149 Background: Metastatic castration-resistant prostate cancer (mCRPrC) is characterized by a loss of androgen receptor (AR) sensitivity and oncogenic activation of the PI3K, AKT, and mTOR (PAM) pathway. Loss of the PI3K regulator, PTEN, is frequent during prostate cancer (PrC) initiation, progression, and therapeutic resistance. Co-targeting PAM/AR pathways is a promising PrC treatment strategy but hampered by complex PAM-AR pathway crosstalk driving either reciprocal negative feedback inhibition or feedback relief. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, potentiating drug resistance depending on the PAM mutation status of patients. AKT-i demonstrated limited efficacy in PTEN-deficient PrC and even less efficacy in PTEN-wild type (wt) in recent clinical studies, suggesting that more comprehensive inhibition of the PI3K isoforms and mTOR may be required. We posited that gedatolisib, a well-tolerated and potent inhibitor of all Class I PI3K isoforms, mTORC1, and mTORC2, would be effective in both PTEN-wt and PTEN-deficient PrC. Methods: A panel of PrC cell lines with different PI3K or PTEN status ( wt: 22RV1, MDA-PCa-2b, DU145; null: LNCaP, PC3, C4-2) were assayed under different conditions for their sensitivity to gedatolisib and other PAM-I ( PI3K: alpelisib, copanlisib; AKT: capivasertib, ipatasertib; mTOR: everolimus; pan-PI3K/mTOR: samotolisib, gedatolisib). A combination of cell viability, growth rate, cytotoxicity and phospho-FACS analytical assays were used. Xenograft studies evaluating gedatolisib with a subset of these models were also performed in castrated mice. Results: We confirmed that AKT-i is more effective in PTEN-null than PTEN-wt PrC. In contrast, the two pan-PI3K/mTOR-i evaluated were effective independent of PI3K or PTEN status. Gedatolisib exhibited superior potency and efficacy compared to each of the other PAM-i for cell proliferation, death, or PAM pathway activation. In vivo, gedatolisib substantively inhibited tumor growth regardless of PTEN or PI3K status. Conclusions: We demonstrated that gedatolisib exerts superior activity regardless of PI3K or PTEN status in all PrC cell lines evaluated compared to the other PAM pathway inhibitors evaluated. Our results indicate potent and simultaneous blockade of all Class I PI3K isoforms, mTORC1, and mTORC2 could circumvent PTEN dependent resistance. Gedatolisib as a single agent and in combination with other therapies reported promising preliminary efficacy and safety in various solid tumor types. In light of the PrC findings reported here, development of gedatolisib may be warranted in PrC patients regardless of their PTEN or PI3K status.

HER2 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. The 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+). HER2−/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. The 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.

e13000 Background: Biological factors other than PIK3CA status, such as aberrant GPCR-linked signaling, may be important to measure when identifying patients eligible for PI3K inhibitors. A new assay, the CELx PI3K test, using an impedance biosensor was developed to measure ex vivo live tumor cell response to specific S1P agonists and PI3K antagonists to diagnose breast tumors with PI3K-involved hyperactive signaling. This study set out to: 1) compare CELx PI3K test results and xenograft results using cell lines with PIK3CA mutations; and 2) assess whether PI3K-involved hyperactive S1P signaling is found in PIK3CA WT breast cancer patient tumors. Methods: A panel of 17 fresh HER2-/PIK3CA WT tumor cells from breast cancer patients and three PIK3CA mutated breast tumor cell lines were obtained. Live cell response to an S1P agonist, PI3K-α antagonist (alpelisib), PI3K-γ antagonist (IPI-549), and a pan-PI3K inhibitor (taselisib) were measured using an xCELLigence RTCA impedance biosensor. From these responses, PI3K-involved signaling was quantified and characterized as normal or abnormal using a previously determined cutpoint. For the xenograft study, 16 NSG mice were injected with HCC1954 PIK3CA mutated breast cancer cells and randomly assigned to either the control or taselisib group (10 mg/kg). Results: Four of the 17 PIK3CA WT tumor cells had abnormal levels of combined PI3K-α and PI3K-γ signaling. Only one of the three PIK3CA mutated breast tumor cell lines (BT20) had abnormal levels of PI3K-α and pan-PI3K involved signaling. The HCC1954 cell line had normal PI3K-α and abnormal pan-PI3K signaling. CAL-51 reported normal PI3K-α and pan-PI3K signaling. The normal levels of PI3K-α signaling found in the HCC1954 and CAL-51 cell lines correlated with previously reported xenograft studies that found alpelisib had no anti-tumor effect. The xenograft study reported here using HCC1954 cells found taselisib induces a significant anti-tumor effect (T/C ratio = 0.21; p = 0.009; t-test). Conclusions: A sub-set of PIK3CA WT patient breast cancer tumors had abnormal PI3K-involved signaling comparable to levels found in PI3KCA mutated cell lines. Abnormal pan-PI3K signaling and normal PI3K-α signaling in the HCC1954 cell line correlated with xenograft results. This study thus suggests that measurement of PI3K-involvement in hyperactive S1P signaling in live patient breast cancer cells may provide a means to identify breast cancer patients who may or may not benefit from treatment with PI3K inhibitors.

Background: Hyperactive c-Met signaling, including cross-talk between c-Met and ErbB family receptors, is suspected of contributing to tumor progression in a variety of cancer types. Clinical trials evaluating c-Met inhibitors alone and in combination with other targeted therapies, have produced mostly negative results in patients with amplified c-Met. This suggests other biological factors, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. To measure the c-Met and ErbB signaling activity of a patient9s live tumor cells, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP test measures a patient9s ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists to diagnose breast cancer tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. In this study, to further elucidate the role of c-Met signaling and its potential involvement with ErbB signaling as a cancer driver, we studied in vivo response to a c-Met inhibitor (tepotinib), an EGFR inhibitor (erlotinib), a pan-HER inhibitor (neratinib), and a combination of these therapies using breast tumor xenograft models. Methods: HCC1954, a HER2+ cell line with hyperactive c-Met and EGFR signaling and normal HER3 and HER2-driven signaling, according to the CELx MP test, was studied. Sixty 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 one of five treatment groups that received either neratinib, tepotinib, erlotinib, erlotinib and tepotinib, or neratinib and tepotinib for 17 days. Results: The most effective treatment was the combination of neratinib plus tepotinib, where the average tumor size reduction relative to the control group was 71% (p=0.0003). The average tumor size in the neratinib plus tepotinib treated group was 37% smaller (p=0.049) than the neratinib treated group, and 67% smaller (p=0.0026) than the tepotinib treated group. In the erlotinib plus tepotinib treatment group, the average tumor size was 51% smaller than the control group tumors, but the difference was not statistically significant (p=0.11). No significant difference in tumor size was found between the control group and the erlotinib or tepotinib treated groups. Conclusions: The results demonstrate that hyperactive and coincident c-Met and EGFR signaling contributes to the progression of certain breast cancers. This breast cancer sub-type is more responsive to treatment with a c-Met inhibitor plus a pan-HER inhibitor than a c-Met inhibitor plus an EGFR inhibitor or any of the single agents studied. This suggests that when hyperactive c-Met signaling and a hyperactive ErbB family member is present in a patient9s tumor, each of the ErbB pathways as well as the c-Met pathway must be inhibited to treat the tumor most effectively. These findings provide strong evidence that HER2-negative breast cancer patients with coincident hyperactive c-MET and ErbB signaling may respond to treatment with a combination of pan-HER and c-Met inhibitors. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies [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 P3-10-15.

Abstract Background: Biological factors other than c-Met status, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. A new assay using an impedance biosensor was developed to measure c-Met and ErbB signaling activity of live tumor cells. The CELx Multi-Pathway Signaling Function (CELx MP) Test measures an individual patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists and antagonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, and c-MET signaling. This study set out to: 1) determine the prevalence of hyperactive c-Met and ErbB family signaling amongst HER2- breast cancer patients; and 2) characterize potential cross-talk between c-Met and ErbB pathways. Methods: For the prevalence study, fresh breast tumor specimens were obtained from 74 HER2- breast cancer patients. The amount of HER1, HER2, HER3, and c-Met for each specimen was determined using FACS. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without ErbB and c-Met antagonists (2C4, a HER2 mAb dimerization inhibitor, tepotinib, a c-Met TKI, or neratinib, a pan-HER TKI) was measured using an xCELLigence RTCA impedance biosensor. From these responses, HER1, HER3, and c-Met signaling initiated by their respective agonists was quantified. The net amount of HER2 participation in EGF and NRG signaling was also quantified. Signaling activity above a previously determined cutpoint was used to identify abnormal levels of HER1, HER2, HER3 and c-Met signaling activity. For the cross-talk study, three primary HER2- breast cancer specimens with hyperactive c-Met and ErbB signaling were obtained. Response to HGF, EGF, and NRG1 alone, with or without tepotinib, was measured for these specimens using an impedance biosensor. Results: The FACS analysis found all 74 tumor samples had normally expressed amounts of HER1, HER2, HER3, and c-Met. Of these samples, 20 of 74 (27.0%; 95% CI=18%-38%) had hyperactive c-Met signaling coincident with hyperactive signaling from at least one ErbB pathway. In each patient sample, neratinib combined with tepotinib inhibited virtually all signaling activity initiated with a combination EGF, NRG1 and HGF. In the cross-talk analysis of the three tumor samples, signaling response to EGF or NRG1 when combined with a c-Met antagonist was 9%-98% higher than signaling response measured with EGF or NRG1 alone. Conclusions: This test found a significant sub-set of HER2- breast cancer patients with coincidental hyperactive c-Met and ErbB signaling tumors that respond ex vivo to a combination of pan-HER and c-Met TKI's. The unexpected increase in EGF and NRG1 signaling in the presence of a c-Met antagonist provides strong evidence that c-Met and ErbB signaling is co-involved and may explain why a c-Met TKI is not an effective antagonistic when c-Met is hyperactive for this patient sub-set. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Sub-group of HER2- breast cancer patients with hyperactive and co-involved c-Met and ErbB pathways identified: Functional signal profiling test identifies patient group that may benefit from c-Met and pan-HER combination therapy [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 P3-10-20.

Abstract Background: To elucidate the role of c-Met signaling and its involvement with ErbB signaling as a cancer driver, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP Test measures ex vivo real-time live cell response to specific ErbB and c-Met agonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. A recent study quantified c-MET and ErbB-driven signaling activity in epithelial cell samples derived from fresh breast tumor specimens obtained from 74 HER2- breast cancer patients. Of the cell samples tested, 20 of 74, (27.0%; 95% CI=18%-38%) had both hyperactive c-MET signaling and at least one hyperactive ErbB-family receptor signaling. Using primary breast cancer cells with hyperactive c-MET and ErbB signaling and the CELx MP test, the current study set out to: 1) determine the IC50 values of six pan-HER and five c-MET inhibitors; and 2) characterize the efficacy of combinations of each pan-HER inhibitor with each c-MET inhibitor. Methods: Epithelial cells from six HER2- tumor specimens with hyperactive c-MET and ErbB-driven signaling were obtained. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without one of six pan-HER antagonists (neratinib, lapatinib, ibrutinib, dacomitinib, sapitinib, poziotinib) or one of five c-MET antagonists (tepotinib, cabozantinib, crizotinib, capmatinib, or savolitinib) was quantified using an xCELLigence RTCA impedance biosensor. Each individual drug IC50 was determined using a 1000-fold, 5-point, dose response curve with a single fixed concentration of a corresponding agonist. For the drug combination efficacy studies, fixed concentrations of the agonist mixture and clinically relevant concentrations of combinations of the antagonists were used to determine the percentage inhibition of the ErbB and c-MET signaling. Results: The IC50 values for the individual c-MET and pan-HER inhibitors ranged from 3.10nM - 28nM and 2.67nM – 137.27nM, respectively. In the drug efficacy studies, an average of at least 80% of the ErbB and c-MET signaling activated by NRG1, EGF, and HGF co-stimulation was inhibited by each combination of c-MET and pan-HER inhibitors. c-Met Inhibitor Resultsc-MET inhibitorIC50 (nM)Avg Inhibition (%) w/diff ErbBi's Capmatinib3.1094Savolitinib3.3298Tepotinib14.7096Cabozantinib27.3699Crizotinib28.21100 Pan-HER Inhibitor ResultsPan-HER inhibitorIC50 (nM)Avg Inhibition (%) w/diff c-METi's Poziotinib2.67100Neratinib4.81100Ibrutinib13.1099Dacomitinib22.06100Sapitinib41.2898Lapatinib137.2780 Conclusions: The CELx MP test using live cells measures IC50 values comparable to those derived using cell-free methods. Every combination of pan-HER and c-MET inhibitors provided comparably high (at least 80%) levels of inhibitory effect ex vivo. This suggests the sub-group of HER2- breast cancer patients diagnosed with coincident hyperactive c-MET and ErbB signaling by the CELx Test may respond to virtually any pan-HER and c-Met inhibitor combination. Studying combinations designed to minimize drug toxicities without sacrificing efficacy should thus be possible. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluation of pan-HER and c-MET inhibitors tested ex vivo in primary HER2- breast cancer cells with hyperactive c-MET and ErbB family signaling [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 P2-05-05.

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.

e24292 Background: Clinical trials evaluating anti-Met therapies have produced mixed results when subjects are selected for c-Met overexpression or gene amplification. To further elucidate the role of c-Met signaling and its potential involvement with ErbB signaling as a cancer driver, a new assay to identify dynamic multi-pathway signaling dysfunction (CELx MP test) was developed. The current study set out to characterize ErbB family and c-Met function in primary HER2-negative breast tumor cells and evaluate in vivo response to a c-Met inhibitor in breast tumor xenograft models. Methods: Tumor specimens were obtained from 74 HER2- breast cancer patients. Live cell response to ErbB and c-Met agonists with or without antagonist (pertuzumab, neratinib, or tepotinib) was quantified using a 96-well biosensor. Signaling activity above a cut-off value of 250 signaling units was used to identify abnormal EGFR, HER2 and c-Met signaling. HCC1954, a HER2+ cell line with normal HER2 signaling, abnormal EGFR signaling, and abnormal c-Met signaling, as determined by the CELx MP test, was injected into 40 female NSG mice. Mice were treated for 16 days with either Captisol, neratinib, tepotinib or a combination (10 mice/arm). Results: The CELx MP test found abnormal signaling sub-groups as follows: HER2 - 16 of 74 (22%; 95% CI = 14%-32%); c-MET - 31 of 74 (41%; 95% CI = 31%-53%); EGFR – 17 of 74 (23%; CI = 15%-34%). For all samples tested, EGFR and HER2 signaling were each strongly correlated to c-Met signaling levels (r = 0.89 and r = 0.85 respectively). The average amount of c-Met signaling activity inhibited in cell samples increased by 75% when tepotinib was combined with neratinib versus tepotinib alone. In the xenograft study, no significant difference in tumor volume between the control and tepotinib-treated arms after 16 days of treatment was recorded. Tumor volumes after treatment were reduced by 17% in the neratinib arm and by 45% in the tepotinib + neratinib arm. Conclusions: These findings suggest that a sub-group of HER2- breast cancer patients have co-activated abnormal ErbB and c-Met signaling tumors and may respond better to ErbB and c-Met inhibitors combined than c-Met inhibitors alone.

Distinct populations of effector memory T cells use different homing receptors to traffic to the skin and gut. Whether tissue-selective T cells are needed for early rejection of a neoplasm growing in these tissues remains an open question. We chose to study an allogeneic tumor model because growth of such a fully mismatched tumor would signify a profound immune deficit. We implanted allogeneic tumor cells in the skin or gut of mice deficient in either α(1,3) fucosyltransferases IV and VII, enzymes critical for generating E-selectin ligands on skin-homing T cells, or β7 integrin, a component of the α4β7 integrin ligand for the mucosal adressin MAdCAM. During the first 9 days after tumor implantation, FucTVII −/− mice showed a profoundly impaired capacity to reject tumors growing in the skin, but readily rejected tumors implanted in the gut. Rejection of tumors in the skin was even more impaired in mice deficient in both FucTIV and FucTVII. This impairment was corrected by infusion of T cells from normal mice. By contrast, β7 integrin −/− mice showed profoundly impaired rejection of tumors in the gut, but no defect in the skin tumor rejection. These differences were unrelated to antigen recognition or effector function of T cells, since all strains of mice were capable of generating tumor-specific CTLs in vitro against the tumor cell line used in vivo. These results demonstrate that T-cell homing defects in vivo impair immune surveillance of peripheral epithelial tissues in a specific and selective fashion.

BACKGROUND:Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies.METHODS:A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor.RESULTS:The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line.CONCLUSIONS:Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.

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.

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

Pseudomonas exotoxin (PE) potently blocks protein synthesis by catalyzing the inactivation of elongation factor-2 (EF-2). Targeted PE-cytotoxins have been used as antitumor agents, although their effective clinical translation in solid tumors has been confounded by off-target delivery, systemic toxicity, and short chemotherapeutic half-life. To overcome these limitations, we have created toxin-resistant stem cells by modifying endogenous EF-2, and engineered them to secrete PE-cytotoxins that target specifically expressed (interleukin-13 receptor subunit alpha-2) or overexpressed (epidermal growth factor receptor) in glioblastomas (GBM). Molecular analysis correlated efficacy of PE-targeted cytotoxins with levels of cognate receptor expression, and optical imaging was applied to simultaneously track the kinetics of protein synthesis inhibition and GBM cell viability in vivo. The release of IL13-PE from biodegradable synthetic extracellular matrix (sECM) encapsulated stem cells in a clinically relevant GBM resection model led to increased long-term survival of mice compared to IL13-PE protein infusion. Moreover, multiple patient-derived GBM lines responded to treatment, underscoring its clinical relevance. In sum, integrating stem cell-based engineering, multimodal imaging, and delivery of PE-cytotoxins in a clinically relevant GBM model represents a novel strategy and a potential advancement in GBM therapy.

e11583 Background: This work reports a dynamic real time analysis of live cells to detect drug attenuation of cell signaling related to the MOA of a targeted therapy. The assay employs an impedance biosensor to detect cellular changes in close proximity to a nanoelectrode caused by specific pathway perturbation. When applied to live patient diseased cells ex vivo, this approach holds great promise to determine a priori whether a targeted drug therapy will clinically benefit that patient. If the targeted therapy is not functional in the patient’s cells ex vivo, then it is not likely to function as intended in the patient. Methods: To demonstrate the utility of the assay, HER2+ and ER+/HER2- live breast cancer cell samples (both cell lines and primary cells from fresh tumor specimens) were tested with 6 targeted breast cancer therapies and specific pathway stimulating ligands corresponding to each drug target. Real time signaling impedance profiles were generated for each live cell sample under 3 different ...

e11597 Background: This report provides evidence that an assay using live primary cells can reliably measure the real-time dynamic signaling of different cells and the attenuated response to therapies targeting the signaling. Characterization of live cells has delivered impressive results in the discovery of new therapeutic compounds. As a first step to translating these discovery results to the administration of the correct drug to the right patient, an assay is presented that measures in real-time the complex changes of viable patient cells growing on a nanoelectrode in a standard 96-well microplate. Since cell health and function modulate adhesion and phenotypic properties leading to changes near the electrode-cell interface, an impedance biosensor is capable of detecting and quantifying changes in cell health and specific pathway perturbation. The electrode impedance measurement is referred to as a cell attachment signal (CAS) and is expressed in ohms. The attachment of the cells via a specific ECM provides a quality control signal as well as coupling the cell communication link to the nanoelectrode. Methods: To confirm the reproducibility of the assay, a HER2+ breast cancer cell line (SKBr3) was tested with NRG1b, EGF, and lapatinib, a dual tyrosine kinase inhibitor which disrupts PI3K and MAPK pathway signaling. Each test measures the live cells’ CAS under three different conditions: 1) cells in buffer; 2) cells exposed first to lapatinib and then to either NRG1b or EGF; and 3) cells exposed only to either NRG1b or EGF. Tests were run over 3 days with 2 runs of tests performed each day. A total of 576 measurements were recorded. This experiment was then repeated using live primary cells extracted from a fresh HER2+ breast cancer specimen. Results: The CV of the CAS measurements taken using SKBr3 cells and the CV of the CAS measurements derived from the primary cells were both less than 10%. Conclusions: These results provide evidence the assay is a suitably robust method to analyze cellular functional pathway signaling activity in primary cells extracted from fresh tumor specimens.