Diagnostic Value of Claudin-4 and EZH2 Immunohistochemistry in Effusion Cytology.

BACKGROUND: Metastatic adenocarcinoma (MAC) accounts for most cases of malignant effusions. Sometimes, it can be difficult to distinguish MAC from reactive mesothelial cells (RMC) in cytologic specimens. Our aim was to assess the diagnostic performance of a novel immunohistochemical panel composed of claudin-4 and EZH2 in differentiating MAC from RMC in effusion cytology. METHODS: A total of 80 cases of serous effusions (48 MAC and 32 RMC) were included. Immunohistochemistry using claudin-4 and EZH2 was performed on cell block sections of these cases. Assessment of staining patterns, intensity and percentage of target cells stained was done. RESULTS: Claudin-4 showed membranous staining in 46/48 of MAC and 1/32 of RMC. High EZH2 (≥ 50% of target cells) was detected in 42/48 MAC and 2/32 RMC. For the discrimination between MAC and RMC, claudin-4 exhibited 95.8% sensitivity and 96.9% specificity, high-EZH2 exhibited 87.5% sensitivity and 93.8% specificity, while the combination of both claudin-4 and high EZH2 showed 100% sensitivity and 90.6% specificity. CONCLUSION: Claudin-4 shows high sensitivity and specificity in differentiation between MAC and RMC in effusion cytology, and might be useful as a solitary marker for MAC. Adding EZH2 to claudin-4 increases the sensitivity to 100%. However, the interpretation of EZH2 results can be challenging due to its focal expression in RMC and inflammatory cells.

Evaluation of solid-supported room-temperature ionic liquids containing crown ethers as media for metal ion separation and preconcentration.

Extraction chromatographic (EXC) resins incorporating an appropriate crown ether in an oxygenated organic solvent such as 1-octanol are well established as sorbents for the analytical-scale separation and preconcentration of radiostrontium from a variety of sample types. Recent solvent extraction studies employing crown ethers in various 1-alkyl-3-methylimidazolium-based (CnC1im(+)) room-temperature ionic liquids (RTILs) indicate that under certain conditions, distribution ratios (DSr) for strontium far in excess of those observed with conventional organic solvents are observed. To determine if this increase in liquid-liquid extraction efficiency will lead to improved strontium sorbents, several EXC resins and sol-gel glasses incorporating di-tert-butylcyclohexano-18-crown-6 (DtBuCH18C6) in either 1-decyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (C10C1imTf2N) or the related hydroxyalkyl-functionalized IL 1-(12-hydroxydodecyl)-3-butylimidazolium bis[(trifluoromethyl)sulfonyl]imide (C12OHC4im Tf2N) were prepared and characterized. Unexpectedly the performance of these materials was not uniformly better than that of a conventional EXC resin, an apparent result of the greater viscosity of the ionic liquids and the lower solubility of the crown ether in ILs versus conventional organic solvents.

Penile agenesis in adult.

Penile agenesis (PA) is an extremely rare congenital anomaly with profound surgical and psychosocial consequences. Only seventy five cases have been reported in the literature, the highest age of presentation known seven years. We present a twenty six years old otherwise normal aphallic male with attraction to female sex and night emission through anus. The patient did not agree to the female gender assignment and functioning phallic reconstruction is practically unavailable. Early female gender assignment and feminizing perineal reconstruction (Vaginoplasty) in neonatal cases is the technically easier goal of treatment. Late presenting cases add difficulty to debated decision making of gender assignment.

Effect of maternal anemia on fetal outcome.

We assess the effect of maternal iron deficiency anemia (MIDA) on cord blood iron status, placental weight and fetal outcome [birth weight, APGAR (appearance, pulse, Grimace, activity, and respiration) scores and birth asphyxia]. We conducted a cross sectional analytic study on fifty hospitalized pregnant women and their neonates over a year in a teaching hospital in the capital city of Bangladesh. Serum and cord hemoglobin concentration [Hb] with ferritin values were determined immediately after delivery, placental weight, gestational age, birth weight, APGAR scores and birth asphyxia were recorded. It was observed that 36 percent of the pregnant women were anemic. Maternal [Hb] and serum ferritin showed a highly significant positive correlation (r=0.92; p<0.001) indicating that iron deficiency was the most dominant factor in the causation of anemia amongst them. The maternal [Hb] showed a significant correlation with placental weight (r=0.40; p<0.001), birth weight (r=0.35; p<0.001), APGAR score (r=0.52; p<0.001), gestational age (r=0.61; p<0.001) and birth asphyxia. Maternal serum ferritin also correlated positively with cord ferritin (r=0.94; p<0.001), placental weight (r=0.26; p<0.001) and birth weight (r=0.27; p<0.001). Iron deficiency anemia (IDA) during pregnancy had significant adverse affect on the foetal outcome.

Wilms tumor in adult.

Wilms Tumor (WT) is a very rare malignancy in adults representing 1% of all renal neoplasms. It is however the most common renal tumor of children and adult patients are treated like pediatric cases. Bilateral tumors occur in 5% of adult cases. The typical presenting features are asymptomatic abdominal mass (most common), hematuria, pain, fever, and hypertension. As clinical presentation of WT is similar to that of renal cell carcinoma (RCC), it tends to be an unsuspected pathological diagnosis in most cases. The diagnosis of the tumor needs positive sonographic and computed tomography (CT) findings with histopathological confirmation. Prognosis of adult WT is relatively poor and resistant to chemotherapy. We present a case of wilms tumor in a 68 years old male patient with right sided non tender abdominal mass and occasional flank pain. The patient was normotensive but hematuric and radiological findings suggested right renal mass with enlarged lymph node and histopathological analysis revealed nephroblastoma associated with lymph node metastases.

Effects of bile acids on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced aberrant crypt foci and DNA adduct formation in the rat colon.

The effects of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci (ACF) in the rat colon were examined. The effect of these bile acids on DNA adduct formation by PhIP in the colon was then analyzed, since the main action of PhIP is the formation of DNA adducts and subsequent gene mutations. For the ACF study, male F344 rats were administered PhIP-HCl (75 mg/kg, 10 doses) by gavage, and a diet containing bile acid (0.4% DCA or UDCA) was provided from 3 days before the first dose of PhIP for 8 weeks. The mean number of ACF per colon of DCA, UDCA and controls were 9.9, 2.4 and 5.5, respectively. The ACF number was significantly increased by DCA and decreased by UDCA (P<0.001). To examine the effect of bile acids on DNA adduct formation, male F344 rats were fed a diet supplemented with bile acids (0.1 or 0.4% of DCA and UDCA) 7 days prior to the PhIP administration. All rats were administered a single dose of PhIP-HCl (50 mg/kg) by gavage and sacrificed 48 hours later. DNA adduct levels of the 0.1% UDCA, 0.1% DCA and controls were 2.93 (adducts/10(7) nucleotides), 2.65 and 1.10, respectively. Those of 0.4% UDCA, 0.4% DCA and controls were 1.64, 1.30 and 1.00, respectively. The PhIP-DNA adduct level was significantly increased by administration of 0.1% UDCA, 0.1% DCA (P<0.05) and 0.4% UDCA (P<0.01). The increasing effect of both DCA and UDCA on PhIP-induced DNA adduct formation was unexpected, and was not directly associated with ACF formation.

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

Identification of hepatic tamoxifen-DNA adducts in mice: alpha-(N(2)-deoxyguanosinyl)tamoxifen and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.

Tamoxifen-DNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifen-DNA adduct, four stereoisomers of alpha-(N:(2)-deoxyguanosinyl)tamoxifen N:-oxide 3'-monophosphate (dG(3'P)-N:(2)-TAM N:-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-alpha-acetoxytamoxifen N:-oxide in addition to four stereoisomers of alpha-(N:(2)-deoxyguano- sinyl)tamoxifen 3'-monophosphate (dG(3'P)-N:(2)-TAM) that was reported previously. Liquid chromatography-electrospray ionization-mass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M - H](-)), which corresponded to dG(3'P)-N:(2)-TAM N:-oxide. The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N:(2)-TAM N:-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/kg/day for 7 days by (32)P-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG(3'P)-N:(2)-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/10(8) nucleotides, which accounted for 53.3% of the total tamoxifen-DNA adducts. Lesser amounts of cis-dG(3'P)-N:(2)-TAM (2.8%) were also observed. An epimer of the trans-dG(3'P)-N:(2)-TAM N:-oxide (3.9 adducts/10(8) nucleotides) was detected as the third biggest adduct (7.2% of the total). The cis-dG(3'P)-N:(2)-TAM N:-oxide (0.4 adducts/10(8) nucleotides) accounted for 0.7% of the total. Thus, dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N:-oxide were identified in tamoxifen-treated mouse liver.

Tamoxifen-DNA adducts formed by alpha-acetoxytamoxifen N-oxide.

DNA adduct formation is assumed to be a major carcinogenic event, leading to the development of endometrial cancer in breast cancer patients taking tamoxifen and healthy women enrolled in a tamoxifen chemopreventive trial. To determine whether DNA adducts were formed by tamoxifen, trans- and cis-alpha-acetoxytamoxifen N-oxides were synthesized as model-activated forms via major tamoxifen metabolites, tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide. When alpha-acetoxytamoxifen N-oxide was reacted with human DNA, at least three DNA adducts were detected by (32)P-postlabeling coupled with HPLC. The total amount of DNA adducts formed by trans-alpha-hydroxytamoxifen N-oxide was 1.5-fold higher than that formed by the cis form. Both trans- and cis-alpha-acetoxytamoxifen N-oxide reacted with 2'-deoxyguanosine, resulting in the formation of three adducts (fr-1, fr-2-1, and fr-2-2). These products were studied using mass spectroscopy and proton magnetic resonance spectroscopy. fr-1 was identified as a mixture of the epimers of trans-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. fr-2-1 and fr-2-2 were determined to be epimers of cis-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.