University of Wisconsin–Madison
Albert Van der Kogel, PhD

Albert van der Kogel, PhD


Department of Human Oncology

During my active career my research has focused on two main areas. The first is on the effects of radiation on normal tissues, particularly the central nervous system and the various factors that determine the tolerance of the spinal cord. The spinal cord is probably the most critical normal tissue in radiation oncology, and my research has largely contributed to optimizing treatment of cancer close to the spine and preventing the development of debilitating complications. In addition to addressing important questions related to clinical radiation treatments, this research has elucidated the mechanisms and cellular basis of various radiation-induced changes in the CNS. My other research has aimed at overcoming radiation resistance mechanisms related to the tumor microenvironment, which are known to be mostly tumor hypoxia and proliferation. To study these aspects in a clinically relevant setting, my lab has developed a panel of patient-derived tumor xenografts that reflect the clinical heterogeneity of individual patients. The overall aim of this research has been to test new therapeutic modalities and develop assays to individualize treatment based on tumor biopsies and molecular imaging. Since my retirement in the Netherlands, I have had a part-time appointment in the UW Department of Human Oncology, where I consult and collaborate on various research projects and teach radiobiology courses for medical physics undergraduates and graduates and radiation oncology residents.


PhD, University of Amsterdam, Radiation Biology (1979)

MS, Free University of Amsterdam, Zoology and Radiation Biology (1971)

Academic Appointments

Professor, Human Oncology (2011–pres.)

Professor and Head of Laboratory (1987-2011), Radiation Oncology, Radboud University Nijmegen (1987–2011)

Associate Professor, University of New Mexico–Albuquerque (and Los Alamos National Laboratory) (1984–1987)

Research Associate, Radiobiological Institute TNO, Rijswijk, Netherlands (1972–1984)

Selected Honors and Awards

ESTRO Lifetime Achievement Award (2013)

ESTRO Honorary Physicist Award (2011)

ICRU Gray Medal (2009)

Honorary member, Polish Society of Radiation Oncology (2004)

Bacq & Alexander Award, European Society of Radiation Biology (2002)

John S. Laughlin Visiting Professor, Memorial Sloan Kettering Cancer Center, New York (2002)

Emmanuel van der Schueren Award, Belgian Society of Radiation Oncology, Leuven, Belgium (2001)

Charles Botstein Memorial Visiting Professor, Albert Einstein College of Medicine and Montefiore Hospital, NY (1997) (1997)

ESTRO Gold Medal, 13th Annual meeting of the European Society of Therapeutic Radiology and Oncology (1994)

Research Focus

Effects of Radiation on Normal Tissue, Particularly the Central Nervous System, Tumor Microenvironment

  • Corrigendum: Potential role of the glycolytic oscillator in acute hypoxia in tumors (2015 <i>Phys. Med. Biol.</i> 60 9215). Phys Med Biol
    Che Fru L, Adamson EB, Campos DD, Fain SB, Jacques SL, van der Kogel AJ, Nickel KP, Song C, Kimple RJ, Kissick MW
    2018 Jun 06; :
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      At the time of publication, our group had performed short tandem repeat (STR) testing on the SCC22B cell line and believed that had been correctly identified. As part of a recent comprehensive process to confirm the identity of cell lines in use in our lab, we repeated STR testing on all cell lines. These results were compared to the ExPASy Cellosaurus database ( One cell line used in this manuscript was a near perfect match for T24 (CVCL_0554), a bladder carcinoma cell line commonly found as a cellular contaminant. Although we are unable to test the exact cells used in this manuscript, we believe that the cells labeled as SCC22B are most likely to actually be T24. The authors believe that neither the results nor the conclusions have been significantly changed on the basis of the specific cell line utilized.

      View details for PubMedID 29873307
  • SU-E-T-06: A Mathematical Explanation to Tumor's Response to Perfusion and Hypoxic Fraction after Radiation. Med Phys
    Yan Y, Kissick M, Bussink J, Jacques S, van der Kogel A, Campos D, Zhao D
    2012 Jun; 39 (6Part9): 3703
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      PURPOSE: To develop a dynamic model that explains oxygen dynamics between the microvascular perfusion and the hypoxic cell population inside a tumor.

      METHODS: Bussink et al (Radiat Res 153(4), p.398 (2000)) observed fast oxygen dynamics, faster than cell-death. Based on a simplified three-compartment-model: the microvasculature, well-oxygenated, and hypoxic tumor cell populations. We applied a first-order differential model for the tumor's transient response as a function of oxygen content within the blood vessels. The sink terms in our model for each compartment are fast changing parameters because radiation rapidly changes the oxygen consumption of the tumor cell in a time scale which is much faster than the population changes of the tumor. Transportation balance condition is also applied for each compartment.

      RESULTS: Our simulation results can explain the experimental data in Bussink et al's (Radiat Res 153(4), p.398 (2000)) paper. We provide an explanation for the relative complex behavior of the microvascular perfusion after radiation that emphasizes the role of dynamic metabolic changes in addition to population changes.

      CONCLUSIONS: A newly developed dynamic model leads our understanding to the interrelationship between microvascular oxygen content within the blood vessels and the hypoxia state of the tumor to a deeper level, which has the potential to provide the theoretical foundation for the patient' specific adaptive radiotherapy.

      View details for PubMedID 28519053
  • SU-E-J-196: In-Vivo Tumor Blood-Oxygen Content Measurement via Interstitial Optical Transmission Spectroscopy. Med Phys
    Campos D, Yan Y, Zhao D, Kissick M, Jacques S, van der Kogel A, Kimple R
    2012 Jun; 39 (6Part9): 3698
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      PURPOSE: To demonstrate the application of in-vivo diffuse optical transmission spectroscopy in quantifying oxygen saturation in interstitial tissue, and to use this technique to examine reoxygenation dynamics in real-time as tumors responds to radiotherapy.

      METHODS: Two 200 micron core fiber optics were threaded through two 21 gauge hypodermic needles: one coupled to an OceanOptics QE65000 spectrometer, and the other to an Ocean Optics HL-2000-HP 20W light source. These needles were fixed approximately 3 mm apart, and inserted into nude mice with human head- and-neck tumor xenografts. The oxygen saturation was then measured as a function of time after irradiation at intervals of 0.5, 1, 2, 6, 12, and 24, to measure the tumors' prompt oxygen saturation response to radiation.

      RESULTS: Blood volume, deoxy and oxy-hemoglobin concentrations were measured through least-squares fitting of transmission spectra. Furthermore, various configurations of interstitial fiber optic probes were explored to optimize signal strength. Improvement of the optical coupling to the biological system and a concurrent increase in source intensity are the main two focuses for boosting signal strength.

      CONCLUSIONS: This work has the potential to give an understanding of the time-scales of hypoxia and reoxygenation in vivo as tumors respond to radiation injury. This technique is of particular interest for hypofractionated therapies particularly treatments of only two or three treatments, where optimizing treatment timing can increase the tumorcidal effect of the remaining fractions.

      View details for PubMedID 28519036
  • SU-E-J-197: A Novel Optical Interstitial Fiber Spectroscopic System for Real-Time Tissue Micro-Vascular Hemodynamics Monitoring. Med Phys
    Zhao D, Campos D, Yan Y, Kimple R, Jacques S, van der Kogel A, Kissick M
    2012 Jun; 39 (6Part9): 3698
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      PURPOSE: To demonstrate a novel interstitial optical fiber spectroscopic system, based on diffuse optical spectroscopies with spectral fitting, for the simultaneous monitoring of tumor blood volume and oxygen tension. The technique provides real-time, minimally-invasive and quantification of tissue micro-vascular hemodynamics.

      METHODS: An optical fiber prototype probe characterizesthe optical transport in tissue between two large Numerical Aperture (NA) fibers of 200μm core diameter (BFH37-200, ThorLabs) spaced 3-mm apart. Two 21-Ga medical needles are used to protect fiber ends and to facilitate tissue penetration with minimum local blunt trauma in nude mice with xenografts. A 20W white light source (HL-2000-HP, Ocean Optics) is coupled to one fiber with SMA adapter. The other fiber is used to collect light, which is coupled into the spectrometer (QE65000 with Spectrasuite Operating software and OmniDriver, Ocean Optics). The wavelength response of the probe depends on the wavelength dependence of the light source, and of the light signal collection that includes considerable scatter, modeled with Monte-Carlo techniques (S. Jacques 2010 J. of Innov. Opt. Health Sci. 2 123-9). Measured spectra of tissue are normalized by a measured spectrum of a white standard, yielding the transmission spectrum. A head-and-neck xenograft on the flank of a live mouse is used for development.

      RESULTS: The optical fiber probe delivers and collects light at an arbitrary depth in the tumor. By spectral fitting of the measured transmission spectrum, an analysis of blood volume and oxygen tension is obtained from the fitting parameters in real time.

      CONCLUSIONS: A newly developed optical fiber spectroscopic system with an optical fiber probe takes spectroscopic techniques to a much deeper level in a tumor, which has potential applications for real-time monitoring hypoxic cell population dynamics for an eventual adaptive therapy metric of particular use in hypofractionated radiotherapy.

      View details for PubMedID 28519022
  • Obituary and Tribute to John "Jack" Francis Fowler, PhD, DSc (1925-2016). Int J Radiat Oncol Biol Phys
    Harari PM, Ritter MA, van der Kogel AJ
    2017 04 01; 97 (5): 886-888
  • Radiation Promptly Alters Cancer Live Cell Metabolic Fluxes: An In Vitro Demonstration. Radiat Res
    Campos D, Peeters W, Nickel K, Burkel B, Bussink J, Kimple RJ, van der Kogel A, Eliceiri KW, Kissick MW
    2016 May; 185 (5): 496-504
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      Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

      View details for PubMedID 27128739
  • Potential role of the glycolytic oscillator in acute hypoxia in tumors. Phys Med Biol
    Fru LC, Adamson EB, Campos DD, Fain SB, Jacques SL, van der Kogel AJ, Nickel KP, Song C, Kimple RJ, Kissick MW
    2015 Dec 21; 60 (24): 9215-25
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      Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation.

      View details for PubMedID 26576743
  • Antitumor Effects of MEHD7945A, a Dual-Specific Antibody against EGFR and HER3, in Combination with Radiation in Lung and Head and Neck Cancers. Mol Cancer Ther
    Li C, Huang S, Armstrong EA, Francis DM, Werner LR, Sliwkowski MX, van der Kogel A, Harari PM
    2015 Sep; 14 (9): 2049-59
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      Human epidermal growth factor receptor family members (EGFR, HER2, HER3, and HER4) play important roles in tumorigenesis and response to cancer therapeutics. In this study, we evaluated the capacity of the dual-target antibody MEHD7945A that simultaneously targets EGFR and HER3 to modulate radiation response in lung and head and neck cancer models. Antitumor effects of MEHD7945A in combination with radiation were evaluated in cell culture and tumor xenograft models. Mechanisms that may contribute to increased radiation killing by MEHD7945A, including DNA damage and inhibition of EGFR-HER signaling pathways, were analyzed. Immunohistochemical analysis of tumor xenografts was conducted to evaluate the effect of MEHD7945A in combination with radiation on tumor growth and microenvironment. MEHD7945A inhibited basal and radiation-induced EGFR and HER3 activation resulting in the inhibition of tumor cell growth and enhanced radiosensitivity. MEHD7945A was more effective in augmenting radiation response than treatment with individual anti-EGFR or anti-HER3 antibodies. An increase in DNA double-strand breaks associated γ-H2AX was observed in cells receiving combined treatment with MEHD7945A and radiation. Immunohistochemical staining evaluation in human tumor xenografts showed that MEHD7945A combined with radiation significantly reduced the expression of markers of tumor proliferation and tumor vasculature. These findings reveal the capacity of MEHD7945A to augment radiation response in lung and head and neck cancers. The dual EGFR/HER3-targeting action of MEHD7945A merits further investigation and clinical trial evaluation as a radiation sensitizer in cancer therapy.

      View details for PubMedID 26141946
  • Hypoxia, metabolism, and growth factor signaling in head and neck squamous cell carcinoma: correlation between primary and xenograft tumors. Head Neck
    Stegeman H, Rademakers SE, Span PN, Takes RP, van der Kogel AJ, Kaanders JH, Bussink J
    2014 Sep; 36 (9): 1288-95
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      BACKGROUND: Hypoxia, metabolism, and growth factor signaling are important prognostic features in most solid tumors. The purpose of this study was to determine whether head and neck squamous cell carcinoma (HNSCC) xenografts show similar biological and molecular characteristics as the primary tumor they originate from.

      METHODS: Eighteen HNSCC primary tumor-xenograft pairs were immunofluorescently stained for pimonidazole (hypoxia), carbonic anhydrase IX (CAIX), glucose transporter-1 (GLUT-1), monocarboxylate transporter-1 (MCT-1), monocarboxylate transporter-4 (MCT-4), epidermal growth factor receptor (EGFR), and phosphorylated protein kinase B (pAKT).

      RESULTS: Although no correlation was found for the amount of hypoxia, significant correlations between primary tumors and xenografts were observed for both the percentage of cells positive for expression and the hypoxia-related expression pattern of CAIX, GLUT-1, and MCT-1. For EGFR and MCT-4, the intensity of expression was correlated. No correlation was observed for pAKT.

      CONCLUSION: Xenografts did not always resemble the primary tumor they originate from, but the xenografts did represent the variability in expression levels and patterns observed in the primary tumors.

      View details for PubMedID 24668936
  • Systematic analysis of 18F-FDG PET and metabolism, proliferation and hypoxia markers for classification of head and neck tumors. BMC Cancer
    Hoeben BA, Starmans MH, Leijenaar RT, Dubois LJ, van der Kogel AJ, Kaanders JH, Boutros PC, Lambin P, Bussink J
    2014 Feb 26; 14: 130
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      BACKGROUND: Quantification of molecular cell processes is important for prognostication and treatment individualization of head and neck cancer (HNC). However, individual tumor comparison can show discord in upregulation similarities when analyzing multiple biological mechanisms. Elaborate tumor characterization, integrating multiple pathways reflecting intrinsic and microenvironmental properties, may be beneficial to group most uniform tumors for treatment modification schemes. The goal of this study was to systematically analyze if immunohistochemical (IHC) assessment of molecular markers, involved in treatment resistance, and 18F-FDG PET parameters could accurately distinguish separate HNC tumors.

      METHODS: Several imaging parameters and texture features for 18F-FDG small-animal PET and immunohistochemical markers related to metabolism, hypoxia, proliferation and tumor blood perfusion were assessed within groups of BALB/c nu/nu mice xenografted with 14 human HNC models. Classification methods were used to predict tumor line based on sets of parameters.

      RESULTS: We found that 18F-FDG PET could not differentiate between the tumor lines. On the contrary, combined IHC parameters could accurately allocate individual tumors to the correct model. From 9 analyzed IHC parameters, a cluster of 6 random parameters already classified 70.3% correctly. Combining all PET/IHC characteristics resulted in the highest tumor line classification accuracy (81.0%; cross validation 82.0%), which was just 2.2% higher (p = 5.2×10-32) than the performance of the IHC parameter/feature based model.

      CONCLUSIONS: With a select set of IHC markers representing cellular processes of metabolism, proliferation, hypoxia and perfusion, one can reliably distinguish between HNC tumor lines. Addition of 18F-FDG PET improves classification accuracy of IHC to a significant yet minor degree. These results may form a basis for development of tumor characterization models for treatment allocation purposes.

      View details for PubMedID 24571588
  • Prognostic value of the proliferation marker Ki-67 in laryngeal carcinoma: results of the accelerated radiotherapy with carbogen breathing and nicotinamide phase III randomized trial. Head Neck
    Rademakers SE, Hoogsteen IJ, Rijken PF, Terhaard CH, Doornaert PA, Langendijk JA, van den Ende P, van der Kogel AJ, Bussink J, Kaanders JH
    2015 Feb; 37 (2): 171-6
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      BACKGROUND: The prognostic and predictive value of the proliferation marker Ki-67 was investigated in a randomized trial comparing accelerated radiotherapy with carbogen breathing and nicotinamide (ARCON) to accelerated radiotherapy in laryngeal carcinoma.

      METHODS: Labeling index of Ki-67 (Li Ki-67) in immunohistochemically stained biopsies and the colocalization with carbonic anhydrase IX (CAIX) were related to tumor control and patient survival.

      RESULTS: On average, node-positive patients had a higher Li Ki-67 (median 14% vs 8%; p < .01). In patients with a high Li Ki-67, the 5-year regional control and metastases-free survival were 79% versus 96% (p < .01) and 71% versus 88% (p = .05) for accelerated radiotherapy and ARCON, respectively. The 5-year local control and disease-specific survival were not significantly different. Patients with low Ki-67 expression had an excellent outcome with accelerated radiotherapy alone.

      CONCLUSION: Patients with laryngeal carcinomas with high proliferative activity are at increased risk of regional and distant metastases formation. This risk can be reduced by treatment with ARCON.

      View details for PubMedID 24347430
  • Combining radiotherapy with MEK1/2, STAT5 or STAT6 inhibition reduces survival of head and neck cancer lines. Mol Cancer
    Stegeman H, Kaanders JH, Verheijen MM, Peeters WJ, Wheeler DL, Iida M, Grénman R, van der Kogel AJ, Span PN, Bussink J
    2013 Nov 05; 12 (1): 133
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      BACKGROUND: Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.

      METHODS: An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.

      RESULTS: The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.

      CONCLUSIONS: Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.

      View details for PubMedID 24192080
  • Molecular radiation biology/oncology at its best: cutting edge research presented at the 13th International Wolfsberg Meeting on Molecular Radiation Biology/Oncology. Radiother Oncol
    Baumann M, Bodis S, Dikomey E, van der Kogel A, Overgaard J, Rodemann HP, Wouters B
    2013 Sep; 108 (3): 357-61
  • On the importance of prompt oxygen changes for hypofractionated radiation treatments. Phys Med Biol
    Kissick M, Campos D, van der Kogel A, Kimple R
    2013 Oct 21; 58 (20): N279-85
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      This discussion is motivated by observations of prompt oxygen changes occurring prior to a significant number of cancer cells dying (permanently stopping their metabolic activity) from therapeutic agents like large doses of ionizing radiation. Such changes must be from changes in the vasculature that supplies the tissue or from the metabolic changes in the tissue itself. An adapted linear-quadratic treatment is used to estimate the cell survival variation magnitudes from repair and reoxygenation from a two-fraction treatment in which the second fraction would happen prior to significant cell death from the first fraction, in the large fraction limit. It is clear the effects of oxygen changes are likely to be the most significant factor for hypofractionation because of large radiation doses. It is a larger effect than repair. Optimal dose timing should be determined by the peak oxygen timing. A call is made to prioritize near real time measurements of oxygen dynamics in tumors undergoing hypofractionated treatments in order to make these treatments adaptable and patient-specific.

      View details for PubMedID 24061351
  • Paralysis following stereotactic spinal irradiation in pigs suggests a tolerance constraint for single-session irradiation of the spinal nerve. Radiother Oncol
    Medin PM, Foster RD, van der Kogel AJ, Meyer J, Sayre JW, Huang H, Öz OK
    2013 Oct; 109 (1): 107-11
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      BACKGROUND AND PURPOSE: Paralysis observed during a study of vertebral bone tolerance to single-session irradiation led to further study of the dose-related incidence of motor peripheral neuropathy.

      MATERIALS AND METHODS: During a bone tolerance study, cervical spinal nerves of 15 minipigs received bilateral irradiation to levels C5-C8 distributed into three dose groups with mean maximum spinal nerve doses of 16.9 ± 0.3 Gy (n=5), 18.7 ± 0.5 Gy (n=5), and 24.3 ± 0.8 Gy (n=5). Changes developing in the gait of the group of pigs receiving a mean maximum dose of 24.3 Gy after 10-15 weeks led to the irradiation of two additional animals. They received mean maximum dose of 24.9 ± 0.2 Gy (n=2), targeted to the left spinal nerves of C5-C8. The followup period was one year. Histologic sections from spinal cords and available spinal nerves were evaluated. MR imaging was performed on pigs in the 24.9 Gy group.

      RESULTS: No pig that received a maximum spinal nerve point dose ≤19.0 Gy experienced a change in gait while all pigs that received ≥24.1 Gy experienced paralysis. Extensive degeneration and fibrosis were observed in irradiated spinal nerves of the 24.9 Gy animals. All spinal cord sections were normal. Irradiated spinal nerve regions showed increased thickness and hypointensity on MR imaging.

      CONCLUSION: The single-session tolerance dose of the cervical spinal nerves lies between 19.0 and 24.1 Gy for this model.

      View details for PubMedID 24060168
  • EGFR-inhibition enhances apoptosis in irradiated human head and neck xenograft tumors independent of effects on DNA repair. Radiat Res
    Stegeman H, Span PN, Cockx SC, Peters JP, Rijken PF, van der Kogel AJ, Kaanders JH, Bussink J
    2013 Oct; 180 (4): 414-21
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      Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.

      View details for PubMedID 24059677
  • Human epidermal growth factor receptor 3 (HER3) blockade with U3-1287/AMG888 enhances the efficacy of radiation therapy in lung and head and neck carcinoma. Discov Med
    Li C, Brand TM, Iida M, Huang S, Armstrong EA, van der Kogel A, Wheeler DL
    2013 Sep; 16 (87): 79-92
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      HER3 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In the present study, we investigated the capacity of the HER3 blocking antibody, U3-1287/AMG888, to modulate the in vitro and in vivo radiation response of human squamous cell carcinomas of the lung and head and neck. We screened a battery of cell lines from these tumors for HER3 expression and demonstrated that all cell lines screened exhibited expression of HER3. Importantly, U3-1287/AMG888 treatment could block both basal HER3 activity and radiation induced HER3 activation. Proliferation assays indicated that HER3 blockade could decrease the proliferation of both HNSCC cell line SCC6 and NSCLC cell line H226. Further, we demonstrated that U3-1287/AMG888 can sensitize cells to radiation in clonogenic survival assays, in addition to increasing DNA damage as detected via λ-H2AX immunofluorescence. To determine if U3-1287/AMG888 could enhance radiation sensitivity in vivo we performed tumor growth delay experiments using SCC6, SCC1483, and H226 xenografts. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation could decrease tumor growth in studies using single or fractionated doses of radiation. Analysis of HER3 expression in tumor samples indicated that radiation treatment activated HER3 in vivo and that U3-1287/AMG888 could abrogate this activation. Immunohistochemistry analysis of SCC6 tumors treated with both U3-1287/AMG888 and a single dose of radiation demonstrated that various cell survival and proliferation markers could be reduced. Collectively our findings suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by increasing DNA damage and cell death. These findings suggest that HER3 may play an important role in response to radiation therapy and blocking its activity in combination with radiation may be of therapeutic benefit in human tumors.

      View details for PubMedID 23998444
  • Dasatinib Inhibits DNA Repair after Radiotherapy Specifically in pSFK-Expressing Tumor Areas in Head and Neck Xenograft Tumors. Transl Oncol
    Stegeman H, Span PN, Rijken PF, Cockx SC, Wheeler DL, Iida M, van der Kogel AJ, Kaanders JH, Bussink J
    2013 Aug; 6 (4): 413-9
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      Src family kinases (SFKs) have been implicated in resistance to both radiation and epidermal growth factor receptor (EGFR) inhibition. Therefore, we investigated whether inhibition of SFK through dasatinib (DSB) can enhance the effect of radiotherapy in two in vivo human head and neck squamous cell carcinoma (HNSCC) models. Response to DSB and/or radiotherapy was assessed with tumor growth delay assays in two HNSCC xenograft models, SCCNij153 and SCCNij202. Effects on EGFR signaling were evaluated with Western blot analysis, and effects on DNA repair, hypoxia, and proliferation were investigated with immunohistochemistry. DSB and radiotherapy induced a significant growth delay in both HNSCC xenograft models, although to a lesser extent in SCCNij202. DSB did not inhibit phosphorylated protein kinase B (pAKT) or phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) but did inhibit (phosphorylated) DNA-dependent protein kinase. Moreover, DSB reduced repair of radiation-induced DNA double-strand breaks as shown by an increase of p53-binding protein 1 (53BP1) staining 24 hours after radiation. This effect on DNA repair was only observed in the cell compartment where phosphorylated SFK (pSFK) was expressed: for SCCNij153 tumors in both normoxic and hypoxic areas and for SCCNij202 tumors only in hypoxic areas. No consistent effects of DSB on hypoxia or proliferation were observed. In conclusion, DSB enhances the effect of radiotherapy in vivo by inhibition of radiation-induced DNA repair and is a promising way to improve outcome in HNSCC patients.

      View details for PubMedID 23908684
  • The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response. Radiother Oncol
    Nagelkerke A, Bussink J, van der Kogel AJ, Sweep FC, Span PN
    2013 Sep; 108 (3): 415-21
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      BACKGROUND AND PURPOSE: Lysosome-associated membrane protein 3 (LAMP3) is induced by the PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the unfolded protein response (UPR) during hypoxia. LAMP3 has prognostic value in breast cancer patients treated with radiotherapy. Here, we specifically investigated the role of the PERK/ATF4/LAMP3-arm in the radiation response of breast cancer cells.

      MATERIAL AND METHODS: Radiosensitivity of breast cancer cells was examined after siRNA-mediated knockdown of PERK, ATF4 and LAMP3. Activation of DNA damage repair proteins was evaluated by Western blotting and immunocytochemistry.

      RESULTS: Knockdown of the PERK/ATF4/LAMP3-arm and chemical inhibition of PERK could radiosensitise MDA-MB-231 cells significantly. Western blot analysis of several DNA damage repair proteins showed that LAMP3 knockdowns had an attenuated DNA damage response after radiation compared to controls. γ-H2AX foci analysis revealed that LAMP3 knockdowns had a reduced number of positive cells after irradiation, indicating that their DNA damage repair signalling response is decreased. In addition, the effect of autophagy inhibition was examined and revealed a radiosensitising effect and the presence of residual γ-H2AX foci.

      CONCLUSIONS: The PERK/ATF4/LAMP3-arm causes radioresistance of breast cancer cells by increasing DNA damage repair signalling. Inhibition of PERK and/or autophagy may sensitise tumours to radiotherapy.

      View details for PubMedID 23891100
  • Epidermal growth factor receptor expression in laryngeal cancer predicts the effect of hypoxia modification as an additive to accelerated radiotherapy in a randomised controlled trial. Eur J Cancer
    Nijkamp MM, Span PN, Terhaard CH, Doornaert PA, Langendijk JA, van den Ende PL, de Jong M, van der Kogel AJ, Bussink J, Kaanders JH
    2013 Oct; 49 (15): 3202-9
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      Accelerated radiotherapy (AR) improves the poor prognosis associated with epidermal growth factor receptor (EGFR) overexpression frequently seen in head and neck carcinomas. Combining AR with carbogen and nicotinamide (ARCON) counteracts enhanced tumour cell proliferation- and hypoxia-related radioresistance. The purpose of this study was to investigate if EGFR expression levels are associated with response to ARCON in patients with carcinoma of the larynx. Patients (N=272) with advanced stage larynx carcinoma were randomised between AR alone and ARCON. Paraffin-embedded biopsies from these patients were processed for immunohistochemical staining of EGFR. EGFR fraction was quantitated by automated image analysis and related to clinical outcome. A large variation was observed in EGFR fraction between tumours with expression levels ranging from 0 to 0.93 (median fraction 0.4). No difference in 5-year locoregional control was found between low and high EGFR expressing tumours in the AR arm (69% versus 75%), which is in line with the established effect of AR in EGFR overexpressing tumours. There was, however, a significant association in the ARCON arm: patients with low EGFR levels had a better 5-year locoregional control (88% versus 72% p=0.02) and disease-specific survival (92% versus 77% p=0.01). ARCON improved locoregional control relative to AR only in patients with low EGFR expression (hazard ratio (HR) 0.34 p=0.009). In conclusion, only in tumours with a low EGFR fraction, adding hypoxia modification to AR has an additive beneficial effect on outcome. EGFR expression is a predictive biomarker for the selection of patients that will or will not respond to ARCON.

      View details for PubMedID 23867129
  • Evaluation and immunohistochemical qualification of carbogen-induced ΔR₂ as a noninvasive imaging biomarker of improved tumor oxygenation. Int J Radiat Oncol Biol Phys
    Baker LC, Boult JK, Jamin Y, Gilmour LD, Walker-Samuel S, Burrell JS, Ashcroft M, Howe FA, Griffiths JR, Raleigh JA, van der Kogel AJ, Robinson SP
    2013 Sep 01; 87 (1): 160-7
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      PURPOSE: To evaluate and histologically qualify carbogen-induced ΔR2 as a noninvasive magnetic resonance imaging biomarker of improved tumor oxygenation using a double 2-nitroimidazole hypoxia marker approach.

      METHODS AND MATERIALS: Multigradient echo images were acquired from mice bearing GH3 prolactinomas, preadministered with the hypoxia marker CCI-103F, to quantify tumor R2 during air breathing. With the mouse remaining positioned within the magnet bore, the gas supply was switched to carbogen (95% O2, 5% CO2), during which a second hypoxia marker, pimonidazole, was administered via an intraperitoneal line, and an additional set of identical multigradient echo images acquired to quantify any changes in tumor R2. Hypoxic fraction was quantified histologically using immunofluorescence detection of CCI-103F and pimonidazole adduct formation from the same whole tumor section. Carbogen-induced changes in tumor pO2 were further validated using the Oxylite fiberoptic probe.

      RESULTS: Carbogen challenge significantly reduced mean tumor R2 from 116 ± 13 s(-1) to 97 ± 9 s(-1) (P<.05). This was associated with a significantly lower pimonidazole adduct area (2.3 ± 1%), compared with CCI-103F (6.3 ± 2%) (P<.05). A significant correlation was observed between ΔR2 and Δhypoxic fraction (r=0.55, P<.01). Mean tumor pO2 during carbogen breathing significantly increased from 6.3 ± 2.2 mm Hg to 36.0 ± 7.5 mm Hg (P<.01).

      CONCLUSIONS: The combined use of intrinsic susceptibility magnetic resonance imaging with a double hypoxia marker approach corroborates carbogen-induced ΔR2 as a noninvasive imaging biomarker of increased tumor oxygenation.

      View details for PubMedID 23849692
  • Pattern of CAIX expression is prognostic for outcome and predicts response to ARCON in patients with laryngeal cancer treated in a phase III randomized trial. Radiother Oncol
    Rademakers SE, Hoogsteen IJ, Rijken PF, Oosterwijk E, Terhaard CH, Doornaert PA, Langendijk JA, van den Ende P, Takes R, De Bree R, van der Kogel AJ, Bussink J, Kaanders JH
    2013 Sep; 108 (3): 517-22
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      BACKGROUND AND PURPOSE: In a phase III trial in patients with advanced stage laryngeal carcinoma comparing ARCON (accelerated radiotherapy with carbogen breathing and nicotinamide) to accelerated radiotherapy alone (AR) the prognostic and predictive value of CAIX, a hypoxia-associated protein, was investigated.

      MATERIAL AND METHODS: 261 Paraffin embedded tumor biopsies and 79 fresh frozen biopsies from patients entered in the trial were immunohistochemically stained for CAIX. CAIX-fraction and CAIX expression pattern were related to tumor control and patient survival.

      RESULTS: Low CAIX-fraction was prognostic for worse regional control and overall survival in patients treated with AR. Patients with a low CAIX-fraction treated with ARCON had better regional control and metastasis-free survival compared to AR (RC 97% vs 71%, p < 0.01 and MFS 92% vs 69%, p = 0.06). Patients with a perinecrotic CAIX staining pattern had a significantly worse local control, metastasis-free and overall survival compared to patients with a diffuse pattern (65% vs 84%, p = 0.01, 70% vs 96%, p < 0.01 and 42% vs 71%, p < 0.01 respectively), and this could not be improved with ARCON. After multivariate analysis CAIX pattern and N-stage emerged as significant predictors for metastasis-free survival and overall survival.

      CONCLUSIONS: ARCON improves regional control and metastasis-free survival only in patients with low CAIX expression. The different patterns of CAIX expression suggest different mechanisms of upregulation and have important prognostic value.

      View details for PubMedID 23719582
  • PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS. Proc Natl Acad Sci U S A
    Rouschop KM, Dubois LJ, Keulers TG, van den Beucken T, Lambin P, Bussink J, van der Kogel AJ, Koritzinsky M, Wouters BG
    2013 Mar 19; 110 (12): 4622-7
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      Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

      View details for PubMedID 23471998
  • Predictive value of hypoxia, proliferation and tyrosine kinase receptors for EGFR-inhibition and radiotherapy sensitivity in head and neck cancer models. Radiother Oncol
    Stegeman H, Kaanders JH, van der Kogel AJ, Iida M, Wheeler DL, Span PN, Bussink J
    2013 Mar; 106 (3): 383-9
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      BACKGROUND AND PURPOSE: EGFR-inhibitor Cetuximab (C225) improves the efficacy of radiotherapy in only a subgroup of HNSCC patients. Identification of predictive tumor characteristics is essential to improve patient selection.

      MATERIAL AND METHODS: Response to C225 and/or radiotherapy was assessed with tumor growth delay assays in 4 HNSCC xenograft models with varying EGFR-expression levels. Hypoxia and proliferation were quantified with immunohistochemistry and the expression of proteins involved in C225-resistance with Western blot.

      RESULTS: EGFR-expression did not predict response to C225 and/or radiotherapy. Reduction of hypoxia by C225 was only observed in SCCNij202, which was highly sensitive to C225. Proliferation changes correlated with response to C225 and C225 combined with radiotherapy, as proliferation decreased after C225 treatment in C225-sensitive SCCNij202 and after combined treatment in SCCNij185, which showed a synergistic effect to combined C225-radiotherapy. Furthermore, C225-resistant SCCNij153 tumors expressed high levels of (activated) HER3 and MET.

      CONCLUSIONS: EGFR-expression is needed for C225-response, but is not sufficient to predict response to C225 with or without radiotherapy. However, basal expression of additional growth factor receptors and effects on proliferation, but not hypoxia, correlated with response to combined C225-radiotherapy treatment and are potential clinically relevant predictive biomarkers.

      View details for PubMedID 23453541
  • Bioeffect modeling and equieffective dose concepts in radiation oncology--terminology, quantities and units. Radiother Oncol
    Bentzen SM, Dörr W, Gahbauer R, Howell RW, Joiner MC, Jones B, Jones DT, van der Kogel AJ, Wambersie A, Whitmore G
    2012 Nov; 105 (2): 266-8
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      The International Commission on Radiation Units and Measurements (ICRU) Report Committee on "Bioeffect Modeling and Biologically Equivalent Dose Concepts in Radiation Therapy" is currently developing a comprehensive and consistent framework for radiobiological effect modeling based on the equieffective dose, EQDX(α/β), a concept encompassing BED and EQD2 as special cases.

      View details for PubMedID 23157980
  • Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck. BMC Cancer
    Stegeman H, Kaanders JH, Wheeler DL, van der Kogel AJ, Verheijen MM, Waaijer SJ, Iida M, Grénman R, Span PN, Bussink J
    2012 Oct 10; 12: 463
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      BACKGROUND: Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.

      METHODS: Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O(2) and treated with MK-2206.

      RESULTS: We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs = 0.77, p = 0.10, rs = 0.89, p = 0.03) and rs = 0.93, p = 0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all r(s)<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs = 0.51, p = 0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.

      CONCLUSIONS: These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.

      View details for PubMedID 23046567
  • Spinal cord tolerance to single-session uniform irradiation in pigs: implications for a dose-volume effect. Radiother Oncol
    Medin PM, Foster RD, van der Kogel AJ, Sayre JW, McBride WH, Solberg TD
    2013 Jan; 106 (1): 101-5
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      BACKGROUND AND PURPOSE: This study was performed to test the hypothesis that spinal cord radiosensitivity is significantly modified by uniform versus laterally non-uniform dose distributions.

      MATERIALS AND METHODS: A uniform dose distribution was delivered to a 4.5-7.0 cm length of cervical spinal cord in 22 mature Yucatan minipigs for comparison with a companion study in which a laterally non-uniform dose was given [1]. Pigs were allocated into four dose groups with mean maximum spinal cord doses of 17.5 ± 0.1 Gy (n=7), 19.5 ± 0.2 Gy (n=6), 22.0 ± 0.1 Gy (n=5), and 24.1 ± 0.2 Gy (n=4). The study endpoint was motor neurologic deficit determined by a change in gait within one year. Spinal cord sections were stained with a Luxol fast blue/periodic acid Schiff combination.

      RESULTS: Dose-response curves for uniform versus non-uniform spinal cord irradiation were nearly identical with ED(50)'s (95% confidence interval) of 20.2 Gy (19.1-25.8) and 20.0 Gy (18.3-21.7), respectively. No neurologic change was observed for either dose distribution when the maximum spinal cord dose was ≤ 17.8 Gy while all animals experienced deficits at doses ≥ 21.8 Gy.

      CONCLUSION: No dose-volume effect was observed in pigs for the dose distributions studied and the endpoint of motor neurologic deficit; however, partial spinal cord irradiation resulted in less debilitating neurologic morbidity and histopathology.

      View details for PubMedID 22985780
  • (64)Cu-ATSM and (18)FDG PET uptake and (64)Cu-ATSM autoradiography in spontaneous canine tumors: comparison with pimonidazole hypoxia immunohistochemistry. Radiat Oncol
    Hansen AE, Kristensen AT, Jørgensen JT, McEvoy FJ, Busk M, van der Kogel AJ, Bussink J, Engelholm SA, Kjær A
    2012 Jun 15; 7: 89
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      BACKGROUND: The aim of this study was to compare (64)Cu-diacetyl-bis(N(4)-methylsemicarbazone) ((64)Cu-ATSM) and (18)FDG PET uptake characteristics and (64)Cu-ATSM autoradiography to pimonidazole immunohistochemistry in spontaneous canine sarcomas and carcinomas.

      METHODS: Biopsies were collected from individual tumors between approximately 3 and 25 hours after the intravenous injection of (64)Cu-ATSM and pimonidazole. (64)Cu-ATSM autoradiography and pimonidazole immunostaining was performed on sectioned biopsies. Acquired (64)Cu-ATSM autoradiography and pimonidazole images were rescaled, aligned and their distribution patterns compared. (64)Cu-ATSM and (18)FDG PET/CT scans were performed in a concurrent study and uptake characteristics were obtained for tumors where available.

      RESULTS: Maximum pimonidazole pixel value and mean pimonidazole labeled fraction was found to be strongly correlated to (18)FDG PET uptake levels, whereas more varying results were obtained for the comparison to (64)Cu-ATSM. In the case of the latter, uptake at scans performed 3 h post injection (pi) generally showed strong positive correlated to pimonidazole uptake.Comparison of distribution patterns of pimonidazole immunohistochemistry and (64)Cu-ATSM autoradiography yielded varying results. Significant positive correlations were mainly found in sections displaying a heterogeneous distribution of tracers.

      CONCLUSIONS: Tumors with high levels of pimonidazole staining generally displayed high uptake of (18)FDG and (64)Cu-ATSM (3 h pi.). Similar regional distribution of (64)Cu-ATSM and pimonidazole was observed in most heterogeneous tumor regions. However, tumor and hypoxia level dependent differences may exist with regard to the hypoxia specificity of (64)Cu-ATSM in canine tumors.

      View details for PubMedID 22704363
  • Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer. Radiat Oncol
    Røe K, Mikalsen LT, van der Kogel AJ, Bussink J, Lyng H, Ree AH, Marignol L, Olsen DR
    2012 May 23; 7: 75
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      BACKGROUND: Radiotherapy (RT) and androgen-deprivation therapy (ADT) are standard treatments for advanced prostate cancer (PC). Tumor vascularization is recognized as an important physiological feature likely to impact on both RT and ADT response, and this study therefore aimed to characterize the vascular responses to RT and ADT in experimental PC.

      METHODS: Using mice implanted with CWR22 PC xenografts, vascular responses to RT and ADT by castration were visualized in vivo by DCE MRI, before contrast-enhancement curves were analyzed both semi-quantitatively and by pharmacokinetic modeling. Extracted image parameters were correlated to the results from ex vivo quantitative fluorescent immunohistochemical analysis (qIHC) of tumor vascularization (9 F1), perfusion (Hoechst 33342), and hypoxia (pimonidazole), performed on tissue sections made from tumors excised directly after DCE MRI.

      RESULTS: Compared to untreated (Ctrl) tumors, an improved and highly functional vascularization was detected in androgen-deprived (AD) tumors, reflected by increases in DCE MRI parameters and by increased number of vessels (VN), vessel density (VD), and vessel area fraction (VF) from qIHC. Although total hypoxic fractions ( HF) did not change, estimated acute hypoxia scores (AHS)--the proportion of hypoxia staining within 50 μm from perfusion staining--were increased in AD tumors compared to in Ctrl tumors. Five to six months after ADT renewed castration-resistant (CR) tumor growth appeared with an even further enhanced tumor vascularization. Compared to the large vascular changes induced by ADT, RT induced minor vascular changes. Correlating DCE MRI and qIHC parameters unveiled the semi-quantitative parameters area under curve (AUC) from initial time-points to strongly correlate with VD and VF, whereas estimation of vessel size (VS) by DCE MRI required pharmacokinetic modeling. HF was not correlated to any DCE MRI parameter, however, AHS may be estimated after pharmacokinetic modeling. Interestingly, such modeling also detected tumor necrosis very strongly.

      CONCLUSIONS: DCE MRI reliably allows non-invasive assessment of tumors' vascular function. The findings of increased tumor vascularization after ADT encourage further studies into whether these changes are beneficial for combined RT, or if treatment with anti-angiogenic therapy may be a strategy to improve the therapeutic efficacy of ADT in advanced PC.

      View details for PubMedID 22621752
  • Accelerated radiotherapy with carbogen and nicotinamide for laryngeal cancer: results of a phase III randomized trial. J Clin Oncol
    Janssens GO, Rademakers SE, Terhaard CH, Doornaert PA, Bijl HP, van den Ende P, Chin A, Marres HA, de Bree R, van der Kogel AJ, Hoogsteen IJ, Bussink J, Span PN, Kaanders JH
    2012 May 20; 30 (15): 1777-83
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      PURPOSE: To report the results from a randomized trial comparing accelerated radiotherapy (AR) with accelerated radiotherapy plus carbogen inhalation and nicotinamide (ARCON) in laryngeal cancer.

      PATIENTS AND METHODS: Patients with cT2-4 squamous cell laryngeal cancer were randomly assigned to AR (68 Gy within 36 to 38 days) or ARCON. To limit the risk of laryngeal necrosis, ARCON patients received 64 Gy on the laryngeal cartilage. The primary end point was local control. Secondary end points were regional control, larynx preservation, toxicity, disease-free survival, and overall survival. In a translational side study, the hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies.

      RESULTS: From April 2001 to February 2008, 345 patients were accrued. After a median follow-up of 44 months, local tumor control rate at 5 years was 78% for AR versus 79% for ARCON (P = .80), with larynx preservation rates of 84% and 87%, respectively (P = .48). The 5-year regional control was significantly better with ARCON (93%) compared with AR (86%, P = .04). The improvement in regional control was specifically observed in patients with hypoxic tumors and not in patients with well-oxygenated tumors (100% v 55%, respectively; P = .01). AR and ARCON produced equal levels of toxicity.

      CONCLUSION: Despite lack of benefit in local tumor control for advanced laryngeal cancers, a significant gain in regional control rate, with equal levels of toxicity, was observed in favor of ARCON. The poor regional control of patients with hypoxic tumors is specifically countered by ARCON treatment.

      View details for PubMedID 22508814
  • Spinal cord tolerance to reirradiation with single-fraction radiosurgery: a swine model. Int J Radiat Oncol Biol Phys
    Medin PM, Foster RD, van der Kogel AJ, Sayre JW, McBride WH, Solberg TD
    2012 Jul 01; 83 (3): 1031-7
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      PURPOSE: This study was performed to determine swine spinal cord tolerance to single-fraction, partial-volume irradiation 1 year after receiving uniform irradiation to 30 Gy in 10 fractions.

      METHODS AND MATERIALS: A 10-cm length of spinal cord (C3-T1) was uniformly irradiated to 30 Gy in 10 consecutive fractions and reirradiated 1 year later with a single radiosurgery dose centered within the previously irradiated segment. Radiosurgery was delivered to a cylindrical volume approximately 5 cm in length and 2 cm in diameter, which was positioned laterally to the cervical spinal cord, resulting in a dose distribution with the 90%, 50%, and 10% isodose lines traversing the ipsilateral, central, and contralateral spinal cord, respectively. Twenty-three pigs were stratified into six dose groups with mean maximum spinal cord doses of 14.9 ± 0.1 Gy (n = 2), 17.1 ± 0.3 Gy (n = 3), 19.0 ± 0.1 Gy (n = 5), 21.2 ± 0.1 Gy (n = 5), 23.4 ± 0.2 Gy (n = 5), and 25.4 ± 0.4 Gy (n = 3). The mean percentage of spinal cord volumes receiving ≥10 Gy for the same groups were 34% ± 1%, 40% ± 1%, 46% ± 3%, 52% ± 1%, 56 ± 3%, and 57% ± 1%. The study endpoint was motor neurologic deficit as determined by a change in gait during a 1- year follow-up period.

      RESULTS: A steep dose-response curve was observed with a 50% incidence of paralysis (ED(50)) for the maximum point dose of 19.7 Gy (95% confidence interval, 17.4-21.4). With two exceptions, histology was unremarkable in animals with normal neurologic status, while all animals with motor deficits showed some degree of demyelination and focal white matter necrosis on the irradiated side, with relative sparing of gray matter. Histologic comparison with a companion study of de novo irradiated animals revealed that retreatment responders had more extensive tissue damage, including infarction of gray matter, only at prescription doses >20 Gy.

      CONCLUSION: Pigs receiving spinal radiosurgery 1 year after receiving 30 Gy in 10 fractions were not at significantly higher risk of developing motor deficits than pigs that received radiosurgery alone.

      View details for PubMedID 22197239
  • Preclinical evaluation and validation of [18F]HX4, a promising hypoxia marker for PET imaging. Proc Natl Acad Sci U S A
    Dubois LJ, Lieuwes NG, Janssen MH, Peeters WJ, Windhorst AD, Walsh JC, Kolb HC, Ollers MC, Bussink J, van Dongen GA, van der Kogel A, Lambin P
    2011 Aug 30; 108 (35): 14620-5
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      Hypoxia has been shown to be an important microenvironmental parameter influencing tumor progression and treatment efficacy. Patient guidance for hypoxia-targeted therapy requires evaluation of tumor oxygenation, preferably in a noninvasive manner. The aim of this study was to evaluate and validate the uptake of [(18)F]HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [(18)F]HX4 was found within rat rhabdomyosarcoma tumors that was significantly (P < 0.0001) higher compared with the surrounding tissues, with temporal increasing tumor-to-blood ratios reaching a plateau of 7.638 ± 0.926 and optimal imaging properties 4 h after injection. [(18)F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [(18)F]HX4 tumor uptake was analyzed based on 16 regions within the tumor according to the different orthogonal planes at the largest diameter. Validation of heterogeneous [(18)F]HX4 tumor uptake was shown by a strong and significant relationship (r = 0.722; P < 0.0001) with the hypoxic fraction as calculated by the percentage pimonidazole-positive pixels. Furthermore, a causal relationship with tumor oxygenation was established, because combination treatment of nicotinamide and carbogen resulted in a 40% reduction (P < 0.001) in [(18)F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [(18)F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.

      View details for PubMedID 21873245
  • Characterization of positron emission tomography hypoxia tracer uptake and tissue oxygenation via electrochemical modeling. Nucl Med Biol
    Bowen SR, van der Kogel AJ, Nordsmark M, Bentzen SM, Jeraj R
    2011 Aug; 38 (6): 771-80
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      PURPOSE: Unique uptake and retention mechanisms of positron emission tomography (PET) hypoxia tracers make in vivo comparison between them challenging. Differences in imaged uptake of two common hypoxia radiotracers, [(61)Cu]Cu-ATSM and [(18)F]FMISO, were characterized via computational modeling to address these challenges.

      MATERIALS AND METHODS: An electrochemical formalism describing bioreductive retention mechanisms of these tracers under steady-state conditions was adopted to relate time-averaged activity concentration to tissue partial oxygen tension (PO(2)), a common metric of hypoxia. Chemical equilibrium constants of product concentration to reactant concentration ratios were determined from free energy changes and reduction potentials of pertinent reactions reported in the literature. Resulting transformation functions between tracer uptake and PO(2) were compared against measured values in preclinical models. Additionally, calculated PO(2) distributions from imaged Cu-ATSM tracer activity concentrations of 12 head and neck squamous cell carcinoma (HNSCC) patients were validated against microelectrode PO(2) measurements in 69 HNSCC patients.

      RESULTS: Both Cu-ASTM- and FMISO-modeled PO(2) transformation functions were in agreement with preclinical measured values within single-deviation confidence intervals. High correlation (r(2)=0.94, P<.05) was achieved between modeled PO(2) distributions and measured distributions in the patient populations. On average, microelectrode hypoxia thresholds (2.5 and 5.0 mmHg) corresponded to higher Cu-ATSM uptake [2.5 and 2.0 standardized uptake value (SUV)] and lower FMISO uptake (2.0 and 1.4 SUV). Uncertainties in the models were dominated by variations in the estimated specific activity and intracellular acidity.

      CONCLUSIONS: Results indicated that the high dynamic range of Cu-ATSM uptake was representative of a narrow range of low oxygen tension whose values were dependent on microenvironment acidity, while FMISO uptake was representative of a wide range of PO(2) values that were independent of acidity. The models shed light on possible causes of these discrepancies, particularly as it pertains to image contrast, and may prove to be a useful methodology in quantifying relationships between other hypoxia tracers. Comprehensive and robust assessment of tumor hypoxia prior to as well as in response to therapy may be best provided by imaging of multiple hypoxia markers that provide complementary rather than interchangeable information.

      View details for PubMedID 21843774
  • Spatial relationship of phosphorylated epidermal growth factor receptor and activated AKT in head and neck squamous cell carcinoma. Radiother Oncol
    Nijkamp MM, Hoogsteen IJ, Span PN, Takes RP, Lok J, Rijken PF, van der Kogel AJ, Bussink J, Kaanders JH
    2011 Oct; 101 (1): 165-70
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      BACKGROUND: Overexpression of EGFR correlates with decreased survival after radiotherapy in head and neck squamous cell carcinoma (HNSCC). However, the contribution of the activated form, pEGFR, and its downstream signaling (PI3-K/AKT) pathway is not clear yet.

      METHODS: Fifty-eight patients with HNSCC were included in the study. pEGFR, pAKT, hypoxia, and vessels were visualized using immunohistochemistry. Fractions (defined as the tumor area positive for the respective markers relative to the total tumor area) were calculated by automated image analysis and related to clinical outcome.

      RESULTS: Both pEGFR (median 0.6%, range 0-34%) and pAKT (median 1.8%, range 0-16%) expression differed between tumors. Also, a large variation in hypoxia was found (median pimonidazole fraction 3.9% 0-20%). A significant correlation between pEGFR and pAKT (r(s) 0.44, p=0.004) was seen, however, analysis revealed that this was not always based on spatial coexpression. Low pAKT expression was associated with increased risk of regional recurrence (p<0.05, log-rank) and distant metastasis (p=0.04).

      CONCLUSION: The correlation between expression of pEGFR and pAKT is indicative of activation of the PI3-K/AKT pathway through phosphorylation of EGFR. Since not all tumors show coexpression to the same extent, other factors must be involved in the activation of this pathway as well.

      View details for PubMedID 21775008
  • Expression of E-cadherin and vimentin correlates with metastasis formation in head and neck squamous cell carcinoma patients. Radiother Oncol
    Nijkamp MM, Span PN, Hoogsteen IJ, van der Kogel AJ, Kaanders JH, Bussink J
    2011 Jun; 99 (3): 344-8
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      PURPOSE: E-cadherin is a transmembrane glycoprotein, involved in cell-cell adhesion and epithelial-mesenchymal transition (EMT). Vimentin is highly expressed in mesenchymal cells and is positively correlated with increased metastasis. Here we set out to determine the expression of E-cadherin and vimentin in head and neck squamous cell carcinomas (HNSCC).

      PATIENTS AND METHODS: Twenty-six patients with primary stage II-IV HNSCC were included. E-cadherin and vimentin were visualised using immunohistochemistry, semi-automatically analysed for expression patterns and correlated with the clinical behaviour of these tumours.

      RESULTS: A large variation in E-cadherin and vimentin expression was observed between tumours (median 17% range 0-51% respectively median 0% range 0-20%). Tumours with low E-cadherin expression showed a significantly higher incidence of metastasis formation compared to tumours with high expression (81% versus 19%, p=0.004). Enhanced expression of vimentin was associated with a trend towards a higher metastatic risk (33% versus 77%) compared to tumours without expression of vimentin. All patients with low E-cadherin and high vimentin expression (an EMT-phenotype) developed distant metastases versus only 44% of the other patients (p=0.008).

      CONCLUSION: Loss of E-cadherin and gain of vimentin may be associated with enhanced migration of tumour cells, leading to higher metastatic risk of HNSCC patients.

      View details for PubMedID 21684617
  • Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction. Radiother Oncol
    Rouschop KM, Dubois L, Schaaf MB, van den Beucken T, Lieuwes N, Keulers TG, Savelkouls KG, Bussink J, van der Kogel AJ, Koritzinsky M, Wouters BG
    2011 Jun; 99 (3): 385-91
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      BACKGROUND AND PURPOSE: Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation.

      MATERIAL AND METHODS: The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells.

      RESULTS: We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation.

      CONCLUSIONS: Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.

      View details for PubMedID 21665307
  • Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4. BMC Cancer
    Rademakers SE, Lok J, van der Kogel AJ, Bussink J, Kaanders JH
    2011 May 12; 11: 167
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      BACKGROUND: The cellular response of malignant tumors to hypoxia is diverse. Several important endogenous metabolic markers are upregulated under hypoxic conditions. We examined the staining patterns and co-expression of HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 with the exogenous hypoxic cell marker pimonidazole and the association of marker expression with clinicopathological characteristics.

      METHODS: 20 biopsies of advanced head and neck carcinomas were immunohistochemically stained and analyzed. All patients were given the hypoxia marker pimonidazole intravenously 2 h prior to biopsy taking. The tumor area positive for each marker, the colocalization of the different markers and the distribution of the markers in relation to the blood vessels were assessed by semiautomatic quantitative analysis.

      RESULTS: MCT1 staining was present in hypoxic (pimonidazole stained) as well as non-hypoxic areas in almost equal amounts. MCT1 expression showed a significant overall correlation (r = 0.75, p < 0.001) and strong spatial relationship with CAIX. LDH-5 showed the strongest correlation with pimonidazole (r = 0.66, p = 0.002). MCT4 and GLUT-1 demonstrated a typical diffusion-limited hypoxic pattern and showed a high degree of colocalization. Both MCT4 and CAIX showed a higher expression in the primary tumor in node positive patients (p = 0.09 both).

      CONCLUSIONS: Colocalization and staining patterns of metabolic and hypoxia-related proteins provides valuable additional information over single protein analyses and can improve the understanding of their functions and environmental influences.

      View details for PubMedID 21569415
  • Spinal cord tolerance to single-fraction partial-volume irradiation: a swine model. Int J Radiat Oncol Biol Phys
    Medin PM, Foster RD, van der Kogel AJ, Sayre JW, McBride WH, Solberg TD
    2011 Jan 01; 79 (1): 226-32
    • More

      PURPOSE: To determine the spinal cord tolerance to single-fraction, partial-volume irradiation in swine.

      METHODS AND MATERIALS: A 5-cm-long cervical segment was irradiated in 38-47-week-old Yucatan minipigs using a dedicated, image-guided radiosurgery linear accelerator. The radiation was delivered to a cylindrical volume approximately 5 cm in length and 2 cm in diameter that was positioned lateral to the cervical spinal cord, resulting in a dose distribution with the 90%, 50%, and 10% isodose lines traversing the ipsilateral, central, and contralateral spinal cord, respectively. The dose was prescribed to the 90% isodose line. A total of 26 pigs were stratified into eight dose groups of 12-47 Gy. The mean maximum spinal cord dose was 16.9 ± 0.1, 18.9 ± 0.1, 21.0 ± 0.1, 23.0 ± 0.2, and 25.3 ± 0.3 Gy in the 16-, 18-, 20-, 22-, and 24-Gy dose groups, respectively. The mean percentage of spinal cord volumes receiving ≥ 10 Gy for the same groups were 43% ± 3%, 48% ± 4%, 51% ± 2%, 57% ± 2%, and 59% ± 4%. The study endpoint was motor neurologic deficit determined by a change in gait during a 1-year follow-up period.

      RESULTS: A steep dose-response curve was observed with a median effective dose for the maximum dose point of 20.0 Gy (95% confidence interval, 18.3-21.7). Excellent agreement was observed between the occurrence of neurologic change and the presence of histologic change. All the minipigs with motor deficits showed some degree of demyelination and focal white matter necrosis on the irradiated side, with relative sparing of the gray matter. The histologic findings were unremarkable in the minipigs with normal neurologic status.

      CONCLUSIONS: Our results have indicated that for a dose distribution with a steep lateral gradient, the pigs had a lower median effective dose for paralysis than has been observed in rats and more closely resembles that for rats, mice, and guinea pigs receiving uniform spinal cord irradiation.

      View details for PubMedID 20934278
  • Longitudinal magnetic resonance imaging-based assessment of vascular changes and radiation response in androgen-sensitive prostate carcinoma xenografts under androgen-exposed and androgen-deprived conditions. Neoplasia
    Røe K, Seierstad T, Kristian A, Mikalsen LT, Mælandsmo GM, van der Kogel AJ, Ree AH, Olsen DR
    2010 Oct; 12 (10): 818-25
    • More

      Prostate cancer (PCa) patients receive androgen-deprivation therapy (ADT) to reduce tumor burden. However, complete eradication of PCa is unusual, and recurrent disease is evident within approximately 2 years in high-risk patients. Clinical evidence suggests that combining ADT with radiotherapy improves local control and disease-free survival in these patients compared with radiotherapy alone. We investigated whether vascularization of androgen-sensitive PCa xenografts changed after ADT and whether such therapy affected radiation response. CWR22 xenografts received combinations of ADT by castration (CWR22-cas) and 15 Gy of single-dose irradiation. At a shortest tumor diameter of 8 mm, vascularization was visualized by dynamic contrast-enhanced magnetic resonance imaging before radiation and 1 and 9 days after radiation. Voxel-wise quantitative modeling of contrast enhancement curves extracted the hemodynamic parameter K(trans), reflecting a combination of permeability, density, and blood flow. Tumor volumes and prostate-specific antigen (PSA) were monitored during the experiment. The results showed that K(trans) of CWR22-cas tumors 36±4 days after ADT was 47.1% higher than K(trans) of CWR22 tumors (P = .01). CWR22-cas tumors showed no significant changes in K(trans) after radiation, whereas K(trans) of CWR22 tumors at day 1 decreased compared with pretreatment values (P = .04) before a continuous increase from day 1 to day 9 followed (P = .01). Total PSA in blood correlated positively to tumor volume (r = 0.59, P < .01). In conclusion, androgen-exposed xenografts demonstrated radiation-induced reductions in vascularization and tumor volumes, whereas androgen-deprived xenografts showed increased vascularization and growth inhibition, but no significant additive effect of radiation.

      View details for PubMedID 20927320
  • Benign uterine uptake of FDG: a case report and review of literature. Neth J Med
    Vriens D, de Geus-Oei LF, Flucke UE, van der Kogel AJ, Oyen WJ, Vierhout ME, van der Meer JW
    2010 Sep; 68 (9): 379-80
  • Effect of cetuximab and fractionated irradiation on tumour micro-environment. Radiother Oncol
    Santiago A, Eicheler W, Bussink J, Rijken P, Yaromina A, Beuthien-Baumann B, van der Kogel AJ, Baumann M, Krause M
    2010 Nov; 97 (2): 322-9
    • More

      BACKGROUND AND PURPOSE: Previous experiments have shown that application of the anti-EGFR monoclonal antibody C225 (cetuximab) improves local tumour control after irradiation in FaDu human squamous cell carcinoma (hSCC) due to the combined effect of decreased repopulation and improved reoxygenation. The present study investigates early changes of the pimonidazole hypoxic fraction of FaDu tumours and the expression and phosphorylation of the EGFR and its downstream signal transduction molecules after treatment with C225 alone or in combination with irradiation.

      MATERIAL AND METHODS: FaDu tumour xenografts were irradiated with up to 3×3Gy with or without additional C225 treatment and excised at different time points. Tumour hypoxia was evaluated using pimonidazole. EGFR expression and phosphorylation and intratumoural distribution of C225 were assessed by immunofluorescence analysis. Western blots were performed to evaluate expression and phosphorylation of EGFR, ErbB2, AKT and MAPK (ERK1/2).

      RESULTS: Hypoxia did not change during the 4days of treatment in the tumours treated with C225 alone or combined with irradiation. C225 treatment led to downregulation of the total EGFR in FaDu tumours, accompanied by a change of the spatial distribution of the receptor favouring the membranous expression. An induction of phosphorylation of the EGFR (tyr992, tyr1173) was observed with C225 alone or combined with irradiation. AKT phosphorylation was decreased, whereas MAPK phosphorylation remained unchanged. C225 membrane staining was homogeneously distributed over the whole tumour with no differences between hypoxic and non-hypoxic tumour cells.

      CONCLUSION: Pimonidazole-hypoxia of FaDu tumours during the initial part of fractionated irradiation is not influenced by C225, indicating that external hypoxia markers may not be promising as biomarkers for tumour response to combined treatment. The downregulation of the total EGFR, but at the same time higher membrane staining, as well as the changes in downstream signal transduction molecules, warrants further investigation in other tumour models.

      View details for PubMedID 20667608
  • Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors. Clin Cancer Res
    Kruser TJ, Wheeler DL, Armstrong EA, Iida M, Kozak KR, van der Kogel AJ, Bussink J, Coxon A, Polverino A, Harari PM
    2010 Jul 15; 16 (14): 3639-47
    • More

      BACKGROUND: Motesanib is a potent inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit receptors. In this report we examine the interaction between motesanib and radiation in vitro and in head and neck squamous cell carcinoma (HNSCC) xenograft models.

      EXPERIMENTAL DESIGN: In vitro assays were done to assess the impact of motesanib on VEGFR2 signaling pathways in human umbilical vein endothelial cells (HUVEC). HNSCC lines grown as tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of motesanib alone and in combination with radiation.

      RESULTS: Motesanib inhibited VEGF-stimulated HUVEC proliferation in vitro, as well as VEGFR2 kinase activity. Additionally, motesanib and fractionated radiation showed additive inhibitory effects on HUVEC proliferation. In vivo combination therapy with motesanib and radiation showed increased response compared with drug or radiation alone in UM-SCC1 (P < 0.002) and SCC-1483 xenografts (P = 0.001); however, the combination was not significantly more efficacious than radiation alone in UM-SCC6 xenografts. Xenografts treated with motesanib showed a reduction of vessel penetration into tumor parenchyma, compared with control tumors. Furthermore, triple immunohistochemical staining for vasculature, proliferation, and hypoxia showed well-defined spatial relationships among these parameters in HNSCC xenografts. Motesanib significantly enhanced intratumoral hypoxia in the presence and absence of fractionated radiation.

      CONCLUSIONS: These studies identify a favorable interaction when combining radiation and motesanib in HNSCC models. The data presented suggest that motesanib reduces blood vessel penetration into tumors and thereby increases intratumoral hypoxia. These findings suggest that clinical investigations examining combinations of radiation and motesanib are warranted in HNSCC.

      View details for PubMedID 20507929
  • Assessment of Blood Hemodynamics by USPIO-Induced R(1) Changes in MRI of Murine Colon Carcinoma. Appl Magn Reson
    Gambarota G, van Laarhoven HW, Philippens M, Peeters WJ, Rijken P, van der Kogel A, Punt CJ, Heerschap A
    2010 Jun; 38 (3): 349-360
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      The objective of this study is to assess whether ultrasmall superparamagnetic iron oxide (USPIO)-induced changes of the water proton longitudinal relaxation rate (R(1)) provide a means to assess blood hemodynamics of tumors. Two types of murine colon tumors (C26a and C38) were investigated prior to and following administration of USPIO blood-pool contrast agent with fast R(1) measurements. In a subpopulation of mice, R(1) was measured following administration of hydralazine, a well-known blood hemodynamic modifier. USPIO-induced R(1) increase in C38 tumors (DeltaR(1) = 0.072 +/- 0.0081 s(-1)) was significantly larger than in C26a tumors (DeltaR(1) = 0.032 +/- 0.0018 s(-1), N = 9, t test, P < 0.001). This was in agreement with the immunohistochemical data that showed higher values of relative vascular area (RVA) in C38 tumors than in C26a tumors (RVA = 0.059 +/- 0.015 vs. 0.020 +/- 0.011; P < 0.05). Following administration of hydralazine, a decrease in R(1) value was observed. This was consistent with the vasoconstriction induced by the steal effect mechanism. In conclusion, R(1) changes induced by USPIO are sensitive to tumor vascular morphology and to blood hemodynamics. Thus, R(1) measurements following USPIO administration can give novel insight into the effects of blood hemodynamic modifiers, non-invasively and with a high temporal resolution.

      View details for PubMedID 20502507
  • Histopathologic validation of 3'-deoxy-3'-18F-fluorothymidine PET in squamous cell carcinoma of the oral cavity. J Nucl Med
    Troost EG, Bussink J, Slootweg PJ, Peeters WJ, Merkx MA, van der Kogel AJ, Oyen WJ, Kaanders JH
    2010 May; 51 (5): 713-9
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      UNLABELLED: Accelerated tumor cell repopulation is an important mechanism adversely affecting therapeutic outcome in head and neck cancer. The noninvasive assessment of the proliferative state of a tumor by PET may provide a selection tool for customized treatment. 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a PET tracer that is phosphorylated by thymidine kinase 1 (TK-1) and, as such, reflects cellular proliferation. Before the use of (18)F-FLT PET for tumor characterization is accepted and introduced into clinical studies, validation against tumor histology is mandatory. The aim of this study was to validate (18)F-FLT PET in squamous cell carcinomas of the oral cavity using immunohistochemical staining for the proliferation marker iododeoxyuridine and for TK-1.

      METHODS: Seventeen patients with primary squamous cell carcinomas of the oral cavity underwent an (18)F-FLT PET/CT scan before surgery, and iododeoxyuridine was administered 20 min before tumor resection. (18)F-FLT PET/CT scans were segmented, and PET/CT volumes and PET signal intensities were calculated (mean standardized uptake value [SUV(mean)] and maximum standardized uptake value [SUV(max)]). Multiple paraffin-embedded tumor sections were immunohistochemically stained for iododeoxyuridine and TK-1. For iododeoxyuridine, labeling indices and optical densities were calculated and correlated with SUV(mean) and SUV(max). TK-1 staining was visually and semiquantitatively assessed.

      RESULTS: All primary tumors were identified with (18)F-FLT PET but with a large range in tracer uptake (mean SUV(max), 5.9; range, 2.2-15.2). Also, there was a large variability in iododeoxyuridine labeling indices (mean, 0.09; range, 0.01-0.29) and optical densities (mean, 28.2; range, 12.6-37.8). The iododeoxyuridine optical densities correlated significantly with SUV(mean) and SUV(max), but the labeling indices did not. In most tumors, TK-1 staining of varying intensity was present but correlated with neither iododeoxyuridine binding nor (18)F-FLT uptake.

      CONCLUSION: The current study demonstrated only a weak correlation between (18)F-FLT uptake and iododeoxyuridine staining intensity in oral cavity tumors. This weak correlation may be explained by differences in biomarker characteristics, resolution, and quantification methods.

      View details for PubMedID 20395329
  • Radiation dose-volume effects in the spinal cord. Int J Radiat Oncol Biol Phys
    Kirkpatrick JP, van der Kogel AJ, Schultheiss TE
    2010 Mar 01; 76 (3 Suppl): S42-9
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      Dose-volume data for myelopathy in humans treated with radiotherapy (RT) to the spine is reviewed, along with pertinent preclinical data. Using conventional fractionation of 1.8-2 Gy/fraction to the full-thickness cord, the estimated risk of myelopathy is <1% and <10% at 54 Gy and 61 Gy, respectively, with a calculated strong dependence on dose/fraction (alpha/beta = 0.87 Gy.) Reirradiation data in animals and humans suggest partial repair of RT-induced subclinical damage becoming evident about 6 months post-RT and increasing over the next 2 years. Reports of myelopathy from stereotactic radiosurgery to spinal lesions appear rare (<1%) when the maximum spinal cord dose is limited to the equivalent of 13 Gy in a single fraction or 20 Gy in three fractions. However, long-term data are insufficient to calculate a dose-volume relationship for myelopathy when the partial cord is treated with a hypofractionated regimen.

      View details for PubMedID 20171517
  • The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. J Clin Invest
    Rouschop KM, van den Beucken T, Dubois L, Niessen H, Bussink J, Savelkouls K, Keulers T, Mujcic H, Landuyt W, Voncken JW, Lambin P, van der Kogel AJ, Koritzinsky M, Wouters BG
    2010 Jan; 120 (1): 127-41
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      Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

      View details for PubMedID 20038797
  • Innovations in radiotherapy planning of head and neck cancers: role of PET. J Nucl Med
    Troost EG, Schinagl DA, Bussink J, Boerman OC, van der Kogel AJ, Oyen WJ, Kaanders JH
    2010 Jan; 51 (1): 66-76
    • More

      Modern radiotherapy techniques heavily rely on high-quality medical imaging. PET provides biologic information about the tumor, complementary to anatomic imaging. Integrated PET/CT has found its way into the practice of radiation oncology, and (18)F-FDG PET is being introduced for radiotherapy planning. The functional information possibly augments accurate delineation and treatment of the tumor and its extensions while reducing the dose to surrounding healthy tissues. In addition to (18)F-FDG, other PET tracers are available for imaging specific biologic tumor characteristics determining radiation resistance. For head and neck cancer, the potential gains of PET are increasingly being recognized. This review describes the current role of PET and perspectives on its future use for selection and delineation of radiotherapy target volumes and for biologic characterization of this tumor entity. Furthermore, the potential role of PET for early response monitoring, treatment modification, and patient selection is addressed in this review.

      View details for PubMedID 20009000
  • Can hypoxia-PET map hypoxic cell density heterogeneity accurately in an animal tumor model at a clinically obtainable image contrast? Radiother Oncol
    Busk M, Horsman MR, Jakobsen S, Hansen KV, Bussink J, van der Kogel A, Overgaard J
    2009 Sep; 92 (3): 429-36
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      BACKGROUND: PET allows non-invasive mapping of tumor hypoxia, but the combination of low resolution, slow tracer adduct-formation and slow clearance of unbound tracer remains problematic. Using a murine tumor with a hypoxic fraction within the clinical range and a tracer post-injection sampling time that results in clinically obtainable tumor-to-reference tissue activity ratios, we have analyzed to what extent inherent limitations actually compromise the validity of PET-generated hypoxia maps.

      MATERIALS AND METHODS: Mice bearing SCCVII tumors were injected with the PET hypoxia-marker fluoroazomycin arabinoside (FAZA), and the immunologically detectable hypoxia marker, pimonidazole. Tumors and reference tissue (muscle, blood) were harvested 0.5, 2 and 4h after FAZA administration. Tumors were analyzed for global (well counter) and regional (autoradiography) tracer distribution and compared to pimonidazole as visualized using immunofluorescence microscopy.

      RESULTS: Hypoxic fraction as measured by pimonidazole staining ranged from 0.09 to 0.32. FAZA tumor to reference tissue ratios were close to unity 0.5h post-injection but reached values of 2 and 6 when tracer distribution time was prolonged to 2 and 4h, respectively. A fine-scale pixel-by-pixel comparison of autoradiograms and immunofluorescence images revealed a clear spatial link between FAZA and pimonidazole-adduct signal intensities at 2h and later. Furthermore, when using a pixel size that mimics the resolution in PET, an excellent correlation between pixel FAZA mean intensity and density of hypoxic cells was observed already at 2h post-injection.

      CONCLUSIONS: Despite inherent weaknesses, PET-hypoxia imaging is able to generate quantitative tumor maps that accurately reflect the underlying microscopic reality (i.e., hypoxic cell density) in an animal model with a clinical realistic image contrast.

      View details for PubMedID 19729214
  • Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia. Eur J Cancer
    Hoogsteen IJ, Lok J, Marres HA, Takes RP, Rijken PF, van der Kogel AJ, Kaanders JH
    2009 Nov; 45 (16): 2906-14
    • More

      Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics. One hundred and three patients with T2-T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0-19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0-34%) was observed. In 67% of the tumours, hypoxia involved 1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak (p=0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) (p=0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage. From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.

      View details for PubMedID 19699082
  • Hypoxia-induced expression of carbonic anhydrase 9 is dependent on the unfolded protein response. J Biol Chem
    van den Beucken T, Koritzinsky M, Niessen H, Dubois L, Savelkouls K, Mujcic H, Jutten B, Kopacek J, Pastorekova S, van der Kogel AJ, Lambin P, Voncken W, Rouschop KM, Wouters BG
    2009 Sep 04; 284 (36): 24204-12
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      Adaptation to tumor hypoxia is mediated in large part by changes in protein expression. These are driven by multiple pathways, including activation of the hypoxia inducible factor-1 (HIF-1) transcription factor and the PKR-like endoplasmic reticulum kinase PERK, a component of the unfolded protein response. Through gene expression profiling we discovered that induction of the HIF-1 target gene CA9 was defective in mouse embryo fibroblasts derived from mice harboring an eIF2alpha S51A knock-in mutation. This finding was confirmed in two isogenic human cell lines with an engineered defect in eIF2alpha phosphorylation. We show that impaired CA9 expression was not due to changes in HIF activity or CA9 mRNA stability. Using chromatin immunoprecipitation we show that the eIF2alpha-dependent translationally regulated gene ATF4 binds directly to the CA9 promoter and is associated with loss of the transcriptional repressive histone 3 lysine 27 tri-methylation mark. Loss or overexpression of ATF4 confirmed its role in CA9 induction during hypoxia. Our data indicate that expression of CA9 is regulated through both the HIF-1 and unfolded protein response hypoxia response pathways in vitro and in vivo.

      View details for PubMedID 19564335
  • Molecular aspects of tumour hypoxia. Mol Oncol
    Rademakers SE, Span PN, Kaanders JH, Sweep FC, van der Kogel AJ, Bussink J
    2008 Jun; 2 (1): 41-53
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      Hypoxia is an important feature of the microenvironment of a wide range of solid tumours. Its critical role in radio- and chemoresistance and its significance as an adverse prognostic factor have been well established over the last decades. On a cellular level, hypoxia evokes a complex molecular response with a central role for the HIF-1 pathway. The cellular processes under control of HIF-1 contain important prognostic information and comprise potential candidates for directing hypoxia-modifying therapies. This review will provide an overview of the current knowledge on the molecular aspects of tumour hypoxia and the link to clinical practice.

      View details for PubMedID 19383328
  • Angiogenesis, hypoxia and VEGF expression during tumour growth in a human xenograft tumour model. Microvasc Res
    Hendriksen EM, Span PN, Schuuring J, Peters JP, Sweep FC, van der Kogel AJ, Bussink J
    2009 Mar; 77 (2): 96-103
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      Tumour growth and spread of tumour cells requires angiogenesis. Incipient angiogenesis is not induced by tumour cell hypoxia but probably by proangiogenic factors. During growth tumours depend on a further induction of vascular development for adequate oxygen and nutrient supply. If the oxygen supply is insufficient, the resulting hypoxia stimulates angiogenesis through upregulation of HIF-1 alpha and VEGF. VEGF upregulation is associated with a poor response to treatment and poor prognosis. The aim of the study was to analyze the interrelationship between hypoxia and angiogenesis during tumour growth. Therefore the tumour vasculature architecture and functional properties of the vessels were studied during subsequent phases of tumour growth in relation to hypoxia and VEGF-expression. Tumours from the human glioblastoma multiforme tumour line E106 were transplanted in athymic mice. Tumours were harvested at 2 days after transplantation and when tumours reached a mean size of 2, 4, 6, 8 and 10 mm. VEGF was present early in the onset of angiogenesis independent of HIF-1 alpha. During tumour growth VEGF increased from 0.94 to 7.27 ng/mg assessed by ELISA. However, there was increasing intratumoural heterogeneity in the architecture of the tumours, even in the largest tumours small well oxygenated areas were detected resembling the relatively well organized architecture of the smallest tumours. The observation that tumour vasculature develops in early phases under normoxic and at later phases under hypoxic conditions with the presence of both conditions in the larger tumours, suggested that anti-angiogenic therapy should be directed towards HIF-1 alpha dependent and HIF 1-alpha independent pathways.

      View details for PubMedID 19118564
  • Bath and shower effect in spinal cord: the effect of time interval. Int J Radiat Oncol Biol Phys
    Philippens ME, Pop LA, Visser AG, Peeters WJ, van der Kogel AJ
    2009 Feb 01; 73 (2): 514-22
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      PURPOSE: To evaluate the time dependency of the sensitizing effect of a large low-dose field on a small high-dose field in the rat cervical spinal cord.

      METHODS AND MATERIALS: Irradiation experiments with a relatively low dose to a large volume (bath, 2 cm, 4 Gy) were combined with high doses to a small volume (shower, 4.7 mm, 26-43 Gy) at intervals of 8 minutes and 3, 12, and 24 hours. Both a functional score defined as motor impairment and a histologic score characterized as white matter necrosis were used as end points.

      RESULTS: Application of the 4-Gy bath dose resulted in a significant decrease in 50% isoeffective dose (ED(50)) from 48.7 Gy (small field) to 40.8 Gy. If the interval was extended, the ED(50) increased to 44.4 (3 hours) and 44.8 Gy (12 hours), whereas a 24-hour interval resulted in a significant increase to 51.9 Gy. If the histologic end point was considered, the ED(50) for all dose-response curves decreased slightly with 0.2 to 2.6 Gy without significantly changing the kinetics.

      CONCLUSIONS: The bath effect as applied in the bath-and-shower experiment lasted for at least 12 hours and disappeared in the 24-hour interval. This time scale clearly deviates from the repair kinetics in spinal cord derived from low-dose-rate and fractionated irradiations.

      View details for PubMedID 19046823
  • Radiation effects in the rat spinal cord: evaluation with apparent diffusion coefficient versus T2 at serial MR imaging. Radiology
    Philippens ME, Gambarota G, van der Kogel AJ, Heerschap A
    2009 Feb; 250 (2): 387-97
    • More

      PURPOSE: To prospectively determine whether apparent diffusion coefficients (ADCs) are more sensitive to radiation-induced changes in the rat spinal cord than T2 relaxation times.

      MATERIALS AND METHODS: The study was approved by the institutional ethical committee on animal welfare. One centimeter of the thoracolumbar spinal cord of six rats was irradiated with 36 Gy. For 3-6 months after irradiation, five 7.0-T magnetic resonance (MR) imaging measurements were performed in each rat until motor impairment developed. Six age-matched rats were examined as controls. Measurements were performed by using diffusion-weighted imaging with five b values and a spin-echo sequence with 20 echoes. ADC and T2 values were calculated, and the spatiotemporal evolution of the radiation-induced lesions was determined semiautomatically. The final MR measurements were compared with the histologic findings.

      RESULTS: Shortly before the neurologic signs appeared, the first radiation effects manifested as well-circumscribed white matter (WM) lesions with a low longitudinal ADC and normal or high T2. WM lesions with high T2 correlated with confluent necrosis at histologic analysis, whereas WM lesions with normal T2 correlated with focal necrosis and demyelination. In the gray matter (GM), lesions with diffusely high T2 were present and were attributed to edema. T2 changes in the GM preceded T2 and ADC changes in the WM.

      CONCLUSION: In the WM, longitudinal ADC was more sensitive for the detection of radiation damage than T2, but in the GM, T2 was more sensitive.

      View details for PubMedID 19037019
  • No detectable hypoxia in malignant salivary gland tumors: preliminary results. Int J Radiat Oncol Biol Phys
    Wijffels KI, Hoogsteen IJ, Lok J, Rijken PF, Marres HA, de Wilde PC, van der Kogel AJ, Kaanders JH
    2009 Apr 01; 73 (5): 1319-25
    • More

      PURPOSE: Hypoxia is detected in most solid tumors and is associated with malignant progression and adverse treatment outcomes. However, the oxygenation status of malignant salivary gland tumors has not been previously studied. The aim of this study was to investigate the potential clinical relevance of hypoxia in this tumor type.

      METHODS AND MATERIALS: Twelve patients scheduled for surgical resection of a salivary gland tumor were preoperatively injected with the hypoxia marker pimonidazole and the proliferation marker iododeoxyuridine. Tissue samples of the dissected tumor were immunohistochemically stained for blood vessels, pimonidazole, carbonic anhydrase-IX, glucose transporters-1 and -3 (Glut-1, Glut-3), hypoxia-inducible factor-1alpha, iododeoxyuridine, and epidermal growth factor receptor. The tissue sections were quantitatively assessed by computerized image analysis.

      RESULTS: The tissue material from 8 patients was of sufficient quality for quantitative analysis. All tumors were negative for pimonidazole binding, as well as for carbonic anhydrase-IX, Glut-1, Glut-3, and hypoxia-inducible factor-1alpha. The vascular density was high, with a median value of 285 mm(-2) (range, 209-546). The iododeoxyuridine-labeling index varied from <0.1% to 12.2% (median, 2.2%). Epidermal growth factor receptor expression levels were mostly moderate to high. In one-half of the cases, nuclear expression of epidermal growth factor receptor was observed.

      CONCLUSION: The absence of detectable pimonidazole binding, as well as the lack of expression of hypoxia-associated proteins in all tumors, indicates that malignant salivary gland tumors are generally well oxygenated. It is unlikely that hypoxia is a relevant factor for their clinical behavior and treatment responsiveness.

      View details for PubMedID 18973979
  • Cellular uptake of PET tracers of glucose metabolism and hypoxia and their linkage. Eur J Nucl Med Mol Imaging
    Busk M, Horsman MR, Jakobsen S, Bussink J, van der Kogel A, Overgaard J
    2008 Dec; 35 (12): 2294-303
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      PURPOSE: Tumour hypoxia and elevated glycolysis (Warburg effect) predict poor prognosis. Each parameter is assessable separately with positron emission tomography, but they are linked through anaerobic glycolysis (Pasteur effect). Here, we compare the oxygenation-dependent retention of fluoroazomycin arabinoside ([(18)F]FAZA), a promising but not well-characterised hypoxia-specific tracer, and fluorodeoxyglucose ([(18)F]FDG) in four carcinoma cell lines.

      METHODS: Cells seeded on coverslips were positioned in modified Petri dishes that allow physically separated cells to share the same tracer-containing medium pool. Following oxic, hypoxic or anoxic tracer incubation, coverslips were analysed for radioactivity ([(18)F]FDG + [(18)F]FAZA) or re-incubated in tracer-free oxygenated medium and then measured ([(18)F]FAZA). Next, we tested the reliability of [(18)F]FDG as a relative measure of glucose metabolic rate. Finally, from two cell lines, xenografts were established in mice, and the tracer distribution between hypoxic and well-oxygenated areas were deduced from tissue sections.

      RESULTS: Three hours of anoxia strongly stimulated [(18)F]FAZA retention with anoxic-to-oxic uptake ratios typically above 30. Three out of four cell lines displayed similar selectivity of [(18)F]FDG versus glucose, but oxic uptake and anoxic-to-oxic uptake ratio of [(18)F]FDG varied considerably. Although less pronounced, [(18)F]FAZA also showed superior in vivo hypoxia specificity compared with [(18)F]FDG.

      CONCLUSIONS: [(18)F]FAZA displays excellent in vitro characteristics for hypoxia imaging including modest cell-to-cell line variability and no binding in oxic cells. In contrast, the usability of [(18)F]FDG as a surrogate marker for hypoxia is questionable due to large variations in baseline (oxic) glucose metabolism and magnitudes of the Pasteur effects.

      View details for PubMedID 18682937
  • Two distinct tumor phenotypes isolated from glioblastomas show different MRS characteristics. NMR Biomed
    Thorsen F, Jirak D, Wang J, Sykova E, Bjerkvig R, Enger PØ, van der Kogel A, Hajek M
    2008 Oct; 21 (8): 830-8
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      We have developed a human brain tumor model in immunodeficient rats that gradually changes its phenotype by serial passages in vivo, from a highly infiltrative, non-angiogenic one with numerous stem cell markers [low-generation (LG) tumor] to a more typical glioblastoma one with extensive angiogenesis and necrosis [high-generation (HG) tumor]. In this study we determined the metabolic properties of these two phenotypes, using (1)H MRS. The LG tumors showed an intact blood-brain barrier and normal vascular morphology, as shown by MRI and Hoechst staining. In contrast, the HG tumors exhibited vascular leakage and necrosis. The animals with HG tumor had raised concentrations of choline and myo-inositol, and decreased concentrations of glutamate and N-acetylaspartate. In the LG tumor group, similar changes in metabolic concentrations were detected, although the alterations were more pronounced. The LG tumors also had higher concentrations of choline, taurine, and lactate. Subdividing the LG and HG tumors into large and small tumors revealed a significant increase in choline and decrease in glutamate as the LG tumors increased in size. Our results show that metabolic profiles produced by (1)H MRS can be used to distinguish between two distinct glioblastoma phenotypes. More pronounced anaerobic metabolism was present in the LG stem-cell-like tumors, suggesting a more malignant phenotype.

      View details for PubMedID 18613001
  • Characterisation of tumour vasculature in mouse brain by USPIO contrast-enhanced MRI. Br J Cancer
    Gambarota G, Leenders W, Maass C, Wesseling P, van der Kogel B, van Tellingen O, Heerschap A
    2008 Jun 03; 98 (11): 1784-9
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      To enhance the success rate of antiangiogenic therapies in the clinic, it is crucial to identify parameters for tumour angiogenesis that can predict response to these therapies. In brain tumours, one such parameter is vascular leakage, which is a response to tumour-derived vascular endothelial growth factor-A and can be measured by Gadolinium-DTPA (Gd-DTPA)-enhanced magnetic resonance imaging (MRI). However, as vascular permeability and angiogenesis are not strictly coupled, tumour blood volume may be another potentially important parameter. In this study, contrast-enhanced MR imaging was performed in three orthotopic mouse models for human brain tumours (angiogenic melanoma metastases and E34 and U87 human glioma xenografts) using both Gd-DTPA to detect vascular leakage and ultrasmall iron oxide particles (USPIO) to measure blood volume. Pixel-by-pixel maps of the enhancement in the transverse relaxation rates (Delta R(2) and Delta R(2)(*)) after injection of USPIO provided an index proportional to the blood volume of the microvasculature and macrovasculature, respectively, for each tumour. The melanoma metastases were characterised by a blood volume and vessel leakage higher than both glioma xenografts. The U87 glioblastoma xenografts displayed higher permeability and blood volume in the rim than in the core. The E34 glioma xenografts were characterised by a relatively high blood volume, accompanied by only a moderate blood-brain barrier disruption. Delineation of the tumour was best assessed on post-USPIO gradient-echo images. These findings suggest that contrast-enhanced MR imaging using USPIOs and, in particular, Delta R(2) and Delta R(2)(*) quantitation, provides important additional information about tumour vasculature.

      View details for PubMedID 18506183
  • Correlation of [18F]FMISO autoradiography and pimonidazole [corrected] immunohistochemistry in human head and neck carcinoma xenografts. Eur J Nucl Med Mol Imaging
    Troost EG, Laverman P, Philippens ME, Lok J, van der Kogel AJ, Oyen WJ, Boerman OC, Kaanders JH, Bussink J
    2008 Oct; 35 (10): 1803-11
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      PURPOSE: Tumour cell hypoxia is a common feature in solid tumours adversely affecting radiosensitivity and chemosensitivity in head and neck squamous cell carcinomas. Positron emission tomography (PET) using the tracer [(18)F]fluoromisonidazole ([(18)F]FMISO) is most frequently used for non-invasive evaluation of hypoxia in human tumours. A series of ten human head and neck xenograft tumour lines was used to validate [(18)F]FMISO as hypoxia marker at the microregional level.

      METHODS: Autoradiography after injection of [(18)F]FMISO was compared with immunohistochemical staining for the hypoxic cell marker pimonidazole in the same tumour sections of ten different human head and neck xenograft tumour lines. The methods were compared: first, qualitatively considering the microarchitecture; second, by obtaining a pixel-by-pixel correlation of both markers at the microregional level; third, by measuring the signal intensity of both images; and fourth, by calculating the hypoxic fractions by pimonidazole labelling.

      RESULTS: The pattern of [(18)F]FMISO signal was dependent on the distribution of hypoxia at the microregional level. The comparison of [(18)F]FMISO autoradiography and pimonidazole immunohistochemistry by pixel-by-pixel analysis revealed moderate correlations. In five tumour lines, a significant correlation between the mean [(18)F]FMISO and pimonidazole signal intensity was found (range, r(2)=0.91 to r(2)=0.99). Comparison of the tumour lines with respect to the microregional distribution pattern of hypoxia revealed that the correlation between the mean signal intensities strongly depended on the microarchitecture. Overall, a weak but significant correlation between hypoxic fractions based on pimonidazole labeling and the mean [(18)F]FMISO signal intensity was observed (r(2)=0.18, p=0.02). For the three tumour models with a ribbon-like microregional distribution pattern of hypoxia, the correlation between the hypoxic fraction and the mean [(18)F]FMISO signal intensity was much stronger and more significant (r(2)=0.73, p<0.001) than for the tumours with a more homogenous, patchy, microregional distribution pattern of hypoxia.

      CONCLUSION: Different patterns of [(18)F]FMISO accumulation dependent on the underlying microregional distribution of hypoxia were found in ten head and neck xenograft tumours. A weak albeit significant correlation was found between the mean [(18)F]FMISO signal intensity and the hypoxic fraction of the tumours. In larger clinical tumours, [(18)F]FMISO-PET provides information on the tumour oxygenation status on a global level, facilitating dose painting in radiation treatment planning. However, caution must be taken when studying small tumour subvolumes as accumulation of the tracer depends on the presence of hypoxia and on the tumour microarchitecture.

      View details for PubMedID 18421457
  • Patterns and levels of hypoxia in head and neck squamous cell carcinomas and their relationship to patient outcome: in regard to Evans et al. (Int J Radiat Oncol Biol Phys 2007;69:1024-1031). Int J Radiat Oncol Biol Phys
    Bussink J, van der Kogel AJ, Kaanders JH
    2008 Apr 01; 70 (5): 1616
  • Phenotypic and genotypic characterization of orthotopic human glioma models and its relevance for the study of anti-glioma therapy. Brain Pathol
    Claes A, Schuuring J, Boots-Sprenger S, Hendriks-Cornelissen S, Dekkers M, van der Kogel AJ, Leenders WP, Wesseling P, Jeuken JW
    2008 Jul; 18 (3): 423-33
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      Most human gliomas are characterized by diffuse infiltrative growth in the brain parenchyma. Partly because of this characteristic growth pattern, gliomas are notorious for their poor response to current therapies. Many animal models for human gliomas, however, do not display this diffuse infiltrative growth pattern. Furthermore, there is a need for glioma models that represent adequate genocopies of different subsets of human gliomas (e.g., oligodendrogliomas). Here, we assessed the intracerebral growth patterns and copy number changes [using multiplex ligation-dependent probe amplification (MLPA)/comparative genomic hybridization (CGH)] of 15 human glioma lines in nude mice. Most xenografts present with compact growing lesions intracerebrally. Only the E98 and, to a lesser degree, E106 xenograft lines (propagated through subcutaneous growth) consistently produced intracerebral tumors, displaying diffuse infiltrative growth in the brain parenchyma. In contrast, four xenograft lines (E434, E468, E473 and E478), established by direct intracerebral inoculation of human glioma cells and serially propagated intracerebrally, consistently showed extensive diffuse infiltration throughout the brain. After several passages, the neoplastic cells still carry typical chromosomal aberrations [(-1p/-19q in oligodendroglioma, +7/-10 in glioblastoma multiforme (GBM)]. Especially these latter four models and the E98 line thus represent adequate geno- and phenocopies of human gliomas and form an attractive platform to investigate different therapeutic approaches in a preclinical setting.

      View details for PubMedID 18371177
  • Aerobic glycolysis in cancers: implications for the usability of oxygen-responsive genes and fluorodeoxyglucose-PET as markers of tissue hypoxia. Int J Cancer
    Busk M, Horsman MR, Kristjansen PE, van der Kogel AJ, Bussink J, Overgaard J
    2008 Jun 15; 122 (12): 2726-34
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      The hypoxia-responsiveness of the glycolytic machinery may allow pretreatment identification of hypoxic tumors from HIF-1 targets (e.g., Glut-1) or [18F]-fluorodeoxyglucose positron emission tomography but results have been mixed. We hypothesized that this discrepancy is an inevitable consequence of elevated aerobic glycolysis in tumors (Warburg effect) as energetics in predominantly glycolytic cells is little affected by hypoxia. Accordingly, we characterized glycolytic and mitochondrial ATP generation in normoxic and anoxic cell lines. Measurements demonstrated that most cancer cells rely largely on aerobic glycolysis as it accounts for 56-63% of their ATP budget, but in the cervical carcinoma SiHa, ATP synthesis was mainly mitochondrial. Moreover, the stimulatory effect of anoxia on glycolytic flux was inversely correlated to the relative reliance on aerobic glycolysis. Next, tumor cells representing a Warburg or a nonglycolytic phenotype were grown in mice and spatial patterns of hypoxia (pimonidazole-stained), Glut-1 expression and (18)F-FDG uptake were analysed on sectioned tumors. Only in SiHa tumors did foci of elevated glucose metabolism consistently colocalize with regions of hypoxia and elevated Glut-1 expression. In contrast, spatial patterns of Glut-1 and pimonidazole staining correlated reasonably well in all tumors. In conclusion, Glut-1's value as a hypoxia marker is not severely restricted by aerobic glycolysis. In contrast, the specificity of (18)F-FDG uptake and Glut-1 expression as markers of regional hypoxia and glucose metabolism, respectively, scales inversely with the intensity of the Warburg effect. This linkage suggests that multi-tracer imaging combining FDG and hypoxia-specific markers may provide therapeutically relevant information on tumor energetic phenotypes.

      View details for PubMedID 18351643
  • Clinical radiobiology in 2008. Radiother Oncol
    Begg A, van der Kogel A
    2008 Mar; 86 (3): 295-9
  • Imaging hypoxia in xenografted and murine tumors with 18F-fluoroazomycin arabinoside: a comparative study involving microPET, autoradiography, PO2-polarography, and fluorescence microscopy. Int J Radiat Oncol Biol Phys
    Busk M, Horsman MR, Jakobsen S, Keiding S, van der Kogel AJ, Bussink J, Overgaard J
    2008 Mar 15; 70 (4): 1202-12
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      PURPOSE: Positron emission tomography (PET) allows noninvasive assessment of tumor hypoxia; however the combination of low resolution and slow tracer clearance from nonhypoxic tissue is problematic. The aim of this study was to examine the in vivo hypoxia selectivity of fluoroazomycin arabinoside ([18F]-FAZA), a promising tracer with improved washout kinetics from oxygenated tissue.

      METHODS AND MATERIALS: Three squamous cell carcinomas and one fibrosarcoma with widely differing spatial patterns of vascularization, hypoxia, and necrosis were grown in mice and evaluated with PET and complementary methods.

      RESULTS: Eppendorf electrode measurements consistently demonstrated median PO2 values<1 mm Hg. In accordance with that, PET revealed that all tumors accumulated [18F]-FAZA in excess of reference tissue. Next the two-dimensional spatial distribution of [18F]-FAZA (from autoradiography) was compared with fluorescence images of the same tumor sections showing localization of the hypoxia marker pimonidazole and the perfusion marker Hoechst 33342. Pixel-by-pixel analysis of co-registered images showed a highly significant co-localization between the two hypoxia markers and an inverse correlation (except for the fibrosarcoma) between the distribution of [18F]-FAZA and Hoechst dye. Moreover intratumoral heterogeneity in tracer distribution was clearly visible on autoradiograms, with a [18F]-FAZA concentration approximately six times higher in poorly oxygenated areas than in vascular hot spots.

      CONCLUSIONS: The distribution of [18F]-FAZA is consistent with hypoxia as the key driving force for tracer tissue retention in a selection of tumors with widely differing physiology.

      View details for PubMedID 18313528
  • Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer. Lancet Oncol
    Bussink J, van der Kogel AJ, Kaanders JH
    2008 Mar; 9 (3): 288-96
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      Activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (AKT) pathway is associated with three major radioresistance mechanisms: intrinsic radioresistance; tumour-cell proliferation; and hypoxia. Monitoring and manipulation of this signal-transduction pathway can have important implications for the management of head and neck cancer, because activation of the PI3-K/AKT pathway is a frequent event in these tumours. PI3-K/AKT signalling regulates cellular processes, including proliferation, invasion, apoptosis, and the upregulation of hypoxia-related proteins. Activation of this pathway can be caused by stimulation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). In clinical trials, a strong and independent association has been noted between expression of activated AKT and treatment outcome. Therefore, the search for molecular predictors of sensitivity to EGFR-directed treatment should be extended to markers of PI3-K/AKT activation. Another strategy might be the direct targeting and inhibition of this pathway. Such inhibition will enhance the efficacy of radiotherapy, by antagonising radiation-induced cellular defense mechanisms, especially in tumours that have activated the PI3-K/AKT cascade. Thus, the activation status of this pathway might be a key element for the prediction of treatment response and for therapeutic targeting in head and neck cancer.

      View details for PubMedID 18308254
  • Dose-volume effects in rat thoracolumbar spinal cord: the effects of nonuniform dose distribution. Int J Radiat Oncol Biol Phys
    Philippens ME, Pop LA, Visser AG, van der Kogel AJ
    2007 Sep 01; 69 (1): 204-13
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      PURPOSE: To investigate dose-volume effects in rat spinal cord irradiated with nonuniform dose distributions and to assess regional differences in radiosensitivity.

      METHODS AND MATERIALS: A total of 106 rats divided into three groups were irradiated with (192)Ir gamma-rays at a high dose rate. The groups were irradiated with one, two, or six catheters distributed around the thoracolumbar spinal cord to create different dose distributions. After irradiation, the animals were tested for motor function for 9 months. The response was defined as motor dysfunction and WM or nerve root necrosis. Dose-response data were analyzed with a probit analysis as function of the dose level at a percentage of the volume (D(%)) and with different normal tissue complication probability models. Additionally, the histologic responses of the individual dose voxels were analyzed after registration with the histologic sections.

      RESULTS: The probit analysis at D(24) (24% of the volume) gave the best fit results. In addition, the Lyman Kutcher Burman model and the relative seriality model showed acceptable fits, with volume parameters of 0.17 and 0.53, respectively. The histology-based analysis revealed a lower radiosensitivity for the dorsal (50% isoeffective dose [ED(50)] = 32.3) and lateral WM (ED(50) = 33.7 Gy) compared with the dorsal (ED(50) = 25.9 Gy) and ventral nerve roots (ED(50) = 24.1 Gy).

      CONCLUSIONS: For this nonuniform irradiation, the spinal cord did not show typical serial behavior. No migration terms were needed for an acceptable fit of the dose-response curves. A higher radiosensitivity for the lumbar nerve roots than for the thoracic WM was found.

      View details for PubMedID 17707274
  • Tumour cell proliferation under hypoxic conditions in human head and neck squamous cell carcinomas. Oral Oncol
    Wijffels KI, Marres HA, Peters JP, Rijken PF, van der Kogel AJ, Kaanders JH
    2008 Apr; 44 (4): 335-44
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      Two mechanisms of radiotherapy resistance of major importance in head and neck cancer are tumour cell repopulation and hypoxia. Hypoxic tumour cells that retain their clonogenic potential can survive radiation treatment and lead to local recurrences. The aim of this study was to quantify this cellular population in a cohort of human head and neck carcinomas and to investigate the prognostic significance. The proliferation marker iododeoxyuridine (IdUrd) and the hypoxia marker pimonidazole were administered intravenously prior to biopsy taking in patients with stage II-IV squamous cell carcinoma of the head and neck. Triple immunohistochemical staining of blood vessels, IdUrd and pimonidazole was performed and co-localization of IdUrd and pimonidazole was quantitatively assessed by computerized image analysis. The results were related with treatment outcome. Thirty-nine biopsies were analyzed. Tumours exhibited different patterns of proliferation and hypoxia but generally the IdUrd signal was found in proximity to blood vessels whereas pimonidazole binding was predominantly at a distance from vessels. Overall, no correlations were found between proliferative activity and oxygenation status. The fraction of IdUrd-labelled cells positive for pimonidazole ranged from 0% to 16.7% with a mean of 2.4% indicating that proliferative activity was low in hypoxic areas and occurring mainly in the well-oxygenated tumour compartments. IdUrd positive cells in hypoxic areas made up only 0.09% of the total viable tumour cell mass. There were no associations between the magnitude of this cell population and local tumour control or survival. Co-localization between proliferating cells and hypoxia in head and neck carcinomas was quantified using an immunohistochemical triple staining technique combined with a computerized simultaneous analysis of multiple parameters. The proportion of cells proliferating under hypoxic conditions was small and no correlation with treatment outcome could be found.

      View details for PubMedID 17689286
  • 18F-FLT PET does not discriminate between reactive and metastatic lymph nodes in primary head and neck cancer patients. J Nucl Med
    Troost EG, Vogel WV, Merkx MA, Slootweg PJ, Marres HA, Peeters WJ, Bussink J, van der Kogel AJ, Oyen WJ, Kaanders JH
    2007 May; 48 (5): 726-35
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      UNLABELLED: Repopulation of clonogenic tumor cells is inversely correlated with radiation treatment outcome in head and neck squamous cell carcinomas. A functional imaging tool to assess the proliferative activity of tumors could improve patient selection for treatment modifications and could be used for evaluation of early treatment response. The PET tracer 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) can image tumor cell proliferation before and during radiotherapy, and it may provide biologic tumor information useful in radiotherapy planning. In the present study, the value of (18)F-FLT PET in determining the lymph node status in squamous cell carcinoma of the head and neck was assessed, with pathology as the gold standard.

      METHODS: Ten patients with newly diagnosed stage II-IV squamous cell carcinoma of the head and neck underwent (18)F-FLT PET before surgical tumor resection with lymph node dissection. Emission (18)F-FLT PET and CT images of the head and neck were recorded and fused, and standardized uptake values (SUVs) were calculated. From all 18 (18)F-FLT PET-positive lymph node levels and from 8 (18)F-FLT PET-negative controls, paraffin-embedded lymph node sections were stained and analyzed for the endogenous proliferation marker Ki-67 and for the preoperatively administered proliferation marker iododeoxyuridine. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for (18)F-FLT PET.

      RESULTS: Primary tumor sites were oral cavity (n=7), larynx (n=2), and maxillary sinus (n=1). Nine of the 10 patients examined had (18)F-FLT PET-positive lymph nodes (SUV(mean): median, 1.2; range, 0.8-2.9), but only 3 of these patients had histologically proven metastases. All metastatic lymph nodes showed Ki-67 and iododeoxyuridine staining in tumor cells. In the remaining 7 patients, there was abundant Ki-67 and iododeoxyuridine staining of B-lymphocytes in germinal centers in PET-positive lymph nodes, explaining the high rate of false-positive findings. The sensitivity, specificity, positive predictive value, and negative predictive value of (18)F-FLT PET were 100%, 16.7%, 37.5%, and 100%, respectively.

      CONCLUSION: In head and neck cancer patients, (18)F-FLT PET showed uptake in metastatic as well as in nonmetastatic reactive lymph nodes, the latter due to reactive B-lymphocyte proliferation. Because of the low specificity, (18)F-FLT PET is not suitable for assessment of pretreatment lymph node status. This observation may also negatively influence the utility of (18)F-FLT PET for early treatment response evaluation of small metastatic nodes.

      View details for PubMedID 17475960
  • The hypoxic tumour microenvironment, patient selection and hypoxia-modifying treatments. Clin Oncol (R Coll Radiol)
    Hoogsteen IJ, Marres HA, van der Kogel AJ, Kaanders JH
    2007 Aug; 19 (6): 385-96
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      Tumour hypoxia has been found to be a characteristic feature in many solid tumours. It has been shown to decrease the therapeutic efficacy of radiation treatment, surgery and some forms of chemotherapy. Successful approaches have been developed to counteract this resistance mechanism, although usually at the cost of increased short- and long-term side-effects. New methods for qualitative and quantitative assessment of tumour oxygenation have made it possible to establish the prognostic significance of tumour hypoxia. The ability to determine the degree and extent of hypoxia in solid tumours is not only important prognostically, but also in the selection of patients for hypoxia-modifying treatments. To provide the best attainable quality of life for individual patients it is of increasing importance that tools be developed that allow a better selection of patients for these intensified treatment strategies. Several genes and proteins involved in the response to hypoxia have been identified as potential candidates for future use in predictive assays. Although some markers and combinations have shown potential benefit and are associated with treatment outcome, their clinical usefulness needs to be validated in prospective trials. A review of published studies was carried out, focusing on the assessment of tumour hypoxia, patient selection and the possibilities to overcome hypoxia during treatment.

      View details for PubMedID 17433637
  • Dynamics of tumor hypoxia measured with bioreductive hypoxic cell markers. Radiat Res
    Ljungkvist AS, Bussink J, Kaanders JH, van der Kogel AJ
    2007 Feb; 167 (2): 127-45
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      Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1alpha and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.

      View details for PubMedID 17390721
  • Tumor microenvironment in head and neck squamous cell carcinomas: predictive value and clinical relevance of hypoxic markers. A review. Head Neck
    Hoogsteen IJ, Marres HA, Bussink J, van der Kogel AJ, Kaanders JH
    2007 Jun; 29 (6): 591-604
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      BACKGROUND: Hypoxia and tumor cell proliferation are important factors determining the treatment response of squamous cell carcinomas of the head and neck. Successful approaches have been developed to counteract these resistance mechanisms although usually at the cost of increased short- and long-term side effects. To provide the best attainable quality of life for individual patients and the head and neck cancer patient population as a whole, it is of increasing importance that tools be developed that allow a better selection of patients for these intensified treatments.

      METHODS: A literature review was performed with special focus on the predictive value and clinical relevance of endogenous hypoxia-related markers.

      RESULTS: New methods for qualitative and quantitative assessment of functional microenvironmental parameters such as hypoxia, proliferation, and vasculature have identified several candidate markers for future use in predictive assays. Hypoxia-related markers include hypoxia inducible factor (HIF)-1alpha, carbonic anhydrase IX, glucose transporters, erythropoietin receptor, osteopontin, and others. Although several of these markers and combinations of markers are associated with treatment outcome, their clinical value as predictive factors remains to be established.

      CONCLUSIONS: A number of markers and marker profiles have emerged that may have potential as a predictive assay. Validation of these candidate assays requires testing in prospective trials comparing standard treatment against experimental treatments targeting the related microregional constituent.

      View details for PubMedID 17252597
  • Microenvironmental transformations by VEGF- and EGF-receptor inhibition and potential implications for responsiveness to radiotherapy. Radiother Oncol
    Bussink J, Kaanders JH, van der Kogel AJ
    2007 Jan; 82 (1): 10-7
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      The microregional distribution and dynamics of tumor cell hypoxia and proliferation are important determinants of tumor aggressiveness and resistance to treatment. Modulation of these elements by biological targeted drugs such as EGFR- and VEGFR-inhibitors may improve the effect of radiotherapy significantly. These combinations are being evaluated in clinical trials and evidence of their effectiveness is accumulating. However, the mechanistic basis of this cooperative effect and the role and behavior of the microregional tumor phenotype under EGF- and VEGF-blockage is poorly understood. Unfolding of these interactions and effects further downstream is necessary to exploit these biological modifiers most profitably to unravel questions such as: (1) can microregional phenotypes be modulated by EGFR- or VEGFR-blockage and how do downstream effects in the signaling pathways relate to these changes? (2) How do the microregional changes induced by EGFR- and VEGF-blockage affect the responsiveness of tumors to ionizing radiation? Answering these questions will improve our understanding of tumor growth related phenotypic transformations at the microregional level and how these can be influenced by modulation of the EGF- and VEGF-signaling pathways. This knowledge can be used to identify and improve therapeutic combinations with the novel biological modifiers and test a variety of biological-based treatment approaches.

      View details for PubMedID 17141899
  • Hedgehog: an attribute to tumor regrowth after chemoradiotherapy and a target to improve radiation response. Clin Cancer Res
    Sims-Mourtada J, Izzo JG, Apisarnthanarax S, Wu TT, Malhotra U, Luthra R, Liao Z, Komaki R, van der Kogel A, Ajani J, Chao KS
    2006 Nov 01; 12 (21): 6565-72
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      PURPOSE: Despite aggressive chemotherapy, radiotherapy, surgery, or combination approaches, the survival rate of patients with esophageal cancer remains poor. Recent studies have suggested that constitutive activation of the Hedgehog (Hh) pathway in cancers of the digestive tract may contribute to the growth and maintenance of cancer. However, the relationship between Hh signaling and therapeutic response is unknown.

      EXPERIMENTAL DESIGN: The expression and temporal kinetics of Hh signaling and proliferation biomarkers after chemoradiotherapy were examined in esophageal tumor xenografts. Additionally, immunohistochemical analysis of Sonic Hh (Shh) and Gli-1 expression were done on residual tumors from patients who received neoadjuvant chemoradiotherapy followed by surgery. The ability of Shh signaling to induce proliferation in esophageal cell lines was determined. Expression of cell cycle checkpoint proteins was analyzed in cells in which Hh signaling was activated or inhibited. We further determined the effect of inhibiting Hh signaling in sensitizing esophageal tumors to radiation.

      RESULTS: We showed that the Shh signaling pathway was extensively activated in esophageal cancer xenografts and residual tumors after chemoradiotherapy and the temporal kinetics of Hh signaling preceded increases in proliferation biomarker expression and tumor size during tumor regrowth. We further showed that Hh pathway activity influences proliferation rates of esophageal cancer cell lines through up-regulation of the G1-cyclin-Rb axis. Additionally, we found that blocking Hh signaling enhanced radiation cytotoxicity of esophageal cancer cells.

      CONCLUSIONS: These results suggest that activation of the Hh pathway may promote tumor repopulation after chemoradiotherapy and contribute to chemoradiation resistance in esophageal cancers.

      View details for PubMedID 17085672
  • Angiogenesis-independent tumor growth mediated by stem-like cancer cells. Proc Natl Acad Sci U S A
    Sakariassen PØ, Prestegarden L, Wang J, Skaftnesmo KO, Mahesparan R, Molthoff C, Sminia P, Sundlisaeter E, Misra A, Tysnes BB, Chekenya M, Peters H, Lende G, Kalland KH, Øyan AM, Petersen K, Jonassen I, van der Kogel A, Feuerstein BG, Terzis AJ, Bjerkvig R, Enger PØ
    2006 Oct 31; 103 (44): 16466-71
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      In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

      View details for PubMedID 17056721
  • Late effects of radiation on the central nervous system: role of vascular endothelial damage and glial stem cell survival. Radiat Res
    Coderre JA, Morris GM, Micca PL, Hopewell JW, Verhagen I, Kleiboer BJ, van der Kogel AJ
    2006 Sep; 166 (3): 495-503
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      Selective irradiation of the vasculature of the rat spinal cord was used in this study, which was designed specifically to address the question as to whether it is the endothelial cell or the glial progenitor cell that is the target responsible for late white matter necrosis in the CNS. Selective irradiation of the vascular endothelium was achieved by the intraperitoneal (ip) administration of a boron compound known as BSH (Na(2)B(12)H(11)SH), followed by local irradiation with thermal neutrons. The blood-brain barrier is known to exclude BSH from the CNS parenchyma. Thirty minutes after the ip injection of BSH, the boron concentration in blood was 100 microg (10)B/ g, while that in the CNS parenchyma was below the detection limit of the boron analysis system, <1 microg (10)B/g. An ex vivo clonogenic assay of the O2A (oligodendrocyte-type 2 astrocyte) glial progenitor cell survival was performed 1 week after irradiation and at various times during the latent period before white matter necrosis in the spinal cord resulted in myelopathy. One week after 4.5 Gy of thermal neutron irradiation alone (approximately one-third of the dose required to produce a 50% incidence of radiation myelopathy), the average glial progenitor cell surviving fraction was 0.03. The surviving fraction of glial progenitor cells after a thermal neutron irradiation with BSH for a comparable effect was 0.46. The high level of glial progenitor cell survival after irradiation in the presence of BSH clearly reflects the lower dose delivered to the parenchyma due to the complete exclusion of BSH by the blood-brain barrier. The intermediate response of glial progenitor cells after irradiation with thermal neutrons in the presence of a boron compound known as BPA (p-dihydroxyboryl-phenylalanine), again for a dose that represents one-third the ED(50) for radiation-induced myelopathy, reflects the differential partition of boron-10 between blood and CNS parenchyma for this compound, which crosses the blood-brain barrier, at the time of irradiation. The large differences in glial progenitor survival seen 1 week after irradiation were also maintained during the 4-5-month latent period before the development of radiation myelopathy, due to selective white matter necrosis, after irradiation with doses that would produce a high incidence of radiation myelopathy. Glial progenitor survival was similar to control values at 100 days after irradiation with a dose of thermal neutrons in the presence of BSH, significantly greater than the ED(100), shortly before the normal time of onset of myelopathy. In contrast, glial progenitor survival was less than 1% of control levels after irradiation with 15 Gy of thermal neutrons alone. This dose of thermal neutrons represents the approximate ED(90-100) for myelopathy. The response to irradiation with an equivalent dose of X rays (ED(90): 23 Gy) was intermediate between these extremes as it was to thermal neutrons in the presence of BPA at a slightly lower dose equivalent to the approximate ED(60) for radiation myelopathy. The conclusions from these studies, performed at dose levels approximately iso-effective for radiation-induced myelopathy as a consequence of white matter necrosis, were that the large differences observed in glial progenitor survival were directly related to the dose distribution in the parenchyma. These observations clearly indicate the relative importance of the dose to the vascular endothelium as the primary event leading to white matter necrosis.

      View details for PubMedID 16953668
  • Imaging hypoxia after oxygenation-modification: comparing [18F]FMISO autoradiography with pimonidazole immunohistochemistry in human xenograft tumors. Radiother Oncol
    Troost EG, Laverman P, Kaanders JH, Philippens M, Lok J, Oyen WJ, van der Kogel AJ, Boerman OC, Bussink J
    2006 Aug; 80 (2): 157-64
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      PURPOSE: Hypoxia is one of the reasons for radiation therapy resistance. Positron emission tomography using (18)F-labeled misonidazole ([(18)F]FMISO) is a non-invasive method of imaging tumor hypoxia. Aim of this study was to validate [(18)F]FMISO against the clinically most widely used hypoxic cell marker pimonidazole under different oxygenation conditions.

      MATERIALS AND METHODS: One human head and neck squamous cell carcinoma (SCCNij3) and two human glioblastoma (E102 and E106) xenograft tumor lines were studied after injection of [(18)F]FMISO and pimonidazole. Control mice were compared with a second group breathing carbogen to reduce tumor hypoxia and with a third group with clamped tumors to increase hypoxia. Tumor sections were analyzed on a phosphor imaging system and consecutively stained immunohistochemically (IHC) for visualization of pimonidazole. Pixel-by-pixel analysis was performed and the hypoxic fraction, obtained after segmentation of the pimonidazole signal, was related to the mean optical density of [(18)F]FMISO and pimonidazole.

      RESULTS: A moderate pixel-by-pixel correlation between [(18)F]FMISO autoradiography and pimonidazole IHC was found for the control tumors, after carbogen breathing and after clamping for SCCNij3. For E102 and E106, mean signal intensities for pimonidazole significantly decreased after carbogen breathing and increased after clamping, mean [(18)F]FMISO signal intensities increased significantly after clamping and a significant correlation between the hypoxic fractions and the mean [(18)F]FMISO signal intensities was found.

      CONCLUSIONS: [(18)F]FMISO autoradiography and pimonidazole immunohistochemistry can both be used to visualize treatment induced changes in tumor hypoxia. However, the response to these modifications differs widely between xenograft tumor lines.

      View details for PubMedID 16905213
  • Colonisation of Clostridium in the body is restricted to hypoxic and necrotic areas of tumours. Anaerobe
    Lambin P, Theys J, Landuyt W, Rijken P, van der Kogel A, van der Schueren E, Hodgkiss R, Fowler J, Nuyts S, de Bruijn E, Van Mellaert L, Anné J
    1998 Aug; 4 (4): 183-8
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      The use of gene therapy is one of the most recent molecular strategies for the treatment of cancer. It is essential, however, to have an efficient transfer system by which the desired gene can be delivered to the correct environment. The experiments described in this report investigate apathogenic Clostridium as a possible vector to transfer a specific gene product into the extracellular microenvironment of the tumour which is hypoxic/necrotic in parts, using WAG/Rij rats with transplantable rhabdomyosarcomas as a model. Our data show that Clostridium, after systemic administration of at least 10(7) spores, specifically colonises the hypoxic/necrotic areas of our tumour model, the most efficient species being C. acetobutylicum (NI-4082) and C. oncolyticum. Although spores were also detected in normal tissues for up to 4 weeks, they did not germinate in these tissues. We conclude that it seems likely that these bacteria can be used as a selective transfer system into the extracellular environment of tumours which have hypoxic regions. This strategy would be more tumour-specific than various other strategies that are currently being investigated in anti-cancer gene therapy.

      View details for PubMedID 16887640
  • Effect of carbogen breathing on the radiation response of a human glioblastoma xenograft: analysis of hypoxia and vascular parameters of regrowing tumors. Strahlenther Onkol
    Schuuring J, Bussink J, Bernsen HJ, Peeters W, van der Kogel AJ
    2006 Jul; 182 (7): 408-14
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      BACKGROUND AND PURPOSE: The aim of these experiments was to study the relationship between the previously demonstrated efficacy of carbogen breathing on tumor oxygenation status and the response to radiation assessed by a growth delay assay. This study was also developed to investigate the microenvironmental changes caused by combined treatment compared to irradiation only in regrowing tumors.

      MATERIAL AND METHODS: A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without carbogen breathing. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after immunohistochemical staining. Tumor growth delay was monitored for up to 120 days after treatment.

      RESULTS: In general, there was no benefit of combined treatment. However, a small subgroup with good response to combined radiation and carbogen treatment was identified showing little hypoxia and mainly necrosis in the regrowing tumors. These microenvironmental characteristics were not seen in tumors of the other treatment groups.

      CONCLUSION: The observations suggest that a subgroup of patients, who could potentially benefit from the combined carbogen and radiation treatment, might be identified. However, the heterogeneous response to treatment illustrates the need for selection of patients before start of treatment.

      View details for PubMedID 16826360
  • Carbogen breathing differentially enhances blood plasma volume and 5-fluorouracil uptake in two murine colon tumor models with a distinct vascular structure. Neoplasia
    van Laarhoven HW, Gambarota G, Lok J, Lammens M, Kamm YL, Wagener T, Punt CJ, van der Kogel AJ, Heerschap A
    2006 Jun; 8 (6): 477-87
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      For the systemic treatment of colorectal cancer, 5-fluorouracil (FU)-based chemotherapy is the standard. However, only a subset of patients responds to chemotherapy. Breathing of carbogen (95% O2 and 5% CO2) may increase the uptake of FU through changes in tumor physiology. This study aims to monitor in animal models in vivo the effects of carbogen breathing on tumor blood plasma volume, pH, and energy status, and on FU uptake and metabolism in two colon tumor models C38 and C26a, which differ in their vascular structure and hypoxic status. Phosphorus-31 magnetic resonance spectroscopy (MRS) was used to assess tumor pH and energy status, and fluorine-19 MRS was used to follow FU uptake and metabolism. Advanced magnetic resonance imaging methods using ultrasmall particles of iron oxide were performed to assess blood plasma volume. The results showed that carbogen breathing significantly decreased extracellular pH and increased tumor blood plasma volume and FU uptake in tumors. These effects were most significant in the C38 tumor line, which has the largest relative vascular area. In the C26a tumor line, carbogen breathing increased tumor growth delay by FU. In this study, carbogen breathing also enhanced systemic toxicity by FU.

      View details for PubMedID 16820094
  • The prognostic value of endogenous hypoxia-related markers for head and neck squamous cell carcinomas treated with ARCON. Radiother Oncol
    Jonathan RA, Wijffels KI, Peeters W, de Wilde PC, Marres HA, Merkx MA, Oosterwijk E, van der Kogel AJ, Kaanders JH
    2006 Jun; 79 (3): 288-97
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      BACKGROUND AND PURPOSE: Hypoxic radioresistance is an important cause for treatment failure in a number of tumor types including head and neck cancers. Recent studies suggest that outcome can be improved by oxygenation modifying treatments such as ARCON. A robust endogenous marker of hypoxia might be a valuable aid to select patients for such treatments. The aim of this investigation was to study associations between the putative endogenous hypoxia markers CA-IX, Glut-1 and Glut-3 and clinical tumor and patient characteristics and to evaluate the prognostic value of these markers.

      PATIENTS AND METHODS: Tumor biopsies from 58 patients treated with ARCON in a phase II trial were included. Tumor sections were immunohistochemically stained for CA-IX, Glut-1 and Glut-3. Sections were scored for relative tumor area stained by the markers (CA-IX and Glut-3) and for intensity of staining (Glut-1 and Glut-3). Further, sections were stained for CD34, an endothelial marker to assess microvascular density.

      RESULTS: Staining of CA-IX and Glut-3 was observed at some distance from vessels and adjacent to necrosis. Glut-1 staining was generally very diffuse. The distribution of clinical characteristics was equal between tumors with high and low marker expression. Significant differences were found for locoregional control (P = 0.04) and for freedom of distant metastases (P = 0.02) in favour of patients with high CA-IX positivity (>25% of tumor area). High Glut-3 expression was associated with a better locoregional control (P = 0.04). Higher Glut-1 intensity was associated with an increased rate of distant metastases (P = 0.0005) and a worse overall survival (P = 0.001).

      CONCLUSIONS: The inconsistent associations with outcome of CA-IX and the glucose transporters indicate that different factors play a role in up-regulation of these markers. Compared to studies with conventional treatment, the correlation between CA-IX expression and Glut-3 expression and outcome was reversed after treatment with ARCON. This does not support the potential of any of these proteins as very specific and robust hypoxia markers.

      View details for PubMedID 16730088
  • Assessment of absolute blood volume in carcinoma by USPIO contrast-enhanced MRI. Magn Reson Imaging
    Gambarota G, van Laarhoven HW, Philippens M, Lok J, van der Kogel A, Punt CJ, Heerschap A
    2006 Apr; 24 (3): 279-86
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      OBJECTIVES: The characterization of tumor vasculature is essential in studying tumor physiology. The aim of this study was to develop a new method - based on water proton MR density measurements, in combination with ultrasmall superparamagnetic iron oxide (USPIO) administration - to measure absolute blood volume (BV) in murine colon carcinoma.

      MATERIALS AND METHODS: MRI experiments were performed at 7 T. CPMG imaging was performed on subcutaneous murine colon carcinoma in six mice before and after administration of an USPIO blood-pool contrast agent. Density maps were obtained from the signal amplitude at TE=0 of the CPMG decay fit. Post-USPIO density maps were subtracted from pre-USPIO density maps to quantitatively yield absolute tumor BV maps. In a separate group of mice (n=6), the relative vascular area (RVA) of tumors was determined by immunohistochemistry.

      RESULTS: Ultrasmall superparamagnetic iron oxide administration resulted in a small decrease in the water proton MR density. The BV averaged over the six tumors was 4.6+/-1.6%. The value of the RVA measured by immunohistochemical staining was equal to 3.9+/-2.2%.

      CONCLUSIONS: After administration of an USPIO blood-pool agent (T(2) relaxivity > 100 mM(-1) s(-1)), the blood water protons become MRI invisible, and pixel-by-pixel BV map can be obtained by subtracting the calculated post-USPIO density map from the pre-USPIO density map. The value of absolute BV obtained with this novel MR approach is in good agreement with the value of the relative vascular measured by immunohistochemical staining.

      View details for PubMedID 16563957
  • Influence of adjacent low-dose fields on tolerance to high doses of protons in rat cervical spinal cord. Int J Radiat Oncol Biol Phys
    Bijl HP, van Luijk P, Coppes RP, Schippers JM, Konings AW, van der Kogel AJ
    2006 Mar 15; 64 (4): 1204-10
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      PURPOSE: The dose-response relationship for a relatively short length (4 mm) of rat spinal cord has been shown to be significantly modified by adjacent low-dose fields. In an additional series of experiments, we have now established the dose-volume dependence of this effect.

      METHODS AND MATERIALS: Wistar rats were irradiated on the cervical spinal cord with single doses of unmodulated protons (150 MeV) to obtain sharp lateral penumbras, by use of the shoot-through technique, which employs the plateau of the depth-dose profile rather than the Bragg peak. Three types of inhomogeneous dose distributions were administered: Twenty millimeters of cervical spinal cord were irradiated with variable subthreshold (= bath) doses (4 and 18 Gy). At the center of the 20-mm segment, a short segment of 2 mm or 8 mm (= shower) was irradiated with variable single doses. These inhomogeneous dose distributions are referred to as symmetrical bath-and-shower experiments. An asymmetrical dose distribution was arranged by irradiation of 12 mm (= bath) of spinal cord with a dose of 4 Gy. The caudal 2 mm (= shower) of the 12-mm bath was additionally irradiated with variable single doses. This arrangement of inhomogeneous dose distribution is referred to as asymmetrical bath-and-shower experiment. The endpoint for estimation of the dose-response relationships was paralysis of the fore limbs or hind limbs and confirmation by histology.

      RESULTS: The 2-mm bath-and-shower experiments with a 4-Gy bath dose showed a large shift of the dose-response curves compared with the 2-mm single field, which give lower ED50 values of 61.2 Gy and 68.6 Gy for the symmetrical and asymmetrical arrangement, respectively, compared with an ED50 of 87.8 Gy after irradiation of a 2-mm field only. If the bath dose is increased to 18 Gy, the ED50 value is decreased further to 30.9 Gy. For an 8-mm field, addition of a 4-Gy bath dose did not modify the ED50 obtained for an 8-mm field only (23.2 and 23.1 Gy).

      CONCLUSIONS: The spinal cord tolerance of relatively small volumes (shower) is strongly affected by low-dose irradiation (= bath) of adjacent tissue. The results of all bath-and-shower experiments show the effect of a low bath dose to be highest for a field of 2 mm, less for 4 mm, and absent for 8 mm. Adding a 4-Gy bath to only 1 side of a 2-mm field still showed a large effect. Because glial progenitor cells are known to migrate over at least 2 to 3 mm, this observation indicates that interference with stem cell migration is not the most likely mechanism of a bath effect.

      View details for PubMedID 16504760
  • Dynamics of hypoxia, proliferation and apoptosis after irradiation in a murine tumor model. Radiat Res
    Ljungkvist AS, Bussink J, Kaanders JH, Wiedenmann NE, Vlasman R, van der Kogel AJ
    2006 Mar; 165 (3): 326-36
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      Proliferation and hypoxia affect the efficacy of radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine adenocarcinoma tumor line (C38). The hypoxia marker pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second hypoxia marker, CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated caspase 3 staining. After immunohistochemical staining, the tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated tumors to 8% at both 8 h and 28 h after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated tumors and 90% after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in tumor oxygenation after irradiation. In addition, a decreased tumor cell density without a significant change in tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.

      View details for PubMedID 16494521
  • Solid tumors "melt" from the inside after successful CD8 T cell attack. Eur J Immunol
    Blohm U, Potthoff D, van der Kogel AJ, Pircher H
    2006 Feb; 36 (2): 468-77
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      Adoptive transfer of tumor-specific T cells represents a promising approach for cancer immunotherapy. Here, we visualized the anti-tumor response of CD8 T cells from P14 TCR-transgenic mice specific for the model antigen GP33 by immunohistology. P14 T cells, adoptively transferred into tumor-bearing hosts, induced regression of established 3LL-A9(GP33) and MCA102(GP33) tumors that express GP33 as a tumor-associated model antigen. Strikingly, the visible effects of P14 T cell attack, such as the destruction of the tumor vasculature and accumulation of granulocytes, were predominantly detected inside the tumor mass. In regressing tumors, P14 T cells were found in the intact rim zone but not in central areas that were infiltrated with granulocytes and lacked CD31(+) endothelial cells. The rim of P14 T cell-treated tumors showed an increase in vascular density and decrease in hypoxia compared to untreated tumors. Hypoxic areas of tumors are known to exhibit decreased sensitivity to radiation therapy or chemotherapy. Thus, our data also imply that adoptive transfer of tumor-specific CD8 T cells might synergize with radiation therapy or chemotherapy in the elimination of solid tumors in vivo.

      View details for PubMedID 16385625
  • Effects of the tumor vasculature targeting agent NGR-TNF on the tumor microenvironment in murine lymphomas. Invest New Drugs
    van Laarhoven HW, Gambarota G, Heerschap A, Lok J, Verhagen I, Corti A, Toma S, Gallo Stampino C, van der Kogel A, Punt CJ
    2006 Jan; 24 (1): 27-36
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      TNF-alpha may improve drug delivery to tumors by alteration of vascular permeability. However, toxicity precludes its systemic administration in patients. NGR-TNF comprises TNF coupled to the peptide CNGRC, which is a ligand for CD13. CD13 is expressed on tumor vasculature. Therefore, to assess the efficacy of NGR-TNF its biological effect on tumor vasculature should be measured rather than its effect on tumor growth. The aim of this study was to assess the effects of a low dose of NGR-TNF (5 ng/kg) on vascular permeability, tumor hypoxia, perfusion and proliferation in lymphoma bearing mice. MRI measurements with blood pool contrast agent showed an increased leakage of the contrast agent from the vasculature in NGR-TNF treated tumors compared with controls (p < 0.05), suggesting NGR-TNF-induced vascular permeability. Immunohistochemical analysis two hours after NGR-TNF treatment showed a decrease in tumor hypoxia (p < 0.1) and an increase in labeling index of the S-phase marker bromodeoxyuridine (p < 0.1), possibly due to an increase in tumor blood flow after NGR-TNF treatment. Although a decrease in tumor hypoxia and an increase in labeling index could have lead to increased tumor growth, in this experiment after one day a decrease in tumor volume was measured. Possibly, the effects on tumor hypoxia and proliferation two hours after treatment are transient and overruled by other, more longlasting effects. For example, the observed increase in vascular permeability may lead to haemoconcentration and increased interstitial pressure, ultimately resulting in an reduction of tumor blood flow and thus a decrease in tumor growth. A beneficial effect of NGR-TNF in combination with other therapeutical agents may therefore critically depend on the sequence and timing of the regimens. Currently, NGR-TNF is being tested in clinical studies.

      View details for PubMedID 16379040
  • Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer. Int J Radiat Oncol Biol Phys
    van Laarhoven HW, Kaanders JH, Lok J, Peeters WJ, Rijken PF, Wiering B, Ruers TJ, Punt CJ, Heerschap A, van der Kogel AJ
    2006 Feb 01; 64 (2): 473-82
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      PURPOSE: To investigate hypoxia measured by pimonidazole binding, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CA-IX) expression, proliferation, and vascularity in liver metastases of colorectal cancer and to compare GLUT1 and CA-IX expression in corresponding primary tumors.

      METHODS AND MATERIALS: Twenty-five patients with liver metastases of colorectal cancer, planned for metastasectomy, were included. The hypoxia marker pimonidazole and proliferation marker iododeoxyuridine were administered before surgery. After immunofluorescent staining of the frozen metastases, pimonidazole binding, vascularity, and proliferation were analyzed quantitatively. Thirteen paraffin-embedded primary tumors were stained immunohistochemically for GLUT1 and CA-IX expression, which was analyzed semiquantitatively in primary tumors and corresponding liver metastases.

      RESULTS: In liver metastases, pimonidazole binding showed a pattern consistent with diffusion-limited hypoxia. The mean pimonidazole-positive fraction was 0.146; the mean distance from vessels to pimonidazole-positive areas was 80 microm. When expressed, often co-localization was observed between pimonidazole binding and GLUT1 or CA-IX expression, but microregional areas of mismatch were also observed. No correlation between the level of pimonidazole binding and GLUT1 or CA-IX expression was observed. In some patients, a large fraction (up to 30%) of proliferating cells was present in pimonidazole-stained areas. Expression of CA-IX in primary tumors and metastases showed a significant correlation, which was absent for GLUT1 expression.

      CONCLUSIONS: Compared with other tumor types, liver metastases of colorectal cancer contain large amounts of hypoxic cells. The lack of correlation with pimonidazole binding brings into question the value of GLUT1 and CA-IX as endogenous markers of hypoxia.

      View details for PubMedID 16242253
  • Oxygen-modifying treatment with ARCON reduces the prognostic significance of hemoglobin in squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys
    Hoogsteen IJ, Pop LA, Marres HA, Merkx MA, van den Hoogen FJ, van der Kogel AJ, Kaanders JH
    2006 Jan 01; 64 (1): 83-9
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      PURPOSE: To evaluate the prognostic significance of hemoglobin (Hb) levels measured before and during treatment with accelerated radiotherapy with carbogen and nicotinamide (ARCON).

      METHODS AND MATERIALS: Two hundred fifteen patients with locally advanced tumors of the head and neck were included in a phase II trial of ARCON. This treatment regimen combines accelerated radiotherapy for reduction of repopulation with carbogen breathing and nicotinamide to reduce hypoxia. In these patients, Hb levels were measured before, during, and after radiotherapy.

      RESULTS: Preirradiation and postirradiation Hb levels were available for 206 and 195 patients respectively. Hb levels below normal were most frequently seen among patients with T4 (p < 0.001) and N2 (p < 0.01) disease. Patients with a larynx tumor had significantly higher Hb levels (p < 0.01) than other tumor sites. During radiotherapy, 69 patients experienced a decrease in Hb level. In a multivariate analysis there was no prognostic impact of Hb level on locoregional control, disease-free survival, and overall survival. Primary tumor site was independently prognostic for locoregional control (p = 0.018), and gender was the only prognostic factor for disease-free and overall survival (p < 0.05). High locoregional control rates were obtained for tumors of the larynx (77%) and oropharynx (72%).

      CONCLUSION: Hemoglobin level was not found to be of prognostic significance for outcome in patients with squamous cell carcinoma of the head and neck after oxygen-modifying treatment with ARCON.

      View details for PubMedID 16213107
  • Gadopentetate dimeglumine and FDG uptake in liver metastases of colorectal carcinoma as determined with MR imaging and PET. Radiology
    van Laarhoven HW, de Geus-Oei LF, Wiering B, Lok J, Rijpkema M, Kaanders JH, Krabbe PF, Ruers T, Punt CJ, van der Kogel AJ, Oyen WJ, Heerschap A
    2005 Oct; 237 (1): 181-8
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      PURPOSE: To examine the in vivo relationship between fluorine 18 fluorodeoxyglucose (FDG) uptake, as measured with positron emission tomography (PET), and functional tumor vasculature, as measured with dynamic contrast material-enhanced magnetic resonance (MR) imaging, in patients with liver metastases of colorectal cancer.

      MATERIALS AND METHODS: All patients provided written informed consent, and the study was approved by the institutional review board. A total of 26 patients (12 men and 14 women; mean age, 59 years) who were suspected of having liver metastases of histologically proved colorectal cancer and underwent work-up for liver metastasectomy were included. Patients underwent whole-body FDG PET, and tumor-to-nontumor ratio of FDG uptake in metastases was calculated. Dynamic contrast-enhanced MR imaging was performed, and the rate constant k(ep) (s(-1)) of gadopentetate dimeglumine uptake in metastases was determined. Pimonidazole was used to determine tumor hypoxia and vascular density of metastases. To assess the relationship between FDG uptake, rate constant k(ep) of gadopentetate dimeglumine uptake, hypoxic fraction, and vascular density, the Pearson correlation coefficient was calculated.

      RESULTS: Negative correlation between tumor-to-nontumor ratio of FDG uptake and rate constant k(ep) was observed (r = -0.421, P = .082). No correlation between tumor hypoxia and tumor-to-nontumor ratio of FDG uptake or rate constant k(ep) was found. A positive correlation was observed between vascular density and rate constant k(ep) (r = 0.458, P = .034) but not between tumor-to-nontumor ratio of FDG uptake.

      CONCLUSION: Negative correlation between tumor-to-nontumor ratio of FDG uptake and rate constant k(ep) suggests that lower values of gadopentetate dimeglumine uptake imply an acutely reduced supply of oxygen, which necessitates a higher uptake of glucose to maintain tumor energy levels. The positive correlation of vascular density with rate constant k(ep), but not with tumor-to-nontumor ratio of FDG uptake, emphasizes the potential of dynamic contrast-enhanced MR imaging to enable measurement of tumor vascularity in vivo and its additional value compared with ex vivo methods.

      View details for PubMedID 16183932
  • ESTRO-Wolfsberg a synergistic approach towards modern radiobiology. Radiother Oncol
    Overgaard J, van der Kogel A
    2005 Aug; 76 (2): 109-11
  • Modulation of hypoxia in murine liver metastases of colon carcinoma by nicotinamide and carbogen. Radiat Res
    van Laarhoven HW, Bussink J, Lok J, Verhagen I, Punt CJ, Heerschap A, Kaanders JH, van der Kogel AJ
    2005 Sep; 164 (3): 245-9
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      There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.

      View details for PubMedID 16137196
  • Erythropoietin receptor is not a surrogate marker for tumor hypoxia and does not correlate with survival in head and neck squamous cell carcinomas. Radiother Oncol
    Hoogsteen IJ, Peeters WJ, Marres HA, Rijken PF, van den Hoogen FJ, van der Kogel AJ, Kaanders JH
    2005 Aug; 76 (2): 213-8
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      BACKGROUND AND PURPOSE: To evaluate erythropoietin receptor (EPOR) expression in human head and neck squamous cell carcinomas and correlate this to the presence of tumor hypoxia and treatment outcome.

      PATIENTS AND METHODS: Eighty-five patients with locally advanced tumors of the head and neck were included. Of these, 34 were given the hypoxia marker pimonidazole i.v. 2 h prior to biopsy taking. Contiguous paraffin embedded biopsies were stained for EPOR expression and, if administered, for pimonidazole binding. Immunohistochemical staining for EPOR was interpreted semiquantitatively according to a composite scale, ranging from 0 to 200. Pimonidazole positivity was quantitatively analyzed in a semiautomatic way.

      RESULTS: Diffuse weak-to-moderate cytoplasmic and membrane EPOR immunostaining was observed in 80 of 85 biopsies (94%) and staining scores ranged from 0 to 198 (median 100). No correlations were found between EPOR expression, and the primary tumor site, T-stage or N-stage. Also, There was no association between EPOR expression and treatment outcome. The degree of tumor hypoxia represented by the relative area of pimonidazole binding varied between 0 and 26% (median 7%). Contiguous biopsy sections showed a lack of colocalization between EPOR and pimonidazole binding.

      CONCLUSION: EPOR expression was demonstrated in the majority of the head and neck tumors. No colocalization was found between EPOR expression and pimonidazole binding indicating that the presence or absence of hypoxia did not necessarily indicate a distinct pattern of EPOR expression. The level of EPOR expression was not of prognostic significance in patients with head and neck cancer, although small effects of EPOR cannot be excluded because of the sample size of this study.

      View details for PubMedID 16112214
  • Decreased repopulation as well as increased reoxygenation contribute to the improvement in local control after targeting of the EGFR by C225 during fractionated irradiation. Radiother Oncol
    Krause M, Ostermann G, Petersen C, Yaromina A, Hessel F, Harstrick A, van der Kogel AJ, Thames HD, Baumann M
    2005 Aug; 76 (2): 162-7
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      BACKGROUND AND PURPOSE: Inhibition of repopulation and enhanced reoxygenation has been suggested to contribute to improvement of local tumour control after fractionated irradiation combined with inhibitors of the epidermal growth factor receptor (EGFR). The present study addresses this hypothesis in FaDu human squamous cell carcinoma. For this tumour model marked repopulation and incomplete reoxygenation during fractionated irradiation has previously been demonstrated. Furthermore, the anti-EGFR monoclonal antibody C225 has been shown to significantly improve the results of fractionated irradiation in this tumour.

      MATERIALS AND METHODS: FaDu tumours in nude mice were irradiated with 18 fractions in 18 days (18f/18d) or 18 fractions in 36 days (18f/36d). Three Gy fractions were given either under ambient or under clamp hypoxic conditions. C225 or carrier was applied four times during the course of treatment. Fractionated irradiations were followed by graded top-up doses to obtain complete dose-response curves for local tumour control. Tumour control dose 50% (TCD50) was determined at day 120 after end of treatment.

      RESULTS: Significant repopulation and reoxygenation occurred during fractionated irradiation of FaDu tumours (P-values between 0.028 and <0.001). Application of C225 significantly decreased TCD50 for 18f/36d under ambient conditions (P=0.04). Bootstrap analysis revealed decreased repopulation and increased reoxygenation after application of C225 (P=0.06 for the combined effect). This was further corroborated by a significant effect of C225 on the 'repopulated' dose under ambient conditions which is influenced by both, reoxygenation and repopulation (P=0.012).

      CONCLUSIONS: Our study provides evidence that both decreased repopulation as well as increased reoxygenation contribute to the improvement of local control after targeting of EGFR by C225 during fractionated irradiation of FaDu tumours.

      View details for PubMedID 16024114
  • Comparison of different methods of CAIX quantification in relation to hypoxia in three human head and neck tumor lines. Radiother Oncol
    Troost EG, Bussink J, Kaanders JH, van Eerd J, Peters JP, Rijken PF, Boerman OC, van der Kogel AJ
    2005 Aug; 76 (2): 194-9
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      PURPOSE: In head and neck cancer, it has been shown that hypoxic tumors respond poorly to therapy. Methods to identify hypoxic tumors are, therefore, of importance to select patients for oxygenation modifying or other intensified treatments. The aim of this study was to compare tumor cell hypoxia assessed by the hypoxic cell marker pimonidazole (PIMO) with expression of the endogenous hypoxia-related marker carbonic anhydrase IX (CAIX) in three human head and neck tumor lines.

      MATERIAL AND METHODS: Forty-five tumors of three human head and neck tumor lines, SCCNij3, SCCNij59 and MEC82, xenografted in athymic mice, were used. CAIX was quantified by biodistribution (% injected dose/g tumor) after injecting 3-5 microl 111In-labeled G250 mouse antibody 3 days prior to euthanizing. In a tissue section from the same tumor, fractions of tumor area positive for PIMO, CAIX and Hoechst 33342 (perfusion marker) were assessed after immunohistochemical staining, using a digital image analysis system.

      RESULTS: SCCNij3 and MEC82 were relatively hypoxic tumor lines with fractions of tumor area positive for pimonidazole of 0.16 and 0.15, respectively. SCCNij59 was a better-oxygenated tumor line with a PIMO-fraction of 0.03. The three tumor lines showed different levels and patterns of CAIX immunohistochemical staining, but only in MEC82 there was a good correlation between PIMO-fraction and CAIX-fraction (r2=0.92, P<0.0001). Correlations between 111In-G250 uptake and CAIX-fraction or PIMO-fraction within tumor lines were weak or absent.

      CONCLUSIONS: Assessment of CAIX expression depends largely on the techniques and tumor lines used. Furthermore, the immunohistochemical staining pattern of CAIX relative to PIMO differs between human tumor lines of similar anatomical origin. Therefore, the use of CAIX as endogenous marker of tumor hypoxia remains questionable.

      View details for PubMedID 16024110
  • Thymidine analogues to assess microperfusion in human tumors. Int J Radiat Oncol Biol Phys
    Janssen HL, Ljungkvist AS, Rijken PF, Sprong D, Bussink J, van der Kogel AJ, Haustermans KM, Begg AC
    2005 Jul 15; 62 (4): 1169-75
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      PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342.

      METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored.

      RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis.

      CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.

      View details for PubMedID 15990022
  • Hypoxic cell turnover in different solid tumor lines. Int J Radiat Oncol Biol Phys
    Ljungkvist AS, Bussink J, Kaanders JH, Rijken PF, Begg AC, Raleigh JA, van der Kogel AJ
    2005 Jul 15; 62 (4): 1157-68
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      PURPOSE: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved.

      METHODS AND MATERIALS: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker).

      RESULTS: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days.

      CONCLUSIONS: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

      View details for PubMedID 15913908
  • Recovery capacity of glial progenitors after in vivo fission-neutron or X irradiation: age dependence, fractionation and low-dose-rate irradiations. Radiat Res
    Philippo H, Winter EA, van der Kogel AJ, Huiskamp R
    2005 Jun; 163 (6): 636-43
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      Previous experiments on the radiosensitivity of O-2A glial progenitors determined for single-dose fission-neutron and X irradiation showed log-linear survival curves, suggesting a lack of accumulation of recovery of sublethal damage. In the present study, we addressed this question and further characterized the radiobiological properties of these glial stem cells by investigating the recovery capacity of glial stem cells using either fractionated or protracted whole-body irradiation. Irradiations were performed on newborn, 2-week-old or 12-week-old rats. Fractionated irradiations (four fractions) were performed with 24-h intervals, followed by cell isolations 16- 24 h after the last irradiation. Single-dose irradiations were followed by cell isolation 16-24 h after irradiation or delayed cell isolation (4 days after irradiation) of the O-2A progenitor cells from either spinal cord (newborns) or optic nerve (2- and 12-week-old rats). Results for neonatal progenitor cell survival show effect ratios for both fractionated fission-neutron and X irradiation of the order of 1.8 when compared with single-dose irradiation. A similar ratio was found after single-dose irradiation combined with delayed plating. Comparable results were observed for juvenile and adult optic nerve progenitors, with effect ratios of the order of 1.2. The present investigation clearly shows that fractionated irradiation regimens using X rays or fission neutrons and CNS tissue from rats of various ages results in an increase in O-2A progenitor cell survival while repair is virtually absent. This recovery of the progenitor pool after irradiation can be observed at all ages but is greatest in the neonatal spinal cord and can probably be attributed to repopulation.

      View details for PubMedID 15913395
  • Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1. Radiother Oncol
    Williams KJ, Telfer BA, Xenaki D, Sheridan MR, Desbaillets I, Peters HJ, Honess D, Harris AL, Dachs GU, van der Kogel A, Stratford IJ
    2005 Apr; 75 (1): 89-98
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      BACKGROUND AND PURPOSE: To test the hypothesis that deficiency in expression of the transcription factor, HIF-1, renders tumours more radioresponsive than HIF-1 proficient tumours.

      PATIENTS AND METHODS: Tumours comprising mouse hepatoma cells lacking HIF-1beta (and thereby HIF-1 function) were grown in nude mice and radiation-induced growth delay compared with that seen for wild-type tumours and tumours derived from HIF-1beta negative cells where HIF-1 function had been restored.

      RESULTS: The xenografts that lack HIF-1 activity take longer to establish their growth and are more radioresponsive than both parental xenografts and those with restored HIF-1 function. Pre-treatment of the HIF-1 deficient xenografts with the hypoxic radiosensitizer misonidazole, had little effect on radioresponse. In contrast this treatment radiosensitized the parental xenografts. In spite of this, no difference in oxygenation status was found between the tumour types as measured by Eppendorf O(2)-electrodes and by binding of the hypoxic cell marker NITP. Admixing wild type and HIF-1 deficient cells in the same tumour at ratios of 1 in 10 and 1 in 100 restores the growth of the mixed tumours to that of a 100% HIF-1 proficient cell population. However, when comparing the effects of radiation on the mixed tumours, radioresponsiveness is maintained in those tumours containing the high proportion of HIF-1 deficient cells.

      CONCLUSIONS: The differences in radioresponse do not correlate with tumour oxygenation, suggesting that the hypoxic cells within the HIF-1 deficient tumours do not contribute to the outcome of radiotherapy. Thus, hypoxia impacts on tumour radioresponsiveness not simply because of the physio-chemical mechanism of oxygen with radiation-induced radicals causing damage 'fixation', but also because hypoxia/HIF-1 promotes expression of genes that allow tumour cells to survive under these adverse conditions. Further, the results from the cell mixing experiments uncouple the growth promoting effects of HIF-1 and the underlying mechanism by which HIF-1 may increase radiation resistance in solid tumours.

      View details for PubMedID 15878106
  • Interleukin-12 has no effect on vascular density, perfusion, hypoxia and proliferation of an implanted human squamous cell carcinoma xenograft tumour despite up-regulation of ICAM-1. Anticancer Res
    van Herpen CM, Bussink J, van der Kogel AJ, Peeters WJ, van der Voort R, van Schijndel A, de Wilde PC, Adema GJ, de Mulder PH
    2005 Mar-Apr; 25 (2A): 1015-21
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      BACKGROUND: Interleukin-12 is an anti-angiogenic and antitumor agent in many transplanted murine tumour models. In a previous clinical study in head and neck squamous cell carcinoma patients treated with rhIL-12 the tumour turned pale, after an initial reddening. The aim of this study was to investigate the effects of rmIL-12 on the vasculature, blood perfusion, hypoxia and proliferation of tumour cells in an implanted human head and neck squamous cell carcinoma xenograft tumour, with a relatively large diameter, in Balb/c nu/nu mice over time.

      MATERIALS AND METHODS: Established human squamous cell carcinoma xenograft tumours were intratumorally injected for 3 days with either 200 ng rmIL-12 or PBA. Mice were sacrificed at 4 different time points (between 8 hours and 8 days after the last injection), after administration of Pimonidazole, BrdUrd and Hoechst 33342. The tumour sections were quantitatively analysed with a semi-automatic method based on a computerised digital image analysis system, after immunohistochemical staining.

      RESULTS: Despite a faster and higher up-regulation of anti-mouse ICAM-1 in the IL-12-treated tumours, no significant differences in vascular density, perfusion fraction, hypoxic fraction and BrdUrd labelling index were detected between IL-12-treated tumour and control tumours.

      CONCLUSION: We suggest that the main reason why the observation made in humans could not be confirmed in this mice study is the combination of a lack of an intact immune system in the Balb/c nu/nu mice and a relatively large tumour with probably a lot of mature vessels.

      View details for PubMedID 15868941
  • Data on dose-volume effects in the rat spinal cord do not support existing NTCP models. Int J Radiat Oncol Biol Phys
    van Luijk P, Bijl HP, Konings AW, van der Kogel AJ, Schippers JM
    2005 Mar 01; 61 (3): 892-900
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      PURPOSE: To evaluate several existing dose-volume effect models for their ability to describe the occurrence of white matter necrosis in rat spinal cord after irradiation with small proton beams.

      METHODS AND MATERIALS: A large number of dose-volume effect models has been fitted to data on the occurrence of white matter necrosis after irradiation with small proton beams. The fitting was done with the maximum likelihood method. For each model, the goodness of fit was calculated. An empirical tolerance dose-volume (eTDV) model was designed to describe data obtained after uniform irradiation.

      RESULTS: The eTDV model, the critical element model, and critical volume model with inclusion of the repair-by-migration principle described by Shirato, were able to describe the data obtained after irradiation with uniform dose distributions of varying sizes. However, none of the models under investigation was able to describe all the data. Extension of the developed empirical model with a repair mechanism with a limited range resulted in a good description of the tolerance doses.

      CONCLUSIONS: In the rat spinal cord, a nonlocal repair mechanism, acting from nonirradiated to irradiated tissue, plays an important role in the (prevention of the) occurrence of white matter necrosis after irradiation. Models that take into account this effect need to be developed.

      View details for PubMedID 15708272
  • Colocalization of carbonic anhydrase 9 expression and cell proliferation in human head and neck squamous cell carcinoma. Clin Cancer Res
    Hoogsteen IJ, Marres HA, Wijffels KI, Rijken PF, Peters JP, van den Hoogen FJ, Oosterwijk E, van der Kogel AJ, Kaanders JH
    2005 Jan 01; 11 (1): 97-106
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      PURPOSE: Tumor cells undergo a variety of biological changes under sustained hypoxic conditions, allowing cells to survive and retain their clonogenic potential. The purpose of this study is to relate the expression of the hypoxia marker carbonic anhydrase 9 (CA9) to the uptake of iododeoxyuridine (IdUrd), a marker of proliferation, in head and neck squamous cell carcinomas. Colocalization of IdUrd and CA9 may identify an important subpopulation of tumor cells that might be responsible for repopulation and disease progression.

      EXPERIMENTAL DESIGN: Expression of CA9, IdUrd labeling, and colocalization between IdUrd and CA9 was examined by immunohistochemistry in biopsies of head and neck squamous cell carcinomas. Biopsies were taken from 51 patients recruited between 1998 and 2001 after administration of the proliferation marker IdUrd.

      RESULTS: A large variation was observed between the tumors in CA9 expression (range 0-39%), IdUrd labeling (range 0-81%), and colocalization between IdUrd and CA9 [FId(CA9); range 0-53%]. FId(CA9), the fraction of IdUrd-labeled cells positive for CA9, was highest at an intermediate distance from the blood vessels (100-150 microm). IdUrd labeling was higher in T4 carcinomas relative to lower stage tumors (P = 0.04). High FId(CA9) correlated with the worst disease-free survival rates (P = 0.04).

      CONCLUSIONS: Colocalization between IdUrd labeling and CA9 expression was observed in head and neck squamous cell carcinomas, suggesting the presence of a population of tumor cells under intermediate hypoxic conditions which still has proliferative capacity. The size of this subpopulation may be indicative of tumor aggressiveness and is associated with the worst disease-free survival rates.

      View details for PubMedID 15671533
  • Regional differences in radiosensitivity across the rat cervical spinal cord. Int J Radiat Oncol Biol Phys
    Bijl HP, van Luijk P, Coppes RP, Schippers JM, Konings AW, van Der Kogel AJ
    2005 Feb 01; 61 (2): 543-51
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      PURPOSE: To study regional differences in radiosensitivity within the rat cervical spinal cord.

      METHODS AND MATERIALS: Three types of inhomogeneous dose distributions were applied to compare the radiosensitivity of the lateral and central parts of the rat cervical spinal cord. The left lateral half of the spinal cord was irradiated with two grazing proton beams, each with a different penumbra (20-80% isodoses): lateral wide (penumbra = 1.1 mm) and lateral tight (penumbra = 0.8 mm). In the third experiment, the midline of the cord was irradiated with a narrow proton beam with a penumbra of 0.8 mm. The irradiated spinal cord length (C1-T2) was 20 mm in all experiments. The animals were irradiated with variable single doses of unmodulated protons (150 MeV) with the shoot-through method, whereby the plateau of the depth-dose profile is used rather than the Bragg peak. The endpoint for estimating isoeffective dose (ED(50)) values was paralysis of fore and/or hind limbs within 210 days after irradiation. Histology of the spinal cords was performed to assess the radiation-induced tissue damage.

      RESULTS: High-precision proton irradiation of the lateral or the central part of the spinal cord resulted in a shift of dose-response curves to higher dose values compared with the homogeneously irradiated cervical cord to the same 20-mm length. The ED(50) values were 28.9 Gy and 33.4 Gy for the lateral wide and lateral tight irradiations, respectively, and as high as 71.9 Gy for the central beam experiment, compared with 20.4 Gy for the homogeneously irradiated 20-mm length of cervical cord. Histologic analysis of the spinal cords showed that the paralysis was due to white matter necrosis. The radiosensitivity was inhomogeneously distributed across the spinal cord, with a much more radioresistant central white matter (ED(50) = 71.9 Gy) compared with lateral white matter (ED(50) values = 28.9 Gy and 33.4 Gy). The gray matter did not show any noticeable lesions, such as necrosis or hemorrhage, up to 80 Gy. All lesions induced were restricted to white matter structures.

      CONCLUSIONS: The observed large regional differences in radiosensitivity within the rat cervical spinal cord indicate that the lateral white matter is more radiosensitive than the central part of the white matter. The gray matter is highly resistant to radiation: no lesions observable by light microscopy were induced, even after a single dose as high as 80 Gy.

      View details for PubMedID 15667978

Contact Information

Albert van der Kogel, PhD

1111 Highland Avenue, 3137 WIMR,
Madison, WI 53705