Differences In Staining Intensity Research Articles

Trophoblast Glycoprotein is Associated With a Favorable Outcome for Mesothelioma and a Target for Antibody Drug Conjugates.

The prognosis for patients with mesothelioma is poor, which prompts the need for the development of better treatment options. Antibody drug conjugates (ADCs) are gaining interest as a therapeutic strategy in mesothelioma. Trophoblast glycoprotein (5T4) is an oncofetal protein overexpressed in mesothelioma with low expression in normal tissue and therefore a good candidate for ADC treatment. Here,we evaluated and manipulated 5T4 as a suitable antigen for ADC targeted therapy in patients with mesothelioma. Expression of the 5T4 antigen is evaluated in (primary) mesothelioma cell lines and biopsy specimens, and correlated with clinical outcome. Internalization was assessed in 5T4 expressing cells. The cytotoxicity of three different 5T4-targeting ADCs was tested on (primary) mesotheliomacells. 5T4 was expressed in 10 of 12 (primary) cell lines. Most biopsy specimens stained positive for the 5T4 antigen, with marked differences in staining intensity and percentage of positive cells. High expression correlated with long progression-free survival. Both free antibody and ADCs targeting 5T4 were internalized and entered lysosomal compartments. Cytotoxicity experiments showed that cell lines with a high expression for 5T4 were sensitive to two of three ADCs. Lack of efficacy for the third ADC could be restored by neutralizing lysosomal compartments with chloroquine. The 5T4 antigen is expressed in mesothelioma and 5T4-based ADCs are internalized in lysosomes. Two of three ADCs were capable of killing the mesothelioma cells; the third ADC required additional lysosomal neutralization for its effect. 5T4-based ADCs would be a selective strategy for the treatment of mesothelioma.

Interobserver variation in CD30 immunohistochemistry interpretation; consequences for patient selection for targeted treatment.

CD30 immunohistochemistry (IHC) in malignant lymphoma is used for selection of patients in clinical trials using brentuximab vedotin, an antibody drug-conjugate targeting the CD30 molecule. For reliable implementation in daily practice and meaningful selection of patients for clinical trials, information on technical variation and interobserver reproducibility of CD30 immunohistochemistry (IHC) staining is required. We conducted a three-round reproducibility assessment of CD30 scoring for categorised frequency and intensity, including a technical validation, a 'live polling' pre- and post-instruction scoring round and a web-based round including individual scoring with additional IHC information to mimic daily diagnostic practice. Agreement in all three scoring rounds was poor to fair (κ = 0.12-0.35 for CD30-positive tumour cell percentage and κ = 0.16-0.41 for staining intensity), even when allowing for one category of freedom in percentage of tumour cell positivity (κ = 0.30-0.61). The first round with CD30 staining performed in five independent laboratories showed objective differences in staining intensity. In the second round, approximately half the pathologists changed their opinion on CD30 frequency after a discussion on potential pitfalls, highlighting hesitancy in decision-making. Using fictional cut-off points for percentage of tumour cell positivity, agreement was still suboptimal (κ = 0.35-0.60). Lack of agreement in cases with heterogeneous expression is shown to influence patient eligibility for treatment with brentuximab vedotin, both in clinical practice and within the context of clinical trials, and limits the potential predictive value of the relative frequency of CD30-positive neoplastic cells for clinical response.

Automatic Search of Spots and Color Classification in ELISPOT Assay.

Accuracy of spot detection and classification plays a critical role in the analysis of ELISPOT data. Differences in staining intensities of spots and their morphological variations make it difficult developing a reliable software application. An image recognition method allowing the automatic detection and classification of round objects (spots) on ELISPOT images independently of the registration conditions was developed. The emphasis is done on objects of elliptical shape, which is typical for a wide range of spots. It can be analyzed by both monochrome and a dual-color version of our software. The method of subdivision of objects into groups is also described which is based on color attributes of spots.

Profiling differential microRNA expression between in situ, infiltrative and lympho-vascular space invasive breast cancer: a pilot study.

Ductal carcinoma in situ (DCIS), invasive breast cancer (IBC) and lympho-vascular invasion (LVI) represent distinct stages in breast cancer progression with different clinical implications. Altered microRNA (miRNA) expression may play a role in mediating the progression of DCIS to IBC and LVI. The aim of this pilot study was to investigate whether differential miRNA expression could play a role in breast cancer progression. Cancer cells from DCIS, IBC and LVI were microdissected from formalin fixed paraffin embedded (FFPE) tissue of five breast cancer samples. MiRNA profiling of extracted RNA was performed using the TaqMan® Array Human MicroRNA Cards A and B v3.0. Candidate miRNAs and gene targets were validated by qPCR. 3D culture of MCF10A, MCF10DCIS.com and T47D cells were used as models for normal, DCIS and IBC. Immunohistochemistry of candidate genes was performed on FFPE 3D cell cultures as well as on tissue microarray which included cores of DCIS and IBC samples. MiR-150, miR-126 and miR-155 were found to be more highly expressed in IBC and LVI compared to DCIS. Gene targets of these miRNAs, RhoA, PEG10 and MYB, were found to be more highly expressed in DCIS compared to IBC by qPCR and in MCF10A and MCF10DCIS.com cells compared to T47D cells by immunohistochemistry. There was no difference in intensity of staining of RhoA by immunohistochemistry in DCIS versus IBC samples on tissue microarray. In this pilot study, we found evidence to support a potential role for variation in miRNA levels in the transition from DCIS to IBC.

Sphingosine-1-Phosphate-Receptor 1 as a Marker for Endothelial Cells in Mouse Xenograft Models of Human Cancer.

The sphingosine-1-phosphate (S1P) 1 receptor (S1P1R) is an important receptor for the modulation of endothelial cell function. We, therefore, wanted to investigate its expression as a blood vessel marker. The expression of the S1P receptor 1 (S1P1R) in mouse blood vessel endothelium was investigated immunohistochemically in normal blood vessel endothelium and blood vessel endothelium of xenografted human tumors. The S1P1 receptor was expressed in the endothelium of most mouse organs. Endothelia of the intestinal tract and of adipose tissue showed the strongest immunoreactivity. However, a difference in staining between larger vessels and fenestrated endothelium was observed, whereas fenestrated endothelium expressed a weaker staining. In addition to normal endothelia, most tumor blood vessel endothelia expressed S1P1R. A particularly strong expression in the endothelium was detected in primary pancreatic and prostate cancer xenografts. In both xenograft tumor entities, no significant difference in staining intensities of the endothelium between arteries, veins and capillaries was observed. As S1P1R is expressed in most blood vessel endothelia and in the tumor blood vessel endothelia, it is an ideal blood vessel marker.

Oxytocin receptors in dioestrous and anoestrous canine uteri.

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)-stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real-time PCR from full-thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non-pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p<.05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real-time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non-pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin-dependent activity during dioestrus than anoestrus.

Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature.

To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature. Immunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed. Over the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. As a "salvage" technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

Immunohistochemical and ultrasonic signs of the disturbance of anti-thrombogenic properties of endothelium in atherosclerosis of the carotid sinus

Introduction. Endothelial dysfunction is currently believed to play a crucial role in pathogenesis of atherosclerosis. Nevertheless, there are only few studies that demonstrate the relationship between changes in parameters of the atherosclerotic process and the main functional properties of endothelium. In addition, the findings are often controversial, which served as an incentive for this study. Materials and methods. Thirteen atherosclerotic plaques resected during carotid endarterectomy were subjected to immunochemical and electron microscopy examination. The intensity of expression of the von Willebrand factor, thrombomodulin, and endothelial nitric acid synthase was assessed semi-quantitatively and compared to the main parameters of activity and the course of atherosclerosis in a plaque (volume of atheromatosis, lipophage aggregation, infiltration of the fibrous cap by monocytes and macrophages). Complicated and uncomplicated plaques were also compared. Results. The level of von Willebrand factor in the carotid sinus endothelium increased as lipids were accumulated in it and as the fibrous cap was infiltrated by monocytes and macrophages (р<0.017), as opposed to the levels of thrombomodulin and endothelial nitric oxide synthase whose expression did not show the activity of the atherosclerotic process and did not correlate with the level of von Willebrand factor. Meanwhile, the expression of thrombomodulin and endothelial nitric oxide synthase correlated with each other (р=0.004). No difference in staining intensity for all three markers was detected between the complicated and uncomplicated plaques. Ultrasonic analysis of endothelium demonstrated the strongly pronounced disturbance of anti-thrombogenicity of the vascular wall as a result of numerous defects of the endothelial stratum, adhesion of blood cells to the arterial surface causing microthrombi formation and activation of endotheliocytes along with dystrophic changes in them, necrosis, and exfoliation into the vascular lumen. Conclusions. The von Willebrand factor is found to have high diagnostic significance in the assessment of activity of the atherosclerotic process in the carotid sinus and the risk of developing complications. Thrombomodulin and endothelial nitric acid synthase are not recommended to be used as biomarkers of progression of atherosclerosis of this localization, since they are not indicative of the intensity of inflammatory response and destructive processes in a plaque.

Is phosphorylated tau unique to chronic traumatic encephalopathy? Phosphorylated tau in epileptic brain and chronic traumatic encephalopathy

Repetitive traumatic brain injury (rTBI) is one of the major risk factors for the abnormal deposition of phosphorylated tau (PT) in the brain and chronic traumatic encephalopathy (CTE). CTE and temporal lobe epilepsy (TLE) affect the limbic system, but no comparative studies on PT distribution in TLE and CTE are available. It is also unclear whether PT pathology results from repeated head hits (rTBI). These gaps prevent a thorough understanding of the pathogenesis and clinical significance of PT, limiting our ability to develop preventative and therapeutic interventions. We quantified PT in TLE and CTE to unveil whether a history of rTBI is a prerequisite for PT accumulation in the brain. Six postmortem CTE (mean 73.3 years) and age matched control samples were compared to 19 surgically resected TLE brain specimens (4 months-58 years; mean 27.6 years). No history of TBI was present in TLE or control; all CTE patients had a history of rTBI. TLE and CTE brain displayed increased levels of PT as revealed by immunohistochemistry. No age-dependent changes were noted, as PT was present as early as 4 months after birth. In TLE and CTE, cortical neurons, perivascular regions around penetrating pial vessels and meninges were immunopositive for PT; white matter tracts also displayed robust expression of extracellular PT organized in bundles parallel to venules. Microscopically, there were extensive tau-immunoreactive neuronal, astrocytic and degenerating neurites throughout the brain. In CTE perivascular tangles were most prominent. Overall, significant differences in staining intensities were found between CTE and control (P<0.01) but not between CTE and TLE (P=0.08). pS199 tau analysis showed that CTE had the most high molecular weight tangle-associated tau, whereas epileptic brain contained low molecular weight tau. Tau deposition may not be specific to rTBI since TLE recapitulated most of the pathological features of CTE.

The expression of β3-adrenoceptors and their function in the human prostate.

Little is known about β3-adrenoceptor (AR) expression and function in human prostate. We examined the expression and distribution of β-AR subtypes in normal prostate and benign prostatic hyperplasia (BPH) tissues, and investigated which selective β-AR subtype agonist was most involved in the relaxation of isolated human prostate strips. Messenger RNA (mRNA) expression for β1-, β2-, and β3 -ARs was investigated using reverse transcriptase-polymerase chain reactions (RT-PCR). Quantitative analysis of mRNA expression of β-AR subtypes between normal prostate and BPH tissues was performed using quantitative RT-PCR (qPCR). Distributions were examined by immunohistochemistry (IHC). Strips of human normal prostate or BPH were suspended in organ baths and exposed to isoproterenol, dobutamine, procaterol, and TRK-380 to investigate their relaxant effects on KCl-induced contractions, and their inhibitory effects on electrical field stimulation (EFS)-induced contractions. We confirmed the presence of mRNA for β1-, β2-, and β3-ARs both in normal prostate and in BPH tissues. For β3-AR, mRNA expression in BPH tissues was significantly higher than in normal prostate tissues, but there was no significant difference in β1- and β2-AR expression between normal and BPH tissues. IHC revealed differences in staining intensity between smooth muscle cells and glandular cells, with different proportions for different β-AR subtypes. Staining of β3-AR was particularly intense in smooth muscle cells as opposed to glandular cells. Isoproterenol and TRK-380 significantly decreased the tone of KCl-induced contractions of the normal prostate strips. The rank order of relaxant effects was isoproterenol > TRK-380 > procaterol > dobutamine. All selective β-AR agonists significantly decreased the amplitude of EFS-induced contractions of the normal prostate strips. The rank order of inhibitory effects was isoproterenol > dobutamine >TRK-380 > procaterol. In BPH strips, all selective β-AR agonists showed no significant relaxant or inhibitory effects on KCl- or EFS-induced contractions. β3 -AR is abundant in human prostate smooth muscle, whose relaxation is mediated by β1- and β3-AR stimulation. β3-AR agonists may have clinical use in the treatment of male non-BPH patients or neurogenic bladder patients with voiding dysfunction.

S100A6 expression in cutaneous smooth muscle neoplasms.

The S100A6 protein is expressed in a variety of tissues and distinct staining patterns in S100A6 immunohistochemistry may be useful in the differential diagnosis of difficult lesions. We evaluated the staining pattern of the S100A6 antibody in 22 cases each of pilar leiomyoma (LM), angioleiomyoma (ALM), and cutaneous leiomyosarcoma (LMS). S100A6 labeled both the nucleus and cytoplasm of myocytes in positive cases. About 64% of LM and 86% ALM had positive staining to the S100A6 antibody but predominantly in a weak staining pattern. In contrast, 95% of the LMS exhibited moderate to strong staining with the S100A6 antibody. The difference in the frequency of positive cases was statistically significant in the LM vs LMS comparison (p=0.025), but we found intensity of staining to be of greatest practical utility. Analysis between the groups taking in to consideration differences in intensity of staining using the nonparametric rank sum (Mann-Whitney U test) demonstrated that there was a statistically significant difference between LM and LMS and between ALM and LMS. Weak or absent S100A6 staining supports a diagnosis of LM, whereas strong positive staining supports a diagnosis of LMS.

Natural (auto)antibodies in calves are affected by age and diet

Background: Natural autoantibodies (N(a)ab) were found in every species tested so far, and are likely important in maintaining homeostasis.Objectives: (1) To determine N(a)ab in Bos taurus calves, (2) evaluate effects of diet and age on N(a)ab binding repertoires in calves, and (3) delineate bovine liver cell lysate (BLL) antigens related with variation in rumen score and body weight.Animals and methods: Effects of age and diet on staining of BLL fragments by IgM and IgG antibodies in serum samples collected at 20 or at 26 weeks of age from bull calves either fed a restricted or ad libitum diet were analyzed using quantitative Western blotting. Correlations between fragments stained and grouping of calves were done by Principal Component Analysis (PCA). Redundancy analysis (RDA) was done to relate rumen score and body weight variation at slaughter at 27 weeks of age with stained BLL fragments.Results: In sera from all calves IgM and IgG antibodies binding BLL antigens were found. Corresponding fragments were stained, but quantitative differences in staining intensities were related to diet and age for both IgM and IgG. PCA revealed that age had a greater influence than diet on BLL fragment staining. RDA suggested that staining by IgM or IgG of specific BLL fragments was related with variation in rumen score and body weight. Conclusions and clinical importance: Analyses of N(a)ab in serum could be a potential tool to estimate the health status of cattle, and be used to evaluate effects of husbandry practices.

Abstract B55: Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer

Abstract Background: TRIM28 is a universal transcriptional co-repressor with pleotropic effects in both normal and tumor cells. We have previously shown that varying TRIM28 levels in epithelial cells and stromal fibroblasts have a prognostic value in colorectal cancer patients. The pathophysiological role of TRIM28 in carcinogenesis may therefore be associated with TRIM28 expression levels and cell type of expression within the tumor microenvironment. In this study we aim to dissect the molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment by isolating individual populations of neoplastic epithelial and stromal cells and investigating their protein signaling networks. Methods: TRIM28 expression was analyzed by immunohistochemistry on FFPE tissue from 19 colorectal cancer patients. TRIM28 staining was evaluated in both epithelial and stromal compartments. IHC scoring of at least 2 units of difference in staining intensity between stromal fibroblasts and epithelial cells was defined as a high TRIM28 expression ratio and a low TRIM28 expression ratio was defined as 1 or 0 units of difference in staining intensity. Reverse-phase protein microarrays (RPMA) were constructed from laser capture micro-dissection (LCM) enriched tumor epithelium and stroma isolated from fresh-frozen tissue of the same patient cohort. The protein signaling networks were measured for 32 downstream signaling endpoints. Spearman rank analysis was used to assess the correlations between individual protein pairs. Correlation coefficient ρ ≥ 0.75 with P ≤ 0.01 was considered significant. Results: Immunohistochemical analysis of the FFPE tissue sections identified 10 TRIM28 high ratio cases and 9 TRIM28 low ratio cases. Proteomic networks were assessed in TRIM28 high and low ratio cases in fresh-frozen tissue using RPMA. Spearman ρ rank correlation analyses for the 32 signaling proteins revealed that 184 highly correlated protein pairs exclusive to the TRIM28 high ratio group, whereas 157 protein pairs were found exclusively in the TRIM28 low ratio group. In addition 191 protein pairs were shared across both TRIM28 high and low ratio groups. The caspases 3 and 7, as well as the cell surface receptor RAGE, were prominent in the high TRIM28 ratio group proteomic network, whereas the Metalloprotease MMP9 and the GTPase Ras-GRF1 were exclusive to the low TRIM28 ratio group. Furthermore we found Survivin and JNK to be prominent in the stromal compartment of the high TRIM28 cases. Conclusions: We characterized molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment. Proteomic analysis revealed distinct protein signaling networks associated with varying TRIM28 expression levels in epithelial and stromal compartments. The study presents a novel way of deciphering molecular crosstalk between the epithelial and stromal compartments within the microenvironment. Citation Format: Seán Fitzgerald, Virginia Espina, Katherine M. Sheehan, Robert Cummins, Anthony O'Grady, Dermot Kenny, Richard O'Kennedy, Lance Liotta, Elaine W. Kay, Gregor Kijanka. Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B55. doi:10.1158/1538-7445.CHTME14-B55

Glomerular Collagen V Codeposition and Hepatic Perisinusoidal Collagen III Accumulation in Canine Collagen Type III Glomerulopathy.

Collagen type III glomerulopathy, also known as collagenofibrotic glomerulopathy, is a rare renal disease of unknown pathogenesis. The disease occurs in humans and animals and is characterized by massive glomerular accumulations of collagen type III. In the present study, we describe a Drever dog litter affected by an early onset variant of this glomerular disease, where 4 of 9 puppies developed renal failure within 50 days of age. Necropsy specimens of kidney from the 4 affected cases were studied by light microscopy, electron microscopy, and immunohistochemistry, and characteristic lesions compatible with a diagnosis of collagen type III glomerulopathy were found. In addition, 2 cases showed atypical epithelium in the collecting ducts of the medulla, so-called adenomatoid change. Immunohistochemistry of renal specimens from collagen type III glomerulopathy-affected dogs (n = 10) originating from two different dog strains, the Drever dogs and a mixed-breed strain, demonstrated that the deposited glomerular collagen is composed of a mixture of collagen III and collagen V. The distribution of the collagen V corresponded to the localization of collagen III; however, differences in staining intensity showed that collagen type III is the dominating component. Immunohistochemistry for collagen III (n = 9) and a transmission electron microscopic study (n = 1) showed hepatic perisinusoidal collagen type III deposition in affected cases from both dog strains. This is the first report documenting glomerular accumulations of collagen type V and perisinusoidal liver collagen III deposition in canine collagen type III glomerulopathy.

Distributional variation of lignin and non-cellulosic polysaccharide epitopes in different pit membranes of Scots pine and Norway spruce seedlings

Microdistribution of non-cellulosic polysaccharides in pit membranes of bordered pits (intertracheid pits between adjacent tracheids), cross-field pits (half bordered pits between tracheids and ray parenchyma cells) and ray pits (simple pits in nodular end walls of ray parenchyma cells) was investigated in mature earlywood of juvenile Scots pine and Norway spruce seedlings using immunocytochemistry combined with monoclonal antibodies specific to (1→4)-β-galactan (LM5), (1→5)-α-arabinan (LM6), homogalacturonan (HG, LM19, LM20), xyloglucan (LM15), xylan (LM10, LM11) and mannan (LM21, LM22) epitopes. Using phloroglucinol-HCl and KMnO4 staining, lignin distribution in pit membranes was also examined. Apart from cross-field pit membranes in Scots pine, all pit membranes observed showed a positive reaction for lignin with differences in staining intensity. Ray pit membranes showed strongest reaction with lignin staining in both species. Intensity of lignin staining in bordered pit membranes was stronger in Norway spruce than in Scots pine. With localization of non-cellulosic polysaccharide epitopes, Scots pine showed differences in cross-field pit membranes (rhamnogalacturonan-I (RG-I), HG and xyloglucan epitopes) from bordered and ray pit membranes (RG-I and HG epitopes). In contrast, Norway spruce showed significant differences in ray pit membranes (RG-I, HG, xyloglucan, xylan and mannan epitopes) from bordered and cross-field pit membranes (HG and no/trace amount of RG-I epitopes). Distributional differences in HG epitopes depending on antibody type/ membrane regions were also observed in cross-field pit membranes between the two species. Together, the results suggest that distribution patterns of lignin and non-cellulosic polysaccharides in pit membranes differ significantly between pit types and between Scots pine and Norway spruce. Compared with the same types of pit membranes in hardwoods, the results for Scots pine and Norway spruce (softwoods) differed significantly.

Abstract 911: Prostate-specific membrane antigen (PSMA) expression as a potential patient selection marker in patients with refractory solid tumors administered BIND-014, a PSMA-targeted nanoparticle containing docetaxel

Abstract Background: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is overexpressed in prostate adenocarcinomas and in non-prostatic tumor neovasculature. As a novel target for therapeutic approaches, the distribution pattern of PSMA in primary and metastatic tumors is of significant interest. In this study we addressed the cellular distribution and heterogeneity of PSMA expression in solid tumors from patients on a phase 1 study of BIND-014, a PSMA-targeted nanoparticle containing docetaxel.Methods: Paraffin-embedded sections of surgical specimens from 1 patient (pt) with prostate carcinoma and 4 pts with non-prostatic tumors (cervical, NSCLC, breast and hepatobiliary) were evaluated. Immunohistochemistry by platelet endothelial cell adhesion molecule (CD31) for the detection of endothelial cells and monoclonal mouse anti-human PSMA (Clone 3E6) for detection of PSMA were used to determine the cellular localization and staining intensity for each tumor type. Results: All pts evaluated experienced some clinical benefit from BIND-014 (including complete response, partial response or stable disease) which is targeted to PSMA. We demonstrated differences in staining intensity and localization of PSMA in neoplastic epithelium and neoplastic tissue-associated blood vessels. PSMA was detected in the epithelium of prostate tissue and in neoplastic tissue-associated vessels of cervical, anal, NSCLC, and hepatobiliary cancers. PSMA was not detected in benign tissue of any specimens evaluated. Conclusions: Our findings support the feasibility of developing this assay as a companion diagnostic to potentially select patients who may benefit from PSMA-directed targeted therapy. Ongoing Phase 2 studies with BIND-014 include further PSMA expression analysis. Citation Format: Susan Low, Daniel Von Hoff, Monica Mita, Howard Burris, Peter Eisenberg, Lowell Hart, Patricia LoRusso, Glen Weiss, Jasgit Sachdev, Alain Mita, Ramesh Ramanathan, Jeff Hrkach, Jason Summa, Gregory Berk. Prostate-specific membrane antigen (PSMA) expression as a potential patient selection marker in patients with refractory solid tumors administered BIND-014, a PSMA-targeted nanoparticle containing docetaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 911. doi:10.1158/1538-7445.AM2014-911

Sa1941 The Candidate Molecular Markers for Evaluating the Risk of Gastric Cancer After Helicobacter pylori Eradication

skin, gastrointestinal and genitourinary tracts, cardiac, smooth and skeletal muscle and immune cells (lymphocytes, mast cells and eosinophils). Dysregulation of NGF expression and signalling have been described in allergy, asthma, inflammation in the gut, chronic constipation, coronary artery disease and some cancers. In prostate cancer NGF stimulates tumour cell growth and metastasis and may drive nerve sprouting and infiltration. Inhibiting NGF by using blocking antibodies has a significant analgesic effect and is proposed to decrease metastatic cancer pain. Aim: ProNGF expression has not been previously described in tumours of the gastrointestinal tract. Materials and Methods: The expression of proNGF was examined by immunocytochemistry of esophageal squamous cancer (SCC) (15 cases), gastric adenocarcinoma (10 cases) and colorectal adenocarcinoma (12 cases) and corresponding normal adjacent tissues in the gut in tumour microarrays (Figure 1). Sections were scored for site of cytoplasmic staining in esophagus and cancer grade. Results: All gut epithelial tissue expressed pro-NGF. In normal oesophageal squamous epithelium expression site was diffuse, superficial or basal (Figure 2), in squamous carcinoma, staining was intense and diffuse. In adenocarcinomas (stomach and colon) staining was diffuse, and in intestinal metaplasia (IM) in the stomach, staining was more intense compared to adjacent gastric epithelium. There was no significant difference in intensity of staining by grade of cancer. Conclusions: There is significant expression of pro-NGF in squamous oesophageal and gastrointestinal columnar epithelium in both normal tissue and cancer. Pro-NGF may be therefore be a target of interest in biomarker exploration and therapeutic development for inhibition of cancer growth and cancer pain.

Diversity of glutelin acidic subunit polypeptides in rice cultivars collected from Northern Vietnam

AbstractThe diversity of glutelin acidic polypeptides in rice cultivars collected from Northern Vietnam was characterized via sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and isoelectric focusing (IEF) electrophoresis. Glutelin acidic subunits were separated into four bands by molecular mass, as α‐1 (39 kDa), α‐2 (38 kDa), α‐3 (37.5 or 37 kDa) and α‐4 (34 or 33 kDa). One hundred and eighty‐five Vietnamese rice cultivars were divided into three types, based on differences in staining intensity and the molecular size of the α‐3 and α‐4 polypeptides derived from SDS‐PAGE analysis. Wide variation was also observed in the isoelectric point (pI) staining intensity, in addition to the absence/presence of pI bands detected via IEF analysis. A total of 16 pI bands, ranging from pI 6.30 to pI 7.52, were identified in the various local rice cultivars. The maximum and minimum of IEF bands detected were 14 and 10, respectively. The genetic variability index (H′) ranged from 0.280 to 0.820, which confirms that local rice cultivars from Northern Vietnam have diverse glutelin seed storage units.

Immunohistochemical detection of cancer stem cell related markers CD44 and CD133 in metastatic colorectal cancer patients.

Aim. The goal of this study was to semiquantitatively detect presence of cancer stem cells markers CD44 and CD133 in immunohistochemically stained paired samples of colorectal cancer (CRC) and colorectal liver metastases (CLM). Level of staining intensity was compared to clinical and pathological characteristics of tumors with the aim to identify impact of CD44 or CD133 expression on tumor behavior. Patients and Methods. Formalin fixed paraffin embedded samples from 94 patients with colorectal tumor and liver metastases were collected at Sikl's Department of Pathology. Samples were stained by antibodies against CD44 and CD133. Presence and intensity of staining was assessed semiquantitatively by three trained researchers. Results. Patients with higher level of CD133 staining in CRC had longer disease free interval (Cox-Mantel P = 0.0244), whereas we found no relation between CD44 expression and overall survival or disease free interval. CD133 expression in CRC and CLM differed based on CRC grading; in case of CD44 we found differences in staining intensity in individual stages of tumor lymph node invasion. Conclusion. Effect of cancer stem cell markers on prognosis of colorectal cancer can vary depending on pathological classification of tumor, and we have shown that CD133, generally considered to be a negative marker, can bear also clinically positive prognostic information in group of patients with colorectal liver metastases.

Orexin expression in different prostate histopathologic examinations: Can it be a marker for prostate cancer? A preliminary result.

The aim of this study was to evaluate the expression of the orexin receptor in different prostate pathologies, including prostate adenocarcinoma, benign prostate hyperplasia and chronic prostatitis. A total of 90 patients (mean age 64.01±7.2 years) were enrolled in the study. The patients were divided into three groups of equal numbers based on their histopathologic findings: prostate cancer (Group 1), benign prostate hyperplasia (Group 2) and chronic prostatitis (Group 3). All the tissues were incubated with a primary antibody recognizing the Orexin receptor. The specific cytoplasmic immunoreactivity of the Orexin receptor was semiquantitatively scored for intensity and distribution based on a grading scale. The staining intensity and orexin expression were evaluated using Pearson χ(2) test. A heterogeneous staining pattern of the Orexin receptor was observed between the groups. The expression rates were 90% (27/30) in Group 1, 53.3% (16/30) in Group 2 and 26.7% (8/30) in Group 3. While 5 patients (9.3%) in Group 1 showed strong staining, all samples from the other 2 groups showed only weak staining. There were significant differences in staining intensity between the three groups. The expression and distribution of the Orexin receptor was more widespread in Group 1 than in the other groups and was higher in patients with poorly differentiated malignancy. However, there was no significant difference based on Gleason score. Orexin receptors are found in human prostate tissues and their expression is widespread in prostate cancer and in patients with a higher Gleason score. Therefore, we believe that Orexin immunoreactivity can be considered to be an indicator of poor prognosis and of poorly differentiated prostate cancer cases.