hypoxia and cancer

1. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: Randomised, double-blind, placebo-controlled trial.

 

Anaemia is associated with poor cancer control, particularly in patients undergoing radiotherapy. We investigated whether anaemia correction with epoetin beta could improve outcome of curative radiotherapy among patients with head and neck cancer. METHODS: We did a multicentre, double-blind, randomised, placebo-controlled trial in 351 patients (haemoglobin <120 g/L in women or <130 g/L in men) with carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx. Patients received curative radiotherapy at 60 Gy for completely (R0) and histologically incomplete (R1) resected disease, or 70 Gy for macroscopically incompletely resected (R2) advanced disease (T3, T4, or nodal involvement) or for primary definitive treatment. All patients were assigned to subcutaneous placebo (n=171) or epoetin beta 300 IU/kg (n=180) three times weekly, from 10-14 days before and continuing throughout radiotherapy. The primary endpoint was locoregional progression-free survival. We assessed also time to locoregional progression and survival. Analysis was by intention to treat. FINDINGS: 148 (82%) patients given epoetin beta achieved haemoglobin concentrations higher than 140 g/L (women) or 150 g/L (men) compared with 26 (15%) given placebo. However, locoregional progression-free survival was poorer with epoetin beta than with placebo (adjusted relative risk 1.62 [95% CI 1.22-2.14]; p=0.0008). For locoregional progression the relative risk was 1.69 (1.16-2.47, p=0.007) and for survival was 1.39 (1.05-1.84, p=0.02). INTERPRETATION: Epoetin beta corrects anaemia but does not improve cancer control or survival. Disease control might even be impaired. Patients receiving curative cancer treatment and given erythropoietin should be studied in carefully controlled trials.

 

2. Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: A survival study.

 

PURPOSE: To evaluate the effect on survival and quality of life of maintaining hemoglobin (Hb) in the range of 12 to 14 g/dL with epoetin alfa versus placebo in women with metastatic breast cancer (MBC) receiving first-line chemotherapy.

 

PATIENTS AND METHODS: Eligible patients were randomly assigned to receive epoetin alfa 40,000 U once weekly or placebo for 12 months. Study drug was initiated if baseline Hb was  13 g/dL or when Hb decreased to  13g/dL during the study. The primary end point was 12-month overall survival (OS).

 

RESULTS: The study drug administration was stopped early in accordance with a recommendation from the Independent Data Monitoring Committee because of higher mortality in the group treated with epoetin alfa. Enrollment had been completed, with 939 patients enrolled (epoetin alfa, n = 469; placebo, n = 470). Most patients had Hb more than 12 g/dL at baseline (median Hb, 12.8 g/dL) or during the study. From the final analysis, 12-month OS was 70% for epoetin alfa recipients and 76% for placebo recipients (P = .01). Optimal tumor response and time to disease progression were similar between groups. The reason for the difference in mortality between groups could not be determined from additional subsequent analyses involving both study data and chart review.

 

CONCLUSION: In this trial, the use of epoetin alfa to maintain high Hb targets in women with MBC, most of whom did not have anemia at the start of treatment, was associated with decreased survival. Additional research is required to clarify the potential impact of erythropoietic agents on survival when the Hb target range is 10 to 12 g/dL.

 

 

3. Severe anemia is associated with poor tumor oxygenation in head and neck squamous cell carcinomas.

 

Abstract

Purpose: To investigate the relationship between tumor oxygenation and the blood hemoglobin (Hb) concentration in patients with squamous cell carcinoma of the head and neck (SCCHN).

 

Methods and Materials: A total of 133 patients with SCCHN underwent pretreatment polarographic pO2 measurements of their tumors. In 66 patients measurements were also made in sternocleidomastoid muscles. The patients were divided into three groups according to their Hb concentration—severe anemia (Hb < 11.0 g/dl), mild anemia (female: Hb 11.0–11.9 g/dl; male: Hb 11.0–12.9 g/dl), and normal Hb concentration (female: Hb ≥12.0 g/dl; male: ≥13.0 g/dl).

 

Results: No significant difference in tumor oxygenation could be detected between mildly anemic patients and patients with a normal Hb level. However, the tumor oxygenation in the severely anemic group was significantly below that of each of the other two groups (p < 0.0001). There was no significant difference between the Hb groups in oxygenation of sternocleidomastoid muscles. In a multivariate analysis including Hb group, tumor volume, smoking habits, gender, T-stage, N-stage, and histologic grade a Hb level < 11 g/dl was found to be the strongest predictor for a poor tumor oxygenation. Smoking also had a marginal influence on median pO2.

 

Conclusion: Our data suggest that a low Hb concentration and cigarette smoking contribute to inadequate oxygenation of SCCHN and thus for increased radioresistance. Consequently, Hb correction and abstinence from smoking may significantly improve tumor oxygenation.

 

4. Impact of hemoglobin levels on tumor oxygenation: The higher, the better?

 

Background and Purpose:  Tumor hypoxia has been linked to tumor progression, the development of treatment resistance, and thus poor prognosis. Since anemia is a major factor causing tumor hypoxia, the association between blood hemoglobin concentration (cHb) and tumor oxygenation status has been examined.

Patients and Methods:  Published data on the relationship between pretreatment cHb values and tumor oxygenation (in terms of median pO2 values, hypoxic fractions) have been summarized. Pretreatment O2 tension measurements were performed in histologically proven experimental tumors, human breast cancers, squamous cell carcinomas of the head and neck, and cancers of the uterine cervix and of the vulva. In order to allow for a comparison between solid tumors and normal tissues, pO2 measurements were also performed in healthy tissue in anemic and nonanemic patients. cHb was determined at the time of the pO2 measurements.

Results:  Based on current information from experimental and clinical studies there is increasing evidence that anemia is associated with a detrimental tumor oxygenation status. Increasing cHb values are correlated with significantly higher pO2 values and lower hypoxic fractions. Maximum tumor oxygenation in squamous cell carcinomas is observed at normal (gender-specific) cHb values (approximately 14 g/dl in women and approximately 15 g/dl in men). Above this “optimal” Hb range, the oxygenation status tends to worsen again. In anemic patients, tumor oxygenation is compromised due to a decreased O2 transport capacity of the blood. At the upper edge of the Hb scale, a substantial increase in the blood’s viscous resistance to flow in “chaotic” tumor microvessels is thought to be mainly responsible for the observed restriction of O2 supply.

Conclusion:  Review of relevant clinical data suggests that a maximum oxygenation status in solid tumors is to be expected in the range 12 g/dl < cHb < 14 g/dl for women and 13 g/dl < cHb < 15 g/dl for men. Considering the “optimal” cHb range with regard to tumor oxygenation, the concept of “the higher, the better” is therefore no longer valid. This finding has potentially far-reaching implications in the clinical setting (e. g., inappropriate erythropoietin treatment of nonanemic tumor patients).

 

 

5. Oxygenation gain factor: A novel parameter characterizing the association between hemoglobin level and the oxygenation status of breast cancers.

 

Tumor hypoxia has been linked to acquired treatment resistance, tumor progression, and poor prognosis. Because anemia is a major causative factor for the development of hypoxia, the association between blood hemoglobin concentration (cHb) and breast cancer oxygenation was examined in this study. In addition, a novel parameter characterizing the relationship between oxygenation status and rising cHb is introduced: the oxygenation gain factor (OGF). In breast cancer patients, median cHb over the range 8.5–14.7 g/dl correlated positively with the median pO2 (3–15 mm Hg), yielding an average OGF of 2 mm Hg·dl/g. In contrast, in normal tissues (normal breast, subcutis, and skeletal muscle) the median pO2 values were substantially higher (52 mm Hg, 51 mm Hg, and 37 mm Hg, respectively) and remained constant irrespective of the hemoglobin level over the range from 10 to 16 g/dl (OGF = 0 in grade I anemia and nonanemic patients). Moderately lower median pO2 values in subcutis and skeletal muscle were only observed in grade II anemia (8 g/dl < cHb 10 g/dl), although this would appear to be of no biological relevance. Conversely, in breast cancers, even mild anemia (grade I anemia) is a major causative factor for the development of hypoxia or anoxia.

 

6. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix.

 

Experimental tumors contain a significant fraction of microregions that are chronically or transiently hypoxic. Experimental evidence showing that hypoxia (and subsequent reoxyenation) may have a profound impact on malignant progression and on responsiveness to therapy is growing. The clinical relevance of tumor oxygenation in human solid malignancies is under investigation.

 

We have developed and validated a clinically applicable method for measurement of tumor oxygenation in locally advanced cancer of the uterine cervix using a computerized polarographic electrode system. Applying this procedure in patients with cervical cancers 3 cm in diameter, who gave informed consent, we have been studying the clinical relevance of tumor oxygenation prospectively since 1989.

 

As of June 1995, 103 patients with advanced cancers of the uterine cervix [Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stages Ib, bulky (n = 13), IIa and IIb (n = 51), IIIa and IIIb (n = 34), and IVa and IVb (n = 5)] had entered the study. Fifty % of the patients had carcinomas with median pO2 readings <10 mm Hg, referred to as hypoxic tumors.

 

Tumor oxygenation was found to be independent of various patient demographics and also of pretreatment tumor characteristics, such as clinical tumor stage and size, histological type, and differentiation. However, histopathological examination of the surgical specimens following radical tumor resection in 47 patients showed that low-pO2 tumors exhibited larger tumor extensions and more frequent (occult) parametrial spread, as well as lymph-vascular space involvement, compared to well-oxygenated tumors of similar clinical stage and size. Forty-two patients completing primary radiation therapy and 47 patients who underwent radical surgery were analyzed for treatment outcome after a median observation period of 28 months (range, 3–76 months). Patients with hypoxic tumors had significantly worse disease-free and overall survival probabilities compared to patients with nonhypoxic tumors. Cox regession analysis identified tumor oxygenation and FIGO stage as the most important independent prognostic factors. The poorer outcome of the patients with hypoxic tumors was mainly due to locoregional failures with and without distant metastases, irrespective of whether surgery or radiation was applied as primary treatment.

 

Tumor oxygenation as measured with a standardized polarographic method proved to be a powerful new pretherapeutic prognostic parameter providing important information on malignant progression in terms of extracervical tumor spread and radioresistance in advanced cervical cancers.

 

7. Anaemia in Cancer. European School of Oncology Scientific Updates. Effects of anaemia and hypoxia on tumor biology.

 

8. Hypoxia in cancer: Significance and impact on clinical outcome.

 

Abstract  Hypoxia, a characteristic feature of locally advanced solid tumors, has emerged as a pivotal factor of the tumor (patho-)physiome since it can promote tumor progression and resistance to therapy. Hypoxia represents a “Janus face” in tumor biology because (a) it is associated with restrained proliferation, differentiation, necrosis or apoptosis, and (b) it can also lead to the development of an aggressive phenotype. Independent of standard prognostic factors, such as tumor stage and nodal status, hypoxia has been suggested as an adverse prognostic factor for patient outcome. Studies of tumor hypoxia involving the direct assessment of the oxygenation status have suggested worse disease-free survival for patients with hypoxic cervical cancers or soft tissue sarcomas. In head & neck cancers the studies suggest that hypoxia is prognostic for survival and local control. Technical limitations of the direct O2 sensing technique have prompted the use of surrogate markers for tumor hypoxia, such as hypoxia-related endogenous proteins (e.g., HIF-1α, GLUT-1, CA IX) or exogenous bioreductive drugs. In many—albeit not in all—studies endogenous markers showed prognostic significance for patient outcome. The prognostic relevance of exogenous markers, however, appears to be limited. Noninvasive assessment of hypoxia using imaging techniques can be achieved with PET or SPECT detection of radiolabeled tracers or with MRI techniques (e.g., BOLD). Clinical experience with these methods regarding patient prognosis is so far only limited. In the clinical studies performed up until now, the lack of standardized treatment protocols, inconsistencies of the endpoints characterizing the oxygenation status and methodological differences (e.g., different immunohistochemical staining procedures) may compromise the power of the prognostic parameter used.

 

9. Hypoxia-inducible factor 1alpha is closely linked to an aggressive phenotype in breast cancer.

10. Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells.

 

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O2) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O2, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.

 

11. Effect of hypoxia on the tumor phenotype: The neuroblastoma and breast cancer models.

The tumor oxygenation status associates with aggressive behavior. Oxygen shortage, hypoxia, is a major driving force behind tumor vascularization, and hypoxia enhances mutational rate, metastatic spread, and resistance to radiation and chemotherapy. We recently discovered that hypoxia promotes dedifferentiation of neuroblastoma and breast carcinoma cells and development of stem cell-like features. In both these tumor forms there is a correlation between low differentiation stage and poor outcome, and we conclude that the dedifferentiating effect of lowered oxygen adds to the aggressive phenotype induced by hypoxia. With neuroblastoma and breast carcinoma as human tumor model systems, we have addressed questions related to hypoxia-induced molecular mechanisms governing malignant behavior of tumor cells, with emphasis on differentiation and growth control. By global gene expression analyses we are currently screening for gene products exclusively expressed or modified in hypoxic cells with the aim to use them as targets for treatment.

 

12. Hypoxia-induced dedifferentiation of tumor cells--a mechanism behind heterogeneity and aggressiveness of solid tumors.

 

Histopathological examination of solid tumors frequently reveals pronounced tumor cell heterogeneity with regards to cell organization, cell morphology, cell size, nuclei morphology, etc. Analyses of gene expression patterns by immunohistochemistry or in situ hybridization techniques further strengthen the actual presence of phenotypic heterogeneity, often demonstrating substantial diversity within a given tumor. The molecular mechanisms underlying the phenotypic heterogeneity are very complex with genetic, epigenetic and environmental components. Hypoxia, shortage in oxygen, greatly influences cellular phenotypes by altering the expression of specific genes, and is an important contributor to intra- and inter-tumor cell diversity as revealed by the pronounced but non-uniform expression of hypoxia driven genes in solid tumors

The oxygen pressure in solid tumors is generally lower than in the surrounding non-malignant tissues, and tumors exhibiting extensive hypoxia have been shown to be more aggressive than corresponding tumors that are better oxygenized [Vaupel P. Oxygen transport in tumors: characteristics and clinical implications. Adv Exp Med Biol 1996;388:341–51; Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol 2001;18:243–59.]. We recently observed that hypoxic neuroblastoma cells and breast cancer cells loose their differentiated gene expression patterns and develop stem cell-like phenotypes [Jögi A, Øra I, Nilsson H, Lindeheim A, Makino Y, Poellinger L, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci USA 2002;99:7021–6; Helczynska K, Kronblad A, Jögi A, Nilsson E, Beckman S, Landberg G, et al. Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ. Cancer Res 2003;63:1441–4.]. As low stage of differentiation in neuroblastoma and in breast cancer is linked to poor prognosis, hypoxia-induced dedifferentiation will not only contribute to tumor heterogeneity but could also be one mechanism behind increased aggressiveness of hypoxic tumors. The effect(s) of hypoxia on tumor cell differentiation status is the focus of this review.

 

13. Angiogenesis in endocrine tumors.

 

Angiogenesis is the process of new blood vessel development from preexisting vasculature. Although vascular endothelium is usually quiescent in the adult, active angiogenesis has been shown to be an important process for new vessel formation, tumor growth, progression, and spread. The angiogenic phenotype depends on the balance of proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and inhibitors, as well as interactions with the extracellular matrix, allowing for endothelial migration. Endocrine glands are typically vascular organs, and their blood supply is essential for normal function and tight control of hormone feedback loops. In addition to metabolic factors such as hypoxia, the process of angiogenesis is also regulated by hormonal changes such as increased estrogen, IGF-I, and TSH levels.

 

By measuring microvascular density, differences in angiogenesis have been related to differences in tumor behavior, and similar techniques have been applied to both benign and malignant endocrine tumors with the aim of identification of tumors that subsequently behave in an aggressive fashion.

 

In contrast to other tumor types, pituitary tumors are less vascular than normal pituitary tissue, although the mechanism for this observation is not known. A relationship between angiogenesis and tumor size, tumor invasiveness, and aggressiveness has been shown in some pituitary tumor types, but not in others. There are few reports on the role of microvascular density or angiogenic factors in adrenal tumors. The mechanism of the vascular tumors, which include adrenomedullary tumors, found in patients with Von Hippel Lindau disease has been well characterized, and clinical trials of antiangiogenic therapy are currently being performed in patients with Von Hippel Lindau disease. Thyroid tumors are more vascular than normal thyroid tissue, and there is a clear correlation between increased VEGF expression and more aggressive thyroid tumor behavior and metastasis. Although parathyroid tissue induces angiogenesis when autotransplanted and PTH regulates both VEGF and MMP expression, there are few studies of angiogenesis and angiogenic factors in parathyroid tumors.

 

An understanding of the balance of angiogenesis in these vascular tumors and mechanisms of vascular control may assist in therapeutic decisions and allow appropriately targeted treatment.

 

14. The role of hypoxia-induced factors in tumor progression.

 

Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with diminished therapeutic response and, more recently, with malignant progression, that is, an increasing probability of recurrence, locoregional spread, and distant metastasis. Emerging evidence indicates that the effect of hypoxia on malignant progression is mediated by a series of hypoxia-induced proteomic and genomic changes activating angiogenesis, anaerobic metabolism, and other processes that enable tumor cells to survive or escape their oxygendeficient environment. The transcription factor hypoxia-inducible factor 1 (HIF-1) is a major regulator of tumor cell adaptation to hypoxic stress. Tumor cells with proteomic and genomic changes favoring survival under hypoxic conditions will proliferate, thereby further aggravating the hypoxia. The selection and expansion of new (and more aggressive) clones, which eventually become the dominant tumor cell type, lead to the establishment of a vicious circle of hypoxia and malignant progression.

 

15. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review.

 

The objective of this review article is to summarize current knowledge of blood flow and perfusion-related parameters, which usually go hand in hand and in turn define the cellular metabolic microenvironment of human malignancies. A compilation of available data from the literature on blood flow, oxygen and nutrient supply, and tissue oxygen and pH distribution in human tumors is presented. Whenever possible, data obtained for human tumors are compared with the respective parameters in normal tissues, isotransplanted or spontaneous rodent tumors, and xenografted human tumors. Although data on human tumors in situ are scarce and there may be significant errors associated with the techniques used for measurements, experimental evidence is provided for the existence of a compromised and anisotropic blood supply to many tumors. As a result, O2-depleted areas develop in human malignancies which coincide with nutrient and energy deprivation and with a hostile metabolic microenvironment (e.g., existence of severe tissue acidosis). Significant variations in these relevant parameters must be expected between different locations within the same tumor, at the same location at different times, and between individual tumors of the same grading and staging. Furthermore, this synopsis will attempt to identify relevant pathophysiological parameters and other related areas future research of which might be most beneficial for designing individually tailored treatment protocols with the goal of predicting the acute and/or long-term response of tumors to therapy.

 

16. Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth.


 

Deficiencies in oxygenation are widespread in solid tumors. The transcription factor hypoxia-inducible factor (HIF)-1 is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors important for solid tumor expansion, including the angiogenic factor vascular endothelial growth factor (VEGF). We have isolated two cell lines nullizygous for HIF-1, one from embryos genetically null for HIF-1, and the other from embryos carrying loxP-flanked alleles of the gene, which allows for cre-mediated excision. The loss of HIF-1 negatively affects tumor growth in these two sets of H-ras-transformed cell lines, and this negative effect is not due to deficient vascularization. Despite differences in VEGF expression, vascular density is similar in wild-type and HIF-1-null tumors. The evidence from these experiments indicates that hypoxic response via HIF-1 is an important positive factor in solid tumor growth and that HIF-1 affects tumor expansion in ways unrelated to its regulation of VEGF expression.

 

 

17. What is the evidence that tumors are angiogenesis dependent?

18. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.

 

• Introduction

• Transgenic Mouse Models Reveal the Angiogenic Switch in Early Stages of Tumor Development

• Angiogenesis in Premalignant Stages of Two Human Cancers

• Emerging Mechanisms of the Angiogenic Switch

• The Angiogenic Inducers

• The Angiogenic Inhibitors

• A New Paradigm of Cryptic Angiogenesis Inhibitors within Larger Proteins

• The Balance Hypothesis for the Angiogenic Switch

• Apoptosis, Clinical Applications, and the Goal to Control the Switch

• Acknowledgements

• References

 

19. Hypoxic stress proteins: Survival of the fittest.

The importance of tumor microenvironment in malignant progression has been largely ignored. Tumor cells protect themselves from changes in the microenvironment due to a decrease in nutrients and oxygen by reducing macromolecular synthesis and inducing genes that will promote angiogenesis and tissue remodeling. This ability of transformed cells to survive fluctuations in oxygen tensions is clinically important, as tumors with high hypoxic fractions respond poorly to many forms of cancer therapy. While it is commonly accepted that the decrease in molecular oxygen in hypoxic cells makes them more refractory to killing by agents such as ionizing radiation which use oxygen radical formation, the cessation of division and loss of apoptotic (cell suicide) potential in hypoxic cells are also important in their resistance to killing by radiotherapy and chemotherapy. In this chapter, we will discuss hypoxia-induced stress proteins in regards to three clinically relevant end points: inhibition of cell proliferation, induction of apoptosis, and regulation of genes modulating angiogenesis. Although these three end points may seem unrelated, in fact, they are intimately linked with each other in the cellular response to hypoxia and malignant progression. One of the goals of this review is to inform both clinican and scientist of these interrelationships and discuss how hypoxia selects for tumors that are clonal expansions of cells that have lost their apoptotic ability and have switched to a proangiogenic phenotype.

 

 

20. Regulation of hypoxia-induced angiogenesis: A chaperone escorts VEGF to the dance.

21. Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

 

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.

 

22. Proteolytic balance and capillary morphogenesis in vitro.

23. Cellular abnormalities of blood vessels as targets in cancer.

 

Tumor blood vessels have multiple structural and functional abnormalities. They are unusually dynamic, and naturally undergo sprouting, proliferation, remodeling or regression. The vessels are irregularly shaped, tortuous, and lack the normal hierarchical arrangement of arterioles, capillaries and venules. Endothelial cells in tumors have abnormalities in gene expression, require growth factors for survival and have defective barrier function to plasma proteins. Pericytes on tumor vessels are also abnormal. Aberrant endothelial cells and pericytes generate defective basement membrane. Angiogenesis inhibitors can stop the growth of tumor vessels, prune existing vessels and normalize surviving vessels. Loss of endothelial cells is not necessarily accompanied by simultaneous loss of pericytes and surrounding basement membrane, which together can then provide a scaffold for regrowth of tumor vessels. Rapid vascular regrowth reflects the ongoing drive for angiogenesis and bizarre microenvironment in tumors that promote vascular abnormalities and thereby create therapeutic targets.

 

24. Vascular-targeted Therapies in Oncology. Abnormal microvasculature and defective microcirculatory function in solid tumors.

 

25. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

 

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia- inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF- 1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.

26. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.

 

 

27. Hypoxia, clonal selection, and the role of HIF-1 in tumor progression.

 

Tumor progression occurs as a result of the clonal selection of cells in which somatic mutations have activated oncogenes or inactivated tumor suppressor genes leading to increased proliferation and/or survival within the hypoxic tumor microenvironment. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to reduced O2 availability, including angiogenesis and glycolysis. Expression of the O2-regulated HIF-1α subunit and HIF-1 transcriptional activity are increased dramatically in hypoxic cells. Recent studies indicate that many common tumor-specific genetic alterations also lead to increased HIF-1α expression and/or activity. Thus, genetic and physiologic alterations within tumors act synergistically to increase HIF-1 transcriptional activity, which appears to play a critical role in the development of invasive and metastatic properties that define the lethal cancer phenotype.

 

 

28. Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins.

 

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR–VN and integrin–VN interactions and that it does so by binding to the uPA present in uPA–uPAR–integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA–uPAR–integrin complexes and their endocytic clearance by the low density lipoprotein receptor–related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA–uPAR–integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA–uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.

 

29. Hypoxia and anemia: Effects on tumor biology and treatment resistance.

 

In locally advanced solid tumors, oxygen (O2) delivery is frequently reduced or even abolished. This is due to abnormalities of the tumor microvasculature, adverse diffusion geometries, and tumor-associated and/or therapy-induced anemia. Up to 50–60% of locally advanced solid tumors may exhibit hypoxic and/or anoxic tissue areas that are heterogeneously distributed within the tumor mass. In approximately 30% of pretreatment patients, a decreased O2 transport capacity of the blood as a result of tumor-associated anemia can greatly contribute to the development of tumor hypoxia. While normal tissues can compensate for this O2 deficiency status by a rise in blood flow rate, locally advanced tumors (or at least larger tumor areas) cannot adequately counteract the restriction in O2 supply and thus the development of hypoxia. Hypoxia-induced alteration in gene expression and thus in the proteome (<1% O2, or <7 mmHg), and/or genome changes (<0.1% O2, or <0.7 mmHg) may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile metabolic microenvironment and to favor unrestricted growth. Sustained hypoxia may thus lead to cellular changes resulting in a more clinically aggressive phenotype. In addition, hypoxia is known to directly or indirectly confer resistance to X- and γ-radiation, and some chemotherapies leading to treatment failures. Whereas strong evidence has accumulated that hypoxia plays a pivotal role in tumor progression and acquired treatment resistance, the mechanism(s) by which treatment efficacy and survival may be compromised by anemia (independent of hypoxia) are not fully understood.

 

30. Detection and characterization of tumor hypoxia using pO2 histography.

Data from 125 studies describing the pretreatment oxygenation status as measured in the clinical setting using the computerized Eppendorf pO2 histography system have been compiled in this article. Tumor oxygenation is heterogeneous and severely compromised as compared to normal tissue. Hypoxia results from inadequate perfusion and diffusion within tumors and from a reduced O2 transport capacity in anemic patients. The development of tumor hypoxia is independent of a series of relevant tumor characteristics (e.g., clinical size, stage, histology, and grade) and various patient demographics. Overall median pO2 in cancers of the uterine cervix, head and neck, and breast is 10 mm Hg with the overall hypoxic fraction (pO2 ≤ 2.5 mm Hg) being approx. 25%. Metastatic lesions do not substantially deviate from the oxygenation status of (their) primary tumors. Whereas normal tissue oxygenation is independent of the hemoglobin level over the range of 8–15 g/dL, hypoxia is more pronounced in anemic patients and above this range in some cancers. Identification of tumor hypoxia may allow an assessment of a tumor's potential to develop an aggressive phenotype or acquired treatment resistance, both of which lead to poor prognosis. Detection of hypoxia in the clinical setting may therefore be helpful in selecting high-risk patients for individual and/or more intensive treatment schedules.

 

31. Effect of recombinant human erythropoietin (rHuEPO) on tumor control in patients with cancer-induced anemia.

It is well recognized that anemia-induced tumor hypoxia is associated with a reduced sensitivity of tumors to radi-ation and some forms of chemotherapy. Thus, the cor-rection of lower hemoglobin (Hb) concentrations with re-combinant human erythropoietin (rHuEPO) can play an essential role by improving tumor oxygenation. Based on evidence from a number of trials, treatment with rHuEPO will effectively ameliorate anemia and improve quality of life. However, one of the most essential prereq-uisites for achieving this benefit is the use of rHuEPO in agreement with the evidence-based ASCO/ASH-guide-lines recommending a target Hb concentration of 12 g/dl (7.44 mmol/l).

 

32. Recombinant human erythropoietin (rHuEPO) therapy in patients with cancer-related anaemia: What have we learned?

 

33. Effects of epoetin alfa on hematologic parameters and quality of life in cancer patients receiving non-platinum chemotherapy: Results of a randomized, double-blind, placebo-controlled trial.

 

PURPOSE: This randomized, double-blind, placebo-controlled clinical trial assessed the effects of epoetin alfa on transfusion requirements, hematopoietic parameters, quality of life (QOL), and safety in anemic cancer patients receiving nonplatinum chemotherapy. The study also explored a possible relationship between increased hemoglobin and survival.

PATIENTS AND METHODS: Three hundred seventy-five patients with solid or nonmyeloid hematologic malignancies and hemoglobin levels 10.5 g/dL, or greater than 10.5 g/dL but 12.0 g/dL after a hemoglobin decrease of 1.5 g/dL per cycle since starting chemotherapy, were randomized 2:1 to epoetin alfa 150 to 300 IU/kg (n = 251) or placebo (n = 124) three times per week subcutaneously for 12 to 24 weeks. The primary end point was proportion of patients transfused; secondary end points were change in hemoglobin and QOL. The protocol was amended before unblinding to prospectively collect and assess survival data 12 months after the last patient completed the study.

RESULTS: Epoetin alfa, compared with placebo, significantly decreased transfusion requirements (P = .0057) and increased hemoglobin (P < .001). Improvement of all primary cancer- and anemia-specific QOL domains, including energy level, ability to do daily activities, and fatigue, was significantly (P < .01) greater for epoetin alfa versus placebo patients. Although the study was not powered for survival as an end point, Kaplan-Meier estimates showed a trend in overall survival favoring epoetin alfa (P = .13, log-rank test), and Cox regression analysis showed an estimated hazards ratio of 1.309 (P = .052) favoring epoetin alfa. Adverse events were comparable between groups.

CONCLUSION: Epoetin alfa safely and effectively ameliorates anemia and significantly improves QOL in cancer patients receiving nonplatinum chemotherapy. Encouraging results regarding increased survival warrant another trial designed to confirm these findings.

 

34. Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors.

Hypoxia exists in most human and rodent solid tumors and has been shown to correlate with poor survival in carcinoma of the cervix, carcinoma of the head and neck, and soft tissue sarcoma. It exists both chronically, due to the poorly organized vasculature of solid tumors, and acutely, due to fluctuations in blood flow. It has been found that tumors that are more hypoxic are more likely to metastasize in humans and in rodent models, and it has been demonstrated that exposure of tumor cells to hypoxia in vitro can transiently enhance their metastatic potential when they are reinjected i.v. into mice. The purpose of the present study was to determine whether experimentally imposed hypoxic stress in vivo, either chronic or acute, affects the process of spontaneous metastasis in tumor-bearing mice. We exposed mice bearing KHT tumors to low oxygen conditions (5–7% O2 breathing) daily during tumor growth in an attempt to induce additional chronic (2 h/day) and acute (12 x 10 min/day) hypoxia in their tumors. By monitoring tumor pO2 levels over the course of treatment, we demonstrated that these treatments produce acute and chronic hypoxia within the tumor tissue. The acute but not the chronic hypoxia treatment significantly increased the number of spontaneous microscopic lung metastases in the mice by a factor of about 2, and the results suggest that this effect was due to the changes induced in the primary tumor. This study describes a novel method for studying the effects of hypoxia in solid tumors and demonstrates that acute and chronic hypoxia can have different effects on tumor cell behavior in vivo.

35. Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma.

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O2 followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2–4) as compared with control mice (1–3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.

 

36. Fluctuating and diffusion-limited hypoxia in hypoxia-induced metastasis.

Purpose: Most tumors develop regions with hypoxic cells during growth, owing to permanent limitations in oxygen diffusion (chronic or diffusion-limited hypoxia) and/or transient limitations in blood perfusion (acute or fluctuating hypoxia). The aim of this study was to investigate the relative significance of chronic and acute hypoxia in the development of metastatic disease.

Experimental Design: D-12 and R-18 human melanoma xenografts were used as models of human cancer. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Fraction of radiobiologically hypoxic cells (HFRad) was measured in individual primary tumors by using a radiobiological assay based on the paired survival curve method. Fraction of immunohistochemically hypoxic cells (HFImm) was assessed in the same tumors by using a pimonidazole-based immunohistochemical assay optimized with respect to achieving selective staining of chronically hypoxic cells. HFImm and the difference between HFRad and HFImm, HFRad − HFImm, were verified to be adequate variables for fraction of chronically hypoxic cells and fraction of acutely hypoxic cells, respectively.

Results: Chronic as well as acute hypoxia were found to promote spontaneous metastasis of D-12 and R-18 tumors. Acute hypoxia influenced metastasis to a greater extent than chronic hypoxia, partly because the fraction of acutely hypoxic cells was larger than the fraction of chronically hypoxic cells in most tumors and partly because acutely hypoxic cells showed a higher metastatic potential than chronically hypoxic cells.

Conclusions: It may be beneficial to focus on fluctuating hypoxia rather than diffusion-limited hypoxia when searching for hypoxia-related prognostic variables and predictive assays.

37. Erythropoiesis-stimulating agents: Favorable safety profile when used as indicated.

Background and Purpose:  Several studies with erythropoiesis-stimulating agents (ESAs) have raised a number of safety issues. Therefore, a discussion of available data in light of the current EORTC guidelines 2006 on the use of ESAs in anemic patients is warranted.

Methods:  Literature is reviewed with respect to experimental and clinical data on the effect of ESA therapy on tumor growth both in the preclinical setting and on patient survival.

Results:  Studies showing an adverse effect of ESA therapy on patient survival generally exhibit considerable methodological deficiencies. Moreover, they investigated treatment situations for which ESAs are not approved and/or did not involve recommended baseline (“intervention”) or target hemoglobin levels.

Conclusion:  When used as indicated, ESAs are valuable and safe drugs for the treatment of anemia and do not negatively affect patient survival. In particular, the data situation confirms the importance and correctness of the EORTC guidelines 2006 and their recently updated version. It is therefore recommended that these guidelines continue to be strictly followed in the treatment of chemotherapy-induced anemia.