Bispecific T-Cell Engager (BiTE) Antibody Construct Blinatumomab for the Treatment of Patients With Relapsed/Refractory Non-Hodgkin Lymphoma: Final Results From a Phase I Study.

PURPOSE: Blinatumomab is a CD19/CD3 BiTE (bispecific T-cell engager) antibody construct for the treatment of Philadelphia chromosome-negative acute B-lymphoblastic leukemia. We evaluated blinatumomab in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: This 3 + 3 design, phase I dose-escalation study determined adverse events and the maximum tolerated dose (MTD) of continuous intravenous infusion blinatumomab in patients with relapsed/refractory NHL. Blinatumomab was administered over 4 or 8 weeks at seven different dose levels (0.5 to 90 µg/m(2)/day). End points were incidence of adverse events, pharmacokinetics, pharmacodynamics, and overall response rate. RESULTS: Between 2004 and 2011, 76 heavily pretreated patients with relapsed/refractory NHL, who included 14 with diffuse large B-cell lymphoma, were enrolled; 42 received treatment in the formal dose-escalation phase. Neurologic events were dose limiting, and 60 µg/m(2)/day was established as the MTD. Thirty-four additional patients were recruited to evaluate antilymphoma activity and strategies for mitigating neurologic events at a prespecified MTD. Stepwise dosing (5 to 60 µg/m(2)/day) plus pentosan polysulfate SP54 (n = 3) resulted in no treatment discontinuations; single-step (n = 5) and double-step (n = 24) dosing entailed two and seven treatment discontinuations due to neurologic events, respectively. Grade 3 neurologic events occurred in 22% of patients (no grade 4/5). Among patients treated at 60 µg/m(2)/day (target dose; n = 35), the overall response rate was 69% across NHL subtypes and 55% for diffuse large B-cell lymphoma (n = 11); median response duration was 404 days (95% CI, 207 to 1,129 days). CONCLUSION: In this phase I study of relapsed/refractory NHL, continuous infusion with CD19-targeted immunotherapy blinatumomab at various doses and schedules was feasible, with an MTD of 60 µg/m(2)/day. Single-agent blinatumomab showed antilymphoma activity.

Darbepoetin-α Promotes Cell Proliferation in Established Extrahepatic Colorectal Tumors after Major Hepatectomy.

BACKGROUND: The glycoprotein hormone erythropoietin and its analogue darbepoetin-α (DPO) have been shown to reduce the risk of acute liver failure after major hepatectomy. However, previous experimental studies have also shown that DPO significantly enhances neovascularization and tumor cell proliferation in established colorectal liver metastasis in hepatectomized and nonhepatectomized mice. The present study now analyzes whether DPO influences cell proliferation and migration as well as vascularization and growth of established colorectal metastasis at extrahepatic sites after major hepatectomy. METHODS: GFP-transfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Five days after tumor cell implantation, the animals received a single dose of DPO (10 µg/kg body weight) or phosphate-buffered saline solution (PBS) intravenously. Additional animals received a 70% hepatectomy and DPO or PBS treatment. Tumor vascularization and growth as well as tumor cell migration, proliferation and apoptosis were studied repetitively over 14 days using intravital fluorescence microscopy, histology and immunohistochemistry. RESULTS: DPO did not influence tumor cell migration and apoptosis. In addition, DPO did not stimulate tumor cell infiltration or vascularization; however, significantly increased tumor cell proliferation was detected in hepatectomized animals. CONCLUSION: DPO increases cell proliferation in established extrahepatic colorectal metastases after major hepatectomy. Thus, DPO may not be recommended to stimulate regeneration of the remnant liver after major hepatectomy for colorectal liver metastasis.

Darbepoetin-α accelerates neovascularization and engraftment of extrahepatic colorectal metastases.

PURPOSE: Erythropoietin and its analogue darbepoetin (DPO)-α have been shown to improve liver function and regeneration after partial hepatectomy (Phx). However, previous experimental studies have also shown that DPO significantly enhances Phx-induced engraftment of colorectal liver metastases by increasing neovascularization and tumor cell proliferation. Therefore, the present study analyzed whether DPO affects engraftment and neovascularization of extrahepatic colorectal metastases after major hepatectomy. METHODS: Green fluorescent protein-transfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Animals received a single dose of DPO (10 µg/kg body weight) at the day of tumor cell implantation (day 0). Phosphate-buffered saline-treated animals served as controls. To study whether the effect of DPO is influenced by Phx, additional animals with and without DPO treatment underwent 70% Phx at day 0. Tumor vascularization and growth as well as tumor cell migration, proliferation and apoptosis were studied repetitively over 14 days using intravital fluorescence microscopy, histology and immunohistochemistry. RESULTS: In nonhepatectomized animals, DPO significantly accelerated tumor cell engraftment and slightly enhanced tumor neovascularization. Tumor cell migration and host tissue infiltration were not affected by DPO. In hepatectomized animals, DPO slightly enhanced tumor growth and significantly accelerated tumor neovascularization, but did not affect tumor cell migration and infiltration. CONCLUSION: The present study indicates that DPO accelerates extrahepatic engraftment of colorectal cancer cells, most probably by stimulating the process of neovascularization.

Interaction of the chemokines I-TAC (CXCL11) and SDF-1 (CXCL12) in the regulation of tumor angiogenesis of colorectal cancer.

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14 days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.

Darbepoetin-α promotes neovascularization and cell proliferation in established colorectal liver metastases.

BACKGROUND: The erythropoietin-analogue darbepoetin-α (DPO) improves liver function and regeneration after hepatectomy (Phx), however, also enhances Phx-induced tumor cell engraftment and neovascularization. Because it is unknown whether DPO also enhances the growth of established tumors, we herein studied the effect of DPO on established colorectal liver metastases after Phx. METHODS: CT26.WT cells were implanted into the liver of BALB/c mice. Five days after tumor establishment, animals underwent 50% Phx and received 10 µg/kgBW DPO or saline. Non-Phx animals with DPO or saline-treatment served as controls. Seven days after Phx tumors were analyzed regarding blood vessel formation, leukocyte adhesion, cell proliferation, apoptotic cell death, and growth using intravital fluorescence microscopy, histology, and immunohistochemistry. RESULTS: The growth of established colorectal liver metastases was slightly stimulated after DPO-treatment in hepatectomized and non-hepatectomized animals. However, tumor vessel formation and tumor cell proliferation were significantly enhanced after DPO-treatment in hepatectomized and non-hepatectomized mice compared with controls. Apoptotic cell death and leukocyte-endothelial cell interaction were significantly reduced after DPO-treatment. CONCLUSION: Our study indicates that DPO-treatment promotes neovascularization and cell proliferation in established colorectal liver metastases of hepatectomized and non-hepatectomized mice. DPO-application in patients with colorectal liver metastases might promote tumor progression and should therefore be avoided.

Bone Marrow Suppression by c-Kit Blockade Enhances Tumor Growth of Colorectal Metastases through the Action of Stromal Cell-Derived Factor-1.

Background. Mobilization of c-Kit(+) hematopoietic cells (HCs) contributes to tumor vascularization. Whereas survival and proliferation of HCs are regulated by binding of the stem cell factor to its receptor c-Kit, migration of HCs is directed by stromal cell-derived factor (SDF)-1. Therefore, targeting migration of HCs provides a promising new strategy of anti-tumor therapy. Methods. BALB/c mice (n = 16) were pretreated with an anti-c-Kit antibody followed by implantation of CT26.WT-GFP colorectal cancer cells into dorsal skinfold chambers. Animals (n = 8) additionally received a neutralizing anti-SDF-1 antibody. Animals (n = 8) treated with a control antibody served as controls. Investigations were performed using intravital fluorescence microscopy, immunohistochemistry, flow cytometry and western blot analysis. Results. Blockade of c-Kit significantly enhanced tumor cell engraftment compared to controls due to stimulation of tumor cell proliferation and invasion without markedly affecting tumor vascularization. C-Kit blockade significantly increased VEGF and CXCR4 expression within the growing tumors. Neutralization of SDF-1 completely antagonized this anti-c-Kit-associated tumor growth by suppression of tumor neovascularization, inhibition of tumor cell proliferation and reduction of muscular infiltration. Conclusion. Our study indicates that bone marrow suppression via anti-c-Kit pretreatment enhances tumor cell engraftment of colorectal metastases due to interaction with the SDF-1/CXCR4 pathway which is involved in HC-mediated tumor angiogenesis.

Darbepoetin-alpha enhances hepatectomy-associated stimulation of colorectal liver metastatic growth.

BACKGROUND: Liver insufficiency after major hepatectomy still represents a serious challenge in liver surgery. Although some previous studies indicate that erythropoietin (EPO) and its analogue darbepoetin-alpha (DPO) may improve liver function and liver regeneration, little is known on their effect on tumor growth after hepatectomy. Because EPO may promote tumor progression, we herein studied the effect of DPO on tumor growth after major hepatectomy. METHODS: CT26.WT colorectal cancer cells were implanted into the left liver lobe of BALB/c mice. Animals underwent 50% hepatectomy (Phx) and received 10 microg/kg DPO-treatment. Additional Phx animals received only saline treatment. Nonhepatectomized animals with DPO-treatment or saline treatment served as controls. One week after hepatectomy angiogenic blood vessel formation, leukocyte-endothelial cell interaction, tumor cell proliferation, apoptotic cell death, and tumor growth were studied using intravital fluorescence microscopy, histology, Immunohistochemistry, and Western blot analysis. RESULTS: Phx significantly enhanced the growth of liver metastases. This was associated with an increase of tumor capillary density and tumor cell proliferation. In nonhepatectomized animals, DPO only slightly affected metastatic growth. In hepatectomized animals, however, DPO significantly enhanced the Phx-induced stimulation of tumor growth. This was associated with an increased tumor capillary density, a decreased leukocyte-endothelial cell interaction, and a reduced cleaved caspase-3 expression of the CT26.WT cells. CONCLUSIONS: : Our data indicate that DPO significantly enhances the hepatectomy-induced stimulation of colorectal liver metastatic growth by increasing neovascularization, suppressing intratumoral leukocyte recruitment, and reducing tumor cell apoptosis. Thus, EPOs may not be used in patients undergoing hepatectomy for malignant tumor resection.

CXCR4 and CXCR7 regulate angiogenesis and CT26.WT tumor growth independent from SDF-1.

Recent studies have shown that the chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4 are involved in the metastatic process of colorectal cancer. The impact of SDF-1 on the stimulated metastatic growth during hepatectomy-associated liver regeneration is unknown. With the use of a heterotopic murine colon cancer model, we analyzed whether blockade of SDF-1 inhibits angiogenesis and extrahepatic growth of colorectal cancer after liver resection. Functional neutralization of SDF-1 by 1 mg/kg body weight anti-SDF-1 antibody only slightly delayed the initial tumor cell engraftment but also did not reduce the size of established extrahepatic tumors compared with controls. Tumor cell apoptosis was increased by anti-SDF-1 treatment only during the early 5-9-day period of tumor cell engraftment, but was found significantly decreased during the late phase of tumor growth. The initial delay of tumor cell engraftment was associated with an increase of tumor capillary density and microvascular permeability. This was associated with an increased vascular endothelial growth factor (VEGF) expression and an enhanced tumor cell invasion of the neighboring tissue. In contrast to the neutralization of SDF-1, blockade of the SDF-1 receptors CXCR4 and CXCR7 significantly reduced tumor capillary density and tumor growth. Thus, our study indicates that neutralization of SDF-1 after hepatectomy is not capable of inhibiting angiogenesis and growth of extrahepatic colorectal tumors, because it is counteracted by the compensatory actions through an alternative VEGF-dependent pathway.

Rapamycin inhibits hepatectomy-induced stimulation of metastatic tumor growth by reduction of angiogenesis, microvascular blood perfusion, and tumor cell proliferation.

BACKGROUND: Liver regeneration after hepatectomy stimulates metastatic tumor growth through the release of potent growth factors. In the signaling cascades of several growth factors, mTOR is a downstream mediator, triggering cell survival and cell cycle progression. The mTOR inhibitor rapamycin (RAPA) has been shown to exhibit potent antitumor activities. However, it is unknown whether RAPA is capable of exerting these effects under the conditions of hepatectomy-associated liver regeneration. We therefore analyzed the effect of RAPA and cyclosporine A (CyA) on tumor growth characteristics after major hepatectomy using a mouse model of colorectal metastasis. METHODS: Tumor growth was studied by using GFP-transfected CT26.WT colorectal cancer cells, which were implanted into the dorsal skinfold chambers of BALB/c-mice after 70% hepatectomy. The animals were treated daily with RAPA (1.5 mg/kg) or CyA (10 mg/kg). Tumors were analyzed for angiogenesis, microvascular blood perfusion, cell proliferation, apoptotic cell death, and tumor growth. RESULTS: RAPA significantly inhibited tumor growth compared with CyA and sham treatment. This was associated with a decreased microvascular density within the tumors and a markedly reduced microvascular blood perfusion. CyA only slightly reduced angiogenesis and tumor growth. The effects of RAPA were associated with a significant reduction of tumor cell proliferation, whereas manifestation of apoptotic cell death was not affected by the immunosuppressive treatment regimen. CONCLUSIONS: RAPA is capable of inhibiting angiogenesis, microvascular blood perfusion, and tumor growth of colorectal metastasis during hepatectomy-associated liver regeneration. Thus, targeting mTOR might represent an interesting strategy to prevent tumor recurrence after hepatectomy for colorectal metastasis.

Liver resection-associated macrophage inflammatory protein-2 stimulates engraftment but not growth of colorectal metastasis at extrahepatic sites.

BACKGROUND: Previous studies have shown that liver resection enhances intrahepatic engraftment of CXCR-2-expressing colorectal cancer cells by the action of the CXC chemokine macrophage inflammatory protein (MIP)-2. Herein we studied how liver resection-associated MIP-2 affects extrahepatic tumor cell engraftment and whether MIP-2 also stimulates the growth of already established metastases. MATERIALS AND METHODS: Green fluorescent protein-transfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Additionally, all animals underwent a 30% hepatectomy. To study MIP-2 in extrahepatic tumor cell engraftment, animals were treated with an anti-MIP-2 antibody, starting at the day of tumor cell implantation. To study MIP-2 in established metastases, anti-MIP-2 treatment was initiated at day 5 after tumor cell implantation. Hepatectomized animals without neutralization of MIP-2 served as controls. Tumor vascularization and growth as well as tumor cell migration, proliferation, apoptosis, and CXCR-2 expression were studied over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. RESULTS: Functional inhibition of MIP-2 significantly delayed extrahepatic tumor cell engraftment but not the growth of established metastases. The initial delay of engraftment was associated with a compensatory stimulation of vascularization and tumor cell migration when compared to controls (P < 0.05). Further, inhibition of tumor cell engraftment by initial anti-MIP-2 treatment was associated with a significant (P < 0.05) reduction of CXCR-2 expression and tumor cell apoptosis. CONCLUSION: Our study indicates that MIP-2 is involved in extrahepatic engraftment of CT.26 colorectal cancer cells. The MIP-2/CXCR-2 signaling pathway may be a promising target for early antitumor therapy in patients undergoing liver resection.

Stromal cell-derived factor-1 promotes cell migration and tumor growth of colorectal metastasis.

UNLABELLED: In a mouse model of established extrahepatic colorectal metastasis, we analyzed whether stromal cell-derived factor (SDF) 1 stimulates tumor cell migration in vitro and angiogenesis and tumor growth in vivo. METHODS: Using chemotaxis chambers, CT26.WT colorectal tumor cell migration was studied under stimulation with different concentrations of SDF-1. To evaluate angiogenesis and tumor growth in vivo, green fluorescent protein-transfected CT26.WT cells were implanted in dorsal skinfold chambers of syngeneic BALB/c mice. After 5 days, tumors were locally exposed to SDF-1. Cell proliferation, tumor microvascularization, and growth were studied during a further 9-day period using intravital fluorescence microscopy, histology, and immunohistochemistry. Tumors exposed to PBS only served as controls. RESULTS: In vitro, > 30% of unstimulated CT26.WT cells showed expression of the SDF-1 receptor CXCR4. On chemotaxis assay, SDF-1 provoked a dose-dependent increase in cell migration. In vivo, SDF-1 accelerated neovascularization and induced a significant increase in tumor growth. Capillaries of SDF-1-treated tumors showed significant dilation. Of interest, SDF-1 treatment was associated with a significantly increased expression of proliferating cell nuclear antigen and a downregulation of cleaved caspase-3. CONCLUSION: Our study indicates that the CXC chemokine SDF-1 promotes tumor cell migration in vitro and tumor growth of established extrahepatic metastasis in vivo due to angiogenesis-dependent induction of tumor cell proliferation and inhibition of apoptotic cell death.

Major but not minor hepatectomy accelerates engraftment of extrahepatic tumor cells.

BACKGROUND: The effect of hepatectomy and hepatic regeneration on intra- and extrahepatic tumor growth is still controversially discussed. Herein we studied the effect of minor (30%) or major (70%) hepatectomy on engraftment of extrahepatic tumor cells, and the role of tumor neovascularization and tumor cell migration. METHODS: Green fluorescent protein (GFP)-transfected CT26.WT colorectal cancer cells were implanted in dorsal skinfold chambers of syngeneic BALB/c mice. Animals underwent 30% (30%Phx, n = 8) or 70% hepatectomy (70%Phx, n = 8). Sham-operated animals served as controls (n = 8). Angiogenesis and neovascularization as well as tumor cell migration, proliferation and growth were studied over 14 days using intravital fluorescence microscopy, histology and immunohistochemistry. RESULTS: After both minor and major hepatectomy tumor proliferating cell nuclear antigen (PCNA) expression increased significantly (P < 0.05) when compared with nonhepatectomized controls. However, only major but not minor hepatectomy accelerated neovascularization (P < 0.05) and tumor cell migration (P < 0.05). This was associated with a significantly (P < 0.05) enhanced tumor growth after 70%Phx when compared with 30%Phx and controls. The rate of apoptotic cell death was not affected by major or minor hepatectomy. CONCLUSION: Regeneration after major hepatectomy accelerates extrahepatic tumor cell engraftment, most probably by acceleration of neovascularization and induction of tumor cell migration.