Angiogenesis imaging in patients with breast carcinoma using PET based Ga-68 NODAGA RGD₂: in-house preparation

Breast cancer accounts for one third of all cancers. Formation of new blood vessels is regulated by integrin α_vβ₃, which are over expressed on various tumours like breast, lung and prostate carcinomas, melanomas and osteosarcomas. α_vβ₃ integrins have sites for the binding of RGD (Arginine-Glycine-Aspartic acid) tripeptide. This study aims to image angiogenesis in breast carcinoma patients using Ga-68 NODAGA-RGD₂ and its correlation with micro vessel density (MVD) data obtained from immunohistochemistry. Radiolabelling for NODAGA-RGD₂ was done in-house. Optimization of labelling was done at various temperatures, reaction time and pH with freshly eluted Ga-68. The characterization of purified Ga-68 NODAGA-RGD₂ was done by HPLC and MALDI-TOF mass spectrometry. Quality control parameters include Radio-ITLC, Gas chromatography, sterility and pyrogen testing. After obtaining ethical clearance from institutional ethics committee, locally advanced breast carcinoma patients (n=30, all female; mean age 51 years; range 29-75 years) were enrolled in this study and informed written consent was taken from all patients. Whole body acquisition was done on a dedicated PET-CT scanner with 90 sec per bed position, 45 min post intravenous administration of 111MBq-185MBq of Ga-68 NODAGA-RGD₂. Anti-CD31 antibody was used for immunohistochemistry. CD31 stained vessels were counted at 40X magnification. Results: Preparation yield at RT was >94% with 40 μg peptide. However >95% radiolabelling yield was achieved by heating 20 μg peptide at 95ºC for 5 and 10 min. The optimum pH for the reaction was 4.0. The RCP of pre-purified and purified Ga-68 NODAGA-RGD₂ was >90 and >99% as determined by ITLC. The residual ethanol content was below the permissible limit (4000 ppm) and was raging from 1946-2495 ppm in all samples. The endotoxins content was ranged between 1.2-3.7 EU/ml, indicating apyrogenic nature of prepared Ga-68 NODAGA-RGD₂. Physiological uptake of radiotracer was seen in liver, spleen, ventricles, thyroid and salivary glands with excretion through the kidneys. Variable radiotracer uptake was noticed in breast lesion in patients with the SUVmax ranging from 2.89-21.12. Metastatic lesions were also detected. Tumour uptake was well correlated to micro vessel density indicating the application of Ga-68 NODAGA-RGD₂ in imaging angiogenesis. Prepared Ga-68 NODAGA-RGD₂ is suitable for injection and showed good tumour to background ratio in carcinoma breast patients which is well correlated with MVD indicating the application of this radiotracer as a potential agent for imaging angiogenesis. (author)