Hypoxia In Human Tumors Research Articles

A novel approach to overcome hypoxic tumor resistance: Cu-ATSM-guided intensity-modulated radiation therapy

Purpose: Locoregional tumor control for locally advanced cancers with radiation therapy has been unsatisfactory. This is in part associated with the phenomenon of tumor hypoxia. Assessing hypoxia in human tumors has been difficult due to the lack of clinically noninvasive and reproducible methods. A recently developed positron emission tomography (PET) imaging-based hypoxia measurement technique which employs a Cu(II)-diacetyl-bis( N 4-methylthiosemicarbazone) (Cu-ATSM) tracer is of great interest. Oxygen electrode measurements in animal experiments have demonstrated a strong correlation between low tumor pO 2 and excess 60Cu-ATSM accumulation. Intensity-modulated radiation therapy (IMRT) allows selective targeting of tumor and sparing of normal tissues. In this study, we examined the feasibility of combining these novel technologies to develop hypoxia imaging (Cu-ATSM)-guided IMRT, which may potentially deliver higher dose of radiation to the hypoxic tumor subvolume to overcome inherent hypoxia-induced radioresistance without compromising normal tissue sparing. Methods and Materials: A custom-designed anthropomorphic head phantom containing computed tomography (CT) and positron emitting tomography (PET) visible targets consisting of plastic balls and rods distributed throughout the “cranium” was fabricated to assess the spatial accuracy of target volume mapping after multimodality image coregistration. For head-and-neck cancer patients, a CT and PET imaging fiducial marker coregistration system was integrated into the thermoplastic immobilization head mask with four CT and PET compatible markers to assist image fusion on a Voxel-Q treatment-planning computer. This system was implemented on head-and-neck cancer patients, and the gross tumor volume (GTV) was delineated based on physical and radiologic findings. Within GTV, regions with a 60Cu-ATSM uptake twice that of contralateral normal neck muscle were operationally designated as ATSM-avid or hypoxic tumor volume ( hGTV) for this feasibility study. These target volumes along with other normal organs contours were defined and transferred to an inverse planning computer (Corvus, NOMOS) to create a hypoxia imaging-guided IMRT treatment plan. Results: A study of the accuracy of target volume mapping showed that the spatial fidelity and imaging distortion after CT and PET image coregistration and fusion were within 2 mm in phantom study. Using fiducial markers to assist CT/PET imaging fusion in patients with carcinoma of the head-and-neck area, a heterogeneous distribution of 60Cu-ATSM within the GTV illustrated the success of 60Cu-ATSM PET to select an ATSM-avid or hypoxic tumor subvolume ( hGTV). We further demonstrated the feasibility of Cu-ATSM-guided IMRT by showing an example in which radiation dose to the hGTV could be escalated without compromising normal tissue (parotid glands and spinal cord) sparing. The plan delivers 80 Gy in 35 fractions to the ATSM-avid tumor subvolume and the GTV simultaneously receives 70 Gy in 35 fractions while more than one-half of the parotid glands are spared to less than 30 Gy. Conclusion: We demonstrated the feasibility of a novel Cu-ATSM-guided IMRT approach through coregistering hypoxia 60Cu-ATSM PET to the corresponding CT images for IMRT planning. Future investigation is needed to establish a clinical-pathologic correlation between 60Cu-ATSM retention and radiation curability, to understand tumor re-oxygenation kinetics, and tumor target uncertainty during a course of radiation therapy before implementing this therapeutic approach to patients with locally advanced tumor.

Semiquantitative immunohistochemical analysis for hypoxia in human tumors

Objective: The goal of this study was to develop a semiquantitative scoring system for measuring hypoxia in human tumors by an immunohistochemical marker approach. Methods and Materials: Eighteen patients diagnosed with squamous cell carcinoma of the uterine cervix or head and neck were infused intravenously with a solution of pimonidazole hydrochloride at a dose of 0.5 gm/m 2. Twenty-four hours later, four biopsies on average from each tumor were fixed in formalin, processed into paraffin blocks, and sectioned. Tissue sections were immunostained for the presence of pimonidazole adducts. Microscopic images (×200) of immunostaining were captured and quantitated by standard image analysis. Images with known amounts of hypoxia spanning ranges of > 0% to 5%, > 5% to 15%, > 15% to 30%, and >30% were assigned scores of +1, +2, +3, and +4, respectively. Three observers then used this calibrated scoring system to analyze hypoxia in tumor sections in a blinded fashion. Results: Excellent interobserver reproducibility was obtained with the calibrated, semiquantitative, immunohistochemical assay for hypoxia in squamous cell carcinomas. Conclusion: The calibrated, semiquantitative assay shows promise as an approach to simplifying the quantitation of human tumor hypoxia by immunohistochemical techniques.

Tumour oxygenation levels correlate with dynamic contrast-enhanced magnetic resonance imaging parameters in carcinoma of the cervix

Background and purpose: The Eppendorf pO 2 histograph is the ‘gold standard’ method for measuring tumour oxygenation. The method is not suitable for widespread application because its use is limited to accessible tumours. A non-invasive imaging technique would be an attractive alternative. Therefore, the relationships between tumour oxygenation and dynamic contrast-enhanced magnetic resonance imaging (MRI) parameters were investigated. Materials and methods: The study comprised 30 patients with carcinoma of the cervix. Tumour oxygenation was measured pre-treatment as median pO 2 and the proportion of values less than 5 mmHg (HP5) using a pO 2 histograph. Repeat measurements were obtained for nine patients following 40–45 Gy external beam radiotherapy giving a total of 39 measurements. Dynamic contrast-enhanced MRI using gadolinium was performed prior to obtaining the oxygenation data. Time/signal intensity curves were generated to obtain two standard parameters: maximum enhancement over baseline (SI−I) and the rate of enhancement (SI−I/s). Results: Using the 39 measurements, there was a significant correlation between SI−I and both median pO 2 ( r=0.59; P<0.001) and HP5 ( r=−0.49; P=0.002). There was a weak, borderline significant correlation between SI−I/s and both median pO 2 ( r=0.29; P=0.071) and HP5 ( r=−0.34; P=0.037). There was a significant relationship between tumour size and SI−I ( r=0.54; P<0.001), but not SI−I/s. In 29 tumours, where data were available, there was no relationship between histological assessment of tumour angiogenesis (intra-tumour microvessel density; IMD) and either MRI parameter. Conclusions: Tumour oxygenation levels measured using a pO 2 histograph correlate with dynamic contrast-enhanced MRI parameters. Therefore, non-invasive dynamic MRI may be a method for measuring hypoxia in human tumours.

Diffusion limited hypoxia estimated by vascular image analysis: comparison with pimonidazole staining in human tumors

Purpose: To assess diffusion limited hypoxia in human tumors using image analysis of vasculature and to compare it with the bioreductive marker pimonidazole as an independent method. Materials and methods: To set up the method, nine rectal adenocarcinomas and ten squamous cell carcinomas were analyzed. To validate the method, ten squamous cell carcinomas of the cervix were analyzed from patients who were injected with pimonidazole and biopsied approximately 24 h later. Sections of the rectal and esophageal tumors were stained for vasculature, while cervix tumor sections were double stained for vasculature and pimonidazole. Tumor areas were delineated on digitized images, and the proportion of tumor tissue greater than a fixed distance from the nearest blood vessel (called diffusion limited fraction, DLF) was then calculated. The proportion of tumor area stained for pimonidazole was also measured. Results: There was a wide variation between tumors in both the vascular-derived DLF and in the pimonidazole-stained fraction. Average DLFs varied between 1.5 and 92% for different tumors, with significant differences between them. The area stained by pimonidazole was significantly smaller than DLF for all tumors. The correlation between pimonidazole area and DLF was significant in three of seven tumors containing ≥3 images. When images from all tumors ( n=123) were analyzed together, the correlation was highly significant ( r=0.47, P<0.0001). Conclusion: The vascular derived DLF correlates significantly with pimonidazole staining, but there was large scatter. Both methods may underestimate perfusion limited hypoxia.

2020 Is analysis of vasculature alone sufficient to estimate human tumor hypoxia?

2020 Is analysis of vasculature alone sufficient to estimate human tumor hypoxia?

Preclinical assessment of hypoxic marker specificity and sensitivity

Purpose: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. Materials and Methods: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of ∼35%. As well, nuclear medicine images were acquired from R3327-AT (HF ≃15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. Results: The HSF for FC-103 and other iodinated markers were higher (5–40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3–4 × higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC® rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. Conclusions: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC®, a perfusion agent with no known hypoxic binding activity.

Proliferation and hypoxia in human squamous cell carcinoma of the cervix: First report of combined immunohistochemical assays

Preliminary data establishes pimonidazole as a useful probe for human tumor hypoxia. Although different in chemical structure and properties, pimonidazole is indistinguishable from other markers in its binding to hypoxic cells in human tumors. Pimonidazole hydrochloride is convenient to administer and is well tolerated when given as single intravenous infusion of 0.5 g/m 2. Comparison of immunostaining for pimonidazole binding and for PCNA or MIB-1 shows an inverse relationship between proliferation and hypoxia.

Quantifying regional hypoxia in human tumors with positron emission tomography of [ 18F]fluoromisonidazole: A pretherapy study of 37 patients

To assess pretreatment hypoxia in a variety of tumors using positron emission tomography (PET) after injection of the hypoxia-binding radiopharmaceutical [18F]fluoromisonidazole ([18F]FMISO). Tumor fractional hypoxic volume (FHV) was determined in 21 nonsmall cell lung cancer patients, 7 head and neck cancer patients, 4 prostate cancer patients, and 5 patients with other malignancies by quantitative PET imaging after injection of [18F]FMISO (0.1 mCi/kg). The FHV was defined as the proportion of pixels in the imaged tumor volume with a tissue:blood [18F] activity ratio > or = 1.4 at 120-160 min postinjection. A FHV > 0 was taken as evidence for tumor hypoxia. Hypoxia was observed in 36 of 37 tumors studied with FMISO PET imaging; FHVs ranged from 0 to 94.7%. In nonsmall cell lung cancers (n = 21), the median FHV was 47.6% and the range, 1.3 to 94.7%. There was no correlation between tumor size and FHV. In the seven head and neck carcinomas, the median FHV was 8.8%, with a range from 0.2 to 18.9%. In the group of four prostate cancers, the median and range were 18.2% and 0 to 93.9%, while in a group of five tumors of different types the median FHV was 55.2% (range: 21.4 to 85.8%). Hypoxia was present in 97% of the tumors studied and the extent of hypoxia varied markedly between tumors in the same site or of the same histology. Hypoxia also was distributed heterogeneously between regions within a single tumor. These results are consistent with O2 electrode measures with other types of human tumors. The intra- and intertumor variability indicate the importance of making oxygenation measures in individual tumors and the necessity to sample as much of the tumor volume as possible.

Modification of hypoxia-induced radioresistance in tumors by the use of oxygen and sensitizers

It is now well established that most animal solid tumors contain oxygen-deficient hypoxic cells and that these cells will influence the response of those tumors to radiation. Identifying hypoxic cells in human tumors has proven more difficult, primarily because most of the direct procedures used in animals are not applicable to humans. However, substantial indirect evidence, which goes back to as early as 1909, clearly indicates the presence of hypoxia in human tumors, although with a considerable heterogeneity among individual tumors. Experimental studies during the last 30 years have shown that this source of radiation resistance can be effectively eliminated by a variety of procedures that include high oxygen-content gas breathing, nitrometric radiation sensitizers, blood transfusions, hemoglobin-oxygen affinity modifiers, and nicotinamide. A number of these procedures have also been tested clinically such that by 1995 over 10,000 patients in 83 randomized trials had undergone treatment designed to modify tumor hypoxia before radiation therapy. Although a number of these trials showed no benefit, an overview analysis showed that modification of tumor hypoxia significantly improved the locoregional tumor control after radiotherapy with an odds ratio of 1.21 (95% confidence interval 1.12-1.30). The treatment benefit could mostly be related to an improved response in head and neck with odds ratio 1.31 (1.19-1.43) and to a lesser extent in bladder tumors; no significant effect was observed in other tumor sites (cervix, lung and esophagus). Similar to the local control benefit, the overall survival rate improved with an overall odds ratio of 1.13 (1.05-1.21). The overall results thus showed that the biological issue related to hypoxia appears to be a sound rationale, which may impact the outcome of radiotherapy, especially with head and neck carcinoma. However, despite this wealth of positive data, "hypoxic modification" still has no impact on general clinical practice.

A modeling approach for quantifying tumor hypoxia with [F-18]fluoromisonidazole PET time-activity data.

[F-18]fluoromisonidazole (FMISO), a positron-emitting nitroimidazole, binds preferentially to hypoxic cells. It has been used to image hypoxia in human tumors with positron emission tomography (PET). In order to quantify tumor oxygenation status from these PET data, a kinetic model of FMISO cellular bioreduction has been developed to relate cellular oxygen concentration to the cellular FMISO reaction rate constant, kappa A. Also, a compartmental model of FMISO transport and metabolism has been developed to compute the volume average kappa A in tissue regions from [F-18]FMISO PET time-activity data. This compartmental model was characterized using Monte Carlo simulations and [F-18]FMISO PET time-activity data. The model performed well in Monte Carlo simulations; performance was enhanced by fixing three of the seven model parameters at physiologically reasonable values. The four parameters optimized were blood flow rate, kappa A for two partial volume/spillover correction factors. The model was able to accurately determine kappa A for a variety of computer-generated time-activity curv including those for hypothetical heterogeneous tissue regions and poorly perfused tissue regions. The model was also able to fit [H-3]FMISO time-activity data from 36B-10 rat tumors as well as [F-18]FMISO PET time-activity data from a human patient with a base of the tongue squamous cell carcinoma. The kappa A values in muscles ROIs were comparable to those in well-oxygenated cell monolayers while kappa A values in tumor ROIs were greater, suggesting the presence of hypoxic cells in the tumor.

Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole

Fluoromisonidazole (FMISO) has been shown to bind selectively to hypoxic cells in vitro and in vivo at radiobiologically significant oxygen levels. When labeled with the positron emitter fluorine-18 (F-18), its uptake in tissue can be detected quantitatively with high precision by positron emission transaxial tomography (PETT). This paper presents the first experiences with PETT imaging of [F-18]FMISO uptake in human malignancies, and describes the development of this technique as a tool for the non-invasive assessment of tumor hypoxia. Eight patients with selected cancers were imaged prior to primary radiotherapy, and 3 returned for follow-up scans, for a total of 11 imaging studies. Six of eight pre-radiotherapy studies revealed retention of [F-18FMISO in tumors that significantly exceeded plasma concentrations by 2 hr after drug injection; all five patients with head and neck primaries had such “positive” scans. An analytic method for the interpretation of [F-18] FMISO PETT images is presented, defining hypoxic elements within a tumor volume as regions with a threshold regional tumor:plasma [F-18]FMISO ratio of ?1.4 by 2 or more hours after injection. Toward the end of a course of fractionated radiotherapy, three repeat studies in patients with initially positive scans showed no tumor accumulation of drug above the threshold ratio of 1.4, suggesting reoxygenation had occurred. Pharmacokinetic and dosimetry data support continued use of [F-18]FMISO as a safe hypoxia probe. Two imaging protocols have been developed for human studies; a long protocol allows for more complete biodistribution and dosimetry information, and a shorter protocol facilitates increased patient accrual by applying a simple, clinically expedient imaging procedure. When correlated with tumor outcome, [F-18]FMISO PETT imaging may be developed as a predictor of tumor response to conventional radiotherapy. The implications of this technique in addressing persistent questions of tumor hypoxia in human oncology is discussed.

Sensitization of Hypoxic Tumour Cells—Clinical Experience

There is substantial evidence for the presence of hypoxia in human tumours. This is documented by histopathological demonstration of vascular insufficiency, direct oxygen measurements in tumours, as well as by physiological imaging and mapping of hypoxic areas. As a consequence, clinical trials have focused on the hypoxia problem for more than 30 years. This includes the use of hyperbaric oxygen, hypoxic cell radiosensitizers, and, more recently, modification of the oxygen-unloading capacity of haemoglobin. Agents directed towards destruction of hypoxic cells have also been applied, such as hyperthermia and bio-reductive drugs. Despite decades of clinical trials, the results are still inconclusive, and although some trials have shown significant benefit, it has become apparent that hypoxia is a complex problem. Hypoxia appears to be especially a problem in certain tumour types (e.g. squamous cell carcinoma), but even within tumours of the same type, site, and stage, hypoxia does not occur to the same extent. Furthermore, there are increasing suggestions that hypoxia may occur in two principally different ways, namely acutely and chronically, yielding varying responses to modifying agents. Although improvement in hypoxic cell radiosensitizers and other agents is under way, a definitive solution to the hypoxia problem will not be found until the tumours in which hypoxia occurs can be identified. This will require detailed analysis of individual tumours and patients' parameters, and better knowledge of the mechanisms of reoxygenation in clonogenic tumour cells.

RSU 1069, a nitroimidazole containing an aziridine group: Bioreduction greatly increases cytotoxicity under hypoxic conditions

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation.