High Staining Research Articles

Peculiar Fibronectin immunohistochemical staining in placental tissue of pathological pregnancies associated with insufficient placentation

Objective: To investigate the role of Fibronectin, an extracellular matrix protein essential in the process of trophoblastic invasion, in the development of pregnancy-related pathologies associated with insufficient placentation. Materials and methods: Retrospective study including 36 early (< 34th gestational age) preeclampsia (PE), 14 late (≥34th gestational age) PE, 6 HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), 18 small for gestational age (SGA) fetuses (under the 10th percentile), 17 early intrauterine growth restriction (IUGR), 35 late IUGR, and 64 controls. Fibronectin localization was evaluated in placental tissue microarray by immunohistochemistry and the amount assessed by intensity semi-quantitative score (3 = strong, 2 = moderate, and 1 = weak). Results: Fibronectin was present in cytoplasm and peri-cytoplasm areas. Moreover, in general Fibronectin cytoplasm staining had a higher intensity score among cases compared to controls. Furthermore, both cytoplasm and peri-cytoplasm Fibronectin intensity was higher in early PE (1, IQR 0 – 1), HELLP (1, IQR 1 – 1), and early IUGR (1, IQR 1 – 1) than in controls of 22 – 34 weeks' gestation (0, IQR 0 – 1) (p < 0.05). Intensity was also greater in late PE (1, IQR 0 – 1) than late IUGR (0, IQR 0 – 0) and controls (0, IQR 0 – 1) of 34 – 42 weeks' gestation, as well as in late PE associated with IUGR (1, IQR 0 – 1) than in IUGR alone (0, IQR 0 – 0) and in early IUGR (1, IQR 1 – 1) than late IUGR (0, IQR 0 – 0) (p < 0.05). In addition, peri-cytoplasmic Fibronectin was found to be significantly more represented in late PE associated with IUGR (2, IQR 1 – 2) than in controls (1, IQR 0 – 1) (p < 0.05). Conclusions: Fibronectin shows higher staining among PE, HELLP and IUGR cases compared to controls, probably reflecting a common underlying pathological mechanism coupled with insufficient placentation. Late IUGR seems to be unaffected by Fibronectin amount, suggesting a peculiar pathogenetic pattern.

FDG PET/CT Scan and Functional Adrenal Tumors: A Pilot Study for Lateralization.

Patients with Cushing's Syndrome (CS) and Conn's Syndrome with bilateral adrenal masses pose a dilemma. Uptake of 18F-FDG by hyperfunctioning adrenal glands has not been previously reported and may help lateralize. The aim was to determine if 18F-FDG PET/CT scan could identify hyperfunctioning adrenal masses and determine a biological basis for uptake. Patients with nonfunctional adenomas (n = 9), CS (n = 11), and Conn's syndrome (n = 4) underwent an 18F-FDG PET/CT scan with a volume of interest circumscribing each mass to obtain a maximal standardized uptake value (SUVmax). Thirty-two adrenal masses were analyzed. Genome-wide expression data from an independent cohort were analyzed in nonfunctioning adenomas (n = 20), Conn's syndrome (n = 29), and CS (n = 24) focusing on GLUT genes. For genes differentially expressed, immunohistochemistry was performed on tissue samples. Cortisol-secreting masses (n = 16) had a higher average SUVmax of 5.9 compared to nonfunctioning masses (n = 11, average SUVmax 4.2) and aldosterone-hypersecreting masses (n = 5, average SUVmax 3.2) (p = 0.007). SUVmax cut-off of 5.33 had 50.0% sensitivity and 81.8% specificity in localizing a cortisol-secreting mass. GLUT3 expression was 2.19-fold higher in patients with CS compared to patients with nonfunctioning adenomas (p = 0.003) and 2.16-fold higher in patients with CS compared to Conn's syndrome (p = 0.006). GLUT3 immunohistochemistry showed 2.2-fold higher staining in CS tumor samples compared to nonfunctioning adenomas. Differential 18F-FDG PET/CT uptake was observed in patients with nonfunctioning, aldosterone-hypersecreting, and cortisol-secreting masses. GLUT3 overexpression in cortisol-secreting tumor likely accounts for the differential uptake. Future larger cohort studies will need to be conducted to determine if 18F-FDG PET/CT uptake can lateralize cortisol-secreting adrenal masses in patients with bilateral adrenal masses.

Sa1851 Prognostic Significance of TGF-β Signaling in Human Hepatocellular Carcinoma

A S L D A b st ra ct s (LCD) including the variants small cell change (SCC), and large cell change (LCC), HCC, and NLC control tissues using Vectra® automated multispectral imaging system, and to evaluate any differential staining features between groups.Design: FOXM1 immunohistochemistry was performed on a liver cancer progression tissue array consisting of 85 paired liver tissue cores representing 20 NDC, 40 LCD consisting of 20 SCC and 20 LCC, 20 HCC, and 5 NLC control tissues in optical density by Vectra® automated multispectral imaging system. Staining was statistically compared between sample groups using a 2 tailed p-value derived from Welch's T Test. Results: The mean FOXM1 nuclear staining in OD was NLC 0.0318 ±0.0107, SCC 0.0741 ±0.0239, NDC 0.0717 ±0.0216, LCC 0.1103 ±0.1210, and HCC 0.1134 ±0.0378. Nuclear staining features by FOXM1 had three distinct groups with the highest staining seen in HCC and LCC, followed by SCC and NDC, and the least in NLC. The nuclear staining features of FOXM1 in HCC and LCC were similar (p=0.9158), as were those seen in SCC and NDC (p=0.7437). NLC showed markedly less staining (p <0.0088) than all other sample groups. SCC and NDC had statistically less staining than HCC (p<0.0003). SCC and NDC did not have statistically different nuclear staining from LCC (p=0.1961 for SCC, and p=0.1677 for NDC). Conclusions: Here, we provide evidence that the hepatocyte nuclear expression of the protein product of human proto-oncogene FOXM1 shows a stepwise increase in NLC and paired liver tissue samples of NDC and SCC, and LCC and HCC by Vectra®automated multispectral imaging system. Further, FOXM1 nuclear expression rises in LCC, before the development of HCCduring hepatocarcinogenesis. Additionally, the FOXM1 nuclear immunolevels did not differ in LCC and HCC.

Stem cell expression profile in hepatocellular carcinoma, small cell dysplasia, and cirrhosis

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with various etiologies and its incidence still continues to increase. Tumors with progenitor/stem cell (P/SC) immunophenotype are currently the main focus of interest. Human hepatic P/SCs are located in ductal plates in fetal and neonatal livers and they remain relatively constant throughout life. Enhanced self-renewal level of normal hepatic P/SCs serves as an early event in hepatocarcinogenesis. Approximately 40% of HCCs arise from P/SCs and these cells have a critical role in development and progression of HCC. Thus, CD133 expression was found to be upregulated during early liver restoration. In spite of the increasing evidence of the impact of cancer stem cells (CSCs) in hepatocarcinogenesis, the role of CSCs in the sequence of cirrhosis (Crh)-dysplastic nodules (DN)-HCC is still not clear. In our recent research, we investigated the expression rate and staining patterns of CD133 and CD90 in Crh, small cell dysplasia, and HCC. We also evaluated the relationship between the expression rates of CSC markers and the etiology of liver diseases. Among the P/SC markers, we like to attract more attention to CD133 and CD90. Both CD133 and CD90 expressions were higher in poorly differentiated HCC cases than well differentiated ones. According to the etiology, we found that the highest staining rate for CD133 in HCC cases developed in a cirrhotic background with chronic hepatitis B and D co-infection. The highest rate for CD90 was determined in HCC cases with chronic hepatitis C. Since Crh is the end-stage of chronic injury with continuing regeneration, detection of accompanying P/SC activation by CSC markers in suspicious nodules may suggest the initiation of early phases of hepatocarcinogenesis. Some other immunophenotypical features such as Glypican 3, heat shock protein-70, and glutamine synthetase stainings are also being used to diagnose precursor lesions. Facing the emerging concept of personalized treatment strategy, it is obvious that the studies in hepatocarcinogenesis related to the theory of CSCs will provide new ideas on genesis, development, and metastasis of HCC and will bring new insights for the diagnosis and treatment of these tumors.

Enhancement of the neuroprotective activity of Hericium erinaceus mycelium co-cultivated with Allium sativum extract

This study investigated the neuroprotective effects of Hericium erinaceus mycelium enriched with garlic extract (HGE) on rat pheochromocytoma nerve cells (PC12). The survival rates of the PC12 nerve cells and the neurite-bearing cells after the addition of HGE were estimated as 3.5 × 103 viable cells/ml and 2.3 × 103 viable cells/ml, respectively, which were 50% and 30% higher, respectively, compared with the untreated group. For the in vivo ischemia experiments, after treatment with the HGE extract, the hippocampal CA1 region was more strongly stained (>20%) than the control group, and the HGE extract also promoted higher staining levels than HFB, HM and HGEF, and even the garlic extract. This result indicates that HGE must have neuroprotective effects. Furthermore, HGE greatly decreased p21 gene expression to approximately 70% of the control and decreased p21 gene expression to even lower levels compared with HM, HGEF and the garlic extract. This work suggests that a synergistic effect of the H. erinaceus mycelium and the garlic extract (mainly allicin) exist because the amount of allicin in HGE (5.81 µg/ml) was lower than the garlic extract itself (6.89 µg/ml).

The prognostic significance of MET overexpression in Chinese patients with gastric adenocarcinoma.

53 Background: MET, receptor for hepatocyte growth factor (HGF), has been proposed as a therapeutic target in gastroesophageal cancer (GEC). The purpose of this study was to evaluate MET expression in Chinese patients with resectable gastric or gastroesophageal junction (GEJ) adenocarcinoma and to correlate expression with clinicopathologic parameters and prognosis. Methods: MET protein expression was assessed by immunohistochemistry (IHC) in paraffin-embedded tissues from resected tumor specimens. MET IHC was conducted using the CONFIRM SP44 anti-MET monoclonal antibody (Ventana Medical Systems Inc., Tucson, AZ). Four MET IHC scores were defined: 3+ (≥50% of tumor cells staining with strong intensity); 2+ (≥50% of tumor cells with moderate or higher staining, but &lt;50% with strong intensity); 1+ (≥50% of tumor cells with weak or higher staining, but &lt;50% with moderate or higher intensity); or 0 (no staining, or &lt;50% of tumor cells with any intensity) (Spigel et al, J Clin Oncol 2011;29:Suppl, Abst 7505). Kaplan-Meier was used to estimate OS and differences between MET subgroups were compared using the log-rank test. Univariate survival analysis was performed using the stratified Cox model. Results: 442 resected gastric adenocarcinoma samples were collected from patients who underwent surgery between Sept 2007 and Feb 2010. After excluding patients with missing survival, MET, tumor location, differentiation status and stage data, 362 samples were analyzed. Stage II and III accounted for 23% and 59%, respectively. MET prevalence by IHC was: 3+, 5.5%; 2+, 19.6%; 1+, 37.0%; 0, 37.9%. At the OS cut-off, 15 Apr 2012, median follow-up was 35.3 months (range 5.9–49.8 months). The group with MET IHC scores of 2+ or 3+ had significantly shorter OS compared to those with scores of 0 or 1+. The 2-year survival rate was 64% in the 2+/3+ group and 73% in the 0/1+ group (p=0.047). The HR for MET 2+/3+ versus 0/1+ was 1.65 (95% CI: 1.13–2.39, p=0.009), stratified by stage, tumor location and differentiation status. Conclusions: MET expression in resected tissue from Chinese patients with GEC was correlated with poor prognosis. This finding further supports the development of anti-MET agents in this patient population.

Reliability of Digital Camera over Spectrophotometer in measuring the Optical Properties of Orthodontic Elastomeric Ligatures

Objective: The aim of this study is to evaluate the reliability of digital camera and spectrophotometer in measuring the optical properties of clear elastomeric ligatures under the influence of different solutions. Materials and methods: Four hundred and thirty-two clear elastomeric modules from three manufacturers (3M, ORMCO, OT) were used and were immersed into six different solution of three segments each. Each solution (200 ml) containing a determined substance, as follows: distilled water (control group), coke, turmeric, sauce (20 ml), tea and coffee. All test specimens were immersed in the solutions and kept in an appropriate receptacle for 7, 14, 21 and 30 days. The differences in color changes depen ding on the type of elastomeric modules, immersion solution, and immersion period were analyzed by using the spectro photometer and digital camera. Results: These results were analyzed statistically using the software SPSS version 11.5. 3M showed the least and OT showed the highest staining with all types of immersion solutions. Coffee showed the highest while coke showed the least amount of discoloration. As the immersion time increases, amount of discoloration of elastomeric modules increases.

THU0245 Phosphodiesterase 4 Expression in Rheumatoid Arthritis Synovium and Effects of Apremilast on Synovial Fibroblasts

BackgroundApremilast (APR), an oral small-molecule inhibitor of phosphodiesterase 4 (PDE4) enzymes, works intracellularly to modulate a network of pro-inflammatory and anti-inflammatory mediators. PDE4 is a cyclic adenosine monophosphate (cAMP)-specific PDE,...

Secreted alkaline phosphatase defined by ms17-57 mab and ectopically expressed on the surface of GI cancer cells as a potential therapeutic target.

e22169 Background: There remains considerable interest in generating monoclonal antibody (mAb) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. In this investigation, mAbs were generated against potentially novel Ags on the cancer cell surface using a flow cytometry-high throughput screening (FACS-HTS) strategy. Methods: Mixed live cells from four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; Sixteen highly positive hybridoma colonies were selected via FACS-HTS using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus normal human PBMC). The purified ms17-57 and control commercial ALP mAbs were used to confirm the target Ags, which were identified as alkaline phosphatases (ALPs) expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). Results: MS identified the Ags recognized by ms17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and Intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules (i.e. dephosphorylation). Immunohistochemical staining using ms17-57 demonstrated significantly higher staining of gastrointestinal (GI; gastric and colorectal) cancer tissues than normal (non-cancer) GI control tissues (P&lt;0.01), and confirmed binding of ms17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assay using the PALP/IALP-expressing GC cell lines demonstrated that ms17-57 inhibited cell growth by 32±8%. Transwell cell migration assay documented that ms17-57 can inhibit PALP/IALP-expressing GI cancer cell migration. Conclusions: Our findings indicate that PALP and IALP can be ectopically expressed in the extracellular matrix of GI cancers, and that ms17-57 directed against PALP/IALP identified through HTS can inhibit GI cancer cell growth and migration. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.

Abstract 5380: Detection of OvCa1 in human malignancies.

Abstract Immunohistochemistry biomarkers are currently being developed to target specific proteins found in cancer cells, which could contribute to a better understanding of specific types of cancers and provide information to develop better treatments for cancer patients. The biomarker and putative tumor suppressor, OvCa1, has a function that is not well characterized. Due to a lack of staining reagents, we developed monoclonal antibodies of OvCa1 to examine multiple human malignancies. The goal of this project was to gain a better understanding of OvCa1 and its role in cancer. Primary cancers with different histological grades as well as metastatic lesions were examined with the monoclonal antibodies. Ovarian cancer tissue samples from the Indiana University Simon Cancer Center Tissue Bank were used for this study. The samples were fixed in neutral buffered formalin and processed into paraffin blocks. The tissues were cut using a microtome and placed on slides. Staining of the OvCa1 antibody was completed using immunohistochemistry (IHC) techniques. Thirty-one samples were evaluated including low grade tumors, medium grade tumors, high grade tumors, and metastatic ovarian carcinomas. All cases revealed a range of staining intensity with the OvCa1 antibody. The results indicated that OvCa1 for the high grade, stage 3/4, ovarian carcinomas had the highest staining intensity. Medium grade tumors had less OvCa1 expression. The metastatic tumors had less staining than any of the other three grades. Immunostaining was observed primarily in the cytoplasm and nucleus of the tumor cells. In addition, we evaluated approximately 20 tumors from various different organs. These included prostate, breast, spleen, lung, colon, stomach, and kidney tumors, which were positive for immunostaining with the OvCa1 antibody. In summary, the results indicated that all histological grades expressed the biomarker, OvCa1, and the staining intensity was highest in the high grade, stage 3/4, tumors. Our preliminary studies demonstrated a further need to delineate OvCa1 as a potential biomarker, which could be used for early detection and diagnosis of ovarian cancer. Citation Format: Jessica L. Jackson, Kristen A. Grothaus, Connie J. Temm, Lindsey D. Mayo, George E. Sanduksy. Detection of OvCa1 in human malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2013-5380

Analysis of cycle Gene Expression in Aedes aegypti Brains by In Situ Hybridization

Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.

Preclinical evaluation of PARP inhibition in breast cancer: Comparative effectiveness of olaparib and iniparib.

1042 Background: The main function of PARP1 is repair of single-strand DNA. Phase I/II clinical trials have shown that the PARP inhibitor, olaparib has efficacy in BRCA1/2-related breast cancer. Due to the similarities between BRCA1/2-associated and triple negative breast cancer (TNBC), we hypothesise that TNBC may also be sensitive to PARP inhibition. In order to assess this we addressed the effects of 2 PARP/PARP-like inhibitors, on a panel of breast cancer cell lines. Methods: PARP1 was measured by immunohistochemistry in 101 TNBC and 116 non-TN cancers. Comparative growth inhibitory capacity of olaparib and iniparib was evaluated using cell viability (MTT) and colony formation assays in 12 breast cancer cell lines (TN=7, non-TN=5). Results: Using immunohistochemistry, PARP1 staining was predominantly nuclear with some cytoplasmic staining. High staining intensity for PARP1 was found more frequently in ER-negative (p = 0.001), in high grade (p = 0.013) and in Ki67-positive ( p = 0.003) samples. Potentially important was the finding that high PARP1 staining intensity was detected more frequently in TN than non-TN samples (p = 0.0001). IC50 concentrations across 12 cell lines ranged from 3.7-31 µM for olaparib and 13-70 µM for iniparib. No difference in sensitivity was observed between the TN and non-TN cell lines (by MTT). Olaparib also reduced the ability of cells to form colonies with IC50 values ranging from &lt;0.01-2.5 µM. Addition of the CDKI inhibitor CDK1i (Calbiochem) to olaparib resulted in formation of significantly fewer colonies compared with either inhibitor alone, in a cell line dependent manner. Conclusions: Our results suggest that although PARP1 is expressed in the majority of breast cancer, significantly higher staining intensity was found in TN than non-TN samples. Furthermore, our work suggests that olaparib is a more potent inhibitor of the in vitro growth of breast cancer cells than iniparib. Combined inhibition of PARP1 with olaparib and CDK1 with CDK1i may be a way forward for the treatment of TNBC. Acknowledgement: The authors thank SFI (SRC award, 08/SRC/B1410 MTCI) for funding this work.

Abstract A37: Low-density protein array reveals inflammation associated serum-autoantibody profile in prostate cancer.

Abstract Background: Chronic prostatic inflammation of the prostate is one of the main reasons for false positive serum PSA tests in prostate cancer screening. In addition there is accumulating evidence that an inflammatory microenvironment may support the development of malignancy and the progression to metastatic disease. Easy accessible and non-invasive indicators for prostate inflammation would be very helpful for a biopsy decision after an elevated PSA test result. However, at present, the histological examination of prostate biopsies remains the only way for identification of prostatic inflammation. The finding that cancer is immunogenic and that patients are able to elicit and potentially amplify an immune response against their tumor antigens suggests that autoantibodies serve as non-invasive serum biomarkers for prostate cancer diagnosis. Consequently, autoantibodies may also be capable of detecting differential disease progression grades -e.g. inflammatory states- enabling a more specific and individualized treatment in cancer therapy. In the present study, we aimed to find new non-invasive biomarkers for the diagnosis of prostatic inflammation in prostate cancer. Methods: Seventy prostate cancer tissue samples derived from radical prostatectomy were classified into two groups (high/low inflammation) according to the amount of tissue infiltrating lymphocytes. Clinical parameters including PSA, fPSA, GSC, prostate weight, prostate volume, tumor size, age and CRP were controlled for equal distribution amongst the two groups. The corresponding blood serum samples were analyzed using a low density protein array (microarray) containing 4012 prostate cancer associated and inflammation related recombinant proteins. Statistical comparison of the serum autoantibody profile in the two groups was used to identify differentially expressed biomarkers. Finally, specificity of candidate autoantibody markers was validated by immunohistochemical detection of corresponding autoantigens in prostate tissue. Results: Autoantibodies against 165 antigens were found to be differentially expressed in the high compared to the low inflammation group. 30 out of these proteins have recently been identified to be associated with prostate cancer. The identified autoantibody profile discriminated between prostate cancer patients bearing high or low inflammation in prostate tissue with a ROC curve AUC of 0.71. Out of the top ranked autoantibodies, three candidate proteins were selected due to their differential expression and significant up-regulation in serum samples of the high inflammation group: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Immunohistochemical analysis showed that targets of all three autoantibody candidates are expressed in epithelial prostate cells. Remarkably, the highest staining intensity was detected in tissue infiltrating lymphocytes in both high and low inflammation samples. Conclusion: The presented data evaluates the possible use of serum autoantibody markers as non-invasive indicators for the diagnosis of chronic inflammation in prostate cancer. The polyclonal immune response to aberrant proteins in heterogeneous diseases such as cancer and autoimmunity favors the development of autoantibody markers with potential specificities to detect disease subtypes and disease progression grades. We provide evidence for an inflammation specific antibody profile and verify the expression of three biomarker candidates in prostate tissue. Citation Format: Bettina Schlick, Petra Massoner, Angelika Luecking, Christof Saifarth, Georg Schaefer, Klaus Marquart, Peter Amersdorfer, Peter Schulz-Knappe, Helmut Klocker. Low-density protein array reveals inflammation associated serum-autoantibody profile in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A37.

Monoclonal Antibodies against the Human Somatostatin Receptor Subtypes 1–5: Development and Immunohistochemical Application in Neuroendocrine Tumors

Background: Activation of somatostatin receptors (sstr1–5) by somatostatin and its analogues exerts an inhibitory effect on hormone secretion and provides the basis for the treatment of a range of endocrine diseases such as acromegaly, Cushing’s disease and neuroendocrine tumors (NET). The lack of well-characterized commercially available sstr subtype-specific antibodies prevents routine identification of the sstr expression profile in patients. Methods: We generated and characterized new mouse monoclonal antibodies (mAbs) targeting the five human sstr subtypes using ELISA and immunohistochemistry, and tested their suitability in formalin-fixed and paraffin-embedded (FFPE) human tissues and archival samples of normal pancreatic tissue and NET. Results: All mAbs were highly specific with no cross-reactivity. The sstr1–5 immunoreactivity in gastrointestinal NET (n = 67) was correlated with clinicopathologic data. With the exception of sstr3, NET were highly positive for all receptor subtypes (42, 63, 6, 32 and 65% of tumors were positive for sstr1, sstr2a, sstr3, sstr4 and sstr5, respectively). sstr1, sstr2a and sstr5 were present at the plasma membrane and in the cytoplasm of tumor cells, whereas sstr3 and sstr4 were almost exclusively cytoplasmic. Immunoreactivity of sstr1, sstr2a and sstr4 tended to decrease as tumor aggressiveness increased. sstr5 showed an opposite pattern, with higher staining in well-differentiated carcinomas compared with well-differentiated tumors. sstr5 immunoreactivity was correlated with the presence of metastases and angioinvasion, suggesting a possible association with more aggressive behavior. Conclusion: Determination of the sstr1–5 by immunohistochemistry using subtype-specific mAbs is feasible in FFPE tissue and may provide a tool for routine clinical practice.

Abstract B33: Transcriptional control of genome surveillance and repair by a metabolic switch

Abstract A hallmark of primitive and more aggressive breast cancer is increased genome instability due to deficits in DNA repair. Recently we described a molecular mechanism through which the NADH regulated protein C-terminal binding protein (CtBP) represses the expression of the Breast Cancer One tumor suppressor gene BRCA1. These initial observations provided a molecular mechanism through which specific aspects of genome repair and stability, mediated by BRCA1, could be controlled by cellular metabolic status. By profiling the global interactions of CtBP with the breast cancer genome via chromatin immuno-precipitation combined with deep sequencing (ChlP-seq), we now reveal that CtBP acts both as a co-transcriptional repressor and activator to integrate distinct pathways that control genome stability, epithelial to mesenchymal transition, and stem cell transcriptional programming. Breast cancer cells depleted of CtBP show increased DNA repair as detected by comet and gamma H2AX foci formation assays. Over-expression of CtBP shows loss of the CD24 luminal surface marker while CtBP depletion increases CD24 expression. Kaplan-Meier survival analysis, based on our CtBP targeted gene lists, distinguishes breast cancer patients with worst outcome/survival in several published gene expression studies. Finally, tissue micro-array studies reveal significantly higher staining of CtBP predominantly in patients with poorly differentiated, triple negative and basal-like morphologic and molecular phenotypes. We propose a model in which CtBP acts at multiple levels to enhance dedifferentiation and genomic instability in malignancies of the breast. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B33.

A quantitative proteomic analysis of FFPE melanoma

A quantitative proteomic analysis of FFPE melanoma

Prognostic impact on FOXP3 expression in triple-negative breast cancer (TNBC).

1089 Background: TNBC, one of the most aggressive breast cancer subtypes, is of pivotal clinical importance given the lack of therapeutic target. Regulatory T cells (Tregs) including FOXP3 are potent mediators of dominant self tolerance for cancer. However, obscurity as to the identity, stability and suppressive function of Tregs has impeded therapeutic use. Some of TNBCs are thought to be related with immune genes. Interestingly, TNBCs were reported to be associated with higher number of FOXP3 (Bohling et al. Mod Pathol 2008). The aim of this study is to investigate the prognostic significance of FOXP3 in TNBC. Methods: We investigated the number of tumor-infiltrating FOXP3(+) Tregs in formalin-fixed TNBC specimens which patients received palliative treatment in Samsung Medical Center between 1999 and 2007. Immunohistochemistry was used to assess the numbers of CD4(+), CD25(+), and FOXP3(+) Tregs in tumors and normal breast tissues from 86 patients. The number of FOXP3(+) Tregs was calculated as the total number of positive cells per 10 high-power fields (HPF). Univariate and multivariate analyses for survival were performed according to the number of FOXP3(+) Tregs. Results: Of the 86 tumor specimens, 22 (25.6%) were found to express over 15 per 10 HPF for FOXP3(+) Tregs in peritumoral area. Within the invasive tumor samples, there was a significant correlation between FOXP3(+) (≥15) and lymphovascular invasion (P=0.012), extensive intraductal component (P=0.001), metastasis to axillary lymph nodes (P=0.048), and metastasis to pleura (P=0.008). Kaplan-Meier survival curves for OS and progression free survival (PFS) according to FOXP3 expression revealed significant better outcomes for patients with higher staining (≥ 15) than those with lower staining (< 15) (median PFS 4.2 vs 3.0 months, P=0.036; median OS 62 vs 18 months, P=0.036). By multivariate analysis, higher FOXP3(+) tumor staining ≥15 was an independent prognostic factor for OS (P=0.036) with hazard ratios of 0.46 (95% CI, 0.24 to 0.97; P=0.040). Conclusions: The metastatic TNBC patients with higher FOXP3(+) showed significant better outcomes than those with lower FOXP3(+) in terms of PFS and OS. The inclusion of FOXP3(+) may improve the prognositication of TNBC.

Expression and role of interleukin-23 in human endometrium throughout the menstrual cycle and early pregnancy

Interleukin-23 (IL-23) is a novel cytokine involved in the regulation of organ-specific immune responses. We hypothesized that expression of IL-23 in the human endometrium is menstrual cycle and pregnancy dependent, and is involved in endometrial immune regulation. IL-23 expression and regulation was investigated in the human endometrium and placenta in vivo using immunohistochemistry and in vitro using Western blot and cell viability analyses. IL-23 immunoreactivity in endometrial glandular cells was highest in the late proliferative and early secretory phases, as compared to other cycle phases and first trimester tissues. Endometrial stromal cells (ESC) showed weak IL-23 immunoreactivity without significant changes in intensity and distribution throughout the menstrual cycle. First trimester decidual cells revealed significantly stronger IL-23 staining compared to ESC from non-pregnant endometrium. Both villous cytotrophoblasts and syncytiotrophoblasts also showed positive IL-23 immunoreactivity, with a higher staining in syncytiotrophoblasts. In the trophoblastic cell line HRT8, IL-23 expression increased in a time-dependent manner, but was undetectable in stromal cells under all treatment conditions. ESC treated with recombinant IL-23 showed significantly decreased IL-8 secretion and cell viability. These results suggest a possible regulatory role for IL-23 in the menstrual cycle and in early pregnancy, although the extent and function of this role are yet to be determined.

The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBαin Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit.

Precursor of prostate‐specific antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: a study of 90 cases

To assess the expression of the precursor of prostate-specific antigen (pro-PSA), a distinct molecular form of serum-free PSA that includes native and truncated forms, in benign epithelium, high-grade prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma. We immunohistochemically evaluated 90 formalin-fixed, paraffin-embedded prostate needle biopsies using monoclonal antibodies against [-2] pro-PSA, native [-5/-7] pro-PSA, prostate-specific membrane antigen (PSMA), PSA and racemase. Staining intensity was recorded using a scale of 0-3 (0, no staining; 3, highest staining). The percentage of immunoreactive cells in benign epithelium, high-grade PIN and adenocarcinoma was estimated in increments of 10%. All cases had [-5/-7] pro-PSA immunoreactivity. There was weak focal perinuclear cytoplasmic immunoreactivity for [-5/-7] pro-PSA in 62% (range 0-90%) of benign epithelial cells, whereas there was strong diffuse cytoplasmic staining in 83% (range 10-90%) of high-grade PIN and 87% (range 40-90%) of cancer cells. Almost all (99%) cases were immunoreactive for [-2] pro-PSA. The median (range) proportion of cells expressing [-2] pro-PSA was lower in benign epithelium, at 17 (0-80)%, than in high-grade PIN, at 55 (0-90)% (P < 0.001) and adenocarcinoma, at 55 (0-100)% (P < 0.001). The intensity of immunoreactivity for both isoforms increased from benign to neoplastic (high-grade PIN and adenocarcinoma) epithelium. A total of 31% of high-grade PIN and 11% of cancer cases with negative racemase staining showed strong staining for [-5/-7] pro-PSA. The expression of [-5/-7] pro-PSA in benign and neoplastic cells might be used in combination with high molecular weight keratin, p63, and racemase to distinguish benign epithelium from high-grade PIN and adenocarcinoma, particularly when racemase staining is negative. Both isoforms are sensitive markers for prostatic epithelium, making them possible candidates for investigating carcinoma with an unknown primary, particularly in cases in which PSA staining is negative and the level of suspicion is high.