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The detection of driver oncogenic variants and the recent identification of tumor-agnostic genomic biomarkers has driven the use of comprehensive genomic profiling (CGP) for disease diagnosis, prognosis, and treatment selection. The Oncomine™ Comprehensive Assay Plus (OCA+) panel uses DNA and RNA to detect single nucleotide variants (SNVs), small insertions/deletions (Indels), and structural variants (SVs) across 501 genes. Moreover, microsatellite instability (MSI), tumor mutational burden (TMB), and homologous recombination deficiency (HRD) status are assessed in a single workflow. Herein, we present the analytical validation and clinical utilization of OCA+. By using commercial reference materials, we found good analytical sensitivity, specificity, and precision for all biomarkers analyzed. The limit of detection (LoD) was validated for SNVs and Indels at 4%, except for Indels located in homopolymeric regions, where the LoD was 10%. An additional set of 81 tumor samples, including cytology smears, were sequenced to assess the clinical utility of the OCA+ across different tumor types. Among the clinical cohort, OCA+ demonstrated 100% accuracy, sensitivity, and specificity for all biomarkers analyzed, except for MSI assessment of endometrial cancer cases, where 83% accuracy and 67% sensitivity were achieved, compared to PCR and IHC. The validation of accuracy and robustness of this assay supports the OCA+’s utility for solid tumor CGP.

Enhanced CD8 T cell cytotoxicity to BPTF KD cells is not due to the OVA or pmel antigens.

Genetic studies in fruit flies have implicated the chromatin remodeling complex nucleosome remodeling factor (NURF) in immunity, but it has yet to be studied in mammals. Here we show that its targeting in mice enhances antitumor immunity in two syngeneic models of cancer. NURF was disabled by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. We found that both CD8+ and CD4+ T cells were necessary for enhanced antitumor activity, with elevated numbers of activated CD8+ T cells observed in BPTF-deficient tumors. Enhanced cytolytic activity was observed for CD8+ T cells cocultured with BPTF-silenced cells. Similar effects were not produced with T-cell receptor transgenic CD8+ T cells, implicating the involvement of novel antigens. Accordingly, enhanced activity was observed for individual CD8+ T-cell clones from mice bearing BPTF-silenced tumors. Mechanistic investigations revealed that NURF directly regulated the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. The PSMB8 inhibitor ONX-0914 reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor antigenicity and its depletion improves antigen processing, CD8 T-cell cytotoxicity, and antitumor immunity, identifying NURF as a candidate therapeutic target to enhance antitumor immunity. Cancer Res; 76(21); 6183–92. ©2016 AACR.

Microarray data analysis of significant genes in 4T1 tumors harvested from NSG mice. List of primer sequences used in this study.

Supplemental figure legends for Mayes et al.

Gene Ontology of microarray data by DAVID analysis.

AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.MethodsCell lines harbouringEML4(13)–ALK(20) andSLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.ResultsFour (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.ConclusionsReference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Calcineurin inhibitors are highly efficacious immunosuppressive agents used in pediatric kidney transplantation. However, calcineurin inhibitor nephrotoxicity (CNIT) has been associated with the development of chronic renal allograft dysfunction and decreased graft survival. This study evaluated 37 formalin-fixed paraffin-embedded biopsies from pediatric kidney transplant recipients using gene expression profiling. Normal allograft samples (n = 12) served as negative controls and were compared to biopsies exhibiting CNIT (n = 11). The remaining samples served as positive controls to validate CNIT marker specificity and were characterized by other common causes of graft failure such as acute rejection (n = 7) and interstitial fibrosis/tubular atrophy (n = 7). MiRNA profiles served as the platform for data integration. Oxidative phosphorylation and mitochondrial dysfunction were the top molecular pathways associated with overexpressed genes in CNIT samples. Decreased ATP synthesis was identified as a significant biological function in CNIT, while key toxicology pathways included NRF2-mediated oxidative stress response and increased permeability transition of mitochondria. An integrative analysis demonstrated a panel of 13 significant miRNAs and their 33 CNIT-specific gene targets involved with mitochondrial activity and function. We also identified a candidate panel of miRNAs/genes, which may serve as future molecular markers for CNIT diagnosis as well as potential therapeutic targets.

AimNext generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow.MethodsThe TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols.ResultsOverall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants.ConclusionThe TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.

Progressive fibrosis of the interstitium is the dominant final pathway in renal destruction in native and transplanted kidneys. Over time, the continuum of molecular events following immunological and non-immunological insults lead to interstitial fibrosis and tubular atrophy (IFTA) and culminate in kidney failure. We hypothesize that these insults trigger changes in DNA methylation (DNAm) patterns which in turn could exacerbate injury and slow down the regeneration processes, leading to fibrosis development and graft dysfunction. Herein, we analyzed biopsy samples from kidney allografts collected after 24-months post transplantation and used an integrative multi-omics approach to understand the underlying molecular mechanisms. The role of DNAm and microRNAs on the graft gene expression was evaluated. Enrichment analyses of differentially methylated CpG sites were performed using GenomeRunner. CpGs were strongly enriched in regions that were variably methylated among tissues implying high tissue specificity in their regulatory impact. Corresponding to this methylation pattern, gene expression data were related to immune response (activated state) and nephrogenesis (inhibited state). Preimplantation biopsies showed similar DNAm patterns to normal allograft biopsies at 2-years post-transplantation. Our findings demonstrate for the first time a relationship among epigenetic modifications and development of interstitial fibrosis, graft function, and inter-individual variation on long-term outcomes.

We describe a mathematical model for the aggregation of starved first-stage C elegans larvae (L1s). We propose that starved L1s produce and respond chemotactically to two labile diffusible chemical signals, a short-range attractant and a longer range repellent. This model takes the mathematical form of three coupled partial differential equations, one that describes the movement of the worms and one for each of the chemical signals. Numerical solution of these equations produced a pattern of aggregates that resembled that of worm aggregates observed in experiments. We also describe the identification of a sensory receptor gene, srh–2, whose expression is induced under conditions that promote L1 aggregation. Worms whose srh–2 gene has been knocked out form irregularly shaped aggregates. Our model suggests this phenotype may be explained by the mutant worms slowing their movement more quickly than the wild type., Author summary Among the most complex of animal behaviors are collective behaviors, in which animals interact with each other so as to produce large-scale organization. Starved first-stage larvae of the nematode Caenorhabditis elegans exhibit such a behavior: they come together to form aggregates of several hundred worms. How and why they do this are unknown. To address these questions, we developed a mathematical model of starved L1 aggregation. This model reproduced the main features of the behavior.

// Kimberly Mayes 1 , Zeinab Elsayed 1 , Aiman Alhazmi 1 , Michael Waters 2 , Suehyb G. Alkhatib 1 , Mark Roberts 1 , Carolyn Song 1 , Kristen Peterson 1 , Vivian Chan 1 , Nikhil Ailaney 1 , Pumoli Malapati 1 , Tana Blevins 3 , Berislav Lisnic 4 , Catherine I. Dumur 3 and Joseph W. Landry 1 1 The Department of Human and Molecular Genetics, Virginia Institute of Molecular Medicine, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA 2 The Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia 23298, USA 3 The Department of Pathology, Virginia Commonwealth University, Richmond, Virginia 23298, USA 4 The Center for Proteomics and Department for Histology and Embryology, University of Rijeka, Faculty of Medicine, 51000 Rijeka, Croatia Correspondence to: Joseph W. Landry, email: joseph.landry@vcuhealth.org Keywords: BPTF, chromatin remodeling, antitumor immunity, NK cell, heparanase Received: January 31, 2017 Accepted: April 26, 2017 Published: May 12, 2017 ABSTRACT Using syngeneic BALB/c mouse breast cancer models, we show that the chromatin remodeling subunit bromodomain PHD finger transcription factor (BPTF) suppresses natural killer (NK) cell antitumor activity in the tumor microenvironment (TME). In culture, BPTF suppresses direct natural cytotoxicity receptor (NCR) mediated NK cell cytolytic activity to mouse and human cancer cell lines, demonstrating conserved functions. Blocking mouse NCR1 in vivo rescues BPTF KD tumor weights, demonstrating its importance for the control of tumor growth. We discovered that BPTF occupies heparanase ( Hpse ) regulatory elements, activating its expression. Increased heparanase activity results in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans (HSPGs). Using gain and loss of function approaches we show that elevated heparanase levels suppress NK cell cytolytic activity to tumor cells in culture. These results suggest that BPTF activates heparanase expression, which in turn reduces cell surface HSPGs and NCR co-ligands, inhibiting NK cell activity. Furthermore, gene expression data from human breast cancer tumors shows that elevated BPTF expression correlates with reduced antitumor immune cell signatures, supporting conserved roles for BPTF in suppressing antitumor immunity. Conditional BPTF depletion in established mouse breast tumors enhances antitumor immunity, suggesting that inhibiting BPTF could provide a novel immunotherapy.

To gain insight into the cellular and molecular interactions mediating the desmoplastic reaction and aggressive malignancy of mass-forming intrahepatic cholangiocarcinoma (ICC), we modeled ICC desmoplasia and progression in vitro . A unique three-dimensional (3D) organotypic culture model was established; within a dilute collagen–type I hydrogel, a novel clonal strain of rat cancer-associated myofibroblasts (TDF SM ) was co-cultured with a pure rat cholangiocarcinoma cell strain (TDE CC ) derived from the same ICC type as TDF SM . This 3D organotypic culture model reproduced key features of desmoplastic reaction that closely mimicked those of the in situ tumor, as well as promoted cholangiocarcinoma cell growth and progression. Our results supported a resident liver mesenchymal cell origin of the TDF SM cells, which were not neoplastically transformed. Notably, 3D co-culturing of TDE CC cells with TDF SM cells provoked the formation of a dense fibrocollagenous stroma in vitro that was associated with significant increases in both proliferative TDF SM myofibroblastic cells and TDE CC cholangiocarcinoma cells accumulating within the gel matrix. This dramatic desmoplastic ICC-like phenotype, which was not observed in the TDE CC or TDF SM controls, was highly dependent on transforming growth factor (TGF)-β, but not promoted by TGF-α. However, TGF-α was determined to be a key factor for promoting cholangiocarcinoma cell anaplasia, hyperproliferation, and higher malignant grading in this 3D culture model of desmoplastic ICC.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n=10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Two classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3-internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor-κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing-defined poor-prognostic cancer hotspot mutations, and CD34(+)/CD38(-)/CD123(+) populations, but not normal CD34(+) progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P < .0001) in AML xenograft models with negligible toxicity, accompanied by pharmacodynamic effects observed in vitro. Collectively, these findings argue that MLN4924 and belinostat interact synergistically by reciprocally disabling the DDR in AML/MDS cells. This strategy warrants further consideration in AML/MDS, particularly in disease with unfavorable genetic aberrations.

Acute myelogenous leukemia (AML) is characterized by frequent aberrations of the PI3K/AKT/mTOR axis steming from diverse mechanisms, including FLT3, Ras, and c-KIT mutations, among multiple others. In a previous report, we demonstrated that dual inhibition of the PI3K/mTOR axis and the BH3-mimetic ABT-737, which inhibits both Bcl-2 and Bcl-xL, but not Mcl-1, exhibits potent anti-leukemia activity both in vitro and in vivo (Cancer Res. 2013;73(4):1340-51). It has also been shown that while TORC1 inhibitors such as rapamycin can elicit rebound activation of AKT in various tumor cells, dual inhibition of TORC1/2 potently inhibits AKT activation at both Thr308 and Ser473 sites. In the present studies, we examined whether concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL would lead to enhanced anti-leukemia activity in AML cells. Notably, dual tetracycline-inducible Bcl-2/Bcl-xL knockdown markedly sensitized acute myeloid leukemia (AML) cells to the dual TORC1/2 inhibitor INK128 (ChemieTek) in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/Bcl-xL antagonist ABT-737 (AbbVie) sharply induced cell death in multiple AML cell lines, including TKI-resistant FLT3-ITD mutants as well as primary AML blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras among others, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38-/CD123+ primitive leukemia progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal micro-environment (i.e., co-culture with HS-5 cells), suggesting that this strategy may circumvent the protective effects of bone marrow stromal cells, which are known to play an important role in leukemia cell survival as well as drug resistance. Notably, INK128 was very effective in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737-mediated apoptosis. Furthermore, ectopic expression of Mcl-1 dramatically attenuated INK128/ABT737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 interactions. In contrast to INK128, rapamycin increased AML cell AKT phosphorylation, and was largely ineffective in down-regulating Mcl-1, decreasing 4EBP1 phosphorylation, or enhancing ABT-737 lethality. Interestingly, addition of a specific AKT inhibitor to the rapamycin/ABT-737 regimen sharply increased apoptosis in association with pronounced AKT inactivation and Mcl-1 down-regulation, analogous to effects observed with INK128/ABT-737. These findings argue that AKT activation may oppose rapamycin/ABT-737 lethality in AML cells. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak shRNA knock-down reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic AML xenograft model (P < 0.0001; log-rank test for combined treatment vs either agent alone). In addition, analysis of subcutaneous AML-derived tumors showed significant declines in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with in vitro results. Collectively, these findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in AML. They also raise the possibility that dual mTORC1/mTORC2 inhibitors may offer advantages over pure mTORC1 inhibitors in this setting. Disclosures No relevant conflicts of interest to declare.

Astrocyte elevated gene-1 (AEG-1) and c-Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG-1 is transcriptionally regulated by c-Myc, and AEG-1 itself induces c-Myc by activating the Wnt/β-catenin-signaling pathway. We now document the cooperation of AEG-1 and c-Myc in promoting hepatocarcinogenesis by analyzing hepatocyte-specific transgenic mice expressing either AEG-1 (albumin [Alb]/AEG-1), c-Myc (Alb/c-Myc), or both (Alb/AEG-1/c-Myc). Wild-type and Alb/AEG-1 mice did not develop spontaneous HCC. Alb/c-Myc mice developed spontaneous HCC without distant metastasis, whereas Alb/AEG-1/c-Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N-nitrosodiethylamine significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG-1/c-Myc, the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG-1/c-Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of prosurvival and epithelial-mesenchymal transition-signaling pathways. RNA-sequencing analysis identified a unique gene signature in livers of Alb/AEG-1/c-Myc mice that was not observed when either AEG-1 or c-Myc was overexpressed. Specifically, Alb/AEG-1/c-Myc mice overexpressed maternally imprinted noncoding RNAs (ncRNAs), such as Rian, Meg-3, and Mirg, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG-1/c-Myc hepatocytes.Our studies reveal a novel cooperative oncogenic effect of AEG-1 and c-Myc that might explain the mechanism of aggressive HCC. Alb/AEG-1/c-Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC.

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

Background Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. Methods Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. Results Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. Conclusion This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPaseold-35), we performed gene expression analysis of HeLa cells in which hPNPaseold-35 was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPaseold-35 was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPaseold-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 hPNPaseold-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.hPNPaseold-35 infected HO-1 melanoma cells and HeLa cells overexpressing hPNPaseold-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 hPNPaseold-35 is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation. J. Cell. Physiol. 229: 1952–1962, 2014. © 2014 Wiley Periodicals, Inc.

Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function. Of 1733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time points after kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early after kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.

Background Ischemia-reperfusion (I/R) injury is a multifactorial phenomenon that occurs during the transplant event and frequently compromises early graft function after liver transplantation (LT). Current comprehension of molecular mechanisms and regulation processes of I/R injury lacks clarity. MicroRNA (miRNA) regulation results critical in several biological processes. Methods This study evaluated gene expression and miRNA expression profiles using microarrays in 34 graft biopsies collected at preimplantation (L1) and at 90 min postreperfusion (L2) from consecutives deceased-donor LT recipients. miRNA profiles were first analyzed. Data integration analysis (gene expression/miRNA expression) aimed to identify potential target genes for each identified miRNA from the L1/L2 differential gene expression profile. Results Pairwise comparison analyses identified 40 miRNAs and 3168 significantly differentially expressed genes at postreperfusion time compared with preimplantation time. Pathway analysis of miRNAs associated these profiles with antiapoptosis, inhibition of cellular proliferation, and proinflammatory processes. Target analysis identified an miRNA-associated molecular profile of 2172 genes involved in cellular growth and proliferation modulation by cell cycle regulation, cell death and survival, and proinflammatory and anti-inflammatory processes. miRNA-independent genes involved proinflammatory molecules. Conclusion We identified a miRNA profile involved in posttranscriptional regulatory mechanisms in I/R injury post-LT. A better understanding of these molecular processes involved in I/R may contribute to develop new strategies to minimize graft injury.

Important progress has been made in improving short term outcomes in solid organ transplantation. However, long-term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response. MicroRNAs (miRNAs) are a class of small single stranded noncoding RNAs that function through translational repression of specific target mRNAs. MiRNA expression has been associated with different diseases and physiological conditions. Moreover, miRNAs have been detected in different biological fluids and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA detection, their disease specificity, and the availability of accurate techniques for detecting and monitoring these molecules has encouraged a pursuit of miRNA biomarker research and the evaluation of specific applications in the transplant field. miRNA expression might develop as excellent biomarkers of allograft injury and function. In this minireview, we summarize the main accomplishments of recently published reports focused on the identification of miRNAs as biomarkers in organ quality, ischemia-reperfusion injury, acute rejection, tolerance and chronic allograft dysfunction emphasizing their mechanistic and clinical potential applications and describing their methodological limitations.

An increase in Rab1b levels induces changes in Golgi size and in gene expression. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element binding protein consensus binding. The results show a Rab1b increase in secretory cells after stimulation and suggest that this increase is required to elicit a secretory response., Rab1b belongs to the Rab-GTPase family that regulates membrane trafficking and signal transduction systems able to control diverse cellular activities, including gene expression. Rab1b is essential for endoplasmic reticulum–Golgi transport. Although it is ubiquitously expressed, its mRNA levels vary among different tissues. This work aims to characterize the role of the high Rab1b levels detected in some secretory tissues. We report that, in HeLa cells, an increase in Rab1b levels induces changes in Golgi size and gene expression. Significantly, analyses applied to selected genes, KDELR3, GM130 (involved in membrane transport), and the proto-oncogene JUN, indicate that the Rab1b increase acts as a molecular switch to control the expression of these genes at the transcriptional level, resulting in changes at the protein level. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element-binding protein consensus binding site in those target promoter regions. Moreover, our results reveal that, in a secretory thyroid cell line (FRTL5), Rab1b expression increases in response to thyroid-stimulating hormone (TSH). Additionally, changes in Rab1b expression in FRTL5 cells modify the specific TSH response. Our results show, for the first time, that changes in Rab1b levels modulate gene transcription and strongly suggest that a Rab1b increase is required to elicit a secretory response.

Genetic studies in fruit flies have implicated the chromatin remodeling complex nucleosome remodeling factor (NURF) in immunity, but it has yet to be studied in mammals. Here we show that its targeting in mice enhances antitumor immunity in two syngeneic models of cancer. NURF was disabled by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. We found that both CD8+ and CD4+ T cells were necessary for enhanced antitumor activity, with elevated numbers of activated CD8+ T cells observed in BPTF-deficient tumors. Enhanced cytolytic activity was observed for CD8+ T cells cocultured with BPTF-silenced cells. Similar effects were not produced with T-cell receptor transgenic CD8+ T cells, implicating the involvement of novel antigens. Accordingly, enhanced activity was observed for individual CD8+ T-cell clones from mice bearing BPTF-silenced tumors. Mechanistic investigations revealed that NURF directly regulated the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. The PSMB8 inhibitor ONX-0914 reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor antigenicity and its depletion improves antigen processing, CD8 T-cell cytotoxicity, and antitumor immunity, identifying NURF as a candidate therapeutic target to enhance antitumor immunity. Cancer Res; 76(21); 6183–92. ©2016 AACR.

Environmental stress triggers multilevel adaptations in animal development that depend in part on epigenetic mechanisms. In response to harsh environmental conditions and pheromone signals, Caenorhabditis elegans larvae become the highly stress-resistant and long-lived dauer. Despite extensive studies of dauer formation pathways that integrate specific environmental cues and appear to depend on transcriptional reprogramming, the role of epigenetic regulation in dauer development has remained unclear. Here we report that BLMP-1, the BLIMP-1 ortholog, regulates dauer formation via epigenetic pathways; in the absence of TGF-β signaling (in daf-7 mutants), lack of blmp-1 caused lethality. Using this phenotype, we screened 283 epigenetic factors, and identified lin-40, a homolog of metastasis-associate protein 1 (MTA1) as an interactor of BLMP-1. The interaction between LIN-40 and BLMP-1 is conserved because mammalian homologs for both MTA1 and BLIMP-1 could also interact. From microarray studies, we identified several downstream target genes of blmp-1: npr-3, nhr-23, ptr-4, and sams-1. Among them S-adenosyl methionine synthase (SAMS-1), is the key enzyme for production of SAM used in histone methylation. Indeed, blmp-1 is necessary for controlling histone methylation level in daf-7 mutants, suggesting BLMP-1 regulates the expression of SAMS-1, which in turn may regulate histone methylation and dauer formation. Our results reveal a new interaction between BLMP-1/BLIMP-1 and LIN-40/MTA1, as well as potential epigenetic downstream pathways, whereby these proteins cooperate to regulate stress-specific developmental adaptations.

Animals change feeding behavior depending on their metabolic status; starved animals are eager to eat and satiated animals stop eating. C. elegans exhibits satiety quiescence under certain conditions that mimics many aspects of post-prandial sleep in mammals. Here we show that this feeding behavior depends on fat metabolism mediated by the SREBP-SCD pathway, an acetyl-CoA carboxylase (ACC) and certain nuclear hormone receptors (NRs). Mutations of the genes in the SREBP-SCD pathway reduce satiety quiescence. An RNA interference (RNAi) screen of the genes that regulate glucose and fatty acid metabolism identified an ACC necessary for satiety quiescence in C. elegans. ACC catalyzes the first step in de novo fatty acid biosynthesis known to be downstream of the SREBP pathway in mammals. We identified 28 NRs by microarray whose expression changes during refeeding after being starved. When individually knocked down by RNAi, 11 NRs among 28 affect both fat storage and satiety behavior. Our results show that the major fat metabolism pathway regulates feeding behavior and NRs could be the mediators to link the feeding behavior to the metabolic changes.

Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and lysine-rich CECAM-1 co-isolated protein (LYRIC), is now established as a positive regulator of tumorigenesis (1). Using immunohistochemistry in a large cohort of patient samples, elevated AEG-1 protein expression has been documented in a variety of cancers (1). AEG-1 expression gradually increases with disease progression and displays a negative correlation with patient survival. The AEG-1 gene is located in human chromosome 8q22, which is amplified in breast and liver cancers (2, 3). AEG-1 is a downstream gene in the Ha-Ras signaling pathway that activates PI3K/Akt and leads to transcriptional upregulation of AEG-1 by c-Myc (4). AEG-1 is a target of miRNA-375, a tumor suppressor in diverse cancers (5). Thus AEG-1 expression might be increased by a variety of mechanisms during carcinogenesis. Gain- and loss-of-function studies in diverse cell lines confirm the importance of AEG-1 in the development and progression of cancer. In multiple cancer cell lines that express low levels of AEG-1 and are poorly aggressive, AEG-1 overexpression results in a significant increase in in vitro proliferation, anchorage-independent growth, migration and invasion and in vivo tumorigenesis, metastasis and angiogenesis in nude mice xenograft models (1). As a corollary, RNAi-mediated inhibition of AEG-1 in aggressive cell lines expressing high levels of AEG-1 significantly inhibits aforementioned in vitro and in vivo oncogenic phenotypes. AEG-1 overexpression results in activation of multiple pro-survival signal transduction pathways, and profoundly contributes to chemoresistance and tumor angiogenesis, major hallmarks of aggressive cancers (1). Thus AEG-1 plays a fundamental role in aggressive progression of the carcinogenic process. The molecular mechanism by which AEG-1 induces these profound changes is gradually being clarified. AEG-1 is a 582 amino acids protein with a transmembrane domain and multiple nuclear localization signals (NLS) (1). In cancer cells, AEG-1 is detected in the cytoplasm as well as on the cell membrane and in the nucleus (2). Depending upon location, AEG-1 interacts with different protein complexes regulating diverse functions. AEG-1 interacts with NF-κB and CBP promoting NF-κB-mediated transcription (6) while it interacts with YY1 along with CBP to repress transcription (7). In the cytoplasm, AEG-1 is a component of the RNA-induced silencing complex (RISC) and assists oncomiR-mediated degradation of tumor suppressor mRNAs (8). AEG-1 facilitates translation of specific mRNAs, such as the mRNA for the multidrug resistance gene (MDR1), which contributes to chemoresistance (9). The membrane-located AEG-1 promotes interaction of cancer cells with lung endothelium thus augmenting metastasis (3). The identification of the diverse interacting partners indicates that AEG-1 may be a scaffold protein mediating formation of multi-protein complexes in different intracellular compartments. AEG-1 plays an important role in hepatocarcinogenesis (2). AEG-1 mRNA and protein overexpression as well as amplification of AEG-1 gene was detected in a large percentage of Hepatocellular carcinoma (HCC) patients (2). To better comprehend the role of AEG-1 in hepatocarcinogenesis and to decipher the underlying molecular mechanism(s) in an in vivo context, we have generated a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG-1). We document that compared to wild-type (WT) mice, the hepatocarcinogenic process is significantly amplified in Alb/AEG-1 mice. We unravel novel aspects of AEG-1, including induction of steatosis, protection from senescence and activation of coagulation pathways, which contribute to its tumor promoting functions. This is the first study analyzing AEG-1 function in vivo and Alb/AEG-1 mouse provides a useful model to further understand the hepatocarcinogenic process and evaluate emerging novel therapies for this invariably fatal disease.

Data are accumulating to support a role for adipose-derived mesenchymal stem cells (MSCs) in breast cancer progression; however, to date most studies have relied on adipose MSCs from non-breast sources. There is a particular need to investigate the role of adipose MSCs in the pathogenesis of basal-like breast cancer, which develops at a disproportionate rate in pre-menopausal African-American women with a gain in adiposity. The aim of this study was to better understand how breast adipose MSCs (bMSCs) contribute to the progression of basal-like breast cancers by relying on isogenic HMT-3255 S3 (pre-invasive) and T4-2 (invasive) human cells that upon transplantation into nude mice resemble this tumor subtype. In vitro results suggested that bMSCs may contribute to breast cancer progression in multiple ways. bMSCs readily penetrate extracellular matrix components in part through their expression of matrix metalloproteinases 1 and 3, promote the invasion of T4-2 cells and efficiently chemoattract endothelial cells via a bFGF-independent, VEGF-A-dependent manner. As mixed xenografts, bMSCs stimulated the growth, invasion and desmoplasia of T4-2 tumors, yet these resident stem cells showed no observable effect on the progression of pre-invasive S3 cells. While bMSCs form vessel-like structures within Matrigel both in vitro and in vivo and chemoattract endothelial cells, there appeared to be no difference between T4-2/bMSC mixed xenografts and T4-2 xenografts with regard to intra- or peri-tumoral vascularity. Collectively, our data suggest that bMSCs may contribute to the progression of basal-like breast cancers by stimulating growth and invasion but not vasculogenesis or angiogenesis.

Aim: Recent studies have suggested that increased α-smooth muscle-actin positive myofibroblastic cells (α-SMA positive CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients. To facilitate investigating cellular and molecular interactions between α-SMA positive CAF and cholangiocarcinoma cells related to ICC progression, we developed a novel 3-D organotypic culture model of cholangiocarcinoma that more accurately mimics the stromal microenvironment, gene expression profile and select pathophysiological characteristics of desmoplastic ICC in vivo. Methods: This unique model was established by co-culturing within a type I collagen gel matrix, a strain of cholangiocarcinoma cells (derived from an ICC formed in syngeneic rat liver following bile duct inoculation of spontaneously-transformed rat cholangiocytes) with varying numbers of clonal α-SMA positive CAF established from the same tumor type. Results: Cholangiocarcinoma cells and α-SMA positive CAF in monoculture each exhibited cell-specific biomarker gene expression profiles characteristic of stromal myofibroblastic cell versus malignant cholangiocyte cell types. In comparison, the gene expression profile and histopathological characteristics exhibited by the organotypic co-culture closely resembled those of whole tissue samples of the parent orthotopic ICC. We further showed α-SMA positive CAF to significantly enhance cholangiocarcinoma cell “ductal-like” growth and cancer cell migration/invasiveness in vitro, as well as to promote upregulated expression of select genes known to be associated with ICC invasion. Conclusion: This novel organotypic model provides an important new resource for studying the effects of microenvironment on cholangiocarcinoma progression in vitro and may have potential as a preclinical model for identifying molecularly targeted therapies.

Object 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. Methods 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. Results 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. Conclusions 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.

Summary Women classified as having triple-negative tumors have a poor prognosis. The importance of CD44 + /CD24 −/low (stem/progenitor cell-phenotype) in breast cancer patients has also been appreciated. However, correlation between triple negativity and CD44 + /CD24 −/low with tumor recurrence remains elusive. In the present study, we evaluated tumor specimens of 50 breast cancer patients with known hormone receptor status for whom we had follow-up information and outcome data available, and performed immunohistochemistry analysis to determine CD44 and CD24 expression. Gene expression arrays were also independently performed on 52 breast cancer specimens with banked frozen tissue. Lastly, we used FVBN202 transgenic mouse model of breast carcinoma and determined the hormone receptor status, the proportion of CD44 + /CD24 −/low breast cancer stem-like cells, and the behavior of the tumor. We determined that patients with triple-negative tumors had significantly higher incidence of recurrence or distant metastasis associated with increased frequency of breast cancer stem cell phenotypes compared with those with non–triple-negative tumors. Preclinical studies in FVBN202 transgenic mice confirmed these findings by showing that relapsed tumors were triple negative and had significantly higher frequency of breast cancer stem cells compared with their related primary tumors. Unlike non–triple-negative primary tumors, relapsed triple-negative tumors were tumorigenic at low doses when inoculated into FVBN202 transgenic mice. These findings suggest that CD44 + /CD24 −/low breast cancer stem-like cells play an important role in the clinical behavior of triple-negative breast cancer and that development of therapeutic targets directed to breast cancer stem-like cells may lead to reduction in the aggressiveness of triple-negative breast cancers.

BACKGROUND Severe liver steatosis is a known risk factor for increased ischemia-reperfusion injury (IRI) and poor outcomes after liver transplantation (LT). This study aimed to identify steatosis-related molecular mechanisms associated with IRI exacerbation after LT. METHODS Paired graft biopsies (n = 60) were collected before implantation (L1) and 90 minutes after reperfusion (L2). The LT recipients (n = 30) were classified by graft macrosteatosis: without steatosis (WS) of 5% or less (n = 13) and with steatosis (S) of 25% or greater (n = 17). Plasma samples were collected at L1, L2, and 1 day after LT (postoperative [POD]1) for cytokines evaluation. Tissue RNA was isolated for gene expression microarrays. Probeset summaries were obtained using robust multiarray average algorithm. Pairwise comparisons were fit using 2-sample t test. P values 0.01 or less were significant (false discovery rate

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 aim of this brief review is to provide an up-to-date view of the role played by α-smooth muscle actin-positive cancer-associated fibroblastic cells in promoting intrahepatic cholangiocarcinoma progression.An increase in α-smooth muscle actin-positive cancer-associated fibroblastic cells in the stroma of intrahepatic cholangiocarcinoma has recently been demonstrated to accelerate cholangiocarcinoma progression. However, our understanding of the evolving cellular and molecular interactions between these stromal cells and cholangiocarcinoma cells in relation to promoting intrahepatic cholangiocarcinoma progression is only just beginning to be elucidated. Imbalances in multifactorial growth factor/cytokine signaling, activation of Hedgehog-GLI signaling and of proteases involved in extracellular matrix remodeling, and matricellular protein-protein and protein-cholangiocarcinoma cell interactions, as well as hypoxia, all appear to factor into the complex and dynamic interactive mechanisms through which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote intrahepatic cholangiocarcinoma progression. Novel three-dimensional organotypic co-culture models are being developed to facilitate relevant studies of cancer-associated fibroblastic cell/cholangiocarcinoma cell interactions that may more accurately mimic physiologically pertinent features of the tumor.Increasing our understanding of critical interactive pathways by which cancer-associated fibroblastic cells crosstalk with cholangiocarcinoma cells to promote tumor progression can lead to the development of novel multitargeting strategies for intrahepatic cholangiocarcinoma therapy.

BACKGROUND: The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. METHODS: FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). RESULTS: Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. CONCLUSIONS: FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies. Cancer (Cancer Cytopathol) 2011;000:000–000. V C 2011 American Cancer Society.

Using syngeneic BALB/c mouse breast cancer models, we show that the chromatin remodeling subunit bromodomain PHD finger transcription factor (BPTF) suppresses natural killer (NK) cell antitumor activity in the tumor microenvironment (TME). In culture, BPTF suppresses direct natural cytotoxicity receptor (NCR) mediated NK cell cytolytic activity to mouse and human cancer cell lines, demonstrating conserved functions. Blocking mouse NCR1 in vivo rescues BPTF KD tumor weights, demonstrating its importance for the control of tumor growth. We discovered that BPTF occupies heparanase (Hpse) regulatory elements, activating its expression. Increased heparanase activity results in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans (HSPGs). Using gain and loss of function approaches we show that elevated heparanase levels suppress NK cell cytolytic activity to tumor cells in culture. These results suggest that BPTF activates heparanase expression, which in turn reduces cell surface HSPGs and NCR co-ligands, inhibiting NK cell activity. Furthermore, gene expression data from human breast cancer tumors shows that elevated BPTF expression correlates with reduced antitumor immune cell signatures, supporting conserved roles for BPTF in suppressing antitumor immunity. Conditional BPTF depletion in established mouse breast tumors enhances antitumor immunity, suggesting that inhibiting BPTF could provide a novel immunotherapy. Citation Format: Kimberly Mayes, Zeinab Elsayed, Aiman Alhazmi, Michael Waters, Suehyb Alkhatib, Mark Roberts, Carolyn Song, Kristen Peterson, Vivian Chan, Nikhil Ailaney, Pumoli Malapati, Tana Blevins, Berislav Lisnic, Catherine Dumur, Joseph Landry. Tumor cell intrinsic BPTF inhibits NK cell activity and the abundance of natural cytotoxicity receptor co-ligands [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A28.

Clinical workup of metastatic malignancies of unknown origin is an arduous and expensive process, which is reported to be unsuccessful in up to 30% of cases. Global gene expression-based molecular testing may offer accurate classification of metastatic tumors in which a primary site has not been identified. Recently, the US FDA cleared the Pathwork((R)) tissue-of-origin test, which is a gene expression microarray-based test that quantifies the molecular similarity of tumor specimens to 15 known tissue types. A blinded, multicenter validation on poorly differentiated and undifferentiated tumors showed 87.8% sensitivity and 99.4% specificity in frozen tissue samples. The availability of ancillary gene expression-based molecular tests for tissue of origin determination represents a milestone in cancer patient management as part of the personalized medicine revolution.

In this review, we will examine various molecular biomarkers for their potential to serve as independent prognostic factors for predicting survival outcome in postoperative patients with progressive intrahepatic cholangiocarcinoma. Specific rodent models of intrahepatic cholangiocarcinoma that mimic relevant cellular, molecular, and clinical features of the human disease are also described, not only in terms of their usefulness in identifying molecular pathways and mechanisms linked to cholangiocarcinoma development and progression, but also for their potential value as preclinical platforms for suggesting and testing novel molecular strategies for cholangiocarcinoma therapy. Last, recent studies aimed at addressing the role of desmoplastic stroma in promoting intrahepatic cholangiocarcinoma progression are highlighted in an effort to underline the potential value of targeting tumor stromal components together with that of cholangiocarcinoma cells as a novel therapeutic option for this devastating cancer.

Background Delayed graft function (DGF) after kidney transplantation (KTx) ranges between 2% and 50%. The mechanisms leading to DGF deserve special interest because DGF exerts negative influences on long-term outcomes. We studied gene expression profiles in deceased donor kidney (DDK) biopsies with and without DGF. Methods Gene expression profiling was performed on donor kidney tissues from 33 DDK with the use of microarrays. DDK were classified as grafts with immediate function (non-DGF; n=21) and grafts with DGF (n=12). DGF was defined as a dialysis requirement in the first week after transplantation. Demographic donor and recipient information was collected. The robust-multiarray average method was used to estimate probe set expression summaries. Logistic regression was used to identify genes significantly associated with DGF development. Results Patients were followed for 3 months after KTx. Thirty-eight probe sets (n=36 genes) were univariably differentially expressed in DDK with DGF when compared with DDK with non-DGF (alpha=0.001). Sixty-nine probe sets (n=65 genes) were differentially expressed in DDK with DGF when compared with DDK with non-DGF after adjusting for cold ischemia time (alpha=0.001). Gene ontology terms classified the overexpressed genes in DDK with DGF as principally related to cell cycle/growth (e.g., IGFBP5, CSNK2A2), signal transduction (e.g., RASGRP3), immune response (e.g., CD83, BCL3, MX1), and metabolism (e.g., ENPP4, GBA3). TNFRSF1B was overexpressed in DDK with DGF. Conclusions Cold ischemia time was a predictor of DGF independently of the preservation method. We identified a set of 36 genes candidates of DGF in DDK, with genes involved in the inflammatory response being the more important.

Previous cross-platform reproducibility studies have compared consistency of intensities as well as consistency of fold changes across different platforms using Pearson's correlation coefficient. In this study, we propose the use of measurement error models for estimating gene-specific correlations. Additionally, gene-specific reliability estimates are shown to be useful in prioritizing clones for sequence verification rather than selecting clones using a simple random sample. The proposed 'disattenuated' correlation may prove useful in a wide variety of studies when both X and Y are measured with error, such as in confirmation studies of microarray gene expression values, wherein more reliable laboratory assays such as real-time polymerase chain reaction are used.