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Evidence of Efficacy: Researchers Investigating Markers for Angiogenesis Inhibitors

Vicki Brower

In June, results of a large phase III trial with the antiangiogenesis drug Avastin (bevacizumab) showed that patients with metastatic colorectal cancer taking the drug with standard chemotherapy lived 4 months longer than those taking chemotherapy alone. And in August, a phase II study demonstrated that patients with metastatic renal cancer who took Avastin had a greater time to tumor progression than those taking a placebo, although there was no difference in survival time between the two groups.

Although antiangiogenesis pioneer Judah Folkman, M.D., called the positive results "a watershed," moving ahead with studies of angiogenesis inhibitors will require developing reliable biomarkers for treatment with antiangiogenesis drugs. "As yet, there are no agreed-upon surrogate markers for angiogenesis inhibitor efficacy," said Folkman, of Children’s Hospital, Boston. "It’s the next big thing."

Traditional chemotherapy drugs are used at the maximum tolerated dose, which is determined by toxicity. However, many angiogenesis inhibitors in development cause little or no toxicity—a blessing for patients, but somewhat of a curse for physicians who need a marker for proper dosing. Moreover, because angiogenesis inhibitors such as endostatin take more time to work and generally arrest, but do not necessarily shrink, tumors, new criteria are needed to judge efficacy of antiangiogenesis drugs. Because most of these drugs are cytostatic rather than cytotoxic, the goal of tumor regression may not be a realistic endpoint for angiogenesis inhibitors, say Folkman and others in the field.

Unlike traditional chemotherapy, which targets the cancer cell, angiogenesis inhibitors such as bevacizumab, endogenous proteins such as endostatin and angiostatin, and low-dose, continuous ("metronomic") chemotherapy all target the more genetically stable endothelial cells that comprise the new blood vessels that feed tumors. And because angiogenesis inhibitors seem to work synergistically with chemotherapy drugs and, in some cases, with each other, determining new surrogate markers and new endpoints that do not rely on toxicity or tumor regression is both necessary and complex.

"Because for antiangiogenic agents there is an uncertain relation between toxicity and response, determining the optimum biological dose of angiogenesis inhibitors depends on a different logic than conventional cytotoxic chemotherapy," said Roy Herbst, M.D., Ph.D., chief of the thoracic medical oncology section at the University of Texas M. D. Anderson Cancer Center, Houston, who has conducted a number of trials with endostatin. "Several clinical studies have shown that angiogenesis inhibitors are optimal at doses well below the maximum dose studied," Herbst said.

There are a number of methods being used to monitor these therapies, but as yet there is no consensus as to which are most accurate. Methods include measuring growth factors and other pro-angiogenic proteins, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular cell-adhesion molecule (VCAM), and matrix metalloproteinase (MMP) enzymes in the blood serum, plasma, or urine; performing biopsies; using radiologic methods; and measuring circulating endothelial cells (CECs) that are recruited to form tumor blood vessels.

Although all of these growth factors and proteins have been found at higher levels in tumors and in metastatic cancer and have been considered as possible surrogate markers, the evidence is mixed on their reliability as markers of drug efficacy and disease progression. Because tumors are highly heterogeneous, the amount of growth factors in one section of a tumor biopsy may not be the same in another section, and therefore measuring growth factor concentration in sections of tumors may not be reliable, noted Herbst.

VEGF is vital to the growth of tumor blood vessels, and as such has long been regarded as a potential surrogate marker of cancer growth and antiangiogenesis drug efficacy. However, many studies of its use as a surrogate marker have not produced clear evidence of its reliability. Many researchers have concluded that VEGF status alone is inadequate as a diagnostic or prognostic marker because it is only one of many pro-angiogenic factors involved in cancer growth and may be more significant to certain cancers at certain stages of tumor growth.

New research on tumor growth markers and drug efficacy is shifting away from VEGF to the cells on which it exerts its effects: the recruitment of hematopoietic and circulating endothelial cells (CECs), and specifically circulating endothelial progenitor cells (CEPs) from the bone marrow to the tumor.

"CEPs are a subset of circulating endothelial cells, which are a subset of the larger circulating endothelial cell population found to be increased in the majority [of]—but not all—human cancers, and in some precancerous conditions," said Francesco Bertolini, M.D., Ph.D., co-director of the Laboratory of Clinical Hematology-Oncology at the European Institute of Oncology in Milan. His group is investigating in which type of human cancers CEPs are systemically increased. Ratios of both types of cells vary depending on what type of cancer one is studying.



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Dr. Francesco Bertolini

 
A number of recent studies from Bertolini’s laboratory indicate that CEC levels may be promising biomarkers. In work that was presented at this year’s annual meeting of the American Association for Cancer Research, Bertolini and colleagues found a switch to increased VEGF, CEC, and VE-cadherin circulating RNA in lymphoma patients (see graph). In another study published in June 2001, Bertolini evaluated the kinetics and viability of CECs as a surrogate angiogenesis marker in an animal model of lymphoma and showed a strong correlation among CECs, tumor volume, and tumor-generated VEGF. When he gave animals high-dose cyclophosphamide, a traditional chemotherapy drug, most of the circulating apoptotic cells were hematopoietic (indicating predictable bone marrow damage), but not endothelial (which would indicate dying tumor blood vessels and hence, antiangiogenesis). But when given the angiogenesis inhibitor endostatin, only apoptotic endothelial cells from the endothelial cell compartment increased, indicating drug activity against new tumor blood vessels.



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This three-dimensional surface plot shows plasma VEGF, CECs, and the ratio between circulating RNA of VE-cadherin versus a reporter gene in a group of non-Hodgkin’s lymphoma patients. A normal distribution was observed in healthy control patients, whereas a switch to increased VEGF, CEC, and VE-cadherin circulating RNA was observed in cancer patients. (Image courtesy of Francesco Bertolini, M.D., Ph.D.)

 
And in new research, Bertolini and Robert Kerbel, Ph.D., of the University of Toronto show that the maximum tolerated dose of chemotherapy and low-dose, continuous chemotherapy have opposite effects on the mobilization and viability of CEPs. Animals receiving maximum chemotherapy similar to the dose and schedule of traditional chemotherapy showed robust CEP mobilization a few days after the end of a chemotherapy cycle, and the rapid development of drug resistance. "But with low-dose ‘metronomic’ chemotherapy, mice showed a consistent decrease in CEP numbers and viability, with more durable tumor growth inhibition," said Kerbel.

CEP mobilization supports and promotes tumor cell growth and drug resistance via the development of new vasculature to feed tumors, whereas CEP depletion acts to down-regulate tumor blood vessel development, Kerbel said. High-dose, intermittent chemotherapy results in drug resistance that is aided in part by new blood vessel formation by CEPs, say antiangiogenesis researchers. But administration of a low-dose, continuous infusion without rest periods results in sustained tumor blood vessel destruction by depriving the body of the vessels’ building blocks, CEPs. "An influx of mobilized CEPs during the rest periods between cycles of [maximum tolerated dose] therapy may replace damaged or killed endothelial cells and explain regrowth of tumors and drug resistance," said Bertolini.

Other research indicating the importance of CECs in angiogenesis has been done by Shahin Rafii, Ph.D., who discovered that by blocking the signaling of both VEGF receptors, it is possible to impair mobilization and recruitment of both hematopoetic and endothelial cells to the tumor vasculature and thereby slow the growth of certain tumors. "The characterization and quantification of these cells in the peripheral blood and VEGF in plasma, as well as MMP-9 and other factors, might lead to their use as surrogate markers for assessing response to therapy or progression of certain malignancies," said Rafii, professor at Cornell University Medical College, New York.

Since the flow of blood and nutrients to tumors is a key principle in angiogenesis, researchers are employing radiologic techniques to view blood flow to tumors, vascular permeability, and tumor metabolism. PET scans, MRI, three-dimensional ultrasound, and laser scanning cytometry of biopsies are being tested. A drawback to most imaging methods is that the highest resolution or best image is on preserved tissue, not live tissue, according to some critics.

Intravital microscopy (IVM)—a multi-photon laser-scanning microscope being developed by Harvard University Medical School professor Rakesh Jain, Ph.D., who has worked on the in vivo technique for 20 years—may change that. "IVM allows continuous non-invasive monitoring of molecular and cellular processes in intact living tissue with a 1-10 µm resolution not possible with non-optical techniques," Jain said.

Jain’s IVM work has shown that blood flow, vascular permeability, and interstitial diffusion change during treatment with angiogenesis inhibitors. Perhaps most interesting is that IVM confirmed that antiangiogenesis drugs can actually normalize tumor vessels, making them more efficient than their regularly leaky state. Jain believes that this could be used against the tumor: antiangiogenic drugs can normalize blood vessels, which can in turn be exploited by delivering chemotherapy more efficiently to the tumor.


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