Preclinical Sarcoma Models Research Articles

HMGA1 regulates trabectedin sensitivity in advanced soft-tissue sarcoma (STS): A Spanish Group for Research on Sarcomas (GEIS) study

HMGA1 is a structural epigenetic chromatin factor that has been associated with tumor progression and drug resistance. Here, we reported the prognostic/predictive value of HMGA1 for trabectedin in advanced soft-tissue sarcoma (STS) and the effect of inhibiting HMGA1 or the mTOR downstream pathway in trabectedin activity. The prognostic/predictive value of HMGA1 expression was assessed in a cohort of 301 STS patients at mRNA (n = 133) and protein level (n = 272), by HTG EdgeSeq transcriptomics and immunohistochemistry, respectively. The effect of HMGA1 silencing on trabectedin activity and gene expression profiling was measured in leiomyosarcoma cells. The effect of combining mTOR inhibitors with trabectedin was assessed on cell viability in vitro studies, whereas in vivo studies tested the activity of this combination. HMGA1 mRNA and protein expression were significantly associated with worse progression-free survival of trabectedin and worse overall survival in STS. HMGA1 silencing sensitized leiomyosarcoma cells for trabectedin treatment, reducing the spheroid area and increasing cell death. The downregulation of HGMA1 significantly decreased the enrichment of some specific gene sets, including the PI3K/AKT/mTOR pathway. The inhibition of mTOR, sensitized leiomyosarcoma cultures for trabectedin treatment, increasing cell death. In in vivo studies, the combination of rapamycin with trabectedin downregulated HMGA1 expression and stabilized tumor growth of 3-methylcholantrene-induced sarcoma-like models. HMGA1 is an adverse prognostic factor for trabectedin treatment in advanced STS. HMGA1 silencing increases trabectedin efficacy, in part by modulating the mTOR signaling pathway. Trabectedin plus mTOR inhibitors are active in preclinical models of sarcoma, downregulating HMGA1 expression levels and stabilizing tumor growth.

1554TiP A randomized phase II study to investigate the efficacy and safety of the tumor-targeting human antibody-cytokine fusion protein L19TNF in previously treated patients with advanced or metastatic soft tissue sarcoma

Although tumor necrosis factor (TNF) is one of the most potent antitumor cytokines, its unacceptable toxicities do not allow for systemic administration of therapeutically active doses. L19TNF is a fully human antibody-cytokine fusion protein, composed of the neovascular targeting antibody fragment scFv(L19) fused to human recombinant TNF, that selectively delivers therapeutically relevant doses of TNF to the tumor tissue while sparing normal tissues. L19TNF has demonstrated substantial therapeutic activity in preclinical models of sarcoma when administered alone or in combination with chemotherapy, leading to hemorrhagic tumor necrosis and immune-mediated tumor rejection.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

Deep-learning and MR images to target hypoxic habitats with evofosfamide in preclinical models of sarcoma.

Rationale: Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial of non-resectable soft tissue sarcomas, TH-302 did not improve survival in combination with doxorubicin (Dox), possibly due to a lack of patient stratification based on hypoxic status. Therefore, we used magnetic resonance imaging (MRI) to identify hypoxic habitats and non-invasively follow therapies response in sarcoma mouse models.Methods: We developed deep-learning (DL) models to identify hypoxia, using multiparametric MRI and co-registered histology, and monitored response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (radiation-induced fibrosarcoma, RIF-1).Results: A DL convolutional neural network showed strong correlations (>0.76) between the true hypoxia fraction in histology and the predicted hypoxia fraction in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (p<0.05), but not in the RIF-1 model, which had a lower volume of hypoxic habitats. Control studies showed that RIF-1 resistance was due to hypoxia and not other causes. Notably, PDX tumors developed resistance to TH-302 under prolonged treatment that was not due to a reduction in hypoxic volumes.Conclusion: Artificial intelligence analysis of pre-therapy MR images can predict hypoxia and subsequent response to HAPs. This approach can be used to monitor therapy response and adapt schedules to forestall the emergence of resistance.

Abstract 2772: Targeting hypoxic habitats with hypoxia pro-drug evofosfamide in preclinical models of sarcoma

Abstract Tumor habitats are phenotypically and spatially distinct intratumoral regions. Hypoxic habitats are associated with reduced accessibility of chemotherapeutics, resulting in resistance. Hypoxia targeted prodrugs (HAPs) may overcome this resistance. However, in a phase III clinical trial, the HAP evofosfamide (TH-302) did not improve survival of sarcomas when used in combination with the standard of care doxorubicin (Dox). This may have been due to a lack of patient stratification based on hypoxic status. Thus, identification of hypoxic tumor habitats (Jardim-Perassi et al. 2019) would allow for pre-therapy patient stratification, and monitoring of habitats post-therapy. Our goal was to use multiparametric (mp) MRI and co-registered histology to classify hypoxic habitats in preclinical models of sarcoma, and to use Habitat Imaging to monitor therapy response. A patient-derived xenograft (PDX) of rhabdomyosarcoma (obtained from St. Jude's) and a syngeneic model of fibrosarcoma (RIF-1) were randomized into four groups, receiving saline (control), Dox, TH-302, or the combination (Dox + TH-302). mpMRI was acquired pre and post therapy, and parameter maps were calculated for T2, T2*, diffusion weighted imaging (DWI), and Dynamic contrast-enhanced (DCE). MRI based 3D printed tumor molds were used to guide MRI and histology co-registration. Machine learning was used to classify MRI into viable, hypoxic and non-viable tumor habitats using their co-registered histological counterparts as ground-truth. TH-302 monotherapy or the Dox + TH-302 combination reduced tumor growth and increased overall survival in the PDX model (p&amp;lt;0.05). The combination therapy was superior, consistent with TH-302 controlling hypoxic habitats and Dox controlling viable normoxic regions. Pimonidazole-positive (hypoxic) areas were reduced with TH-302 monotherapy (p&amp;lt;0.05) but not with the Dox + TH-302 combination. This suggests that TH-302 decreases the hypoxic area, concurrently as Dox may have increased this habitat, possibly as a resistance mechanism in tumors undergoing prolonged therapy. Notably, the RIF-1 model was resistant to both Dox and TH-302. This is not due to inherent resistance, as RIF-1 cells in vitro were sensitive to TH-302 under hypoxia and to alkylating agents, indicating that resistance is dependent on an intact tumor microenvironment. Co-registration of MRI and histology allowed spatial comparison of imaging modalities to develop a machine learning method for classification of these tumor microenvironments. In conclusion, the use of HAPs to target hypoxic tumor habitats shows superiority over conventional chemotherapy in rhabdomyosarcoma, but was without effect in RIF-1. Imaging habitats have the potential to classify different microenvironments within a tumor, and this concept may allow the choice of therapy to be based on habitat distribution. Citation Format: Bruna Victorasso Jardim Perassi, Wei Mu, Dominique F. Abrahams, Michal R. Tomaszewski, Jan Poleszczuk, Jack Higgins, Gary V. Martinez, Robert J. Gillies. Targeting hypoxic habitats with hypoxia pro-drug evofosfamide in preclinical models of sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2772.

Abstract 3699: Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma

Abstract Sarcomas are rare malignancies with generally poor prognosis, for which current therapies have shown limited efficacy. In order to improve the prognosis of patients, there is an urgent medical need to identify new molecular targets that can be exploited for the development of innovative treatment approaches. Inhibition of histone deacetylases (HDACs) has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and HDAC inhibitors (HDACi) have been reported as potent and specific anticancer molecules in preclinical and clinical studies. Here we investigated the molecular and functional effects of a new generation HDACi, ITF2357, in preclinical models of soft tissue and bone sarcomas. A wide panel of sarcomas cells, both established and patient derived cell lines, were used. For in vitro experiments Western Blot, RT-PCR, flow cytometry and conservative isobologram analysis have been performed. In vivo efficacy of ITF2357/doxorubucin combination was evaluated in nude mice bearing sarcoma xenografts. Using in vitro and in vivo sarcoma models, we demonstrate that ITF2357 decreases cell viability, activates apoptosis and increases the growth inhibitory efficacy of doxorubicin. The molecular mechanisms of action of ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by the increase of pro-apoptotic BH3-only proteins, and both caspase and bcl-2 dependent cell death. We also found that ITF2357 transcriptionally upregulated the pro-apoptotic BH3-only genes Bax, Puma, Noxa and BIM in cells harbouring mutant or wild type p53, thus indicating a p53 independent mechanism. Both bcl-2 overexpression and caspases inhibition strongly reduced apoptosis, corroborating the evidence that activation of mitochondrial pathway occurs upstream and before caspases activation. We also found that ITF2357 induces a canonical autophagic process, and that inhibition of autophagy increased apoptosis induced by ITF2357, indicating that in our models autophagy shows a prosurvival effect. We also reported that ITF2357 induced FOXO1/3a up-regulation, FOXO proteins nuclear accumulation, and transcriptional activation of FOXO-target genes, such as BH3-only proteins Puma, Noxa and Bim. Knockdown experiments demonstrated that reduction of both FOXO1 and 3a did not inhibit the induction of Bim expression in response to ITF2357, and consequently did not protect sarcoma cells against ITF2357-induced apoptosis, independently from p53 status. Notably, ITF2357 synergized with doxorubicin to induce in vitro cell death and to reduce in vivo tumor growth. Therefore ITF2357 may represent an important therapeutic agent against human sarcoma regardless p53 status, and the pharmacological combination of ITF2357 with doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma. Citation Format: Maria Grazia Tupone, Marta Di Martile, Marianna Desideri, Simonetta Buglioni, Barbara Antoniani, Carlotta Mastroiorio, Rita Falcioni, Virginia Ferraresi, Nicola Baldini, Roberto Biagini, Michele Milella, Daniela Trisciuoglio, Donatella Del Bufalo. Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3699.

Histone deacetylase inhibitor ITF2357 leads to apoptosis and enhances doxorubicin cytotoxicity in preclinical models of human sarcoma

Sarcomas are rare tumors with generally poor prognosis, for which current therapies have shown limited efficacy. Histone deacetylase inhibitors (HDACi) are emerging anti-tumor agents; however, little is known about their effect in sarcomas. By using established and patient-derived sarcoma cells with different subtypes, we showed that the pan-HDACi, ITF2357, potently inhibited in vitro survival in a p53-independent manner. ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by induction of pro-apoptotic BH3-only proteins and a caspases-dependent mechanism. ITF2357 also induced autophagy, which protected sarcoma cells from apoptotic cell death. ITF2357 activated forkhead box (FOXO) 1 and 3a transcription factors and their downstream target genes, however, silencing of both FOXO1 and 3a did not protect sarcoma cells against ITF2357-induced apoptosis and upregulated FOXO4 and 6. Notably, ITF2357 synergized with Doxorubicin to induce cell death of established and patient-derived sarcoma cells. Furthermore, combination treatment strongly impaired xenograft tumor growth in vivo, when compared to single treatments, suggesting that combination of ITF2357 with Doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma. Overall, our study highlights the therapeutic potential of ITF2357, alone or in rational combination therapies, for bone and soft tissue sarcomas management.

Redox active metals and H2O2 mediate the increased efficacy of pharmacological ascorbate in combination with gemcitabine or radiation in pre-clinical sarcoma models

Soft tissue sarcomas are a histologically heterogeneous group of rare mesenchymal cancers for which treatment options leading to increased overall survival have not improved in over two decades. The current study shows that pharmacological ascorbate (systemic high dose vitamin C achieving ≥ 20mM plasma levels) is a potentially efficacious and easily integrable addition to current standard of care treatment strategies in preclinical models of fibrosarcoma and liposarcoma both in vitro and in vivo. Furthermore, enhanced ascorbate-mediated toxicity and DNA damage in these sarcoma models were found to be dependent upon H2O2 and intracellular labile iron. Together, these data support the hypothesis that pharmacological ascorbate may represent an easily implementable and non-toxic addition to conventional sarcoma therapies based on taking advantage of fundamental differences in cancer cell oxidative metabolism.

Antitumor efficacy of the heparan sulfate mimic roneparstat (SST0001) against sarcoma models involves multi-target inhibition of receptor tyrosine kinases.

The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Hypoxia-activated chemotherapeutic TH-302 enhances the effects of VEGF-A inhibition and radiation on sarcomas.

Background:Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. trimodality therapy).Methods:Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines.Results:In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11–13% and 13–20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia.Conclusions:The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity.

A randomized phase Ib/II study evaluating the safety and efficacy of olaratumab (IMC-3G3), a human anti-platelet-derived growth factor α (PDGFRα) monoclonal antibody, with or without doxorubicin (Dox), in advanced soft tissue sarcoma (STS).

10501 Background: Options for patients (pts) with unresectable/metastatic STS are limited. While Dox is standard of care, Dox combinations have yet to improve overall survival (OS) over Dox alone. Olaratumab (IMC-3G3), a fully human monoclonal antibody, selectively binds PDGFRα (a signaling pathway implicated in STS), blocks ligand binding, and enhances Dox activity in preclinical sarcoma models. Methods: We conducted a phase Ib, open-label, randomized phase II study of Dox ± olaratumab in unresectable/metastatic STS (NCT01185964). Pts received Dox (75 mg/m2 Day 1) with (Arm A) or without (Arm B) olaratumab (15 mg/kg Days 1 and 8, every 21 days), a dose and schedule confirmed safe in phase Ib, for up to 8 cycles. In Arm A, olaratumab monotherapy continued after Dox until disease progression; in Arm B, pts could cross over to olaratumab at progression. The primary endpoint was progression-free survival (PFS; target hazard ratio [HR] = 0.67, 80% power, α = 0.2) with OS as a secondary endpoint. Results: Of 1...

Novel histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in sarcoma cancer stem cells.

Musculoskeletal sarcomas are aggressive malignancies of bone and soft tissues often affecting children and adolescents. Histone deacetylase inhibitors (HDACi) have been proposed to counteract cancer stem cells (CSCs) in solid neoplasms. When tested in human osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma stem cells, the new HDACi MC1742 (1) and MC2625 (2) increased acetyl-H3 and acetyl-tubulin levels and inhibited CSC growth by apoptosis induction. At nontoxic doses, 1 promoted osteogenic differentiation. Further investigation with 1 will be done in preclinical sarcoma models.

Abstract 4526: BIBF1120, an investigational triple angiokinase inhibitor, in combination with inhibitors of mTOR signaling shows potent antitumor activity in preclinical models of sarcoma

Abstract BIBF1120 (Nintedanib), an investigational potent small molecule angiokinase inhibitor, selectively binds to and blocks receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptor, (VEGFR), fibroblast growth factor receptor (FGFR) and platelet derived growth factor receptor (PDGFR). Nintedanib has shown promising results in preclinical as well as clinical studies of non-small cell lung cancer, colorectal, ovarian cancer and other solid tumors. We tested the in vitro anti-proliferative activity of BIBF1120 in a panel of sarcoma cell lines including malignant peripheral nerve sheath tumor (MPNST), synovial (SYO-I), Ewing's (CHP100), osteo (SaOS2) and liposarcoma (LS141). Our results indicate that when compared to other potent RTK inhibitors such as Imatinib and Pazopanib, BIBF1120 is highly efficient at nanomolar to low micromolar concentrations in blocking proliferative activity of sarcoma cell lines, specifically, SYO-I and MPNST, which are highly dependent on PDGFR signaling. Western blot analysis to test the efficacy of BIBF1120 confirmed significant blockade of RTK signaling pathways. Ras activation and/or activation of mitogen activated protein kinase (MAPK) pathway has been shown to be critical in sarcomas including MPNST and synovial sarcomas. Unlike Imatinib and Pazopanib, BIBF1120 also blocked MAPK signaling pathway as demonstrated by p-ERK1/2 blockade. In addition to the RTKs, mTOR (mammalian Target of Rapamycin) protein plays a key role in AKT activation and downstream survival signaling. Combination of RTK blockade by BIBF1120 and mTOR blockade by TORC1 inhibitor rapamycin resulted in significantly stronger inhibition of in vitro cellular proliferation compared to single agent treatments. Taken together, our data strongly supports further clinical research of BIBF1120 in sarcoma. Combining potent inhibitors of tyrosine kinase signaling, MAPK signaling and inhibitors of mTOR pathway such as rapamycin could provide a new therapeutic approach in this disease. Citation Format: Parag P. Patwardhan, Elgilda Musi, Kathryn S. Ivy, Natalie Giovino, Gary K. Schwartz. BIBF1120, an investigational triple angiokinase inhibitor, in combination with inhibitors of mTOR signaling shows potent antitumor activity in preclinical models of sarcoma. [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 4526. doi:10.1158/1538-7445.AM2014-4526

Phase II study of gemcitabine (GEM) plus sirolimus (SIR) in previously treated patients with advanced soft tissue sarcoma (STS): A Spanish Group for Research on Sarcomas (GEIS) study.

10594 Background: Combination of chemotherapy and targeted agents could be a strategy to improve efficacy. In preclinical sarcoma models, combination of GEM with SIR enhances apoptosis in vitro and...

Phase I/II study of TH-302 combined with doxorubicin in soft tissue sarcoma.

10036 Background: TH-302 is a 2-nitroimidazole prodrug of the DNA alkylator, bromo-isophosphoramide mustard. TH-302 is activated in severe hypoxia, releasing Br-IPM. In preclinical sarcoma models, TH-302 was active alone and enhanced the activity of doxorubicin (Dox). A phase I/II study was performed to investigate the safety and activity of TH-302 in combination with Dox in soft tissue sarcoma (STS). Methods: Eligible patients (pts) had ECOG ≤1, advanced or metastatic STS (AJCC grade 3 or 4), ≤2 prior chemotherapies, evaluable by RECIST, and acceptable hematologic, liver, renal and cardiac function. TH-302 was administered IV over 30-60 minutes on Day 1 and 8 and full dose Dox (75 mg/m2) as IV bolus 2 hours later on Day 1 of a 21-day cycle. A standard 3+3 design was used with phase II expansion in first-line pts at the combination MTD. TH-302 starting dose was 240 mg/m2. Results: 22 pts with STS (6 liposarcoma, 6 leiomyosarcoma, 5 pleomorphic NOS, 2 synovial, 3 other) were treated at TH-302 doses of 240 (6 pts), 300 (12) or 340 (4). 18 pts were first-line; 4 pts had received prior chemo. Median age: 58 (range 19-85); 10 M/12 F. 8 pts are ongoing, 6 completed study (4 received extra cycles TH-302 alone), 8 discontinued prior to Cycle 6 (AE/QOL-4, PD-3, pt decision-1). After Gr4 neutropenia at 240 mg/m2, all later pts received growth factor (GCSF) on Day 8. DLTs were Gr4 thrombocytopenia and Gr3 infection with Gr4 neutropenia at 340 mg/m2. The combination MTD of TH-302 is 300 mg/m2. 12 pts were treated at MTD with 1 DLT (Gr3 febrile neutropenia). Skin toxicities were reported in 11 pts (one Gr 3 cellulitis); mucosal toxicities were reported in 12 pts (one Gr 3 vaginitis). Nonhematologic AEs are Gr 1/2: fatigue (59%), nausea (45%), alopecia (41%). Gr3/4 hematologic toxicity: neutropenia (45%), thrombocytopenia (23%) and anemia (32%). There was no PK interaction between TH-302 and Dox. Best response assessments in 20 pts: 5 PRs (25%), 12 SDs (60%) and 3 PDs; excluding pt with post cycle 6 pathology confirmed enlarging necrotic tumor, 6 month progression-free rate is 56%. Conclusions: TH-302 may be administered in combination with full dose Dox in STS. Myelosuppression is dose limiting with the effects partially obviated by use of GCSF. Antitumor activity is encouraging. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Threshold Pharmaceuticals Threshold Pharmaceuticals Threshold Pharmaceuticals Threshold Pharmaceuticals

Abstract C250: The pericyte as a novel stromal element and therapeutic target in sarcoma

Abstract Background: Mesenchymal Stem Cells (MSCs) have been proposed as the cell of origin of sarcoma. In soft tissues, the microvascular pericyte has been recently shown to have MSC properties (Crisan et al, Cell Stem Cell 3:301). We have previously reported the isolation of benign mesenchymal cultures from sarcoma surgical samples. These cells have the immunophenotype (CD45-CD31- CD73+CD105+CD90+) and in vitro differentiation potential characteristic of MSCs. We hypothesized that these sarcoma-associated MSCs (SA-MSCs) are derived from pericytes associated with the sarcoma vasculature. Methods: We examined whether benign SA-MSCs have surface markers characteristic of pericytes and cooperate with endothelial cells in tube formation assays. We also examined expression of CD146, a pericyte marker, and CD105, an MSC marker, in sarcoma archived tissue by IHC. Results: Benign SA-MSCs indeed demonstrated properties of pericytes, such as characteristic cytoplasmic projections at low density, surface expression of pericyte markers (CD146, PDGFR-beta, NG2 and endosialin) and cooperation with endothelial cells in tube formation assays in matrigel. To demonstrate the existence of pericytes in sarcoma archived tissue, we used CD146 that has been shown to be restricted to the vasculature in sarcoma, but thought to be an endothelial marker (Shih et al, CCR 2:569). We demonstrate for the first time the existence of CD146+ pericytes in diverse sarcoma subtypes. In addition, we examined sarcoma archived material for expression of CD105 (endoglin), an MSC marker and a known tumor-specific endothelial marker in carcinoma. We found that CD105 was expressed in sarcoma endothelium similarly to its expression on carcinoma endothelium. In addition, by three-color immunofluorescence, CD105 was expressed in a subset of sarcoma pericytes. It was not expressed in normal surrounding tissues. Conclusion: We show that SA-MSC cultures have properties of pericytes. Using a novel combination of markers we demonstrate that pericytes are abundant in sarcoma. Being in contact with both the endothelial cells and the malignant sarcoma cells, pericytes represent a novel stromal element in sarcoma and a potential therapeutic target. Based on these findings we are evaluating endosialin (CD248) and CD105 antibodies, both of which are in clinical development, for effectiveness in preclinical models of sarcoma. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C250.