Wenny J.M. Peeters

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.

Preclinical evaluation and validation of [18F]HX4, a promising hypoxia marker for PET imaging

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 [18F]HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [18F]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. [18F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [18F]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 [18F]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 [18F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [18F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.

Radiolabeled cetuximab: Dose optimization for epidermal growth factor receptor imaging in a head‐and‐neck squamous cell carcinoma model

Noninvasive imaging of the epidermal growth factor receptor (EGFR) in head‐and‐neck squamous cell carcinoma could be of value to select patients for EGFR‐targeted therapy. We assessed dose optimization of 111Indium‐DTPA‐cetuximab (111In‐cetuximab) for EGFR imaging in a head‐and‐neck squamous cell carcinoma xenograft model. 111In‐cetuximab slowly internalized into FaDu cells in vitro, amounting to 1.0 × 104 molecules cetuximab per cell after 24 hr (15.8% of added activity). In nude mice with subcutaneous FaDu xenograft tumors, a protein dose escalation study with 111In‐cetuximab showed highest specific accumulation in tumors at protein doses between 1 and 30 μg per mouse (mean tumor uptake 33.1 ± 3.1%ID/g, 3 days postinjection (p.i.)). The biodistribution of 111In‐cetuximab and 125I‐cetuximab was determined at 1, 3 and 7 days p.i. at optimal protein dose. Tumor uptake was favorable for 111In‐cetuximab compared to 125I‐cetuximab. With pixel‐by‐pixel analysis, good correlations were found between intratumoral distribution of 111In‐cetuximab as determined by autoradiography and EGFR expression in the same tumor sections as determined immunohistochemically (mean r = 0.74 ± 0.14; all correlations p < 0.0001). Micro Single Photon Emission Computed Tomography (MicroSPECT) scans clearly visualized FaDu tumors from 1 day p.i. onward and tumor‐to‐background contrast increased until 7 days p.i. (tumor‐to‐liver ratios 0.58 ± 0.24, 3.42 ± 0.66, 8.99 ± 4.66 and 16.33 ± 11.56, at day 0, day 1, day 3 and day 7 p.i., respectively). Our study suggests that, at optimal cetuximab imaging dose, 111In‐cetuximab can be used for visualization of EGFR expression in head‐and‐neck squamous cell carcinoma using SPECT.

Parametric mapping of immunohistochemically stained tissue sections; a method to quantify the colocalization of tumor markers

BackgroundAutomated analysis of immunohistochemically stained tissue sections is of great importance in cancer research to detect tumor-specific prognostic markers and make therapy decisions. Here, an automated quantitative analysis is presented to assess the colocalization of CAIX, a membrane-bound hypoxic marker and Ki-67, a nuclear proliferation marker.MethodsTissue sections of 104 biopsies from 89 patients were stained for CAIX and Ki-67 with diaminobenzidine and haematoxylin counterstain. Image scans of whole tumor sections were recorded and image maps were created with parametric mapping to quantify the markers and assess the colocalization.ResultsThe fraction of CAIX showed a range of 0–93%. The interobserver correlation and the correlation between manual scores and automated analysis were both very strong (rs=0.96, p <0.0001, and rs=0.97, p <0.0001). The labelling index of Ki-67 exhibited a range of 0–42% with less strong interobserver and manual to automated analysis correlations (rs=0.90, p <0.0001, and rs=0.71, p <0.0008). The relative tumor area positive for both markers varied from 0 – 76%.ConclusionParametric mapping of immunohistochemically stained tumor sections is a reliable method to quantitatively analyze membrane-bound proteins and assess the colocalization of various tumor markers in different subcellular compartments.

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.

Combining radiotherapy with MEK1/2, STAT5 or STAT6 inhibition reduces survival of head and neck cancer lines

BackgroundKinases 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.MethodsAn 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.ResultsThe 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.ConclusionsKinases 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.

Bath and Shower Effect in Spinal Cord: The Effect of Time Interval

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.

Effect of carbogen breathing on the radiation response of a human glioblastoma xenograft : Analysis of hypoxia and vascular parameters of regrowing tumors

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.Hintergrund und Ziel:In der Vergangenheit wurde die Effektivität von Carbogenbeatmung auf den Oxygenationsstatus von Tumoren nachgewiesen. Ziel dieser Experimente war es, den Zusammenhang zwischen o. g. Carbogenbeatmung und der Reaktion auf Bestrahlung mit Hilfe der Wachstumsverzögerung zu analysieren. Des Weiteren wurde diese Studie entwickelt, um durch die Kombinationsbehandlung mit Bestrahlung und Carbogen verursachte Veränderungen des Mikromilieus im Vergleich zu alleiniger Strahlenbehandlung zu untersuchen.Material und Methodik:Eine humane Glioblastom-Xenotransplantat-Tumorlinie wurde in Nacktmäuse implantiert. Die Bestrahlungsdosis betrug 10 Gy oder 20 Gy mit und ohne Carbogenbeatmung. Verschiedene Parameter des Mikromilieus (Tumorzellhypoxie, Tumordurchblutung, vaskuläres Volumen und mikrovaskuläre Dichte) wurden nach immunhistochemischer Färbung analysiert. Die Tumorwachstumsverzögerung wurde bis zu 120 Tage nach Behandlung beobachtet.Ergebnisse:Im Allgemeinen hatte die kombinierte Behandlung keinen Vorteil. Eine kleine Untergruppe wies allerdings eine gute Reaktion auf Kombinationsbehandlung mit Bestrahlung und Carbogenbeatmung auf; diese erneut wachsenden Tumoren zeigten nur sehr wenig Hypoxie und waren größtenteils nekrotisch. Diese Eigenschaften im Mikromilieu wurden nicht in den Tumoren gesehen, die schnell gewachsen waren.Schlussfolgerung:Die Beobachtungen suggerieren, dass eine Untergruppe von Patienten, die möglicherweise von der Kombinationsbehandlung profitieren, identifiziert werden könnte. Die unterschiedliche Reaktion auf Behandlung veranschaulicht allerdings die Notwendigkeit einer Patientenauswahl vor Behandlungsbeginn.

Effect of Carbogen Breathing on the Radiation Response of a Human Glioblastoma Xenograft

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.Hintergrund und Ziel:In der Vergangenheit wurde die Effektivität von Carbogenbeatmung auf den Oxygenationsstatus von Tumoren nachgewiesen. Ziel dieser Experimente war es, den Zusammenhang zwischen o. g. Carbogenbeatmung und der Reaktion auf Bestrahlung mit Hilfe der Wachstumsverzögerung zu analysieren. Des Weiteren wurde diese Studie entwickelt, um durch die Kombinationsbehandlung mit Bestrahlung und Carbogen verursachte Veränderungen des Mikromilieus im Vergleich zu alleiniger Strahlenbehandlung zu untersuchen.Material und Methodik:Eine humane Glioblastom-Xenotransplantat-Tumorlinie wurde in Nacktmäuse implantiert. Die Bestrahlungsdosis betrug 10 Gy oder 20 Gy mit und ohne Carbogenbeatmung. Verschiedene Parameter des Mikromilieus (Tumorzellhypoxie, Tumordurchblutung, vaskuläres Volumen und mikrovaskuläre Dichte) wurden nach immunhistochemischer Färbung analysiert. Die Tumorwachstumsverzögerung wurde bis zu 120 Tage nach Behandlung beobachtet.Ergebnisse:Im Allgemeinen hatte die kombinierte Behandlung keinen Vorteil. Eine kleine Untergruppe wies allerdings eine gute Reaktion auf Kombinationsbehandlung mit Bestrahlung und Carbogenbeatmung auf; diese erneut wachsenden Tumoren zeigten nur sehr wenig Hypoxie und waren größtenteils nekrotisch. Diese Eigenschaften im Mikromilieu wurden nicht in den Tumoren gesehen, die schnell gewachsen waren.Schlussfolgerung:Die Beobachtungen suggerieren, dass eine Untergruppe von Patienten, die möglicherweise von der Kombinationsbehandlung profitieren, identifiziert werden könnte. Die unterschiedliche Reaktion auf Behandlung veranschaulicht allerdings die Notwendigkeit einer Patientenauswahl vor Behandlungsbeginn.

Characterization of late radiation effects in the rat thoracolumbar spinal cord by MR imaging using USPIO.

The aim of this study was to detect late radiation effects in the rat spinal cord using MR imaging with ultra-small particles of iron oxide (USPIO) contrast agent to better understand the development of late radiation damage with emphasis on the period preceding neurological signs. Additionally, the role of an inflammatory reaction was assessed by measuring macrophages that internalized USPIO. T2-weighted spin echo MR measurements were performed at 7T in six rats before paresis was expected (130–150 days post-irradiation, early group), and in six paretic rats (150–190 days post-irradiation, late group). Measurements were performed before, directly after and, only in the early group, 40 h after USPIO administration and compared with histology. In the early group, MR images showed focal regions in grey matter (GM) and white matter (WM) with signal intensity reduction after USPIO injection. Larger lesions with contrast enhancement were located in and around edematous GM of three animals of the early group and five of the late group. Forty hours after injection, additional lesions in WM, GM and nerve roots appeared in animals with GM edema. In the late paretic group, MR imaging showed WM necrosis adjacent to areas with large contrast enhancement. In conclusion, detection of early focal lesions was improved by contrast administration. In the animals with extended radiation damage, large hypo-intense regions appeared due to USPIO, which might be attributed to blood spinal cord barrier breakdown, but the involvement of blood-derived iron-loaded macrophages could not be excluded.