Richard Nicoletti

Neuregulin 1 Expression Is a Predictive Biomarker for Response to AV-203, an ERBB3 Inhibitory Antibody, in Human Tumor Models

Purpose: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. Experimental Design: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. Results: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. Conclusions: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203. Clin Cancer Res; 21(5); 1106–14. ©2014 AACR.

Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients.

Cancer‐related weight loss is associated with increased inflammation and decreased survival. The novel inflammatory mediator growth differentiation factor (GDF)15 is associated with poor prognosis in cancer but its role in cancer‐related weight loss (C‐WL) remains unclear. Our objective was to measure GDF15 in plasma samples of cancer subjects and controls and establish its association with other inflammatory markers and clinical outcomes.

MAP3K11/GDF15 axis is a critical driver of cancer cachexia

Cancer associated cachexia affects the majority of cancer patients during the course of the disease and thought to be directly responsible for about a quarter of all cancer deaths. Current evidence suggests that a pro‐inflammatory state may be associated with this syndrome although the molecular mechanisms responsible for the development of cachexia are poorly understood. The purpose of this work was the identification of key drivers of cancer cachexia that could provide a potential point of intervention for the treatment and/or prevention of this syndrome.

23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity

Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit. Mol Cancer Ther; 14(8); 1858–67. ©2015 AACR.

Abstract 4650: From bench to bedside: are cytokines still relevant biomarkers for staging cancer cachexia.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: We have provided initial evidence1 on the clinical usefulness of the cancer cachexia stages (CCS) proposed by Fearon et al2. However it is still unclear3 if particular molecular phenotypes are also associated with these stages, as well with relevant clinical outcomes. Methods: A candidate list of cytokines (Activin A, Eotaxin, FGF, G-CSF, GDF15, GM-CSF, IFN-g, IL-10, IL-12\_p70, IL-13, IL-15, IL-17, IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1\_MCAF, MIP-1a, MIP-1b, PDGF-bb, RANTES, TNF-a, VEGF) was measured in 210 blood samples from patients with advanced lung and gastrointestinal cancers, via Luminex and ELISA methods. Non-parametric t-test, Kaplan-Meier and Kruskal-Wallis analyses were used to test the association between cytokines levels with CCS, Patient-Generated Subjective Global Assessment scores (PG-SGA) and survival. Results: Using non-cachectic patients as controls, Activin A and GDF15 were significantly up-regulated in pre-cachectic (p<0.01), cachectic (p<0.05) and refractory cachectic (p<0.001) patients. IL-6, IL-8 and VEGFa were significantly up-regulated only in refractory cachectic (p<0.001) patients. Activin A (p<0.001), GDF15 (p<0.001) and IL-8 (P<0.001) plasma levels correlated with PG-SGA. Quartiles of GDF15 plasma levels identified better survival curves as compared to Activin A, IL-6 and IL-8 quartiles. Conclusions: Activin A and GDF15 appear to be useful aids for the diagnosis of all cachexia stages in advanced cancer. Because of their correlation with nutritional and survival outcomes, these cytokines may also represent useful targets in the development of new compounds for the treatment of cancer cachexia. 1. Vigano A, Del Fabbro E, Bruera E, Borod M. The cachexia clinic: from staging to managing nutritional and functional problems in advancer cancer patients. Critical Reviews in Oncogenesis, 2012 17(3), 293-304 2. Fearon K,Strasser F,Anker SD,Bosaeus I,Bruera E,Fainsinger RL,Jatoi A, Loprinzi C, MacDonald N, Mantovani G, Davis M, Muscaritoli M, Ottery F, Radbruch L, Ravasco P, Walsh D, Wilcock A, Kaasa S, Baracos VE. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011; 12:489-495. 3. Scheede-Bergdahl, C., Watt, H.L., Trutschnigg, B., Kilgour, R.D., Haggarty, A., Lucar, E., Vigano, A. 2011. Is IL-6 the best pro-inflammatory biomarker of clinical outcomes of cancer cachexia? Clinical Nutrition 31: 85-8. Citation Format: Antonio Vigano, Lorena Lerner, Nianjun Tao, Brian Krieger, Bin Feng, Richard Nicoletti, Qing Liu, Ailin Bai, Zhigang Weng, Thierry Alcindor, Domenico Fuoco, Jeno Gyuris, Maria Isabel Chiu. From bench to bedside: are cytokines still relevant biomarkers for staging cancer cachexia. [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 4650. doi:10.1158/1538-7445.AM2013-4650

Abstract 1153: Effective treatment of cancer associated cachexia by AV-380, a GDF15 inhibitory antibody

Background: One of the most lethal and debilitating effects of cancer is the development of cancer-related anorexia-cachexia syndrome (CACS). It affects the majority of advanced cancer patients and is thought to be responsible for up to ∼30% of all cancer deaths. CACS is a complex metabolic syndrome associated with malnutrition and severe involuntary weight loss due to the loss of muscle and fat tissue, as well as the clinical manifestation of anemia, inflammation and suppression of immune functions. The precise molecular mechanisms responsible for cancer cachexia are poorly understood, thus limiting the development of effective therapeutics. Current evidence suggests that a pro-inflammatory state may be responsible for many of the symptoms associated with CACS. Growth Differentiation Factor 15 (GDF15) is a pro-inflammatory cytokine whose circulating levels are significantly increased in cachectic cancer patients and several animal models of cancer cachexia. Here we demonstrate that the inhibition of GDF15 function results in the compete reversion of the phenotypic and metabolic changes associated with CASC. Methods: Mice bearing HT-1080 human fibrosarcoma xenografts have increased plasma GDF15 levels and develop cachexia were used in this study. Tumor bearing animals were treated either with AV-380, a GDF15 neutralizing antibody or IgG1 control. The effect on body weight, muscle/fat mass and organ sizes were assessed. Metabolic changes induced by the treatment were measured by a comprehensive laboratory animal monitoring system (CLAMS). Results: Treatment with AV-380 restored body weight, muscle and fat mass as well as normal organ sizes. Analyses of the CLAMS data demonstrated that mice receiving AV-380 reversed the phenotypic and metabolic changes associated with the cachexia induced by this tumor model. Moreover, AV-380 treated mice showed an increase in locomotor activity and energy expenditure, achieving levels similar to non tumor bearing control animals. Conclusions: Inhibition of GDF15 function completely reverted body weight loss, restored normal body composition and triggered a catabolic to anabolic metabolic switch in this cancer cachexia model. The data highlights the therapeutic potential of the GDF15 inhibitory antibody AV-380 for the treatment for CACS. Citation Format: Lorena Lerner, Nianjun Tao, Brian Krieger, Richard Nicoletti, Bin Feng, Nesreen Ismail, William Winston, Yanyu Zhang, Jinwei Jiang, Solly Weiler, Jeno Gyuris. Effective treatment of cancer associated cachexia by AV-380, a GDF15 inhibitory antibody. [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 1153. doi:10.1158/1538-7445.AM2015-1153

Relationship of hypoxia signature with variant subgroup of clear cell renal cell carcinoma (ccRCC) and its association with clinical activity on tivozanib hydrochloride.

361 Background: TIVO-1, a randomized Phase III trial in first-line targeted therapy for patients (pts) with ccRCC, demonstrated significant improvement in progression-free survival (PFS) in pts receiving tivozanib hydrochloride (T) vs. sorafenib (S) (11.9 vs. 9.1 months [12.7 vs. 9.1 in treatment-naïve pts]). To further characterize molecular ccRCC subtypes and assess relationships between subtypes and vascular endothelial growth factor tyrosine kinase inhibitor activity, we characterized available molecularly annotated datasets from TIVO-1. METHODS Tumor subtypes were established using hierarchical clustering and evaluated in two microarray ccRCC datasets using gene set enrichment analysis with 51 signatures representing a set of molecular phenotypes. A 9-gene signature comprising genes associated with hypoxia-inducible factor (HIF) transcription was quantified by RT-PCR on all available (69/517) formalin-fixed, paraffin-embedded material from patients using a predefined classifier score and cutoff. RESULTS Hierachical clustering generated 3 distinct tumor classes. Molecular clusters defining HIF gene expression (low), endothelial cell content (low), extracellular matrix (low), proliferation (high) epithelial cell phenotype (high), and metabolism (high) were differentially expressed in cluster 3 tumors, which represented approximately 15% of the populations. Based on predefined analysis, the hypoxia signature was significantly associated with better PFS on T using a previously established classifier (Table). The hypoxia signature was not significantly associated with PFS on S. There was no significant correlation to single largest diameter for either agent. CONCLUSIONS A novel molecular subtype of ccRCC is characterized by a distinct molecular profile and can be classified by a low hypoxia signature. This hypoxia gene signature may help identify T responders. This signature is seen in subsets of other solid tumors supporting the broad exploration of this candidate T response biomarker. CLINICAL TRIAL INFORMATION NCT01030783. [Table: see text].

Abstract 3504: Plasma growth differentiating factor-15 (GDF-15) and other inflammatory markers are associated with weight loss and poor prognosis in cancer patients.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Cachexia is associated with increased inflammatory markers and decreased survival in cancer. Also, elevated GDF-15 has been associated with poor prognosis in several cancer types but its role in cachexia is not well-understood. Methods: We measured body weight change, appetite, plasma GDF-15, and other inflammatory markers in 62 males with cancer-cachexia (CC), 72 non-cachectic cancer subjects (CNC) and 64 non-cancer controls (Co) matched by age, gender and pre-illness body weight. In a subset of patients we also measured grip strength (HGS), appendicular lean body mass (aLBM), ECOG and KPS. Results: GDF-15, IL-6 and IL-8 were increased in CC vs. other groups. Activin and G-CSF were significantly upregulated in CC vs. Co. A subset analyses showed that GDF-15, Activin A and IL-8 were increased in CC vs. CNC in lung cancer patients and that GDF-15, IL-6 and IL-8 were increased in CC patients treated with platinum-based chemotherapy. GDF15, IL-6 and IL-8 levels significantly correlated with 6-month weight loss and with IL-6, IL-1ra, IL-2, IL-4, IL-9, IL-10, IFN, MCP-10, MIP-1a, MIP-1b, TNF-a, VGEF and activin in cancer patients. Analysis in a subset of patients showed that CC had lower grip strength, aLBM, and fat mass; and that ECOG and KPS were lower in CC and CNC compared to controls. GDF-15 and IL-8 correlated negatively with aLBM, HGS and fat mass. Activin correlated negatively with aLBM. Survival analysis showed that GDF-15 and IL-8 predicted survival adjusting for stage and weight change (Cox regression p<0.01, HR 3). Conclusion: GDF-15 and other inflammatory markers are associated with weight loss, decreased muscle mass and strength and poor survival in cancer patients. GDF-15 may serve as a prognostic indicator in cancer patients and be a novel therapeutic target for cancer cachexia. Citation Format: Lerner Lorena, Teresa Hayes, Nianjun Tao, Brian Krieger, Bin Feng, Richard Nicoletti, Ailin Bai, Zhigang Weng, Qing Liu, Maria Isabel Chiu, Jeno Gyuris, Jose M. Garcia. Plasma growth differentiating factor-15 (GDF-15) and other inflammatory markers are associated with weight loss and poor prognosis in cancer patients. [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 3504. doi:10.1158/1538-7445.AM2013-3504

Abstract 5063: Preclinical evaluation of an EMT multi-gene biomarker index for predicting response to inhibitors of EGFR and IGF1R

In oncology, the goal of personalized medicine is to improve patient outcome by tailoring therapy to the biochemical signaling within the individual9s tumor. This requires identifying effective predictors of response that can be measured in biopsy material. High expression of the epithelial marker E-cadherin is associated with increased sensitivity in cultured cells and an improved survival benefit in response to erlotinib in patients. Downregulation of E-cadherin is a critical event in epithelial to mesenchymal transition (EMT) and is associated with decreased sensitivity to erlotinib. In order to determine if a more complete characterization of the EMT state would better predict sensitivity to erlotinib as well as other epithelial-targeting drugs, we developed an 88-gene signature that can be used to calculate a numerical index value which represents the EMT state of cells or tumors. In a panel of human tumor cell lines, the EMT index predicted erlotinib sensitivity correctly in 21 out of 24 lines. The index also predicted sensitivity to the IGF1R inhibitor OSI-906. While the EMT index marginally improved sensitivity prediction in vitro compared to E-cadherin, the numerical index allowed for a comparison of relative EMT states that was not possible with E-cadherin alone. Using a microarray database of human tumor sections, we computed EMT index values across 8 solid tumor types in order to compare their relative EMT states. Breast and lung tumors had a fairly even distribution between epithelial and mesenchymal tumors, while colon tumors were more epithelial, and kidney tumors were more mesenchymal. When comparing EMT index values to E-cadherin mRNA levels, some tumor types showed good agreement between E-cadherin and the EMT index (colon, kidney, lung) while others showed less agreement (breast, pancreas). We next compared our EMT signature to other published signatures of EMT and EGFR inhibitor response. Interestingly, most signatures were equally predictive of erlotinib sensitivity in vitro, even though less than 20% of the genes overlaped between any two signatures. We also analyzed signatures developed for individual characteristics of EMT such as invasion or stem cells. These did not correlate with erlotinib sensitivity, suggesting the protection from current therapeutics is not controlled solely by these specific aspects of EMT. Finally, we characterized sub-types of lung cancer for EMT state to determine if the EMT classification might correlate with established response rates to erlotinib. Adenocarcinomas, which tend to respond well, had more epithelial index values while squamous cell carcinomas, which tend to be less sensitive, had more mesenchymal values. This suggests that more epithelial index values might predict for erlotinib response in patients, potentially providing clinicians with a tool to more effectively treat patients based on the EMT status of their tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2011-5063

Population-based engineered tumor models as a platform for biomarker discovery in cancer

This abstract is being presented as a short talk in Plenary Session 1. A full abstract is printed in the Proffered Abstracts section (PR1) of the Conference Proceedings.