Discovery of CRN04894: A Novel Potent Selective MC2R Antagonist.

A novel class of nonpeptide melanocortin type 2 receptor (MC2R) antagonists was discovered through modification of known nonpeptide MC4R ligands. Structure-activity relationship (SAR) studies led to the discovery of 17h (CRN04894), a highly potent and subtype-selective first-in-class MC2R antagonist, which demonstrated remarkable efficacy in a rat model of adrenocorticotrophic hormone (ACTH)-stimulated corticosterone secretion. Oral administration of 17h suppressed ACTH-stimulated corticosterone secretion in a dose-dependent manner at doses ≥3 mg/kg. With its satisfactory pharmaceutical properties, 17h was advanced to Phase 1 human clinical trials in healthy volunteers with the goal of moving into patient trials to evaluate CRN04894 for the treatment of ACTH-dependent diseases, including congenital adrenal hyperplasia (CAH) and Cushing's disease (CD).

A Comparison of Variable Versus Fixed Insulin Infusion Rate on Resolution of Diabetic Ketoacidosis.

BACKGROUND: Diabetic ketoacidosis (DKA) is a well-known, potentially fatal complication of diabetes. The American Diabetes Association hyperglycemic crises guidelines suggest the use of intravenous insulin in patients presenting with DKA, along with a recommended rate of glucose reduction of 50-75 mg/dL/h. However, no specific guidance is provided regarding how to best achieve this rate of glucose decline. STUDY QUESTION: Is there a difference in time to DKA resolution between a variable intravenous insulin infusion strategy and a fixed infusion strategy in the absence of an institutional protocol? STUDY DESIGN: Single-center, retrospective cohort study of DKA patient encounters in 2018. MEASURES AND OUTCOMES: Insulin infusion strategy was considered to be variable if the infusion rate changed within the first 8 hours of therapy or was considered fixed if the rate remained unchanged for the same period. The primary outcome was time to resolution of DKA. Secondary outcomes were hospital length of stay, intensive care unit length of stay, hypoglycemia, mortality, and DKA recurrence. RESULTS: The median time to resolution of DKA was 9.3 hours in the variable infusion group compared with 7.8 hours in the fixed infusion group (HR, 0.82; 95% CI, 0.43-1.5, P = 0.5360). Severe hypoglycemia was observed in 13% of patients in the variable infusion group and in 50% of patients in the fixed infusion group ( P = 0.006). CONCLUSIONS: In this analysis, insulin infusion strategy (variable vs. fixed) was not associated with a significant difference in the time to resolution of DKA in the absence of an institutional protocol. The fixed infusion strategy was associated with a higher incidence of severe hypoglycemia.

Evaluation of intravenous lorazepam dosing strategies and the incidence of refractory status epilepticus.

INTRODUCTION: Status epilepticus (SE) is a neurological emergency associated with high mortality if not identified and treated promptly. For the emergent treatment of SE, the recommended intravenous (IV) lorazepam dose is 0.1 mg/kg/dose, up to a maximum of 4 mg. It has been shown that lorazepam is commonly under dosed in SE, but there is conflicting data on whether this has a negative impact on patient outcomes. This study assessed any dose less than 4 mg to help identify the effects of under dosing lorazepam in SE. METHODS: This was a retrospective cohort study of patients admitted to a quaternary health system between October 1, 2017 and September 30, 2019 that experienced SE and were initially treated with IV lorazepam. Patients were divided into two cohorts, less than 4 mg or 4 mg, based on the initial one-time dose of lorazepam received. The primary outcome was the proportion of patients that progressed to refractory status epilepticus (RSE) that received an initial IV lorazepam dose of 4 mg compared to less than 4 mg for the treatment of SE. Secondary outcomes evaluated include length of stay, mortality, time in SE, number of seizures, cumulative lorazepam dose prior to urgent therapy, number of lorazepam doses prior to urgent therapy, time to urgent therapy, appropriately dosed urgent therapy, and number of antiepileptic drugs given in SE. RESULTS: One hundred twenty patients were included in this study (107 patients received less than 4 mg and 13 patients received 4 mg). All patients included in the study were greater than 40 kg. The primary outcome of progression to RSE was observed in a significantly greater proportion of patients in the less than 4 mg group compared to the 4 mg group (93 [87%] vs. 8 [62%], p = 0.03). There was no difference in hospital or intensive care unit length of stay. However, there was an increased rate of in-hospital mortality in patients who received 4 mg compared to less than 4 mg (5 [39%] vs. 12[11%], p = 0.02). DISCUSSION: The majority of patients in the study received less than the recommended dose of IV lorazepam for SE. Patients who received less than 4 mg experienced an increased progression to RSE, which supports current guideline recommended dosing. While there was an increased rate of mortality in patients who received 4 mg compared to less than 4 mg, time in SE was prolonged in the patient population and severity of illness was only available for a limited number of patients included.

Discovery of 4-(3-aminopyrrolidinyl)-3-aryl-5-(benzimidazol-2-yl)-pyridines as potent and selective SST5 agonists for the treatment of congenital hyperinsulinism.

SST5 receptor activation potently inhibits insulin secretion from pancreatic ß-cells, and an orally available nonpeptide selective SST5 agonist may be used to effectively manage the blood glucose levels of congenital HI patients to avoid severe hypoglycemia. Our medicinal chemistry efforts have led to the discovery of 4-(3-aminopyrrolidinyl)-3-aryl-5-(benzimidazol-2-yl)-pyridine analogs as potent SST5 agonists. This class of molecules exhibits excellent human SST5 potency and selectivity against SST1, SST2, SST3 and SST4 receptors. Leading compound 3-{4-[(3S)-3-aminopyrrolidin-1-yl]-5-(4-methyl-1H-1,3-benzodiazol-2-yl)pyridin-3-yl-5-fluorobenzonitrile (28, CRN02481) showed limited off-target activity and good pharmacokinetic profiles in both male Sprague Dawley rats and Beagle dogs to advance into further preclinical evaluations.

Evaluation of levocarnitine, lactulose, and combination therapy for the treatment of valproic acid-induced hyperammonemia in critically ill patients.

INTRODUCTION: Critically ill patients treated with valproic acid are at risk for hyperammonemic encephalopathy. Both levocarnitine and lactulose, either alone or in combination, have been used for the treatment of hyperammonemia associated with valproic acid, however they have not been directly compared in the literature. The aim of this study was to compare the effect of levocarnitine, lactulose, and combination therapy for the treatment of valproic acid-induced hyperammonemia in critically ill patients. METHODS: This was a retrospective, system-wide, cohort study of critically ill patients who received valproic acid and levocarnitine, lactulose, or combination therapy from January 1, 2012 to October 31, 2019. The primary outcome of the study was the change in ammonia level from baseline to the lowest point within the first 48 h of treatment. Secondary outcomes included the change in ammonia levels within the first 7 days, the incidence of a clinically significant reduction, ICU length of stay, hospital length of stay, and hospital mortality. RESULTS: A total of 371 charts were reviewed and 114 patients (levocarnitine [n = 15], lactulose [n = 72], and combination [n = 27]) were included. No difference in the primary outcome was observed (levocarnitine [11umol/L] vs. lactulose [20 umol/L] vs. combination [23 umol/L], p = 0.605). The incidence of a clinically significant reduction in ammonia levels at 48 h did not differ between groups, nor did mortality. CONCLUSION: In critically ill patients with valproic acid-induced hyperammonemia, there was no significant difference in the reduction in ammonia levels in the first 48 h of treatment between levocarnitine, lactulose, and combination therapy.

Incidence and Risk Factors Associated with Multi-Drug-Resistant Pathogens in a Critically Ill Trauma Population: A Retrospective Cohort Study.

Background: Multi-drug resistance is considered a serious health threat particularly in the intensive care unit (ICU) setting. Studies evaluating multi-drug-resistant (MDR) pathogens in critically ill trauma patients are limited. The objectives were to describe the incidence of MDR, extensive-drug-resistant (XDR), and pan-drug-resistant (PDR) organism growth in ICU patients admitted with traumatic injuries and to identify any risk factors associated with MDR growth. Patients and Methods: This was a retrospective single-center cohort study of all ICU adult patients identified via the institution's trauma registry from January 1, 2016 to August 31, 2017. Patients were included if they had positive culture growth with susceptibility data taken during the index hospitalization. Patients were excluded if their cultures were drawn within 48 hours of emergency department triage. Study groups were defined based on the presence of at least one MDR pathogen during the index hospitalization. Results: A total of 2,578 charts were reviewed and 95 patients (mean age, 60 years; 66 males [69%]) with 201 total cultures were included. The majority of positive cultures were from respiratory (69%) and urinary (16%) sources. Of the 201 positive cultures, the majority of species identified was Enterobacteriaceae (47%), Staphylococcus (32%), Enterococcus (7%), Acinetobacter (5%), and Pseudomonas (3%). Of the 95 patients with positive cultures, the incidence of MDR, XDR, and PDR organisms was found to be 31%, 17%, and 0%, respectively. Augmented renal clearance (ARC) was the only risk factor associated with an increased risk for MDR organism growth (adjusted odds ratio 9.78, 95% confidence interval [CI] 2.56-37.41; p = 0.001). Conclusions: In this cohort of critically ill trauma patients, the incidence of an MDR pathogen occurred in 31% of patients. This is the first study to find an association of ARC and multi-drug resistance, which should be further validated as a potential cause for MDR organisms.

Loss of the trpc4 gene is associated with a reduction in cocaine self-administration and reduced spontaneous ventral tegmental area dopamine neuronal activity, without deficits in learning for natural rewards.

Among the canonical transient receptor potential (TRPC) channels, the TRPC4 non-selective cation channel is one of the most abundantly expressed subtypes within mammalian corticolimbic brain regions, but its functional and behavioral role is unknown. To identify a function for TRPC4 channels we compared the performance of rats with a genetic knockout of the trpc4 gene (trpc4 KO) to wild-type (WT) controls on the acquisition of simple and complex learning for natural rewards, and on cocaine self-administration (SA). Despite the abundant distribution of TRPC4 channels through the corticolimbic brain regions, we found trpc4 KO rats exhibited normal learning in Y-maze and complex reversal shift paradigms. However, a deficit was observed in cocaine SA in the trpc4 KO group, which infused significantly less cocaine than WT controls despite displaying normal sucrose SA. Given the important role of ventral tegmental area (VTA) dopamine neurons in cocaine SA, we hypothesized that TRPC4 channels may regulate basal dopamine neuron excitability. Double-immunolabeling showed a selective expression of TRPC4 channels in a subpopulation of putative dopamine neurons in the VTA. Ex vivo recordings of spontaneous VTA dopamine neuronal activity from acute brain slices revealed fewer cells with high-frequency firing rates in trpc4 KO rats compared to WT controls. Since deletion of the trpc4 gene does not impair learning involving natural rewards, but reduces cocaine SA, these data demonstrate a potentially novel role for TRPC4 channels in dopamine systems and may offer a new pharmacological target for more effective treatment of a variety of dopamine disorders.

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIß.

Type III phosphatidylinositol 4-kinase (PI4KIIIß) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIß. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIß inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIß bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIß inhibitors, which may play significant roles as antiviral therapeutics.

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIß with Rab11.

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIß) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIß in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIß-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.

WIDE AWAKE mediates the circadian timing of sleep onset.

How the circadian clock regulates the timing of sleep is poorly understood. Here, we identify a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. We propose that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.

Drosophila TRP channels and animal behavior.

Multiple classes of cell surface receptors and ion channels participate in the detection of changes in environmental stimuli, and thereby influence animal behavior. Among the many classes of ion channels, Transient Receptor Potential (TRP) cation channels are notable in contributing to virtually every sensory modality, and in controlling a daunting array of behaviors. TRP channels appear to be conserved in all metazoan organisms including worms, insects and humans. Flies encode 13 TRPs, most of which are expressed and function in sensory neurons, and impact behaviors ranging from phototaxis to thermotaxis, gravitaxis, the avoidance of noxious tastants and smells and proprioception. Multiple diseases result from defects in TRPs, and flies provide an excellent animal model for dissecting the mechanisms underlying "TRPopathies." Drosophila TRPs also function in the sensation of botanically derived insect repellents, and related TRPs in insect pests are potential targets for the development of improved repellents to combat insect-borne diseases.

Coexisting follicular and mantle cell lymphoma with each having an in situ component: A novel, curious, and complex consultation case of coincidental, composite, colonizing lymphoma.

A diagnosis of composite lymphoma is typically prompted by the observation of morphologic discordance. We present a case of a spleen revealing histologic features of follicular lymphoma, without any indication of a second lymphoma. Immunohistochemical stains supported this diagnosis and showed the follicular lymphoma to be BCL2-. However, these studies revealed 2 additional unexpected findings: cyclin D1+ mantle zone cells surrounding neoplastic and reactive follicles (indicative of in situ mantle cell lymphoma) and BCL2-bright, histologically nonneoplastic follicles (indicative of in situ follicular lymphoma). ImmunoFISH and microdissection and polymerase chain reaction analysis documented the clonal nature of the cyclin D1+ mantle zones and illustrated clonal independence from the follicular lymphoma. This case illustrates an uncommon and unusual composite follicular and mantle cell lymphoma, with the follicular lymphoma accompanied by an in situ component, whereas the only manifestation of the mantle cell lymphoma was in situ.

Dopamine modulates an mGluR5-mediated depolarization underlying prefrontal persistent activity.

The intrinsic properties of neurons that enable them to maintain depolarized, persistently activated states in the absence of sustained input are poorly understood. In short-term memory tasks, individual prefrontal cortical (PFC) neurons can maintain persistent action potential output during delay periods between informative cues and behavioral responses. Dopamine and drugs of abuse alter PFC function and working memory, possibly by modulating intrinsic neuronal properties. Here we used patch-clamp recording of layer 5 PFC pyramidal neurons to identify a postsynaptic depolarization that was evoked by action potential bursts and mediated by metabotropic glutamate receptor 5 (mGluR5). This depolarization occurred in the absence of recurrent synaptic activity and was reduced by a dopamine D1 receptor (D1R) protein kinase A pathway. After behavioral sensitization to cocaine, the depolarization was substantially diminished and D1R modulation was lost. We propose that burst-evoked intrinsic depolarization is a form of short-term cellular memory that is modulated by dopamine and cocaine experience.

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain.

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

Parenting skills and family support programs for drug-abusing mothers.

Children born to drug-using mothers can suffer from fetal alcohol or drug syndrome (FAS/FDS) or fetal alcohol or drug effect (FAE/FDE). Such children have a greater likelihood of developing acute or chronic physical, cognitive and behavioral problems. In-utero exposure to tobacco, alcohol or drugs impact on the developing fetus and, after birth, the family environment and family system exert effects on the infants and children of substance-abusing parents. Evidence-based prevention and maternal drug treatment programs focus on enhancing parental childcaring abilities, supporting parent-child attachment and encouraging family support systems to improve children's health and cognitive outcomes. FAS/FDS prevention programs, as well as selective and indicated prenatal and postnatal interventions, can improve the support given both to mother and to child, and evidence-based, in-home parenting and family-skills-training approaches are particularly useful.

A role for the subiculum in the brain motivation/reward circuitry.

The ventral subiculum (vSUB) is an interface between the hippocampal formation and structures in the brain reward circuitry, such as the nucleus accumbens (NAc) and prefrontal cortex (PFC). The vSUB powerfully activates the dopamine system, particularly in response to novelty. This activity is both necessary and sufficient to elevate nucleus accumbens dopamine levels triggered by a novel stimulus. Direct stimulation of the vSUB increases the population of active dopamine neurons in the ventral tegmental area and dopamine levels in the accumbens via a multisynaptic route relayed through the nucleus accumbens. Furthermore, activity in the vSUB is correlated with drug-seeking behaviour such that vSUB inhibition attenuates cocaine-primed reinstatement of drug-seeking, while brief vSUB activation triggers reinstatement cocaine-seeking. We report that acute cocaine alters vSUB pyramidal neuron activity by inducing a frequency-dependent output mode transition from bursting to single-spiking. We suggest that under normal conditions bursting and output mode switching (bursting to single-spiking) may be needed for proper routing of information in and out of the vSUB. We propose that psychostimulants disrupt bursting and output mode switching leading to inappropriate dopamine/novelty signaling that is necessary to set motivational states and direct attention and ultimately, behaviour.

Malignant mixed tumors of the salivary gland: a study of loss of heterozygosity in tumor suppressor genes.

Carcinosarcomas and carcinoma ex pleomorphic adenoma of the salivary glands are rare tumors that fit into the broader category of malignant mixed tumors. Although most evidence has suggested that the different morphologic components arise from a common clonal origin, there are very few studies that have provided molecular evidence for this clonality. In this study, we examined a set of seven carcinosarcomas and four carcinomas ex pleomorphic adenoma for tumor suppressor gene loss of heterozygosity, in order to assess the clonal patterns in the varying components. Microdissection was performed to obtain each morphological component and tumor suppressor gene loci on 3p, 5q, 9p, 17p, 17q, and 18q were analyzed. The fractional allelic loss (FAL) was calculated for each area, and the different targets were compared for their molecular profile. The overall mean FAL of the malignant targets was 42%. In carcinosarcomas, the sarcomatous targets had a higher mean FAL than the carcinomatous targets (68 vs 46%, respectively) and in carcinomas ex pleomorphic adenoma, the mean FAL in the benign component was 11 vs 46% seen in the carcinomatous component. The most frequently lost genetic loci were p53 (17p13, 73%), nm23-H1 (17q21, 55%), and DCC (18q21, 50%). Loss of heterozygosity of 17q21 and 9p21 only occurred in carcinosarcomas and not in carcinomas ex pleomorphic adenoma. Within the carcinosarcomas, the mutational profiles were conserved between epithelial and sarcomatous areas. In carcinomas ex pleomorphic adenoma, loss of heterozygosity was uncommon in the benign component, but the mutations were conserved in the corresponding malignant areas. These results support the hypothesis that the carcinomatous and sarcomatous components of carcinosarcomas are clonally related. Furthermore, these data support prior studies that suggest a common clonal origin for the benign and malignant components of carcinomas ex pleomorphic adenoma.

Prenatal detection of deletion 6q13q15 in a complex karyotype.

OBJECTIVES: Prenatal diagnosis of a pregnancy with elevated maternal serum alpha-fetoprotein identified a karyotype with a complex chromosomal rearrangement, a Robertsonian translocation and a 6q deletion involving bands q13q15. Sonography identified mild IUGR, polyhydramnios and micrognathia. The infant presented with multiple congenital anomalies, primarily limited to the head and neck, including hypertelorism, broad nose, micrognathia, cleft palate, microglossia and low-set ears with microtia. METHODS: Amniocytes of the fetus and blood of the patient and her parents were analyzed by cytogenetics and fluorescence in situ hybridization. RESULTS: The karyotype on the fetus was 45,XX,t(3;21;20)(p12;q11.2;p11.2), del(6)(q13q15),der(13;14) (q10;q10)mat. CONCLUSION: The 13;14 Robertsonian translocation was inherited from the mother and the three-way translocation appeared to be balanced. The patient had facial dysmorphology similar to that which has been described in 6 previously reported cases with the same deletion involving 6q13q15. There was no recognizable abnormality of limbs or digits, and the autopsy did not identify defects involving the internal organs.

Tumor suppressor gene allelic loss profiles of the variants of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTCa) is a relatively common, indolent tumor that usually has an excellent prognosis. While the diagnosis of conventional PTCa is relatively straightforward, encapsulated tumors with follicular growth pattern and unusual or incomplete cytologic features of papillary carcinoma can be diagnostically challenging. Encapsulated, noninvasive tumors are particularly controversial as the differential diagnosis includes a nonneoplastic nodule, a benign follicular adenoma, and papillary carcinoma. In this study, we performed molecular genotyping to identify loss of heterozygosity of tumor suppressor genes in conventional PTCa and in several different morphologic variants, including follicular variant, tall cell variant, and oncocytic variant. Our data demonstrate that conventional PTCas have the lowest frequency of allelic loss (7%), as compared with follicular, oncocytic, and tall cell variants (19%, 34%, and 20%, respectively). Frequency of allelic loss increased with increasing size of the tumors, but did not correlate with age, gender, extrathyroidal extension, or lymph node metastases. Though it is unlikely that these results will enable the distinction between different variants of papillary carcinoma, the finding of significant rates ofallelic loss in the variants of PTCa provides additional evidence of malignancy and may be useful in distinguishing encapsulated tumors from nonneoplastic or benign nodules.

Comparative molecular analysis of loss of heterozygosity in adenocarcinoma in bile duct brushings and corresponding surgical pathology specimens.

BACKGROUND: Bile duct brushing is the procedure of choice for the assessment of neoplasia of the biliary and pancreatic ducts. Conventional cytopathologic evaluation has been reported to have high specificity but relatively low sensitivity. Although a number of molecular studies regarding biliary tract tissue specimens have been performed, to the authors' knowledge their precise applicability to cytopathology specimens has not been critically analyzed. METHODS: Bile duct brushing specimens with the cytopathologic diagnosis of "suspicious" or "positive for malignant cells" along with corresponding surgical pathology specimens demonstrating adenocarcinoma were searched for in the files of UPMC-Presbyterian Hospital for the years 1990-1996. Tumor cells from representative cytopathology and histology slides were microdissected and analyzed for loss of heterozygosity (LOH) in a panel of microsatellite markers. The results obtained from cytopathologic and surgical pathology specimens were compared. RESULTS: Eight paired surgical and cytopathology cases of adenocarcinoma involving the biliary tract were identified. The fractional allelic loss (FAL) for the surgical specimens (FAL-S) ranged from 12.5-71.4% and the FAL for the cytopathology specimens (FAL-C) ranged from 25-71.4%. However, when evaluating the actual loci of LOH, the concordance rate of the surgical and cytopathology specimens ranged from 71.4-100% (mean, 88.6%). Only 3 of the 8 cases (37.5%) were found to have identical matching of the LOH loci. CONCLUSIONS: Although the overall concordance rate of LOH in biliary cytology and surgical specimens by molecular analysis is relatively high, the issue of molecular tumoral heterogeneity must be considered if clinical decisions are to be based exclusively on cytopathologic analysis.