Response In HER2-positive Breast Cancer Research Articles

Abstract PO3-01-01: Dysregulation of Phenylalanine-tyrosine Metabolic Signaling Pathway and Neoadjuvant Response in HER2-positive Breast Cancer

Abstract Background: Although trastuzumab and tyrosine inhibitors combined with neoadjuvant chemotherapy could significantly improve patients' pathological complete response (pCR) rate, nearly half of the patients could not achieve complete remission. Our objective was to explore the relationship between metabolic signaling pathway changes and therapeutic efficacy in patients receiving neoadjuvant therapy for HER2-positive breast cancer. Methods: Core needle biopsy specimens from HER2-positive breast cancer patients who underwent neoadjuvant therapy and radical mastectomy at Fudan University Shanghai Cancer Center between January 2017 and December 2021 were collected for RNA sequencing. According to pathological evaluation, samples (n=133) were divided into pCR and non-pCR groups. Gene differences between the two groups were further evaluated for KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. The TCGA (The Cancer Genome Atlas, n=1091) and the HAN ethnic GEO datasets (GSE162228, GSE206, GSE48391, n=305) were used to analyze the metabolic differences between tumors and normal tissues with patients’ prognosis. Consensus clustering analysis was performed to divide patients for low and high phenylalanine-tyrosine metabolism dysregulation to analyze patients’ outcomes. The IC50 value of cell lines was tested using CCK-8 experiments. Western blot was performed to analyze the protein level changes in mTOR and glycolysis pathway. Results: We found that phenylalanine-tyrosine metabolism was significantly enriched in HER2-positive breast cancer patients. Consensus clustering analysis of TCGA and the HAN ethnic GEO datasets showed that patients with low phenylalanine-tyrosine catabolism had a significantly worse survival outcome than patients with high tyrosine metabolism (P= 0.017). In our cohort, phenylalanine-tyrosine pathway metabolic enzyme DDC was upregulated while MAOA was downregulated in non-pCR patients compared to pCR patients, suggesting the increased production of dopamine and reduced catabolism of dopamine (DA) in non-pCR tumors. In vitro experiments showed that dopamine and the knockdown of MAOA decreased cell sensitivity to pyrotinib. In addition, dopamine and clorgyline- an MAOA inhibitor, promoted the proliferation of HER2-positive cell lines under the treatment of pyrotinib. The GSEA analysis of our neoadjuvant cohort and TCGA datasets revealed that MAOA expression was associated with an inverse enrichment of the mTORC1 and glycolysis pathways. Western blot experiment results showed that dopamine or the knockdown of MAOA could activate the mTOR and glycolysis pathway Conclusions: Phenylalanine-tyrosine metabolism was significantly enriched in HER2-positive breast cancer patients. The phenylalanine-tyrosine signaling pathway may be dysregulated in HER2-positive breast cancer and patients with low phenylalanine-tyrosine catabolism have a worse prognosis. Low expression of MAOA promotes tumor resistance to HER2-targeted drugs and could serve as a therapeutic biomarker in breast cancer. Citation Format: Chih Wan Goh, Bingqiu Xiu, Jingyan Xue, Yayun Chi, Jiong Wu. Dysregulation of Phenylalanine-tyrosine Metabolic Signaling Pathway and Neoadjuvant Response in 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 PO3-01-01.

Abstract PO1-01-01: ECOG-ACRIN EA1211: Interim FDG-PET/CT for predicting response of HER2-positive breast cancer to neoadjuvant therapy (DIRECT Trial)

Abstract Background: Strategies to optimize treatment decision-making in early stage HER2-positive breast cancer are a current priority for the oncology and patient advocacy community. Recent studies suggest that early metabolic changes on FDG-PET/CT (imaging biomarker) predict response to HER2-directed therapy. The TBCRC026 trial showed that participants not obtaining a 40% reduction in SULmax by cycle 1 day 15 (C1D15) following neoadjuvant trastuzumab/pertuzumab (HP) were unlikely to obtain pCR [negative predictive value (NPV) 91%]. The ECOG-ACRIN EA1211/DIRECT trial aims to validate FDG-PET/CT as a neoadjuvant interim (niFDG-PET/CT) imaging integral biomarker in patients treated with standard HER2-directed regimens (NCT05710328). Design: EA1211/DIRECT is a multicenter, single-arm, primary imaging phase 2 study enrolling patients with stage II/III HER2-positive breast cancer. Patients undergo standard skull base-thigh FDG-PET/CT at baseline and C1D15 and receive standard of care pertuzumab-based neoadjuvant therapy followed by surgery. Eligibility: Age ≥18 years, ECOG performance status 0-2, stage IIa-IIIc, untreated HER2-positive breast cancer with known hormone receptor status, suitable to undergo FDG-PET/CT imaging and neoadjuvant therapy. Methods: ΔSULmaxD15 will be computed as: (D15 SULmax – baseline SULmax)/baseline SULmax. The primary objective is to estimate the NPV of niFDG-PET/CT for pCR using ΔSULmaxD15 of the primary breast cancer at a threshold of 40%, in patients treated with neoadjuvant HER2-directed therapy. NPV is defined as the probability that pCR will not be achieved by participants with ΔSULmaxD15 < 40%. Secondary endpoints include evaluating the sensitivity, specificity, and positive predictive value of ΔSULmaxD15 of the primary breast cancer at a threshold of 40% and the ability of the niFDG-PET/CT biomarker to predict 3-year event free survival (EFS). Sample Size: We expect that 50% of patients will not reach the ΔSULmax threshold of 40% and a pCR rate of 40-60%. The proposed sample size is 210 participants, adjusted to 235 to account for missing data in 10% of cases. Computation of exact, two-sided 95% confidence intervals (CI) for NPV determined the lower limit of the CI is at least 80% when the true NPV is at least 88%. The lower limit is 84% when the true NPV has the value of 91%, which was estimated in the TBCRC026 study. Current Status: EA1211/DIRECT was activated in May 2023 across the NCI National Clinical Trials Network (NCTN), and accrual is anticipated to complete in March 2025. Those interested in the clinical trial can email ea1211team@ecog-acrin.org. Conclusion: The EA1211/DIRECT trial is the first step in implementing a Response-Guided Treatment Strategy by validating the ΔSULmax threshold of 40% as the optimum cut point across standard of care HER2-directed neoadjuvant regimens. If EA1211/DIRECT meets its objectives, the results will be used to design clinical utility studies, and thus hoping to change practice. Citation Format: Heather Jacene, Constantine Gatsonis, Roisin Connolly, Brian Burnette, Erica Stringer-Reasor, Maeve Hennessy, Justin Romanoff, Alexander Taurone, Ciara O'Sullivan, H. T. Carisa Le-Petross, Courtney Lawhn Heath, Vered Stearns, Amy Fowler, Shou-Ching Tang, Karla A Sepulveda, Angela DeMichele, David Mankoff, Antonio Wolff. ECOG-ACRIN EA1211: Interim FDG-PET/CT for predicting response of HER2-positive breast cancer to neoadjuvant therapy (DIRECT 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 PO1-01-01.

Abstract LB275: Reactivation of mitotic arrest upon targeting TACC3 restores immunogenic cell death and T-DM1 response in HER2-positive breast cancer

Abstract Immunogenic cell death (ICD) is a specific form of cell death that is characterized by the release of danger-associated molecular patterns (DAMPs), such as cell surface exposure of calreticulin, ATP and HMGB1 release, and can be induced by several different anti-cancer therapies. This, in turn, evokes a strong anti-tumor immune response, leading to reduced tumor growth. Despite being the first and one of the most successful antibody-drug conjugates (ADCs) approved for refractory HER2-positive breast cancer, little is known if response and resistance to trastuzumab emtansine (T-DM1) involves ICD modulation that can be leveraged to enhance T-DM1 response. Here we report that T-DM1 induces spindle assembly checkpoint (SAC)-dependent ICD in sensitive cells, mediated by its payload by inducing eIF2α phosphorylation, surface exposure of calreticulin, ATP and HMGB1 release, and secretion of ICD-related cytokines, all of which are lost in resistance. Accordingly, ICD-related gene signatures correlate with clinical response to T-DM1-containing therapy in pre-treatment samples, and increased infiltration of anti-tumor CD8+ T cells is correlated with better T-DM1 response in post-treatment samples. We found that transforming acidic coiled-coil containing 3 (TACC3) is overexpressed in T-DM1 resistant cells, and that T-DM1 responsive patients have reduced TACC3 protein while the non-responders exhibited increased TACC3 expression during T-DM1 treatment. Notably, genetic or pharmacological inhibition of TACC3 revives T-DM1-induced SAC activation and induction of ICD markers in vitro. TACC3 inhibition elicits ICD in vivo as shown by vaccination assay. It also potentiates T-DM1 in the human HER2-overexpressing MMTV-huHER2#5 (Fo5) transgenic model by activating ICD markers, release of cytokines and inducing dendritic cell (DC) maturation and enhancing infiltration of cytotoxic T cells. Together, our results show that ICD is a key mechanism of action of T-DM1 which is lost in resistance, and that targeting TACC3 restores T-DM1-mediated ICD and overcomes resistance. Citation Format: Ozge Saatci, Mustafa Emre Gedik, Nathaniel Oberholtzer, Meral Uner, Ozge Akbulut, Metin Cetin, Mertkaya Aras, Kubra Ibis, Burcu Caliskan, Erden Banoglu, Stefan Wiemann, Aysegul Uner, Sercan Aksoy, Shikhar Mehrotra, Ozgur Sahin. Reactivation of mitotic arrest upon targeting TACC3 restores immunogenic cell death and T-DM1 response in HER2-positive breast cancer [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 LB275.

Targeting TACC3 Induces Immunogenic Cell Death and Enhances T-DM1 Response in HER2-Positive Breast Cancer.

Loss of induction of immunogenic cell death in response to T-DM1 leads to resistance that can be overcome by targeting TACC3, providing an attractive strategy to improve the efficacy of T-DM1.

Cuproptosis-related genes predict prognosis and trastuzumab therapeutic response in HER2-positive breast cancer

Breast cancer is the most common diagnosed cancer, the HER2-positive subtype account for 15% of all breast cancer. HER2-targeted therapy is the mainstay treatment for HER2-positive breast cancer. Cuproptosis is a novel form of programmed cell death, and is caused by mitochondrial lipoylation and destabilization of iron-sulfur proteins triggered by copper, which was considered as a key player in various biological processes. However, the roles of cuproptosis-related genes in HER2-positive breast cancer remain largely unknown. In the present study, we constructed a prognostic prediction model of HER2-positive breast cancer patients using TCGA database. Dysregulated genes for cells resistant to HER2-targeted therapy were analyzed in the GEO dataset. KEGG pathway, GO enrichment and GSEA was performed respectively. The immune landscape of DLAT was analyzed by CIBERSORT algorithm and TIDE algorithm. HER2-positive breast cancer patients with high CRGs risk score showed shorter OS. DLAT was downregulated and correlated with better survival of HER2-positive breast cancer patients (HR = 3.30, p = 0.022). High expressed DLAT was associated with resistant to HER2-targeted therapy. Knocking down DLAT with siRNA increased sensitivity of breast cancer to trastuzumab. KEGG pathway and GO enrichment of DEGs indicated that DLAT participates in various pathways correlated with organelle fission, chromosome segregation, nuclear division, hormone-mediated signaling pathway, regulation of intracellular estrogen receptor signaling pathway, condensed chromosome and PPAR signaling pathway. There was a negative correlation between TIDE and DLAT expression (r = − 0.292, p < 0.001), which means high DLAT expression is an indicator of sensitivity to immunotherapy. In conclusion, our study constructed a four CRGs signature prognostic prediction model and identified DLAT as an independent prognostic factor and associated with resistant to HER2-targeted therapy for HER2-positive breast cancer patients.

Differential response of HER2-positive breast cancer to anti-HER2 therapy based on HER2 protein expression level

BackgroundIncreasing data indicate that HER2-positive (HER2 + ) breast cancer (BC) subtypes exhibit differential responses to targeted anti-HER2 therapy. This study aims to investigate these differences and the potential underlying molecular mechanisms.MethodsA large cohort of BC patients (n = 7390) was utilised. The clinicopathological characteristics and differential gene expression (DGE) of HER2+ immunohistochemical (IHC) subtypes, specifically HER2 IHC 3+ and IHC 2 + /Amplified, were assessed and correlated with pathological complete response (pCR) and survival in the neoadjuvant and adjuvant settings, respectively. The role of oestrogen receptor (ER) status was also investigated.ResultsCompared to HER2 IHC 3+ tumours, BC patients with IHC 2 + /Amplified showed a significantly lower pCR rate (22% versus 57%, P < 0.001), shorter survival regardless of HER2 gene copy number, were less classified as HER2 enriched, and enriched for trastuzumab resistance and ER signalling pathway genes. ER positivity significantly decreased response to anti-HER2 therapy in IHC 2 + /Amplified, but not in IHC 3 + BC patients.ConclusionIn HER2 + BC, overexpression of HER2 protein is the driver of the oncogenic pathway, and it is the main predictor of response to anti-HER2 therapy. ER signalling pathways are more dominant in BC with equivocal HER2 expression. personalised anti-HER2 therapy based on IHC classes should be considered.

126MO HER2DX and pathological complete response in HER2-positive breast cancer: A combined analysis of 4 neoadjuvant studies

The optimal neoadjuvant therapy for HER2-positive breast cancer patients is subject to ongoing debate and the use of biomarkers can help to personalise treatment decision. The 27-gene HER2DX test predicts pathological complete response (pCR) following neoadjuvant trastuzumab-based chemotherapy. Here, we evaluated the value of HER2DX to predict increases in pCR rate when 1) a second anti-HER2 agent is added to trastuzumab and chemotherapy, and 2) multi-agent chemotherapy is used instead of a single agent taxane in the context of dual HER2 blockade. We conducted a combined analysis of four neoadjuvant cohorts with HER2DX and clinical data (i.e., DAPHNe, GOM-HGUGM-2018-05, CALGB-40601 and ISPY-2). All patients were treated with neoadjuvant trastuzumab (n=568) in combination with multi-agent chemotherapy (n=282), a single taxane (n=286), pertuzumab (n=264), lapatinib (n=103) or without a second anti-HER2 drug (n=201). Interaction tests were performed to evaluate the predictive capacity of HER2DX to identify patients who benefit most from each treatment approach. HER2DX pCR as continuous score was significantly associated with pCR in all patients (odds-ratio [OR]=1.62, 95% CI 1.43-1.85; AUC=0.75), in patients treated with trastuzumab and chemotherapy (OR=1.48, 1.18-1.86) and in patients treated with dual HER2 blockade and chemotherapy (OR=1.69, 1.50-1.91). A statistically significant increase in pCR rate due to dual HER2 blockade over trastuzumab-only was observed in HER2DX pCR-high tumors treated with chemotherapy (OR=4.10, 1.85-9.07; interaction test p=0.03). A statistically significant increase in pCR rate due to multi-agent chemotherapy over a single taxane was observed in HER2DX pCR-medium tumors treated with dual HER2 blockade (OR=2.52, 1.18-5.52; interaction test p=0.01). The pCR rates in HER2DX pCR-low tumors were ≤30.0% regardless of type of treatment. HER2DX might help identify patients with HER2-positive breast cancer who benefit from neoadjuvant dual HER2 blockade in combination with a single taxane.

Abstract P4-09-05: Characterization and validation of biologically-driven HER2-positive breast cancer subgroups in the ALTTO and NeoALTTO clinical trials

Abstract Background: Tumor and microenvironment features, including luminal phenotype as well as metabolic, immune and stroma activation, impact prognosis and treatment response in HER2-positive breast cancer. Here, we aimed to identify subgroups depicting biological processes associated with prognosis in patients receiving adjuvant trastuzumab in the phase III ALTTO trial, and to validate their biological and prognostic characteristics in the phase III neoadjuvant NeoALTTO trial. Methods: By applying a case-control approach (1:2), we selected from the ALTTO trastuzumab arm 134 and 268 patients with and without a distant relapse respectively, matched for clinicopathological characteristics. In ALTTO, RNA was obtained from FFPE tumor cores from surgical samples. RNA sequencing (RNAseq) data available from 254 frozen pre-treatment samples were used for validation in patients receiving trastuzumab and/or lapatinib in NeoALTTO. The Absolute Intrinsic Molecular Subtyping (AIMS) was used to compute PAM50 subtypes. Prognostic genes were identified using a multivariable Cox proportional hazard model (controlling for clinicopathological characteristics and PAM50 HER2-enriched [HER2-E] vs others) for distant relapse-free survival (DRFS). Clusters were identified using non-negative matrix factorization (NMF) and k-means clustering, and characterized with gene expression profiling and Gene Set Variation Analysis (GSVA, hallmark gene sets from MSigDB). Aiming at identifying the subgroups in external cohorts, we identified relevant genes to develop group-specific signatures with LASSO regression, and used the derived scores to build a multinomial classifier. Results: The case-control cohort includes a high-risk population, with higher proportions of &amp;gt;2cm, node positive and G3 tumors compared to the whole ALTTO trastuzumab arm. RNAseq data were generated for 386/402 patients. NMF and k-means clustering performed on genes associated with DRFS (false discovery rate &amp;lt; 0.05) identified 4 groups with distinct biological characteristics and prognosis: immune-enriched (IM, N = 69), proliferative/metabolic (P/M, N = 87, characterized by glycolysis, cholesterol homeostasis and proliferation pathways), mesenchymal/stroma-enriched (M/S, N = 76, characterized by epithelial-mesenchymal transition, angiogenesis and TGF-beta signaling), and hormone receptor positive-enriched (N = 154), further divided into PAM50 HER2-E (N = 91) and non-HER2-E (N = 63, LUM, mainly luminal A/B tumors). The IM and LUM groups presented better DRFS (91% and 87% 5-year DRFS, respectively) compared to the others (5-year DRFS of 72%, 58% and 51% for HER2-E, M/S and P/M, respectively). The gene expression-based classifier identified the same 5 groups in NeoALTTO. GSVA analysis and comparisons among the 5 groups showed similar results in the two studies. Of interest, we observed significant differences in event-free survival in NeoALTTO (P = 0.035 and P = 0.041 in the whole population and in the subgroup with residual disease, respectively), with IM and LUM presenting better outcomes compared to the other groups. Sensitivity to neoadjuvant treatment as described by pathological complete response (pCR, ypT0/is ypN0) differed across the groups, with LUM presenting lower pCR rates (8%) compared to HER2-E (40%), IM (35%), P/M (32%), and M/S (32%). Conclusions: Five biologically-driven HER2-positive breast cancer subgroups were identified in ALTTO, highlighting the heterogeneity of this disease. Of note, their biological features and clinical behavior were validated in the NeoALTTO population, suggesting the robustness of our findings. IM and LUM tumors could be considered for treatment de-escalation approaches. Additional validation in cohorts receiving standard (neo)adjuvant therapies is warranted. Citation Format: Mattia Rediti, David Venet, Andrea Joaquin Garcia, Dominique Agbor-Tarh, Marion Maetens, Delphine Vincent, Samira Majjaj, Sarra El-Abed, Takayuki Ueno, Serena Di Cosimo, Martine Piccart, Lajos Pusztai, Sherene Loi, Roberto Salgado, Giuseppe Viale, Françoise Rothé, Christos Sotiriou. Characterization and validation of biologically-driven HER2-positive breast cancer subgroups in the ALTTO and NeoALTTO clinical trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-05.

Abstract P4-06-08: HER2-protein expression is a predictive marker for treatment response in patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with dual HER2-blockade

Abstract Background In HER2-positive breast cancer, pathologic complete response (pCR) has been known to be a surrogate marker for favorable prognosis after neoadjuvant chemotherapy. We aimed to identify the clinico-pathologic factors related to pCR in patients with HER2-positive breast cancer treated with neoadjuvant dual HER2-targeted therapy. Methods Two-hundred ninty-five patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) were included in this retrospective analysis. We assessed the association between age, clinical T, N stage, pathologic factors such as histologic grade, hormone receptor (HR), tumor-infiltrating lymphocytes, the membranous expression of HER2 protein evaluated by immunohistochemistry (IHC) and pCR. The fluorescent in situ hybridization (FISH) was performed in a tumor with HER2 score of 2+. Results Of the 295 patients, 195 (66.1%) achieved pCR (ypT0/is and ypN0). Besides, 240 (81.4%) patients were HER2 score of 3+, and 55 (18.6%) patients were HER2 score of 2+. The pCR was frequently observed in patients with HER2 score of 3+ (64 of 176 [73.3%]) than in those with HER2 score of 2+ (19 of 55 [34.5%]), regardless of HR status. After adjusting other clinicopathologic factors, the high HER2 protein expression was only an independent factor for pCR (adjusted OR 3.85, 95% CI, 1.66-8.91, p=0.002). Conclusions Our results suggest that high expression of HER2 protein assessed by IHC is important in predicting neoadjuvant TCHP response in HER2-positive breast cancer. Citation Format: Soong June Bae, Ji Soo Jang, Yoonwon Kook, Seung Ho Baek, Jee Hung Kim, Sung Gwe Ahn, Joon Jeong. HER2-protein expression is a predictive marker for treatment response in patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with dual HER2-blockade [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-06-08.

Tumor-Infiltrating Lymphocytes and Immune Response in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer accounts for 15 to 25% of breast cancer cases. Although therapies based on the use of monoclonal anti-HER2 antibodies present clinical benefit for a subtype of patients with HER2-positive breast cancer, more than 50% of them are unresponsive to targeted therapies or they eventually relapse. In recent years, reactivation of the adaptive immune system in patients with solid tumors has emerged as a therapeutic option with great potential for clinical benefit. Since the approval of the first treatment directed against HER2 as a therapeutic target, the range of clinical options has expanded greatly, and, in this sense, cellular immunotherapy with T cells relies on the cytotoxicity generated by these cells, which ultimately leads to antitumor activity. Lymphocytic infiltration of tumors encompasses a heterogeneous population of immune cells within the tumor microenvironment that exhibits distinct patterns of immune activation and exhaustion. The prevalence and prognostic value of tumor-infiltrating lymphocyte (TIL) counts are associated with a favorable prognosis in HER2-positive breast cancers. This review discusses emerging findings that contribute to a better understanding of the role of immune infiltrates in HER2-positive breast cancer. In addition, it summarizes the most recent results in HER2-positive breast cancer immunotherapy and anticipates which therapeutic strategies could be applied in the immediate future.

Molecular Assessment of HER2 to Identify Signatures Associated with Therapy Response in HER2-Positive Breast Cancer.

Simple SummaryThe HER2 status of breast cancers is accurately determined by measuring HER2 protein overexpression and gene amplification. However, these clinical diagnostic tests cannot predict the response to therapy. Single molecule imaging approaches can quantify molecular features of HER2, such as receptor nano-organization, with exquisite spatial resolution and sensitivity. The aim of our study was to assess how the molecular features of HER2 varied with the therapy response. According to our results in cultured cell lines and six patient specimens, the therapy response was associated with high detected HER2 densities and clustering. This advanced imaging approach can thus provide key data to complement the current diagnostic standards.Trastuzumab, the prototype HER2-directed therapy, has markedly improved survival for women with HER2-positive breast cancers. However, only 40–60% of women with HER2-positive breast cancers achieve a complete pathological response to chemotherapy combined with HER2-directed therapy. The current diagnostic assays have poor positive-predictive accuracy in identifying therapy-responsive breast cancers. Here, we deployed quantitative single molecule localization microscopy to assess the molecular features of HER2 in a therapy-responsive setting. Using fluorescently labeled trastuzumab as a probe, we first compared the molecular features of HER2 in trastuzumab-sensitive (BT-474 and SK-BR-3) and trastuzumab-resistant (BT-474R and JIMT-1) cultured cell lines. Trastuzumab-sensitive cells had significantly higher detected HER2 densities and clustering. We then evaluated HER2 in pre-treatment core biopsies from women with breast cancer undergoing neoadjuvant therapy. A complete pathological response was associated with a high detected HER2 density and significant HER2 clustering. These results established the nano-organization of HER2 as a potential signature of therapy-responsive disease.

Molecular subtype to predict pathologic complete response in HER2-positive breast cancer in the I-SPY2 trial.

510 Background: HER2-positive breast cancer (bc) is a very heterogenous disease. We hypothesized that molecular subtype may predict disease response to investigational agents in HER2+ bc. Here, we report the pathologic complete response (pCR) rate in the first six agents tested in HER2+ bc in the I-SPY 2 trial for the full HER2+ cohort, by molecular subtype, and by disease receptor status. Methods: Women with HER2+ tumors which were &gt; 2.5 cm were eligible. The I-SPY2 platform trial tests novel agents given neoadjuvantly with a backbone of taxol (T) and trastuzumab (H) followed by doxorubicin and cyclophosphamide. Agents investigated in HER2+ bc were TH (control), MK2206, AMG386, pertuzumab (P), neratinib (N (given in place of H), and TDM1+P (given in place of TH). An investigational arm graduated if there was &gt;85% chance of success compared to control in a 300-person phase 3 neoadjuvant trial. Further details of the I-SPY2 methods have been previously published. Molecular subtyping based on gene expression was utilized to categorize tumors into 5 response predictive subtypes (RPS) (HER2-/Immune-/DRD (DNA repair deficiency)-, HER2-/Immune+, HER2-/Immune-/DRD+, HER2+/Her2_or_Basal and HER2+/Luminal). Results: For the full HER2+ cohort (N=245) pCR rate was higher in all investigational arms than control (Table). By tumor receptor status, HER2+/HR- tumors (N=89) had a higher pCR rate than HER2+/HR+ tumors (N=156; 63% vs 37%, p = 0.0001). In HER2+/HR- tumors N, MK2206, P and TDM1/P graduated. In HER2+/HR+ tumors P and TDM1/P graduated. 76% (185/245) of I-SPY 2 HER2+ patients were classified as HER2+/Her2_or_Basal and 24% (60/245) were HER2+Luminal. pCR rate was significantly higher in the HER2+/Her2_or_Basal group than in the HER2+/Luminal group (57% vs 15%, p &lt; 0.0001). All agents, except for MK2206, where numbers were small, showed greater efficacy in the HER2+/Her2_or_Basal group than in the HER2+/Luminal group. HER2+/Luminal appeared to be more sensitive to the AKT inhibitor MK2206 than to targeted HER2 agents, though numbers are small. Conclusions: pCR rates for patients with HER2+ bc treated with investigational agents, particularly dual HER2-blockade, were promising. Molecular response predictive subtype classification provides insight on how to better target therapy. The HER2+/Luminal group had low pCR rates with dual HER2-blockade but may have higher pCR rate with the addition of an AKT inhibitor and identifies a subgroup of HER2+ tumors in need of novel approaches. AKT inhibition for HER2/Luminal is being tested in I-SPY 2.2. Clinical trial information: NCT01042379. [Table: see text]

Immune Checkpoint Blockade in HER2-Positive Breast Cancer: What Role in Early Disease Setting?

Simple SummaryThis work aims to discuss how an anti- or pro-tumor immune response could be manipulated through immune checkpoint blockade in patients with early stage HER2-positive breast cancer. By summarizing previously published evidence in the field, authors present their personal view on how immune checkpoint blockade could be implemented in the neoadjuvant setting in this patient population. The hypothesis being presented is that an appropriate and effective administration of immune checkpoint blockade could assure a lasting control of the disease, by preventing relapses. One of the research priorities should be the identification of the patients who could benefit more by this strategy.The present commentary synthesizes the current evidence on the role of the immune response in HER2-positive breast cancer. It points out the strengths and weaknesses of the findings observed so far, particularly in the early setting, including the clinical significance of scoring tumor-infiltrating lymphocytes. A figure proposing research hypotheses for the implementation of immune checkpoint blockade use for patient candidates to neoadjuvant treatment is presented.

Immunohistochemical HER2 score correlates with response to neoadjuvant chemotherapy in HER2-positive primary breast cancer.

The accuracy of biomarker assessment in breast cancer (BC) is paramount for therapy decisions and informs prognosis. We investigated neoadjuvant chemotherapy (NAC) response in HER2-positive BC with respect to immunohistochemistry (IHC) and in situ hybridization (ISH) results. We aimed to determine the role of HER2 protein expression in predicting NAC response and long-term outcome in two HER2-positive groups: IHC 3 + versus IHC 2 + ISH amplified groups. This retrospective study included 192 consecutive HER2 + primary BCs diagnosed from 2007 to 2019 treated with NAC and HER2-targeted agent (NACH). There were 158 HER2 3 + and 34 HER2 2 + ISH + cases. Clinicopathological parameters and long-term outcomes were analyzed. The Pathological Complete Response (pCR) rate was 85.7% (72/84) in ER-/HER2 + BCs and was lower in ER + /HER2 + BCs (42.6%, 46/108). The pCR was 55.1% (86/156) in the HER2 3 + group and was only 17.6% in HER2 2 + ISH + group (p < 0.001). Patients who achieved pCR in HER2 2 + ISH + group did not show a significantly higher HER2/CEP17 ratio or HER2 copy number. The overall survival (OS) and progression-free survival (PFS) were significantly higher in pCR compared to non-pCR cases (p = 0.011 and p = 0.015, respectively). There is significant heterogeneity in response to the NACH regimens in HER2 + cases. Our findings indicate that HER2 IHC score and ER expression determine NACH response in HER2 + BC. We recommend considering HER2 protein expression and ISH value to better select patients and assess the response for HER2-targeted therapy.

Abstract 1913: Soluble TNFα blockade overcomes lapatinib resistance and induces an innate immune response in HER2-positive breast cancer

Abstract Lapatinib (LAP), a dual EGFR/HER2 tyrosine kinase inhibitor, is used as second-line therapy in women with HER2+ breast cancer (BC), but less than 25% of the patients achieve an objective response. An alternative therapeutic approach is needed to overcome LAP resistance in women with metastatic HER2+ BC, particularly in patients with central nervous system (CNS) metastasis where large biological molecules are not effective. Previously, we reported that women with HER2+ tumors that express transmembrane glycoprotein mucin 4 (MUC4) have worse survival, and that HER2+/MUC4+ cell lines resistant to trastuzumab (T) express higher levels of tumor necrosis factor α (TNF) than T-sensitive cell lines. In addition, we proved that inhibition of soluble TNF (sTNF) decreases the expression of MUC4 and reverses T resistance. The aim of this work is to evaluate the participation of sTNF and transmembrane TNF (tmTNF) in LAP resistance in vivo and in the anti-tumor innate immune response. We used the LAP-resistant human BC cell line JIMT-1 and compared etanercept (E), a fusion protein that non-selectively blocks both sTNF and tmTNF, with the dominant negative-TNF protein INB03 (DN), that neutralizes sTNF without affecting tmTNF. Nude mice bearing JIMT-1 tumors (~50 mm3), received LAP (100 mg/kg) daily by oral gavage and IgG (5 mg/kg), E (5 mg/kg), DN (10 mg/kg), LAP+E or LAP+DN, twice a week i.p. Tumor volume was monitored routinely. At the end of the experiment, tumor-infiltrating immune cells were evaluated by immunofluorescence and analyzed by flow cytometry. DN or E treatments did not exhibit any anti-tumor effect alone, but in combination with LAP (LAP+DN and LAP+E) tumor growth decreased in a 54% and 34% vs. IgG, respectively (p&amp;lt;0.0001). LAP+DN combination was significantly more effective in decreasing JIMT-1 tumor growth than LAP+E (p&amp;lt;0.05). Analysis of tumor-infiltrating immune cells showed that tumor growth inhibition was accompanied by an increase in NK cell activation and degranulation, and a decrease in monocytic-myeloid-derived suppressor cells in the tumor bed of LAP+E and LAP+DN treated groups (p&amp;lt;0.01, vs. IgG). This is the first report to show that TNF blockade is able to overcome LAP resistance. In addition, TNF neutralization together with LAP treatment unleashes an anti-tumor innate immune response. These data, combined with previous results, suggest that MUC4 expression in patients with HER2+ BC could act not only as a biomarker of T resistance but also of LAP resistance. Women with HER2+/MUC4+ tumors undergoing treatment with LAP would benefit from the combined administration of LAP with the selective sTNF inhibitor DN to help overcome resistance; a hypothesis to be tested in a clinical trial. This therapeutic strategy may be particularly useful in patients with CNS metastasis because LAP and DN cross the blood-brain-barrier. Citation Format: Sofia Bruni, Florencia L. Mauro, Mara De Martino, Agustina Roldán Deamicis, María F. Mercogliano, Patricia V. Elizalde, Roxana Schillaci. Soluble TNFα blockade overcomes lapatinib resistance and induces an innate immune response in HER2-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1913.

HER2-enriched subtype and pathological complete response in HER2-positive breast cancer: A systematic review and meta-analysis

HER2-enriched subtype and pathological complete response in HER2-positive breast cancer: A systematic review and meta-analysis

Serum microRNA-21 predicted treatment outcome and survival in HER2-positive breast cancer patients receiving neoadjuvant chemotherapy combined with trastuzumab

The purpose of this study was to evaluate the expression of ser-miRNAs at different periods during treatment and analyze their relationship with therapeutic response and prognosis in HER2-positive breast cancer patients receiving neoadjuvant chemotherapy combined with trastuzumab (NCCT). Venous blood was drawn from patients at different periods during NCCT. The expression of ser-miRNAs was assessed by qRT-PCR and their relation to treatment response and survival was analyzed. The results showed the expression of miR-10b, -21, -34a, -125b, -145, -155, and -373 in patients before the start of treatment was significantly higher, ser-miR-210 was lower, and ser-miR-122 was comparable to the levels in healthy controls. Changes in ser-miR-21 levels during NCCT were significantly correlated to clinical response and survival and, however, were not associated with pathology response. The expression levels of ser-miR-21 were decreased from the start of NCCT to the end of the second cycle and from the start to the end of NCCT in clinical responders; however, there was no significant difference in non-responders. The patients with decreased ser-miR-21 expression from the start to the end of the second cycle and from the start to the end of NCCT had better overall survival (OS) and disease-free survival (DFS) than those with elevated ser-miR-21 expression. These results showed that changes in ser-miR-21 levels were significantly related to NCCT clinical response and prognosis. Ser-miR-21 may serve as a non-invasive biomarker to predict NCCT response in HER2-positive breast cancer.

Imaging and Clinicopathologic Features Associated With Pathologic Complete Response in HER2-positive Breast Cancer Receiving Neoadjuvant Chemotherapy With Dual HER2 Blockade

BackgroundIn human epidermal growth factor receptor 2-positive (HER2+) breast cancer, the incorporation of a dual HER2 blockade into neoadjuvant chemotherapy (NAC) has been shown to induce a higher rate of pathologic complete response (pCR). The purpose of this study was to investigate whether pretreatment imaging and clinicopathologic features show any association with pCR in HER2+ breast cancer receiving NAC plus dual blockade. Materials and MethodsThis retrospective study evaluated 94 consecutive patients (mean age, 49.8 ± 9.9 years) with HER2+ breast cancer who underwent NAC plus dual blockade with trastuzumab and pertuzumab between April 2016 and June 2018. All patients underwent mammography, ultrasound, and magnetic resonance imaging prior to NAC. Clinicopathologic and imaging features acquired before NAC were evaluated for their ability to predict the pathologic response after surgery. Multivariate analysis was used to identify independent predictors of pCR. ResultsFifty patients (53.2%) showed pCR and 44 (46.8%) did not. According to a univariate analysis, fine pleomorphic/fine linear or linear-branching calcification morphology on mammography, parallel orientation on ultrasound, intratumoral high signal intensity on T2-weighted magnetic resonance imaging, progesterone receptor negativity, and high levels of tumor-infiltrating lymphocytes were associated with pCR. On multivariate analysis, fine pleomorphic/fine linear or linear-branching calcification morphology on mammography (odds ratio [OR], 7.23), progesterone receptor negativity (OR, 6.76), and a high tumor-infiltrating lymphocyte level (OR, 5.92) remained significant independent factors associated with pCR. ConclusionSeveral pretreatment imaging and clinicopathologic features were shown to be independent variables predicting pCR in patients with HER2+ breast cancer receiving NAC with dual blockade.

Elevated WBP2 Expression in HER2-positive Breast Cancers Correlates with Sensitivity to Trastuzumab-based Neoadjuvant Therapy: A Retrospective and Multicentric Study.

Trastuzumab-based chemotherapy has shown remarkable clinical benefits for patients with HER2-positive breast cancer. However, treatment regimens involving trastuzumab had little or no effect for a subset of patients. Preliminary studies revealed WW-binding protein 2 (WBP2), an oncogenic transcription coactivator, to be coamplified with HER2 in 36% of HER2-positive breast cancers. We hypothesize that WBP2 regulates and correlates with the response of HER2-positive breast cancer to trastuzumab. The coexpression of WBP2 and HER2 in breast tumors was validated using IHC. The role and mechanism of WBP2 in regulating breast cancer response to trastuzumab was elucidated using in vitro, patient-derived xenograft and murine xenograft models. A multicenter retrospective study involving 143 patients given neoadjuvant trastuzumab-based chemotherapy was conducted to determine whether WBP2 expression correlates with pathologic complete response (pCR). Elevated expression of WBP2 significantly enhanced breast cancer's response to trastuzumab by augmenting trastuzumab-induced HER2 downregulation and cell-cycle arrest via inhibition of cyclin D expression. High level of WBP2 correlated with better pCR (67.19%) compared with low WBP2 level (26.58%). The highest response was observed in subgroups of patients with high WBP2-expressing tumors also aged below 50 years (77.78%) or were premenopausal in status (73.33%). Retrospectively, WBP2 demonstrated sensitivity of 80% to 81% and specificity of 76.5% to 80% in discriminating between patients showing pCR and non-pCR. WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab-based neoadjuvant chemotherapy.

Integrated Analysis of RNA and DNA from the Phase III Trial CALGB 40601 Identifies Predictors of Response to Trastuzumab-Based Neoadjuvant Chemotherapy in HER2-Positive Breast Cancer

Purpose: Response to a complex trastuzumab-based regimen is affected by multiple features of the tumor and its microenvironment. Developing a predictive algorithm is key to optimizing HER2-targeting therapy.Experimental Design: We analyzed 137 pretreatment tumors with mRNA-seq and DNA exome sequencing from CALGB 40601, a neoadjuvant phase III trial of paclitaxel plus trastuzumab with or without lapatinib in stage II to III HER2-positive breast cancer. We adopted an Elastic Net regularized regression approach that controls for covarying features within high-dimensional data. First, we applied 517 known gene expression signatures to develop an Elastic Net model to predict pCR, which we validated on 143 samples from four independent trials. Next, we performed integrative analyses incorporating clinicopathologic information with somatic mutation status, DNA copy number alterations (CNA), and gene signatures.Results: The Elastic Net model using only gene signatures predicted pCR in the validation sets (AUC = 0.76). Integrative analyses showed that models containing gene signatures, clinical features, and DNA information were better pCR predictors than models containing a single data type. Frequently selected variables from the multiplatform models included amplifications of chromosome 6p, TP53 mutation, HER2-enriched subtype, and immune signatures. Variables predicting resistance included Luminal/ER+ features.Conclusions: Models using RNA only, as well as integrated RNA and DNA models, can predict pCR with improved accuracy over clinical variables. Somatic DNA alterations (mutation, CNAs), tumor molecular subtype (HER2E, Luminal), and the microenvironment (immune cells) were independent predictors of response to trastuzumab and paclitaxel-based regimens. This highlights the complexity of predicting response in HER2-positive breast cancer. Clin Cancer Res; 24(21); 5292-304. ©2018 AACR.