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Existing predictive biomarkers for immune checkpoint blockade (ICB) therapies that rely on characterization of tumor genomics or immunohistological assessment of the tumor microenvironment have demonstrated mixed success, with no single biomarker proving to be universally predictive of ICB treatment outcome. To complement these existing approaches, we present a workflow that relies on high-resolution single-cell mass measurements curated with CNN-based image classification to characterize T cell activation and functional response to treatment with ICB therapies. As a highly integrative measure of cellular phenotype, cell mass can be used as a label-free biomarker for characterizing immune cell function. For instance, upon activation with anti-CD3/CD28, T cells isolated from human PBMC showed a rapid and significant increase in cell mass. However, in T cells that had been repeatedly stimulated in vitro to generate a PD-1+ exhausted-like phenotype, this mass response to activation was blunted. We hypothesized that this variability in activation-induced mass response was the result of an altered balance of stimulatory and inhibitory “signal two” cues seen by the T cells. Consistent with this hypothesis, purified T cells stimulated with anti-CD3 alone (no co-stimulation) demonstrated a significantly smaller increase in cell mass when compared with those stimulated with both anti-CD3 and anti-CD28. Furthermore, the T cell response measured when unpurified PBMC were activated with anti-CD3 demonstrated an intermediate mass increase, suggesting contributions of both stimulatory and inhibitory signaling from other cells in the milieu. To explore how ICB treatment disrupts this co-stimulatory/inhibitory signaling balance, we measured T cell mass responses for samples treated with anti-CD3 alone or in combination with pembrolizumab, atezolizumab, nivolumab, or dostarlimab. These samples included PBMC isolated from blood and tumor/immune cell milieus isolated from malignant fluids (pleural effusions or abdominal ascites) collected from patients with advanced cancer. We found that T cells isolated from samples treated with both anti-CD3 and an ICB drug often showed a significantly greater mass increase than the same samples treated with anti-CD3 alone. This mass response showed significant heterogeneity across patients, with many samples showing no significant treatment effect. Despite all four of these therapies targeting ostensibly the same PD-1/PD-L1 signaling axis, we also found significant response heterogeneity to these different treatments within individual samples.These results suggest that mass response measurements capture biological heterogeneity associated with ICB response and may serve as a valuable complementary readout to existing biomarkers. Citation Format: Robert Kimmerling, Mark Stevens, Selim Olcum, Anthony Minnah, Madeleine Vacha, Rachel LaBella, Katie Katsis, Audrey Guo, Juanita Fujii, Zayna Shaheen, Srividya Sundaresan, Anobel Tamrazi, Cliff Reid. A cell mass-based platform for characterizing immune cell function and response to checkpoint blockade in primary blood and malignant fluid samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2176.

The authors seek to evaluate hemodynamic parameters as potential clinical markers of real-time clinical improvement among patients with massive pulmonary embolism (PE) in correlation with post-thrombolytic pulmonary arterial pressure improvement and overall clinical outcome. Thirteen patients with submassive or massive PE were admitted to the interventional radiology service and treated with catheter-directed thrombolysis. Among the four patients who qualified as massive PE, systolic blood pressure (BP) and vasopressor dependence suggested meaningful trends toward clinical improvement, after only 26.4% of treatment course/dose. Hemodynamic parameters such as systolic BP and inotropic vasopressor dependence may be considered in future treatment protocols as early indicators of treatment response.

Our study sought to compare the overall survival in patients with hepatocellular carcinoma (HCC) and portal venous thrombosis (PVT), treated with either conventional trans-arterial chemoembolization (cTACE) or drug-eluting beads (DEB) TACE. This retrospective analysis included a total of 133 patients, treated without cross-over and compared head-to-head by means or propensity score weighting. Mortality was compared using survival analysis upon propensity score weighting. Adverse events and liver toxicity grade ≥3 were recorded and reported for each TACE. In order to compare with historical sorafenib studies, a sub-group analysis was performed and included patients who fulfilled the SHARP inclusion criteria. The median overall survival (MOS) of the entire cohort was 4.53 months (95 % CI, 3.63–6.03). MOS was similar across treatment arms, no significant difference between cTACE (N = 95) and DEB-TACE (N = 38) was observed (MOS of 5.0 vs. 3.33 months, respectively; p = 0.157). The most common adverse events after cTACE and DEB- TACE, respectively, were as follows: post-embolization syndrome [N = 57 (30.0 %) and N = 38 (61.3 %)], diarrhea [N = 3 (1.6 %) and N = 3 (4.8 %)], and encephalopathy [N = 11 (5.8 %) and N = 2 (3.2 %)]. Our retrospective study could not reveal a difference in toxicity and efficiency between cTACE and DEB-TACE for treatment of advanced stage HCC with PVT. • Conventional TACE (cTACE) and drug-eluting-beads TACE (DEB-TACE) demonstrated equal safety profiles. • Survival rates after TACE are similar to patients treated with sorafenib. • Child-Pugh class and tumor burden are reliable predictors of survival.

Purpose: To review the safety of hepatic radioembolization (RE) in patients with high (>= 10%) hepatopulmonary shunt fraction (HPSF) using various prophylactic techniques. Materials and Methods: A review was conducted of 409 patients who underwent technetium 99m-labeled macroaggregated albumin scintigraphy before planned RE. Estimated pulmonary absorbed radiation doses based on scintigraphy and hepatic administered activity were calculated. Outcomes from dose reductions and adjunctive catheter-based prophylactic techniques used to reduce lung exposure were assessed. Results: There were 80 patients with HPSF >= 10% who received RE treatment (41 resin microspheres for metastases, 39 glass microspheres for hepatocellular carcinoma). Resin microspheres were used in 17 patients according to consensus guideline-recommended dose reduction; 38 patients received no dose reduction because the expected lung dose was

To evaluate the feasibility, risks, and techniques of percutaneous removal of permanent TrapEase and Simon Nitinol IVC filters. Between August 2011 and August 2015, 12 patients (5 women, 7 men; age range, 26–75 years) underwent an attempt at percutaneous removal of permanent TrapEase (10) and Simon Nitinol (2) IVC filters due to a history of IVC filter complications or need for lifelong anticoagulation due to the filter. Medical records were reviewed for filter dwell time, presence of iliocaval deep venous thrombosis, procedural technique, and complications. Filter dwell times ranged from 7 days to 15 years (mean 5.1 years). Successful removal of permanent IVC filters was possible in 11 of 12 patients (91.6 %). In 1 patient, a chronically thrombosed IVC filter could not be removed despite laser sheath assistance, but was successfully recanalized with the PowerWire RF guidewire. In the failed retrieval attempt, a stent was placed through the chronically thrombosed IVC filter with restoration of in-line flow. One major complication of large venous groin hematoma was encountered. In carefully selected patients, percutaneous removal of permanent IVC filters can be performed safely despite prolonged filter dwell times. Extraction of chronically embedded permanent IVC filters may be facilitated by jugular and femoral approaches, often with laser sheath assistance. Chronic filter thrombosis and caval scarring may increase the risk of retrieval failure.

Biological sealants are being increasingly used in a variety of surgical specialties for their hemostatic and sealing capabilities. However, their use in interventional radiology has not been widely reported. The authors describe a case of duodenal perforation occurring after 15 years of gastric bypass surgery, in whom surgical diversion was unsuccessfully attempted and the leakage was successfully controlled using percutaneous administration of a combination of biological and organic sealants.

To determine the impact, if any, of the 2010 FDA safety communication on the rate of inferior vena cava filter (IVCF) placement over time.The Nationwide Inpatient Sample was interrogated for the most recent years preceding and after the FDA safety communication-from 2005 to 2014. IVCF placements and associated diagnoses were identified using corresponding International Classification of Diseases, version nine codes. Trends in number of IVCF placement were evaluated in aggregate and by associated diagnoses, both of which were further stratified by hospital geographic cluster, hospital teaching status, and patient demographics. Generalized linear regression models were used to determine statistical significance of trends over time.IVCF placements steadily increased between 2005 and 2010 (100,434 in 2005 versus 129,614 in 2010, growth rate 5.81%). Aggregate IVCF placements subsequently declined between 2010 and 2014 (96,005 in 2014, decline rate -6.48%). IVCF placements peaked in 2010, the year of the FDA advisory. The proportion of filter placements for therapeutic indication of venous thromboembolism increased significantly during the study period (69.8% in 2005 versus 80.4% in 2014, P.001). Neither trend varied significantly by patient demographics or hospital characteristics.IVCF placements have declined significantly since 2010, when the FDA advisory was released. The proportion of IVCFs placed in patients with venous thromboembolism, as opposed to prophylactic indications, is increasing.

Background Perioperative management of patients on anticoagulant therapy prior to interventional pain procedures creates a challenge when balancing the risk of bleeding against thromboembolic events. Case Report We report a case of epidural hematoma formation in the cervical spine following interlaminar epidural steroid injection in an elderly woman with chronic neck and arm pain, who was on clopidogrel therapy. Conclusions This is the first reported case of hematoma formation immediately following an epidural steroid injection possibly associated with clopidogrel, even though established guidelines on the timing of the discontinuation of clopidogrel prior to the procedure were exceeded. Severe pain appears to be the first symptom of hematoma formation, and therefore immediate diagnostic workup and evacuation of hematoma are essential in preventing neurological damage. It may be advisable to carry out a test specific for clopidogrel such as the P2Y12 to ensure that there is no residual action on platelet aggregation function, particularly in patients who may be slow metabolizers of clopidogrel. Caution is advised prior to administering analgesics with antiplatelet effects such as ketorolac.

OBJECTIVE. The purpose of our study was to evaluate image quality in a 3D spoiled gradient-recalled echo (SPGR) sequence that was modified to incorporate respiratory navigation to limit the deleterious effects of respiratory motion and to compare it with conventional scanning during breath-holding and free breathing.CONCLUSION. Respiratory navigation of 3D SPGR sequences is technically feasible, and image quality is modestly improved over free breathing acquisitions using conventional 3D SPGR sequences. This may represent a promising imaging alternative for patients who cannot hold their breath.

We describe an incidental finding of intradural lumbar disc herniation diagnosed radiographically during discography. A patient was referred to our center for discography with symptoms of worsening, intractable low back pain radiating to both hips and the left leg which was exacerbated when standing and walking. Magnetic resonance imaging of the lumbar spine revealed multiple disc bulges and lumbar facet arthroses with ligamentum flavum hypertrophy producing moderate central canal and lateral recess stenosis. Discography was performed at three levels (L3-4, L4-5, L5-S1). During fluoroscopically guided injection into L4-5 it was noted that contrast was not contained within the disc and spread intrathecally with a myelographic appearance. Computerized tomography confirmed accurate needle placement and a spread of contrast into the intrathecal space. To the best of our knowledge, this is the first report describing a finding of intradural disc herniation while performing discography. Physicians should be aware of this potential finding while performing this technique.

The direct regulation of gene transcription by nuclear receptors, such as the estrogen receptor (ER), involves not just ligand and DNA binding but the recruitment of coregulators. Typically, recruitment of p160 coactivator proteins to agonist-liganded ER is considered to be unidirectional, with ligand binding stabilizing an ER ligand binding domain (LBD) conformation that favors coactivator interaction. Using fluorophore-labeled ERα-LBDs, we present evidence for a pronounced stabilization of ER conformation that results from coactivator binding, manifest by decreased ER sensitivity to proteases and reduced conformational dynamics, as well as for the formation of a novel coactivator-stabilized (costabilized) receptor conformation, that can be conveniently monitored by the generation of an excimer emission from pyrene-labeled ERα-LBDs. This costabilized conformation may embody features required to support ER transcriptional activity. Different classes of coactivator proteins combine with estrogen agonists of different structure to elicit varying degrees of this receptor stabilization, and antagonists and coactivator binding inhibitors disfavor the costabilized conformation. Remarkably, high concentrations of coactivators engender this conformation even in apo- and antagonist-bound ERs (more so with selective ER modulators than with pure antagonists), providing an in vitro model for the development of resistance to hormone therapy in breast cancer.

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.

Clinical application efforts for the hepatocyte-specific MRI contrast agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) have mainly been directed toward detection and characterization of various hepatic masses in the adult population. Here we report our initial experience with Gd-EOB-DTPA for evaluating congenital and acquired hepatobiliary pathologies in the pediatric population. Twenty-one consecutive children receiving Gd-EOB-DTPA for functional hepatobiliary evaluation at our institution were retrospectively identified with IRB approval. The use of Gd-EOB-DTPA was classified in each case as definite, potential, or no clinical utility, focusing on the clinical value gained beyond traditional noncontrast fluid-sensitive MR cholangiopancreatography (FS-MRCP) and other imaging modalities. Definite added value of Gd-EOB-DTPA was found in 12 patients, with potential value in 4 patients, and no value in 5 patients. Benefit was seen in cases of iatrogenic and non-iatrogenic biliary strictures, perihepatic fluid collections for biliary leak, hepatobiliary dysfunction in the absence of hyperbilirubinemia, and in the functional exclusion of cystic duct occlusion that can be seen in acute cholecystitis. This is the first reported series of children with Gd-EOB-DTPA and this early work suggests potential pediatric applications.

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.

Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.

The ligand-induced conformation of a nuclear receptor ligand-binding domain (LBD) is a principal factor leading to transcriptional activity and determining the pharmacological response. Using the estrogen receptor (ER) LBD-labeled site specifically with a fluorophore, we demonstrate that effects of ligand binding on the conformation and dynamics of this domain can be studied directly, in a quantitative and convenient fashion, by various fluorescence methods. Estrogen ligands of different pharmacological character-agonists, selective ER modulators (SERMs), and pure antagonists-each produce distinctive spectroscopic signatures, characteristic of the conformational or dynamic features of their ER-LBD complexes. We can directly follow the equilibrium of helix 12 positions through the degree of local fluorophore rotational freedom and receptor helicity near the C terminus of helix 11. We observe differences even between ligands within a specific pharmacological class, such as the SERMs raloxifene and trans-4-hydroxytamoxifen, highlighting the ability of these fluorescent receptor sensors to detect unique ER conformations induced even by closely related ligands, yet ones that produce distinctive biological activities in estrogen target cells. Fluorophore-labeled LBDs can serve as versatile molecular sensors predictive of ligand pharmacological character and should be broadly applicable to other members of the nuclear receptor superfamily.

Publisher Summary The estrogen receptor (ER) is a ligand-regulated transcription factor that belongs to the nuclear receptor (NR) superfamily and acts as a dimeric species. There are two subtypes of ER—ERα and ERβ—which are both mainly regulated by the endogenous estrogen—estradiol (E2). ER modulation is involved in the development and regulation of reproductive, cardiovascular, and bone health, in addition to controlling the various aspects of cognitive function. Ligands that bind to the ER can be categorized into three pharmacological classes: agonists, mixed agonist—antagonists, and pure antagonists. The mixed agonist–antagonists are also referred to as “selective estrogen receptor modulators (SERMs)” because of their tissue-selective agonist or antagonist activities. Nuclear receptors (NRs) form strong dimers that are essential for their functioning as transcription factors. NRs that bind steroid ligands (i.e., ER) typically function as dimers, either homodimers between identical receptor monomers or between closely related subtypes. NRs that bind nonsteroidal ligands, however, typically function as heterodimers with the retinoid X receptor (RXR). Using fluorescence resonance energy transfer (FRET) and fluorescent-labeled ERα-LBDs, the regulation of homodimer stability by ligands and coactivator peptides is monitored. This technique provides structure–activity relationships (SARs) of ligands in terms of ER dimer stability and can be used as a functional assay to identify the pharmacological character of novel synthetic or environmental ER ligands. Fluorescent-labeled ERβ-LBDs can be used to study homo- and heterodimers of both ER subtypes and the manner in which SERMs and ER subtype-selective ligands (both agonists and antagonists) modulate differential homo- and heterodimer stability. Site-specific fluorescent ERs can also be used for the development of other types of assays to characterize receptor conformation, conformational dynamics, and ligand or coregulator interactions.

Nuclear receptors form strong dimers that are essential for their function as transcription factors, and it is thought that ligand binding can affect dimer stability. In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of dimers of the estrogen receptor-alpha ligand-binding domain (ERalpha-LBD). We have developed receptors that are chemically labeled with a single fluorophore in a site-specific manner. These fluorophore-labeled ERs are functional and can be used to measure directly the affinity and stability of ERalpha-LBD dimers. Our results indicate that unliganded ERalpha-LBDs exist as very stable dimers and that the dissociation rate of these dimers is slow (t(1/2)=39 +/- 3 min at 28 C) and is further slowed (< or =7-fold) by the addition of various ligands. Estrogen antagonists provide greater kinetic stabilization of the ER dimers than agonists. In addition, coactivator peptides containing the LXXLL motif selectively stabilize agonist-bound ERalpha-LBD dimers. These fluorescence-based assays for measuring the kinetic and thermodynamic stability of ER dimers provide a functional in vitro method for assessing the agonist or antagonist character of novel ligands.

Purpose Consensus guidelines dictate that patients with high hepatopulmonary shunt fractions should not undergo Y-90 radioembolization (RE), or should have administered activity reduced 20-40% to decrease the risk of radiation pneumonitis. We reviewed our experience of managing and treating patients with high shunt fractions. Materials and Methods We retrospectively reviewed 247 patients treated from 2004-11. We calculated pulmonary radiation dose based on technetium macroaggregated albumin (TcMAA) scintigraphy, and reviewed dose calculations and reductions as well as adjunctive prophylactic techniques utilized to reduce lung exposure during the delivery of RE microspheres. Results A total of 50/247 patients (20%) had shunt fractions > 10%. Glass microspheres were used to treat 20 and resin to treat 30 patients. 18 patients were treated with recommended dose reduction only. 4 patients with high shunt fractions (25-44%) were treated prior to RE with bland embolization and/or sclerosis, and repeat TcMAA scintigraphy demonstrated median shunt reduction of 34% (range 0 - 51%). 15 patients (including these 4) were treated using various prophylactic techniques such as hepatic vein balloon occlusion, variceal embolization, and bland arterial embolization before or during RE delivery, and 10/15 received full dose without reduction. An additional 17 patients received no dose reduction or adjuvant maneuvers because the calculated lung dose was well below 30 Gy. Of the 50 patients, only 7 would have received calculated lung exposures > 30 Gy without dose adjustment. Calculated median expected lung dose for the prophylactic treatment group was 16 Gy (range 8-68 Gy). In follow up, 1 heavily pretreated patient developed radiation pneumonitis requiring corticosteroid therapy (shunt 28%, lung exposure 16 Gy). Conclusion Standard recommendations for RE dose reductions for high hepatopulmonary shunt fractions are not mathematically justifiable, may compromise therapeutic efficacy in the liver, and are most often avoidable without substantial increase in risk. Shunting high enough to exceed the 30 Gy pulmonary threshold can be reduced by adjunctive techniques.

We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.