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First published online October 1, 2007
British Journal of Radiology (2007) 80, 934-938
© 2007 British Institute of Radiology
doi: 10.1259/bjr/25046649
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Short communication

Hypoxia can be detected in irradiated normal human tissue: a study using the hypoxic marker pimonidazole hydrochloride

C B Westbury, MRCP, FRCR 1,2 A Pearson, BSc 1 A Nerurkar, MD 3 J S Reis-Filho, MD, PhD, MRCPath 2 D Steele, BSc 2 C Peckitt, BSc, MSc 4 G Sharp, BSc 1 and J R Yarnold, MRCP, FRCR 1

1 Department of Radiotherapy, The Royal Marsden Hospital, Sutton, Surrey, 2 The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London, 3 Department of Pathology, The Royal Marsden Hospital, London,, 4 Clinical Trials and Statistics Unit, Section of Clinical Trials, Institute of Cancer Research, Sutton, UK

Correspondence: Professor John R Yarnold, The Royal Marsden Hospital, Downs Road, Sutton, Surrey, SM2 5PT, UK. E-mail: john.yarnold{at}icr.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
 References
 
Chronic tissue hypoxia may play a role in the pathogenesis of late radiation fibrosis. In order to investigate this hypothesis, the immunohistochemical distribution of pimonidazole hydrochloride (n = 14 patients) and carbonic anhydrase IX (CAIX) (n = 38 patients) was studied in samples of previously irradiated normal human tissue. One sample of irradiated breast tissue, which also showed marked histological features of radiation injury, stained positive for pimonidazole hydrochloride. No CAIX staining was seen in irradiated tissue other than some evidence of physiological hypoxia in the epidermis of two samples of irradiated skin; both were positive for pimonidazole and one was focally positive for CAIX. Pimonidazole hydrochloride staining of tissue with morphological changes of radiation injury could support a role for hypoxia in the pathogenesis of late normal tissue fibrosis in humans.


   Introduction
Top
 Abstract
 Introduction
Methods and materials
 Results
 Discussion
 Conclusions
 References
 
Radiotherapy plays an important curative role in common malignancies, and unavoidable inclusion of normal tissues into the high-dose treatment volume results in late adverse effects in a proportion of patients. Fibrosis is a clinically important component of these effects and, although interesting molecular and cellular mechanisms have been proposed, much remains to be clarified [1, 2]. Tissue ischaemia caused by radiation-induced vascular endothelial injury plays an important role in the pathogenesis of normal tissue atrophy [3] and may also be relevant to the pathogenesis of fibrosis. Hypoxia has been shown to directly stimulate production of profibrotic cytokines and collagen deposition in vitro [4–8] and to be implicated in wound healing and radiation injury in animal models [9, 10]. The only human studies testing for hypoxia after therapeutic radiation employed transcutaneous oxygen microelectrodes to measure oxygen tension in heavily irradiated skin; in these studies, oxygen tension reduced to approximately 50% of that in non-irradiated skin [11, 12]. Not all studies have reproduced these findings [13] but, if confirmed, the presence of tissue hypoxia would support its role in the pathogenesis of fibrosis. Furthermore, a mechanistic framework would be provided for the therapeutic effects of hyperbaric oxygen in selected radiation injuries [14, 15].

In the present study, immunohistochemical staining of pimonidazole hydrochloride and carbonic anhydrase IX (CAIX) were applied to test for hypoxia in normal human tissue removed at surgery several years after high-dose radiotherapy. Pimonidazole hydrochloride was adopted as the gold standard immunohistochemical marker of hypoxia, and CAIX was selected as one of a number of endogenous markers. Positive staining for pimonidazole hydrochloride was seen in the only breast sample with pathological features of severe radiation injury. This isolated finding demonstrates proof-of-principle that hypoxia can be present in tissues showing evidence of severe radiation damage, and justifies further investigation of its role in fibrosis.


   Methods and materials
Top
 Abstract
 Introduction
 Methods and materials
Results
 Discussion
 Conclusions
 References
 
Tissue collection
The study was approved by the research ethics committees of the participating centres, and all patients gave informed written consent. Eligible patients were those undergoing salvage surgery for cancer recurrence or new primary cancer after prior radical radiotherapy. A subset of patients undergoing surgery for locally recurrent breast cancer were asked to donate three subcutaneous core biopsies of the contralateral normal breast whilst under general anaesthetic. Patients consenting to pimonidazole hydrochloride administration (Hypoxyprobe; NPI Inc., Belmont, MA) received an infusion of 500 mg m2 over 20 min, 18–24 h before surgery. After surgery, tissue of interest from inside the irradiated volume, at least several centimetres from the tumour, was selected by the pathologist (A.N.) and fixed in formalin. When available, a specimen of normal tissue outside the irradiated volume was collected as an internal control; otherwise, a contralateral breast biopsy was used. Additional breast samples were used from a tissue bank of patients who had given written consent for research into late normal tissue injury. The samples were collected using an identical method but these patients were not offered pimonidazole hydrochloride. One patient was recruited from the Royal Surrey County Hospital NHS Trust and a second from Mayday Healthcare NHS Trust. The remaining patients were recruited from the Royal Marsden Hospital NHS Foundation Trust.

Immunohistochemistry
3 µm thick sections were cut from representative formalin-fixed paraffin-embedded blocks. Sections were dried overnight at 37°C and subsequently dewaxed and rehydrated through a series of alcohols to water. Endogenous peroxidase was quenched by incubating the sections with 0.03% hydrogen peroxide containing sodium azide for 10 min. For CAIX staining, no epitope retrieval pre-treatment was performed. For pimonidazole staining, epitope retrieval was carried out using 0.01% pronase at 37°C for 10 min. Immunohistochemistry was performed as previously described [16, 17]. Briefly, all sections were treated for 5 min with protein block (Dako Serum Free Protein Block; Dako Glostrup, Denmark) prior to 30 min incubation with either rabbit anti-CAIX 1:1000 (Novus Biologicals, Inc, Littleton, CO) or mouse anti-pimonidazole IgG1 monoclonal antibody 1:100 (Hypoxyprobe-1MAB1 NPI, Inc, Belmont, MA) at room temperature. Detection and visualization were achieved using the Envision System (Dako, Glostrup, Denmark). Positive controls (clear cell renal cell carcinoma for CAIX staining and pleomorphic sarcoma for pimonidazole staining) and negative controls (primary antibody omitted) were included in each slide run. For CAIX, only membrane staining was considered specific, whereas for pimonidazole only cytoplasmic staining was considered specific. Positive controls consistently showed strong membrane staining for CAIX and cytoplasmic staining for pimonidazole hydrochloride (Figure 1Go).


Figure 1
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  		Figure 1. (a–c) CAIX staining and (d–f) pimonidazole hydrochloride staining. (a) Strong membranous staining of CAIX is seen in clear cell renal cell carcinoma control tissue; (b) focal expression of CAIX can be seen in the basal layer and stratum spinosum of the epidermis in irradiated skin. (c) Membranous staining of CAIX in irradiated breast tissue is absent. (d) Strongly positive cytoplasmic staining of pimonidazole hydrochloride is seen adjacent to areas of necrosis in pleomorphic sarcoma tissue control. (e) Pimonidazole hydrochloride staining is shown in the stratum spinosum of epidermis and sweat glands (arrowheads). (f) Positive pimonidazole hydrochloride staining of luminal cells in irradiated breast tissue is associated with (g) cell atypia, atrophy and sclerosis of the TDLU.

 
For both CAIX and pimonidazole hydrochloride, the section was scored as "positive" or "negative". Staining was considered to be positive where there was specific staining in any area of the section in cell types from either stromal or epithelial compartments. Assessment was performed by one observer (A.N.), and slides were scored blind to patient identity or prior tissue exposure to radiation.

Histological examination
For breast tissue specimens, the following features were analysed by one observer (A.N.) using a four-tier semi-quantitative scoring system (0 = negative, 1 = mild, 2 = moderate, 3 = severe): epithelial atypia, sclerosis and atrophy of the terminal duct lobular unit (TDLU), vascular endothelial atypia and fibroblast atypia. When evaluating these features, nuclear enlargement, chromatin pattern, presence of nucleoli and cytoplasmic changes were considered. Stromal collagen was scored as comprising <50% or >50% of the section.

Statistical methods
The analysis is all descriptive. The main statistic used is proportion of positively stained samples with a 95% confidence interval giving a measure of accuracy of these estimates. Analysis was performed using STATA version 8.2 (STATA Corp, College Station, TX).


   Results
Top
 Abstract
 Introduction
 Methods and materials
 Results
Discussion
 Conclusions
 References
 
Patients and tissue characteristics
Table 1Go summarizes the characteristics of the 39 patients, including prior radiation dose, years since irradiation, the nature of the tissues collected, and the number receiving pimonidazole. The median age of the group was 55 years (range 44–62 years). The majority of patients (37/39) had breast surgery and contributed breast tissue samples (Table 2Go). Pimonidazole hydrochloride was administered to 12 patients undergoing breast surgery prior to tissue retrieval (Table 2Go). Specimens were collected from an additional 25 breast patients not receiving this extrinsic marker. Of the patients undergoing breast surgery, 11 donated paired samples of irradiated and non-irradiated tissue and, for one patient, paired tissue samples were collected from simultaneous bilateral mastectomy specimens following prior radiotherapy on both sides. One sample of skin was collected from a single patient undergoing breast surgery. Tissue was collected from two additional patients who both received pimonidazole hydrochloride: one undergoing surgery to the lower limb from whom both irradiated and non-irradiated tissue was collected (muscle and skin), and the other undergoing surgery to the pharynx who donated irradiated tissue only (muscle and mucosa).


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  		Table 1. Patient characteristics(n = 39)

 

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  		Table 2. Characteristics of breast tissue samples(n = 49)

 
Immunohistochemistry
Figure 1Go shows examples of immunohistochemical staining for pimonidazole hydrochloride and CAIX in samples of irradiated breast and irradiated skin. Of the irradiated samples of breast tissue, 1 of 12 (8.3%, 95% confidence interval (CI) 0–38.5) was positive for pimonidazole hydrochloride and none out of 37 (0%, 95% CI 0–9.5) was positive for CAIX (Table 3Go). No non-irradiated samples of breast tissue stained positive for either pimonidazole hydrochloride or CAIX. The one case of irradiated breast tissue staining positive for pimonidazole hydrochloride and negative for CAIX was the only sample showing obvious pathological features characteristic of late radiation injury within the TDLU, including cellular atypia, enlargement of luminal cells associated with cytoplasmic blebbing, and sclerosis. Luminal epithelial cells in the TDLU of this sample showed widespread positive staining for pimonidazole, but the adjacent myoepithelial cells stained negative. Intra- and inter-lobular stromal fibroblasts were heterogeneous in their positive staining. In both samples of irradiated skin, staining for pimonidazole hydrochloride was observed in the stratum spinosum of the epidermis, in eccrine sweat glands and in the infundibular region of hair follicles (skin is known to be hypoxic in rodents) [9, 18]. Additionally, in one sample, patchy staining for CAIX was seen in the stratum spinosum of the epidermis and in the infundibular region of the hair follicles. Weak cytoplasmic staining with CAIX was occasionally seen in perivascular smooth muscle cells; this was considered non-specific. The remaining microanatomical structures were negative in all cases. The two irradiated muscle samples were negative for both markers.


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  		Table 3. Results of immunohistochemical staining for breast tissue samples

 
Histological characteristics
Using a semi-quantitative scoring system in breast tissue, we were unable to score features of late radiation injury and pathological fibrosis owing to sample heterogeneity. Indeed, breast tissue samples showed considerable histological variation across organs in terms of both cell content and density of structures (i.e. ducts, lobules and vessels). In addition, architectural changes, although present, were subtle and unevenly distributed, and cytological abnormalities in particular were patchy. Interestingly, the one breast sample with overt features of radiation-induced changes stained positive for pimonidazole. Although not formally scored, certain findings were noted in the non-breast specimens. Pathological fibrosis was seen focally in the pharyngeal muscle underlying an area of ulceration, and therefore is likely to be related to local inflammation. Fibrosis was also seen in muscle from the limb specimen. This caused disruption of the muscle bundles and resulting atrophy of myocytes. Additional features specific to radiation injury were not seen in either tissue. Neither fibrosis nor other features of radiation injury were seen in the specimens of skin.


   Discussion
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
Conclusions
 References
 
Early and persistent endothelial and vascular injury in irradiated normal tissues is a characteristic feature of response to radiation. Tissue hypoxia is likely to be a consequence of therapeutic radiation in humans [19]. The exact role of hypoxia in the development of late normal tissue injury is, however, uncertain. It is possible that early tissue hypoxia initiates the fibrotic response. Alternatively, hypoxia could be involved in the progression or maintenance of the disorder after radiation injury and fibrosis is established.

Pimonidazole hydrochloride and CAIX have been widely used as immunohistochemical markers of hypoxia in tumours. The main limitation of pimonidazole hydrochloride is that it needs to be administered intravenously to patients prior to surgical resection. Animal studies using pimonidazole hydrochloride have shown the presence of hypoxia both in normal non-neoplastic tissues and in wound healing and other models of normal tissue injury [9, 10, 18, 20]. CAIX is a membrane-bound carbonic anhydrase involved in cell acid–base balance and cell adhesion and has been used extensively for detection of hypoxia in tumours. Expression in normal human breast tissue (the majority of samples in our study) is uncommon [17, 21], although variation of expression of CAIX in normal tissues may occur in relation to acid–base balance [22, 23]. Its usefulness in detecting hypoxia in normal tissue, in which it is not usually expressed, may be limited. Both immunohistochemical markers may be further limited in the presence of mild hypoxia below the threshold of detection, i.e. 20 mmHg for CAIX and 10 mmHg for pimonidazole hydrochloride.

Immunohistochemical markers have not previously been used to test for hypoxia in irradiated human normal tissues. In this small series, staining was positive for pimonidazole hydrochloride but negative for CAIX in a single sample of breast tissue. This sample was also the only one from 12 breast cancer patients given pimonidazole to show marked histological features of radiation injury in breast. The patient received two doses of pimonidazole: the first was given 24 h before a planned operation that was cancelled at short notice, and the second the day before surgery 5 weeks later. It is not thought likely that the double exposure to drug influenced the distribution or intensity of staining. All other tissue samples, apart from two, were negative for both pimonidazole hydrochloride and CAIX staining. The exceptions were the two samples of irradiated skin (one from breast and one from lower leg) showing widespread positive epidermal staining for pimonidazole hydrochloride and, in the breast skin only, focal positive staining for CAIX. Despite prior radiotherapy, the hypoxia is likely to be physiological, and is consistent with both animal data for pimonidazole hydrochloride [9, 18] and human data for CAIX [21]. As strong staining was seen in our positive controls, we consider it highly unlikely that the generally negative results for CAIX reflect technical problems with the staining process.


   Conclusions
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
References
 
In conclusion, a single case of irradiated breast tissue (out of 14 specimens) sampled showed clear evidence of pimonidazole staining, suggesting hypoxia with an oxygen tension <10 mm. The lack of positive staining with CAIX does not exclude moderate hypoxia (>10 mm to <20 mm oxygen tension), as it may seldom be upregulated under conditions of hypoxia in non-cancerous tissues. The single positive example justifies further investigation into the possible role of hypoxia in radiation-induced fibrosis, given that it occurred in the only sample of 14 that showed obvious morphological changes of radiation injury, including fibrosis.

Dr Charlotte Westbury receives funding from Cancer Research UK


   Acknowledgments
 
We would like to thank our patients for their participation, as well as the following colleagues who collaborated in this study: Mr Gerald Gui, Mr Uccio Querci della Rovere, Mr William Allum, Mr Stephen Ebbs, Dr Abed Arnout, Mr Meirion Thomas, Mr Peter Rhys Evans, Mr Adam Searle, Mr Peter Meagher and Mr Mark Kissin. Professor James Raleigh (UNC School of Medicine, USA) and Frances Daley (Gray Cancer Institute, UK) are thanked for their advice.

Received for publication February 5, 2007. Revision received April 11, 2007. Accepted for publication April 18, 2007.


   References
Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Conclusions
 References
 

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