Abnormal HER2 Research Articles

A comparative study of gastric adenocarcinoma HER2 IHC phenotype and mass spectrometry-based quantification.

Gastric cancer is a highly heterogeneous malignant tumor of the digestive system. Anti-HER2 treatment can inhibit downstream signaling pathways and improve clinical treatment and outcomes in patients with HER2 protein overexpression. Currently, two standard methods for evaluating HER2 expression status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these low-throughput assays often produce discordant or equivocal results. In this study, we presented a new HER2 protein detection method based on mass spectrometry selected reaction monitoring (MS-SRM) and validated the method. We conducted a retrospective study on 118 formalin-fixed paraffin-embedded (FFPE) tissues from patients with advanced gastric adenocarcinoma in northern China, and we compared the MS-SRM results with those from IHC and correlated them with FISH. We established and validated the upper and lower detection limits (300-700 amol/μg) for abnormal HER2 protein expression in advanced gastric cancer. We also found that, among samples with mixed Lauren subtypes, those with a high level of HER2 expression had typical intestinal type features in pathology. This study demonstrated that the MS-SRM method can overcome the limitations and deficiencies of IHC, directly quantify the expression of HER2 protein in tumor cells and be used as a supplement to IHC. It has the potential to be used as a companion diagnosis for new drugs used to treat advanced gastric cancer. Large-scale clinical validation is required.

Anticancer Activities of Tetrasubstituted Imidazole-Pyrimidine-Sulfonamide Hybrids as Inhibitors of EGFR Mutants.

A new series of tetrasubstituted imidazole derivatives carrying pyrimidine sulfonamide pharmacophores has been synthesized and evaluated for their anticancer activities. In-vitro screening of these hybrids against a full 60-cell-line panel at a single dose of 10 μM showed significant growth inhibition of up to 95 %. The most active compound showed in-vitro anticancer activities against (i) abnormal HER2 and (ii) two mutants for EGFR. Apoptotic gene expression revealed that lead compounds induced MCF-7 cell line apoptosis together with considerable change in the Bax/Bcl-2 expression ratio. One lead compound led to a significant cell-cycle S-phase arrest, while another blocked the cell cycle at G1/S-phase causing the accumulation of cells. Docking analysis of these two hybrids adopted the orientation and binding interactions with a higher liability to enter the active side pocket of HER2, L858R, and T790 M, preferable to that of co-crystallized ligands. Modelling simulation was consistent with the acquired biological evaluation.

A study of Her-2/Neu oncogene expression in benign and malignant endometrial lesions and its association with clinicopathologic variables

Introduction: Endometrial carcinoma is commonly diagnosed gynaecological malignancy in women and the incidence of endometrial cancer and associated mortality rates have increased in recent years. Endometrial hyperplasia is recognized precursor lesion of endometrial cancer and its timely detection offers an opportunity to prevent the development of endometrial cancer. Her-2/neu oncogene amplification has been implicated in the development and progression of many tumours and its overexpression may be involved in tumorigenesis of endometrial carcinoma as well. The present study was aimed to evaluate Immunohistochemical (IHC) expression of Her-2/neu oncogene in Benign and Malignant Endometrial Lesions and its correlation with clinicopathological features in order to define its potential prognostic significance. Material and Methods : A total of 62 cases including 20 cases of endometrial hyperplasia and 42 cases of Endometrial Carcinoma were enrolled in the study. Clinical and pathologic information was obtained and recorded. Her-2/neu expression was evaluated by immunohistochemistry (IHC) on paraffin embedded tissue sections with Her-2/neu antibody. Overexpression was defined as complete, membrane, moderate to strong intensity staining in greater than 10% of cells. Results: Positive HER-2/neu expression is seen in 45% cases of Endometrial Hyperplasia and in 54.76% cases of endometrial carcinoma. The Her-2/neu overexpression was observed in patients with advanced stage (p Conclusion: In the present study Abnormal high Her-2/neu expression was observed in Endometrial carcinoma, correlating with High FIGO stage, high grade, lymph node metastasis, myometrial invasion and cervical invasion, indicating Her-2/neu expression analysis could be an important prognostic factor of Endometrial carcinoma. Keywords: Endometrial carcinoma, Endometrial Hyperplasia, HER­2/neu, prognostic factor

Abstract OT-28-04: Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test

Abstract Background: While HER2 targeted therapeutics have significantly improved outcomes of patients with HER2 overexpressed advanced breast cancer (ABC), c-Met pathway activation serves as a resistance mechanism in about 60% of patients. Neratinib is an irreversible tyrosine kinase inhibitor of the ErbB family of receptors (EGFR, HER2, and HER4) and tepotinib is a tyrosine kinase inhibitor of c-Met. From live extracted cells from patient biopsies, the CELsignia Pathway Activity Test measures in real-time ErbB and c-Met signaling activity and the dynamic response to pathway inhibition by targeted therapy ex vivo. The CELsignia test may identify patients with ABC who would benefit from the combination of neratinib and tepotinib regardless of the molecular breast cancer subtype. Trial Design: We designed a phase Ib/II multi-center, non-randomized, open-label study of combination of neratinib with tepotinib in patients with advanced breast cancer including those with inflammatory breast cancer. The study sites include UT MD Anderson Cancer Center, City of Hope, and The Ohio State University Cancer Center. Four dose levels of neratinib, and two dose levels of tepotinib will be studied in phase Ib to determine the maximum tolerated dose (MTD). Phase II portion of the study will test the early efficacy of neratinib and tepotinib combination in patients with HER2-/HR+ ABC with abnormal HER2 and c-Met signaling activity determined by the CELsignia test. Eligibility: All patients with IBC/ABC will be eligible for the phase Ib. Eligible patients must have measurable metastatic disease per RECIST v1.1 or unresectable disease amenable for biopsy and have had at >1 line of systemic therapy for advanced disease. Patients will have an Eastern Cooperative Oncology Group performance status of 0 to 2. For the phase II component, patients must have hyperactive HER2 and c-Met signaling determined by CELsignia test. Aims: The primary objective of the phase Ib component is to determine the MTD of the combination in previously treated IBC/ABC of any molecular subtype. The primary objective of the phase II component is to determine the overall response rate (ORR) in patients with HER2-/HR+ ABC who have a positive CELsignia test result (described in the Eligibility). Secondary objectives include progression free and overall survival, clinical benefit rate, duration of response, and safety and tolerability. Exploratory objectives will be to assess HER2 and c-Met driven signaling pathways as either hyperactive or abnormally active by CELsignia test. Statistical Methods: The phase I study will be conducted using a Bayesian optimal interval (BOIN) design to establish the MTD and the recommended phase II dose and assess early efficacy of the combination with a maximum of 21 patients. Two dose levels of tepotinib, and four dose levels of neratinib will be studied. The phase II study will assess the ORR using Bayesian optimal phase 2 (BOP2) design to rule out a 10% ORR in favor of a 30% target ORR, with a power of 80% and two-sided 5% significance level. Accrual: Once MTD is determined, up to 29 evaluable CELsignia test positive subjects will be enrolled to the phase II component of the study and an interim analysis will be performed when the total number of enrolled patients reaches 15. We hope to complete the accrual of patients by July 2022 and to schedule the last patient visit by July 2023. Contact information: For more information or to refer a patient, email blim@mdanderson.org, eahobbs@mdanderson.org Citation Format: Evthokia Hobbs, Eric Lindquist, Brian Sullivan, Esther Yoon, Ying Yuan, Angela Marx, Jie Willey, Huiming Sun, Rachel Layman, Daniel Stover, Yuan Yuan, Bora Lim. Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-28-04.

Abstract 3198: Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy

Abstract Background: Sub-groups of breast, lung, and colon cancer patients with genetic variants associated with dysregulated ErbB signaling benefit from treatment with ErbB targeted therapies. No sub-groups of ovarian cancer patients with actionable ErbB genetic variants have yet been found. Measurement of biological factors other than genetic variants, such as ErbB signaling activity, may identify ovarian cancer patients likely to benefit from ErbB targeted therapies. We have previously reported studies detecting dysregulated ErbB and c-Met signaling activity in 20%-25% of HER2-negative breast cancer patient tumors using the CELx Multi-Pathway (MP) Signaling Function Test. To determine whether ErbB or c-Met signaling dysregulation is involved in ovarian cancer, the CELx MP test was adapted to analyze ovarian tumor cells. The current study set out to: 1) characterize c-Met and ErbB family signaling activity in ovarian patient tumor cells and ovarian tumor cell lines; 2) evaluate in vivo response to pan-HER and c-Met inhibitors in an ovarian tumor xenograft model. Methods: For the ex vivo studies, a set of fresh tumor specimens from 15 ovarian cancer patients (90% high-grade serous, 10% endometrioid) and a set of 12 ovarian cancer cell lines was obtained. Cell samples were cultured from each patient specimen. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) with or without an antagonist (2C4, a HER2 dimerization inhibitor or tepotinib, a c-Met kinase inhibitor) was measured and quantified using an xCELLigence impedance biosensor (Agilent Technologies). Signaling activity above a previously established cut-off value was used to identify abnormal levels of EGFR, HER2 and c-Met signaling activity. For the xenograft study, OVCAR-4, an ovarian cancer cell line, determined to have abnormal HER2, EGFR, and c-Met signaling with the CELx MP Test, was studied. 40 female NSG mice were injected with two million OVCAR-4 cells. Mice were randomly assigned to either a control group that received Captisol excipient or a treatment group that received neratinib, tepotinib, or neratinib and tepotinib for 16 days. Results: Of the patient cell and cell lines samples tested ex vivo with the CELx MP test, 4 of 27 (15%; 95% CI=6%-32%) had hyperactive HER2 and c-Met signaling pathways. In the xenograft study, there was no significant difference in tumor volume between the control and tepotinib-treated or neratinib groups after 16 days of treatment. Tumor volumes relative to tumor size before treatment were reduced by 40% in the tepotinib + neratinib treated group. Conclusions: These findings suggest that a significant sub-group of ovarian cancer patients have abnormal ErbB and c-Met signaling activity that may respond to treatment with a combination of ErbB and c-Met inhibitors. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Stefano Rossetti, Catherine Kuzmicki, Aaron Broege, Joanna Sabat, Salmaan Khan, Ian MacNeil, Brian Sullivan, Lance Laing. Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy [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 3198.

Test identifies ovarian cancer patients with hyperactive c-Met and ErbB signaling tumors who may benefit from c-Met and pan-HER combination therapy.

e18038 Background: No sub-groups of ovarian cancer patients with clinically actionable ErbB genetic variants have yet been found. Measurement of ErbB signaling activity rather than genetic variants may identify ovarian cancer patients likely to benefit from ErbB targeted therapies. We have previously reported studies detecting dysregulated ErbB and c-Met signaling activity in 20%-25% of HER2-negative breast cancer patient tumors using the CELx Signaling Function Test. To determine whether ErbB or c-Met signaling dysregulation is involved in ovarian cancer, the CELx Test was adapted to analyze ovarian tumor cells. The current study set out to: 1) characterize c-Met and ErbB family signaling activity in ovarian patient tumor cells and tumor cell lines; 2) evaluate in vivo response to pan-HER and c-Met inhibitors in an ovarian tumor xenograft model. Methods: For the ex vivo studies, a set of fresh tumor specimens from 15 ovarian cancer patients and a set of 12 ovarian cancer cell lines was obtained. Cell samples were cultured from each. Live cell response to ErbB and c-Met agonists (NRG1b, EGF, or HGF) with or without an antagonist (2C4, a HER2 dimerization inhibitor or tepotinib, a c-Met kinase inhibitor) was measured using an xCELLigence impedance biosensor (Agilent Technologies). Signaling activity above a previously established cut-off value was used to identify abnormal levels of EGFR, HER2 and c-Met signaling activity. For the xenograft study, OVCAR-4, an ovarian cancer cell line, determined to have abnormal HER2, EGFR, and c-Met signaling with the CELx Test, was studied. 40 female NSG mice were each injected with two million cells. Mice were randomly assigned to either a control group or a treatment group that received neratinib, tepotinib, or neratinib and tepotinib for 16 days. Results: Of the patient cell and cell lines samples tested ex vivo with the CELx Test, 4 of 27 (15%; 95% CI = 6%-32%) had hyperactive HER2 and c-Met signaling pathways. In the xenograft study, tumor volume change was 40% less in the tepotinib + neratinib treated group than in the control arm change. There was no significant difference in tumor volume between the control and tepotinib or neratinib groups. Conclusions: These findings suggest that a significant sub-group of ovarian cancer patients have abnormal ErbB and c-Met signaling activity that may respond to treatment with a combination of ErbB and c-Met inhibitors. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted.

New HER2-negative breast cancer subtype responsive to anti-HER2 therapy identified

PurposeHER2 signaling functional activity may be important to measure in addition to HER2 protein quantification when identifying patients eligible for HER2 therapies. A HER2 Signaling Function (CELx HSF) Test for HER2-negative patients uses patient’s live tumor cells on a biosensor to identify patients with abnormally high HER2-related signaling (HSFs+) likely to respond to anti-HER2 therapies.MethodsThe CELx HSF test was employed to: (1) characterize the sensitivity and specificity of the test to detect abnormal levels of HER2 signaling; (2) evaluate the inhibitory effectiveness of five different anti-HER2 therapies; (3) assess the correlation between CELx HSF test detection of abnormal HER2 signaling and response to HER2 therapy using xenograft models; and (4) confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients (HER2−/HSFs+).ResultsHER2−/HSFs+ breast cancer patient samples were identified and showed sensitivity to five approved anti-HER2 therapies. Xenograft studies using both HER2+ and HER2− cell lines confirmed that CELx HER2 signaling status better predicts HER2 inhibitor efficacy than HER2 receptor status. In a study of 114 HER2-negative breast tumor patient samples, 27 (23.7%; 95% CI = 17–32%) had abnormal HER2 signaling (HSFs+). A ROC curve constructed with this dataset projects the CELx HSF Test would have greater than 90% sensitivity and specificity to detect the HER2−/HSFs+ patient population.ConclusionsThe CELx HSF test is a well-characterized functional biomarker assay capable of identifying dynamic HER2-driven signaling dysfunction in tumor cells from HER2-negative breast cancer patients. This test has demonstrated efficacy of various HER2 targeted therapies in live tumor cells from the HSFs+ population and correlated the test result to HER2 drug response in mouse xenograft studies. The proportion of HER2-negative breast cancer patients found to have abnormal HER2 signaling in a 114 patient sample study, 20–25%, is significant. A clinical trial to evaluate the efficacy of anti-HER2 therapies in this patient population is warranted.

Abstract P2-09-22: Use of a functional signal profiling test with high sensitivity and specificity to determine the prevalence of abnormal HER2-driven signaling activity in the HER2-negative breast cancer patient population: New patient group may benefit from HER2 therapy

Abstract Background: Biological factors, such as HER2 signaling activity, may be important to measure in addition to expression and amplification of HER2 when identifying patients eligible for HER2 therapies. The CELx HER2 Signaling Function (CELx HSF) Test measures HER2 signaling activity in live tumor cells using a label-free impedance biosensor to identify HER2-negative breast cancer patients likely to be responsive to treatment with anti-HER2 therapies. Previous studies quantified HER2-driven signaling activity in a training set (N=34) of primary tissue samples from HER2-negative breast cancer patients and found 21% of the samples had abnormal HER2 signaling. Other studies confirmed that anti-HER2 therapies, such as trastuzumab, pertuzumab, afatinib, and neratinib, are as effective in inhibiting HER2-driven signaling activity in HER2- tumor cells as they are in HER2+ tumor cells. This study set out to confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients in a larger sample (N=114) and to characterize the sensitivity and specificity of the CELx HSF Test. Methods: A validation set of de-identified fresh breast tumor specimens were obtained from 114 HER2- breast cancer patients. Real time live cell response to specific HER2 agonists (NRG1b or EGF) with or without an antagonist (HER2 dimerization inhibitor) was measured using an impedance biosensor. From these responses, the net amount of HER2 participation in HER2 signaling initiated by the HER2 agonists was quantified. Samples with HER2 signaling activity levels above a previously determined cut-off value were identified as abnormal. Results: Of the HER2- breast tumor cell samples tested, 27 of 114 patients (23.7%; 95% CI=17%-32%) had abnormal HER2 signaling activity. Little or no correlation was found between a patient's HER2 signaling activity and their estrogen receptor status or tumor grade. To compare the results obtained from the training set of 34 patients and the current set of 114 patients, the Kolmogorov-Smirnov two-sample test was applied (D=0.17, P-value 0.45) and found no significant difference between the training and validation sets. A normal mixture model was fitted to the new 114 patient data set and found that HER2- breast cancer patients fall into three distinct groups (abnormal, normal, low). Patients falling into the abnormal group had mean HER2 signaling scores 4.5 standard deviations above the mean score of the normal group. A ROC curve constructed with this data projects that both the sensitivity and specificity of the CELx HSF Test would be greater than 90%. Conclusions: These results confirm that a clinically relevant proportion of HER2- breast cancer patients, approximately 20%, have tumors with abnormal HER2-signaling activity and may benefit from HER2 therapy. With high specificity and sensitivity, the CELx HSF test may be suitable as a companion diagnostic to identify new patients eligible to receive HER2 therapies. An interventional trial to evaluate the efficacy of trastuzumab and pertuzumab in HER2- patients selected with the CELx HSF test is underway. Citation Format: Laing LG, Burns DJ, MacNeil IA, Rich BE, Myhre S, Soltani S, Sullivan BF. Use of a functional signal profiling test with high sensitivity and specificity to determine the prevalence of abnormal HER2-driven signaling activity in the HER2-negative breast cancer patient population: New patient group may benefit from HER2 therapy [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 P2-09-22.

Measuring functional HER2-driven signaling status ex vivo to predict response to HER2 therapy: Results from a mouse breast tumor xenograft study.

e23203 Background: Clinical trials indicate a weak correlation between HER2 expression levels and HER2 targeted therapy benefit. Other biological factors, such as HER2 signaling activity, may be important to measure when identifying patients for treatment with HER2 therapies. To measure the HER2 signaling activity of a patient’s live tumor cells, a new assay using an impedance biosensor, the CELx HER2 Signaling Function (CELx HSF) Test, was developed. In this study, we evaluated whether functional HER2 signaling status was more predictive of response to a dual-HER2 kinase inhibitor than HER2 receptor or gene amplification status, using breast tumor xenograft in vivo models. Methods: Two breast cancer cell lines were studied: HCC1954, a HER2+ cell line with normal HER2 signaling according to the CELx HSF Test, and BT483, a HER2- cell line with abnormally high HER2 signaling according to the CELx HSF Test. Thus, the HER2 receptor status of each cell line was opposite its HER2 signaling status. For each cell line, twenty 4-5 week old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or a treatment group that received lapatinib at a dose of 20mg/kg daily for 16 days. Results: There was no significant difference in tumor volume between the control and lapatinib-treated groups in the mice injected with the HCC1954 (HER2+, normal HER2 signaling) cells. Average tumor sizes reached 1028.7 ± 166.9 mm3 for the control group and 893.8 ± 111.5 mm3 for the lapatinib-treated group (P = 0.285) by the end of the study. With the BT483 (HER2-, abnormal HER2 signaling) cells, lapatinib treatment significantly slowed down the increase in tumor size. Average tumor sizes reached 328.3 ± 54.9 mm3 for the control group and 192.4 ± 19.4 mm3 for the lapatinib-treated group (P = 0.0049). Conclusions: The results demonstrate that functional HER2-driven signaling status in live tumor cells is more correlative to response to lapatinib in mouse xenograft tumors than HER2 expression level. These findings provide strong evidence that HER2- breast cancer patients with abnormal HER2 signaling may respond to anti-HER2 therapies.

Abstract P6-07-14: New method to measure functional HER2-driven signaling activity in primary tumor cells identifies HER2-negative breast cancers with abnormal HER2 signaling activity: New group of patients may benefit from anti-HER2 therapy

Abstract Background: Clinical trials have indicated a weak correlation between HER2 expression levels and HER2 targeted therapy benefit. Other biological factors, such as HER2 signaling activity, may be important to measure, in addition to expression and amplification of HER2, when identifying patients eligible for HER2 therapies. To measure the HER2-driven signaling activity of a patient's tumor cells, a new assay using an impedance biosensor, the CELx HER2 Signaling Profile (CELx HSP) Test, was developed. This study set out to provide an initial assessment of the CELx HSP Test, specifically to: 1) quantify HER2-driven signaling activity (HER2S) in cell lines and primary epithelial cells; 2) assess the correlation between HER2 expression levels and HER2 signaling activity; 3) define a preliminary cut-point between normal and abnormal HER2 signaling; and 4) estimate the proportion of HER2- primary breast cancer tumors with abnormal HER2 signaling. Methods: A training set of de-identified fresh breast tissue specimens was obtained from 50 patients, 34 with HER2- breast cancer (IHC 0 or 1+) and 16 healthy patients. Cell samples were comprised of epithelial cells extracted and cultured from each specimen. Reference human breast cancer cell lines (9 HER2+, 10 HER2-) were also tested, including two cell lines used as controls in IHC HER2 tests. Real time live cell response to specific HER2 agonists (NRG1b or EGF) and with or without an antagonist (pertuzumab, an FDA-approved HER2 dimerization inhibitor) was measured and quantified using an xCELLigence RTCA impedance biosensor (ACEA Biosciences, San Diego, CA). From these responses, the net amount of HER2 participation in HER2 signaling initiated by the HER2 agonists (“HER2S”) was determined. Fluorescence cytometry was used to measure HER2 expression levels of each cell sample. Results: Of the HER2– breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI=10%-37%) had net HER2 signaling activity that was greater than the median HER2S of the HER2+ cell lines. There was no categorical correlation between HER2 IHC status (+ or -) and HER2 signaling activity (abnormal or normal) (Pearson's Chi-Square = 3.68; Phi Max = -0.78, Contingency Coefficient 0.28). The median HER2S, or net HER2 signaling activity, was comparable for the HER2- tumor, HER2- cell line, and the healthy patient samples (Md = 100, 117, 77, respectively). The median HER2S in HER2+ cell lines (Md = 248) is approximately 2.5-3.0 fold greater than the median of the other groups. A HER2S above 250 was considered abnormal or test positive, and was defined as the cut-point. The HER2S for the two IHC HER2 test control cell lines, SKBR3 for HER2+ and MDA-MB231 for HER2-, was 544 and 0. Conclusions: These findings provide strong evidence that measurement of HER2 signaling activity may provide clinically relevant information, particularly for HER2- breast cancer patients. These results suggest a new group of HER2- breast cancer patients with abnormal HER2-driven signaling may benefit from anti-HER2 therapy. Additional studies are underway to confirm these findings and to analytically validate the CELx HSP test. Citation Format: Laing L, Burns D, MacNeil I, Rich B, Huang Y, Myhre S, Soltani S, Sullivan B. New method to measure functional HER2-driven signaling activity in primary tumor cells identifies HER2-negative breast cancers with abnormal HER2 signaling activity: New group of patients may benefit from anti-HER2 therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-14.

Abstract P4-12-06: Quantification of HER2-driven signaling (HER2S) inhibition of four different anti-HER2 drugs tested ex vivo in live primary HER2-negative breast cancer cell samples with abnormal HER2 signaling activity

Abstract Background: A new functional cellular analysis platform, the CELx HER2 Signaling Profile (CELx HSP) Test, uses a label-free impedance biosensor to measure HER2 signaling activity in live tumor cells. A recently completed study quantified HER2-driven signaling activity in epithelial cell samples extracted and cultured from fresh breast tissue specimens obtained from 34 patients with HER2-negative breast cancer (DAKO 0 or 1+). Of the cell samples tested, 7 of 34 HER2-negative breast tumor patients (20.5%; 95% CI=10%-37%) were found to have abnormal HER2 signaling activity (HER2S+). The current study set out to: 1) evaluate the primary cells with abnormal HER2-driven signaling with four HER2 signal inhibitors - pertuzumab, lapatinib, neratinib, afatinib; and 2) evaluate the same four HER2 signal inhibitors with 9 HER2-positive cell lines. The objective was to quantify the percentage of HER2-driven signaling activity each drug could inhibit ex vivo in the primary cell samples and cell lines. Comparing the results between the HER2-negative primary cells and the HER2-positive cell lines was also of interest. The anti-HER2 drug, trastuzumab, was not studied because its primary mechanism of action does not appear to be direct mediation of HER2-driven signaling. Methods: Epithelial cells from the 7 HER2-negative tumor specimens with abnormal HER2-driven signaling (HER2S+) and the 9 HER2-positive cell lines were obtained. Real time live cell response to NRG1, a specific HER2/HER3 agonist, with or without a HER2 targeted drug (pertuzumab, lapatinib, neratinib, afatinib) was measured and quantified using an xCELLigence RTCA impedance biosensor (ACEA Biosciences, San Diego, CA). Clinically relevant concentrations of the HER2 drugs were used. From these responses, the percentage inhibition of the HER2-driven signaling initiated by NRG1 by the HER2 drugs was determined. Results: Each of the HER2 drugs inhibited an average of at least 69% of the HER2-driven signaling activated by NRG1 stimulation in the HER2-negative primary cell samples; the highest level of inhibition was found with the two irreversible covalent dual RTKi's, afatinib and neratinib. All of the HER2 drugs inhibited a greater percentage of HER2-driven signaling in the HER2-negative primary tumor cells than in the HER2-positive cell lines. table 1 Avg. % NRG1 Inhibition Cell LinesPrimariesHER2 DrugsMechanism of Action(HER2+)(HER2-)PertuzumabHER2 dimerization inhibitor46%78%LapatinibReversible Dual RTKi (HER2, EGF)15%69%AfatinibIrreversible Covalent Dual RTKi (HER2, EGF)47%93%NeratinibIrreversible Covalent Dual RTKi (HER2, EGF)95%100% Conclusions: These findings provide strong evidence that HER2 signal inhibitors are effective in blocking abnormal levels of HER2-driven signaling (HER2S+) ex vivo in live primary cells from breast cancer patients with normal expression levels of HER2. These results suggest a new group of breast cancer patients, HER2-negative with abnormal HER2 signaling (HER2-/HER2S+), may benefit from the addition of HER2 signal inhibitors to current combination therapeutic regimens. Additional studies to confirm these findings are underway. Citation Format: Laing L, Burns D, Huang Y, MacNeil I, Rich B, Myhre S, Soltani S, Sullivan B. Quantification of HER2-driven signaling (HER2S) inhibition of four different anti-HER2 drugs tested ex vivo in live primary HER2-negative breast cancer cell samples with abnormal HER2 signaling activity [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-06.

Incidence of abnormal HER2 values in the serum of breast cancer patients: influence of disease stage and HER2 status.

e23053Background: This translational study sought to elucidate the incidence of abnormal sHER2 values in a real-world population of breast cancer (BC) patients (pts) with primary and recurrent dise...

PD28-09 ABNORMAL HER2 AND HER3 SIGNALLING IN PROSTATE CANCER: POTENTIALS FOR CLINICAL APPLICATION.

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I1 Apr 2016PD28-09 ABNORMAL HER2 AND HER3 SIGNALLING IN PROSTATE CANCER: POTENTIALS FOR CLINICAL APPLICATION. Kasturi Rao, Massar Alsamraae, Luke Gaughan, Craig Robson, and Stuart McCracken Kasturi RaoKasturi Rao More articles by this author , Massar AlsamraaeMassar Alsamraae More articles by this author , Luke GaughanLuke Gaughan More articles by this author , Craig RobsonCraig Robson More articles by this author , and Stuart McCrackenStuart McCracken More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.396AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES HER family members (EGFR, HER2, HER3 and HER4) have been associated with the development of castrate-resistant prostate cancer. Previous research has focussed on targeting EGFR and HER2 receptors, however the role of HER3 in driving PI3K/Akt signalling and mediating drug resistance has shifted the attention towards this signalling partner. The use of pan-inhibitors targeting multiple members of the HER receptor family represents an attractive therapeutic approach in prostate cancer. METHODS We performed immunohistochemistry on human prostate samples, plus immunoblotting with nuclear-cytoplasmic extractions, luciferase reporter assays and proliferation/migration/invasion assays on prostate cancer cell lines. Utilized prostate cell line models included: androgen sensitive disease (LNCaP); an androgen independent derived line (LNCaP-AI); and cell line models of acquired resistance to Lapatinib (LNCaP-LapR) and Enzalutamide (LNCaP-EnzR). We have also developed HER2 and HER3 overexpressing stable cell lines using lentiviral technology. RESULTS We analysed the expression of HER2 and HER3 receptors and their downstream targets using tissue microarrays. We have shown strong correlations between HER2 and HER3 cytoplasmic and nuclear expression with poor prognostic disease. Part of the analysis consists of matched paired patient tissue samples (before and after relapse on conventional anti-androgen treatment). We have also shown in our cell line models that HER2/HER3 signalling: increases androgen receptor activity; increases downstream MAP Kinase and PI3 Kinase signalling activity; and increases cell proliferation, migration and invasion. The addition of a pan-HER family inhibitor inhibited all of these activities and also reduced nuclear translocation of HER2 and HER3, with prevention of chromatin binding. Finally, both Lapatinib and Enzalutamide resistant cell lines demonstrated increased HER2 and HER3 activity, subsequently shown to be sensitive to pan-HER inhibition. CONCLUSIONS HER2 and HER3 play a prominent role in advanced prostate cancer. We have also demonstrated the reduced activity of androgen receptor, plus PI3K and MAPK pathways downstream of the HER2/3 signalling pathways by the use of a novel pan-HER inhibitor. This pan-HER inhibitor has also reduced cellular activities important for cancer progression. The increased expression of HER2 and HER3 in both the Lapatinib and Enzalutamide resistant lines suggests that combination therapy of novel anti-androgens with pan-HER inhibitiors may delay cancer progression in prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e657 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Kasturi Rao More articles by this author Massar Alsamraae More articles by this author Luke Gaughan More articles by this author Craig Robson More articles by this author Stuart McCracken More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The HER2- and Heregulin β1 (HRG)–Inducible TNFR Superfamily Member Fn14 Promotes HRG-Driven Breast Cancer Cell Migration, Invasion, and MMP9 Expression

HER2 overexpression occurs in 15% to 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2-HER3 heterodimers and drives breast cancer cell growth and metastasis. In a previous study, we found that fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, was frequently overexpressed in human HER2+ breast tumors. We report here that HER2 and Fn14 are also coexpressed in mammary tumors that develop in two different transgenic mouse models of breast cancer. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with a HER2 expression plasmid increased Fn14 protein levels. Also, HRG1-β1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2- and HRG1-β1-induced increase in Fn14 expression in MCF7 cells as well as basal Fn14 expression in HER2 gene-amplified AU565 cells could be reduced by HER2 kinase inhibition with lapatinib or combined HER2 and HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-β1-stimulated cell migration, invasion, and matrix metalloproteinase (MMP)-9 expression. Together, these results indicate that Fn14 may be an important downstream regulator of HER2/HER3-driven breast cancer cell migration and invasion.

Interference-Free HER2 ECD as a Serum Biomarker in Breast Cancer.

Over-expression of the HER2/neu receptor occurs in 20 to 30 percent of breast tumors and is linked to poorer prognosis. The HER2/neu expression status determines whether or not patient will receive trastuzumab-based treatment. In clinical practice, over-expression of HER2/neu is routinely identified using Immunohistochemistry (IHC) or Fluorescence in Situ Hybridization (FISH), both of which are invasive approaches requiring tissue samples. Serum assays for the Extra Cellular Domain of HER2/neu receptor (HER2 ECD) have been reported but the use is very limited due to serum interference factors (e.g. human anti-animal immunoglobulin antibodies) that lead to false test results and inconsistency with tissue Her2 status. We have developed an ELISA based approach using an MBB buffer to eliminate false results and to obtain more accurate assessment of HER2 ECD levels. Using this refined assay we retroactively measured HER2/neu levels from breast cancer patients and controls. Abnormal HER2 ECD levels were detected in about 32% of invasive breast cancer patients but not in controls or patients with benign diseases. In addition, we also showed that patients with elevated serum HER2 levels appeared to have worse survival regardless of treatments. In a small group of 12 Ductal Carcinoma in situ (DCIS) patients who received HER2/neu peptide vaccination and surgery, only one patient showed constantly rising HER2 levels after treatment and this patient had recurrence of HER2 positive tumor within 5 years. Our studies indicate that once the serum interference issue is resolved, serum HER2 ECD can have potential clinical utility to supplement the tissue based tests.

Identifying HER2 Inhibitors from Natural Products Database

The relationship between abnormal HER2 expression and cancer is important in cancer therapeutics. Formation and spread of cancer cells may be restricted by inhibiting HER2. We conducted ligand-based and structure-based studies to assess the potency of natural compounds as potential HER2 inhibitors. Multiple linear regression (MLR) and support vector machine (SVM) models were constructed to predict biological activities of natural compounds, and molecular dynamics (MD) was used to assess their stability with HER2 under a dynamic environment. Predicted bioactivities of the natural compounds ranged from 6.014–9.077 using MLR (r2 = 0.7954) and 5.122–6.950 using SVM (r2 = 0.8620). Both models were in agreement and suggest bioactivity based on candidate structure. Conformation changes caused by MD favored the formation of stabilizing H-bonds. All candidates had higher stability than Lapinatib, which may be due to the number and spatial distribution of additional H-bonds and hydrophobic interactions. Amino acids Lys724 and Lys736 are critical for binding in HER2, and Thr798, Cys805, and Asp808 are also important for increased stability. Candidates may block the entrance to the ATP binding site located within the inner regions and prevent downstream activation of HER2. Our multidirectional approach indicates that the natural compounds have good ligand efficacy in addition to stable binding affinities to HER2, and should be potent candidates of HER2 inhibitors. With regard to drug design, designing HER2 inhibitors with carboxyl or carbonyl groups available for H-bond formation with Lys724 and Lys736, and benzene groups for hydrophobic contact with Cys805 may improve protein-ligand stability.

Outcome of Patients With Early-Stage Breast Cancer Treated With Doxorubicin-Based Adjuvant Chemotherapy As a Function of HER2 and TOP2A Status

Amplification and deletion of the TOP2A gene have been reported as positive predictive markers of response to anthracycline-based therapy. We determined the status of the HER2 and TOP2A genes in a large cohort of breast cancer patients treated with adjuvant doxorubicin (A) and cyclophosphamide (C). TOP2A/CEP17 and HER2/CEP17 fluorescent in situ hybridization (FISH) were performed on tissue microarrays (TMAs) constructed from 2,123 of the 3,125 women with moderate-risk primary breast cancer who received equivalent doses of either concurrent adjuvant chemotherapy with A plus C (n = 1,592) or sequential A followed by C (n = 1,533). An abnormal TOP2A genotype was identified for 153 (9.4%) of 1,626 patients (4.0% amplified; 5.4% deleted). An abnormal HER2 genotype was identified for 303 (20.4%) of 1,483 patients (18.8% amplified; 1.6% deleted). No significant differences in either overall survival (OS) or disease-free survival (DFS) were identified for TOP2A. In univariate analysis, OS and DFS rates were strongly and adversely associated only with higher levels of HER2 amplification (ratio > or = 4.0). Survival was not associated with low-level HER2 amplification (ratio > or = 2; OS hazard ratio [HR], 1.14; P = .39; DFS HR, 1.07; P = .62), but it was associated for a ratio > or = 4 (OS HR, 1.45; P = .03; DFS HR, 1.38; P = .033), in which analysis was adjusted for menopausal status, hormone receptor status, treatment, number of positive nodes, and tumor size. In this population of patients with early-stage breast cancer who were treated with adjuvant AC chemotherapy, TOP2A abnormalities were not associated with outcome. HER2 high-level amplification was a prognostic marker in anthracycline-treated patients.

Study of serum EGFR, HER-2/neu,VEGF in metastatic breast cancer patients treated with taxanes

757 Background: Novel drugs with activity against EGFR,Her-2/neu,VEGF are used in clinical practice. Previous studies have examined the possible prognostic and predictive role of soluble EGFR, Her-2/neu,VEGF, in metastatic breast cancer(MBC) with various results.We examine whether their combined measurement has any prognostic or predictive role. Methods: Retrospective analysis of frozen serum from women with MBC who received first line taxane based chemotherapy. Analysis was performed centrally with the use of commercially available ELISA. Specifically for EGFR and Her-2/neu a Microtiter ELISA (Bayer Diagnostics) and for VEGF a Microliter ELISA (R &D System). Normal values were calculated according to previous published literature. Results: Demographics: Total 41 women with a median age of 54, 54% premenopausal,51% postmenopausal,61% were ER positive 39% negative. Serum was drawn prior to chemotherapy. All patients received first line treatment: 22 Paclitaxel /Epirubicin,8 Paclitaxel /Carboplatin,8 Paclitaxel /Trastuzumab,3 Paclitaxel/Gemcitabine/Trastuzumab. Complete response had 10% partial response 49%.After a median follow-up of 46,7(3,2–62,8) months 38 patients have relapsed and 28 have died yielding a median TTP of 13,4 mo and a median OS of 34,8 mo. EGFR was abnormal in 51%,Her-2/neu in 61% and VEGF in 54%. 6/41 patients over-expressed all three markers and 7/41 none. OS was statistically worse for patients who had all abnormal (p=0.027 long rank) compared to those with all normal with median overall survival of 44,1 vs 10 months. Elevated Her-2/neu and elevated VEGF showed a trend for decreased OS ( p=0.0564,p=0.0519 respectively ). Patients with abnormal Her-2/neu who received trastuzumab based therapy had a better TTP (p=0.043). Conclusions: In this small retrospective analysis the group of MBC patients with elevated serum EGFR, Her -2/neu and VEGF seem to have a worse prognosis and trastuzumab based therapy seems to benefit patients with abnormal serum Her-2/neu level. Further study in larger groups of patients with MBC is warranted along with serial measurement during targeted therapy. No significant financial relationships to disclose.

Study of serum EGFR, HER-2/neu,VEGF in metastatic breast cancer patients treated with taxanes

757 Background: Novel drugs with activity against EGFR,Her-2/neu,VEGF are used in clinical practice. Previous studies have examined the possible prognostic and predictive role of soluble EGFR, Her-2/neu,VEGF, in metastatic breast cancer(MBC) with various results.We examine whether their combined measurement has any prognostic or predictive role. Methods: Retrospective analysis of frozen serum from women with MBC who received first line taxane based chemotherapy. Analysis was performed centrally with the use of commercially available ELISA. Specifically for EGFR and Her-2/neu a Microtiter ELISA (Bayer Diagnostics) and for VEGF a Microliter ELISA (R &D System). Normal values were calculated according to previous published literature. Results: Demographics: Total 41 women with a median age of 54, 54% premenopausal,51% postmenopausal,61% were ER positive 39% negative. Serum was drawn prior to chemotherapy. All patients received first line treatment: 22 Paclitaxel /Epirubicin,8 Paclitaxel /Carboplatin,8 Paclitaxel /Trastuzumab,3 Paclitaxel/Gemcitabine/Trastuzumab. Complete response had 10% partial response 49%.After a median follow-up of 46,7(3,2–62,8) months 38 patients have relapsed and 28 have died yielding a median TTP of 13,4 mo and a median OS of 34,8 mo. EGFR was abnormal in 51%,Her-2/neu in 61% and VEGF in 54%. 6/41 patients over-expressed all three markers and 7/41 none. OS was statistically worse for patients who had all abnormal (p=0.027 long rank) compared to those with all normal with median overall survival of 44,1 vs 10 months. Elevated Her-2/neu and elevated VEGF showed a trend for decreased OS ( p=0.0564,p=0.0519 respectively ). Patients with abnormal Her-2/neu who received trastuzumab based therapy had a better TTP (p=0.043). Conclusions: In this small retrospective analysis the group of MBC patients with elevated serum EGFR, Her -2/neu and VEGF seem to have a worse prognosis and trastuzumab based therapy seems to benefit patients with abnormal serum Her-2/neu level. Further study in larger groups of patients with MBC is warranted along with serial measurement during targeted therapy. No significant financial relationships to disclose.

Benign Myoepithelial Tumors of the Breast Have Immunophenotypic Characteristics Similar to Metaplastic Matrix-Producing and Spindle Cell Carcinomas

We immunohistochemically compared benign myoepithelial tumors (adenomyoepitheliomas [AMEs]) and metaplastic matrix-producing (MMP-CA) and spindle cell (MSC-CA) carcinomas of the breast to identify helpful diagnostic markers. Normal myoepithelial cells (MECs) consistently expressed cytokeratin, α-smooth muscle actin (SMA), myosin, S-100, CD10, and maspin. They were variably positive for vimentin and negative for epithelial membrane antigen (EMA), steroid receptors, p53, and HER-2/neu. MECs in AMEs less frequently expressed CD10 (4/8 [50%]) and myosin (6/8 [75%]) but frequently acquired characteristics of luminal cells, such as expression of EMA (5/8 [63%]) and steroid receptors (5/8 [63%]). No abnormal p53 or HER-2/neu expression was seen in AMEs. MMP-CA and MSC-CA were similar to AMEs in cytokeratin, vimentin, S-100, maspin, and HER-2/neu expression. MMP-CAs expressed less α-SMA (2/8 [25%]) and myosin (2/7 [29%]) and lacked estrogen receptor (0/9 [0%]). MSC-CAs were consistently CD10+ (4/4 [100%]) yet failed to express myosin (0/3 [0%]). p53 overexpression was seen frequently in MMP-CAs (4/8 [50%]) and MSC-CAs (1/3 [33%]). Benign myoepithelial mammary tumors differ immunophenotypically from normal MECs; a panel of immunohistochemical markers may be required to establish their myoepithelial origin. A similarly altered myoepithelial phenotype also is characteristic of metaplastic mammary carcinomas. The abnormal expression of oncogenes or antioncogenes, such as p53, may be more useful for distinguishing between those entities than the expression of the classic myoepithelial markers.