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Original Articles

Comparison Between Pimonidazole Binding,Oxygen Electrode Measurements, and

Expression of Endogenous Hypoxia Markersin Cancer of the Uterine Cervix

B. Jankovic,1 C. Aquino-Parsons,1 J. A. Raleigh,2 E. J. Stanbridge,3 R. E. Durand,1 J. P. Banath,1

S. H. MacPhail,1 and P. L. Olive1*1Medical Biophysics Department, British Columbia Cancer Agency Research Centre, Vancouver, British Columbia, Canada

2Department of Radiation Oncology, University of North Carolina, Chapel Hill, North Carolina, USA 3Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA

Background: Although tumor hypoxia has been associated with a more aggressive phenotype and lowercure rate, there is no consensus as to the method best suited for routine measurement. Binding of thechemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1a andCAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advancedcarcinoma of the cervix.

Methods: Two biopsies were taken one day after administration of pimonidazole and were analyzed forpimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1a expression anddegree of colocalization were measured in sequential antibody-stained sections. Patient subsets wereexamined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1a expression over the course of treatment.

Results: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1a, or CAIX. TheCAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significantcorrelations 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 withthe other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with thehypoxic fraction measured using the three hypoxia markers. HIF-1a levels tended to decrease with time af-ter 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 patternthan is HIF-1a, and high CAIX expression in the absence (or low levels) of HIF-1a may indicate a differentbiology. q 2006 International Society for Analytical Cytology

Key terms: hypoxia markers; oxygen electrode; CAIX; HIF-1a; pimonidazole; cervical cancer

Hypoxia that develops in many solid tumors is a criticalfactor limiting the success of conventional radiation ther-apy. Hypoxic cells are also less accessible to nutrients anddrugs, more likely to be noncycling, and therefore resist-ant to many forms of chemotherapy (1). Hypoxia hasbeen shown to be a driving force in tumor angiogenesisand has been implicated in promotion of metastasis andgenomic instability (2–6). The weight of evidence indicat-ing the importance of hypoxia in tumor development,progression, and response to treatment is now undeni-able. Yet in spite of its obvious importance, the presenceof hypoxia in individual human solid tumors is not rou-

tinely measured. This can be attributed in large part tothe limitations associated with the application of prospec-tive, invasive methods such as oxygen microelectrodes

*Correspondence to: Peggy L. Olive, Medical Biophysics Department,

British Columbia Cancer, Research Centre, 675 W. 10th Ave., Vancouver,

British Columbia V5Z 1L3, Canada.

E-mail: [email protected]

Received 19 July 2005; Accepted 17 October 2005Published online 2 February 2006 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cyto.b.20086

Cytometry Part B (Clinical Cytometry) 70B:45–55 (2006)

q 2006 International Society for Analytical Cytology


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and extrinsic hypoxia markers. Endogenous protein markershave been identified that have the potential to allow for theimplementation of routine measurement of tumor hypoxiain the clinic (7). However, application of these markers re-quires careful validation against established methods, espe-cially when many other factors could complicate the use of hypoxia responsive gene expression as an indication of tu-mor hypoxia (8,9).

The expression of more than 70 genes is altered under hypoxic conditions as a result of the change in stability of a critical transcription factor called hypoxia inducible fac-tor-1 (HIF-1) (10). The HIF-1a subunit of this heterodimer undergoes stabilization when the oxygen concentrationdrops below about 2%. Once stabilized, HIF-1 upregulatesexpression of genes involved in oxygen delivery, glycolysis,and angiogenesis. Retrospective analysis of tumor biopsieshave typically shown that higher expression for HIF-1a or its downstream targets carbonic anhydrase 9 (CAIX) andglucose transporters 1 and 3 are correlated with tumor ag-gressiveness and a worse prognosis (7). However, of the

almost three dozen or so retrospective clinical studiesexamining expression of endogenous markers in relation toprognosis, only a few have compared expression of theseendogenous markers with an established method for mea-surement of tumor hypoxia, such as oxygen partial pressure(pO2 ) or binding of chemical hypoxia markers.

Although a trend was observed between high levels of pimonidazole binding and low pO2 measurements in 86cervical carcinoma, this relation was not found to be sig-nificant (11). A study in 28 brain tumors comparing pO2to EF5 binding also failed to identify a correlation be-tween the two measures of hypoxia (12). The lack of cor-relation between extrinsic hypoxia markers and oxygenelectrode results is of concern, since it raises the question

whether the proper comparison for endogenous hypoxiamarkers is pO2 measurements or measurements of extrin-sic hypoxia marker binding. Tumor hypoxia measured

with oxygen microelectrodes is associated with a poorer outcome (overall survival and/or local control) for many tumor types including cervical cancer (13), so perhapsthis is the proper comparison. However, chemical hypoxi-a markers detect hypoxia at the level of the individual celland could be viewed as the more appropriate comparison

with hypoxia-regulated gene expression patterns meas-ured in individual cells. It has been suggested that the na-ture of tumor hypoxia, chronic or transient, may influ-ence chemical markers and electrode measurements dif-

ferently (14), and that necrotic regions may influenceoxygen electrode measurements but not hypoxia marker binding (11). Of course, sampling variability as a result of tumor heterogeneity for both oxygen electrodes and hy-poxia markers can be substantial (15–17). However, incomparing the extent of binding and colocalization of dif-ferent hypoxia marker staining patterns on sequential sec-tions, the sampling problem is less important.

A small number of studies have compared endogenousmarker expression with either oxygen microelectrodemeasurements or hypoxia marker binding. Airley et al.(14) compared pimonidazole binding with GLUT-1 and

CAIX expression in 42 patients with cancer of the cervixusing a semi-quantitative scoring method. They concludedthat GLUT-1 correlated with pimonidazole binding, butthere was only a borderline correlation between pimonida-zole and CAIX. In other studies, no correlation was foundbetween oxygen electrode measurements and GLUT-1 ex-pression (18) or HIF-1a expression (15). Recent results indi-cate that erythropoietin expression was correlated with pimonidazole binding ( r ¼ 0.74) (19). Modest correlationsbetween pO2 and expression of HIF-1a ( r ¼ 0.4, P < 0.01)(20) and CAIX ( r ¼ 0.43, P < 0.001) (21) have beenreported. Unlike HIF-1a expression (20), CAIX expression

was found to have prognostic value in terms of overall survival (21). In the latter study, the measured correlationbetween GLUT-1 and pO2 was weak ( r ¼ 0.28, P ¼ 0.04)(21). In 21 patients with bladder cancer, a strong correla-tion was observed between pimonidazole and CAIX ( r ¼0.86) as well as between pimonidazole and GLUT-1 ( r ¼0.91) (22). Although the markers did not predict for localcontrol, both CAIX and GLUT-1 were independent prog-

nostic factors for overall survival. A general conclusionfrom these studies has been that endogenous markers offer promise for the routine measurement of tumor hypoxia,but they may not provide the same information as pO2measurements using microelectrodes or binding of chemi-cal hypoxia markers.

Treatment outcome is determined by various patient,tumor and treatment-related factors. In addition to well-established clinical factors including tumor size and nodalstatus, tumor proliferation and hypoxia are recognized tobe independent and potentially complementary predic-tive assays in cervical cancer (23). Since the early work by Fertil and Malaise, intrinsic radiosensitivity is also knownto be an important and measurable property of both tumorsand normal tissues (24). The challenging approach to apply functional assays to measure several properties of tumors,taken by some groups (25–27) will undoubtedly increasethe likelihood of developing a useful predictive assay. How-ever, each one of these tumor characteristics will need tobe individually selected and validated. Toward this goal,

we compared several methods for their ability to detecthypoxic regions within tumors. A quantitative digital imageanalysis method was applied to tiled images of sequentialsections from tumor biopsies stained for pimonidazole,HIF-1a and CAIX. Patients were also examined for tumor oxygen tension using the Eppendorf oxygen microelec-trode and biopsies from a subset of these patients were

examined for S phase DNA content. Therapy outcomes will be reported separately.

METHODS

Patient Selection and Treatment Protocol

Ethical approval for this study was granted by the British Columbia Cancer Agency Ethics Board and the University of British Columbia Ethics committee. Over the course of the study, more than 100 patients received pimonidazoleas an intravenous infusion before the study was closed at

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the end of 2004. Seventy-eight patients with advancedcervical carcinoma that participated in this project weretreated at the Vancouver Cancer Centre between Septem-ber 1999 and June 2003.

All patients underwent initial clinical evaluation in ac-cordance with local protocol, including a complete history and physical, CT, or MRI of the abdomen and pelvis, chest

X-ray, liver function tests, complete blood count, and serumcreatinine and electrolytes. Staging was according to FIGOguidelines. Suitable candidates for this study had a clinically

visible and histologically confirmed invasive carcinoma of the cervix: squamous-cell carcinoma, adenocarcinoma, ade-nosquamous carcinoma, or carcinoma not otherwise speci-fied, and gave informed consent. Patients were ineligible if they had liver enzyme tests greater than twice the normallaboratory values, serum creatinine 150 mmol/l, or a his-tory of a peripheral neuropathy.

Seventy-four patients with cervical carcinoma (FIGOstages Ia to IVb) were treated with radical radiotherapy,48 of these patients received adjuvant chemotherapy (cis-

platin), and 60 patients inhaled carbogen (95% oxygen, 5%carbon dioxide) 4 min before and during the daily fractionsof external beam radiation therapy. Three patients under-

went palliative treatment for recurrent tumors, and onepatient refused treatment.

All patients received a 20 min i.v. infusion of 0.5 g/m2

of Hypoxyprobe-1 (pimonidazole hydrochloride; NaturalPharmaceuticals International Inc., Research Triangle Park,NC) dissolved in 100 ml of 0.9% sterile saline at room tem-perature. The following day, 24 h later (about 4 plasmahalf-lives), the patients underwent tumor oxygen meas-urements using the Eppendorf electrode, after which twoincisional biopsies ( 150 mg) were acquired. Oxygenelectrode measurements as well as all biopsies were takenfrom clinically representative areas of the tumor. Areas of obvious necrosis were avoided. Biopsies were transportedto the laboratory immediately after excision and disaggre-gated into single cells for analysis of pimonidazole bind-ing using flow cytometry, and DNA content. Another biop-sy was fixed in formalin and embedded in paraffin. Sequen-tial sections were prepared by the Pathology Department atthe Vancouver Cancer Centre.

All of the patients were eligible for marker correlation stud-ies, regardless of the therapy they received. Measurementof the fluctuations in HIF-1a over the course of chemo-radiotherapy was performed on 15 patients. For each patient,in addition to one pretreatment biopsy, multiple biopsies

(1–4 biopsies) obtained during treatment were available for analysis.

Oxygen Microelectrode Measurements

Measurements of pO2 were performed pretreatmentusing an Eppendorf pO2 histograph-6650 with sterile, po-larographic probes 250 mm in diameter (Hamburg, Ger-many). The location of, the number, and length of thetracks for each site was at the discretion of the clinician,and were dependent on the clinical size and location of the lesion, the tolerance of the patient to the procedure,

and the clinical suspicion of any measurement artifactsthat may have occurred as previously described (28). Typ-ically 4 tracks and >80 measurements were obtained per tumor. Median pO2 as well as the percentages of pO2 val-ues 2.5 or 5 mmHg were calculated.

Pimonidazole Analysis by Flow Cytometry

Tumor biopsies were finely minced with scalpels anddisaggregated into single cells using trypsin, collagenase,and DNase as previously described (29). Ethanol-fixedcells were rinsed in phosphate-buffered saline (PBS) andresuspended in PST (PBS containing 4% FBS and 0.1% triton

X-100). A fluorescein isothiocyanate (FITC)-conjugated(1:1000 dilution) anti-pimonidazole primary antibody wasincubated with 2 106 alcohol-fixed cells for 2 h at 378C.Samples were rinsed with PST and resuspended for DNA staining in 1 ml PBS containing 1 mg/ml 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Single cellsuspensions were analyzed using a Coulter Epics Elite ESP

3-laser, 6-color cyometer (Coulter Corp. Hialeah, FL) for theintensity of FITC-anti-pimonidazole staining and DNA con-tent. Approximately 100,000 cells were acquired for theanalysis. A sample of antibody-stained cells was also ana-lyzed microscopically in a blinded fashion, and the percent-age of brightly stained cells was recorded from a sample of 500 cells.

Univariate histograms from flow cytometry analysis,plotted as cell number versus logarithm of fluorescentanti-pimonidazole antibody intensity, were analyzed by aleast-squares approach for three Gaussian distributionsrepresenting aerobic, intermediately oxygenated, and hy-poxic tumor cell populations. No constraints on the posi-tions of the distribution means were imposed. Cells consid-

ered hypoxic were, on average, 10 times more fluorescentthan well-oxygenated cells within the population (29)

Measurement of S Phase Fraction

A third incisional biopsy was taken after pimonidazoleadministration and disaggregated to produce a single cellsuspension. Cells were fixed in 70% ethanol, and analyzedusing cells stained for DNA content using DAPI and for presence of epithelial cells using SIGMA pan cytokeratinmonoclonal antibody (clone 11; 1:100 dilution). List modefiles were collected and S phase DNA content was deter-mined using MODFIT software (Veristy Software House,Inc., Topsham, ME). For hyperdiploid tumors, S phase

content was determined using the tumor population only.For diploid adenocarcinoma, all cytokeratin positive cells

were included in the analysis. For squamous-cell cancers,all cells were analyzed. More details have been providedelsewhere (30).

Antibody Staining of Sections

Immunoperoxidase with diaminobenzidine tetrahydro-chloride (DAB) substrate was performed to detect hypoxicregions indicated by the presence of pimonidazole, CAIX,or HIF-1a. For dewaxing, 5 mm paraffin-embedded sec-

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tions were incubated at 508C for 1.5 h and immersed inxylene to complete the dewaxing process. The slides

were hydrated in graded alcohols, and rinsed in distilled water and PBS. Sections were treated with 3% hydrogenperoxide in methanol for 10 min to eliminate the endoge-nous peroxidase activity and thereby prevent nonspecificreactions with DAB substrate. After rinsing, each section

was treated with 50 ml of protease (1:100 v/v in PBS) for 30 min at 378C to degrade the protein cross-links formedby formalin fixation and expose the antigenic sites. Whenstaining for HIF-1a, the application of protease was sub-stituted by treatment with high pH target retrieval solu-tion from DAKO (Carpinteria, CA), as this method wasfound to have improved efficacy for nuclear antigen expo-sure compared to protease treatment. The slides were sub-merged for 30 min in a preheated solution inside a 958C

water bath. Subsequently, they were taken out of the bath and allowed to cool at room temperature for 15 min. They

were then rinsed several times with PBS. To reduce thebackground staining, a blocking agent, PTN, containing

1% BSA (w/v) and 0.2% Tween 20 (v/v) in PBS, wasapplied to the sections for 20 min. Fifty to sixty microli-ters (depending on the size of the tissue section) of anti-pimonidazole or anti-HIF-a (diluted 1:100 in PTN) wasapplied to each drained section for 2 h (pimonidazole) or 1 h (HIF-1a ). After washing three times (two times with PBS and once with PTN) for 5 min, the sections wereincubated in rabbit anti-mouse biotinylated secondary antibody (1:200 in PTN) for 30 min and subsequently

washed in PBS for 5 min. Eosin (HIF-1a sections) or hem-atoxlin (pimonidazole sections) were used as counter-stains. Staining for CAIX was performed at UC Irvine aspreviously described (31) (32).

Quantitative Analysis of Antibody-Stained Sections

A Zeiss Axioplan 2 microscope with an attached mono-chrome 12 bit CCD camera was used for acquisition of images of stained tumor sections. Composite images of the entire tumor tissue were prepared by electronically tiling up to 200 individual frames. Images of pimonida-zole- and CAIX-stained sections were focused and cap-tured in 8-bit grayscale, while the images of HIF-1astained sections were captured in 24-bit RGB using anRGB color filter. Image artifacts, such as bubbles createdupon application of the mounting medium were removedin NIH image Pieces of tissue present in one section and

absent in a sequential section (stained for a different anti-gen) were deleted or disregarded. Digitized images wereoptimized to obtain maximum contrast between back-ground and stained regions. Thresholding was performedtwice to differentiate tissues from background andstained tissue from the rest of the image (Fig. 1). The twohighlighted areas were measured and the hypoxic frac-tion was calculated by dividing the stained tissue area by the total tissue area. The selected threshold intensities

were adapted to the intensity parameters of each imageanalyzed to differentiate between the unstained andstained tissue as accurately as possible. The threshold in-

tensities depended on the intensity of the staining, themarker being detected, the extent of the counterstainabsorption, the brightness of the image, and the noise inthe image. Upon completion of the image processingsteps, the images were analyzed for the fractions of tumor sections stained for pimonidazole, CAIX, and HIF-1a andfor marker colocalization. Images were thresholded andanalyzed independently by three observers for marker positive fractions and results were averaged.

Although care was taken to maintain consistency inconcentrations and duration of treatment by antibodies,

ABC and DAB reagents between the experiments, the ac-tivity of these reagents, and the resulting intensity of thesignal were subject to variation. For this reason, the exactrelationship between the staining intensity and the amountof marker present could not be precisely identified or assumed consistent between batches. All of the antibodies

yielded strong signals when present, or no signal relative tobackground, and therefore only the percentage of ‘‘stained’’pixels as a fraction of total tumor tissue, and not the stain-ing intensity, was assessed.

Qualitative Analysis of Hypoxia Marker Colocalization

A qualitative colocalization analysis was performed onall images to calculate the extent of colocalization

between pairs of markers. A discrete scoring system wasused that assigned scores from 0 to 4 to each pair of stained sections. Pairs of sections with scores greater thanor equal to 2 were considered moderately to highly co-localized. As analysis was applied to marker staining in se-quential sections, and there were differences in intracellu-lar localization or oxygen dependency of binding, precisecolocalization was not expected. Instead, pimonidazole-la-beled regions that fell within regions of HIF-1a or CAIX staining (but not necessarily extending to the limits of staining by those antibodies) were considered completely co-localized. Similarly, lack of HIF-1a staining in perine-

FIG. 1. Analysis of hypoxia marker binding in antibody-stained sec-tions and single cells. Panel (a) show a representative tiled tumor sec-tion stained for CAIX. In panel (b), this is converted to a gray scaleimage. Panel (c) shows the total area of the section and panel (d) showsthe marker positive regions. The ratio of the number of red pixels in (d)divided by the number of red pixels in (c) gives the percentage of the tu-mor that is CAIX positive.

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crotic regions (33) was not taken into account in mea-surement of degree of colocalization.

Statistical Analysis

Linear least squares regression analysis was used todetermine the degree of correlation between the fractions

of tissue sections labeled for pimonidazole, CAIX, andHIF-1a. Bivariate Pearson correlations were performed toidentify pairs of clinical and hypoxia parameters which correlated significantly. To examine the differences in hy-poxic fraction between populations with different valuesof clinical parameters, independent Student t tests wereperformed. A P -value less than or equal to 0.05 was con-sidered statistically significant.

RESULTS

Patient characteristics and percentage of cells consideredhypoxic are given in Table 1. Frequencies of adenocarcino-ma and squamous-cell carcinoma in this study reflected the

incidence of these two types of cancer in the North Ameri-can population (34,35). Similar ranges, means, and median

values for hypoxic fraction were found for pimonidazole,CAIX, and HIF-1a staining. As in many previous studies(36–38), hypoxic fraction measured by three hypoxiamarkers did not correlate with the well-established clinicalprognostic factors (including FIGO stage, maximum clinicaldiameter, presenting hemoglobin, nodal status, and tumor grade) (results of statistical analyses not shown).

Figures 1a–1d illustrates the method used to determinethe percentage of tumor that was positive for each hypoxiamarker after antibody staining. Colored images were ob-tained by a x-y stage automatic tiling of entire sectionsunder 10 magnification (Fig. 1a). Tiled images were con-

verted to gray scale images in Figure 1b. These underwentthresholding to identify the entire area of the tumor (Fig. 1c)as well as the marker positive region (Fig. 1d). Areas of obvious necrosis or tissue folds were not included in theanalysis but no attempt was made to eliminate normal tis-sue components. Before comparing the staining patternsfor different hypoxia markers, it was important to estab-lish whether immunohistochemical staining of sequentialsections was reproducible and consistent between sec-tions. Fractions of the tumor stained for pimonidazole intwo sequential sections from 16 tumors were compared.Results indicated a strong correlation ( r ¼ 0.99, slope ¼1.1, data not shown).

In addition to immunohistochemical analysis, another method was employed to measure pimonidazole binding.

A second biopsy was taken at the same time and within30 min of biopsy, it was disaggregated with enzymes, andfixed in ethanol. Flow cytometry evaluation was per-formed with anti-pimonidazole antibodies. The percent-age of hypoxic cells was calculated by fitting flow histo-grams to three normal distributions representing aerobic,intermediate, and hypoxic populations (29). For 30% of the tumor samples, DNA content could be used to dis-criminate between diploid cells and hyperdiploid tumor cells (Fig. 2). By gating on DNA content, hypoxic fraction

T a b l e 1

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H P 2 . 5

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7 3

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S q u a m o u s c e l l c a r c i n o m a

5 5

7 5 . 3

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( 0 . 4 – 2 7 . 8

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5 . 4

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A d e n o c a r c i n o m a

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2 1 . 9

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( 0 . 6 – 8 . 9

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2 . 9

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( 1 . 0 – 2 3 . 5

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( 0 . 0 – 8 0 . 0

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9 . 6

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( 6 . 6 – 8 . 2

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6 4 . 2

( 6 4 . 2 – 6 4 . 2

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( 7 0 . 4 – 7 0 . 4

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4 1 . 5

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3 8 . 5

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6 0 . 0

( 0 . 0 – 1 0 0 . 0

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could be determined independently for tumor and ‘‘nor-mal’’ cells. Invariably, the diploid population showed alower hypoxic fraction than the tumor cells, suggestingthat these cells are closer to functional blood vessels. A reduced ability of diploid cells to metabolize and bindpimonidazole was also observed, consistent with a

reduced nitroreductase activity. To compare hypoxic frac-tion measured by flow cytometry with hypoxic fractionusing image analysis of stained sections, DNA content

was not used to discriminate tumor cells from normalcells even in those cases where this was feasible.

The percentage of hypoxic cells determined by this flow cytometry approach agreed well with the much simpler approach of visually scoring the fraction of brightly stainedcells cytospun onto slides after pimonidazole staining (Fig.3a). Image analysis of pimonidazole-stained tumor sectionsfrom a separate biopsy taken at the same time also showeda significant correlation with the flow cytometry method.

However, because two different biopsies were comparedusing two different methods, tumor heterogeneity and cellloss resulted in a poorer correlation (Fig. 3b).

The mean and range of marker positive fractions weresimilar for pimonidazole-, HIF-1a-, and CAIX-stained se-quential sections (Fig. 4a). The comparison shown in Fig-

ure 4b gives confidence that the markers are identifyingthe same property of the tumors. However, correlationsbetween the individual pairs, although significant, are lessconvincing with the exception of the correlation be-tween pimonidazole and CAIX (Table 2, Figs. 5a–5c). Fivetumors showed low expression of HIF-1a but high expression of CAIX ( >3.5 times higher). Not surprisingly,the correlations between pimonidazole and CAIX or CAIX and HIF-1a improved significantly when these five tumors

were removed from analysis (data not shown). Interest-ingly, preliminary outcome analysis indicates that none of these tumors has progressed.

FIG. 2. Representative flow cytometric analyses of pimonidazole binding in cells from cervical carcinoma biopsies. Single cells prepared from twopatient samples were fixed and stained for pimonidazole adducts and for DNA content. The diploid are hyperdiploid cell populations are evident in pan-els (a) and (b), and the relation between DNA content and pimonidazole antibody binding is shown in panels (c) and (d). Panels in (e) indicate the distri-bution of pimonidazole antibody binding for diploid and hyperdiploid populations for each tumor, and the application of a curve fitting algorithm to cal-culate hypoxic fraction. The hypoxic cell population (black) is on average 10 times more fluorescent than the aerobic population (gray).

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The degree of colocalization between stained regions isalso critical in their evaluation as hypoxia markers. Morethan 70% of the stained regions showed extensive coloc-alization when two markers were compared, and thedegree of colocalization was again greatest for pimonida-zole and CAIX (Figs. 5d–5f). A comparison between thesemi-quantitative colocalization score and the marker pos-itive fraction was used to estimate the percentage of themarker positive regions that were occupied by one mark-er, two markers, or all three markers (Table 3). Few areasshowed pimonidazole binding in the absence of HIF-1a

and CAIX staining, and in this analysis, 64–77% of regionsexpressed all three markers. Figure 6 shows two exam-ples where pimonidazole and HIF-1a were located in thesame tumor cords. However, pimonidazole-labeled cellsappeared to be associated within the necrotic area, whileHIF-1a labeled cells were located in adjoining regions thatappeared viable.

There was no relationship between Eppendorf oxygenelectrode measurements and hypoxia marker binding. Fig-ures 7a–7c show the comparison between the percentageof pO2 readings 2.5 mmHg and the mean percentage of hypoxic cells calculated based on pimonidazole, HIF-1a,and CAIX antibody staining. Similar results were obtainedusing the median pO2 or percentage of readings 5 mmHg(data not shown). For a smaller subset of these patients ana-lyzed for S phase fraction, there was no correlation be-tween S phase content and mean percentage of hypoxiccells (Figs. 7d–7f).

The change in hypoxic fraction over the course of ther-apy may provide a useful measure of tumor response to

therapy and could extend the usefulness of this approach.Between two and five tumor biopsies taken over thecourse of treatment from a subset of 15 patients wereavailable for examination of HIF-1a expression. The aver-age time between the initial pretreatment biopsy to thelast biopsy obtained was 39.3 days. Although no consist-ent pattern was observed, in 11/15 patients, HIF-1a de-creased within two weeks after the first radiotherapy treatment (Fig. 8). In 6 out of these 11 patients, HIF-1a de-creased by more than 2-fold. Preliminary outcome analysisindicated that all of the patients who progressed (5/15)experienced a decrease in HIF-1a during the first two

weeks of treatment. Although HIF-1a levels have been re-ported to increase between 48 and 72 h after irradiationof xenograft tumors (39), this was not evident in the tumorsof these patients while undergoing weekly chemotherapy

FIG. 3. Comparison of pimonidazole binding determined by flow cy-tometry, visual analysis of cytospun cells, or image analysis of stainedsections. Single cells prepared from biopsies were fixed and labeled forpimonidazole antibody binding. In panel (a), coded samples were ana-lyzed by flow cytometry using the curve fitting approach in Fig. 2 or byvisual counting of brightly stained cells after cytospinning. In panel ( b),

results from the flow cytometry analysis of pimonidazole binding fromone biopsy were compared to the analysis of pimonidazole positive pix-els from a second biopsy taken at the same time. Linear best-fit linesare drawn.

FIG. 4. Percentage of pimonidazole, HIF-1a,and CAIX stained pixels in sequential sections ofcervical cancer biopsies. Panel (a) shows hypoxicfraction distributions for 67 cervical tumors. Panel(b) compares the percentage of marker positivepixels for all three hypoxia markers.



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and daily radiation treatment. This is likely explained by the fact that biopsies taken during treatment were obtainedon a weekly basis.

DISCUSSION

The search for a practical and robust method for meas-uring tumor hypoxia in the clinic has been a major catalystin the development of chemical and endogenous hypoxia

markers. The main goal of this study was to perform aquantitative analysis for three hypoxia markers examinedon sequential stained sections from pretreatment biopsies.The assessment of patient outcome related to markers wasa secondary objective and will be reported later. Previousstudies have shown positive correlations between variousendogenous markers and established methods for meas-uring hypoxia (9). Nevertheless, some studies have eluci-dated the shortcomings and the differences between themethods. Possible causes for marker mismatch patterns in-

clude differences in marker sensitivity with respect todegree and duration of hypoxia as well as effects of factorsother than hypoxia on expression of endogenous markers(18). Critical analysis of marker mismatch patterns is essen-tial because colocalization could provide additional infor-mation on the nature of hypoxia (Table 3). Although bestaccomplished using multiple antibodies in a single section,analysis using stained sequential sections provides some

information relevant to this question. To our knowledge,no previous study has quantified spatial correlations be-tween the staining of endogenous markers and chemicalhypoxia marker binding in a patient cohort that has alsobeen characterized for pO2. Janssen et al (8) analyzed coloc-alization and hypoxia marker binding in relation to blood

vessels in several patients with head and neck cancers.Their more quantitative approach indicated a poor correla-tion between pimonidazole and HIF-1a stained regions andrelatively low colocalization.

Table 2Pearson’s Correlation Coefficients Between Pairs of Hypoxia Measurement Techniques

Markerstatistics

Pimo Pimo-FC CAIX HIF-1a HP2.5 HP5

Pearson P Pearson P Pearson P Pearson P Pearson P Pearson P

Pimo 1 0 0.45* 0 0.60* 0 0.34* 0 0.12 0.91 0.02 0.89Pimo-FC 1 0 0.36* 0 0.13 0.31 0.07 0.59 0.04 0.76CAIX 1 0 0.42* 0 0.03 0.83 0.04 0.79HIF-1a 1 0 0.02 0.91 0.04 0.75HP2.5 1 0 0.85* 0HP5 1 0

Asterisk indicate that the correlation is significant at the 0.01 level (2-tailed).

FIG. 5. Comparisons between hypoxia marker expres-sion in cervical cancer biopsies. The percentage ofmarker positive pixels are compared pair-wise in panels(a)–(c), and linear best-fit lines are shown. Panels ( d)–(f) present histograms of colocalization frequency be-

tween pairs of markers.

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None of the three markers correlated with Eppendorf oxygen electrode measurement (median, HP 2.5 mmHg or HP 5 mmHg). This is in spite of the fact that tumors with high oxygen partial pressure (pO2 ) have been shown to ex-hibit decreased pimonidazole binding (40) and lower HIF-1a expression (20). The relationship between marker ex-pression and oxygen electrode measurements is a complexone, as these methods do not sample the same tumor microenvironment or provide directly comparable meas-ures of hypoxia. There are now reports of a lack of corre-lation between oxygen electrode measurements and GLUT-1expression (18), CAIX expression (15), and pimonidazolebinding (11).

Significant correlations were observed between thethree ‘‘single-cell’’ measures of tumor hypoxia. Pimonida-zole binding, HIF-1a, and CAIX expression all indicatedan average hypoxic fraction of about 6% in these tumors.The mean/median values are similar to those previously reported by Kaanders et al. for CAIX and pimonidazole inhead and neck cancers (6.4 and 6% respectively) (41),and for pimonidazole binding in cervical cancers as re-ported by Azuma et al (4.5 6 4.8%) (42). They are higher than the median value of 2% measured for HIF-1a in cervi-cal cancers by Haugland et al (20). Other groups have useda semi-quantitative scoring system making comparisonsmore difficult. Underestimates of HIF-1a and CAIX stainingas a result of localization to nucleus or membrane, respec-

tively, might be expected, and the dynamic range of stainingof tissue sections by the endogenous markers was typically lower than for pimonidazole staining. However, the per-centage of stained area is only one aspect of a comparisonbetween markers. Marker colocalization at a microregionallevel was high in most cases, confirming that hypoxia islikely to be the major factor in the expression of HIF-1a andCAIX in these tumors. Lack of marker colocalization at a cel-lular level could be a useful indicator of the nature of tumor hypoxia and at least some regions of some tumors showedmismatch between these markers. Although HIF-1a stabili-zation under hypoxia is largely responsible for expression

of CAIX, once formed, CAIX is typically lost only upon celldeath or division. Conversely, HIF-1a is lost within minutesupon reoxygenation (33). Therefore, it was interesting to

FIG. 6. Comparison between HIF-1a and pimonidazole antibody stain-ing patterns in sequential sections from three biopsies. Note that pimoni-dazole, unlike HIF-1a, appears to occur within necrotic regions in all threeof these tumors.

Table 3Estimates of Marker Mismatch and Possible Explanations for Variation in Colocalization of Markers

Pattern observedFrequency

(% of stained regions) Possible explanation for mismatch

Pimo only


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identify five tumors with high expression of CAIX but low levels of HIF-1a. This pattern is consistent with transienthypoxia, or more accurately, recent reoxygenation. It is alsoconsistent with the reported lack of HIF-1a expression inperinecrotic regions that maintain CAIX expression becauseof its long half-life (33).

Pimonidazole was administered to patients 18–20 h be-fore biopsy. This timing was based on the reported pimo-nidazole plasma half-life of 4.8 h for women and 5.4 h for men (43), and the requirement that no ‘‘free’’ pimonida-zole be present at the time of biopsy when any unmeta-bolized drug would be rapidly bound under hypoxia.However, during this 18–20 h, hypoxic cells borderingnecrosis will die, so the cells labeled by pimonidazole willnot include those recently hypoxic but could includesome that are now necrotic. The movement of pimonida-zole-stained cells into necrotic regions has been quanti-fied in human tumor xenografts by using comparisons

with HIF-1a expression over time (33) or by expressionof a second hypoxia marker (44). In frozen sections of SiHa cervical carcinoma xenografts examined 90 min after pimonidazole injection, 80% of pimonidazole-labeled cells

expressed HIF-1a. However, 48 h later, only 32% of pimo-nidazole-labeled cells still expressed HIF-1a (33). In our sequential DAB-stained sections, we also detected evi-dence of pimonidazole binding extending into necroticregions (Fig. 6). Although this degree of ‘‘mismatch’’ wasnot taken into consideration in our colocalization analysis,it does contribute to observed differences between marker expression or binding at the cellular level (Table 3). Thispotential problem was previously recognized by Denekampand Dasu (45).

The possibility that tumors that express high levels of CAIX but low amounts of HIF-1a are less likely to progress

suggests that combining information from these twomarkers could be a more informative approach than relyingon results from a single endogenous marker. Similarly, analy-sis of response to treatment using a second biopsy wouldimprove the prognostic value of hypoxia measurement.Ultimately, information from protein chips that includethese markers for hypoxia, as well as indicators of prolifera-tion and repair capacity, are expected to improve the ability to predict response to therapy or at least to select patientslikely to respond to hypoxia-directed treatments (e.g. tira-pazamine). Factors that continue to limit this approach aretumor heterogeneity (no single biopsy can be completely representative) and the value of pretreatment indicators asopposed to measures of responses to specific therapies.

FIG. 7. Comparison between hypoxia marker bindingand Eppendorf oxygen electrode measurements (a)–(c) orS phase fraction (d)–(f). Linear best-fit lines are shown.

FIG. 8. Change in expression of HIF-1a as a function of time after thestart of radiochemotherapy. Sequential biopsies from 15 patients wereanalyzed for the percentage of HIF-1a antibody-positive area.

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ACKNOWLEDGMENTS

This work was supported by the Canadian Institutes of Health Research. The assistance of Genevieve Law duringa BCCRC summer studentship is gratefully acknowledged.

LITERATURE CITED1. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical,

biologic, and molecular aspects. J Natl Cancer Inst 2001;93:266–276.2. De Jaeger K, Kavanagh MC, Hill RP. Relationship of hypoxia to meta-

static ability in rodent tumours. Br J Cancer 2001;84:1280–1285.3. Dunn T. Oxygen and cancer. N C Med J 1997;58:140–143.4. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW,

Giaccia AJ. Hypoxia-mediated selection of cells with diminished apo-ptotic potential in solid tumours. Nature 1996;379:88–91.

5. Vaupel P, Kelleher DK, Hockel M. Oxygen status of malignanttumors: pathogenesis of hypoxia and significance for tumor therapy.Semin Oncol 2001;28:29–35.

6. Young SD, Marshall RS, Hill RP. Hypoxia induces DNA over-replica-tion and enhances metastatic potential of murine tumor cells. ProcNatl Acad Sci USA 1988;85:9533–9537.

7. Bussink J, Kaanders JH, van der Kogel AJ. Tumor hypoxia at themicro-regional level: clinical relevance and predictive value of exoge-nous and endogenous hypoxic cell markers. Radiother Oncol 2003;67:3–15.

8. Janssen HL, Haustermans KM, Sprong D, Blommestijn G, Hofland I,Hoebers FJ, Blijweert E, Raleigh JA, Semenza GL, Varia MA, Balm AJ, van

Velthuysen ML, Delaere P, Sciot R, Begg AC. HIF-1A, pimonidazole, andiododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors. Int J Radiat Oncol Biol Phys 2002;54:1537–1549.

9. Vordermark D, Brown JM. Endogenous markers of tumor hypoxiapredictors of clinical radiation resistance? Strahlenther Onkol 2003;179:801–811.

10. Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecu-lar level. Physiology (Bethesda) 2004;19:176–182.

11. Nordsmark M, Loncaster J, Aquino-Parsons C, Chou SC, Ladekarl M,Havsteen H, Lindegaard JC, Davidson SE, Varia M, West C, Hunter R,Overgaard J, Raleigh JA. Measurements of hypoxia using pimonida-zole and polarographic oxygen-sensitive electrodes in human cervixcarcinomas. Radiother Oncol 2003;67:35–44.

12. Evans SM, Judy KD, Dunphy I, Jenkins WT, Nelson PT, Collins R, Wileyto EP, Jenkins K, Hahn SM, Stevens CW, Judkins AR, Phillips P,Geoerger B, Koch CJ. Comparative measurements of hypoxia in humanbrain tumors using needle electrodes and EF5 binding. Cancer Res2004;64:1886–1892.

13. Milosevic M, Fyles A, Hedley D, Hill R. The human tumor microenvir-onment: invasive (needle) measurement of oxygen and interstitialfluid pressure. Semin Radiat Oncol 2004;14:249–258.

14. Airley RE, Loncaster J, Raleigh JA, Harris AL, Davidson SE, Hunter RD, West CM, Stratford IJ. GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: relationship to pimonidazolebinding. Int J Cancer 2003;104:85–91.

15. Hedley D, Pintilie M, Woo J, Morrison A, Birle D, Fyles A, Milosevic M,Hill R. Carbonic anhydrase IX expression, hypoxia, and prognosis inpatients with uterine cervical carcinomas. Clin Cancer Res 2003;9:5666–5674.

16. Thrall DE, Rosner GL, Azuma C, McEntee MC, Raleigh JA. Hypoxiamarker labeling in tumor biopsies: quantification of labeling variationand criteria for biopsy sectioning. Radiother Oncol 1997;44:171–176.

17. Wong RK, Fyles A, Milosevic M, Pintilie M, Hill RP. Heterogeneity of polarographic oxygen tension measurements in cervix cancer: anevaluation of within and between tumor variability, probe position,and track depth. Int J Radiat Oncol Biol Phys 1997;39:405–412.

18. Mayer A, Hockel M, Wree A, Vaupel P. Microregional expression of glu-

cose transporter-1 and oxygenation status: lack of correlation in locally advanced cervical cancers. Clin Cancer Res 2005;11:2768–2773.

19. Arcasoy MO, Amin K, Chou SC, Haroon ZA, Varia M, Raleigh JA.Erythropoietin and erythropoietin receptor expression in head andneck cancer: relationship to tumor hypoxia. Clin Cancer Res 2005;11:20–27.

20. Haugland HK, Vukovic V, Pintilie M, Fyles AW, Milosevic M, Hill RP,Hedley DW. Expression of hypoxia-inducible factor-1alpha in cervicalcarcinomas: correlation with tumor oxygenation. Int J Radiat OncolBiol Phys 2002;53:854–861.

21. Loncaster JA, Harris AL, Davidson SE, Logue JP, Hunter RD, Wycoff CC, Pastorek J, Ratcliffe PJ, Stratford IJ, West CM. Carbonic anhydrase(CA IX) expression, a potential new intrinsic marker of hypoxia: cor-relations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res 2001;61:6394–6399.

22. Hoskin PJ, Sibtain A, Daley FM, Wilson GD. GLUT1 and CAIX asintrinsic markers of hypoxia in bladder cancer: relationship with vascu-larity and proliferation as predictors of outcome of ARCON. Br J Cancer 2003;89:1290–1297.

23. Tsang RW, Fyles AW, Milosevic M, Syed A, Pintilie M, Levin W, Man-chul LA. Interrelationship of proliferation and hypoxia in carcinomaof the cervix. Int J Radiat Oncol Biol Phys 2000;46:95–99.

24. Fertil B, Malaise EP. Inherent cellular radiosensitivity as a basic con-cept for human tumor radiotherapy. Int J Radiat Oncol Biol Phys

1981;7:621–629.25. Bussink J, Kaanders JH, Rijken PF, Peters J P, Hodgkiss RJ, Marres HA, van der Kogel AJ. Vascular architecture and microenvironmental pa-rameters in human squamous cell carcinoma xenografts: effects of carbogen and nicotinamide. Radiother Oncol 1999;50:173–184.

26. Eschwege F, Bourhis J, Girinski T, Lartigau E, Guichard M, Deble D,Kepta L, Wilson GD, Luboinski B. Predictive assays of radiationresponse in patients with head and neck squamous cell carcinoma: areview of the Institute Gustave Roussy experience. Int J Radiat OncolBiol Phys 1997;39:849–853.

27. Hill RP, Fyles W, Milosevic M, Pintilie M, Tsang RW. Is there a relation-ship between repopulation and hypoxia/reoxygenation? Results fromhuman carcinoma of the cervix. Int J Radiat Biol 2003;79:487–494.

28. Aquino-Parsons C, Green A, Minchinton AI. Oxygen tension in prima-ry gynaecological tumours: the influence of carbon dioxide concen-tration. Radiother Oncol 2000;57:45–51.

29. Olive PL, Durand RE, Raleigh JA, Luo C, Aquino-Parsons C. Compari-son between the comet assay and pimonidazole binding for meas-uring tumour hypoxia. Br J Cancer 2000;83:1525–1531.

30. Durand RE, Aquino-Parsons C. Predicting response to treatment inhuman cancers of the uterine cervix: sequential biopsies duringexternal beam radiotherapy. Int J Radiat Oncol Biol Phys 2004;58:555–560.

31. Liao SY, Brewer C, Zavada J, Pastorek J, Pastorekova S, Manetta A,Berman ML, DiSaia PJ, Stanbridge EJ. Identification of the MN antigen asa diagnostic biomarker of cervical intraepithelial squamous and glandu-lar neoplasia and cervical carcinomas. Am J Pathol 1994;145:598–609.

32. Olive PL, Aquino-Parsons C, MacPhail SH, Liao SY, Raleigh JA, LermanMI, Stanbridge EJ. Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res 2001;61:8924–8929.

33. Sobhanifar S, Aquino-Parsons C, Stanbridge EJ, Olive P. Reducedexpression of hypoxia-inducible factor-1alpha in perinecrotic regionsof solid tumors. Cancer Res 2005;65:7259–7266.

34. Duarte-Franco E, Franco EL. Cancer of the uterine cervix. BMC Wom-en’s Health 2004;4(Suppl 1):S13.

35. Liu S, Semenciw R, Mao Y. Cervical cancer: the increasing incidenceof adenocarcinoma and adenosquamous carcinoma in younger wom-

en. CMAJ 2001;164:1151–1152.36. Fyles A, Milosevic M, Hedley D, Pintilie M, Levin W, Manchul L, Hill RP.Tumor hypoxia has independent predictor impact only in patients with node-negative cervix cancer. J Clin Oncol 2002;20:680–687.

37. Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Associa-tion between tumor hypoxia and malignant progression in advancedcancer of the uterine cervix. Cancer Res 1996;56:4509–4515.

38. Stone JE, Parker R, Gilks CB, Stanbridge EJ, Liao SY, Aquino-ParsonsC. Intratumoral oxygenation of invasive squamous cell carcimoma of the vulva is not correlated with regional lymph node metastasis. Eur

J Gynaecol Oncol 2005;26:31–35.39. Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to

regulate vascular radiosensitivity in tumors: role of reoxygenation,free radicals, and stress granules. Cancer Cell 2004;5:429–441.

40. Nordsmark M, Loncaster J, Chou SC, Havsteen H, Lindegaard JC,Davidson SE, Varia M, West C, Hunter R, Overgaard J, Raleigh JA.Invasive oxygen measurements and pimonidazole labeling in humancervix carcinoma. Int J Radiat Oncol Biol Phys 2001;49:581–586.

41. Kaanders JH, Wijffels KI, Marres HA, Ljungkvist AS, Pop LA, van den

Hoogen FJ, de Wilde PC, Bussink J, Raleigh JA, van der Kogel AJ.Pimonidazole binding and tumor vascularity predict for treatmentoutcome in head and neck cancer. Cancer Res 2002;62:7066–7074.

42. Azuma Y, Chou SC, Lininger RA, Murphy BJ, Varia MA, Raleigh JA. Hy-poxia and differentiation in squamous cell carcinomas of the uterine cer-

vix: pimonidazole and involucrin. Clin Cancer Res 2003;9:4944–4952.43. Saunders MI, Anderson PJ, Bennett MH, Dische S, Minchinton A,

Stratford MR, Tothill M. The clinical testing of Ro 03-8799–pharmacoki-netics, toxicology, tissue and tumor concentrations. Int J Radiat OncolBiol Phys 1984;10:1759–1763.

44. Ljungkvist AS, Bussink J, Kaanders JH, Rijken PF, Begg AC, Raleigh JA, van der Kogel AJ. Hypoxic cell turnover in different solid tumor lines.Int J Radiat Oncol Biol Phys 2005;62:1157–1168.

45. Denekamp J, Dasu A. Inducible repair and the two forms of tumour hypoxia–time for a paradigm shift. Acta Oncol 1999;38:903–918.


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