Phase I study of noninvasive imaging of adenovirus-mediated gene expression in the human prostate

Abstract

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTK(SR39)rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na(99 m)TcO(4) in infected cells using single photon emission-computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 x 10(12) virus particles (vp) but not at 1 x 10(11) vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 x 10(12) vp in 1 cm(3), GE volume (GEV) increased to a mean of 6.6 cm(3), representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1-2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.

Figure 1. Treatment schema

Patients received an intraprostatic injection of the Ad5-yCD/mutTK_SR39rep-hNIS adenovirus on day 1 at a dose of 1 × 10¹¹ virus particles (vp) (cohort 1) or 1 × 10¹² vp (cohort 2). Two days later (day 3), patients were administered 5-fluorocytosine (5-FC) + valganciclovir (vGCV) prodrug therapy for 3 weeks (weekdays only) concomitant with a standard course (38 × 2 Gy for 76 Gy) of intensity-modulated radiation therapy (IMRT). Patients 1–9 underwent two nuclear imaging sessions. The first (baseline scan) was within 30 days before the adenovirus injection. The second was following the adenovirus injection on day 2 or 4. Patients 10–12 underwent multiple imaging sessions on days 2, 3, 4 and 8. Na^99mTcO₄ was not administered, and scheduled imaging sessions were not performed, if no gene expression was detected in the prostate in the previous scan. Toxicity assessments were taken once a week through day 90. hNIS, human sodium iodide symporter.

Figure 2. Patient 10 treatment planning and imaging results

(a) Three-dimensional reconstruction of the prostate (gray) with approximate location of the cancer (red) based on 12-core biopsy. The bulk of the cancer with Gleason 7 pattern resided in the right midgland and apex regions, and only a few malignant glands of Gleason 6 were noted on the left side. (b) A single, transverse transrectal ultrasound image of the midgland/apex region acquired immediately before adenovirus injection. The bulky tumor on the right side appears as a hypoechoic region (dashed oval). The urethra (U), rectum (R), right and left sides of the patient are indicated. (c,d), Coronal and sagittal single photon emission–computed tomography images, respectively, of the pelvic region 2 days following the adenovirus injection. The color bar on the right indicates the relative activity. The location of the prostate is indicated by the dotted oval. Activity in the urinary bladder (UB) and penis (Pe) is indicated. The activity in the penis is due to blood flow through that organ and is seen in the baseline scans. The dotted yellow line in c indicates the midline. The right, left, anterior and posterior sides of the patient are indicated. The inserts show gene expression in the prostate at higher magnification. RM, right midgland; RA, right apex.

Figure 3. Gene expression in the prostate for patients 4, 6, 8, 9 and 12

Shown are fused computerized tomography/single photon emission–computed tomography images of a single transverse section at the maximum intensity of gene expression. For patient 4, the pretreatment (pre) and post-treatment (post) scans of the same transverse section are shown for comparison. The prostate and rectum are indicated by yellow dashed and red dotted ovals, respectively. Activity in the femoral arteries (FA) and penis (Pe) can be seen in some patients owing to blood flow through those structures. The activity seen at the posterior of all patients is due to leakage of radioactive urine during the imaging session. In patients 4, 6, 8, and 9, activity in the urinary bladder (UB) is not seen because the transverse section shown is well below the bladder. In patient 12, the maximum level of gene expression was found in the left base region and, therefore, the high activity in the UB is seen. The color bar on the right indicates the relative activity.

Figure 4. Kinetics and persistence of gene expression (GE) in the prostate in patient 10

The Ad5-yCD/mutTK_SR39rep-hNIS adenovirus was injected on day 1 at 1 × 10¹² virus particles (vp). a, Fused computerized tomography/single photon emission–computed tomography images of a single transverse section at the maximum intensity of GE 1 (D2), 2 (D3), 3 (D4), and 7 (D8) days after adenovirus injection. In the day 2 panel, the prostate is indicated by a dashed yellow oval and the rectum is labeled R. The color bar on the right indicates the relative activity. (b) The volume (circles) and total amount (squares) of GE on each day of imaging are plotted. Actual values are shown in the table along with the mean pixel intensity. (c) PCR of Ad5-yCD/mutTK_SR39rep-hNIS viral DNA in blood. Pre-Tx unspiked, pretreatment blood with no added adenovirus. Pre-Tx spiked, pretreatment blood spiked with 1 × 10⁴ vp of Ad5-yCD/mutTK_SR39rep-hNIS. The day of each blood draw is indicated above the lanes with day 1 being the day of the adenovirus injection. (+) Control, purified Ad5-yCD/mutTK_SR39rep-hNIS viral DNA. (–) Control, no DNA. The PCR signal reflects the amount of adenoviral DNA present in 20 μl of blood. To calculate the total adenoviral load, use the standard curve to estimate the viral particles and multiply by 250,000 (50 to account for volume of blood used in the PCR assay and 5,000 to account for the average human blood volume). At its peak (day 15), the viral load was ~2.5 × 10⁸ vp, or ~0.025% of the injected adenovirus dose. The number above each lane in the standard curve indicates the amount of adenoviral DNA in vp. The sensitivity of the PCR assay is at least 10 vp. hNIS, human sodium iodide symporter.

Figure 5. Whole-body imaging

Shown are whole-body planar scans before (preinjection) and 3 days after (post-injection) the adenovirus injection for patient 7. Activity in the salivary glands (Sal), lachrymal glands (Lac), thyroid gland, and stomach is due to endogenous human sodium iodide symporter expression. The activity observed in the penis (Pen) and testicles (Tes) is due to blood flow through those structures. Na^99mTcO₄ is secreted through the urinary tract, accounting for the activity in the urinary bladder. The intravenous ^99mTcO₄ injection site can be seen in the post-injection scan.