Normalisation to Blood Activity Is Required for the Accurate Quantification of Na/I Symporter Ectopic Expression by SPECT/CT in Individual Subjects

Abstract

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used 99mTc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to ‘noisy’, and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.

Endocrine Scintigraphy with Hybrid SPECT/CT

“…SPECT/CT has been used in the research setting to develop novel applications for study of endocrine disorders (288). For Graves eye disease, 99m Tc-EDDA/HYNIC-Tyr3-octreotide (TOC) SPECT/CT was used to localize radiotracer uptake to the orbits.…”

Adrenal Gland Infection by Serotype 5 Adenovirus Requires Coagulation Factors

“…As such a signal is absent in SPECT/CT images of mice administered intravenously with the same adenovirus [31] or in the contra-lateral kidney (not shown), this set of data suggests that the intra-renal route of administration of adenovirus is more efficient than the intravenous one to transduce this organ. A similar imaging protocol involving intra-renal administration of 5×10 8 PFU of Ad-CMV-rNIS in mice pre-treated with warfarin resulted in a lack of signal in the adrenal glands area (Figure 5B), confirming that adrenal gland infection depends on blood factors.…”

“…Forty-eight hours later, the animals were administered intra-peritoneally with the tracer 99m TcO 4 − . This tracer accumulates selectively in organs expressing the Na/I symporter (NIS) [31], i.e the thyroid, stomach and salivary glands that express NIS endogenously as well as the organs/tissues transduced by the adenovector. The animals were then anaesthetized and positioned into a SPECT/CT scanner.…”

“…Five hours later, the animals were anaesthetized and scanned. In both cases ( 99m Tc pertechnetate and 99m Tc-DMSA) SPECT/CT scans were performed using a micro-SPECT-CT (eXplore speCZT CT120, General Electric), using a previously published protocol [31]. Image analyses and quantification were performed using the ‘AMIDE’ software [34].…”

“…This modest level of transgene expression is illustrated by the fact that gene transfer in the adrenal glands cannot be visualized by sensitive, whole-body imaging techniques such as PET or even SPECT using the Na/I symporter (NIS) as a reporter gene, while transgene expression in the liver can easily be detected and quantified using either of these methodologies [30], [31]. Two strategies could be envisaged to restrict and/or increase transgene expression in the adrenal gland: i) selective transduction of the adrenal glands, but, for this strategy to be efficient, the mechanism of infection of the two organs would have to be different and ii) a more localized type of injection (i.e.…”