Intense Nuclear Staining Research Articles

Abstract 4636: Increased expression of CSE1L/hCAS in colorectal cancer: Correlation with tumor progression

Abstract Introduction. The chromosome segregation and human cellular apoptosis susceptibility gene (CSE1L/hCAS) has been implicated in cancer and has been shown to be amplified in several human maligancies, including colorectal cancer. In this work, we sought to evaluate the gene expression of CSE1L/hCAS in colorectal cancer cell lines and tumor tissue and to examine CSE1L/hCAS expression in colorectal cancer progression. Methods. In order to examine CSE1L/hCAS at the gene expression level, we surveyed three different datasets from studies in which Affymetrix U133+ gene chips had been performed on tissue samples from patients with colorectal cancer, as well as patients with adenomas and normal individuals. We examined relative gene expression levels for 3 probe sets on the U133+ gene chip that closely align to the RefSeq for the CSE1L/hCAS gene. In order to examine CSE1L/hCAS at the level of protein expression, we utilized a tissue microarray (TMA) that had been previously created at the Moffitt Cancer Center from primary colorectal tumors of various stages, adenomas and normal tissues and performed immunohistochemical (IHC) staining for the CSE1L/hCAS protein. Results. CSE1L/hCAS showed increased gene expression in all of the adenoma and colorectal cancer patient samples tested by microarray, compared to tissue samples from normal individuals. However, there was not a significant difference in CSE1L/hCAS mRNA levels between patients with adenoma and those with invasive cancer, nor between patients compared by cancer stage, grade, vascular invasion, sex or age. IHC staining also demonstrated intense nuclear and/or cytoplasmic staining for the CSE1L/hCAS gene product in adenomas and invasive colorectal cancers. Furthermore, later stage specimens showed more intense nuclear staining, while early stage lesions, and even fibroblasts in nearby normal tissue, showed predominately cytoplasmic staining for CSE1L/hCAS. Conclusions. These results demonstrate that increased expression of the CSE1L/hCAS gene, while not specific for invasive cancer cells, might be a good marker for early detection of colorectal cancer. This gene appears to be expressed early and across all stages of cancer development. Because of its early expression and potential involvement in a variety of important cellular processes, we believe that this gene product might represent a potential target for prevention and/or treatment of early colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4636.

2056 ANDROGEN RECEPTOR EXPRESSION IS DECREASED IN MATCHED PROSTATE CANCER LYMPH NODE METASTASES

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 20102056 ANDROGEN RECEPTOR EXPRESSION IS DECREASED IN MATCHED PROSTATE CANCER LYMPH NODE METASTASES George N. Thalmann, Carla Rocha, Sylviane Schobinger, Roland Seiler, and Achim Fleischmann George N. ThalmannGeorge N. Thalmann More articles by this author , Carla RochaCarla Rocha More articles by this author , Sylviane SchobingerSylviane Schobinger More articles by this author , Roland SeilerRoland Seiler More articles by this author , and Achim FleischmannAchim Fleischmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2103AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen receptors (AR) play a central role in prostate development and prostate cancer. Differential AR expression in the primary tumor and matched lymph node metastases has never been evaluated systematically. Histologically a heterogeneous distribution with AR negative areas is a constant finding in prostate cancer tissues. AR expression profile in the different Gleason patterns (GP) of primary tumors and corresponding nodal metastases is unknown and the prognostic potential of AR expression is controversial. We analysed AR expression in a consecutive series of patients with that underwent radical prostatectomy with extended pelvic lymph node dissection for prostate cancer and were found to be lymph node positive. METHODS A tissue microarray (TMA) was constructed from consecutive 119 hormone-naïve lymph node positive patients surgically treated for prostate cancer. The TMA contains tissues from all GP present in every primary tumor and the matched metastases. Immunohistochemical staining for AR (monoclonal mouse antibody, Novocastra, UK) was performed with positive and negative controls. Percentage of positive neoplastic cells and their nuclear staining intensity (1-3) were determined. RESULTS By immunohistochemical detection AR expression was up-regulated in primary tumors as compared to normal glands. AR expression profile in metastases was significantly lower than in the primary tumor. AR scores in primary tumors for the different GPs was GP3=134.8, GP4=157.7, GP5=123.5 (p=0.016) and in lymph node metastases GP3=70.5; GP 4= 88.7; GP5=71.7 (p<0.001). AR expression in the primary tumor and the lymph node metastases did not predict survival nor did it correlate with age, prostate cancer volume, number and total size of metastases, nor tumor stage, Gleason Score of the primary tumor and metastases. CONCLUSIONS AR expression is decreased in lymph node metastases suggesting a selection of less hormone-sensitive clones or decreased androgen dependence. AR expression varies according to Gleason pattern. AR expression is not prognostic in node positive disease. Bern, Switzerland© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e799 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information George N. Thalmann More articles by this author Carla Rocha More articles by this author Sylviane Schobinger More articles by this author Roland Seiler More articles by this author Achim Fleischmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B40: Derangement HNF4α gene expression as a candidate marker of hepatocellular carcinoma progression

Abstract Hepatocellular carcinoma (HCC) is one of the world's most common cancers. The development of malignant phenotype is considered to be a multistep process that results from the accumulation of genetic alterations. Mechanisms of epithelial carcinogenesis are determined by the specific properties of tumor progenitor tissue. According to our investigations, HCC progression is associated with the dysfunction of hepatocyte nuclear factors (HNFs), which control liver-specific gene expression, and particularly HNF4α, a nuclear receptor playing a key role in hepatic differentiation. This gene is transcribed from two distinct promoters, which activity results in producing two groups of isoforms and reflects the level of cell differentiation. HNF4αP2 isoforms are synthesized in embryonic liver and normally disappear after birth, while HNF4αP1 become dominant in mature hepatocytes. Reexpression of HNF4αP1 reversed the malignant phenotype of dedifferentiated mouse HCC variants. Studies on the collection of chemically induced mouse HCCs of independent origin revealed strict correlation of HNF4α expression with tumor differentiation state. Using semiquantitative RT-PCR we have analyzed the expression of 46 genes in 21 samples of human HCCs. 17 of studied cases were not associated with Hepatitis viruses infections, while 4 of patients were infected with hepatitis B virus. The most frequent event revealed in HCC samples compared to corresponding non-tumor tissues was the complete loss (in 7 of 17 non-hepatitis HCCs) or significant decrease (in 5 of 17 non-hepatitis HCCs) of HNF4αP1 transcription. Decrease of HNF4α expression appeared to be a poor prognostic factor: patients with highly expressed HNF4αP1 had 100% 3-years survival, and 83% of them didn't have any disease progression, while only 33% of patients with low HNF4αP1 expression showed no disease progression. HCCs which were associated with hepatitis infection didn't show relevant alterations in HNF4αP1 expression. We suppose that hepatitis-associated hepatocarcinogenesis might develop through HNF4α1-independent mechanism. Intracellular localization and protein expression level of HNF4α isoforms on the paraffine sections of human HCCs was investigated immunohistochemically using specific mouse monoclonal antibodies for HNF4αP1 (K9218; R&amp;D systems; 1:6000) or HNF4αP2 (H6939; R&amp;D systems; 1:3000) groups of isoforms. HNF4α isoforms immunoreactivity was detectable in all studied tumors except severely dedifferentiated variants. Intense nuclear staining for HNF4αP2 was observed in hepatocytes, but nuclear staining for HNF4αP1 showed decreased intensity. We haven't revealed synthesis of any HNF4α isoforms in dedifferentiated tumors. All this data corroborate with RT-PCR analysis. We suppose that HCC progression is accompanied with activation of HNF4αP2 isoform, decreased synthesis of HNF4αP1 isoform while in severely dedifferentiated tumors expression of both groups of HNF4α isoforms is repressed. We assume that induction of HNF4αP2 expression might serve a marker of early hepatocarcinogenesis, while decrease and subsequent loss of HNF4αP1 and lately HNF4αP2 marks HCC progression. This provides a new data important for further development of approaches for HCC diagnostics and prognosis. Expanded analysis of human HCCs archival samples and further investigation of correlation between tumor differentiation state and patients postoperative survival rate with the pattern of HNF4α isoforms expression would allow to estimate the usefulness of the analysis of HNF4α isoforms expression for the long-term outlook for this tumor type. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B40

RNA Microarray Analysis in Prenatal Mouse Cochlea Reveals Novel IGF-I Target Genes: Implication of MEF2 and FOXM1 Transcription Factors

BackgroundInsulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness.Methodology/Principal FindingsA combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1−/− null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1−/− and Igf1+/+ transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1+/+ mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27Kip1.Conclusions/SignificanceWe have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.

Nuclear expression of sox11 is highly associated with mantle cell lymphoma but is independent of t(11;14)(q13;q32) in non-mantle cell B-cell neoplasms

Sox11 is a transcription factor involved in embryonic neurogenesis and tissue remodeling. Its role in lymphopoiesis is presently unknown. Recent studies have shown the nuclear expression of sox11 in mantle cell lymphoma, which raises the question about its possible association with t(11;14)(q13;q32), the genetic hallmark of mantle cell lymphoma leading to the overexpression of cyclin D1. In this study, we examined sox11 expression in 211 cases of B-cell neoplasms by immunohistochemistry, and evaluated its association with t(11;14) and overexpression of cyclin D1. Nuclear staining of sox11 was observed in 54 of 57 (95%) mantle cell lymphomas, including 52 of 53 (98%) classical and 2 of 4 variant types. Two of the three mantle cell lymphomas negative for nuclear sox11 staining were analyzed and were positive for t(11;14). All other B-cell lymphomas (114 cases) showed variable positive staining in the cytoplasm, but no nuclear positivity. Sox11 was then examined in plasma cell myeloma and hairy cell leukemia as a subset of plasma cell myeloma carry t(11;14) and overexpress cyclin D1, and cyclin D1 is overexpressed in a subset of hairy cell leukemia independent of t(11;14). We found no nuclear staining of sox11 in 30 plasma cell myelomas examined, including 12 cases with t(11;14)(q13;q32). It is interesting that intense nuclear staining of sox11 was present in a subset of hairy cell leukemias (5 of 10), and was associated with the overexpression of cyclin D1. Our results indicate that the nuclear expression of sox11 is highly associated with mantle cell lymphoma, but is independent of t(11;14)(q13;q32) in non-mantle cell B-cell neoplasms. Its association with the overexpression of cyclin D1 in hairy cell leukemia suggests that sox11 may be involved in the upregulation of cyclin D1 in this leukemia.

Correlation of Oncotype DX Recurrence Score with Cyclin D1 and ErbB2.

Abstract Background: The Oncotype DX assay predicts the risk of recurrence in patients with stage I-II ER+, node negative breast cancer treated with tamoxifen. It is not understood if the Oncotype DX assay predicts the natural aggressiveness of an individual breast cancer or if it identifies a subtype of tamoxifen resistant breast cancer. In clinical practice, a high recurrence score (RS) on Oncotype DX is interpreted as a more aggressive tumor and is used to justify the use of chemotherapy. However, if the RS was actually predictive of tamoxifen resistance, patients may benefit from the use of an aromatase inhibitor, and chemotherapy may be unnecessary. Cyclin D1 and ErbB2 are two biomarkers shown to predict tamoxifen resistance.Cyclin D1 is overexpressed in approximately 35% of breast cancers. The Austrian Breast and Colorectal Cancer Study Group assessed expression of Cyclin D1 in patients taking tamoxifen within the ABCSG trial 05 and ABCSG trial 06. In both trials, Cyclin D1 overexpression correlated with a lower relapse free survival and overall survival compared to patients without Cyclin D1 overexpression.Erb2 is overexpressed in 15-30% of breast cancers. In the Gruppo Universitario Napoletano 1 trial, ER+ patients with early stage node negative breast cancer who overexpressed ErbB2 had no improvement in progression free survival and overall survival with 2 years of adjuvant tamoxifen therapy. Additional retrospective studies have supported initial reports of an association between overexpression of ErbB2 and tamoxifen resistance.Methods: 69 patients who had the Oncotype DX assay performed and had unstained pathology slides available were assessed for ErbB2 and Cyclin D1 expression. ErbB2 overexpression status was also obtained in another 74 patients who had the Oncotype DX assay performed. ErbB2 overexpression was determined from a review of medical records where ErbB2 was defined as being positive if immunohistochemical (IHC) staining intensity was 3+ with &amp;gt;90% of cells expressing ErbB2 or FISH revealed an amplification of &amp;gt;2.0. IHC analysis of Cyclin D1 was performed according to standard protocol and using commercially available antibodies. Scoring of slides for Cyclin D1 staining were performed by blinded pathologists who assessed both extent and intensity of nuclear staining for Cyclin D1.Results: The median Oncotype Dx RS within ErbB2+ patients was significantly higher than ErbB2- patients (36.5 vs. 18 p&amp;lt;0.0001), and approximately 50% of patients within each RS grouping (high, intermediate, and low) overexpressed cyclin D1.Conclusion: ErbB2 overexpression among high RS patients suggests the Oncotype DX assay may predict tamoxifen resistance and other markers for tamoxifen resistance need to be correlated with the RS. Although preliminary analysis of the IHC staining for Cyclin D1 does not correlate with a high RS, high Cyclin D1 expression among patients within the low RS subgroup is concerning since this subgroup may have an increased likelihood of disease relapse when treated with adjuvant tamoxifen alone. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3035.

Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen–glucose deprivation

Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen-glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5-10 min of HS and decreased to baseline levels or lower by 30-60 min. Decrease in nuclear CHIP at 30-60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen-glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute changes in subcellular localization of CHIP in response to cellular stress suggest that cellular changes that occur shortly after exposure to stress ultimately impact on the capacity and capability of a cell to recover and survive.

Secondary Resistance to Lenalidomide in Del(5q) MDS Is Associated with CDC25C & PP2A Overexpression.

Abstract 292 Background:Allelic deficiency for the RPS14 gene impairs differentiation and survival of erythroid progenitors in del(5q) MDS (Nature 2008; 451:335). Nucleolar stress arising from disruption of ribosome assembly fosters MDM2 sequestration by free ribosome components resulting in p53 stabilization and erythroid hypoplasia (Nat Cell Biol 2009; 11:501). We recently reported that reduced gene dosage of the lenalidomide (LEN) inhibitable, haplodeficient phosphatases CDC25C and PP2Acα is a key determinant of drug sensitivity in del(5q) MDS (PNAS 2009; 106: 12974). We now show that shRNA suppression of these genes to levels commensurate with haplodeficiency reinforces p53 accumulation, and that treatment with LEN promotes MDM2-mediated p53 degradation to transition del(5q) clones to G2/M arrest. We hypothesized that emergence of resistance to LEN in del(5q) MDS arises from two possible mechanisms: (1) up-regulation of haplodeficient drug targets or compensatory isotypes, or (2) inactivating mutations of the TP53 or CDC25C genes. Methods:To investigate mechanisms of LEN resistance, we studied sequential bone marrow (BM) specimens obtained at baseline (BL), response to treatment (TR) and treatment failure (TF) from 12 LEN treated patients with Low/INT-1 risk, transfusion-dependent del(5q) MDS. Eleven patients achieved clonal suppression and transfusion independence; 7 patients developed clinical drug resistance with primary clonal recovery. Immunohistochemical (IHC) staining for cdc25-C, -A and -B; PP2A–Ca and p53 were performed using a biotin-streptavidin-horseradish peroxidase method and compared to 6 age-matched controls; intensity of cytoplasmic or nuclear staining in hematopoietic elements was recorded after blinded review. DNA and RNA were extracted from cryopreserved BM mononuclear cells (BM-MNC) or fixed paraffin blocks from BM clot and biopsy sections. Expression of CDC25C splice variants was assessed by RT-PCR and total gene expression by real time (QT)-PCR. Exonic DNA encoding the catalytic [exons 8–14] and nuclear export domains [exon 11] of CDC25C and the DNA-binding domain of TP53 [exons 4–9] was sequenced for gene mutation analysis. Differences in mean values were compared by paired t-test. Results:P53 immunostaining was significantly higher in del(5q) BL specimens compared to controls ( relative expression [RE] 9.6 vs. 0.25; P =0.007). An admixture of nuclear and cytoplasmic staining for p53 and each cdc25 isotype was observed at BL that was largely restricted to erythroid precursors, whereas at TR cdc25-C and -A expression was primarily cytoplasmic, consistent with drug-induced nuclear exclusion. At TR, RE of only cdc25C (BL, 75 vs. TR, 49; P=0.05) and PP2A (29.2 vs. 12.3; P=0.025) was significantly reduced; whereas at TF cdc25C (TR, 43 vs. TF, 166; P=0.003), cdc25A (42.4 vs. 150; P=0.006), PP2A (7.3 vs. 65.6; P=0.028) and p53 (0.92 vs. 25.4; P=0.024) RE significantly increased. Nuclear localization of cdc25C and p53 but not cdc25A predominated at TF, consistent with escape from cdc25C inhibition. QT-PCR confirmed transcriptional up-regulation of CDC25C at TF with a mean 8.8-fold increase in gene expression vs. BL. DNA sequencing revealed no acquisition of somatic mutations within the CDC25C and TP53 exons studied [n=5]. Conclusions:Secondary resistance to LEN in del(5q) MDS is associated with over-expression and activation of the haplodeficient drug-inhibitable phosphatases, cdc25C and PP2A, with consequent restoration of wt-p53 activation. Absence of gene mutations within the coding exons analyzed suggests that transcriptional compensation alone is responsible for drug resistance. Novel agents targeting transcriptional repression of CDC25C may restore LEN sensitivity and merit investigation in drug resistant del(5q) MDS. Disclosures:List:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Komrokji:Celgene: Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. Maciejewski:Esai: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Further immunohistochemical characterization of BRD1 a new susceptibility gene for schizophrenia and bipolar affective disorder

We have recently shown that the gene BRD1 is associated with schizophrenia and bipolar affective disorder and that the BRD1 protein (BRD1) which is expressed in neurons may occur in a short and a long variant. The aim of the study was to generate polyclonal antibodies against new BRD1 epitopes enabling discrimination between the long and short BRD1 variants, and elucidate the BRD1 distribution in several human tissues, including the CNS. Polyclonal rabbit antibodies were raised against three different BRD1 epitopes. One (67) was specific for the long BRD1 variant, whereas the two others (63/64 and 65/66) like the original monoclonal mouse antibody (K22) were predicted to stain both variants. Immunohistochemical staining procedures were subsequently performed on paraffin-embedded human cerebral cortex and microarray slides containing 30 different human tissues. Western blotting confirmed the predicted specificity of the developed antibodies. K22, 63/64 and 65/66 displayed a similar neuronal staining pattern characterized by a distinct but weak nuclear staining, while the surrounding cytoplasm and proximal dendrites were more intensely stained. Interestingly, staining with 67 generated in contrast primarily an intense nuclear staining. The new antibodies resulted, furthermore, in a prominent neuroglial reaction characterized by staining of cell bodies, nuclei and glial processes. The tissue microarray analysis revealed that BRD1 was widely distributed in human tissues. The particular expression profile, e.g., the degree of nuclear and/or cytoplasmatic staining, seemed, however, to be highly tissue dependent. These results suggest a general role of BRD1 in the cell and stress that the two BRD1 variants may play different roles in the etiology of psychiatric disease.

A Comparison of Estrogen Receptor SP1 and 1D5 Monoclonal Antibodies in Routine Clinical Use Reveals Similar Staining Results

Clinical therapies for breast cancer are guided by estrogen receptor (ER) status determined by immunohistochemical analysis. A previous retrospective study comparing the recently generated rabbit SP1 monoclonal antibody (MAb) with the conventionally used mouse 1D5 MAb reported that 8% of breast carcinomas were SP1+/1D5- (correlating with good outcomes), and 2% were SP1-/1D5+ (correlating with poorer outcomes). This study on mostly previously frozen tissue implied that 1D5 fails to identify some women who may benefit from endocrine therapy. The current prospective study compared SP1 and 1D5 immunostaining on routinely processed consecutive cases of breast carcinoma. ER was classified using the same positive threshold used in the prior study (<1% negative; > or = 1% positive). Of 508 carcinomas, 2 were SP1+/1D5-, and none were SP1-/1D5+. Although SP1 is our preferred antibody, with more intense nuclear staining, both MAbs give similar results in tissue from routine clinical samples with discrepant results in fewer than 0.5% of cases.

ALK Expression in Rhabdomyosarcomas: Correlation with Histologic Subtype and Fusion Status

Immunohistochemical staining for anaplastic lymphoma kinase (ALK) has been described in rhabdomyosarcomas (RMS), especially the alveolar subtype. Previous studies have yielded conflicting results regarding the pattern of staining (nuclear versus cytoplasmic), and there has been no correlation with PAX3-7/FKHR fusion status. This study was undertaken to evaluate ALK receptor protein expression in a large series of RMS; to correlate these results with fusion status; and to investigate the possibility of 2p23 amplification or translocation using fluorescence in situ hybridization (FISH). Sixty-nine cases of RMS were examined and classified as alveolar RMS (ARMS), embryonal RMS (ERMS), or unclassifiable RMS (URMS) subtypes. Anaplastic lymphoma kinase immunohistochemistry was performed using anti-human CD246 antibody; cases were considered positive when more than 50% of cells had moderate or intense cytoplasmic and/or nuclear staining. There were 30 ARMS, 37 ERMS, and 2 URMS subtypes. Reverse transcription-polymerase chain reaction for PAX3/PAX7-FKHR fusion analysis had been done in all cases of ARMS, in 27 of 37 cases of ERMS, and in both URMS cases. Anaplastic lymphoma kinase staining was positive in 16 of 30 ARMS (53%) and 9 of 39 nonalveolar RMS (23%) cases (P < 0.05). Of the 21 ARMS cases with PAX3-FKHR fusion, 10 of 21 (48%) were positive for ALK staining; of the 6 ARMS cases with PAX7-FKHR fusion, 3 of 6 (50%) were positive for ALK staining; and 3 of 3 (100%) of the fusion-negative ARMS were positive with ALK staining. When comparing each of the ARMS subtypes, statistical significance was not reached. All positive cases showed dot-like cytoplasmic staining; nuclear staining was not seen. Of a subset of 6 ALK-positive ARMS submitted for break-apart FISH for the ALK locus, there was no evidence of a translocation; 1 case had ALK amplification and 2 had low-level gains of the ALK gene. We conclude that there is ALK overexpression in RMS, more commonly in ARMS than in ERMS, most likely independent of fusion status. Amplification or upregulation of ALK may underlie ALK protein overexpression.

Estrogen Receptor-β Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ERbeta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ERalpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that ERbeta expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17beta-estradiol led to an ERbeta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ERbeta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ERbeta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients.

Prognostic significance of HPV and p16 status in patients with oropharyngeal cancer treated on a large international phase III trial

6004 Background: Previous studies have reported that in patients with oropharyngeal cancer (OPC) the presence of human papilloma virus (HPV) is associated with an improved prognosis. We sought to determine the prognostic importance of HPV and p16 in patients with OPC treated with concurrent chemoradiation on a large international phase III trial. Methods: Patients with previously untreated Stage III or IV head and neck squamous cell cancer were randomized to receive definitive radiotherapy concurrently with either cisplatin or cisplatin plus tirapazamine. In this substudy, analyses were restricted to patients with OPC who received &gt; 60 Gy and did not have major radiation deviations predicted to impact on tumor control. HPV 16/18 were detected by in situ hybridization and scored as detected or undetected. p16 was detected by immunohistochemistry. Nuclear and cytoplasmic staining intensity of tumor cells was scored as grade 0–3, with grade 2 and 3 called positive. Log rank and Cox regression used for survival analyses. p values were 2-sided . Results: 384 out of 861 patients had OPC and met the eligibility criteria. Slides were available for HPV assay in 195 and for p16 in 186, and for both in 173. 54/195 (28%) were HPV positive, 107/186 (58%) were p16 positive. HPV pos tumors were associated with better 2-year overall survival (OS) (94 v 77%, p = 0.007) and better failure-free survival (FFS) (86 v 75%, p = 0.035) compared to HPV neg tumors. Similarly p16 pos tumors were associated with better 2-year OS (92 v 75%, p = 0.004) and FFS (87 v 72%, p = 0.003) compared to p16 neg . After adjustment for stage, Hb and ECOG PS, HPV pos had better OS than HPV neg (HR 0.29, p = 0.018), and p16 pos had better OS than p16 neg (HR 0.39, p = 0.013). When the HPV and p16 results were combined the relative HRs for OS were: HPVpos/p16pos 0.35 (45 patients, 26% of cases), HPVpos/p16neg 0 (3pts, 2%), HPVneg/p16pos 0.73 (58pts, 33%), HPVneg/p16neg 1.79 (67 pts, 39%). Conclusions: Our results confirm the prognostic significance of tumor HPV status in oropharyngeal cancer treated with chemoradiation, but also show that p16 identifies a larger group with an improved prognosis. The HPV neg/p16 pos population has a better prognosis compared to patients with HPV neg/p16 neg tumors. No significant financial relationships to disclose.

Thymidylate synthase polymorphisms and immunohistochemistry in non-small cell lung cancer

11101 Background: Recently, pemetrexed has been introduced into the treatment of non-small cell lung cancer (NSCLC). There's evidence that pemetrexed appears to be more efficient in non squamous NSCLC including adenocarcinoma (AC) and large cell carcinoma (LCC). TS represents a major target enzyme of pemetrexed. We evaluated the expression of TS among different histological types of NSCLC and its association with functional genetic polymorphisms of the TS gene. Methods: Archived tumor samples from 312 individuals with NSCLC were analyzed. Immunohistochemistry (IHC) was performed using a mouse monoclonal antibody to TS. TS stained sections were scored for cytoplasmic and nuclear localization. For each compartment, the tumor was evaluated for the estimated percentage of positive cells with the greatest intensity within the tumor. Intensity was scored on a scale of 0–4. A score of 3–4 was classified as high expression. Genotyping of TS-VNTR including the G/C SNP and TS1394del6 was performed by PCR based RFLP techniques. Genotypes supposed to be associated with low expression were grouped (group A: 2R/2R or 2R/3RC or 3RC/3RC + -6/-6) and compared to genotype groups associated with high expression (group B: 2R/3RG or 3RC/3RG or 3RG/3RG + -6/+6 or +6/+6). Results: IHC and polymorphisms could be performed successfully in 312 (100%) and 287 (92%), respectively. Distribution of histology was as follows: 50% AC, 42% squamous cell carcinoma (SCC), 3% LCC and 5% mixed or other histological subtypes. Group B genotypes were significantly more present in SCC than in AC (49% vs 23%, p=.002). Tumors with group B genotypes were more likely to display high nuclear staining intensity than group A tumors (30% vs. 17%, p=.077). A similar but not significant trend was seen for cytoplasmic staining intensity. There was no significant difference between histological subtypes of NSCLC with respect to TS protein expression. AC were more common than SCC to show a G1 differentiated tumors (8% vs 1%, p&lt;.001). No difference between genotype groups were observed with respect to grading. Conclusions: TS polymorphisms are significantly associated with histology subtypes in NSCLC. These results represent a potential explanation for efficacy differences of pemetrexed in NSCLC and warrants further investigations. [Table: see text]

Expression of immunoreactivity and genetic mutation in eosinophilic and ciliated metaplastic changes of endometrial glandular and stromal breakdown: cytodiagnostic implications

Various metaplastic changes may be present in endometrium, in which also cellular atypias may often be observed. Particularly, eosinophilic and ciliated changes (ECCs) occur in both nonneoplastic and neoplastic endometrium. This may cause confusion in the cytodiagnosis. This study was enterprised to investigate the possible help of immunocytochemical and cytogenetic study in the diagnostic and biologic assessment of ECC cells. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the material consists of 40 cases of cytologic smears examined by direct sampling of the endometrial cavity comprising 30 cases of ECC in endometrial glandular and stromal breakdown (EGBD) and 10 cases of well-differentiated adenocarcinoma (G1). After cytodiagnosis, immunostaining for p53 protein, Ki-67, and cyclin A was performed on multiple wet-fixed slides from each single case to evaluate the immunoreactivity, intensity of nuclear staining, and nuclear labeling index (N-LI). The intensity of nuclear staining was scored as negative (0), weak (1), moderate (2), or strong (3), and the N-LI was scored as less than 10% (0), from 10% to 25% (1), from 26% to 50% (2), or more than 50% (3), and the final score was calculated by adding both partial scores. A statistical significance test was performed by using Mann-Whitney U test, and the result was judged as significant when the P value was less than .05. For genetic mutation analysis of p53, the material comprised 6 cases of EGBD in which a high score was measured with immunocytochemistry for p53 protein, and the presence of ECC was confirmed on the hematoxylin and eosin. The ECC cells on paraffin-embedded specimens were captured using laser capture microdissection technology. Mutations in p53 gene (exons 5-8) were examined using DNA sequencing analysis. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the proportions of immunoreactive cells for p53 were statistically higher in ECC compared with those of G1 (P < .05). The proportions of the immunoreactive cells for Ki-67 and cyclin A were statistically higher in G1 compared with those of ECC (P < .05). (2) In genetic mutation analysis of p53, DNA sequencing of p53 in 6 cases revealed no mutations. The percentage of immunoreactive cells for p53 protein were higher in ECC than in G1, but the mutation point was not confirmed in genetic mutation analysis. The differential expression of these biologic parameters in ECC cells could be considered of possible relevance to the cytopathologic diagnosis in the future.

Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer

Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

Relationship among Oxidative Stress, DNA Damage, and Proliferative Capacity in Human Corneal Endothelium

To determine whether human corneal endothelial cells (HCECs) exhibit signs of oxidative DNA damage and to test whether oxidative stress affects the proliferative capacity of HCECs. Donor human corneas were divided into two age groups: young (<30 years) and older (>50 years). An 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA assay was used to quantify oxidative DNA damage in HCECs freshly isolated from ex vivo corneas. 8-OHdG immunostaining localized the sites of oxidative DNA damage in corneal wholemounts and cultured HCECs. To test whether oxidative stress induces oxidative DNA damage, HCECs cultured from young donors were treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) and immunostained for 8-OHdG. To test the effect of oxidative stress on proliferative capacity, HCECs cultured from young donors were treated with H(2)O(2) and cell numbers determined by WST-8 assay. 8-OHdG levels were significantly higher (P = 0.0031) in the central endothelium of older donors than of young donors. Intense nuclear staining for 8-OHdG was observed in central endothelium of older, but not young, donors. The relative intensity of 8-OHdG in the nuclei of cultured HCECs was similar to that observed in ex vivo corneas. Treatment of cultured HCECs from young donors with increasing concentrations of H(2)O(2) resulted in a dose-dependent increase in nuclear 8-OHdG staining and a decrease in proliferative capacity similar to that observed in untreated HCECs from older donors. Age-dependent and topographical decreases in proliferative capacity observed in HCECs resulted, at least in part, from nuclear oxidative DNA damage.

Effects of Short-Term Finasteride on Apoptotic Factors and Androgen Receptors in Prostate Cancer Cells

We explored the molecular correlates of the effect of finasteride on prostate tissue in patients undergoing radical prostatectomy. Patients undergoing radical prostatectomy for localized prostate cancer were eligible for study. After providing informed consent patients were randomized to receive 5 mg finasteride or placebo daily for at least 30 days before surgery. At surgery prostate tissue was harvested from the surgical specimen and sent for analysis. Tissue samples were analyzed for the pro-apoptotic factors caspase-3, caspase-7 and IGFBP-3. Samples were also analyzed for the tumor suppressor/proto-oncoproteins bcl-2, p53 and p21. Finally, tissues were analyzed for androgen receptor density and insulin growth factor-1. A total of 22 study and 20 placebo samples were collected and analyzed. Negligible staining for bcl-2 or caspase-3 was noted in each group. Statistical differences were not observed for bcl-2, p53, p21 or insulin growth factor-1 between the groups. There was a statistically significant difference in caspase-7 and IGFBP-3. A mean of 77% and 99.9% of cells stained for caspase-7 in the treatment and placebo groups, respectively (p = 0.007). In 3 patients caspase-7 staining disappeared completely and it was decreased by 70% and 50% in 1 patient each. Mean intensity staining for IGFBP-3 was 1.03 in the treatment group and 1.54 in the placebo group (p = 0.005). The staining intensity of nuclear androgen receptors on benign and cancerous cells was not significantly different between the treatment and placebo groups. However, there was a significant difference in androgen receptor staining between benign and cancer cells in the 2 populations. Mean nuclear androgen receptor staining intensity in all cancer and all benign tissue samples was 119.3 and 151.8, respectively (0.001). Finasteride administered 30 days before surgery appears to decrease the apoptotic factors caspase-7 and IGFBP-3 in cancer cells, while having little to no effect on caspase-3, insulin growth factor-1, bcl-2, p53 and p21. This short-term study may have interesting implications for interpreting Prostate Cancer Prevention Trial data on the molecular level. No differences were noted between the treatment and placebo groups in the expression of nuclear androgen receptor. However, decreased expression of androgen receptors was present in cancer cells compared to that in benign prostate cells in the 2 groups.

6 EFFECT OF TRICHOSTATIN A ON HISTONE ACETYLATION AND METHYLATION, EMBRYO DEVELOPMENT, AND IN VIVO VIABILITY OF INTER-SPECIES BLACK FOOTED CAT CLONED EMBRYOS

Aberrant DNA methylation and histone acetylation patterns following somatic cell nuclear transfer (SCNT) may disrupt the expression of imprinted and pluripotency-related genes, resulting in improper embryo development and fetal abnormalities. Treatment of mouse, pig and rabbit intra-species cloned embryos with the histone deacetylase inhibitor trichostatin A (TSA) has been reported to improve embryo development to the blastocyst stage (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Zhang et al. 2007 Cloning Stem Cells 9, 357–363; Shi et al. 2008 J. Anim. Sci. 86, 1106–1113). TSA has also increased the number of mouse live offspring after embryo transfer (Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have evaluated the effect of TSA on the ability of domestic cat (DSH, Felis catus) cytoplasts to support nuclear reprogramming and embryo development using black-footed cat (BFC, Felis nigripes) donor nuclei. To observe the effect of TSA on the covalent modification of histone H3, BFC-DSH reconstructed embryos were treated with 0, 5, 50, and 100 nm concentrations of TSA for 2 h before activation and 4 h and 16 h following activation. Levels of trimethylation and acetylation at lysine 9 of histone 3 (H3K9) were assessed by immunofluorescence and confocal microscopy. Average nuclear staining intensity was calculated for each antibody and compared between TSA treatments for each time point. Two to three oocytes were analyzed per group. H3K9 methylation was low or non-detectable for all treatments with or without TSA. H3K9 acetylation levels were not affected at 2 h for each of the TSA concentrations v. control. At 4 h and 16 h post-activation, H3K9 acetylation increased with exposure to TSA in a concentration-dependent manner. Next, BFC-DSH cloned embryos were treated with 50 or 100 nm TSA for 6 h or 24 h to determine the effect of TSA on in vitro development to the blastocyst stage (n = 157) and in vivo development after embryo transfer into domestic cat recipients (n = 255). TSA exposure did not enhance embryo cleavage, and among TSA treated embryos, blastocyst development was observed only in cybrids that were treated with 50 nm TSA for 24 h. The development rate of 2.8% for this group (n = 38) was similar to that of untreated embryos (2.0 to 3.3%, n = 502; P &gt; 0.05). One pregnancy was established after transfer of TSA treated embryos, using a 24 h, 50 nm TSA treatment. The pregnancy rate (50%) and percentage of embryos that implanted following transfer (4.4%, n = 68) corresponded to those of untreated BFC-DSH cloned embryos (57.1% and 4.2%, n = 213; P &gt; 0.05). Similar to untreated controls, all TSA treated embryos that implanted in DSH recipients developed an amorphous fetal mass and started reabsorption by Day 30 of gestation. In summary, administration of TSA did have an effect on the acetylation level of histone H3K9, but did not improve embryo development to the blastocyst stage or in vivo viability of inter-species black-footed cat cloned embryos.

CXCL12 and CXCR4 in adenocarcinoma of the lung: Association with metastasis and survival

Although the chemokine CXCL12 and its receptor CXCR4 have been implicated in metastasis of non-small cell lung carcinoma, the prognostic significance of these molecules is poorly defined. This study aimed to determine whether expression of these molecules is associated with clinicopathologic features and disease-free survival in non-small cell lung carcinoma. Immunohistochemical staining for CXCL12 and CXCR4 was performed on 154 primary non-small cell lung carcinomas. Staining intensity was compared with tumor histotype, TNM stage, and disease-free survival; correlation was assessed by using the Fisher's exact test, and Kaplan-Meier and Cox multivariate proportional hazards regression analysis. Intense CXCL12 immunostaining was associated with nodal metastasis, although no difference in survival was observed. The prognostic relevance of CXCR4 was dependent on its subcellular location: in univariate analysis intense nuclear staining was significantly associated with lower T classification and improved disease-free survival in patients with adenocarcinoma, whereas cytomembranous staining was associated with distant metastasis and decreased disease-free survival. On multivariate analysis, cytomembranous CXCR4 expression conferred a significantly worse disease-free survival (relative risk, 2.8; 95% confidence interval, 1.4-5.7; P = .004). Cytomembranous expression of the chemokine receptor CXCR4 in adenocarcinoma of the lung is an independent risk factor associated with worse disease-free survival, whereas nuclear staining confers a survival benefit. These findings are consistent with a model in which CXCR4 promotes tumor cell proliferation and metastasis when present in the cytoplasm or cell membrane, whereas localization of this molecule in the nucleus prevents it from exerting these effects.