Paul F.J.W. Rijken

Tumor vascular architecture and function evaluated by non-invasive susceptibility MRI methods and immunohistochemistry.

To investigate the physiological origins responsible for the varying blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) responses to carbogen (95% O2/5% CO2) breathing observed with different tumor types.

Spatial relationship between hypoxia and the (perfused) vascular network in a human glioma xenograft: a quantitative multi-parameter analysis.

PURPOSE To quantitatively study the spatial distribution of tumor hypoxia in relation to the perfused vasculature. METHODS AND MATERIALS Using a human glioma xenograft model, nude mice were administered two different hypoxia markers (NITP or pimonidazole) and the perfusion marker Hoechst 33342. Frozen tumor sections were sequentially scanned for perfusion, hypoxia, and vasculature, respectively, to quantitate perfusion, vasculature, and hypoxia parameters in the same section. RESULTS All tumors showed incomplete perfusion. Both NITP and pimonidazole stained the same hypoxic tumor areas. No statistically significant differences between the two markers were observed. The density of the perfused vessels was inversely related to the hypoxic fraction. At critical distances from perfused vessels, hypoxia occurred. These data suggest that predominantly diffusion-limited hypoxia was detected, based on the spatial distribution of nearby vessels. Also, the proportion of hypoxia distributed over arbitrary zones of 50 microm around perfused vessels was calculated. The largest proportion of hypoxia was found at distances beyond 100 microm from perfused vessels. CONCLUSION With the multiple staining and functional microscopic imaging technique described here, the spatial relationship between perfused vessels and hypoxia was quantified in whole tumor cross-sections. The usefulness of this histologically-based method to quantitate morphological and physiological aspects of the tumor microenvironment was evaluated.

PET of Hypoxia with 89Zr-Labeled cG250-F(ab′)2 in Head and Neck Tumors

Hypoxic tumor cells are resistant to radiotherapy and various chemotherapeutic agents. The pretherapeutic assessment of intratumoral hypoxia may allow selection of patients for intensified treatment regimens. Carbonic anhydrase IX (CAIX) is an endogenous hypoxia-related protein involved in pH regulation and is upregulated in many tumor types. Radionuclide imaging using a monoclonal antibody against CAIX, such as cG250, may allow noninvasive PET of hypoxia in these tumor types. The aims of this study were to investigate whether 89Zr-labeled cG250-F(ab′)2 allowed visualization of tumor hypoxia using small-animal PET and whether the tracer showed spatial correlation to the microscopic distribution of CAIX-expressing cells in a human head and neck xenograft tumor model. Methods: Athymic mice with subcutaneous human head and neck carcinoma xenografts (SCCNij3) were imaged with small-animal PET after injection of 89Zr-cG250-F(ab′)2. PET images were parameterized in terms of standardized uptake values (SUVs). After injection with the nitroimidazole hypoxia marker pimonidazole and the perfusion marker Hoechst 33342, the animals were sacrificed, tumors excised, and CAIX- and pimonidazole-marked hypoxia and blood perfusion were analyzed immunohistochemically. 89Zr-cG250-F(ab′)2 tumor uptake was analyzed by ex vivo activity counting and by autoradiography of tumor sections. Results: As early as 4 h after administration, accumulation of 89Zr-cG250-F(ab′)2 in the tumor had occurred and tumors were clearly visualized by PET, with reduced uptake by 24 h after injection. Pixel-by-pixel analysis showed a significant positive spatial correlation between CAIX expression and 89Zr-cG250-F(ab′)2 localization (r = 0.57–0.74; P < 0.0001). Also, significant correlations were found between pimonidazole staining intensity and 89Zr-cG250-F(ab′)2 activity concentration, although less strong (r = 0.46–0.68; P < 0.0001). Tumor maximum SUV correlated significantly with tumor uptake determined ex vivo (r = 0.93; P = 0.0067), as did fractions of CAIX and pimonidazole in tumor sections (r = 0.75; P = 0.03 and r = 0.78; P = 0.02, respectively). Conclusion: 89Zr-labeled cG250-F(ab′)2 small-animal PET showed rapid accumulation in a head and neck xenograft tumor model with good correlation to CAIX expression on a microscopic level.

Characterization and validation of noninvasive oxygen tension measurements in human glioma xenografts by 19F-MR relaxometry.

PURPOSE The aim of this study was to characterize and to validate noninvasive 19F-magnetic resonance relaxometry for the measurement of oxygen tensions in human glioma xenografts in nude mice. The following three questions were addressed: 1. When perfluorocarbon compounds (PFCs) are administrated intravenously, which tumor regions are assessed by 19F-MR relaxometry? 2. Are oxygen tension as detected by 19F-MR relaxometry (pO2/relaxo) comparable to Eppendorf O2-electrode measurements (pO2/electrode)? 3. Can 19F-MR relaxometry be used to detect oxygen tension changes in tumor tissue during carbogen breathing? METHODS AND MATERIALS Slice-selective 19F-MR relaxometry was carried out with perfluoro-15-crown-5-ether as oxygen sensor. The PFC was injected i.v. 3 days before the 19F-MR experiments. Two datasets were acquired before and two after the start of carbogen breathing. The distribution of PFCs and necrotic areas were analyzed in 19F-Spin Echo (SE) density MR images and T2-weighted 1H-SE MR images, respectively. One day after the MR investigations, oxygen tensions were measured by oxygen electrodes in the same slice along two perpendicular tracks. These measurements were followed by (immuno)histochemical analysis of the 2D distribution of perfused microvessels, hypoxic cells, necrotic areas, and macrophages. RESULTS The PFCs mainly became sequestered in perfused regions at the tumor periphery; thus, 19F-MR relaxometry probed mean oxygen tensions in these regions throughout the selected MR slice. In perfused regions of the tumor, mean PO2/relaxo values were comparable to mean PO2/electrode values, and varied from 0.03 to 9 mmHg. Median pO2/electrode values of both tracks were lower than mean pO2/relaxo values, because low pO2 electrode values that originate from hypoxic and necrotic areas were also included in calculations of median pO2/electrode values. After 8-min carbogen breathing, the average PO2/relaxo increase was 3.3 +/- 0.8 (SEM) mmHg and 2.1 +/- 0.6 (SEM) after 14 min breathing. CONCLUSIONS We have demonstrated that PFCs mainly became sequestered in perfused regions of the tumor. Here, mean PO2/relaxo values were comparable to mean PO2electrode values. In these areas, carbogen breathing was found to increase the PO2/relaxo values significantly.

Noninvasive Assessment of the Functional Neovasculature in 9L-Glioma Growing in Rat Brain by Dynamic 1H Magnetic Resonance Imaging of Gadolinium Uptake

Pathophysiologic parameters of the functional neovasculature and the blood-brain barrier of 9L-glioma in rat brain were measured noninvasively by dynamic 1H magnetic resonance imaging studies of gadolinium (Gd)-DTPA uptake. Changes of apparent [Gd-DTPA] uptake in time (CT[t]) were analyzed in a slice through the center of 10 9L-gliomas using fast T1 measurements. The distribution of the contrast agent was spatially correlated with the distribution of perfused microvessels as determined by immunohistochemical analysis. This method permits a distinction between perfused and nonperfused microvessels with a disrupted blood-brain barrier. In transverse slices of the whole tumor, a spatial correlation was observed between CT maps and the two-dimensional distribution of perfused microvessels. In the next step, Gd-DTPA uptake rates were spatially related to the perfused microvessel density (Np) or vascular surface area (Sp). In tumor voxels with perfused microvessels, a linear correlation was found between Gd-DTPA uptake rate constants (k values) and Np or Sp. No correlation was observed between k values and the total microvessel density. These are the first data that show a relation between Gd-DTPA uptake rates and parameters of the functional neovasculature in 9L-glioma growing in rat brain. Now that Gd-DTPA uptake studies can be related to parameters of the functional neovasculature, they may be used more efficiently as a prognostic tool before or during therapy.

Assessment of the neovascular permeability in glioma xenografts by dynamic T(1) MRI with Gadomer-17.

The uptake of Gadomer‐17, as probed by fast dynamic T1 measurements, was used to assess the vascular permeability surface‐area product per leakage volume of tissue (kTofts) of human glioma xenografts implanted in mice. With this approach we could discriminate between two types of glioma xenograft lines with a known difference in the perfused vascular architecture and degree of hypoxia. The T1 data were analyzed according to the Tofts‐Kermode compartment model. The fast‐growing E102 tumor demonstrated a homogeneous distribution of the vascular permeability surface area across the tumor (mean kTofts value = 0.18 ± 0.05 min−1). The slowly growing E106 tumor showed a more heterogeneous pattern. Three perfused tumor areas with differences in vascular permeability surface area could be distinguished: a well‐perfused periphery with high kTofts values (0.24 ± 0.04 min−1), perfused capillaries inside the tumor with low kTofts values (0.108 ± 0.026 min−1), and perfused capillaries adjacent to necrotic regions with high kTofts values (0.29 ± 0.10 min−1). On a different series of tumors, the hypoxic fractions were measured, and these were significantly higher in E106 tumors (0.14 ± 0.05) compared to tumors of the E102 line (0.03 ± 0.02). Magn Reson Med 47:305–313, 2002.

Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia.

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.

Effect of cetuximab and fractionated irradiation on tumour micro-environment

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.

Spatial relationship of phosphorylated epidermal growth factor receptor and activated AKT in head and neck squamous cell carcinoma

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.

Combretastatin A-4 Phosphate Affects Tumor Vessel Volume and Size Distribution as Assessed Using MRI-Based Vessel Size Imaging

Purpose: Combretastatin A-4 disodium phosphate (CA4P) is a promising vascular disrupting agent (VDA) in clinical trials. As CA4P acts on dividing endothelial cells, we hypothesize that CA4P affects vessels of certain sizes. The aim of this study was to evaluate the effect of CA4P by the MRI-based vessel size imaging (VSI). Experimental Design: C3H mammary carcinomas were grown to 200 mm3 in the right rear foot of female CDF1 mice. A control group of mice received no treatment, and a treatment group had CA4P administered intraperitoneally at a dose of 250 mg/kg. VSI was conducted on a 3 Tesla MR scanner to estimate the tumor blood volume (ζ0) and mean vessel radius (R). Vascularization was also estimated histologically by endothelial and Hoechst 33342 staining. Results: ζ0 and R showed different spatial heterogeneity. Tumor median and quartile values of ζ0 were all significantly reduced by about 35% in the CA4P-treated group as compared with the control group, and the median and upper quartile of R were significantly increased. Histograms of ζ0 and R showed a general decrease in ζ0 following treatment, and values of R in a certain range (≈20–30 μm) were decreased in the treatment group. The drug-induced change in ζ0 was in agreement with histology and our previous dynamic contrast enhanced MRI (DCE-MRI) data. Conclusions: Tumor blood volume and mean vessel radius showed a clear response following treatment with CA4P. VSI may prove valuable in estimation of tumor angiogenesis and prediction of response to VDAs. Clin Cancer Res; 18(23); 6469–77. ©2012 AACR.