A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT
© Chan et al; licensee Springer. 2011
Received: 6 July 2011
Accepted: 17 August 2011
Published: 17 August 2011
Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT.
Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies.
111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (K d = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab.
We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab.
The human epidermal growth factor receptor-2 (HER2) is overexpressed in 20% to 25% of breast cancers (BC) and is the target for treatment with trastuzumab (Herceptin), a humanized IgG1 monoclonal antibody (mAb) [1, 2]. HER2 amplification is normally assessed ex vivo in a primary tumor biopsy by immunohistochemical (IHC) staining for HER2 protein or by fluoresecence in situ hybridization to detect increased HER2 gene copy number . However, discordance in HER2 expression between primary and metastatic BC has been found in 20% to 30% of cases [4, 5] and thus, it would be useful to have an imaging technique to assess HER2 phenotype in situ in BC lesions. Several investigators have shown that HER2 expression can be imaged in human BC xenografts in athymic mice by single photon emission computed tomography (SPECT) using trastuzumab or its Fab fragments labeled with 111In or 99 mTc [6–9]. These studies have been extended to imaging HER2-positive BC in patients using 111In-labeled trastuzumab IgG [2, 10]. More recently, positron-emission tomography (PET) using trastuzumab labeled with 89Zr has shown promise for imaging HER2 expression in tumor xenograft mouse models and in patients with metastatic BC [11, 12]. Imaging also offers an opportunity to detect response to HER2-targeted therapies in BC . We previously reported that SPECT with 111In-labeled pertuzumab (anti-HER2) detected early response to treatment with trastuzumab (Herceptin) in athymic mice bearing s.c. MDA-MB-361 BC xenografts . Smith-Jones et al. demonstrated that PET with 68Ga-labeled trastuzumab F(ab')2 fragments identified response of HER2-positive BT-474 human BC tumors in mice to treatment with heat shock protein (Hsp90) inhibitors .
PET offers several potential advantages compared to SPECT for imaging tumors because it has higher intrinsic sensitivity, is more easily quantified, and in some instances offers higher spatial resolution. Despite these apparent benefits, few studies have reported a comparison of PET and SPECT for imaging HER2-positive tumors using the same agent labeled with a single photon-emitter or positron-emitter. Dijkers et al. compared 89Zr- and 111In-labeled trastuzumab in mice bearing s.c. SK-OV-3 human ovarian cancer xenografts and reported no significant differences in tumor and normal tissue uptake . MicroPET with 89Zr-labeled trastuzumab visualized these tumors, but the corresponding microSPECT images with 111In-labeled trastuzumab were not presented.
In this study, we compared microSPECT/CT and microPET/CT for imaging s.c. human tumor xenografts expressing a wide range of HER2 density in athymic mice using trastuzumab Fab fragments modified with 1,4,7,10-tetraazacyclododecane N, N', N″, N'″-tetraacetic acid (DOTA) for complexing 111In or 64Cu. 64Cu decays with a half-life of 12.7 h by positron emission [Eβ+ = 0.65 MeV (17.4%)], β- emission [Eβ = 0.58 MeV (39%)] and electron capture (43.6%). 111In decays by electron capture with a half-life of 2.8 days emitting Auger electrons and two γ-photons [Eγ = 171 keV (90%) and 245 keV (94%)]. DOTA was selected as a chelator because both 111In and 64Cu form thermodynamically stable complexes with DOTA (Kd = 1024 and 1023 M-1, respectively) [16, 17]. 64Cu complexed to DOTA and linked to mAbs and peptides has been widely studied for PET imaging of tumors [15, 18–23]. Fab fragments were selected for these studies because their pharmacokinetics of tumor uptake and elimination from the blood and normal tissues is compatible with the half-lives of 64Cu and 111In .
Materials and methods
Preparation of Fab fragments
Trastuzumab (Herceptin) and rituximab (anti-CD20; Rituxan) are humanized IgG1 mAbs and were obtained from Roche Pharmaceuticals Ltd. (Mississauga, ON, Canada). Fab fragments were prepared by digestion with immobilized papain (Pierce Chemical Co., Rockford, IL, USA) and purified as reported [7, 25]. Fab purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% Tris HCl gradient mini-gel (BioRad, Mississauga, ON, Canada) and by size-exclusion high performance liquid chromatography (HPLC). For SDS-PAGE, Fab (10 μg) were electrophoresed under non-reducing and reducing [dithiothreitol (DTT)] conditions. The gel was stained with Coomassie R-250 brilliant blue (Bio-Rad). Size-exclusion HPLC was performed on a BioSep SEC-2000 column (Phenomenex, Torrance, CA, USA) eluted with 100 mM NaH2PO4 buffer (pH 7.0) at a flow rate of 0.8 mL/min in line with a diode array detector (PerkinElmer, Wellesley, MA, USA) monitoring at 280 nm. Fab fragments were concentrated and buffer-exchanged into 50 mM NaHCO3 buffer (pH 7.5) on an Amicon Ultracel 30 K device (M r cut-off = 30 kDa; Millipore Corp., Billerica, MA, USA). Trace metals were removed from all buffers using Chelex-100 cation-exchange resin (BioRad). The final Fab fragments concentration was measured spectrophotometrically [E 280 nm = 1.45 (mg/mL)-1 cm-1]  and was adjusted to 5 mg/mL with 50 mM NaHCO3 buffer, pH 7.5.
DOTA conjugation and radiolabeling of Fab fragments
Trastuzumab or rituximab Fab fragments were modified with DOTA for complexing 111In or 64Cu by reaction of 1.5 mg of Fab in 300 μL of NaHCO3 buffer (pH 7.5) with a 60- or 90-fold excess, respectively, of the N-hydroxysuccinimidyl ester of 1,4,7,10-tetraazacyclododecane tetraacetic acid (NHS-DOTA; Macrocyclics, Dallas, TX, USA). The conjugation reaction was performed at 4°C for 18 h. DOTA-conjugated Fab were purified from excess DOTA by transferring to an Amicon Ultracel 30 K device, diluting to 12.0 mL with 1 M CH3COONH4 buffer, pH 6.0 and centrifuging at 4,000 × g for 15 min. This purification step was repeated six times. Finally, purified DOTA-Fab were recovered and the concentration determined spectrophotometrically [E 280 nm = 1.45 (mg/mL)-1 cm-1]. The final concentration was adjusted to 5 mg/mL with 1 M CH3COONH4 buffer, pH 6.0.
Radiolabeling was performed by incubating 50 μg of DOTA-Fab in 10 μL of CH3COONH4 buffer, pH 6.0 with 360 MBq of 111InCl3 (> 7 GBq/mL; MDS-Nordion, Kanata, ON, Canada) or 216 MBq of 64CuCl2 (> 4 GBq/mL; MDS-Nordion) for 3 h at 46°C. 111In- or 64Cu-labeled DOTA-Fab were purified on an Amicon Ultracel 30 K device. The final radiochemical purity was measured by instant thin layer-silica gel chromatography (ITLC-SG; Pall Life Sciences, Ann Arbor, MI, USA) developed in 100 mM sodium citrate, pH 5.0 or by size-exclusion HPLC using a flow-through radioactivity detector (FSA; PerkinElmer). The R f values for 111In- or 64Cu-DOTA-Fab on ITLC were 0.0 and those for 111In- or 64Cu-DOTA or free radionuclides were 1.0. The DOTA substitution level of the Fab fragments (chelators/molecule) was measured by labeling a 10 μL aliquot of the unpurified conjugation reaction with 111In, then determining the proportion of 111In-DOTA-Fab vs. free 111In-DOTA by ITLC-SG and multiplying this fraction by the molar ratio used in the reaction .
HER2 binding affinity of 111In- and 64Cu-DOTA-trastuzumab Fab
The HER2 binding affinity of 111In- and 64Cu-DOTA-trastuzumab Fab was determined by direct (saturation) radioligand binding assays using SKBR-3 human BC cells (1.3 × 106 HER2/cell) . Briefly, increasing concentrations (0 to 600 nmol/L) of 111In- or 64Cu-DOTA-trastuzumab Fab were incubated with 1 × 105 cells in 24-well plates at 4°C for 3 h. Unbound radioactivity was removed and the dishes were rinsed two times with phosphate-buffered saline. The cells were dissolved in 100 mM NaOH, recovered, and the total cell-bound radioactivity (TB) was measured in a γ-counter (PerkinElmer Wizard 3). The assay was repeated in the presence of 16 μmol/L of unlabeled trastuzumab IgG to measure non-specific binding (NSB). Specific binding (SB; nanomoles per liter) was calculated by subtracting NSB from TB and was plotted vs. the concentration of 111In- or 64Cu-DOTA-trastuzumab Fab (nanomoles per liter) added. The resulting curve was fitted by non-linear regression to a one-site receptor-binding model by Prism Ver. 4.0 software (GraphPad, San Diego, CA, USA). The dissociation constant (K d) and maximum number of receptors per cell (B max) were calculated and compared for 111In- and 64Cu-DOTA-trastuzumab Fab.
Tumor and normal tissue distribution studies
The tumor and normal tissue distribution of 111In- or 64Cu-DOTA-trastuzumab Fab were determined at 24-h post-intravenous (tail vein) injection (p.i.) in athymic mice with s.c. human tumor xenografts with a wide range of HER2 density. This time point was selected due to the short physical half-life of 64Cu (12.7 h) and because high tumor uptake [> 5 percent injected dose per gram (% i.d./g)] and tumor/blood (T/B) ratios (> 4:1) were previously found for 111In-DTPA-trastuzumab Fab at 24 h p.i. . Tumors were established in female athymic (CD1-nude) mice by s.c. inoculation of 1 × 107 MDA-MB-231, BT-20, or MDA-MB-361 BC cells expressing 5.4 × 104, 1.6 × 105, or 5.1 × 105 HER2/cell, respectively, or with SK-OV-3 ovarian cancer cells displaying 1.2 × 106 HER2/cell . At 4 to 7 weeks post-inoculation, when tumors were well established (5 to 15 mm in diameter), groups of mice (n = 4) were injected i.v. (tail vein) with 12 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 18 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab. To determine if tumor uptake was specific, control groups of mice (n = 4) with MDA-MB-231, BT-20, or MDA-MB-361 xenografts were injected i.v. with 12 MBq (10 μg) of irrelevant 111In-DOTA-rituximab Fab or 18 MBq (10 μg) of 64Cu-DOTA-rituximab Fab. Mice were euthanized by cervical dislocation under general anaesthesia. Tumor and normal tissue uptake of radioactivity was measured in a γ-scintillation counter (Wizard 3, PerkinElmer, Waltham, MA) was expressed as percent injected dose per gram and as tumor/normal tissue (T/NT) ratios. The relationship between tumor/blood (T/B) ratios and HER2 density was examined. The uptake of 111In- or 64Cu-DOTA-trastuzumab Fab fragments in small (5 to 10 mm diameter) vs. larger (10 to 15 mm diameter) tumor xenografts was compared.
MicroSPECT and microPET imaging
MicroSPECT was performed at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 111In-DOTA-rituximab Fab in athymic mice with s.c. HER2-positive tumor xenografts. Anaesthesia was induced and maintained by inhalation of 2% isoflurane in O2. MicroSPECT was performed on a NanoSPECT/CT tomograph (Bioscan, Washington, DC, USA) equipped with four NaI scintillation detectors fitted with 1.4-mm multi-pinhole collimators [full-width half-maximum (FWHM) = 1.2 mm]. A total of 24 projections were acquired in a 256 × 256 matrix with a minimum of 80,000 counts per projection. MicroSPECT image acquisition time was 85 to 120 mins. MicroSPECT images were reconstructed using an ordered-subset expectation maximization (OSEM) algorithm (nine iterations). Prior to microSPECT imaging, cone-beam CT images were acquired (180 projections, 1 s/projection, 45 kVp) on the NanoSPECT/CT system. Co-registration of microSPECT and CT images was performed using InvivoScope software (Bioscan).
MicroPET was performed at 24 h p.i. of 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or 64Cu-DOTA-rituximab Fab on a Focus 220 microPET tomograph (Siemens Preclinical Solutions, Knoxville, TN, USA). Images were acquired for 20 mins and reconstructed using OSEM, followed by a maximum a posteriori probability reconstruction algorithm with no correction for attenuation or partial-volume effects. The FWHM resolution of the microPET tomograph was 1.6 mm. Immediately after imaging, CT was performed on an eXplore Locus Ultra Preclinical CT scanner (GE Healthcare, Mississauga, ON, Canada) with routine acquisition parameters (80 kVp, 70 mA, and voxel size of 150 × 150 × 150 mm). MicroPET and CT images were coregistered using Inveon Research Workplace software (Siemens). All animal studies were conducted under a protocol (no. 989.9) approved by the Animal Use Committee at the University Health Network following Canadian Council on Animal Care guidelines.
Statistical significance of comparisons were assessed by Student's t test (P < 0.05).
Preparation of 111In and 64Cu-labeled DOTA-Fab fragments
HER2 binding affinity of 111In- and 64Cu-DOTA-trastuzumab Fab
Tumor and normal tissue distribution studies
Tumor and normal tissue distribution at 24 h post-injection of 111In- or 64Cu-DOTA-trastuzumab Fab
Percent injected dose/g a, b, c
0.42 ± 0.08
1.40 ± 0.16
0.92 ± 0.06
2.42 ± 0.39
0.80 ± 0.14
4.86 ± 0.57
3.13 ± 0.16
8.52 ± 0.81
62.85 ± 6.45
57.00 ± 7.09
0.58 ± 0.05
3.41 ± 0.22
0.61 ± 0.06
4.76 ± 0.41
2.25 ± 0.13
5.00 ± 0.27
0.78 ± 0.31
0.75 ± 0.06
4.00 ± 0.91
5.01 ± 1.20
Tumor/normal tissue (T/NT) ratios at 24 h post-injection of 111In- or 64Cu-DOTA-trastuzumab Fab
T/NT Ratio a, b, c
111 In-DOTA-trastuzumab Fab
64 Cu-DOTA-trastuzumab Fab
9.73 ± 2.46
3.56 ± 0.62
4.31 ± 0.83
2.09 ± 0.56
5.06 ± 1.12
1.03 ± 0.19
1.27 ± 0.26
0.59 ± 0.16
0.06 ± 0.02
0.09 ± 0.02
6.99 ± 2.05
1.48 ± 0.37
6.61 ± 1.78
1.05 ± 0.22
1.79 ± 0.48
1.00 ± 0.20
6.12 ± 3.33
6.78 ± 1.90
MicroSPECT/CT and microPET/CT imaging
Our results revealed that both microSPECT/CT and microPET/CT with 111In- or 64Cu-DOTA-trastuzumab Fab fragments were able to image s.c. human tumor xenografts in mice with low, intermediate, or high HER2 expression. The range of HER2 expression examined (5.4 × 104 to 1.2 × 106 receptors/cell) corresponded to HER2 scores of 0 to 3+ assessed clinically in BC specimens by IHC staining . There was no apparent increased ability of microPET/CT with 64Cu-DOTA-trastuzumab Fab compared to microSPECT/CT with 111In-DOTA-trastuzumab Fab to visualize MDA-MB-231 tumors with low HER2 density (1.6 × 105 receptors/cell; Figures 4a and 5a). In addition, there was no increased ability of microPET/CT using 64Cu-DOTA-trastuzumab Fab to image small (5 to 10 mm diameter) or larger (10 to 15 mm diameter) MDA-MB-361 tumors with intermediate HER2 expression (5.1 × 105 HER2/cell; Figure 6). The intensity of the tumor signal was dependent on HER2 expression with tumors with intermediate (MDA-MB-361) or high (SK-OV-3) HER2 density most readily imaged by microSPECT/CT (Figure 4) or microPET/CT (Figure 5). However, a threefold higher dose of radioactivity was administered for microSPECT/CT than microPET/CT (70 vs. 22 MBq) and image acquisition times were up to sixfold longer for microSPECT/CT (85 to 20 vs. 20 min, respectively). Thus, the photon detection efficiency (i.e., intrinsic sensitivity) was much higher for microPET/CT than microSPECT/CT. Nonetheless, our results revealed that provided that the administered dose of radioactivity was sufficient and image acquisition times were long enough to yield good counting statistics, microSPECT/CT with 111In-DOTA-trastuzumab Fab was able to image tumors with the similar HER2 density and size as microPET/CT with 64Cu-DOTA-trastuzumab. These results agree with those reported by Cheng et al. who noted that s.c. HER2-positive SUM190 tumor xenografts were imaged by either microSPECT or microPET using trastuzumab conjugated to biotinylated 99 mTc- or 18F-labeled phosphodiamidate morpholinos (MORFs) through a streptavidin linker . The doses of 99 mTc or 18F used in their study were 13 and 0.22 MBq, respectively. Phantom studies revealed that microPET was 15-fold more sensitive in terms of photon detection, but the spatial resolution of microSPECT was superior to that of microPET (1.2 vs. 2.4 mm). The results are also in concordance with those reported by Wong et al. , who showed that s.c. epidermal growth factor receptor-positive LS174T human colon cancer xenografts could be imaged using panitumomab F(ab')2 fragments labeled with 111In or 86Y. However, they compared low resolution planar γ-camera imaging with microPET. In our study, we used similar quality high resolution and high sensitivity small animal imaging technologies, namely microSPECT/CT (NanoSPECT; Bioscan) and microPET (Siemens Focus 220) systems for these comparisons.
T/B ratios were used to compare the tumor localization of 111In- and 64Cu-DOTA-trastuzumab Fab vs. HER2 density because we previously found that there are differences in perfusion between different tumor xenografts which can affect the uptake of radioimmunoconjugates . Use of T/B ratios minimizes these effects by normalizing for blood concentrations which then reveals the relationships between HER2 density and tumor accumulation. Moreover, the T/B ratios are important for discriminating tumors that have different HER2 expression on the images. There was a strong and direct association between the T/B ratios for 111In-DOTA-trastuzumab and tumor HER2 density (Figure 3a). In addition, the T/B ratios for 111In-DOTA-trastuzumab Fab were significantly greater than those of irrelevent control 111In-DOTA-rituximab Fab for MDA-MB-231, BT-20, and MDA-MB-361 xenografts, demonstrating specific localization in tumors with low or intermediate HER2 expression. In contrast, specific uptake of 64Cu-DOTA-trastuzumab Fab was shown in MDA-MB-361 tumors with intermediate HER2 density but not for tumors with lower HER2 expression (Figure 3b). Tumor uptake was not significantly different for 64Cu- and 111In-DOTA-trastuzumab Fab, but blood radioactivity was threefold lower for 111In-DOTA-trastuzumab Fab (Table 1). Thus, T/B ratios were threefold lower for 64Cu- than 111In-DOTA-trastuzumab (3.6:1 vs. 10:1; Table 2) in mice with MDA-MB-361 tumors. The increased circulating radioactivity for 64Cu-DOTA-trastuzumab Fab may be due to kinetic instability of the 64Cu-DOTA complex with transchelation of released 64Cu to copper binding proteins (e.g., albumin, ceruloplasmin, or superoxide dismutase) . These 64Cu-labeled proteins may non-specifically localize in tumors, disrupting the association between T/B ratios and HER2 density, especially for tumors with low HER2 expression (i.e., MDA-MB-231 and BT-20).
DOTA forms thermodynamically stable complexes with copper (K d = 1023 M-1) but kinetic instability of 64Cu-DOTA complexes in vivo can lead to loss of radiometal resulting in high blood radioactivity and liver and spleen uptake . In addition to the higher levels of blood radioactivity, we found that the liver and spleen uptake for 64Cu-DOTA-trastuzumab Fab were three- and twofold greater, respectively, than 111In-DOTA-trastuzumab Fab (Table 1). In order to improve the kinetic stability of 64Cu complexes, more thermodynamically stable cross-bridged (CB-DO2A) or sarcophagine (SarAr) chelators have been synthesized [30, 31]. A comparison of 64Cu complexed to DOTA or CB-DO2A (but not conjugated to mAbs) showed fourfold lower radioactivity in the blood and twofold lower liver accumulation at 24 h p.i. in rats . Voss et al. noted that ch14.18 mAbs labeled with 64Cu through the extremely stable SarAr chelator for PET imaging of neuroblastoma or melanoma xenografts in mice exhibited low liver uptake (5% to 10% i.d./g) but no comparison with other chelators was provided . Dearling et al. recently compared the tumor and normal tissue distribution of these same 64Cu-labeled ch14.18 mAbs using a variety of chelators including DOTA and SarAr in mice bearing M21 melanoma xenografts . Unexpectedly, no significant differences in tumor or liver uptake were found for ch14.18 labeled with 64Cu using DOTA or the much more stable SarAr chelator. They suggested that in addition to 64Cu-chelator stability, factors such as the net charge on the chelators may play an important role in sequestration of radioactivity by tissues. In our study, tumor uptake was not significantly different between 111In- and 64Cu-DOTA-trastuzumab Fab in mice with MDA-MB-361 tumors, despite the apparent instability of 64Cu-DOTA-trastuzumab Fab as evidenced by higher levels of radioactivity in the blood, liver, and spleen (Table 1). The use of more stable chelators such as CB-DO2A or SarAr may diminish blood radioactivity and improve the association between tumor HER2 density and T/B ratios for 64Cu-labeled trastuzumab Fab. The CB-DO2A and SarAr chelators are unfortunately not yet commercially available in a chemically reactive form for conjugation to mAbs for 64Cu labeling.
Provided that administered doses of radioactivity and acquisition times were sufficient to yield good counting statistics, we conclude that either microSPECT/CT with 111In-DOTA-trastuzumab Fab or microPET/CT with 64Cu-DOTA-trastuzumab Fab visualized small (5 to 10 mm diameter) or larger (10 to 15 mm diameter) s.c. tumor xenografts with low, intermediate, or high HER2 expression in athymic mice. However, due to the higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab, no association between HER2 density and T/B ratios was established. In contrast, there was a strong direct association between T/B ratios and HER2 density of these tumors for 111In-DOTA-trastuzumab Fab. Thus, 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors with low HER2 density. The use of more stable CB-DO2A or SarAr chelators for 64Cu may potentially diminish blood radioactivity, provide a stronger association between T/B ratios and tumor HER2 density, and improve the specificity of imaging with 64Cu-labeled trastuzumab Fab.
This study was supported by a grant from the Ontario Institute for Cancer Research (1 mm Challenge) with funds from the Province of Ontario. Parts of this study were presented at the European Association of Nuclear Medicine Congress, Barcelona, Spain, October 9 to 13, 2009.
- Revillion F, Bonneterre J, Peyrat JP: ERBB2 oncogene in human breast cancer and its clinical significance. Eur J Cancer 1998, 34: 791–808. 10.1016/S0959-8049(97)10157-5PubMedView ArticleGoogle Scholar
- Behr TM, Béhé M, Wörmann B: Trastuzumab and breast cancer. N Engl J Med 2001, 345: 995–996.PubMedView ArticleGoogle Scholar
- Owens MA, Horten BC, Da Silva MM: HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer 2004, 5: 63–69. 10.3816/CBC.2004.n.011PubMedView ArticleGoogle Scholar
- Munzone E, Nolé F, Goldhirsch A, Botteri E, Esposito A, Zorzino L, Curigliano G, Minchella I, Adamoli L, Cassatella MC, Casadio C, Sandri MT: Changes in HER2 status in circulating tumor cells compared with the primary tumor during treatment for advanced breast cancer. Clin Breast Cancer 2010, 10: 392–397. 10.3816/CBC.2010.n.052PubMedView ArticleGoogle Scholar
- Pestrin M, Bessi S, Gallardi F, Truglia M, Biggeri A, Biagioni C, Cappadona S, Biganzoli L, Giannini A, Di Leo A: Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients. Breast Cancer Res and Treatment 2009, 118: 523–530. 10.1007/s10549-009-0461-7View ArticleGoogle Scholar
- Lub-de Hooge MN, Kosterink JG, Perik PJ, Nijnuis H, Tran L, Bart J, Suurmeijer AJH, de Jong S, Jager PL, de Vries EGE: Preclinical characterisation of 111 In-DTPA-trastuzumab. Br J Pharmacol 2004, 143: 99–106. 10.1038/sj.bjp.0705915PubMed CentralPubMedView ArticleGoogle Scholar
- Tang Y, Wang J, Scollard DA, Mondal H, Holloway C, Kahn HJ, Reilly RM: Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using 111 In-trastuzumab (Herceptin) Fab fragments. Nucl Med Biol 2005, 32: 51–58. 10.1016/j.nucmedbio.2004.08.003PubMedView ArticleGoogle Scholar
- Tang Y, Scollard D, Chen P, Wang J, Holloway C, Reilly RM: Imaging of HER2/neu expression in BT-474 human breast cancer xenografts in athymic mice using 99 m Tc-HYNIC-trastuzumab (Herceptin) Fab fragments. Nucl Med Commun 2005, 26: 427–432. 10.1097/00006231-200505000-00006PubMedView ArticleGoogle Scholar
- McLarty K, Cornelissen B, Scollard DA, Done SJ, Chun K, Reilly RM: Associations between the uptake of 111 In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts. Eur J Nucl Med Mol Imaging 2009, 36: 81–93. 10.1007/s00259-008-0923-xPubMedView ArticleGoogle Scholar
- Perik PJ, Lub-de Hooge MN, Gietema JA, van der Graaf WTA, de Korte MA, Jonkman S, Kosterink JG, van Veldhuisen DJ, Sleijfer DT, Jager PL, de Vries EGE: Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol 2006, 24: 2276–2282. 10.1200/JCO.2005.03.8448PubMedView ArticleGoogle Scholar
- Dijkers EC, Oude Munnink TH, Kosterink JG, Brouwers AH, Jager PL de Jong R, van Dongen GA, Lub-de Hooge MN, de Vries EGE: Biodistribution of 89 Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther 2010, 87: 586–592. 10.1038/clpt.2010.12PubMedView ArticleGoogle Scholar
- Dijkers ECF, Kosterink JGW, Rademaker AP, Perk LR, van Dongen GA, Bart J, de Jong R, de Vries EGE, Lub-de Hooge MN: Development and characterization of clinical-grade 89 Zr-trastuzumab for HER2/ neu immunoPET imaging. J Nucl Med 2009, 50: 974–981. 10.2967/jnumed.108.060392PubMedView ArticleGoogle Scholar
- McLarty K, Reilly RM: Molecular imaging as a tool for personalized and targeted anticancer therapy. Clin Pharmacol Ther 2007, 81: 420–424. 10.1038/sj.clpt.6100096PubMedView ArticleGoogle Scholar
- McLarty K, Cornelissen B, Scollard D, Reilly RM: Micro-SPECT/CT with 111 In-DTPA-pertuzumab sensitively detects trastuzumab-mediated HER2 downregulation and tumor response in athymic mice bearing MDA-MB-361 human breast cancer xenografts. J Nucl Med 2009, 50: 1340–1348. 10.2967/jnumed.109.062224PubMedView ArticleGoogle Scholar
- Smith-Jones PM, Solit DB, Akhurst T, Afroze F, Rosen N, Larson SM: Imaging the pharmacodynamics of HER-2 degradation in response to Hsp90 inhibitors. Nat Biotechnol 2004, 22: 701–706. 10.1038/nbt968PubMedView ArticleGoogle Scholar
- Garcia R, Kubicek V, Drahos B, Gano L, Santos IC, Campello P, Paulo A, Toth E, Santos I: Synthesis, characterization and biological evaluation of In(III) complexes anchored by DOTA-like chelators bearing a quinazoline moiety. Metallomics 2010, 2: 571–580. 10.1039/c004797jPubMedView ArticleGoogle Scholar
- Dearling JLJ, Voss SD, Dunning P, Snay E, Fahey F, Smith SV, Huston JS, Meares CF, Treves ST, Packard AB: Imaging cancer using PET - the effect of the bifunctional chelator on the biodistribution of a 64 Cu-labeled antibody. Nucl Med Biol 2011, 38: 29–38. 10.1016/j.nucmedbio.2010.07.003PubMed CentralPubMedView ArticleGoogle Scholar
- Olafsen T, Kenanova VE, Sundaresan G, Anderson AL, Crow D, Smith SV, Huston JS, Meares CF, Treves ST, Packard AB: Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res 2005, 65: 5907–5916. 10.1158/0008-5472.CAN-04-4472PubMed CentralPubMedView ArticleGoogle Scholar
- Cai W, Chen K, He L, Cao Q, Koong A, Chen X: Quantitative PET of EGFR expression in xenograft-bearing mice using 64 Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur J Nucl Med Mol Imaging 2007, 34: 850–858. 10.1007/s00259-006-0361-6PubMedView ArticleGoogle Scholar
- Niu G, LI Z, Cao Q, Chen X: Monitoring therapeutic response of human ovarian cancer to 17-DMAG by non-invasive PET imaging with 64 Cu-DOTA-trastuzumab. Eur J Nucl Med Mol Imaging 2009, 36: 1510–1519. 10.1007/s00259-009-1158-1PubMed CentralPubMedView ArticleGoogle Scholar
- Anderson CJ, Dehdashti F, Cutler PD, Schwartz SW, LaForest R, Bass LA, Lewis JS, McCarthy DW: 64 Cu-TETA-octreotide as a PET imaging agent for patients with neuroendocrine tumors. J Nucl Med 2001, 42: 213–221.PubMedGoogle Scholar
- Rogers BE, Bigott HM, McCarthy DW, Manna DD, Kim J, Sharp TL, Welch MJ: MicroPET imaging of a gastrin-releasing peptide receptor-positive tumor in a mouse model of human prostate cancer using a 64 Cu-labeled bombesin analogue. Bioconjug Chem 2003, 14: 756–763. 10.1021/bc034018lPubMedView ArticleGoogle Scholar
- Thakur ML, Aruva MR, Gariepy J, Acton P, Rattan S, Prasad S, Wickstrom E, Alavi A: PET imaging of oncogene overexpression using 64 Cu-vasoactve intestinal peptide (VIP) analog: comparison with 99 m Tc-VIP analog. J Nucl Med 2004, 45: 1381–1389.PubMedGoogle Scholar
- Reilly RM, Sandhu J, Alvarez-Diez TM, Gallinger S, Kirsh J, Stern H: Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clin Pharmacokinet 1995, 28: 126–142. 10.2165/00003088-199528020-00004PubMedView ArticleGoogle Scholar
- Scollard DA, Chan C, Holloway CMB, Reilly RM: A kit to prepare 111 In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER-2 positive breast cancer. Nucl Med Biol 2011, 38: 129–136. 10.1016/j.nucmedbio.2010.06.010PubMedView ArticleGoogle Scholar
- Reilly RM: The radiochemistry of monoclonal antibodies and peptides. In Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer. Edited by: Reilly RM. Hoboken, NJ: John Wiley & Sons; 2010:39–100.View ArticleGoogle Scholar
- Orlova A, Tolmachev V, Pehrson R, Lindborg M, Tran T, Sandström M, Nilsson FY, Wennborg A, Abrahmsén L, Feldwisch J: Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer Res 2007, 67: 2178–2186. 10.1158/0008-5472.CAN-06-2887PubMedView ArticleGoogle Scholar
- Cheng D, Wang Y, Liu X, Pretorius PH, Liang M, Rusckowski M, Hnatowich DJ: Comparison of 18 F PET and 99 m Tc SPECT imaging in phantoms and in tumored mice. Bioconjug Chem 2010, 21: 1565–1570. 10.1021/bc1001467PubMed CentralPubMedView ArticleGoogle Scholar
- Wong KJ, Baidoo KE, Nayak TK, Garmestan K, Brechbiel MW, Milenic DE: In vitro and in vivo pre-clinical analysis of a F(ab') 2 fragment of panitumumab for molecular imaging and therapy of HER1-positive cancers. EJNMMI Research 2011, 1: 1–15. 10.1186/2191-219X-1-1PubMed CentralPubMedView ArticleGoogle Scholar
- Boswell CA, Sun X, Niu W, Wesiman GR, Wong EH, Rheingold AL, Anderson CJ: Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes. J Med Chem 2004, 47: 1465–1474. 10.1021/jm030383mPubMedView ArticleGoogle Scholar
- Voss SD, Smith SV, DiBartolo N, McIntosh LJ, Cyr EM, Bonab AA, Dearling JL, Carter EA, Fischman AJ, Treves ST, Gillies SD, Sargeson AM, Huston JS, Packard AB: Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64 Cu-SarAr immunoconjugates. Proc Natl Acad Sci USA 2007, 104: 17489–17493. 10.1073/pnas.0708436104PubMed CentralPubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.