PDGFR Pathways Research Articles

Preclinical Evaluation of Nintedanib, a Triple Angiokinase Inhibitor, in Soft-tissue Sarcoma: Potential Therapeutic Implication for Synovial Sarcoma.

Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329-40. ©2018 AACR.

Abstract P2-06-01: Molecular characterization of human malignant phyllodes tumors reveals potential targeted approaches

Abstract Malignant phyllodes tumor (MPT) which belong to the fibroepithelial neoplasm spectrum is a rare type of breast malignancy, and currently there is no effective targeted approach available for MPT. In this study, we tried to identify key genomic alterations and biologic pathways in MPT by whole exome and RNA sequencing of nine MPT tissues. Whole exome sequencing revealed somatic alterations in EGFR, MED12, PIK3CA, PIK3R1, PDGFRA, PDGFRB, PTEN, and TP53. Transcriptome sequencing showed dysregulation of ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling in MPTs when compared to normal breast or invasive breast cancer tissues. Based on the transcriptome profiles, the MPTs were classified into two subtypes; fibrous subtype with upregulation of stromal genes such as collagens and epithelial subtype with upregulation of E-cadherin and Claudins. The molecular classification of fibrous and epithelial subtypes was validated in 28 paraffin-embedded MPT tissues. The fibrous subtype showed higher mitotic index and increased risk for recurrence when compared to the epithelial subtype. We established a patient-derived xenograft model from one fibrous subtype MPT which harbored somatic mutation in PIK3R1 and PDGFRB. In that model, targeted treatment against PIK3CA/mTOR and PDGFR pathways effectively suppressed the tumor growth in vivo. Our data provide insights on the biologic understanding of MPT and suggest a clinically relevant molecular classification. Furthermore, we show that developing effective targeted approaches in MPT can be possible with genomic profiles and patient-derived xenograft models. The clinical efficacy of targeting PDGFR and PIK3CA/mTOR pathways in MPT should be tested in future clinical trials. Citation Format: Moon H-G, Yun J, Hong BS, Lee E, Lee H-B, Han W, Kim J-I, Noh D-Y, Heo W, Hur S, Kang W, Lee C. Molecular characterization of human malignant phyllodes tumors reveals potential targeted approaches [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-06-01.

Abstract LB-A28: Super-enhancer signatures of pancreatic ductal adenocarcinoma and associated fibroblasts

Abstract Therapeutic targeting of super-enhancers (SEs) has emerged as a promising approach for cancer treatment. To understand how SEs regulate the gene expression of tumors and their microenvironments, we sought to profile the SE landscape in pancreatic ductal adenocarcinoma (PDAC) cell lines and cancer-associated fibroblast (CAF) cells that were derived from different PDAC patients. The global profiling of the H3K27Ac signals in 6 PDAC and 6 CAF cell lines by chromatin immunoprecipitation coupled to sequencing (ChIP-seq) were performed to identify the SE landscapes in each cell line. Our data showed distinct SE landscapes in PDACs and CAFs. A total of 1025 SEs were identified in the PDAC cell lines. Signaling pathways that were significantly enriched among the genes associated with these SEs included targets of c-MYC, PDGFR, EGFR, AP1, uPA and uPAR pathways. Interestingly, wound-healing genes were highly enriched in the PDAC SE analysis; these genes are well known for their roles in cancer stemness and metastasis. 330 SEs were present in more than one PDAC cell line, but only 11 SEs were present in all 6 of the cell lines. Despite this low number of common SEs, the biological pathways of SE-associated genes, including ‘hallmark TNFA signaling via NFkB’ and ‘response to growth factor’, were significantly enriched in all of the PDAC cell lines. The profiling of the CAF cell lines identified a total of 1720 SEs, with 985 SEs present in more than one of the CAF cell lines and 99 SEs were present in all 6 of the CAF cell lines. As expected, the SE-associated genes in CAFs were involved in focal adhesion, extracellular matrix interaction and organization, and platelet activation. Interestingly, the biological pathways of ‘hallmark TNFA signaling via NFkB’ and ‘response to growth factor’ were present in all of the PDAC and CAF cell lines, while the ‘hallmark epithelial mesenchymal transition’ pathway was found only in the 6 CAF cell lines. RNA-Seq analysis of the same cell lines found that the gene expression intensities of the SE-associated genes had a pattern similar to that of the H3K27Ac signals of the SEs, indicating a high correlation between SE activity and gene expression. To functionally validate the identified SEs and the level of enhancement of gene activity by SEs, we are investigating the biological effects of suppressing the SEs associated with critical oncogenes using CRISPR interference. Overall, our study has identified important biological pathways that are controlled by SEs in PDAC cells and CAFs. These observations offer us a means to study the consequences of perturbing the SE network on PDAC and its microenvironment. Citation Format: Shaimaa Hussein, Pawan Noel, Kenian Chen, Lauren Metang, Daniel Von Hoff, Haiyong Han, Yin Lin. Super-enhancer signatures of pancreatic ductal adenocarcinoma and associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A28.

Recent Advances in Targeted Therapy for Glioma.

Gliomas are the most common primary malignant brain tumors, which have a universally fatal outcome. Current standard treatment for glioma patients is surgical removal followed by radiotherapy and adjuvant chemotherapy. Due to therapeutic resistance and tumor recurrence, efforts are ongoing to identify the molecules that are fundamental to regulate the tumor progression and provide additional methods for individual treatment of glioma patients. By studying the initiation and maintenance of glioma, studies focused on the targets of tyrosine kinase receptors including EGFR, PDGFR and other crucial signal pathways such as PI3K/AKT and RAS/RAF/MAPK pathway. Furthermore, recent advances in targeting immunotherapy and stem cell therapy also brought numerous strategies to glioma treatment. This article reviewed the researches focused on the advanced strategies of various target therapies for improving the glioma treatment efficacy, and discussed the challenges and future directions for glioma therapy.

A phase 1b, open label, single institution trial of nintedanib in combination with bevacizumab in patients with advanced solid tumors.

TPS2614 Background: Vascular endothelial growth factor (VEGF) is a potent factor in inducing angiogenesis. VEGF inhibitors have produced demonstrable but limited and transient clinical benefit for various cancers. One mechanism of resistance includes revascularization secondary to up-regulation of alternative pro-angiogenic signals such as platelet derived growth factor receptor (PDGF) and fibroblast growth factor receptor (FGFR) pathway. Nintedanib is an oral triple kinase inhibitor that blocks the VEGFR, PDGFR and FGFR pathways. Our study is using combination of Nintedanib (Nin) and Bevacizumab (Bev) which will block VEGF as well as salvage pathway of angiogenesis (PDGFR and FGFR). Phase I dose selection studies revealed that Nin is generally well tolerated (Clin Can Res 16:47, 2010). LUME-Lung 1 phase 3, international, double blind, placebo controlled trial using Nin and docetaxel in non-small cell lung cancer (NSCLC) showed significant improvement in progression free survival (PFS) regardless of histology and improvement in overall survival (OS) in lung adenocarcinoma (Lancet oncology 15:2, 2014). Methods: This is a phase 1b, open label, single institution trial with standard 3+3 design. Primary objective is to evaluate the safety and tolerability of combination of Nin and Bev. The secondary objective is to determine clinical efficacy (objective response), PFS, and evaluation of plasma levels of angiogenic and anti-angiogenic biomarkers like VEGF, PDGF, VEGF-R and FGF. Patients (pts) in cohort I will be treated with Bev 15 mg/kg day 1 intravenously every 3 weeks and Nin 150 mg orally (PO) twice daily (BID) from day 2-21. In the absence of dose limiting toxicities, Nintedanib dose will be increased to 200 mg PO BID in cohort II. Major inclusion criteria includes advanced solid tumors for which Bev has an indication (non-squamous, NSCLC, colon, ovarian, cervical and renal cancer), progression after at least 1 line of systemic treatment, and measurable disease. Pts with prior treatment with Bev can be enrolled. We will enroll 18 patients. Cohorts I has been completed without DLT (n = 3). Cohort II has enrolled 10 patients. Clinical trial information: NCT02835833.

MTOR, VEGF, PDGFR, and c-kit signaling pathway activation in Kaposi sarcoma

Kaposi sarcoma (KS) is a locally progressive, intermediate-grade vascular neoplasm with no known cure, high recurrence rates, and potential for wide dissemination. Low efficacy and high toxicity limit current therapeutic options for advanced disease. Activation of mammalian target of rapamycin (mTOR), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and c-kit signaling pathways has been implicated in KS pathogenesis and may suggest a role for targeted inhibitors. KS cases were retrospectively retrieved (N=274), most (90%) associated with human immunodeficiency virus. Tissue microarray slides were stained with human herpes virus-8, Friend leukemia integration 1 transcription factor, CD117 (c-kit), phospho-S6 (pS6), PDGF receptor-β, VEGF, and phospho-mTOR. Both intensity and extent of staining were scored. Multiplying these scores for each core yielded total staining H-scores. Human herpes virus-8 was positive in 87% and Friend leukemia integration 1 transcription factor in 95.7% of cases. Most were also VEGF+ (97.6%), pS6+ (95.7%), CD117+ (92.5%), and PDGFRB+ (87.4%). Approximately half (55.6%) were phospho-mTOR+. There was no significant difference in staining among patients with low (<500 cells/mm3) or preserved CD4 T-cell counts. Immunohistochemistry confirms upregulation of the mTOR, PDGF, VEGF, and c-kit pathways in a large cohort of KS samples. Of proteins tested, pS6, downstream of mTOR, demonstrated the highest proportion of strong positivity (67.1%). These results support the possibility of using targeted inhibitors in KS. Overexpression was independent of CD4 count, suggesting that even patients with low counts may be targeted therapy candidates.

Characterizing and targeting PDGFRA alterations in pediatric high-grade glioma.

Pediatric high-grade glioma (HGG, WHO Grade III and IV) is a devastating brain tumor with a median survival of less than two years. PDGFRA is frequently mutated/amplified in pediatric HGG, but the significance of this finding has not been fully characterized. We hypothesize that alterations of PDGFRA will promote distinct prognostic and treatment implications in pediatric HGG. In order to characterize the impact of PDGFR pathway alterations, we integrated genomic data from pediatric HGG patients (n=290) from multiple pediatric datasets and sequencing platforms. Integration of multiple human datasets showed that PDGFRA mutation, but not amplification, was associated with older age in pediatric HGG (P= <0.0001). In multivariate analysis, PDGFRA mutation was correlated with worse prognosis (P = 0.026), while PDGFRA amplification was not (P = 0.11). By Kaplan-Meier analysis, non-brainstem HGG with PDGFRA amplification carried a worse prognosis than non-brainstem HGG without PDGFRA amplification (P = 0.021). There were no pediatric patients with PDGFRA-amplified HGG that survived longer than two years. Additionally, we performed paired molecular profiling (germline / tumor / primary cell culture) and targeting of an infant thalamic HGG with amplification and outlier increased expression of PDGFRA. Dasatinib inhibited proliferation most effectively. In summary, integration of the largest genomic dataset of pediatric HGG to date, allowed us to highlight that PDGFRA mutation is found in older pediatric patients and that PDGFRA amplification is prognostic in non-brainstem HGG. Future precision-medicine based clinical trials for pediatric patients with PDGFRA-altered HGG should consider the optimized delivery of dasatinib.

Abstract 5055: SPEN stimulates primary cilia formation, cell migration and is associated with metastasis in breast cancer

Abstract The primary cilium (PC) is a microtubule-based and non-motile organelle that functions as animal cells’ antennae, regulating a number of signalling pathways, including the mTOR, PDGFR and Wnt pathways, in addition to being essential for activation of the canonical Hedgehog (Hh) pathway. In breast cancer, the PC is a structure that is frequently lost due to transcription- and mutation-driven alterations in cilium-related genes. In a study aiming to delineate the hormone-independent transcriptional program regulated by SPEN, a gene that we have identified as a tumour suppressor gene in estrogen receptor (ER)-positive breast cancers, we found that SPEN was co-expressed (R&amp;gt;0.95) with a number of genes involved in ciliary biology (P = 1.64E-19), including the ciliogenic transcription factors RFX2, RFX3 and FOXJ1. Interestingly, the overexpression of SPEN resulted in the restoration of the PC in T47D cells, which have previously been shown to lack this structure. Consistent with the re-establishment of a PC in T47D cells, SPEN-overexpressing T47D cells had decreased mTOR signalling, hyperactivation of PC-dependent pathways, including the Hh and PDGFR pathways, and increased migration. Silencing SPEN expression in the non-tumorigenic MCF10A cells, which express high endogenous levels of SPEN and show a high prevalence of PC (36%), considerably decreased PC levels in MCF10A cells and was accompanied with increased mTORC1 activity as well as attenuated cell migration. We also found that SPEN silencing in MCF10A cells decreased the expression of the ciliogenic transcription factor RFX3 and that SPEN interacts with DNA upstream of RFX3 gene transcription start site in chromatin immunoprecipitation experiments. Collectively, these observations indicate that SPEN may mediate its effects, at least in part, through the transcriptional regulation of RFX3. Accordingly, RFX3 expression levels were strongly positively correlated (R = 0.55, P&amp;lt;0.0001) with SPEN levels in a cohort of 82 clinical samples from triple negative breast cancer patients. Notably, high SPEN RNA levels were also predictive of early metastasis in two independent cohorts of 77 (HR = 2.25, P = 0.03) and 170 (HR = 2.23, P = 0.004) ER-negative breast cancer patients. Together, our findings highlight a role for SPEN in the regulation of PC formation and support important functions in the inhibition of mTORC1 and activation of Hh and PDGFR pathways as well as cell migration, a process that may contribute to early metastasis in ER-negative breast cancers. Citation Format: Stéphanie Légaré, Catherine Chabot, Mark Basik. SPEN stimulates primary cilia formation, cell migration and is associated with metastasis in breast 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 5055.

Cancer Stem-like Cells Act via Distinct Signaling Pathways in Promoting Late Stages of Malignant Progression.

Cancer stem-like cells (CSC) play key roles in long-term tumor propagation and metastasis, but their dynamics during disease progression are not understood. Tumor relapse in patients with initially excised skin squamous cell carcinomas (SCC) is characterized by increased metastatic potential, and SCC progression is associated with an expansion of CSC. Here, we used genetically and chemically-induced mouse models of skin SCC to investigate the signaling pathways contributing to CSC function during disease progression. We found that CSC regulatory mechanisms change in advanced SCC, correlating with aggressive tumor growth and enhanced metastasis. β-Catenin and EGFR signaling, induced in early SCC CSC, were downregulated in advanced SCC. Instead, autocrine FGFR1 and PDGFRα signaling, which have not been previously associated with skin SCC CSC, were upregulated in late CSC and promoted tumor growth and metastasis, respectively. Finally, high-grade and recurrent human skin SCC recapitulated the signaling changes observed in advanced mouse SCC. Collectively, our findings suggest a stage-specific switch in CSC regulation during disease progression that could be therapeutically exploited by targeting the PDGFR and FGFR1 pathways to block relapse and metastasis of advanced human skin SCC.

Nintedanib, a novel triple angiokinase inhibitor for the treatment of non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Recent advances involving targeted therapies are promising, but most patients do not have an "oncogene addicted" disease. A platinum doublet chemotherapy regimen has been the mainstay of therapy since 1997. The addition of antiangiogenic agents to traditional chemotherapy has improved survival in patients with non-small cell lung cancer. However, these agents are limited by serious adverse events such as thromboembolism, bowel perforation and hemorrhage and by the development of resistance. Nintedanib is a novel, orally available triple angiokinase inhibitor that targets three important pathways involved in the initiation and propagation of angiogenesis in tumors, the VEGF, FGF and PDGFR pathways. Phase I and II trials have identified the maximum tolerated dose in monotherapy and in combination with traditional chemotherapy. The toxicity profile is tolerable and reversible, dominated by transaminitis and gastrointestinal side effects. The phase III LUME-lung 1 study (NCT00805194) compared docetaxel, a standard treatment in the second line, with docetaxel in combination with nintedanib. Progression-free survival was 3.4 months in the combination group compared to 2.7 months in the docetaxel group, (HR 0.79, 95% CI 0.68-0.92, P = 0.0019). There was a significant improvement in overall survival in adenocarcinoma patients, 12.6 vs. 10.3 months (HR 0.83, 95% CI 0.7-0.99, P = 0.036). Based on the results of this study, nintedanib has been approved by the EMA in Europe, as a second-line treatment in patients with adenocarcinoma. It is a promising, well-tolerated therapy that is currently being investigated in multiple different tumor types.

Abstract 3000: Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma

Abstract Background. Sunitinib inhibits VEGFR/PDGFR signaling in cancer cells and reduces tumor blood flow, leading to tumor starvation, hypoxia, and necrosis. The aim of this study was to investigate mechanisms associated with adaptive resistance to sunitinib in vitro and in vivo in renal cancer models (RCC). Materials and Methods: CAKI1 (VHL+) and 786-0 (VHL-) RCC cells were exposed in vitro to stepwise increasing doses of sunitinib for &amp;gt;6 months. In vitro, vascular mimicry was assessed by pseudo-tube formation assay using Matrigel. Pseudo-tube networks were quantified by “angiogenesis analyzer” for Image J, assessing the meshes number. CAKI1 or 786-0 cells were engrafted in nude mice and treated at 60 mg/kg/day of sunitinib until progression. Results. We confirmed that the VHL pathway was functional in CAKI1 but not in 786-0 cells. In vivo, sunitinib significantly delay tumor growth with a median time-to-tumor progression of 50 days versus 17 days (p&amp;lt;0.001) in CAKI and over 60 days versus 22 days (p&amp;lt;0.01) in 786-0 xenogratfs. In CAKI1 and 786-0, quantitative immunohistochemistry and fluorescence analyses at the time of sunitinib progression showed patterns of angiogenesis recovery with tumor blood vessel sprouting associated with reduction of hypoxic and necrotic areas. In these sunitinib-resistant tumors, cancer cells displayed stem cell and mesenchymal differentiation markers such as CD133 vimentin and CXCR4. Vessels sprouting under sunitinib exposure contained CD10/CD31/von Willebrand co-stained cells embedded in their walls suggesting vascular mimicry. We further investigated whether cancer cell differentiation into endothelial cell was related to chemical inhibition of VEGFR/PDGFR by sunitinib. We first characterized CAKI and 786 cells for VEGFR and PDGFR expression, showing that CAKI expressed high level of PDGFR, whereas 786 expressed low levels of VEGFR and PDGFR. In normally oxygenated culture conditions, 786-0 cells formed spontaneously more meshes than CAKI1 cells (22 versus 0.33, p&amp;lt;0.05). Interestingly, chronic exposure of CAKI1 cells to sunitinib led to a 32-fold increase in the number of meshes (10.6 versus 0.33, p&amp;lt;0.01), which could be related to the inhibition of the PDGFR pathway. Conclusions. Our data showed that chronic exposure to subtoxic doses of sunitinib might force cancer stem cells to differentiate into endothelial-like cells in vitro and in vivo independently of the VHL status of tumor cells. This phenomenon known as vasculogenic mimicry is associated with poor prognosis in renal cell carcinoma. VHL-mutated 786-0 cells appeared more prompt to form vessels and can spontaneously form meshes in normoxic condition in vitro. Interestingly, this plastic capacity of forming vessels in normoxic condition can be acquired in wild-type VHL CAKI1 when treated chronically with subtoxic doses of sunitinib. Citation Format: Annemilai Tijeras-Raballand, Célia Dos Santos, Maria Serova, Matthieu Martinet, Sandrine Faivre, Armand de Gramont, Eric Raymond. Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma. [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 3000. doi:10.1158/1538-7445.AM2014-3000

19IN - Molecular Targeted, Systemic Therapies for Meningiomas

ABSTRACT Standard treatments for meningiomas comprise surgical resection and radiotherapy. In the recurrent setting after exhaustion of all local treatment options no effective therapies are known and several drugs failed to show efficacy. Novel compounds may offer hope for better disease control. Upregulation of pro-angiogenic molecules and dysregulatoin of some signaling cascades such as mTor and PDGFR pathways are recurrently found in meningiomas. Furthermore, some evidence indicates that trabectedin may be an effective therapy for high-grade meningioma and a prospective trial is underway. Recent phase II trials investigating sunitinib pasireotide LAR and vatalinib have been completed. Still, there is a lack of conclusive clinical trials on medical therapy of meningioma. There remains a significant need for more basic investigations and clinical trials in meningioma. Promising novel therapeutics include antiangiogenic agents, molecular inhibitors of signaling cascades and trabectedin. Disclosure: The author has declared no conflicts of interest.

Imatinib Mesylate Exerts Anti-Proliferative Effects on Osteosarcoma Cells and Inhibits the Tumour Growth in Immunocompetent Murine Models

Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients.

Elevated PDGFRB gene copy number gain is prognostic for improved survival outcomes in resected malignant pleural mesothelioma.

PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis, and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome. Eighty-eight archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRβ, p-PDGFRβ) and fluorescence in situ hybridization analysis of PDGFRB gene CNG. Spearman rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome. Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in >10% of tumor cells had lower cytoplasmic p-PDGFRβ (P=.029), while PDGFRB gene CNG in >40% of tumor cells had a higher cytoplasmic PDGFRβ (P=.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P=.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed. From a retrospective review of archived tissue specimens from patients with resected malignant pleural mesothelioma tumors, we show that patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P=.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients.

Phase II trial of sorafenib in esophageal (E) and gastroesophageal junction (GEJ) cancer: Response and prolonged stable disease observed in adenocarcinoma.

91 Background: Sorafenib is a tyrosine kinase inhibitor targeting VEGFr, PDGFr, Raf and other pathways. Encouraging response and survival were observed in a phase II trial of sorafenib with chemotherapy in GE cancer (J Clin Oncol 27:2947;2010). We performed a phase II trial of single-agent sorafenib with the primary endpoint of progression-free survival (PFS). Secondary endpoints include response and toxicity. Methods: Patients (Pts) with measurable metastatic E and GEJ cancer with ≤3 prior chemotherapy regimens were treated with sorafenib 400 mg BID. CT scans were performed monthly for the first 2 months, then every 2 months. Results: Thirty-five patients have been accrued, with 34 evaluable; median age 62, 31 male, 4 female, median KPS 80%, E 25, GEJ 10, adenocarcinoma (AC) 30 squamous 5, median no. of prior therapies 2. Of 31 response-evaluable Pts, 1 (3%) ongoing complete response was noted (34+ months) in a Pt with E AC with biopsy-proven lymph node recurrence after chemoradiation and surgery; 23 Pts (74%) had stable disease. Median PFS is 3.7 months (95% CI 1.9 to 4 months), with median overall survival 8.9 months (95% CI 6.9 to 11.6 months). Four patients remain on treatment. Significant grade 3 toxicities included hand foot reaction (3 Pts), rash (1 Pt), dehydration (3 Pts) and fatigue (2 Pts). Twenty-seven of 33 tumors (82%) tested positive for phospho-ERK by immunohistochemistry. Conclusions: Encouraging activity in terms of PFS has been noted in this heavily pre-treated population. Updated data will be presented. Supported by a grant from Bayer. Clinical trial information: NCT00917462.

Dasatinib Therapy Affects Bone Homeostasis in Patients with Chronic Myelogenous Leukemia in Chronic Phase Independently of Molecular Response

AbstractAbstract 1682 BackgroundWith the success of targeted oral tyrosine kinase inhibitor (TKI) therapy, long-term/life-long therapy for patients with chronic myelogenous leukemia (CML) can now be envisaged. Dasatinib is a second generation TKI, which in a phase I clinical trial was found to be 100–300 times more potent at inhibiting tyrosine kinases than imatinib. In vitro data suggest that dasatinib therapy also may have a direct effect on bone metabolism by interfering with c-FMS, c-SRC and the PDGF-R pathways (Boufker et al, BMC Cancer 2010, 10:298). In this study, we evaluated the effects of dasatinib monotherapy on bone homeostasis of CML patients treated at our center between 2006 and 2011. The levels of traditional biomarkers of bone health, namely serum bone specific alkaline phosphatase isoenzyme (BAP) and osteoprotegerin (OPG) were measured and were correlated with morphometric assessment of bone marrow biopsy specimens (BM Bx). We also assessed for any correlations between bone homeostasis and the results of conventional cytogenetics and molecular analysis with respect to treatment response. MethodsThe study group included 23 patients with chronic phase CML without evidence of clonal evolution who were treated with dasatinib. The median age was 51 years (range, 25–63) with a male to female ratio of 1.5: 1. Four different treatment groups were assessed (20mg/day, 40 mg/day, 80 mg/day and 100 mg/day). We compared paired BM specimens, obtained at initial diagnosis and 12–51 months (mos) after commencing dasatinib therapy. We performed whole slide imaging (Philips) on routinely stained BM Bx and specimen compartments were quantified using an area pixel count algorithm (Image Pro Plus system Version 6.3, MediaCybernetics, Bethesda, MD, USA) to calculate trabecular bone volume as a percentage area. The morphometric results were correlated with the results of conventional cytogenetics and molecular analysis obtained every 3 mos after enrollment. We also performed competitive inhibition ELISA assays to measure serum levels of OPG (baseline and after 12/18 mos) and BAP (every 4 mos), respectively. ResultsWith a median follow up of 24 mos (range 12–51mo), a significant increase in trabecular bone volume (TBV) was noted (p=0.022, Figure 1); 74% (17 pts) showed an increase in TBV on therapy, with an absolute increase of 5.2% (mean). Twelve of 16 pts (75%) evaluated had a decrease in OPG after 18 mos, which correlated in 90% of pts with an increase in TBV using a linear regression model (p= 0.0017, Figure 2). No significant difference was detected in TBV changes among the four dose groups. There was no significant trend in serum BAP levels, thus no statistical correlation was performed at this time point. All patients treated with dasatinib showed cytogenetic and molecular BCR-ABL1 response. There was no correlation between the degree of TBV change and either cytogenetic or molecular response (Figure 3) Conclusions:Our results indicate that within the therapeutic range for CML patients, dasatinib therapy promotes bone formation. However, there was no correlation between the dose levels studied and degree of bone formation and no correlation between TBV change and either cytogenetic or molecular response. The combination of digital image analysis and bone biomarkers enhances our understanding of bone homeostasis in patients on dasatinib. Further studies in a larger cohort of patients will be helpful to further validate our results.Figure 1: mean and standard deviation of TBV for each visit.Figure 2: correlation between “percent change TBV between visit 1 and visit 2” and 18 months OPG levels (linear regression model R2 =0.292, p= 0.0017)Figure 3: association between “percent change in TBV between visit 1 and visit 2” and “Rate of PCR response change between 3 and 12 months” (Spearman correlation coefficient = −0.05; p =0.81) [Display omitted] [Display omitted] [Display omitted] Disclosures:Cortes:Ariad: research support and consulting, research support and consulting Other; Pfizer: research support and consulting, research support and consulting Other; Novartis: research support and consulting, research support and consulting Other; BMS: research support and consulting Other.

Stromal and antitumor effects of BIBF 1120 (nintedanib) in a preclinical lung cancer model.

e13563 Background: BIBF 1120 is an angiogenic receptor tyrosine kinase inhibitor that potently inhibits VEGFR, PDGFR and FGFR kinase activity in in vitro enzymatic assays. This study investigated the effect and mechanism of BIBF 1120, in vitro and in vivo, as a single agent and in combination with chemotherapy in preclinical models of lung cancer. Methods: Anti-tumor effects of BIBF 1120 in vitro were assessed using cell proliferation assays on five lung cancer lines (A549, Calu-3, Calu-6, H1993, H1703) with BIBF 1120 as a single agent and in combination with gemcitabine and cisplatin. To demonstrate anti-tumor activity in vivo, NOD/SCID mice bearing subcutaneous A549 grafts were treated daily with BIBF 1120 or BIBF 1120 plus chemotherapy. Perfusion studies were conducted using labeled dextrans. Ex vivo tumor tissues were assessed for architecture, microvessel density, pericyte coverage, proliferation, and apoptosis. Results: In vitro, BIBF 1120 did not show anti-proliferative effects at pharmacologically achievable concentrations (IC50&gt;20μM) as a single agent; nor did it sensitize tumor cells to chemotherapy. However, BIBF 1120 inhibited primary tumor growth as a single agent and in combination therapy in subcutaneous endpoint studies with A549 xenografts. There was no significant difference in tumor architecture between BIBF 1120 and control-treated animals. Microvessel density (CD31,endomucin) and pericyte-covered vessels (NG2) were significantly decreased in BIBF 1120-treated animals compared to the control and chemotherapy only groups. Proliferation (phospho-histone 3) was decreased and apoptosis (cleaved caspase 3) was increased in BIBF 1120-treated animals compared to the control and chemotherapy only groups. Finally, BIBF 1120 significantly decreased perfusion in A549 xenografts. Conclusions: BIBF 1120 demonstrated potent anti-tumor and anti-angiogenic activity in a preclinical model of lung cancer. Because A549 was previously demonstrated to be an anti-VEGFR therapy resistant cell line, this study highlights the importance of FGFR and PDGFR pathways in the treatment of lung cancer.

Elevated PDGFRB gene copy number gain as prognostic in resected malignant pleural mesothelioma.

7078 Background: PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) and PDGFR pathway protein expression by immunohistochemistry (IHC) in the tumor cell cytoplasm, membrane, nucleus, and stroma, and correlate it to patient clinical outcome. Methods: 88 archived tumor blocks from resected MPM with full clinical information were used to perform the analyses. IHC biomarkers for PDGFRα,β and p-PDGFRβ, and fluorescence in situ hybridization were performed for analysis of PDGFRB gene CNG. Spearman’s rank correlation, Wilcoxon rank-sum test or Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to assess the biomarkers and their correlation to clinical outcome. Results: There were several correlations identified between the IHC biomarkers; however, none associated with patient demographics or histology subtype, with the exception of high cytoplasmic PDGFRα occurring in patients with no prior known asbestos exposure (p=0.029). In the CNG analysis, PDGFRB gene CNG in &gt; 10% of tumor cells had lower cytoplasmic p-PDGFRβ (p=0.029), while PDGFRB gene CNG in &gt; 40% of tumor cells had a higher cytoplasmic PDGFRβ (p=0.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. Patients with PDGFRB CNG &gt; 40% of tumor cells had an improved relapse-free survival (RFS) [HR 0.25 (95% CI 0.09, 0.72), p=0.0096]. In the patients with PDGFRB CNG &gt; 40% of cells, the addition of chemotherapy appeared to also improve RFS (p=0.017). In the multi-covariate analyses for RFS, there was no association with any IHC biomarker. In the overall survival (OS) analysis, having PDGFRB gene CNG &gt; 40% of tumor cells correlated with an improved OS [HR 0.32 (95% CI 0.11, 0.89), p=0.029] and the addition of peri-operative chemotherapy led to a trend towards improved OS (p=0.089). Conclusions: PDGFRB CNG &gt; 40% of tumor cells is a potential prognostic biomarker in surgically resected MPM tumors. Adding chemotherapy to patients with PDGFRB CNG &gt; 40% of cells improved RFS and led to a trend towards improved OS. Future validation of this biomarker in prospective trials is needed.

Relevance of angiogenesis in neuroendocrine tumors

While traditional cytotoxic drugs have shown limited efficacy in neuroendocrine tumors (NETs), their biological features have been characterized and can be exploited therapeutically. Their most prominent trait is an extraordinary vascularization in low-grade NETs and an hypoxia-dependent angiogenesis in high-grade NETs, which is associated to a significant expression of many proangiogenic molecules. Therefore, several antiangiogenic compounds have been tested in these malignancies, and among these, sunitinib has demonstrated activity in pancreatic NET patients by dually targeting the VEGFR and PDGFR pathways. In spite of these efficacious clinical results, apparent resistance to antiangiogenic therapies has been described in NET animal models and in clinical trials. Therefore, overcoming antiangiogenic resistance is a crucial step in the subsequent development of antiangiogenic therapies. Several strategies have been postulated to fight resistance, but preclinical studies and clinical trials will investigate and address these therapeutic approaches in the coming years in order to overcome resistance to antiangiogenic therapies in NETs.

Abstract 2706: Phase 2 study with pharmacokinetics (PK) analysis of sorafenib and vinorelbine in metastatic breast cancer

Abstract Background Sorafenib (S) is an oral inhibitor of the Raf, VEGFR and PDGFR pathways that contribute to chemoresistance in cancer cells. The combination of S with chemotherapy agents (capecitabine, paclitaxel) in metastatic breast cancer (BC) resulted in improved progression-free survival. In preclinical models of BC, favorable effects were noted combining S and vinorelbine (V). S and V are both metabolized through hepatic isoenzymes CYP3A, with the potential of PK interactions. A previous phase 1b study documented the feasibility of this combination. We aimed to define the activity and safety of this regimen in advanced BC and to investigate PK interactions of the 2 drugs at full doses. Methods Eligible patients were affected by Her2 negative, chemotherapy-naive metastatic BC, with measurable (RECIST) disease, adequate renal, liver and bone marrow functions and LVEF Δ50%. Treatment included V (30 mg/m2 days 1, 8 every 21) + S (400 mg bid continuously), with dose reductions for grade 2-4 toxicities. Patients still benefiting after 8 cycles of treatment could continue on single-agent S. Six patients underwent PK analysis, starting treatment with S on day 4 of the first cycle, allowing for comparison of PK of each agent (V and S) administered alone versus during concomitant treatment with the other agent. Results 27 evaluable patients started on treatment, median age 57 (35-78); they received a median of 8 cycles to date (1-28), with 4 patients still on treatment. 44% of patients required at least one dose reduction for toxicity. One patient died of complicated sepsis after febrile neutropenia. Other significant toxicities included: febrile neutropenia (8%), uncomplicated Grade 3 (G3)-G4 neutropenia (60%); G3 fatigue (20%), hand-foot syndrome (12%), diarrhea (4%), enteritis (4%), hypertension (4%), asymptomatic increase of AST/ALT (16%), amylase (12%), lipase (8%), GGT (4%); G2 decrease in LVEF (4%), rash (16%), alopecia (20%). 33% of patients had a partial response, 85% experienced a clinical benefit (including stable disease after at least 4 cycles of treatment). Median progression-free survival was 5.5 months (95% CI 4.6-7.5). PK analysis showed no statistically significant interaction between S and N. Also the conversion of S to its metabolite S-N-oxide was not affected by the concomitant treatment with V. A non-significant trend was noticed for higher Cmax/AUC of V in the first 30 minutes after infusion when administered with ongoing treatment with S. Conclusions Combining S with V is feasible, but not devoid of toxicity, for which frequent dose reductions are necessary. A PK interaction could not be ruled out with the small sample size analyzed. More frail patients should start at a lower dose of both agents and we recommend close monitoring for the first 2 cycles, including amylase and lipase. Promising efficacy of this combination was observed, with a very high rate of disease control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2706. doi:1538-7445.AM2012-2706