Next generation of radiotracers for sentinel lymph node biopsy: What is still necessary to establish new imaging paradigms? / Nueva generación de radiotrazadores para la biopsia del ganglio centinela: ¿qué es necesario para establecer nuevos paradigmas de imagen?

Sentinel lymph node (SLN) biopsy is now the standard of care for regional staging in several solid tumors. The interstitial administration of a radiotracer around the primary tumor provide the possibility to sequentially obtain images with a gamma camera and visualize lymphatic mapping and the SLN. There is, however, a large geographical variability in those radiotracers and nanocolloids ranging from 15-100nm which are most widely employed in Europe, while filtered and unfiltered 99mTc-sulfur colloid (range 20-1000nm) is usually used in the USA with different drawbacks in its use. The new radiotracer 99mTc-Tilmanocept, designed specifically for the identification of SLNs and recently becoming commercially available in USA and Europe, appears to have the potency to overcome the shortcomings described for the conventional radiotracers used until now for SLN biopsy and at the same time to transform current imaging paradigms. After delineating the challenges for the next generation of radiotracers, this paper discusses the properties of 99mTc-Tilmanocept, its validation process for SLN biopsy and its emerging clinical applications in various malignancies.

Radiation doses in the surroundings of patients undergoing nuclear medicine diagnostic studies.

Dose rate measurements were performed at 0, 0.5, and 1 m from the external surface of 79 patients corresponding to the most frequent studies: 99mTc-cardiac with reinjection, 99mTc-cardiac single injection, 99mTc-bone scan, 99mTc-lung studies, and cardiac studies using 201Tl. Doses to staff, nearby patients, and the collective effective doses were estimated for the different working shifts and hospital areas. The estimated dose for nurses for 1 y was 518 microSv in the cardiology section and 338 microSv in the short stay section. For patients, the mean dose per stay was calculated to be 8.5 microSv in the cardiology section. The maximum dose that a patient could receive from a radioactive patient is 499 microSv for a double injection cardiac patient study. The maximum collective effective dose for the whole hospital was calculated to be 0.063 person-Sv. The probability of staff receiving doses higher than the limits for a working day is negligible. Maximum doses for staff and patients are far below dose limits for the public and therefore no additional radiological protection is needed.

[(99m)Tc]MAG(3)-mannosyl-dextran: a receptor-binding radiopharmaceutical for sentinel node detection.

Technetium-99m-labeled benzoyl-mercaptoacetylglycylglycyl-glycine-mannosyl-dextran ([(99m)Tc]MAG(3)-mannosyl-dextran) is a receptor-binding radiotracer that binds to mannose-binding protein, a receptor expressed by recticuloendothelial tissue. This agent is composed of a 10.5-kilodalton molecule of dextran and multiple units of mannose, and benzoyl-mercaptoacetylglycylglycyl-glycine (BzMAG(3)). The tetraflorophenol-activated ester of BzMAG(3) and the imidate of thiomannose were used to covalently attach BzMAG(3) and mannose to an amino-terminated conjugate of dextran. This yielded a 19-kilodalton macromolecule consisting of 3 BzMAG(3) and 21 mannose units per dextran. Dynamic light scattering was used to measure a mean diameter of 5.5 nanometers for BzMAG(3)-mannosyl-dextran and 0.28 microns for filtered Tc-99m sulfur colloid. A preliminary sentinel node detection study employing right fore and hind footpad injections of [(99m)Tc]MAG(3)-mannosyl-dextran and left fore and hind footpad injections of filtered Tc-99m sulfur colloid demonstrated greater sentinel lymph node uptake by the receptor-binding agent.

A synthetic macromolecule for sentinel node detection: (99m)Tc-DTPA-mannosyl-dextran.

UNLABELLED: We report the synthesis and preliminary biologic testing of a synthetic macromolecule, (99m)Tc-diethylenetriaminepentaacetic acid (DTPA)--mannosyl-dextran, for sentinel node detection. METHODS: Synthesis started with a 2-step process that attaches a high density of amino-terminated leashes to a dextran backbone. Allyl-bromide was reacted with pharmaceutical-grade dextran to yield allyl-dextran. After diafiltration with water, filtration, and lyophilization, the product was reacted with aminoethanethiol and ammonium persulfate. The resulting amino-conjugated dextran was dialyzed, filtered, and lyophilized. The mixed anhydride method was used to attach DTPA; after dialysis, filtration, and lyophilization, 2-imino-2-methoxyethyl-1-D-mannose was used to attach the receptor substrate. The molecular diameter was measured by dynamic light scattering. Amino, mannose, and DTPA densities were measured by trinitrobenzene sulfonate assay, sulfuric acid/phenol assay, and inductively coupled plasma spectroscopy of gadolinium-DTPA-mannosyl-dextran, respectively. Receptor affinity was measured by Scatchard assay of rabbit liver. Axillary, popliteal, and iliac lymph nodes and each injection site were assayed for radioactivity at 1 and 3 h after injection of approximately 3.7 MBq (0.050 mL) (99m)Tc-DTPA-mannosyl-dextran (0.22 nmol) or filtered (99m)Tc-sulfur colloid into the foot pads. Four animals were studied at each time point. RESULTS: DTPA-mannosyl-dextran had a molecular weight of 35,800 g/mol and a molecular diameter of 7.1 nm. The final amine, mannose, and DTPA densities were 23, 55, and 8 mol per dextran. Labeling yields were in excess of 98% and stable for 6 h. Specific activities of 74 x 10(6) GBq/mol were achieved. The equilibrium dissociation constant for binding to the mannose-terminated glycoprotein receptor was 0.12 +/- 0.07 nmol/L. The popliteal extraction at both 1 h and 3 h was significantly (P < 0.05) higher for (99m)Tc-DTPA-mannosyl-dextran (90.1% +/- 10.7% and 97.7% +/- 2.0%, respectively) than for filtered (99m)Tc-sulfur colloid (78.8 +/- 6.5 and 67.4% +/- 26.8%, respectively). (99m)Tc-DTPA-mannosyl-dextran exhibited significantly faster injection site clearance than did filtered (99m)Tc-sulfur colloid. The (99m)Tc-DTPA-mannosyl-dextran percentage injected dose (%ID) for the front and rear paws was 52.6 +/- 10.5 and 52.3 +/- 8.0 at 1 h and 45.7 +/- 8.5 and 43.6 +/- 8.2 at 3 h after administration. The filtered (99m)Tc-sulfur colloid %ID for the front and rear paws was 70.4 +/- 11.0 and 66.3 +/- 15.1 at 1 h and 55.5 +/- 7.8 and 66.9 +/- 8.5 at 3 h. Lymph node accumulation of each agent at either 1 or 3 h was not significantly different. CONCLUSION: (99m)Tc-DTPA-mannosyl-dextran is a receptor-based sentinel node radiotracer that exhibits the desired properties of rapid injection site clearance and low distal node accumulation. This molecule is the first member of a new class of diagnostic agents based on a macromolecular backbone with a high density of sites for the attachment of substrates and imaging reporters.

Receptor measurements via Tc-GSA kinetic modeling are proportional to functional hepatocellular mass.

UNLABELLED: Kinetic modeling of 99mTc-diethylenetriaminepentaacetic acid galactosyl human serum albumin (Tc-GSA) measures the total amount of asialoglycoprotein receptor within a subject's liver. This study tested the hypothesis that the amount of asialoglycoprotein receptor measured by Tc-GSA modeling provides a valid index of functional liver mass. METHODS: Twenty-two patients with cirrhosis, 18 patients with chronic hepatitis, and 9 patients with normal liver parenchyma were studied with Tc-GSA using a 30-min dynamic imaging protocol. The total amount of hepatic receptor was measured by kinetic modeling of the Tc-GSA time-activity data. The total number of viable hepatocytes was calculated using standard morphometric measurements of liver biopsy samples and liver volume measurements through CT. RESULTS: The total receptor amount strongly correlated with the total hepatocyte number (r = 0.803; P < 0.0001). CONCLUSION: Tc-GSA measurement of the total receptor amount is proportional to the number of viable hepatocytes and therefore provides a valid assessment of functional liver mass.

Portal-Systemic shunts reduce asialoglycoprotein receptor density in rats.

UNLABELLED: The clinical usefulness of quantitative functional imaging techniques that use asialoglycoprotein receptor (ASGP-R) binding is based on the correlation between ASGP-R density and hepatic functional reserve. Portal-systemic shunting (PSS) is common in patients with cirrhosis and portal hypertension-the same group that is most frequently considered for such imaging. PSS occurs spontaneously through collateral vessels and from the creation of surgical shunts or placement of transjugular intrahepatic portal-systemic shunts (TIPS). Understanding the physiologic relationship between PSS and ASGP-R activity may aid in the interpretation of quantitative clinical imaging. This study was conducted to determine the relationship between PSS and ASGP-R density in the absence of parenchymal disease. METHODS: Sprague-Dawley rats with end-to-side portal-systemic shunts and sham-operated control rats were imaged with 99mTC-diethylenetriaminepentaacetic acid galactosyl-neoglycoalbumin. Pharmacokinetic modeling of the liver and heart time-activity data was used to measure ASGP-R concentration, as well as hepatic plasma volume and flow. RESULTS: The mean ASGP-R density (nmol/g of liver) was significantly decreased in the shunted rats. Blood ammonia was significantly elevated, whereas hepatic plasma flow, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase levels were unaltered. Liver histology was normal in both groups. CONCLUSION: A significant change in the ASGP-R density occurs with PSS in the absence of parenchymal disease. PSS appears to be an independent variable affecting ASGP-R activity. This could prove clinically important during interpretation of quantitative imaging from patients with varying degrees of PSS based on underlying disease or the presence of a surgical shunt or TIPS device.

TIPS reduces hepatic asialoglycoprotein receptor concentration in healthy pigs.

PURPOSE: Technetium-99m-labeled diethylenetriamine pentaacetic acid galactosyl human serum albumin (TcGSA), a new agent for liver scintigraphy, is selectively attached to asialoglycoprotein receptors on liver cell membranes. A direct correlation exists between asialoglycoprotein receptor concentration, [R]o and hepatic functional reserve. The purpose of this study was to determine the effects of transjugular intrahepatic portosystemic shunting (TIPS) on hepatic asialoglycoprotein receptor concentration in pigs without liver parenchymal disease. MATERIALS AND METHODS: TIPS was performed in eight pigs with use of a single 10-mm-wide x 68-mm-long Wallstent dilated to 10 mm. TcGSA dynamic imaging studies were performed before and twice after (7 and 14 days) TIPS. To be included in the study, pigs had to have a patent TIPS at 1 week of follow-up. Liver function tests were drawn parallel to the TcGSA studies. Liver biopsies were performed at 2 weeks when the animals were killed. RESULTS: Five of the eight pigs had open shunts at 1 week and were included in the study. There was a significant (P < .0001, Student t test) difference between the mean [R]o of the pre-TIPS studies ([R]o = 1.12+/-0.04 microM) and the mean of the post-TIPS studies at 7 days ([R]o = 0.40+/-0.04 microM) and 14 days ([R]o = 0.51+/-0.06 microM, P < .01). The only blood test that was abnormal after TIPS was ammonia (mean, 129.0+/-42.7). Liver biopsies were normal. CONCLUSIONS: TIPS reduces asialoglycoprotein receptor concentration in normal pigs.

Recovery of hepatic asialoglycoprotein receptors after major hepatic resection.

UNLABELLED: Although morphological restoration of the hepatic mass after partial hepatectomy has been well studied, fewer reports have appeared on the change of functional hepatic capacity during liver regeneration. Asialoglycoprotein receptor (ASGP-R) is a hepatic cell surface receptor specific for galactose-terminated glycoprotein. Kinetic modeling of 99mTc-labeled diethylenetriamine pentaacetic acid-galactosyl-human serum albumin (TcGSA) time-activity data yields estimates of ASGP-R concentration [R]o and amount R0, which are directly related to functional liver mass. We have investigated the changes in ASGP-R status as well as liver volume in regenerating human liver after major hepatic resection. METHODS: Twenty-two patients (18 noncirrhotic, 4 cirrhotic) had a TcGSA study before and 3 wk after major hepatic resection, with a mean hepatic parenchymal resection rate of 36.0%. RESULTS: [R]0 was significantly decreased from 0.683+/-0.024 micromol/L to 0.565+/-0.032 micromol/L (P < 0.001) after resection. The decrease in [R]0 was more prominent in cirrhotic patients. Recovery of ASGP-R was observed as a significantly increased R0 3 wk after the operation. Subsequent (long-term) restoration of ASGP-R appeared to be slower when compared with the volume restoration. CONCLUSION: ASGP-R concentration of the liver significantly decreased after major hepatic resection. Subsequent recovery of ASGP-R amount was shown by TcGSA study. By estimating hepatic functional reserve expressed by ASGP-R amount and concentration, one may detect a delayed or impaired liver regeneration with higher sensitivity.

Sentinel node imaging via a nonparticulate receptor-binding radiotracer.

UNLABELLED: Technetium-99m-labeled polydiethylenetriamine pentaacetic acid polymannosyl polylysine (DTPA-man-PL) was synthesized and tested for lymph node scintigraphy by subcutaneous administration. The agent was designed for receptor-mediated uptake by mannosebinding protein, which resides on the plasma membrane of reticuloendothelial cells. METHODS: Subcutaneous injections of a 99mTc-labeled agent having 18 DTPA and 82 mannosyl groups attached to a polylysine of 100 units ([99mTc]DTPA18-man82-PL100) were made at the level of the metacarpus and metatarsus of three healthy rabbits. Images were acquired at 1, 6, 12 and 24 hr. Popliteal and axillary nodes were then assayed for percent of injected dose (%ID). A negative control study was performed in three normal rabbits with [99mTc]DTPA18-PL100. RESULTS: Significant differences in mean 24-hr %ID between the receptor specific and nonspecific agents were observed for both the popliteal (p < 0.006) and axillary (p < 0.012) nodes. Popliteal percent injected dose at 24 hr was 3.00 +/- 0.72% for [99mTc]DTPA-man-PL and 0.13 +/- 0.08% for [99mTc] DTPA-polylysine. Axillary accumulation at 24 hr was 2.84 +/- 0.83% for [99mTc]DTPA-mannosyl-polylysine and 0.22 +/- 0.12% for [99mTc] DTPA-polylysine. Percent injected dose of the receptor-specific agent was highest (4%) during the 6-hr scan. Accumulation of the nonspecific agent by the popliteal and axillary nodes at 6-hr postinjection was approximately 0.5%. CONCLUSION: This study provides proof of principle for lymphoscintigraphy by receptor-mediated delivery of a nonparticulate imaging agent.

Automatic preparation of radiopharmacokinetic data for in vivo estimation of receptor biochemistry.

UNLABELLED: We present a fully automated region of interest (ROI) and motion correction program for the generation of heart and liver time-activity data resulting from a hepatic functional imaging study using [99mTc-]galactosyl-neoglycoalbumin (99mTc-NGA). METHODS: The program automatically draws heart and liver ROI and corrects for lateral movement of the subject. Eighty-four 99mTc-NGA studies, consisting of 32 healthy subjects and 52 patients with liver disease, were processed and submitted to an automated kinetic analysis that estimates the subject's asialoglycoprotein receptor concentration [R]o. RESULTS: When compared to time-activity data generated by operator-drawn ROIs without motion correction, the average reduced Chi-square of the kinetic analysis decreased significantly (p < 0.001) from 2.20 to 1.37 and the number of studies that satisfied quality control increased from 74 to 81 studies. Receiver operating characteristic of [R]o resulted in greater detectability (0.984 +/- 0.012 compared with 0.965 +/- 0.020) when automatic ROI generation was employed. Using the test criteria of 0.65 microM, the sensitivity of [R]o increased from 0.88 to 0.92 and the specificity increased from 0.96 to 0.97. CONCLUSION: Automated definition of liver and heart ROIs with motion correction, that reduces observational noise, increased the success rate of the radiopharmacokinetic analysis from 88% to 96%.

Diagnostic performance of a receptor-binding radiopharmacokinetic model.

UNLABELLED: The aim of this study was to determine which measurement obtained from a radiopharmacokinetic model of a receptor-binding radiotracer provides the highest diagnostic performance for the detection of diffuse hepatocellular disease. METHODS: Twenty-seven healthy subjects and 46 patients with diffuse hepatocellular disease were studied with the receptor-binding radiopharmaceutical, 99mTc-galactosyl-neoglycoalbumin. A radiopharmacokinetic model was used to produce estimates of receptor concentration [R]o, the scaled forward-binding rate constant Kb, hepatic plasma volume, Vh, extrahepatic plasma volume, Ve and hepatic plasma flow, F. Receiver operating characteristic analysis of each model estimate was conducted. RESULTS: Receptor concentration [R]o and the metrics [R]o/tbw and kb[R]o[R]o/tbw provided the best discrimination between healthy and diseased liver. The forward-binding rate constant kb and the metrics F/Ve and Vh/tbw provided no discrimination. CONCLUSION: Based on simplicity and higher measurement precision, [R]o was selected as the most accurate index of hepatic function.

Tumor imaging with a macromolecular paramagnetic contrast agent: gadopentetate dimeglumine-polylysine.

RATIONALE AND OBJECTIVES: We evaluated magnetic resonance (MR) contrast enhancement of tumor tissue following injection of the macromolecular conjugate, gadopentetate dimeglumine-polylysine. METHODS: T1-weighted MR imaging scans were performed on female Fisher-344 rats with subcutaneously implanted mammary adenocarcinoma tumors. Following the baseline scan, gadopentetate dimeglumine-polylysine or gadopentetate dimeglumine was injected at a dose of 0.1 mmol gadolinium per kilogram. RESULTS: Gadopentetate dimeglumine-polylysine injection resulted in a maximum enhancement of tumor contrast of 310 +/- 60% (n = 7). Tumor tissue remained enhanced and well defined for several days after gadopentetate dimeglumine-polylysine injection. Gadopentetate dimeglumine injection at the same dose resulted in a 70 +/- 25% (n = 4) maximal tumor enhancement and a corresponding 25 +/- 4% muscle enhancement. CONCLUSION: Gadopentetate dimeglumine-polylysine provides higher, more sustained tumor contrast than does gadopentetate dimeglumine for the same dosage of gadolinium.

A molecular receptor-binding contrast agent for magnetic resonance imaging of the liver.

RATIONALE AND OBJECTIVES: A gadolinium complex of polydiethylenetriamine pentaacetic acid polyneogalactosyl polylysine (Gd-DTPA-gal-PL) was developed and tested as a paramagnetic contrast agent for magnetic resonance (MR) imaging of the liver. The agent was designed for receptor-mediated uptake by the asialoglycoprotein receptor (ASGP-R), which is unique to hepatocytes and exhibits high specificity for galactose-terminated glycoconjugates. METHODS: Polylysine was alkylated with a mixed anhydride of diethylenetriamine pentaacetic acid. This product was complexed with gadolinium and N-alkylated with 3-oxopropyl-1-thio-beta-D-galactopyranoside. With this reaction sequence, we prepared a gadolinium complex consisting of 2284 galactose groups and 858 chelators per polylysine having 2136 amino groups. Hepatic enhancement was tested by MR imaging of nine rats with liver-implanted mammary adenocarcinoma before and after injection of 20 x 10(-9) mol/kg Gd-DTPA858-gal2284-PL2136. The conjugate was labeled with technetium-99m and tested (1.5 x 10(-10) mol/kg) for hepatic specificity via nuclear imaging. RESULTS: Mean hepatic enhancement was 86% within 10 min and remained constant for 25 min. Hepatic relative intensity exceeded preinjection intensities by at least four times the standard deviation of the preinjection values (p < .01). The tumors, which are devoid of ASGP-R, did not exhibit significant enhancement (p > .1). The liver accumulated 90% of the technetium-99m-labeled conjugate. CONCLUSION: A molecular paramagnetic ligand to the asialoglycoprotein receptor has been developed for hepatocyte-specific MR contrast enhancement.

Arabinogalactan-coated superparamagnetic iron oxide: effect of particle size in liver MRI.

OBJECTIVE: We evaluated the effect of particle size on MR contrast-enhancing properties of arabinogalactan-coated superparamagnetic iron oxide (AG-SPIO) in tumor-bearing rats. MATERIALS AND METHODS: T2*-weighted gradient-recalled echo MR studies were performed on rats with surgically implanted liver tumors before and after AG-SPIO administration. Contrast-to-noise ratio (CNR) and percent contrast enhancement (PCE) were calculated for animals given small (15.7 +/- 9.5 nm: n = 6), medium (49.1 +/- 19.7 nm; n = 4), and large (86.9 +/- 27.5 nm; n = 4) particles intravenously (10 mumol Fe/kg). RESULTS: Postcontrast CNRs were 15.8 +/- 6.9, 8.9 +/- 4.1, and 10.0 +/- 1.8 for small, medium, and large particle groups, respectively. The PCE was -60.0 +/- 3.3, -75.5 +/- 7.9, and -80.5 +/- 1.2%. There was a significant difference in preversus postcontrast CNR for all particle sizes (p < 0.001) and in PCE for small particles as compared with the two larger sizes (p < 0.001). There was no between-group statistical difference in postcontrast CNR for any particle size. CONCLUSION: Larger AG-SPIO particles slightly improve liver contrast enhancement, but have no significant effect on hepatic lesion detection as assessed by CNR.

Evaluation of hepatocellular function by way of receptor-mediated uptake of a technetium-99m-labeled asialoglycoprotein analog.

UNLABELLED: We have developed a quantitative functional imaging study of the liver using a radiolabeled asialoglycoprotein analog, Tc-galactosyl-neoglycoalbumin. Heart and liver time-activity data can be transformed by automated kinetic analysis into asialoglycoprotein hepatocyte receptor concentration. Twenty-eight healthy controls, 46 patients with noncholestatic chronic liver injury and 11 patients with primary biliary cirrhosis were studied. Liver function was also assessed by Pugh modified-Child-Turcotte criteria, 14C-aminopyrine breath test and indocyanine green clearance (24 patients). RESULTS: (a) In normal controls with a Child-Turcotte criteria score of 5, receptor concentration ranged from 0.63 to 1.19 mumol/L, with a mean 0.83 +/- 2 S.D. 0.06 mumol/L, which was significantly higher (p < 0.001) than that of the patient group (mean receptor concentration = 0.44 +/- 2 S.D. 0.04 mumol/L). In cirrhotic patients with Child-Turcotte criteria score of 5, the mean receptor concentration was 0.60 +/- 2 S.D. 0.07 mumol/L, which was significantly lower than controls (p < 0.01). In end-stage cirrhosis (Child-Turcotte criteria score 11 to 15), a group in which patients died or required orthotopic liver transplantation within 1 yr, the mean receptor concentration was 0.35 +/- 2 S.D. to 0.07 mumol/L. The sensitivity and specificity for receptor concentration in relation to liver disease, with values above 0.65 mumol/L being normal, were 0.96 and 0.88, respectively. Receptor concentration correlated well with Child-Turcotte criteria score (r = 0.78, p = < 0.001), with aminopyrine breath test (r = 0.75, p = < 0.001) and with indocyanine green clearance (r = 0.88, p = < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Local identifiability of a receptor-binding radiopharmacokinetic system having measured parameters of known uncertainty.

Local identifiability was determined for a receptor-binding radiopharmacokinetic system that included measured parameters of known uncertainty. Healthy subjects and patients with severe liver disease were studied with [99mTc] galactosylneoglycoalbumin (TcNGA). Measurements during the 30-min dynamic imaging study included the count rate over liver and heart, the quantity of TcNGA injected Lo, and the fraction-of-injected dose per liter of sampled plasma f. Typical relative standard deviations for these measurements were 1, 0.2, and 5 percent, respectively. A four-state nonlinear model describing the hepatic and plasma time-activity data was then used to calculate the standard error se(pj) for model parameters representing receptor concentration [R]o, the TcNGA-receptor forward binding rate constant kb, extrahepatic plasma volume Ve, hepatic plasma volume Vh, and hepatic plasma flow F. Accounting for the measurement uncertainties of Lo and f did not significantly increase the standard errors for parameters [R]o, kb, Ve, Vh and F. When the relative errors of Lo and f were increased to 40%, the change in se(pj) ranged from 10 to 100%, with parameter Vh being the most sensitive. The exception was se(kb), the increase of which was less than 1%. Imaging studies with reduced [R]o, typically associated with patients with liver disease, resulted in greater increases in all estimated parameter errors except se(kb) which had a lower increase. Lastly, the error propagation introduced by direct measurement of the liver observational coefficients sigma 12 and sigma 13 was investigated by simulating changes in the relative standard deviation in parameters sigma 12 and sigma 13 from 0 to 40%. Imaging studies from healthy subjects showed no increase in se(pj).(ABSTRACT TRUNCATED AT 250 WORDS)