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Under normal conditions, hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase (TK), is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand activated receptor internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis and tumor progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis. The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programs. Pathway inhibitors can be divided broadly into biologicals and low molecular weight synthetic TK inhibitors; of these, the latter now outnumber all other inhibitor types. We review here Met structure and function, the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development, as well as the latest clinical trial results. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents and model systems available concerning HGF/Met oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (

Purpose The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively) are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma. Patients and Methods Foretinib safety and tolerability, and objective response rate (ORR) were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks) or daily (80 mg/day) dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity. Results From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25–88); 93% had received prior therapy. Best response was stable disease (SD) in 10 (23%) patients receiving intermittent dosing and five (20%) receiving daily dosing; SD duration was 1.9–7.2 months (median 3.2 months). Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15%) and elevated aspartate aminotransferase (23% vs. 8%) were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET), the pMET:total MET protein ratio decreased with foretinib treatment. Conclusion These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in unselected patients with metastatic gastric cancer. Trial Registration ClinicalTrials.gov NCT00725712

Background: Standard treatment for muscle-invasive bladder cancer (MIBC) includes chemotherapy with either gemcitabine-cisplatin (GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC). These regimens have similar clinical complete response rates of approximately 30%. While no targeted treatment has been approved for bladder cancer, clinical trials have identified biochemical markers that predict the chemoresponsiveness of MIBC tumors to specific chemotherapeutic agents. For example, patients with high hENT1 and low RRM1 expression responded better to GC-based chemotherapy than their counterparts, and HER2 overexpression predicted resistance to cisplatin-based therapy. To quantify targets that are known indicators of tumor behavior, we used targeted proteomic analysis to assess 30 different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) MIBC tumor tissue. Methods: Twelve FFPE MIBC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area from 1 or 2 slides. Following laser microdissection of the marked areas, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins in each patient sample. Results: Of the 12 patient samples, 7 (58%) expressed high levels of hENT1 and 11 (92%) expressed low levels of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. A single patient expressed high levels of RRM1, an indication for non-gemcitabine based therapy. All 12 patients expressed TUBB3, a marker of resistance to taxane (vinblastine) and 10 patients (83%) lacked expression of FR-alpha, a marker of sensitivity to methotrexate. The majority of patients expressed a marker of sensitivity to anthracycline (TOPO2A) and did not express a resistance biomarker for platinum therapy (ERCC1). Of 3 patients whose tumors expressed HER2, 2 had overexpression (>750 amol/ug) and would thus be eligible for HER2 basket trials. Further, multiplex-targeted proteomics discovered patients expressing FGFR1 (17%), cMET (33%), Axl (17%) and IDO1 (25%), which would make them eligible for clinical trials of targeted or immunotherapies. Conclusion: MIBC is heterogeneous and expresses a wide range of proteins. Yet, it continues to be treated with only 2 chemotherapeutic regimens. Multiplexed proteomics is currently being used in clinical practice to inform personalized patient care decisions with identification and the relative quantities of actionable proteins known to predict tumor behavior. Citation Format: Fabiola Cecchi, Sheeno Thyparambil, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Henry Frierson, Daniel Theodorescu. Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 449.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Well differentiated HCC can sometimes be extremely difficult to be distinguished from other hepatocellular mass lesions such as hepatocellular adenoma and dysplastic liver nodule due to considerable morphologic overlaps. Immunohistochemical markers may have a limited utility, especially on core biopsy. Choice of treatment depends on the extent and location of the cancer, and the overall health of the patient. For patients who are healthy enough to undergo surgery and who have early-stage cancer confined to the liver, treatment may involve hepatic resection; however many patients develop a cancer recurrence, which is the main cause of death in long-term evaluations. Recurrence rates after treatment with resection are high, highlighting the importance of finding effective adjuvant treatments and markers that aid in determining differences between hepatocellular lesions. To explore differences between hepatocellular lesions that could be indicators of tumor behavior, we used targeted proteomic analysis to assess different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) HCC tumor tissue Twenty-two FFPE HCC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area. Following laser microdissection, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins. As anticipated, well differentiated HCC, hepatocellular adenoma and dysplastic nodule expressed high rates of P-glycoprotein and the majority expressed multi-drug resistance gene protein (MDR1). Such markers may account in part for the chemotherapy refractory nature of HCC. All 22 patients expressed high levels of hENT1 and lacked expression of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. All 22 patients lacked expression of marker of sensitivity to anthracycline (TOPO2A) and the majority of patients did not express a marker of resistance to platinum therapy (ERCC1). Of the 22 patients whose tumors expressed EGFR, 5 had expression above the 75%ile and would thus be eligible for EGFR small molecule inhibitor therapy. Dysplastic liver nodule patients did not expressed significant level of EGFR. Further, multiplex-targeted proteomics discovered patients expressing cMET and IDO1, which indicate eligibility for clinical trials of targeted therapies or immunotherapies. 60% of dysplastic liver nodule patients did not expressed cMET at any level This study retrospectively evaluates HCC patients in an attempt to identify predictors of tumor behavior. Proteomic and genomic screening should be performed to identify differences between various hepatocellular lesions. Prospective evaluation of molecular and genetic profile is warranted in HCC patients to be distinguished from other hepatocellular mass lesions. Citation Format: Fabiola Cecchi, Nam Ku, Hanlin Wang, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jiaoti Huang. Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4935.

e23237Background: Traditional methods for HER2 assessment such as IHC and FISH are semi-quantitative and often limited by reproducibility issues. This may be a problem in tumors with borderline HER...

The overexpression and overactivation of hepatocyte growth factor receptor (Met) in various cancers has been linked to increased proliferation, progression to metastatic disease, and drug resistance. Developing a PET agent to assess Met expression would aid in the diagnosis and monitoring of responses to Met-targeted therapies. In these studies, onartuzumab, the experimental therapeutic 1-armed monoclonal antibody, was radiolabeled with 76Br or 89Zr and evaluated as an imaging agent in Met-expressing cell lines and mouse xenografts. Methods:89Zr-desferrioxamine (df)-onartuzumab was synthesized using a df-conjugate; 76Br-onartuzumab was labeled directly. Met-binding studies were performed using the human tumor–derived cell lines MKN-45, SNU-16, and U87-MG, which have relatively high, moderate, and low levels of Met, respectively. Biodistribution and small-animal PET studies were performed in MKN-45 and U87-MG xenografts. Results:76Br-onartuzumab and 89Zr-df-onartuzumab exhibited specific, high-affinity Met binding (in the nanomolar range) that was concordant with established Met expression levels. In MKN-45 (gastric carcinoma) xenografts, both tracers cleared slowly from nontarget tissues, with the highest uptake in tumor, blood, kidneys, and lungs. 76Br-onartuzumab MKN-45 tumor uptake remained relatively constant from 18 h (5 percentage injected dose per gram of tissue [%ID/g]) to 48 h (3 %ID/g) and exhibited tumor-to-muscle ratios ranging from 4:1 to 6:1. In contrast, 89Zr-df-onartuzumab MKN-45 tumor uptake continued to accumulate from 18 h (10 %ID/g) to 120 h (23 %ID/g), attaining tumor-to-muscle ratios ranging from 20:1 to 27:1. MKN-45 tumors were easily visualized in imaging studies with both tracers at 18 h, but after 48 h 89Zr-df-onartuzumab image quality improved, with at least 2-fold-greater tumor uptake than nontarget tissues. MKN-45 tumor uptake for both tracers correlated significantly with tumor mass and Met expression and was not affected by the presence of plasma shed Met. Conclusion:89Zr-df-onartuzumab and 76Br-onartuzumab specifically targeted Met in vitro and in vivo; 89Zr-df-onartuzumab achieved higher tumor uptake and tumor-to-muscle ratios than 76Br-onartuzumab at later times, suggesting that 89Zr-df-onartuzumab would be better suited to image Met for diagnostic and prognostic purposes.

Under normal conditions, hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase (TK), is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand-activated receptor internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis and tumor progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis.The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programs. The authors review Met structure and function, the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development, as well as the latest clinical trial results.Clinical trials with HGF/Met pathway antagonists show that as a class these agents are well tolerated. Although widespread efficacy was not seen in several completed Phase II studies, promising results have been reported in lung, gastric, prostate and papillary renal cancer patients treated with these agents. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment are optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents, and model systems available concerning HGF/Met oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

Under normal conditions, hepatocyte growth factor (HGF)-induced Met tyrosine kinase (TK) activation is tightly regulated by paracrine ligand delivery, ligand activation at the target cell surface, and ligand activated receptor internalisation and degradation. Despite these controls, HGF/Met signalling contributes to oncogenesis and tumour progression in several cancers and promotes aggressive cellular invasiveness that is strongly linked to tumour metastasis. The prevalence of HGF/Met pathway activation in human malignancies has driven rapid growth in cancer drug development programmes. Pathway inhibitors can be divided broadly into biologicals and low molecular weight synthetic TK inhibitors; of these, the latter now outnumber all other inhibitor types. We review here the basic properties of HGF/Met pathway antagonists now in preclinical and clinical development as well as the latest clinical trial results. The main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of optimal therapy combinations. The wealth of basic information, analytical reagents and model systems available concerning HGF/Met oncogenic signalling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control.

Glycosaminoglycans in normal human plasma, mainly represented by chondroitin sulfates and heparan sulfates/heparin (HSGAGs), show a specific distribution in the Cohn-Oncley fractions of human plasma. In the present study we investigated their effects on coagulation. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated for extraction of glycosaminoglycans after extensive proteolysis; the anticoagulant activity in the extracted samples was measured by activated partial thromboplastin time (APTT). The effects of the samples containing HSGAGs on factor II and factor X activities, before and after treatment with heparinase I, were also measured. The molecular weight of HSGAGs was determined by polyacrylamide gel-electrophoresis. Cryoprecipitate and fraction I, fraction II+III, and fraction IV-1 (the fractions containing HSGAGs) prolonged the APTT, whereas fractions IV-4 and V had no effect on the APTT. Fractions containing HSGAGs showed effects on factor II and factor X activities that were sensitive to heparinase I treatment. The molecular weight of HSGAGs recovered in cryoprecipitate and fraction I was 15-18 kDa; that of HSGAGs recovered in fraction IV-1 was 12.0 kDa. In conclusion, these results demonstrate that HSGAGs of different molecular weight, endowed with anticoagulant activity, circulate in normal human plasma in association with specific proteins involved in the regulation of hemostasis; and that endogenous HSGAGs play a role in maintaining the antithrombotic/hemostatic balance in normal human plasma.

Background: Despite improvements in the treatment of HER2+ breast cancer (BC), almost all patients (pts) progress in the metastatic setting. Three examples of resistance mechanisms are: PI3K mutations, lack of ADCC, or low expression of HER2. We recently showed that among 237 pts who had HER2 amplifications, 49% had normal or low levels of HER2 RNA. In addition, quantification of HER2 protein by selected reaction monitoring mass spectrometry (SRM-MS) accurately predicted HER2 expression status compared with IHC (3+)/ISH (≥2.0). Here we report a comprehensive panomic approach that integrates whole genome sequencing (WGS), RNASeq, quantitative proteomics, and pathway analysis to determine associations between tumor molecular profiles and prognosis among HER2+ pts. Methods: Matched tumor-normal samples (FFPE tumors and blood) were obtained from 58 pts with HER2+ BC who had received standard adjuvant chemotherapy and trastuzumab. Pts were divided into 2 groups: those who had no recurrence after 5 years and those who had developed metastases. The HER2 status of each pt was previously determined using IHC/FISH. Samples underwent WGS and RNASeq according to NantOmics CLIA-approved assay specifications. WGS data were processed using Contraster; RNASeq data confirmed the presence of gene mutations and was used to identify mutational and transcript abundance. PARADIGM was used to reveal associations between gene mutations and pathway levels. SRM-MS was used for proteomics analysis of a panel of 53 proteins. Tumor areas from FFPE tissue sections were analyzed after laser microdissection. Absolute protein quantitation was accomplished through simultaneous detection of endogenous target and synthetic labeled heavy peptide identical to analytical targets. Genetic alterations in germline and tumor DNA were compared in pts with vs without recurrence. Results: There was no statistically significant difference in the mean concentration of HER2 in the tumors of pts with vs without recurrence: 2.34 fmol/µL vs 2.56 fmol/µL. Other analyzed proteins did not appear to be associated with recurrence; however, expected correlations between pt and tumor characteristics and protein expression were found. With regard to clinically relevant mutations, we found one germline BRCA2 mutation in a pt with no family history of this mutation. The most commonly found somatic mutations were in TP53 (11 pts), AMBRA1 (11 pts), MORC4 (10 pts), SETD2 (8 pts), CDC27 (6 pts), BCLAF1 (5 pts), ZNF479 (4 pts) , PIK3CA (3 pts), PIK3R1 (3 pts), RUNX1 (3 pts), and GATA3 (3 pts). Conclusion: Whereas HER2 expression status was predictive of OS and PFS in pts treated with trastuzumab (Nuciforo et al. Mol Onc. 2015), in this small exploratory study of HER2+ BC pts, HER2 expression status was not predictive of recurrence. To better understand the molecular mechanisms driving recurrence beyond HER2 status alone, genomic sequencing may define a signature of recurrence after anti-HER2 therapy. Citation Format: Benz SC, Rabizadeh S, Cecchi F, Beckman MW, Brucker SY, Hartmann A, Golovato J, Hembrough T, Janni W, Rack B, Sanborn JZ, Schneeweiss A, Vaske CJ, Soon-Shiong P, Fasching PA. Integrating whole genome sequencing data with RNAseq, pathway analysis, and quantitative proteomics to determine prognosis after standard adjuvant treatment with trastuzumab and chemotherapy in primary breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-14.

Background: The use of next-generation sequencing has significantly advanced personalized medicine for patients (pts) with breast cancer. Despite this technological advancement, there remains the challenge of understanding how and if tumor heterogeneity can confound molecular analysis and treatment decisions. It has been shown that the expression of ER, PR, and HER2 can vary widely within different areas of the same tumor and between matched primary and metastatic lesions. The "Intensive Trial of OMics in Cancer"-001 (ITOMIC-001; NCT01957514) enrolls pts with metastatic TNBC who are platinum-naive and scheduled to receive cisplatin. Multiple biopsies of up to 7 metastatic sites are performed prior to cisplatin and repeated upon completion of cisplatin and following subsequent therapies. A subset of specimens is chosen for DNA sequencing, RNA sequencing, and quantitative proteomics. We explored the discordance of genomic and proteomic alterations for intrapatient and temporal heterogeneity in pts with TNBC, and the potential benefit of panomic analysis to better inform treatment decisions. Methods: Between 7 and 107 tumor samples/biopsy specimens were obtained from each pt from 1-23 different time points. Blood samples were collected for matched tumor-normal genomic analysis. DNA sequencing data were processed using Contraster; RNASeq data confirmed the presence of gene mutations and was used to identify mutational and transcript abundance. PARADIGM was used to determine associations between gene mutations and signaling pathways. Selected reaction monitoring-mass spectrometry (SRM-MS) was used for proteomics analysis. Results: Almost all pts had loss of TP53 (common in TNBC), and 5 pts had germline BRCA1/2 events, some exhibiting a signature of mutations corresponding to a mismatch repair defect in ≥1 pt. FGFR1/2/3 mutations/amplifications occurred in 5 pts. Three of 12 pts (25%) achieved partial responses after receiving treatments (post cisplatin) based on the molecular profile of their tumor: 1 pt with two FGFR2 activating mutations treated with ponatinib, 1 with a germline BRCA2 mutation treated with veliparib, and 1 with highly expressed Gpnmb treated with an antibody drug conjugate against Gpnmb. Tumor samples showed increased mutational and rearrangement burdens over time but shared mutational characteristics that were unique to each pt. Through the shared alterations across time points for 3 pts, it was possible to reconstruct the clonal history and heterogeneity of the tumors as various therapeutic approaches were attempted. Conclusions: Here we show in TNBC, intrapatient and temporal heterogeneity that may lead to a lack of response to identified targeted therapies. Tumor samples taken over time from the same pt become enriched for more complex genomic structures post therapy but share mutational characteristics, indicating the presence of recurrent tumor populations. This study enabled us to reconstruct the clonal history and heterogeneity of tumors across space (metastatic vs primary at t=0) and time, illustrating the need for comprehensive molecular analysis and combination/multi-targeted therapeutics for optimal treatment in TNBC. Citation Format: Soon-Shiong P, Rabizadeh S, Benz S, Cecchi F, Hembrough T, Mahen E, Burton K, Song C, Senecal F, Schmechel S, Pritchard C, Dorschner M, Blau S, Blau A. Integrating whole exome sequencing data with RNAseq and quantitative proteomics to better inform clinical treatment decisions in patients with metastatic triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-08.

74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.

Foretinib is an oral multi-kinase inhibitor targeting MET, vascular endothelial growth factor receptor (VEGFR)-2, RON, KIT, and AXL kinases. In this Phase 1, open-label, non-randomized study, foretinib was administered once daily at doses of 60 mg, 80 mg, 100 mg, or 120 mg for 28 days. The primary objectives were to determine the maximum tolerated dose (MTD) and assess the safety and tolerability of the daily oral administration schedule. Secondary objectives included pharmacokinetics, pharmacodynamics, and assessment of tumor response. Patients had histologically confirmed metastatic or unresectable solid tumors for which no standard treatments existed and all received oral foretinib once daily. Dose escalation was planned as a conventional "3+3" design with an expansion at the MTD for collection of additional safety and pharmacokinetic information. Thirty-seven patients were treated across four dose levels. The MTD was established as 80 mg foretinib. Dose-limiting toxicities were hypertension, dehydration, and diarrhea. The most common adverse events included fatigue, hypertension, nausea, and diarrhea. Twenty-three of 31 patients (74 %) had a best response of stable disease. No patient had a confirmed partial or complete response. At the MTD, steady state was achieved by approximately 2 weeks, with average post-dose time to maximum concentration, peak concentration, and trough concentration of 4 h, 46 ng/mL, and 24 ng/mL, respectively. In patients treated at the MTD, soluble MET and VEGF-A plasma levels significantly increased (P

Background: Antibody-Drug Conjugates (ADCs) are poised to become an extremely important class of therapeutics in oncology. By conjugating cytotoxic payloads to antibodies that target proteins found primarily on cancer cells, ADCs represent a novel mechanism for directing extremely toxic small molecules specifically to tumor cells. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also screening for the presence of any markers of resistance or response to the payload. Several proteins, such as multi-drug effluxers and tubulin-beta 3, have been implicated in resistance to small molecule cytotoxins and microtubule inhibitor drugs. OncoPlex Diagnostics has built a multiplexed ADC biomarker panel that simultaneously quantifies the levels of the antibody target and putative resistance markers for several known payloads, such as maytansinoids, auristatins and taxanes, as well as response markers for the topoisomerase inhibitor payloads SN-38 and doxorubicin. Methods: Liquid Tissue-Selected Reaction Monitoring (LT-SRM) is a multiplexed, quantitative method that uses mass spectrometry to quantify proteins based on a unique sequence of amino acids, and thus does not have the same limitations as traditional antibody-based, semi-quantitative protein detection methods, such as immunohistochemistry. We developed a LT-SRM assay to quantify protein levels of EGFR, FRalpha, Her2, CD30 and Mesothelin (antibody targets) and MCL1, MDR, MRP1, tubulin-beta3, Topo1 and Topo2a (payload response and resistance markers) simultaneously from FFPE biopsies. Calibration curves for all the proteins in the ADC panel are linear over 5-orders of magnitude, with limits of detection for each analyte between 25 and 400 amol/ug of tissue. Results: Analysis of FFPE tumor tissues show a broad range of expression for the ADC proteins, with some tissues showing no detectable levels of some payload markers. Clinical analysis of FRalpha showed a range of expression from Conclusions: The OncoPlexDx ADC panel can determine of a cutoff for expression levels of the antibody-target protein necessary for ADC response as well as identify markers of payload response or resistance to further understand how these markers affect therapeutic efficacy. This panel can be used to predict which patients will derive the most benefit from ADC therapy based on the specific biology of their tumor. Citation Format: Adele Blackler, Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Fabiola Cecchi, Marlene Darfler, Todd Hembrough, Jon Burrows. Development of a mass spectrometry based antibody-drug conjugate biomarker panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3398. doi:10.1158/1538-7445.AM2015-3398

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example amplification of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies nine proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-qualified, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. Because of the importance of TOPO2A in anthracycline-based therapies, which are commonly prescribed in breast cancer patients, sixty-two breast cancer biopsies were retrospectively reviewed. Of the 62 samples, 41 were positive for TOPO2A expression; ranging from 233-1750amol/ug. Of the primary biopsies, 80% of them expressed TOPO2A; however only two of seven liver metastases were positive for TOPO2A. These data suggest that anthracycline-based therapy might not be as efficacious in metastatic sites due to the lack of TOPO2A. Also of interest, quantification of FRalpha, a biomarker for folate-targeted therapies, showed a 50-fold range of expression in NSCLC cases, and TOPO1, the target of irinotecan and topotecan, showed a 10-fold range of expression in various indications, with 4% of biopsies having no detectable TOPO1. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually. Citation Format: Fabiola Cecchi, Adele Blackler, Heather Jordan, Marlene Darfler, Todd Hembrough, Michael Stocum, Jon Burrows. Clinical Survey of 19 actionable proteins in multiple indications using multiplex mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2015-3395

BackgroundImmune check point proteins play a pivotal role in immune evasion by the tumor. Recent trials involving inhibitors of the immune checkpoint protein pairs, PD-1 and PD-L1 have demonstrated anti-tumor activity. Measuring the levels of immune check point proteins and other members of the immunological synapse will help clinicians personalize therapy. Currently, immunohistochemistry (IHC) is the preferred diagnostic to assess PD-L1 status; however, PD-L1 positivity varies based on the antibody that is used. Additionally, PD-L1 negative patients by IHC have responded to anti-PD-L1 therapy implicating disconnect between PD-L1 diagnostics and response. We have developed and clinically validated a quantitative mass spectrometric technique that not only quantitates PD-L1 in formalin fixed paraffin embedded (FFPE) tissue but can concurrently quantitate other members (B7H3, B7.1, B7.2, OX40L) of the immunological synapse using the same tissue section. MethodRecombinant PD-L1 protein was used to identify optimal quantitative peptides for PD-L1 assay. Standard curves were generated using labeled and unlabeled peptides. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was then run on archived FFPE sections from in 9 normal tissues, 21 early staged (stage 1 and 2) and 4 advanced staged (stage 3) NSCLC patients. In addition PD-L1 was also assayed in bladder, breast and gastric cancer. Results PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis demonstrated moderate correlation (R2 = 0.8894). Normal lung tissue did not express PD-L1; ∼24% of early stage (5/21) and 50% of advanced stage NSCLC (2/4) expressed measurable PD-L1 protein. PD-L1 was detected more frequently in squamous cell carcinoma than adenocarcinoma. We are currently assessing the levels of PD-L1 and other targets of the immunological synapse using multiplex mass spectrometry and comparing it with IHC in 100 cholangiocarcinoma and possible inclusion of PD-L1diagnostics in clinical trials. DiscussionThe need to characterize expression levels of druggable targets in small biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Initial PD-L1 screening using clinical NSCLC samples suggests that more advanced NSCLC patients are more likely to be PD-L1 positive compared to early stage NSCLC patients. Laser microdissection (LMD) can be used to specifically microdissect the immunological synapse. Additional quantitative assays for both lymphocyte (CD8, CD68) and immunotargets (B7-H3,B.1, B7.2 etc) have been developed for assessing the ‘immune profile’ in tumor associated stroma via LMD. This immuno-proteomic assay of the key immunological synapse members within tumor and/or stroma may lead to improved personalized immunotherapy. Citation Format: Sheeno P. Thyparambil, Fabiola Cecchi, Eunkyung An, Wei-Li Liao, Jon Burrows, Todd Hembrough, Daniel Catenacci. Development and clinical validation of a quantitative mass spectrometric assay for immuno-oncology targets in FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4255. doi:10.1158/1538-7445.AM2015-4255

BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/μg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (1500 amol/μg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC. Citation Format: Fabiola Cecchi, Wei-Li Liao, Sheeno Thyparambil, Marlene Darfler, David Krizman, Todd Hembrough, Jon Burrows, Don Bottaro, Daniel V.T. Catenacci. A novel clinical tool that provides quantitative and accurate measurement of Met protein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3397. doi:10.1158/1538-7445.AM2015-3397

1045 Background: The folate pathway is a critical nucleotide biosynthetic pathway in many tumor cells, and blockade of this pathway with antifolates has demonstrated clinical utility in NSCLC and m...

4050 Background: Trastuzumab-based chemotherapy is standard treatment for HER2-positive advanced gastric cancer (AGC). Although increased HER2 gene amplification by fluorescent in situ hybridizatio...

In clinical practice heparin has to be administered by injection with obvious disadvantages; thus, transdermal delivery by electrically assisted methods have been studied. In this study we evaluated the efficacy of a Food and Drug Administration-approved pulsed current iontophoresis system in delivering heparin through living rat skin. Fluorescent and radioactive heparin as well as a commercial heparin preparation were delivered through rat skin via a pulsed current iontophoresis system. Pulsed current iontophoresis allowed fluorescent heparin to cross the stratum corneum localizing in epidermis and dermis. Unfractionated, high-, and low molecular weight fraction pools, obtained by fractionating [35S]-unfractionated heparin on a molecular weight sieve, were then separately tested. Pulsed current iontophoresis elicited the transdermal delivery of low molecular weight heparin, but not that of high molecular weight heparin. Finally, pulsed current iontophoresis of an unfractionated pharmaceutical heparin preparation significantly decreased plasmatic factor Xa activity. We hypothesize that this technique could be used to administer low molecular weight heparin in a cost-efficient and safe manner without the need for syringes and needles.

Heparin and heparan sulfate proteoglycans (HSPGs) are structurally diverse biopolymers that modulate many important protein-protein interactions. As ubiquitous components of extracellular matrices, the extended conformation, inherent complexity and high negative charge density of HSPGs enhance tissue integrity and provide selective yet substantial protein binding capacity [1]. Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate development and homeostasis, and drive tumorigenesis, tumor angiogenesis and metastasis in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases (RTKs) and HSPGs on target cell surfaces. Their respective RTKs also directly bind HSPGs, enabling the formation of ternary ligand-HSPG-RTK complexes with enhanced stability and signaling capacity [1, 2]. In a recent report, Cecchi et al. compared the HSPG binding sites on HGF and VEGF and found that critical basic HSPG binding residues provided similar surface charge distributions without underlying sequence or structural similarity, suggestive of convergent evolutionary adaptation to the common binding partner [3]. Prior studies to assess the impact of HSPG on HGF or VEGF signaling using alanine substitutions at HSPG binding residue positions showed some functional perturbation, but not to an extent consistent with the importance of HSPG in VEGF and HGF signaling gleaned from a variety of other studies. To reconcile this apparent discrepancy, Cecchi et al. used another approach to assess the impact of HSPG binding, wherein three opposite charge substitutions were combined in these sites in the HGF isoform NK1 (NK1 3S) or the VEGF isoform VEGF165 (VEGF 3S), with the goal of repelling HSPG from the ligand-RTK complex [3]. Remarkably, the substituted proteins were devoid of biological activity but retained native RTK binding affinity, and thus competitively antagonized their respective signaling pathways in normal and tumor-derived cells in vitro and in vivo [3]. NK1 3S and VEGF 3S antagonism of HGF and VEGF signaling, respectively, occurs through the combination of charge-based repulsion of HSPG and competitive displacement of the endogenous ligand from its RTK. Interestingly, NK1 3S retains optimal HGF receptor (Met) binding not only because its Met binding residues are unaltered, but also because HSPG-HGF binds more tightly to Met than HGF alone, suggesting that the acidic substitutions in NK1 3S mimic bound HSPG in this regard [3]. They fail to mimic bound HSPG, however, for the purpose of Met activation. Only HSPG polymers capable of binding multiple HGF molecules and enhancing HGF:HGF aggregation enabled Met signaling in HSPG-negative cells [3]. Together with prior studies, these results suggest that HSPG promotes HGF-Met complex clustering and, in turn, Met-Met interactions needed for kinase activation. In the VEGF receptor KDR, VEGF binds to IgG-like domains (D) 1-3; D2 contains primary contacts and D1 and D3 contribute to binding affinity and specificity [4]. HSPG functions similarly in the activation of Met and KDR. Like NK1, HSPG is required for high affinity binding of VEGF165 to KDR ectopically expressed in HSPG-negative cells [5]. Similar to Met [6] and fibroblast growth factor (FGF) receptors [2], KDR also interacts directly with HSPG, through at least one site located between D6 and D7 [5]. As shown for other family members and related receptors [7], a series of binding events may promote and incrementally stabilize HSPG-VEGF-KDR aggregates capable of signaling: VEGF-HSPG binding to KDR D2 stabilizes weaker VEGF-D1 and -D3 interactions, with additional stability gained through HSPG-VEGF-KDR bridging at D6/D7. These events, in turn, induce and/or stabilize conformational changes that enable homotypic D7 interactions and finally, TK domain interaction and transactivation [4]. VEGF 3S binds KDR at D2, but by repelling HSPG from the complex, is likely to destabilize some subsequent weaker interactions and ultimately, conformation changes leading to TK activation. Structural and functional studies of ligand-RTK interactions over the last decade highlight the importance of multiple binding events and associated conformational changes in RTK ectodomains that are required for kinase activation. These events vary in strength, and even weak interactions appear to provide necessary increments of increased stability to a signal transduction process whose complexity we are only beginning to appreciate. The acquisition of competitive antagonism by 3S forms of HGF and VEGF exposes the susceptibility of HSPG-facilitated events to selective disruption, and functionally defines the importance of HSPG in the ternary HS-ligand-RTK complex in normal and oncogenic signaling [3]. By tethering multiple long chain HS polymers, HSPGs stabilize ligand-RTK binding events by binding to both; beyond this, HSPGs have the potential to stabilize the aggregation of ligand-RTK complexes that is likely to accompany larger scale processes, such as endocytosis and endosomal sorting, that coincide with mitogenic and motogenic signaling and that have been shown to significantly influence the nature and magnitude of these cellular responses [8]. Future studies of HSPGs in sustaining endosomal RTK signaling should address, for example, whether HSPG fragmentation in early endosomes negatively regulates RTK signaling, and whether HSPG association affects the fate of ligand-RTK complexes to recycling vs. lysosomal endpoints, as shown for FGF-FGFR [9], and likely to be true for HGF-Met [10]. Further defining the role of HSPG in these large scale signaling processes should help identify cancer-associated aberrations and new opportunities for targeting this system to control disease progression.

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example high expressive of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-accredited, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually.

Signaling by hepatocyte growth factor (HGF) via its cell surface receptor, Met, drives cell survival, growth and motility, as well as tumor growth and metastasis in several prevalent human cancers. HGF and Met have become highly sought targets for cancer drug development: 165 clinical trials of 21 experimental agents have been completed or are in progress. We reported previously that a selective HGF antagonist failed to block growth of the human prostate adenocarcinoma derived cell line PC3M as primary tumor xenografts in mice, but potently blocked spontaneous PC3M tumor metastasis, as well as cell motility and invasion in vitro. Thus partial activation of the Met pathway in PC3M cells by murine HGF (mHGF) is capable of driving invasion but not proliferation, whereas human HGF (hHGF) is capable of driving both activities. To identify the signaling and gene modulation events critical for Met-driven cell invasion and to distinguish them from Met-driven proliferation, mRNA expression array and pathway profiling analyses of hHGF and mHGF-treated PC3M cells were performed. Ingenuity Pathway Analysis (IPA) of 344 hHGF-specific statistically significant gene modulation events of 2-fold or greater showed significant overlap with cell cycle progression and proliferation pathways, cancer and upstream activation of the cyclin D1 and choriogonadotropin pathways. IPA analysis of 520 gene modulation events associated with motility and invasion showed significant overlap with cell migration and invasion pathways, upstream activation of the MEK/MAPK and mir-155 pathways and upstream inhibition of PI3K and pro-inflammatory cytokine pathways. HGF-specific blockade of spontaneous PC3M metastasis in our model prompted us to further compare significant gene modulation events related to cell invasion with those reported for prostate cancer tumor samples as part of The Cancer Genome Atlas Project (TCGA; MSKCC, 2010; 216 cases). Of 520 altered genes in PC3M, 345 were also interrogated in TCGA: 150 of these (47%) were similarly modulated in prostate cancer patient samples. Twenty-four gene modulation events (7%) were associated with significant change in disease-free survival (DFS) in 88% of cases: 1 gene with increased DFS (logrank p: 0.002747, 56% of cases affected) and 23 with decreased DFS (logrank p: 0.007542; 65% of cases affected). Together these findings identify critical differences in intracellular pathways downstream of HGF/Met interaction leading to cell proliferation vs invasion and reveal that a substantial fraction of prostate cancer patients who show gene modulation events associated with increased tumor cell invasion and metastasis in the PC3M model described here also experience significantly accelerated disease progression. Citation Format: Fabiola Cecchi, Jason Lih, Young Lee, William Walsh, Mickey Williams, Donald P. Bottaro. Gene expression array and pathway profiling analyses distinguish HGF/Met pathways driving cell proliferation from invasion and identify events correlated with prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5259. doi:10.1158/1538-7445.AM2014-5259

16 Background: Overexpression of MET in gastroesophageal cancer (GEC) is associated with poor prognosis and potentially predictive of anti-MET therapeutic benefit. IHC has been the method chosen to quantify MET expression to date. However, IHC is semi-quantitative, suffers from cross-reactivity, and is low throughput. Moreover, MET IHC is hampered by antigenic instability in FFPE sections, limiting its utility to recently cut FFPE sections. Increasing recognition of the importance of other biomarkers in GEC suggests that ‘economic’ testing of scarce samples will be required. We sought to develop a MET quantitative assay within our ‘GEC-plex’ Liquid-Tissue-selected reaction monitoring (LT-SRM) MS test. Methods: We used trypsin digestion mapping of rMET to identify unique peptides for MS assay development. The assay was pre-clinically validated in 5 cell lines, where electrochemiluminescence (ECL) immunoassay measurement of MET was also performed. To assess the MET MS assay stability from archival FFPE sections, freshly cut FFPE tissue sections were immediately microdissected, processed and analyzed, while adjacent sections were processed and analyzed one year after cutting, to compare temporal quantification from the same FFPE samples (n=33). MET expression was assessed in GEC cases (n=121), and compared to IHC and FISH in select cases. Results: Tryptic digestion mapping of rMET showed that peptide TEFTTALQR was optimal for MET quantification. The LLOD for this peptide was 150 amol with CV2=0.99). The MET MS assay demonstrated temporal stability of serial sections cut from 33 samples analyzed one year apart: CVs2=0.75. Analysis of 121 GEC FFPE tissues showed a broad range of MET expression levels (

11083 Background: Developing an imaging agent to assess Met expression would aid in diagnosis and monitoring tumor response to Met-targeted therapies. Onartuzumab (MetMAb), a Met selective humanized one-armed monoclonal antibody, has been studied in Phase I-II clinical trials in which it was generally well tolerated and has shown the most benefit in patients with MET positive tumors. Methods: Studies to assess Met-binding were executed using the human gastric carcinoma cell line MKN-45 which exhibits a high level of Met expression. Murine PET studies and biodistribution assays were performed using MKN-45 xenografts. Results: Plasma shed Met concentration is directly related to total tumor burden (p < 0.001). The absence of a positive correlation between shed Met and %ID in blood indicates that binding of tracer to shed Met present in plasma is unlikely. There are positive correlations between tumor mass, Met abundance, and phosphoMet content and uptake of 89Zr-df-onartuzumab in MKN-45 mouse xenografts. Lastly, tumor mass, Met, pMet and 89Zr-df-onartuzumab uptake were all significantly decreased by drug treatment. Conclusions: MKN-45 tumor uptake of 89Zr-df-onartuzumab correlated significantly with tumor mass and Met abundance. Blood tracer uptake did not positively correlate with the presence of plasma shed Met. The amounts of Met, pMet, as well as 89Zr-df-onartuzumab image intensity correlated significantly with tumor size (all Spearman p values < 0.001).Tumor volumes and Met content were significantly decreased in TKI treated versus control mice, which correlated with imaging results.89Zr-df-onartuzumab has potential utility for imaging Met to identify patients for treatment with Met-targeted therapeutics and to identify the emergence of Met-driven acquired resistance to other molecularly targeted cancer therapies.

Interspecies cross-reactivity is an important consideration when interpreting the results of tissue xenograft studies. For many paracrine signaling pathways, murine growth factors may not activate their orthologous human receptors in tumor xenografts, thereby failing to recapitulate a potentially relevant human condition in the mouse model. Unrecognized or poorly defined species-specific differences of this type can seriously hinder basic research progress as well as the development of therapeutic or diagnostic agents. For example, the contributions of both tumor- and stromal-cell derived vascular endothelial growth factor (VEGF)-A to the vascularization of human tumor xenografts in immunodeficient mice prevented the direct comparison of anti-VEGF antibodies with different abilities to block host VEGF, and led to the production of mice expressing humanized VEGF with biological activity comparable to both human and mouse VEGF-A. Hepatocyte growth factor (HGF) and its receptor, Met, are also highly sought targets in cancer drug and diagnostic development. HGF/Met signaling drives tumor cell growth and motility, tumor invasiveness and metastasis. However, the failure of murine HGF to stimulate the growth of human tumor xenografts has restricted preclinical drug studies to a handful of human tumor cell lines that possess autocrine pathway activation or other more rare Met activation mechanisms, and ultimately forced the costly development of human HGF transgenic and knock-in mice so that preclinical studies could begin to encompass the known breadth of the pathway's involvement across human cancers. We report here that despite the failure of murine HGF to drive human tumor xenograft growth, mouse HGF potently stimulates human Met autophosphorylation, increased cell motility and matrix invasion, but not proliferation. As a result, the growth of xenografted PC3M human prostate adenocarcinoma cells as primary tumors in mice was not inhibited by a selective HGF antagonist, but spontaneous tumor metastasis was completely blocked. These results improve our understanding of the cross-reactivity between human and murine HGF/Met signaling pathways and suggest that the preclinical evaluation of agents for their ability to block HGF-driven tumor metastasis may incorporate cell lines expressing human Met but not HGF, i.e. a wide variety of human carcinoma-derived cell lines, without the need for using mice engineered to express human HGF. Citation Format: Fabiola Cecchi, Chih-Jian Lih, Young H. Lee, William D. Walsh, Michael P. Williams, Donald P. Bottaro. Experimental metastasis by the prostate adenocarcinoma-derived cell line PC3M is driven by partial activation of the human Met pathway. [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 4085. doi:10.1158/1538-7445.AM2013-4085

Acquired drug resistance is a long-standing problem of cancer therapeutics. The issue has become even more vexing with the development of highly selective agents; resistance to gefitinib or erlotinib is acquired frequently in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop strategies to prevent or circumvent occurrence. Through its receptor tyrosine kinase Met, hepatocyte growth factor (HGF) regulates mitogenesis, motogenesis, and morphogenesis during development and adulthood. HGF/Met signaling also contributes to cancer progression in many malignancies, including glioblastoma. Rilotumumab is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and glioblastoma, as well as combination trials in gastric, colorectal, small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, an HGF/Met dependent human glioblastoma-derived cell line (U87-MG) was grown in rilotumumab (600 nM) for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences were normal in both parental and resistant cells. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor, suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where most cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling via multiple pathways lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: Fabiola Cecchi, Karen Rex, Joanna Schmidt, Daniel Baker, Michael A. Damore, Angela Coxon, Teresa L. Burgess, Donald P. Bottaro. A cellular model of acquired resistance to rilotumumab (AMG 102) in glioblastoma. [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 5637. doi:10.1158/1538-7445.AM2013-5637

4543 Background: Mounting evidence supports Met as a therapeutic target in urothelial cancer (UC). Activated Met can promote angiogenesis and tumor growth by upregulating VEGF and may play a role in UC pathogenesis. Cabozantinib inhibits VEGFR2 and Met pathways. In this study, we assessed shed Met (sMet) levels in the urine and serum of UC patients (pts) and cabozantinib’s effects on HGF-driven UC cell growth and invasion. Methods: sMet levels in serum and urine samples from 31 pts with UC (23 metastatic, 8 muscle-invasive) were correlated with stage, presence of visceral metastases and urinary source. The effects of cabozantinib on 4 human UC-derived cell lines were studied in vitro. Intact RT4, TCC-SUP, T24M2 and T24M3 cells at 80% confluence were serum deprived 16 h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of Met, phospho- (p)Met, pAkt, Akt, pMAPK and MAPK by immunoassay or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion, proliferation and soft agar growth were measured. Results: Median serum Met levels were modestly higher in pts with metastatic versus muscle-invasive disease. Urinary Met levels were clearly higher in pts with visceral metastasis (P=0.0111) and in urine from ileal conduits and neobladders compared to normally voided urine, regardless of stage (P=0.0489). Met content in UC cell lines was low in RT4 and higher in T24M2, T24M3 and TCC-SUP. Basal pMet content was universally low, increased significantly by HGF and this was reversed by cabozantinib. HGF-driven increases in pAkt/Akt and pMAPK/MAPK in all 4 cell lines were reversed by cabozantinib, as were HGF-enhanced UC cell invasion, proliferation and anchorage independent growth. Conclusions: Median urinary sMet is significantly higher in pts with visceral metastasis and in specimens from ileal conduits and neobladders relative to normally voided urine. UC cell Met content in culture increased with disease grade; HGF stimulated activation of Met and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. These data support evaluation of cabozantinib in pts with metastatic UC.

Acquired drug resistance is a long-standing problem of cancer therapeutics, for example chemoresistance to temozolomide occurs in >90% of recurrent gliomas. The issue has become even more vexing with the development of highly selective targeted agents; e.g. agents targeting the epidermal and hepatocyte growth factor (EGF and HGF, respectively) pathways; resistance to gefitinib and erlotinib has already been frequently demonstrated in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop clinical strategies to prevent or circumvent its occurrence. HGF, through its receptor tyrosine kinase Met, regulates mitogenesis, motogenesis, and morphogenesis in a range of cellular targets during development and homeostasis. HGF/Met signaling also contributes to oncogenesis and tumor progression in many prevalent human malignancies, including glioblastoma. Rilotumumab (AMG102) is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and Glioblastoma, as well as combination trials in gastric, colorectal and small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, the HGF/Met dependent human glioblastoma-derived cell line U87-MG, was grown in continuous exposure to 600 nM rilotumumab for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences and the HGF promoter DATE region, where truncation reportedly increases HGF expression level, were found to have normal sequence and length in both parental and resistant cell lines. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where nearly all cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling by ARAF, CRAF, IGFR1, PDGFR, and other sources lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A204.

10528 Background: Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. Methods: To understand the role of HS in ligand-receptor interaction and biological signaling, we identified three putative HS binding residues in N domain (K60, K62 and R73), generated opposite charge mutations at these positions in NK1, and characterized the recombinantly expressed proteins biochemically and biologically. Results: Nuclear magnetic resonance analysis of mutant and wild type (WT) proteins revealed that no significant structural alterations were introduced by the mutations. The binding of each of the mutant protei...

10632 Background: Oncogenic signaling via the hepatocyte growth factor (HGF)/Met pathway promotes tumor cell proliferation, migration, invasion and survival. High levels of Met are poorly prognosti...

Signal Transduction via the hepatocyte growth factor (HGF)/Met receptor kinase pathway is critical for normal embryogenesis and adult homeostasis, and aberrant HGF/Met signaling contributes to tumorigenesis and metastasis in many prevalent forms of cancer. By defining the basic molecular mechanisms of pathway activation at the target cell surface, we hope to develop novel strategies to antagonize oncogenic signaling as well as methods to assess pathway status in cancer patients. These methods may help identify those most likely to benefit from HGF/Met targeted therapies and help monitor treatment response. Distinct domains of HGF bind to the Met receptor tyrosine kinase and to cell-surface heparan sulfate (HS) proteoglycans. The latter are widely expressed, structurally diverse biopolymers that modulate many important protein-protein interactions, but their importance in eliciting HGF biological responses is controversial. We have identified basic (positively charged) amino acid residues in HGF that form the primary binding site for (negatively charged) HS proteoglycans. HS binding by these residues increases the affinity of ligand-receptor binding and, with HS polymers of sufficient length, facilitates ligand oligomerization. Ligand oligomerization, in turn, controls Met kinase activation, cell motility and proliferation under normal conditions. Opposite (negative) charge amino acid substitutions at these critical HS binding residues in HGF can mimic bound HS in promoting high affinity receptor binding, but antagonize the binding of long HS polymers on the cell surface, thereby antagonizing ligand oligomerization and its consequences. Engineering these substitutions in the context of a short HGF isoform containing only the primary HS and Met binding domains yields a potent and selective competitive inhibitor of oncogenic HGF signaling in vivo. These findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development that may be applicable to other HS-binding growth factors. The Met receptor kinase is among many transmembrane proteins that are proteolytically released from the cell surface by a process known as ectodomain shedding. Consistent with the concept that Met ectodomain shedding is a normal physiological process, we found that healthy human volunteers (n > 100) display soluble Met (sMet) plasma concentrations in the range of 100 - 300 ng/ml and urinary sMet concentrations that are approximately 1000-fold lower. We hypothesized that aberrantly high Met expression, a frequent occurrence in many forms of cancer, and/or dysregulated proteolytic activity, another common feature of aggressive malignancies, could lead to increased ectodomain shedding and that sMet levels could be a useful biomarker of tumorigenesis, tumor progression and overall tumor burden. In prior studies we found that increased Met shedding correlated with malignancy in several cell lines, and that urinary sMet levels were significantly elevated in metastatic prostate cancer patients relative to those with organ-confined cancer or no evidence of disease. In an ongoing investigation of sMet as a diagnostic and/or prognostic biomarker of bladder cancer, the mean urinary sMet level among 150 patients with pathologically confirmed disease was significantly higher than that obtained from a group of 50 individuals with no evidence of disease, whether mean values were normalized to urine creatinine concentration or not (two-tailed Mann-Whitney t-test p-value We have also measured plasma sMet levels in patients participating in a phase II clinical trial of Foretinib (formerly GSK1363089/XL880), a small molecule multikinase inhibitor targeting Met and vascular endothelial cell growth factor-2 (VEGFR-2) Axl and Ron, designed to evaluate the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single therapeutic agent in patients with metastatic gastric carcinoma (GC). Plasma sMet concentrations, as well as plasma VEGF-A, HGF and soluble VEGFR-2 (sVEGFR-2) levels were measured before and during treatment with foretinib to measure potential pharmacodynamic (PD) effects and/or the modulation by foretinib of Met and VEGFR-2 pathways. Marker changes from baseline were analyzed at days 5, 15, 29 and 47 using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (sum of longest diameter [SLD] of tumor as a surrogate for tumor shrinkage, progression-free survival [PFS], and RECIST) were also examined. In GC patients on the intermittent dosing schedule, plasma sMet and VEGF-A concentrations tended to increase during the treatment periods compared with the drug holidays, suggestive of a direct short-term drug effect on sMet and VEGF-A levels. Although mean tumor burden did not change significantly from baseline to week 8, plasma levels of sMet and VEGF-A correlated positively with the change in SLD over this period, suggesting that the increases of sMet and VEGF-A may be reflective of increased GC tumor burden. Citation Format: Fabiola Cecchi, Deborah Pajalunga, Brian McNeil, Daniel Rabe, Andrew Fowler, Yuan Liu, C. Robert Gagnon, Howard Kallender, Manish A. Shah, Robert Getzenberg, Mark Schoenberg, Anne-Marie Martin, Ramaprasad Srinivasan, W. Marston Linehan, R Andrew Byrd, Donald P. Bottaro. Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY23-03

The MET tyrosine kinase receptor is important during human development for normal cell growth and migration. Activating alterations of MET have also been identified in several carcinomas. Previous immunohistochemistry studies in our laboratory found high levels of MET in 85% of osteosarcoma tumor samples. In addition, pharmacologic modulation of MET resulted in a migratory decrease in some osteosarcoma cell lines. This led us to hypothesize that MET is necessary for osteosarcoma metastasis, and genetic down-regulation of MET expression would reduce the metastatic phenotype of osteosarcoma cells. We used lentiviral shRNA constructs to knockdown MET expression in several osteosarcoma cell lines. These included MNNG-HOS, a metastatic chemically transformed line that expresses constitutively activated MET, MG63.2, a highly metastatic variant of poorly metastatic MG63, AI and LR, which are two newly derived cell lines that have extremely high MET levels. We quantified levels of MET expression using a sensitive and quantitative electrochemiluminescence (ECL) assay and assessed in vitro parameters of metastasis including motility, migration, and invasion. MET expression was assessed by ECL in protein lysates from frozen osteosarcoma tumor samples and cell lines, and 15-20% of samples showed extremely high levels of total MET. AI, LR and MG63.2 had the highest levels of MET expression. MNNG-HOS has activation of MET due to translocation of the 3′end of MET with the 5′end of TPR. We utilized these lines for shRNA knock down of MET. ECL quantification of total MET levels revealed greater than 70% decrease in MET expression with shRNA knockdown in all four cell lines, compared with cells infected with scramble shRNA controls. As predicted for MNNG-HOS, shRNA constructs targeting the 3’ tyrosine kinase domain of MET resulted in marked decreased motility, compared to intermediate decreases using shRNA constructs targeting the 5’ region. Knock down of MET in MG63.2 resulted in inhibition of both motility and migration. AI and LR assays are in progress. Taken together, these data are the first evidence that genetic down-regulation of MET results in a decreased metastatic phenotype in osteosarcoma. This is a promising finding, as several small molecule tyrosine kinase inhibitors targeting MET are in development. We are currently testing our hypothesis in a mouse model to determine the ability of the above genetically knocked-down MET cell lines to decrease metastasis in vivo. Concurrently, in preparation for translational studies, we are also assessing MET specific tyrosine kinase inhibitors utilizing the parental cell lines described above. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3401.

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth. We now show that HGF signaling requires specific HS binding interactions and that targeted disruption of these HS binding sites in HGF yields a potent and selective competitive inhibitor of normal and oncogenic HGF signaling. Our findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development which may be applicable to other HS-binding growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 342.

Foretinib (formerly GSK1363089) is a small molecule multikinase inhibitor which includes inhibition of MET and VEGFR2. The ongoing phase II study, MET111643, is evaluating the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single agent in pts with metastatic GC. An additional component of the study is to collect patient plasma samples before and during treatment with foretinib, to explore the potential pharmacodynamic effect and/or the modulation by foretinib of MET and VEGFR2. Plasma samples were collected from 42 pts on the intermittent 5 days on/9 off foretinib schedule at baseline and prior to dosing on days 5, 15, 29, and 47. The plasma levels of sMET, HGF, sVEGFR2, and VEGFA were measured using the Meso Scale Discovery platform (MSD). These marker changes from baseline were analyzed at each time point using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (progression free survival [PFS] and RECIST response) were also examined. Statistically significant increases from baseline of sMET at days 5, 15, 29, and 47 (p Changes in sMET, VEGFA, and sVEGFR2 levels were observed for both intermittent and daily dosing schedules in the gastric cancer study. Although SLD at 8 weeks did not change significantly from baseline in the GC study, plasma levels of sMET and VEGFA correlated positively with the week 8 magnitude of SLD. Hence plasma levels of sMET and VEGFA may reflect biological changes following foretinib treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B210.

Foretinib is a potent, orally available, small-molecule inhibitor of MET and VEGFR2. Significant tumor cell growth inhibition was observed preclinically following treatment in multiple tumor models. Antitumor activity was observed in a previous phase I study in which foretinib was dosed intermittently on days 1–5 every 14 days. This phase I dose-escalation study to evaluate daily oral administration of foretinib was conducted in adults with solid tumors. Foretinib was orally administered until disease progression or toxicity mandated removal from the study. Dose-limiting toxicities (DLTs) that occurred during the first 28 days of treatment were used to determine the maximum tolerated dose (MTD). Safety, tolerability, pharmacodynamics (PD), and pharmacokinetics (PK) were evaluated. Tumor volume was assessed every 8 weeks by RECIST. A total of 37 patients (pts) were treated across 4 dose levels in the following order: 60 mg/d (6 pts), 80 mg/d (12 pts), 120 mg/d (3 pts), 100 mg/d (3 pts), and 80 mg/d (13 pts, PK expansion). Reported DLTs were hypertension and dehydration at 120 mg/d and diarrhea at 100 mg/d. The MTD of foretinib was determined to be 80 mg. With chronic dosing, 12 of 25 pts (48%) on 80 mg required a dose reduction to 60 mg between 21 days and 4 months on treatment. Frequent AEs associated with foretinib were hypertension (62%), fatigue (51%), nausea (43%), diarrhea (41%), proteinuria (30%), increased lactate dehydrogenase (27%), vomiting (24%), anorexia (22%), increased aspartate transaminase (19%), rash (16%), increased GGT (16%), and increased lipase (16%), primarily CTCAE grades 1 and 2. The foretinib plasma PK (at MTD) was characterized by a tmax of ≈4 hours, a steady-state oral clearance (CL/F) of ≈83 L/h, and ≈3.8-fold accumulation at steady state (achieved by ≈day 15). At steady state, values of Cmax and AUC0–24 were ≈45.7 ng/mL and ≈805 h · ng/mL, respectively, and the Cmin was ≈52% of Cmax. Plasma PD markers were measured during cycle 1 in 19 pts treated at the MTD. Shed Met (sMET) and VEGF showed increases, whereas sVEGFR2 showed a decrease. No pts had confirmed partial response or complete response, but stable disease (SD) (range, 2.1–18.1 months) was seen in 23 pts (74.2%). 11 pts had tumor regression from baseline, with tumor shrinkage ranging from 1%–21%. Disease progression was seen in 8 pts (25.8%). Overall, 35.5% of pts were event-free at 6 months, and 12.9% of pts (diagnoses included medullary thyroid, hepatocellular, and papillary renal cell carcinomas, and alveolar soft part sarcoma) were event-free at 12 months. 62% percent of pts withdrew due to progressive disease, 8% due to AEs, 5% due to death, and 24% due to consent withdrawal/investigator discretion/other. Foretinib can be safely administered at the oral daily dose of 80 mg (MTD); however, with chronic administration, dose reductions were not uncommon. The safety profile observed with daily dosing of foretinib is similar to that seen with intermittent dosing. Plasma PK results indicate that exposure to foretinib was well maintained over the daily dosing interval. PD results are similar to that seen with intermittent dosing and indicate that sMET, VEGF, and sVEGFR2 are promising markers for monitoring biological activity of foretinib. Prolonged SD and tumor regression in pts with various cancers suggest that foretinib has promise as an anticancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A8.

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth, and inhibition of these activities by 3M NK1 validates a novel strategy for developing antagonists of the HGF pathway and potentially those of other HS-binding ligands Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B228.

Background Among genitourinary malignancies, bladder cancer (BCa) ranks second in both prevalence and cause of death. Biomarkers of BCa for diagnosis, prognosis and disease surveillance could potentially help prevent progression, improve survival rates and reduce health care costs. Among several oncogenic signaling pathways implicated in BCa progression is that of hepatocyte growth factor (HGF) and its cell surface receptor, Met, now targeted by 25 experimental anti-cancer agents in human clinical trials. The involvement of this pathway in several cancers is likely to preclude the use of urinary soluble Met (sMet), which has been correlated with malignancy, for initial BCa screening. However, its potential utility as an aid to disease surveillance and to identify patients likely to benefit from HGF/Met-targeted therapies provide the rationale for this preliminary retrospective study comparing sMet levels between benign conditions and primary BCa, and in BCa cases, between different disease stages. Methods Normally voided urine samples were collected from patients with BCa (Total: 183; pTa: 55, pTis: 62, pT1: 24, pT2: 42) and without BCa (Total: 83) on tissue-procurement protocols at three institutions and sMet was measured and normalized to urinary creatinine. Normalized sMet values grouped by pathologic stage were compared using non-parametric tests for correlation and significant difference. ROC analyses were used to derive classification models for patients with or without BCa and patients with or without muscle-invasive BCa (MIBCa or NMIBCa). Results Urinary sMet levels accurately distinguished patients with BCa from those without (p