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Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models.Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized.Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

XLS file, 33K, Summary of RG7212 monotherapy and combination antitumor efficacy.

PDF file, 306K, RG7212 antitumor efficacy and characterization of tumor models used to assess RG7212 antitumor efficacy.

Supplemental Figure S1 and Table S1. Figure S1: RG7212 phase I study flow diagram and treatment schema. Table S1. Summary of Fn14 Testing and Positivity Rate (IHC{greater than or equal to}1+) for the Most Commonly Screened Solid Tumors

PDF file, 771K, TWEAK promotes proliferation and survival signaling with minimal effect on cell viability.

Purpose: Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand–receptor pair frequently overexpressed in solid tumors. TWEAK:Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFκB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14.Experimental Design: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of TWEAK:Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics.Results: In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment.Conclusion: RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced TWEAK:Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258–66. ©2014 AACR.

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XLS file, 193K, Sorted Affymetrix gene array data.

PDF file, 29K, Lead antibodies have identical binding properties and neutralizing activities.

PDF file, 176K, Assessment of baseline gene expression in RG7212 responders and non-responders in qPCR array.

Acute myeloid leukaemia (AML) is uniquely sensitive to p53 activation 1, 2 as ≈90% of patients carry wild-type TP53 and frequent MDM2 overexpression.3 MDM2 blocks p53 transactivation and targets p53 for ubiquitin-dependent degradation.4, 5 Nutlins have been characterized as potent and selective small-molecule MDM2 antagonists.1, 6–8 RG7112 was the first such MDM2 antagonist to undergo clinical assessment in solid tumors and leukaemia trials.1, 2, 9 As not all patients with functional p53 will respond to MDM2 antagonists, diagnostic tools may identify patients likely to respond.

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

We report a proof-of-mechanism study of RG7112, a small-molecule MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were eligible for resection.Patients with well-differentiated or dedifferentiated liposarcoma were enrolled at four centres in France. Patients received up to three 28-day neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a patient progressed at any point after the first cycle, the lesion was resected or, if unresectable, an end-of-study biopsy was done. The primary endpoint was to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation (P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and apoptosis). All analyses were per protocol. This trial is registered with EudraCT, number 2009-015522-10.Between June 3, and Dec 14, 2010, 20 patients were enrolled and completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations, assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR 4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day 8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline. The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR -12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of MIC-1 (p0·0001) and haematological toxicity. One patient had a confirmed partial response and 14 had stable disease. All patients experienced at least one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia (nine patients), diarrhoea (nine patients), and thrombocytopenia (eight patients). There were 12 serious adverse events in eight patients, the most common of which were neutropenia (six patients) and thrombocytopenia (three patients).MDM2 inhibition activates the P53 pathway and decreases cell proliferation in MDM2-amplified liposarcoma. This study suggests that it is feasible to undertake neoadjuvant biopsy-driven biomarker studies in liposarcoma.F Hoffmann-La Roche.

Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (±20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase β Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3αβ Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.

David H Geho1, Alessandra Luchini1,3, Enrico Garaci2, Claudio Belluco3, Emanuel Petricoin1 & Lance A Liotta1† †Author for correspondence 1Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, VA 20110, USA Tel.: +1 703 993 9444; Fax: +1 703 993 4288; E-mail: lliotta@gmu.edu 2Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy 3CRO-IRCCS National Cancer Institute, Aviano, Via Pedemontana Occidentale, 12, 33081 Aviano, Italy ‘...the actual trend in biomedical research is moving from the study of cultured cells to the analysis of freshly collected cells, tissue samples and biofluids...’

Reverse phase protein microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling proteins within diseased cells. One limitation of protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for proteins. Enhancement of protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a protein-binding substrate comparable to nitrocellulose.

Vignette 1: current practice A 45-year-old female presents at a clinic with a complaint of shortness of breath. After a thorough cardiovascular evaluation that reveals no evidence of heart disease, a computed tomography (CT) scan of the thorax reveals several large nodules in the right lung. An endoscopically-obtained tissue biopsy is taken and fixed in formalin for morphological analysis by a pathologist. Under a microscope, the pathologist examines the tissue section that has been stained with hematoxylin and eosin. Based on experience, the pathologist classifies the tissue as a poorly differentiated adenocarcinoma of the lung. A subsequent staging imaging study is performed revealing likely significant nodal involvement. Based on years of clinical correlations and clinical trials, this patient begins a treatment regimen including chemotherapy. The oncologist communicates to the patient that a range of patient responses to the proposed chemotherapies has been observed, from good to poor response. Patient concerns over the side effects of therapy are balanced against the hope of a good response. At present, based on morphological assessments, the type of response cannot be predicted.

Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.

A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.

Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously.

Background & Aims: Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder characterized by oculocutaneous albinism and platelet dysfunction, results from mutations in 1 of at least 7 different genes. Some patients develop a fatal pulmonary fibrosis and others a disabling colitis. This study aimed to document the occurrence of colitis among HPS patients, characterize gastrointestinal tract involvement in HPS, and analyze the distribution of colitis among HPS genotypes. Methods: Of the 122 HPS patients followed at the National Institutes of Health Clinical Center between 1993 and 2005, 24 were evaluated by endoscopy for gastrointestinal complaints. The histology of gastrointestinal biopsies was retrospectively examined to assess for inflammatory changes, granulomata, and pigmented macrophages. These data were compared with symptoms and HPS genetic subtypes. Results: At colonoscopy, 7 of 23 patients (30%) had endoscopic mucosal abnormalities, including nodularity, erythema, petechiae, or erosions. Six of these 7 patients (86%) had findings of colitis on biopsy. Of the 16 patients with normal-appearing colonic mucosa, 2 patients (12%) had colitis on biopsy. Pigmented macrophages were also observed in the colonic lamina propria in 16 of the 23 patients (70%). Of the 8 patients with confirmed colitis, 7 had the HPS-1 subtype, and 1 had the HPS-4 subtype. Conclusions: There is an increased frequency of colitis in our population of 122 HPS patients (8/122, 7%) and in HPS patients referred specifically for symptom evaluation (8/24, 33%). Colitis was found in patients with HPS-1 and HPS-4 genotypes.

Molecular diagnostics research within the field of cancer is increasingly focused on detecting low-abundance protein endpoints that can be used to define a patient's disease more completely. Protein microarrays represent an important Clinical Proteomics tool for directly measuring protein endpoints in samples extracted from patient tissues. By combining laser capture microdissection, arraying devices, validated isoform-specific antibodies and advanced reporter technology tools, Clinical Proteomics laboratories are currently generating molecular portraits of cancer cells harvested from patient biopsies.

Recent evidence suggests that each patient's cancer has a unique subset of molecular pathogenetic derangements. These derangements may both genetic and proteomic alterations. Genomic and proteomic research tools enable genome-wide assessment of gene expression as well as kinase driven cell signaling events. These tools are illuminating the molecular derangements of individual tumors, even if these tumors have similar morphological characteristics. A combination of laser capture microdissection with multiplexed phosphoproteomic analysis using reverse phase protein microarray technology is being used to identify protein molecular signatures of individual tumors. The in vivo state of multiple kinase driven signal pathways may be evaluated by reverse phase protein microarray with a panel of specific antibodies developed based upon our knowledge of biological processes. Molecular profiling of individual patient's tumors is currently being evaluated in clinical trials at the National Institutes of Health, National Cancer Institute for monitoring Epidermal Growth Factor (EGF) cell signaling events for patients with breast and ovarian cancer.

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomic endpoints in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states. One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor microenvironment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination, clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.

Abstract 675 Background: Activation of the p53 pathway by inhibition of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. We report the final results of the Phase 1 leukemia trial of RG7112, a small molecule antagonist of MDM2 which activates p53 by disrupting the p53-MDM2 interaction. Methods: The trial was designed as a multi-center, open-label Phase 1 MAD study of patients with relapsed/refractory leukemia treated with escalating doses of single agent RG7112 in two strata: A:AML, ALL, CML-BC; and B:CLL and S-CLL. RG7112 was administered orally daily for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT; secondary objectives to assess PK, PD and clinical responses. Enrollment is complete. 4 patients remain on study, and final data is being compiled. Peripheral blood was collected for PK and biomarkers pre-treatment, at multiple time points during the first 48 hours, and on d10. Bone marrow was collected pre-treatment, d10 and d28. Blood and marrow were purified by MACS sorting (CD19, 33 or 34 Ab). AmpliChip analysis of p53 mutational status (single nucleotide alterations exons 2–11) was performed. MIC-1 (macrophage inhibitory cytokine-1), a p53 transcriptional target, was analyzed in serum by ELISA as a pharmacodynamic marker of p53 activation. Baseline MDM2 mRNA levels, and changes from baseline, as a marker of p53 activation and feedback on MDM2, were measured by RT-PCR. A panel of 24 genes was assessed serially by TLDA (q-PCR). Results: 116 patients (84 AML,10 ALL, 2 CML, 20 CLL/sCLL) were treated with RG7112 doses ranging from 20 – 2430 (1215 BID) mg/m2/day × 10 days. The dose-exposure relationship (Cmax, AUC, and Ctrough) was approximately linear with a median half-life of 1–1.5 days. 43 patients with acute leukemia were dosed at the MTD of 1500 mg BID (flat dose) × 10 days. Major adverse events(> 20%) were nausea, diarrhea and vomiting. Other major (>10%) AEs were complications of leukemia (e.g. neutropenic fever). Clinical activity was noted during dose escalation (ASH 2011), including apoptosis in AML and CLL, CR in an AML patient (360mg/m2) stable for 6 cycles (10 months) and one patient with CLL/Richter's transformation with stable disease (40 mg/m2) for >25 cycles. New data focuses on AML patients treated at MTD. This cohort received a median of 3 prior treatments, 16 were refractory to last treatment. 17 (40%) had evidence of hematologic improvement (greater than 50% drop in peripheral and/or BM blasts by d28). Of the 31 patients who completed cycle 1 dosing, 5 patients (16%) had a CR (2 CR, 2 CRi. 1 CR without recovery). Two CR patients have proceeded to allotransplant. All CR patients' blasts were p53wt. Of the 12 additional patients with single-agent hematologic activity, 3 had biochemical evidence of tumor lysis syndrome, 10 had p53status evaluable, and mutations were detected in 3: (exon 7 missense, exon 9 frameshift, intron 7 splice). p53 activation by RG7112 was demonstrated by MIC-1 induction in serum. MIC-1 levels were elevated at RG7112 plasma concentrations >2 μg/mL or AUC24h >50 μg · h/mL and were highly correlated (0.5629) with exposure to RG7112 (AUC24h) at steady state following a sigmoidal Emax model. Preliminary data review suggests a positive correlation between exposure to RG7112 and changes in MDM2 gene expression at day 2. In addition, q-PCR analysis demonstrated exposure-dependent induction of key p53 transcriptional targets including MDM2, p21, and PUMA. p53 protein increase by WB was demonstrated at the MTD in selected patients. These findings are consistent with the proposed mechanism of action of RG7112. Conclusions: We report evidence of single agent clinical activity in patients with leukemia treated with RG7112, including 6 AML patients with CR, 5 of 31 (16%) at the MTD, and additional patients with significant decreases in blasts. Concentration-related pharmacodynamic biomarker activity of the p53 pathway was demonstrated by increases in MIC-1 levels, and induction of p53 target genes including mdm2, p21 and PUMA. 4 patients remain on treatment and final results will be reported. We provide proof of mechanism and evidence for single-agent clinical activity of MDM2 antagonism in leukemia by demonstrating complete remission, lysis of leukemic blasts and activation of p53 pathway targets in leukemic cells from patients treated with RG7112. Clinical studies of RG7112 in combination are underway. Disclosures: Andreeff: Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other. Yee:Roche: Membership on an entity's Board of Directors or advisory committees. Assouline:Roche: travel funding Other. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy; Ariad: Consultancy. Drummond:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Geho:Roche: Employment. Blotner:Roche: Employment. Cheng:Roche: Employment. Vassilev:Roche: Employment. Ding:Roche: Employment. Zhi:Roche: Employment. Middleton:Roche: Employment. Nichols:Roche: Employment.

Within this review we discuss two methodologies used in tissue proteomics, namely mass-spectrometry (MS)-based protein pattern diagnostics and protein microarrays. Further, we describe current goals within the field of tissue proteomics and suggest points of departure for designing nanotechnology-based tools that will enhance the role of molecularly based diagnostics and therapeutics development in clinical medicine.

Many protein functions are conferred by posttranslational modifications, which allow proteins to perform specific cellular tasks. Protein microarrays enable specific detection of posttranslational modifications not attainable by gene arrays. Reverse-phase protein microarrays have been widely adopted for use with clinical biopsy specimens because they have many advantages including highly reproducible printing of cellular lysates onto array surfaces, buit-in dilution curves, and direct detection using one antibody per analyte. This results in high-sensitivity, broad dynamic range, and favorable precision. Reverse-phase arrays have been restricted to a one slide/one antibody format. Although this is suitable for analyzing treatment effects over populations of samples, it is not well suited to individual patient assessments. One means of reaching this goal is the sector array format. Through the sector array, multiple antibody probes can be multiplexed on a single slide containing replicate immobilized aliquots from one patient. Thus, on one slide, a complete set of analytes can be characterized and used to support a therapy decision. This article describes a method for constructing sector arrays and demonstrates feasibility and adequate sensitivity applied to apoptosis related pathways.

Vessel wall extracellular matrix, which underlies the endothelium, is a potent stimulator of platelet adhesion and activation. Exposure of this matrix can result from damage incurred by vascular interventions, such as saphenous vein bypass grafting and angioplasty. Fibrillar collagens are an important component of the thrombogenic extracellular matrix. Herein we describe a means of targeting poly(ethylene glycol) (PEG)-mediated blockade directly to platelet-binding ECM molecules, such as type I collagen, thereby selectively blocking platelet adhesion to vascular matrix. Purified fibronectin (FN), a matrix protein that interacts with fibrillar collagens and platelets, was selectively pegylated to generate a targeted molecular shielding reagent that masked ECM ligands from platelet recognition and adhesion. This approach protects the functions of other vascular proteins, including surface proteins on intact endothelium. To mask the platelet-binding site of FN, PEG-propyl moieties (5000 Da) were covalently appended to lysine residues on the surface of FN, generating FNPEG-5K. To preserve the collagen-binding function of FN, it was pegylated while bound to a gelatin agarose matrix. We demonstrate that FNPEG-5K blocks platelet adhesion to purified type I collagen. Moreover, the same preparation blocks platelet adhesion to vascular wall components, including collagens.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b · DAF and CD1c · DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c · DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b · DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c · DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b · DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.

At a functional level, cancer is a proteomic disease [(1)][1] . Although DNA is the information archive, proteins do all of the work of the cell. A pathogenetic defect is selected out during somatic genetic progression because the defect ultimately alters the protein network to generate a selective

Inelastic electron tunneling spectroscopy (IETS) is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nanoelectronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices.

Cancer is usually diagnosed and treated when it is too late for local therapeutic strategies and most patients already harbour occult or overt metastasis. Metastatic dissemination can occur via lymphatic and vascular systems and represents the final stage of a disease process that can evolve over 5–20 years. Unrestrained growth by itself does not cause metastasis; additional genetic mutations over and above those that cause uncontrolled proliferation are needed. Less than 0.05% of circulating tumour cells are successful in initiation and development of a metastatic process. Metastatic distribution varies widely with histological type and anatomical location of the primary tumour. The organ preference for metastatic colonization is strongly influenced by communications between the circulating tumour cells and the target host tissue through a variety of molecular mechanisms. Moreover the presence of the new malignant cells within the invaded host tissue is associated with a local reorganization of the stroma, blood vessels, lymphatics, and epithelial morphology. Host cells contribute to enzymes and cytokines that promote tumour cell survival and invasion. Significant progress has been made in our understanding of the molecular cross-talk between tumour cells and host cells at the invasion front. Several molecules including cytokines, growth and motility factors, matrix components, and degradative enzymes are thought to be required for the invasion process. Based on the cellular and extracellular context of the microenvironment, cytokines, stromal elements, and immune factors may promote or retard cancer pathogenesis. Invasion is dependent on the coordinated activity of a series of interacting proteins extending from the inside of the cell to the cell surface and the adjacent host cellular and extracellular microenvironment. Cellular adhesion, local proteolysis at the cell surface and of the extracellular matrix, and motility are the triad of necessary functions that mediate invasion. Normal tissue morphology and organ architecture are tightly regulated by a communication reciprocity between the tissue cells and the extracellular matrix and/or basement membrane. Instead of being autonomous, malignant cells communicate extensively with other cells and the extracellular matrix. Moreover, the transition from normal epithelium to invasive carcinoma is sturdily associated with activation of local host vascular elements, stromal fibroblasts, and matrix turnover. Locally activated vascular channels at the invasive edge of the tumour are highly permeable and offer a reduced barrier for intravasation. The transformation of epithelial cells into mesenchymal elements plays an important role in the promotion of the invasive phenotype. Tumour cells that successfully invade and metastasize are selected out because somatic genetic progression has resulted in an altered communication circuit that continues to call up and support invasion and survival. Keywords: metastasis; neoangiogenesis; cell motility; invasion; cellular adhesion; extracellular matrix; protein kinases; membrane proteins; motility; microenvironment

Reverse-phase protein microarrays (RPPMAs) enable heterogeneous mixtures of proteins from cellular extracts to be directly spotted onto a substrate (such as a protein biochip) in minute volumes (nanoliter-to-picoliter volumes). The protein spots can then be probed with primary antibodies to detect important posttranslational modifications such as phosphorylations that are important for protein activation and the regulation of cellular signaling. Previously, we relied on chromogenic signals for detection. However, quantum dots (QDs) represent a more versatile detection system because the signals can be time averaged and the narrow-emission spectra enable multiple protein targets to be quantified within the same spot. We found that commercially available pegylated, streptavidin-conjugated QDs are effective detection agents, with low-background binding to heterogeneous protein mixtures. This type of test, the RPPMAs, is at the forefront of an exciting, clinically-oriented discipline that is emerging, namely tissue or clinical proteomics.

Numerous previously uncharacterized molecules resident within the low molecular weight circulatory proteome may provide a picture of the ongoing pathophysiology of an organism. Recently, proteomic signatures composed of low molecular weight molecules have been identified using mass spectrometry combined with bioinformatic algorithms. Attempts to sequence and identify the molecules that underpin the fingerprints are currently underway. The finding that many of these low molecular weight molecules may exist bound to circulating carrier proteins affords a new opportunity for fractionation and separation techniques prior to mass spectrometry-based analysis. In this study we demonstrate a method whereby nanoporous substrates may be used for the facile and reproducible fractionation and selective binding of the serum-based biomarker material, including subcellular proteins found within the serum. Aminopropyl-coated nanoporous silicon, when exposed to serum, can deplete serum of proteins and yield a serum with a distinct, altered MS profile. Additionally, aminopropyl-coated, nanoporous controlled-pore glass beads are able to bind a subset of serum proteins and release them with stringent elution. The eluted proteins have distinct MS profiles, gel electrophoresis profiles, and differential peptide sequence identities, which vary based on the size of the nanopores. These material surfaces could be employed in strategies for the harvesting and preservation of labile and carrier-protein-bound molecules in the blood.

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.

Objective The rapid diagnosis of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (HCT) is important for optimizing the management of this life-threatening complication. Current diagnostic techniques are time-consuming and require invasive tissue sampling. We investigated serum protein pattern analysis using surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry as a tool to diagnose GVHD. Patients and Methods Eighty-eight serum samples were obtained from 34 patients undergoing HCT either pretransplant (n = 28 samples) or at various time points posttransplant (n = 60 samples), including 22 samples obtained on the day of onset of acute GVHD symptoms. Serum proteomic spectra generated from a "training set" of known samples were used to identify distinct proteomic patterns that best categorized a sample as either pretransplant, posttransplant non-GVHD, or GVHD; these distinct proteomic signatures were subsequently used to classify samples from a masked "test" sample set into the appropriate diagnostic category. Results Proteomic pattern analysis accurately distinguished GVHD samples from both posttransplant non-GVHD samples and pretransplant samples (100% specificity and 100% sensitivity in both cases). Furthermore, distinct serum proteomic signatures were identified that distinguished pretransplant from posttransplant non-GVHD samples (100% specificity and 94% sensitivity). Conclusion These preliminary data suggest a potential application of SELDI-TOF-based proteomic analysis as a rapid and accurate method to diagnose acute GVHD.

Protein microarray technologies provide a means of investigating the proteomic content of clinical biopsy specimens in order to determine the relative activity of key nodes within cellular signaling pathways. A particular kind of protein microarray, the reverse-phase microarray, is being evaluated in clinical trials because of its potential to utilize limited amounts of cellular material obtained through biopsy. Using this approach, cellular lysates are arrayed in dilution curves on nitrocellulose substrates for subsequent probing with antibodies. To improve the sensitivity and utility of reverse-phase microarrays, we tested whether a new reporter technology as well as a new detection instrument could enhance microarray performance. We describe the use of an inorganic fluorescent nanoparticle conjugated to streptavidin, Qdot 655 Sav, in a reverse-phase protein microarray format for signal pathway profiling. Moreover, a pegylated form of this bioconjugate, Qdot 655 Sav, is found to have superior detection characteristics in assays performed on cellular protein extracts over the nonpegylated form of the bioconjugate. Hyperspectral imaging of the quantum dot microarray enabled unamplified detection of signaling proteins within defined cellular lysates, which indicates that this approach may be amenable to multiplexed, high-throughput reverse-phase protein microarrays in which numerous analytes are measured in parallel within a single spot.

Recent advances in understanding the complex biology of the microenvironment that underlies tumor invasion and migration have revealed novel and promising therapeutic targets. Pharmacological blockade of intra- and extracellular signaling events that regulate migration and survival of multiple cell types may disrupt the host-tumor conspiracy that allows escape from normal developmental regulation.

Recent evidence suggests that each patient's cancer has a unique subset of molecular pathogenetic derangements. These derangements may both genetic and proteomic alterations. Genomic and proteomic research tools enable genome-wide assessment of gene expression as well as kinase driven cell signaling events. These tools are illuminating the molecular derangements of individual tumors, even if these tumors have similar morphological characteristics. A combination of laser capture microdissection with multiplexed phosphoproteomic analysis using reverse phase protein microarray technology is being used to identify protein molecular signatures of individual tumors. The in vivo state of multiple kinase driven signal pathways may be evaluated by reverse phase protein microarray with a panel of specific antibodies developed based upon our knowledge of biological processes. Molecular profiling of individual patient's tumors is currently being evaluated in clinical trials at the National Institutes of Health, National Cancer Institute for monitoring Epidermal Growth Factor (EGF) cell signaling events for patients with breast and ovarian cancer.

Mass spectrometry–based diagnostics has the potential to revolutionize molecular medicine. Using modern mass-spectrometer technologies, clinical tests can be developed that are practical, robust, accurate, and inexpensive. Serum proteomic pattern profiling couples mass spectrometry with adaptive artificial-intelligence–based bioinformatics, which can now be employed to detect pathological states reflected in the serum proteome. With this approach, rapid and cost-effective tests with exquisite clinical sensitivity and specificity are emerging. These tools may dramatically change how disease is detected, monitored, and managed.

CD1 proteins present various glycolipid antigens to T cells, but the cellular mechanisms that control which particular glycolipids generate T cell responses are not understood. We show here that T cell recognition of glucose monomycolate antigens with long (C(80)) alkyl chains involves the delivery of CD1b proteins and antigens to late endosomes in a process that takes several hours. In contrast, analogs of the same antigen with shorter (C(32)) alkyl chains are rapidly, but inefficiently, presented by cell surface CD1b proteins. Dendritic cells (DCs) preferentially present long-chain glycolipids, which results, in part, from their rapid internalization and selective delivery of antigens to endosomal compartments. Nonprofessional antigen-presenting cells, however, preferentially present short-chain glycolipids because of their lack of prominent endosomal presentation pathways. Because long alkyl chain length distinguishes certain microbial glycolipids from common mammalian glycolipids, these findings suggest that DCs use a specialized endosomal-loading pathway to promote preferential recognition of glycolipids with a more intrinsically foreign structure.

e13508 Background: Tumor size change from baseline (RECIST) is often used to assess antitumor activity of investigational agents in phase 1 trials, but this measure does not take into account the tumor growth rate (TGR) prior to treatment. TGR could be highly variable in a phase 1 ‘all-comers’ patient population, with a high TGR potentially masking a meaningful treatment effect (TE). Assessing the change in TGR (Mehrara et al, BJC 2011) using historical tumor burden assessments (Gomez-Roca et al, Eur J Cancer 2011) may provide higher sensitivity to true TE (TTE). The objectives of the current study are to formalize a Growth Rate Based Method (GRBM) and to compare, using simulated patients, the ability of GRBM and RECIST assessments to detect and quantify TTE. Methods: The exponential tumor growth model (Claret et al, J Clin Oncol. 2009) was used to simulate the sum of the longest diameters (SLD) individual time courses of 2000 virtual patients under different TGR scenarios: slow, medium, fast and ‘all comers’ (highly variable TGR as often encountered in phase 1). Different sampling designs were simulated wherein tumor assessments with measurement errors were obtained, ranging from 16 to 4 weeks prior to treatment initiation (TI), immediately before TI, and 8 and 16 weeks after TI. TTE was defined as the difference between the simulated SLDs at 16 weeks with and without treatment. GRBM response was defined as the model-predicted difference between the SLDs with and without treatment, as estimated from the simulated samples. Sensitivity (Se) of RECIST or GRBM was defined as the probability of classifying a patient as a RECIST or GRBM >30% reduction when the TTE >30%, and specificity (Sp) as the probability of RECIST or GRBM reduction

e13600^ Background: RG7112 is a small molecule MDM2 antagonist, designed to non-genotoxically activate p53. A phase I dose escalation (DE) was performed, followed by a sarcoma biomarker extension (EXT) in pts with wild type TP53. Methods: 106 pts (58M, 48 F), median age 57.9 yrs (range 22-84) participated; 76 in 9 cohorts (DE) from 20 to 1800 mg/m2, orally QD x 10 q28 days. 30 pts with sarcoma were treated at MTD (2500 mg) (EXT) with pre-and on dose (d5+/-2) biopsies. Analyses included TP53 mutation (AmpliChip), MDM2 amplification (ISH), p53 and p21 IHC, MDM2 RT-PCR, Ki-67, TUNEL and [18F]-FLT-PET. Blood was obtained for PK and MIC-1, a PD marker of p53 activation. Results: DE: MTD was1440 mg/m2/d (2500 mg flat dose). PK was ~dose linear (t½ 1-1.5 d) with high variability (CV~70%) in AUC and Cmax . Adverse events included nausea/GI and exposure-related neutropenia/thrombocytopenia. 3 DLTs: diarrhea, pancytopenia, hyponatremia occurred (at ≥ 640 mg/m2). Evidence of activity included: 1) concentration dependent increase in plasma MIC-1 (% baseline), 2) decrease in [18F]-FLT PET and PR each in liposarcoma pts at 1800 mg/m2 and 1440 mg/m2, respectively. EXT: Grade3/ 4 cytopenias at MTD precluded subsequent cycles in 6/8 pts (10 day schedule). Dosing was changed to 5 days, and only 3 patients had Gr3/4 cytopenias. 8/22 pts remained on study for >4 cycles, including 2 pts with SD for 7 and 9 cycles respectively. 3 of 4 EXT pts had decreased [18F]-FLT-PET activity. Biopsies (pre- and on treatment) demonstrated: 1) increase in p53 (median 1.5 fold change (X) by IHC, n=15); 2) increase in p21 (median 2.7X by IHC, n=14); 3) increase in MDM2 (median 2.5X by RT-PCR, n = 27); 4) decrease in % Ki-67(+) cells (median % change from baseline -65.4%, range -91% to +275%, n=17 ); 5) increase in TUNEL(+) cells of 9.0 (density of + cells/mm2, range -26.2 to +45.5, n=22). These results were seen both in tumors with and without MDM2 gene amplification, and in multiple sarcoma subtypes. Conclusions: RG7112 has manageable AEs (GI ) and cytopenias correlating with AUC. Single agent disease control, and biomarker activity was seen in both MDM2 amplified and non-amplified, heavily pretreated soft tissue sarcoma pts, with changes reflecting activation of p53-related pathways.

Abstract 1545 Background: The tumor suppressor p53 plays a critical role in protecting cells from malignant transformation by inducing target gene transcription, cell cycle arrest, and apoptosis. MDM2 is the primary regulator of p53, targeting p53 for proteosomal degradation. The small molecule antagonist RG7112, which activates p53 by antagonism of the p53-MDM2 interaction, is currently in clinical trials. Clinical data from the Phase 1 program in leukemias has previously been presented (Andreeff et al., ASH 2010). Here we present pharmacodynamic biomarker data from the ongoing leukemia clinical trial. Methods: Patients with relapsed/refractory leukemia were treated with escalating doses of study drug in two separate strata: A- AML, ALL, CML-BC; and B - CLL and S-CLL. Peripheral blood was collected for biomarkers pre-treatment and at multiple time points during the first 48 hours and on day 10. Bone marrow was collected pre-treatment, and on day 10 and 28. Specimens of blood and marrow were subjected to RBC lysis and MACS sorting (CD19, 33 or 34 Ab). After purification, the leukemic cells were divided for preparation of RNA, DNA and protein, or fresh frozen. The validated p53 transcriptional target and secreted protein, MIC-1 (macrophage inhibitory cytokine-1), was analyzed as a pharmacodynamic marker of p53 activation. Flow cytometric analysis was performed for assessment of apoptosis (CMxRos, AnnexinV) and differentiation, (CD34, CD19). Protein immunoblots for MDM2 and p53, and real-time PCR, after reverse transcription, for quantitation of 24 customized p53 targets by TaqMan low density arrays (TLDA) (Applied Biosystems) were undertaken. AmpliChip analysis of p53 mutational status was also performed. The AmpliChip p53 test detects single nucleotide substitutions and deletions in the entire coding region and splice sites of p53 (exons 2–11) by comparative analysis of probe hybridization patterns between sample and wild-type reference DNA. Results: To date, 68 patients (42 AML, 5 ALL, 1 CML-BC, 19 CLL/sCLL, 1 T-PLL) have been treated with RG7112, with doses ranging from 20 – 2430 (1215 mg BID) mg/m2/day x 10 days. P53 mutational status has been completed in 29 Arm A and 15 Arm B patients. Preliminary results reveal that 4/29 AML patients had mutations (3 MISSENSE, 1 SPLICE). 6 of 15 patients with CLL/SLL had mutations, including a patient with a 2bp deletion in exon 6, confirmed by deep sequencing, who has stable disease on therapy for 2 years. An increase in serum MIC-1 was seen at RG7112 concentrations of >2 μg/mL and was highly correlated (R2=0.4312) with exposure to RG7112 (AUC24h) at steady state. Consistent changes in p53 target genes were observed for PUMA, p21 and FDXR (ferrodoxin reductase) as early as 6 hours after the first dose; levels were found to be significantly (p Conclusions: We report evidence of concentration-related pharmacodynamic biomarker activity on the p53 pathway in blood and marrow specimens from patients with leukemia receiving the MDM2 antagonist, RG7112. The trial is ongoing with additional dose escalation. Further clinical and biomarker analyses are in progress and will be reported. We provide proof of mechanism for MDM2 antagonism by demonstrating p53 stabilization, activation of p53 targets and the p53 pathway in leukemic cells from patients treated with RG7112. Disclosures: Andreeff: Roche: Research Funding. Younes:Genentech: Honoraria, Research Funding. Wu:Roche: Employment. Jukofsky:Roche: Employment. Vassilev:Roche: Employment. Zhi:Roche: Employment. Geho:Roche: Employment. Nichols:Hoffmann-LaRoche: Employment.

Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.

10514 Background: Preclinical data demonstrate p53-dependent MIC-1 activation by both D and the MDM2 inhibitor RG7112. This study evaluated the tolerability of combining D with RG7112 in ASTS patients (pts) and pharmacokinetic (PK) and pharmacodynamic (PD) parameters of the combination. Methods: A phase 1b 3+3 dose escalation study was designed. Pts with ASTS in whom D was considered appropriate were eligible. D was administered IV at either 50 mg/m2 or 60 mg/m2on day 1 with RG7112 administered orally 500 or 1000 mg QD for 3 or 5 days (d1-3 or d1-5) in 28-d treatment cycles. Safety, PK and PD, including serum MIC-1 (an indicator of p53 activation), were assessed. Results: As of January 15, 2013, enrollment was complete with 23 pts with ASTS accrued; safety data for cycle 1 was available for preliminary review in 20 pts. Growth factor support was mandated following the first dosing group (D 60 mg/m2and RG7112 500 mg x 5d) due to febrile neutropenia or grade 4 neutropenia. Of the 20 pts for which cycle 1 safety data is available, 13 pts reported neutropenia (12 grade 3/4); 5 reported grade 3/4 febrile neutropenia (3 following mandatory GCSF prophylaxis); 12 pts developed thrombocytopenia (9 grade 3/4). PK parameters of the combination therapy are similar to single agent values and did not demonstrate evidence of drug-drug interactions. Serum MIC-1 rapidly increased to levels greater than for either agent alone. Best response to date is stable disease in 8/16 pts who have had interim evaluations. Conclusions: Combination therapy with D and RG7112 resulted in a high rate of grade 3/4 neutropenia (60%) or thrombocytopenia (45%). PK analysis does not suggest this is due to changes in the metabolism of either drug. This combination demonstrates apparent potentiation of p53 activation demonstrated by increased MIC-1 levels greater than additive effects of the single agents. Biomarker analyses, safety, PK, and MIC-1 data will be presented. Clinical trial information: NCT01605526. [Table: see text]