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Background. Liver transplantation (LT) represents a curative treatment for small hepatocellular carcinoma (HCC). Potentially curable higher-stage HCC patients are denied LT due to the lack of cancer markers that predict progression and recurrence. Methods. Thirty-eight candidates for LT with hepatitis C virus (HCV) cirrhosis and HCC were studied. Gene expression (Gexp) analysis of tumor samples was performed using microarrays. Results. Twenty patients underwent transplantation, 13 progressed while waiting for transplantation, 4 are alive awaiting transplantation, and 1 died without progression while waiting for LT. Differences in GExp among patients who underwent LT or did not progress (n=25) versus those whose disease progressed while waiting for LT (n= 13) were assessed. Thus, 54 probe sets (Pset) were significantly differentially expressed. Among LT patients, 10 Psets were differentially expressed between LT patients with the same explanted stage that recurred (n= 5) versus LT patients who did not recur (n=5). Ninety-eight Psets were significantly associated with survival at the α=0.005 level. Conclusions. Here, we have identified genes associated with HCC progression in HCV-HCC patients awaiting LT transplantation. A limited number of genes were related to overall survival and cancer-free survival after LT. Incorporation of these molecular markers could help to improve organ allocation for HCV-HCC patients.

Gene expression analysis of plasmablastic lymphoma identifies downregulation of B-cell receptor signaling and additional unique transcriptional programs

To elucidate cancer-associated fibroblast (CAF) origins and diversity within intrahepatic cholangiocarcinoma (ICC), we analyzed the phenotypic, cytogenetic, gene expression profiles, growth propert...

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double-stranded break (DSB) repair. Here, we show that depletion of CHD4 in acute myeloid leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ataxia-telangiectasia mutated pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic agent-induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34(+) hematopoietic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor-forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.

Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. ey- and βH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix® array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Liver transplantation represents the potentially curative treatment for small HCC. Recurrence after surgical resection and liver transplantation remains one of the major obstacles in further prolonging survival of patients with HCC. In the new liver, HCC might be of recipient or donor origin. One approach for investigating this question is by performing human identification and/or engraftment analysis. Distinction between recurrent and de novo HCC after orthotopic liver transplantation could allow for the development of important clinical and therapeutic strategies. Polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA sequences, gene expression profiling, and fluorescence in situ hybridization were applied in a patient who developed a second HCC after orthotopic liver transplantation from an opposite gender donor. These techniques provided consistent evidence that the second HCC was a recurrence of the primary tumor.

Breast cancer drug development costs nearly $610 million and 37 months in preclinical mouse model trials with minimal success rates. Despite these inefficiencies, there are still no consensus breast cancer preclinical models. Murine mammary adenocarcinoma 4T1-luc2 cells were implanted subcutaneous (SQ) or orthotopically percutaneous (OP) injection in the area of the nipple, or surgically into the chest 2nd mammary fat pad under direct vision (ODV) in Balb/c immunocompetent mice. Tumor progression was followed by in vivo bioluminescence and direct measurements, pathology and survival determined, and tumor gene expression analyzed by genome-wide microarrays. ODV produced less variable-sized tumors and was a reliable method of implantation. ODV implantation into the chest 2nd mammary pad rather than into the abdominal 4th mammary pad, the most common implantation site, better mimicked human breast cancer progression pattern, which correlated with bioluminescent tumor burden and survival. Compared to SQ, ODV produced tumors that differentially expressed genes whose interaction networks are of importance in cancer research. qPCR validation of 10 specific target genes of interest in ongoing clinical trials demonstrated significant differences in expression. ODV implantation into the chest 2nd mammary pad provides the most reliable model that mimics human breast cancer compared from subcutaneous implantation that produces tumors with different genome expression profiles of clinical significance. Increased understanding of the limitations of the different preclinical models in use will help guide new investigations and may improve the efficiency of breast cancer drug development .

Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.

We have previously demonstrated that expansion of activated tumor-sensitized T cells in interleukin (IL)-7/15 results in greater expansion and antitumor activity than expansion in IL-2. We sought to determine whether T cells exposed to IL-2 versus IL-7/15 exhibited distinct gene expression patterns. Lymphocytes were harvested from Pmel-1 mice immunized with B16-GMCSF melanoma cells, activated in vitro, and cultured in IL-2 or IL-7/15 for 1, 3 or 6 days. T cells were harvested and analyzed using microarray, real-time quantitative polymerase chain reaction (RT-QPCR) or sorted into T-cell subsets and analyzed. We found significant differences in gene expression for T cells cultured in IL-2 versus IL-7/15, starting on day 3. This was not a function of subset differentiation; when T cells were divided into subsets, the central memory (T(CM)), effector memory (T(EM)) and effector (T(E)) T cells cultured in the IL-2 more closely resembled each other than the identical phenotypic subset exposed to IL-7/15. Thus, the differences in gene expression induced by culture in IL-2 versus IL-7/15 do not merely reflect differences in the frequency of T(CM) versus T(EM) versus T(E) cells, but rather reflect that the gene expression levels of those T-cell subsets when exposed to different cytokines are fundamentally different.

Next-generation sequencing (NGS) has become an important tool for identifying clinically relevant variants in both inherited disorders and oncology. Variants annotation that enables the creation of meaningful clinical reports often requires mining multiple publicly available databases. There are a number of such resources that have been designed to catalog and mine a plethora of germline variants or mutations. However, when analyzing tumor specimens in clinical settings, one may need to use different or ancillary resources that are specific for somatic variants or actionable mutations that may have clinical or treatment implications. The purpose of this review is to recapitulate the state of the art of somatic variation databases, which can aid in the clinical interpretation of NGS-based assays in oncology. In addition, the current need for collating various annotation sources into one-stop solutions to facilitate faster query execution and better integration into existing laboratory information systems are discussed.

The molecular basis of calcineurin inhibitor toxicity (CNIT) in kidney transplantation (KT) and its contribution to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA) were evaluated by: (1) identifying specific CNIT molecular pathways that associate with allograft injury (cross-sectional study) and (2) assessing the contribution of the identified CNIT signature in the progression to CAD with IF/TA (longitudinal study). Kidney biopsies from well-selected transplant recipients with histological diagnosis of CNIT (n = 14), acute rejection (n = 13) and CAD with IF/TA (n = 10) were evaluated. Normal allografts (n = 18) were used as controls. To test CNIT contribution to CAD progression, an independent set of biopsies (n = 122) from 61 KT patients collected at 3 and ~12 months post-KT (range = 9-18) were evaluated. Patients were classified based on 2-year post-KT graft function and histological findings as progressors (n = 30) or nonprogressors to CAD (n = 31). Molecular signatures characterizing CNIT samples were identified. Patients classified as progressors showed an overlap of 7% and 22% with the CNIT signature at 3 and at ~12 months post-KT, respectively, while the overlap was

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPase(old-35)), we performed gene expression analysis of HeLa cells in which hPNPase(old-35) was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPase(old-35) was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPase(old-35) expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPase(old-35) expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPase(old-35) infected HO-1 melanoma cells and HeLa cells overexpressing hPNPase(old-35) under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPase(old-35) is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation.

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA–protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the death domain region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential death domain in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.

We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Right ventricular failure (RVF) is the most frequent cause of death in patients with pulmonary arterial hypertension (PAH); however, specific therapies targeted to treat RVF have not been developed. Chronic treatment with carvedilol has been shown to reduce established maladaptive right ventricle (RV) hypertrophy and to improve RV function in experimental PAH. However, the mechanisms by which carvedilol improves RVF are unknown. We have previously demonstrated by microarray analysis that RVF is characterized by a distinct gene expression profile when compared with functional, compensatory hypertrophy. We next sought to identify the effects of carvedilol on gene expression on a genome-wide basis. PAH and RVF were induced in male Sprague-Dawley rats by the combination of VEGF-receptor blockade and chronic hypoxia. After RVF was established, rats were treated with carvedilol or vehicle for 4 wk. RNA was isolated from RV tissue and hybridized for microarray analysis. An initial prediction analysis of carvedilol-treated RVs showed that the gene expression profile resembled the RVF prediction set. However, a more extensive analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes in pathways involved in cardiac hypertrophy, mitochondrial dysfunction, and protein ubiquitination. Genes encoding proteins in the cardiac hypertrophy and protein ubiquitination pathways were downregulated in the RV by carvedilol, while genes encoding proteins in the mitochondrial dysfunction pathway were upregulated by carvedilol. These gene expression changes may explain some of the mechanisms that underlie the functional improvement of the RV after carvedilol treatment.

TWO MOST COMMONLY USED ANIMAL MODELS FOR STUDYING BREAST CANCER LUNG METASTASIS ARE: lung metastasis after orthotopic implantation of cells into the mammary gland, and lung implantations produced after tail vein (TV) injection of cells. Tail vein injection can produce lung lesions faster, but little has been studied regarding the differences between these tumors, thus, we examined their morphology and gene expression profiles.Syngeneic murine mammary adenocarcinoma, 4T1-luc2 cells, were implanted either subcutaneously (Sq), orthotopically (OS), or injected via TV in Balb/c mice. Genome-wide microarray analyses of cultured 4T1 cells, Sq tumor, OS tumor, lung metastases after OS (LMet), and lung tumors after TV (TVt) were performed 10 days after implantation.Bioluminescence analysis demonstrated different morphology of metastases between LMet and TVt, confirmed by histology. Gene expression profile of cells were significantly different from tumors, OS, Sq, TVt or LMet (10,350 probe sets; FDR≤1%; P0.0001). Sq tumors were significantly different than OS tumors (700 probe sets; FDR≤15%; P0.01), and both tumor types (Sq and OS) were significantly different than LMet (1,247 probe sets;1.5-fold-change; P0.01), with no significant difference between TVt and LMet.There were significant differences between the gene profiles of cells in culture and OS versus LMet, but there were no differences between LMet versus TVt. Therefore, the lung tumor generated by TVt can be considered genetically similar to those produced after OS, and thus TVt is a relevant model for breast cancer lung metastasis.

Human Polynucleotide Phosphorylase (hPNPase(old-35) or PNPT1) is an evolutionarily conserved 3'→ 5' exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPase(old-35) in cellular physiology, we knocked-down and overexpressed hPNPase(old-35) in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPase(old-35) resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPase(old-35) caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPase(old-35) knockdown and overexpression datasets allowed us to identify 77 potential "direct" and 61 potential "indirect" targets of hPNPase(old-35) which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPase(old-35) expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs., Author Summary When oxygen levels fall below normal, cells are said to be in a hypoxic state. Once in hypoxia, dramatic changes are induced that allow for adaptation. In particular, energetic metabolism and transcription are highly affected. HIF (hypoxia inducible factor) is a highly conserved factor that is the driving force behind many hypoxia-induced changes—it is inactive in normal conditions and becomes active in hypoxia. Using the fruit fly as a model system, we show that hypoxic responses consist of HIF and non-HIF-dependent pathways. These response programs counteract the impacts of low oxygen by broadly influencing different cellular processes such as the breakdown of sugars, but only at appropriate developmental times. We provide evidence that HIF- and non-HIF-dependent pathways are complemented by the actions of the steroid hormone receptor estrogen-related receptor (ERR), which we show is also essential in hypoxia. Our results place new emphasis on the actions of HIF and suggest that alternative HIF-independent pathways play a more prominent role than previously thought.

Diagnosis and prediction of the severity of hepatitis C virus recurrence (HCVrec) after liver transplantation (LT) remain a challenge. MicroRNAs have been recently recognized as potential disease biomarkers. Archival liver biopsy samples from 43 HCV+ LT recipients were collected at clinical HCVrec time and at 3 years post-LT. Patients were classified as progressors (P = F0/F1) or nonprogressors (NP = F3/F4) according to the severity of fibrosis on the 3-year biopsy. Training (n = 27) and validation (n = 16) sets were defined. RNA was isolated from all biopsies at clinical HCVrec time, labeled and hybridized to miRNA-arrays. Progressors versus nonprogressors were compared using the two-sample t-test. A p-value ≤0.01 was considered significant. The ingenuity pathway analysis tool was used for microRNA and miRNA:mRNA ontology data integration. Nine microRNAs were differentially expressed between groups. A supervised cluster analysis separated samples in two well-defined groups (progressors vs. nonprogressors). Pathway analysis associated those microRNAs with hepatitis, steatosis, fibrosis, cirrhosis and T cell-related immune response. Data integration identified 17 genes from a previous genomic study as 9-microRNAs signature targets. Seven microRNAs were successfully validated in the validation set using QPCR. We have identified a 9-microRNA signature able to identify early post-LT patients at high risk of severe HCVrec during long-term follow-up.

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.

Because of knowledge gained in the field of cancer biology, clinicians are currently witnessing an explosion of molecular tests as companion diagnostics to targeted therapies against growth factor receptors and their signaling pathways. Such tests are being applied increasingly to cytology specimens as essential components of genomic medicine, because less invasive diagnostic procedures are becoming the norm. The objective of this review was to present an overview of the current and future role of cytopathology in molecular diagnostics, including the adequacy of cytology specimens for such studies. The authors also discuss the critical methodologic aspects of the molecular assays used for the selection of tyrosine kinase treatment for oncology patients.

Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR-3, CAOV-3; patient samples malignant 1 and 2) and resistant (DOV-13, SKOV-3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70-fold with malignant 2, 43-fold with OVCAR-3). Using agonists, antagonists, and shRNA-mediated knockdown of dsRNA receptors, we show that TLR3, RIG-I, and mda5 coordinated a caspase 8/9- and interferon-dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5- to 7.5-fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4–0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI=1.5–2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA-sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.—Van, D. N., Roberts, C. F., Marion, J. D., Lepine, S., Harikumar, K. B., Schreiter, J., Dumur, C. I., Fang, X., Spiegel, S., Bell, J. K. Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics.

The Wilms tumor 1 (WT1) protein-a developmentally regulated transcription factor-is aberrantly expressed in gliomas and promotes their malignant phenotype. However, little is known about the molecular allies that help it mediate its oncogenic functions in glioma cells.The authors used short interfering RNA (siRNA) to suppress WT1 expression in glioblastoma (GBM) cells and evaluated the effect of this on GBM cell invasiveness. Gene expression analysis was then used to identify the candidate genes that were altered as a result of WT1 silencing. One candidate target, CD97, was then selected for further investigation into its role by suppressing its expression using siRNA silencing, followed by proliferation and invasion assays.WT1 levels were reliably and reproducibly suppressed by siRNA application. This resulted in a significant decrease in cellular invasiveness. Microarray analyses identified the gene products that were consistently downregulated (27) and upregulated (11) with WT1 silencing. Of these, CD97 expression was consistently suppressed across the 3 different GBM cell lines studied and was found on further investigation to significantly impact GBM cell invasiveness.Although CD97 expression in gliomas has not been described previously, we conclude that the possible upregulation of CD97 mediated by WT1 promotes cellular invasiveness-one of the most characteristic and challenging aspects of glial tumor cells. Further studies are needed to clarify the nature of this regulation and its impact, as CD97 could represent a novel target for antiglioma therapies.

Background The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys. Methodology/Principal Findings 99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (range = 24–62). The PPP group included 60 biopsies (cold ischemia time (CIT) = 1,367+/−509 minutes) and the CSP group included 39 biopsies (CIT = 1,022+/−485 minutes) (P

Gene expression-based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.

The current model predicts that MDM2 is primarily overexpressed in cancers with wild-type (WT) p53 and contributes to oncogenesis by degrading p53. Following a correlated expression of MDM2 and NF-κB2 transcripts in human lung tumors, we have identified a novel transactivation function of MDM2. Here, we report that in human lung tumors, overexpression of MDM2 was found in approximately 30% of cases irrespective of their p53 status, and expression of MDM2 and NF-κB2 transcripts showed a highly significant statistical correlation in tumors with WT p53. We investigated the significance of this correlated expression in terms of mechanism and biological function. Increase in MDM2 expression from its own promoter in transgenic mice remarkably enhanced expression of NF-κB2 compared with its non-transgenic littermates. Knockdown or elimination of endogenous MDM2 expression in cultured non-transformed or lung tumor cells drastically reduced expression of NF-κB2 transcripts, suggesting a normal physiological role of MDM2 in regulating NF-κB2 transcription. MDM2 could up-regulate expression of NF-κB2 transcripts when its p53-interaction domain was blocked with Nutlin-3, indicating that the MDM2-p53 interaction is dispensable for up-regulation of NF-κB2 expression. Consistently, analysis of functional domains of MDM2 indicated that although the p53-interaction domain of MDM2 contributes to the up-regulation of the NFκB2 promoter, MDM2 does not require direct interactions with p53 for this function. Accordingly, MDM2 overexpression in non-transformed or lung cancer cells devoid of p53 also generated a significant increase in the expression of NF-κB2 transcript and its targets CXCL-1 and CXCL-10, whereas elimination of MDM2 expression had the opposite effects. MDM2-mediated increase in p100/NF-κB2 expression reduced cell death mediated by paclitaxel. Furthermore, knockdown of NF-κB2 expression retarded cell proliferation. Based on these data, we propose that MDM2-mediated NF-κB2 up-regulation is a combined effect of p53-dependent and independent mechanisms and that it confers a survival advantage to lung cancer cells.

Nuclear factor I-X3 (NFI-X3) is a newly identified splice variant of NFI-X that regulates expression of several astrocyte-specific markers, such as glial fibrillary acidic protein. Here, we identified a set of genes regulated by NFI-X3 that includes a gene encoding a secreted glycoprotein YKL-40. Although YKL-40 expression is up-regulated in glioblastoma multiforme, its regulation and functions in nontransformed cells of the central nervous system are widely unexplored. We find that expression of YKL-40 is activated during brain development and also differentiation of neural progenitors into astrocytes in vitro. Furthermore, YKL-40 is a migration factor for primary astrocytes, and its expression is controlled by both NFI-X3 and STAT3, which are known regulators of gliogenesis. Knockdown of NFI-X3 and STAT3 significantly reduced YKL-40 expression in astrocytes, whereas overexpression of NFI-X3 dramatically enhanced YKL-40 expression in glioma cells. Activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes, whereas expression of a dominant-negative STAT3 had a suppressive effect. Mechanistically, NFI-X3 and STAT3 form a complex that binds to weak regulatory elements in the YKL-40 promoter and activates transcription. We propose that NFI-X3 and STAT3 control the migration of differentiating astrocytes as well as migration and invasion of glioma cells via regulating YKL-40 expression.

The clinical significance of tumor-infiltrating immune cells has been reported in a variety of human carcinomas including breast cancer. However, molecular signature of tumor-infiltrating immune cells and their prognostic value in breast cancer patients remain elusive. We hypothesized that a distinct network of immune function genes at the tumor site can predict a low risk versus high risk of distant relapse in breast cancer patients regardless of the status of ER, PR, or HER-2/neu in their tumors. We conducted retrospective studies in a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival. The RNAs were extracted from the frozen tumor specimens at the time of diagnosis and subjected to microarray analysis and real-time RT-PCR. Paraffin-embedded tissues were also subjected to immunohistochemistry staining. We determined that a network of immune function genes involved in B cell development, interferon signaling associated with allograft rejection and autoimmune reaction, antigen presentation pathway, and cross talk between adaptive and innate immune responses were exclusively upregulated in patients with relapse-free survival. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel. These data suggest that neoadjuvant immunotherapy in patients with high risk of relapse may reduce tumor recurrence by inducing the immune function genes.

KLF2 is a Kruppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell and erythroid development. KLF2−/− mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. In KLF2−/− embryos, β-like globin gene expression is reduced, and E10.5 erythroid cells exhibit abnormal morphology. In this study, other genes regulated by KLF2 were identified by comparing E9.5 KLF2−/− and wild-type (WT) yolk sac erythroid precursor cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression between KLF2−/− and WT; eighty-nine of these are downregulated in KLF2−/−. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. The SOX2 gene, encoding a pluripotency factor, is regulated by KLF2 in both ES and embryonic erythroid cells. Previous work had identified genes with erythroid-enriched expression in the yolk sac. The erythroid-enriched genes reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2−/− compared to WT and are therefore candidates for controlling primitive erythropoiesis. Each of these genes contains a putative KLF2 binding site(s) in its promoter and/or an intron. Reelin has an established role in neuronal development. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter in erythroid cells, validating this approach to identify KLF2 target genes.

Previously, we described an orthotopic cholangiocarcinoma model based on bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells) compared to high grade malignant erbB-2/neu- transformed BDE1 cholangiocytes (BDEneu cells) into the livers of syngeneic rats, which closely mimics clinical features of early versus advanced stages of the human cancer. We now used gene expression microarray together with quantitative real-time RT-PCR to profile genes differentially expressed in highly tumorigenic BDEneu cells and corresponding tumors compared to less aggressive tumorigenic BDEsp cells and tumors. Genes identified as being commonly overexpressed in parent BDEneu cells, tumors, and in a BDEneu tumor- derived cholangiocarcinoma cell line included Sox17, Krt20, Erbb2, and Sphk1 when respectively compared to BDEsp cells, tumors, and tumor-derived BDEsp cholangiocarcinoma cells. Muc1 was also prominently overexpressed in BDEneu cells and tumor-derived cholangiocarcinoma cells over that expressed in corresponding BDEsp cell lines. Periostin and tenascin-C, which were produced exclusively by cholangiocarcinoma-associated fibroblastic cells, were each significantly overexpressed in BDEneu tumors compared to BDEsp tumors. Interestingly, amphiregulin was representative of a gene found to be significantly underexpressed in vitro in BDEneu cells compared to BDEsp cells, but significantly overexpressed in BDEneu tumors compared to BDEsp tumors, and correlated with BDEneu cholangiocarcinoma progression in vivo. Our data support a unique animal model that recapitulates important molecular features of human cholangiocarcinoma progression, and may serve as a potentially powerful preclinical platform for identifying and rapidly testing novel molecular targeting strategies for cholangiocarcinoma therapy and/or prevention.

Primary malignant brain tumors are highly lethal cancers and vary in their histology, due to the many cell types in the brain and also the multipotent cells of origin (Singh et al. 2003, 2004). The most notorious among these is glioblastoma multiforme (GBM), the most aggressive glioma subtype, carrying the highest World Health Organization (WHO) grade of IV. As the name suggests, GBM can have features of several cell types, which can be regionally distinct, depending on the evolution of the tumor from CNS precursor cells. The varied cellular composition of these tumors can give rise to differences in phenotype, which alter therapeutic response. To better characterize the varied molecular phenotype of malignant brain tumors, we devised methodology which utilizes preoperative MR imaging to define areas of interest for stereotactic biopsy, image guided biopsy, followed by nucleic acid extraction with an “in-process” histopathological scoring during tissue processing. The procedure is followed by molecular phenotyping using any highly reliable oligonucleotide microarray platform for molecular characterization. We have used Affymetrix gene expression microarrays due to the high reproducibility of these assays. The global gene expression profiling approach also allows a highly robust analysis to be performed on individual tumors, revealing a much more complete assessment of the overall molecular phenotype.

Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.

Background Kruppel-like factor 6 (KLF6) is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes. Methodology/Principal Findings In this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patient's survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X2 p = 0.005; Fisher p = 0.003). Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes. Conclusions/Significance Data are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.

Purpose Malignancies found in unexpected locations or with poorly differentiated morphologies can pose a significant challenge for tissue of origin determination. Current histologic and imaging techniques fail to yield definitive identification of the tissue of origin in a significant number of cases. The aim of this study was to validate a predefined 1,550-gene expression profile for this purpose. Methods Four institutions processed 547 frozen specimens representing 15 tissues of origin using oligonucleotide microarrays. Half of the specimens were metastatic tumors, with the remainder being poorly differentiated and undifferentiated primary cancers chosen to resemble those that present as a clinical challenge. Results In this blinded multicenter validation study the 1,550-gene expression profile was highly informative in tissue determination. The study found overall sensitivity (positive percent agreement with reference diagnosis) of 87.8% (95% CI, 84.7% to 90.4%) and overall specificity (negative percent agreement with reference diagnosis) of 99.4% (95% CI, 98.3% to 99.9%). Performance within the subgroup of metastatic tumors (n = 258) was found to be slightly lower than that of the poorly differentiated and undifferentiated primary tumor subgroup, 84.5% and 90.7%, respectively (P = .04). Differences between individual laboratories were not statistically significant. Conclusion This study represents the first adequately sized, multicenter validation of a gene-expression profile for tissue of origin determination restricted to poorly differentiated and undifferentiated primary cancers and metastatic tumors. These results indicate that this profile should be a valuable addition or alternative to currently available diagnostic methods for the evaluation of uncertain primary cancers.

Objectives: This is a pilot study designed to identify gene expression profiles able to stratify head and neck squamous cell carcinoma (HNSCC) tumors that may or may not respond to chemoradiation or radiation therapy. Study Design: We prospectively evaluated 14 HNSCC specimens, arising from patients undergoing chemoradiotherapy or radiotherapy alone with curative intent. A complete response was assessed by clinical evaluation with no evidence of gross tumor after a 2-year follow-up period. Methods: Residual biopsy samples from eight complete responders (CR) and six nonresponders (NR) were evaluated by genome-wide gene expression profiling using HG-U133A 2.0 arrays. Univariate parametric t-tests with proportion of false discoveries controlled by multivariate permutation tests were used to identify genes with significantly different gene expression levels between CR and NR cases. Six different prediction algorithms were used to build gene predictor lists. Three representative genes showing 100% crossvalidation support after leave-one-out crossvalidation (LOOCV) were further validated using real-time QRT-PCR. Results: We identified 167 significant probe sets that discriminate between the two classes, which were used to build gene predictor lists. Thus, 142 probe sets showed an accuracy of prediction ranging from 93% to 100% across all six prediction algorithms. The genes represented by these 142 probe sets were further classified into different functional networks that included cellular development, cellular movement, and cancer. Conclusions: The results presented herein offer encouraging preliminary data that may provide a basis for a more precise prognosis of HNSCC, as well as a molecular-based therapy decision for the management of these cancers. Laryngoscope, 119:91–101, 2009

The utilization of genome-wide gene expression microarray technology in tumor stratification has proven a powerful tool to identify gene expression signatures associated with cancer prognosis and is currently under evaluation in clinical laboratories. Standardized protocols, including tumor tissue postoperatively handling guidelines are yet to be defined. We aimed at assessing a systematic effect of devitalization in ovarian tumors' gene expression profiling, using high-density oligonucleotide microarrays, under a standardized protocol following strict quality control criteria. Residual tissue from the surgical pathology specimen was divided into 5 samples. Half of each was immediately snap frozen in liquid nitrogen. The remaining halves were kept at room temperature for 0, 15, 30, 60, and 120 minutes, at which time the tissue was snap frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. The entire process from RNA extraction through feature intensity distribution was rigorously monitored for quality. Identification of altered gene expression among each pair of snap frozen and devitalized samples per ovarian tumor specimen was assessed by using the Significance score (S-score) method. We identified only 4 probe sets that seemed to correlate with devitalization time in one of the ovarian tumor specimens, suggesting that they are not likely to have an impact on gene expression profiling tumor stratification. Our study suggests that with proper sample handling and rigorous quality control procedures for RNA extraction and microarray analysis, tumor classification based on global gene expression data will not be adversely affected if devitalization times are kept within a 120-minute window.

Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin-1, and muscleblind-like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis. Developmental Dynamics 237:436–446, 2008. © 2008 Wiley-Liss, Inc.

Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.

Background Recent research examining cross-platform correlation of gene expression intensities has yielded mixed results. In this study, we demonstrate use of a correction factor for estimating cross-platform correlations. Results In this paper, three technical replicate microarrays were hybridized to each of three platforms. The three platforms were then analyzed to assess both intra- and cross-platform reproducibility. We present various methods for examining intra-platform reproducibility. We also examine cross-platform reproducibility using Pearson's correlation. Additionally, we previously developed a correction factor for Pearson's correlation which is applicable when X and Y are measured with error. Herein we demonstrate that correcting for measurement error by estimating the "disattenuated" correlation substantially improves cross-platform correlations. Conclusion When estimating cross-platform correlation, it is essential to thoroughly evaluate intra-platform reproducibility as a first step. In addition, since measurement error is present in microarray gene expression data, methods to correct for attenuation are useful in decreasing the bias in cross-platform correlation estimates.

Induction of tumor-specific immune responses results in the inhibition of tumor development. However, tumors recur because of the tumor immunoediting process that facilitates development of escape mechanisms in tumors. It is not known whether tumor escape is an active process whereby anti-tumor immune responses induce loss or downregulation of the target antigen in the antigen-positive clones. To address this question, we used rat neu-overexpressing mouse mammary carcinoma (MMC) and its relapsed neu antigen-negative variant (ANV). ANV emerged from MMC under pressure from neu-specific T cell responses in vivo. We then cloned residual neu antigen-negative cells from MMC and residual neu antigen-positive cells from ANV. We found marked differences between these neu-negative clones and ANV, demonstrating that the residual neu-negative clones are probably not the origin of ANV. Since initial rejection of MMC was associated with the presence of IFN-gamma-secreting T cells, we treated MMC with IFN-gamma and showed that IFN-gamma could induce downregulation of neu expression in MMC. This appears to be due to methylation of the neu promoter. Together, these data suggest that neu antigen loss is an active process that occurs in primary tumors due to the neu-targeted anti-tumor immune responses.

Protein degradation via the ubiquitin proteasome system (UPS) is regulated by cullin-RING E3-ligases (CRLs). The first-in-class investigational Nedd8-activating enzyme (NAE) inhibitor MLN4924 (pevonedistat) blocks CRL activity by inhibiting their neddylation, thereby disrupting the turnover of a subset of proteins with broad functions associated with cancer cell growth and survival pathways, including NF-κB, DNA replication, and DNA damage checkpoints, among others. Such findings provide a rationale for combining MLN4924 with HDAC inhibitors (HDACIs e.g., the recently FDA-approved belinostat) that interrupt DNA replication, checkpoints and repair. Notably, both single-agent MLN4924 and HDACIs (e.g., pracinostat) have induced CRs in AML patients. Consequently, interactions between MLN4924 and belinostat were investigated in AML. MLN4924/belinostat co-administration synergistically induced apoptosis in AML cells with various genetic backgrounds (e.g., wt- or mutant- p53 or FLT3-ITD), while p53 knock-down or enforced FLT3-ITD expression increased sensitivity to this regimen. MLN4924 blocked belinostat-induced NF-κB activation and belinostat co-administration up-regulated Bim, while Bim shRNA knockdown abrogated lethality. Following MLN4924/belinostat co-exposure, an RT2 Array revealed a distinct expression profile of DNA damage response (DDR) network genes. Significantly, belinostat diminished MLN4924-induced ATR/Chk1/Wee1 activation and Cdt1 up-regulation, abrogating the intra-S checkpoint. Notably, shRNA knockdown of Chk1 or Wee1 significantly sensitized cells to MLN4924 in the absence of belinostat. Moreover, belinostat also blocked MLN4924-mediated up-regulation of multiple proteins involved in HR (e.g., CtIP, BRCA1 S1524 and S1423 phosphorylation, FANCD2-L), while co-administration increased H3K56 and H4K16 acetylation, indicating defective NHEJ repair. As a consequence, co-treatment induced robust DSBs, chromosome pulverization, and apoptosis. This regimen sharply induced apoptosis in primary MDS/AML blasts (n = 47) bearing various poor-prognostic mutations, defined by NGS using the Cancer Hotspot Panel v2, as well as CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells (n = 25), but was minimally toxic to normal CD34+ cells. Finally, MLN4924/belinostat co-treatment profoundly diminished tumor burden and prolonged survival in both s.c. and i.v. AML xenograft models, with minimal toxicity, via the PD events observed in vitro. Collectively, these findings indicate that MLN4924 and HDACIs interact reciprocally to disable the DDR and promote DNA damage and cell death in AML cells, including those with unfavorable genetic features and primitive progenitors, while sparing their normal counterparts. This strategy warrants further consideration in AML. Citation Format: Liang Zhou, Shuang Chen, Yu Zhang, Yun Leng, Lihong Li, Hui Lin, Maciej Kmieciak, Kathryn A. Rizzo, Catherine I. Dumur, Andrea Ferreira-Gonzalez, Yun Dai, Steven Grant. The NAE inhibitor pevonedistat (MLN4924) interacts with the HDAC inhibitor belinostat via disruption of the intra-S checkpoint and both HR and NHEJ DNA repair in AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2015-LB-258

Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

A S L D A b st ra ct s sham or bile duct ligation (BDL) and were then treated with chloroquine (an inhibitor of autophagy; 60 mg/kg/day), rapamycin (an activator of autophagy; 30 mg/kg/day) or recombinant PGRN (5 nmol/kg/day) via intraperitoneal implanted minipump for 3 days. Cholangiocyte proliferation and intrahepatic biliary mass were assessed in vivo by immunohistochemistry using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. In vitro, a parental mouse cholangiocyte cell line and cells overexpressing the pro-autophagic effector Sirt1 were treated with chloroquin and rapamycin and PGRN for up to 48 hr and proliferation assessed using MTS assays. Expression of the autophagy markers, Beclin, ATG5, ATG7 and LAMP1, and Sirt1 were assessed by qPCR, immunoblotting and immunohistochemistry in livers and cholangiocytes after the above-mentioned treatments. Autophagy was also assessed in vitro in mouse cholangiocyte cell lines using a commercially available lysotracker staining kit. Results: After BDL, markers of autophagy were increased, specifically in cholangiocytes. Strategies to inhibit autophagy (chloroquine) increased intrahepatic biliary mass and induced cholangiocyte proliferation in vivo and in vitro, whereas strategies to stimulate autophagy (rapamycin) reduced intrahepatic biliary mass after BDL and reduced cholangiocyte proliferation in vivo and in vitro. Treatment with recombinant PGRN reduced the expression of autophagy markers in cholangiocytes and increased cholangiocyte proliferation. Associated with these effects was a PGRN-induced suppression of the pro-autophagic molecule, Sirt1. Indeed, the inhibitory effect of PGRN on autophagy was alleviated in cholangiocytes overexpressing Sirt1. Conclusion: Our data suggest that autophagy is upregulated during hyperplastic cholangiocyte proliferation, presumably as a compensatory mechanism to prevent overt proliferation. Furthermore, PGRN may induce cholangiocyte proliferation by inhibiting autophagy via the suppression of Sirt1 expression.