A.J. van der Kogel

18F-FLT PET Does Not Discriminate Between Reactive and Metastatic Lymph Nodes in Primary Head and Neck Cancer Patients

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′-18F-fluorothymidine (18F-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 18F-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 18F-FLT PET before surgical tumor resection with lymph node dissection. Emission 18F-FLT PET and CT images of the head and neck were recorded and fused, and standardized uptake values (SUVs) were calculated. From all 18 18F-FLT PET-positive lymph node levels and from 8 18F-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 18F-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 18F-FLT PET-positive lymph nodes (SUVmean: 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 18F-FLT PET were 100%, 16.7%, 37.5%, and 100%, respectively. Conclusion: In head and neck cancer patients, 18F-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, 18F-FLT PET is not suitable for assessment of pretreatment lymph node status. This observation may also negatively influence the utility of 18F-FLT PET for early treatment response evaluation of small metastatic nodes.

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas

Tumour oxygenation and vasculature are determinants for radiation treatment outcome and prognosis in patients with squamous cell carcinomas of the head and neck. In this study we visualized and quantified these factors which may provide a predictive tool for new treatments. Twenty-one patients with stage III–IV squamous cell carcinomas of the head and neck were intravenously injected with pimonidazole, a bioreductive hypoxic marker. Tumour biopsies were taken 2 h later. Frozen tissue sections were stained for vessels and hypoxia by fluorescent immunohistochemistry. Twenty-two sections of biopsies of different head and neck sites were scanned and analysed with a computerized image analysis system. The hypoxic fractions varied from 0.02 to 0.29 and were independent from T- and N-classification, localization and differentiation grade. No significant correlation between hypoxic fraction and vascular density was observed. As a first attempt to categorize tumours based on their hypoxic profile, three different hypoxia patterns are described. The first category comprised tumours with large hypoxic, but viable, areas at distances even greater than 200 μm from the vessels. The second category showed a typical band-like distribution of hypoxia at an intermediate distance (50–200 μm) from the vessels with necrosis at greater distances. The third category demonstrated hypoxia already within 50 μm from the vessels, suggestive for acute hypoxia. This method of multiparameter analysis proved to be clinically feasible. The information on architectural patterns and the differences that exist between tumours can improve our understanding of the tumour micro-environment and may in the future be of assistance with the selection of (oxygenation modifying) treatment strategies.

Effect of X rays and neutrons on repair and regeneration in the rat spinal cord

Clinical and experimental results of neutron irradiation have shown higher RBE values for the central nervous system (CNS) than for most other normal tissues. This is because of a considerable impairment of the large capacity of the CNS to repair subeffective damage induced by low LET radiation. Decreasing the dose per fraction of X rays increases the CNS tolerance significantly; this has no effect for neutrons. In the cervical spinal cord and the brain, two types of delayed damage can be described, so-called early and late. Different target cells are assumed to be involved, oligodendroglial cells in the early, and vascular endothelium in the late type. In the lumbar cord, the main lesion is nerve root necrosis, with the Schwann cell as the most probable target. These target cells show differences in response to X rays and neutrons, resulting in different RBE values. The highest RBE is obtained for cervical white matter necrosis. In addition to cellular repair of subeffective damage, long-term tissue regeneration is observed in the spinal cord, beginning at different times for the various types of damage. With neutrons, the rate of long-term regeneration is at least similar, or even more pronounced than for X rays.

Angiogenesis, hypoxia and VEGF expression during tumour growth in a human xenograft tumour model.

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.

Changes in Blood Perfusion and Hypoxia after Irradiation of a Human Squamous Cell Carcinoma Xenograft Tumor Line

Abstract Bussink, J., Kaanders, J. H. A. M., Rijken, P. F. J. W., Raleigh, J. A. and Van der Kogel, A. J. Changes in Blood Perfusion and Hypoxia after Irradiation of a Human Squamous Cell Carcinoma Xenograft Tumor Line. The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.

Vascularity and perfusion of human gliomas xenografted in the athymic nude mouse

The vascularisation and perfusion of seven subcutaneously xenografted human glioma lines established from surgical specimens has been analysed using an anti-collagen type IV antibody to visualise the vascular walls in combination with a perfusion marker (Hoechst 33342). A computer-based digital image processing system was employed for quantitative analysis of the parameters. The vascular architecture of individual tumours belonging to the same tumour line showed a consistent similarity, while substantial differences occurred between the various tumour lines derived from different patients. Despite the presence of a large inter-tumour variation in vascular area as a proportion of the tumour area, this vascular parameter clearly showed tumour line-specific characteristics. The perfused fraction of the tumour vessels also showed a large inter-tumour variation for all tumour lines ranging from 20% to 85%, but the majority of tumours of all lines had perfusion fractions of more than 55%. Despite large variation, the perfused vascular area as a proportion of the tumour cross-sectional area exhibited clear tumour line-specific tendencies. These observations suggest that consistent differences in vascular parameters are present between glioma xenograft lines, although the tumour lines all originated from histologically similar human high-grade gliomas. These differences may have important consequences for treatment and clinical behaviour of this type of tumour.

Dynamics of hypoxia, proliferation and apoptosis after irradiation in a murine tumor model.

Abstract Ljungkvist, A. S. E., Bussink, J., Kaanders, J. H. A. M., Wiedenmann, N. E., Vlasman, R. and van der Kogel, A. J. Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model. Radiat. Res. 165, 326– 336 (2006). 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.

Multiparameter analysis of vasculature, perfusion and proliferation in human tumour xenografts

A method is presented in this report for concurrent analysis of vascular architecture, blood perfusion and proliferation characteristics in whole-tumour cross-sections of human larynx carcinoma and glioblastoma xenografts. Tumours were implanted subcutaneously in nude mice. After i.v. injection with Hoechst 33342 and bromodeoxyuridine (BrdUrd) as perfusion and proliferation markers, animals were killed. An antiendothelial antibody (9F1) was used to delineate vascular structures. Cross-sections were analysed by a multistep immune staining and a computer-controlled microscope scanning method. Each tumour section was stained and scanned four times (Hoechst, 9F1, BrdUrd and Fast Blue for all nuclei). When these images were combined, vasculature, perfusion and proliferation parameters were analysed. The labelling index (LI) was defined as the ratio of the BrdUrd-labelled area to the total nuclear area. The LI based on manual counting and the LI calculated by flow cytometry (FCM) were in good agreement with the LI based on surface analysis. LI decreased at increasing distance from its nearest vessel. In the vicinity of perfused vessels, the LI was 30-70% higher than near non-perfused vessels. This method shows that both vasculature/perfusion and proliferation characteristics can be measured in the same whole-tumour section in a semiautomatic way. This could be applied in clinical practice to identify combined human tumour characteristics that predict for a favourable response to treatment modifications.

Innovations in radiotherapy planning of head and neck cancers: role of PET.

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 18F-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 18F-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.

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours.

Overwhelming clinical and experimental data demonstrate that tumour hypoxia is associated with aggressive disease and poor treatment outcome as hypoxic cells are refractive to radiotherapy and some forms of chemotherapy. However, hypoxia is rare in physiologically normal tissues representing a tumour-specific condition. To selectively target this therapeutically refractive cell population, we have combined bioreductive chemotherapy with hypoxia-directed gene therapy. We have transfected the human fibrosarcoma cell line, HT1080, with a hypoxia-regulated expression vector encoding the human flavoprotein cytochrome c P450 reductase (HRE-P450R). This conferred hypoxia-dependent sensitivity to the alkylating nitroimidazole prodrug RSU1069 in vitro, with a greater than 30-fold increase in oxic/hypoxic cytotoxicity ratio compared with controls. Xenografts of both the HRE-P450R and empty vector transfectants had comparable hypoxic fractions and were refractive to single dose radiotherapy of up to 15 Gy. However, combining a prodrug of RSU1069 with a reduced radiotherapy dose of 10 Gy represents a curative regimen (50% tumour-free survival; day 100) in the HRE-P450R xenografts. In complete contrast, 100% mortality was apparent by day 44 in the empty vector control xenografts treated in the same way. Thus, an oxygen-sensitive gene-directed enzyme prodrug therapy approach may have utility when incorporated into conventional radiotherapy and/or chemotherapy protocols for loco-regional disease in any tissue where hypoxia is a contra-indication to treatment success.