Mieko Yanagisawa

Bevacizumab improves the delivery and efficacy of paclitaxel

It has been reported that bevacizumab in combination with paclitaxel significantly prolongs progression-free survival compared with paclitaxel alone in the initial treatment for metastatic breast cancer. To understand how bevacizumab enhances the efficacy of paclitaxel, we investigated the mechanism in a MX-1 human breast cancer xenograft model. The antitumor activity of bevacizumab at 5 mg/kg in combination with paclitaxel at 20 or 30 mg/kg was significantly higher than that of either agent alone. First, we measured the paclitaxel concentration in tumor to see whether bevacizumab enhances the activity by increasing the tumor concentration of paclitaxel. When given in combination with bevacizumab, the levels of paclitaxel in the tumor increased. Paclitaxel at 30 mg/kg with bevacizumab showed a similar tumor concentration as paclitaxel alone at either 60 or 100 mg/kg, with a similar degree of tumor growth inhibition. In contrast, no remarkable differences in paclitaxel concentration in the plasma or liver were observed between the paclitaxel monotherapy group and the paclitaxel plus bevacizumab group. An increase in paclitaxel concentration by bevacizumab was also found in another model, A549. In the same MX-1 model, vascular permeability in the tumor was significantly decreased by treatment with bevacizumab. There was no difference in microvessel density between the bevacizumab alone group and the combination group. Results suggest that the synergistic antitumor activity of paclitaxel and bevacizumab in combination may be a result of the increase in paclitaxel concentration in tumor resulting from the downregulation of vascular permeability when co-administered with bevacizumab.

Erlotinib inhibits osteolytic bone invasion of human non-small-cell lung cancer cell line NCI-H292

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.

Biomarkers for antitumor activity of bevacizumab in gastric cancer models

BackgroundBevacizumab is a humanized monoclonal antibody to human vascular endothelial cell growth factor (VEGF) and has been used for many types of cancers such as colorectal cancer, non-small cell lung cancer, breast cancer, and glioblastoma. Bevacizumab might be effective against gastric cancer, because VEGF has been reported to be involved in the development of gastric cancer as well as other cancers. On the other hand, there are no established biomarkers to predict the bevacizumab efficacy in spite of clinical needs. Therefore, we tried to identify the predictive markers for efficacy of bevacizumab in gastric cancer patients by using bevacizumab-sensitive and insensitive tumor models.MethodsNine human gastric and two colorectal cancer mouse xenografts were examined for their sensitivity to bevacizumab. We examined expression levels of angiogenic factors by ELISA, bioactivity of VEGF by phosphorylation of VEGFR2 in HUVEC after addition of tumor homogenate, tumor microvessel density by CD31-immunostaining, and polymorphisms of the VEGF gene by HybriProbe™ assay.ResultsOf the 9 human gastric cancer xenograft models used, GXF97, MKN-45, MKN-28, 4-1ST, SC-08-JCK, and SC-09-JCK were bevacizumab-sensitive, whereas SCH, SC-10-JCK, and NCI-N87 were insensitive. The sensitivity of the gastric cancer model to bevacizumab was not related to histological type or HER2 status. All tumors with high levels of VEGF were bevacizumab-sensitive except for one, SC-10-JCK, which had high levels of VEGF. The reason for the refractoriness was non-bioactivity on the phosphorylation of VEGFR2 and micro-vessel formation of VEGF, but was not explained by the VEGF allele or VEGF165b. We also examined the expression levels of other angiogenic factors in the 11 gastrointestinal tumor tissues. In the refractory models including SC-10-JCK, tumor levels of another angiogenic factor, bFGF, were relatively high. The VEGF/bFGF ratio correlated more closely with sensitivity to bevacizumab than with the VEGF level.ConclusionsVEGF levels and VEGF/bFGF ratios in tumors were related to bevacizumab sensitivity of the xenografts tested. Further clinical investigation into useful predictive markers for bevacizumab sensitivity is warranted.

Impact of bevacizumab in combination with erlotinib on EGFR-mutated non-small cell lung cancer xenograft models with T790M mutation or MET amplification.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non–small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression‐free survival in patients with EGFR‐mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR‐mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR‐mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug‐cessation period. In the HCC827‐EPR model (DEL+T790M) and HCC827‐vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI‐H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL‐resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation‐ or MET amplification‐positive tumors as long as their growth remained significantly suppressed by ERL.

Bevacizumab counteracts VEGF-dependent resistance to erlotinib in an EGFR-mutated NSCLC xenograft model

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Sequential Treatment with Irinotecan and Doxifluridine: Optimal Dosing Schedule in Murine Models and in a Phase I Study for Metastatic Colorectal Cancer

Background: Irinotecan (CPT-11) and doxifluridine (5′-DFUR) are active agents against colorectal cancer. Each drug, however, has the possibility of causing diarrhea. Methods and Results: First, we determined the optimal dosing regimen in murine models. CPT-11 (i.v., q2d ×3) and 5′-DFUR (p.o., qd ×14) were administered to mice bearing a human colorectal cancer xenograft model. Diarrhea was stronger in the simultaneously administered schedule but not much stronger in the sequentially administered schedule compared with monotherapies. Both schedules yielded similar antitumor efficacies. Next, we conducted a phase I study combining CPT-11 on days 1 and 15, and 5′-DFUR on days 3–14 and 17–28 every 5 weeks in 19 patients with metastatic colorectal cancer. The doses of CPT-11 ranged from 80 to 150 mg/m2 and those of 5′-DFUR from 800 to 1,200 mg. Diarrhea of grade 3/4 developed in only 1 patient at 100 mg/m2/800-mg doses. Dose-limiting toxicities were hyperbilirubinemia and skipping doses due to fatigue at 150 mg/m2/1,200-mg doses. Conclusion: For the phase II study, the recommended dose was set at CPT-11 150 mg/m2 and 5′-DFUR 800 mg.

Abstract 4256: Antitumor activity of capecitabine in combination with bevacizumab and cyclophosphamide in triple-negative breast cancer xenograft model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective: Triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) is associated with a poor prognosis. Combination therapy with capecitabine (Cape) and bevacizumab (BV) has been reported to improve the progression-free survival of breast cancer patients (Robert NJ et al. ASCO 2009; RIBBON-1). In addition, Cape plus cyclophosphamide (CPA) combination therapy is considered to be effective for metastatic breast cancer patients (Tanaka M et al. Anticancer Drugs 2010). These findings prompted us to examine the antitumor efficacy of Cape+BV+CPA combination therapy in a TNBC xenograft model. Materials and Methods: MX-1 human TNBC cells were subcutaneously inoculated into the right flank of BALB-nu/nu mice. Treatment began (day 1) when tumor volume reached approximately 200 mm3. The mice were given Cape (359 mg/kg) and CPA (10 mg/kg) orally, 14 days on and 7 days off. BV (5 mg/kg) was administered intraperitoneally every 7 days for 3 weeks. Tumor volumes (mm3) were monitored twice a week and antitumor activity was evaluated on day 22. Six mice were allotted to each group (Cape+BV+CPA, Cape+BV, Cape+CPA, CPA, BV, control). Thymidine phosphorylase (TP: U/mg protein) levels in the tumor were measured by ELISA methods on day 15. Microvessel density (MVD, %) in tumor tissue was determined using CD31 immunohistochemical staining method on day 15. MVD% was calculated as the ratio of the CD31-positive staining area to the total observation area in the viable region. Results and Discussion: The antitumor activity of Cape+BV+CPA was significantly higher than that of Cape+BV or CPA (p≤0.05) and was also significantly stronger than that of Cape+CPA or BV (p≤0.05). The tumor volume on day 22 was as follows: control, 3969±1026; BV group, 2219±410; CPA group, 2149±962; Cape+BV group, 1270±458; Cape+CPA group, 1534±133; Cape+BV+CPA group, 536±261. The TP levels of the CPA (11.6±2.62), BV (6.21±2.59) and BV+CPA (13.1±2.70) groups were significantly increased compared to the control group (3.77±0.883, p≤0.05). The TP levels of the BV+CPA group were significantly higher than those of the BV group. (p≤0.05). The MVD% of BV (1.92±0.385), Cape+BV (1.26±0.347) and Cape+BV+CPA (1.00±0.160) was significantly decreased compared to the control group (3.57±0.343, p≤0.05). The MVD of the Cape+BV+CPA group was significantly depressed compared to that of the Cape+CPA (3.27±0.578), p≤0.05) group. These results indicate that 5-FU converted from Cape was increased by up-regulated TP with CPA, that it caused antitumor activity of Cape+BV+CPA superior to Cape+BV. BV significantly reduced MVD either alone or in the triplet combination (Cape+BV+CPA) and it is considered to result in the greatest effect in the triplet combination. Conclusion: Our preclinical results suggest that the Cape+BV+CPA (XAC) combination may be a rational therapy to be explored in TNBC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4256. doi:10.1158/1538-7445.AM2011-4256