Affiliations of authors: L. Hlatky, P. Hahnfeldt, Dana-Farber Cancer Institute and the Department of Radiation Oncology, Harvard Medical School, Boston, MA; J. Folkman, Laboratory of Surgical Research, Department of Surgery, Children's Hospital, Boston, and Departments of Cell Biology and Surgery, Harvard Medical School.
Correspondence to: Lynn Hlatky, Ph.D., JFB-523, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115 (e-mail: Lynn_Hlatky{at}dfci.harvard.edu).
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INTRODUCTION |
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MICROVESSEL DENSITY IS A USEFUL PROGNOSTIC INDICATOR |
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MICROVESSEL DENSITY MAY NOT BE AN INDICATOR OF ANTIANGIOGENIC TREATMENT EFFICACY |
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1) Microvessel Density Is Not a Measure of the Angiogenic Dependence of a Tumor. To a Large Degree, It Reflects the Metabolic Burden of the Supported Tumor Cells.
Microvessel density varies widely with tumor type. The degree of variability in microvessel density among tumor types is often misconstrued to mean that some tumors depend on angiogenesis whereas others do not. All tumor types, even those with low microvessel densities, depend on a therapeutically targetable angiogenic process. This angiogenic dependence is based on the inviolable demand of a growing tumor for sufficient levels of nutrients and oxygen exchange (72). These metabolic requirements are in addition to any dependence of the tumor cells on paracrine factors provided by the endothelial cells (73,74). Even leukemia, considered to be a `liquid tumor,' has now been shown to induce angiogenesis in the bone marrow to support its growth (75). Evidence of the importance of angiogenesis in hematologic malignancies, in general, is rapidly accruing. Both myeloid and lymphoid disorders may be accompanied by an increase in the microvessel density of the bone marrow (6671,75).
Contrary to common belief, microvessel density does not reflect the angiogenic activity or angiogenic dependence of a tumor. Microvessel density is a measure of the number of vessels per high-power (microscope) field and, as such, reflects intercapillary distance. Intercapillary distances are determined at the local level by the net balance between angiogenic factors that stimulate and those that inhibit vessel growth in each micro-region, as well as by nonangiogenic factors, such as the oxygen and nutrient consumption rates of the tumor cells. Oxygen and nutrient consumption limit how far away from the vasculature tumor cells can remain viable and, thus, the number of tumor cells that can squeeze between capillaries before some become necrotic (Fig. 1). The metabolic needs of cancer cells vary with the tissue of origin and change with tumor progression. Thus, the number of tumor cells that can be supported by a vessel varies, influencing, in turn, the vascular density of the tumor. As was pointed out by Thomlinson and Gray (76), one can think of the supported tumor cells as forming a viable cuff around a vessel, with cuff size being roughly indicative of the metabolic burden of the cancer cells. This is well illustrated in the Dunning rat model of prostate carcinoma, shown in Fig. 2
, wherein tumor cells within approximately 110 µm of the vasculature form a viable cuff around a functional tumor vessel. Outside this radius of oxygen and nutrient support, tumor cells cannot survive and an abrupt shift to necrosis is observed.
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2) The Measurement of Microvessel Density Is Not Predictive of Tumor Response Under Antiangiogenic Treatment and Therefore Is Not Useful for Stratifying Patients for Clinical Trials. Low Microvessel Density Does Not Portend a Poor Response to Antiangiogenic Therapy.
In recent years there has been a trend toward individualizing tumor treatment by using predictive assays to select patients who are the most likely to respond to specific cancer therapies (8284). Predictive assays are used to evaluate to what degree an individual patient's tumor exhibits the specific attributes [e.g., the level of tumor hypoxia (84)] that a specialized cancer therapy (e.g., neutron radiotherapy) targets. It is a common misconception that antiangiogenic treatment can be applied only to cancers that have high microvessel densities. This thinking promotes the further misconceptions that the measurement of microvessel density is the relevant predictive assay for antiangiogenic therapy and that the level of microvascular density within untreated tumors forecasts the efficacy of tumor response to antiangiogenic treatment. Thus, it is widely assumed that tumors with high microvessel densities are good candidates for clinical trials of antiangiogenic therapies, whereas tumors that typically have low microvessel densities (e.g., astrocytomas) are thought to be poor candidates for such clinical trials (85). However, experimental evidence does not support this method of patient stratification, but rather shows that both poorly vascularized and highly vascularized tumors can respond to antiangiogenic therapy. For example, bladder tumors that had less vascularity were found to be treatable by lower doses of an antiangiogenic agent than were those that had more vascularity (86). Because virtually all tumors are angiogenesis-dependent (72,87), low microvessel counts within tumors are not sufficient criteria to exclude patients from treatment with angiogenesis inhibitors.
3) Tumor Microvessel Density May Not Vary in Accordance With the Tissue or Blood Levels of any Single Proangiogenic Factor.
Many studies have investigated the relationship between microvessel density and the levels of single angiogenic factors such as vascular endothelial growth factor (VEGF) (27,32). It is now clear that individual tumors can make a wide variety of proangiogenic factors, and the relative expressions of these factors can change over time. For example, when breast cancers were screened for their expression of a panel of angiogenic factors, the majority originally expressed only a single positive factor, either VEGF or acidic fibroblast growth factor (aFGF), but evolved over time to express up to six angiogenic stimulators (88). The net angiogenic influence of the tumor microenvironment should be thought of as the sum of the positive and negative regulators of angiogenesis that arise from both the tumor cells (89) and host tissues (90). Human breast cancer fibroblasts, for example, exhibit high levels of VEGF (90). Because it is the net effect of total angiogenic stimulation and inhibition that determines the level of neovascularization, the levels of single angiogenic factors are not, as a general rule, strictly associated with measures of tumor vascularization. This principle has been borne out by investigations showing no correlation between tumor microvessel density measurements and levels of VEGF and aFGF or basic fibroblast growth factor (bFGF) (9194). Such findings need to be considered when proposing antiangiogenic cancer therapies that seek to block a single proangiogenic signal. Recent work from the Kerbel group (95) showed that, when the proangiogenic factor VEGF is targeted by antiangiogenic therapy, tumors that express p53 respond more rapidly than tumors that do not. They interpreted the reduced response in the absence of p53 as resistance. More likely, p53-null status does not confer resistance. Rather, p53-null status is proangiogenic (96) through increased expressions of VEGF, interleukin 8 (IL-8), and bFGF, the suppression of which requires a suitably inclusive antiangiogenic counterattack.
4) A Minimum Vessel Density Is Determined by Tumor Cell Metabolic Demand, but Vessel Density Can Exceed the Metabolic Requirements of a Tumor.
Although normally functioning tissues rarely overvascularize, tumors can engage in angiogenic activity beyond that dictated by their metabolic needs, thus leading to overvascularization. As previously discussed, a major factor contributing to vessel density is metabolic demand. Metabolic demand places a lower limit on the density of vessels within the tissue required to maintain tissue viability. If vascular density should fall below the required minimum, the flux of oxygen and glucose to the cells will be inadequate, triggering just enough tumor cell death to restore the minimal vascular density. Attesting to this process, regions of necrotic tumor cells adjacent to, and resulting from, oxygen or nutrient shortages are seen in many human tumors (97) (see Fig. 2). In normal tissues, by contrast, the level of microvascular density fairly accurately reflects the metabolic demands of the cells. This is because evolutionary pressures have forced a tight and efficient coupling between vascular supply and metabolic need. In the genetically unstable tumor, the close coupling between vascular density and oxygen or nutrient consumption may be loosened. In tumors, angiogenic factor expression often becomes uncoupled from normal regulatory controls and, consequently, some angiogenic factors are constitutively expressed at high levels. A prime example is the dissociation of VEGF expression from its regulation by oxygen concentration; in normal tissues, VEGF expression is increased and VEGF mRNA is stabilized under conditions of hypoxia or ischemia (90,98,99), whereas in tumor cells, VEGF is often constitutively expressed at high levels regardless of the ambient oxygen tension (37,100,101). Notable examples of the decoupling of oxygen tension from VEGF expression occur in p53-null tumors (102) and in renal cell carcinomas from individuals that carry a mutation in the von Hippel-Lindau (VHL) gene (103). Both p53 and VHL genes are associated with the degradation of hypoxia-inducible factor-1
, an upstream regulator of VEGF.
5) Rapid Tumor Growth Does Not Imply High Vascular Density. The Microvessel Density of a Tumor Need Not Be Higher, and Is Often Lower, Than That of its Corresponding Normal Tissue, Which Is Experiencing no Net Growth.
Whereas microvessel density frequently increases during tumor progression to accommodate an increased metabolic demand or a decoupling of net angiogenic stimulator expression with metabolic need, microvessel density levels do not reflect growth rate. In fact, Eberhard et al. (78) have shown that for five of the six human cancers examined (prostate carcinoma being the exception), the microvessel density of the tumor was lower than that of the corresponding nongrowing normal tissue. In lung carcinoma, for example, microvessel density was found to be only 29% that of normal lung tissue. In glioblastoma, an exceptionally highly vascularized tumor, microvessel density was found to be 78% that of normal brain tissue. This apparent paradox can be partially explained by the lower oxygen consumption rate of tumor cells (77). In addition, tumor cells are known to tolerate oxygen deprivation and to be resistant to apoptosis under hypoxic conditions (104). Because tumor cells can remain viable at lower oxygen concentrations, they can exist at greater distances from the vasculature than can their normal-cell counterparts. Both the lowered oxygen consumption of tumor cells and their tolerance of hypoxic conditions promote increased intercapillary distance in tumors relative to their normal tissue counterparts.
Although the amount of total tumor vascularization (an extrinsic variable applicable to the tumor as a whole) must increase rapidly in fast-growing tumors to support a rapidly increasing tumor mass, the density of the vessels (an intrinsic variable that is locally defined) need not be high. In addition, the efficacy of antiangiogenic therapy would not be expected a priori to vary with the growth rate of the tumor. The lack of growth rate dependence for antiangiogenic effect stands in marked contrast to what is seen under standard chemotherapy. Chemotherapy targets proliferating cells directly and thus exerts a more demonstrable effect for fast-growing tumors than it does for slow-growing tumors.
6) The Efficacy of Antiangiogenic Agents Cannot Be Simply Visualized by Alterations in Microvessel Density During Treatment.
There is an urgent need to assess the clinical activity of the numerous antiangiogenic agents that are now in patient trials (105,106). Because antiangiogenic therapy suppresses tumor cell growth indirectly by inhibiting endothelial cell growth, reductions in tumor sizes or growth rates under antiangiogenic therapy are likely to occur over longer time frames than they would when standard chemotherapy is used (89). It follows that rapid tumor shrinkage, which is the classic end point scored for in clinical trials of chemotherapeutic agents, cannot be used to reliably discriminate between a null and a positive response to antiangiogenic agents. Instead, an appropriate measure of vascular inhibition is needed to assess the efficacy of an antiangiogenic agent at an early stage in a trial (i.e., before tumor growth inhibition is observed).
A common, albeit erroneous, impression is that measurements of microvessel density made during antiangiogenic therapy may be used to evaluate the therapeutic response to antiangiogenic agents. Numerous clinical protocols for antiangiogenic therapy include the periodic taking of biopsies to evaluate tumor microvessel density (107). However, the efficacy of an antiangiogenic agent cannot be evaluated by measuring the changes in microvessel density that the agent induces. This is because changes in microvessel density do not independently measure vascular inhibition, but rather, reflect the changing ratio of the vascular component of the tumor to its tumor-cell component. Under antiangiogenic therapy, capillary inhibition or elimination occurs first, followed by tumor-cell elimination, and both influence microvessel density. The tightness of the coupling of these two tumor components determines how much the vascular density fluctuates. Consequently, microvessel density is a time-dependent measure that varies in a nonmonotonic manner during antiangiogenic treatmentfirst decreasing in response to capillary inhibition or elimination, then decreasing more slowly or even increasing somewhat as tumor cells drop out. Finally, in those cases where the angiogenic inhibitor is cleared from the bloodstream following bolus dosing (89), vessel density may asymptotically approach the pretreatment microvessel density level if treatment does not alter the metabolic demand or the angiogenic factor production of the tumor cells. The time required to partially or fully restore microvessel density to its pretreatment level following the clearance of an administered bolus dose of an angiogenic inhibitor would depend on the tightness of the coupling between vessel dropout and tumor-cell dropout. This would vary for different tumor types. If the restoration time is less than the typical times between biopsies, one might expect that most measures of microvessel density that are made over the course of treatment would yield a value little changed from the pretreatment value.
Although decreases in vascular density certainly provide a rough indication of the activity of an antiangiogenic agent, the nonmonotonic nature of microvessel density under antiangiogenic therapy renders microvessel density unsuitable as a stand-alone measure to monitor antiangiogenic effect. The concept that measures of vascular density alone do not reflect the efficacy of angiogenic inhibitors is demonstrated in Fig. 3. An untreated tumor (control) and two tumors treated with the angiogenic inhibitor endostatin are shown. Despite the fact that the inhibitor substantially inhibited growth in both of the treated tumors, the post-treatment levels of vascularization of the two tumors vary substantially. Compared with the control tumor, vessel density was substantially lower in one treated tumor and slightly higher in the second. Thus, detection of a decrease in microvessel density during treatment with an antiangiogenic agent suggests that the agent is active. However, the absence of a drop in microvessel density does not indicate that the agent is ineffective.
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All tumor vessels are not equal in their ability to provide oxygen and nutrients to the tumor cells they support. Vessels may be inefficient for several reasons. Tumor vessels can themselves be hypoxic and carry little oxygen, or they can have oscillating rather than directed blood flows and thus be ineffective at transporting oxygen and nutrients. Clearly, inhibiting hypoxic vasculature would shrink a tumor mass less than would inhibiting oxygen-rich vessels. In addition, the vascularization of a tumor may be greater than necessary to support its metabolic needs, thereby creating a state of overvascularization. Inhibiting redundant vasculature would likewise be expected to shrink a tumor mass less than would inhibiting the more effective vessels. In these cases, however, there is benefit to extending treatment. For example, in an effectively overvascularized p53-null tumor, there may be an initial period during antiangiogenic therapy when there is little tumor suppression because the excess vasculature is being targeted. As therapy continues and more critical vasculature becomes the target, a shift to more marked suppression would be observed. This appears to be what is happening in the study by Yu et al. (95), which looked at the response of p53-null colon carcinoma to antiangiogenic therapy using vinblastine and DC101, an antibody specific to VEGF receptor-2 expressed on endothelium.
The disconnect between vascular reduction and tumor response points to the lack of a simple quantitative relationship between therapeutic knockout of capillaries, reduction of microvessel density, and measurable tumor regression. In current theoretical treatments of therapeutic response to antiangiogenic agents, the inability to attribute tumor response to local vessel densities because of inhomogeneities in transport capacities is being dealt with by considering only the "effective vasculature" feeding the tumor. By focusing specifically on the functional vessels in a tumor, the underlying dynamics connecting vessel inhibition and tumor regression are likely to be clarified (89).
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LOOKING BEYOND MEASUREMENTS OF MICROVESSEL DENSITY |
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Although microvessel density per se would not influence the responsiveness of a tumor to antiangiogenic treatment, the detailed composition of the tumor vessels [e.g., the fraction of proliferating endothelial cells they contain (78) or whether pericytes are present (114)] might well modify the action of a particular antiangiogenic agent. VEGF neutralization (e.g., with VEGF-specific antibodies) may not affect mature vessels but has been shown to lead to the regression of immature vessels that lack smooth muscle cells (115). Assays that quantify the maturation state of vessels might also provide some indication of the susceptibility of the existing vasculature in the tumor bed to specific antiangiogenic agents. These assays include those that measure the ratio of concentrations of the angiopoietins Ang-2 and Ang-1 (116), or the presence or absence of an epitope recognized by the monoclonal antibody LH39 that is expressed in the lamina lucida and is associated with vessel maturity (117). Currently, there is a flurry of investigation into the fundamental biology of angiogenesis (118121) that will inevitably yield further insights into the effective use of antiangiogenic agents (122). With antiangiogenic therapy, the challenge comes not in restricting the patient pool, but in providing the proper guidelines for using combination therapies [e.g., those that use several antiangiogenic agents in combination or combined with radiation (123,124) or chemotherapy (125,126)]; delivering the agents [e.g., extended or continuous versus bolus dosing schemes (89,125129)]; classifying the different types of antiangiogenic agents according to target (115), mode of action, or stage of cancer most amenable to antiangiogenic agent attack (130); unveiling and exploiting the serendipitous antiangiogenic effects of classic chemotherapeutic agents (125,126,131136); and accurately assessing patient response, including the identification of appropriate surrogate end points. Recognizing the limitations of microvessel density as a surrogate end point will no doubt improve our resolve to explore supplementary assays for evaluating the efficacy of antiangiogenic agents.
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NOTES |
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We thank Dipak Panigrahy and Samuel Singer for human liposarcoma xenografts, Lloyd Hutchinson for Dunning rat prostate carcinoma xenografts, and Clare Lamont and Kristin Gullage for figure graphics.
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Manuscript received September 11, 2001; revised April 5, 2002; accepted April 11, 2002.
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