Daniel J. Dunphy, PA-C– 06.30.2009

The long-awaited era of personalized genetic medicine is finally dawning for people with cancer. A little-known reality of current “standard of care” in oncology is that genetic analysis of a person’s individual cancer is an effective, but unusual, method of cancer treatment.

Having researched cancer testing for the last five years both in Europe and the US, the author would like to share his insights with fellow health care practitioners. At present, a European lab offers a blood test which filters and isolates circulating tumor cells (a.k.a. micrometastatic cells) from a patient’s blood, genetically fingerprints them, then pharmacogenetically tests the cells for the effectiveness of various medical therapies.

Therapies tested include traditional medical chemotherapeutic agents and new generation targeted therapies, such as monoclonal antibodies and tyrosine kinase inhibitors, hormone blocking therapies, and an array of over forty nutrient, biological and herbal therapies.

The results not only help the practitioner and patient choose an effective combination of therapies, but also helps them understand which cancer genes (oncogenes) are blocked by which treatments. It is important to block the activity of multiple genes to successfully regulate cancer expression because single therapies are extremely rare in cancer care, as they often allow the cancer genome to adapt and become more aggressive.

Genetic tumor analysis is also available. Ideally, a frozen tumor sample is preserved at the time of biopsy. The sample can then be used for oncogene analysis, as well as creating an autologous (made from the patient’s own tissue) vaccine. A growing number of labs in the US and abroad offer this level of testing. However, only a few places worldwide provide autologous vaccine development. With regards to cancer care, it often pays to think and to act outside of the box.

One of the major labs that provide individual genetic analysis is Research Genetic Cancer Centre Labs in Greece (www.rgcc-genlab.com). I am unaware of any lab in the States doing this full multi-level analysis of micrometastatic cells. However, a relatively new blood test is available in the US, called CellSearch® Circulating Tumour Cell Test (Tel: 303 933-9785).

This test filters circulating tumor cells from a patient’s blood sample and gives a numerical probability of metastasis for colon, breast, prostate and ovarian adenocarcinomas. The cost of the RGCC test is about €1200 or $1800 USD. Return time is two weeks. Treating a cancer without oncogenetic (cancer gene) analysis is like trying to tell where a ship is coming from when it is thirty miles from the shore, without a telescope.

You know the ship is there, but where it’s actually coming from and whether it’s friendly or hostile are complete guesses. Individual oncogene analysis provides the appropriate quantum of analysis to pinpoint a cancer patient’s specific needs for care. The goal is to gather as much data as possible before therapies are selected. If a patient is treated with a wrong or inadequate therapy, the cancer will grow more aggressive in time. Similarly, trying to annihilate the cancer often annihilates the patient in the process, whereas containing the cancer over time usually allows the patient to live longer and enjoy a good quality of life, in spite of the cancer.

Think about how a bacterial infection is treated. If a doctor treats a systemic infection without first doing a culture and sensitivity test to determine which bacteria is involved and which antibiotics the bacteria is sensitive to, then he might prescribe the wrong treatment and thereby strengthen the infection. It is the same for the treatment and regulation of cancer.

Cancer can be viewed as a genetic organism with specific needs, strengths and weaknesses. It has an archaic metabolism similar to yeast, growing rapidly in low oxygen environments, and making only two ATP energy molecules from a molecule of sugar, then throwing off waste which creates a moat of toxicity and protects the cancer cells from the body’s defenses.

In normal cells in the body, one molecule of glucose sugar is made into 37 ATP energy molecules. This is an oxygen-based, highly efficient process, unlike cancer cell metabolism. Cancer cells thrive where other cells suffer. A tumor is highly inefficient and dies as fast, if not faster then it can grow; therefore it must seed itself in other areas of the body to survive.

It is a stealth-like parasitic organism which, unlike most evolutionary life forms, has evolved to thrive on inefficiency. It is important to understand that cancer is a systemic illness and that a tumor is NOT the cancer but only a manifestation of it.

Cancer is a disease which occurs at the level of the genes. While arguably an environmental illness, it occurs in individuals; as such, individual genetic analysis is a more precise tool to develop successful treatments for our patients.

Today’s “standard of care” for cancer is simply unacceptable, given the advances in biological sciences.

It is all too often a “standard of carelessness.”

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Daniel has been doing onco genetic testing for his patients since 2005 using Dr. Michael Giesing’s TIRNA (Tumor Induced RNA) blood test.* Currently, Daniel uses the RGCC lab test for circulating tumor cells (aka micrometastatic cells). In this test circulating tumor cells are isolated from the patient’s blood then genetically finger printed. The isolated cancer “seeds” are then pharmaco genetically tested for the effectiveness of commonly used chemotherapeutic agents, gene targeted therapies , monoclonal antibodies and tyrosine kinase inhibitors, as well as over 40 nutrient and herbal based natural therapies. This allows the patient and doctors to more objectively determine the potential effectiveness of therapies before they are used and helps reduce creating an even more aggressive tumor expression often caused by using the wrong agents. You may call Daniel at 415 971-3733 to schedule this test. Please write [email protected] to order your test kit.

* see Daniel Dunphy’s Video page for segments of a lecture given by Dr. Giesing regarding his TIRNA test and its efficacy. See the -Micrometastatic circulating tumor cell- page on this website for more on Dr. Giesing’s research.

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And here is a low dose approach to regulating cancer growth which is less likely to destroy the bone marrow and immune system, helps to regulate the caner genome for an individual and allows time to work on targeting immune function as well as detoxification and tonification of organs and adaptive immune system.

THE ANTI-ANGIOGENIC BASIS OF METRONOMIC CHEMOTHERAPY

Robert S. Kerbel 1&Barton A. Kamen 2 about the authors

1. Molecular and Cellular Biology Research, Sunnybrook and Women’s College Health Sciences Centre, S-217, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.
2. Cancer Institute of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08901, USA.

correspondence to: Robert S. Kerbel [email protected]

In addition to proliferating cancer cells and various types of normal cells, such as those of the bone marrow, conventional cytotoxic chemotherapeutics affect the endothelium of the growing tumour vasculature. The anti-angiogenic efficacy of chemotherapy seems to be optimized by administering comparatively low doses of drug on a frequent or continuous schedule, with no extended interruptions — sometimes referred to as ‘metronomic’ chemotherapy. In addition to reduced acute toxicity, the efficacy of metronomic chemotherapy seems to increase when administered in combination with specific anti-angiogenic drugs. Gaining better insight into the mechanisms of these effects could lessen or even eliminate the empiricism used to determine the optimal dose and schedule for metronomic chemotherapy regimens.

For almost half a century, systemic therapy of cancer has been dominated by the use of cytotoxic chemotherapeutics. Most of these drugs are DNA-damaging agents or microtubule inhibitors that are designed to inhibit or kill rapidly dividing cells. They are often administered in single doses or short courses of therapy at the highest doses possible without causing life-threatening levels of toxicity — this is referred to as the ‘maximum tolerated dose’ (MTD). MTD therapy requires prolonged breaks (generally of 2–3 weeks in duration) between successive cycles of therapy. Despite the number of such chemotherapeutics and the huge number of clinical trials that have been undertaken to test them, progress has been modest in terms of curing or significantly prolonging the lives of patients with cancer — particularly those with advanced-stage or metastatic disease 1,2. Moreover, the progress that has been made in treating certain types of malignancy often comes at a high price, given the toxic side effects that are frequently associated with MTD-based chemotherapy. These include acute myelosuppression, hair loss, damage to the intestinal mucosa, nausea and mucositis, as well as the long-term cardiac, renal, neurological and reproductive consequences. Indeed, many of the recent pharmacological advances in oncology treatment involve growth factors and anti-nausea drugs, which are administered to patients with cancer to minimize the severity of, or accelerate recovery from, chemotherapy-induced toxicities. Such ‘supportive-care drugs’ can significantly add to the financial burden of cancer chemotherapy, and have their own side effects.

A reappraisal of the best ways of administering chemotherapy is underway. Instead of only using short bursts of toxic MTD chemotherapy interspersed with long breaks to allow recovery from the harmful side effects, there is now a shift in thinking towards the view that more compressed or accelerated schedules of drug administration using much smaller individual doses than the MTD would be more effective — not only in terms of reducing certain toxicities, but perhaps even improving antitumour effects as well 3-6 . Moreover, some of these dosing/scheduling strategies are ideally suited to combining chemotherapeutics with many of the new targeted and relatively non-toxic anticancer drugs that have been or are being developed. The most recent refinement of this concept is called ‘metronomic’ chemotherapy 3, which refers to the frequent, even daily, administration of chemotherapeutics at doses significantly below the MTD, with no prolonged drug-free breaks.

Metronomic therapy

There are many different factors that have contributed to the line of reasoning that for chemotherapy, ‘the more frequent the better’ and that ‘less is more’. First, the opposite approach — using ‘high-dose’ chemotherapy with autologous bone-marrow stem-cell transplants (to replace the destroyed bone-marrow-derived stem cells) — has not provided the kind of survival benefits expected, at least when this treatment strategy is used for patients with metastatic breast cancer 7,8. This approach is also very expensive and highly toxic. Furthermore, ‘dose-dense’ chemotherapy, in which one or more chemotherapeutic is administered at more frequent intervals (that is, every other week), has shown clear benefits in randomized Phase III clinical trials 9-11 . This strategy is usually designed to administer at least the same amount or, more commonly, even a greater amount of drug in total over time. 9

So, if every other week is better than every 3 weeks, then why not administer weekly or even daily treatment? Indeed, it is becoming more common to administer taxane drugs to patients with certain types of cancer, such as breast cancer , on a weekly schedule 12-15 . Such dose density — which is allowable because of exogenously administered supportive-care growth factors, antibiotics and transfusion medicine — seems in some respects to be conforming to the metronomic-therapy theme, as discussed below.

METRONOMIC CHEMOTHERAPY can be viewed as a variation of dose-dense therapy with the exception that the cumulative dose with metronomic therapy might be significantly less than with MTD-based chemotherapy 15 ,16 . As metronomic therapy reduces the level of toxicity, it lessens or even removes the need for growth-factor support to accelerate recovery from myelosuppression. Moreover, despite lower cumulative doses of drug administration, the antitumour effects of this approach, in terms of prolonging survival times, might actually be superior to conventional MTD regimens, especially in some preclinical models 17-19 . Support for metronomic therapy also comes from mathematical modelling studies 20 ,21 . Unlike dose-dense chemotherapy, the main targets of which are presumed to be proliferating tumour cells, the main targets of frequent or continuous metronomic chemotherapy are the endothelial cells of the growing vasculature of a tumour 22 . In essence, chemotherapeutics are used as anti-angiogenic agents, therefore “redefining the target of chemotherapy”, to cite Miller et al .23 . This is also the reason that Browder et al .22 coined the term ‘anti-angiogenic chemotherapy’ to describe this treatment strategy.

Another advantage of metronomic chemotherapy is the possibility of combining it with anti-angiogenic drugs, as well as other types of targeted therapies — such those that target specific signal-transduction molecules — or with antitumour vaccines. It is ironic that targeted therapies were originally designed with the goal of replacing chemotherapy, to reduce the serious morbidities associated with standard MTD or high-dose chemotherapy. However, although they are less toxic, most of these rationally designed drugs were found to have very modest efficacy, at least when used as single agents in treating patients with advanced disease. They have therefore mainly been used in combination with standard chemotherapy or radiation protocols. An example of this is bevacizumab (Avastin) — a humanized monoclonal antibody against vascular endothelial cell growth factor ( VEGF ) — which is used in combination with 5-fluorouracil (5-FU)/leucovorin/irinotecan for the treatment of metastatic colorectal cancer 24 ,25 . Another example is trastazumab (Herceptin) — a humanized monoclonal antibody against the ERBB2 oncoprotein — which is combined with an alkylating agent or paclitaxel for the treatment of metastatic breast cancer 26 .

One of the proposed benefits of targeted therapies was reduced toxicity and improved quality of life. When these drugs are combined with MTDs of chemotherapy, however, these benefits are not realized. As it is likely that chemotherapy will continue to be the mainstay for systemic cancer therapy for many years to come, designing more effective ways of administering and combining such drugs with the newest generation of molecularly targeted drugs will become increasingly crucial.

Anti-angiogenic effects

Chemotherapeutics do not specifically target tumour cells, but rather interfere with cell division, such as by inhibiting enzymes involved DNA replication or metabolism (for example, topoisomerases and thymidylate synthase), or microtubules. These drugs therefore also damage the normal dividing cells of rapidly regenerating tissues, such as those of the bone marrow and gut mucosa, and hair-follicle cells. Host toxicity is therefore often only marginally less than antitumour efficacy, so creating a narrow therapeutic index.

But perhaps there is a silver lining in this otherwise dark cloud, in that dividing endothelial cells are present in the growing blood vessels that are found in tumours 27 and, like other normal dividing cells, should be susceptible to chemotherapeutics 28 . Elimination of these dividing endothelial cells, or inhibition of their division, would presumably lead to an anti-angiogenic effect. Moreover, as host vascular endothelial cells are assumed to be genetically stable and lack the diverse genetic defects characteristic of cancer cells that lead to drug resistance, the putative effects of chemotherapy might be more durable in the face of continued therapy. By way of example, successive cycles of MTD-based chemotherapy can cause myelosuppression each time, the extent of which does not change appreciably 28 . If normal bone-marrow-cell progenitors acquired resistance to chemotherapy in the same way that genetically unstable, highly mutable cancer cells do, myelosuppression would gradually decline and disappear. So, the cancer cells that are resistant to a particular chemotherapeutic agent might indirectly respond to that same drug through a ‘side effect’ — loss of or damage to its associated vasculature, as first proposed in 1991 (Ref. 29 ). Literature dating back to the mid-1980s shows that virtually every class of chemotherapeutic has anti-angiogenic effects or antivascular effects in various in vitro and in vivo assays 23 .

Many tumours, however, are intrinsically drug resistant or rapidly acquire resistance after showing initial responsiveness to chemotherapy regimens. So it would seem that chemotherapy has minimal or negligible anti-angiogenic effects. Why is this? Perhaps the proportion of dividing endothelial cells in tumour-associated blood vessels is simply too low for chemotherapy to have a significant therapeutic impact. Alternatively, the endothelial cells might be protected from chemotherapy-induced cell death by high local concentrations of endothelial-cell survival factors such as VEGF, basic fibroblast growth factor ( bFGF ) and angiopoietin 1 (Refs 30 ,31 ). A third explanation, uncovered in a pioneering study from Judah Folkman’s laboratory 22 , is that the anti-angiogenic effects of chemotherapy are both masked and marginalized by the way chemotherapy is usually administered. In this case, the long breaks between drug administration that are necessary to allow the patient to recover from the harmful side effects of the MTD chemotherapy, especially from myelosuppression, reduce the anti-angiogenic effects of the drugs.

Timothy Browder and colleagues evaluated the anti-angiogenic and antitumour effects of the alkylating agent cyclophosphamide in immune-competent syngeneic mice that had been injected subcutaneously with various tumour types 22 . They found that this drug, when administered at the MTD, caused apoptosis of endothelial cells in the newly formed tumour microvessels 22 . A detailed temporal analysis showed that the endothelial cells were the first in the tumour to undergo apoptosis 22 . This anti-angiogenic effect did not, however, translate into a significant therapeutic benefit, apparently because the damage to the vasculature of the tumour was largely repaired during the long (2–3-week) rest/recovery periods between successive cycles of MTD-based therapy.

It was therefore proposed that if cyclophosphamide was given more frequently ( Fig. 1 ), such as once or more per week with no extended breaks, there would be significantly less opportunity for repair of the damaged endothelium and the anti-angiogenic effects of the chemotherapy would irreversibly accumulate. This, of course, necessitates lowering the dose of the drug administered with each injection. Browder et al . showed that this more frequent, regular, lower-dose therapy, which was administered at one-third of the MTD, had impressive anti-angiogenic and antitumour effects when tested on several mouse tumour cell lines grown subcutaneously in syngeneic mice 22 . This approach allowed even very large established subcutaneous tumours, previously selected in vivo for acquired cyclophosphamide resistance using a conventional MTD regimen, to respond to the same drug and almost completely regress. In short, a state of acquired drug resistance could be reversed simply by apparently shifting the focus of the treatment away from the drug-resistant cancer-cell population to the drug-sensitive tumour endothelium 3,22 .

Figure 1 | Different therapeutic regimens.

Metronomic chemotherapy regimens differ from the standard maximum tolerated dose (MTD) chemotherapy regimens that have been common practice in medical oncology for decades. a| In standard chemotherapy, a drug is typically given in a single bolus injection or infusion at the MTD, interspersed by a long break — for example, 3 weeks — before the next course of this therapy is administered. Doses that exceed the MTD (‘high-dose’ chemotherapy) must be accompanied by an autologous bone-marrow stem-cell transplant and supportive-care growth-factor drugs to prevent lethal bone-marrow failure. In band c, examples of metronomic chemotherapy regimens are shown where, for example, the chemotherapy drug is administered more frequently, such as weekly ( b) or daily ( c), with no prolonged drug-free interruptions. Drugs that can be administered orally, such as cyclophosphamide, capecitabine, etoposide (VP-16), UFT (uracil plus tegafur, a fluoropyrimidine antimetabolite), would be ideal for prolonged daily administration schedules. Omission of prolonged drug-free periods is a key aspect of the basis for the anti-angiogenic effects of low-dose metronomic chemotherapy regimens, as these breaks allow repair and recovery from the anti-angiogenic effects of chemotherapy drugs on developing tumour blood vessels.

These results have been confirmed by others 32 ,33 and have also been modified with daily oral administration of the drug through drinking water, which seems to be less toxic than the weekly regimen 19 ,34 . Indeed, a recent detailed analysis showed that long-term daily low-dose cyclophosphamide therapy did not cause significant toxicity to tissues or cells normally affected by MTD regimens of the same drug 35 ; lymphopaenia was the only toxic side effect noted 35 .

Clinical precedents for metronomic therapy

In retrospect, these preclinical results actually have many intriguing clinical precedents 4,36 . For example, 40% of patients with non-small-cell lung cancer (NSCLC) who showed no response to standard doses of intravenous etoposide administered intermittently did respond — that is, their tumours shrank by 50% of more in volume — to the same drug when it was given orally at a much lower dose using a much more frequent basis (every day or every other day), with only a 1-week break every month 37 . Similar results have been shown in patients who have been given other drugs, such as microtubule-inhibiting taxanes, for treatment of advanced metastatic breast or ovarian cancer . In these patients, weekly regimens of drug administration are being increasingly adopted, often using only 30–40% of the MTD given once every 3 weeks 38 . In women who had stopped responding to the MTD of paclitaxel or docetaxel given once every 3 weeks, tumours were found to respond in a high proportion of cases to a regimen of approximately 30–40% the MTD once every week 13 ,36 ,38-40 . However, for the most part, these are not standard-of-care regimens and their benefits remain to be validated in randomized prospective Phase III clinical trials.

Metronomic therapy is also similar in many ways to the various long-term ‘maintenance’ chemotherapy regimens 41-45 that are used to treat children with certain types of cancer, such as acute lymphoblastic leukaemia . Maintenance therapy in this case involves the administration of low doses of oral methotrexate on a weekly basis and 6-mercaptopurine on a daily basis for up to 3 years. This treatment follows short-term remission-inducing chemotherapy using standard regimens and higher doses of various chemotherapeutic drugs 42 ,45 . Several studies have indicated that the drugs used in this type of maintenance therapy have anti-angiogenic effects 46-48 . In the case of methotrexate, low doses have been shown to cause anti-endothelial/anti-angiogenic effects in in vitro and in vivo assays 46 ,47 . Furthermore, the maintenance therapy used to treat patients with acute lymphoblastic leukaemia has been shown to have anti-angiogenic effects in the bone marrow, reducing the number of blood vessels in this tissue compartment 49-51 . The success of following standard MTD ‘remission-induction’ chemotherapy with long-term metronomic therapy regimens highlights the possibility that these two types of dosing regimens are not mutually exclusive, but can be used sequentially in a beneficial and harmonious manner. This approach should also be considered for adults, especially when combined with a cytostatic agent for long-term therapy.

Combination with anti-angiogenic drugs

Clinical trials are underway to determine whether metronomic chemotherapy can prolong survival when compared with standard MTD regimens in patients with various cancers, including advanced prostate and ovarian carcinomas, as well as certain types of haematological malignancies 6,49-52 . Relapses, however, will undoubtedly occur in most patients who initially show some benefit from metronomic therapy 49 . It was partly for this reason that the metronomic-chemotherapy protocol of Browder et al . has been modified and combined with endothelial-cell-specific angiogenesis inhibitors such as anti-VEGF receptor 2 ( VEGFR2 , also known as KDR or FLK1) antibodies or TNP-470, a fumigillin analogue 22 ,53 . This combination approach might improve efficacy without significantly increasing host toxicity 16 .

The rationale for this strategy was based on several considerations. VEGF-receptor tyrosine kinases are expressed preferentially by endothelial cells of the growing neovasculature of a tumour, and VEGF is a key survival (anti-apoptotic) factor for the endothelial cells of newly formed vessels 54 ,55 . There are several signalling pathways and molecular effector mechanisms by which VEGF can inhibit apoptosis in endothelial cells 31 ,56-60 . For example, signalling through VEGFR2 can activate the phosphatidylinositol 3-kinase (PI3K)–AKT pro-survival signalling pathway. This or other pathways lead to subsequent upregulation of several anti-apoptotic effectors, including BCL2, A1, XIAP and survivin 31 .

There is evidence that the anti-proliferative 30 or pro-apoptotic actions of pacitaxel 31 , vinblastine, cisplatinum and adriamycin 31 , as well as of several other types of cytotoxic substances, on human endothelial cells in culture are suppressed by the presence of VEGF 30 ,31 . High local concentrations of VEGF in the tumour microenvironment might therefore induce or promote multidrug resistance, by inducing a highly specific chemoprotective effect towards the VEGFR2-positive endothelial cells of the tumour 30 ,31 ,61 . Chemotherapy itself might also induce or upregulate the expression of VEGF and other endothelial-cell pro-survival growth factors in tumour cells 62 . So, the combination of a chemotherapeutic agent with a drug that blocks VEGF or its receptor (VEGFR2) should selectively amplify the pro-apoptotic effects of the chemotherapeutic against activated endothelial cells, but presumably not against other types of dividing normal cells 16 . This would improve the therapeutic index.

Previous studies have shown that anti-angiogenic drugs can improve the effects of some standard chemotherapy regimens 63 ,64 , and these findings have been validated in the clinic 24 ,25 . For example, in a large, randomized, placebo-controlled Phase III clinical trial, the combination of a standard, approved chemotherapy regimen for metastatic colorectal cancer — consisting of 5-FU/leucovorin and irinotecan — with bevacizumab (the anti-VEGF antibody), caused a statistically significant prolongation of survival, compared with patients treated with only the chemotherapy regimen 25 . So, one might anticipate that anti-angiogenic drugs should also improve the efficacy of continuous low-dose chemotherapy regimens, for which the side effects would be much more tolerable, and that the two types of drug could be administered together for long time periods.

Experiments were undertaken to evaluate this combination treatment concept using xenograft models of neuroblastoma 16 ,melanoma , breast, prostate and colon cancers 19 ,65 . In one of these studies, a very low dose of vinblastine was administered twice weekly — which was about 1/10–1/20 the MTD for mice and therefore represents low-dose chemotherapy 66 ,67 — in combination with an anti-VEGFR2 blocking monoclonal antibody called DC101 (Ref. 70 ). This combination caused complete and sustained regression of large, established — but localized — neuroblastoma xenografts in severe combined immunodeficient (SCID) mice. The metronomic vinblastine treatment was preceded by a 3-week remission-induction schedule of higher cumulative doses of the drug 16 to reduce the large tumour burden. This combination treatment, in which the two different drugs were given twice a week with no breaks, could be maintained for these exceptionally long periods (7 months) because they were not toxic to the mice 16 . By contrast, treatment with either the vinblastine regimen or DC101 alone, although non-toxic, resulted in significant but short delays in the growth of the tumour in the growth followed by relapse, and the animals only survived 1–2 months after initiation of treatment.

Were the low-dose chemotherapy regimens used in these studies anti-angiogenic and, if so, was this was the only antitumour mechanism? To address this question, a quantitative in vivo blood-vessel PERFUSION ASSAY showed that the low-dose vinblastine protocol itself could significantly inhibit angiogenesis 16 . Similarly, Browder et al . tested the weekly low-dose cyclophosphamide regimen using a CORNEAL MICROPOCKET ASSAY to show that this metronomic therapy protocol inhibited angiogenesis 22 .

Other studies involved 65 human breast cancer cell lines that had been previously selected for resistance to agents such as paclitaxel, doxorubicin and vinblastine, as a result of overexpression of the multidrug resistance 1 ( MDR1 ) gene, which encodes the P-glycoprotein drug-efflux pump 65 . In some cases, these cells were resistant to 50–100-fold higher concentrations of drugs than normal cells 65 . In xenograft studies, the combination of DC101 plus metronomic chemotherapy slowed tumour growth, whereas DC101 treatment or metronomic chemotherapy alone resulted in only temporary tumour responses — or even no apparent primary tumour responses, as measured by decreases in tumour volume. Cancer-cell-specific effects of drugs are not sufficient to overcome this level of drug resistance, so some alternative mechanism, such as anti-angiogenesis, must be involved 65 . Furthermore, Browder et al . administered a combination of the angiogenesis inhibitor TNP470 with weekly doses of cyclophosphamide to treat large, established transplanted mouse tumours that were previously selected for resistance to cyclophosphamide. They found that the combined treatment could gradually cause marked and sustained regressions, if not complete disappearance of such tumours 22 .

The results of Browder et al . and Klement et al . have now been confirmed by many different groups using various empirical continuous or frequent low-dose chemotherapy regimens. These regimens included many different chemotherapeutic drugs, as well as several different anti-angiogenic agents 32 ,68-73 . A summary of the drugs, drug combinations and tumour models studied is shown in Table 1 (Refs 18 ,19 ,73 ,74 ). In some of these studies, an empirical metronomic dosing schedule was compared head-to-head with the respective MTD regimen of the same chemotherapeutic drug 17 ,19 ,20 ,71 ,72 . In all cases, the MTD regimens were found to be inferior to the metronomic treatments in terms of either toxicity or survival, or both. This also held for situations in which an anti-angiogenic drug was added to the MTD, compared with respective metronomic chemotherapy regimens 17 ,19 ,20 .

Table 1 | Preclinical examples of antitumour efficacy of metronomic chemotherapy

The number of such studies, however, is limited and these findings need to be confirmed using different chemotherapeutic agents. Moreover, as discussed above, there might be additional benefits to using standard MTD chemotherapy followed by a subsequent long-term metronomic regimen — especially when treating exceptionally large tumours that are known to be responsive to certain chemotherapy drugs administered in the MTD fashion. This approach was used in preclinical studies by Klement et al .16 and Bocci et al .34 . Whereas some of the preclinical studies reported exceptionally long-term tumour responses, and in some cases mice were even cured 16 ,22 , most mice eventually relapsed 75 ,76 . This indicates that some forms of acquired resistance occur — either at the host level (such as through altered drug metabolism), the tumour-cell level, (such as through selection for mutant tumour cells that can survive under the hypoxic conditions created by inhibition of angiogenesis) 75 or at the level of the endothelial cells or blood vessels (such as vascular remodelling into more mature vessels that are less responsive to anti-angiogenic treatment) 76 .

Anti-angiogenic mechanisms

Much evidence, mostly in vitro , indicates that the ‘activated’ endothelial cells of newly forming blood-vessel capillaries are highly and selectively sensitive to very low doses of various chemotherapeutic drugs 47 ,77-81 . For example, several studies have been undertaken to test the antiproliferative, migration-inhibitory and sometimes cytotoxic effects of picomolar concentrations of chemotherapeutic drugs on various human cell types, including fibroblasts, lymphocytes, tumour cells, epithelial cells from various tissues, and microvascular or macrovascular endothelial cells. A summary of some of these studies is shown in Table 2 . Some of the most interesting studies involve various microtubule inhibitors, such as vinblastine, paclitaxel and docetaxel. In these experiments, ultra-low concentrations of these drugs were reported to inhibit proliferation or migration of endothelial cells, but not of other cell types examined. For example, Wang et al . reported that 10–100,000-fold higher concentrations of paclitaxel were required to inhibit proliferation and migration of human astrocytes, fibroblasts, mammary epithelial cells, keratinocytes, prostate epithelial cells or smooth-muscle cells, compared with epithelial cells 80 . Taxanes, however, must be formulated in certain vehicles for injection, to prevent their binding to serum proteins. Clinically relevant concentrations of these vehicles or binding proteins can significantly dampen the anti-angiogenic activity of taxanes, meaning that higher doses of such injectable taxanes would have to be used in vivo to induce anti-angiogenic effects 82 .

Table 2 | Sensitivity of human vascular endothelial cells to metronomic therapy

Most of these studies reported no cytotoxic effects, although we have found that if endothelial cells are continuously exposed to a low concentration of drug such as paclitaxel over a 6 day period (replicating metronomic therapy), endothelial cells, but not dermal fibroblasts or tumour cells, undergo apoptosis within about 5 days 78 . This delay in cytotoxicity indicates that the pro-apoptotic effects of low-dose metronomic chemotherapy on endothelial cells might not be direct, but could instead be a secondary result of some other process that is specific to the vascular endothelial cell. This concept is illustrated in Fig. 2 .

Figure 2 | Possible mechanisms of the anti-angiogenic basis of metronomic chemotherapy.

There are two routes by which metronomic chemotherapy could lead to growth arrest or apoptosis of endothelial cells in the tumour neovasculature. A ‘direct’ pathway (left) assumes that activated, differentiated endothelial cells are intrinsically sensitive to low-dose chemotherapy, for which there is some evidence 80-85 ; the same might be true for circulating endothelial progenitor cells 17 . The ‘indirect’ pathway (right) assumes that the levels of metronomically administered drugs are too low to induce growth arrest or apoptosis of endothelial cells. Instead, an endogenous inhibitor of angiogenesis, such as thrombospondin 1, is induced in certain cells by low-dose chemotherapy. This inhibits tumour angiogenesis and vasculogenesis, leading to a reduction in tumour neovascularization in the absence of side effects such as myelosuppression, hair loss, and nausea or vomiting.

Two recent studies implicate thrombospondin 1 ( TSP1 ) as a potential mediator of the effects of metronomic cyclophosphamide 33 ,34 . In one study, 5 days of exposure to low concentrations of various chemotherapeutic drugs caused a marked increase in TSP1 mRNA and protein levels in vascular endothelial cells in vitro (other cells were not tested). TSP1, a component of the extracellular matrix that can also be secreted and found in the circulation, is a well known endogenous inhibitor of angiogenesis 83 ,84 . It seems to act primarily by binding to CD36 receptors, which are expressed by endothelial cells 85 . This interaction blocks proliferation and induces apoptosis in endothelial cells 86 ,87 , but would not be expected to occur in CD36-negative cells, such as most bone-marrow-derived haematopoietic stem cells or hair-follicle cells. TSP1 can also bind and sequester VEGF, and therefore block its pro-angiogenic activity 88 .

Further evidence to implicate TSP1 as a secondary mediator of the anti-angiogenic properties of metronomic chemotherapy was obtained in experiments that compared the anti-angiogenic and antitumour effects of MTD cyclophosphamide with a continuous daily low-dose regimen of the same drug in wild-type or Tsp1 -null mice 34 . In these experiments, the drug was administered continuously in the drinking water 19 . The antitumour efficacy (tested on subcutaneously transplanted Lewis lung carcinoma tumours) and anti-angiogenic effects were lost in the Tsp1 -null mice, but not in the wild-type controls 34 ,78 . Raghu Kalluri and collaborators have also shown that weekly administration of low doses of cyclophosphamide leads to loss of the antitumour activity of this drug against the B16 mouse melanoma grown in Tsp1-deficient mice 33 . By contrast, the chemotherapy regimen retained its efficacy in mice that were unable to produce either endostatin or tumstatin — two other endogenous inhibitors of angiogenesis 33 . It therefore seems that these molecules are not involved in the anti-angiogenic effects of metronomic treatment with cylophosphamide. TSP1, however, is induced in the melanoma cells and infiltrating host (stromal) cells of the treated tumours 33 .

So, metronomic chemotherapy might not necessarily act directly on endothelial cells, but might instead act by inducing endothelial-cell-specific inhibitors, such as TSP1. This could explain why metronomic chemotherapy regimens do not increase the usual harmful side effects of chemotherapy, such as myelosuppression, despite the elimination or shortening of long, drug-free break periods. It is also interesting to note that other anticancer drugs that have anti-angiogenic ‘side effects’ could also work by a similar mechanism. For example, in 1997 trastazumab, a monoclonal antibody against ERBB2, was implicated to have anti-angiogenic properties 89 , as it inhibits VEGF expression. Furthermore, it can induce TSP1 in tumour cells 90 .

As previously discussed, one crucial aspect and advantage of metronomic therapy is that it prevents the repair to the tumour vasculature that occurs between sessions of MTD therapy. What is the basis of this apparently robust and highly specific repair process? Part of the answer might lie in the effects of metronomic chemotherapy regimens on the mobilization, levels and viability of bone-marrow-derived circulating endothelial progenitor cells (CEPs). These cells contribute to some forms of angiogenesis, such as development of the vasculature in early embryonic development, as well as tumour angiogenesis, essentially constituting a form of ‘systemic’ vasculogenesis and angiogenesis, in contrast to the local division of differentiated endothelial cells in pre-existing vessels. Until 1997, it was thought that all new endothelial cells were derived through the latter process 91 , but there are reports that claim that up to 50% of the endothelial cells in newly forming blood vessels come from CEPs 92 . These cells can be mobilized from the bone marrow by growth factors such as VEGF, and then enter the peripheral-blood circulation. There, they can migrate to sites of ongoing angiogenesis and differentiate into mature endothelial cells 20 ,91 ,93 .

Bertolini et al .17 showed that in immune-deficient mice that were previously injected subcutaneously with human lymphoma cells, the increased levels of CEPs detected in the blood circulation of the mice sharply declined shortly after the mice were treated with a cycle of MTD cyclophosphamide. The number of these cells quickly and sharply rebounded during the drug-free break period, presumably as the result of a compensatory haematopoiesis-like effect 17 . By contrast, when cyclophosphamide was administered at lower doses on a weekly basis or continuously in drinking water, the numbers of CEPs gradually declined, as did their viability, and no compensatory rebound was observed 17 . If CEPs make a significant contribution to tumour angiogenesis — and this remains a point of continuing debate 94 ,95 — the ‘rebound’ of these cells during the long break periods after MTD chemotherapy could contribute, at least in part, to the repair process that occurs in the damaged tumour endothelium. This would explain the inability of MTD chemotherapy to inhibit tumour angiogenesis in a sustained, and therefore therapeutically effective, manner.

These studies call into question the use of growth factors as supportive measures to accelerate recovery from the myelosuppression-inducing effects of high-dose, standard MTD or dose-dense chemotherapy regimens. For example, both erythropoietin and granulocyte colony-stimulating factor ( G-CSF ) are often given to patients to help them recover from anaemia and myelosuppression, respectively, which are induced by MTD chemotherapy regimens. This is because they promote the mobilization of marrow progenitor cells into the peripheral circulation, where they can differentiate into mature white blood cells such as neutrophils or red blood cells, and have been shown to increase the mobilization of CEPs 96 ,97 . This, in turn, can stimulate vasculogenesis/angiogenesis, leading to tumour growth 98 ,99 , and could provide one explanation for the fact that treatment of patients with recombinant erythropoietin after standard chemotherapy is associated with a worse outcome, in terms of survival, in some clinical trials 100 .

Clinical trials of metronomic chemotherapy

Phase II clinical trials have been initiated to test the possible benefits of metronomic chemotherapy regimens — particularly when these are combined with an anti-angiogenic drug. Several of these trials are summarized in Table 3 . Most of these involve chemotherapy regimens in which cyclophosphamide is administered orally on a daily basis, sometimes for up to 2 years, with no break periods. In some cases, oral low-dose methotrexate is also given on two consecutive days on a weekly basis. The targeted drugs that are used include a cyclooxygenase-2 ( COX2 )-specific inhibitor such as celecoxib, which is administered on a daily basis, or a humanized anti-VEGF monoclonal antibody (such as bevacizumab), which is administered intravenously every 2 weeks. Celecoxib was selected for inclusion in the trial because of its commercial availability, ease of administration, excellent side-effect profile and putative anti-angiogenic effects 101 ,102 .

Table 3 | Clinical trials involving metronomic chemotherapy

The combination of cyclophosphamide and methotrexate has already been tested in a clinical trial in Italy, and has spurred additional trials that are underway 49 . Sixty-four women with progressive, advanced and refractory breast cancer received low doses of oral cyclophosphamide on a daily basis and oral methotrexate was given twice per week. Most of the patients had progressive metastatic disease when the trial began and had also previously received first-, second- or third-line treatments. An overall response rate of 32% was observed, which included two complete responders, 10 partial responders and 12 patients with stable disease lasting 6 months or longer 49 . No high-grade adverse events were reported, despite the fact that many patients had previously been treated with chemotherapy. This compares favorably with the standard third-line chemotherapy regimens used in this treatment setting, at least in terms of toxicity. The estimated cost of this outpatient therapy was about US$10 per month 49 .

In another recently reported trial, Glode et al . treated 32 patients with advanced androgen-independent metastatic prostate cancer. These patients received daily oral doses of cyclophosphamide and dexamethasone 52 . Dexamethasone, in addition to other properties, has been reported to have some anti-angiogenic effects 52 . In this small study, almost 70% of the patients showed a decrease in serum prostate-specific antigen levels of 50% or more. Although such preliminary results are encouraging, they need to be confirmed in much larger, controlled and prospective clinical trials 103 .

It is also very difficult to determine conclusively whether these therapies have anti-angiogenic effects that contribute to their putative antitumour efficacy. In this regard, Colleoni et al . reported that serum VEGF levels declined in patients who responded to therapy 49 . Bertolini et al . reported a reduced number of bone-marrow-derived CEPs in the blood of patients with lymphoma and breast cancer who received daily low-dose cyclophosphamide therapy 104 , similar to that observed in patients with rectal carcinoma who are treated with the anti-VEGF monoclonal antibody bevacizumab 105 . Nevertheless, the therapies obviously have other effects that contribute to their efficacy — this is especially true for drugs such as celecoxib and dexamethasone. For example, COX2, which is inhibited by celecoxib, can be expressed by tumour cells as well as by activated endothelial cells 101 . It is also known that low-dose chemotherapy, can stimulate the immune system in some cases, 106 ,107 , making it a potentially useful addition in combination with tumour vaccines or other types of immune-therapy approaches. Indeed, some studies indicate that metronomic chemotherapy using cyclophosphamide can increase the efficacy of immunotherapeutic vaccines in preclinical models 108 ,109 .

Metronomic therapy might also have some direct effects on tumour cells, such as induction of cell differentiation, although there is not yet any evidence for this. Continuous-chemotherapy regimens could also prevent a rebound in tumour-cell division known as ‘repopulation’, which can take place during the rest periods between cycles of MTD chemotherapy. Repopulation kinetics can become increasingly more aggressive with successive cycles of MTD pulsatile chemotherapy 110 , so shortening or eliminating the drug-free break periods might prevent this ‘kinetic drug resistance’.

Several successful paediatric chemotherapy approaches resemble metronomic therapy, as discussed above, in that they involve daily administration of low doses of cytotoxic drugs over prolonged periods of time, as so-called ‘maintenance’ therapies, and have minimal toxicity ( Table 4 ). Paediatric oncologists have shown that daily adminstration of cyclophosphamide along with weekly administration of Vinca alkaloid drugs, such as vincristine or vinblastine, is effective in treating patients with diseases such as neuroblastoma 111 . Weekly administration of Vinca alkaloids is also a key component of therapy for patients with Wilms’ tumour and rhabdomyosarcoma . Paediatric ‘CHOP’ (cyclophosphamide, adriamycicin, vincristine and prednisone) therapy, which is used to treat patients with non-Hodgkin’s lymphoma , is also conceptually similar to metronomic therapy. Aggressive fibromatosis can also be controlled with low-dose vinblastine and methotrexate treatment 112 . Other low-dose chemotherapy regimens that are being tested in children, with some hints of success, include low-dose cyclophosphamide treatment and low-dose vincristine therapy to treat infants with localized, unresectable neuroblastoma 111 .

Table 4 | Metronomic chemotherapy in paediatric oncology

The oral fluoropyrimidine 113 agent, UFT (uracil plus tegafur) — a prodrug mixture that is metabolized to 5-FU and two other metabolites, -butyrolactone (GBL) and -hydroxybutyric acid (GHB) — represents another chemotherapeutic that might be applied metronomically 74 ,111 ,114 . This drug is approved for treatment of certain cancers in Japan and throughout Europe, but not in the United States 115 . Recent randomized Phase III ADJUVANT clinical trials have been undertaken in patients with resected early-stage NSCLC of the adenocarcinoma variety. These patients were given daily low doses of oral UFT for 2 years, resulting in both a survival benefit and very little toxicity, despite the chronic nature of the treatment 116 . 5-FU, GBL and GHB have all been shown to have anti-angiogenic activity in an in vivo assay 74 ,117 , as well as to inhibit growth and migration of endothelial cells in culture 74 ,117 — especially when they are administered continuously at low doses 74 . Could the anti-angiogenic effects of this drug contribute to its clinical efficacy? Could this efficacy be increased by combination therapy with a targeted anti-angiogenic drug? Further studies are necessary to answer these questions. The trial of UFT in patients with lung cancer, however, shows the potential of oral long-term metronomic chemotherapy as an adjuvant therapy to treat patients who could have microscopic, early-stage recurring tumours.

Further experiments are required to determine whether other chemotherapeutics that can be orally administered on daily schedules for extended periods of time, such as the topoisomerase enzyme inhibitor etoposide (VP-16) or the alkylating agent temozolamide 118 , also have anti-angiogenic effects 119 that contribute to their antitumour efficacy. There are several other situations in which prolonged oral administration (4–5 weeks) of relatively low doses of chemotherapeutics is already in use, such as administration of etoposide 37 ,120 , razoxane 121 or temozolamide 122 ,123 to treat malignant melanoma and non-Hodgkin’s lymphoma. These are palliative-like regimens that are less toxic and are sometimes used to treat elderly patients, who are less able to cope with toxic MTD chemotherapy. It will be important to determine if anti-angiogenic effects contribute to the efficacy of such protocols — there is already some preliminary evidence that this is the case 119 ,121 . These studies could also accelerate the development of oral taxanes and other microtubule inhibitors for metronomic therapy 124 . Another approach involves the use of injectable chemotherapeutics that are incorporated into endothelial-targeted liposomes to increase the half-life of the drug in the circulation 125 .

Other types of metronomic therapy

The metronomic approach is not only used with chemotherapy, but also with radiation therapy. Sometimes, radiotherapy is administered at lower than normal doses, known as ‘hyperfractionated radiation’ 92 . In this regard, Garcia-Barros et al . have shown that the antitumour effects of irradiation can be mediated through a primary event that involves damage or destruction of the neovasculature of a tumour, followed by the death of tumour cells that surround the affected vessels 92 . This could help to explain the increased therapeutic efficacy of combining radiation therapy with endothelial-cell-specific anti-angiogenic drugs such as angiostatin or anti-VEGF antibodies 126 ,127 .

The cytokine intereferon- (IFN- ) also has anti-angiogenic effects, and is used effectively to treat paediatric patients with haemangiomas or giant-cell tumours when administered in small daily doses over prolonged periods of time 128 ,129 . This might be considered to be another example of the efficacy of anti-angiogenic metronomic therapy. Preclinical studies from Isiah Fidler’s group have also shown that metronomic administration of IFN- therapy is significantly more effective as an anti-angiogenic and antitumour treatment strategy in mouse xenograft models of cancer 130 . They have shown that daily administration of 10,000 units of IFN- was more effective than 70,000 units administered once a week as an anticancer therapy 130 .

Future directions

Although we have explained the potential benefits of metronomic chemotherapy regimens — especially when used in a combination-therapy context with targeted anti-angiogenic agents — there are several significant challenges that must be overcome to increase the chances of success in the clinic. Foremost among these is the current empiricism associated with trying to determine the optimal dose and schedule for administration of chemotherapeutics. Other medical subspecialties have defined minimally inhibitory concentrations, such as those used to treat patients with infectious diseases or epilepsy.

It seems that there are two main approaches to administering chemotherapy. Perhaps a useful analogy is to compare these approaches to radio airwaves. ‘Amplitude modulation’ (AM) involves increasing the dose, but this requires increasing the time between the doses, whereas ‘frequency modulation’ (FM), involves decreasing the amplitude (the unit dose/time). There are some obvious advantages to FM, such as reduced acute toxicity and the ability to combine the drug with targeted therapeutics for prolonged periods. The challenge therefore is to find the smallest dose that will control the target cells and then the most frequent dosing that will maximize this control. This type of problem is obviously not unique, as many molecularly targeted drugs do not produce their maximum therapeutic effects at the MTD, and some do not even have dose-limiting toxicities 131 ,132 . Similarly, determining the biological activity of such agents in the absence of acute tumour regression can be difficult.

These problems might eventually be overcome through the discovery and application of novel molecular or functional surrogate markers, to guide dose selection and to monitor antitumour activity. The same could be the case for metronomic chemotherapy. So, detecting changes in levels of circulating TSP1 levels in serum or plasma after administration of various low doses of chemotherapeutics might be useful in determining the optimal low dose for a drug such as cyclophosphamide 34 . Another possibility is the application of functional imaging approaches, such as those used to detect changes in tumour blood flow. For example, Walter Wolf and colleagues recently reported that administration of docetaxel on either a once-every-week schedule, or a once-every-3-weeks schedule, resulted in reductions in tumour blood flow in several patients with breast cancer, as determined by dynamic-contrast magnetic-resonance imaging. Moreover, there seemed to be a strong correlation between this change and tumour response 133 . This is strikingly similar to the results of a clinical study involving administration of an anti-angiogenic drug called PTK787 — a small-molecule antagonist that blocks several receptor tyrosine kinases, including VEGFR2 in patients with colorectal carcinoma 134 . So, chemotherapeutics could have significant antivascular properties, which in some cases might be their primary effector function in terms of tumour destruction.

Another promising approach to determining the optimal low dose for a given metronomic chemotherapy regimen is evaluation of the activities of CEPs or circulating endothelial cells (CECs). As previously discussed, CEPs mobilization from the bone marrow into the peripheral circulation is strongly inhibited by low-dose cyclophosphamide, as is CEP viability 17 . So, there could be a direct relationship between the relative efficacy of different (low) doses of metronomic chemotherapy and the ability of these doses to reduce levels of CEPs in the peripheral circulation (Y. Shaked & R.S.K., unpublished observations). Furthermore, assays have been developed to detect the presence of CECs and CEPs in blood samples, such as by performing RT-PCR to detect the mRNA for the vascular endothelial cell-adhesion molecule VE-cadherin 135 . It is therefore encouraging that infusion of bevacizumab in patients with rectal carcinoma was recently found to lead to a rapid decline of both CECs and CEPs in the peripheral blood, as these assays might be exploited not only to monitor anti-angiogenic drug or treatment activity, but to help determine the optimal dose.

A second challenge is the prospect of delayed side effects, including secondary neoplasms, when administering protracted regimens of DNA-damaging agents or other types of genotoxic agents, although it might be argued that these are not a serious concern when treating patients with advanced-stage cancers. But it is certainly a concern in adjuvant-therapy settings for patients with early-stage disease, many of whom are already cured.

As for toxic side effects, although these might be delayed, they might nevertheless be significant. We have found that mice treated with protracted daily oral low-dose cyclophosphamide can eventually develop lymphopaenia 35 . Similarly, patients treated with extended low-dose temozolamide for successive cycles of 6 weeks with 2-week breaks can develop immunosuppressive T-cell lymphopaenia 136 . In this regard, the minimal-toxicity profile observed in patients with early-stage resected NSCLC who were given low doses of UFT every day for 2 years is encouraging — grade 3 toxic effects occurred in only 10/482 (2%) of patients 116 . This is particularly important because anti-angiogenic drugs and metronomic chemotherapy regimens might work best, and sometimes only, in patients with low-volume disease burden. This is true for almost all anticancer drugs and treatment strategies, for various reasons. These include reduced drug access to larger tumours, as well as reduced oxygen levels (hypoxia), which can attenuate the toxic effects of radiation or chemotherapy. It might be possible to treat early-stage disease with long-term combination therapies, but this clearly necessitates the use of non-toxic and less expensive drugs.

Hopefully, some of the clinical trials that are underway, especially those that are prospective and randomized 103 , will better indicate the potential promise of this therapeutic strategy — particularly when such metronomic chemotherapy regimens are integrated with new molecularly targeted drugs. In particular, there is a need to learn more about which chemotherapeutics are the most effective for metronomic dosing regimens, what combinations and sequences might be best to use, and what mechanisms of resistance might develop over time 75 ,76 . Furthermore, it will be important to determine the types of cancer that might be the most responsive to these therapeutic approaches. There is already some data available for the treatment of breast cancer 137 and tumours of the central nervous system 138 using this approach. As scientists carrying out basic research perform more preclinical studies this approach and begin to work more closely with clinical investigators that are leading metronomic chemotherapy-based clinical trials, there should be significant progress towards answering these questions over the next few years.

Links

DATABASES

Cancer.gov: acute lymphoblastic leukaemia |breast cancer |colorectal cancer |melanoma |neuroblastoma |non-Hodgkin’s lymphoma |non-small-cell lung cancer |ovarian cancer |prostate cancer |rhabdomyosarcoma
Entrez Gene: angiopoietin 1 |bFGF |CD36 |COX2 |ERBB2 |G-CSF |IFN- |MDR1 |TSP1 |VE-cadherin |VEGF |VEGFR2
OMIM: Wilms’ tumour

FURTHER INFORMATION

Angiogenesis Foundation

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Acknowledgements

We are grateful to C. Cheng for her excellent secretarial and editorial assistance. We thank U. Emmenegger for critical reading of the manuscript. R.S.K. is a Canada Research Chair in Molecular Medicine whose research is supported by grants from the National Institutes of Health (USA), the National Cancer Institute of Canada, and the Canadian Institutes of Health Research. B.A.K. is an Amercian Cancer Society Clinical Research professor. This review is dedicated to T. Browder, whose pioneering studies in the laboratory of J. Folkman opened up the area of anti-angiogenic metronomic chemotherapy.

Competing interests statement. The authors declare competing financial interests .

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