J. Bussink

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.

Carbonic anhydrase-9 expression levels and prognosis in human breast cancer: association with treatment outcome

Here, we set out to assess CA9 expression levels by real-time quantitative RT–PCR in breast cancer tissue samples obtained from 253 patients, and correlated those with relapse-free (RFS) survival. The median follow-up time was 75 months (range 2–168 months). CA9 expression was mainly found in high-grade, steroid receptor negative cancer tissues. CA9 levels were not significantly associated with RFS (P=0.926, hazard ratio (HR)=0.99, 95% CI=0.80–1.22) in the total cohort of 253 patients. In multivariate analysis with other clinicopathological factors, CA9 (P=0.018, HR=0.77, 95% CI=0.62–0.96), the interaction of adjuvant chemotherapy with CA9 (P=0.009, HR=1.31, 95% CI=1.07–1.61) and the interaction of adjuvant endocrine therapy with CA9 (P<0.001, HR=1.41, 95% CI=1.20–1.66) all contributed significantly to the final model. These results indicate that patients with low CA9 levels benefit more from adjuvant treatment than do patients with high levels. Thus, the determination of CA9 levels could aid in the selection of patients who will not benefit from adjuvant therapy, and whose prognosis will more likely improve with other treatment modalities.

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.

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.

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.

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.

Optical Sensor-Based Oxygen Tension Measurements Correspond with Hypoxia Marker Binding in Three Human Tumor Xenograft Lines

Abstract Bussink, J., Kaanders, J. H. A. M., Strik, A. M., Vojnovic, B. and van der Kogel, A. J. Optical Sensor-Based Oxygen Tension Measurements Correspond with Hypoxia Marker Binding in Three Human Tumor Xenograft Lines. Hypoxia has a negative effect on the outcome of radiotherapy and surgery and is also related to an increased incidence of distant metastasis. In this study, tumor pO2 measurements using a newly developed time-resolved luminescence-based optical sensor (OxyLite™) were compared with bioreductive hypoxia marker binding (pimonidazole). Single pO2 measurements per tumor were compared to hypoxia marker binding in tissue sections using image analysis. Both assays were performed in the same tumors of three human tumor lines grown as xenografts. Both assays demonstrated statistically significant differences in the oxygenation status of the three tumor lines. There was also a good correlation between hypoxia marker binding and the pO2 measurements with the OxyLite™ device. A limitation of the OxyLite™ system is that it is not yet suited for sampling multiple sites in one tumor. An important strength is that continuous measurements can be taken at the same position and dynamic information on the oxygenation status of tumors can be obtained. The high spatial resolution of the hypoxia marker binding method can complement the limitations of the OxyLite™ system. In the future, a bioreductive hypoxic cell marker for global assessment of tumor hypoxia may be combined with analysis of temporal changes in pO2 with the OxyLite™ to study the effects of oxygenation-modifying treatment on an individual basis.

High NOTCH activity induces radiation resistance in non small cell lung cancer

BACKGROUND AND PURPOSE Patients with advanced NSCLC have survival rates <15%. The NOTCH pathway plays an important role during lung development and physiology but is often deregulated in lung cancer, making it a potential therapeutic target. We investigated NOTCH signaling in NSCLC and hypothesized that high NOTCH activity contributes to radiation resistance. MATERIALS AND METHODS NOTCH signaling in NSCLC patient samples was investigated using quantitative RT-PCR. H460 NSCLC cells with either high or blocked NOTCH activity were generated and their radiation sensitivity monitored using clonogenic assays. In vivo, xenograft tumors were irradiated and response assessed using growth delay. Microenvironmental parameters were analyzed by immunohistochemistry. RESULTS Patients with high NOTCH activity in tumors showed significantly worse disease-free survival. In vitro, NOTCH activity did not affect the proliferation or intrinsic radiosensitivity of NSCLC cells. In contrast, xenografts with blocked NOTCH activity grew slower than wild type tumors. Tumors with high NOTCH activity grew significantly faster, were more hypoxic and showed a radioresistant phenotype. CONCLUSIONS We demonstrate an important role for NOTCH in tumor growth and correlate high NOTCH activity with poor prognosis and radioresistance. Blocking NOTCH activity in NSCLC might be a promising intervention to improve outcome after radiotherapy.

Clinical Outcome and Tumour Microenvironmental Effects of Accelerated Radiotherapy with Carbogen and Nicotinamide

Experimental studies have shown an almost 2-fold increase in effectiveness if accelerated radiotherapy combined with carbogen and nicotinamide (ARCON) was compared with standard radiotherapy. This combination was chosen in order to overcome repopulation of clonogens during radiotherapy and to minimize tumour hypoxia. Analysis of microenvironmental parameters is required to identify tumours that can benefit from these new treatment approaches. In this study 124 patients with stage III or IV head and neck squamous cell carcinomas received ARCON treatment. Vascular architecture, perfusion, proliferation and oxygenation were studied in two human laryngeal squamous cell carcinoma xenograft lines and the effects of carbogen and nicotinamide were analysed. Loco-regional control for stage III-IV larynx carcinomas was 85%, for hypopharynx carcinomas 50% and for oral cavity and oropharynx carcinomas 65%. In the experimental studies, carbogen treatment resulted in one tumour line in a decrease of blood perfusion, which was reversed if nicotinamide was added. The other tumour line showed no perfusion changes after carbogen or nicotinamide treatment. Both tumour lines showed a drastic reduction of hypoxia after carbogen breathing only or carbogen breathing plus nicotinamide. The ARCON schedule results in high loco-regional tumour control rates. Analysis of tumour microenvironmental parameters showed differences in response to carbogen and nicotinamide between different tumour lines of similar histology and site of origin. This indicates that it may be advantageous to base the selection of patients for oxygenation modifying treatment on microenvironmental tumour characteristics.

Histopathologic Validation of 3′-Deoxy-3′-18F-Fluorothymidine PET in Squamous Cell Carcinoma of the Oral Cavity

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′-18F-fluorothymidine (18F-FLT) is a PET tracer that is phosphorylated by thymidine kinase 1 (TK-1) and, as such, reflects cellular proliferation. Before the use of 18F-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 18F-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 18F-FLT PET/CT scan before surgery, and iododeoxyuridine was administered 20 min before tumor resection. 18F-FLT PET/CT scans were segmented, and PET/CT volumes and PET signal intensities were calculated (mean standardized uptake value [SUVmean] and maximum standardized uptake value [SUVmax]). 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 SUVmean and SUVmax. TK-1 staining was visually and semiquantitatively assessed. Results: All primary tumors were identified with 18F-FLT PET but with a large range in tracer uptake (mean SUVmax, 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 SUVmean and SUVmax, but the labeling indices did not. In most tumors, TK-1 staining of varying intensity was present but correlated with neither iododeoxyuridine binding nor 18F-FLT uptake. Conclusion: The current study demonstrated only a weak correlation between 18F-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.