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Title: An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance

Abstract

Purpose: To investigate the presence of hypoxia in human prostate carcinoma by using pimonidazole immunohistochemical labeling in radical prostatectomy specimens. Methods and Materials: Forty-three patients (median age, 69 years; range, 49-83 years) with localized prostate adenocarcinoma received 0.5 gm/m{sup 2} i.v. pimonidazole 16-24 h before radical prostatectomy. Hypoxia was detected with a monoclonal antibody directed against pimonidazole and scored in formalin-fixed, paraffin-embedded sections. Median and maximal vessel counts were measured with CD34. Results: Thirty-seven patients completed the study. Pimonidazole binding was present in prostate carcinomas in 34 of 37 patients (92%) and in benign prostatic hyperplasia in 35 of 37 patients (95%). A positive correlation of 3+ pimonidazole binding with Gleason score was demonstrated (Spearman's rank, p = 0.044). Vascularity scores did not correlate with hypoxic status or clinical prognostic parameters. Conclusion: Prostate carcinoma and benign prostatic hyperplasia have significant areas of hypoxia; greater hypoxia scores are seen with more aggressive prostate cancer. It is postulated that a hypoxic microenvironment within the prostate might be responsible for the promotion of secondary genetic alterations and angiogenic stimulation, leading to malignant progression, a more aggressive cell phenotype, and greater radioresistance. Modification of radiation regimens to specifically target hypoxia might improve local tumormore » control.« less

Authors:
 [1];  [2];  [3];  [4];  [5];  [4]
  1. Marie Curie Research Wing, Mount Vernon Hospital, Northwood, Middlesex (United Kingdom). E-mail: peterhoskin@nhs.net
  2. Department of Histopathology, Mount Vernon Hospital, Northwood, Middlesex (United Kingdom)
  3. Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex (United Kingdom)
  4. Marie Curie Research Wing, Mount Vernon Hospital, Northwood, Middlesex (United Kingdom)
  5. Department of Human Oncology, University of Wisconsin, Madison, WI (United States)
Publication Date:
OSTI Identifier:
20793461
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Radiation Oncology, Biology and Physics; Journal Volume: 65; Journal Issue: 1; Other Information: DOI: 10.1016/j.ijrobp.2005.11.044; PII: S0360-3016(05)03078-6; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; ANOXIA; CARCINOMAS; FORMALDEHYDE; LABELLING; MONOCLONAL ANTIBODIES; PARAFFIN; PATIENTS; PHENOTYPE; PROSTATE; RADIOSENSITIVITY; STIMULATION

Citation Formats

Carnell, Dawn M., Smith, Rowena E., Daley, Frances, Saunders, Michele I., Bentzen, Soren M., and Hoskin, Peter J. An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance. United States: N. p., 2006. Web. doi:10.1016/J.IJROBP.2005.1.
Carnell, Dawn M., Smith, Rowena E., Daley, Frances, Saunders, Michele I., Bentzen, Soren M., & Hoskin, Peter J. An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance. United States. doi:10.1016/J.IJROBP.2005.1.
Carnell, Dawn M., Smith, Rowena E., Daley, Frances, Saunders, Michele I., Bentzen, Soren M., and Hoskin, Peter J. Mon . "An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance". United States. doi:10.1016/J.IJROBP.2005.1.
@article{osti_20793461,
title = {An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance},
author = {Carnell, Dawn M. and Smith, Rowena E. and Daley, Frances and Saunders, Michele I. and Bentzen, Soren M. and Hoskin, Peter J.},
abstractNote = {Purpose: To investigate the presence of hypoxia in human prostate carcinoma by using pimonidazole immunohistochemical labeling in radical prostatectomy specimens. Methods and Materials: Forty-three patients (median age, 69 years; range, 49-83 years) with localized prostate adenocarcinoma received 0.5 gm/m{sup 2} i.v. pimonidazole 16-24 h before radical prostatectomy. Hypoxia was detected with a monoclonal antibody directed against pimonidazole and scored in formalin-fixed, paraffin-embedded sections. Median and maximal vessel counts were measured with CD34. Results: Thirty-seven patients completed the study. Pimonidazole binding was present in prostate carcinomas in 34 of 37 patients (92%) and in benign prostatic hyperplasia in 35 of 37 patients (95%). A positive correlation of 3+ pimonidazole binding with Gleason score was demonstrated (Spearman's rank, p = 0.044). Vascularity scores did not correlate with hypoxic status or clinical prognostic parameters. Conclusion: Prostate carcinoma and benign prostatic hyperplasia have significant areas of hypoxia; greater hypoxia scores are seen with more aggressive prostate cancer. It is postulated that a hypoxic microenvironment within the prostate might be responsible for the promotion of secondary genetic alterations and angiogenic stimulation, leading to malignant progression, a more aggressive cell phenotype, and greater radioresistance. Modification of radiation regimens to specifically target hypoxia might improve local tumor control.},
doi = {10.1016/J.IJROBP.2005.1},
journal = {International Journal of Radiation Oncology, Biology and Physics},
number = 1,
volume = 65,
place = {United States},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
}
  • Purpose: To investigate the ability of blood oxygen level-dependent (BOLD) MRI to depict clinically significant prostate tumor hypoxia. Methods and Materials: Thirty-three patients with prostate carcinoma undergoing radical prostatectomy were studied preoperatively, using gradient echo sequences without and with contrast medium enhancement, to map relative tissue oxygenation according to relaxivity rates and relative blood volume (rBV). Pimonidazole was administered preoperatively, and whole-mount sections of selected tumor-bearing slices were stained for pimonidazole fixation and tumor and nontumor localization. Histologic and imaging parameters were independently mapped onto patient prostate outlines. Using 5-mm grids, 861 nontumor grid locations were compared with 237 tumormore » grids (with >50% tumor per location) using contingency table analysis with respect to the ability of imaging to predict pimonidazole staining. Results: Twenty patients completed the imaging and histologic protocols. Pimonidazole staining was found in 33% of nontumor and in 70% of tumor grids. The sensitivity of the MR relaxivity parameter R{sub 2}* in depicting tumor hypoxia was high (88%), improving with the addition of low rBV information (95%) without changing specificity (36% and 29%, respectively). High R{sub 2}* increased the positive predictive value for hypoxia by 6% (70% to 76%); conversely, low R{sub 2}* decreased the likelihood of hypoxia being present by 26% (70% to 44%) and by 41% (71% to 30%) when combined with rBV information. Conclusion: R{sub 2}* maps from BOLD-MRI have high sensitivity but low specificity for defining intraprostatic tumor hypoxia. This together with the negative predictive value of 70% when combined with blood volume information makes BOLD-MRI a potential noninvasive technique for mapping prostatic tumor hypoxia.« less
  • 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-103Fmore » 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 for the greater extent of pimonidazole binding.« less
  • Purpose: Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways. Methods and Materials: The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed.more » Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay. Results: RCC cells exhibited elevated SHH-GLI1 activities under hypoxia, which was mediated by HIF2α. Hypoxia induced GLI1 activation through SMO-independent pathways that could be ablated by PI3K inhibitor or MEK inhibitor. Remarkably, the SHH-GLI1 pathway also upregulated HIF2α expression in normoxia. Apparently, there was a positive correlation between HIF2α and GLI1 expression in RCC patients. The combination of sh-HIF2α and GLI1 inhibitor significantly sensitized RCC cells to IR. Conclusions: Cross-talk between the HIF2α and SHH-GLI1 pathways was demonstrated in RCC. Cotargeting these 2 pathways, significantly sensitizing RCC cells to IR, provides a novel strategy for RCC treatment.« less
  • Purpose: The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma marker of tumor hypoxia. However, the association of immunohistochemical OPN expression in tumor sections with tumor oxygenation parameters (HF5, median pO{sub 2}), the hypoxia-related markers hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and carbonic anhydrase IX (CAIX), or hemoglobin and systemic vascular endothelial growth factor (VEGF) levels has not been investigated. Methods and Materials: Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radiotherapy were assessed by immunochemistry for the expression of OPN, HIF-1{alpha}, and CA IX. Relationship of OPN expression with tumor oxygenation parameters (HF5, median pO{sub 2}), HIF-1{alpha}more » and CA IX expression, hemoglobin and serum VEGF level, and clinical parameters was studied. Results: Bivariate analysis showed a significant correlation of positive OPN staining with low hemoglobin level (p = 0.02), high HIF-1{alpha} expression (p = 0.02), and high serum vascular endothelial growth factor level (p = 0.02) for advanced head-and-neck cancer. Furthermore, considering the 31 Stage IV patients, the median pO{sub 2} correlated significantly with the OPN expression (p = 0.02). OPN expression alone had only a small impact on prognosis. However, in a univariate Cox proportional hazard regression model, the expression of either OPN or HIF-1{alpha} or CA IX was associated with a 4.1-fold increased risk of death (p = 0.02) compared with negativity of all three markers. Conclusion: Osteopontin expression detected immunohistochemically is associated with oxygenation parameters in advanced head-and-neck cancer. When the results of OPN, HIF-1{alpha}, and CA IX immunohistochemistry are combined into a hypoxic profile, a strong and statistically significant impact on overall survival is found.« less
  • Purpose: Although hypoxia is a known prognostic factor, its effect will be modified by the rate of reoxygenation and the extent to which the cells are acutely hypoxic. We tested the ability of exogenous and endogenous markers to detect reoxygenation in a xenograft model. Our technique might be applicable to stored patient samples. Methods and Materials: The human colorectal carcinoma line, HT29, was grown in nude mice. Changes in tumor hypoxia were examined by injection of pimonidazole, followed 24 hours later by EF5. Cryosections were stained for these markers and for carbonic anhydrase IX (CAIX) and hypoxia-inducible factor 1{alpha} (HIF1{alpha}).more » Tumor hypoxia was artificially manipulated by carbogen exposure. Results: In unstressed tumors, all four markers showed very similar spatial distributions. After carbogen treatment, pimonidazole and EF5 could detect decreased hypoxia. HIF1{alpha} staining was also decreased relative to CAIX, although the effect was less pronounced than for EF5. Control tumors displayed small regions that had undergone spontaneous changes in tumor hypoxia, as judged by pimonidazole relative to EF5; most of these changes were reflected by CAIX and HIF1{alpha}. Conclusion: HIF1{alpha} can be compared with either CAIX or a previously administered nitroimidazole to provide an estimate of reoxygenation.« less