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James A Raleigh - One of the best experts on this subject based on the ideXlab platform.

  • Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia.
    PloS one, 2015
    Co-Authors: Nathan A. Koonce, James A Raleigh, Joseph W. Levy, Matthew E. Hardee, Azemat Jamshidi-parsian, Kieng B. Vang, S. Sharma, Ruud P.m. Dings, Robert J. Griffin
    Abstract:

    Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-Pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker Pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-Pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of Pimonidazole-injected mice than in non-Pimonidazole injected tumor bearing mice or non-targeted microbubbles in Pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be ‘created’ on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.

  • Pimonidazole labelling and response to fractionated irradiation of five human squamous cell carcinoma (hSCC) lines in nude mice: The need for a multivariate approach in biomarker studies
    Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 2006
    Co-Authors: Ala Yaromina, James A Raleigh, Daniel Zips, Howard D. Thames, Wolfgang Eicheler, Mechthild Krause, Andrea Rosner, Michael Haase, Cordula Petersen, Verena Quennet
    Abstract:

    Abstract Objective To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches. Material and methods Five human squamous cell carcinoma lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, and UT-SCC-5) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. Histological material was collected from 60 unirradiated tumours after injection of Pimonidazole. The relative hypoxic area within the viable tumour area (Pimonidazole hypoxic fraction, pHF) was determined in seven serial 10 μm cross-sections per tumour by fluorescence microscopy and computerized image analysis. Local tumour control was evaluated in a total of 399 irradiated tumours at 120 days after 30 fractions given within 6 weeks with total doses between 30 and 115 Gy. Results Tumour lines showed pronounced heterogeneity in both pHF and TCD 50 . Mean pHF values varied between 5% and 37%, TCD 50 values between 47 and 130 Gy. A Cox Proportional Hazards model of time to recurrence with two covariates, dose and pHF, yielded significant contributions of both parameters on local control ( p Conclusions This study confirms that tumour hypoxia measured using Pimonidazole in untreated tumours is a significant determinant of local control after fractionated irradiation. The data support the use of multivariate approaches for the evaluation of a single prognostic biomarker such as Pimonidazole, and more generally, suggest that they are required to establish accurate prognostic factors for tumour response.

  • Comparison between Pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix
    Cytometry. Part B Clinical cytometry, 2006
    Co-Authors: B. Jankovic, James A Raleigh, Ralph E. Durand, C. Aquino-parsons, Eric J. Stanbridge, Judit P. Banáth, S. H. Macphail, Peggy L. Olive
    Abstract:

    Background: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, Pimonidazole, and expression of the endogenous hypoxia markers HIF-1a and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. Methods: Two biopsies were taken one day after administration of Pimonidazole and were analyzed for Pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1a expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF1a expression over the course of treatment. Results: Approximately 6% of the tumor area stained positive for Pimonidazole, HIF-1a, or CAIX. The CAIX positive fraction correlated with the Pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between Pimonidazole and HIF-1a (r = 0.31) and CAIX and HIF-1a (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1a levels tended to decrease with time after the start of therapy. Conclusions: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of Pimonidazole. CAIX staining pattern is a better match to the Pimonidazole staining pattern than is HIF-1a, and high CAIX expression in the absence (or low levels) of HIF-1a may indicate a different

  • A comparison of oral and intravenous Pimonidazole in canine tumors using intravenous CCI-103F as a control hypoxia marker
    International journal of radiation oncology biology physics, 2005
    Co-Authors: Miriam Kleiter, Shu Chuan Chou, David Y.w. Lee, Donald E. Thrall, David E. Malarkey, James A Raleigh
    Abstract:

    Purpose: Pimonidazole HCl is widely used in immunohistochemical analyses of hypoxia in normal and malignant tissues. The present study investigates oral administration as a means of minimizing invasiveness. Methods and Materials: Twelve dogs with confirmed malignancy received 0.5 g/m{sup 2} of Pimonidazole HCl: 6 by mouth and 6 by i.v. infusion. All dogs received i.v. CCI-103F as a control. Plasma levels of Pimonidazole, Pimonidazole N-oxide, and CCI-103F were measured. Tumor biopsies were formalin fixed, paraffin embedded, sectioned, immunostained, and analyzed for Pimonidazole and CCI-103F binding. pH dependence for Pimonidazole and CCI-103F binding was studied in vitro. Results: Pimonidazole and CCI-103F binding in carcinomas and sarcomas was strongly correlated for both oral and i.v. Pimonidazole HCl (r {sup 2} = 0.97). On average, the extent of Pimonidazole binding exceeded that for CCI-103F by a factor of approximately 1.2, with the factor ranging from 1.0 to 1.65. Binding of both markers was pH dependent, but Pimonidazole binding was greater at all values of pH. Conclusions: Oral Pimonidazole HCl is effective as a hypoxia marker in spontaneously arising canine tumors. Selective cellular uptake and concomitant higher levels of binding in regions of hypoxia at the high end of pH gradients might account formore » the greater extent of Pimonidazole binding.« less

  • Erythropoietin and Erythropoietin Receptor Expression in Head and Neck Cancer: Relationship to Tumor Hypoxia
    Clinical Cancer Research, 2005
    Co-Authors: Murat O. Arcasoy, Khalid Amin, Zishan A. Haroon, Shu Chuan Chou, Mahesh A. Varia, James A Raleigh
    Abstract:

    Purpose: Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis. Experimental Design: Twenty-one patients received an i.v. infusion of the hypoxia marker Pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and Pimonidazole binding. Results: EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and Pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and Pimonidazole binding. Conclusions: The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

Mahesh A. Varia - One of the best experts on this subject based on the ideXlab platform.

  • Erythropoietin and Erythropoietin Receptor Expression in Head and Neck Cancer: Relationship to Tumor Hypoxia
    Clinical Cancer Research, 2005
    Co-Authors: Murat O. Arcasoy, Khalid Amin, Zishan A. Haroon, Shu Chuan Chou, Mahesh A. Varia, James A Raleigh
    Abstract:

    Purpose: Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis. Experimental Design: Twenty-one patients received an i.v. infusion of the hypoxia marker Pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and Pimonidazole binding. Results: EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and Pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and Pimonidazole binding. Conclusions: The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

  • Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: Pimonidazole and involucrin.
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2003
    Co-Authors: Yoshihiro Azuma, Shu Chuan Chou, Mahesh A. Varia, Ruth A. Lininger, Brian J. Murphy, James A Raleigh
    Abstract:

    Purpose: Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia. Experimental Design: Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with Pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 μm. Qualitative and quantitative analyses for involucrin expression, Pimonidazole binding, and human MT-IIa mRNA expression were performed. Results: No overall correlation between the extent of involucrin expression and Pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and Pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests. Conclusions: Colocalization of immunostaining for involucrin and Pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

  • Hypoxia and Differentiation in Squamous Cell Carcinomas of the Uterine Cervix
    2003
    Co-Authors: Yoshihiro Azuma, Shu Chuan Chou, Mahesh A. Varia, Ruth A. Lininger, Brian J. Murphy, James A Raleigh
    Abstract:

    Purpose: Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia. Experimental Design: Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with Pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 μm. Qualitative and quantitative analyses for involucrin expression, Pimonidazole binding, and human MT-IIa mRNA expression were performed. Results: No overall correlation between the extent of involucrin expression and Pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and Pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests. Conclusions: Colocalization of immunostaining for involucrin and Pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

  • Measurements of hypoxia using Pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas.
    Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 2003
    Co-Authors: Marianne Nordsmark, Shu Chuan Chou, Mahesh A. Varia, Juliette A Loncaster, Hanne Havsteen, Jacob Christian Lindegaard, Susan E Davidson, Christina Aquino-parsons, Morten Ladekarl, Catharine M L West
    Abstract:

    Abstract Background and purpose: The measurement of tumour oxygenation using Eppendorf oxygen-sensitive needle electrodes can provide prognostic information but the method is limited to accessible tumours that are suitable for electrode insertion. In this paper the aim was to study the relationship between such physiological measurements of tumour hypoxia and the labelling of tumours with the hypoxia-specific marker Pimonidazole. Materials and methods: Assessment of tumour oxygen partial pressure ( p O 2 ) using an Eppendorf p O 2 histograph and immunohistochemical Pimonidazole labelling was carried out in 86 patients with primary cervix carcinomas. Pimonidazole was given as a single injection (0.5 g/m 2 i.v.) and 10–24 h later p O 2 measurements were made and biopsies taken. Tumour oxygenation status was evaluated as the median tumour p O 2 and the fraction of p O 2 values ≤10 mmHg (HP 10 ), ≤5 mmHg (HP 5 ) and ≤2.5 mmHg (HP 2.5 ). Hypoxia was detected by immunohistochemistry using monoclonal antibodies directed against reductively activated Pimonidazole. Pimonidazole binding was scored using a light microscope. Each tumour was evaluated by the relative area Pimonidazole at highest score and the accumulated area of Pimonidazole labelling from score 1 to 4. Necrosis was measured in HE stained sections. Results and conclusions : The degree of hypoxia assessed by either Pimonidazole binding or invasive electrode measurements varied significantly between tumours. There was a trend that the most hypoxic tumours measured by oxygen electrodes had the highest score of necrosis, and no or little Pimonidazole binding. However, this observation was not consistent and there was no correlation between Pimonidazole staining expressed in this way and oxygen electrode measurements of hypoxia.

  • HIF-1A, Pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors.
    International journal of radiation oncology biology physics, 2002
    Co-Authors: Hilde Janssen, James A Raleigh, Karin Haustermans, Frank Hoebers, Debbie Sprong, Gerard Blommestijn, Ingrid Hofland, Elke Blijweert, Gregg L. Semenza, Mahesh A. Varia
    Abstract:

    PURPOSE: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. METHODS: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with Pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, Pimonidazole, IdUrd, and HIF-1alpha. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1alpha and Pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. RESULTS: Measurement of all four parameters (diffusion-limited fraction, Pimonidazole fraction, HIF-1alpha fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and Pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of Pimonidazole and HIF-1alpha was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1alpha than Pimonidazole. There was no significant correlation between the Pimonidazole and HIF-1alpha fractions in the 10 tumors studied. CONCLUSIONS: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1alpha and Pimonidazole indicate that HIF-1alpha might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.

Albert J. Van Der Kogel - One of the best experts on this subject based on the ideXlab platform.

  • 64Cu-ATSM and 18FDG PET uptake and 64Cu-ATSM autoradiography in spontaneous canine tumors: Comparison with Pimonidazole hypoxia immunohistochemistry
    Radiation oncology (London England), 2012
    Co-Authors: Anders Elias Hansen, Morten Busk, Annemarie T. Kristensen, Jesper T. Jørgensen, Fintan J. Mcevoy, Albert J. Van Der Kogel, Johan Bussink, Svend Aage Engelholm, Andreas Kjaer
    Abstract:

    The aim of this study was to compare 64Cu-diacetyl-bis(N4-methylsemicarbazone) (64Cu-ATSM) and 18FDG PET uptake characteristics and 64Cu-ATSM autoradiography to Pimonidazole immunohistochemistry in spontaneous canine sarcomas and carcinomas. Biopsies were collected from individual tumors between approximately 3 and 25 hours after the intravenous injection of 64Cu-ATSM and Pimonidazole. 64Cu-ATSM autoradiography and Pimonidazole immunostaining was performed on sectioned biopsies. Acquired 64Cu-ATSM autoradiography and Pimonidazole images were rescaled, aligned and their distribution patterns compared. 64Cu-ATSM and 18FDG PET/CT scans were performed in a concurrent study and uptake characteristics were obtained for tumors where available. Maximum Pimonidazole pixel value and mean Pimonidazole labeled fraction was found to be strongly correlated to 18FDG PET uptake levels, whereas more varying results were obtained for the comparison to 64Cu-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 64Cu-ATSM autoradiography yielded varying results. Significant positive correlations were mainly found in sections displaying a heterogeneous distribution of tracers. Tumors with high levels of Pimonidazole staining generally displayed high uptake of 18FDG and 64Cu-ATSM (3 h pi.). Similar regional distribution of 64Cu-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 64Cu-ATSM in canine tumors.

  • autoradiography in spontaneous canine tumors: comparison with Pimonidazole hypoxia immunohistochemistry
    2012
    Co-Authors: Anders Elias Hansen, Morten Busk, Annemarie T. Kristensen, Fintan J. Mcevoy, Albert J. Van Der Kogel, Johan Bussink, Svend Aage Engelholm, Jesper Jørgensen, Andreas Kjaer
    Abstract:

    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 CuATSM. 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 CuATSM (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.

  • can hypoxia pet map hypoxic cell density heterogeneity accurately in an animal tumor model at a clinically obtainable image contrast
    Radiotherapy and Oncology, 2009
    Co-Authors: Morten Busk, Michael R. Horsman, Steen Jakobsen, Albert J. Van Der Kogel, Johan Bussink, K Hansen, Jens Overgaard
    Abstract:

    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.

  • Hypoxia in larynx carcinomas assessed by Pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia
    European journal of cancer (Oxford England : 1990), 2009
    Co-Authors: I.j. Hoogsteen, Albert J. Van Der Kogel, Jasper Lok, P.f.j.w. Rijken, Henri A. M. Marres, Robert P. Takes, Johannes H.a.m. Kaanders
    Abstract:

    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.

  • Tumour cell proliferation under hypoxic conditions in human head and neck squamous cell carcinomas.
    Oral oncology, 2007
    Co-Authors: Karien I.e.m. Wijffels, Albert J. Van Der Kogel, P.f.j.w. Rijken, Henri A. M. Marres, J.p.w. Peters, Johannes H.a.m. Kaanders
    Abstract:

    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.

Johannes H.a.m. Kaanders - One of the best experts on this subject based on the ideXlab platform.

  • Hypoxia in larynx carcinomas assessed by Pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia
    European journal of cancer (Oxford England : 1990), 2009
    Co-Authors: I.j. Hoogsteen, Albert J. Van Der Kogel, Jasper Lok, P.f.j.w. Rijken, Henri A. M. Marres, Robert P. Takes, Johannes H.a.m. Kaanders
    Abstract:

    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.

  • Correlation of [^18F]FMISO autoradiography and pimonodazole immunohistochemistry in human head and neck carcinoma xenografts
    European Journal of Nuclear Medicine and Molecular Imaging, 2008
    Co-Authors: Esther G.c. Troost, Johannes H.a.m. Kaanders, Peter Laverman, Marielle E.p. Philippens, Wim J.g. Oyen, Otto C. Boerman, Albert J. Kogel, Johan Bussink
    Abstract:

    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 [^18F]fluoromisonidazole ([^18F]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 [^18F]FMISO as hypoxia marker at the microregional level. Methods Autoradiography after injection of [^18F]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 [^18F]FMISO signal was dependent on the distribution of hypoxia at the microregional level. The comparison of [^18F]FMISO autoradiography and Pimonidazole immunohistochemistry by pixel-by-pixel analysis revealed moderate correlations. In five tumour lines, a significant correlation between the mean [^18F]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 [^18F]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 [^18F]FMISO signal intensity was much stronger and more significant ( r ^2 = 0.73, p  

  • Tumour cell proliferation under hypoxic conditions in human head and neck squamous cell carcinomas.
    Oral oncology, 2007
    Co-Authors: Karien I.e.m. Wijffels, Albert J. Van Der Kogel, P.f.j.w. Rijken, Henri A. M. Marres, J.p.w. Peters, Johannes H.a.m. Kaanders
    Abstract:

    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.

  • Imaging hypoxia after oxygenation-modification: comparing [18F]FMISO autoradiography with Pimonidazole immunohistochemistry in human xenograft tumors
    Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 2006
    Co-Authors: Esther G.c. Troost, Albert J. Van Der Kogel, Johannes H.a.m. Kaanders, Jasper Lok, Peter Laverman, Marielle E.p. Philippens, Wim J.g. Oyen, Otto C. Boerman, Johan Bussink
    Abstract:

    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.

  • Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.
    International journal of radiation oncology biology physics, 2005
    Co-Authors: Hanneke W. M. Van Laarhoven, Johannes H.a.m. Kaanders, Jasper Lok, Wenny J.m. Peeters, P.f.j.w. Rijken, Bastiaan Wiering, Theo J.m. Ruers, Cornelis J. A. Punt, Arend Heerschap, Albert J. Van Der Kogel
    Abstract:

    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 μm. 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.

Ralph E. Durand - One of the best experts on this subject based on the ideXlab platform.

  • The fate of hypoxic (Pimonidazole-labelled) cells in human cervix tumours undergoing chemo-radiotherapy.
    Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 2006
    Co-Authors: Ralph E. Durand, Christina Aquino-parsons
    Abstract:

    Abstract Background and purpose A subset of patients in a clinical study where sequential biopsies were to be obtained during multifraction radiotherapy received Pimonidazole prior to initiating treatment, allowing a unique opportunity of following hypoxic cells in situ during therapy. Material and methods After institutional ethics review and with informed consent, women expecting to undergo radical treatment for cancer of the cervix received Pimonidazole hydrochloride, with a biopsy approximately 24h later. Therapy was then started, and weekly biopsies were obtained. In the laboratory, the biopsies were reduced to single cell suspensions for flow cytometry analysis of DNA content, Pimonidazole, and proliferation markers. Results Pre-treatment Pimonidazole-positive cells were largely in G 0 /G 1 . Pimonidazole-labelled cells, though expected to be radioresistant, were markedly decreased even early into treatment, and continued to disappear with a half-time of about 3 days. Concurrently, the cell cycle distribution of the previously hypoxic cells changed from predominantly quiescent to mostly proliferating. Conclusions While a part of the rapid apparent loss of hypoxic cells was certainly due to loss of Pimonidazole adducts through repair and dilution by cell division, the speed with which this occurred suggests that many labelled cells could rapidly re-enter the proliferative pool, a result consistent with many of those Pimonidazole-labelled human cervix tumour cells being cyclically, rather than continuously, hypoxic.

  • Comparison between Pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix
    Cytometry. Part B Clinical cytometry, 2006
    Co-Authors: B. Jankovic, James A Raleigh, Ralph E. Durand, C. Aquino-parsons, Eric J. Stanbridge, Judit P. Banáth, S. H. Macphail, Peggy L. Olive
    Abstract:

    Background: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, Pimonidazole, and expression of the endogenous hypoxia markers HIF-1a and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. Methods: Two biopsies were taken one day after administration of Pimonidazole and were analyzed for Pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1a expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF1a expression over the course of treatment. Results: Approximately 6% of the tumor area stained positive for Pimonidazole, HIF-1a, or CAIX. The CAIX positive fraction correlated with the Pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between Pimonidazole and HIF-1a (r = 0.31) and CAIX and HIF-1a (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1a levels tended to decrease with time after the start of therapy. Conclusions: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of Pimonidazole. CAIX staining pattern is a better match to the Pimonidazole staining pattern than is HIF-1a, and high CAIX expression in the absence (or low levels) of HIF-1a may indicate a different

  • Orally Administered Pimonidazole to Label Hypoxic Tumor Cells
    Cancer research, 2002
    Co-Authors: Kevin L. Bennewith, James A Raleigh, Ralph E. Durand
    Abstract:

    Pimonidazole, a “hypoxia marker” normally delivered i.v. or i.p., was instead administered in the drinking water of tumor-bearing mice. As Pimonidazole exposure was increased from 3–96 h ad libitum, both the fraction of hypoxic tumor cells and the relative number of Pimonidazole adducts in those cells increased. Furthermore, the sustained ingestion of Pimonidazole revealed a larger hypoxic fraction than did a single injection of an alternative hypoxia marker, CCI-103F. The “additional” hypoxia seen with longer-term oral administration apparently reflects the inclusion of transiently hypoxic tumor cells. Thus, in addition to its convenience and versatility when compared with hypoxia marker injection, oral administration of Pimonidazole appears to permit identification of all of the physiologically and therapeutically relevant hypoxic tumor cells.

  • Comparison between the comet assay and Pimonidazole binding for measuring tumour hypoxia.
    British journal of cancer, 2000
    Co-Authors: Peggy L. Olive, James A Raleigh, Ralph E. Durand, Chenmei Luo, C. Aquino-parsons
    Abstract:

    Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define ‘positive’ cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying Pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for Pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 ± 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to Pimonidazole. Hypoxic fraction estimated by the Pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of Pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m–2 Pimonidazole also suggest the suitability of this approach for human tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com

  • Identification of Nonproliferating but Viable Hypoxic Tumor Cells in Vivo
    Cancer research, 1998
    Co-Authors: Ralph E. Durand, James A Raleigh
    Abstract:

    We have used the combination of Pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, Pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that Pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that Pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.