Andreas Loening

All Publications

• Comparison of End-Expiration Versus End-Inspiration Breath-Holds With Respect to Respiratory Motion Artifacts on T1-Weighted Abdominal MRI. AJR. American journal of roentgenology Vu, K., Haldipur, A. G., Roh, A. T., Lindholm, P., Loening, A. M. 2019: 1–6

  Abstract

  OBJECTIVE: The purpose of this study was to compare respiratory motion artifact and diagnostic image quality between end-inspiration and end-expiration breath-holding techniques on unenhanced and contrast-enhanced axial T1-weighted MRI of the liver.MATERIALS AND METHODS: This retrospective observational study included 50 consecutive subjects undergoing axial T1-weighted liver MRI, with unenhanced images acquired with both end-inspiration and end-expiration breath-holding techniques, and with contrast-enhanced images acquired for 47 of the subjects with either the end-inspiration or the end-expiration breath-holding technique. Three radiologists performed blinded independent evaluations of each unenhanced sequence, contrast-enhanced sequence, and subtraction (contrast-enhanced minus unenhanced) image, using a scale ranging from 1 point (denoting nondiagnostic imaging) to 5 points (denoting no artifacts). Blinded side-by-side assessment of each pair of unenhanced sequences was also performed. Two-tailed Wilcoxon signed rank and Wilcoxon rank sum tests were used to assess statistical significance.RESULTS: A significant improvement in motion scores was noted for sequences acquired in end-expiration, compared with those acquired in end-inspiration, for unenhanced sequences (mean, 3.35 vs 2.80; p < 0.00001), contrast-enhanced sequences (mean, 4.02 vs 3.46; p = 0.0003), and subtraction images (mean, 3.67 vs 2.41; p < 0.00001). Severe degradation of image quality or nondiagnostic image quality was noted for 15% of unenhanced images (23/150), 0% of contrast-enhanced images, and 8% (5/63) of subtraction images acquired on end-expiration, whereas it was noted for 36% (54/150) of unenhanced images, 13% (10/78) of contrast-enhanced images, and 59% (46/78) of subtraction images acquired on end-inspiration. When side-by-side assessment of paired unenhanced sequences was performed, images acquired in end-expiration were significantly favored in 59% of paired sequences (88/150) (p < 0.00001), and no difference between images acquired with both breath-hold techniques was noted for 21% (32/150) of paired sequences.CONCLUSION: The end-expiration breath-holding technique leads to significant decreases in respiratory motion artifacts, compared with the end-inspiration technique, on unenhanced and contrast-enhanced T1-weighted liver MRI.

  View details for DOI 10.2214/AJR.18.20239

  View details for PubMedID 30835515

• View-Sharing Artifact Reduction With Retrospective Compressed Sensing Reconstruction in the Context of Contrast-Enhanced Liver MRI for Hepatocellular Carcinoma (HCC) Screening. Journal of magnetic resonance imaging : JMRI Shaikh, J., Stoddard, P. B., Levine, E. G., Roh, A. T., Saranathan, M., Chang, S. T., Muelly, M. C., Hargreaves, B. A., Vasanawala, S. S., Loening, A. M. 2018

  Abstract

  BACKGROUND: View-sharing (VS) increases spatiotemporal resolution in dynamic contrast-enhanced (DCE) MRI by sharing high-frequency k-space data across temporal phases. This temporal sharing results in respiratory motion within any phase to propagate artifacts across all shared phases. Compressed sensing (CS) eliminates the need for VS by recovering missing k-space data from pseudorandom undersampling, reducing temporal blurring while maintaining spatial resolution.PURPOSE: To evaluate a CS reconstruction algorithm on undersampled DCE-MRI data for image quality and hepatocellular carcinoma (HCC) detection.STUDY TYPE: Retrospective.SUBJECTS: Fifty consecutive patients undergoing MRI for HCC screening (29 males, 21 females, 52-72 years).FIELD STRENGTH/SEQUENCE: 3.0T MRI. Multiphase 3D-SPGR T1 -weighted sequence undersampled in arterial phases with a complementary Poisson disc sampling pattern reconstructed with VS and CS algorithms.ASSESSMENT: VS and CS reconstructions evaluated by blinded assessments of image quality and anatomic delineation on Likert scales (1-4 and 1-5, respectively), and HCC detection by OPTN/UNOS criteria including a diagnostic confidence score (1-5). Blinded side-by-side reconstruction comparisons for lesion depiction and overall series preference (-3-3).STATISTICAL ANALYSIS: Two-tailed Wilcoxon signed rank tests for paired nonparametric analyses with Bonferroni-Holm multiple-comparison corrections. McNemar's test for differences in lesion detection frequency and transplantation eligibility.RESULTS: CS compared with VS demonstrated significantly improved contrast (mean 3.6 vs. 2.9, P<0.0001) and less motion artifact (mean 3.6 vs. 3.2, P=0.006). CS compared with VS demonstrated significantly improved delineations of liver margin (mean 4.5 vs. 3.8, P=0.0002), portal veins (mean 4.5 vs. 3.7, P<0.0001), and hepatic veins (mean 4.6 vs. 3.5, P<0.0001), but significantly decreased delineation of hepatic arteries (mean 3.2 vs. 3.7, P=0.004). No significant differences were seen in the other assessments.DATA CONCLUSION: Applying a CS reconstruction to data acquired for a VS reconstruction significantly reduces motion artifacts in a clinical DCE protocol for HCC screening.LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018.

  View details for PubMedID 30390358

• Conical ultrashort echo time (UTE) MRI in the evaluation of pediatric acute appendicitis. Abdominal radiology (New York) Roh, A. T., Xiao, Z., Cheng, J. Y., Vasanawala, S. S., Loening, A. M. 2018

  Abstract

  PURPOSE: Magnetic resonance imaging (MRI) sequences with conical k-space trajectories are able to decrease motion artifacts while achieving ultrashort echo times (UTE). We assessed the performance of free-breathing conical UTE MRI in the evaluation of the pediatric pelvis for suspected appendicitis.METHODS: Our retrospective review of 84 pediatric patients who underwent MRI for suspected appendicitis compared three contrast-enhanced sequences: free-breathing conical UTE, breath-hold three-dimensional (3D) spoiled gradient echo (BH-SPGR), and free-breathing high-resolution 3D SPGR (FB-SPGR). Two radiologists performed blinded and independent evaluations of each sequence for image quality (four point scale), anatomic delineation (four point scale), and diagnostic confidence (five point scale). Subsequently, the three sequences were directly compared for overall image quality (-3 to +3 scale). Scores were compared using Kruskal-Wallis and Wilcoxon signed-rank tests.RESULTS: UTE demonstrated significantly better perceived signal-to-noise ratio (SNR) and fewer artifacts than BH-SPGR and FB-SPGR (means of 3.6 and 3.4, 3.4 and 3.2, 3.1 and 2.7, respectively; p<0.0006). BH-SPGR and FB-SPGR demonstrated significantly better contrast than UTE (means of 3.6, 3.4, and 3.2, respectively; p<0.03). In the remaining categories, UTE performed significantly better than FB-SPGR (p<0.00001), while there was no statistical difference between UTE and BH-SPGR. Direct paired comparisons of overall image quality demonstrated the readers significantly preferred UTE over both BH-SPGR (mean +0.5, p<0.00001) and FB-SPGR (mean +1.2, p<0.00001).CONCLUSIONS: In the evaluation of suspected appendicitis, free-breathing conical UTE MRI performed better in the assessed metrics than FB-SPGR. When compared to BH-SPGR, UTE demonstrated superior perceived SNR and fewer artifacts.

  View details for DOI 10.1007/s00261-018-1705-y

  View details for PubMedID 30066168

• Gallium 68 PSMA-11 PET/MR Imaging in Patients with Intermediate- or High-Risk Prostate Cancer. Radiology Park, S. Y., Zacharias, C., Harrison, C., Fan, R. E., Kunder, C., Hatami, N., Giesel, F., Ghanouni, P., Daniel, B., Loening, A. M., Sonn, G. A., Iagaru, A. 2018: 172232

  Abstract

  Purpose To report the results of dual-time-point gallium 68 (68Ga) prostate-specific membrane antigen (PSMA)-11 positron emission tomography (PET)/magnetic resonance (MR) imaging prior to prostatectomy in patients with intermediate- or high-risk cancer. Materials and Methods Thirty-three men who underwent conventional imaging as clinically indicated and who were scheduled for radical prostatectomy with pelvic lymph node dissection were recruited for this study. A mean dose of 4.1 mCi ± 0.7 (151.7 MBq ± 25.9) of 68Ga-PSMA-11 was administered. Whole-body images were acquired starting 41-61 minutes after injection by using a GE SIGNA PET/MR imaging unit, followed by an additional pelvic PET/MR imaging acquisition at 87-125 minutes after injection. PET/MR imaging findings were compared with findings at multiparametric MR imaging (including diffusion-weighted imaging, T2-weighted imaging, and dynamic contrast material-enhanced imaging) and were correlated with results of final whole-mount pathologic examination and pelvic nodal dissection to yield sensitivity and specificity. Dual-time-point metabolic parameters (eg, maximum standardized uptake value [SUVmax]) were compared by using a paired t test and were correlated with clinical and histopathologic variables including prostate-specific antigen level, Gleason score, and tumor volume. Results Prostate cancer was seen at 68Ga-PSMA-11 PET in all 33 patients, whereas multiparametric MR imaging depicted Prostate Imaging Reporting and Data System (PI-RADS) 4 or 5 lesions in 26 patients and PI-RADS 3 lesions in four patients. Focal uptake was seen in the pelvic lymph nodes in five patients. Pathologic examination confirmed prostate cancer in all patients, as well as nodal metastasis in three. All patients with normal pelvic nodes in PET/MR imaging had no metastases at pathologic examination. The accumulation of 68Ga-PSMA-11 increased at later acquisition times, with higher mean SUVmax (15.3 vs 12.3, P < .001). One additional prostate cancer was identified only at delayed imaging. Conclusion This study found that 68Ga-PSMA-11 PET can be used to identify prostate cancer, while MR imaging provides detailed anatomic guidance. Hence, 68Ga-PSMA-11 PET/MR imaging provides valuable diagnostic information and may inform the need for and extent of pelvic node dissection.

  View details for PubMedID 29786490

• Structured Reporting of Multiphasic CT for Hepatocellular Carcinoma: Effect on Staging and Suitability for Transplant. AJR. American journal of roentgenology Poullos, P. D., Tseng, J. J., Melcher, M. L., Concepcion, W., Loening, A. M., Rosenberg, J., Willmann, J. K. 2018: 1–9

  Abstract

  The purpose of this study is to evaluate whether use of a standardized radiology report template would improve the ability of liver transplant surgeons to diagnose stage T2 hepatocellular carcinoma (HCC) and determine patient suitability to undergo orthotopic liver transplant (OLT).In this retrospective study, a standardized template was devised, and its use was mandated for reporting of liver CT findings for patients with cirrhosis and HCC. Two surgeons analyzed 200 reports (100 before and 100 after template implementation) for descriptions of cirrhosis, portal hypertension, lesion enhancement characteristics, tumor thrombus, portal and superior mesenteric vein patency, and Organ Procurement Transplantation Network (OPTN) class. Ability to determine Milan criteria and surgeon satisfaction were also assessed. Data obtained before and after template implementation were statistically analyzed using the Cochran-Mantel-Haenszel test.Template implementation increased the percentage of reports documenting the presence or absence of portal hypertension (74% to 88% for surgeon 1 and 86% to 87% for surgeon 2; p = 0.042); lesion number (76% to 88% for surgeon 2 [no change for surgeon 1]; p = 0.038), size (95% to 96% for surgeon 1 and 82% to 93% for surgeon 2; p = 0.03), and enhancement (93% to 94% for surgeon 1 and 80% to 91% for surgeon 2; p = 0.049); presence of tumor thrombus (10% to 57% for surgeon 1 and 31% to 63% for surgeon 2; p < 0.001); and OPTN class (8% to 82% for surgeon 1 and 2% to 81% for surgeon 2; p < 0.001). The surgeons were significantly more able to determine the presence of T2 disease and qualification for exception points after implementation of the template (increasing from 80% to 94%; p = 0.025). Satisfaction with reports also improved (p < 0.0001).The reporting template improved determination of patient suitability to undergo transplant according to the Milan criteria.

  View details for DOI 10.2214/AJR.17.18725

  View details for PubMedID 29470153

• The impact of computed high b-value images on the diagnostic accuracy of DWI for prostate cancer: A receiver operating characteristics analysis. Scientific reports Ning, P., Shi, D., Sonn, G. A., Vasanawala, S. S., Loening, A. M., Ghanouni, P., Obara, P., Shin, L. K., Fan, R. E., Hargreaves, B. A., Daniel, B. L. 2018; 8 (1): 3409

  Abstract

  To evaluate the performance of computed high b value diffusion-weighted images (DWI) in prostate cancer detection. 97 consecutive patients who had undergone multiparametric MRI of the prostate followed by biopsy were reviewed. Five radiologists independently scored 138 lesions on native high b-value images (b = 1200 s/mm2), apparent diffusion coefficient (ADC) maps, and computed high b-value images (contrast equivalent to b = 2000 s/mm2) to compare their diagnostic accuracy. Receiver operating characteristic (ROC) analysis and McNemar's test were performed to assess the relative performance of computed high b value DWI, native high b-value DWI and ADC maps. No significant difference existed in the area under the curve (AUC) for ROCs comparing B1200 (b = 1200 s/mm2) to computed B2000 (c-B2000) in 5 readers. In 4 of 5 readers c-B2000 had significantly increased sensitivity and/or decreased specificity compared to B1200 (McNemar's p < 0.05), at selected thresholds of interpretation. ADC maps were less accurate than B1200 or c-B2000 for 2 of 5 readers (P < 0.05). This study detected no consistent improvement in overall diagnostic accuracy using c-B2000, compared with B1200 images. Readers detected more cancer with c-B2000 images (increased sensitivity) but also more false positive findings (decreased specificity).

  View details for PubMedID 29467370

• Detection of Recurrent Prostate Cancer Using Ga-68-RM2 PET/MRI in Patients with Negative Conventional Imaging Harrison, C., Sonni, I., Loening, A., Vasanawala, S., Lagaru, A. SOC NUCLEAR MEDICINE INC. 2017

  View details for Web of Science ID 000404949903111

• Increased Speed and Image Quality for Pelvic Single-Shot Fast Spin-Echo Imaging with Variable Refocusing Flip Angles and Full-Fourier Acquisition. Radiology Loening, A. M., Litwiller, D. V., Saranathan, M., Vasanawala, S. S. 2017; 282 (2): 561-568

  Abstract

  Purpose To assess image quality and speed improvements for single-shot fast spin-echo (SSFSE) with variable refocusing flip angles and full-Fourier acquisition (vrfSSFSE) pelvic imaging via a prospective trial performed in the context of uterine leiomyoma evaluation. Materials and Methods Institutional review board approval and informed consent were obtained. vrfSSFSE and conventional SSFSE sagittal and coronal oblique acquisitions were performed in 54 consecutive female patients referred for 3-T magnetic resonance (MR) evaluation of known or suspected uterine leiomyomas. Two radiologists who were blinded to the image acquisition technique semiquantitatively scored images on a scale from -2 to 2 for noise, image contrast, sharpness, artifacts, and perceived ability to evaluate uterine, ovarian, and musculoskeletal structures. The null hypothesis of no significant difference between pulse sequences was assessed with a Wilcoxon signed rank test by using a Holm-Bonferroni correction for multiple comparisons. Results Because of reductions in specific absorption rate, vrfSSFSE imaging demonstrated significantly increased speed (more than twofold, P < .0001), with mean repetition times compared with conventional SSFSE imaging decreasing from 1358 to 613 msec for sagittal acquisitions and from 1494 to 621 msec for coronal oblique acquisitions. Almost all assessed image quality and perceived diagnostic capability parameters were significantly improved with vrfSSFSE imaging. These improvements included noise, sharpness, and ability to evaluate the junctional zone, myometrium, and musculoskeletal structures for both sagittal acquisitions (mean values of 0.56, 0.63, 0.42, 0.56, and 0.80, respectively; all P values < .0001) and coronal oblique acquisitions (mean values of 0.81, 1.09, 0.65, 0.93, and 1.12, respectively; all P values < .0001). For evaluation of artifacts, there was an insufficient number of cases with differences to allow statistical testing. Conclusion Compared with conventional SSFSE acquisition, vrfSSFSE acquisition increases 3-T imaging speed via reduced specific absorption rate and leads to significant improvements in perceived image quality and perceived diagnostic capability when evaluating pelvic structures. (©) RSNA, 2016 Online supplemental material is available for this article.

  View details for DOI 10.1148/radiol.2016151574

  View details for PubMedID 27564132

  View details for PubMedCentralID PMC5283873

• Prostate Magnetic Resonance Imaging Interpretation Varies Substantially Across Radiologists. European urology focus Sonn, G. A., Fan, R. E., Ghanouni, P., Wang, N. N., Brooks, J. D., Loening, A. M., Daniel, B. L., To'o, K. J., Thong, A. E., Leppert, J. T. 2017

  Abstract

  Multiparametric magnetic resonance imaging (mpMRI) interpreted by experts is a powerful tool for diagnosing prostate cancer. However, the generalizability of published results across radiologists of varying expertise has not been verified.To assess variability in mpMRI reporting and diagnostic accuracy across radiologists of varying experience in routine clinical care.Men who underwent mpMRI and MR-fusion biopsy between 2014-2016. Each MRI scan was read by one of nine radiologists using the Prostate Imaging Reporting and Data System (PIRADS) and was not re-read before biopsy. Biopsy histopathology was the reference standard.Outcomes were the PIRADS score distribution and diagnostic accuracy across nine radiologists. We evaluated the association between age, prostate-specific antigen, PIRADS score, and radiologist in predicting clinically significant cancer (Gleason ≥7) using multivariable logistic regression. We conducted sensitivity analyses for case volume and changes in accuracy over time.We analyzed data for 409 subjects with 503 MRI lesions. While the number of lesions (mean 1.2 lesions/patient) did not differ across radiologists, substantial variation existed in PIRADS distribution and cancer yield. The significant cancer detection rate was 3-27% for PIRADS 3 lesions, 23-65% for PIRADS 4, and 40-80% for PIRADS 5 across radiologists. Some 13-60% of men with a PIRADS score of <3 on MRI harbored clinically significant cancer. The area under the receiver operating characteristic curve varied from 0.69 to 0.81 for detection of clinically significant cancer. PIRADS score (p<0.0001) and radiologist (p=0.042) were independently associated with cancer in multivariable analysis. Neither individual radiologist volume nor study period impacted the results. MRI scans were not retrospectively re-read by all radiologists, precluding measurement of inter-observer agreement.We observed considerable variability in PIRADS score assignment and significant cancer yield across radiologists. We advise internal evaluation of mpMRI accuracy before widespread adoption.We evaluated the interpretation of multiparametric magnetic resonance imaging of the prostate in routine clinical care. Diagnostic accuracy depends on the Prostate Imaging Reporting and Data System score and the radiologist.

  View details for PubMedID 29226826

• Relative value of three whole-body MR approaches for PET-MR, including gadofosveset-enhanced MR, in comparison to PET-CT. Clinical imaging Obara, P., Loening, A., Taviani, V., Iagaru, A., Hargreaves, B. A., Vasanawala, S. 2017; 48: 62–68

  Abstract

  Evaluate MR protocol for PET-MR including coronal DWI (cDWI), fat-suppressed T2 (T2w), and gadofosveset-enhanced T1 (CE).18 patients underwent same-day PET-CT and PET-MR. Image quality and performance of each sequence, and combination of all three sequences, was evaluated with respect to PET-CT.Lesion conspicuity was best on cDWI, while delineation was best on CE. Considering all three sequences combined, both readers showed good sensitivity and specificity (>80%). Relative sensitivity was highest on CE and lowest on T2w.Whole-body MR performed well in detecting malignant lesions compared to PET-CT. CE showed overall highest performance.

  View details for DOI 10.1016/j.clinimag.2017.09.016

  View details for PubMedID 29031209

• Variable refocusing flip angle single-shot fast spin echo imaging of liver lesions: increased speed and lesion contrast. Abdominal radiology (New York) Hicks, R. M., Loening, A. M., Ohliger, M. A., Vasanawala, S. S., Hope, T. A. 2017

  Abstract

  To evaluate acquisition time and clinical image quality of a variable refocusing flip angle (vrf) single-shot fast spin echo (SSFSE) sequence in comparison with a conventional SSFSE sequence for imaging of liver lesions in patients undergoing whole-body PET/MRI for oncologic staging.A vrfSSFSE sequence was acquired in 43 patients with known pancreatic neuroendocrine tumors undergoing68Ga-DOTA-TOC PET on a simultaneous time-of-flight 3.0T PET/MRI. Liver lesions ≥1.5 cm with radionucleotide uptake were analyzed. Contrast-to-noise ratios (CNRs) were measured, and four blinded radiologists assessed overall image quality. Differences in repetition time and CNR were assessed using a paired Student's t test with p < 0.05 considered statistically significant. Inter-reader variability was assessed with Fleiss' kappa statistic.53 eligible lesions in 27 patients were included for analysis. vrfSSFSE demonstrated higher mean lesion CNR compared to SSFSE (9.9 ± 4.1 vs. 6.7 ± 4.1, p < 0.001). Mean repetition time (TR) was 679 ± 97 ms for the vrfSSFSE sequence compared to 1139 ± 106 ms for SSFSE (p < 0.0001), corresponding to a 1.7-fold decrease in acquisition time. Overall quality of liver lesion and common bile duct images with the vrfSSFSE sequence was graded as superior than or equivalent to the SSFSE sequence for 59% and 67% of patients, respectively.Compared to conventional SSFSE, vrfSSFSE resulted in improved lesion contrast on simultaneous PET/MRI in patients with liver metastases. Due to decreased SAR demands, vrfSSFSE significantly decreased TR, allowing coverage of the entire liver in a single twenty-second breath hold. This may have important clinical implications in the setting of PET/MRI, where scan time is limited by the necessity of whole-body image acquisition in addition to bed specific imaging.

  View details for PubMedID 28689221

• Prospective Evaluation of 68Ga-RM2 PET/MRI in Patients with Biochemical Recurrence of Prostate Cancer and Negative Conventional Imaging. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Minamimoto, R., Sonni, I., Hancock, S., Vasanawala, S., Loening, A., Gambhir, S. S., Iagaru, A. 2017

  Abstract

  Purpose:68Ga-labeled DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (68Ga-RM2) is a synthetic bombesin receptor antagonist that targets gastrin-releasing peptide receptors (GRPr). GRPr proteins are highly overexpressed in several human tumors, including prostate cancer. We present data from the use of 68Ga-RM2 in patients with biochemical recurrence (BCR) of prostate cancer (PC) and negative conventional imaging (CI). Methods: We enrolled 32 men with BCR PC, 59-83 year-old (mean±standard deviation (SD): 68.7±6.4). Imaging started at 40-69 minutes (mean±SD: 50.5±6.8) after injection of 133.2-151.7 MBq (mean±SD: 140.6±7.4) of 68Ga-RM2 using a time-of-flight (TOF)-enabled simultaneous positron emission tomography (PET) / magnetic resonance imaging (MRI) scanner. T1-weighted (T1w), T2-weighted (T2w) and diffusion-weighted images (DWI) were acquired. Results: All patients had rising prostate specific antigen (PSA) (range: 0.3-119.0 ng/mL; mean±SD: 10.1 ± 21.3) and negative CI (CT or MRI, and 99mTc MDP bone scan) prior to enrollment. The observed 68Ga-RM2 PET detection rate was 71.8%. 68Ga-RM2 PET identified recurrent prostate cancer in 23 of the 32 participants, while the simultaneous MRI scan identified findings compatible with recurrent prostate cancer in 11 of the 32 patients. PSA velocity (PSAv) values were 0.32±0.59 ng/ml/year (range: 0.04-1.9) in patients with negative PET scans and 2.51±2.16 ng/ml/year (range: 0.13-8.68) in patients with positive PET scans (P: 0.006). Conclusion:68Ga-RM2 PET can be used for assessment of GRPr expression in patients with BCR PC. High uptake in multiple areas compatible with cancer lesions suggests that 68Ga-RM2 is a promising PET radiopharmaceutical for localization of disease in participants with BCR PC and negative CI.

  View details for PubMedID 29084827

• Pilot Comparison of Ga-68-RM2 PET and Ga-68-PSMA-11 PET in Patients with Biochemically Recurrent Prostate Cancer JOURNAL OF NUCLEAR MEDICINE Minamimoto, R., Hancock, S., Schneider, B., Chin, F. T., Jamali, M., Loening, A., Vasanawala, S., Gambhir, S. S., Iagaru, A. 2016; 57 (4): 557-562

  Abstract

  Glu-NH-CO-NH-Lys-(Ahx)-[(68)Ga(HBED-CC)] ((68)Ga-PSMA-11) is a PET tracer that can detect prostate cancer relapses and metastases by binding to the extracellular domain of PSMA.(68)Ga-labeled DOTA-4-amino-1-carboxymethyl-piperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 ((68)Ga-RM2) is a synthetic bombesin receptor antagonist that targets gastrin-releasing peptide receptors. We present pilot data on the biodistribution of these PET tracers in a small cohort of patients with biochemically recurrent prostate cancer.Seven men (mean age ± SD, 74.3 ± 5.9 y) with biochemically recurrent prostate cancer underwent both(68)Ga-PSMA-11 PET/CT and(68)Ga-RM2 PET/MRI scans. SUVmaxand SUVmeanwere recorded for normal tissues and areas of uptake outside the expected physiologic biodistribution.All patients had a rising level of prostate-specific antigen (mean ± SD, 13.5 ± 11.5) and noncontributory results on conventional imaging.(68)Ga-PSMA-11 had the highest physiologic uptake in the salivary glands and small bowel, with hepatobiliary and renal clearance noted, whereas(68)Ga-RM2 had the highest physiologic uptake in the pancreas, with renal clearance noted. Uptake outside the expected physiologic biodistribution did not significantly differ between(68)Ga-PSMA-11 and(68)Ga-RM2; however,(68)Ga-PSMA-11 localized in a lymph node and seminal vesicle in a patient with no abnormal(68)Ga-RM2 uptake. Abdominal periaortic lymph nodes were more easily visualized by(68)Ga-RM2 in two patients because of lack of interference by radioactivity in the small intestine.(68)Ga-PSMA-11 and(68)Ga-RM2 had distinct biodistributions in this small cohort of patients with biochemically recurrent prostate cancer. Additional work is needed to understand the expression of PSMA and gastrin-releasing peptide receptors in different types of prostate cancer.

  View details for DOI 10.2967/jnumed.115.168393

  View details for PubMedID 26659347

• High temporal resolution dynamic MRI and arterial input function for assessment of GFR in pediatric subjects. Magnetic resonance in medicine Yoruk, U., Saranathan, M., Loening, A. M., Hargreaves, B. A., Vasanawala, S. S. 2016; 75 (3): 1301-1311

  Abstract

  To introduce a respiratory-gated high-spatiotemporal-resolution dynamic-contrast-enhanced MRI technique and a high-temporal-resolution aortic input function (HTR-AIF) estimation method for glomerular filtration rate (GFR) assessment in children.A high-spatiotemporal-resolution DCE-MRI method with view-shared reconstruction was modified to incorporate respiratory gating, and an AIF estimation method that uses a fraction of the k-space data from each respiratory period was developed (HTR-AIF). The method was validated using realistic digital phantom simulations and demonstrated on clinical subjects. The GFR estimates using HTR-AIF were compared with estimates obtained by using an AIF derived directly from the view-shared images.Digital phantom simulations showed that using the HTR-AIF technique gives more accurate AIF estimates (RMSE = 0.0932) compared with the existing estimation method (RMSE = 0.2059) that used view-sharing (VS). For simulated GFR > 27 mL/min, GFR estimation error was between 32% and 17% using view-shared AIF, whereas estimation error was less than 10% using HTR-AIF. In all clinical subjects, the HTR-AIF method resulted in higher GFR estimations than the view-shared method.The HTR-AIF method improves the accuracy of both the AIF and GFR estimates derived from the respiratory-gated acquisitions, and makes GFR estimation feasible in free-breathing pediatric subjects. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

  View details for DOI 10.1002/mrm.25731

  View details for PubMedID 25946307

• Ga-68-DOTA-Bombesin (Ga-68-RM2 or Ga-68-Bombesin) PET versus Ga-68-PSMA PET: A pilot prospective evaluation in patients with biochemical recurrence of prostate cancer. Iagaru, A., Minamimoto, R., Hancock, S., Mittra, E., Loening, A., Vasanawala, S. AMER SOC CLINICAL ONCOLOGY. 2016

  View details for DOI 10.1200/jco.2016.34.2_suppl.331

  View details for Web of Science ID 000378109100329

• Increased Speed and Image Quality in Single-Shot Fast Spin Echo Imaging Via Variable Refocusing Flip Angles JOURNAL OF MAGNETIC RESONANCE IMAGING Loening, A. M., Saranathan, M., Ruangwattanapaisarn, N., Litwiller, D. V., Shimakawa, A., Vasanawala, S. S. 2015; 42 (6): 1747-1758

  Abstract

  To develop and validate clinically a single-shot fast spin echo (SSFSE) sequence utilizing variable flip angle refocusing pulses to shorten acquisition times via reductions in specific absorption rate (SAR) and improve image quality.A variable refocusing flip angle SSFSE sequence (vrfSSFSE) was designed and implemented, with simulations and volunteer scans performed to determine suitable flip angle modulation parameters. With Institutional Review Board (IRB) approval/informed consent, patients referred for 3T abdominal magnetic resonance imaging (MRI) were scanned with conventional SSFSE and either half-Fourier (n = 25) or full-Fourier vrfSSFSE (n = 50). Two blinded radiologists semiquantitatively scored images on a scale from -2 to 2 for contrast, noise, sharpness, artifacts, cardiac motion-related signal loss, and the ability to evaluate the pancreas and kidneys.vrfSSFSE demonstrated significantly increased speed (∼2-fold, P < 0.0001). Significant improvements in image quality parameters with full-Fourier vrfSSFSE included increased contrast, sharpness, and visualization of pancreatic and renal structures with higher bandwidth technique (mean scores 0.37, 0.83, 0.62, and 0.31, respectively, P ≤ 0.001), and decreased image noise and improved visualization of renal structures when used with an equal bandwidth technique (mean scores 0.96 and 0.35, respectively, P < 0.001). Increased cardiac motion-related signal loss with full-Fourier vrfSSFSE was seen in the pancreas but not the kidney.vrfSSFSE increases speed at 3T over conventional SSFSE via reduced SAR, and when combined with full-Fourier acquisition can improve image quality, although with some increased sensitivity to cardiac motion-related signal loss. J. Magn. Reson. Imaging 2015.

  View details for DOI 10.1002/jmri.24941

  View details for Web of Science ID 000368258100032

• Prospective Comparison of 99mTc-MDP Scintigraphy, Combined 18F-NaF and 18F-FDG PET/CT, and Whole-Body MRI in Patients with Breast and Prostate Cancer. Journal of nuclear medicine Minamimoto, R., Loening, A., Jamali, M., Barkhodari, A., Mosci, C., Jackson, T., Obara, P., Taviani, V., Gambhir, S. S., Vasanawala, S., Iagaru, A. 2015; 56 (12): 1862-1868

  Abstract

  We prospectively evaluated the combined (18)F-NaF/(18)F-FDG PET/CT in patients with breast and prostate cancers, and compared the results to (99m)Tc MDP bone scintigraphy (BS) and whole-body MRI (WBMRI).30 patients (15 women with breast cancer and 15 men with prostate cancer) referred for standard of care BS were prospectively enrolled in this study. (18)F-NaF/(18)F-FDG PET/CT and WBMRI were performed following BS. WBMRI protocol consisted of both non-contrast enhanced and contrast enhanced sequences. Lesions detected with each test were tabulated and the results were compared.For extra skeletal lesions, (18)F-/(18)F-FDG PET/CT and WBMRI had no statistically significant differences in sensitivity (92.9% vs 92.9%, P = 1.00), PPV (81.3% vs 86.7%, P = 0.68) and accuracy (76.5% vs 82.4%, P = 0.56). However, (18)F-/(18)F-FDG PET/CT showed significantly higher sensitivity and accuracy than WBMRI (96.2% vs 81.4%, P<0.001, 89.8% vs 74.7%, P = 0.01) and BS (96.2% vs 64.6%, P<0.001, 89.8% vs 65.9%, P<0.001) for the detection of skeletal lesions. Overall, (18)F-/(18)F-FDG PET/CT showed higher sensitivity and accuracy than WBMRI (95.7% vs 83.3%, P<0.002, 87.6% vs 76.0%, P< 0.02), but not statistically significant when compared to a combination of WBMRI and BS (95.7% vs 91.6%, P = 0.17, 87.6% vs 83.0%, P = 0.53). (18)F-/(18)F-FDG PET/CT showed no significant difference with a combination of (18)F-/(18)F-FDG PET/CT and WBMRI. No statistically significant differences in PPV were noted among the 3 examinations.The (18)F NaF/(18)F FDG PET/CT is superior to WBMRI and (99m)Tc-MDP scintigraphy for evaluation of skeletal disease extent. Further, (18)F NaF/(18)F FDG PET/CT and WBMRI detected extra-skeletal disease that may change the management of these patients. The (18)F NaF/(18)F FDG PET/CT provide similar diagnostic ability with combination of WBMRI and BS in patients with breast and prostate cancers. Larger cohorts are needed in order to confirm these preliminary findings, ideally using the newly introduced simultaneous PET/MRI scanners.

  View details for DOI 10.2967/jnumed.115.162610

  View details for PubMedID 26405167

• Faster pediatric 3-T abdominal magnetic resonance imaging: comparison between conventional and variable refocusing flip-angle single-shot fast spin-echo sequences. Pediatric radiology Ruangwattanapaisarn, N., Loening, A. M., Saranathan, M., Litwiller, D. V., Vasanawala, S. S. 2015; 45 (6): 847-854

  Abstract

  Single-shot fast spin echo (SSFSE) is particularly appealing in pediatric patients because of its motion robustness. However radiofrequency energy deposition at 3 tesla forces long pauses between slices, leading to longer scans, longer breath-holds and more between-slice motion.We sought to learn whether modulation of the SSFSE refocusing flip-angle train could reduce radiofrequency energy deposition without degrading image quality, thereby reducing inter-slice pauses and overall scan times.We modulated the refocusing flip-angle train for SSFSE to minimize energy deposition while minimizing blurring and motion-related signal loss. In a cohort of 50 consecutive patients (25 boys, mean age 5.5 years, range 1 month to 17 years) referred for abdominal MRI we obtained standard SSFSE and variable refocusing flip-angle (vrfSSFSE) images and recorded sequence scan times. Two readers independently scored the images in blinded, randomized order for noise, tissue contrast, sharpness, artifacts and left lobe hepatic signal uniformity on a four-point scale. The null hypothesis of no difference between SSFSE and vrfSSFSE image-quality was assessed with a Mann-Whitney U test, and the null hypothesis of no scan time difference was assessed with the paired t-test.SSFSE and vrfSSFSE mean acquisition times were 54.3 and 26.2 s, respectively (P-value <0.0001). For each reader, SSFSE and vrfSSFSE noise, tissue contrast, sharpness and artifacts were not significantly different (P-values 0.18-0.86). However, SSFSE had better left lobe hepatic signal uniformity (P < 0.01, both readers).vrfSSFSE is twice as fast as SSFSE, with equivalent image quality with the exception of left hepatic lobe signal heterogeneity.

  View details for DOI 10.1007/s00247-014-3227-2

  View details for PubMedID 25433510

  View details for PubMedCentralID PMC4449830

• Imaging patients with breast and prostate cancers using combined F-18 NaF/F-18 FDG and TOF simultaneous PET/MRI Iagaru, A., Minamimoto, R., Jamali, M., Barkhodari, A., Obara, P., Loening, A., Taviani, V., Mittra, E., Gambhir, S., Vasanawala, S. SOC NUCLEAR MEDICINE INC. 2015

  View details for Web of Science ID 000358738803075

• Prospective evaluation of Tc-99m MDP scintigraphy, F-18 NaF/F-18 FDG PET/CT and WBMRI in patients with breast and prostate cancers Iagaru, A., Minamimoto, R., Mosci, C., Jamali, M., Barkhodari, A., Loening, A., Taviani, V., Mittra, E., Gambhir, S., Vasanawala, S. SOC NUCLEAR MEDICINE INC. 2015

  View details for Web of Science ID 000358738802275

• Prospective evaluation of combined NaF/FDG PET/CT and whole-body MRI in patients with breast and prostate cancer Iagaru, A., Mosci, C., Jamali, M., Loening, A., Mittra, E., Gambhir, S., Vasanawala, S. SOC NUCLEAR MEDICINE INC. 2014

  View details for Web of Science ID 000361438102203

• Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy GENE THERAPY Chen, I. Y., Gheysens, O., Ray, S., Wang, Q., Padmanabhan, P., Paulmurugan, R., Loening, A. M., Rodriguez-Porcel, M., Willmann, J. K., Sheikh, A. Y., Nielsen, C. H., Hoyt, G., Contag, C. H., Robbins, R. C., Biswal, S., Wu, J. C., Gambhir, S. S. 2010; 17 (7): 827-838

  Abstract

  Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

  View details for DOI 10.1038/gt.2010.30

  View details for PubMedID 20237511

• A red-shifted Renilla luciferase for transient reporter-gene expression NATURE METHODS Loening, A. M., Dragulescu-Andrasi, A., Gambhir, S. S. 2010; 7 (1): 5-6

  View details for DOI 10.1038/nmeth0110-05

  View details for Web of Science ID 000273128300003

  View details for PubMedID 20038949

• BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals FASEB JOURNAL De, A., Ray, P., Loening, A. M., Gambhir, S. S. 2009; 23 (8): 2702-2709

  Abstract

  Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (approximately 85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3-based assays will allow imaging of protein-protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.

  View details for DOI 10.1096/fj.08-118919

  View details for PubMedID 19351700

• Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase METABOLIC ENGINEERING Goerke, A. R., Loening, A. M., Gambhir, S. S., Swartz, J. R. 2008; 10 (3-4): 187-200

  Abstract

  Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.

  View details for DOI 10.1016/j.ymben.2008.04.001

  View details for PubMedID 18555198

• Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis JOURNAL OF MOLECULAR BIOLOGY Loening, A. M., Fenn, T. D., Gambhir, S. S. 2007; 374 (4): 1017-1028

  Abstract

  Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

  View details for DOI 10.1016/j.jmb.2007.09.078

  View details for PubMedID 17980388

• Red-shifted Renilla reniformis luciferase variants for imaging in living subjects NATURE METHODS Loening, A. M., Wu, A. M., Gambhir, S. S. 2007; 4 (8): 641-643

  Abstract

  The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.

  View details for DOI 10.1038/NMETH1070

  View details for PubMedID 17618292

• An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects CANCER RESEARCH De, A., Loening, A. M., Gambhir, S. S. 2007; 67 (15): 7175-7183

  Abstract

  Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP(2) acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals.

  View details for DOI 10.1158/0008-5472.CAN-06-4623

  View details for PubMedID 17671185

• Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging JOURNAL OF NUCLEAR MEDICINE Deroose, C. M., De, A., Loening, A. M., Chow, P. L., Ray, P., Chatziioannou, A. F., Gambhir, S. S. 2007; 48 (2): 295-303

  Abstract

  Recent developments have established molecular imaging of mouse models with small-animal PET and bioluminescence imaging (BLI) as an important tool in cancer research. One of the disadvantages of these imaging modalities is the lack of anatomic information. We combined small-animal PET and BLI technology with small-animal CT to obtain fusion images with both molecular and anatomic information.We used small-animal PET/CT and BLI to detect xenografts of different cell lines and metastases of a melanoma cell line (A375M-3F) that had been transduced with a lentiviral vector containing a trimodality imaging reporter gene encoding a fusion protein with Renilla luciferase, monomeric red fluorescent protein, and a mutant herpes simplex virus type 1 thymidine kinase.Validation studies in mouse xenograft models showed a good coregistration of images from both PET and CT. Melanoma metastases were detected by 18F-FDG PET, 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) PET, CT, and BLI and confirmed by ex vivo assays of Renilla luciferase and mutant thymidine kinase expression. 18F-FHBG PET/CT allowed detection and localization of lesions that were not seen on CT because of poor contrast resolution and were not seen on 18F-FDG PET because of higher background uptake relative to 18F-FHBG.The combination of 18F-FHBG PET, small-animal CT, and BLI allows a sensitive and improved quantification of tumor burden in mice. This technique is potentially useful for the study of the biologic determinants of metastasis and for the evaluation of novel cancer treatments.

  View details for PubMedID 17268028

• Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors PROTEIN ENGINEERING DESIGN & SELECTION Venisnik, K. M., Olafsen, T., Loening, A. M., Iyer, M., Gambhir, S. S., Wu, A. M. 2006; 19 (10): 453-460

  Abstract

  An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.

  View details for DOI 10.1093/protein/gzl030

  View details for Web of Science ID 000240544900003

  View details for PubMedID 16882674

• Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output PROTEIN ENGINEERING DESIGN & SELECTION Loening, A. M., Fenn, T. D., Wu, A. M., Gambhir, S. S. 2006; 19 (9): 391-400

  Abstract

  Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

  View details for DOI 10.1093/protein/gzl023

  View details for Web of Science ID 000240544600001

  View details for PubMedID 16857694

• Self-illuminating quantum dot conjugates for in vivo imaging NATURE BIOTECHNOLOGY So, M. K., Xu, C. J., Loening, A. M., Gambhir, S. S., Rao, J. H. 2006; 24 (3): 339-343

  Abstract

  Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.

  View details for DOI 10.1038/nbt1188

  View details for PubMedID 16501578

• HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Zhang, Y., So, M., Loening, A. M., Yao, H., Gambhir, S. S., Rao, J. 2006; 45 (30): 4936-4940

  View details for DOI 10.1002/anie.200601197

  View details for PubMedID 16807952

• Creating self-illuminating quantum dot conjugates NATURE PROTOCOLS So, M., Loening, A. M., Gambhir, S. S., Rao, J. 2006; 1 (3): 1160-1164

  Abstract

  Semiconductor quantum dots are inorganic fluorescent nanocrystals that, because of their unique optical properties compared with those of organic fluorophores, have become popular as fluorescent imaging probes. Although external light excitation is typically required for imaging with quantum dots, a new type of quantum dot conjugate has been reported that can luminesce with no need for external excitation. These self-illuminating quantum dot conjugates can be prepared by coupling of commercially available carboxylate-presenting quantum dots to the light-emitting protein Renilla luciferase. When the conjugates are exposed to the luciferase's substrate coelenterazine, the energy released by substrate catabolism is transferred to the quantum dots through bioluminescence resonance energy transfer, leading to quantum dot light emission. This protocol describes step-by-step procedures for the preparation and characterization of these self-illuminating quantum dot conjugates. The preparation process is relatively simple and can be done in less than 2 hours. The availability of self-illuminating quantum dot conjugates will provide many new possibilities for in vivo imaging and detection, such as monitoring of in vivo cell trafficking, multiplex bioluminescence imaging and new quantum dot-based biosensors.

  View details for DOI 10.1038/nprot.2006.162

  View details for PubMedID 17406398

• AMIDE: a free software tool for multimodality medical image analysis. Molecular imaging Loening, A. M., Gambhir, S. S. 2003; 2 (3): 131-137

  Abstract

  Amide's a Medical Image Data Examiner (AMIDE) has been developed as a user-friendly, open-source software tool for displaying and analyzing multimodality volumetric medical images. Central to the package's abilities to simultaneously display multiple data sets (e.g., PET, CT, MRI) and regions of interest is the on-demand data reslicing implemented within the program. Data sets can be freely shifted, rotated, viewed, and analyzed with the program automatically handling interpolation as needed from the original data. Validation has been performed by comparing the output of AMIDE with that of several existing software packages. AMIDE runs on UNIX, Macintosh OS X, and Microsoft Windows platforms, and it is freely available with source code under the terms of the GNU General Public License.

  View details for PubMedID 14649056

• Whole-body skeletal imaging in mice utilizing microPET: optimization of reproducibility and applications in animal models of bone disease EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Berger, F., Lee, Y. P., Loening, A. M., Chatziioannou, A., Freedland, S. J., Leahy, R., Lieberman, J. R., Belldegrun, A. S., Sawyers, C. L., Gambhir, S. S. 2002; 29 (9): 1225-1236

  Abstract

  The aims were to optimize reproducibility and establish [(18)F]fluoride ion bone scanning in mice, using a dedicated small animal positron emission tomography (PET) scanner (microPET) and to correlate functional findings with anatomical imaging using computed tomography (microCAT). Optimal tracer uptake time for [(18)F]fluoride ion was determined by performing dynamic microPET scans. Quantitative reproducibility was measured using region of interest (ROI)-based counts normalized to (a) the injected dose, (b) integral of the heart time-activity curve, or (c) ROI over the whole skeleton. Bone lesions were repetitively imaged. Functional images were correlated with X-ray and microCAT. The plateau of [(18)F]fluoride uptake occurs 60 min after injection. The highest reproducibility was achieved by normalizing to an ROI over the whole skeleton, with a mean percent coefficient of variation [(SD/mean) x 100] of <15%-20%. Benign and malignant bone lesions were successfully repetitively imaged. Preliminary correlation of microPET with microCAT demonstrated the high sensitivity of microPET and the ability of microCAT to detect small osteolytic lesions. Whole-body [(18)F]fluoride ion bone imaging using microPET is reproducible and can be used to serially monitor normal and pathological changes to the mouse skeleton. Morphological imaging with microCAT is useful to display correlative changes in anatomy. Detailed in vivo studies of the murine skeleton in various small animal models of bone diseases should now be possible.

  View details for DOI 10.1007/s00259-002-0850-1

  View details for Web of Science ID 000178150400020

  View details for PubMedID 12418463

• Host metalloproteinases in Lyme arthritis ARTHRITIS AND RHEUMATISM Hu, L. T., Eskildsen, M. A., Masgala, C., STEERE, A. C., Arner, E. C., Pratta, M. A., GRODZINSKY, A. J., Loening, A., Perides, G. 2001; 44 (6): 1401-1410

  Abstract

  To assess the role of matrix metalloproteinases (MMPs) in cartilage and bone erosions in Lyme arthritisWe examined synovial fluid from 10 patients with Lyme arthritis for the presence of MMP-2, MMP-3, MMP-9, and "aggrecanase" activity using gelatinolytic zymography and immunoblot analysis. We developed an in vitro model of Lyme arthritis using cartilage explants and observed changes in cartilage degradation in the presence of Borrelia burgdorferi and/or various protease inhibitors.Synovial fluid from patients with Lyme arthritis was found to contain at least 3 MMPs: gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9). In addition, there was evidence in 2 patients of "aggrecanase" activity not accounted for by the above enzymes. Infection of cartilage explants with B. burgdorferi resulted in induction of MMP-3, MMP-9, and "aggrecanase" activity. Increased induction of these enzymes by B. burgdorferi alone was not sufficient to cause cartilage destruction in the explants as measured by glycosaminoglycan (GAG) and hydroxyproline release. However, addition of plasminogen, which can act as an MMP activator, to cultures resulted in significant GAG and hydroxyproline release in the presence of B. burgdorferi. The MMP inhibitor batimastat significantly reduced the GAG release and completely inhibited the collagen degradation.MMPs are found in synovial fluids from patients with Lyme arthritis and are induced from cartilage tissue by the presence of B. burgdorferi. Inhibition of MMP activity prevents B. burgdorferi-induced cartilage degradation in vitro.

  View details for Web of Science ID 000171751100023

  View details for PubMedID 11407701

• A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants JOURNAL OF BIOMECHANICS Frank, E. H., Jin, M., Loening, A. M., Levenston, M. E., GRODZINSKY, A. J. 2000; 33 (11): 1523-1527

  Abstract

  We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 microm and sinusoidal rotations as small as 0.01 degrees. Axial resolution is 50 nm for applying sinewaves as low as 10 microm (or 1% based on a 1 mm thickness) or as large as 100 microm. Rotational resolution is 0.0005 degrees. The machine is small enough (30 cm high x 25 cm x 20 cm) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of (35)S-sulfate and (3)H-proline) over controls held at the same static offset compression.

  View details for Web of Science ID 000089948900023

  View details for PubMedID 10940414

• Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Loening, A. M., James, I. E., Levenston, M. E., Badger, A. M., Frank, E. H., Kurz, B., Nuttall, M. E., Hung, H. H., Blake, S. M., Grodzinsky, A. J., Lark, M. W. 2000; 381 (2): 205-212

  Abstract

  A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.

  View details for Web of Science ID 000089557300004

  View details for PubMedID 11032407