall, 228 (34.1%) patients were noted to have at least one grade 3 or 4 event, 100(30.1%) in the nab-paclitaxel group and 128 (38.0%) in the docetaxel plus carboplatin group (P=0·032). The most common grade 3 or 4 events overall were nausea (98[14.6%] of 669 patients), diarrhea (80 [12.0%]), and neuropathy (51 [7.6%]).However, no treatment-associated deaths were identified. Conclusions: In neoadjuvant treatment of HER2-positive EBC, 18 weeks of nab-paclitaxel monotherapy combined with dual HER2 blockade has shown significantly higher pCR rates and improved tolerability compared to the standard docetaxel plus carboplatin and dual HER2 blockade regimen. These findings could potentially reshape preferences for neoadjuvant therapy in HER2-positive EBC. Patient Characteristics pCR by clinical-pathological variables Abbreviation:Nab-PHP,nanoparticle albumin-bound paclitaxel plus trastuzumab and pertuzumab; TCbHP,docetaxel plus carboplatin ,trastuzumab and pertuzumab; HR,hormone receptor; IHC,immunohistochemistry. *estrogen and/or progesterone receptor positive is classified as hormone receptor positive;estrogen and progesterone receptor negative is classified as hormone receptor negative Adverse events Abbreviation:Nab-PHP,nanoparticle albumin-bound paclitaxel plus trastuzumab and pertuzumab; TCbHP,docetaxel plus carboplatin ,trastuzumab and pertuzumab;ALT,alanine aminotransferase;AST,aspartate transaminase. Citation Format: Zhenzhen Liu, Xiuchun Chen, Jianghua Qiao, Jiao Dechuang, Chengzheng Wang, Xianfu Sun, Zhenduo Lu, Lianfang Li, Chongjian Zhang, Min Yan, Ya Wei, Chen Bo, Yueqing Feng, Miao Deng, Mingde Ma. De-escalated neoadjuvant weekly nab-paclitaxel with trastuzumab and pertuzumab in HER2-positive early breast cancer (HELEN-006):a randomized,phase 3 trial [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 PO1-27-05.

Neoadjuvant–adjuvant pertuzumab in HER2-positive early breast cancer: final analysis of the randomized phase III PEONY trial

The randomized, multicenter, double-blind, placebo-controlled, phase III PEONY trial (NCT02586025) demonstrated significantly improved total pathologic complete response (primary endpoint) with dual HER2 blockade in HER2-positive early/locally advanced breast cancer, as previously reported. Here, we present the final, long-term efficacy (secondary endpoints: event-free survival, disease-free survival, overall survival) and safety analysis (62.9 months’ median follow-up). Patients (female; n = 329; randomized 2:1) received neoadjuvant pertuzumab/placebo with trastuzumab and docetaxel, followed by adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, then pertuzumab/placebo with trastuzumab until disease recurrence or unacceptable toxicity, for up to 1 year. Five-year event-free survival estimates are 84.8% with pertuzumab and 73.7% with placebo (hazard ratio 0.53; 95% confidence interval 0.32–0.89); 5-year disease-free survival rates are 86.0% and 75.0%, respectively (hazard ratio 0.52; 95% confidence interval 0.30–0.88). Safety data are consistent with the known pertuzumab safety profile and generally comparable between arms, except for diarrhea. Limitations include the lack of ado-trastuzumab emtansine as an option for patients with residual disease and the descriptive nature of the secondary, long-term efficacy endpoints. PEONY confirms the positive benefit:risk ratio of neoadjuvant/adjuvant pertuzumab, trastuzumab, and docetaxel treatment in this patient population.

Correlation between trophoblast cell-surface antigen-2 (Trop-2) expression and pathological complete response in patients with HER2-positive early breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab.

The prognostic and predictive role of trophoblast cell-surface antigen-2 (Trop-2) overexpression in human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer is currently unknown. We retrospectively analyzed Trop-2 expression and its correlation with clinicopathologic features and pathological complete response (pCR) in HER2-positive early breast cancer (EBC) patients treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab in the PHERGain study. Trop-2 expression at baseline was determined in formalin-fixed, paraffin-embedded primary tumor biopsies by immunohistochemistry and was first classified into expressing (Trop-2-positive) or not-expressing (Trop-2-negative) tumors. Then, it was classified by histochemical score (H-score) according to its intensity into low (0-9), intermediate (10-49), and high (≥ 50). The association between clinicopathologic features, pCR, and Trop-2 expression was performed with Fisher's exact test. Forty-one patients with tissue evaluable for Trop-2 expression were included, with 28 (68.3%) Trop-2-positive tumors. Overall, 17 (41.46%), 14 (34.15%), and 10 (24.40%) tumors were classified as low, intermediate, and high, respectively. Trop-2 expression was significantly associated with decreased pCR rates (50.0% vs. 92.3%; odds ratio [OR] 0.05; 95% CI, 0.002-0.360]; p adjusted = 0.01) but was not correlated with any clinicopathologic features (p ≥ 0.05). Tumors with the highest Trop-2H-score were less likely to obtain a pCR (OR 0.03; 95% CI, 0.001-0.290, p adjusted < 0.01). This association was confirmed in univariate and multivariate regression analyses. These findings suggest a potential role of Trop-2 expression as a biomarker of resistance to neoadjuvant chemotherapy plus dual HER2 blockade and may become a strategic target for future combinations in HER2-positive EBC patients.

Short-term efficacy and safety of neoadjuvant pyrotinib plus taxanes for early HER2-positive breast cancer: a single-arm exploratory phase II trial.

The effectiveness and safety of pyrotinib have been substantiated in human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer (BC). However, the role of pyrotinib as a single HER2 blockade in neoadjuvant setting among BC patients has not been studied. The objective of this study was to evaluate the efficacy and tolerability of pyrotinib plus taxanes as a novel neoadjuvant regimen in patients with HER2-positive early or locally advanced BC. In this single-arm exploratory phase II trial, patients with treatment-naïve HER2-positive BC (stage IIA-IIIC) received pyrotinib 400 mg once daily and taxanes [docetaxel 75 mg/m2 or nanoparticle albumin-bound (nab)-paclitaxel 260 mg/m2 every 3 weeks, or paclitaxel 80 mg/m2 weekly] for a total of four 21-day cycles before surgery. Efficacy assessment was based on pathological and clinical measurements. The primary endpoint of this study was the total pathological complete response (tpCR) rate. The secondary endpoints included breast pCR (bpCR) rate, investigator-assessed objective response rate (ORR) and adverse events (AEs) profiles. From 1 September 2021 to 30 December 2022, a total of 31 patients were enrolled. One patient was withdrawn due to unbearable skin rash after the second cycle of neoadjuvant therapy. The majority of the intention-to-treat (ITT) population was premenopausal (54.8%), had large tumors (90.3%) and metastatic nodes (58.1%) at diagnosis and hormone-receptor positive tumors (64.5%). Most participants used nab-paclitaxel (74.2%) and received mastectomy (67.7%) after neoadjuvant treatment. The tpCR and bpCR rates were 48.4% [95% confidence interval (CI): 30.8-66%] and 51.6% (95% CI: 34-69.2%), respectively. Grade ≥3 treatment-related AEs were observed in 16.1% (5/31) of the ITT population, including diarrhea (n=2, 6.5%), hand and foot numbness (n=1, 3.2%), loss of appetite (n=1, 3.2%), and skin rash (n=1, 3.2%). AE related dose reduction or pyrotinib interruption was not required. In female patients with HER2-positive non-metastatic BC, neoadjuvant pyrotinib monotherapy plus taxanes appears to show promising clinical benefit and controllable AEs [Chinese Clinical Trial Registry (ChiCTR2100050870)]. The long-term efficacy and safety of this regime warrant further verification.

263 (PB-079) Poster - Multiomic profiling reveals predictive molecular characteristics of response to neoadjuvant antibody-drug conjugate versus chemotherapy and dual HER2 blockade in HER2 positive breast cancer

263 (PB-079) Poster - Multiomic profiling reveals predictive molecular characteristics of response to neoadjuvant antibody-drug conjugate versus chemotherapy and dual HER2 blockade in HER2 positive breast cancer

Anti-HER2 therapy response assessment for guiding treatment (de-)escalation in early HER2-positive breast cancer using a novel deep learning radiomics model

ObjectivesAnti-HER2 targeted therapy significantly reduces risk of relapse in HER2 + breast cancer. New measures are needed for a precise risk stratification to guide (de-)escalation of anti-HER2 strategy.MethodsA total of 726 HER2 + cases who received no/single/dual anti-HER2 targeted therapies were split into three respective cohorts. A deep learning model (DeepTEPP) based on preoperative breast magnetic resonance (MR) was developed. Patients were scored and categorized into low-, moderate-, and high-risk groups. Recurrence-free survival (RFS) was compared in patients with different risk groups according to the anti-HER2 treatment they received, to validate the value of DeepTEPP in predicting treatment efficacy and guiding anti-HER2 strategy.ResultsDeepTEPP was capable of risk stratification and guiding anti-HER2 treatment strategy: DeepTEPP-Low patients (60.5%) did not derive significant RFS benefit from trastuzumab (p = 0.144), proposing an anti-HER2 de-escalation. DeepTEPP-Moderate patients (19.8%) significantly benefited from trastuzumab (p = 0.048), but did not obtain additional improvements from pertuzumab (p = 0.125). DeepTEPP-High patients (19.7%) significantly benefited from dual HER2 blockade (p = 0.045), suggesting an anti-HER2 escalation.ConclusionsDeepTEPP represents a pioneering MR-based deep learning model that enables the non-invasive prediction of adjuvant anti-HER2 effectiveness, thereby providing valuable guidance for anti-HER2 (de-)escalation strategies. DeepTEPP provides an important reference for choosing the appropriate individualized treatment in HER2 + breast cancer patients, warranting prospective validation.Clinical relevance statementWe built an MR-based deep learning model DeepTEPP, which enables the non-invasive prediction of adjuvant anti-HER2 effectiveness, thus guiding anti-HER2 (de-)escalation strategies in early HER2-positive breast cancer patients.Key Points• DeepTEPP is able to predict anti-HER2 effectiveness and to guide treatment (de-)escalation.• DeepTEPP demonstrated an impressive prognostic efficacy for recurrence-free survival and overall survival.• To our knowledge, this is one of the very few, also the largest study to test the efficacy of a deep learning model extracted from breast MR images on HER2-positive breast cancer survival and anti-HER2 therapy effectiveness prediction.

Abstract IA21: Mechanisms of Resistance to HER2-Targeted Therapies in HER2-positive Breast Cancer

Abstract Recent advancements in HER2-targeted therapies for HER2+ BC have revolutionized patient outcome. Yet, intrinsic and acquired resistance, especially in the advanced setting remains a clinical challenge. In the era of precision oncology, better understanding of these mechanisms of resistance is key to identifying new strategies to overcome resistance and for developing personalized treatment approaches. Our recent studies suggest that a subset of HER2+ BCs, associated with high and homogeneous levels of HER2 gene amplification, protein, and activity, are addicted to HER2 and may therefore benefit from chemotherapy-sparing HER2-targeted regimens. However, even in such tumors, constitutive activation of the PI3K/AKT pathway due to deregulations in the downstream PI3K pathway can lead to resistance. In all HER2+ BCs, failure to achieve a comprehensive blockade of the HER receptor layer is one of the major mechanisms of resistance due to the functional redundancy of the HER receptors and compensatory signaling within the pathway. But, in some cases, effective inhibition of HER receptors might be challenged by molecular masking of the receptors (e.g., MUC4) or by epi/genetic or post-translational alterations in the receptors itself (e.g., HER2 L755S, p95HER2). Indeed, we recently reported that acquisition of HER2 L755S, either alone or together with PIK3CA mutation, and hyperactive EGFR signaling due to acquired EGFR amplification or excess HER ligands are associated with resistance to HER2-targeted therapy, especially tyrosine kinase inhibitors. Likewise, clinically, HER mutations have been reported to be further enriched in the metastatic vs. primary HER2+ tumors. When HER2 does remain sustainably inhibited by HER2-targeted agents, resistance may arise due to the emergence of alternative signaling pathways transmitting compensatory proliferative and survival signals, such as ER, receptor tyrosine kinases, or downstream/intracellular signaling. Further, the emergence of metabolic pathways as alternative pathways of resistance have also been documented. We recently reported the activation of the cholesterol biosynthetic mevalonate (MVA) pathway as an escape mechanism that provides alternative signals through the YAP/TAZ-mTORC1-survivin axis to evade HER2 blockade. This resistance could be overcome using MVA pathway inhibitors (e.g., statins). Additionally, activation of the cell cycle regulatory complex cyclin D1/CDK4 has been shown to be associated with HER2-targeted therapy resistance, which could be exploited as a potential therapeutic vulnerability by using CDK4/6 inhibitors. Finally, response to HER2-targeted therapy could potentially be also modulated by the tumor microenvironment itself, including components of the host immune system and extracellular matrix. Together, a better understanding of the underlying mechanisms of resistance in the context of tumor and host biology, and treatment setup is fundamental to develop new treatment strategies and to guide patient stratification for personalized treatments and improved patient outcomes. Citation Format: Jamunarani Veeraraghavan, Fu-Tien Liao, Lanfang Qin, Tia Gordon, Alekya Raghavan, Caroline Sabotta, Rachel Kaplan, Carolina Gutierrez, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Mechanisms of Resistance to HER2-Targeted Therapies in HER2-positive Breast Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA21.

Neoadjuvant trastuzumab deruxtecan (T-DXd) with response-directed definitive therapy in early stage HER2-positive breast cancer: a phase II study protocol (SHAMROCK study)

BackgroundThe current standard of care in the neoadjuvant setting for high-risk HER2-positive (HER2 +) breast cancer is to combine systemic chemotherapy with dual HER2 blockade, trastuzumab and pertuzumab. Targeted therapies have significantly improved outcomes for patients with HER2-positive breast cancer. To improve treatment-associated toxicity, chemotherapy-sparing approaches are currently being investigated. Trastuzumab deruxtecan (T-DXd) is an HER2-directed antibody–drug-conjugate (ADC) with promising results in the metastatic setting for HER2-positive breast cancer. The SHAMROCK study investigates neoadjuvant T-DXd in early stage HER2-positive breast cancer, using pathological complete response (pCR) rate as the primary endpoint.MethodsThis is a phase II open-label, single arm, adaptive multi-centre trial of T-DXd in the neoadjuvant setting in stage 2–3 HER2-positive breast cancer. Eligible patients will receive 5.4 mg/kg of T-DXd intravenously every 3 weeks for up to 6 cycles. A repeat biopsy will performed after 2 cycles for the RNA disruption index (RDI) score assessment. According to their likelihood of pCR, as determined by the RDI score, patients will either undergo 4 or 6 cycles of T-DXd prior to imaging. Patients with imaging complete response (iCR) after either 4 or 6 cycles will proceed to surgery. Patients who do not achieve iCR will either undergo further systemic therapy or proceed to surgery.DiscussionThe SHAMROCK study is a chemotherapy-sparing approach to curative intent treatment, investigating neoadjuvant T-DXd. We hypothesise that neoadjuvant T-DXd will have a high pCR rate and be associated low toxicity in early stage HER2-positive breast cancer.Trial registrationEudraCT Number: 2022–002485-32; ClinicalTrials.gov identifier: NCT05710666; Cancer Trials Ireland study number: CTRIAL-IE 22–01.

Pyrotinib plus docetaxel as first-line treatment for HER2-positive metastatic breast cancer: the PANDORA phase II trial

The role of pyrotinib in the treatment of HER2-positive metastatic breast cancer (MBC) has been well-established. This multicenter, single-arm phase II trial (NCT03876587) aimed to assess the benefit of pyrotinib plus docetaxel as a first-line treatment for HER2-positive MBC. Women with HER2-positive MBC who had not undergone HER2 blockade or chemotherapy for metastatic disease were enrolled in the study and received daily oral pyrotinib 400 mg plus intravenous docetaxel 75 mg/m2 every 3 weeks. The primary endpoint was the objective response rate (ORR), secondary endpoints included progression-free survival (PFS), duration of response (DoR), clinical benefit rate (CBR), overall survival (OS) and safety. From June 2019 to June 2021, 79 patients were enrolled. The confirmed ORR was 79.7% (95% confidence interval [CI], 70.8-88.6), and the CBR was 87.3% (95%CI, 80.0-94.6) in the intention-to-treat population. The pre-specified primary endpoint was met. The median DoR was 15.9 months (interquartile range, 8.3-19.5); the median PFS was 16.0 months (95% CI, 11.2-20.8), and the median OS was not reached. The most common grade ≥3 treatment-related adverse events observed were leukopenia (29.1%), neutropenia (27.8%), and diarrhea (21.5%). This study demonstrates that pyrotinib plus docetaxel show an acceptable safety profile and promising antitumor activity as a first-line treatment option for patients with HER2-positive MBC.

Abstract A039: Carnitine palmitoyltransferase IA: an emerging potential metabolic target to counteract HER2-targeted therapy resistance in HER2-positive breast cancer

Abstract HER2 overexpression/amplification (HER2+) occurs in 15-20% of breast cancer (BC) and identifies a clinically aggressive BC subtype. Although the introduction of effective anti-HER2 drugs remarkably improved the prognosis of HER2+ BC patients, primary and acquired tumor resistance to anti-HER2 treatments underscores the need for novel therapies for HER2+ BC patients. Tumor cells exhibit unique metabolic alterations that are responsible for primary or acquired tumor resistance to standard therapies, and which are increasingly emerging as potential targets for novel treatment strategies. In particular, the dysregulation of fatty acid β-oxidation (FAO), a catabolic process that produces energy (ATP and NADPH production) to sustain cancer growth, survival and aggressiveness, is associated with therapy resistance in diverse malignancies including BC. Specifically, carnitine palmitoyltransferase 1A (CPT1A), the key rate-limiting enzyme of mitochondrial FAO, facilitates cancer metabolic adaptation, thus representing a promising target for cancer therapy. In this context, gene set enrichment analysis of profiled matched HER2+ BC samples (n=33) collected before and after trastuzumab-based neo-adjuvant biochemotherapy (trastuzumab plus taxanes) revealed that the Fatty Acids (FA) metabolism gene set was one of the most significantly Hallmark enriched pathway (NES=1.57; FDR=0.0039) in post- compared with pre-treated HER2+ BC samples, implying that trastuzumab plus chemotherapy treatment induces an enhancement of FAO activity. Accordingly, CPT1A transcript levels were found to be inversely associated with lapatinib (L) susceptibility in HER2+ BC cell lines and patient-derived organoids resistant to anti-HER2 therapy versus L-sensitive cell models. Additionally, Seahorse Analysis revealed higher baseline oxidation of the FA palmitate in intrinsically L-resistant HER2+ BC cells as compared with L-sensitive cells, thus supporting the hypothesis that FAO could represent the major lipid metabolic pathway sustaining the energetic needs of HER2+ BC cells resistant to anti-HER2 therapy for survival and growth. Further, the pharmacological dual blockade of HER2 and CPT1A significantly increases cytotoxicity of L-resistant cell models versus single agents, corroborating the candidacy of CPT1A as a potential metabolic vulnerability to be targeted to overcome refractoriness to HER2-targeted therapy in HER2+ BC. Our data support the hypothesis that the inhibition of FAO sensitizes HER2+ BC cells to anti-HER2 drugs, thus representing a potential strategy to overcome drug resistance in HER2+ BC and paving the way to investigate CPT1A inhibitors with anti-HER2 drugs to improve the clinical outcomes of HER2+ BC patients with acquired resistance to standard anti-HER2 therapies. Citation Format: Alma Franceschini, Lorenzo T Castagnoli, Tiziana IINDT Triulzi, Paola Antonia Corsetto, Matteo Dugo, Antonino Belfiore, Andrea Vingiani, Lorena Signati, Serena Mazzucchelli, Fabio Corsi, Francesca Ligorio, Elda Tagliabue, Claudio Vernieri, Serenella M. Pupa. Carnitine palmitoyltransferase IA: an emerging potential metabolic target to counteract HER2-targeted therapy resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A039.

Immune microenvironment dynamics of HER2 overexpressing breast cancer under dual anti-HER2 blockade.

The clinical prognosis of the HER2-overexpressing (HER2-OE) subtype of breast cancer (BC) is influenced by the immune infiltrate of the tumor. Specifically, monocytic cells, which are promoters of pro-tumoral immunosuppression, and NK cells, whose basal cytotoxic function may be enhanced with therapeutic antibodies. One of the standards of care for HER2+ BC patients includes the combination of the anti-HER2 antibodies trastuzumab and pertuzumab. This dual combination was a breakthrough against trastuzumab resistance; however, this regimen does not yield complete clinical benefit for a large fraction of patients. Further therapy refinement is still hampered by the lack of knowledge on the immune mechanism of action of this antibody-based dual HER2 blockade. To explore how the dual antibody challenge influences the phenotype and function of immune cells infiltrating the HER2-OE BC microenvironment, we developed in vitro 3D heterotypic cell models of this subtype. The models comprised aggregates of HER2+ BC cell lines and human peripheral blood mononuclear cells. Cells were co-encapsulated in a chemically inert alginate hydrogel and maintained in agitation-based culture system for up to 7 days. The 3D models of the HER2-OE immune microenvironment retained original BC molecular features; the preservation of the NK cell compartment was achieved upon optimization of culture time and cytokine supplementation. Challenging the models with the standard-of-care combination of trastuzumab and pertuzumab resulted in enhanced immune cytotoxicity compared with trastuzumab alone. Features of the response to therapy within the immune tumor microenvironment were recapitulated, including induction of an immune effector state with NK cell activation, enhanced cell apoptosis and decline of immunosuppressive PD-L1+ immune cells. This work presents a unique human 3D model for the study of immune effects of anti-HER2 biologicals, which can be used to test novel therapy regimens and improve anti-tumor immune function.

Tracking circulating PD-L1-positive cells to monitor the outcome of patients with gastric cancer receiving anti-HER2 plus anti-PD-1 therapy

Dual blockade of HER2 and PD-1/PD-L1 is the most promising regimen for HER2-positive patients with gastric cancer (GC); PD-L1 combined positive score, rather than HER2 status, indicates potential benefit. Circulating tumor cells (CTCs) and circulating endothelial cells (CECs) derived from the tumor microenvironment provide platforms for the dynamic evaluation of PD-L1 expression. Whether PD-L1 positive CTCs/CECs (PD-L1+CTCs/CECs) can serve as biomarkers for evaluating the efficacy of combination therapy remains unknown. Therefore, this study investigated PD-L1 expression and heterogeneous karyotypic features of CTCs/CECs and their involvement in the clinical response to treatment in 72 patients with advanced GC by applying a pre-established surface molecule-independent subtraction enrichment (SE)-iFISH strategy. In the captured PD-L1 positive cells, there were 42.80% and 57.20% of CTCs and CECs, respectively. PD-L1+ CTCs were pre-therapeutically detected in 0% (0/11) of HER2-negative patients and 14.75% (9/61) of HER2-positive patients. The presence of baseline PD-L1+CTCs was relevant to inferior prognosis (mPFS: 14.40 months vs 5.00 months, P = 0.065); post-treatment PD-L1+ CECs were associated with longer irPFS (immunotherapeutic-related PFS) (mPFS: 15.57 months vs 6.73 months, P = 0.053). Further dynamic karyotype-based profiling of PD-L1+ CTCs/CECs indicated that multiploidy and triploidy were the dominant subtypes of baseline PD-L1+ CTCs, and that triploidy was specifically associated with therapeutic resistance. Intratherapeutically detected multiploid PD-L1+ CECs demonstrated a superior clinical response; triploidy and tetraploidy contributed to acquired resistance. The karyotypic features of PD-L1+CTCs/CECs should be dynamically profiled in patients with GC treated with anti-HER2 plus anti-PD-1 therapy. Triploid-PD-L1+ CTCs and multiploid-PD-L1+ CECs are potential indicators of therapeutic response.

Pembrolizumab plus trastuzumab and chemotherapy for HER2-positive gastric or gastro-oesophageal junction adenocarcinoma: interim analyses from the phase 3 KEYNOTE-811 randomised placebo-controlled trial

Evidence for the efficacy of combined PD-1 and HER2 blockade with chemotherapy on progression-free and overall survival in HER2-positive gastro-oesophageal cancer is scarce. The first interim analysis of the randomised, phase 3 KEYNOTE-811 study showed a superior objective response with pembrolizumab compared with placebo when added to trastuzumab plus fluoropyrimidine and platinum-based chemotherapy. Here, we report results from protocol-specified subsequent interim analyses of KEYNOTE-811. The randomised, phase 3 KEYNOTE-811 trial involved 168 medical centres in 20 countries worldwide. Patients aged 18 years or older with locally advanced or metastatic HER2-positive gastro-oesophageal junction adenocarcinoma, without previous first-line treatment, were randomly assigned (1:1) by an integrated interactive voice-response and web-response system to intravenous pembrolizumab 200 mg or placebo, both to be combined with standard chemotherapy (fluoropyrimidine and platinum-based therapy) plus trastuzumab every 3 weeks for up to 35 cycles or until disease progression, unacceptable toxic effects, or investigator or participant-initiated withdrawal. Randomisation used a block size of four and was stratified by region, PD-L1 status, and chemotherapy. Dual primary endpoints were progression-free and overall survival, analysed by intention to treat. Safety was assessed in all randomly assigned patients who received at least one dose of study treatment according to the treatment received. KEYNOTE-811 is registered with ClinicalTrials.gov (NCT03615326) and is active but not recruiting. Between Oct 5, 2018, and Aug 6, 2021, 698 patients were assigned to pembrolizumab (n=350) or placebo (n=348). 564 (81%) were male and 134 (19%) were female. At the third interim analysis, 286 (82%) of 350 patients in the pembrolizumab group and 304 (88%) of 346 in the placebo group who received treatment had discontinued treatment, mostly due to disease progression. At the second interim analysis (median follow-up 28·3 months [IQR 19·4-34·3] in the pembrolizumab group and 28·5 months [20·1-34·3] in the placebo group), median progression-free survival was 10·0 months (95% CI 8·6-11·7) in the pembrolizumab group versus 8·1 months (7·0-8·5) in the placebo group (hazard ratio [HR] 0·72, 95% CI 0·60-0·87; p=0·0002). Median overall survival was 20·0 months (17·8-23·2) versus 16·9 months (15·0-19·8; HR 0·87 [0·72-1·06]; p=0·084). At the third interim analysis (median follow-up 38·4 months [IQR 29·5-44·4] in the pembrolizumab group and 38·6 months [30·2-44·4] in the placebo group), median progression-free survival was 10·0 months (8·6-12·2) versus 8·1 months (7·1-8·6; HR 0·73 [0·61-0·87]), and median overall survival was 20·0 months (17·8-22·1) versus 16·8 months (15·0-18·7; HR 0·84 [0·70-1·01]), but did not meet prespecified criteria for significance and will continue to final analysis. Grade 3 or worse treatment-related adverse events occurred in 204 (58%) of 350 patients in the pembrolizumab group versus 176 (51%) of 346 patients in the placebo group. Treatment-related adverse events that led to death occurred in four (1%) patients in the pembrolizumab group and three (1%) in the placebo group. The most common treatment-related adverse events of any grade were diarrhoea (165 [47%] in the pembrolizumab group vs 145 [42%] in the placebo group), nausea (154 [44%] vs 152 [44%]), and anaemia (109 [31%] vs 113 [33%]). Compared with placebo, pembrolizumab significantly improved progression-free survival when combined with first-line trastuzumab and chemotherapy for metastatic HER2-positive gastro-oesophageal cancer, specifically in patients with tumours with a PD-L1 combined positive score of 1 or more. Overall survival follow-up is ongoing and will be reported at the final analysis. Merck Sharp & Dohme.

Liquid biopsy kinetics and detection of ERBB2 amplification/HER2-positivity in refractory hepatocellular carcinoma

Hepatocellular Carcinoma (HCC) is the most common primary liver carcinoma, accounting for 75–85% of all cases. For patients with advanced unresectable and/or metastatic HCC, treatment options primarily include immunotherapy combinations and/or oral tyrosine kinase inhibitors. For patients with alpha-fetoprotein (AFP) levels above 400 ng/ml, ramucirumab, an anti-VEGF agent, is also approved in the second-line setting. However, none of these therapies are based on a specific molecular marker and the role of molecular testing is limited. In fact, HCC is unique in the sense that a biopsy may not be pursued if imaging findings are classic for HCC, in the appropriate clinical context. Herein, we report a case of a patient with metastatic HCC and a huge disease burden where a circulating tumor DNA (ctDNA) liquid biopsy done as part of our standard of care serendipitously revealed a very high ERBB2 copy number amplification (>78). This was corroborated by HER2 immunohistochemical staining, which demonstrated strong diffuse membranous HER2 3+ expression; tissue next-generation sequencing also identified an ERBB2 unadjusted copy number gain of 183. Furthermore, we were able to demonstrate the feasibility of using both ctDNA and circulating tumor cells (CTCs) to determine the heterogeneity of HER2 and response to dual HER2 blockade trastuzumab/pertuzumab (MyPathway regimen). In addition to the utility of liquid biopsies increasing opportunities for precision medicine, this n-of-1 precision medicine case illustrates and reiterates the need for comprehensive panel-based NGS testing that employs both DNA/RNA for all patients with advanced or metastatic cancers. It also supports the agnostic potential of ERBB2/HER2 as an actionable marker.

Negative Hyperselection of Patients with HER2+ and RAS Wild-Type Metastatic Colorectal Cancer Receiving Dual HER2 Blockade: the PRESSING-HER2 Study.

To demonstrate the negative prognostic impact of a panel of genomic alterations (PRESSING-HER2 panel) and lack of HER2 amplification by next-generation sequencing (NGS) in patients with HER2+, RAS wild-type metastatic colorectal cancer receiving dual HER2 blockade. The PRESSING-HER2 panel of HER2 mutations/rearrangements and RTK/MAPK mutations/amplifications was assessed by NGS. HER2 amplification was confirmed by NGS if copy-number variation (CNV) was ≥ 6. With a case-control design, hypothesizing 30% and 5% PRESSING-HER2 positivity in resistant [progression-free survival (PFS) <4 months and no RECIST response] versus sensitive cohorts, respectively, 35 patients were needed per group. PRESSING-HER2 alterations included HER2 mutations/rearrangements, EGFR amplification, and BRAF mutations and had a prevalence of 27% (9/33) and 3% (1/35) in resistant versus sensitive patients (P = 0.005) and 63% predictive accuracy. Overall, HER2 nonamplified status by NGS had 10% prevalence. Median PFS and overall survival (OS) were worse in PRESSING-HER2+ versus negative (2.2 vs. 5.3 months, P < 0.001; 5.4 vs. 14.9 months, P = 0.001) and in HER2 nonamplified versus amplified (1.6 vs. 5.2 months, P < 0.001; 7.4 vs. 12.4 months, P = 0.157). These results were confirmed in multivariable analyses [PRESSING-HER2 positivity: PFS HR = 3.06, 95% confidence interval (CI), 1.40-6.69, P = 0.005; OS HR = 2.93, 95% CI, 1.32-6.48, P = 0.007]. Combining PRESSING-HER2 and HER2 CNV increased the predictive accuracy to 75%. PRESSING-HER2 panel and HER2 nonamplified status by NGS warrant validation as potential predictive markers in this setting. See related commentary by Raghav et al., p. 260.

Anthracyclines versus No Anthracyclines in the Neoadjuvant Strategy for HER2+ Breast Cancer: Real-World Evidence.

Deescalation strategies omitting anthracyclines (AC) have been explored in early human epidermal growth factor receptor 2-positive breast cancer (HER2+ EBC), showing similar efficacy regarding pathological complete response (pCR) and long-term outcomes as AC-containing regimens. The standard treatment for this tumor subtype is based on chemotherapy and dual HER2 blockade with trastuzumab and pertuzumab, with AC-containing regimens remaining a frequent option for these patients, even in non-high-risk cases. The primary aim of this study was to assess and compare the effectiveness of neoadjuvant regimens with and without AC used in the treatment of HER2+ EBC in the clinical practice according to the pCR achieved with each. This retrospective multicentric study included patients with HER2+ EBC from Portuguese, Spanish, and Chilean hospitals (January 2018-December 2021). Patients receiving neoadjuvant therapy (NAT) with dual HER2 blockade (trastuzumab and pertuzumab), followed by surgery, were included. Statistical analysis used chi-squared/Fisher's exact test for associations, multivariate logistic regression for pCR, and Kaplan-Meier method for event-free survival (EFS). IBM SPSS Statistics 29.0 analyzed the data. The study included 371 patients from eight hospitals. Among them, 237 received sequential AC and taxane-based chemotherapy with 4 cycles of trastuzumab and pertuzumab, while 134 received 6 cycles of TCHP (docetaxel, carboplatinum, trastuzumab, and pertuzumab). The average age of the patients was 52.8 years and 52.7 years, respectively. Omitting AC from the neoadjuvant approach did not preclude achieving pCR [p=0.246, 95% confidence interval (CI) 0.235-0.257] and was safe regardless of patient characteristics. Relapse rates were 6.8% (16 patients) in the AC group and 4.5% (6 patients) in the TCHP group. Over a median follow-up of 2.9 years, the estimated 3-year EFS was 92.5% in the AC group and 95.4% in the TCHP group (hazard ratio 0.602, 95% CI 0.234-1.547, p=0.292, favoring TCHP). This study reports real-world evidence showing similar pCR and EFS outcomes with treatment regimens with and without AC and raises awareness of possible overtreatment and long-term toxicity in some patients with HER2+ EBC with the use of AC.

The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer.

HER2 is amplified or overexpressed in around 20% of breast cancers (BC). HER2-targeted therapies have significantly improved the prognosis of patients with HER2+ BC, however, de novo and acquired resistance to anti-HER2 treatment is common. Activating mutations in the PIK3CA gene are reported in ∼30% of HER2+ BC and are associated with resistance to anti-HER2 therapies and a poor prognosis. Here, we investigated the in vitro and in vivo antitumor efficacy of the alpha-specific PI3K inhibitor alpelisib alone or in combination with anti-HER2 therapy using a panel of HER2+ BC cell lines. We also generated models of acquired resistance to alpelisib to investigate the mechanisms underlying resistance to alpha-specific PI3K inhibition. PIK3CA mutant (HCC1954, KPL4 and JMT1) and wild-type (BT474 and SKBR3) HER2+ BC cell lines were used. The HCC1954 and KPL4 cells were chronically exposed to increasing concentrations of alpelisib or to alpelisib + trastuzumab in order to generate derivatives with acquired resistance to alpelisib (AR) and to alpelisib + trastuzumab (ATR). The transcriptomic profiles of HCC1954, KPL4 and their AR and ATR derivatives were determined by RNA sequencing. Cell growth was assessed by MTT assay. Changes in the protein levels of key PI3K pathway components were assessed by Western blotting. Gene expression, cellular and patients' data from the Cancer Dependency Map (DepMap) and KMPlot datasets were interrogated. HER2+ BC cell lines harboring activating mutations in PIK3CA were less sensitive to single or dual anti-HER2 blockade compared to PIK3CA wild-type cells. Alpelisib treatment resulted in dose-dependent inhibition of the growth of cells with or without PIK3CA mutations and enhanced the antitumor efficacy of anti-HER2 therapies in vitro. In addition, alpelisib greatly delayed tumor growth of HCC1954 xenografts in vivo. Functional annotation of the significantly differentially expressed genes suggested the common activation of biological processes associated with oxidation reduction, cell proliferation, immune response and RNA synthesis in alpelisib-resistant models compared with native cells. Eight commonly upregulated genes (log2 fold-change >1, False Discovery Rate [FDR] <0.05) in models with acquired resistance to alpelisib or alpelisib + trastuzumab were identified. Among these, AKR1C1 was associated with alpelisib-resistance in vitro and with a poor prognosis in patients with HER2+ BC. Our findings support the use of an alpha-selective PI3K inhibitor to overcome the therapeutic limitations associated with single or dual HER2 blockade in PIK3CA-mutant HER2+ breast cancer. Future studies are warranted to confirm the potential role of candidate genes/pathways in resistance to alpelisib.