Screening, docking, and molecular dynamics analysis of Mitragyna speciosa (Korth.) compounds for targeting HER2 in breast cancer

Trastuzumab Beyond Progression in Advanced Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer: UK Practice now and in the Future

Trastuzumab beyond progression: a challenge to translational oncology?

This issue of the Journal contains two important articles that discuss human epidermal growth factor receptor 2 (HER2), also known as c-erbB2 or neu, as a therapeutic target in breast cancer (1,2). The first details preclinical studies that examine the combination of trastuzumab with a number of chemotherapeutic agents. The second applies the knowledge learned in the preclinical studies to the clinical setting and the treatment of patients with HER2-positive metastatic breast cancer. These studies are important with regard to what they tell us about HER2positive breast cancer and its treatment and about a style of research. All have important implications. HER2 is a member of a family of transmembrane receptor tyrosine kinases. To summarize more than two decades of research, HER2 lacks a functioning ligand-binding domain yet represents the preferred dimerization partner for other members of the EGFR family (3,4). Since the late 1980s, HER2 has been known to be overexpressed in the tumors of approximately 20% of patients with breast cancer. Overexpression is typically a consequence of amplification at the DNA level (measurable in the clinic by fluorescence in situ hybridization [FISH]) and is associated with an increased risk of relapse and death for patients with early-stage breast cancer (5). The poor prognosis is the clinical manifestation of the many biologic actions of HER2: increased proliferation, increased cell survival, increased invasion and metastasis, and increased angiogenic activity (6,7). In addition, amplification of HER2 results in impaired response to hormonal manipulations through cross-talk with the estrogen receptor complex (8,9). Although not the subject of this editorial, co-blockade of the estrogen receptor and of either HER2 or EGFR is currently being tested in clinical trials as a potential means of abrogating or preventing hormonal resistance. Similarly, given the role of HER2 as an upstream regulator of vascular endothelial growth factor (VEGF), co-blockade of HER2 and VEGF is also currently under exploration in clinical trials (10). Recognition of the clinical importance of HER2 in breast cancer led to the development in the 1990s of agents that target HER2, in particular, to the development of the humanized monoclonal antibody trastuzumab. In 1998, trastuzumab was approved by the U.S. Food and Drug Administration for clinical use largely on the basis of a randomized clinical trial (11) that compared chemotherapy to chemotherapy plus trastuzumab as a front-line treatment for patients with metastatic breast cancer. This randomized trial demonstrated unequivocally that the addition of trastuzumab to chemotherapy with either doxorubicin plus cyclophosphamide or paclitaxel resulted in increased survival for women with HER2-positive metastatic breast cancer (11). The introduction of any new agent into the breast cancer arena is typically followed by a “feeding frenzy” in which clinical trialists scurry to combine the new agent with existing agents. Such “toothpaste A toothpaste B” combinations litter the medical literature, rarely have any biologic basis, and arguably retard rather than propel the rational development of the new agent. Empiric combination therapy has specific perils beyond mere wastefulness. Indeed, as the HER2 story demonstrates, the combination of trastuzumab with doxorubicin-based chemotherapy was associated with an unacceptably heightened risk of congestive cardiomyopathy (11). This interaction, not detected in initial preclinical toxicology studies, was later found to have a sound biologic basis. HER2 proved to have an important anti-apoptotic role for normal cardiac myocytes, interruption of which leads to increased stress-related cardiac damage (12). This is where the first article by Pegram et al. (1) in this issue of the Journal comes in. At UCLA, Pegram et al. (1) carefully analyzed the combination of trastuzumab with numerous chemotherapeutic agents, using multiple drug effect/combination index isobologram analysis. This approach allows for the demonstration of synergy in the preclinical setting and, as applied in their study, demonstrated synergy in an in vitro model for the two-drug combinations of trastuzumab with carboplatin, 4-hydroxycyclophosphamide (the active metabolite of cyclophosphamide), docetaxel, or vinorelbine. Of these four synergistic compounds, carboplatin was of particular interest. The same authors had previously demonstrated a synergistic interaction between cisplatin and the murine precursor of trastuzumab 4D5 (13). Platinating agents have known single-agent activity in metastatic breast cancer (14–16), combine readily with taxanes (17), and lack the cardiotoxic effects of anthracyclines. Previous work (18) demonstrated that antibodies to the HER2/neu receptor block DNA repair after exposure to cisplatin in HER2-positive human breast cancer cells. Pegram et al. (1) similarly now demonstrate that trastuzumab markedly reduces unscheduled DNA synthesis (a marker of DNA repair) in carboplatin-treated HER2-positive SK-BR-3 breast cancer cells. In addition, they show that the combination of carboplatin with docetaxel was markedly synergistic in the combination index analysis. These in vitro studies led directly to two human in vivo studies, one performed by the Breast Cancer International Research Group (BCIRG) and the other by the UCLA Oncology Research Network (UCLA-ORN) (2). The former trial combined

Screenning Bioactive Compounds from Allium sativum as HER2 Inhibitors Targeting Breast Cancer by Docking Methods

IntroductionThe Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives.MethodsEpidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study.ResultsOf kConFab's first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFab's fresh tissue bank includes 253 specimens of breast or ovarian tissue – both normal and malignant – including 126 from carriers of BRCA1 or BRCA2 mutations.ConclusionThese kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected.

Purpose Basal-like breast cancer, as defined by gene expression profiling, is associated with aggressive phenotype and poor clinical outcome. Recent immunohistochemical validation suggested that basal-like subtype could be characterized by staining for cytokeratin (CK) 5/6 and epidermal growth factor receptor (EGFR) in triple negative breast cancers (TNBCs). Most of studies evaluated surrogate immunopannel of biomarkers to define basal-like breast cancer subtypes only in the TNBCs, although not all basal-like breast cancers are triple negative breast cancers (TNBCs). The significance of basal marker expression in other than triple negative breast cancer remains to be evaluated. To define prognostic impact of basal marker expression in HER2 positive breast cancer, we investigated cytokeratin (CK) 5/6 and epidermal growth factor receptor (EGFR) expression in patients with HER2 positive early breast cancer. Patients and Methods: Biomarker evaluation was performed using five immunohistochemical surrogate panel of estrogen receptor (ER), progesterone receptor (PR), HER2, CK 5/6 and EGFR in HER2 positive early breast cancers. Amplification of HER2 was confirmed by fluorescent in situ hybridization. HER2-positive breast cancer was classified by expression of basal markers (either EGFR or CK5/6) as “basal HER2- positive” (patients with HER2- positive disease who express basal markers) and “non-basal HER2” (patients with HER2-positive disease who did not express basal markers). We compared the prognostic significance of the basal marker expression between two groups. RESULTS: HER2 overexpression was found in 24.8% of early breast cancers with available tissue specimens from the primary tumor (236 of 952 cases). Basal marker co-expression was identified in 12.7% of HER2 positive early breast cancers. (30 of 236 patients) Basal HER2 positive breast cancer was significantly associated with age greater than 50 years (P=0.012), absence of ER (P < 0.001) and PR (P=0.004). The basal marker co-expression in patients with HER2 amplified early breast cancers demonstrated poorer overall survival (basal positive vs. basal negative, 85.6 months [95% confidence interval (CI), 70.8- 100.3 months] vs. 122.3 [C.I. 133.7-139.9], P=0.001) and disease free survival (DFS) (44.6 months [95% CI, 14.7-74.8] vs 110.7 months [95% CI, 96.4-123.9]; P=. 008) respectively. In lymph node positive group, basal marker expression retained its statistical significance at the multivariate level (P=0.047) HER2 positive breast cancer with lymph node involvement with basal marker expression showed substantially poorer overall survival with 2.1-fold (95% CI, 1.0-4.2) risk for death. CONCLUSION: Considerable number of HER2 positive breast cancer co-expressed basal markers. Our data demonstrated that simultaneous basal marker expression in HER2 positive early breast cancer is associated with poor clinical outcome. The molecular significance of basal marker expression in HER2 positive breast cancer needs to be further investigated. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-03.

Background: Human epidermal growth factor receptor-2 (HER2)-positive breast cancer is a rapidly growing cancer that has poor outcomes if left untreated. Generally, this cancer requires treatment with aggressive chemotherapy and HER2-targeting therapies. However, there is increasing evidence in randomized clinical trials (RCTs) that chemotherapy-free regimens can improve outcomes of HER2-positive early breast cancer while maintaining high oncogenic safety. Objective: To examine the efficacy and safety of chemotherapy-free regimens in patients with HER2-positive early breast cancer. Methods: A systematic literature search for randomized trials published until July 2024 was conducted on PubMed, CENTRAL, Google Scholar, and Embase. Trials were eligible if they compared chemotherapy-free HER2-targeted therapies to HER2-targeted therapies combined with chemotherapy in women with early-stage HER2-positive breast cancer. Conversely, trials that examined chemotherapy-free regimens in advanced HER2-positive breast cancer and observational studies were excluded. The primary endpoint of our study was pathological complete response (pCR), and the secondary outcomes were event-free survival (EFS), invasive Disease-free survival (iDFS), and discontinuation from treatment due to drug-related adverse events. EFS encompassed both relapse-free survival and progression-free survival. All outcomes were pooled together using the random-effects model. Moreover, subgroup analysis was performed to outline the effect of each chemo-free therapy on pCR and assess survival outcomes over different assessment periods. Furthermore, we examined the impact of hormone receptor (HR) status on pCR in patients receiving chemo-free therapies. Results: Six randomized trials, including 1976 patients with early-stage HER2-positive breast cancer, met the inclusion criteria for this meta-analysis. The pooled results showed that about 35.5% (269/757) of patients receiving chemo-free therapies achieve pCR. However, the subgroup analysis revealed that patients treated with trastuzumab plus pertuzumab and those treated with trastuzumab emtansine plus pertuzumab had significantly inferior pCR than those receiving chemotherapy combined with anti-HER2 drugs (OR: 0.22, p=0.0002 and OR: 0.69, p=0.04). Additionally, the pooled results showed that among patients receiving chemo-free therapies, those with HR-positive disease had inferior pCR than those with HR-negative disease (OR: 2.92, p=0.05). The subgroup analyses also demonstrated that patients receiving chemo-free therapies have high 3-year and 5-year iDFS (96.84% and 87%) and EFS (94.75% and 81.13%). Regarding safety, the pooled results revealed that the discontinuation rates due to adverse events were statistically similar between the two groups (OR: 0.69, p=0.55). Discussion/Conclusion: Chemotherapy-free regimens demonstrate promising responses and survival outcomes in patients with HER2-positive early breast cancer. However, combining anti-HER2 drugs with chemotherapy still demonstrates better clinical outcomes. Therefore, omitting chemotherapy outside clinical trials might expose patients to unnecessary risks. Moreover, pCR was more significant in patients with HR-negative disease, suggesting that chemo-free therapies might be more beneficial in these patients. Citation Format: Zaheer Qureshi, Abdur Jamil, Faryal Altaf, Rimsha Siddique, Mohsin Ahmad, Moazzam Shahzad. Efficacy and Safety of Chemo-Free Regimens in HER2-Positive Early Breast Cancer: A Comprehensive Systematic Review and Meta-Analysis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-11-13.

Breast cancer metastasis, when cancer cells move and establish tumors in distant organs, confounds treatment options. Therefore, there is an unmet need for targeted therapeutics to address metastasis, especially in the intractable HER2 positive (+) breast cancer type. HER2 (+) breast cancer is treated with the monoclonal HER2 antibody trastuzumab and the HER2/EGFR targeting small molecule lapatinib. However, ˜20% of early stage breast cancer patients and ˜70% of patients with metastatic disease are resistant to trastuzumab. Trastuzumab-resistant breast cancers circumvent HER2 inhibition via bypass signaling, which includes the PI3-K/Akt/mTOR pathway. HER2/EGFR and PI-3K/Akt signaling activate the Rho GTPases Rac and Cdc42, and overexpression of Rac or its downstream effector p21-activated kinase (PAK) significantly diminishes response to anti HER2 therapy and patient survival. Therefore, targeting Rac and Cdc42, pivotal regulators of cancer cell migration/invasion, and thus, metastasis, is a viable option to overcome HER2/EGFR therapy resistance. First, we designed and developed the Rac inhibitor Ehop-016 (US patents 8,884,006 B2, 9,278,956B1), which inhibits Rac activation with an IC50 of 1 μM and reduces breast cancer growth and metastasis in mouse models of trastuzumab resistant HER2 (+) breast cancer. A more potent dual Rac and Cdc42 inhibitor, MBQ-167 (US Patent 9,981,980 B2), inhibits Rac and Cdc42 activation with IC50s of 103 nM and 78 nM, respectively. In vivo, MBQ-167 inhibits trastuzumab resistant HER2 (+) mammary tumor growth and metastasis by ˜90-100% (Humphries-Bickley et al. 2017, Mol Cancer Therap). Therefore, Rac/Cdc42 inhibition blocks cell proliferation, cell cycle progression, migration, and induces apoptosis to ultimately impede tumor growth and metastasis in trastuzumab resistant HER2 (+) breast cancer models. To further investigate the role of Rac/Cdc42 inhibitors in overcoming HER2 therapy resistance, we developed a lapatinib resistant variant of the SKBR3 HER2 (+) breast cancer cell line and found that Rac was overexpressed and over activated in the therapy resistant variant compared to the parental cells. Accordingly, the Rac/Cdc42 inhibitors overcame lapatinib resistance by decreasing cell viability and inducing apoptosis in parental and SKBR3 therapy resistant variants. Pharmacokinetic analysis in mouse plasma demonstrated that the bioavailability of EHop-016 and MBQ-167 is ˜30% with a half-life of 3-4 h. Therefore, to increase the bioavailability of Rac/Cdc42 inhibitors and to facilitate sensitization of trastuzumab therapy, we designed and developed novel nanoliposomal formulations containing Rac/Cdc42 inhibitors with trastuzumab on the outer surface to target HER2 (+) breast cancer cells. This data demonstrates the utility of developing Rac/Cdc42 inhibitors as mono or combined therapy with current targeted therapeutics for HER2 (+) breast cancer. Citation Format: Rivera-Robles MJ, Medina-Vlazquez JI, Borrero-Garcia LD, Velazquez-Vega LE, Maldonado MdM, Ruiz-Calderon J, Castillo-Pichardo L, Vivas-Mejia P, Dharmawardhane S. Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-14.

e13026 Background: The vascular endothelial growth factor (VEGF) pathway plays a crucial part in tumor angiogenesis by enhancing the creation of new blood vessels that supply oxygen and nutrients to malignancies. Breast cancer cells with overexpressed human epidermal growth factor receptor 2 (HER2) usually produce high levels of VEGF because HER2 signaling upregulates VEGF expression, which generates a feedback loop that promotes tumor progression. Therefore, this cross-talk between VEGF and HER2 pathways suggests the need for dual inhibition to prevent tumor growth signals and angiogenesis simultaneously. Methods: To investigate the clinical benefit of VEGF inhibitors combined with HER2 inhibitors in the treatment of breast cancer. A systematic search for records authored in English and published from January 1990 until January 2025 was conducted in PubMed, Web of Science, MEDLINE, Scopus, and Google Scholar. Clinical trials that evaluated the outcomes of VEGF inhibitors combined with HER2 inhibitors alone without chemotherapy and other cancer treatments were included. The primary endpoint of the present review was the overall response rate (ORR), and the secondary endpoints were complete response (CR) and partial response (PR). Results: Five distinct clinical trials enrolling 307 women with HER2-positive breast cancer were included in the present meta-analysis. The trials included adult women with advanced/metastatic HR+ breast cancer, locally advanced stage III/IV HR+ breast cancer, as well as women with relapsed or refractory HER2 positive breast cancer. Out of the Her2-positive cohort, 47% were HR+, and 53% were HR-. The pooled analysis revealed that the ORR of breast cancer patients treated with anti-HER2 combined with anti-VEGF was 31.9% (95% confidence interval (CI): 21.6% to 44.2%). Moreover, 4.9% of patients treated with anti-HER2 combined with anti-VEGF achieved CR, and 32.6% achieved PR. Data from two included trials also showed that patients treated with lapatinib and pazopanib had significantly higher response rates than patients receiving lapatinib alone (OR: 2.21; 95% CI: 1.15 to 4.22; p = 0.017). Conclusions: Overall, dual inhibition of HER2 and VEGF without chemotherapy is a promising therapeutic strategy for patients with breast cancer, especially those with HER2-overexpressing breast cancer.

Background HER3 (human epidermal growth factor receptor 3) is a member of HER family, and overexpressed in breast cancer, NSCLC, melanoma, gastric cancer and pancreatic cancer patients` tissues. U3-1402a is an antibody-drug conjugate (ADC) comprised of a fully human anti-HER3 monoclonal immunoglobulin G1 (IgG1) antibody (U3-1287) covalently conjugated via a cleavable peptide linker to exatecan derivative (DXd). The DXd is released after internalization of U3-1402a and leads to apoptosis of the target tumor cells by the inhibition of topoisomerase I. This ADC achieves a high drug-to-antibody-ratio (DAR 7 to 8) with homogeneous conjugation with the topoisomerase I inhibitor. The aim of this study was to preclinically evaluate the efficacy of U3-1402a in breast cancer models. Materials and methods In order to evaluate the pharmacological potential of U3-1402a, in vitro and in vivo studies were performed. In vitro growth inhibition assay evaluated the sensitivity of U3-1402a in HER3-positive human breast cancer cell line (HCC1569) and HER3-negative human cervical carcinoma cell line (C33A). Cells were treated with U3-1402a or MAAA-1181 (payload of U3-1402a) depending on its concentration (U3-1402a: 0.153 to 10 000 ng/mL, MAAA-1181: 2.44 to 160,000 pg/mL). In vivo growth inhibition study evaluated the dose-dependent sensitivity of U3-1402a in HER3-positive breast cancer xenograft model, MDA-MB-453. In addition, several xenograft models with different HER3 expression were tested with its sensitivity to U3-1402a. These models were HCC1569 (human breast cancer cell line, HER3 IHC score 3+), MDA-MB-453 (human breast cancer cell line, HER3 IHC score 2+), NIBIO-G016 (human gastric cancer patient-derived xenograft, HER3 IHC score 1+) and MDA-MB-231 (human breast cancer cell line, HER3 IHC score 0). R esults In vitro study, U3-1402a exhibited anti-tumor killing activity in HER3-positive human breast cancer cell line, HCC1569. C-33A human cervical carcinoma cell line was not sensitive to U3-1402a even MAAA-1181 itself exhibited anti-tumor killing activity to this cell line. In vivo study, U3-1402a showed dose-dependent anti-tumor killing activity in a HER3-positive breast cancer MDA-MB-453 xenograft model. Finally, in vivo tumor regression was only observed in HER3 2+ and 3+ models. Conclusions U3-1402a preclinically exhibited its efficacy in breast cancer model in vitro and in vivo. In vivo efficacy was correlated with HER3 expression. These studies suggest that U3-1402a, a novel HER3-targeting ADC, would be efficacious in a broader patient population with HER3 expression like breast cancer, melanoma, NSCLC, gastric cancer and pancreatic cancer. Citation Format: Suguru Ueno, Kenji Hirotani, Reimar Abraham, Sabine Blum, Birgit Frankenberger, Mauricio Redondo-Muller, Johannes Bange, Yusuke Ogitani, Akiko Zembutsu, Koji Morita, Takashi Nakada, Shuji Majima, Yuki Abe, Toshinori Agatsuma. U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2017-3092

<p dir="ltr">The biomarker status in breast cancer is crucial for treatment decisions in breast cancer diagnostics. In clinical routine, estrogen receptor (ER), progesterone receptor (PR), Ki67 proliferation index, and human epidermal growth factor receptor 2 (HER2) are routinely analyzed. This thesis focuses on HER2, with in-depth investigations of diagnostic procedures, how to refine future HER2 routine diagnostics, investigations on new methods for HER2 scoring, and research about biological patterns in HER2-positive breast cancer.</p><p dir="ltr">In paper I, the importance of repeated testing of HER2 and other biomarkers were investigated retrospectively by reviewing core needle results compared to the corresponding surgical specimens. As a result of this study, the Swedish breast cancer guidelines issued a new recommendation to perform an assessment of HER2 biomarkers on both core needle biopsies and surgical specimens.</p><p dir="ltr">In paper II, breast tumor diagnostics and treatment data were compared within and between pathology departments in Sweden. Results generally showed good agreement between laboratories for HER2, ER, and PR, but minor discrepancies between HER2 and ER are of clinical importance since those biomarkers are predictive of therapy. There was a variability for Ki67 and tumor grade. The results showed that refined Ki67 assessment might support Ki67 scoring, including image analysis. This paper showed that following pathology reporting results over time might reveal variability that could be fast-detected and thereby reduce the risk of variances in treatment between different departments in Sweden. Even small differences between laboratories and pathologists are of high relevance for HER2-positive patients.</p><p dir="ltr">In Paper III, we manually investigated HER2 biomarker levels in re-stained and re-scored HER2 in situ hybridization (ISH) slides with manual scoring and a digital image analysis algorithm. In addition, we investigated the ERBB2 mRNA levels using RT-PCR. The biomarker levels were examined continuously using Cutoff Finder and STEPP analysis, and the material was divided into three subgroups: low, intermediate, and high biomarker levels of HER2. We found significant differences in prognosis between patients with tumors with different levels of HER2. Those with tumors with low and high levels had a worse prognosis than those with intermediate levels, which showed that these patients might need additional or more effective targeted treatment.</p><p dir="ltr">In Paper IV, we investigated biological patterns in HER2-positive breast cancer and related them to prognosis. By performing unsupervised agglomerative hierarchical clustering, we found four differentially enriched tumor pattern subgroups in the cohort. The subgroup with low stromal markers and low immune markers had a worse prognosis. These results showed that the tumor microenvironment plays a crucial role in treatment response, which should be considered in future studies of HER2-positive breast cancer.</p><p dir="ltr">In conclusion, all papers have addressed HER2 diagnostics in breast cancer but from different angles. We provided important information for improving HER2 diagnostics and revealed patterns in HER2 tumor biology to consider in future studies.</p><h3>List of scientific papers</h3><p dir="ltr">I. Robertson S, <b>Rönnlund C,</b> de Boniface J, Hartman J. Re-testing of predictive biomarkers on surgical breast cancer specimens is clinically relevant. Breast Cancer Research and Treatment. 2019 Apr;174(3):795-805. <a href="https://doi.org/10.1007/s10549-018-05119-2" rel="noreferrer" target="_blank">https://doi.org/10.1007/s10549-018-05119-2</a></p><p dir="ltr">II. Acs B*, Fredriksson I*, <b>Rönnlund C,</b> Hagerling C, Ehinger A, Kovács A, Røge R, Bergh J, Hartman J. Variability in Breast Cancer Biomarker Assessment and the Effect on Oncological Treatment Decisions: A Nationwide 5-Year Population-Based Study. Cancers. 2021 Mar;13(5):1166. *Shared first authorship, equal contributions. <a href="https://doi.org/10.3390/cancers13051166" rel="noreferrer" target="_blank">https://doi.org/10.3390/cancers13051166</a></p><p dir="ltr">III. <b>Rönnlund C,</b> Sifakis EG, Schagerholm C, Yang Q, Karlsson E, Chen X, Foukakis T, Weidler J, Bates M, Fredriksson I, Robertson S*, Hartman J *. Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer. Breast Cancer Research. 2024 Feb;26(1):24. *Shared last authorship, equal contributions. <a href="https://doi.org/10.1186/s13058-024-01779-9" rel="noreferrer" target="_blank">https://doi.org/10.1186/s13058-024-01779-9</a></p><p dir="ltr">IV. <b>Rönnlund C*</b>, Yang Q*, Schagerholm Stanev C, Chen X, Foukakis T, Fredriksson I, Robertson S, Sifakis EG ** , Hartman J **. Prognostic potential of molecular-derived subgroups in early HER2-positive breast cancer. *Shared first authorship, equal contributions. ** Shared last authorship, equal contributions. [Manuscript]</p>

<p dir="ltr">The biomarker status in breast cancer is crucial for treatment decisions in breast cancer diagnostics. In clinical routine, estrogen receptor (ER), progesterone receptor (PR), Ki67 proliferation index, and human epidermal growth factor receptor 2 (HER2) are routinely analyzed. This thesis focuses on HER2, with in-depth investigations of diagnostic procedures, how to refine future HER2 routine diagnostics, investigations on new methods for HER2 scoring, and research about biological patterns in HER2-positive breast cancer.</p><p dir="ltr">In paper I, the importance of repeated testing of HER2 and other biomarkers were investigated retrospectively by reviewing core needle results compared to the corresponding surgical specimens. As a result of this study, the Swedish breast cancer guidelines issued a new recommendation to perform an assessment of HER2 biomarkers on both core needle biopsies and surgical specimens.</p><p dir="ltr">In paper II, breast tumor diagnostics and treatment data were compared within and between pathology departments in Sweden. Results generally showed good agreement between laboratories for HER2, ER, and PR, but minor discrepancies between HER2 and ER are of clinical importance since those biomarkers are predictive of therapy. There was a variability for Ki67 and tumor grade. The results showed that refined Ki67 assessment might support Ki67 scoring, including image analysis. This paper showed that following pathology reporting results over time might reveal variability that could be fast-detected and thereby reduce the risk of variances in treatment between different departments in Sweden. Even small differences between laboratories and pathologists are of high relevance for HER2-positive patients.</p><p dir="ltr">In Paper III, we manually investigated HER2 biomarker levels in re-stained and re-scored HER2 in situ hybridization (ISH) slides with manual scoring and a digital image analysis algorithm. In addition, we investigated the ERBB2 mRNA levels using RT-PCR. The biomarker levels were examined continuously using Cutoff Finder and STEPP analysis, and the material was divided into three subgroups: low, intermediate, and high biomarker levels of HER2. We found significant differences in prognosis between patients with tumors with different levels of HER2. Those with tumors with low and high levels had a worse prognosis than those with intermediate levels, which showed that these patients might need additional or more effective targeted treatment.</p><p dir="ltr">In Paper IV, we investigated biological patterns in HER2-positive breast cancer and related them to prognosis. By performing unsupervised agglomerative hierarchical clustering, we found four differentially enriched tumor pattern subgroups in the cohort. The subgroup with low stromal markers and low immune markers had a worse prognosis. These results showed that the tumor microenvironment plays a crucial role in treatment response, which should be considered in future studies of HER2-positive breast cancer.</p><p dir="ltr">In conclusion, all papers have addressed HER2 diagnostics in breast cancer but from different angles. We provided important information for improving HER2 diagnostics and revealed patterns in HER2 tumor biology to consider in future studies.</p><h3>List of scientific papers</h3><p dir="ltr">I. Robertson S, <b>Rönnlund C,</b> de Boniface J, Hartman J. Re-testing of predictive biomarkers on surgical breast cancer specimens is clinically relevant. Breast Cancer Research and Treatment. 2019 Apr;174(3):795-805. <a href="https://doi.org/10.1007/s10549-018-05119-2" rel="noreferrer" target="_blank">https://doi.org/10.1007/s10549-018-05119-2</a></p><p dir="ltr">II. Acs B*, Fredriksson I*, <b>Rönnlund C,</b> Hagerling C, Ehinger A, Kovács A, Røge R, Bergh J, Hartman J. Variability in Breast Cancer Biomarker Assessment and the Effect on Oncological Treatment Decisions: A Nationwide 5-Year Population-Based Study. Cancers. 2021 Mar;13(5):1166. *Shared first authorship, equal contributions. <a href="https://doi.org/10.3390/cancers13051166" rel="noreferrer" target="_blank">https://doi.org/10.3390/cancers13051166</a></p><p dir="ltr">III. <b>Rönnlund C,</b> Sifakis EG, Schagerholm C, Yang Q, Karlsson E, Chen X, Foukakis T, Weidler J, Bates M, Fredriksson I, Robertson S*, Hartman J *. Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer. Breast Cancer Research. 2024 Feb;26(1):24. *Shared last authorship, equal contributions. <a href="https://doi.org/10.1186/s13058-024-01779-9" rel="noreferrer" target="_blank">https://doi.org/10.1186/s13058-024-01779-9</a></p><p dir="ltr">IV. <b>Rönnlund C*</b>, Yang Q*, Schagerholm Stanev C, Chen X, Foukakis T, Fredriksson I, Robertson S, Sifakis EG ** , Hartman J **. Prognostic potential of molecular-derived subgroups in early HER2-positive breast cancer. *Shared first authorship, equal contributions. ** Shared last authorship, equal contributions. [Manuscript]</p>

Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor2 (HER2) have been reported to lead to resistance to HER2-targeted therapies in HER2-positive breast cancer, while activating mutations of HER2 have been described in HER2-negative breast cancer. The prevalence, clinicopathological characteristics, and phenotypes of HER2 mutations are not well established, thus we sought to describe the HER2 mutation profile of Chinese breast cancer patients. Methods: DNA samples were gathered from breast cancer patients undergoing neoadjuvant (N=102) or adjuvant therapy (N=498) at Fudan University Shanghai Cancer Center between January 1, 2006 and December 31, 2012. Sanger sequencing was performed to analyze all exons of HER2 to identify somatic mutations. To determine the phenotypes of novel HER2 mutations, in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments were conducted. Results: 10 HER2 somatic mutations were observed in 17 patients (17/600, 2.83%). 7 novel HER2 mutations were uncovered, 4 in the transmembrane domain and 3 in the kinase domain. Kinase domain mutations L768S and V773L were detected in HER2-negative tumors, while K753E was found in HER2-positive disease. In vitro kinase assays found that L768S and V773L exhibited a significant increase of tyrosine kinase-specific activity, while Western blots showed that L768S and V773L strongly increased phosphorylation of all signaling proteins in both MCF10A and MCF7cell lines, indicating that they were activating mutations. In Matrigel cultures, L768S and V773L formed acini when seeded in vehicle, but maintained spherical morphology when seeded in culture containing trastuzumab. The addition of lapatinib in Matrigel culture inhibited the growth of all except K753E, which was successfully inhibited by neratinib. Similarly, L768S, V773L and K753E increased the number of cell colonies formed in soft agar, trastuzumab and lapatinib treatment decreased the number of colonies formed by L768S and V773L, but only neratinib could inhibit the colony growth of K753E. Xenograft showed L768S and V773L displayed a more rapid growth, while K753E showed resistance to lapatinib in vivo. MCF10A cells bearing K753E mutation were found to be resistant to lapatinib (IC50>10,000 nmol/L), but could be inhibited by neratinib, though requiring a relatively higher dosage (IC50 of 32 nmol/L) than HER2 WT (IC50 of 480 nmol/L for lapatinib, <2 nmol/L for neratinib) and other HER2 mutations. Meanwhile, clinical follow-up showed that the 2 patients with K753E mutation who received adjuvant trastuzumab treatment presented with either brain or bone metastasis, in their 3rd and 5th year after initial cancer diagnosis, suggesting K753E mutation may have a role in trastuzumab resistance as well. Conclusions: HER2 somatic mutations were found in 2.83% of patients in this study. HER2-positive tumors harboring certain HER2 kinase domain resistance mutations may not benefit from trastuzumab or lapatinib treatment, and neratinib may offer an alternative treatment option for these patients. HER2-negative disease with activating mutations may benefit from HER2-targeted therapies, and may be of interest in prospective clinical trials. Citation Format: Wen-Jia Zuo, Yi-Zhou Jiang, Ke-Da Yu, Zhi-Ming Shao. Activating HER2 mutations promote oncogenesis and resistance to HER2-targeted therapies in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-04.

Introduction: Poziotinib is a novel, pan-HER kinase inhibitor which showed potent anti-tumor activities through irreversible inhibition of HER family tyrosine kinases in preclinical and early clinical studies. Recent the open-label, multicenter phase II trial of poziotinib monotherapy evaluated that poziotinib is a new promising option for patients with HER2-positive metastatic BC who have failed more than two HER2 targeted therapy (NCT02418689). We evaluated genetic profiles of HER2-positive metastatic BC and investigated potential biomarkers of poziotinib for HER2-positive metastatic BC (MBC). Methods: All participants were diagnosed as HER2-positive BCs according to American Society of Clinical Oncology/College of American Pathologists HER2 guideline and provided tissue specimens that would be possible to extract DNA and RNA for next generation sequencing. We performed targeted deep sequencing with a customized 381 cancer gene panel (CancerSCAN™) and analyzed the relationship among the sequencing data, immunohistochemistry and clinical outcome. Results: From Apr 2015 to Feb 2016, 106 patients were enrolled in the trial from 7 institutes in Korea. Of 106 patients, biomarker data were available for 79 patients. TP53 was the most frequently mutated gene (70.8%) followed by PIK3CA (45.6%). HER2 single nucleotide variant (SNV) was detected in 13 BCs (16.5%) and HER3 SNV was in 9 (11.4%). The score of HER2 immunohistochemistry (IHC) was 3+ in 68 BCs and 2+ with positive in situ hybridization in 11 BCs. In copy number variant (CNV) analysis, HER2 amplification (86.1%) was most frequently observed and followed by CDK12 amplification (58.2%) and APOBEC3B deletion (30.4%). IHC score of HER2 was positively correlated to copy number (CN) of HER2 (P=0.001) but 11 breast cancer tissue did not have copy number amplification of HER2 (13.9%) (Six of HER2 IHC score 2+ and 5 of 3+). The median progression free survival (PFS) was 4.04 months (95% CI, 2.96 - 4.40) for patients who treated with poziotinib in this study. PIK3CA activating mutations were associated with short PFS compared to wild type (WT) and other SNVs (Median PFS of activating mutations vs. WT and others: 2.66 vs. 4.40 (months), P=0.009). HER2 CN amplification was positively correlated to duration of PFS (Median PFS of no amplification vs. 4 ≤ CN < 16 vs. 16 ≤ CN: 2.56 vs. 3.02 vs. 4.86 (months), P=0.032). HER2 SNVs prolonged duration of PFS without statistical significance (Median PFS of HER2 SNVs vs. WT: 4.24 vs. 3.19 (months), P=0.114), but 10 of 13 BCs with HER2 SNV (76.9%) had clinical benefit from poziotinib and 5 BCs (38.5%) had durable response more than 6 months. Conclusion: In this biomarker analysis, SNV of HER2 was frequently observed in HER2 positive MBCs and HER2 CN amplification was detected not in all. High CN amplification of HER2 derived longer PFS than those with low CN. To contrary to this, activating PIK3CA mutations shorten PFS compared to those with WT. In addition, HER2 SNVs might be a potential biomarker of poziotinib in HER2-positive MBC. Further functional study would be warranted. Citation Format: Kim J-Y, Lee E, Park K, Jung HH, Park W-Y, Lee K-H, Sohn JH, Lee KS, Jung KH, Kim J-H, Lee KH, Im S-A, Park YH. Molecular alterations and poziotinib, a pan-HER inhibitor efficacy in human epidermal growth factor receptor 2(HER2) positive breast cancers: Combined exploratory biomarker analysis from phase II clinical trial of poziotinib for refractory HER2 positive breast cancer(BC) patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-21.

Background: Amphiregulin is a ligand for the epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor 2 (HER2) shares common signal pathways and forms a heterodimer with EGFR. In this study, we investigated the effect of amphiregulin on trastuzumab therapy in HER2-positive breast cancer. Methods: We analyzed serum amphiregulin levels by enzyme-linked immunosorbent assay (ELISA) from baseline serum samples obtained from HER2-positive metastatic breast cancer patients who received first-line trastuzumab plus taxane chemotherapy. In addition, in vitro experiments were performed to elucidate the biologic mechanism of clinical findings related to amphiregulin using SK-BR-3 and BT-474 cell lines. Results: Between October 2004 and July 2009, a total of 50 women with HER2-positive metastatic breast cancer were consecutively enrolled. The median age was 47 years (range, 27-72 years). Eighteen patients (36.0%) received weekly paclitaxel plus trastuzumab, 24 patients (48.0%) tri-weekly paclitaxel plus trastuzumab, and 8 patients (16.0%) tri-weekly docetaxel plus trastuzumab. Among 43 patients with measurable lesions, the response rate (RR) was 76.7%. The median follow-up duration was 29.2 months (range, 0.7-63.3 months). The median progression-free survival (PFS) was 17.6 months (95% confidence interval (CI), 13.4-21.9 months). The median overall survival (OS) was 47.0 months (95% CI, 35.3-58.6 months). The median serum amphiregulin level was 1.0 ng/mL with a maximum level of 4.4 ng/mL. Patients with high serum amphiregulin levels (≥0.5 ng/mL) had significantly shorter PFS (p=0.018) along with a tendency toward lower RR (p=0.237) and shorter OS (p=0.529) than the others. The in vitro colony forming assay demonstrated that the addition of amphiregulin resulted in increased proliferation of both SK-BR-3 and BT-474 cells. In addition, the anti-proliferative effect of trastuzumab was decreased in the presence of amphiregulin in both SK-BR-3 and BT-474 cells. The Western blot analysis showed that amphiregulin increased the phosphorylation of Akt and its downstream molecules in both SK-BR-3 and BT-474 cells. In addition, in the presence of amphiregulin, sustained phosphorylation of Akt and its downstream molecules was observed after trastuzumab treatment in both SK-BR-3 and BT-474 cells. Conclusions: High serum amphiregulin levels (≥0.5 ng/mL) predicted disease progression after first-line trastuzumab plus taxane chemotherapy in patients with HER2-positive metastatic breast cancer. Amphiregulin promoted the proliferation of HER2-positive breast cancer cells in vitro and induced trastuzumab resistance by activating PI3K/Akt pathway. Our results suggest that the measurement of serum amphiregulin levels by ELISA may provide additional information for the clinical outcome of trastuzumab-based chemotherapy in patients with HER2-positive breast cancer. Citation Format: Ji-Won Kim, Young Seok Joung, Ahrum Min, Hyun-Jin Nam, Jee Hyun Kim, Seock-Ah Im, Kyung-Hun Lee, Jin-Soo Kim, Tae-Yong Kim, Sae-Won Han, Yoon Kyung Jeon, Do-Youn Oh, Tae-You Kim, In Ae Park. Amphiregulin confers trastuzumab resistance by activating PI3K/Akt pathway in HER2-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2013-2386