Orthotopic Pleural Mesothelioma in Mice: SPECT/CT and MR Imaging with HER1- and HER2-targeted Radiolabeled Antibodies

The utility of radiolabeled antibodies for human epidermal growth factor 1 assessment and tumor monitoring with MR imaging was demonstrated in different pleural mesothelioma models in this study.

Abstract

Purpose:

To evaluate the potential of anti–human epidermal growth factor receptor (HER)1– and anti-HER2–targeted radiolabeled antibodies and magnetic resonance (MR) imaging for imaging of orthotopic malignant pleural mesothelioma (MPM) in mouse models.

Materials and Methods:

Animal studies with 165 mice were performed in accordance with National Institutes of Health guidelines for the humane use of animals, and all procedures were approved by the institutional Animal Care and Use Committee. Flow cytometry studies were performed to evaluate HER1 and HER2 expression in NCI-H226 and MSTO-211H mesothelioma cells. Biodistribution and single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in mice (four or five per group, depending on tumor growth) bearing subcutaneous and orthotopic MPM tumors by using HER1- and HER2-targeted indium 111 (¹¹¹In)- and iodine 125 (¹²⁵I)-labeled panitumumab and trastuzumab, respectively. Longitudinal MR imaging over 5 weeks was performed in three mice bearing orthotopic tumors to monitor tumor growth and metastases. SPECT/CT/MR imaging studies were performed at the final time point in the orthotopic models (n = 3). The standard unpaired Student t test was used to compare groups.

Results:

Orthotopic tumors and pleural effusions were clearly visualized at MR imaging 3 weeks after tumor cell inoculation. At 2 days after injection, the mean ¹¹¹In-panitumumab uptake of 29.6% injected dose (ID) per gram ± 2.2 (standard error of the mean) was significantly greater than the ¹¹¹In-trastuzumab uptake of 13.6% ID/g ± 1.0 and the ¹²⁵I-panitumumab uptake of 7.4% ID/g ± 1.2 (P = .0006 and P = .0001, respectively). MR imaging fusion with SPECT/CT provided more accurate information about ¹¹¹In-panitumumab localization in the tumor, as the tumor was poorly visualized at CT alone.

Conclusion:

This study demonstrates the utility of radiolabeled anti-HER1 antibodies in the imaging of MPM in preclinical models.

© RSNA, 2013

Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12121021/-/DC1

Introduction

Malignant pleural mesothelioma (MPM) affects the lung pleura and is a highly lethal cancer, with survival rates ranging from 4 to 17 months from diagnosis (1–3). MPM is primarily caused by the inhalation of asbestos fibers, which then settle in the lungs and become imbedded in the pleura, causing chronic inflammation that eventually leads to malignant transformation (1,3).

Imaging plays an essential role in the diagnosis, staging, and follow-up of disease in patients with MPM. Computed tomography (CT) is used as the primary imaging modality for diagnosis, staging, and monitoring of therapeutic response in MPM (4,5). However, the complex anatomic morphology of a pleural-based tumor combined with nonmalignant features of scarring and fibrosis limit the efficacy of CT (4,5). More recently, magnetic resonance (MR) imaging has been used but suffers similar limitations. MR imaging may be helpful in selected patients with potentially resectable disease to determine the local extent of tumor. Positron emission tomography (PET) with fluorine 18 fluorodeoxyglucose (FDG) has gained popularity for imaging MPM because of its superior specificity for tumors (5,6). These imaging modalities help in triaging patients to the most appropriate treatment options. However, benign pleural plaques or pleural thickening from asbestos exposure may mimic MPM at CT and MR imaging, and high FDG uptake can also be observed in benign inflammatory pleural disease (1,4).

Previous asbestos exposure has been shown to result in increased expression of the cell surface human epidermal growth factor receptor (HER) HER1, which then initiates cell signaling cascades that lead to asbestos-induced carcinogenesis (7–9). Results of recent clinical studies (10–13) have shown that there is an overexpression of HER1 in MPM, which is often linked with poor prognosis and a more aggressive form of the disease. On the basis of these findings, HER1-tyrosine kinase inhibitors such as gefinitib and erlotinib have been investigated for therapeutic efficacy in patients with MPM (14–16). Cetuximab combined with cisplatin or carboplatin and pemetrexed as first-line treatment in patients with MPM is currently being investigated in a multicenter phase II study (clinicaltrials.gov identification number: NCT00996567).

Owing to the overexpression of HER1 in MPM, radioimmunotherapy by using yttrium 86 (⁸⁶Y)/⁹⁰Y-labeled antibodies has been proposed as a possible treatment (17). On the basis of these encouraging results, in this study, we evaluated anti-HER1 and HER2-targeted radiolabeled antibodies and MR imaging for imaging of orthotopic MPM in mice.

Materials and Methods

Cell Lines and Tissue Culture

Human epithelioid mesothelioma cells, NCI-H226 (CRL-5826), and human biphasic mesothelioma cells, MSTO-211H (CRL-2081), were purchased from American Type Culture Collection (Manassas, Va). NCI-H266 and MSTO-211H were selected owing to their different HER1 expression profiles and histologic features. All cell lines were grown as a monolayer at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were cultured in RPMI-1640 media containing 2 mmol/L l-glutamine, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate. All media were additionally supplemented with 10% FetalPlex (Gemini Bio-Products, Woodland, Calif). Media and supplements were obtained from Invitrogen (Carlsbad, Calif) and Lonza (Walkersville, Md).

Flow Cytometric Analysis

HER1 and HER2 expression of the mesothelioma cell lines was evaluated by using standard flow cytometric techniques. Briefly, cells were trypsinized, pelleted at 1500g for 10 minutes, and resuspended in phosphate-buffered saline (PBS) (pH, 7.2) containing 1% bovine serum albumin (BSA). The cells (1 × 10⁶cells in 100 µL of 1% BSA in PBS) were added to 12 × 75-mmol/L polypropylene tubes (Falcon Labware, Franklin Lakes, NJ) along with 1 µg of trastuzumab (Herceptin; F. Hoffmann La Roche, Nutley, NJ) or panitumumab (Vectibix; Amgen, Thousand Oaks, Calif) in 100 µL. The cells were incubated for 1 hour at 4°C and were washed three times by adding 2 mL of 1% BSA in PBS, pelleting the cells at 1000g for 5 minutes, and decanting the supernatant. Following the last wash, 100 µL of fluorescein isothiocyanate–labeled goat antihuman immunoglobulin G (50 µg/mL, Kirkegaard and Perry, Gaithersburg, Md) was added to the cells and incubated for an additional 1 hour at 4°C. The cells were washed three times as before, and mean fluorescence intensity (MFI) was measured and analyzed (10 000 events) by using a FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif) with CellQuest software (BD Biosciences). HuM195, an anti-CD33 monoclonal antibody, kindly provided by Michael McDevitt, PhD, at Memorial Sloan-Kettering Cancer Center (New York, NY), served as a control monoclonal antibody. MFI was used as a parameter to observe differences in HER1 and HER2 between MSTO-211H and NCI-H226 cells.

Preparation of Radioimmunoconjugates

Panitumumab and trastuzumab were conjugated with the bifunctional chelator CHX-A″-DTPA as previously described (18,19). Radiolabeling and quality control of iodine 125 (¹²⁵I)- or indium 111 (¹¹¹In)-panitumumab and ¹²⁵I- or ¹¹¹In-trastuzumab conjugates were performed as previously described (11,19).

Animal and Tumor Models

All animal studies were approved by the institutional Animal Care and Use Committee. One hundred ten 6–8-week-old female athymic nu/nu mice were injected subcutaneously with 2–4 × 10⁶ MSTO-211H or 6–10 × 10⁶ NCI-H226 cells in 200-µL medium containing 20% Matrigel for biodistribution studies and SPECT/CT studies (Fig E1 [online]). Tumor growth was monitored externally biweekly by using Vernier calipers. Tumor volume was calculated with the following formula: (L · W²) · 0.4, where L is length and W is width in millimeters. Mice with tumor volumes of between 200 and 350 mm³ were selected for biodistribution and single photon emission computed tomography (SPECT)/CT imaging studies. Orthotopic tumor xenograft models were also used in this study, as the vasculature and microenvironment in the orthotopic model were more closely related to those of mesothelioma tumors in humans than subcutaneous tumor xenografts in mice. For orthotopic models, 2 × 10⁶ MSTO-211H or 4 × 10⁶ NCI-H226 cells in 50 µL of corresponding medium were directly injected into the pleural cavities of 55 mice by advancing the needle approximately 5 mm through the fourth intercostal space into the right lateral thorax.

Biodistribution and Pharmacokinetic Studies

Tumor-bearing female athymic mice (four or five per group, depending on the tumor growth) were given intravenous injections via the tail vein of 0.4–0.6 MBq (<2 µg) of ¹¹¹In-panitumumab mixed with 0.4 MBq (<5 µg) of ¹²⁵I-trastuzumab or 0.4–0.6 MBq (<2 µg) of ¹¹¹In-trastuzumab mixed with 0.4 MBq (<5 µg) of ¹²⁵I-panitumumab. One, 2, 3, 4, and 7 days after injection, the animals in each of the respective time-point groups were sacrificed by means of CO₂ inhalation. In all the biodistribution studies, the injected amount of each antibody was less than 0.2 mg per kilogram of body weight. Tumor, blood, and selected organs were harvested and wet weighed, and the radioactivity was measured in a gamma counter (Wizard 1480; PerkinElmer, Shelton, Conn). The percentage injected dose (ID) of tissue was calculated by comparison with standards representing 10% of the ID per animal. Percentage ID per gram was obtained by dividing the percentage ID by the weight of the collected tissues. Noncompartmental pharmacokinetic analysis was performed to determine the area under the curve (AUC), expressed as percentage ID ·day · g⁻¹.

SPECT/CT Imaging

Small-animal SPECT/CT studies were performed by using a microSPECT/CT system (NanoSPECT/CT; Bioscan, Washington, DC) at the National Institutes of Health (Bethesda, Md) to determine the tumor uptake and targeting (and delineation from normal tissues).

Whole-body imaging studies (total acquisition time, approximately 1 hour per mouse, 60–100 seconds per projection) were performed in mice anesthetized with 1.5%–1.7% isoflurane on a temperature-controlled platform. CT images were acquired with 180 projections (pitch, 1.0; energy, 45 kVp). Tumor-bearing female athymic mice (n = 3 per group) were intravenously injected with 2.0 MBq (<5 µg) of ¹¹¹In-panitumumab and were scanned. SPECT/CT imaging in subcutaneous tumor xenograft–bearing mice was performed once the tumor reached 200–350 mm³. SPECT/CT and MR imaging in orthotopic tumor xenografts was performed when tumors were clearly visualized at MR imaging. In all the imaging studies, the injected amount of each antibody was less than 0.2 mg/kg. SPECT and CT images were reconstructed, fused, and analyzed with InVivoScope (Bioscan).

MR Imaging

Mice with orthotopic tumors (n = 3 per group) were imaged weekly with a 3.0-T clinical imaging unit (Achieva; Philips Medical System, Best, the Netherlands) to monitor tumor progression after injection. To optimize sensitivity and resolution, a custom-designed 70-mm-long saddle-shaped receiver coil on a 44-mm outer diameter acrylic tube was used. Each mouse was placed on a bed with a built-in nose cone supplied with 1.5%–2.5% isoflurane in oxygen and was inserted into a custom 70-mm-long 44-mm-diameter saddle-shaped receiver coil. The mouse was kept warm by circulating heated perfluorcarbon (FC-770; 3M, St Paul, Minn) around the coil tube. A T2-weighted multisection turbo spin-echo sequence with respiratory triggering to reduce breathing artifacts (pixel resolution, 0.1 × 0.1 × 0.5 mm³; respiration rate, 50–60 breaths per minute; repetition time, four breaths; echo time, 65 msec; turbo factor, 11; number of signals acquired, four) and a three-dimensional T1-weighted fast-field-echo sequence (repetition time, 10.4 msec; echo time, 2.5 msec; flip angle, 20°) of the chest in the coronal view were performed after preliminary survey acquisitions had been performed.

For sequential SPECT/MR imaging, mice were sacrificed by means of isoflurane overdose immediately after the final SPECT/CT study, and, without moving the mouse, the same bed was transferred into the MR imaging unit. Coronal and axial T2-weighted turbo spin-echo and T1-weighted fast-field-echo sequences were performed. SPECT, CT, and MR images were fused by using InVivoScope and the tiepoint coregistration method (Bioscan). MR and SPECT/MR images were interpreted by a medical physicist specializing in MR imaging (M.B., with more than 10 years of experience) and a pharmacologist specializing in animal models and nuclear imaging (T.K.N., with more than 7 years of experience) working under the supervision of consulting radiologist (P.C., with more than 20 years of experience).

Statistical Analysis

All numeric data were expressed as means ± standard errors of the mean. Statistical software (Prism, version 5; Graphpad, San Diego, Calif) was used for statistical analysis. A standard unpaired Student t test was performed. P < .05 was considered to indicate a statistically significant difference. Data were not adjusted for multiple comparisons.

Results

Flow Cytometric Analysis

Flow cytometric analysis revealed high levels of HER1 expression and low levels of HER2 expression for the NCI-H226 and MSTO-211H mesothelioma cells. For HER1 expression, NCI-H226 had higher MFI than the MSTO-211H cells (364 ± 21 vs 206 ± 17, P= .004). For HER2 expression, the MFI for NCI-H226 and MSTO-211H were 78 ± 11 and 64 ± 09, respectively. The MFIs of 45 ± 13 and 36 ± 07 for NCI-H226 and MSTO-211H, respectively, were similar (P = .57) when studies were performed with the negative antibody.

Radioimmunochemistry

Indium 111–labeled panitumumab and trastuzumab were successfully prepared, with radiochemical yields ranging from 80% to 90% and specific activity exceeding 2 GBq/mg. For ¹²⁵I-labeled panitumumab and trastuzumab, the radiochemical yields ranged from 50% to 60%, with a specific activity of approximately 1GBq/mg. The ¹¹¹In- and ¹²⁵I-labeled antibodies demonstrated total cell binding of over 75% and specific cell binding of over 90%, as previously described (18,19).

Subcutaneous Tumor Xenograft Models

In mice bearing the MSTO-211H subcutaneous tumor xenograft, the level of ¹¹¹In-panitumumab in the blood decreased from 15.26% ID/g ± 1.96 at 1 day to 2.43% ID/g ± 0.43 at 7 days after injection (Fig 1, A). In mice bearing NCI-H226 subcutaneous tumor xenografts, ¹¹¹In-panitumumab tumor uptake was greater than ¹¹¹In-trastuzumab tumor uptake at all time points (Fig 1 and Table 1). A similar trend was observed in MSTO-211H tumor xenografts, except for the day 7 time point, where there was no significant difference between ¹¹¹In-panitumumab and ¹¹¹In-trastuzumab. In mice bearing MSTO-211H tumors, the peak tumor uptake of ¹¹¹In-panitumumab (26.43% ID/g ± 1.34) was 1.7-fold greater than that of ¹¹¹In-trastuzumab (15.01% ID/g ± 1.51). As expected, because of the internalizing nature of HER1 and HER2, radioiodinated panitumumab and trastuzumab were rapidly cleared from the tumor because of catabolism of ¹²⁵I after internalization of the complex (20). At day 2, the MSTO-211H tumor uptake for ¹¹¹In-panitumumab was significantly greater than that for ¹²⁵I-panitumumab (23.55% ID/g ± 1.10 vs 7.41% ID/g ± 1.23; P = .0001; Fig E2 [online]).

Figure 1:

A, B, Bar graphs show biodistribution of ¹¹¹In-CHX-A″-DTPA–panitumumab in selected organs of female athymic (NCr) nu/nu mice bearing, A, human MSTO-211H and, B, NCI-H226 subcutaneous tumor xenografts. C, D, Bar graphs show biodistribution of ¹¹¹In-CHX-A″-DTPA–trastuzumab in selected organs of female athymic (NCr) nu/nu mice bearing, C, human MSTO-211H and, D, NCI-H226 subcutaneous tumor xenografts. Data are means ± standard errors of the mean from at least four determinations.

Table 1.

Tumor Uptake of ¹¹¹In-CHX-A″-DTPA–Panitumumab and ¹¹¹In-CHX-A″-DTPA–Trastuzumab in Female Athymic (NCr) nu/nu Mice Bearing NCI-H226 and MSTO-211H Subcutaneous Tumor Xenografts

Note.—Data are mean percentage IDs per gram ± standard errors of the mean.

A similar relationship between the HER1- and HER2-targeting antibodies was observed in mice bearing NCI-H226 subcutaneous tumor xenografts. The ¹¹¹In-panitumumab attained higher levels in the NCI-226 tumor than ¹¹¹In-trastuzumab (Fig 1, B and D). Additionally, compared with that in the MSTO-211H tumor xenografts, the tumor uptake of ¹¹¹In-panitumumab was greater in the NCI-H226 tumor xenografts. Four days after injection of the ¹¹¹In-panitumumab, the percentage ID per gram in the NCI-H226 tumor was 32.12% ID/g ± 0.56, significantly greater than what was observed in the MSTO-211H tumors (20.83% ID/g ± 2.55) (P = .002). The MSTO-211H tumor AUC from 0 to 7 days (AUC_[0→7]) for ¹¹¹In-panitumumab was significantly greater than that for ¹¹¹In-trastuzumab (75.7% ID · day · g⁻¹ ± 5.0 vs 47.0% ID · day · g⁻¹ ± 5.3, P = .004). Similarly, the NCI-H226 tumor AUC_[0→7] for ¹¹¹In-panitumumab was significantly greater than that for ¹¹¹In-trastuzumab (118.8% ID · day · g⁻¹ ± 7.0 vs 43.9% ID · day · g⁻¹ ± 3.7, P = .0001). Comparing the tumor AUC_[0→7] calculated for ¹¹¹In-panitumumab with that calculated for ¹¹¹In-trastuzumab, panitumumab demonstrated 1.6- and 2.7-fold greater targeting than trastuzumab in the MSTO-211H and NCI-H226 tumors, respectively.

Biodistribution studies in subcutaneous tumor xenograft models revealed higher uptake of ¹¹¹In-panitumumab than ¹¹¹In-trastuzumab, confirming higher expression of HER1 than HER2 in mesothelioma models. SPECT/CT imaging was performed in mice bearing NCI-H226 and MSTO-211H subcutaneous mesothelioma tumor xenografts (Fig 2). The tumor was clearly visualized in both tumor models at 1 and 3 days after injection of the ¹¹¹In-panitumumab. The radioactivity predominately localized in the tumor xenograft, with some blood pool visible. At 1 day, the blood activity was greater in mice bearing MSTO-211H tumors than NCI-H226 tumors, which is in agreement with the biodistribution data (Fig 1). After 3 days, the amount of radioactivity in the NCI-H226 tumors was 34.6% ID/cm³ ± 3.1, compared with 25.7% ID/cm³ ± 2.8 for the MSTO-211H tumors.

Figure 2:

Representative coregistered SPECT/CT maximum intensity projections in female athymic (NCr) nu/nu mice bearing (top) NCI-H226 and (bottom) MSTO-211H subcutaneous tumor xenografts. Mice were given intravenous injections via tail vein of 2.0 MBq of ¹¹¹In-CHX-A″-DTPA–panitumumab, and images were acquired 1 day and 3 days after injection. Scale = percentage of maximum and minimum threshold intensity (80% for 1 day and 70% for 3 days).

Orthotopic MPM

MR imaging.—No tumors were visualized at MR imaging at week 1 or 2 for either model of mice injected with MSTO-211H or NCI-H226 cells into the pleural cavity or mice injected with medium only. For NCI-H226 model, at week 3, a mass was observed in the diaphragmatic pleura, which spread to the costal pleura by week 4 (Fig 3). At week 5, the disease had spread to the cervical and the mediastinal pleura with a pleural effusion (Fig 3). Similar observations were found for the MSTO-211H model in terms of spread of the cancerous mass. As expected, no cancerous mass or effusion was observed in mice injected with medium without cells (Fig 3).

Figure 3:

Representative coronal MR imaging sections obtained 3, 4, and 5 weeks after cell inoculation in (top) female athymic (NCr) nu/nu mouse bearing orthotopic NCI-H226 and (bottom) non–tumor-bearing mouse. Arrows = tumors.

Biodistribution and SPECT/CT and MR imaging.—The blood pool activity for ¹¹¹In-panitumumab in NCI H226 tumors decreased approximately 70% over the 7 day study period (10.53% ID/g ± 1.53 at 1 day to 3.45% ID/g ± 0.54 at 7 days, P = .005) (Fig 4). The tumor percentage ID per gram was 29.53% ID/g ± 2.18 at 1 day and 22.68% ID/g ± 1.14 at 7 days, with a peak tumor uptake of 30.86% ID/g ± 3.47 at 3 days. The tumor-to-blood ratio increased more than fourfold, from 1.6 at 1 day to 6.4 at 7 days after injection (P = .003). Two days after injection, the tumor uptake for ¹¹¹In-panitumumab was 2.2-fold greater than that for ¹¹¹In-trastuzumab (29.62% ID/g ± 2.18 vs 13.63% ID/g ± 1.00, P = .0006). Similarly, 4 days after injection, the tumor uptake of 24.76% ID/g ± 3.28 for ¹¹¹In-panitumumab was significantly greater than the tumor uptake of 12.36% ID/g ± 1.52 for ¹¹¹In-trastuzumab (Table 2).

Figure 4:

Bar graph shows biodistribution of ¹¹¹In-CHX-A″-DTPA–panitumumab in selected organs of female athymic (NCr) nu/nu mice bearing orthotopic NCI-H226 MPM. Data are means ± standard errors of the mean from at least four determinations.

Table 2.

Biodistribution of ¹¹¹In-CHX-A″-DTPA–Panitumumab and ¹¹¹In-CHX-A″-DTPA–Trastuzumab in Orthotopic MPM

Note.—Data are mean percentage IDs per gram ± standard errors of the mean.

SPECT/CT imaging performed 2 days after injection revealed high levels of ¹¹¹In-panitumumab at the site of tumor in the pleural cavity, with localization in the cervical and mediastinal pleura (Fig 5). CT images revealed thickening of the pleura, indicative of tumor (Fig 5). Two days after injection, most of the radioactivity was localized in the cervical and the mediastinal pleura. Concomitantly, lower blood pool activity was noted after 5 days, providing better tumor-to-background ratio (Fig 6). Coregistered SPECT, CT, and MR imaging findings confirmed the localization of the radiolabeled antibody in the orthotopic MPM (Fig 6).

Figure 5:

Top: Representative CT images in female athymic (NCr) nu/nu mouse bearing orthotopic NCI-H226 MPM. Bottom: Corresponding coregistered SPECT/CT images in same mouse injected intravenously via tail vein with 2.0 MBq of ¹¹¹In-CHX-A″-DTPA–panitumumab. Left-to-right: Maximum intensity projections, sagittal sections, coronal sections, and transverse sections. Images were acquired 2 days after the injection of radiolabeled panitumumab.

Figure 6:

Representative coronal sections in female athymic (NCr) nu/nu mouse bearing orthotopic NCI-H226 MPM injected intravenously via tail vein with 2.0 MBq of ¹¹¹In-CHX-A″-DTPA–panitumumab. Images were acquired 5 days after the injection of radiolabeled panitumumab.

Discussion

Chemotherapy, immunotherapy, radiation therapy, and surgery remain the mainstays of therapy for MPM (1,2). Collectively, these therapeutic options have had disappointing results, and survival remains poor (1,2). Occupational exposure to asbestos remains the major risk factor for MPM (3,21). Such exposure has been demonstrated to be associated with increased HER1 activation and expression (3,8,9).

Imaging can play a fundamental role in the management of MPM as a means of staging MPM for nodal and metastatic sites and for monitoring the effects of therapy (4,5). In our study, the anatomic information obtained from CT and MR imaging and molecular information obtained from radiolabeled antibodies have been combined for characterization of MPM.

In this study, by using MR imaging, the progression of disease was monitored from the site of cell inoculation in the pleural cavity to the pleura of the diaphragm, to the mediastinal pleura, and throughout the cavity. This approach may also provide a valuable tool for the evaluation of anticancer drugs for MPM in mice bearing orthotopic tumors, in which monitoring of tumor growth inhibition is more difficult than in subcutaneous tumors.

Previous studies have demonstrated HER1 overexpression in 56%–100% of MPM with epithelioid histologic features (12,13,15). In this context, HER1-targeted imaging can play a complimentary role in a better understanding of asbestos-induced mesothelioma and probably personalized therapy with anti-HER1 agents. In mice bearing subcutaneous tumors, the human epithelioid mesothelioma model (NCI H226) had greater accumulation of HER1-targeted ¹¹¹In-panitumumab than the human biphasic mesothelioma model (MSTO-211H). The calculated AUC_[0→7] for the NCI-H226 tumor was 1.5-fold greater than that for the MSTO-211H tumor. The in vitro binding of panitumumab was 1.7-fold greater for NCI-H226 than MSTO-211H; however, it is difficult to predict uptake of radiolabeled antibodies solely on the basis of target expression, as other physiologic factors such as vasculature and interstitial fluid pressure can play a role in the accumulation of antibodies in tumor tissue. Nevertheless, in agreement with in vitro data, HER1-targeted ¹¹¹In-panitumumab tumor accumulation was greater than HER2-targeted ¹¹¹In-trastuzumab tumor accumulation in both MPM models evaluated. As expected, due to the internalizing nature of HER1 and HER2, the ¹¹¹In-labeled antibodies demonstrated more favorable biodistribution and pharmacokinetics than the ¹²⁵I-labeled antibodies. In mice bearing orthotopic MPM tumors, ¹¹¹In-panitumumab uptake was significantly greater than the ¹¹¹In-trastuzumab uptake observed in the subcutaneous model. No significant differences were observed in tumor accumulation of ¹¹¹In-labeled antibody when the subcutaneous and orthotopic models were compared (P = .47). The subcutaneous tumor AUC_[0→7] was similar to the orthotopic tumor AUC_[0→7](118.8% ID/g · day · g⁻¹ ± 7.0 vs 109.2% ID/g · day · g⁻¹ ± 10.7, P = .46). Surprisingly, unlike the results of our study, significant differences in tumor accumulation of zirconium 89–panitumumab in subcutaneous and metastatic models of colorectal cancer have been observed (22), highlighting the complexity of antibody distribution in different biologic systems. This discrepancy also illustrates the necessity for performing the appropriate characterization studies with each radioimmunoconjugate.

In this study, good tumor targeting and localization were achieved. Nevertheless, there are multiple challenges for clinical translation of these radiolabeled antibodies. The anti-HER1 and anti-HER2 antibodies used in this study do not cross react with murine antigen, and therefore minimal uptake in normal organs is observed, which may not be the case in humans because of cross reactivity. Additionally, the HER1 expression in epithelioid mesothelioma is variable and therefore poses challenges in data interpretation; hence, successful application of anti-HER1 antibodies requires further investigations.

In conclusion, molecular imaging with radiolabeled antibodies and MR imaging for noninvasive imaging of MPM were demonstrated. With successful clinical translation, the combination of HER1 assessment by using radiolabeled antibodies and tumor monitoring by using MR imaging offers an attractive diagnostic and prognostic tool for the treatment of patients with MPM.

Orthotopic tumors were visualized with SPECT/CT and MR imaging. These modalities provided valuable anatomic and molecular information about the MPM disease state. In combination, these imaging modalities can potentially be used for therapeutic interventions with HER1-targeted immunotherapies, and potentially, radioimmunotherapy.

Advance in Knowledge.

• • In an orthotopic mesothelioma model, the tumor lesions were visualized with SPECT/CT and MR imaging, and 4 days after injection, the mean tumor uptake of 24.76% injected dose (ID) per gram ± 3.28 (standard error of the mean) for human epidermal growth facto receptor (HER) 1-targeted indium 111 (¹¹¹In)-panitumumab was significantly greater than that (12.36% ID/g ± 1.52) for HER2-targeted ¹¹¹In-trastuzumab (P = .01).

Implication for Patient Care.

• • The combination of MR imaging and molecular imaging performed with radiolabeled antibodies can provide information on disease status and alterations in tumor burden and characteristics during a therapy regimen and may potentially assist in selecting patients for anti-HER1 therapies, particularly for therapies where anti-HER1 antibody acts as a carrier to deliver toxins.

Disclosures of Conflicts of Interest: T.K.N. No relevant conflicts of interest to disclose. M.B. No relevant conflicts of interest to disclose. D.E.M. No relevant conflicts of interest to disclose. P.L.K. No relevant conflicts of interest to disclose. M.W.B. No relevant conflicts of interest to disclose.