Mark D. Starr

Prognostic and Predictive Blood-Based Biomarkers in Patients with Advanced Pancreatic Cancer: Results from CALGB80303 (Alliance)

Purpose: CALGB80303 was a phase III trial of 602 patients with locally advanced or metastatic pancreatic cancer comparing gemcitabine/bevacizumab versus gemcitabine/placebo. The study found no benefit in any outcome from the addition of bevacizumab to gemcitabine. Blood samples were collected and multiple angiogenic factors were evaluated and then correlated with clinical outcome in general (prognostic markers) and with benefit specifically from bevacizumab treatment (predictive markers). Experimental Design: Plasma samples were analyzed via a novel multiplex ELISA platform for 31 factors related to tumor growth, angiogenesis, and inflammation. Baseline values for these factors were correlated with overall survival (OS) using univariate Cox proportional hazard regression models and multivariable Cox regression models with leave-one-out cross validation. Predictive markers were identified using a treatment by marker interaction term in the Cox model. Results: Baseline plasma was available from 328 patients. Univariate prognostic markers for OS were identified including: Ang2, CRP, ICAM-1, IGFBP-1, TSP-2 (all P < 0.001). These prognostic factors were found to be highly significant, even after adjustment for known clinical factors. Additional modeling approaches yielded prognostic signatures from multivariable Cox regression. The gemcitabine/bevacizumab signature consisted of IGFBP-1, interleukin-6, PDGF-AA, PDGF-BB, TSP-2; whereas the gemcitabine/placebo signature consisted of CRP, IGFBP-1, PAI-1, PDGF-AA, P-selectin (both P < 0.0001). Finally, three potential predictive markers of bevacizumab efficacy were identified: VEGF-D (P < 0.01), SDF1 (P < 0.05), and Ang2 (P < 0.05). Conclusion: This study identified strong prognostic markers for pancreatic cancer patients. Predictive marker analysis indicated that plasma levels of VEGF-D, Ang2, and SDF1 significantly predicted for benefit or lack of benefit from bevacizumab in this population. Clin Cancer Res; 19(24); 6957–66. ©2013 AACR.

Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Correlation of angiogenic biomarker signatures with clinical outcomes in metastatic colorectal cancer patients receiving capecitabine, oxaliplatin, and bevacizumab

A novel combination of capecitabine, oxaliplatin, and bevacizumab was evaluated in colorectal cancer patients enrolled in a phase II clinical trial. In this retrospective analysis, plasma samples from patients receiving capecitabine, oxaliplatin, and bevacizumab were analyzed to investigate biomarkers of clinical benefit. Forty‐one protein biomarkers were tested in 38 patients at baseline and after two cycles of drug administration. Correlations among analytes were evaluated by Spearman analysis. Analyte levels at baseline and changes on‐treatment were correlated with progression‐free survival (PFS) and overall survival (OS) by univariate analysis. Multivariate analyses were determined using the Cox proportional hazard model. Time to event analyses were evaluated by Kaplan–Meier analysis and compared by log‐rank test. Baseline levels of vWF and Ang‐2 significantly correlated with PFS, while levels of VCAM‐1, vWF, TSP‐2, IL‐8, MMP‐2, and Ang‐2 correlated with OS (P < 0.05). The fold change of IGF‐1 levels from baseline to the end of cycle 2 was correlated with PFS, while fold changes of Ang‐2, TSP‐2, and TGF‐β2 correlated with OS. A baseline signature of Ang‐2, IGFBP‐3, IL‐6, and VCAM‐1 identified a low‐risk and high‐risk group of patients (OS: 33.9 months vs. 18.1 months, respectively, P = 0.016). For treatment‐related changes, a signature consisting of Ang‐2, E‐Cadherin, IL‐6, MCP‐1, OPN, and TGF‐β1 was able to stratify patients into high‐ and low‐risk groups (PFS: 7.7 months vs. 15.5 months, P = 0.004). Multiplex analysis of patient plasma in this trial identified several baseline‐ and treatment‐related biomarkers associated with clinical outcome. These findings merit further exploration in larger, controlled clinical trials.

Dasatinib (BMS-35482) has synergistic activity with paclitaxel and carboplatin in ovarian cancer cells

PURPOSE To explore the activity of dasatinib alone and in combination with paclitaxel and carboplatin in ovarian cancer cells and to determine if dasatinib activity can be predicted based on evaluation of the SRC pathway. EXPERIMENTAL DESIGN Microarray analysis was performed for IGROV1, OVCAR3, A2780 and SKOV3 ovarian cancer cells and the status of the genomic SRC signature pathway was determined. Cells were treated with carboplatin, paclitaxel and dasatinib individually and in combination. Pre- and post-treatment phospho-SRC (pSRC) and SRC protein expression was determined. Dose-response curves were constructed, and drug interaction was assessed by the Combination Index (CI) method. RESULTS SRC protein expression levels reflected the SRC pathway genomic signature in the cell lines with the lowest (SKOV3) and highest (IGROV1) pathway expression, but not in those with intermediate expression (OVCAR3, A2780). Dasatinib treatment caused loss of pSRC in all cell lines, with 50% growth inhibition for IGROV1 at 70 nM, OVCAR3 at 34 nM, A2780 at 4.1 μM and SKOV3 at 530 nM. Dasatinib combined with cytotoxics yielded a synergistic effect (CI=0.46 to 0.79) in all cell lines except SKOV3. CONCLUSION Dasatinib in combination with standard chemotherapeutic agents appears to interact in a synergistic manner in some ovarian cancer cell lines. Further research is needed to evaluate tumor cell characteristics which predict response to dasatinib.

Ectodomain shedding of TβRIII is required for TβRIII-mediated suppression of TGF-β signaling and breast cancer migration and invasion

The type III transforming growth factor β (TGF-β) receptor (TβRIII), also known as betaglycan, is the most abundantly expressed TGF-β receptor. TβRIII suppresses breast cancer progression by inhibiting migration, invasion, metastasis, and angiogenesis. TβRIII binds TGF-β ligands, with membrane-bound TβRIII presenting ligand to enhance TGF-β signaling. However, TβRIII can also undergo ectodomain shedding, releasing soluble TβRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TβRIII on TGF-β signaling and breast cancer biology, we defined TβRIII mutants with impaired (ΔShed-TβRIII) or enhanced ectodomain shedding (SS-TβRIII). Inhibiting ectodomain shedding of TβRIII increased TGF-β responsiveness and abrogated TβRIIIs ability to inhibit breast cancer cell migration and invasion. Conversely, expressing SS-TβRIII, which increased soluble TβRIII production, decreased TGF-β signaling and increased TβRIII-mediated inhibition of breast cancer cell migration and invasion. Of importance, SS-TβRIII-mediated increases in soluble TβRIII production also reduced breast cancer metastasis in vivo. Taken together, these studies suggest that the ratio of soluble TβRIII to membrane-bound TβRIII is an important determinant for regulation of TβRIII- and TGF-β-mediated signaling and biology.

A phase I study of ABT-510 plus bevacizumab in advanced solid tumors.

Targeting multiple regulators of tumor angiogenesis have the potential to improve treatment efficacy. Bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor and ABT‐510 is a synthetic analog of thrombospondin, an endogenous angiogenesis inhibitor. Dual inhibition may result in additional benefit. We evaluated the safety, tolerability, and efficacy of the combination of bevacizumab plus ABT‐510 in patients with refractory solid tumors. We also explored the effects of these agents on plasma‐based biomarkers and wound angiogenesis. Thirty‐four evaluable subjects were enrolled and received study drug. Therapy was well tolerated; minimal treatment‐related grade 3/4 toxicity was observed. One patient treated at dose level 1 had a partial response and five other patients treated at the recommended phase II dose had prolonged stable disease for more than 1 year. Biomarker evaluation revealed increased levels of D‐dimer, von Willebrand factor, placental growth factor, and stromal‐derived factor 1 in response to treatment with the combination of bevacizumab and ABT‐510. Data suggest that continued evaluation of combination antiangiogenesis therapies may be clinically useful.

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-β (TGF-β) induces the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad2/3 signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-β-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

Modulation of circulating protein biomarkers following TRC105 (anti-endoglin antibody) treatment in patients with advanced cancer

TRC105 is an endoglin‐targeting drug that possesses anti‐angiogenic and antitumor potential. Analysis of the initial phase I trial of TRC105 demonstrated good tolerability and efficacy in cancer patients. In this report, we analyzed multiple circulating biomarkers at baseline, cycle 2 day 1 (C2D1), and end of study (EOS) for each patient. The baseline level and the fold change from baseline to both C2D1 and EOS for each marker were statistically analyzed. At C2D1, seven markers were significantly downregulated (angiopoietin‐2 [Ang‐2], insulin‐like growth factor‐binding protein‐3 [IGFBP‐3], plasminogen activator inhibitor‐1 [PAI‐1] total, platelet‐derived growth factor [PDGF]‐AA, PDGF‐BB, thrombospondin‐1 [TSP‐1], and vascular endothelial growth factor [VEGF]‐D). Meanwhile, seven markers were upregulated by C2D1 (E‐Cadherin, soluble Endoglin [sEnd], E‐Selectin, interleukin‐6 [IL‐6], osteopontin [OPN], TSP‐2, and von Willebrand factor [vWF]). At EOS, seven markers were upregulated including Ang‐2, C‐reactive protein (CRP), intercellular adhesion molecule‐1 (ICAM‐1), IGFBP‐1, IL‐6, TSP‐2, and vascular cell adhesion molecule‐1 (VCAM‐1). A statistical trend was also seen for increases of VEGF‐A and placenta growth factor (PlGF) at EOS. Throughout treatment, sEnd levels significantly increased, an observation that was recapitulated in cultured endothelial cells. This is the first report of plasma‐based biomarkers in patients receiving TRC105. TRC105 treatment by C2D1 was associated with decreases in several angiogenic factors, including Ang‐2, PDGF isoforms, and VEGF isoforms, offering insight into the mechanisms underlying TRC105s anti‐angiogenic, antitumor function. Increases in sEnd were the most significant of all observed biomarker changes and may reflect direct drug effects. Additionally, biomarker changes in response to TRC105 are distinct from those seen in patients treated with VEGF‐targeting drugs, suggesting the possible utility of combining these two classes of angiogenesis inhibitors in patients.

A phase Ib study of combined VEGFR and mTOR inhibition with vatalanib and everolimus in patients with advanced renal cell carcinoma.

BACKGROUND Vatalanib is an oral vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI), whereas everolimus inhibits mammalian target of rapamycin (mTOR). Combination therapy with VEGFR and mTOR inhibitors has not been well tolerated to date but may have efficacy in renal cell carcinoma (RCC). PATIENTS AND METHODS A phase Ib study of vatalanib and everolimus was performed in patients with advanced solid tumors to determine the maximum tolerated dose (MTD), safety, and tolerability of the combination. A dose-expansion cohort of 20 patients with metastatic RCC was studied to further define toxicity and preliminary efficacy in patients with RCC. RESULTS We evaluated 32 patients over 3 dose levels and a dose-expansion cohort. The most common toxicities of any grade were proteinuria, fatigue, hypertriglyceridemia, nausea, and vomiting. Dose-limiting toxicities (DLTs) included severe hypertension, diarrhea, neutropenia, mucositis, and fatigue. The MTD for the combination was vatalanib 1000 mg daily and everolimus 5 mg daily. In all patients, median overall survival (OS) was 16.3 months. In patients with RCC, median progression-free survival (PFS) was 5.8 months, and OS was 16.5 months. OS was significantly better in treatment-naive patients (25.1 months) compared with patients who had received previous vascular endothelial growth factor (VEGF)-targeted therapy (6.3 months). Seven of 24 (29.2%) evaluable patients demonstrated a partial response, and an additional 15 patients exhibited stable disease. Long-term tolerability (> 1 year) was demonstrated in 19% of patients. CONCLUSION Relevant doses of vatalanib and everolimus were achieved in combination, with expected toxicities. A substantial number of patients with RCC achieved an objective response in the treatment-naive setting, with prolonged tolerability and survival. Further comparative phase II/III studies of specifically targeted VEGF and mTOR inhibitor combinations may be warranted in patients with RCC.

Role of TGF-β receptor III localization in polarity and breast cancer progression.

TβRIII is basolaterally localized in polarized breast epithelial cells. The disruption of TβRIII targeting by mutation of proline 826 results in global loss of cell polarity through enhanced EMT. The mistargeting of TβRIII results in enhanced proliferation, migration, and invasion in vitro and enhanced tumor formation and invasion in vivo.