Synopsis

Quantification of tumour R₂* and oxygen-induced ΔR₂* and ΔR₁ are being investigated as potential biomarkers of tumour hypoxia, but their relationship is complex and not well understood. Here, we used a validated R₁ biomarker (oxygen refractory fraction, termed “Oxy-R”) to segment tumours into hypoxic and non-hypoxic sub-regions. This revealed a clear relationship between hypoxic status and native R₂* and hyperoxia-induced ΔR₂*. Preclinical findings were replicated in clinical data from patients with renal carcinoma. These data highlight the importance of heterogeneity-based analysis of tumours and provide further validation of Oxy-R as a biomarker of tumour hypoxia.

Purpose

Several MRI strategies are being investigated for delivering readily translational biomarkers of hypoxia¹. Intrinsic susceptibility imaging (also termed blood oxygenation level dependent (BOLD) imaging) focuses on quantifying native effective transverse relaxation rate (R₂*), or change in R₂* (ΔR₂*) induced by respiratory challenge with hyperoxic gas. Here, paramagnetic deoxyhemoglobin molecules in erythrocytes create magnetic susceptibility perturbations around blood vessels, increasing local native R₂*. Tumour R₂* decreases following inhalation of a hyperoxic gas, due to increased saturation of haemoglobin molecules with oxygen².

In distinction, tumour R₁ increases due to the presence of paramagnetic oxygen molecules (O₂) dissolved in blood plasma or other tissue fluid. R₁ change (ΔR₁) is reported in well-oxygenated tissues following challenge with hyperoxic gas³. However in hypoxic tissue, inhaled O₂ molecules bind preferentially to deoxygenated haemoglobin molecules and do not increase R₁. Recent work suggests that perfused voxels refractory to oxygen challenge (defined as no increase in R₁ and termed “Oxy-R”) identify and map tumour hypoxia⁴.

To attempt to understand the spatial relationship between native R₂*, ΔR₂* and ΔR₁, we measured these biomarkers in a renal cell carcinoma xenograft model, where Oxy-R has already been established as a hypoxia biomarker⁴. We then sought to demonstrate clinical applicability by applying the same analysis to data from patients with renal cancer.

Methods

Preclinical study

Experiments were performed in compliance with licenses issued under the UK Animals (Scientific Procedures) Act 1986. Tumour cells were established from a sunitinib refractory subcutaneous 786-0 RCC xenograft (786-0-R). Tumours were propagated by injecting 3x10⁶ cells in 100µl of sterile PBS into the flanks of 8 week old female C.B17-scid mice. Tumours were typically used for experimentation at a volume of approximately 400mm³. Anaesthesia was induced with a 10 ml/kg intraperitoneal injection of fentanyl citrate plus fluanisone, midazolam and sterile water. Gas delivery (medical air or 100% oxygen) was continuous at 2 l/min through a nosepiece. Data were acquired on a 7T Bruker magnet.

Clinical study

Research ethics and local R&D review were obtained. All patients gave fully informed written consent. Patients with surgically resectable renal cell carcinoma (stage T1-3, N0, M0) were recruited. Patients were scanned supine with a body transit and receive coil. Gas delivery (medical air or 100% oxygen) was at 15 l/min through a non-rebreathing mask. Data were acquired on a 1.5T Philips Achieva magnet.

Data analysis

In both preclinical and clinical data, paired R₂* measurement (breathing air, then 100% oxygen) was performed using multiple gradient echo acquisitions, either side of a dynamic inversion recovery R₁-weighted oxygen-enhanced MRI (OE-MRI) acquisition. Gas switch was performed mid OE-MRI. ΔR₁ and ΔR₂* were calculated, where ∆R_x = R_x (O₂) – R_x (air). Finally, DCE-MRI was used to define vascular enhancement. For perfused tumour the OE-MRI voxels were then classified as “Oxy-R” or as enhancing to oxygen challenge (termed “Oxy-E”).

Results

Preclinical data from nine 786-0-R tumours showed significant but weak relationships between ΔR₁ and ΔR₂* (both p<0.001; Figure 1; Table 1). When voxels were categorized by OE-MRI and DCE-MRI data – to define Oxy-R which identifies hypoxic tissue in 786-0-R tumours 4 – faster native R2* and greater negative ΔR₂* were seen in the hypoxic sub-regions (both p<0.001). In all, 5815 voxels were included and analyzed, of which 9.8% were non-perfused, 79.8% had a normoxic profile and 10.4% had a hypoxic profile.

We then examined if equivalent findings were observed in clinical data. First, we evaluated the ΔR₁ in the renal cortex as a positive control, since positive ΔR₁ has been reported consistently in multiple studies of renal OE-MRI³. One patient failed to inhale the gas sufficiently to generate signal change in the renal cortex (only 3.0% of voxels were oxygen enhancing). This patient failed quality assurance. All other patients had significant positive renal cortex ΔR₁ with 83.7 to 100% (average 95.4%) of voxels being Oxy-E (Figure 2).

Tumours from the six patients who passed quality assurance were analysed. Equivalent relationships were found between imaging biomarkers. Hypoxic tumour (identified as Oxy-R) had faster native R₂* and greater negative ΔR₂* (both p<0.001). Pathology H-score from Glut-1 analysis stratified patients into low and high hypoxic fractions (p=0.003) (Figure 3).

Conclusions

Acknowledgements

References

1. Tatum, J.L., Kelloff, G.J., Gillies, R.J., Arbeit, J.M., Brown, J.M., Chao, K.S. et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol 82, 699-757 (2006).

2. Howe, F.A., Robinson, S.P., Rodrigues, L.M. & Griffiths, J.R. Flow and oxygenation dependent (FLOOD) contrast MR imaging to monitor the response of rat tumors to carbogen breathing. Magn Reson Imaging 17, 1307-18 (1999).

3. O'Connor, J.P.B., Jackson, A., Buonaccorsi, G.A., Buckley, D.L., Roberts, C., Watson, Y. et al. Organ-specific effects of oxygen and carbogen gas inhalation on tissue longitudinal relaxation times. Magn Reson Med 58, 490-6 (2007).

4. O'Connor, J.P., Boult, J.K., Jamin, Y., Babur, M., Finegan, K.G., Williams, K.J. et al. Oxygen-Enhanced MRI Accurately Identifies, Quantifies, and Maps Tumor Hypoxia in Preclinical Cancer Models. Cancer Res 76, 787-95 (2016).