A pipeline for facile cloning of antibody Fv domains and their expression, purification, and characterization as recombinant His-tagged IgGs.

We report a functional pipeline for facile conversion of variable Fv domains, typically discovered in antibody discovery programs, into chimeric monoclonal antibodies (mAbs). Often, in initial screenings, a set of candidate mAbs is produced in small volumes and purified from supernatant for testing. Our pipeline also simplifies purification of mAbs by using an extended histidine tag (His-10) fused to the C-terminus of the light chain. Both the length of the His-10 and its location have been shown to affect the efficacy of mAb purification using an inexpensive nickel-based resin at neutral pH. Our antibody cloning and purification pipeline, when followed together with detection and affinity measurements, can be smoothly incorporated into an antibody discovery workflow.

Coil or Plug-Assisted Ethylene Vinyl Alcohol Copolymer (EVOH) Thoracic Duct Embolization in the Treatment of Postoperative Chylothorax.

Eleven patients (5 men, 6 women) with post-operative thoracic duct injuries and high output chylothorax were treated with thoracic duct embolization (TDE). Six patients underwent intraprocedural thoracic duct ligation at the time of original procedure. In all cases, the pleural fluid demonstrated high triglyceride levels (414 mg/dL; interquartile range [IQR], 345 mg/dL). Median daily (IQR) chest tube outputs before and after TDE were 900 mL (1,200 mL) and 325 mL (630 mL), respectively. Coil- or plug-assisted ethylene vinyl alcohol (EVOH) copolymer was used as embolic agent in all patients. Technical and clinical success rates were 100% and 82%, respectively. Nontarget venous embolization of EVOH copolymer was not identified on subsequent imaging.

A Multicenter Phase 1 Trial Evaluating Nanoliposomal Irinotecan for Heated Intraperitoneal Chemotherapy Combined with Cytoreductive Surgery for Patients with Peritoneal Surface Disease.

BACKGROUND: Nanoliposomal irinotecan (nal-IRI) is a promising novel hyperthermic intraperitoneal chemotherapy (HIPEC) agent given its enhanced efficacy against gastrointestinal tumors, safety profile, thermo-synergy, and heat stability. This report describes the first in-human phase 1 clinical trial of nal-IRI during cytoreductive surgery (CRS) and HIPEC. METHODS: Patients with peritoneal surface disease (PSD) from appendiceal and colorectal neoplasms were enrolled in a 3 + 3 dose-escalation trial using nal-IRI (70-280 mg/m2) during HIPEC for 30 min at 41 ± 1 °C. The primary outcome was safety. The secondary outcomes were pharmacokinetics (PK) and disease-free survival. Adverse events (AEs) categorized as grade 2 or higher were recorded. The serious AEs (SAEs) were mortality, grade ≥ 3 AEs, and dose-limiting toxicity (DLT). Irinotecan and active metabolite SN38 were measured in plasma and peritoneal washings. RESULTS: The study enrolled 18 patients, who received nal-IRI during HIPEC at 70 mg/m2 (n = 3), 140 mg/m2 (n = 6), 210 mg/m2 (n = 3), and 280 mg/m2 (n = 6). No DLT or mortality occurred. The overall morbidity for CRS/HIPEC was 39% (n = 7). Although one patient experienced neutropenia, no AE (n = 131) or SAE (n = 3) was definitively attributable to nal-IRI. At 280 mg/m2, plasma irinotecan and SN38 measurements showed maximum concentrations of 0.4 ± 0.6 µg/mL and 3.0 ± 2.4 ng/mL, a median time to maximum concentration of 24.5 and 26 h, and areas under the curve of 22.6 h*µg/mL and 168 h*ng/mL, respectively. At the 6-month follow-up visit, 83% (n = 15) of the patients remained disease-free. CONCLUSIONS: In this phase 1 HIPEC trial (NCT04088786), nal-IRI was observed to be safe, and PK profiling showed low systemic absorption overall. These data support future studies testing the efficacy of nal-IRI in CRS/HIPEC.

Radiologists Help Each Other to Improve Efficiency by Decreasing the Time to Look Up a Patient's Relevant Clinical History in the Electronic Medical Record.

The study aims to prove that it takes less time to look up relevant clinical history from an electronic medical record (EMR) if the information is already provided in a specific space in the EMR by a fellow radiologist. Patients with complex oncological and surgical histories need frequent imaging, and every time a radiologist may spend a significant amount of time looking up the same clinical information as their peers. In collaboration with ACMIO and Radiant Epic team, a space labeled "Specialty Comments" was added to the SNAPSHOT of patient's chart in EMR. For our research purpose, the specialty comment was labeled as boxed history as a variable for data analysis. If the history was not provided in that particular space, it was labeled as without boxed history. Inclusion criteria included outpatients with complex oncological histories undergoing CT chest, abdomen, and pelvis with IV contrast. The time to look up history (LUT) was documented in minutes and seconds. Two assistant professors from Abdominal Imaging provided LUT. A total of 85 cases were included in the study, 39 with boxed history and 46 without boxed history. Comparing averages of the individual reader means for history, mean LUT differed by 2.03 min (without boxed history) versus 0.57 min (with boxed history), p < 0.0001. The t-test and the nonparametric Wilcoxon tests for a difference in the population means were highly significant (p < 0.0001). A history directed to radiologist's needs resulted in a statistically significant decrease in time spent by interpreting radiologists to look through the electronic medical records for patients with complex oncological histories. Availability of history pertinent to radiology has wide-ranging advantages, including quality reporting, decrease in turnaround time, reduction in interpretation errors, and radiologists' continued learning. The space for documenting clinical history may be reproduced, or some similar area may be developed by optimizing the electronic medical records.

Peritumoral Hyperintensity at Hepatobiliary Phase Gadoxetic Acid-enhanced MRI in Hepatic Neuroendocrine Tumors.

BACKGROUND/AIM: To identify the imaging and clinical features of hepatic neuroendocrine tumors (NETs) associated with peritumoral hyperintensity in the hepatobiliary phase of gadoxetic acid-enhanced magnetic resonance (MR) imaging. PATIENTS AND METHODS: Fifty-seven patients with hepatic NETs were enrolled. Based on the degree of peritumoral hyperintensity, patients were divided into three groups: group 0 (no peritumoral hyperintensity), group 1 (lower peritumoral hyperintensity), and group 2 (higher peritumoral hyperintensity). The imaging and clinical findings were compared among the three groups. RESULTS: Apparent diffusion coefficient (ADC) values of group 2 were significantly lower than those of group 0 and group 1. Atypical (cholangiocarcinoma-like) enhancement pattern in the arterial phase was significantly more frequently observed in group 2 as compared to that in group 0 and group 1. Group 2 patients showed significantly poorer progression-free survival than group 0 patients. CONCLUSION: Hepatic NETs with greater peritumoral hyperintensity exhibit greater malignant potential.

Use of 64Cu-DOTA-Trastuzumab PET to Predict Response and Outcome of Patients Receiving Trastuzumab Emtansine for Metastatic Breast Cancer: A Pilot Study.

We hypothesized that functional imaging with 64Cu-DOTA-trastuzumab PET/CT would predict the response to the antibody-drug conjugate trastuzumab-emtansine (T-DM1). Methods: Ten women with metastatic human epidermal growth factor receptor 2-positive breast cancer underwent 18F-FDG PET/CT and 64Cu-DOTA-trastuzumab PET/CT on days 1 and 2 before treatment with T-DM1. Results: T-DM1-responsive patients had higher uptake than nonresponsive patients. Day 1 minimum SUVmax (5.6 vs. 2.8, P < 0.02), day 2 minimum SUVmax (8.1 vs. 3.2, P < 0.01), and day 2 average SUVmax (8.5 vs. 5.4, P < 0.05) for 64Cu-DOTA-trastuzumab all favored responding patients. Tumor-level response suggested threshold dependence on SUVmax Patients with a day 2 minimum SUVmax above versus below the threshold had a median time to treatment failure of 28 mo versus 2 mo (P < 0.02). Conclusion: Measurement of trastuzumab uptake in tumors via PET/CT is promising for identifying patients with metastatic breast cancer who will benefit from T-DM1.

Change in Apparent Diffusion Coefficient Is Associated With Local Failure After Stereotactic Body Radiation Therapy for Non-Small Cell Lung Cancer: A Prospective Clinical Trial.

PURPOSE: Response assessment with computed tomography after stereotactic body radiation therapy (SBRT) for non-small cell lung cancer (NSCLC) is challenging because myriad anatomic changes can occur after treatment. Diffusion-weighted magnetic resonance imaging (MRI) may provide additional data to guide therapy response. The primary objective was to evaluate the effect of SBRT on the mean apparent diffusion coefficient (ADC). METHODS AND MATERIALS: This is a prospective clinical study of patients with NSCLC who received SBRT to the primary lung lesion. Patients underwent MRI scans before and at 1 month after completion of SBRT. MRI consisted of T1- and T2-weighted sequences, along with postcontrast, dynamic-contrast, and diffusion-weighted sequences with construction of ADC maps. Two blinded radiologists generated the ADC. SBRT was given over 5 fractions. RESULTS: A total of 13 patients were enrolled. Twelve patients were eligible for analysis. An average increase of 50% and 46% in mean single-plane ADC was observed after treatment by readers 1 and 2, respectively (P < .01, both reviewers). There was good interobserver agreement of single-plane ADC values between the 2 radiologists (Pearson correlation of 0.85 [baseline] and 0.89 [1-month post-SBRT], P < .001 for both). There was also a significant 18% increase in mean volumetric ADC on the 1-month scan (Wilcoxon P = .02). Two patients developed a local failure after SBRT, 1 at 6 months and the other at 34 months. Using a threshold of volumetric ADC increase of greater than 40%, 2 of 2 patients demonstrated local failure compared with 0 of 10 patients below this limit. CONCLUSIONS: A statistically significant increase in ADC was observed 1 month after treatment. An ADC increase of 40% at 1 month was associated with a higher rate of local failure. This pilot study provides impetus for studying ADC as a radiomic biomarker in patients receiving lung SBRT for NSCLC.

Tumor Uptake of 64Cu-DOTA-Trastuzumab in Patients with Metastatic Breast Cancer.

The goal of this study was to characterize the relationship between tumor uptake of 64Cu-DOTA-trastuzumab as measured by PET/CT and standard, immunohistochemistry (IHC)-based, histopathologic classification of human epidermal growth factor receptor 2 (HER2) status in women with metastatic breast cancer (MBC). Methods: Women with biopsy-confirmed MBC and not given trastuzumab for 2 mo or more underwent complete staging, including 18F-FDG PET/CT. Patients were classified as HER2-positive (HER2+) or -negative (HER2-) based on fluorescence in situ hybridization (FISH)-supplemented immunohistochemistry of biopsied tumor tissue. Eighteen patients underwent 64Cu-DOTA-trastuzumab injection, preceded in 16 cases by trastuzumab infusion (45 mg). PET/CT was performed 21-25 (day 1) and 47-49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Radiolabel uptake in prominent lesions was measured as SUVmax Average intrapatient SUVmax (pt) was compared between HER2+ and HER2- patients. Results: Eleven women were HER2+ (8 immunohistochemistry 3+; 3 immunohistochemistry 2+/FISH amplified), whereas 7 were HER2- (3 immunohistochemistry 2+/FISH nonamplified; 4 immunohistochemistry 1+). Median pt for day 1 and day 2 was 6.6 and 6.8 g/mL for HER 2+ and 3.7 and 4.3 g/mL for HER2- patients (P < 0.005 either day). The distributions of pt overlapped between the 2 groups, and interpatient variability was greater for HER2+ than HER2- disease (P < 0.005 and 0.001, respectively, on days 1 and 2). Conclusion: By 1 d after injection, uptake of 64Cu-DOTA-trastuzumab in MBC is strongly associated with patient HER2 status and is indicative of binding to HER2. The variability within and among HER2+ patients, as well as the overlap between the HER2+ and HER2- groups, suggests a role for 64Cu-DOTA-trastuzumab PET/CT in optimizing treatments that include trastuzumab.

Quantification of hemodynamics in abdominal aortic aneurysms during rest and exercise using magnetic resonance imaging and computational fluid dynamics.

Abdominal aortic aneurysms (AAAs) affect 5-7% of older Americans. We hypothesize that exercise may slow AAA growth by decreasing inflammatory burden, peripheral resistance, and adverse hemodynamic conditions such as low, oscillatory shear stress. In this study, we use magnetic resonance imaging and computational fluid dynamics to describe hemodynamics in eight AAAs during rest and exercise using patient-specific geometric models, flow waveforms, and pressures as well as appropriately resolved finite-element meshes. We report mean wall shear stress (MWSS) and oscillatory shear index (OSI) at four aortic locations (supraceliac, infrarenal, mid-aneurysm, and suprabifurcation) and turbulent kinetic energy over the entire computational domain on meshes containing more than an order of magnitude more elements than previously reported results (mean: 9.0-million elements; SD: 2.3 M; range: 5.7-12.0 M). MWSS was lowest in the aneurysm during rest 2.5 dyn/cm(2) (SD: 2.1; range: 0.9-6.5), and MWSS increased and OSI decreased at all four locations during exercise. Mild turbulence existed at rest, while moderate aneurysmal turbulence was present during exercise. During both rest and exercise, aortic turbulence was virtually zero superior to the AAA for seven out of eight patients. We postulate that the increased MWSS, decreased OSI, and moderate turbulence present during exercise may attenuate AAA growth.

Molecular imaging: a primer for interventionalists and imagers.

The characterization of human diseases by their underlying molecular and genomic aberrations has been the hallmark of molecular medicine. From this, molecular imaging has emerged as a potentially revolutionary discipline that aims to visually characterize normal and pathologic processes at the cellular and molecular levels within the milieu of living organisms. Molecular imaging holds promise to provide earlier and more precise disease diagnosis, improved disease characterization, and timely assessment of therapeutic response. This primer is intended to provide a broad overview of molecular imaging with specific focus on future clinical applications relevant to interventional radiology.

Molecular imaging: a primer for interventionalists and imagers.

The characterization of human diseases by their underlying molecular and genomic aberrations has been the hallmark of molecular medicine. From this, molecular imaging has emerged as a potentially revolutionary discipline that aims to visually characterize normal and pathologic processes at the cellular and molecular levels within the milieu of living organisms. Molecular imaging holds promise to provide earlier and more precise disease diagnosis, improved disease characterization, and timely assessment of therapeutic response. This primer is intended to provide a broad overview of molecular imaging with specific focus on future clinical applications relevant to interventional radiology.

Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

Promising techniques for breast cancer detection, diagnosis, and staging using non-ionizing radiation imaging techniques.

Traditional imaging for the diagnosis and staging of breast cancer has relied on the tissue morphology of cancers in the background of normal patterns of fibroglandular breast tissue. X-ray mammography and ultrasound have been the primary modalities for the diagnosis and the work-up of breast cancer. New modalities have been validated including magnetic resonance imaging (MRI) and positron emission tomography (PET). New pulse sequences in MRI combined with contrast enhancement kinetic perfusion curves have greatly enhanced detection of mammographically occult cancers. New modalities on the horizon include optical imaging, exploiting again the differential perfusion properties of cancers in a background of normal glandular tissue. Even more specificity can be ach eved with the addition of ductal or intravenous introduction of optical probes specific to tumor associated antigens such as the HER-2/neu receptor in aggressive breast cancers. Quantum dots and other fluorescent dyes coupled to peptides or other probes will greatly enhance our ability to detect cancers earlier and without ionizing radiation.

Reproducibility of 3'-deoxy-3'-(18)F-fluorothymidine microPET studies in tumor xenografts in mice.

UNLABELLED: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been used to image tumor proliferation in preclinical and clinical studies. Serial microPET studies may be useful for monitoring therapy response or for drug screening; however, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FLT microPET studies. METHODS: C6 rat glioma xenografts were implanted into nude mice (n = 9) and grown to mean diameters of 5-17 mm for approximately 2 wk. A 10-min acquisition was performed on a microPET scanner approximately 1 h after (18)F-FLT (1.9-7.4 MBq [50-200 muCi]) was injected via the tail vein. A second microPET scan was performed approximately 6 h later on the same day after reinjection of (18)F-FLT to assess for reproducibility. Most of the mice were studied twice within the same week (for a total of 17 studies). Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility after subtraction of the estimated residual tumor activity from the first (18)F-FLT injection. RESULTS: The coefficient of variation (mean +/- SD) for %ID/g values between (18)F-FLT microPET scans performed 6 h apart on the same day was 14% +/- 10%. The difference in %ID/g values between scans was -0.06% +/- 1.3%. Serum thymidine levels were mildly correlated with %ID/g values (R(2) = 0.40). Tumor size, mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, controlling body temperature, the time of imaging after injection, and the ROI size. CONCLUSION: (18)F-FLT microPET mouse tumor xenograft studies are reproducible with moderately low variability. Serial studies may be performed to assess for significant changes in therapy response or for preclinical drug development.

Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials.

PURPOSE: To critically assess the accuracy and reproducibility of human epidermal growth factor receptor type 2 (HER-2) testing in outside/local community-based hospitals versus two centralized reference laboratories and its effect on selection of women for trastuzumab (Herceptin)-based clinical trials. EXPERIMENTAL DESIGN: Breast cancer specimens from 2,600 women were prospectively evaluated by fluorescence in situ hybridization (FISH) for entry into Breast Cancer International Research Group (BCIRG) clinical trials for HER-2-directed therapies. RESULTS: HER-2 gene amplification by FISH was observed in 657 of the 2,502 (26%) breast cancers successfully analyzed. Among 2,243 breast cancers with central laboratory immunohistochemistry (10H8-IHC) analysis, 504 (22.54%) showed overexpression (2+ or 3+). Outside/local laboratories assessed HER-2 status by immunohistochemistry in 1,536 of these cases and by FISH in 131 cases. Overall, the HER-2 alteration status determined by outside/local immunohistochemistry showed a 79% agreement rate [kappa statistic, 0.56; 95% confidence interval (95% CI), 0.52-0.60], with FISH done by the central laboratories. The agreement rate comparing BCIRG central laboratory 10H8-IHC and outside/local laboratory immunohistochemistry was 77.5% (kappa statistic, 0.51; 95% CI, 0.46-0.55). Finally, HER-2 status, determined by unspecified FISH assay methods at outside/local laboratories, showed a 92% agreement rate (kappa statistic, 0.83; 95% CI, 0.73-0.93), with FISH done at the BCIRG central laboratories. CONCLUSIONS: Compared with the HER-2 status determined at centralized BCIRG reference laboratories, these results indicate superiority of FISH to accurately and reproducibly assess tumors for the HER-2 alteration at outside/local laboratories for entry to clinical trials.

Solitary pulmonary nodule diagnosis on CT: results of an observer study.

RATIONALE AND OBJECTIVES: To investigate the performance of observers with different levels of experience in distinguishing between benign and malignant solitary pulmonary nodules (SPN) on CT, and to determine the effects on interpretation of three different conditions: image data alone, the addition of clinical data, and the addition of output from a computer-aided diagnosis (CAD) system. MATERIALS AND METHODS: 28 thin-section CT datasets of SPNs with proven diagnoses (15 malignant and 13 benign) were used to measure observer performance. Readers were categorized according to their experience and read the cases in random order. For each case readers were asked to assign a level of confidence on a scale from 0.0-1.0 (0.0 benign, 1.0 malignant) for the diagnosis of the nodule. Each reader scored the cases based on review of image data alone (phase 1), then with limited clinical data (phase 2), and finally with CAD output (phase 3). To assess performance, multiple reader multiple case (MRMC) receiver operating characteristic (ROC) analysis was used. RESULTS: 2 thoracic radiologists, 1 thoracic radiology fellow, 2 nonthoracic radiologists, and 3 radiology residents read the cases. The average area under the ROC curve for all readers (A(z)) at each stage was 0.68, 0.75, and 0.81, for image data alone, with clinical data, and with CAD output respectively. The difference in performance between phases (2 and 3) and (1 and 3) was significantly different (P = 0.018 and P = 0.020). However, the difference between phases (1 and 2) was not significantly different (P = 0.155). CONCLUSION: Diagnostic performance increased significantly with the addition of CAD output. With further validation CAD output may play a significant role in SPN management.

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting.

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.

Internet consultations from a remote Pacific island: impact of digitized radiologic images on referral decisions.

A study was carried out to determine whether digitized radiologic images added valuable information to Internet consultations from a remote Pacific Island. Chuuk State Hospital (Federated States of Micronesia) has limited film screen radiology, minimal ultrasound capability, and no radiologist. Providers initiate Web-based referrals for consultation or patient transfer. Digitized images (via low-cost digital camera or flatbed scanner) were uploaded to a Web site. Images were assessed for impact on referral decisions. A radiologist scored image quality and confidence (scale: 1-7). Of 97 referrals with images that were reviewed, 74 (76%) image sets were abnormal, 20 (20%) were normal, and 3 (4%) were indeterminate. Median scores were 4 for image quality and 5 for diagnostic confidence. In most cases with abnormal radiology (52/74, 70%), images were considered valuable. Radiologic images digitized with a low-cost camera or flatbed scanner provided valuable information for decision making in an Internet-based consultation and referral process from a remote, impoverished Pacific Island jurisdiction, despite relatively low image quality.

Antibody-mediated targeting of replication-competent retroviral vectors.

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.