Urban Emmenegger

Increased plasma vascular endothelial growth factor (VEGF) as a surrogate marker for optimal therapeutic dosing of VEGF receptor-2 monoclonal antibodies

A major obstacle compromising the successful application of many of the new targeted anticancer drugs, including angiogenesis inhibitors, is the empiricism associated with determining an effective biological/therapeutic dose because many of these drugs express optimum therapeutic activity below the maximum tolerated dose, if such a dose can be defined. Hence, surrogate markers are needed to help determine optimal dosing. Here we describe such a molecular marker, increased plasma levels of vascular endothelial growth factor (VEGF), in normal or tumor-bearing mice that received injections of an anti-VEGF receptor (VEGFR)-2 monoclonal antibody, such as DC101. Rapid increases of mouse VEGF (e.g., within 24 hours) up to 1 order of magnitude were observed after single injections of DC101 in non–tumor-bearing severe combined immunodeficient or nude mice; similar increases in human plasma VEGF were detected in human tumor-bearing mice. RAFL-1, another anti-VEGFR-2 antibody, also caused a significant increase in plasma VEGF. In contrast, increases in mouse VEGF levels were not seen when small molecule VEGFR-2 inhibitors were tested in normal mice. Most importantly, the increases in plasma VEGF were induced in a dose-dependent manner, with the maximum values peaking when doses previously determined to be optimally therapeutic were used. Plasma VEGF should be considered as a possible surrogate pharmacodynamic marker for determining the optimal biological dose of antibody drugs that block VEGFR-2 (KDR) activity in a clinical setting.

Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia.

Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti-vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1alpha expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased.

Strategies for Delaying or Treating In vivo Acquired Resistance to Trastuzumab in Human Breast Cancer Xenografts

Purpose: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivo tumorigenic models. Experimental Design: We derived an erbB-2–positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2–negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response. Results: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti–epidermal growth factor receptor antibody (cetuximab) was effective as was the anti–vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors. Conclusions: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab.

A Comparative Analysis of Low-Dose Metronomic Cyclophosphamide Reveals Absent or Low-Grade Toxicity on Tissues Highly Sensitive to the Toxic Effects of Maximum Tolerated Dose Regimens

The survival benefits of traditional maximum tolerated dose (MTD) cytotoxic therapy have been modest for the treatment of most types of metastatic malignancy and, moreover, often come with increased acute and chronic toxicity. Recent studies have demonstrated that the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks [low-dose metronomic (LDM) chemotherapy], may not only be at least as efficient as MTD therapy but also less toxic. This coincides with an apparent selectivity for “activated” endothelial cells of the tumor vasculature. However, the impact of LDM chemotherapy on the most sensitive target cell populations normally affected by MTD therapy (i.e., bone marrow progenitors, gut mucosa, and hair follicle cells) has not been analyzed in experimental detail. Therefore, we compared effects of LDM and MTD cyclophosphamide (CTX) on bone marrow and gut mucosa. Furthermore, we studied the potential impact of LDM CTX on angiogenesis in the context of wound healing and evidence of organ toxicity. We show absent or moderate hematologic and intestinal toxicity of LDM as opposed to MTD CTX. Of note was the finding of sustained lymphopenia, which is not unexpected given the use of CTX as immunosuppressive drug. There was no negative impact on wound healing or evidence of organ toxicity. LDM offers clear safety advantages over conventional MTD chemotherapy and therefore would appear to be ideal for long-term combination therapy with targeted antiangiogenic drugs.

Metronomic Low-Dose Chemotherapy Boosts CD95-Dependent Antiangiogenic Effect of the Thrombospondin Peptide ABT-510: A Complementation Antiangiogenic Strategy

Blocking angiogenesis is a promising approach in cancer therapy. Natural inhibitors of angiogenesis and derivatives induce receptor-mediated signals, which often result in the endothelial cell death. Low-dose chemotherapy, given at short regular intervals with no prolonged breaks (metronomic chemotherapy), also targets angiogenesis by obliterating proliferating endothelial cells and circulating endothelial cell precursors. ABT-510, a peptide derivative of thrombospondin, kills endothelial cell by increasing CD95L, a ligand for the CD95 death receptor. However, CD95 expression itself is unaffected by ABT-510 and limits its efficacy. We found that multiple chemotherapy agents, cyclophosphamide (cytoxan), cisplatin, and docetaxel, induced endothelial CD95 in vitro and in vivo at low doses that failed to kill endothelial cells (cytoxan > cisplatin > docetaxel). Thus, we concluded that some of these agents might complement each other and together block angiogenesis with maximal efficacy. As a proof of principle, we designed an antiangiogenic cocktail combining ABT-510 with cytoxan or cisplatin. Cyclophosphamide and cisplatin synergistically increased in vivo endothelial cell apoptosis and angiosuppression by ABT-510. This synergy required CD95, as it was reversible with the CD95 decoy receptor. In a mouse model, ABT-510 and cytoxan, applied together at low doses, acted in synergy to delay tumor take, to stabilize the growth of established tumors, and to cause a long-term progression delay of PC-3 prostate carcinoma. These antitumor effects were accompanied by major decreases in microvascular density and concomitant increases of the vascular CD95, CD95L, and apoptosis. Thus, our study shows a “complementation” design of an optimal cancer treatment with the antiangiogenic peptide and a metronomic chemotherapy.

Phase I/II Trial of Metronomic Chemotherapy With Daily Dalteparin and Cyclophosphamide, Twice-Weekly Methotrexate, and Daily Prednisone As Therapy for Metastatic Breast Cancer Using Vascular Endothelial Growth Factor and Soluble Vascular Endothelial Growth Factor Receptor Levels As Markers of Response

PURPOSE Preclinical studies indicate that metronomic chemotherapy is antiangiogenic and synergistic with other antiangiogenic agents. We designed a phase I/II study to evaluate the safety and activity of adding dalteparin and prednisone to metronomic cyclophosphamide and methotrexate in women with measurable metastatic breast cancer (MBC). PATIENTS AND METHODS Patients received daily dalteparin and oral cyclophosphamide, twice-weekly methotrexate, and daily prednisone (dalCMP). The primary study end point was clinical benefit rate (CBR), a combination of complete response (CR), partial response (PR), and prolonged stable disease for > or = 24 weeks (pSD). Secondary end points included time to progression (TTP), duration of response, and overall survival (OS). Biomarker response to treatment was assessed by using plasma vascular endothelial growth factor (VEGF) and soluble VEGF receptors (sVEGFRs) -1 and -2. Results Forty-one eligible patients were accrued. Sixteen (39%) had no prior chemotherapy for MBC; 15 (37%) had two or more chemotherapy regimens for MBC. Toxicities were minimal except for transient grade 3 elevation of liver transaminases in 11 patients (27%) and grade 3 vomiting in one patient (2%). One patient (2%) had CR, six (15%) had PR, and three (7%) had pSD, for a CBR of 10 (24%) of 41 patients. Median TTP was 10 weeks (95% CI, 8 to 17 weeks), and median OS was 48 weeks (95% CI, 32 to 79 weeks). VEGF levels decreased but not significantly, whereas sVEGFR-1 and -2 levels increased significantly after 2 weeks of therapy. There was no correlation between response and VEGF, sVEGFR-1, or sVEGFR-2 levels. CONCLUSION Metronomic dalCMP is safe, well tolerated, and clinically active in MBC.

Low-Dose Metronomic Daily Cyclophosphamide and Weekly Tirapazamine: A Well-Tolerated Combination Regimen with Enhanced Efficacy That Exploits Tumor Hypoxia

The recent clinical successes of antiangiogenic drug-based therapies have also served to highlight the problem of acquired resistance because, similar to other types of cancer therapy, tumors that initially respond eventually stop doing so. Consequently, strategies designed to delay resistance or treat resistant subpopulations when they arise have assumed considerable importance. This requires a better understanding of the various possible mechanisms for resistance. In this regard, reduced oxygenation is thought to be a key mediator of the antitumor effects of antiangiogenic therapies; accordingly, increased hypoxia tolerance of the tumor cells presents a potential mechanism of resistance. However, hypoxia can also be exploited therapeutically through the use of hypoxic cell cytotoxins, such as tirapazamine. With this in mind, we measured the oxygenation of PC-3 human prostate cancer xenografts subjected to chronic low-dose metronomic (LDM) antiangiogenic chemotherapy using cyclophosphamide given through the drinking water. We found that LDM cyclophosphamide impairs the oxygenation of PC-3 xenografts even during relapse, coinciding with reduced microvessel density. Combination of LDM cyclophosphamide with tirapazamine results in significantly improved tumor control in the PC-3, HT-29 colon adenocarcinoma, and MDA-MB-231 breast cancer human xenograft models without having a negative effect on the favorable toxicity profile of LDM cyclophosphamide. These results provide further evidence that reduced vascular dependence/increased hypoxia tolerance may be a basis for eventual resistance of tumors exposed to long-term LDM chemotherapy.

Pharmacodynamic and pharmacokinetic study of chronic low-dose metronomic cyclophosphamide therapy in mice

Prolonged, frequently administered low-dose metronomic chemotherapy (LDM) is being explored (pre)clinically as a promising antiangiogenic antitumor strategy. Although appealing because of a favorable side effect profile and mostly oral dosing, LDM involves new challenges different from conventional maximum tolerated dose chemotherapy. These include possible altered pharmacokinetic characteristics due to long-term drug exposure potentially resulting in acquired resistance and increased risk of unfavorable drug interactions. We therefore compared the antitumor and antivascular effects of LDM cyclophosphamide (CPA) given to mice that had been pretreated with either LDM CPA or normal saline, obtained blood 4-hydroxy-CPA (activated CPA) concentrations using either gas chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry in mice treated with LDM CPA, and measured hepatic and intratumoral activity of enzymes involved in the biotransformation of CPA and many other drugs [i.e., cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase]. Exposure of mice to LDM CPA for ≥8 weeks did not compromise subsequent activity of LDM CPA therapy, and biologically active 4-hydroxy-CPA levels were maintained during long-term LDM CPA administration. Whereas the effects on CYP3A4 were complex, aldehyde dehydrogenase activity was not affected. In summary, our findings suggest that acquired resistance to LDM CPA is unlikely accounted for by altered CPA biotransformation. In the absence of reliable pharmacodynamic surrogate markers, pharmacokinetic parameters might become helpful to individualize/optimize LDM CPA therapy. LDM CPA-associated changes of CYP3A4 activity point to a potential risk of unfavorable drug interactions when compounds that are metabolized by CYP3A4 are coadministered with LDM CPA. [Mol Cancer Ther 2007;6(8):2280–9]

Comparative Impact of Trastuzumab and Cyclophosphamide on HER-2–Positive Human Breast Cancer Xenografts

Purpose: Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2–positive human breast cancer models. Experimental Design: Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2–positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell–secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival. Results: Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity. Conclusions: Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2–positive metastatic breast cancer. (Clin Cancer Res 2009;15(20):6358–66)

Tumor-Associated Fibroblasts as "Trojan Horse" Mediators of Resistance to Anti-VEGF Therapy

While targeting VEGF has shown success against a number of human cancers, drug resistance has resulted in compromised clinical benefits. In this issue of Cancer Cell, Crawford et al. (2009) report that tumors resistant to anti-VEGF therapy stimulate tumor-associated fibroblasts to express proangiogenic PDGF-C, implicating it as a potential therapeutic target.